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Acknowledgement

All praises and compliments to the supreme ruler of the universe and almighty

Allah for the blessing bestowed upon the successful accomplishment of my initial

research study.

I have the honor to express my heartfelt thanks and earnest gratitude to my

honorable teacher and supervisor Dr. Mamun Rashid Chowdhury, Professor and

Chairman, Department of Biochemistry and Molecular Biology, University of Dhaka, for

his enthusiastic guidance, affectionate inspiration, passionate supervision and endearing

company. I owe his a debt of gratitude for his invaluable advice, indispensable

cooperation and constructive criticism throughout my thesis work and during

preparation of this dissertation.

I feel proud to express my heartfelt gratitude to my reverend research

supervisor, Dr. S. M. Faruque, Scientist and Head, Molecular Genetics Laboratory,

Laboratory Science Division, ICDDR,B, for allowing me to use the facilities and for his

valuable suggestion to carry out the work during my thesis under his supervision. I feel

greatly honoured to have the opportunity of having a scientist like him as my supervisor.

I would like to convey my gratitude to Kazi shafi Ahmed and especially to Dr.

Kamruzzaman for their encouragement and valuable suggestions.

My cordial thanks to Lincoln bhai, Rajiv bhai, Nishat bhai, Sumon bhai, Javed

bhai, Shohag bhai for their continuous advice, encouragement and proper guidance. It

might be almost impossible for me to complete this thesis work without their supreme

help.

I am thankful to Farid bhai ,Sajib bhai and Wahed bhai for their assistance. I

would like to express my special appreciation to Afjal bhai for his help, suggestion and

delightful company.

I extend my heartiest thanks and special gratefulness to my beloved friends Adil,

Nayeem, Zia bhai, Mohasin, Roy, Rakib, Sajal for their help and delightful company.

Finally, I want to express my utmost gratitude to my parents and my younger

brother for their unremitting support.

The Author

Department of Biochemistry and Molecular Biology January, 2010

University of Dhaka

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ABSTRACT

Aeromonads are ubiquitous bacteria that occur as natural inhabitants of aquatic

environment. Motile Aeromonas spp. have been reported to occur in drinking water,

surface water and also in both raw and processed foods. They cause serious infections

in both poikilothermic and endothermic animals, including humans. Though

Aeromonads have been recognized for many years, but only during the past three

decades their role in a variety of human illness has been documented. The role of

Aeromonas species in bacterial infections is not yet clearly understood owing to a

paucity of long-term studies. Prevalence of different species of Aeromonas is likely to

vary with geographical locations. To determine the prevalence of Aeromonas species

in Dhaka, the capital of Bangladesh, 256 surface water samples from in and outskirts

of the city were analyzed. Aeromonas caviae and Aeromonas media were found to

being the dominant species in environment. No significant correlation between

prevalence of Aeromonas and seasonality was observed in this study. A total of 30

strains of different species of Aeromonas were isolated. Their antibiogram and

ribotyping study were done as well. At least 3 different patterns of antibiotic

resistance were found and consistency of antibiotic pattern between clinical and

environmental isolates was also observed. Studies of bactriophages have been of

historical interest and with the rise of drug resistant bacteria; the therapeutic potential

of phages has received renewed attention. Bactriophages are known to occur in

natural reservoirs of bacteria which prompted to search for new Aeromonas specific

phages and examine their reservoir in the environmental resources. Study was also

carried out on the prevalence of phages of those water samples and no seasonal effect

on prevalence of phages was observed. But inverse correlation between prevalence of

Aeromonas and their phages was observed in this study. Nine novel phages with

distinct genetic characteristics and host specificity were isolated. The phages were

characterized by their lytic pattern and restriction profile analysis. All the phages were

found to be genus specific. Southern blot hybridization showed that isolated phages

were genetically distant. No lysogenic host of the phages was found in this study.

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Table of Contents

Chapter 1

Introduction 1-37

1.1 General information about Aeromonas spp. .......................................................... 2

1.2 Prevalence of Aeromonas spp. ................................................................................ 4

1.2.1 Occurrence in Human Population .................................................................... 5

1.2.2 Occurrence in Water ........................................................................................ 5

1.2.2.1 Surface Waters .......................................................................................... 5

1.2.2.2 Ground Waters .......................................................................................... 6

1.2.2.3 Drinking Water ........................................................................................... 6

1.2.2.4 Bottled Water ............................................................................................ 7

1.2.2.5 Wastewaters .............................................................................................. 7

1.2.3 Occurrence in Food .......................................................................................... 8

1.3 Health Effect of Aeromonads in Humans ............................................................. 10

1.3.1 Clinical Symptoms of Aeromonas Infection ................................................... 11

1.3.2 Virulence Mechanism of Aeromonads .......................................................... 12

1.3.3 Gastrointestinal Infection by Aeromonas spp. ............................................... 13

1.4 Isolation of Aeromonads ....................................................................................... 15

1.4.1 Isolation From Environmental Samples ......................................................... 17

1.4.2 Biochemical characteristics of Aeromonas spp. ............................................. 17

1.5 Antimicrobial susceptibility of aeromonads ......................................................... 18

1.6 Bacteriophages ...................................................................................................... 19

1.6.1 Diversity of Bacteriophages ........................................................................... 20

1.6.2 Lysogen and Lysogenic Phages ....................................................................... 22

1.6.3 Viral Life-cycle ................................................................................................ 22

1.6.4 The Lytic Cycle of Bacteriophages .................................................................. 23

1.6.5 The Lysogenic Cycle of Bacteriophages.......................................................... 26

1.6.6 Pseudo-lysogeny or the Unstable Carrier-State ............................................. 28

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1.6.7 Plaque Morphology ........................................................................................ 28

1.6.8 Horizontal Gene Transfer and Evolution of Bacteria ..................................... 29

1.6.9 Other Modes Of Horizontal Gene Transfer .................................................... 30

1.6.10 Evolution of Bacteriophages ........................................................................ 30

1.6.11 Role of Phages in Bacterial Evolution through Phage-mediated Gene

Transfer .................................................................................................................... 31

1.6.12 Bacteriophages of Aeromonas: .................................................................... 33

1.6.13 Restriction and Modification ........................................................................ 34

1.7 Objectives of the Study ......................................................................................... 37

Chapter 2

Methods & Materials 38-57

2.1 Laboratory Apparatus ........................................................................................... 38

2.2 Laboratory Chemicals and Reagents ..................................................................... 38

2.2.1 Stock Solutions ................................................................................................ 38

2.2.2 Preparation of Antibiotic Solution ................................................................. 40

2.3 Microbiological Media .......................................................................................... 41

2.4 Bacterial Strains Used In This Study ...................................................................... 43

2.5 Enzymes used in this Study ................................................................................... 43

2.6 Collection of Environmental Surface Water of Dhaka .......................................... 43

2.7 Isolation of Aeromonas spp from Environmental Surface Water ......................... 45

2.8 Biochemical Identification methods : ................................................................... 46

2.9 Determination of Antimicrobial Resistance Pattern by Disc Diffusion Method .... 49

2.10 Isolation of total DNA from bacteria : ................................................................. 50

2.11 Determination of rRNA gene restriction pattern (Ribotyping): ........................... 51

2.11.1 Method of southern transfer of digested DNA : ........................................... 51

2.11.2 Preparation of probe for Ribotyping : ........................................................... 51

2.11.3 Method of hybridization,Washing and Autoradiography: ............................ 52

2.12 Detection and Isolation of Bacteriophages......................................................... 52

2.12.1 Preparation of Plating Bacteria ..................................................................... 52

2.12.2 Aeromonas Phage Plaque Generation .......................................................... 53

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2.12.3 Picking Up of Plaques .................................................................................... 53

2.12.4 Preparing Stock Phages from a Single Plaque ............................................... 53

2.12.5 Preservation of Bacteriophages ................................................................... 54

2.13 Calculation of Titre of Bacteriophages ................................................................ 54

2.14 Preparation of Phage DNA .................................................................................. 55

2.14.1 Large Scale Preparation of Phage................................................................. 55

2.14.2 Isolation of Phage DNA ................................................................................. 55

2.15 Analysis of Phage DNA ........................................................................................ 56

2.15.1 Restriction Profile Analysis ........................................................................... 56

2.15.2 Southern Hybridization Using Total Phage DNA as Probe ............................ 56

2.16 Preparation of Colony Blots: ............................................................................... 57

Chapter 3

Results 58-83

3.1 Prevalence and Isolation of Aeromonas Species From Environmental Surface

Water ........................................................................................................................... 58

3.1.1 Modification of Media Composition .............................................................. 58

3.1.2 Prevalence of Aeromonas .............................................................................. 58

3.1.3 Isolation of Aeromonas .................................................................................. 61

3.2 Characterization of Isolated Aeromonas Spp........................................................ 61

3.2.1 Biochemical Characteristics of Isolated Aeromonas spp. .............................. 61

3.2.2 Ribotyping of Isolated Aeromonas spp .......................................................... 62

3.2.3 Antibiogram of Isolated Aeromonas Species ................................................. 66

3.3 Seasonal Variation study of the phages of Aeromonas spp in Environmental

Surface Water .............................................................................................................. 68

3.3.1 Isolation of Aeromonas phages from Environmental Surface Water Samples

.................................................................................................................................. 68

3.3.2 Prevalence of Aeromonas Phages in Environmental Surface Water .............. 69

3.4 An Inverse Correlation Exist Between Prevalence of Aeromonas and Their Phages

...................................................................................................................................... 72

3.5 All new phages Were Specific for Aeromonas Species: ........................................ 73

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3.6 Plaque Morphology of the isolated phages on Lawns of Respective Indicator

Strains .......................................................................................................................... 75

3.7 Restriction Profile Analysis of Isolated Phages ..................................................... 77

3.8 Genomic Size Determination of the Isolated Phages ........................................... 80

3.9 Genotyping of Aeromonas Phages By Cross Hybridization Study ........................ 82

3.10 No Lysogenic Host Found for Aec-2 and Aec-4 Phage ........................................ 83

Chapter 4

Discussions 84-88

Chapter 5

Conclusion 89

Chapter 6

Reference 90-103

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LIST OF FIGURES

Figures Title Page

Fig 1.1 Aeromonas hydrophila electron micrograph 1

Fig 1.2 Aeromonas hydrophila adhering to human epithelial cells. 1

Fig 1.3 Electron micrograph of Aeromonas salmonicida. 2

Fig 1.4 Phylogenetic relationships of described Aeromonas

genomospecies as determined by a continuous 1502-

nucleotide 16S rDNA sequence comparison using the

neighbor-joining method

4

Fig 1.5 Source of Aeromonas contaminations in food. 9

Fig 1.6 Aeromonas colonies in ADA plate 16

Fig 1.7 Schematic diagram of bacteriophage 20

Fig 1.8 Step wise life cycle of bacteriophage. 24

Fig 1.9 Release of phage particles after lysis of bacteria. 26

Fig 1.10 Life cycle of temperate bacteriophage 27

Fig 1.11 Electron micrograph of Aeromonas phage 31. 34

Fig 1.12 Activity of the restriction enzyme EcoRI and restriction

modification enzyme EcoRI methylase. Arrows indicate the

site of EcoRI cleavage and arrowheads indicate the site of

methylation by EcoRI methylase

36

Fig 2.1 Sites for isolation of Aeromonas and their bacteriophages

around surface water of Dhaka

45

Fig 3.1 Distinct colonies of Aeromonas from environmental sample

in ADA plate.

60

Fig 3.2 Fluctuation of weakly mean counts of Aeromonas species in

the environmental surface water of Dhaka city during the

study period.

60

Fig 3.3 A typical biochemical reaction set for identification of

Aeromonas.

62

Fig: 3.4 Ribotyping of environmental and clinical Aeromonas

species by hybridization with PKK 3535 probe

63

Fig 3.5 Ribotyping of environmental and clinical Aeromonas 64

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species by hybridization with PKK 3535 probe (continued).

Fig 3.6 Dendrogram of environmental and clinical isolates of

Aeromonas species summarizing ribotyping band profile

generated by UPGMA

65

Fig 3.7 Disc diffusion method of antibiogram 66

Fig 3.8 Fluctuation of weekly mean Aeromonas phages

concentration during the study period considering total

phage count of all eight sites under study.

71

Fig 3.9 Comparison of frequency of Aem and Acm phages during

the study period.

71

Fig 3.10 Comparison of prevalence of Aeromonas strains and their

phages in the environmental surface water during the study

period

72

Fig 3.11 Plaque morphology of Aec phages: Aec-1, Aec-2, Aec-3,

Aec-4, Aec-5, and Aec-6. Respective plaques are indicated

with arrow.

76

Fig 3.12 Plaque morphology of Aem phages: Aem-1, Aem-2, and

Aem-3. Respective plaques are indicated with arrow.

77

Fig 3.13 Restriction pattern analysis of the DNA of isolated

Aeromonas phages.

78

Fig 3.14 Aprroximate genomic size of the isolated phages 80

Fig 3.15 Approximate size of the Aec-5 and Aec-6 phages from

restriction fragment by Hind III

81

Fig 3.16 Southern hybridization of Aeromonas phage DNAs using

Aec-2 total DNA as probe

82

Fig 3.17 Southern hybridization of Aeromonas phage DNAs using

Aec-4 total DNA as probe

83

Fig 3.18 Colony blot hybridization of 200 different strains. 84

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LIST OF TABLES

Tables Title Page

Table 1.1 The current genomospecies and phenospecies within the

genus Aeromonas

3

Table 1.2 Aeromonas Population Shift in Activated Sludge 8

Table 1.3 Occurrence of Aeromonas spp. in Foods 9

Table 1.4 Virulence Factors of Aeromonas species 13

Table 1.5 Selective and differential media for Aeromonas spp. 16

Table 1.6 Classification of Bacteriophages by International

Committee on Taxonomy of Viruses (ICTV) based on

morphology and nucleic acid

21

Table 1.7 Bacteriophages of Aeromonas. 33

Table 2.1 Enzymes used in this study 44

Table 2.2 Biochemical characteristics of Aeromonas species 48

Table 2.3 Name of antibiotic and their zone of inhibition. 49

Table 3.1 Prevalence of Aeromonas spp. In different environmental

surface water sample from April, 2009 to November, 2009.

59

Table 3.2 Isolates of Aeromonas species during study period 61

Table 3.3 Antibiotic resistance patterns of both clinical and

environmental isolates.

67

Table 3.4 Name and primary host of the isolated novel phages during

the study period

68

Table 3.5 Prevalence of Aeromonas phages isolated from

environmental surface water sample from May, 2009 to

December, 2009

70

Table 3.6 Host specificity of different Aeromonas phages isolated

from water sample.

74

Table 3.7 Intra-genus host range of Aeromonas phages isolated from

water sample.

75

Table 3.8 Susceptibility of phage DNAs to different restriction

endonucleases.

79

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Chapter 1 Introduction

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I n t r o d u c t i o n

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Bacteria resembling motile Aeromonas species were first isolated from water

and diseased animals over 100 years ago. Aeromonas spp. are Gram-negative, rod

shaped, mainly motile, facultative anaerobic bacteria. Recently they have been

transferred from Vibrionaceae to their own family Aeromonadaceae (1). These

bacteria have a broad host spectrum, with both cold-and warm-blooded animals,

including human. Aeromonas spp. has been involved in wound infections, sepsis,

outbreaks of water and food-borne gastroenteritis (2). Aeromonads are ubiquitous

organisms found in aquatic environments including groundwater and chlorinated

drinking water; food items, including meat, fish, and vegetables; and the intestines of

apparently healthy humans and humans with diarrhea (3). As they are ubiquitous in

the environment, so there are multiple opportunities for transmission to humans

through food, water, animal contact, and direct human contact. There is circulation

and transmisson of strains between humans and the environment. Aeromonas species

in the environment should be considered a threat to public health, since infections

caused by these pathogens are generally the result of ingestion of contaminated water

or food (4) (5). For that reason, Aeromonas species in the environment should be

considered a threat to public health (6) and water quality regulatory agencies of some

countries including USEPA have adopted aeromonad counts as an additional indicator

of water quality.

Having been implicated in clinical cases of diarrhoea, where Aeromonads

were isolated as the sole pathogen, these bacteria are considered as emerging

pathogens (7) (8). The high prevalence of Aeromonas in the environment leds support

to the hypothesis that infections are mainly acquired through the consumption of food

and water (9), and also a number of reports have shown the implication of these

Figure 1.1: Aeromonas hydrophila electron

micrograph.

Figure 1.2: Aeromonas hydrophila

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opportunistic bacteria in some well documented cases of food-borne or water-borne

outbreaks in France, Japan, Norway, Sweden and Libya (10) (11) (12) (13) (14).

Besides diarrhoea, Aeromonas spp. have been known to cause other infections such as

septicemia, wound infections, endocarditis, meningitis, hemolytic-uremic syndrome,

and pneumonia (7) (8). While originally thought to be an opportunistic pathogen in

immunocompromised humans (15) (16) (17), an increasing number of cases of

intestinal and extraintestinal disease documented worldwide suggest that it is an

emerging human pathogen irrespective of the host’s immune status (18).

1.1 General information about Aeromonas spp.

Until the late 1970s, aeromonads were divided into two groups, based upon

physiological properties and host range. Motile aeromonads that grew at 35-37º C and

were recognized to cause human infections were called A. hydrophila. Non-motile

aeromonads that grew at 22-28º C and infected fish were called A. salmonicida. The

genus Aeromonas has undergone a number of taxonomic and nomenclature revisions

over the past 20 years. Although originally placed in the family Vibrionaceae (19),

which also included the genera Vibrio, Photobacterium, and Plesiomonas, subsequent

phylogenetic investigations indicated that the genus Aeromonas is not closely related

to vibrios but rather forms a monophyletic unit in the γ-3 subgroup of the class

Proteobacteria (20) (21). These conclusions necessitated the removal of Aeromonas

from the family Vibrionaceae and transfer to a new family, the Aeromonadaceae (1)

(22).

Figure 1.3: Electron micrograph of Aeromonas salmonicida.

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The current taxonomy of the genus Aeromonas is based upon DNA-DNA

hybridization and 16S ribosomal DNA relatedness studies (Fig. 1.4). The genera of

the family Aeromonadaceae now include Aeromonas, Oceanimonas, Oceanisphaera,

and Tolumonas (incertae sedis) (23). The current genomospecies and phenospecies

within the genus Aeromonas are shown in table1.1.

DNA Hybridization Group (HG) Genomospecies Phenospecies

1 A. hydrophila A. hydrophila

1 A. hydrophila subsp. dhakensis A. hydrophila subsp. dhakensis

1 A. hydrophila subsp. ranae A. hydrophila subsp. ranae

2 A. bestiarum A. hydrophila-like

3 A. salmonicida A. salmonicida subsp. salmonicida

3 A. salmonicida A. salmonicida subsp. achromogenes

3 A. salmonicida A. salmonicida subsp. masoucida

3 A. salmonicida A. salmonicida subsp. smithia

3 unnamed A. hydrophila-like

4 A. caviae A. caviae

5A A. media A. caviae-like

5B A. media A. media

6 A. eucrenophila A. eucrenophila

7 A. sobria A. sobria

8X A. veronii A. sobria

8Y A. veronii A. veronii biovar sobria

9 A. jandaei A. jandaei

10 A. veronii biovar veronii A. veronii biovar veronii

11 unnamed Aeromonas spp. (ornithine positive)

12 A. schubertii A. schubertii

13 Aeromonas Group 501 A. schubertii-like

14 A. trota A. trota

15 A. allosaccharophila A. allosaccharophila

16 A. encheleia A. encheleia

17 A. popoffii A. popoffii

Unassigned A. culicicola A. culicicola

The comparative analysis of the 16S rRNA gene sequences for Aeromonas

species generally correlates with species designations derived from DNA–DNA

hybridization studies (24). While there is some lack of congruence between DNA–

DNA hybridization studies and 16S rDNA sequencing results, the overall

differentiation between groups is very similar. Figure 1.4 is a phylogenetic tree

showing the relationships between described Aeromonas species.

Table 1.1 : The current genomospecies and phenospecies within the genus Aeromonas

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1.2 Prevalence of Aeromonas spp.

Aeromonas spp. are found worldwide in surface water (25) (26), ground water

(26), non-chlorinated drinking water (27), chlorinated drinking water (27) (28), and

bottled mineral water (29). Aeromonads are found in a wide variety of foods (30).

They are found in the intestinal tract of humans and animals (7), raw sewage (31),

sewage effluents (31), activated sludge (32), and sewage-contaminated waters (33).

Aeromonads have been shown to grow in foods held at refrigerator temperatures (34)

(35) (36). However, while aeromonads have been isolated from fish, shellfish, meats,

dairy products, and fresh vegetables, few foodborne outbreaks have been reported.

While Aeromonas spp. are not considered fecal bacteria, they are present in the feces

of healthy animals and humans, presumably as the result of ingestion of food and

water containing these organisms (31) (37).

Figure 1.4: Phylogenetic relationships of described Aeromonas genomospecies as determined by a

continuous 1502-nucleotide 16S rDNA sequence comparison using the neighbor-joining method

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1.2.1 Occurrence in Human Population

Humans carry Aeromonas spp. in their gastrointestinal tract both in the

presence and absence of disease. The rates of fecal carriage in asymptomatic persons

in developed countries range from 0% to 4.0% (38) (39) (40) (41), while the isolation

rate from persons with diarrheal illness ranges from 0.8 to 7.4% (39) (42). In

Southeast Asia, asymptomatic carriage rates as high as 27.5% and recovery rates from

patients with diarrhea as high as 34% have been reported (43). In Bangladesh

Aeromonas spp. are significantly isolated from childhood diarrhea patients (44).

Pazzaglia et al. reported that 23.1% of newborns in Peru demonstrated transitory

gastrointestinal colonization with Aeromonas spp. during the first days of life (43).

The isolation rates for human fecal specimens vary widely, as geographical areas,

patient populations, food habits, level of sanitation, and culture methods influence the

recovery rates (45).

Saad et al. observed that the frequency of recovery of Aeromonas spp. from

stools corresponded to the warm summer months when Aeromonas growth reached

their maximum and postulated that fresh vegetables may be a source (46). No

corresponding increase in the number of aeromonads in water was evident, suggesting

food as the primary contributor to human carriage of Aeromonas spp. The relationship

between presence of Aeromonas spp. in human fecal specimens and clinical

manifestations of disease continues to challenge epidemiologists.

1.2.2 Occurrence in Water

1.2.2.1 Surface Waters

Aeromonads are found in all aqueous environments except thermal springs,

hypersaline lakes, and extremely polluted waters (47). Maalej et al. studied the

seasonal occurrence of aeromonads in urban effluents and the costal marine

environment (48). In urban sewage effluents, presence of aeromonads exhibited a

seasonal cyclic distribution similar to fecal coliforms, with the highest numbers

(29x106 CFU/100mL) in winter months and the lowest levels in summer months. In

coastal waters, aeromonads reached highest levels (56 CFU/100mL) in summer

months. Lowest levels of aeromonads occurred under conditions of maximal solar

irradiation and minimum turbidity. The lack of correlation between fecal indicator

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bacteria and aeromonads suggests that the former group of organisms is not predictive

of the presence of aeromonads in ambient waters.

Bonadonna et al. studied the occurrence of bacteria of anthropomorphic

(human) origin and those of autochthonous (natural) origin using model systems for

prediction of public health risk to marine bathers (49). The resulting model used

salinity, total coliforms, fecal coliforms, E. coli, and location as predictive variables

for presence of aeromonads. Presence of E. coli and fecal coliforms were associated

with lower Aeromonas counts, while predominance of total coliform was associated

with higher Aeromonas counts. Fecal coliforms and increased salinity was associated

with higher Aeromonas counts. Aeromonas counts ranged from < 100 CFU/100mL to

> 105 CFU/100mL. The complexity of the association between anthropomorphic and

autochthonous bacteria confounds development of a predictive model for estimating

public health risk of recreational exposure to marine waters.

1.2.2.2 Ground Waters

Anoxic groundwater may support growth of aeromonads. Massa et al. studied

occurrence of aeromonads in natural mineral water and well water in Italy (50).

Aeromonads were not detected in the 60 natural mineral waters examined.

Aeromonads were found in 5 of 20 well samples with counts ranging from 26-1,609

CFU/250 mL.

Villari et al. examined 103 isolates obtained over 3 years from natural mineral

water and surface streams within the watershed of the wells from which the mineral

water samples were collected (51). Evidence of clonal identity was found in the A.

caviae isolates and among A. hydrophila strains in the mineral water samples using

PFGE. Aeromonads from surface waters did not show clonal identity. Biofilm was

thought to be responsible for the clonal nature of well water isolates.

1.2.2.3 Drinking Water

Aeromonas spp. have been isolated from chlorinated drinking water supplies

in several countries (25) (52) (53) (54) (18). Aeromonads grow in water distribution

systems (55) (56). They occur as biofilms in distribution system where they may be

protected from disinfection (57) (58). Multiple strains are frequently found in water

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sources (59) (60). The presence of aeromonads in distribution system water indicates

neither fecal pollution nor treatment failure; however, a large number of aeromonads

present in distribution water suggests that water conditions support growth. Because

of the prevalence of aeromonads in foods, water appears to be an incidental source of

colonization of the human gastrointestinal tract. A drinking water standard of 200

CFU/100 mL at 25º C has been established in the Netherlands (55).

Gavriel et al. studied a drinking water distribution system in Scotland for the

presence of Aeromonas spp. and their relationship to chlorine concentration, pH,

temperature, and rainfall (27). They isolated aeromonads from chlorinated reservoirs

and suggested a relationship between rainfall and increased recovery, probably due to

increased organic load caused by formation of chloramines.

Several species of aeromonads have been isolated from drinking water. The

longer survival rate of A. caviae compared to A. hydrophila may explain its frequent

isolation from drinking water (3) and its high concentrations in treated sewage (31).

1.2.2.4 Bottled Water

Aeromonas spp. have been cultured from bottled mineral water (61) (62) (63)

(55) (64) (29). Isolations rates as high as 35.5% and cell concentrations greater than 3

log10 CFU/mL have been reported. The behavior of aeromonads in bottled mineral

waters under various conditions of temperature and nutrient levels was studied (65). It

was also demonstrated that growth in polyethylene bottles at 10º C reached peak cell

densities of 4.47 log10 CFU/100mL in 28 days. It took 60 days to reach this cell

density at 20º C (65).

1.2.2.5 Wastewaters

Aeromonads are widespread in wastewater treatment processes (Poffe and Op

de Beeck 1991; Kampfer et al., 1996). Microbial populations in activated sludge were

studied over time using phenotyping and ribotyping (66). The initial variety of

hybridization groups was reduced and replaced by dominant hybridization groups.

Over the course of a year, Aeromonas populations shifted as shown in table 1.2.

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Table 1.2: Aeromonas Population Shift in Activated Sludge.

Initial Populations Altered Populations

A. caviae group 54.5% A. enucrenophila 47.1%

A. hydrophila group 22.7% A. caviae group 41.2%

A. sobria group 18.2% A. hydrophila group 5.4%

A. eucrenophila 4.5% A. sobria group 5.8%

1.2.3 Occurrence in Food

Aeromonads have been isolated from pork, chicken, beef, milk, dairy

products, shellfish, fish, fish eggs and fresh vegetables (Fig. 1.5) (8). Most of the

foodborne outbreaks attributed to Aeromonas spp. resulted from ingestion of fish or

shellfish. While aeromonads have been isolated from these food items, few foodborne

outbreaks have been reported. A growing body of epidemiological evidence supports

the possibility of aeromonads causing foodborne gastroenteritis. While a plethora of

putative virulence factors has been postulated and demonstrated in food isolates, the

exact role and mechanism of aeromonads in causing diarrheal illness has not been

elucidated. Evidence suggests that a high infective dose is necessary to produce

gastrointestinal disease in a susceptible host, and the fact that aeromonads may

survive and grow at refrigerator temperatures provides a reservoir of bacteria that may

achieve an infective dose when foods are mishandled.

A variety of foods have been shown to harbor aeromonads (Table 1.3) (67).

Ibrahim and MacRae reported aeromonads present in beef (60%), lamb (58%), pork

(74%), and milk (26%) samples (68). Krovacek et al. found aeromonads in 43% of

random samples from retail food outlets in Sweden (13). It was also reported

aeromonads in fish and fresh salads (69), and aeromonds have been isolated from

lamb (70), oysters (71), cheese and raw milk (72), and fish and seafood (73). Szabo et

al. isolated Aeromonas spp. from 70 of 120 samples of lettuce in Australia (74).

Aeromonads are found in ready to eat foods, including seafoods (75) (71). Studies

published before 1990 relied upon phenotypic identification, while several studies

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published after that time identified isolates to hybridization group. While

hybridization groups containing virulence factors are found in environmental samples

and foods, aeromonads only cause gastroenteritis when their presence exceeds an

infective dose for a susceptible host (76). Strain variability and undetermined host

susceptibility factors have made it impossible to determine a nominal infective dose,

however anecdotal evidence suggests that the infective dose is highly strain and host

dependent, and probably exceeds 6 log10 CFU/g. Such high doses are unlikely to be

ingested in drinking water, since ambient water concentrations rarely exceed 4 log10

CFU/mL except in sewage-polluted waters.

Food Cell Density (log10CFU/g) Reference

Vegetables < 2 to > 6 McMahon and Wilson 2001

Fish 2-4 Pin et al., 1995

Seafood < 2 to > 5 Hanninen et al., 1997

Cheese 3-5 Pin et al., 1995

Meats (beef, pork, lamb) 2-4 Pin et al., 1995

Poultry 2-4 Pin et al., 1995

Milk (pasteurized) 4-5 Pin et al., 1995

Figure 1.5 : Source of Aeromonas

contaminations in food.

Table 1.3: Occurrence of Aeromonas spp. in Foods (179) (180) (73).

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Agarwal et al. isolated aeromonads from fish (22%), snails (6.25%), and quail

eggs (18%), buffalo milk (2.8%), and goat meat 8.9% – all foods of animal origin in

India (77). These findings are consistent with those of Tsai and Chen, who found

22.2% of fish samples contained aeromonads (71), and Glunder and Siegmann, who

reported finding aeromonads in birds and poultry eggs (78). Neyts et al. cultured 68

food samples quantitatively to determine the presence of mesophilic Aeromonas spp.

Aeromonads were found in 26% of vegetable samples, 70% of meat and poultry

samples, and 72% of fish and shrimp samples at numbers from < 2 log10 CFU/g to >

5 log10 CFU/g (76).

1.2.4 Geographical variation of occurrence

The prevalence of different species of Aeromonas is to be expected to vary

with geographical locations. A. hydrophila and A. veroniibv. sobria are the dominant

species in Australia and Thailand (15). European and American studies have revealed

that the majority of isolates were A. caviae (15). However, A. hydrophila and A.

veronii bv. sobria were also isolated in significant numbers. A study in southern India

has revealed that A. hydrophila is the predominant species (79). In Bangladesh, A.

trota was isolated from a large number of diarrhoeal patients (42). However, this

species was not found in hospitalized diarrhoeal cases in Kolkata. It can be said that

variation in geographical distribution may, to a certain extent, reflect the tentativeness

of Aeromonas taxonomy (80). But no study has yet conducted to determine which

Aeromonas species are prevalent in the environment of Bangladesh.

1.3 Health Effect of Aeromonads in Humans

Some Aeromonas spp. are opportunistic pathogens of humans, causing a wide

variety of extra-intestinal infections and occasionally associated with gastrointestinal

disease. Aeromonas infections occur in four broad groups of patients:

I. Persons with impared immune function or serious underlying disease,

especially cirrhosis or hematologic malignancy.

II. Persons with hospital-acquired postoperative infections or infections

associated with health care.

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III. Previously healthy persons with community-acquired infection

following trauma and/or exposure to contaminated water.

IV. Previously healthy persons, especially children, who ingest

contaminated foods and subsequently develop gastrointestinal illness.

The frequency of Aeromonas infections remained stable over the past 15

years, so that Aeromonas infection should not be considered an “emerging” problem.

Aeromonas spp. reportedly cause cellulitis, abscess, wound infection, necrotizing

fasciitis, myonecrosis, pneumonia, empyema, septicemia, septic arthritis,

osteomyelitis, endocarditis, meningitis, gastroenteritis, appendicitis, peritonitis, acute

suppurative cholangitis, and corneal ulcer.

1.3.1 Clinical Symptoms of Aeromonas Infection

Clinical symptoms of Aeromonas infection depend upon the site and severity

of infection. Wound infections frequently result in cellulitis and rarely necrotizing

fasciitis. Septicemia may follow wound infection, or may be secondary to systemic

diseases such as cancer, cirrhosis, diabetes, biliary disease, or diseases resulting in

gastrointestinal perforation. Dissemination may result in meningitis or endocarditis.

Pneumonia is rare and it is usually associated with aspiration, such as in near

drowning. Gastroenteritis symptoms range from mild self-limiting to dysentery or

cholera-like illness.

Patients present with a spectrum of disease symptoms from mild self-limiting

diarrhea to acute, severe diarrhea with abdominal cramps, vomiting, and fever.

Bloody stools occur with some strains. Adults have chronic diarrhea and abdominal

cramps, whereas children 12 years or younger are likely to have more acute and

severe illness. A. caviae and A. hydrophila have been associated with chronic diarrhea

lasting up to one year (81). A syndrome resembling ulcerative colitis has been

observed by endoscopy, and segmented colitis has also been reported (82).

Colonoscopy reveals exudates, superficial ulcerations, erythema, and friability of the

mucosa, as well as loss of vascular pattern and overlying mucus.

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1.3.2 Virulence Mechanism of Aeromonads

Virulence of aeromonads is incompletely understood despite decades of

intense investigation (83). Many putative virulence factors have been described,

including toxins, enterotoxins, proteases, hemolysins, lipases, adhesins, agglutinins,

hydrolytic enzymes, outer membrane proteins, S-layer, flagella, and pili (84).

Expression of virulence factors is multifactorial and host susceptibility dependent

(85).

Aeromonads possess all of the requirements of pathogenic bacteria (86).

Attachment and entry into host cells is facilitated through production of flagella, pili,

and adhesins. Multiplication in host tissue is aided by production of siderophores and

outer membrane proteins, while resistance to host defenses is conferred by production

of capsule, S-layer, lipopolysaccharide and porins. Enterotoxins, proteases,

phospholipases, and hemolysins effect damage to host cells leading to cell death.

Structural factors of bacteria promote attachment (pili, flagella) colonization

(adhesins, outer membrane proteins) and protect cells from host response (S-layer,

lipopolysaccharide (LPS), capsule). Long wavy flexible fimbriae and afimbrial

adhesions are associated with colonization of A. hydrophila. Removal of surface

structures reduces adherence in HEp-2 cells by up to 80% (87) (88). A. caviae and A.

veronii biovar sobria were found to adhere better than A. hydrophila. Most adherence

studies were done with clinical isolates and little is known about adherence of

environmental strains. Differences in pili of clinical and environmental strains was

reported (89). Adhesion to HEp-2 cells correlated with clinical strains possessing low

numbers of thin flexible long L/W type pili, while environmental strains expressed a

larger number of short rigid pili termed the S/R type.

Nonfilamentous adhesins include S-layer, LPS, and outer membrane proteins.

Collagen-binding protein is found extracelluarly and in loose association with cells,

and it is thought to have adherence properties. Surface array proteins are thought to

have antiphagocytic properties (90). The role of LPS is not clear, but it is thought to

be associated with colonization. It was also suggested that it might confer resistance

to complement-mediated lethal effects (91).

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Several exotoxins and enzymes from Aeromonas spp. with putative virulence

properties have been characterized (92). The cytotoxic group of extracellular products

includes hemolysin, aerolysin, and phospholipase. The enterotoxin group is comprised

of heat-labile enterotoxin, heat-stable enterotoxin, non-cholera toxin cross reactive,

cholera toxin cross reactive, aerolysin (Asaotoxin, β hemolysin), and non-channel

forming hemolysin (HlyA). The protease group contains thermostable metalloprotease

(38 kDa), thermolabile serine protease (68kDa), thermostable serine protease (22

kDa), and zinc protease (19 kDa). The hydrolase group includes DNAse, gelatinase,

acetylcholinesterase, amylase, lipase, and chitinase. Other non-enzyme proteins such

as histone-like protein, multidrug-resistance protein, and collagen-binding protein are

thought to play a role in virulence.

1.3.3 Gastrointestinal Infection by Aeromonas spp.

The role of aeromonads as causative agents of diarrheal disease is problematic

and frustrating. While some stains of Aeromonas undoubtedly cause diarrhea, the

ability to unequivocally demonstrate cause and effects continues to elude

investigators, who must rely upon clinical and epidemiological associations rather

than conclusive evidence. This dilemma results from the inability to find an animal

model system that replicates the pathogenesis of gastroenteritis in humans.

While the production of extra-intestinal disease in humans is incontrovertible, the

role of mesophilic Aeromonas spp. as agents causing gastroenteritis is controversial.

Much of the controversy results from the inconclusive human volunteer feeding study,

where ingestion of Aeromonas strains in concentrations as high as 9 log10 CFU/mL failed

to produce disease (93). Because no animal model has been identified to fulfill Koch’s

Cell-Associated Virulence Factors Extracellular Virulence Factors

Pili (fimbriae) Hemolysin

Flagella Enterotoxin

Outer membrane proteins Cytotoxin

A or S layer Protease

Lipopolysaccharide Glycerophospholipid cholesterol

Capsule Other hydrolytic enzymes

Table 1.4: Virulence Factors of Aeromonas species (112).

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postulates for gastrointestinal disease in humans, the role of aeromonads as agents of

gastroenteritis has been extrapolated from anecdotal case reports, case-control studies,

and a handful of outbreaks epidemiologically associated with food or water ingestion

(30).

Though the role of aeromonads as agents of gastroenteritis remains

controversial, several microbiological, epidemiological, and clinical/immunological

investigations indicate that some strains of Aeromonas are enteric pathogens (15)

(94). Gastroenteritis has been linked with but not necessarily caused by Aeromonas

spp. worldwide (15). The association is strongest in children under the age of 2 years,

adults over 50 years of age, and the immunocompromised (95) (96) (97). A summer

peak for isolation of aeromonads from stools corresponds with their increased

presence in the environment (52) (39) (98). While strains possess virulence properties

such as the ability to produce enterotoxin, cytoxtoxin, hemolysins, adhesins, invasins,

and an array of hydrolytic enzymes, not all strains possessing these properties cause

disease in humans, and the host factors predisposing to colonization and disease are

unknown (99). While A. hydrophila and A. veronii biovar sobria are generally

recognized as agents of gastroenteritis, the causal role of A. caviae was considered

controversial despite the fact that a causal role was first proposed by Fritsch et al.

thirty four years ago (100). Today, most investigators acknowledge A. caviae as the

cause of gastrointestinal disease (101). Aeromonas spp. have been established as an

enteric pathogen (42), but the mechanisms of pathogenicity remain elusive.

Aeromonads may be present in the gastrointestinal tract of humans, and most

epidemiological studies show higher numbers in stools of patients with gastroenteritis

than in asymptomatic individuals. Acute self-limiting diarrhea occurs in children, and

chronic gastroenteritis or enterocolitis may occur in children and the elderly. The

presentation of gastroenteritis caused by aeromonads includes various combinations

of fever, vomiting, and increased fecal leucocytes or erythrocytes (102).

Aeromonas spp. is considered to be enteric pathogens. Certain strains of

aeromonads ingested at a high inoculum levels (probably > 8 log10 CFU) may

produce diarrheal disease in susceptible hosts (102). It was suggested that aeromonads

may be associated with up to 13% of gastroenteritis cases in the U.S (103). O:11 and

O:34 serotypes are common in gastroenteritis (104) (105) (106) (107). The majority

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of aeromonads associated with gastroenteritis are A. veronii biovar sobria (HG-8/10),

A. hydrophila (HG-1), and A. caviae (HG-4), though A. veronii biovar veronii (HG-

8/10), A. trota (HG-13), and A. jandaei (HG-9) occur occasionally (108).

Gastroenteritis attributed to A. sobria was characterized by acute watery diarrhea,

vomiting abdominal pain, and fever (14). Goldsweig and Pacheco (2001) reviewed

infection colitis caused by Aeromonas spp (109). Picard et al. suggested the intestinal

tract as the source of invasive infections (110), but Outin et al. observed that the

source of gastrointestinal infection with Aeromonas is rarely proven (111).

Only certain strains of aeromonads are associated with gastroenteritis in

humans, though humans continually ingest aeromonads in food and water (59).

Despite demonstration of virulence factors such as enterotoxins, cytotoxins,

hemolysins, aerolysins, proteases, hemagglutinins, and invasins, it has not been

possible to predict pathogenicity for the human gastrointestinal tract based upon

presence and production of recognized virulence factors alone (112). Expression of

virulence factors is multifactorial and host susceptibility dependent (85). The difficulty

in unequivocally determining the pathogenesis of aeromonads results from their

extreme strain heterogeneity and the lack of a suitable animal model system (42).

While some investigators refuse to accept aeromonads as a cause of diarrheal

disease, the available evidence supports the conclusion that some strains do cause

either acute or chronic gastrointestinal illness in susceptible hosts.

1.4 Isolation of Aeromonads

No unified medium have yet established for isolation and recovery of

aeromonads. A large number of selective and differential culture media have been

developed for recovery of Aeromonas spp. from different types of sample. But the

matter of proper selective medium for isolation of Aeromonas spp. is still

controversial (23) (113). Comparative studies suggest that no single medium results in

optimum recovery of aeromonads. The matter of the proper incubation temperature is

controversial as well, with some researchers finding better growth at 25ºC than at

37ºC (114), but others finding no difference at all between these two temperatures

(115). Combinations of media and methods are frequently employed for increasing

efficacy and fidelity of isolation method depending on source of specimens.

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Medium Selective compound(s)

(concn [mg/ml]) Differential agent(s) Reference

AD Ampicillin (10) Dextrin Havelaar et al. (116)

mA Ampicillin (10), sodium

deoxycholate, ethanol

Trehalose Rippey and Cabelli (22)

MIX Ampicillin (20), bile salts,

citrate

Xylose, meso-

inositol

Cunliffe and Adcock (7)

PBG Sodium lauryl sulfate Glycogen McCoy and Pilcher (16)

RS Novobiocin (5), citrate,

sodium

Lysine, ornithine,

maltose

Shotts and Rimler (24)

SA Ampicillin (10) Starch Palumbo et al. (19)

m-Endo Bile salts Lactose American Public Health

Association (2)

DFS Dextrin Schubert 1987

BGBSS Bile salts, oxgall Starch, lactose Modified from the

method of Difco

EXA

Ampicillin (30), Ecosan-2 Xylose Modified from the

method of Rogol et al.

(23)

Figure 1.6: Aeromonas colonies in

ADA plate

Table 1.5: Selective and differential media for Aeromonas spp. ©

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1.4.1 Isolation From Environmental Samples

Though Aeromonas spp. is ubiquitous in environment, their isolation is not so

straightforward. Moreover isolation of Aeromonas spp. from environmental samples

is problematic because of the presence of competing background microflora and the

possibility of sample matrix interference with sample preparation and culture methods

(117). The use of dilution schemes and enrichment media facilitate isolation of

aeromonads from heavily contaminated environmental water samples. Most

frequently used selective media for isolation of aeromonads are listed in table 1.5.

Several culture media for isolation and enumeration of aeromonads from

environmental water samples and concluded that ampicillin dextrin agar (ADA)

produced the best overall results with sufficient selectivity for colonial morphology

differentiation in mixed cultures (Fig. 1.6). (118) (119). In this media, ampicillin is

used as a selective agent which inhibit growth of background microflora with

sufficient growth of Aeromonas spp.. Almost 94.9% aeromonads are resistant to

ampicillin (120). Only controversy of this media includes several species of clinically

significant Aeromonas, most notably A. trota are susceptible to ampicillin (121) (122).

Since A. trota strain is rarely found in environment and addition of ampicillin

increases resolution of the medium, it is considered as best media for isolation of

Aeromonas spp. from environment. It has been shown that the effect of ampicillin on

Aeromonas spp. is generally negligible (116). With some modification and

standardization of composition depending on source of specimens and reagents, ADA

shows outstanding performance in enumeration and colony differentiation of

aeromonads from environmental water.

1.4.2 Biochemical characteristics of Aeromonas spp.

Aeromonas spp. are facultatively anaerobic, catalase positive, oxidase positive,

chemoorganotrophic bacteria that exhibit both oxidative and fermentative metabolism

on carbohydrates. Aeromonas spp. produce a wide variety of extracellular hydrolytic

enzymes such as arylamidases, amylase, deoxyribonuclease, esterases, peptidases,

elastase, chitinase, and lipase (123). Aeromonas spp. grow optimally within a

temperature range between 22-35º C, but growth occurs in a temperature range from

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0-45º for some species (124). Some species, including most nonmotile A. salmonicida

strains, do not grow at 35º C (23). They tolerate a pH range from 4.5 to 9.0, but the

optimum pH range is from 5.5 to 9.0 (8), and optimum sodium chloride concentration

range is from 0 to 4%.

The genus Aeromonas is differentiated from Plesiomonas and Vibrio by its

resistance to O/129 (150 Fg) and variable presence of ornithine decarboxylase (23).

Other key differential characteristics include its inability to grow in the presence of

6.5% sodium chloride, gelatin liquefaction, inability to ferment i-inositol, and a

negative String Test. Additional useful but variable phenotypic characteristics include

an inability to grow on thiosulfate citrate bile salts sucrose agar (TCBS), and ability of

most but not all Aeromonas species to ferment D-mannitol, and sucrose (117).

Variability in biochemical characteristics is the basis of identification of

different species. Abbott et al reviewed the biochemical characteristics of all

described species of Aeromonas. (125).

1.5 Antimicrobial susceptibility of aeromonads

Most motile aeromonads are generally resistant to penicillin, ampicillin,

carbenicillin and ticarcillin, with variable resistance to cephalexin (76.3%),

trimethoprim (37.3%), tetracycline (11.9%), cefuroxime (5.1%), and ceftazidime

(1.7%). They are typically susceptible to second and third generation cephalosporins,

aminoglycosides, carbapenems, chloramphenicol, tetracyclines, trimethoprim-

sulfamethoxazole, and quinolones (126) (7). Most aeromonads produce an inducible

chromosomal β-lactamase (127). Aeromonas trota has a unique susceptibility to

ampicillin, with up to 30% of some A. caviae isolates being susceptible as well (121)

(128). Antibiotic resistance to streptomycin, chloramphenicol, tetracycline, cephalexin,

cefoxitin, erythromycin, furazolidone, and sulfathiazole is mediated by plasmids (129).

Antimicrobial resistance to tetracycline, trimethoprim-sulfamethoxazole, some

extended-spectrum cephalosporins, and aminoglycosides seems to be increasing

among clinical aeromonad isolates in Taiwan, as compared to isolates from Australia

and the United States (130). A study of the spectrum of extraintestinal disease due to

Aeromonas species in Queensland, Australia found that in nine cases, the empirical

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antibiotic regimen prescribed did not adequately cover infection due to Aeromonas

(130). This suggests that identification to the species level may play a role in the

selection of appropriate antimicrobial therapy for infection with motile Aeromonas

species.

1.6 Bacteriophages

A bacteriophage (from 'bacteria' and Greek phagein, 'to eat') is any one of a

number of viruses that infect bacteria. The term is commonly used in its shortened

form, phage. Phages are estimated to be the most widely distributed and diverse

entities in the biosphere (131). Phages are ubiquitous and can be found in all

reservoirs populated by bacterial hosts, such as soil or the intestine of animals. One of

the densest natural sources for phages and other viruses is seawater, where up to 109

virions per milliliter have been found at the surface, and up to 70% of marine bacteria

may be infected by phages (132). They are also found in drinking water and in some

foods, including fermented vegetables and meats e.g. pickles, salami, where they

serve the function of controlling any growth of bacteria.

Bacteriophages are discovered indepentdently by British bacteriologist

Frederick Twort, superintendent of the Brown Institution of London in 1915 and

French-Canadian microbiologist Félix d'Hérelle, working at the Pasteur Institute in

Paris, in 1917 (132).

Since ancient times, there have been documented reports of river water having

the ability to cure infectious diseases, such as leprosy. Twort and D'Herelle began to

use phages in treating human bacterial diseases such as bubonic plague and cholera.

Phage therapy was not successful and after the discovery of antibiotics in the 1940s it

was virtually abandoned. With the rise of drug-resistant bacteria in the 1990s,

however, the therapeutic potential of phages has received renewed attention (132).

Typically, like all viruses, phages are simple nucleoprotein particle that consist

of a core of genetic material (nucleic acid) surrounded by a protein capsid (Fig. 1.7).

There are three basic structural forms of phage: an icosahedral (twenty-sided) head

with a tail, an icosahedral head without a tail, and a filamentous form. The genetic

material can be ssRNA (single stranded RNA), dsRNA, ssDNA, or dsDNA between 5

and 500 kilo base pairs long with either circular or linear arrangement.

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Bacteriophages are much smaller than the bacteria they destroy - usually between 20

and 200 nm in size (133).

During infection a phage attaches to a bacterium and inserts its genetic

material into the cell. After this a phage follows one of two life cycles, lytic (virulent)

or lysogenic (temperate). Lytic phages take over the machinery of the cell to make

phage components. They then destroy or lyse the cell, releasing new phage particles.

Lysogenic phages incorporate their nucleic acid into the chromosome of the host cell

and replicate with it as a unit without destroying the cell. Under certain conditions

lysogenic phages can be induced to follow a lytic cycle.

1.6.1 Diversity of Bacteriophages

Among the most numerous objects in the biosphere, phages show enormous

diversity in morphology and genetic content. There are at least 12 distinct groups of

bacteriophages, which are very diverse structurally and genetically (Table-1.6). The

dsDNA tailed phages, or Caudovirales, account for 95% of all the phages reported in

the scientific literature, and possibly make up the majority of phages on the planet

(131).

Figure 1.7 : Schematic diagram of bacteriophage

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However, there are other phages that occur abundantly in the biosphere, phages with

different virions, genomes and lifestyles. Phages are classified by the International

Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic

acid (132).

Family or Group Genera Type Member

Particle

Morphology

Envelope Genome

Corticoviridae Corticovirus PM2 isometric No supercoiled d/s

DNA

Cystoviridae Cystovirus Ø6 isometric Yes 3 segments d/s

RNA

Inoviridae

Inovirus coliphage fd

rod No circular s/s DNA

Plectrovirus Acholeplasma phage

Leviviridae

Levivirus coliphage MS2

icosahedral No 1 (+)strand RNA

Allolevirus coliphage Qbeta

Lipothrixviridae Lipothrixvirus Thermoproteus phage

1 rod Yes linear d/s DNA

Microviridae

Microvirus coliphage ØX174

icosahedral No circular s/s DNA Spirovirus Spiroplasma phages

Mac-1 phage

Myoviridae coliphage T4 tailed phage No linear d/s DNA

Plasmaviridae Plasmavirus Acholeplasma phage pleiomorphic Yes Circular d/s DNA

Podoviridae coliphage T7 tailed phage No linear d/s DNA

Siphoviridae

(Vibriophage)

Lambda phage

group coliphage lambda tailed phage No linear d/s DNA

Sulpholobus

shibatae virus SSV-1 lemon-shaped No circular d/s DNA

Tectiviridae Tectivirus phage PRD1 icosahedral No linear d/s dna

Table 1.6: Classification of Bacteriophages by International Committee on Taxonomy of Viruses (ICTV) based on

morphology and nucleic acid ©

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1.6.2 Lysogen and Lysogenic Phages

A bacterial cell whose chromosome harbors the genome of a temperate

bacteriophage (contains integrated viral DNA) is known as lysogen. And lysogenic

phage is a phage that does not go into a lytic cycle, instead integrates its genome into

the host bacterial chromosome and persists in a latent state and may be perpetuated

indefinitely without affecting the host. Only one phage gene cI is then active that

codes for repression of the lytic action, inhibiting the expression of genes that code

for phage replication (132).

There are two outstanding features of lysogenic bacteria: one is they possess

the potentiality to produce and release phage as stable, heritable trait and secondly,

they are immune to infection by the same or closely related phages (134). Lysogeny is

not limited to the carriage of one type of phage by single kind of bacteria. It is

reported that double, triple lysogeny is possible i.e, one strain of staphylococcus has

claimed to carry as many as five different phages type (135). It is important to note

that lysogenic conversion affect bacterial fitness, at least in five different ways (i) as

anchor points for genome rearrangements, (ii) via gene disruption, (iii) by protection

from lytic infection, (iv) by lysis of competing strains through prophage induction,

and (v) via the introduction of new fitness factors (lysogenic conversion,

transduction).

1.6.3 Viral Life-cycle

Bacteriophages may have a lytic cycle or a lysogenic cycle, but a few viruses

are capable of carrying out both. With lytic phages such as the T4 phage, bacterial

cells are broken open (lysed) and destroyed after immediate replication of the virion.

As soon as the cell is destroyed, the new bacteriophages viruses can find new hosts. In

contrast, the lysogenic cycle does not result in immediate lysing of the host cell.

Those phages able to undergo lysogeny are known as temperate phages (133). Their

viral genome will integrate with host DNA and replicate along with it fairly

harmlessly, or may even become established as a plasmid. The virus remains dormant

until host conditions deteriorate, perhaps due to depletion of nutrients, then the

endogenous phages (known as prophages) become active.

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At this point they initiate the reproductive cycle resulting in lysis of the host

cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce,

the virus is reproduced in all of the cell’s offspring (132).

Sometimes prophages may provide benefits to the host bacterium while they

are dormant by adding new functions to the bacterial genome in a phenomenon called

lysogenic conversion. A famous example is the conversion of a harmless strain of

Vibrio cholerae by a phage into a highly virulent one, which causes cholera

1.6.4 The Lytic Cycle of Bacteriophages

In lytic cycle the virus must induce a living host cell to synthesize all essential

components needed to make more virus particles. These components must then be

assembled into the proper structure, and the new virus must escape from the host and

infect other host cells (Fig. 1.8). This involved the following steps-

Attachment

The first step in infection of a bacterial host by the phage is attachment. There

is a high specificity in the interaction between virus and host. The virus particle itself

has one or more proteins on the outside that interact with specific cell surface

component called receptor. The receptors on the cell surface are normal surface

components of the host, such as proteins, polysaccharides, glycoproteins and

lipoprotein-polysaccharide complexes, teichoic acids or even flagella to which the

virion attaches (131). As for example, CTXФ, a filamentous vibriophage, uses TCP

(toxin-coregulated pilus) as a receptor to infect Vibrio cholerae (136). This specificity

means that a bacteriophage can only infect certain bacteria bearing receptors that they

can bind to, which in turn determines the phage's host range. In the absence of the

receptor site the virus cannot adsorb and hence cannot infect. If the receptor is altered

the host becomes resistant to virus infection. In general, virus receptors carry out

normal functions in the host cell. For example, in bacteria some phage receptors are

pilli or flagella, others are cell envelope components and transport binding proteins

(131).

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Penetration

The means by which the virus particles penetrate into the host cell depends on

the nature of the host, especially on its surface structure. Bacterial cells are infected in

a different manner than animal host that have the most complicated penetration

mechanism. If too many virus particles are attaching to the same host and penetrate it,

there may be premature lysis, which is not accompanied by the production of new

virus. The actual penetration of virus into the host is mechanical, but it may be

facilitated by localized digestion of certain cell surface structures either by phage

enzyme carried out on the tail of the phage or viral activation of the host degradative

enzyme (131).

Complex bacteriophages use a syringe-like motion to inject their genetic

material into the cell. After making contact with the appropriate receptor, the tail

fibers bring the base plate closer to the surface of the cell. Once attached completely,

Aeromonas spp.

Adsorption &

Penetration

Replication of

phage particles

New phage after

lysis of host cell

Figure 1.8 Step wise life cycle of bacteriophage.

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the tail contracts, possibly with the help of ATP present in the tail, injecting genetic

material through the bacterial membrane (132).

Synthesis of Proteins and Nucleic Acid

New copies of viral genome must be replicated, and virus specific proteins

must be synthesized in order for virus multiplication to occur. Typically at least some

viral proteins production begins very early after the viral genome has been taken up

by the cells. In order for this to happen, virus specific mRNA must first be made.

Exactly how the virus brings about new mRNA synthesis depends on the type of virus

and on the structure of its genome (132).

Within minutes, however, bacterial ribosomes start translating viral mRNA

into protein. For RNA-based phages, RNA replicase is synthesized early in the

process. Proteins modify the bacterial RNA polymerase so that it preferentially

transcribes viral mRNA. The host’s normal synthesis of proteins and nucleic acids is

disrupted, and it is forced to manufacture viral products instead. These products go on

to become part of new virions within the cell, helper proteins which help assemble the

new virions, or proteins involved in cell lysis (132).

Assembly

Within the bacterial host when sufficient copies of coat proteins and nucleic

acids are available for assembly. These viruses are then able to infect the new host

and begin the cycle over again. In some cases the construction of new virus particles

involves the assistance of helper proteins. The base plates are assembled first, with the

tails being built upon them afterwards. The head capsids, constructed separately, will

spontaneously assemble with the tails. The DNA is packed efficiently within the

heads. The whole process takes about 15 minutes (132).

Release of Virions

Phages may be released via cell lysis or by host cell secretion. In the case of

the T4 phage, in just over twenty minutes after injection upwards of three hundred

phages will be released via lysis within a certain timescale. This is achieved by an

enzyme called endolysin which attacks and breaks down the peptidoglycan. In

contrast, "lysogenic" phages do not kill the host but rather become long-term parasites

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and make the host cell continually secrete more new virus particles. The new virions

bud off the plasma membrane, taking a portion of it with them to become enveloped

viruses possessing a viral envelope (Fig. 1.9). All released virions are capable of

infecting a new bacterium (132).

1.6.5 The Lysogenic Cycle of Bacteriophages

All infectious bacterial cells do not proceed toward the lysis of the host cell to

produce more viral particles. In a small proportion of the infected cells, the lytic cycle

is aborted and an alternative pathway is activated that leads to the integration of the

temperate phage (such as bacteriophage λ) DNA into the genome of the bacterial host

in the form of a prophage. In this situation the bacterium metabolizes and

reproduces

Figure 1.9 Release of phage particles after lysis of bacteria.

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Figure 1.10 Life cycle of temperate bacteriophage

normally, the viral DNA being transmitted to each of daughter cell through all

successive generations. The choice between the lytic and lysogenic pathways depends

on an intricate balance of a number of host and bacteriophage factors that are present

in the infected cell. In case of bacteriophage λ, two phage gene products are essential

to establish lysogeny. One of the products is a repressor of early transcription and

consequently blocks the early gene expression. The other product recognizes

sequences at the att sites in the bacterial and bacteriophage genome and catalyzes a

breaking and joining event that leads to insertion of viral DNA into the host

chromosome. Bacteriophages mutated in repressor genes are unable to lysogenize;

they therefore, form clear plaques. At intervals, the prophage retrieves its genome

Bacterial cellChromosome

Bacteriophage

Attachment &

DNA injection

Integration

into host DNA

Cell division

Induction

event

Protein

synthesis

DNA replication

& protein coat

assembly

Packaging

of virus

Cell lysis &

release

LYTIC PATHWAY

Integrated DNA replicate with host DNA

PROPHAGE PATHWAY

DNA become

circularized

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from the host, replicates and finally breaks open the cell, releasing new viruses. The

cycle of a lysogenic virus infection extends over several replications of the infected

host cell. Occasionally, parts of bacterial DNA are carried along with the viral

genome during the cutting-out process. This ‘transduction’ plays an important role in

bacterial genetic exchange, spreading, for example, information for resistance

properties (137) (138) (139) (140). The lysogenized prophage can also be induced

into lytic growth, most commonly by cleavage of the repressor gene product by host

recA gene product, following exposure to agents that damage DNA (Fig. 1.10). Once

the repressor activity is destroyed, transcription of the early genes resumes. One of

these early gene products causes the att sites to recombine and detaches the prophage

DNA from the host chromosome. The lytic cycle then follows its usual course. The

lysogenized bacterium can be detected by the fact that it is “immune” to the same or

related phages and that it has the potential of making phage when induced by DNA

damaging agents, such as, UV irradiation or mytomycin C (141).

1.6.6 Pseudo-lysogeny or the Unstable Carrier-State

In addition to the two well-established life-styles of bacteriophages, lysis and

lysogeny, recently a third type, pseudolysogeny or the unstable carrier-state has been

described. This unusual state has been found to occur in many of the lytic

bacteriophage. Pseudolysogeny is defined as an unstable coexistence of a

bacteriophage in a host bacterium without a constant inheritance of the phage

genome. This leads to both infected and phage sensitive progeny in the same culture.

The pseudolysogens are more resistant to ultraviolet light and hydrogen peroxide.

Some of the pseudolysogeny-associated changes are pigment production, nutritional

changes, and toxin production. These new phenotypes could play an important role in

the survival of the bacteria and phage in the natural ecosystem (142).

1.6.7 Plaque Morphology

When a host bacterial lawn is inoculated with respective specific phages, clear

or turbid zones relative to the control plate are produced. These morphologically

different zones are known as plaques of those phages. The clear plaques,

characteristics of lytic phages, are produced due to the lysis of the bacterial hosts by

the phages. Whereas the turbid plaques, characteristics of the temperate phages, are

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produced not due to the lysis of the bacterial hosts but due to the slow growth rate of

the lysogenized bacteria. Plaques of different phages vary in size, shape, and

morphology. In some occasions plaques of same phage can even vary in size, shape,

and morphology when propagated on different susceptible hosts. Plaques can be with

sharp circular or slightly irregular shape or even with bigger or smaller diameter,

depending on the phages and their respective bacterial hosts. A lytic phage can be

mutated to a temperate phage producing turbid plaques, while a temperate one can be

mutated to a lytic one producing clear plaques.

1.6.8 Horizontal Gene Transfer and Evolution of Bacteria

During bacterial evolution, the ability of Bacteria to adapt to new

environments most often results from the acquisition of new genes through horizontal

transfer rather than by the alteration of gene functions through numerous point

mutations. Horizontal gene transfer is defined as the movement of genetic material

between bacteria other than by descent in which information travels through the

generations as the cell divides. Bacterial species have acquired several mechanisms by

which they exchange genetic materials.

Transformation

Transformation is a common mode of horizontal gene transfer that can

mediate the exchange of any part of a chromosome. This mode of transferring genetic

information involves uptake and incorporation of the naked DNA that is free in the

environment into host genome by homologous recombination. Typically only short

DNA fragments are exchanged. Transformation is the molecular basis of penicillin

resistance in pneumococci and Neisseria (143).

Conjugation or Mating

The transfer of genetic material from one bacterial cell to another by direct

contact through a sex pilus or bridge is termed conjugation. This mode of DNA

transfer is mediated by conjugal plasmids or conjugal transposons. The process can

occur between distantly related bacteria or even bacteria and eukaryotic cells and can

transfer long fragments of DNA.

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Transduction

Transduction is acquisition of bacterial DNA by a host bacterium from a

bacteriophage that has incorporated DNA from a previous host bacterium within its

outer coat. Transduction that is the transfer of DNA by phage requires that the donor

and recipient share cell surface receptors for phage binding and thus is usually limited

to closely related bacteria. The length of DNA transferred is limited by the size of the

phage head.

1.6.9 Other Modes Of Horizontal Gene Transfer

Another way that bacteria can adapt to new environmental conditions is via

the acquisition of large contiguous fragments of DNA. Such large DNA fragments

may be acquired in several ways:

� Inheritance of a plasmid which may either remain as autonomous replicons or

recombine into the chromosome;

� Integration of a lysogenic phage into the chromosome;

� Insertion of a linear DNA fragment into the chromosome (usually by

transposition or recombination with flanking homologous sequences) (144)

(145) (146) (147) (148).

1.6.10 Evolution of Bacteriophages

We simply do not know when bacterial viruses evolved as phages have no

fossil record and no molecular clock. Some phages may have originated from

assemblages of host genes that split billions of years ago from bacterial genomes,

escaped from cellular control, and now lead a selfish life. Other phages might have

originated recently. Most importantly, there is ample evidence for continued exchange

of genetic elements between phages, bacterial genomes, and various other mobile

genetic elements. Today it is widely accepted that phages evolve newly, mainly via

the exchange of modules, point mutation, deletion, recombination and from existing

phage as well.

Among recombination, illegitimate recombination takes place at random sites

between different phages. However, most of these recombination events occur within

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open reading frames, changing the phage genome size beyond useful limits, or

disrupting gene clusters, rendering the recombinant phage nonfunctional. Only a few

recombination events lead to viable phage known as productive recombination. These

‘productive’ recombinations most probably occur in intergenic regions, which do not

disrupt functional modules. There are many other environmental factors that lead to

mutation in bacteriophages genome. Point mutation is such an important alternation

that helps to emergence of new phages even from the existing one.

1.6.11 Role of Phages in Bacterial Evolution through Phage-mediated

Gene Transfer

Horizontal gene transfer can be regarded as the fast mode of evolution. New

sets of genes are acquired by transduction, transposition, transformation, and last but

not least, lysogenization with phages. The effect of this quick process on the long-

term evolution of bacteria is less certain. Apparently only very small amounts of

prophage DNA are fixed in the bacterial chromosome. Usually, phage-mediated

horizontal gene transfer occurs via transduction or lysogenic conversion.

Comparative genomics demonstrated that the chromosomes from bacteria and

their viruses (bacteriophages) are coevolving.

This process is most evident for

bacterial pathogens where the

majority contains prophages or phage remnants

integrated into the bacterial DNA. Many prophages from bacterial pathogens encode

virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-

producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum

depend on a specific prophage-encoded toxin for causing a specific disease, whereas

Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar

Typhimurium harbor a multitude of prophages and each phage-encoded virulence or

fitness factor makes an incremental contribution to the fitness of the lysogen. These

prophages behave like "swarms" of related prophages. Prophage diversification seems

to be fueled by the frequent transfer of phage material by recombination with

superinfecting phages, resident prophages, or occasional acquisition of other mobile

DNA elements or bacterial chromosomal genes. Prophages also contribute to the

diversification of the bacterial genome architecture. In many cases, they actually

represent a large fraction of the strain-specific DNA sequences. In addition, they can

serve as anchoring points for genome inversions. The current review presents

the

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available genomics and biological data on prophages from bacterial pathogens in an

evolutionary framework (149).

In transduction DNA is transferred from one cell to other cell through the

agency of viruses. Genetic transfer of host genes by virus can occur by two ways:

generalized transduction (150) and specialize transduction (151).

Generalized Transduction

When the phage infects a sensitive cell, the phage DNA replicates within the

bacterial cell and produces a number of copies of phage genome. Expression of phage

gene produces the empty heads that stabilized by scaffolding proteins and the viral

genome is packed into the empty head by a highly specific manner. Here host DNA

can be derived from any part of host genome (phage mediated degradation of the host

chromosome) that occasionally becomes a part of the mature virus particle.

These transducing phage particle are capable to infect the sensitive cell and the

nucleic acid injected into the new host cell. Except the abortive transduction this

transferred DNA is then integrated into recipient chromosome. Here if the donor

genes do not undergo homologous recombination with the recipient bacteria it will be

lost as they cannot replicate independently and are not part of viral genome. At first

Zinder and Lederberg observed the generalized transduction in Salmonella with

temperate phage P-22 (150).

Specialize Transduction

Specialize transduction, occur only in some temperate virus when lysogeny

breaks down and the phage enters the lytic cycle. It is excised from the chromosome

by recombination between sequences at each end of the integrated prophage (152) and

if any malrecombination happens then DNA from a specific region of host cell

chromosome is integrated directly into the virus genome usually replacing some of the

viral genes. The progeny phage produced containing bacterial gene will therefore be

transduced at very high frequency. The DNA transferred is limited to a very small

region of the chromosome and therefore the phenomenon is known as specialize

transduction.

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1.6.12 Bacteriophages of Aeromonas:

Though Aeromonas spp. have been studied by many investigetors, relatively

few studies have been conducted on their phages. Only 12 lytic and 1 temperate

bacteriophages of Aeromonas species have yet reported (table 1. ). Most of them are

T4 like bacteriophages (153). Within them only Aeromonas hydrophila phage Aeh-1

is thoroughly studied. On the basis of its morphology, Aeh1 was classified as one of

the few then known T4-like phages that infected nonenterobacterial hosts. Subsequent

analyses of its sequences (154) revealed that Aeh1 belonged to another subgroup of

T4-like phages that have diverged more from T4 than the PseudoT-evens.

Table 1.7 : Bacteriophages of Aeromonas.

Phage Primary Host Genome Size(bp) Type

Aeh-1 A. hydrophila 233,234 Lytic

Aeh-2 A. hydrophila - Lytic

PM-2 A. hydrophila - Lytic

PM-3 A. hydrophila - Lytic

PM-4 A. hydrophila - Lytic

PM-5 A. veronii biovar

sobria

- Lytic

PM-6 A. veronii biovar

sobria

- Lytic

Φ 018P A. media - Lysogenic

Aeromonas Phage-

25

A. salmonicida 161,475 Lytic

Aeromonas Phage-

31

A. salmonicida 172,963 Lytic

Aeromonas Phage-

65

A. salmonicida 235,000 Lytic

Aeromonas Phage-44RR A. salmonicida 173,591 Lytic

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1.6.13 Restriction and Modification

Lytic phages are virulent to their hosts and temperate phages are also virulent

to their hosts at least at some stage of their life cycle. So bacteria have evolved

different means to get rid of phages at almost every stages of their life cycle.

Prevention of a productive contact between the phage and the host bacteria is the most

effective of the different ways. Mutational change of the phage receptors or secretion

of a protective barrier such as slime layer or a capsule prevents the phages from

approaching toward infection (155). In a study it was found that after adsorption the

next phase of infection had been blocked due to the inability of that phage to inject its

DNA into the host bacteria (156) (157). Once the DNA is injected the phage usually

takes over the control of the host metabolic machineries. Before the phage can do so

the bacteria can still survive depending on the availability of one of the two

mechanisms. The host bacteria can produce some inhibitors of phage life cycle and/or

the host bacteria can destroy the phage DNA using one of its weapons, restriction

endonucleases. Bacteria have evolved restriction endonucleases to protect themselves

against invasion by foreign DNA’s. These enzymes recognize specific base sequences

on the DNAs of the intruders and crave them into pieces. The bacterial own genomic

DNA remains protected because bacteria have evolved restriction modification

systems as well. Restriction endonucleases come paired with restriction modification

enzymes. These modification enzymes have the same recognition sites as their partner

Figure1.11: Electron micrograph of Aeromonas phage 31.

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endonucleases. But the modification enzymes (a methylase) add a methyl group onto

one of the deoxyribonucleotides at the recognition site (Fig. 1.11).

Bacteriophages can become adapted to their host cells on which the phage last

propagated by host-controlled modification (158). These specifically modified phages

gain improved ability to replicate in cells of the same strain or different strains with

same restriction modification system, but their ability to replicate in strains with

different restriction modification system become restricted. These modifications are

distinct from mutations, as it is obvious from the reversibility of the phenotypic

change by one growth cycle of the phage in another host strain (159). A bacteriophage

is restricted and thus specifically cleaved by host restriction endonuclease when its

DNA is not specifically modified by methylation, and protected when its DNA is

specifically modified- this is the classical mechanism of restriction and modification

of bacteriophage (160) (161) (162) (163) (164) (165). Bacteriophages can also

overcome its host restriction by a protein modification mechanism, where the

bacteriophages acquire a protein modification from its previous host, which in tern

help the phages to adsorb to a new host cell (166). T2, T4, and T6 phages are resistance

to all of the restriction enzymes during their infection to host bacteria due to the

presence of an unusual base in their DNA (hydroxymethyl cytosine in place of

cytosine) and a post synthetic modification (glucosylation) of their DNA. T2 and T4

phages also encode a DNA adenine methyl transferase, which methylates a fraction of

the adenine residues in the phage DNA to 6-methyl amino purine, this further

contribute to the protection against restriction enzymes (167).

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Figure 1.12: Activity of the restriction enzyme EcoRI and restriction modification enzyme EcoRI methylase.

Arrows indicate the site of EcoRI cleavage and arrowheads indicate the site of methylation by

EcoRI methylase.

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1.7 Objectives of the Study

Aeromonads have been found in almost every aquatic environment. These

bacteria have a broad host spectrum, with both cold and warm-blooded animals,

including humans. Aeromonads are widely isolated from clinical, environmental and

food samples, where they can survive and multiply even at low temperatures. The

occurrence of different species of Aeromonas is expected to vary with geographical

locations (15). But no study has yet conducted to determine which species of

Aeromonas are prevalent in environment of Bangladesh.

In the recent time bacteriophages specific for vibrio cholerae were found to

occur amazingly large number in the environment of Bangladesh (168), which

prompted to search for new Aeromonas specific phages and examine their reservoir in

the environmental resources. It was also known that there is an inverse relationship

between prevalence of V. cholerae and vibriophages in environmental water (168)

(169). This finding led to study the relationship between prevalence of Aeromonas

and their phages. With this end of view, the present investigation was designed for the

following objectives:

� Monitoring of the occurrence of Aeromonas spp. in environmental

surface water in and outskirts of Dhaka city.

� Isolation of different Aeromonas spp. from environmental surface

water and determination of species which are prevalent in environment

under study.

� Antibiogram analysis and 16s rRNA hybridization study of the isolated

strains.

� Monitoring of the prevalence of Aeromonas phages in the

environmental surface water

� Isolation of Aeromonas phages from the surface water and their

characterization on the basis of both host specificity and restriction

profile.

� Examination of clonal relationship and sequence homology among

isolated Aeromonas phages

� Identification of lysogenic host of the isolated Aeromonas phages.

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Chapter 2 Methods and

Materials

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2.1 Laboratory Apparatus

Petri dishes and plastic wares used in the experiments were provided by

Sterilin (UK). Eppendorf tubes and micropipette tips were taken from Sigma

(Germany), and were sterilized by autoclaving at 121°C for 20 minutes. The gel kits

used for agarose gel electrophoresis and the apparatus for southern blot preparation

were obtained from Gibco-BRL Life Technologies (Rockville, MD). Mini scale

centrifugations were carried out in an Eppendorf microfuge and large-scale

centrifugation were carried out in a Sorvall RC-5C super speed centrifuge using the

SS-34 rotor. Other laboratory equipments were of standard types and are described in

the relevant sections.

2.2 Laboratory Chemicals and Reagents

The sources of reagents were described in the relevant method section.

Standard laboratory chemicals were of Analar Grade and supplied by either Sigma or

Gibco BRL Life Technologies.

2.2.1 Stock Solutions

1M Tris-Cl: 121.1 g tris base [tris (hydroxymethyl) aminomethane] was

dissolved in 800 ml of distilled water. The pH was adjusted to the desired value by

adding HCl, and the final volume was made up to 1 liter with distilled water. The

solution was sterilized by autoclaving and stored at room temperature.

TE buffer (pH 7.4): For preparation of TE buffer, 5.0ml 2M tris-Cl (pH 7.4)

was added with 2.0ml 0.5M EDTA (pH 8.0) and made the volume 1 liter with

distilled water so that the final concentration of Tris-HCl was 10 mM and EDTA was

1 mM. It was stored at 4°C.

3M NaCl: 175.32 g of NaCl was dissolved in distilled water to a final volume

of 1 liter. The solution was sterilized by autoclaving and stored at room temperature.

0.5M EDTA: 186.1g of Na2EDTA.2H2O (disodium ethylene diamine tetra

acetate) and 20g NaOH pellets were added to 800 ml distilled water and dissolved by

stirring on a magnetic stirrer. The pH was adjusted to 8.0 with 10M NaOH and the

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final volume was made 1 liter with distilled water. The solution was sterilized by

autoclaving and stored at room temperature.

3M sodium acetate: 40.81 g of Na2 (CH3COO). 3H2O was dissolved in 80

ml distilled water. The pH was adjusted to 5.2 with glacial acetic acid. The final

volume was adjusted to 100 ml with distilled water and the solution was sterilized by

autoclaving. It was stored at 4°C.

TES buffer (pH 8.0): TES was prepared by diluting stock solutions to a final

concentration of 20 mM Tris-Cl, 10 mM NaCl, and 0.1 mM EDTA.

Buffer-saturated phenol: Buffer saturated phenol was taken from Invitrogen

Life technology. Before use it was melted at 68°C and 0.1 g of 8-hydrixy-quinoline

was added per 100 ml of phenol. The phenol was stored under equilibration buffer

(TE, pH 8.0) at 4°C in the dark for a maximum period of 2 months.

Phenol-chloroform-isoamyl alcohol: Buffer saturated phenol, chloroform

and isoamyl alcohol (25:24:1) was taken from Invitrogen Life technology. Before use

it was melted at 68°C and 0.1 g of 8-hydrixy-quinoline was added per 100 ml of

phenol. The phenol was stored under equilibration buffer (TE, pH 8.0) in the dark at

4°C for a maximum period of 2 months.

Electrophoresis buffer [10X tris-borate-EDTA (TBE) buffer] (pH 8.0): 54

g tris base, 27.5 g boric acid and 3.7 g Na2EDTA.2H2O was dissolved in distilled

water to a final volume of 1 liter and stored at room temperature.

Gel loading buffer: 6X concentrated gel loading buffer was prepared by

adding 0.025% bromophenol blue, 0.025% xylene cyanol, and 2.5% Ficoll (type 400)

in water. It was stored at room temperature.

Ethidium bromide solution: Ethidium bromide was dissolved in distilled

water at a concentration of 10 mg/ml and stored at 4°C in the dark.

Phosphate buffer saline (PBS, pH 7.2-7.3): PBS of 10X concentration (stock

solution) was prepared by dissolving 10.96 g Na2HPO4 and 3.15 g NaH2PO4.H2O in 1

liter of distilled water. 1X PBS (Working solution) was prepared by diluting the stock

and adding 8.5 g NaCl in 1 liter of working solution.

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Saline sodium citrate (SSC): 20X concentrated SSC were prepared by

dissolving 175.3 g of NaCl and 88.2 g of tri-Sodium citrate in distilled water. The pH

was adjusted to 7.0 and the final volume was made 1 liter. It was sterilized by

autoclaving and stored at room temperature.

Denhardt’s solution: 50X concentrated Denhardt’s solution was prepared by

dissolving 5.0 g of Ficoll (type 400), 5.0 g polyvinyl pyrrolidone, and 5.0 g bovine

serum albumin (Pentax Fraction V, Sigma) in distilled water to make a final volume

of 500 ml. This solution was stored at -20°C.

Denatured Salmon sperm DNA: Sodium salt of salmon sperm DNA (type

III, Sigma) was dissolved in distilled water at a concentration of 10 mg/ml by stirring

vigorously on a magnetic stirrer. The DNA was sheared by passing several times

through an 18-gauge hypodermic needle. It was denatured by boiling for 10 minutes

followed by quick chilling on ice and was stored at -20°C.

SM buffer: SM buffer was prepared by dissolving 5.8 g NaCl, 2.0 g

MgCl.7H2O, 50.0 ml of 1 M Tris-Cl (pH 7.5), and 5.0 ml of 2% gelatin in water to a

final volume of 1 liter and finally sterilized by autoclaving. SM buffer was stored at

room temperature and was used for phage storage and dilution.

RNase A solution: Molecular biology grade pancreatic RNase (RNase A,

Sigma) was dissolved at a concentration of 10 mg/ml in 10 mM Tris-Cl (pH 7.5), 15

mM NaCl. The solution boiled for 10 minutes to remove any contaminated DNase,

and allowed to cool slowly to room temperature and stored at -20°C.

Proteinase K solution: Proteinase K (type XI, Sigma) was dissolved in sterile

distilled water and made the concentration 20 mg/ml. It was stored at -20°C.

2.2.2 Preparation of Antibiotic Solution

Antibiotic solutions were prepared in the following concentration and

preserved as such:

Ampicillin: Water solution of ampicillin was prepared at concentration of 75

mg/ml. The solution was sterilized by passing through a 0.22 µm Millipore filter and

stored at -20°C.

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Streptomycin: It was prepared at concentration of 50 mg/ml in water and

stored at –20˚c.

Sulfamethoxazole: It was prepared at concentration of 23.75 mg/ml in

absolute ethanol and stored at –20˚c.

Trimethoprim: It was prepared at concentration of 16 mg/ml in 50 % ethanol;

wrapped with aluminium foil and stored at room temperature.

2.3 Microbiological Media

ADA(Ampicillin Dextrin Agar) Medium

Tryptose 5.0 g

Yeast extract 2.0 g

Dextrin weiss 10.0 g

NaCl 3.0 g

KCI 2.0 g

MgSO4.7 H2O 0.2 g

FeCI3 .6 H2O 0.1 g

Bromothymolblue solution 0.08 g

These were dissolved in 1000 ml of distilled water, and the pH was adjusted to

8.0 with a few drops of 10 M NaOH and then 15.0g of agar was added and dissolved

by gentle boiling. The medium was sterilized by autoclaving at 121°C for 20 minutes

and then 10 mL of ampicillin-sodium deoxycholate solution (ampicillin 10 mg and

sodium deoxycholate 100 mg were dissolved in 10 ml water and sterilized by

filtration through a 0.22 pm membrane) is added.

LB (Luria-Bertani) medium:

Bacto-tryptone 10.0 g

Bacto-yeast extracts 5.0 g

NaCl 10.0 g

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These were dissolved in 1000 ml of distilled water, and the pH was adjusted to

7.4 with a few drops of 10 M NaOH. The medium was sterilized by autoclaving at

121°C for 20 minutes and stored at 4°C.

LA (Luria agar) medium:

Bacto-tryptone 10.0 g

Bacto-yeast extract 5.0 g

Agar 15.0 g

NaCl 10.0 g

These were dissolved in 1000 ml of distilled water, and the pH was adjusted to

7.4 with 10 M NaOH. The medium was sterilized by autoclaving at 121°C.

Soft agar (Nutrient broth containing 0.8%Bacto agar):

Bacto-tryptone 10.0 g

Bacto-yeast extract 5.0 g

Agar 8.0 g

NaCl 10.0 g

These were dissolved in 1000 ml of distilled water, and the pH was adjusted to

7.4 with a few drops of 10 M NaOH. The medium was sterilized by autoclaving at

121°C. This medium was stored at 4°c for several weeks and could be melted by

simple heating at 47°c.

GA (Gelatin Agar) medium:

Tryptone 10.0 g

NaCl 10.0 g

Gelatin 30.0 g

Agar 15.0 g

These were dissolved in 1000 ml of distilled water, and the pH was adjusted to

7.4 (±0.2) with 10 M NaOH. The medium was sterilized by autoclaving at 121°C.

Tauracholate Tellurite Gelatin Agar (TTGA or Monnsur’s medium):

Tryptone 10.0 g

NaCl 10.0 g

Sodium taurocholate 5.0 g

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Sodium carbonate 1.2-1.3 g

Gelatin 30.0 g

Agar 16.0 g

These were dissolved in 1 liter of distilled water, and the pH was adjusted to

8.5-9.0 with a few drops of 1M NaOH. The medium was sterilized by autoclaving at

121°C. Then 2.5mL of tellurite was added before use.

2.4 Bacterial Strains Used In This Study

In addition to isolated Aeromonas species, some previous isolates of clinical

Aeromonas, some clinical and environmental isolates of Vibrio Cholerae, E. coli,

Shigella spp. were used in this study. These strains were provided by Molecular

Genetics Laboratory, ICDDR’B and Media and Lyophilization Department of

ICDDR’B.

2.5 Enzymes used in this Study

Many enzymes were used in this study (Table 2.1), such as restriction

endonucleases, DNase, RNase, protease, and bacterial cell wall degrading enzyme

lysozyme etc. These enzymes were taken from Invitrogen Life technology

(Carlsbad,USA), Takara Biomedicals (Japan), New England Biolabs (UK) and

GibcoBRL Life technology (Rockville, MD) with respective buffer.

2.6 Collection of Environmental Surface Water of Dhaka

To study the prevalence of Aeromonas spp.and their phages in the

environment, water samples were collected from 8 different sites (Fig. 2.1) in two

major rivers (the Turag and the Buriganga) and a lake (Gulshan lake) in and around

Dhaka city was tested. Water samples were collected from each of these sites once a

week throughout the study period. All water samples were obtained in sterile

containers, and initial processing of the samples for detection of Aeromonas phages

was done within 3 hours of collection and stored at 4°C for further analysis. The

sampling sites are following:

� Tongi Bridge

� Gabtoli Bridge

� Aminbazar Bridge

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� Swarighat

� Kamrangirchar

� Khilkhet

� Rampura Bridge

� Mohakhali Lake

Enzymes Incubation temperature Storing temperature

Alu-I 37°°°°C

-20°°°°C

Asc-I 37°°°°C

Ava-I 37°°°°C

BamH-I 37°°°°C

Ban-II 37°°°°C

Bgl-I 37°°°°C

Bgl-II 37°°°°C

BstX-I 55°°°°C

Cla-I 37°°°°C

DNase-I 37°°°°C

Dra-I 37°°°°C

EcoR-I 37°°°°C

EcoR-V 37°°°°C

Hae-III 37°°°°C

Hinc-II 37°°°°C

Hind-III 37-55°°°°C

Hinf-I 37°°°°C

Kpn-I 37°°°°C

Lysozyme 40°°°°C

Mlu-I 37°°°°C

Mung Bean

Nuclease

30°°°°C

Not-I 37°°°°C

Proteinase K 65°°°°C

Pst-I 37°°°°C

RNase A 37°°°°C

Sac-I 37°°°°C

Sau3A-I 37°°°°C

Sma-I 30°°°°C

Xba-I 37°°°°C

Table2.1: Enzymes used in this study

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2.7 Isolation of Aeromonas spp from Environmental Surface Water

Aeromonads are isolated from environmental surface water with a three steps

screening method.

Step 1:

Environmental surface waters from different sites were spread on ADA plate

at several dilutions. After overnight incubation at 37°C colonies were selected on the

basis of their morphology. On ADA plate Aeromonas spp. were grown as yellow

colonies with characteristic deep color at the centre of them.

Figure 2.1: Sites for isolation of Aeromonas and their bacteriophages around

surface water of Dhaka.

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Step 2:

Selected colonies from ADA plate were streaked onto TTGA plate and

incubated at 37°C. Colonies were selected for second times on the basis of their

morphology. On TTGA plate Aeromonas spp. were grown as water like transparent

colonies with characteristic dark color at the centre of them due to reduction of

potassium tellurite. Gelatinase action was observed as clear zone around the colony.

Step 3:

Selected colonies at stage 2 were finally identified on the basis of their

biochemical characteristics. Colonies of Aeromonas were determined upto species

level.

2.8 Biochemical Identification methods :

Following biochemical tests were performed for identification of Aeromonas

upto species level:

Oxidase test: A portion of the bacterial colony was picked up from gelatin

agar plate with a sterile wooden toothpick and streaked onto the filter paper soaked

with oxidase reagent. Formation of a deep purple color within 5-10 seconds indicated

positive result for oxidase test.

Gelatinase test: Colonies were streaked onto gelatin agar plate. After

overnight incubation gelatinase action was observed. A clear zone around the colony

(due to hydrolysis of gelatin) was seen if the bacteria possessed gelatinase activity.

0/129 disc test: Aeromonads are resistant to vibriostatic compound 0/129. So

no clear zone around 0/129 discs was observed following overnight incubation of the

bacterial lawn.

Kligler’s iron agar (KIA) test: The test was performed to assess the mode of

dextrose utilization in oxidative/fermentative test. Inoculate a portion of the suspected

colony (fresh culture) was inoculated in a tube containing KIA media by stabbing the

butt and streaking the slant. After incubation at 37oC for 18-24 hours, observations

were made for changes in color of the butt and slant, H2S or other gas production.

Formation of acid from dextrose in fermentative mode was indicated by yellowing of

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the butt, whereas the yellowing of the slant was indicated the oxidative mode.

Aeromonas give acid butt and alkaline slant. Blackening of the medium was due to

hydrogen sulphide (H2S) production and bubble formation in the tube was due to the

gas production.

Carbohydrate fermentation test: The test organisms were inoculated into

medium with different carbohydrate (glucose, sucrose, mannose, arabinose etc).

Bromophenol blue was used as PH indicator. Glucose containing test tube also

contained inverted Durham’s tube for indication of gas production. Following

incubation at 37°C for 24 hrs, change in color indicated acid production while

formation of bubble in the Durham’s tube indicated gas production.

Lysine, arginine and ornithine utilization test: The test organism was

inoculated into medium enriched with amino acid. Phenol red was used as PH

indicator. If enzyme acted the PH became alkaline indicating development of orange

or red color (positive reaction).

Salts test: Suspected strains were allowed to grow in a set of nutrient broth

containing 0 %, 3%, 6.5 %, 8.5 %, and 10.0 % NaCl. After overnight incubation

growth of the test bacteria was observed.

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Trait Glucose+Gas

Su

cro

se

L-A

rab

ino

se

Ma

nn

ose

Ma

lto

se

AMINO ACIDS

Ind

ole

Esc

uli

n

Ge

lati

n

Cit

rate

NaCl

Glu

cose

Ga

s

LDC

AD

H

OD

C

0 % 3 %

A. hydrophila + + + V + + + + - + + + + + + A.bestiarum + V + + + + V + - + V V - + + A.salmonicida + V + + + + V V - + + + + + +

A.caviae + - + + V + - + - V V V + + + A.media + - + + + + - V - + V V V + + A.eucrenophila + V V V + + - V - + V + - + +

A.sorbia + V + - + + + - - + - - + + - A.veronii HG- 8 + + + - + + + + - + - + V + + A.veronii HG- 10 + + + - + + + - + + + + + + +

A.jandaei + + - - + + + + - + - + + + + A.schubertii + - - - + + V + - - - V V + + A.trota + V V - + + + + - + - + + + +

A.encheleia + V V - + + - V - + V - - + + A.

allosaccharophila + + + V + + + V V + V + V + +

A.popoffii + + - V + + - + - V - + + + +

Table2.2 : Biochemical characteristics of Aeromonas species. ‘+’ for positive test; ‘- ‘for negative test and V for variable characteristics.

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2.9 Determination of Antimicrobial Resistance Pattern by Disc

Diffusion Method

Bacterial susceptibility to antimicrobial agents was determined by disk

diffusion method as recommended by the National Committee for clinical Laboratory

Standards (the National Committee for clinical Laboratory Standards, 1990) with

commercial antimicrobial disk (Oxoid Ltd., Basingstoke, Hampshire,UK). For this

purpose isolated single colony was picked from culture plate and then given into

Mueller-Hinton broth that was incubated for 3-4 hours at 37 °C. Then using soft steak

the culture was spread on Mueller-Hinton plate, and standard antibiotic disks (Oxoid

Ltd., Basingstoke, Hampshire,UK) were placed on the plate. After over night

incubation the zones of complete inhibition were measured in millimeter. The zone

diameters for individual antimicrobial agents was then translated into susceptible,

intermediate resistant, or resistant categories by according to the interpretation table

(Table2.3).

Name of Antibiotic Zone of Inhibition

Sensitive Moderately Sensitive Resistant

Ampicillin >16 14-16 ≤14

Streptomycin >14 12-14 ≤12

SXT >15 11-15 ≤11

Chloramphenicol ≥18 13-17 ≤13

Tetracyclin ≥18 15-17 ≤15

Erythromycin ≥22 14-21 ≤14

Kanamycin ≥18 14-17 ≤14

Table2.3 : Name of antibiotic and their zone of inhibition.

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2.10 Isolation of total DNA from bacteria :

Mini preparations of total DNA were made according to Maniatis et., al

Reagents :

a. Solution-I

b. 0.5 M EDTA

c. 0.5 M Tris-HCl

d. Lysozyme

e. Proteinase K

f. Proteinase K buffer

g. Phenol: Chloroform: Isoamyl alchohol

h. TE

i. RNase A

DNA isolation Method:

A 1.5 ml alequets of overnight cultures were taken in microcentrifuge tube

and centrifuged at 14000 rpm to harvest bacterial cell. Cell pellets were dissolved in

200ul of solution-I.Then 150 ul of lysozyme solution was added and mixed well by

gentle pipetting. Tubes were incubated at 450C for 5 minutes. After incubation 140 ul

proteinase K buffer and 20µl proteinase K solutions were added and again incubated

at 550 C for 1hr. Then the preparations were extracted with phenol and then with

phenol-chloroform-isoamyl alcohol followed by a precipitation with double volume

of pre-chilled absolute ethanol. DNA was then washed by 70% ethanol and then

centrifuged at 10,000 rpm for 5 minutes. Then the pellet was dried in a vacuum drier

and dissolved in 400µl TE buffer.

Purification of DNA:

The preparation was mixed with 15µl RNase solution and incubated for 2 hrs

at 37 0C.Then contents of the tubes were extracted with phenol, followed by phenol-

chloroform-isoamyl alcohol and finally DNA samples were precipited by double

volume of cold ethanol. Then DNA was washed in 70% ethanol and then centrifuged

at 10,000 rpm for 5 minutes. Pellet was dried and then dissolved in 40 µl TE buffer.

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2.11 Determination of rRNA gene restriction pattern (Ribotyping):

Reagents, Enzymes and Other Requirments:

a) Bgl1 and HindIII restriction enzymes

b) The rRNA probe was a 7.5 kb BamHI fragments of pKK3535

c) Eletroelution kit for probe preparation

d) Other Requirments described under section

e) Bovine Serum Albumin :1 mg/ml solution

f) 10X React Buffer for restriction enzymes

g) Denaturing Solution

h) Neutralizing Solution

i) Nylon Membrane :Hybond- N, Amersham Internatioal Plc,

Aylesbury, United kingdom

j) Random priming DNA labeling system (Bethesda Research

Laboratory) with [ -P32

] deoxycitidine triphosphate (3000 Ci / m

mol, Amersham

k) 20X SSC

l) 20X Denhardt’s Solution

m) Sodium Dodecyl Sulfate (SDS)

n) Salmon Sperm DNA : 100 mg/ml solution

o) Fuji X-ray Film

2.11.1 Method of southern transfer of digested DNA :

Southern blot hybridization was performed by using the restriction digestion

of high molecular weight chromosomal DNA as described by Maniatis. The digested

DNAs were then electrophoresed in 0.8 % agarose gels. After denaturation and

neutralization of the gel digested DNAs were transferred onto nylon membrane

(Hybond- N, Amersham Internatioal Plc, Aylesbury, United kingdom) by southern

blotting.

2.11.2 Preparation of probe for Ribotyping :

Southern blot hybridization with the rRNA gene probe was performed by

using the restriction enzymes Bgl1 and Hind III. The rRNA probe was a 7.5 kb

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BamHI fragments of pKK3535, which is a pBR322 derived recombinant plasmid

containing an E.coli rRNA operon consisting of one copy each of the genes coding

for 5S rRNA , 16S rRNA, 23S rRNA and t RNAGlu .

The recombinant plasmid was

prepared and digested with BamHI, and thereafter the insert was purified by

electroelution from agarose gels as described by Maniatis et al.

2.11.3 Method of hybridization,Washing and Autoradiography:

Southern blots were hybridized, washed, and autoradiographed as described

(Maniatis 1982). In short , blots were prehybridized at 65 0 C for 2 hrs in a solution

containing 20X SSC, 10X Denhardt’s solution, 10% SDS, and 100 ug of freshly

prepared salmon sperm DNA per ml . After 2 hrs of prehybridization, freshly

denatured labeled probe was added to solution and hybridized at 65 0

C for 18 hrs.

hybridized blots were washed once in 2X SSC for 15 min, once in 2X SSC for 30 min

and once in 2 X SSC with 0.1 % SDS for 30 minutes. Thereafter the blots were

washed in 0.1X SSC for 15 min. followed by a second washing in 0.1X SSC with

0.1% SDS for 15 min. After washing the blots were rinsed in 2X SSC and exposed to

Fuji X-ray film at –70 0 C for 24 hours. Then autoradiographs were developed.

2.12 Detection and Isolation of Bacteriophages

Collected environmental surface water samples were first filtered using the

0.22 µm pore size filter (Millipore) which allowed the phage particles to pass through

and made bacteria free. These sample filtrates containing phages were immediately

used for the experiment. Eight samples of filtrates were inoculated on bacterial lawn

and incubated for a certain period at 37°C. Lytic or lysogenic plaques indicated the

presence of phages, and the number of plaques indicated the phage count in that water

sample.

2.12.1 Preparation of Plating Bacteria

A single colony of bacteria was inoculated into rich medium (i.e, LB

medium). These bacteria were grown for 3 hrs at 37ºc with 120 rpm and logarithmic

phage bacterial cells were used for lawn preparation.

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2.12.2 Aeromonas Phage Plaque Generation

For detection of plaques 500µl of logarithmic bacteria were mixed with 3.5 ml

aliquots of soft agar medium (the nutrient broth having 0.8% bactoagar) at 47ºc. The

bactoagar-bacterial suspension were then overlaid on nutrients agar plates and placed

in laminar air flow for drying. An aliquot of pre-filtered bacteria-free sample water

were inoculated on the plates and the plates were incubated at 37ºc for 16 hours. A

water sample was considered as phage positive when one or more plaques obtained on

the bacterial lawn.

2.12.3 Picking Up of Plaques

A Pasture pipette equipped with rubber bulb was used to pick the plaques. The

pipette was stabbed through the chosen plaque in to the hard agar beneath and mild

suction was applied so that the plaque with agar drawn in to the pipette. This fragment

of agar with plaque was placed in to polypropylene tube containing SM buffer and left

for sometime in room temperature. A little vortex is applied to diffuse out the phage

particles from agar. This phage containing SM buffer is then filtered with 0.22 µm

pore size filter (Millipore) and stored at 4ºc. This picking of single plaque also ensures

the purity and an average plaque yields 106 to 10

7 phage particles.

2.12.4 Preparing Stock Phages from a Single Plaque

Two different techniques were used for preparing stock phages and both

methods yields approximately equal amount of phage particles.

a) Plate lysate method

b) Small scale liquid culture method.

a) Plate Lysate Method :

105 to 10

6 plaque forming unit of bacteriophages was mixed with 0.5ml

logarithmic phage bacteria and incubated for 10 to 20 minutes at 37ºc. This mixture

was then mixed with 3.5 ml of soft agar and poured on nutrients agar. To obtain the

confluent lysis, each plate was incubated for12-16 hours at 37ºc. 5ml of SM buffer

was added to the plate and stored at room temperature with intermittent shaking. After

gentle scarping of the top agar, the buffer with little of dissolved soft agar was

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harvested by Pasture pipette. Recovered mixture was centrifuge at 8000 rpm for 5

minutes at 4ºc. The supernatant was filtered by 0.22µm pore size filter (Millipore) and

stored at 4°C as the stock.

b) Small Scale Liquid Culture Method

500µl of overnight bacterial culture was mixed with approximately 106 phage

particles in in a two-drum vial and incubate for half an hour at 37ºc which allowed the

phages to be absorbed. 3.0 ml of LB medium was added and incubated incubated for

6-8 hours at 37ºc with 120 rpm until complete lysis occurred. The suspension was

centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was recovered and

filtered using 0.22 µm filter. The filtrate was then stored at 4°C as the stock

2.12.5 Preservation of Bacteriophages

SM buffer was used for preparation of phage in both plate lysate and small

scale liquid culture method. Aliquot of phages were taken in eppendorf tube and a

very small amount of chloroform added in order to prevent the bacterial growth. This

stock was stored at 4ºc for several weeks to months without loss of viability but

prolong stored may loss the titer of phages.

2.13 Calculation of Titre of Bacteriophages

The titer of bacteriophages was determined by enumeration of the plaque

forming units (pfu/ml). It was determine by using double layer plaque method. The

concentrated phages were diluted 105,10

6,10

7,10

8 times in SM buffer. 100µl of diluted

phage was added to 500µl of sensitive strains present in the logarithmic phase. The

suspension was mixed with 3.2ml soft agar and poured on LA plate and incubated

O/N at 37ºc in an inverted position. The plaque appeared as clear zone in the opaque

layer of bacterial growth. The plaque were counted and scored.

The number of plaque (clear zone) that appears in the bacterial lawn of LA

plate indicated the number of plaque forming units in used amount of phage mixture

assayed. For instance, if there are 15 plaques in 100µl of 108

diluted phage mixtures,

the titer was calculated as:

Plaque counted x dilution factor

Vol. of diluted phage used for infection

pfu / ml

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2.14 Preparation of Phage DNA

2.14.1 Large Scale Preparation of Phage

Two methods were followed for the preparation of large quantities of phage.

I. Liquid culture method, and

II. Plate lysate method.

Liquid Culture Method

3.0 ml of LB in a two-drum vial was inoculated with a single colony of an

appropriate bacterial host and incubated overnight at 37°C with 120 rpm. 0.2 ml of

the turbid culture was taken into a fresh 50.0 ml LB medium, and incubated at 37°C

with 120 rpm for 3-4 hours. . The bacteria reaches to the logarithmic phage and

approximately 3x106 phage particles were added into the cell suspension and

incubated at 37°C for 9-12 hours with 120 rpm. This resulted compete lysis of the

cells. The lysed culture containing considerable number of bacteriophages, bacterial

debris was transferred into sorval tube and the debris was removed by centrifugation

at 8000 rpm for 35 minutes. Supernatant was harvested then passed through 0.22µm

filter to to exclude debris and any unlysed cell. This filtrate was then used for phage

DNA preparation.

Plate Lysate Method

105 PFU of phage was mixed with 0.5 ml of plating bacteria and incubated at

37°C for 20 minutes. 3.5 ml of soft agar was added to the mixer, votexed and poured

onto a labeled plate containing hardened bottom agar. The plate was incubated at

37°C for 8-12 hours, until confluent lysis occurred. 5.0 ml of SM was added on the

plate and kept at 37°C for several hours with intermittent, gentle shaking. The SM

was harvested and centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant

was filtered using a 0.22 µm filter. The filtrate of 8-10 such plates was used for phage

DNA preparation.

2.14.2 Isolation of Phage DNA

One-fourth volume of a solution, PEG (containing 20% polyethylene glycol

6000 and 10% 2.5 M NaCl ) was then added to the filtrate, vortexed, and allowed to

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stand for at least 1 hour at 4°C. The phage particles was precipitated and recovered by

centrifugation at 15000 rpm for 50 minutes at 4ºc. The supernatant was discarded and

the phage pellet was dried. The pellet was dissolved in 0.5 ml of React 2 buffer

containing 20 mM Tris-Cl (pH 7.5), 60 mM KCl, 10 mM MgCl, and 10 mM NaCl

and transferred into a fresh eppendrof tube. The solution was treated with 2µl of

pancreatic DNase I and 15µl of RNase A at 37°C for 11/2 hours to remove the

bacterial DNA and RNA. Proteinase K buffer ( 0.5% EDTA and 0.5% SDS) followed

by 15µl Proteinase K enzyme was added into the mixture and incubated at 37ºc for

11/2 hours to remove the phage coat protein and to expose the phage DNA.

An equal volume of equilibrated phenol was added and mixed by inverting the

tube for 10 minutes. The two phases were separated by centrifugation at 14, 000 rpm

for 10 minutes at 4°C. The aqueous phase was transferred to a fresh tube. The solution

was similarly extracted with phenol-chloroform-isoamyl alcohol (25:24:1). Phage

DNA was precipitated with double volume of absolute ethanol. The precipitated DNA

was spooled out and washed with 70% ethanol to remove any salt and centrifuged at

14, 000 rpm for 10 minutes at 4°C. The precipitated DNA was dried and dissolved in

1X TE, which can be stored at 4°C.

2.15 Analysis of Phage DNA

2.15.1 Restriction Profile Analysis

Restriction endonucleases were used to analyze the DNA pattern of the

bacteriophages. Desired amount of DNA was mixed with digestion mixture

containing appropriate amount React buffer, Bovine Serum Albumin (BSA), water

and enzyme. This mixture was then incubated at 37ºc for overnight in water bath.

Reaction was stopped by adding 0.5M EDTA (pH 7.5). The digested product was

analyzed by agarose gel electrophoresis.

2.15.2 Southern Hybridization Using Total Phage DNA as Probe

Southern transfer was used to identify the localization of particular sequence

of DNA fragments that were separated by gel electrophoresis. and denatured followed

by membrane transfer.

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Probe Labeling

The probes were labeled by random priming with a random primer DNA

labeling kit (BRL) and [α-32

P] dCTP (3,000 Ci/mmol) (Amersham). Southern blots

and colony blots were hybridized with the labeled probes.

Hybridization, Washing and Autoradiography:

Southern blots were prehybridized at 65°C for 2 hours in a solution containing

5X SSC (1X SSC is 0.15 M NaCl plus 0.15 M sodium citrate), 5X Denhardt’s

solution (1X Denhardt’s solution is 0.02 % polyvinylpyrrolidone, 0.02 % Ficoll 400,

and 0.02 % bovine serum albumin), 0.5 % sodium dodecyl sulfate (SDS), and 100 mg

of freshly denatured sheared salmon sperm DNA per ml. The blots were then

hybridized with freshly denatured labeled probe at 65°C for 18 hours. Hybridized

blots were washed at first in 2X SSC for 15 minutes and then for 30 minutes, in 2X

SSC–0.1% SDS for 30 minutes, in 0.1X SSC for 15 minutes, in 0.1X SSC–0.1% SDS

for 15 minutes at 65°C temperature and finally washed with 2X SSC.

Autoradiographs were developed from the hybridized filters by using Fuji X-ray film.

In order to clearly visualize both the faint and the dark bands, two autoradiographs

were developed from each hybridized filter by exposing the films once for 24 hours

and then for 48 hours with intensifying screens.

2.16 Preparation of Colony Blots:

For the preparation of colony blot strains were cultured in LA plate at 37˚C for

over night and then spotted on another LA plate using grid of 100 with sterile tooth

pick. Then the cells were allowed to grow at 37˚C for overnight. The nylon filter

(Hybond; Amersham International PLC, Ayelesbury, United Kingdom) was pressed

on the cultured plate and allowed to stand sometimes so that the cells transferred to

the nylon membrane. Then the nylon membrane was processed by a standard method

(Maniatis). Briefly, colonies were lysed with denaturing solution (0.5 M NaOH and

1.5 M NaCl) and neutralized in neutralizing solution (0.5 M Tris- HCl [pH 8.0] and

1.5 M NaCl) and the liberated DNA was fixed to the nylon membrane by exposure to

UV light for 3 minutes in accordance with the supplier’s instructions. The

hybridization procedure was same as for Southern hybridization.

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Chapter 3 Results

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3.1 Prevalence and Isolation of Aeromonas Species From

Environmental Surface Water

3.1.1 Modification of Media Composition

No unified medium have yet established for isolation and recovery of

Aeromonas spp. regardless of source. Modification and standardization of media and

methods are frequently employed depending on the source. In this study Ampicillin

Dextrin Agar (ADA) medium was modified and standardized for isolation of

Aeromonas spp. from environmental water samples. The sites of collection of

environmental surface water were highly polluted, thus higher incidence of

background microflora reduce recovery rate of aeromonads. So, the composition of

media was modified from that originally described by Havelaar et al. (116).

Use of a relatively higher dose of ampicillin upto 1.4mg/100ml in Ampicillin

Dextrin Agar (ADA) resulted higher recovery rate of aeromonas from polluted

environmental water. As all of the species of Aeromonas except A. trota and some

A.caviae are resistant to ampicillin, it effectively inhibits the growth of background

microflora with sufficient growth of Aeromonas spp..

3.1.2 Prevalence of Aeromonas

High prevalence of Aeromonas species was observed in the environmental

surface water. Water samples collected from the sites mentioned in previous chapter

were directly spread at several dilutions on ADA plate (Fig-3.1). After overnight

incubation count of yellow or honey colored colonies with dark center were taken.

Sampling was done weekly during the study period from March, 2009 to October

2009. Distribution of Aeromonas spp. in the environmental surface water in and

outskirts of Dhaka city is represented in table 3.1. In this study, it was observed that

occurrence of Aeromonas spp. was fairly constant throughout the study period and no

significant seasonal variation was observed (Fig-3.2). No significant correlation was

found between prevalence of Aeromonas species and diarrhea epidemic.

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Table 3.1: Prevalence of Aeromonas spp. In different environmental surface water sample from

April, 2009 to November, 2009.

WEEK

Count of Aeromonas spp. (cfu/ml)

To

ng

i B

rid

ge

Kh

ilk

he

t

Ra

mp

ura

Bri

dg

e

Mo

ha

kh

ali

Sw

ari

gh

at

Ka

mra

ng

irch

ar

Ga

bto

li

Am

inb

aza

r

Me

an

co

un

t

of

Ae

rom

on

ad

s

Mar-Wk-1 8.2×104

4.2×104 9.8×10

2 4.6×10

2 2.0×10

3 3.9×10

2 2.3×10

3 4.0×10

2 1.63×10

4

Mar-Wk-2 9.9×103

5.6×104 1.2×10

3 2.5×10

2 2.9×10

4 2.2×10

4 5.2×10

3 9.3×10

2 7.30×10

3

Mar-Wk-3 7.9×104

5.2×103 5.6×10

2 8.2×10

2 2.0×10

3 3.9×10

2 2.3×10

3 4.0×10

2 9.05×10

3

Mar-Wk-4 7.3×104

6.3×104 2.9×10

2 5.0×10

2 1.9×10

4 6.7×10

4 8.1×10

3 4.5×10

2 9.08×10

3

Mar-Wk-5 1.9×104

2.1×104 1.6×10

2 3.8×10

2 1.8×10

4 5.0×10

4 2.1×10

3 3.9×10

2 9.06×10

3

Apr-Wk-1 8.9×104

5.2×104 9.6×10

2 5.2×10

2 4.0×10

4 4.2×10

4 6.2×10

3 4.7×10

2 1.24×10

4

Apr-Wk-2 7.9×104

2.5×104 1.0×10

3 7.2×10

2 5.1×10

4 4.9×10

4 6.9×10

3 5.2 ×10

2 1.51×10

4

Apr-Wk-3 8.2×104

3.1×104 9.9×10

2 6.7×10

2 5.3×10

4 6.2×10

4 7.8×10

3 6.3×10

2 1.55×10

4

Apr-Wk-4 8.9×104

5.2×104 9.6×10

2 5.2×10

2 4.0×10

4 4.2×10

4 6.2×10

3 4.7×10

2 1.56×10

4

May-Wk-1 7.8×104

5.2×104 9.6×10

2 5.2×10

2 5.2×10

2 4.2×10

4 6.2×10

3 4.7×10

2 2.42×10

4

May-Wk-2 5.6×104

5.1×104 3.2×10

2 2.9×10

2 8.9×10

3 1.9×10

4 4.2×10

3 3.1×10

2 1.78×10

4

May-Wk-3 5.5×104

4.6×104 9.6×10

2 3.0×10

2 2.1×10

4 1.2×10

4 2.6×10

3 6.5×10

2 1.78×10

4

May-Wk-4 5.5×104

4.6×104 9.6×10

2 3.0×10

2 2.1×10

4 1.2×10

4 2.6×10

3 6.5×10

2 1.78×10

4

June-Wk-1 6.9×103

7.9×103 1.2×10

2 1.9×10

2 1.9×10

4 6.7×10

4 8.1×10

3 4.5×10

2 1.45×10

4

June-Wk-2 7.4×104

6.9×104 3.7×10

2 6.1×10

2 2.9×10

4 5.2×10

4 5.6×10

3 3.4×10

2 1.18×10

4

June-Wk-3 5.2×104

3.4×104 7.8×10

2 6.5×10

2 3.0×10

4 5.8×10

4 6.3×10

3 6.1×10

2 1.14×10

4

June-Wk-4 6.2×104

4.0×104 5.6×10

2 6.5×10

2 2.1×10

4 3.6×10

4 5.4×10

3 7.3×10

2 1.13×10

4

June-Wk-5 6.9×104

1.9×103 2.8×10

2 3.9×10

2 5.3×10

4 1.0×10

4 3.1×10

3 1.1×10

2 1.06×10

4

July-Wk-1 7.2×104

3.4×104 7.8×10

2 6.5×10

2 3.0×10

4 5.8×10

4 6.3×10

3 6.1×10

2 1.78×10

4

July-Wk-2 3.1×103 2.1×10

2 2.1×10

2 3.9×10

3 3.0×10

3 2.9×10

3 7.8×10

3 9.1×10

2 1.78×10

4

July-Wk-3 6.1×104

1.9×104 6.2×10

2 3.0×10

2 4.5×10

4 8.0×10

4 2.6×10

3 5.7×10

2 1.77×10

4

July-Wk-4 2.3×104

2.9×104 8.6×10

2 5.9×10

2 2.0×10

4 2.9×10

4 5.8×10

3 3.9×10

2 1.99×10

4

Aug-Wk-1 9.0×104

3.2×104 9.6×10

2 4.2×10

2 1.7×10

3 4.8×10

3 1.9×10

3 3.6×10

2 2.82×10

4

Aug-Wk-2 4.3×104

6.9×104 8.2×10

2 6.3×10

2 5.0×10

4 8.2×10

4 3.9×10

3 2.6×10

2 2.89×10

4

Nov-Wk-1 5.9×104

7.8×104 5.2×10

2 1.1×10

2 3.9×10

4 1.2×10

4 3.1×10

3 6.1×10

2 1.63×10

4

Nov-Wk-2 7.9×104

8.1×104 2.1×10

2 6.9×10

2 3.0×10

4 2.4×10

4 3.9×10

3 5.2×10

2 7.30×10

3

Nov-Wk-3 8.2×104

5.4×104 3.1×10

2 5.2×10

2 5.1×10

4 2.9×10

4 1.5×10

3 6.0×10

2 9.05×10

3

Nov-Wk-4 2.4×104

3.2×104 1.8×10

2 2.0×10

2 1.8×10

4 1.0×10

4 1.9×10

3 3.2×10

2 9.08×10

3

Dec-Wk-1 9.8×104 8.8×10

3 1.6×10

2 2.3×10

2 8.5×10

3 7.1×10

3 4.3×10

2 1.6×10

2 9.06×10

3

Dec-Wk-2 7.8×103

8.2×103 3.2×10

2 1.1×10

2 8.2×10

3 6.9×10

3 6.2×10

2 1.4×10

2 1.24×10

4

Dec-Wk-3 8.7×103 9.8×10

3 1.1×10

2 1.2×10

2 5.8×10

4 8.5×10

4 5.3×10

2 1.2×10

2 1.51×10

4

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Figure 3.2: Fluctuation of weakly mean counts of Aeromonas species in the environmental surface water

of Dhaka city during the study period. (Considering mean of all eight sites under study.)

Distinct yellow/honey colored

colony of Aeromonas spp.

with dark center

Figure 3.1 : Distinct colonies of Aeromonas from environmental sample in ADA plate.

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3.1.3 Isolation of Aeromonas

Aeromonas strains were isolated from environmental surface water samples by

newly modified three steps screening method described in previous chapter. Surface

water samples collected from different sites were directly spread on ADA plate.

Morphologically Aeromonas like colonies were chosen randomly from ADA plate,

screened further on TTGA plate and finally confirmed by biochemical test. A total of

30 Aeromonas strains were isolated during the study period (April 2009 to October

2009) from different environmental samples and further characterized to species level

via biochemical test. Numbers of different isolated species of Aeromonas genus are

represented in table 3.2. Among the strains isolated during the study period 60 %(18

out of 30) of the isolates were A. caviae and 26.67% (8 out of 30) isolates were A.

media. This data suggests that A. caviae and A. media are the prevalent strains in the

environment under study.

Species No. of Strains Isolated

A. hydrophila 1

A. caviae 18

A. media 8

A. sobria 2

A. schubertii 1

3.2 Characterization of Isolated Aeromonas Spp.

Environmental isolates of aeromonas spp. are characterized by biochemical

test and rRNA hybridization. Antibiogram of isolated strains were also performed.

3.2.1 Biochemical Characteristics of Isolated Aeromonas spp.

The isolated strains were tested for their biochemical characteristics for

confirmation and species level identification. Strains were primarily selected for

positive test (+) for fermentation of glucose and maltose and growth in peptone water

with 0% NaCl. Their identification to complex level was done by Moeller reaction

(125) and further identified to species level according to Table 2.2.

A typical biochemical reaction set of Aeromonas spp. is depicted in fig 3.1.

Table 3.2: Isolates of Aeromonas species during study period

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3.2.2 Ribotyping of Isolated Aeromonas spp

Isolated environmental strains of Aeromonas species were subjected to

ribotyping. Some clinical Aeromonas isolates and a reference strain ATCC-7966 were

also included in ribotyping analysis. Different types of band pattern were observed

b)

a)

Figure 3.3: A typical biochemical reaction set for identification of Aeromonas. (a) Reaction set

before inoculation, (b) Reaction set after inoculation. Positive citrate, glucose, arabinose, base,

arginine, indole, growth in 0% NaCl and negative gas, sucrose, mannose tests indicate that the

inoculated strain was Aeromonas caviae.

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upon hybridization with PKK 3535 (Fig 3.4 and Fig 3.5). Distant ribotyping pattern

was observed among environmental isolates of Aeromonas. To simplify the

ribotyping patterns dendrogram was generated from band profile of ribotyping by the

unweighted pair group method using arithmetic mean (UPGMA) (Fig 3.6). From the

dendrogram, the diverse clonal relationship among the environmental Aeromonas

isolates can be easily demonstrated. No cluster of closely related ribotypes was seen

for the environmental isolates. There were some pairs of ribotypes with >80%

similarity, but when joined to the closest neighbouring ribotype they counted for

Figure 3.4: Ribotyping of environmental and clinical Aeromonas species by hybridization with

PKK 3535 probe. Lanes: M, 1 kb plus Marker (Invitrogen); 1, ASc 3513/2; 2, AM 3654/1; 3, AC

3658/2; 4, AC 3655/2; 5, AC 3660/2; 6, AC 3673/2; 7, AM 3673/3; 8, AC 3679/1; 9, AC 3684/1;

10, AC 3684/2; 11, AC 3689/3; 12, AS 3688/3; 13, AS 28676; 14, AC 26553; 15, AC 27181; 16, AC

26538; 17, AH ATCC-7966. Lane 1 to 12 include environmental isolates and Lane 13 to 16

include clinical isolates. (AC, Aeromonas caviae; AH, Aeromonas hydrophila; AS, Aeromonas

sorbia; ASc, Aeromonas schubertii;)

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~60% similarity among them. Two clinical A. caviae isolates were indistinguishable

from the ribotype and showed 100% similarity in the dendrogram. It is seen that most

of the environmental strains are distantly related with the clinical strains. This result is

consistent with the report that most of the environmental Aeromonas can’t cause

disease in human (117).

Figure 3.5: Ribotyping of environmental and clinical Aeromonas species by hybridization with

PKK 3535 probe (continued). Lanes: M, 1 kb plus Marker (Invitrogen); 18, AS 3585/1; 19, AC

3595/1; 20, AC 3605/2; 21, AC 3608/3; 22, AC 3610/1; 23, AC 3615/1; 24, AM 3618/1; 25, AC

3624/1; 26, AC 3621/1; 27, AM 3629/2; 28, AM 3639/2; 29, AM 3642/2. Lane 18 to 29 include

environmental isolates. (AC, Aeromonas caviae; AH, Aeromonas hydrophila; AS, Aeromonas

sorbia; ASc, Aeromonas schubertii;)

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Figure 3.6: Dendrogram of environmental and clinical isolates of Aeromonas species

summarizing ribotyping band profile generated by UPGMA. Entries on branches of the tree give

name of the strain, source of isolation, strain ID. (AC, Aeromonas caviae; AH, Aeromonas

hydrophila; AS, Aeromonas sorbia; ASc, Aeromonas schubertii; Env, Environmental isolates; Clin,

Clinical isolates.

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3.2.3 Antibiogram of Isolated Aeromonas Species

Antibiotic resistance pattern of all 30 isolated aeromonas spp. were

determined by disc diffusion method (Fig 3.7). For comparison with clinical isolates,

five clinical isolates of Aeromonas strains provided by Media and Lyophilization

department of ICCDDR’B were included in the antibiogram study. Aeromonas strains

are naturally resistant to ampicillin. Intermediary resistance to erythromycin was also

found in almost all environmental isolates (Table-3.3). At least 3 different patterns of

antibiotic resistance were observed among the environmental isolates. Resemblance

of antibiotic pattern between clinical and some environmental isolates was also found.

Figure 3.7: Disc diffusion method of antibiogram. a) and b) two Aeromonas starins sensitive to

Kanamycin, Streptomycin, Chloramphenicol and resistant to Ampicillin, Sulfamethoxazole-

Trimethoprim (SXT) and vibriostatic agent 0129.

a) b)

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Table-3.3 : Antibiotic resistance patterns of both clinical and environmental isolates.

Species Strain ID

Name of antibiotics

SXT Tet Chl Ery Kan Cip Amp Str

A.

cav

iae

3595/1 R S S I S S R R

3605/2 S S S I S S R S

3608/3 S S S I S S R S

3610/1 S S S I S S R S

3615 S S S I S S R S

3624/1 S S S I S S R S

3621/1 S S S S S S R S

3650/1 S S S I S S R S

3658/2 S S S I S S R S

3655/2 R S S I I S R I

3660/2 S S S I S S I S

3673/2 S S S S S S R S

3679/1 S S S I S S R S

3684/1 R S I I S S R R

3684/2 R S I I S S R R

3689/3 S S S I S S R S

3709/2 S S S I S S R S

3715/1 S S S I S S R S

AV-26553 R S S I S S R R

AT-27181 S S S I S S R S

AU-26538 R S S I S S R R

A.

me

dia

3618 S S S I S S R S

3629/2 S S S I S S R S

3639/2 S S S I S S R S

3642/2 S S S I S S R S

3654/1 S S S I S S R S

3673/3 S S S I S S R S

3718/2 S S S I S S R S

3720/1 S S S I S S R S

A.sorbia

3585/1 S S S I S S R S

3713/1 S S S S S S R S

AT-28676 S S S I S S R S

A.

hydrophila

3689/2 S S S I S S R S

ATCC-7966 S S S S S S R S

A.schubertii 3513/2 S S S I S S R S

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Note: SXT, Sulfamethoxazole-Trimethoprim; Tet,Tetracycline; Chl, Chloramphenicol; Ery,

Erythromycin; Kan, Kanamycin; Cip,Ciprofloxacin; Amp,Ampiciliin; Str,Streptomycin; S, Sensitive; R,

Resistant; I, Intermediary Resistant.

3.3 Seasonal Variation study of the phages of Aeromonas spp in

Environmental Surface Water

3.3.1 Isolation of Aeromonas phages from Environmental Surface

Water Samples

The water samples collected from sites mentioned in the previous chapter

were analyzed to study the presence of phages specific for Aeromonas species. A

number of 144 water samples were analyzed. A number of 30 Aeromonas isolates,

which were isolated at the beginning period of this study used as indicator strains for

the presence of phages. 20µl of pre-filtered (pore size 0.22µm) water sample was

applied directly on the bacterial lawn and phage positive samples were determined by

plaque forming unit (pfu) observed on the bacterial lawn. The concentration of

bacteriophages was estimated by counting the plaques. A total of 28 plaques of

Aeromonas phages were isolated and screening of lytic pattern followed by DNA

restriction profile analysis resulted that these plaques occurred from nine novel

phages. The identity of isolated new phages and their primary hosts are listed in table-

3.4:

Name of the

Phage

Primary Host

Species Strain ID Date of first

isolation

Aec-1 Aeromonas caviae 3605/2 05/05/09

Aec-2 Aeromonas caviae 3624/1 10/06/09

Aec-3 Aeromonas caviae 3624/1 17/06/09

Aec-4 Aeromonas caviae 3658/2 10/06/09

Aec-5 Aeromonas caviae 3608/3 10/06/09

Aec-6 Aeromonas caviae 3610/1 10/06/09

Aem-1 Aeromonas media 3618 10/06/09

Aem-2 Aeromonas media 3629/2 05/05/09

Aem-3 Aeromonas media 3654/1 10/06/09

Table-3.4: Name and primary host of the isolated novel phages during the study period

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3.3.2 Prevalence of Aeromonas Phages in Environmental Surface

Water

Samples collected weekly from May, 2009 to December, 2009 were routinely

tested for prevalence of Aeromonas phages. A panel of 30 different isolates of

different Aeromonas species that isolated earlier in this study was used as potential

indicator host strains to detect the presence of Aeromonas phages in environmental

surface water.

The concentration of Aeromonas phages was determined as the PFU/mL of

filtrate inoculated on lawns of indicator strains. One or few of the indicator strains

under study were lysed by each and every isolated phage. The weekly concentration

of these phages in the environment was found to vary during the study period. At the

beginning of the prevalence study, first week of May, 2009 only two phages (Aem-1

and Aec-2) were detected and isolated. Other seven phages were not present in

detectable concentration in environmental water sample until second week of July

2009. Although phage Aem-1 and Aec-2 were found in almost all samples analyzed at

variable concentrations, incidence of other phages was varied highly from week to

week sampling (Fig 3.8).

The fluctuation of phage concentration during the study period was

determined by considering total number of phages from all 8 sampling sites (Table

3.5). Mean concentrations of all Aeromonas caviae and Aeromonas media phages

were also analyzed separately (Fig 3.9). No significant correlation between prevalence

of Aeromonas phages and seasonality has been established by considering data of this

study.

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Table-3.5: Prevalence of Aeromonas phages isolated from environmental surface water sample from

May, 2009 to December, 2009

WEEK

Phage Titer ( pfu/mL )

Ae

c-1

Ae

c-2

Ae

c-3

Ae

c-4

Ae

c-5

Ae

c-6

Ae

m-1

Ae

m-2

Ae

m-3

May-Wk-1 250 0 0 0 0 0 0 450 0

May-Wk-2 450 0 0 0 0 0 0 350 0

May-Wk-3 350 0 0 0 0 0 0 300 0

May-Wk-4 550 0 0 0 0 0 0 200 0

June-Wk-1 650 0 0 0 0 0 0 600 0

June-Wk-2 50 250 0 150 50 300 50 100 0

June-Wk-3 200 50 150 300 150 150 100 50 300

June-Wk-4 50 0 250 50 0 250 50 300 150

June-Wk-5 400 0 100 0 0 150 250 0 100

July-Wk-1 50 0 150 150 50 150 100 50 100

July-Wk-2 400 350 0 100 0 100 50 350 50

July-Wk-3 100 150 50 50 0 50 0 150 100

July-Wk-4 50 100 0 200 0 0 250 200 150

Aug-Wk-1 50 50 0 100 0 150 100 100 200

Aug-Wk-2 0 0 50 50 0 0 0 0 0

Nov-Wk-1 150 0 50 150 50 300 0 100 300

Nov-Wk-2 300 150 50 150 150 250 0 350 50

Nov-Wk-3 0 0 200 50 0 150 0 50 0

Nov-Wk-4 250 200 0 50 0 100 150 150 50

Dec-Wk-1 100 50 0 50 0 50 0 200 100

Dec-Wk-2 250 250 100 100 100 250 0 250 50

Dec-Wk-3 100 300 0 100 50 300 50 50 250

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Figure 3.8: Fluctuation of weekly mean

total phage count of all eight sit

0

100

200

300

400

500

600

700

Ma

y-W

k-1

Ma

y-W

k-2

Ma

y-W

k-3

Ma

y-W

k-4

Ph

ag

e c

on

cen

tra

tio

n

Pfu

/mL

Seasonal Variation of

0

200

400

600

800

1000

1200

Ma

y-W

k-1

Ma

y-W

k-2

Ma

y-W

k-3

Ph

ag

e C

on

cen

tra

tio

n

Pfu

/mL

Comparison of Frequency of Aec and Aem

Figure 3.9: Comparison of frequency of Aem and Acm phages during the study period. The total of Aem and

Acm phages from 8 sites plotted separately.

Fluctuation of weekly mean Aeromonas phages concentration during the study period considering

of all eight sites under study.

Ma

y-W

k-4

Jun

e-W

k-1

Jun

e-W

k-2

Jun

e-W

k-3

Jun

e-W

k-4

Jun

e-W

k-5

July

-Wk

-1

July

-Wk

-2

July

-Wk

-3

July

-Wk

-4

Au

g-W

k-1

Au

g-W

k-2

No

v-W

k-1

No

v-W

k-2

No

v-W

k-3

No

v-W

k-4

De

c-W

k-1

Time Period

Seasonal Variation of Aeromonas Phages

Ma

y-W

k-3

Ma

y-W

k-4

Jun

e-W

k-1

Jun

e-W

k-2

Jun

e-W

k-3

Jun

e-W

k-4

Jun

e-W

k-5

July

-Wk

-1

July

-Wk

-2

July

-Wk

-3

July

-Wk

-4

Au

g-W

k-1

Au

g-W

k-2

No

v-W

k-1

No

v-W

k-2

No

v-W

k-3

No

v-W

k-4

De

c-W

k-1

Time Period

Comparison of Frequency of Aec and Aem

Phages

: Comparison of frequency of Aem and Acm phages during the study period. The total of Aem and

Acm phages from 8 sites plotted separately.

R e s u l t s

71 | P a g e

concentration during the study period considering

De

c-W

k-1

De

c-W

k-2

De

c-W

k-3

Phages

Aec-1

Aec-2

Aec-3

Aec-4

Aec-5

Aec-6

Aem-1

Aem-2

Aem-3

De

c-W

k-1

De

c-W

k-2

De

c-W

k-3Comparison of Frequency of Aec and Aem

Aec

Aem

: Comparison of frequency of Aem and Acm phages during the study period. The total of Aem and

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3.4 An Inverse Correlation Exist Between

and Their Phages

There was no

Aeromonas and their phages.

aeromonas and their phages it was found that

between occurrences

increases in environment, prevalence of phages of aeromonas decr

time and vice-versa.

0

500

1000

1500

2000

2500

3000

3500

Ma

y-W

k-1

Ma

y-W

k-2

Ma

y-W

k-3

Ma

y-W

k-4

Pre

va

len

ce

Comparison of Prevalence of Aeromons

Aeromonas

Phages

Figure 3.10: Comparison of prevalence of Aeromonas strains and their phages in the

environmental surface water during the study period (considering mean of Aeromonas and total

number of phages occurred in all 8 sites

Inverse Correlation Exist Between Prevalence

here was no significant seasonal effect found on prevalence of both

Aeromonas and their phages. But by considering data of prevalence o

aeromonas and their phages it was found that there have an inverse correlation

of them (Fig 3.10). When prevalence of aeromonas strain

in environment, prevalence of phages of aeromonas decr

Ma

y-W

k-4

Jun

e-W

k-1

Jun

e-W

k-2

Jun

e-W

k-3

Jun

e-W

k-4

Jun

e-W

k-5

July

-Wk

-1

July

-Wk

-2

July

-Wk

-3

July

-Wk

-4

Au

g-W

k-1

Au

g-W

k-2

No

v-W

k-1

No

v-W

k-2

No

v-W

k-3

Time Period

Comparison of Prevalence of Aeromons

and Their phages

Aeromonas

: Comparison of prevalence of Aeromonas strains and their phages in the

mental surface water during the study period (considering mean of Aeromonas and total

number of phages occurred in all 8 sites under study).

R e s u l t s

72 | P a g e

Prevalence of Aeromonas

on prevalence of both

But by considering data of prevalence of both

there have an inverse correlation

. When prevalence of aeromonas strains

in environment, prevalence of phages of aeromonas decreases at the same

No

v-W

k-3

No

v-W

k-4

De

c-W

k-1

De

c-W

k-2

De

c-W

k-3

Comparison of Prevalence of Aeromons

: Comparison of prevalence of Aeromonas strains and their phages in the

mental surface water during the study period (considering mean of Aeromonas and total

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3.5 All new phages Were Specific for Aeromonas Species:

Randomly picked plaques from the lawns of indicator strains were purified

and enriched. The host specificity of the phages was examined by using a panel of 30

environmental isolates and 5 clinical isolates belonging to five species of Aeromonas

genus (Table 3.6). The primary host strains from which the phage plaque was first

picked, was used as a positive control for the representative phage and a classical

Vibrio cholerae strain designated S224 was used as negative control in host

specificity test. Production of single lytic plaque in soft agar overlay assay was

considered as positive susceptibility to the phage.

In the lytic pattern study it was found that most of the newly isolated

Aeromonas phages were highly host species specific (table 3.7). In this study, about

75 V. cholera of different biotypes and serotypes, 20 E. coli and 26 Shigella strains

belonging to all 4 Shigella species were used to test intra-genus host specificity of

these phages. But, the phages were not lytic to any of them. That is why it was

concluded that these phages were Aeromonas specific.

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Host Strains Sensitivity*

Species Strain ID

Ae

c-1

Ae

c-2

Ae

c-3

Ae

c-4

Ae

c-5

Ae

c-6

Ae

m-1

Ae

m-2

Ae

m-3

A. caviae

3595/1 - - - - - - - - - 3605/2 + - + - - - + - - 3608/3 + - - - + + + - - 3610/1 - - - - - + - - -

3615 + - + - - - + + + 3624/1 + + + - - - + + + 3621/1 - - + - - - + - + 3650/1 - - - - - - - - - 3658/2 - - + + - - - + + 3655/2 - - - - - - + - - 3660/2 + - + - - - + + - 3673/2 + - + - - - + + - 3679/1 - - + - - - + - - 3684/1 - - - - - - - - + 3684/2 - - - - - - - - - 3689/3 - - - - - - + - + 3709/2 - - - - - - - - - 3715/1 - - - - - - - - -

AV-26553 AT-27181 - - - - - - - - - AU-26538 - - - - - - - - -

A. media

3618 - - + - - - + + + 3629/2 - - - - - - - + - 3639/2 - - + - - - + - - 3642/2 + - + - - + - + + 3654/1 - - + - - - - - + 3673/3 + - + - - - - + + 3718/2 - - - - - - - - - 3720/1 - - - - - - - - -

A.sorbia

3585/1 - - - - - - - - - 3713/1 - - - - - - - - -

AT-28676 - - - - - - - - -

A. hydrophila 3689/2 - - - - - - - - -

ATCC-7966 - - + - - - - - - A.schuberti

i

3513/2 - - - - - - - - -

Table 3.6: Host specificity of different Aeromonas phages isolated from water sample.

*’+’ sign indicates that the strain designated was susceptible to that phage and ‘-‘ sign indicates that the

strain designated was resistance to that phage.

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3.6 Plaque Morphology of the isolated phages on Lawns of Respective

Indicator Strains

All 9 phages produced clear plaques on respective control hosts. This means

that these phages were lytic and virulent for their respective hosts. Aem-1 and Aec-2

were found to make turbid plaques characteristics of temperate phages on some

strains other than their primary host. Most of these phages produced plaques with

diameter ranging from 0.75 mm to 2.5 mm, on the lawns of respective hosts.

Genus

Species

&

types

No.of

Starins

Checked

Sensitivity*

Ae

c-1

Ae

c-2

Ae

c-3

Ae

c-4

Ae

c-5

Ae

c-6

Ae

m-1

Ae

m-2

Ae

m-3

Vibrio

Vibrio

cholerae

(0139) 5 - - - - - - - - -

Vibrio

cholerae

(01) El Tor 20 - - - - - - - - -

Vibrio

cholerae

O1

(Classical)

20 - - - - - - - - -

Vibrio

cholerae

Non O1 30 - - - - - - - - -

Shigella

Shigella

boydii 4 - - - - - - - - -

Shigella

sonnei 9 - - - - - - - - -

Shigella

dysenteriae 7 - - - - - - - - -

Shigella

flaxinery 6 - - - - - - - - -

Escherichia E. coli 20 - - - - - - - - -

Table 3.7: Intra-genus host range of Aeromonas phages isolated from water sample.

*’-‘ minus sign indicates that the strain designated was resistance to that phage.

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Aec-1 Aec-2

Aec-3 Aec-4

Aec-5 Aec-6

Figure 3.11: Plaque morphology of Aec phages: Aec-1, Aec-2, Aec-3, Aec-4, Aec-5, and Aec-6.

Respective plaques are indicated with arrow.

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3.7 Restriction Profile Analysis of Isolated Phages

Phages with different host specificity were subjected to phage DNA

extraction. Phage DNAs were then digested with at least 20 different restriction

endonucleases to obtain a comparable profile following agarose gel electrophoresis.

But no suitable enzyme was found to make a comparable restriction profile for all

phages (Table 3.8). DNA of Aec-5 and Aec-6 phages were digested by many

restriction enzymes used in this analysis but DNA of rest 7 phages were digested by

only Dra-I. So two sets of restriction profile were made:

Aem- 1 Aem- 2

Aem-3

Figure 3.12: Plaque morphology of Aem phages: Aem-1, Aem-2, and Aem-3. Respective plaques are

indicated with arrow.

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1. DNA of Aec-5 and Aec-6 were digested by Hind-III (Fig 3.11b)

2. DNA of rest 7 phages were digested by Dra-I (Fig 3.11a)

By analyzing these two sets of restriction profile it was confirmed that all 9

phages isolated were genetically different.

a) b)

Figure 3.13: Restriction pattern analysis of the DNA of isolated Aeromonas phages. (a)All 9

phages were digested with Dra-I but Aec-5 and Aec-6 remained undigested. (b)Restricrtion

digestion of Aec-5 and Aec-6 by Hind-III. 1 kb plus marker (Invirogen) used as size standard.

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Restriction

Endonucleases

Phage designation

Ae

c-1

Ae

c-2

Ae

c-3

Ae

c-4

Ae

c-5

Ae

c-6

Ae

m-1

Ae

m-2

Ae

m-3

Alu-I - - - - + + - - -

Asc-I - - - - - - - - -

Ava-I - - - - - - - - -

BamH-I - - - - + + - - -

Ban-II - - - - + + - - -

Bgl-I - - - - + + - - -

Bgl-II - - - - + + - - -

BstX-I - - - - + + - - -

Cla -I - - - - + + - - -

Dra-I + + + + - - + + +

EcoR-I - - - - + + - - -

EcoR-V - - - - - - - - -

Hae-III - - - - + + - - -

Hinc-II - - - - + + - - -

Hind-III - - - - + + - - -

Hinf-I - - - - + + - - -

Kpn-I - - - - + + - - -

Mlu-I - - - - + + - - -

Not-I - - - - + + - - -

Pst-I - - - - - - - - -

Sac-I - - - - + + - - -

Sau3A-I - - - - + + - - -

Sma-I - - - - + + - - -

Xba-I - - - - + + - - -

Note: ‘+’ sign indicates that the phage DNA was susceptible to that restriction endonuclease digestion and

‘-‘ sign indicates that the phage designated was resistant to that restriction endonuclease digestion.

Table 3.8: Susceptibility of phage DNAs to different restriction endonucleases.

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3.8 Genomic Size Determination of the Isolated Phages

Genomic size of the isolated Aeromonas phages were estimated by taking sum

of all fragments of the phage DNA produced by respective restriction endonuclease

digestion. The genomic size of the phages was approximated. There was no restriction

enzyme found in this study that can generate fragments only within the range of

marker. So approximate size of the phage genome was determined by ‘Alpha

Imager’software(Alpha Innotech) (Fig 3.12 and Fig 3.13).

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3.9 Genotyping of Aeromonas Phages By Cross Hybridization Study

Cross-hybridization study was conducted to identify any genetic relationship

among the isolated Aeromonas phages. DNAs of all phages except Aec-5 and Aec-6

were digested with Dra-I. DNAs of Aec-5 and Aec-6 were digested with Hind III.

Digestion product was fractionated on a 0.8% agarose gel and transferred to nylon

membrane. Southern blot hybridization was done using radio-labeled Dra-I digested

Aec-2 and Aec-4 phage DNA as probe (Fig 3.14 and Fig 3.15). But no cross

hybridization was occurred with the labeled phages DNA. From cross hybridization

study it has been confirmed that Aec-2 and Aec-4 are genetically distant from other

phages.

Figure 3.16: Southern hybridization of Aeromonas phage DNAs using Aec-2 total DNA as probe (a)

Restriction digestion profile of all Aeromonas phages DNA, Lane M, 1 kb plus marker (Invirogen) as

size standard; 1, Aec-1; 2, Aem-1; 3, Aec-2; 4, Aec-3; 5, Aem-2; 6, Aem-3; 7, Aec-4; 8, Aec-5; 9, Aec-6;

10, Aec-5; 11, Aec-6. Lane 1 through 9, Phage DNA was digested with Dra-I; Lane 10 and 11, Phage

DNA was digested with Hind III; (b) Autoradiograph generated from hybridization. Lane of the

labeled phage is indicated in both cases.

a) b)

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3.10 No Lysogenic Host Found for Aec-2 and Aec-4 Phage

A very common characteristic of bacteriophage is to undergo lytic to

lysogenic conversion and vice-versa. Keeping this fact in mind almost 200 colonies of

environmental and clinical strains of Aeromonas, Vibrio cholerae, E. coli, Shigella

spp. was used in making colony blots. Aec-2 and Aec-4 Phage DNA was used

seperately as probe in respective cases. But no lysogenic host was found for the

phages as there were no hybridization occurred in both cases.

Figure 3.17: Southern hybridization of Aeromonas phage DNAs using Aec-4 total DNA as probe (a)

Restriction digestion profile of all Aeromonas phages DNA, Lane M, 1 kb plus marker (Invirogen) as

size standard; 1, Aec-1; 2, Aem-1; 3, Aec-2; 4, Aec-3; 5, Aem-2; 6, Aem-3; 7, Aec-4; 8, Aec-5; 9, Aec-6;

10, Aec-5; 11, Aec-6. Lane 1 through 9, Phage DNA was digested with Dra-I; Lane 10 and 11, Phage

DNA was digested with Hind III; (b) Autoradiograph generated from hybridization. Lane of the

labeled phage is indicated in both cases.

a) b)

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From this observation it apparently could be surmised that phage Aec-2 and

Aec-4 were lytic phage in nature. They might not be able to undergo to lysogenic

state. So they might be susceptible to adverse condition and might not be able to

survive without a suitable lytic host. However, as the study could not consider all

sorts of many more strains, so it was not possible to draw a conclusion regarding the

lytic or lysogenic nature.

Figure 3.18: Colony blot hybridization of 200 different strains. Arrows showing the black spots

indicate the positive controls.

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Chapter 4 Discussion

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D i s c u s s i o n

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Aeromonads have been recognized occasionally, but only during the past three

decades has their role in a variety of human illness been documented. Bacteria of the

genus Aeromonas are considered as potential pathogen for many years. Though they

are primarily considered as aquatic organism, they have enormous host range.

Aeromonads have been reported as not only human pathogen, but also pathogens of

fish, amphibians, and reptiles. 15% of child and upto 26% of adult diarrhea are caused

by arromonas associated infection (44). Ubiquity of this organism facilitates its

exposure. Exposure to Aeromonas spp. through ingestion of food and water is

continuous. But fortunately most environmental strains do not produce

gastrointestinal disease in normal humans (117). Human immune system can resist

some degree of Aeromonas infection (15) (170). But this organism is still causing life

threatening infection to immune compromised patients and wound infection cases

(102). Chronic exposure to high numbers of aeromonads via foods and drinking water

can cause severe illness (117).

Different species of Aeromonas is likely to vary with geographical locations

(171). A. hydrophila and A. veronii. bv. sobria are the dominant species in Australia

and Thailand (15). European and American studies have revealed that the majority of

isolates were A. caviae (15). No studies have yet conducted to determine the

prevalence of aeromonas species before this study. This study demonstrates that A.

caviae and A.media are the dominant species in Dhaka, Bangladesh. However, A.

hydrophila and A. sobria were also isolated in significant numbers.

In third world countries like Bangladesh, prevalence of Aeromonas is a

concerning problem. Because most of the peoples of this country live in unhygienic

condition and they have also lack of basic health knowledge. From this study, it was

found that there is a high prevalence of Aeromonas in aquatic environment of Dhaka

throughout the year but no significant seasonality was observed. Though these water

resources are not directly used as drinking water, a huge portion of people use these

raw water for their bathing, household activities and in food processing. So they are

chronically exposed to Aeromonas continuously. Consistent to this study, in a study of

ICDDR’B it was reported that patients of gastroenteritis due to Aeromonas infection

were frequently occurred throughout the year (44). Though only a few diarrheal

outbreaks have been attributable to aeromonads, its infection is of concern because of

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ubiquity of occurrence. Aeromonas spp. as significant human pathogens is also causing a

variety of extra-intestinal infections (15) (117).

Distant ribotyping band pattern was observed among the environmental

Aeromonads isolates. From the dendrogram, the diverse clonal relationship among the

environmental Aeromonas isolates can be easily demonstrated which is consistent to

the previous reports as well. Almost all of the environmental isolates show high clonal

variation with the clinical strains. So it can be predicted that most of the

environmental isolates are not clinically significant.

Antibiotic resistance among bacteria is increasing both in developing as well

as in developed countries (172). During the last few decades, the frequency and

spectrum of antibiotic resistant infections have increased worldwide (173).

Aeromonas spp. are inherently resistant to ampicillin. In this study a different pattern

of antibiotic resistance among Aeromonas spp.have been found. Resistance to SXT

(Sulphomethoxazole-Trimethoprim) which is occurred due to presence of SXT

element common in Vibrio cholerae O1 serotype was observed among some

environmental isolates of aeromonads. Same pattern of antibiotic resistance was also

found in clinical strains of Aeromonas which is a matter of concern. Genus Vibrio and

Aeromonas have close phylogenetic relationship. Multiple antibiotic resistant Vibrio

have been reported in several studies. So there may have chance of transfer of

antibiotic resistance from Vibrio to Aeromonas.

Bacteriophages have played an important role in laboratory research in the 20th

century. Studies of pahges brought revolutionary changes in our understanding of

molecular genetics over the last few decades. But only a few studies have been

conducted on phages of Aeromonas and only a little is known about these. From the

studies of phages of other micro-organisms it has known that phages have important

role in evolution of bacteria (174). So, studies of pahges of Aeromonas have been a

subject of great interest. In the recent time bacteriophages specific for vibrio cholerae

were found to occur amazingly in large number in the environment of Bangladesh

(168) (169) (175), which prompted to search for new Aeromonas specific phages and

examine their reservoir in the environmental resources.

Nine Aeromonas phages had been isolated from environmental surface water

of Dhaka city which were designated as Aec-1, Aec-2, Aec-3, Aec-4, Aec-5, Aec-6,

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Aem-1, Aem-2 and Aem-3. During study period concentration of all nine phages

varied sharply from month to month. Isolated nine phages were different and it was

confirmed by their lytic pattern, host range and genetic variation (restriction profile)

and genomic size.

Prevalence of Aeromonas phages were found to be independent of seasonality.

This result was consistent to the prevalence of Aeromonas spp. in environment. Since

lytic phages are amplified in their respective bacterial host in proper environment and

kill their hosts, so there must be a relationship between the concentration of

Aeromonas and Aeromonas phages in the environmental waters. By comparing the

prevalence of both Aeromonas spp.and Aeromonas phages it can be speculated that an

inverse correlation exist between their occurrences. Monitoring Aeromonas phage

concentration in the environmental water as an indirect tool to trace the presence of

Aeromonas spp. is much easier than the conventional methods.

The genomes of seven out of 9 isolated phages were found to be resistant to

many restriction enzymes. These genomes were not digested by 23 out of 24

restriction enzyme used in this study. Many of these restriction enzymes were even 4

cutter. This data suggests that the genome of these phages either somehow acquired

both ‘dam’ and ‘dcm’ methylase activity or frequent occurrence of unusual bases in

their genome.

From the lytic pattern study of the isolated phages it was found that all the

phages are genus specific. So from this data it can be speculated that phages of

Aeromonas differs sufficiently from phages of other species.

Studies of phages of other organism indicate that bacteriophages have

important role in horizontal gene transfer. For example: CT and TCP pathogenecity

island are acquired by Vibrio cholerae by a filamentus phage, named CTXφ (176). In

this study it was found that the genomes of the isolated phages were larger compared

to other bacteriophages. So these phages may have role in phage mediated gene

transfer.

All nine Aeromonas phages formed unique plaques on their respective host

strains (Fig 3.11 and Fig 3.12).These also help to distinguish the isolated phages

visually. Plaque morphology of these phages is very simple identification

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characteristics. Among the isolated Aeromonas phages Aem-1 nad Aec-2 produce

lytic plaque on primar host strain and also formed turbid plaques on some of other

Aeromonas strains It was already demonstrated that lysogenic bacteriophages form

turbid plaques on their respective host strains, since it was assumed that Aem-1 and

Aec-2 phages have the capability of lysogenization in those bacteria.

Bacterial resistance to antibiotics has become a serious medical problem.

Multiple antibiotic resistance in Aeromonas have been reported to many studies (117)

(171). Although the spread of antibiotic resistance has long been known as a

worldwide phenomenon, research seems to have reached a dead end. During the last

30 years, no new classes of antibiotics have been found. Pharmaceutical companies

have mainly focused on the development of new products derived from the known

classes of antibiotics.

Several reports showed that lytic phages could be used to cure severe bacterial

infections (177) (178). As aeromonads are primarily aqutic species, lytic phages also

could be used to control Aeromonas spp in aquatic reservoir. Each kind of bacterium

hosts only its own, specific phages. These phages can be found from wherever that

particular bacterium grows. Thus, phages can be selected and isolated as an antidote

from sewage, feces, soil, or springs. Further processing of bacteriophages depends on

the intended treatment. As phage therapy is safe and highly effective so therapy with

specific bacteriophages can be used to treat bacterial infections, in which a routine

antibiotic therapy is failed. Phage therapy may be applied to all patients from whom

isolated bacterial strains show full sensitivity to specific phages.

Unlike antibiotics, bacteriophages are self-replicating as well as self-limiting.

Bacteriophages replicate exponentially as long as the specific bacteria are available in

abundance. With a decreasing amount of bacteria, the number of phages declines too

and they are gradually eliminated from the patient and from the environment. Bacteria

will develop resistance to phages too. However, since phages have a higher mutation

and replication rate, they can overcome the adaptation of the bacteria, and

development of resistance is therefore limited.

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Chapter 5 Conclusion

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In conclusion it had been revealed that Aeromonas species are highly

prevalent in the surface water in and around Dhaka city. Their prevalence was

fluctuated with time throughout the year and no significant seasonality was observed.

The environmental isolates of Aeromonas species are genetically distant from

clinical isolates. So, most of the environmental isolates are not sufficiently pathogenic

to human. Multiple antibiotic resistant Aeromonas species found to be occurred in

both environmental and clinical cases.

Phages of Aeromonas species also occur highly in environment. An inverse

correlation exists between prevalence of Aeromonas species and their phages in

environmental surface water. So, prevalence of Aeromonas species in environment

can be easily monitored by monitoring prevalence of phages.

The isolated phages are highly genus specific. So these phages can be used to

design phage therapy in Aeromonas infections.

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Chapter 6 Rreference

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Appendices

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LIST OF ABBREVIATION

µl Microliter

AC Adenylate cyclase

ADA Ampicillin Dextrin Agar

ADP Adenosine Diphosphate

ADPR Ribosyl Adenosine Diphosphate

ATP Adenosine Triphosphate

bp Base pairs

BSA Bovine serum albumin

cAMP Cyclic Adenosine monophosphate

º C Degree Celsius

DNA Deoxy ribonucleic acid

DNase Deoxy ribonuclease

EDTA Ethylene diamine tetra acetate

GA Gelatin agar

GM Ganglioside

gm gram

HCl Hydrochloric acid

hrs Hours

ICDDR,B International Centre for Diarrhoeal Disease Research,

Bangladesh

kb Kilobase pairs

L Litre

LA Luria agar

LB Luria Bertani

LSD Laboratory Science Division

M Molar

mins minutes

mg Milligram

ml Milliliter

mM Milli molar

NaCl Sodium chloride

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NaOH Sodium hydroxide

O/N Over night

ORF Open Reading Frame

PBS Phosphate buffer saline

pfu plaque forming unit

RNase Ribonuclease

rRNA Ribosomal RNA

rpm Rotation per minutes

RE Restriction endonuclease

R/T Room temperature

SDS Sodium dodecyl sulfate

SSC Saline sodium citrate

SM Sodium- Magnesium

TBE Tris borate EDTA

TCP Toxin - coregulated pilus

TE Tris EDTA

TES Tris EDTA sodium

TTGA Taurocholate Tellurite Gelatin Agar

USEPA United States Environmental Protection Agency

UV Ultra Violet

V. Cholerae Vibrio Cholerae

V. Vibrio

WHO World Health Organization

% percent

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