Dr d p rajani

SCREENING, CHARACTERIZATION AND RESISTOGRAMSTUDIES OF PATHOGENIC MICRO-ORGANISMS

FROM DIFFERENT LABORATORY SPECIMENS

PRESENTED BYDHANJI P. RAJANI

GUIDEDR. Y. A. SHELAT

Ex - Head, Research Guide & P. G Associate Professor of Microbiology,Ex. Co-ordinator: P.G.D.M.L.T Microbiology Department,

Sir P.P Institute of Science,Bhavnagar .

INTRODUCTIONMicrobes may be the most significant life forms sharing this planet with humans because of their pervasive presence and their utilization of any available food source, including humans whose defenses may be breached.

Infectious diseases are still major cause of morbidity and mortality worldwide.

According to WHO’s report of more than 17 million out of 52 million deaths were due to infectious diseases. On the contrary 30 new infectious diseases were recognized in the world during last 20 years, (WHO. 2010).

Yet the high death toll from these infectious diseases is only a part of story. Ingoing ill-health is one of the main reasons why poor stay poor.

There are diverse group of organisms causing various infections of human anatomical systems like central nervous system, respiratory system, gastro intestinal system, reproductive system, urinary system and even skin infections.

Numerous clinical specimens like urine, blood, sputum etc. containing enormous amount of infectious microbial flora, which are causative agents of dangerous communicable diseases.

These samples are tested in microbiological laboratories, to present data which indeed is helpful in the prevention, diagnosis and treatment of human diseases. Particularly for nosocomial infections these type of testing is useful in cases of antibiotic treatment failure against resistant microorganism. Resistance to antimicrobial agents has been recognized since the dawn of antibiotic era. Paul Ehrlich, the father of modern chemotherapy, observed that, during treatment of trypanosome infection, organism sometimes emerged were resistant to agents being used.

Ehrlich observed that resistance, once acquired, was stably inherited and in 1908 proposed that resistance was due to “reduced avidity of chemoreceptor so that they are no longer able to take up” the drug (Ehrlich et al. 1909).

Earlier, resistance was categorized as either natural or acquired. For example, natural resistance to gentian violet was a property of Gram negative as compared to Gram positive organisms

The natural resistance of Gram negative agents was attributed to an outer membrane barrier. (Nikaido 1996). Acquired resistance properly involved reduced susceptibility of an organism that was previously more sensitive to drug.

Plasmids carry genes for resistance to many other antimicrobial agents. Some genes codes for enzymes that modify or inactivate the agents, others for enzymes that alter drug targets in the cell or provide alternate biosynthetic pathways. Genes for antibiotic efflux (chloramphenicol, tetracycline) were also found to be plasmid determined.

Antibiotics resistant mechanism.

Antibiotic resistance is an inevitable consequence of antibiotic use. Surveys have shown that as much as 50% of all antimicrobial use is inappropriate (Livermore , 2009).

Determining the drug resistant in terms of quantity and quality is a real need of village, city or district.

In short, our country needs to (i) Spread knowledge regarding antibiotics from authoritative sources rather than

commercial pamphlets. (ii) Standardize antibiotic resistance testing to ensure comparability.(iii) Have mandatory institutional mechanisms to regular antibiotic prescription and control

drug resistance.(iv) Have a national policy for treating community infections.(v) Establish a National Institute to study antibiotic resistance in nation as a whole.

AIMS & OBJECTIVES

To find out the prevalence of pathogens in various clinical samples.

To study the resistogram of pathogenic micro organisms.

To study the prevalence of infection caused by pathogenic bacteria including Mycobacteria.

To study the identification and cultivation of pathogens

To study the drug resistance pattern of routinely isolated organism from laboratory specimen.

To study incidence of fungus infections on the basis of fungal isolates from various clinical specimens.

METHODOLOGY Present study was carried out between August 2007 to August 2009We had analyzed totally 6372 different samples such as urine, blood, pus, sputum, body fluids, stool, semen and swabs from wound and throat.All clinical samples used for the study were collected as per WHO guideline and as per mentioned by the standard book (Isenberg, 2nd edition. 2007.)Before screening for their morphological characteristics the samples urine, stool, sputum, pus, body fluids and semen were analyzed for their physical characteristics. The screening of samples was carried out by Gram’s staining technique reported their characteristics.

Sample Collection Tray & Container

All samples were streaked on differential media and moderately selective or highly selective media i.e. Mac-conkey’s agar, sheep blood agar, Cysteine Lactose Electrolyte-Deficient Agar, brain heart infusion, Thioglycollate broths , chocolate agar, Xylose lysine deoxycholate agar, tellurite agar, selenite F broth , alkaline peptone water, TCBS agar.

Then incubate at 37 C for 24 to 48 hrs aerobically.

The isolates collected from various selective and differential medium were further characterized. Isolates were identified by morphological and biochemical characteristics as per standard guidelines.

Antibiotic Susceptibility test:

Antibiotic susceptibility tests were carried out by Kirbey-Bauer disk diffusion technique.

The plates were then incubated at 370C for 24 hours. After 24 hour’s incubation, each plate was examined and the diameters of the zone of inhibition were noted using a zone reader scale for antimicrobial disc and result interpreted as per CLSI guidelines.

MRSA detection: A direct colony suspension of each S. aureus isolate was prepared to a 0.5 McFarland standard and plated on Mueller-Hinton agar containing 2-4% NaCl. An oxacillin (1 µg) and 5µg methicillin discs were placed on the surface and incubated at 35°C for 24 hours. Zone of inhibition were measured following incubation.

ESBL DETECTION: All the gram negative lactose fermentor isolates under study were also tested for their extended spectrum β-Lactamase production. Combination Disk diffusion method was used to confirm ESBL production by gram negative lactose fermentor isolates. Plates are inoculated as per the standard disc diffusion method as recommended in CLSI guidelines).Zone of inhibition were measured following overnight incubation aerobically at 37°C.The test organism was regarded as an ESBL producer if the zone of inhibition around the combination disc is at least 5mm larger than that of the cephalosporin alone, or if the zone diameter is expanded by 50% in the presence of the clavulanic acid regardless of zone diameters.

Isolation and cultivation of fungi: Samples suspected to be having fungal pathogen were collect in sterile containers.They were inoculated into the Sabouraud dextrose agar.The inoculated plates were sealed with gas permeable tape and incubated at 22 0 c

for up to 15 days. Primary plates were read daily for the first week and every other day for the second

week. When growth appears, differentiation between yeast and filamentous forms was done by microscopic examination. (Isenberg. 2007).

Isolation and cultivation of Mycobacterium tuberculosis: We have analyzed sputum, pus, urine and some fluid samples suspected to be

collected from patients suffering from tuberculosis. The smears prepared from untreated samples were subjected to ZNCF stain and

results were noted as per RNTCP gradation. The sputum and pus samples were subjected to pretreatment for digestion and

decontamination by Petroff’s method (Paramshivam CN, 1998), and cultured on the L.J medium. (Khatri GR et al., 2002).

They were incubated at 370 c for up to 8 week. Cultures were examined starting from 1st week up to 8th week.

Isolated colonies were studied in detail for their morphological, cultural and biochemical characteristics. All positive cultures were further tested for drug susceptibility towards first line drugs (rifampicin, isoniazid, streptomycin, ethambutol,) by 1% proportion method. (SOP for Mycobacteriology laboratory, 2007).

Sr.no Sample No. of Samples

1 Urine 24522 Blood 22763 Pus 10334 Wound swab 575 Body fluid 2036 Sputum 1417 Stool 918 Abscess 719 Throat swab 3710 Semen 11

Total 6372

Distributions of number of sample

Sr.no

Sample Male Female Total

1 Urine 1466 986 24522 Blood 1024 1252 22763 Pus 658 375 10334 Body fluid 118 85 2035 Sputum 90 51 1416 Stool 54 37 917 Abscess 39 32 718 Throat swab 22 15 379 Wound Swab 21 36 5710 Semen 11 0 11

Distribution based on Gender wise and sample type

Urine

Blood Pu

s

Body

fluid

Sput

umSt

ool

Absce

ss

Thro

at swab

Wou

nd sw

ab

Sem

en0

10

20

30

40

50

60

70

80

90

100

% male%female

38%

36%

16%

1%

3% 2% 1% 1%1% 0%

Urine Blood Pus Swab Body fluid Sputum Stool

Abscess Throat swab Semen

Sr. no.

Sample Pure Mix No growth

1 Urine 1437 29 986

2 Blood 574 0 1702

3 Body fluid 25 3 175

4 Semen 7 0 4

Screening of pathogen isolated from sterile clinical samples

Urine Blood Fluid Semen0

102030405060708090

% pure%Mix% no growth

Sr.no. Sample Pure Mix No growth

1 Pus 663 10 364

2 Sputum 96 38 7

3 Stool 84 5 2

4 Abscess 17 0 54

5 Throat swab 31 6 0

6 Wound Swab 30 0 23

Screening of pathogen isolated from non sterile clinical samples

Pus

Sput

umSt

ool

Absce

ss

Thro

at swab

Wou

nd S

wab

0

10

20

30

40

50

60

70

80

90

100

% pure%Mix% no growth

Sr.no

Sample Gram-veGram

+veAcid fast

bacilliFungal*

Total

1 Urine 1282 124 0 60 14662 Blood 347 173 0 54 5743 Pus 260 285 7 14 5624 Body fluid 23 27 4 1 525 Sputum 50 67 25 24 1486 Stool 83 0 0 6 897 Abscess 5 12 0 0 17

8 Throat swab 5 31 0 1 379 Swab 13 11 0 3 2710 Semen 1 6 0 0 7

Morphological characteristic of bacterial isolates from diverse clinical samples

(*) Gram resistant yeast culture or fungal spore.

UrineBlood

PusBody fluid

SputumStool

AbscessThroat swab

SwabSemen

0% 20% 40% 60% 80% 100%

Gram-ve

Gram +ve

Acid fast bacilli

Fungal

Sr.no Organism Urine

1 Escerichia coli 945

2 Klebsiella spp. 229

3Pseudomonas aeruginosa

90

4 Proteus spp. 18

5 Group D Enterococci 69

6 Staphylococcus aureus 53

7 Streptococci spp. 2

8 Candida spp. 60

Distribution of pathogens isolated from urine samples

Esce

richi

a co

li

Klebs

iella

spp

.

Psed

omon

as a

erug

inos

a

Prot

eus sp

p.

Group

D E

nter

ococ

ci

Stap

hylo

cocc

us a

ureu

s

Stre

ptoc

occi

spp.

Candi

da sp

p.0

10

20

30

40

50

60

70

Sr.no Organism Blood1 Escherichia coli 1912 Klebsiella spp. 1163 Pseudomonas aeruginosa 244 Salmonella typhi 25 Acinetobacter spp. 146 Staphylococcus aureus 1437 Coagulase negative staphylococci 23

8Methicillin resistant staphylococcus aureus 7

9 Candida spp. 54

Distribution of pathogens isolated from blood samples

Distribution of pathogens isolated from body fluid samples

Sr.no Organism Fluid

1 Escherichia coli 10

2 Pseudomonas aeruginosa 9

3 Klebsiella spp. 4


5 Candida spp. 1

36%

32%

14%

14%

4%

Escherichia coliPseudomonas aeruginosaKlebsiella spp.Staphylococcus au-reusCandida spp.

Distribution of pathogens isolated from sputum samples

Sr. no Organism Sputum






6 Candida spp. 23

7 Aspergillus spp. 1

17%

11%

8%

43%

4%

16%

1%

Escherichia coliPseudomonas aerug-inosaKlebsiella spp.Strptococci spp.Staphylococcus au-reusCandida sppAspergillus spp

Distribution of pathogens isolated from stool samples

Sr.no Organism Stool



3 Salmonella typhi 2

4 Candida spp. 662%

29%

2%7%

Escherichia coliKlebsiella spp.Salmonella typhiCandida spp.

Distribution of pathogens isolated from abscess samples

Sr.no Organism Abscess


2 Klebsiella spp. 2


4 Acinetobacter spp, 1


6 Coagulase negative staphylococci 1


6%

12%

6%

59%

6%

6%6%

Escherichia coliKlebsiella spp.Pseudomonas aerug-inosaStaphylococcus au-reusStreptococci spp.Coagulase negative staphylococciAcinatobactor spp.

Distribution of pathogens isolated from throat swab samples

Sr.no Organism Throat swab


2 Klebsiella spp. 1



5 Candida spp. 1

11%3%

81%

3% 3%

Pseudomonas aeruginosa

Klebsiella spp.

Streptococci spp.

Staphyloccous aureus

Candida spp.

Distribution of pathogens isolated from semen samples

Sr. no Organisms Semen



Distribution of pathogens isolated from pus samples

Sr. no Organisms Pus




4 Proteus mirabilis 1

5 Proteus vulgaris 1



8 Coagulase negative staphylococci 13

9 Methicillin resistant staphylococcus aureus 5

10 Candida spp. 14

Escherichia coli

Klebsiella spp.

Proteus vulgaris

Staphylococcus aures

Methicillin resistant staphylococcus aureus

0 5 10 15 20 25 30 35 40 45

Distribution of pathogens isolated from wound and other swab samples

Sr. no Organisms Swab


2 Klebsiella spp. 2




6 Methicillin resistant staphylococcus aureus 1

7 Candida spp. 3

33%

7%7%

30%

7%

4%11% Escherichia coli Klebsiella spp.

Pseudomonas aerug-inosa

Staphyloccous au-reus

Streptococcia spp. Methicillin resistant staphylococcus au-reus

Candida spp.

Results of the ESBL detection from all isolated microorganisms

Name of Antibiotic

Symbol Positive Negative Positive (%) Negative (%)

Ceftazidime/Clavulanic acid CAC

558 1269 30.54 69.46

Cephotaxime/Clavulanic acid CEC

608 1219 33.27 66.73

Results of the MRSA detection from all Staphylococcus aureus isolated organisms

Name of Antibiotic Symbol Resistant Sensitive Resistant (%) Sensitive (%)

Methicillin M 13 528 2.4 97.6

Antibiogram/resistogram study of Escherichia coli isolated from all clinical samples

Genta

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Co-tri

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Norflo

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0

20

40

60

80

100

120

% S% R

Genta

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Netro

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Amikac

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Co-tri

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azole

Nitrof

urad

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Norflo

xacin

0

20

40

60

80

100

120

% S % R

Antibiogram/resistogram study of Pseudomonas aeruginosa isolated from all clinical samples

Antibiogram/resistogram study of Klebsiella spp. isolated from all clinical samples

Genta

myc

in

Netro

myc

in

Amikac

in

Tobr

amyc

in

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Mer

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cin

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Doxyc

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Minoc

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l

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l.

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Amox

y+cla

vu.

Co-tri

mox

azole

Nitrof

urad

entin

e

Norflo

xacin

0

20

40

60

80

100

120

% S

% R

Genta

myc

in

Netro

myc

in

Amikac

in

Tobr

amyc

in

Imep

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Mer

open

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Cefaz

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Ampicil

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Cipro

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cin

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cin

Lom

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Doxyc

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Minoc

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mox

azole

Nitrof

urad

entin

e

Norflo

xacin

0

10

20

30

40

50

60

70

80

90

100

% S% R

Antibiogram/resistogram study of Salmonella typhi isolated from all clinical samples

Antibiogram/ resistogram study of Proteus spp. isolated from all clinical samples

Genta

myc

in

Netro

myc

in

Amikac

in

Tobr

amyc

in

Imep

enem

Mer

open

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Cefaz

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Cefap

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0

10

20

30

40

50

60

70

80

90

100

% S

% R

Genta

myc

in

Netro

myc

in

Amikac

in

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amyc

in

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open

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y+cla

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mox

azole

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urad

entin

e

Norflo

xacin

0

10

20

30

40

50

60

70

80

90

100

% S

% R

Antibiogram/ resistogram study of Acinetobacter spp. isolated from all clinical samples

Antibiogram/resistogram study of Staphylococcus aureus isolated from all clinical samples

Genta

myc

in

Imep

enem

Mer

open

em

Cefur

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e

Cefot

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e

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lin g

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y+cla

vu.

Met

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cin

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ycin

Clinda

myc

in

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urad

entin

e

Norflo

xacin

0

20

40

60

80

100

120

% S% R

Genta

myc

in

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Mer

open

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Cefur

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e

Cefot

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Clinda

myc

in

Nitrof

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e

Norflo

xacin

0

20

40

60

80

100

% S% R

Antibiogram/resistogram study of Streptococci spp. isolated from all clinical samples

Antibiogram/resistogram study of Coagulase negative staphylococci isolated from all clinical samples

Genta

myc

in

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enem

Mer

open

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Cefot

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lin g

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Amox

y+cla

vu.

Met

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Cipro

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cin

Ofloxa

cin

Lom

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acin

Levo

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Doxyc

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Minoc

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Tetra

cycli

ne

Teico

panin

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ine

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olid

Rifam

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Azith

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ycin

Clarit

hrom

ycin

Clinda

myc

in

Nitrof

urad

entin

e

Norflo

xacin

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

% S% R

Genta

myc

in

Imep

enem

Mer

open

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Cefur

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e

Cefot

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e

Ceftri

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lin g

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Amox

y+cla

vu.

Met

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Cipro

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cin

Ofloxa

cin

Lom

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Levo

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Doxyc

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Minoc

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ne

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ine

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Rifam

picin

Azith

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ycin

Clarit

hrom

ycin

Clinda

myc

in

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

% S% R

Antibiogram/resistogram study of Methicillin resistant staphylococcus aureus isolated from all clinical samples

Antibiogram/resistogram study of Group D Enterococci isolated from all clinical samples

Genta

myc

in

Imep

enem

Mer

open

em

Cefur

oxim

e

Cefot

axim

e

Ceftri

xone

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Penicil

lin g

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lin

Amox

y+cla

vu.

Met

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lin

Cipro

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cin

Ofloxa

cin

Lom

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acin

Levo

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cin

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acin

Doxyc

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Minoc

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Tetra

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ne

Teico

panin

Vanc

omyc

ine

Linez

olid

Rifam

picin

Azith

rom

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Clarit

hrom

ycin

Clinda

myc

in

Nitrof

urad

entin

e

Norflo

xacin

0

20

40

60

80

100

120

% S% R

Sr. no Sample Yeast Mould01 Sputum 20 302 Pus 29 103 Urine 60 004 Body fluid 1 005 Blood 54 006 Stool 6 007 Throat Swab 1 0

Distribution of fungal pathogen isolated from Clinical samples

Distribution of Mycobacteria tuberculosis isolation from clinical samples

Sr.no Sample No. of sample

01 Sputum 43

02 Pus 30

03 Urine 09

04 Body fluid 08SPUTUM PUS URINE FLUID

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

PERCENTAGE [%]

Acid fast bacilli gradation wise distribution as per Revised National Tuberculosis Control Programme

Sr. no Gradation No. of samples01 1+ 1502 2+ 1503 3+ 605 Total 36

1+

2+

3+

SCANTY

0.00%5.00%

10.00%

15.00%

20.00%

25.00%

30.00%

35.00%

40.00%

45.00%

PERCENTAGE (%)

Distribution of Mycobacterium spp. based on biochemical test

Organism No. of culture Biochemical test

PNB CATALASE NIACIN

Mycobacterium tuberculosis

32 - - +

Mycobacteria other than tuberculosis

01 + + -

M.tuberculosisMOTT

Sensitivity pattern of Mycobacterium tuberculosis

Sr.no

Group No. of strain

01 Multi Drug Resistant 13

02 Sensitive to all Drugs 02

03 Mono Drug & Other than multi drug resistant

18MDR

SENSITIVE TO ALL DRUGS

MONO DRUG & OTHER THAN MDR

0 2 4 6 8 10 12 14 16 18

NO.OF STRAIN

Multi Drug Resistant patterns (13 strains)

Drugs No. of strains

HR 4

HSR 3

HER 2

HESR 4

TOTAL 13HR HSR HER HESR

0.00%

5.00%

10.00%

15.00%

20.00%

25.00%

30.00%

35.00%

PERCENTAGE(%)

CONCLUSION We can conclude that various gram negative organisms causing urinary tract infection, respiratory tract infection, genital tract infection, blood stream infection, gastro intestinal infection, pyogenic infection and cerebrospinal and other effusion were reported.

We found staphylococcus and streptococci spp. as gram positive isolates causing various infections in our body. Our result shows that the gram negative bacteria were highest among all bacterial pathogens where as the fungal pathogens were meager in number.

In case of uropathogens majority of them were gram negative bacteria and Escherichia coli found to be prevalent. Samples collected in respiratory tract infection like sputum shows majority of gram positive organisms i.e. Streptococci spp.

In the present study the pathogens isolated from blood causing bacteremia were several bacteria as well as fungi .The prevalence found among them was of Escherichia coli but several fungal pathogens like Candida spp. was also reported. Stool samples collected in gastro intestinal infection shows the dominating organism was Escherichia coli.

In our study we found Escherichia coli and Pseudomonas aeruginosa were highest in number from body fluid samples. The only prevalent organism found in case of abscess and semen was gram positive isolate i.e. Staphylococcus aureus but Streptococci spp. was found more in number in case of throat swab samples.

In pyogenic infection gram positive dominating isolates was Staphylococcus aureus, while in case of wound and other swab gram negative bacteria Escherichia coli was prevalent.

Finally we can conclude that Escherichia coli were found to be the most prevalent isolates among various clinical specimens.

The main objective of present study was to find out resistogram among the microbial pathogen. We found Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus Klebsiella and Streptococci spp. showing highest resistance among all clinical samples from their resistogram study.

While testing the antibiotic susceptibility for various drugs recommended by Clinical Laboratory Standard Institute (CLSI), ampicillin was found to be resistant for majority of gram negative organisms.

In our study among gram negative isolates we found Escherichia coli was highest resistant for ampicillin, amoxycillin+clavunic acid, co-trimoxazole and tetracycline group, whereas Pseudomonas aeruginosa was resistant for 1st generation of cephalosporin and 2nd generation of cephalosporin , co trimoxazole and tetracycline group.

Klebsiella spp. was more resistant to penicillin group, 1st generation of cephalosporin, amoxycillin+clavunic and co-trimoxazole, while in case of Salmonella typhi only one drug was resistant i.e. ampicillin.

We have observed that for Proteus spp. more resistant drugs were ampicillin, co-trimoxazole and tetracycline group. Acinetobacter isolates were more resistant for ampicillin, tetracycline group, amoxycillin+clavunic acid, aztreonam and Cefazolin.

From gram positive pathogens Staphylococcus aureus was highest resistant for penicillin G, ampicillin, ciprofloxacin and Azithromycin, while Streptococci spp. was resistant to only one antibiotic i.e. ciprofloxacin.

Methicillin resistant Staphylococcus aureus shows more resistant to cephalosporin, tetracycline, Azithromycin group and ciprofloxacin.

In case of Group D Enterococci ciprofloxacin, Ofloxacin, Lomefloxacin, clindamycin and Azithromycin group were highest resistant.

In our study we found ESBL positive isolates were 32.% when tested with CE/CEC and CA/CAC combination. We have observed low rate of ESBL production by Enteropathogens.We also concluded that among Staphylococcus aureus majority of them were methicillin sensitive i.e. 97.6%, only 2.5% of them were methicillin resistant.

In our study we found fungal pathogen from various clinical samples and majority was Candida spp.

The mould infections were found only in case of sputum samples (13.04% ) and pus (3.33%) where as the infection with yeast was found to be nearly 100% in all other samples like urine, body fluid, stool, blood and throat swabs.

The Mycobacterium infection was found to be highest in case of sputum samples suggesting the prevalence of pulmonary tuberculosis.

REFERENCES

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Health & Medicine

Dr d p rajani