IDENTIFICATION, CHARACTERISATION AND ANTIMICROBIAL RESISTANCE PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM

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 “

is the bonafide original work done by

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

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 “

is a bonafide research work done by

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

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

Department of Microbiology, Government. Kilpauk Medical College and Hospital, Chennai, under the guidance and supervision of,

Professor & H.O.D of Microbiology,

Professor of Microbiology Department of Microbiology and

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

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

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

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 aeruginosa^5,8, 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 expressinnate 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 foundto be sensitive to aminoglycosides, anti pseudomonal penicillin, fluoroquinolones, and third generation cephalosporins. Amikacin andceftazidime were found to be highly effective.⁸⁰ The incidence of meropenem– resistant Pseudomonas aeruginosa is also increasing among nosocomially infectedpatients in ICU.⁸¹ The potential risk factors are previous antimicrobial drugexposure. A growing number of multidrug resistant (MDR) Pseudomonas aeruginosa producing metallo betalactamases (MBL) is also reported. Such strains areresistant to most broad spectrum beta-lactams, aminoglycosides andfluoroquinolones 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) fluids^83,86

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

Amipicillin Pipercillin Tazobctum efotaxime Cefipime Aztreonam Meropenem Polymixin B Aminoglycoside Cotrimoxazole ceftriaxone chloramphenicol

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.¹⁴A 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; bla_SHV, bla_TEM, bla_VEB, bla_PER and bla_OXA types) and carbapenemases (bla_GES, bla_KPC, bla_IMP, bla_SPM, bla_VIM and bla_NDM)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.¹⁹The 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 MEDIUM⁶

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