IDENTIFICATION, CHARACTERISATION AND

ANTIMICROBIAL RESISTANCE PATTERN OF

NON FERMENTING GRAM NEGATIVE BACILLI FROM

VARIOUS CLINICAL ISOLATES

Dissertation Submitted To

THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY

CHENNAI

In partial fulfillment of the regulations

For the award of the degree of

M.D. (MICROBIOLOGY)

BRANCH IV

GOVT. KILPAUK MEDICAL COLLEGE

CHENNAI

May 2018

CERTIFICATE

This is to certify that this dissertation entitled “IDENTIFICATION,

CHARACTERISATION AND ANTIMICROBIAL RESISTANCE

PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM

VARIOUS CLINICAL ISOLATES” is the bonafide original work done by

Dr.R.SHARANYA, Post graduate in Microbiology, under my overall

supervision and guidance in the Department of Microbiology, Govt. Kilpauk

Medical College, Chennai, in partial fulfillment of the regulations of The

Tamil Nadu Dr. M.G.R. Medical University for the award of M.D Degree

in Microbiology (Branch IV).

Dr.K.V.LEELA, M.D.,DGO.,

Professor & H.O.D

Department of Microbiology

Govt. Kilpauk Medical College

Chennai-600010

Dr.P.VASANTHAMANI M.D.,DGO.,

The Dean

Govt. Kilpauk Medical College

Chennai-600010.

CERTIFICATE

This is to certify that the dissertation entitled “IDENTIFICATION,

CHARACTERISATION AND ANTIMICROBIAL RESISTANCE

PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM

VARIOUS CLINICAL ISOLATES” is a bonafide research work done by

Dr.R.SHARANYA Post graduate in Microbiology, under my guidance in

the Department of Microbiology, Govt. Kilpauk Medical College, Chennai,

in partial fulfillment of the regulations of The Tamil Nadu

Dr.M.G.R.Medical University for the award of M.D Degree in

Microbiology (Branch IV).

Dr. THYAGARAJAN RAVINDER,M.D.,

Professor

Department of Microbiology

Govt. Kilpauk Medical College

Chennai-600010

CERTIFICATE

This is to certify that this dissertation work titled “IDENTIFICATION,

CHARACTERISATION AND ANTIMICROBIAL RESISTANCE

PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM

VARIOUS CLINICAL ISOLATES” of the candidate Dr.R. SHARANYA

with registration number 201414152 for the award of M.D Degree in

Microbiology (Branch IV). I personally verified the urkund.com website for the

purpose of plagiarism check. I found that the uploaded thesis file contains from

introduction to conclusion pages and result shows 6 percentage of plagiarism in

the dissertation.

Dr. THYAGARAJAN RAVINDER,M.D.,

Professor

Department of Microbiology

Govt. Kilpauk Medical College

Chennai-600010

DECLARATION

I solemnly declare that this dissertation “IDENTIFICATION,

CHARACTERISATION AND ANTIMICROBIAL RESISTANCE

PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM

VARIOUS CLINICAL ISOLATES” is the bonafide work done by me at the

Department of Microbiology, Government. Kilpauk Medical College and

Hospital, Chennai, under the guidance and supervision of,

Dr.K.V.LEELA, M.D., DGO., Professor & H.O.D of Microbiology,

Dr.THYAGARAJAN RAVINDER, M.D., Professor of Microbiology

Department of Microbiology and Dr .M. KAVITHA.M.D., Associate

Professor, Department of Microbiology Govt. Kilpauk Medical College,

Chennai - 600 010. This dissertation is submitted to The Tamil Nadu Dr.

M.G.R. Medical University, Chennai in partial fulfillment of the University

regulations for the award of Degree of M.D. Branch IV Microbiology

examinations to be held in May 2018.

Place : Chennai.

Date : Dr.R.SHARANYA

ACKNOWLEDGEMENT

My heartfelt thanks and deepest sense of gratitude to

Dr.VASANTHAMANI, M.D.,DGO., Dean, Government Kilpauk Medical

College and Hospital for giving me permission to carry out my dissertation

work and also to avail all the facilities available in the department.

I owe my gratitude to Dr.K.V.LEELA,M.D.DGO, Professor and

H.O.D., Department of Microbiology for her relentless efforts, valuable

advice, excellent guidance and encouragement given to me throughout this

study.

I am immensely grateful to Dr. THYAGARAJAN RAVINDER,

M.D., Professor, and Department of Microbiology for his constant

motivation and guidance extended to me during my study.

My sincere thanks to Dr.M. KAVITHA, M.D., Associate Professor,

Department of Microbiology for her timely advice, guidance and

encouragement in this study.

I extend my sincere thanks to Dr.K.LAVANYA, M.D

Dr.M.SUGANTHI, M.D., Dr.S. HEMALATHA,M.D., Dr.B.RAVICHANDRAN,

M.D., Dr.C.AMUTHA, M.D., Assistant Professors, Department of

Microbiology for their help, support, interest and valuable suggestions.

I also thank all my department colleagues for their timely help,

cooperation and moral support. I express many thanks to all the technical

staffs and other staff members of the Department of Microbiology for their

kind co-operation to carry out this work successfully.

I also extend my thanks to all the patients who participated in my

study. I also thank my family members for their selfless love and moral

support.

SL.NO. TITLE PAGE NO.

1. INTRODUCTION 1

2. AIMS AND OBJECTIVES 4

3. REVIEW OF LITERATURE 5

4. MATERIALS AND METHODS 34

5. RESULTS 51

6. DISCUSSION 69

7. SUMMARY 80

8. CONCLUSION 83

9. ANNEXURES

I) PROFORMA

II) APPENDIX

III) BIBLIOGRAPHY

IV) MASTER CHART

INTRODUCTION

The Nonfermentative gram-negative bacilli are a group of aerobic, non-

sporing bacilli that do not either use carbohydrates as a source of energy or

degrade them through metabolic pathways other than fermentation. Non-

fermentative gram negative bacilli account for ≥ 15% of isolates from most

clinical specimens 1 Hospital acquired infections in the acute care units are major

threat to patient safety.

Even after a decade, four nonfermenting gram-negative bacilli (NFGNB)

continue to be recognised as notorious multidrug-resistant organisms. These are

Pseudomonas aeruginosa, Acinetobacter calcoaceticus-baumannii complex,

Stenotrophomonas maltophilia and Burkholderia cepacia complex (BCC).22

Pseudomonas aeruginosa is implicated in a wide spectrum of nosocomial

infections, including bacteremia, secondary meningitis, wound infection, severe

sepsis, ocular and urinary tract infection2 These organisms seem to have a

remarkable ability to acquire antibiotic resistance genes, to persist in the hospital

environment and to spread easily from patient to patient.2. Acinetobacter causes a

wide variety of illnesses in debilitated and hospitalized patients, especially in the

intensive care units (ICUs)23

. Burkholderia Cepacia Complex, a devastating

pulmonary pathogen in Cystic fibrosis and chronic granulomatous disease (CGD)

patients, has also been reported as a cause of bacteraemia, particularly in patients

with indwelling catheters, urinary tract infection, septic arthritis, peritonitis and

respiratory tract infection22

Antimicrobial resistance is on the rise and it is a major public health

problem across the world, and especially in developing countries like India.

Infections caused by bacterial pathogens with multi drug resistant (MDR),

extremely drug resistant(XDR) and pan drug resistant phenotypes (PDR) are

challenging and difficult to treat. 9 Pseudomonas aeruginosa resistant to

carbapenem, currently the most effective treatment option is being increasingly

reported.

Resistance to carbapenems is often mediated by production of Metallo-

Beta-Lactamase (MBL), a class B type of beta-lactamases that require bivalent

metal ions, usually zinc for their activity.3 Pseudomonas aeruginosa, producing

MBLs, was first reported from Japan in 1991 and since then has been described

from various parts of the world, including Asia, Europe, Australia, South

America, and North America.3. Prompt detection and recognition of the MBLs is

important to implement adequate counter-measures to control the spread of the

organisms bearing these enzymes, and proper treatment of infections caused by

MBL-producing microorganisms.4

MBL production is a significant problem in hospital isolates of

Pseudomonas aeruginosa28

the accurate identification and reporting of MBL-

producing Pseudomonas aeruginosa will aid infection control practitioners in

preventing the spread of these multidrug-resistant isolates. Many phenotyping

methods have performed to search MBL enzymes of Pseudomonas aeruginosa

strains. All these methods are based on the ability of metal chelators, such as

EDTA and thiol-based compounds, to inhibit the activity of MBL.4

Thus,

identification, characterisation and antimicrobial resistance pattern of non

fermenting gram negative bacilli from various clinical isolates and finding MBL

is of prime importance.

AIM

To identify, characterise and detect antimicrobial resistance pattern of non

fermenting gram negative bacilli from various clinical isolates

OBJECTIVES

1. To isolate and speciate the non fermenting Gram negative bacilli

2. To characterise the non-fermenting Gram negative bacilli isolated

3. To find out the antimicrobial resistance pattern of the non-fermenting

Gram negative bacilli isolated.

4. To detect the production of extended spectrum of betalactamases.

5. To detect the acquired metallo betalactamases(MBL) by phenotypic

method of detection.

6. To identify the genes responsible for acquired MBL production

REVIEW OF LITERATURE

Nonfermenting Gram Negative Bacilli (NFGNB) are a group of

taxonomically diverse organisms growing significantly under aerobic conditions.

They all share the common phenotypic feature of failing to acidify the butt of

Triple sugar iron agar (TSI) or Kligler iron agar (KIA) agar or of oxidative-

fermentative (OF) media.7 Nonfermenters are cosmopolitan in their distribution

inhabiting soil, water, plants and animals. Their medical importance derives

principally from their being opportunistic pathogens and clinical diseases they

cause are nosocomial in origin.

Approximately 15% of all gram negative clinical isolates are nonglucose

fermenting gram negative rods. Of these, more than 2/3rds are Pseudomonas

aeruginosa5,8

, Large group of these nonfermenters have undergone confusing

taxonomic changes for many years. New definitions of species and genera using

modern genotyping analysis, together with reliable identification methods have

resulted in a better knowledge of these bacteria and a significantly increased

awareness of their pathogenic role in hospitals and in rare cases of community

acquired infections.5,6

The major genera of nonfermenting Gram negative bacilli

have been classified into atleast 15 families in addition to a number of clinically

important nonfermenters with uncertain taxonomic positions. Medically important

nonfermenters can be grouped on the basis of presence / absence of motility and

the type of flagella present in strains that are motile.6

NFGNB, normally a saprophyte, causes serious infections in

immunocompromised and hospitalized patients especially those admitted to

intensive care units (ICU). These bacteria survive for a long time in the hospital

environment and thereby the opportunities for cross infection between patients are

enhanced.23,40

Because of frequent resistance to aminoglycosides,

fluoroquinolones, ureidopenicillins and third-generation cephalosporins,

carbapenems are important agents for managing these infections. 23,43

Carbapenem

resistance is also being increasingly reported in Pseudomonas aeruginosa and

Acinetobacter baumannii.41,42,

These organisms further worsen the situation by

virtue of their multidrug resistance and thus limit therapeutic options. 30, 31

MOTILE WITH POLAR FLAGELLA

Family : Pseudomonadaceae Family Xanthomonadaceae

Genus Pseudomonas Genus stenotrophomonas

Family Burkholderiaceae Family Sphingomonadaceae

Genus Burkholderia Genus sphingomonas

Genus cupriavidus Family Oceanospirillaceae

Family Comamonadaceae Genus Balneatrix

Genus Comamonas Family Alteromonadaceae

Genus Acidovorax Genus Alishewanella

Genus Delftia Genus Shewanella

Family Caulobacteraceae Family Oxalobacteraceae

Genus Brevundimonas Genus Herbaspirillum

Family Methylobacteriaceae

Genus Methylobacterium

MOTILE WITH PERITRICHOUS FLAGELLA

Family Alcaligenaceae

Genus Achromobacter

Genus Alcaligenes

Genus Bordetella

Family Rhizobiaceae

Genus Rhizobium

Family Brucellaceae

Genus Ochrobactrum

Family Halomonadaceae

Genus Halomonas

NONMOTILE, OXIDASE NEGATIVE

Family Moraxellaceae

Genus Acinetobacter

Family Alcaligenaceae

Genus Bordetella

Organisms Whose Taxonomlc

Position is Uncertain

CDC group NO-1

CDC group EO-5

NONMOTILE, OXIDASE POSITIVE

Family Flavobacteriaceae

Genus Flavobacterium

Genus Bergeyella

Genus Chryseobacterium

Genus Weeksella

Family Sphingobacteriaceae

Genus Sphingobacterium

Family Moraxellaceae

Genus Moraxella

Genus Psychrobacter

Family Neisseriaceae

Genus Neisseria

INITIAL CLUES THAT AN UNKNOWN ISOLATE IS A

NONFERMENTER

Lack of evidence for glucose fermentation.

Positive cytochrome oxidase test.

CHARACTERISTICS OF INDIVIDUAL ORGANISMS

PSEUDOMONADS

The Genus pseudomonas and closely related genera which were formerly

placed in the Genus pseudomonas are referred to as pseudomonads.

Pseudomonads are straight or slightly curved, aerobic, gram negative bacilli

motile by means of polar flagella and utilize glucose and other carbohydrates

oxidatively and are usually cytochrome oxidase positive. 6

Molecular analysis led to revised taxonomic classification and many

species have been reallocated to new genera which includes Burkholderia,

Comamonas, Stenotrophomonas, Ralstonia and Brevundimonas.32

Palleroni

separated pseudomonads into five ribosomal RNA homology groups based on

rRNA-DNA homology studies. Gilardi on the other hand separated pseudomonads

into seven major groups based on phenotypic characteristics

Fluorescent

Stutzeri

Alcaligenes

Pseudomallei

Facilis-delafieldii

Acidovorans

Diminuta

Pseudomonas species included in rRNA group 1 includes 3 groups

Fluorescent group

Stutzeri group

Alcaligenes group

Fluorescent Group

The species within this group are characterized by the production of water-

soluble pigment pyoverdin that fluoresces white to blue-green under UV light.

This group includes Pseudomonas aeruginosa, Pseudomonas fluorescens and

P.putida. Although all 3 species produce pyoverdin, only Pseudomonas

aeruginosa produces the distinctive blue water-soluble pigment pyocyanin.6

Pseudomonas aeruginosa is the species most commonly associated with human

disease.32

there are several reasons for the prominence of Pseudomonas

aeruginosa as a human pathogen.

Its adaptability

Its innate resistance to many antibiotics and disinfectants

Its armoury of putative virulence factors

An increasing supply of patient’s compromised by age, underlying

diseases or immunosuppressive therapy. 32

Pseudomonas aeruginosa produces a characteristic appearance on Blood

agar plate (BAP) and the colonies have an alligator skin appearance and exhibits a

metallic sheen with beta-hemolysis. Rapid identification in culture can be made

by

Typical colony morphology

Production of diffusible pigments

Presence of fruity odour

Positive oxidase.

Pseudomonas aeruginosa infection is prevalent among patients with burns,

cystic fibrosis, acute leukemia, organ transplantation and intravenous drug

addicts.10

Infection commonly occurs at any site where moisture tends to accumulate

tracheotomies, indwelling catheters, burns, external ear and weeping cutaneous

wounds. Pseudomonas aeruginosa also causes urinary tract infections and lower

respiratory tract infections, the later can be severe and life threatening in

immunocompromised patients.6 The organism also causes keratitis. Analysis of

bacterial keratitis reveals that Pseudomonas species is the second most important

cause of bacterial keratitis in India after gram-positive bacteria.78

Pseudomonas aeruginosa produces several substances that are thought to

enhance the colonization and infection of host tissues. These substances, together

with the variety of virulence factors including lipopolysaccharide, exotoxin A,

leucocidin, extracellular slime, proteases, phospholipases and several other

enzymes make Pseudomonas aeruginosa the most clinically significant bacteria

among NFB. 6 An unususal mucoid morphotype of Pseudomonas aeruginosa is

recovered from respiratory secretions of patients with cystic fibrosis which is due

to the production of large amounts of polysaccharide called alginate. The

production of alginate is associated with poor prognosis and high mortality rates

among patients with cystic fibrosis. 33

Pseudomonas fluorescens and P.putida occur in water and soil and may

exist in water sources in hospital environment. Both may exist as normal

pharyngeal flora and are rare opportunistic pathogens. Both the species fail to

grow at 42ᴼC as Pseudomonas aeruginosa. They produce only pyoverdin and not

pyocyanin. Another character in which they differ from Pseudomonas aeruginosa

is that they do not deaminate acetamide.

These two species differ from each other in gelatine hydrolysis where

P.flourescens gives a positive reaction; P.putida gives a negative reaction.6

P.putida has been reported to cause catheter-related sepsis in patients with cancer

and septic arthritis.35

Treatment of Pseudomonas aeruginosa infection is difficult

because it express innate resistance to many antibiotics. An alarming increase in

resistance to various antimicrobial agents has been reported from India and

abroad.80

Increased use of broad–spectrum antibiotics, intubation of respiratory,

gastrointestinal or urinary tract and intravascular catheterization are significant

predisposing factors for development of antibiotic resistance.79

They are found to

be sensitive to aminoglycosides, anti pseudomonal penicillin, fluoroquinolones,

and third generation cephalosporins. Amikacin and ceftazidime were found to be

highly effective.80

The incidence of meropenem–

resistant Pseudomonas

aeruginosa is also increasing among nosocomially infected patients in ICU.

81 The

potential risk factors are previous antimicrobial drug exposure. A growing number

of multidrug resistant (MDR) Pseudomonas aeruginosa

producing metallo

betalactamases (MBL) is also reported. Such strains are resistant to most broad

spectrum beta-lactams, aminoglycosides and fluoroquinolones and the traditional

antipseudomonal antimicrobials.77

The

common form of drug resistance is

mediated by lack of drug penetration (porin mutation and efflux pump) and/or

cabapenem–hydrolysing betalactamases.

Based on molecular studies, carbapenem-hydrolysing enzymes are

classified into four groups A,B,C,D. The metallo betalactamases are enzymes

requiring divalent cations as cofactors for enzyme activity, being inhibited by the

action of a metal ion chelator.70

There are reports of MBL production in

Pseudomonas aeruginosa from various countries like Brazil, Korea, Singapore

and France. MBL was first reported as a zinc dependent enzyme in Bacillus

cereus in mid 1960s. A few decades later, meropenem hydrolyzing

metalloenzymes were found in Aeromonas hydrophila and Bacteroides fragilis.

All these enzymes were produced by chromosomal genes and at first

recorded only from single clinical isolates. In 1991, Japan reported the first

plasmid mediated MBL in Pseudomonas aeruginosa. Apart from Pseudomonas

aeruginosa, other bacteria like Serratia, Klebsiella pneumonia, Escherichia coli,

Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Proteus

vulgaris, P.putida, Acinetobacter and Alcaligenes xylosoxidans were also shown

to produce MBL. These carbapenems may be class B MBL(VIM,IMP) or class D

oxacillinases(OXA-23 to OXA-27) or class A clavulanic acid inhibitory

enzymes(SME,NMC,IMI,KPC). They may be chromosomally or plasmid

mediated and therefore possess a threat of spread of resistance by gene transfer

among GNB. 30

Since carbapenem resistance is mediated by several mechanisms,

cross-resistance is commonly seen among related antibiotics.

Although there are various specific tests to detect the underlying

mechanism of carbapenem resistance, Kirby-bauer disc diffusion test is a simple,

easy to perform and cost-effective test which can be conveniently used to screen

carbapenem resistance. These strains also remain resistant to several other

antibiotics including penicillins, cephalosporins, quinolones, aminoglycosides and

third generation cephalosporins including ceftazidime and cefotaxime.

Thus, they may be ESBL producers as well. These MBLs effectively

hydrolyse all betalactams except Aztreonam in vitro. This disturbing situation

could be attributed to the increased use of antibiotics which has to be controlled

by strict antibiotic policy. Various strategies such as strict infection control

measures, judicious prescribing of antibiotics, antibiotic resistance surveillance

programs and antibiotic cycling must be tried. Therefore, detection of MBL-

producing gram negative bacilli especially Pseudomonas aeruginosa is crucial for

the optimal treatment of patients particularly in critically ill and hospitalized

patients and to control the spread of resistance

PSEUDOMONAS AERUGINOSA

Pseudomonas aeruginosa is the most common organism isolated among

the nonfermenters from the clinical specimens, more often than all other

Pseudomonas species especially in teaching hospitals with more than 500 beds.10

They are ubiquitous organisms widely distributed in nature. They have emerged

as a major hospital pathogens because of their ability to grow in a variety of

environments with minimal nutritional requirements.82

Intensive care units,

Immunosuppressants, invasive procedures and antibiotic usage have provided

opportunities for emergence, persistence and transmission of Pseudomonas

between patients, from patients to staff and to inanimate reservoirs. 11

Many

carriage sites like respiratory tract, genitourinary tract and skin serve as source of

dissemination.12

The virulence is multifactorial including loss of host defence

mechanisms like immunosuppression, loss of mucosal barrier, cellular factors,

toxins elaborated by Pseudomonas aeruginosa like endotoxins, exotoxin A,

enzymes like elastases, alkaline protease and hemolysins are responsible for many

of the systemic manifestations of Pseudomonas disease.12

.In addition, the

colonies of the organism form biofilms within which they are protected from host

defenses and antimicrobial agents and communicate with each other through

complex system of cell to cell signaling called Quorum sensing.The production of

alginate and epithelial cell tropism in cystic fibrosis is associated with poor

prognosis and high mortality.10

In the National Nosocomial Infection Surveillance (NNIS) survey from the

Centres for Disease Control and Prevention (CDC), it is the fourth most common

cause of nosocomial infection and leading cause of hospital acquired infections.It

is the most common cause of wound infection caused by gram negative bacteria

with an isolation rate of upto 62%.Urinary tract infections caused by these

organisms are mostly hospital acquired and isolations range from 12%-30%. It

causes life threatening bacteremia especially in intensive care settings at a rate of

10%. Pseudomonas aeruginosa is the leading cause of pneumonia in ICU patients

with a mortality of 80 -100% Other infections caused by Pseudomonas

aeruginosa are osteochondritis, chronic suppurative otitis media, external ear

infections, meningitis following trauma and surgery, endochondritis and

peritonitis 7

IDENTIFICATION 6

Pseudomonas aeruginosa produces large flat colonies with spreading and

serrated edges witha metallic sheen. Various diffusible pigments are produced like

pyoverdin andpyocyanin. It is betahemolytic on blood agar It produces nonlactose

fermenting colonies on MacConkey agar. They are motile organisms. It is oxidase

positive, catalase positive, indole negative, citrate and urease variable. It oxidizes

glucose in OF media, reduces nitrates to nitrites, arginine is decarboxylated,

acetamide positive, ONPG negative, sensitive to Polymixin B and grows at 42ᴼ C

which differentiates it from Pseudomonas fluorescens and Pseudomonas putida.

Characteristics of fluorescent group

Test Pseudomonas

aeruginosa

Pseudomonas

fluorescens

Pseudomonas

putida

Oxidase + + +

Motility + + +

Pyoverdin + + +

Pyocyanin + - -

OF glucose A A A

Acetamide V + +

Growth at 42c + - -

Nitrate reduction V(74%) V(19%) -

Arginine + + +

+ positive, - negative ,V- variable, A - acid reaction / ( ) numbers in the

parenthesis are % of strains giving positive reactions.

ANTIBIOTIC SENSITIVITY

They are sensitive to semisynthetic penicillins like Piperacillin/Ticaricillin,

third generation cephalosporins (ceftazidime), carbapenems (imipenem and

meropenem), monobactams, aminoglycosides and fluroquinolones.13

It is

intrinsically resistant to ampicillin, amoxycillin and amoxicillin-clavulanic acid

due to an inducible chromosomal AmpC beta lactamase.14

, Multiple resistance in

these organisms is frequent, leading to the development of multidrug and pandrug

resistant Ps.aeruginosa strains caused by mutations & or production of

betalactamases ranging from extended spectrum of betalactamases to

metallobetalactamases.7

ACINETOBACTER BAUMANNI

Acinetobacter are strictly aerobic, gram negative coccobacillary rods,

widely distributed in nature and hospital environments 17,

7 They are second most

commonly isolated nonfermenters in human specimens next to Pseudomonas

aeruginosa with a prevalence of 10% of all gram negative isolates.7 They are

generally considered as nonpathogic but cause serious infections in debilitated

patients. The species most frequently isolated is Acinetobacter baumannii It is

most often responsible for hospital acquired infections.7 They are the most

common gram-negative organisms to be isolated from the hands of medical

personnel.

A study conducted by CDC has reported Acinetobacter baumannii to be

the cause of 1% nosocomial blood stream infections(CDC) A mortality of 17-

46% is associated with nosocomial bacteremia by these organisms.20

Analysis of

data from the NNIS system showed that the proportion of ICU pneumonia

episodes range from 4% -7%.14

These organisms have high rate of colonization of the trachea. Respiratory

tract is the most common site for Acinetobacter baumannii infections in ICU with

a mortality rate approaching 70%.12

Traumatic wounds, burns and postoperative surgical site infections are also

common with multidrug resistant strains being observed.16

Several reviews have described these organisms in 2-6% of nosocomially

acquired urinary tract infections. 16,

17

IDENTIFICATION6

Colonies are translucent to opaque, convex and entire with a diameter

between 0.5 and 2mm. It produces nonlactose fermenting colonies on MacConkey

agar with a pinkish tint. It is oxidase negative, nonmotile, catalase positive, citrate

positive and urease negative. It oxidizes glucose and 10% lactose and dextrose in

OF media. It does not reduce nitrates to nitrites. It deaminates arginine,

acetamide negative, ONPG negative and grows at 44ᴼC.

Characteristics of Acinetobacter

TEST A.baumannii A,iwoffi

Oxidase - -

Motility - -

Growth on Macconkey + +

OF glucose A -

Nitrate reduction - -

Citrate + v

10% Lactose + -

+ positive , - negative ,A – acid reaction.

ANTIBIOTIC SUSCEPTIBILITY 16

,6,14

They are universally resistant to penicillin, ampicillin and

chloramphenicol. They show variable susceptibility to second and third generation

cephalosporins. Recently extended spectrum of betalactamases and

carbapenemase resistance is reported in nosocomial infections.

PSEUDOMONAS FLUORESCENS

P.fluorescens is a psychrophilic organism which favours its presence in

blood products. Outbreaks of bacteremia, respiratory tract infections in cystic

fibrosis patients, wound infections, urinary tract infections and rare cases of

community acquired pneumonia have been reported. They behave as opportunistic

pathogens in immunocompromised patients.6

IDENTIFICATION 6

Colonies are large with spreading edges forming nonlactose fermenting

colonies on MacConkey agar and hemolytic colonies on blood agar. It is oxidase

positive,catalase positive, motile, oxidizing glucose, deaminating arginine,

reducing nitrates to nitrites, ONPG negative, acetamide negative, sensitive to

polymyxin B and do not grow at 42ᴼC.

STENOTROPHOMONAS MALTOPHILIA

Originally classified as Pseudomonas maltophilia, it is an obligate aerobe

and an ubiquitous organism It is an emerging opportunistic pathogen. It is the

third most common encountered nonfermenter in clinical laboratory next to

Pseudomonas and Acinetobacter.6

It is an important nosocomial pathogen associated with substantial

morbidity and mortality especially in immunosuppressed patients.It is one among

the most common causes of wound infections due to trauma. It is frequently

isolated from patients with ventilatory support in ICU. It is an important pathogen

in cystic fibrosis patients. It produces proteolytic enzymes, deoxyribonucleases,

ribonucleases, hemolysins, hyaluronidase and mucinase etc. which contribute to

its severity in immunosuppressed patients. The rate of infections caused by

Stenotrophomonas maltophilia is increased in recent years and are being isolated

from wound infections, bacteremia, pneumonia, urinary tract infections,

meningitis and peritonitis. A significant feature of Stenotrophomonas maltophilia

is its ability to adhere to plastics and form bacterial films (biofilms).

Stenotrophomonas maltophilia has been identified on the surfaces of materials

used in intravenous (i.v.) cannulae, prosthetic devices, dental unit waterlines, and

nebulizers 96,97,98,99,100,101,102

IDENTIFICATION6

Colonies formed are pale yellow / lavender green with good growth on

Blood agar and MacConkey agar. It is oxidase negative, motile, catalase positive,

indole negative, citrate variable, urease negative. It oxidizes glucose and maltose,

decarboxylates lysine, ONPG positive, with variable nitrate reduction.

Characteristics of Stenotrophomonas maltophilia and Burkholderia

cepacia complex

Test

Stenotrophomonas

maltophilia

Burholderia

cepacia complex

Oxidase - +(93)

Motility + +

Growth on Mac conkey agar + +

OF glucose A Weak A

Nitrate reduction V(42%) V(37%)

Nitrate to gas - -

Lysine + +

Polymyxin B S R

+ positive , - negative , V-variable , A – acid reaction / ( ) numbers in the

parenthesis are % of strains giving positive/ S- Susceptible / R-Resistant

ANTIBIOTIC SUSCEPTIBILITY

Therapy for Stenotrophomonas maltophilia infections is problematic

because of the broad antibiotic resistance that typifies this organism. The most

active agents are trimethoprimsulphamethoxazole, colistin and quinolones. Like

other nonfermenters it is intrinsically resistant to many common antibiotics like

aminoglycosides, carbapenems and many betalactam agents.6

BURKHOLDERIA CEPACIA

It is a motile free living phytopathogen identified as both endemic and

epidemic nosocomial pathogen.Its detection rates are low, in the range of 1%-16%

of clinical samples.It belongs to rRNA group Ie. It produces virulence factors like

proteases, lipases, exopolysaccharides and lipopolysaccharides.

A few case reports have described serious infections, including severe

pneumonia, invasive otitis and sepsis in cystic fibrosis patients. Diabetes mellitus

is a potential risk factor for development of infections.by Burkholderia cepacia .6ia

Burkholderia cepacia is also an important pathogen among patients with chronic

granulomatous disease. Like other nonfermenters, it can contaminate disinfectant

solutions The major importance of this organism lies in its role as opportunistic

agent of pneumonia in cystic fibrosis patients seeded in sputum samples.

Burkholderia cepacia complex is ambiguously reported as a non-fermenting

Gram-negative bacilli (NFGNB). Hence, there is a need for molecular

confirmation of Burkholderia cepacia complex.84

Burkholderia cepacia complex

has emerged as a serious nosocomial pathogen worldwide, due to its high intrinsic

resistance to most antibiotics, acquired resistance to fluoroquinolones and

antiseptics, besides its ability to survive in the environment for prolonged periods

with limited nutrition.23

Members of Burkholderia cepacia complex family are the

most common contaminants of many finished pharmaceutical products and

environment in which pharmaceutical products are manufactured.85

Burkholderia

cepacia complex survives, multiplies and may persist for long periods in moist

hospital environment, including detergent solutions and intravenous (IV)

fluids83,86

The spectrum of infections by these organisms includes wound infections,

bacteremia, UTI, pneumonia, meningitis, peritonitis, and endocarditis.

IDENTIFICATION6

Colonies are smooth and glistening, forming non-lactose fermenting

colonies on MacConkey agar and yellow pigmented colonies on blood agar. It is

weakly oxidase positive, catalase positive, motile, oxidizes all sugars,

decarboxylates lysine, ONPG negative, acetamide negative and resistant to

Polymixin B. Nitrate reduction is variable.

ANTIBIOTIC SUSCEPTIBILITY

As with other nonfermenters intrinsic antibiotic resistance typifies

Burkholderia cepacia and greatly complicates treatment. Trimethoprim-

sulfamethoxazole has historically been the drug of choice. Most active agents are,

ceftazidime, meropenem, ciprofloxacin and other quinolones.

ANTIBIOTIC SUSCEPTIBILITY 14

,6

It is sensitive to antipseudomonal penicillins like Piperacillin, betalactam

agents and carbapenems. It is resistant to penicillins.

WEEKSELLA VIROSA 6

Flavobactericeae comprises indole positive organisms like

Chyseobacterium, Empedobacter, Spingobacterium and Weeksella.6

Weeksella

virosa are associated with urinary tract infections.34

IDENTIFICATION6

Weeksella virosa form yellow colonies on blood agar. They are oxidase

positive and nonmotile, form indole, citrate variable and urease negative, do not

oxidizes glucose and maltose. They are nitrate negative. Weeksella is sensitive to

penicillin and polymyxin B.

SHEWANELLA PUTREFACIENS 87,88

Shewanella is a marine bacteria rarely implicated as a human pathogen.

Two species of importance are Shewanella algae and Shewanella putrefaciens. It

is an oxidase-positive, hydrogen sulphide producing Gram negative bacilli. It was

infrequently recovered from clinical specimens probably because of inadequate

processing of non-fermenting oxidase-positive gram-negative bacilli.87

Most

human isolates of S. putrefaciens occur as part of a mixed bacterial flora, clouding

their clinical significance. However, a number of monomicrobic illnesses due to S.

putrefaciens have been documented and include bacteremia, soft tissue infections,

and otitis media .93, 94, 95

IDENTIFICATION

Isolates that were motile, had oxidative metabolisms, were oxidase and

catalase positive, ornithine decarboxylase positive, and DNase positive, and

produced H2S on triple sugar iron slants within 72 h of incubation were identified

as belonging to the phenospecies S.putrefaciens.All other reactions were

uniformly positive for the Shewanella strains studied. 6

The biovar and biotype of

each isolate were determined according to Gilardi90

and Weyant et al91

respectively, based on acid production from sucrose and maltose, growth on SS

agar, and growth in the presence of 6.5% NaCl. Species designations were

determined by the criteria of Nozue et al. 92

by using the following tests:

hemolysis on sheep blood agar, growth at 42°C, growth on nutrient agar

containing 6.5% NaCl, growth on SS agar, and acid production from maltose and

l-arabinose.

Methods for Identification Using Automated Identification Systems

The Vitek Legacy System

The Vitek Legacy System (BioMérieux), , has also been used with success

in the identification of the nonfermenters most frequently encountered in the

clinical laboratory.

The Vitek 2 System

The original Vitek 2 card for gram-negative bacteria identification has been

redesigned to improve the identification of fermenting and nonfermenting bacilli.

The new card contains 47 tests (26 that had been included in the previous card and

21 new tests), compared with 41 in the established Vitek 2 ID-GNB card. The

database for the new card has been expanded to 159 taxa compared with only 101

for the original Vitek 2 card.

The Microscan Walkaway-96, Walkaway-40, and Autoscan-4 Systems

These three systems (manufactured by Beckman Coulter, West

Sacramento, CA),all have an extensive database that includes many species of

NFBs. Tenover and colleagues1064 evaluated the Walkaway-96 (formerly called

the autoSCAN-W/A) for its ability to identify 310 well-characterized non–

glucosefermenting gram-negative bacilli. In their study, two types of

identification panels were tested: the dried colorimetric Neg ID type 2 panel

(DCP) and the rapid fluorometric Neg ID panel (RFP). Problems in identifying

relatively common nonfermentative bacilli, such as Pseudomonas fluorescens, P.

putida and Stenotrophomonas maltophilia were reported with the DCP panel. The

researchers reported better results with the RFP panels.The RFP panels were

available as early as 2 hours; thus, if an organism cannot be identified, additional

biochemical tests can be inoculated on the same day, and less time is lost in

identifying the organisms.

The Sensititre AP80 System6

The Sensititre AP80 Identification panels (TREK Diagnostic Systems,

Cleveland, OH) can be inoculated and incubated offline and then read in the

Sensititre Autoreader, or can be inoculated and placed in the ARIS (Automated

Reading and Incubation System) Instrument. The AP80 panel identifies gram-

negative bacilli as early as 5 hours,

The Phoenix System

The Phoenix Automated Microbiology System (Becton Dickinson

Microbiology Systems) is a fully automated, identification and antimicrobial

susceptibility test system. 6

Methods for Identification Using Molecular Systems

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass

Spectrometry

An overview of this new technology and modern applications in the

clinical microbiology laboratory are described in a recent review. Overall

performance of MALDI-TOF MS has been reported to be significantly better than

commercially available systems for identification of the NFBs although overall

performance is still less than satisfactory. 44, 45, 46, 6

Discrepancies were refereed with 16S rRNA sequencing or whole genome

sequencing (WGS) using Illumina’s MiSeq technology. Correct identification to

the species level for Bruker RUO, Vitek RUO, and Vitek IVD was 62.1%, 48%,

and 54.3%, respectively.

Both systems gave a low number (<5%) of incorrect IDs; however, the

ability to identify NFBs correctly to species level was low for both systems.

Improvements are needed in the databases used for identification of NFBs with

both systems for accurate identification of NFBs to the species level.

16S rRNA Gene Sequencing

Due to the poor performance of commercially available systems for the

identification of NFBs and sometimes less than satisfactory performance of

MALDI-TOF MS to identify NFBs at the species level, laboratories have

increasingly turned to sequencing methods such as 16S rRNA gene sequencing to

determine the identification of clinically relevant isolates. 50,51,6

16S rRNA is a

component of the 30S small subunit of prokaryotic ribosomes.

16S rRNA gene sequences contain hypervariable regions that can provide

species-specific signature sequences useful for identification of bacteria. As a

result, 16S rRNA gene sequencing has become prevalent in medical microbiology

as a rapid and inexpensive alternative to phenotypic methods of bacterial

identification.

Phenotypic testing was performed by conventional phenotypic and

commercial methods in use at each of the participating laboratories and included

the Vitek or API 20NE systems (bioMèrieux, Durham, NC) or the MicroScan

system (Beckman Coulter, Sacramento, CA). 52,49,6

Using 16S rRNA sequencing,

92% of the isolates were assigned to species level and 8% to genus level (100%

combined). Using API 20 NE, 54% of the isolates were identified to the species

level, and 7% to the genus level (61% combined), and 39% of the isolates could

not be identified. For Vitek-2, 53% could be identified to the species level, 1% to

the genus level (54% combined), and 46% could not be identified.48,6

Resolution of 16S rRNA Gene Sequencing.

Although 16S rRNA gene sequencing is highly useful in regards to

bacterial classification, it has low phylogenetic power at the species level and

poor discriminatory power for some genera. With the NFB this is particularly true

for members of the Burkholderia cepacia complex, the Acinetobacter

calcoaceticus–Acinetobacter baumannii complex, and some members of the

genus Pseudomonas, the genus Achromobacter, the genus Bordetella, and the

genus Ralstonia.47, 48, 6

As for any identification method, limitations for 16S rRNA gene

sequencing exist and students and laboratorians should be aware of these pitfalls

when using gene sequencing for bacterial identification in the diagnostic

laboratory.47, 49, 6

ANTIBIOTIC SUSCEPTIBILITY6

They are resistant to aminoglycosides, third generation cephalosporins,

Imipenem and erythromycin. They are sensitive to ciprofloxacin and

betalactamase inhibitors.

INTRINSIC RESISTANCE15

ANTIMICROBIAL

AGENT

Am

ipic

illi

n

Pip

erci

llin

Tazo

bct

um

efo

tax

ime

Cef

ipim

e

Azt

reo

na

m

Mer

op

enem

Po

lym

ixin

B

Am

ino

gly

cosi

de

Co

trim

ox

azo

le

ceft

ria

xo

ne

chlo

ram

ph

enic

ol

Acinetobacter

baumannii

R R R

Burkholderia

cepacia complex

R R R R R R R R

Pseudomonas

aeruginosa

R R R R R

Stenotrophomonas

maltophila

R R R R R R R

MULTIDRUG RESISTANCE IN NONFERMENTING GRAM NEGATIVE

BACILLI

Nonfermenting Gram Negative Bacilli pose a particular difficulty for

healthcare community because they represent the problem of multidrug resistance

to the maximum6. They are resistant to three or more drugs and important

members of this group are Pseudomonas aeruginosa, Acinetobacter baumannii,

Stenotrophomonas maltophilia and Burkholderia cepacia 21

. They use several

mechanism of resistance including intrinsic and rapidly acquired resistance.

Intrinsic resistance is due to relative impermeability of outer membrane proteins

compared to that of other gram negative bacteria (ten fold times lower). Efflux

system also contributes to intrinsic resistance Acquired resisitance is by

mutational changes and acquisition of exogenous genetic material. Lastly

resistance may also develop during therapy turning an initially susceptible isolate

into a resistant one.13

Pseudomonas aeruginosa exhibits multidrug resistance to 4

antibiotic classes -ceftazidime, imipenem, gentamicin, and a fluroquinolone. The

increase in multidrug resistant strains suggests that therapy with compounds like

polymyxinB or colistin must be considered.14

A report in Germany revealed

multidrug resistant profiles in Acinetobacter to drugs like cefepime, ciprofloxacin

and amikacin 14

Stenotrophomonas maltophilia and Burkholderia cepacia are associated

with intrinsic drug resistance. Multidrug antibiotic resitance negatively affects

outcomes of the patients.14

. Intrinsic includes over-expression of efflux pumps

(mexAB, mexCD, mexEF and mexXY), chromosomal hyper ampC producers and

loss of porins (OprD); extrinsic includes acquisition of resistance genes such as

extended spectrum beta-lactamases (ESBLs; blaSHV, blaTEM, blaVEB, blaPER and

blaOXA types) and carbapenemases (blaGES, blaKPC, blaIMP, blaSPM, blaVIM and

blaNDM)9

EXTENDED SPECTRUM OF BETALACTAMASES

ESBL are a group of betalactamases which share the ability to hydrolyse

third generation cephalosporins and are inhibited by clavulanic acid. They are

plasmidcoded. Carbapenems are treatment of choice for serious infections due to

ESBL producing organisms. ESBLs in nonfermenters are Ambler class A. These

enzymes are SHV type, TEM type, TEM 1 and 2, CTX-M type, OXA- type ,

PER- type, VEB, BES – types and others. Screening tests for ESBL producers are

disk diffusion and dilution susceptibility testing methods. The phenotypic

confirmatory tests for ESBL production are

1. Cephalosporin / clavulanate combination disks15

2. E tests 15,21

CARBAPENEMASES AND METALLOBETALACTAMASES

Carbapenemases are betalactamases with versatile hydrolytic capacities.

They have the ability to hydrolyze penicillins, cephalosporins, monobactams, and

carbapenems. Bacteria producing these betalactamases may cause serious

infections in which the carbapenemases activity renders many betalactams

ineffective. They are members of molecular class A, B and D betalactamases.

Class A and D have serine based hydrolytic mechanisms while class B are

metallobetalactamases that contain zinc in the active site. Class D carbapenemases

consist of OXA type betalactamases frequently detected in Acinetobacter

baumanni. The metallobetalactamases belong to IMP, VIM, SPM, GIM and SIM

families and have been detected primarily in Pseudomonas aeruginosa.

Nonfermenters especially Pseudomonas aeruginosa and Acinetobacter baumannii

have acquired metallobetalactamases through genetic elements (plasmids/

transposons) and Scan be transmitted to other bacteria. These enzymes confer

resistance to all carbapenems (Imipenems, Meropenems, Ertapenems), all

betalactams, aminoglycosides and quinolones. The dissemination is thought to be

driven by regional consumption of ESBLs. Stenotrophomonas maltophilia is

naturally resistant to imipenem and meropenem because of chromosomally

mediated carbapenemase production.19

The families and subgroups of

carbapenemases known till now are IMP-1&2, VIM-1&2, SPM-1, GIM-1, and

SIM-1.

IMP was first discovered in Ps.aeruginosa in Japan.and this has spread to

other gram negative bacteria and reports show their detection in Acinetobacter

baumannii, Serratia and Klebsiella. Currently IMP family members number upto

18 in the published literature. The second dominant group of acquired MBLs is

the VIM type enzymes. It was first described in Verona Italy, from Pseudomonas

aeruginosa isolate. This family currently consists of 14 members and seen mostly

in Pseudomonas aeruginosa. It has dubious distinction of being the most reported

metallo-beta-lactamase worldwide. These genes are easily transferred on mobile

elements among species. While considered by some to be rare, reports of their

occurrence have increased.

DETECTION OF CARBAPENEMASES

1. Raise in MIC of carbapenems in the range of 8 >128 μgm / ml.

2. Microbiological test with inhibitors:

a. Disc approximation test with EDTA

b. Combined disc method: Imipenem with EDTA 18

c. E test strips with Imipenem and Imipenem EDTA combination

d. Modified Hodge test 18

Of these tests, studies conducted showed that both combined disc test and

E test were more sensitive and equally effective for MBL detection.

MOLECULAR METHOD

In this study, PCR was used to determine the gene for MBL production in

Pseudomonas aeruginosa isolates that were resistant to carbapenems PCR was

done using primers specific for MBL genes.

Cell lysates of the isolates were used as DNA template for colony lysate

PCR. Around 5 – 10 colonies were suspended in 100ml of Milli Q water & boiled

for 5 minutes. It is then centrifuged at 10,000 rpm for 10 minutes. The supernatant

provided templates for PCR reactions.

Forty amplification cycles were performed with an automated thermocycler

according to the following format: Initial denaturation for 5 min at 94ᴼC, 30

cycles of DNA denaturation for 30 s at 94.c, annealing for 30 s at 55.c and

extension for 1.5 min at 72ᴼc. The final cycle was followed by an additional 5 min

at 72ᴼc to complete partial polymerizations. Amplified products were run using

horizontal 1.5 % agarose gel electrophoresis. The gel was visualized using a UV

transilluminator. The amplified PCR products and 100 base pair DNA molecular

markers were seen as bright fluorescent bands.

INTERPRETATION

A 261 bp corresoponds to VIM and 587 bp corresponds to IMP

gene.specific oligonucleotides.

MATERIALS & METHODS

STUDY PERIOD

This cross sectional study was conducted from January 2015 to January 2016

PLACE OF STUDY

Govt Kilpauk Medical College and Hospital, Chennai.

ETHICAL CONSIDERATION

The study was approved by our Institutional Ethical Committee and Ethical

clearance was obtained

STATISTICAL ANALYSIS

All statistical analyses were carried out using SPSS for Windows. Odds

ratios (ORs) and 95% confidence intervals (CIs) were calculated. P values were

calculated using the chi-square test. A p value of < 0.05 was considered

significant.

SAMPLE

A total of 200 nonfermenting bacteria isolated from various clinical

specimens like pus, urine, blood, bronchoalveolar lavage, endotracheal aspirations

and cerebrospinal fluids collected from both outpatients and inpatients of Govt

kilpauk Medical college and hospital, Chennai were studied.

SAMPLE PROCESSING

The samples were processed according to standard procedures. The

collected samples were subjected to direct Gram stain and all specimens were

inoculated onto nutrient agar, 5% sheep blood agar and MacConkey’s agar

medium. Urine samples were also inoculated onto Cystine Lactose Electrolyte

Deficient agar (CLED) in addition.

All the catalase positive, oxidase positive and negative, nonlactose

fermenting colonies on Mac Conkey agar were provisionally identified by colony

morphology and pigment production. They were inoculated in Triple sugar iron

(TSI) agar slope. The colonies which failed to acidify the TSI agar were

considered as nonfermenters and subjected to the following tests.(annexure)

Motility, Indole, Citrate, Urease, Nitrate reduction, growth at 42ᴼ c and 44ᴼc,

Sensitivity to Polymyxin B and following special biochemical tests and grouped

according to P.C.Schreckenberger scheme6

TESTS USED FOR IDENTIFICATION OF NON-FERMENTERS6.

Positive Cytochrome Oxidase Reaction

Any colony of a GNB growing on blood agar or any other primary

isolation media that is cytochrome oxidase positive can be suspected of belonging

to NF group. The cytochromes are iron-containing hemoproteins that act as the

last link in the chain of aerobic respiration by transferring electrons (hydrogen) to

oxygen, with the formation of water.6

The cytochrome oxidase test uses certain reagent dyes, such as p-

phenylenediamine dihydrochloride, that substitute for oxygen as artificial electron

acceptors. In the reduced state, the dye is colorless; however, in the presence of

cytochrome oxidase and atmospheric oxygen, p-phenylenediamine is oxidized,

forming indophenol blue:

1. Positive control: Pseudomonas aeruginosa

2. Negative control: Escherichia coli

The test is commonly performed by one of two methods:

1. The direct plate technique, in which two to three drops of reagent are

added directly to isolated bacterial colonies growing on plate medium; and

2. The indirect paper strip procedure, in which either a few drops of the

reagent are added to a filter paper strip or commercial disks or strips

impregnated with dried reagent are used. The tetramethyl derivative of p-

phenylenediamine is recommended because the reagent is more stable in

storage and is more sensitive to the detection of cytochrome oxidase and is

less toxic than the dimethyl derivative. In either method, a loopful of

suspected colony is smeared into the reagent zone of the filter paper.

Bacterial colonies having cytochrome oxidase activity develop a deep blue

color at the inoculation site within 10 seconds. Any organism producing a blue

color in the 10- to 60-second period is considered negative.6

Lack Of Evidence For Glucose Fermentation

Acid produced by NFs are considerably weaker than mixed acids derived

from fermentative bacteria, thus the pH in fermentation test media in which a NF

is growing may not drop sufficiently to convert the pH indicator. The initial clue

that an unknown organism is a NF is usually the lack of acid production in either

Triple sugar iron (TSI) or Kligler iron agar(KIA) media, manifested as an alkaline

slant and an alkaline deep.6

Motility6

The hanging drop preparation may be more accurate in detecting motility

of NFGNB. A loopful of 6 to 24 hr, actively growing broth culture that has been

incubated at 37ᴼC is placed in the center of No-1 coverslip that is inverted and

suspended over the concavity of depression slide. True motility must be

differentiated from Brownian movement. Motile bacteria show directional

movement and change in position relative to each other; when Brownian

movement is the cause of motion, they maintain the same relative position.

Motility B medium with tetrazolium also used for demonstrating motility of

NFGNB. Flagellar stains can also be used to demonstrate motility.

Pigment Production6

Pseudomonas produces water-soluble and diffusible pigments like

fluorescein (pyoverdin), pyocyanin, pyorubin, pyomelanin that discolor the

culture media. “Tech” and “Flo” media were developed to enhance the formation

of water-soluble pigments pyoverdin and pyocyanin. These media have special

peptones and an increased concentration of magnesium and sulfate ions to

enhance pigment production. Pigment production also enhanced by growing the

organism in gelatin, potato or milk-containing media and by incubating them at 25

–30ᴼC.

Nitrate Reduction6

The ability of the organisms to reduce nitrate to nitrite is an important

characteristic used in the identification and speciation of many microorganisms.

Organisms demonstrating nitrate reduction have the capability of extracting

oxygen from nitrate to form nitrite and other reduction products. The presence of

nitrite in the test medium is detected by the addition of alpha-naphthylamine and

sulphanilic acid which leads to the development of red color. If red color do not

develop, either nitrate has not been reduced or reduction is beyond the nitrite stage

to the formation of other compounds or to nitrogen gas (denitrification). The

appearance of red color on addition of small quantity of zinc dust indicates the

residual presence of nitrate, denoting a negative test; absence of color indicates

nitrate has been reduced beyond nitrite, indicating the original test was positive.

Denitrification of Nitrates And Nitrites6

Certain nonfermenters have the capability of reducing nitrate or nitrite or

both to gaseous nitrogen. Nitrate-nitrite broth with an inverted Durham tube may

be used. Because the media contains no carbohydrate, any gas that is formed is

derived from nitrate or nitrite, indicating a positive reaction.

Indole Production6

An enriched tryptophan –containing media, usually heart infusion broth

may be needed. Because only small quantities of Indole are formed by some NFs,

extraction of culture media by layering a small quantity of xylene or chloroform

on the surface may be helpful. The appearance of fuchsia red color at the surface

of medium with the reagent (kovac or Ehrlich reagent) indicates indole formation

and a positive test. One organism, Delftia acidovorans, produces a distinctive

“pumpkin orange” indole reaction owing to the formation of anthranilic acid

rather than indole from tryptophan.

Citrate Utilisation6

A well isolated colony is picked from the surface of a primary inoculation

plate and inoculated as a single streak on the slant surface of Simmon’s citrate

medium and incubated at 35ᴼC for 24 to 48 hours. Development of blue color

indicates a positive test.

Hydrolysis Of Urea6

Christensen’s urea agar slants used. Bacterial species like Bordetella

bronchiseptica produce a red color change within 4hours; weak reactors may

require up to 48 hours

SPECIAL BIOCHEMICAL TESTS USED FOR IDENTIFICATION

OF NON FERMENTERS

1. HUGH – LEIFSON OXIDATION - FERMENTATION MEDIUM

6

Two tubes were required for the test, each inoculated with the unknown

organism, using a straight needle stabbing the medium three to four times half

way to the bottom of the tube. One tube of each pair was covered with a 1cm layer

of sterile mineral oil (or) melted paraffin, leaving the other open to the air. Both

tubes were incubated at 35ᴼC and examined daily for several days.

In case of oxidative metabolism, yellow color appears along the upper one

fourth of the medium and in the tube where no oil overlay was done. In case of

fermentative organisms yellow color develops in both the tubes.

CONTROL

Glucose fermentation: Escherichia coli

Glucose oxidation: Pseudomonas aeruginosa

Non saccharolytic: Alcaligenes species.

2. DECARBOXYLATION OF LYSINE, ARGININE ORNITHINE 6

Decarboxylases are a group of specific enzymes which react with carboxyl

portion of aminoacid forming alkaline reacting amines. The reaction is

decarboxylation. Each enzyme is specific for Lysine, Arginine and Ornithine.

Amino Acid Positive Control Negative Control

Lysine Enterobacter aerogenes Enterobacter cloacae

Ornithine Enterobacter cloacae Klebsiella pneumoniae

Arginine Enterobacter cloacae Enterobacter aerogenes

Procedure

From a well-isolated colony of the test organism previously recovered on

primary isolation agar, inoculate two tubes of Moller decarboxylase medium, one

containing the amino acid to be tested and the other to be used as a control tube

devoid of amino acid. Overlay both tubes with sterile mineral oil to cover about 1

cm of the surface and incubate at 35°C for 18–24 hours.

Conversion of the control tube to a yellow color indicates that the organism

is viable and that the pH of the medium has been lowered sufficiently to activate

the decarboxylase enzymes. Reversion of the tube containing the amino acid to a

blue-purple color indicates a positive test owing to the formation of amines from

the decarboxylation reaction

3. O–NITROPHENYL β - D GALACTOPYRANOSIDE 6

A dense suspension of the test organism grown in TSI agar was prepared in

saline.About 1 drop of toluene was added to the suspension and ONPG disc was

added to the suspension and incubated at 37ᴼC b-galactosidase producing

organism show yellow color after 1 hour or 18-24 hours incubation.

.

4. GELATIN LIQUEFACTION TEST6

Gelatin breakdown can be demonstrated by incorporating it in a buffered

nutrient agar, growing the culture and then flooding the medium with tannic acid

that differentially precipitates either gelatin or its breakdown products.causing

opacity in the medium with clear zones around gelatin-liquefying colonies

ANTIBIOTIC SENSITIVITY15

Antibiotic susceptibility pattern was done on Mueller Hinton Agar by

Kirby- Bauer disc diffusion method as recommended by Clinical and Laboratory

Standards Institute(CLSI).Himedia discs were used for disc diffusion testing.

Antibiotic Discs Contents

Amikacin - 30µg

Gentamicin - 10µg

Cephotaxime - 30µg

Ceftazidime - 30µg

Cefepime - 30µg

Ciprofloxacin - 5µg

Ofloxacin - 5µg

Piperacillin - 100µg

Piperacillin – Tazobactum 100/10µg

Imipenem - 10µg

Meropenem - 10µg

Aztreonam - 30µg

colistin - 10µg

polymyxin - 300 U

The control strains used were E.coli ATCC 25922 and Pseudomonas

aeruginosa ATCC 27853 Overnight broth culture compared to 0.5 McFarland’s

was used as inoculum. After incubation at 37ᴼC for 16-18 hrs, zone of inhibition

was noted. Results were interpreted according to CLSI standard.

Multidrug resistant (MDR) isolates of the nonfermenters were estimated

MDR isolate was defined as resistant to three or more drugs of therapeutic

relevance.

DETECTION OF EXTENDED SPECTRUM OF β -LACTAMASES 15

All nonfermenters that were resistant to cefotaxime and or ceftazidime

were tested for Extended Spectrum of β-Lactamases.by the following methods:

Phenotypic confirmation test with Cephalosporin / clavulanate combination

disks.15, 75

This was done as recommended by CLSI guidelines. Mueller Hinton Agar

plates were swabbed with test organism having the turbidity equivalent to 0.5 Mc

Farland’s standard. Aseptically cefotaxime disk (30µg), cefotaxime – clavulanic

acid (30µg/10µg) ceftazidime (30µg) & ceftazidime clavulanic acid (30µg/10µg)

were placed on surface of agar. The plates were incubated at 35ᴼC for 16-18 hours

and diameter of zone of inhibition produced was recorded. A 5mm increase in

zone diameter for combination disc than that when tested alone confirmed the

presence of ESBL production. ATCC Escherichia coli 25922 & Klebsiella

pneumoniae ATCC 700603 were used as negative and positive control

respectively.

DETECTION OF ESBL BY E-TEST15

It is a plastic drug – impregnated strips, one end of which contains a

gradient of ceftazidime (MIC test range 0.5 to 32 μg/ml) and the other with a

gradient of ceftazidime plus a constant concentration of clavulanate (4 μg/ml).

Similar strips were used for cefotaxime and cefotaxime / clavulanate. A 0.5 Mc

Farland turidity standard of the organism was inoculated as a lawn culture on

Mueller Hinton agar. E strips were placed on the agar surface and plate were

incubated at 35°C for 16-18 hours.

INTERPRETATION

A > 8 fold reduction in cephalosporin MICs in the presence of clavulanate

is taken as positive for ESBL.

Amp C disk test: AmpC disk test was also done for the meropenem resistant

strains for detection of AmpCβ-lactamases. On a MHA plate, lawn culture of

E.coli ATCC 25922 was made from an overnight culturesuspension adjusted to

0.5 McFarland standard12. A 30μg cefoxitin disk was kept on the surface of the

agar. A blank disk (6 mm in diameter, Whatmann filter paper no.1) was moistened

with sterile saline and inoculated with a few colonies of the test strain. The

inoculated disk was then placed beside the cefoxitin disk almost touching it. The

plate was incubated overnight at 37ᴼC. A flattening or indentation of the cefoxitin

inhibition zone in the vicinity of the disk with test strain was interpreted as

positive for the production of AmpC β-lactamase. An undistorted zone was

considered as negative54

SCREENING FOR METALLOBETALACTAMASE PRODUCTION

Screening for metallobetalactamse production was done in isolates of

nonfermenters that were resistant to Imipenem and or Meropenem. Due to

intrinsic resistance to carbapenems mediated by resident MBL production, isolates

of Stenotrophomonas maltophilia were not considered eligible for MBL

screening. The methods used were:

MODIFIED HODGE TEST 18

The indicator organism, Escherichia coli ATCC 25922, at a turbidity of 0.5

McFarland Standard was used to inoculate the surface of a Mueller - Hinton agar

plate supplemented with zinc sulfate at a concentration of 70μgm/ml.and the test

strain was heavily streaked from the centre to the plate periphery. After the plate

was allowed to stand for 15 min at room temperature, 10 mg Imipenem disk was

placed at the center and the plate was incubated overnight. The presence of a

distored inhibition zone was interpreted as a positive result for carbapenem

hydrolysis screening.

IMIPENEM – EDTA DOUBLE DISC SYNERGY TEST 18

Test organism was adjusted to the 0.5 Mc Farland Standard and used to

inoculate Muller Hinton agar plates. 10mg Imipenem disk was placed on the plate

and a blank filter paper disk was place at a distance of 10mm (edge to edge). To

the blank disk, 10ml of 0.5M EDTA solution (1,900 mg of disodium salt,

dihydrate) was added. After overnight incubation, the presence of even a small

synergistic zone was interpreted as positive.

IMIPENEM – EDTA COMBINED DISK TEST 75

Test organisms of 0.5 Mc Farland Standard were inoculated onto plates of

Mueller– Hinton agar. A 0.5M EDTA solution was prepared. Two 10mg

Imipenem discs and Meropenem discs were placed on the plate. 5ml of EDTA

solution was added to one of the disc. The inhibition zones of the Imipenem and

Imipenem + EDTA discs were compared after 16-18 hrs of incubation at 37ᴼC.

An increase in inhibition zone of ≥ 7mm in combined disc than imipenem disc

alone was considered as positive

MBL E-test

The E Test MBL strip containing a double sided seven-dilution range of

IPM (4 to 256 μg/mL) and IPM (1 to 64 μg/mL) in combination with a fixed

concentration of EDTA has been reported to be the most sensitive format for

MBL detection. The E-test was done according to manufacturer's instructions.

MIC ratio of IP (Imipenem)/IPI (Imipenem-EDTA) of >8 or >3 log 2 dilutions

indicates MBL production24

DETERMINATION. OF METALLOBETALACTAMASE GENE BY PCR

METHOD

When the presence of a carbapenemase is suspectd, PCR is the fastest way

to determine which family of betalactamase is present. Ultimately the

identification of the betalactamase gene requires sequencing of the entire coding

region.

Molecular identification of AntibioticResistance Gene

PureFast Bacterial DNA minispin purification kit [Kit contains Lysozyme,

Lysozyme digestion buffer, Proteinase-K, Binding buffer, Wash Buffer-1, Wash

Buffer-2, Spin columns with collection tube and elution buffer. 2X ReDdye PCR

Master Mix, Agarose gel electrophoresis consumables and blaKPC Primers are

used.

2X Master Mix:

It contains 2U of Taq DNA polymerase, 10X Taq reaction buffer, 2mM

MgCl2, 1μl of 10mM dNTPs mix and RedDye PCR additives.

Agarose gel electrophoresis:

Agarose, 50X TAE buffer, 6X gel loading buffer and Ethidium bromide

are used.

PCR:

Ready to use VIM gene Primer mix - 5μl/reaction

Product size: 480bp

Bacterial DNA Purification

1. 1ml of overnight culture centrifuged at 6000rpm for 5min

2. Supernatant discarded

3. Pellet is suspended in 0.2ml PBS.

4. 180μl of Lysozyme digestion buffer and 20μl of Lysozyme [10mg/ml]

added.

5. Incubated at 37C for 15min.

6. 400μl of Binding buffer, 5μl of internal control template and 20μl of

Proteinase K added,

Mixed well by inverting several times.

7. Incubate at 56ºC for 15min.

8. Added 300μl of Ethanol and mixed well.

9. Transferred entire sample into the PureFast® spin column. Centrifuged for

1 min. Discard the flow-through and place the column back into the same

collection tube.

10. Added 500μl Wash buffer-1 to the PureFast® spin column. Centrifuge for

30-60 seconds and discard the flow-through. Place the column back into

the same collection tube.

11. Added 500μl Wash buffer-2 to the PureFast® spin column. Centrifuge for

30-60 seconds and discard the flow-through. Place the column back into

the same collection tube.

12. Discard the flow-through and centrifuge for an additional 1 min. This step

is essential to avoid residual ethanol.

13. Transferred the PureFast® spin column into a fresh 1.5 ml micro-

centrifuge tube.

14. Added 100μl of Elution Buffer to the center of PureFast® spin column

membrane.

15. Incubate for 1 min at room temperature and centrifuge for 2 min.

16. Discard the column and store the purified DNA at -20°C. Quality and

Quantity of extracted DNA is checked by loading in 1% agarose gel and

5μl of extracted DNA is used for PCR amplification.

PCR Procedure:

1. Reactions set up as follows;

Components Quantity

RedDye PCR Master mix 10μl

Ready to use - VIM gene primer mix 5μl

Purified Bacterial DNA 5μl

1. Total volume 20μl

2. Mixed gently and spin down briefly.

3. Place into PCR machine and program it as follows;

Initial Denaturation: 94ºC for 5 min

Denaturation: 94ºC for 30sec

Annealing: 58ºC for 30sec 35 cycles

Extension: 72ºC for 30sec

Final extension: 72º C for 5 min

Loading:

1. Prepared 2% agarose gel. [2gm of agarose in 100ml of 1X TAE buffer]

2. Run electrophoresis at 50V till the dye reaches three fourth distances and

observe the bands in UV Transilluminator.

Agarose gel electrophoresis:

1. Prepared 2% agarose. (2gm agarose in 100ml of 1X TAE buffer and

melted using micro oven)

2. When the agarose gel temperature was around 60ºC, added 5μl of Ethidium

bromide.

3. Poured warm agarose solution slowly into the gel platform.

4. Kept the gel set undisturbed till the agarose solidifies.

5. Poured 1XTAE buffer into submarine gel tank.

6. Carefully placed the gel platform into tank. Maintained the tank buffer

level 0.5cm above than the gel.

7. PCR Samples are loaded after mixed with gel loading dye along with 10μl

100bp DNA Ladder. [100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp,

800bp, 900bp, 1000bp and 1500bp]

8. Run electrophoresis at 50V till the dye reaches three fourth distance of the

gel.

9. Gel viewed in UV Transilluminator and observed the bands pattern.

RESULTS

Table:1 Age Distribution of Patients (n=200)

Age group No of patients Percentage

0-10 7 3.5

11-20 19 9.5

21-30 47 23.5

31-40 57 28.5

41-50 26 13

51-60 28 14

>60 16 8

Two hundred nonfermenting gram negative bacilli were isolated from all

age groups, it ranged from less than one year to 80 years, the youngest was 1 year

old child and the oldest was of 80 years age. the Table 1( Agewise distribution )

gives higher incidence of infection by NFGNB was seen in the age group of 31-40

years (28.5%) followed b 21-30 years age group(23.5%)

Table: 2. Gender wise distribution (n=200)

As per our study in table -2, the total numbers of males were 121(60.5%)

and females were 79(39.5%). The male to female ratio was 3.1:1.

Gender Number Percentage

Male 121 60.5%

Female 79 39.5%

46%

27%

9%

11%

7%

Sample Distribution (n=200)

Pus(wound infection)

Urine

Blood

Sputum

Body Fluids

0

10

20

30

40

50

60

Age Distribution of Patients (n-200)

No of patients Percentage

0-10 11-20 21-30 31-40 41-50 51-60 >60

TABLE 1

TABLE : 2

Table:3. Sample Distribution (n=200)

According to this table, most samples were obtained from pus 93

samples(46.5%) followed by urine53 samples(26.5%),Blood 18((9%) sputum

22(11%) and body fluids 14(7%)

Table: 4. Wardwise Distribution (n=200)

Sample Total No Percentage

Pus(wound

infection)

93 46.5%

Urine 53 26.5%

Blood 18 9%

Sputum 22 11%

Body Fluids 14 7%

Speciality No of cases Percentage

Burns Ward 73 36.5

Otorhinolaryngology 34 17

Surgery 22 11

Chest Medicine 19 9.5

Medicine 15 7.5

Intensive care unit 13 6.5

Urology 9 4.5

Labour Ward 9 4.5

Nephrology ward 6 3

As per the table 4(wardwise distribution), most samples are obtained from

burns ward 73(36.5%) followed by otorhinolaryngology 34 (17%), Surgery

22(11%), Medicine 15(7.5), chest Medicine 19(9.5%), Intensive care unit

13(6.5%), Urology 9(4.5%), Labour ward 9(4.5%) Nephrology 6(3%)

Table: 5. Risk Factors Associated With Infections By Nonfermenters (n=200)

According to table no 5 (Risk factors associated with infections by

nonfermenters) Diabetes Melitus 26 (13%) Road traffic accident (contaminated

wound) 21(10.5), Indwelling Catheter 13(6.5%), Intensive care unit stay

13(6.5%), Chronic renal disease 6(3%) Post renal transplantation 2(1%)

Risk Factor Number Percentage

Diabetes Melitus 26 13

Post operative wound infection 21 10.5

Indwelling catheter 13 6.5

Intensive care unit stay 13 6.5

Chronic renal disease 6 3

Post renal transplantation 2 1

0

10

20

30

40

50

60

70

80

Wardwise Distribution (n=200)

No of cases

Percentage

Table : 4

Table:5

0

5

10

15

20

25

30

Risk Factors Associated With Infections By Nonfermenters (n=200)

Number

Percentage

Table: 6. Grouping of Non Fermenting Gram Negative Bacilli (n=200)

According to Table no 6 (Grouping of Non fermenting gram negative

bacilli) Oxidase positive and motile is 129(64.5%) which includes Pseudomonas

aeruginosa Pseudomonas fluorescens, Burkholderia cepacia, Shewanella

putrefaciens, oxidase positive and non motile is 2(1%) includes Weeksella virosa,

Oxidase negative and motile is 3(1.5%) includes Stenotrophomonas maltophilia,

Oxidase negative and nonmotile is 66(33%) includes Acinetobacter baumannii,

Acinetobacter lwoffii

Group Number Percentage

Oxidase positive and Motile 129 64.5%

Oxidase positive and non motile 2 1%

Oxidase negative and Motile 3 1.5%

Oxidase negative and Nonmotile 66 33%

Total 200 100%

Table:7. Identification of Non Fermenting Gram Negative Bacilli Isolated

(n=200)

As per the table 7, predominant organism is Pseudomonas aeruginosa

117(58.5%) followed by Acinetobacter baumannii 60(30%), Pseudomonas

fluorescens 8(4%), Acinetobacter iwoffi 6(3%) Burkholderia cepacia 3(1.5%)

and Stenotrophomonas maltophilia 3(1.5%) and Weeksella virosa 2(1%).

Clinical Isolates Number Percentage

Pseudomonas aeruginosa 117 58.5%

Acinetobacter baumaunnii 60 30%

Pseudomonas fluorescens 8 4%

Acinetobacter lwoffii 6 3%

Burkholderia cepacia 3 1.5%

Stenotrophomonas maltophilia 3 1.5%

Weeksella virosa 2 1%

Shewanella putrefaciens 1 0.5%

Table: 8. Drug Susceptibility of Pseudomonas Aeruginosa (n=117)

According to Table no8, Pseudomonas aeruginosa shows 100% sensitive to

colistin, it shows higher sensitivity Pipercillin tazobactem 115(98.29%),

Imipenem 100(98.29%), Amikacin 81(69.23%), Ceftazidime 73(62.39%),

Gentamicin51 (43.58%), Ciprofloxacin 44 (3.60%), Levofloxacin 43 (36.75%),

Cefoperazone sulbactam 39 (33.3%), Cotrimoxazole 37(31.62%)

Drug Susceptible Percentage of susceptibility

Gentamicin 51 43.58%

Amikacin 81 69.23%

cotrimoxazole 37 31.62%

Ceftazidime 73 62.39%

Cefoperazone

sulbactam

39 33.3%

Pipercillin

tazobactem

115 98.29%

Imipenem 100 85.47%

Ciprofloxacin 44 37.60%

levofloxacin 43 36.75%

Colistin 117 100%

Table: 9. Drug Susceptibility of Acinetobacter Baumannii (n=60)

According to Table no 9, Acinetobacter baumannii shows 100% sensitive

to colistin(60), it shows higher sensitivity Pipercillin tazobactem 53(88.33%),

Imipenem 64(90%), Amikacin 48(80%), Ceftazidime 40(66.6%),

Gentamicin34(56.66%), Ciprofloxacin 40(66.6%), Levofloxacin 30(50%),

Cefoperazone sulbactam 40(66.6%), Cotrimoxazole 32(53.33%)

Drug Susceptible Percentage of susceptible

Gentamicin 34 56.66%

Amikacin 48 80%

Cotrimoxazole 32 53.33%

Imipenem 54 90%

Ceftazidime 40 66.6%

Ceftriaxone 52 86.6%

Cefoperazone

sulbactam

40 66.6%

Pipercillin

tazobactem

53 88.33%

Ciprofloxacin 40 66.6%

Levofloxacin 30 50%

Colistin 60 100%

Table: 10. Drug Susceptibility of other NFGNB

According to Table no 10, Drug susceptibility of different NFGNB, All

Burkholderia cepacia and Stenotrophomonas maltophilia shows sensitive to

cotrimoxazole and resistant to Amikacin. Weeksella virosa and Shewanella

putrefaciens shows sensitive to almost all drugs

Organisms Pseudomonas

fluorescens

(n=8)

Acinetobacter

lwoffii (n=6)

Burkholderia

cepacia (n=3)

Stenotrop

homonas

maltophilia (n=3)

Weeksella

virosa

(n=2)

Shewanella

putrefaciens

(n=1)

Amikacin 5 4 0 0 2 1

Ceftazidime 7 6 3 3 2 1

cotrimoxazole 3 2 3 3 1 0

Pipercillin

tazobactem 8 6 3 3 2 1

Imipenem 8 6 3 3 2 1

Ceftriaxone 6 4 2 2 2 1

Colistin 8 6 3 3 2 1

Table:11 Multidrug Resistant Nonfermenting gram negative bacilli (>2 drugs

resistant) (n=200)

According to the Table no 11, Multi drug Resistant among NFGNB that is

resistant to more than two drugs. Among 117 Pseudomonas aeruginosa ,86

organisms shows multi drug resistant, 49 MDR in Acinetobacter baumannii, 6

MDR in Pseudomonas fluorescens,5 MDR in Acinetobacter lwoffi, 1 MDR in

case of Burkholderia cepacia and Stenotrophomonas maltophilia each, 1MDR in

Shewanella putrefaciens and no MDR in Weeksella virosa

Organisms Total No MDR

Pseudomonas aeruginosa 117 86

Acinetobacter baumaunnii 60 49

Pseudomonas fluorescens 8 6

Acinetobacter lwoffii 6 5

Burkholderia cepacia 5 1

Stenotrophomonas maltophilia 3 1

Weeksella virosa 2 0

Shewanella putrefaciens 1 1

0

20

40

60

80

100

120

140

Multidrug Resistant Nonfermenting gram negative bacilli

(>2 drugs resistant) (n=200)

Total No

MDR

Table :10

Table :11

0

1

2

3

4

5

6

7

8

9Drug Susceptibility of Other - NFGNB

Pseudomonas fluorescens (n=8) Acinetobacter lwoffii (n=6)

Burkholderia cepacia (n=3) Stenotrophomonas maltophilia (n=3)

Weeksella virosa (n=2) Shewanella putrefaciens (n=1)

1. Amikacin

2. Ceftazidime

3. Cotrimoxazole

4. Pipercillin

Tazobactem

5. Imipenem

6. Ceftriaxone

7. Colistin

1 2 3 4 5 6 7

Table: 12 ESBL producers in NFGNB (n=200)

Clinical Isolates Total no ESBL Production

Pseudomonas aeruginosa 117 24

Acinetobacter baumaunnii 60 12

Pseudomonas fluorescens 8 2

Acinetobacter lwoffii 6 0

Burkholderia cepacia 5 0

Stenotrophomonas

maltophilia

3 0

Shewanella putrefaciens 1 0

Total 200 38

Table: 12

Chi-Square Test Value p-Value

Fisher's Exact Test 0.328 0.899

According to Table no 12, ESBL producers in non fermenting gram

negative bacilli, Twenty four of 117 Pseudomonas aeruginosa are ESBL

producers , twelve of 60 Acinetobacter baumannii, two of 8 Pseudomonas

fluorescens, no ESBL producers Acinetobacter lwoffii, Burkholderia cepacia,

Stenotrophomonas maltophilia , Shewanella putrefaciens. p value is insignificant

( p value>0.05) ie insignificant association between organisms and ESBL

production

Table: 13.ESBL Production in Sample (n=200)

Table 13

Chi-Square Test Value p-Value

Fisher's Exact Test 9.617 0.039

According to Table no 13, Sample wise ESBL producers, 18 ESBL

producers are from Pus sample, 5 from Urine sample, 4 from Blood sample, 9

from Sputum sample, 2 from Body fluids. p value is significant (p value<0.05) ie

significant association between samples and ESBL production

Sample Total No ESBL Percentage

Pus 93 18 19.3

urine 53 5 9.4

Blood 18 4 22.2

Sputum 22 9 40.9

Body Fluids 14 2 14.2

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5

ESBL Production in Sample (n=200)

Total No ESBL Percentage

1. Pus 2. urine 3. Blood 4. Sputum 5.Body Fluids

0

50

100

150

200

250

1 2 3 4 5 6 7 8

ESBL producers in NFGNB (n=200)

Total no ESBL Producer

1.Pseudomonas aeruginosa 2. Acinetobacter baumaunnii 3. Pseudomonas fluorescens 4. Acinetobacter lwoffii 5. Burkholderia cepacia 6.Stenotrophomonas maltophilia 7. Shewanella putrefaciens 8. Total organism

Table :12

Table :13

Table:14. MBL screening by Imipenem Resistance (n=200)

Clinical Isolates Total no Carbapenem

resistant Isolates

Percentage

Pseudomonas aeruginosa 117 17 14.5

Acinetobacter baumaunnii 60 8 13.3

Pseudomonas fluorescens 8 0 0

Acinetobacter lwoffii 6 0 0

Burkholderia cepacia 2 0 0

Stenotrophomonas maltophilia 1 0 0

Shewanella putrefaciens 1 0 0

Table 14

Chi-Square Test Value p-Value

Pearson Chi-Square 0.047 0.829

According to Table no 14 carbapenem resistant isolates in NFGNB,

seventeen of 117 Pseudomonas aeruginosa are imipenem resistant, eight of 60

isolates of Acinetobacter baumannii are imipenem resistant. No imipenem

resistant in Pseudomonas fluorescens, Acinetobacter lwoffii, Burkholderia

cepacia, Stenotrophomonas maltophilia, Shewanella putrefaciens. p value is

insignificant (p value>0.05) ie insignificant association between organisms and

carbapenem resistance.

Table: 15. MBL production in Non fermenters by Imipenem EDTA E Strip

Clinical Isolates Total no Carbapenem

resistant Isolates

MBL

Pseudomonas

aeruginosa

117 17 8

Acinetobacter

baumaunnii

60 8 5

Table 15

Chi-Square Test Value p-Value

Fisher's Exact Test 0.250 0.912

According to Table no 15,

MBL production in Non fermenters by

Imipenem EDTA E Strip ,Eight MBL producers (6.83%) in 17 Imipenem resistant

Pseudomonas aeruginosa (8MBL/117 total Pseudomonas aeruginosa), five MBL

producers (8.33%) in 8 Imipenem resistant Acinetobacter baumannii (8 MBL/ 60

total Acinetobacter baumannii). No MBL producers in other organisms

(Pseudomonas fluorescens, Acinetobacter lwoffii, Burkholderia cepacia,

Stenotrophomonas maltophilia, Shewanella putrefaciens) . p value is insignificant

(p value>0.05)) ie insignificant association between organisms and MBL

production.

117

175

60

8

8

0

20

40

60

80

100

120

140

Total no Carbapenam resistant Isolates

MBL

MBL production in Non fermenters by Imipenem EDTA

E Strip

Pseudomonas aeruginosa

Acinetobacter baumaunnii

Table :14

Table :15

0

20

40

60

80

100

120

140

1 2 3 4 5 6 7

MBL screening by Imipenem Resistance (n=200)

Total no Carbapenem resistant Isolates Percentage

1.Pseudomonas aeruginosa 2. Acinetobacter baumaunnii 3. Pseudomonas fluorescens 4. Acinetobacter lwoffii 5. Burkholderia cepacia 6.Stenotrophomonas maltophilia 7.Shewanella putrefaciens

Table:16. Comparison of MBL detection by different methods

Clinical Isolates Modified

Hodge Test

Double disc

synergy test

Combined

disc test

Imp EDTA

E strip

Pseudomonas

aeruginosa (n=17)

5 7 8 8

Acinetobacter

baumaunnii (n=8)

3 3 4 4

Table:16

Chi-Square Test Chi-Square Test Value p-Value

Pseudomonas

aeruginosa (n=17) Pearson Chi-Square 1.457 0.692

Acinetobacter

baumaunnii (n=8) Fisher's Exact Test 0.688 0.999

According to Table no 16, Comparison of MBL detection by different

methods such as Modified Hodge test, Double disc synergy test (DDST),

combined disc diffusion test (CDDT), Imipenem EDTA E strip method. p value is

insignificant (p value>0.05) for both Pseudomonas aeruginosa and Acinetobacter

baumaunnii ) ie insignificant association between organisms and MBL

production.

Table: 17.MBL production in samples (n=200)

Sample Total No MBL Percentage

Pus 93 9 9.6

urine 53 2 3.7

Blood 18 0 0

Sputum 22 1 4.5

Body Fluids 14 0 0

Table:18

Chi-Square Test Value p-Value

Fisher's Exact Test 3.014 0.496

According to Table no 17, sample wise distribution of MBL producers, 9

MBL producers from Pus sample, 2 from Urine sample and one from sputum

sample. No MBL producers in blood, body fluids sample. p value is insignificant

(p value>0.05) ie insignificant association between samples and MBL production.

Table: 18. Result of PCR in MBL Pseudomonas aeruginosa (n=8)

MBL Gene Total No of Cases Percentage

Positive 4 50%

Negative 4 50%

According to table 18, four of 8 (50%) MBL-positive isolates were

confirmed to be positive for MBL by PCR

Table: 19. Pattern of MBL Gene in Pseudomonas Aeruginosa n=8

Type of MBL gene Total number cases Percentage

Bla-VIM 4 50%

Bla-IMP - 0%

According to Table no 19, pattern of MBL gene in Pseudomonas

aeruginosa, Four of 8 (50%) MBL-positive isolates were confirmed to be positive

for MBL for the gene Bla VIM by PCR and no Bla IMP was found in the 8 MBL

producers

Table:20. AmpC βlactamase production in NFGNB

Clinical Isolates Total no Amp C Production

Pseudomonas

aeruginosa

117 52

Acinetobacter

baumaunnii

60 18

Pseudomonas

fluorescens

8 0

Acinetobacter lwoffii 6 0

Burkholderia cepacia 5 0

Stenotrophomonas

maltophilia

3 0

Shewanella putrefaciens 1 0

Total 200 70

Table -20

Chi-Square Test Value p-Value

Person Chi-Square 3.461 0.063

According to Table no 20, Amp C producers in NFGNB, Fifty two Amp C

producers (44.4%) in (Pseudomonas aeruginosa52/117 )and 18 AmpC

producers(30%) in Acinetobacter baumannii (18/60). No Amp C producers on

other NFGNB. p value is insignificant (p value>0.05) ie insignificant association

between organisms and Amp C production.

Table:17

Table:20

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5

Total No MBL Percentage

1. Pus 2. urine 3. Blood 4. Sputum 5.Body Fluids

0

50

100

150

200

250

1 2 3 4 5 6 7 8

Amp C βlactamase production in NFGNB

Total no Amp C Producer

1.Pseudomonas aeruginosa 2. Acinetobacter baumaunnii 3. Pseudomonas fluorescens 4. Acinetobacter lwoffii 5. Burkholderia cepacia 6.Stenotrophomonas maltophilia 7.Shewanella putrefaciens8. Total organism

Table:21. Amp C β lactamase producers in samples

Sample Total No Amp C

producers

Percentage

Pus 93 36 38.7

Urine 53 21 39.6

Blood 18 4 22.2

Sputum 22 6 27.2

Body Fluids 14 3 21.4

Table-21

Chi-Square Test Value p-Value

Fisher's Exact Test 3.789 0.438

According to Table no 21, Sample wise distribution of Amp C producers,

maximum Amp C producers seen in urine 21(39.6%), followed by Pus 36(38.7%),

sputum 6(27.2%), blood 4(22.2%) and body fluids 3(21.4%).p value is

insignificant (p value>0.05 ) ie insignificant association between samples and

Amp C production.

DISCUSSION

Two hundred isolates of non fermenting gram negative bacilli were taken

from various clinical samples like pus, urine, blood, sputum, body fluids and

evaluated for their role in infections in hospitalised patients including the

characteristics of their drug resistance.

Two hundred nonfermenting gram negative bacilli were isolated from all

age groups, study gives higher incidence of infection by NFGNB was seen in the

age group of 31-40 years (28.5%) followed b 21-30 years age group(23.5%)

which is in contrast with Usha Kalawat, Sumathi I, Jaya Prada, Satish Kumar

Reddy et al where 61-70 year age group(19%) and 51- 60 year age group(18%)

are common26

. This may be due to the reason that in our study most of the cases

are from burns ward(73/200), NFGNB infections are more common in the age

groups 21-30 years(23.5%) and 31-40(28.5%) years which is in concordance with

the study Enayatollah kalantar et al where 24.7% seen in 21-30 age groups and

19.8% seen in 31-40 age group 65.

The total numbers of males were 121(60.5%) and females were 79(39.5%).

The male to female ratio was 3.1:1. It shows higher incidence in male (60.5%)

compared to females(39.5%) which is in concordance with P. A. Shiny1, S.

Rajendran et al 25

.

Non fermenting gram negative bacilli obtained from various clinical

samples were collected, most samples were obtained from pus 93 samples(46.5%)

followed by urine53 samples(26.5%),Blood 18((9%) sputum 22(11%) and body

fluids 14(7%) which is concordance with study N sinha, J Agarwal , S srivastava,

M singh et al where maximum isolates pus 52/140 samples(37.14%) followed by

blood 32/140 samples(22.85%) and urine 19/140(13.57%)36

and also according to

N Ozkalay Yilmaz1, N Agus

1, E Bozcal

2, A Uzel

2 et al, Strains were dominantly

isolated from the pus (n: 16, 42.1%), also blood cultures (n: 10, 26.3%), urinary

tract (n: 6, 15.8%) and tracheal aspirate specimens (n: 6, 15.8%)4

Most samples are obtained from burns ward 73(36.5%) followed by

otorhinolaryngology 34 (17%), Surgery 22(11%), Medicine 15(7.5), chest

Medicine 19(9.5%), Intensive care unit 13(6.5%), Urology 9(4.5%), Labour ward

9(4.5%) Nephrology 6(3%) in contrast to N sinha, J Agarwal , S srivastava, M

singh et al where maximum cases are from ICU(22.14%) followed by

paediatrics(20.71%), neurosurgery(15.71%) and general surgery wards (12.85%)36

and also in the study N Ozkalay Yilmaz1, N Agus

1, E Bozcal

2, A Uzel

2 et

al,Bacterial strains were isolated mainly from intensive care unit (n: 18, 47%),

general surgery (n: 12, 32%) and internal medicine (n: 8, 21%).4

Most common risk factors associated with infections by nonfermenters

were Diabetes Melitus 26( 13%) followed by Post op wound infection, 21(10.5),

Indwelling Catheter 13(6.5%), Intensive care unit stay 13(6.5%), Chronic renal

disease 6(3%) Post renal transplantation 2(1%), which is in concordance with

Sherertz .R.J and F A Sarubbi 1983 et al where Post op wound infection infection

rate is 2.08%, Intensive care unit stay (22.9%) (especially Lower respiratory

tract) 53

which is also in concordance with Meharwal SK , Taneja N, Sharma SK,.

et al, where Postoperative wound infection accounts for 42.6%, chronic renal

failure(5.3%), indwelling wrinary catheter (2.6%), Diabetes mellitus(1.3%), Post

renal transplant(8.1%)54

Grouping of Non fermenting gram negative bacilli were Oxidase positive

and motile organisms were 129(64.5%) which includes Pseudomonas aeruginosa,

Pseudomonas fluorescens, Burkholderia cepacia, Shewanella putrefaciens ,

oxidase positive and non motile organisms were 2(1%) includes Weeksella virosa,

Oxidase negative and motile organisms were 3(1.5%) includes Stenotrophomonas

maltophilia, Oxidase negative and nonmotile organisms were 66(33%) includes

Acinetobacter baumannii, Acinetobacter lwoffii

Most predominant organism was Pseudomonas aeruginosa 117(58.5%)

followed by Acinetobacter baumannii 60(30%), Pseudomonas fluorescens

8(4%), Acinetobacter iwoffi 6(3%) Burkholderia cepacia 3(1.5%) and

Stenotrophomonas maltophilia 3(1.5%) and Weeksella virosa 2(1%). Which is in

contrast with S Arora, V Gautam, P Ray at al, 18% (1781/9662) grew NFGNBs.

Acinetobacter spp. (62%) was the most common followed by Pseudomonas

aeruginosa (18%), Burkholderia cepacia complex (5%) and S. maltophilia (3%).

12% (221/1781) of the NFGNBs could not be identified. 29ia

and in concordance

with ziad et al,

Pseudomonas aeruginosa (72.9%%) was the most common

followed by Acinetobacter spp (25.3%), S. maltophilia(1.8%)

Pseudomonas aeruginosa shows 100% sensitive to colistin, it shows

higher sensitivity Pipercillin tazobactem 115(98.29%), Imipenem 100(98.29%),

Amikacin 81(69.23%),Ceftazidime 73(62.39%), Gentamicin51(43.58%),

Ciprofloxacin 44(3.60%), Levofloxacin 43(36.75%), Cefoperazone sulbactam

39(33.3%), Cotrimoxazole 37(31.62%) which is in concordance with B Behera1, P

Mathur2, A Das

3, A Kapil

3, V Sharma

1 et al of the 91 isolates of Pseudomonas

aeruginosa, 64 (70%) were resistant to ceftazidime, 68 (75%) to piperacillin, 54

(59%) to piperacilin/tazobactam, 58 (63%) to ticarcillin/clavulanic acid, 75 (82%)

to cefoperazone, 67(74%) to amikacin, 74 (81%) to cefepime, 65 (71%) to

levofloxacin, and 72 (79%) to ciprofloxacin by the disc diffuse ion (CLSI)

method.24

Acinetobacter baumannii shows 100% sensitive to colistin(60), it shows higher

sensitivity Pipercillin tazobactem 53 (88.33%), Imipenem 64 (90%), Amikacin

48(80%),Ceftazidime 40 (66.6%), Gentamicin34 (56.66%), Ciprofloxacin 40

(66.6%), Levofloxacin 30 (50%), Cefoperazone sulbactam 40 (66.6%),

Cotrimoxazole 32 (53.33%) which is in concordance with Reza Mirnejad ,

Somayeh Vafaei et al where highest antibiotic resistance in 100 isolates of the

Acinetobacter baumannii was related to antibiotics namely: cefepime (100%),

ceftriaxone (95%), amikacin (95%), imipenem (76%), piperacillin - tazobactam

(70%), meropenem (69%), gentamicin (63%), tobramycin (56%), tetracycline

(51%), ampicillin - sulbactam (49%) and the lowest resistance to polymyxin B73

Drug susceptibility of different NFGNB, all Burkholderia cepacia and

Stenotrophomonas maltophilia shows sensitive to cotrimoxazole (3/3) and

resistant to Amikacin (0/3) each which is in concordance with Malavalli

Venkatesh Bhavana, Sangeetha Joshi et al where Burkholderia cepacia shows

100% sensitive to cotrimoxazole and Stenotrophomonas maltophilia shows 90%

sensitive to cotrimoxazole. Cotrimoxazole exhibited very good susceptibility

against both the isolates. According to the SENTRY Antimicrobial Surveillance

Program, which monitors the predominant community-acquired and nosocomial

pathogens including their antimicrobial resistance globally, cotrimoxazole was

found to have an excellent susceptibility against both these organisms. In the

Indian scenario, Gautam et al. from North India have reported susceptibility rates

of 75%–80% among Burkholderia cepacia complex isolates and 70%–90%

among Stenotrophomonas maltophilia isolates to cotrimoxazole.72

As per a study

conducted in a tertiary care centre of coastal Karnataka by Chawla et al., 86.7%

susceptibility to cotrimoxazole for Stenotrophomonas maltophilia was seen.71

Weeksella virosa and Shewanella putrefaciens shows sensitive to almost all drugs

Multi drug Resistant among NFGNB that is resistant to more than two

drugs. Among 117 Pseudomonas aeruginosa ,86 organisms shows multi drug

resistant, 49 MDR in 60 Acinetobacter baumannii, 6 MDR in Pseudomonas

fluorescens,5 MDR in Acinetobacter lwoffi, 1 MDR in case of Burkholderia

cepacia and Stenotrophomonas maltophilia each, 1MDR in Shewanella

putrefaciens and no MDR in Weeksella virosa. This is higher than the studies in

India reported previously in the year 1998 (Vijaya et al) and 2000.(Veenu et al) at

31% & 62% respectively and comparable to the study .by Patwardhan et el in

2008 who showed >68% of Pseudomonas isolates and >90% Acinetobacter

isolates were multidrug resistant.60,59,58

While Stenotrophomonas maltophilia is

intrinsically resistant to antibiotics, Pseudomonas aeruginosa & Acinetobacter

baumannii acquire resistance by many different mechanism like Extended

spectrum of betalactamases (ESBL) and Metallobetalactamases(MBL). This is of

concern as efficacious antimicrobial options are limited.61

The present study

showing Acinetobacter as more multidrug resistant than Pseudomonas correlated

with the studies by Tanya et al (53%and49%) and Homer et al(62% and 58%)56, 57

ESBL producers in non fermenting gram negative bacilli, Twenty four of

117 Pseudomonas aeruginosa are ESBL producers, twelve of 60 Acinetobacter

baumannii, two of 8 Pseudomonas fluorescens, no ESBL producers in

Acinetobacter lwoffii, Burkholderia cepacia, Stenotrophomonas maltophilia,

Shewanella putrefaciens. While Stenotrophomonas maltophilia and Burkholderia

cepacia show intrinsic resistance , ESBL production by Pseudomonas aeruginosa

and Acinetobacter baumannii is significant 64

All the ESBL producers were

multidrug resistant. Maximum sensitivity among the ESBL producers was seen

with Imipenem (82%) followed by piperacillin – Tazobactam (60%) as seen in

the studies conducted by Brauers et al and Freshteh et al62,63

Sample wise ESBL producers, 18 ESBL producers are from Pus sample, 5

from Urine sample, 4 from Blood sample, 9 from Sputum sample, 2 from Body

fluids which is in concordance with Shahanara Begum Md Abdus Salam et al,

where out of 31 ESBL producers, 18 ESBL producers from pus and swab,12 from

urine and 8 from sputum74

Carbapenem resistant isolates in NFGNB, seventeen of 117 Pseudomonas

aeruginosa are imipenem resistant, eight of 60 isolates of Acinetobacter

baumannii are imipenem resistant. No imipenem resistant in Pseudomonas

fluorescens, Acinetobacter lwoffii, Burkholderia cepacia, Stenotrophomonas

maltophilia, Shewanella putrefaciens. This carbapenem resistant was tested with

imipenem in our study, in which out of 17 imipenem resistant screening for MBL

production, only 7 were found to be MBL producers phenotype method and in

that only 4 turned to be positive by genotype method (4 blaVIM, no bla IMP ) by

PCR shows that imipenem resistant screening test is not that sensitive for MBL

production detection . This is in concordance with S Buchunde et al where Out of

326 Pseudomonas aeruginosa strains studied, 63 (19.3%) were resistant to

meropenem, 63 (19.3%) to CAZ and 58 (17.8%) to Imipenem (screen-test

positive). IPM failed to pick up five isolates that were found resistant to

meropenem and Ceftazidime. meropenem DDST and Ceftazidime DDST

confirmed all the 63 screen-test positives, while Imipenem DDST 88.9% of the

screen-test positives. Using criteria by Yong D et al. 69

for Disc potentiation test

(DPT-1), meropenem confirmed 100% screen-test positive isolates, Imipenem

85.7% and Ceftazidime 76.2% ( p>0.05). However, when criteria given by

Hemlatha et al. 70

for Ceftazidime DPT was used (DPT-2), Ceftazidime was able

to confirm additional 11 (17.4%) isolates, thereby raising the positivity of

Ceftazidime DPT to 59 (93.7%). All the 63 screen-test positive isolates, including

those not picked by Imipenem, were found to be positive for the presence of bla

using PCR.68

MBL production in Non fermenters by Imipenem EDTA E Strip ,Eight

MBL producers(6.83%) in 17 Imipenem resistant Pseudomonas aeruginosa

(8MBL/117 total Pseudomonas aeruginosa), five MBL producers(8.33%) in 8

Imipenem resistant Acinetobacter baumannii(8 MBL/ 60 total Acinetobacter

baumannii). No MBL producers in other organisms (Pseudomonas fluorescens,

Acinetobacter lwoffii, Burkholderia cepacia, Stenotrophomonas maltophilia ,

Shewanella putrefaciens. Out of 117 total Pseudomonas aeruginosa isolates, 17

were resistant for Imipenem and screened for MBL production. 8 were positive

for MBL by both DDST and MIC reduction test which is in concordance with G

Agrawal et al, out of 174 total isolates, 18 were resistant for Imipenem and

screened for MBL production. 14 were positive for MBL by both DDST and MIC

reduction test66

Higher MBL production is seen in Acinetobacter baumannii (8.33%) than

Pseudomonas aeruginosa (6.83%) which is in concordance with s John el al

where higher MBL production is seen in Acinetobacter baumannii (27.7%) than

Pseudomonas aeruginosa (14.8%)64.

Comparison of MBL detection by different methods such as Modified

Hodge test, double disc synergy test (DDST), combined disc diffusion test

(CDDT), Imipenem EDTA E strip method. In Pseudomonas aeruginosa, among

17 Imipenem resistant isolates MBL producers are identified by Modified Hodge

test 5, Double disc synergy test (DDST) 7, combined disc diffusion test (CDDT)

8, Imipenem EDTA E strip method 8. In case of Acinetobacter baumannii, MBL

producers are identified by as Modified Hodge test 3, Double disc synergy test

(DDST) 3, combined disc diffusion test (CDDT) 4, Imipenem EDTA E strip

method 4 . This is in concordance with B Behera1, P Mathur

2, A Das

3, A Kapil

3, V

Sharma et al Of the 63-imipenem resistant isolates, 56 isolates were tested for

MBL production. Forty-eight of these 56 isolates shows positive in the combined

disc test, whereas 36 isolates gave positive result by DDST. MBL E test was done

in 30 isolates (in 26 of which, both DDST and combined disc method

demonstrated MBL production and four isolates were randomly selected from the

12 which gave a positive combined disc result and negative DDST result36.

Sample wise distribution of MBL producers, 9 MBL producers from Pus

sample, 2 from Urine sample and one from sputum sample. No MBL producers in

blood, body fluids sample. This is in concordance with N Ozkalay Yilmaz et al,

where among 38 imipenem resistant isolates, maximum isolated from pus sample

16(42.1%), blood culture 10(26.3%), urinary tract 6(15.8%) and tracheal aspirate

6(15.8%) and majority of MBL production is from pus sample4

According to our study,the MBL enzyme was found positive in 8 of 17

(47.05%) IMP resistant Pseudomonas aeruginosa isolates by DDST and Imipenem

EDTA E strip. And according to table 18, Four of 8 (50%) MBL-positive isolates

were confirmed to be positive for MBL by PCR. According to N Ozkalay

Yilmaz1, N Agus

1, E Bozcal

2, A Uzel et al, MBL enzyme was found positive in 27

of 38 (71.05%) IMP resistant Pseudomonas aeruginosa isolates by DDST. Eight

of 38 (21.05%) MBL-positive isolates were confirmed to be positive for MBL by

PCR. 28,4

Pattern of MBL gene in Pseudomonas aeruginosa, Four of 8 (50%) MBL-

positive isolates were confirmed to be positive for MBL for the gene Bla VIM by

PCR and no Bla IMP was found in the 8 MBL producers where is in concordance

with AK Pragasam1, S Vijayakumar

1, YD Bakthavatchalam

1, A Kapil

2, BK Das et

al in which among the class B carbapenemases (MBLs), blaIMP, blaVIM, blaNDM

was identified except blaSPM. Within that, blaVIM were detected in 24%–57%,

blaNDM were 8%–19% and blaIMP were 4%–5%, respectively.9

and also this is

concordance with M Purohit1, DK Mendiratta

1, VS Deotale

1, M Madhan

2, A

Manoharan2, P Narang et al, we found bla-VIM MBL gene only in 7 (16.28%) of

the 43 screen test positive isolates. None of our isolates showed presence of bla-

IMP gene39

AmpC producers in NFGNB, Fifty two AmpC producers (44.4%) in

(Pseudomonas aeruginosa 52/117 )and 18 Amp producers(30%) in Acinetobacter

baumannii(18/60). No AmpC producers on other NFGNB. Higher incidence of

AmpC production is seen in Pseudomonas aeruginosa (44.4%) than

Acinetobacter baumannii (30%) which is in contrast with with Noyal et al where

Higher incidence of AmpC production is seen in Acinetobacter baumannii

(67.4%) than Pseudomonas aeruginosa (46.9%)55

Samplewise distribution of Amp C producers, maximum Amc C producers

seen in urine 21(39.6%), followed by Pus 36(38.7%), sputum 6(27.2%), blood

4(22.2%) and bodyfluids 3(21.4%) which is in concordance with S Upadhyay1, S

Mishra1, MR Sen

1, T Banerjee

1, A Bhattacharjee et al where Strains harbouring

chromosomal and plasmidic AmpC gene (i.e., n0 = 64) were predominantly

cultured from pus ( n = 26), urine ( n = 13), burn wound ( n = 09), blood ( n = 06),

endotracheal tube (ETT) aspirates ( n = 07) and other body sites/fluids ( n = 03).36

SUMMARY

Two hundred nonfermenting gram negative bacilli were isolated from all

age groups, The total numbers of males were 121(60.5%) and females were

79(39.5%). The male to female ratio was 3.1:1.

Most samples were obtained from pus 93 samples (46.5%) followed by

urine 53 samples (26.5%). Most samples are obtained from burns ward

73(36.5%) followed by otorhinolaryngology 34 (17%), Surgery 22(11%).

Risk factors associated with infections by nonfermenters are Diabetes

Mellitus 26 ( 13%), Post surgical wound infection 21(10.5).

Most predominant organism is Pseudomonas aeruginosa 117(58.5%)

followed by Acinetobacter baumannii 60(30%), Pseudomonas fluorescens

8(4%), Acinetobacter lwoffi 6(3%) Burkholderia cepacia 3(1.5%) and

Stenotrophomonas maltophilia 3(1.5%) and Weeksella virosa 2(1%).

Pseudomonas aeruginosa shows higher sensitivity Pipercillin tazobactem

115(98.29%), Imipenem 100(85.47%), Amikacin 81(69.23%),Ceftazidime

73(62.39%) and least to Cotrimoxazole 37(31.62%). It shows 100%

sensitive to colistin

Acinetobacter baumannii shows higher sensitivity Pipercillin tazobactem

53(88.33%), Imipenem 64(90%), Amikacin 48(80%),Ceftazidime

40(66.6%). It shows 100% sensitive to colistin (60),

Drug susceptibility of different NFGNB are that all Burkholderia cepacia

and Stenotrophomonas maltophilia shows sensitive to cotrimoxazole and

resistant to Amikacin. Weeksella virosa and Shewanella putrefaciens

shows sensitive to almost all drugs

Multi drug Resistant among NFGNB that is resistant to more than two

drugs. Among 117 Pseudomonas aeruginosa ,86 organisms shows multi

drug resistant, 49 MDR in Acinetobacter baumannii, 6 MDR in

Pseudomonas fluorescens,5 MDR in Acinetobacter lwoffi, 1 MDR in case

of Burkholderia cepacia, Stenotrophomonas maltophilia and Shewanella

putrefaciens

ESBL producers in non fermenting gram negative bacilli, Twenty four of

117 Pseudomonas aeruginosa are ESBL producers, twelve of 60

Acinetobacter baumannii, two of 8 Pseudomonas fluorescens, no ESBL

producers in other NFGNB.

Carbapenem resistant isolates in NFGNB are seventeen of 117

Pseudomonas aeruginosa are imipenem resistant, eight of 60 isolates of

Acinetobacter baumannii are imipenem resistant. No imipenem resistant in

other NFGNB.

MBL production in Non fermenters by Imipenem EDTA E Strip , Eight

MBL producers (6.83%) in 17 Imipenem resistant Pseudomonas

aeruginosa (8MBL/117 total Pseudomonas aeruginosa), five MBL

producers(8.33%) in 8 Imipenem resistant Acinetobacter baumannii (8

MBL/ 60 total Acinetobacter baumannii). No MBL producers in other

organisms

Comparison of MBL detection by different methods such as Modified

Hodge test , Double disc synergy test (DDST), combined disc diffusion

test(CDDT), Imipenem EDTA E strip method were done and found

Combined disc diffusion test (CDDT) and Imipenem EDTA E strip method

were more sensitive for MBL screening than Modified Hodge test , Double

disc synergy test(DDST) .

Four of 8 (50%) MBL-positive isolates were confirmed to be positive for

MBL by PCR for the gene Bla VIM by PCR and no Bla IMP was found in

the 8 MBL producers.

AmpC producers in NFGNB are Fifty two Amp C producers(44.4%) in

(Pseudomonas aeruginosa52/117 )and 18 Amp C producers(30%) in

Acinetobacter baumannii(18/60). No Amp C producers on other NFGNB.

Early diagnosis of Nonfermenting gram negative bacilli and administration

of specific antibiotic therapy, based on the antibiogram of the isolated

pathogen will reduce the complication as well as the emergence of drug

resistance, there by hasten the recovery of patients. This will lead to better

outcome and effective patient care

CONCLUSION

In our study, 200 nonfermenting gram negative bacilli were isolated from

various clinical samples. Higher incidence of infection by NFGNB was

seen in the age group of 31-40 years with male preponderance

Maximum samples were obtained from pus samples especially from Burns

ward and the maximum risk factors associated with infections by

nonfermenters are Diabetes Mellitus.

Most predominant organism is Pseudomonas aeruginosa followed by

Acinetobacter baumannii

Pseudomonas aeruginosa and Acinetobacter baumannii shows higher

sensitivity to Pipercillin tazobactem and Imipenem. Stenotrophomonas

maltophilia and Burkholderia cepacia shows sensitive to cotrimoxazole

and resistant to Amikacin. Weeksella virosa and Shewanella putrefaciens

shows sensitive to almost all drugs. All NFGNB were 100% sensitive to

colistin.

In Pseudomonas aeruginosa, Acinetobacter baumannii ESBL producers,

MBL producers and Amp C producers were seen.

MBL-positive isolates were confirmed to be positive for the gene Bla VIM

and no Bla IMP.

The present study showed emerging resistance of Nonfermenting gram

negative bacilli especially Pseudomonas aeruginosa and Acinetobacter

baumannii which may lead to increase in morbidity & mortality that can

be controlled by strict enforcement of antibiotic policy coupled with strict

adherence to infection control measures to prevent further emergence and

spread of antibiotic resistance.

PROFORMA

Name: Dept / Ward:

Age/Sex IP.No / OP No:

Address: Lab No:

Occupation: Date of Admission:

Present Complaints: Diagnosis:

Past History & Treatment:

Type of Clinical Sample:

Microscopic Finding:

Gram Staining:

Culture Characteristics:

Blood Agar:

MacConkey Agar:

CLED Agar:

Biochemical Characteristics:

Antibiotic Susceptibility Pattern:

Signature of Investigator Signature of Guide

APPENDIX

PEPTONE WATER

Peptone - 10g

Sodium chloride - 5g

Distilled water - 1 litre

Dissolve the ingredients in warm water, adjust the pH to 7.4 -7.5 and filter.

Distribute as required and autoclave at 121 degree Celsius for 15 mins.

BLOOD AGAR

Sterile sheep blood - 50 ml

Peptone - 10 g

Beef extract - 3g

Sodium chloride - 5 g

Distilled water - 1000 ml

Autoclave the nutrient agar base at 121º C for 15 minutes and blood with sterile

precautions and distribute in Petri dishes.

MAC CONKEY AGAR

Peptic digest of animal tissue - 17g

Proteose peptone - 3g

Lactose - 10g

Bile salts - 1.5g

Sodium chloride - 5g

Neutral red - 0.03g

Agar - 15g

Distilled water - 1000 ml

Final pH at (25º C) 7.1±0.2.

Suspend 51.53 grams in 1000 ml of distilled water. Heat to boiling to

dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure

(121ºC) for 15 minutes. Mix well and pour into Petri dish plates.

NUTRIENT AGAR

Peptic digest of animal tissue - 5g

Beef extract - 1.5g

Yeast extract - 5g

Agar - 15g

Distilled water - 1000ml

Dissolve the contents in water and mix by heating. Autoclave at 121° C for

15 minutes. Adjust pH to 7.4 + 0.2. Pour 20-25 ml of 9 cm diameter. Petri dishes

to give 4 mm thickness

1. OXIDASE TEST 6

This test is mainly done to differentiate organisms lacking cytochrome

oxidase enzyme, mainly the members of enterobactericeae. This enzyme helps in

the transfer of electrons to oxygen, with formation of water. The dye tetramethyl

paraphenylene diamine dihydrochloride is substituted for oxygen as artificial

electron acceptor. In the reduced state, it is colorless and in the presence of

cytochrome oxidase and oxygen it forms indophenol, which is purple. Strips

impregnated with dried reagents were used. The colony was taken on a wooden

stick and smeared onto the strip. The appearance of purple color within 10 sec.

was taken as positive.

2. CATALASE TEST 6

Organisms’ possessing the enzyme catalase splits hydrogen peroxide into

water and oxygen. The evolution of oxygen appears as bubbles. Tube catalse test

was performed 3ml of 3% Hydrogen peroxide was taken in a test tube. The colony

of the organism to be tested was taken on a glass rod and introduced into the tube.

Appearance of brisk effervescence was taken as positive.

3. MOTILITY6

Motility was done by hanging drop method. A drop of saline with the test

organism was placed on the coverslip. The hanging drop slide is inverted over the

coverslip where wax had been applied on the corners. The slide is turned quickly

so that drop is in centre of the concavity. The edge of the drop is focused on the

low power and high power objectives. Motility was observed.

4. INDOLE TEST6

Indole is a benzyl pyroll which is a metabolic product of tryptophan.

Bacteria which possess tryptophanase hydrolyse trytophan to indole. The

organism was inoculated in peptone water medium, incubated at 370C for 18-24

hours, 1ml of zylene was added and Erlichs’reagent (Para dimethyl amino

benzaldehyde) was added drop by drop. Formation of fuchsia red ring was

positive. It is a red complex of indole and paradimethylaminobenzaldehyde.

5. TRIPLE SUGAR IRON MEDIUM 6

Triple sugar iron medium was taken and the organism was stabbed to the

butt as well streaked on the surface. It was incubated for 18-24hours at 370C, and

then looked for the presence of growth and change in pH Growth with no change

in PH (slant and butt) indicated the organism to be nonfermenting.

6. CITRATE UTILIZATION TEST 6

Sodium citrate is a salt of citric acid seen in metabolism in Krebs cycle.

Some bacteria utilize citrate as the sole source of carbon and it is detected by the

production of alkaline by products Christensen’s citrate media was used. The

organisms were streaked on the surface of the slant and incubated at 370C for 18-

24hrs. Development of deep blue color of the medium with growth was taken as

positive.

8. UREASE TEST 6

Urea is a diamide carbonic acid which when hydrolyzed releases ammonia

and carbon dioxide. Urease is an enzyme when present hydrolyses urea and

release ammonia changing the medium to alkaline, PH.

The organism was streaked on the surface of the slant and incubated at

370C for 18-24 hours. Development of magenta pink of the medium along with

growth was taken as positive.

9. NITRATE REDUCTION TEST 6

The capacity of an organism to reduce nitrates to nitrites is shown by this

test. The presence of nitrate in the medium is detected by addition of a

naphthalamine and sulphanilic acid.

The organism was inoculated in nitrate broth and incubated at 370C for 18-

24 hours and observed for gas production by Durham’s tube. One ml each of

reagents a naphthalamine & sulphanilic acid were added simultaneously and

looked for the development of red color.Development of red color was taken as

positive and when it was negative zinc dust was added. When the red color

developed after adding zinc the test was taken as negative because it indicated the

presence of residual nitrates.

10. GROWTH AT 42ᴼC 6

The organism was plated in nutrient agar plate and incubated at 420C for

18-24 hours and looked for growth. Presence of uniform growth indicated positive

results.

11. GROWTH AT 44ᴼC 6

The organism was plated in nutrient agar plate and incubated at 440C for

18-24 hours and looked for growth. Presence of uniform growth indicated positive

results.

12. POLYMYXIN B SENSITIVITY 6

The organism was plated on nutrient agar plate and a polymyxin B disc of

300U was kept. It was incubated at 370C for 18-24 hrs and looked for zone of

inhibition

SPECIAL BIOCHEMICAL TESTS USED FOR IDENTIFICATION OF

NON FERMENTERS

1. HUGH – LEIFSON OXIDATION - FERMENTATION MEDIUM6

Peptone: 2g

Sodium Chloride: 5g

D-Glucose: 10g

Bromothymol blue: 0.03g

Agar: 3.0g

Dipotassium Phosphate: 0.3g

Distilled water: 1 litre

pH: 7%

Medium was sterilized by autoclaving. After cooling the medium to 450C,

filter sterilized carbohydrate solution was added to get a final concentration of 1%

Carbohydrates used for the study were glucose, maltose, xylose, fructose and

mannitol. Of medium was poured as a butt and without a slant into tubes. Two

tubes were required for the test, each inoculated with the unknown organism,

using a straight needle stabbing the medium three to four times half way to the

bottom of the tube. One tube of each pair was covered with a 1cm layer of sterile

mineral oil (or) melted paraffin, leaving the other open to the air. Both tubes were

incubated at 350C and examined daily for several days.

In case of oxidative metabolism, yellow color appears along the upper one

fourth of the medium and in the tube where no oil overlay was done. In case of

fermentative organisms yellow color develops in both the tubes.

CONTROL

Glucose fermentation: Escherichia coli

Glucose oxidation: Pseudomonas aeruginosa

Non saccharolytic: Alcaligenes species.

2. DECARBOXYLATION OF LYSINE, ARGININE & ORNITHINE 6

Decarboxylases are a group of specific enzymes which react with carboxyl

portion of aminoacid forming alkaline reacting amines. The reaction is

decarboxylation. Each enzyme is specific for Lysine, Arginine and Ornithine.

INGREDIENTS

Yeast extract: 5g

Peptone: 5g

Glucose: 0.5g

Pyridoxal: 5mg

Bromocresol Purple: 5ml

Cresol Red: 2.5ml

Distilled water: 1 ltr.

All aminoacids were added individually to a final concentration of 1% pH

adjusted at 6.0 & autoclaved at 15lbs for 15 minutes.

PROCEDURE

The organism was inoculated in four tubes. One having the basal medium

without aminoacid for control. Other three tubes having lysine, arginine and

ornithine each. All tubes were overlaid with liquid paraffin. All were incubated at

370C for 24 hours.

The control tube turned yellow indicating that the organism is viable and

the test medium turning blue purple indicating positive result.

3. O–NITROPHENYL β - D GALACTOPYRANOSIDE 6

Reagent

Sodium Phosphate buffer 1M pH 7.0 – 5ml

O – Nitrophenyl β - D – galactopyranoside – 80mg

Distilled water – 15ml.

A dense suspension of the test organism grown in TSI agar was prepared in

saline.About 1 drop of toluene was added to the suspension and 0.2ml of ONPG

solution was added to the suspension and incubated at 370C β -galactosidase

producing organism show yellow color after 1 hour or 18-24 hours incubation.

CONTROL

Positive Control – Escherichia coli

Negative control – Proteus species.

4. ACETAMIDE AGAR

Ingredients

Magnesium Sulphate: 0.2g

Ammonium dihydrogen Phosphate: 1g

Pottasium monohydrogen phosphate: 1g

Sodium Chloride: 5g

Acetamide: 10g

Bromothymol blue solution: 6.4ml

Agar: 15g

Final pH: 6.9

Distilled water: 1 litre

The ingredients are mixed and pH adjusted to 6.9, dispensed into screw cap

tubes and sterilized at 1210C for 15 min. The medium was allowed to cool in a

slant. The slant was inoculated with a portion of isolated colony and incubated at

370C overnight and was observed for color change. Tubes with negative result

were further incubated for 7days.

Control

Positive control: Pseudomonas aeruginosa

Negative Control: Stenotrophomonas maltophilia

5. GELATIN LIQUEFACTION TEST 6

Gelatin breakdown can be demonstrated by incorporating it in a buffered

nutrient agar, growing the culture and then flooding the medium with mercuric

chloride that differentially precipitates either gelatin or its breakdown products

causing opacity in the medium with clear zones around gelatin-liquefying colonies

CONTROL

Positive control: Pseudomonas aeruginosa

Negative Control: Stenotrophomonas maltophilia

PCR Primers and procedure

Two sets of primers were used for multiplex PCR. The primers used are

given below

IMP FORWARD – 5’ CTA CCG CCG CAG CAG AGT CTT TG -3’

REVERSE 5’-AAC CAG TTT TGC CTT ACC AT-3’

VIM FORWARD 5’- AGT GGT GAG TAT CCG ACA G -3’

REVERSE 5’-ATG AAA GTG CGT GGA GAC -3

PCR was conducted with 1μl of boiled bacterial suspensions, 0.5μl of each

primer, 2μl 5mM of dNTP, 2.μl 10mM Tris-HCL (pH 8.3), 0.75 μl 50mM Mgcl2

and 2U of Taq DNA polymerase in a total volume of 20μl

ABBREVIATIONS

NFGNB - Nonfermenting gram negative bacilli

NF - Nonfermenters

MDR - Multidrug resistant

PDR - Pandrug resistant

XDR - Extremely drug resistant

ESBL - Extended Spectrum of Betalactamases

MBL - Metallo betalactamases

ONPG - O–Nitrophenyl β - D Galactopyranoside

ATCC - American Type Culture Collection

CAPD - Chronic Ambulatory Peritoneal Dialysis

CFU - Colony Forming Units.

CLED - Cystine Lactose Electrolyte Deficient

CLSI - Central Laboratory Standards Institute

ELISA - Enzyme Linked Immunosorbent Assay

E- TEST - Epsilometer Test

FDA - Food and Drug Administration (United States

USFDA)

ICU - Intensive Care Unit

OPD - Out Patient Department

MALDI –TOF - Matrix Assisted Laser Desorption/Ionisation –

Time of Flight.

DDST - Double Disc Synergy Test

CDDT - Combined Disc Diffusion Test

DPT - Disc Potentiation Test

MHA - Mueller Hinton Agar

MIC - Minimum Inhibition Concentration

PCR - Polymerase Chain Reaction

PFGE - Pulsed-Field Gel Electrophoresis

SPSS - Statistical Package for Social Science

KEY TO MASTER CHART

M - Male patient

F - Female patient

IP - Inpatient

OP - Outpatient

LBW - Labour Ward

ICU - Intensive Care Unit

ENT - Otorhinolaryngology

UTI - Urinary tract infection

DM - Diabetes Mellitus

RTA - Road Traffic Accident

P - Positive

N - Negative

S - Susceptible

R - Resistant

AMP - Ampicillin

AMX - Amoxicillin

CN - Cephalexin

CTR - Ceftriaxone

CAZ - Ceftazidime

AK - Amikacin

GEN - Gentamicin

CIP - Ciproflaxacin

CFS - Cefoperazone Sulbactam

COT - Cotrimoxazole

PIT - Piperacillin tazobactam

CX - Cefoxitin

IMP - Imipenem

LE - Levofloxacin

NIT - Nitrofurantoin

LZ - Linezolid

CL - Colisitin

ESBL - Extended spectrum of betalactamases

MBL - Metallobetalactamases

Amp C - AmpC betalactamases

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S.NO sex Age Lab

no IP.No Risk factor ward sample Organism AMP AMX CN CTR CAZ AK GEN CIP CFS COT PIT CX IMP LF CL AmpC ESBL MBL

PCR -

Genotype

for MBL

Bla-

VIM

Bla-

IMP

1 F 42 1606 7888 ENT Pus Pseudomonas

aeruginosa

S R R R S S S R R S S S S R S N N N

2 F 21 1610 176 Burns Pus Acinetobacter

baumannii

R R R S S S S S S S S S S S S N N N

3 F 24 1615 9072 ENT Pus Pseudomonas

aeruginosa

S R R R S S S R S R S S S S S N N N

4 M 43 1616 9247 Chest

Medicine

Sputum Pseudomonas

aeruginosa

S R R R S R S R S R S R S R S P N N

5 F 29 1618 9296 Burns Pus Acinetobacter

baumannii

R S R S S S R S S R S R S S S P N N

6 M 61 1619 9252 ICU stay IMCU Blood Pseudomonas

aeruginosa

S R R S S R S R R R S R S S S P N N

7 M 34 1621 9016 Burns Pus Pseudomonas

fluorescens

S S S S S S S S S S S S S S S N N N

8 F 13 1623 8975 ICU stay IMCU Blood Pseudomonas

aeruginosa

S R R R S R R R R S S R S S S P N N

9 M 44 1624 9122 RTA Surgery Pus Acinetobacter

baumannii

R S R S S R S S R S R R S R S P N N

10 M 29 1625 9022 RTA Surgery Pus Pseudomonas

aeruginosa

S R R R S S R R S R S R S R S P N N

11 M 16 1626 8028 DM, ICU stay IMCU Blood Acinetobacter

baumannii

S S R S S S S R S S S S S S S N N N

12 M 23 1637 8765 RTA Surgery Pus Pseudomonas

fluorescens

S S S S S S S S S S S S S S S N N N

13 M 46 1648 9072 DM Chest

Medicine

Sputum Pseudomonas

aeruginosa

S R R R R R R R S R S R S S S P N N

14 M 55 1650 9321 ICU stay IMCU Blood Acinetobacter

baumannii

R R R R R S R S R R S R S R S P N N

15 F 8 1651 9302 RTA Surgery Pus Pseudomonas

aeruginosa

S R R R S S R R R S S R R R S P N N

16 M 72 1663 9147 DM Medicine Ascitic fluid Acinetobacter

baumannii

R R R S S R S S S S S R R S S P N N

17 M 9 1667 9446 Urology Urine Pseudomonas

aeruginosa

R R R S R S R R S R S R S S S P P N

18 M 48 1670 9209 ICU stay IMCU Blood Pseudomonas

aeruginosa

R R R R S S R R R S S R S S S P N N

19 F 24 1676 8358 Indwelling

catheter

Urology Urine Pseudomonas

aeruginosa

R R R R S R R R R R S R S R S P N N

20 M 18 1690 9761 Chest

Medicine

Sputum Acinetobacter

baumannii

S S R S S S S S S S S S S S S N N N

21 F 56 1697 8527 DM, ICU stay IMCU Blood Pseudomonas

aeruginosa

R R R R R S R R S R S R R R S P P N

22 M 41 1700 9702 RTA Surgery Pus Pseudomonas

fluorescens

R S R S S S S S S S S S S R S N P N

23 M 43 1567 9929 Burns Pus Pseudomonas

aeruginosa

R S S S S S S S S S S R S S S P N N

24 M 80 1710 8542 Indwelling

catheter

Urology Urine Acinetobacter

baumannii

R R R S R S R R S S R S S R S N P N

25 M 29 1714 9748 RTA Surgery pus Pseudomonas

aeruginosa

R R R R S S R R R S R R S S S P N N

26 M 5 1721 7315 Indwelling

catheter

Urology Urine Acinetobacter

baumannii

R S R S S R S S R R S R S S S P N N

27 M 29 1748 75636 RTA Surgery Pus Pseudomonas

aeruginosa

R R R R S S R R S R S R S R S P N N

28 M 44 1751 9945 Medicine Pleuralfluid Acinetobacter

baumannii

R S R R S S S S S S S S S R S N N N

29 F 7 1764 9801 Burns urine Acinetobacter

baumannii

R R R S S R R R S S S S S S S N N N

30 M 22 1767 505/15 Burns urine Pseudomonas

aeruginosa

R R R S R S R R S R S R S S S P P N

31 M 65 1769 10308 Burns urine Pseudomonas

aeruginosa

R R R R S R R R R S S R S R S P N N

32 F 53 1772 10279 RTA Surgery Pus Acinetobacter

baumannii

S R S S R S S S S R S R S S S P P N

33 F 8 1776 9774 Chronic renal

disease

Medicine Ascitic fluid Pseudomonas

aeruginosa

R R R R S S R R R R S R S S S P N N

34 F 49 1780 41427 DM, ICU stay IMCU Pus Pseudomonas

aeruginosa

R R R R S S R R R R S R R R S P N P N N N

35 F 23 1784 10353 ICU stay IMCU Pus Pseudomonas

aeruginosa

R R R S S S R R S R S S S R S N N N

36 F 55 1785 8204 Indwelling

catheter

Burns urine Pseudomonas

aeruginosa

R R R R R R R R S S S S S S S N P N

37 M 11 1787 9133 Burns urine Acinetobacter

baumannii

R S R S S S S R S R R S S R S N N N

38 M 3 1793 10205 Burns urine Pseudomonas

aeruginosa

R R R R S S R R R R S S S S S N N N

39 M 25 1801 8692 Burns urine Acinetobacter

baumannii

R S R R S S R S S S S S S S S N N N

40 F 19 1808 100634 RTA Surgery urine Pseudomonas

aeruginosa

R R R R S R R R R R S S S R S N N N

41 F 45 1813 10048 Indwelling

catheter

Urology urine Acinetobacter

baumannii

R R R S S R S S R R S S S R S N N N

42 M 67 1815 9989 Surgery urine Pseudomonas

aeruginosa

R R R R R R R R R S S S S S S N P N

43 F 6 1816 10353 Chest

Medicine

Sputum Acinetobacter

baumannii

S S S S R S R R S S S R S S S P P N

44 M 51 1824 10440 DM Chest

Medicine

Sputum Acinetobacter

baumannii

R R R S S S S S S R S S S R S N N N

45 F 26 1834 10601 DM, ICU stay IMCU Pus Acinetobacter

lwoffii

S S S S S S S S S S S S S S S N N N

46 M 13 1836 9900 Medicine Pleuralfluid Pseudomonas

aeruginosa

R R R R S R R R S R S S S R S N N N

47 M 22 1840 10666 DM, ICU stay IMCU Pus Pseudomonas

aeruginosa

R R R R S R R R S R S S R S S N N N

48 M 57 1850 10509 Burns Pus Acinetobacter

baumannii

S R S S S R S S S R S S S S S N N N

49 F 17 1859 91443 Burns urine Acinetobacter

baumannii

R S R R R S R R R S S R S R S P P N

50 M 48 1875 11046 Indwelling

catheter

Burns urine Pseudomonas

aeruginosa

R R R R R S R R R S S S S S S N P N

51 M 55 1890 11228 ENT Pus Pseudomonas

aeruginosa

R R R R R S R R S R S S S R S N P N

52 M 27 1924 8743 ICU stay IMCU Blood Acinetobacter

lwoffii

S S S S S S S S S S S S S S S N N N

53 F 13 1943 DM ENT Pus Pseudomonas

aeruginosa

R R R R S R R R R R S S S S S N N N

54 F 55 1953 11220 Burns Pus Pseudomonas

fluorescens

R R R R S S S R R R S S S S S N N N

55 M 41 1958 11275 Chronic renal

disease

Nephro urine Acinetobacter

baumannii

S S R S S S R S S R S S R S S N N N

56 F 12 1960 11548 ENT Pus Pseudomonas

aeruginosa

R R R R S R R R S S S S S R S N N N

57 F 29 1974 Burns Pus Acinetobacter

baumannii

R R S S S R S S S R S S S R S N N N

58 M 28 1983 12013 ENT urine Pseudomonas

aeruginosa

R R R R S R R R R R S S S S S N N N

59 F 44 1994 12043 Medicine Pleuralfluid Acinetobacter

baumannii

R S R S R S R R S S S R S S S P N N

60 M 23 2001 11611 RTA Surgery Pus Pseudomonas

aeruginosa

R R R R R S R S S R S S R R S N P N

61 M 47 2024 9893 DM Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R R R S S R R S S R S S S P N N

62 M 49 2033 11952 Surgery Pus Pseudomonas

aeruginosa

R R R R S S R R S R S R S R S P N N

63 F 59 2034 12104 Burns Pus Acinetobacter

baumannii

S R R S S S R S R R S S S R S N N N

64 M 43 2036 11117 Burns Pus Acinetobacter

baumannii

R R S S S R S S S R S S S S S N N N

65 M 29 2040 12105 ENT Pus Pseudomonas

aeruginosa

R R R R S R R R S R S R S S S P N N

66 M 47 2043 12334 Indwelling

catheter

Nephro urine Acinetobacter

baumannii

R S R R S S R R S S S S R S S N N P N N N

67 M 32 2050 12708 Urology urine Pseudomonas

aeruginosa

R R R R S R R S R R S R S R S P N N

68 M 46 2065 91026 Indwelling

catheter

Urology urine Pseudomonas

aeruginosa

R R R R S S R S R S S S S R S N N N

69 M 52 2066 12080 Indwelling

catheter

Urology urine Pseudomonas

aeruginosa

R R R R R S R S S R S R S R S P P N

70 F 36 2067 12043 Burns Pus Acinetobacter

baumannii

S R R S R S R S S R R R S R S P N N

71 M 37 2068 12334 Medicine Pleuralfluid Pseudomonas

aeruginosa

R R R R S R S R R R S S S S S N N N

72 F 42 2081 12104 DM ENT Pus Pseudomonas

aeruginosa

R R R R S R S R R S S R R S S P N P N N N

73 F 51 2100 11927 Burns Pus Acinetobacter

baumannii

R R S S S R R S R R S S S R S N N N

74 M 69 2108 12257 Urology urine Pseudomonas

aeruginosa

R R R R S R S S R S S S S R S N N N

75 F 31 2117 12830 Burns urine Pseudomonas

aeruginosa

R R R R R R S R R S S S S R S N P N

76 M 22 2118 12823 Chronic renal

disease

Nephro urine Acinetobacter

baumannii

R R R S S S S S S S S R S S S P N N

77 M 47 2119 12104 DM Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R R S R S R R R S R S S S P N N

78 M 67 2124 471/15 Burns Pus Acinetobacter

baumannii

S S R S R S R R S R S S S R S N P N

79 M 53 2125 12145 ENT Pus Pseudomonas

aeruginosa

R R R R S R R S R S S S S R S N N N

80 F 24 2126 12134 ENT Pus Pseudomonas

aeruginosa

R R R R S R R R R R S R S R S P N N

81 F 44 2128 12167 Burns Pus Acinetobacter

baumannii

R R S R S S R S S S S R R R S P N P N N N

82 F 33 2130 12105 Burns Pus Acinetobacter

baumannii

S R R S S S S S R R S S S S S N N N

83 M 70 2131 12146 DM Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R R R R R R R R S R S R S P N N

84 F 55 2132 12147 ENT Pus Pseudomonas

aeruginosa

R R R R S R R S R S S S R S S N N P P P N

85 F 27 2133 12149 Burns Pus Acinetobacter

baumannii

R S R S R S R R S R S R S R S P P N

86 M 41 2134 12165 Indwelling

catheter

Burns urine Pseudomonas

aeruginosa

R R R R S R R R R R S S S S S N N N

87 F 28 2135 12164 Chest

Medicine

Sputum Acinetobacter

baumannii

R S S S S R R S R S S S S R S N N N

88 M 43 2137 12161 Burns Pus Acinetobacter

baumannii

S R R R R S S S S S S R S S S P N N

89 F 75 2139 12169 DM Medicine Pleuralfluid Pseudomonas

aeruginosa

R R R S S R R R R S S R S R S P N N

90 F 57 2140 12157 Burns Pus Acinetobacter

baumannii

R R R S R S S R R R R S R R S N N P N N N

91 M 44 2143 12198 Chronic renal

disease

Nephro urine Acinetobacter

baumannii

S S S S S R R S R S S S S R S N N N

92 M 21 2145 12164 ENT Pus Pseudomonas

aeruginosa

R R R R S R S S R R S S S R S N N N

93 M 37 2146 12178 ENT Pus Pseudomonas

aeruginosa

R R R R R R S R R R S S S S S N P N

94 M 42 2147 12189 Burns Pus Acinetobacter

baumannii

R S R S R S S S S R S S S S S N P N

95 M 59 2148 12187 RTA Surgery urine Pseudomonas

aeruginosa

R R R R R R S R S R S R S S S P P N

96 F 39 2150 12168 Chest

Medicine

Sputum Acinetobacter

lwoffii

R R S R S S S R S R S S S S S N N N

97 M 69 2152 12197 ENT Blood Pseudomonas

aeruginosa

R R R R S R S R R R S S R R S N N P P P N

98 M 58 2154 12179 DM, ICU stay IMCU Blood Acinetobacter

baumannii

S R R S R S S R S S S R S R S P N N

99 M 26 2156 12181 RTA Surgery Pus Pseudomonas

aeruginosa

R R R S R R R R R R S R S R S P N N

100 F 45 2157 12183 Burns Blood Acinetobacter

baumannii

S R R S S S R S R S S S R R S N N P N N N

101 M 31 213 9893 RTA Surgery Pus Pseudomonas

aeruginosa

R R R R S R R R R R S S S S S N N N

102 M 23 137 11611 Burns Pus Acinetobacter

baumannii

R S S S S S S S R R R R S S S P N N

103 F 81 159 11952 Surgery urine Pseudomonas

aeruginosa

R R R R S R S R S R S S S R S N N N

104 F 51 165 12104 Burns Pus Acinetobacter

baumannii

S R R S R R S R S S S S S R S N P N

105 F 49 168 1117 Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R S R R S S R R S R S S P N N

106 F 16 185 12105 Burns Pus Acinetobacter

baumannii

R R R S S S R S R S S R S R S P N N

107 M 22 192 12334 Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R R R S R S R R S S R S S N P N

108 M 34 207 12708 Burns Pus Acinetobacter

baumannii

S S S S S S S S S R S S R S S N N N N N N

109 F 53 698 91026 Burns Blood Pseudomonas

aeruginosa

R R R R S S R R R R S S S S S N N N

110 F 36 221 12080 Burns Blood Acinetobacter

baumannii

R R R S R S S R R S S S S R S N P N

111 M 12 262 12043 RTA Surgery Pus Pseudomonas

aeruginosa

R R R S R S R R S R S R S R S P P N

112 F 25 273 11927 RTA Surgery urine Pseudomonas

aeruginosa

R R R R R S R S R R S S S R S N P N

113 M 32 286 12257 Burns Pus Pseudomonas

fluorescens

S S S R R S S S S R S S S S S N N N

114 M 24 296 12830 Burns urine Pseudomonas

aeruginosa

R R R R S S R S R R S S S R S N N N

115 M 11 310 12823 DM Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R S R S R S S R S R R S S P N P N N N

116 F 21 311 12104 RTA Surgery Pus Pseudomonas

aeruginosa

R R R R R S R S R R S S S R S N P N

117 F 83 322 471/15 RTA Surgery Pus Pseudomonas

aeruginosa

R R R R R S R S R S S S S R S N N N

118 F 18 324 13158 Burns urine Pseudomonas

aeruginosa

R R R S S S R S S R R R S R S P N N

119 F 33 325 11568 RTA Surgery Pus Pseudomonas

aeruginosa

R R R R S S R S R R S S S S S N N N

120 M 27 329 13297 Burns Pleuralfluid Acinetobacter

baumannii

S R R S S S S S S S S R S S S P N N

121 F 33 330 12982 DM Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R R R S S S R R S R S R S P P N

122 F 12 338 13473 ENT Pus Pseudomonas

aeruginosa

R R R S R S S S S R S S S R S N N N

123 F 31 342 119730 Burns urine Pseudomonas

aeruginosa

R R R R R S S S R S S S R R S N P N

124 M 11 354 13066 ENT Pus Pseudomonas

aeruginosa

R R R R S S S S R R S R S R S P N N

125 M 65 358 13312 DM ENT Pus Pseudomonas

aeruginosa

R R S S S S S S R R S S S S S N N N

126 M 39 359 588416 Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R R R S S R S R S S S R S N N N

127 M 23 362 1922 DM Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R R R S S S R S S R S R S P N N

128 F 39 365 13020 Burns Pus Acinetobacter

baumannii

R R R S S S R S R R S S S R S N N N

129 M 38 369 2977 DM ENT Pus Pseudomonas

aeruginosa

R R R S R S S R R S S S R R S N P P P P N

130 M 26 379 3726 Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R R R S S S S S S S R S S S P N N

131 F 38 41 2832 Burns Pus Acinetobacter

baumannii

S S S S R S S R S S S S S S S N N N

132 M 34 432 304246 ENT Pus Pseudomonas

aeruginosa

R R R R S S S R R R S S S R S N N N

133 M 35 437 577744 Medicine Pleuralfluid Acinetobacter

baumannii

R R R S S S S S S S S S S S S N N N

134 F 23 439 661 ENT Pus Pseudomonas

aeruginosa

R R R S R S S S R S S S S R S N N N

135 M 62 454 58 burns Pus Acinetobacter

baumannii

R R S S S S R R R R S S S R S N N N

136 M 35 494 85 Burns urine Pseudomonas

aeruginosa

R R S R S S S R S R S R S R S P N N

137 M 33 517 799 burns Pus Acinetobacter

lwoffii

R R R S S S S S R S S S S R S N N N

138 F 29 520 288 DM Medicine Pleuralfluid Pseudomonas

aeruginosa

R R R S R S S R R R S S S S S N P N

139 F 32 527 43801 ENT Pus Pseudomonas

aeruginosa

R R R R S S S S R S S R S R S P N N

140 M 31 531 705 burns Pus Pseudomonas

fluorescens

R R R S S R S R R R S S S R S N N N

141 M 37 541 584601 Indwelling

catheter

Burns urine Pseudomonas

aeruginosa

R R R S R S S R S R S S R R S N N N

142 M 22 543 15423 Burns urine Pseudomonas

aeruginosa

R R R R S S S R R R S R S R S P N N

143 F 38 547 939 ENT Pus Pseudomonas

aeruginosa

R R R R S S S S R S S S S S S N N N

144 F 39 1172 698 burns Pus Stenotrophomonas

maltophilia

R S S S S S S S S S S S S S S N N N

145 F 23 1240 381 ENT Pus Pseudomonas

aeruginosa

R R R S R S S R S R S R S R S P P N

146 F 38 1318 575077 Burns urine Pseudomonas

aeruginosa

R R R R R S S S R R S S S R S N P N

147 M 56 1337 18781 DM ENT Pus Pseudomonas

aeruginosa

R R R R S S S R R S S S S R S N N N

148 F 34 1358 1547 burns Pus Pseudomonas

fluorescens

R R R S S R R S S R S S S R S N N N

149 M 22 1367 2100 DM Medicine Pleuralfluid Pseudomonas

aeruginosa

R R R S S S S R S R S S R S S N N P P P N

150 M 56 132 590167 burns Pus Acinetobacter

baumannii

S R R S R S S R S S R S S S S N P N

151 M 88 1447 2268 Burns urine Pseudomonas

aeruginosa

R R R R S S S R R R S R S R S P N N

152 F 14 1470 2518 burns urine Acinetobacter

baumannii

R S S S S S S S S S S S S R S N N N

153 M 18 1511 2225 ENT Pus Pseudomonas

aeruginosa

R R R R S S S R R S S S S R S N N N

154 F 33 1568 27281 Burns urine Pseudomonas

aeruginosa

R R R R R S S R S R S R S R S P N N

155 F 23 163 5875 burns urine Acinetobacter

baumannii

S R R S S S R S R R S S S R S N N N

156 F 31 1788 2339 Medicine Ascitic fluid Acinetobacter

baumannii

R R R S R S S R S S S S S S S N N N

157 F 58 5 50827 RTA Surgery Pus Pseudomonas

aeruginosa

R R R S R S S S R R S S S S S N P N

158 F 33 9 585581 burns Pus Pseudomonas

fluorescens

S S S S S S S S S R S S S S S N N N

159 M 27 16 591920 Chest

Medicine

Sputum Pseudomonas

aeruginosa

R R S R S S S S R S S R S R S P N N

160 M 39 50 1948 RTA Surgery Pus Acinetobacter

baumannii

R R R R S S S S S R S S R R S N N N

161 F 19 71 16 Indwelling

catheter

Burns urine Pseudomonas

aeruginosa

R R R R S S S S R R S R S R S P N N

162 M 51 94 1948 burns Pus Acinetobacter

baumannii

S S R R S S S S S R S S S R S N N N

163 F 35 107 1622 Surgery Pus Pseudomonas

aeruginosa

R R R S S S S S R S S S S R S N N N

164 M 25 109 1518 Indwelling

catheter

Nephro urine Acinetobacter

baumannii

R S R S S S R S R R S S S S S N N N

165 F 11 122 51227 DM, ICU stay IMCU Ascitic fluid Pseudomonas

aeruginosa

R R R R R S S S S R S R S S S P N N

166 F 33 41 1869 Chest

Medicine

Sputum Acinetobacter

baumannii

R S R S R S S R S R S S S R S N P N

167 M 56 57 2588 Burns urine Pseudomonas

aeruginosa

R R R R S S R R R S S R R S N P N

168 F 28 65 52978 Chronic renal

disease

Nephro urine Acinetobacter

baumannii

S S R S S S R S S S S S R S S N N N

169 M 32 66 49886 Medicine Sputum Acinetobacter

baumannii

R R R S S S S S R S S S S R S N N N

170 M 38 72 3115 Burns Pus Pseudomonas

aeruginosa

R s R S S S S S R R S R R R S P N P N N N

171 F 36 73 50421 Burns urine Pseudomonas

aeruginosa

R R R R S S S S S S S S S R S N N N

172 F 22 77 3007 Burns Pus Shewanella

putrifaciens

S S S S S S S S S R S S S S S N N N

173 F 34 81 3106 Post renal

transplantation

Nephro Urine Burkholderia

cepacia

R S R R S R S S S S S S S S S N N N

174 M 65 82 1788 Burns Pus Stenotrophomonas

maltophilia

R S R R S S S S R S S S S R S N N N

175 M 34 118 3763 DM Medicine Sputum Pseudomonas

aeruginosa

R R R R S S S R R R S S S S S N N N

176 M 22 127 2564 Burns Pus Pseudomonas

aeruginosa

R s R R S S S S R R S R S R S P N N

177 M 55 128 51030 Burns Pus Acinetobacter

lwoffii

R R R R S R R R S R S S S S S N N N

178 M 27 129 3103 Burns urine Pseudomonas

aeruginosa

R R S S R S S S S S S S S R S N N N

179 M 51 115 3949 Post renal

transplantation

Nephro urine Weekseilla virosa S S S S S S S S S R S S S S S N N N

180 F 32 221 2924 Chronic renal

disease

Nephro urine Weekseilla virosa S S S S S S S S S S S S S S S N N N

181 M 31 276 5443 DM Medicine Sputum Pseudomonas

aeruginosa

R R R R S S S R R R S R S R S P N N

182 M 32 229 38117 Burns Pus Pseudomonas

aeruginosa

R s R S S S S S R R S R S S S P N N

183 F 24 321 5699 Burns Urine Burkholderia

cepacia

R S R S S R R R S S S S S R S N N N

184 M 58 333 6110 ENT Pus Pseudomonas

aeruginosa

R R R R S S S S S S S S S R S N N N

185 M 33 376 1252 ENT Pus Pseudomonas

aeruginosa

R R S R R S S R R R S S S R S N N N

186 M 22 399 2464 Chest

Medicine

Blood Pseudomonas

aeruginosa

R s R R R S S S R R S S S R S N N N

187 M 36 367 169 Burns Blood Pseudomonas

aeruginosa

R R R R S S S R S S S S S S S N N N

188 M 37 397 472 ENT Pus Pseudomonas

aeruginosa

R R R R S S S S R R S S S R S N N N

189 F 37 323 44320 Burns Urine Burkholderia

cepacia

R S R S S R S S S S S S S S S N N N

190 M 39 334 472 Burns Pus Pseudomonas

aeruginosa

R R R R S S S R R S S S S R S N N N

191 M 39 367 44320 ENT Pus Pseudomonas

aeruginosa

R s R R S S S S S R S S S R S N N N

192 M 45 345 7219 Burns Pus Stenotrophomonas

maltophilia

R S R S S S S R R S S S S R S N N N

193 F 38 390 2225 ENT Pus Pseudomonas

aeruginosa

R R R S R S S R R R S S S S S N N N

194 M 34 312 8207 Burns urine Pseudomonas

aeruginosa

R s R R S S S R R S S S S R S N N N

195 M 35 311 3857 Burns Pus Acinetobacter

lwoffii

S S S S S R R S S S S S S R S N N N

196 M 33 344 51810 RTA Surgery Ascitic fluid Pseudomonas

aeruginosa

R R S R R S S S S R S S S R S N N N

197 M 31 370 52626 ENT Pus Pseudomonas

aeruginosa

R R R R S S S S R S S S S S S N N N

198 M 32 307 1125 ENT Pus Pseudomonas

aeruginosa

R s R R S S S R R R S S S R S N P N

199 M 37 309 5043 Burns Blood Acinetobacter

baumannii

R S R S R S R R S R S S S S S N P N

200 M 55 366 319 Burns Blood Pseudomonas

aeruginosa

R R R R R S S R R R S S S R S N N N