Study on the Prevalence of Beta Lactamases in Gram Negative Clinical Isolates

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CERTIFICATE

Certified that this dissertation entitled “STUDY ON THE PREVALENCE OF BETA LACTAMASES IN GRAM NEGATIVE CLINICAL ISOLATES” is a bona fide work done by Dr. S.Sarasa, Post graduate student, Karpaga Vinayaga Institute of Medical Sciences, Madurantagam, during the academic year 2013 – 2016.

Dr .A.R. Chakravarthy ^{MD DGO,} Dean,

Karpaga Vinayaga Institute of Medical Sciences and Research Centre

Madurantagam Taluk, Kancheepuram Dist-603308 Tamil Nadu, India.

Dr T.S.Vijayalakshmi M.D Professor &Head

Dept. of Microbiology,

Karpaga Vinayaga Institute of Medical Sciences and Research Centre

Madurantagam Taluk, Kancheepuram Dist-603308, Tamil Nadu, India.

DECLARATION

I declare that the dissertation entitled “Study on the prevalence of beta lactamases in gram negative clinical isolates” submitted by me for the Degree of M.D is the record work carried out by me during the period of April 2014 to March 2015 under the guidance of Dr .T.S Vijayalakshmi M.D. PROFESSOR and H.O.D of Microbiology, Karpaga Vinayaga Institute of Medical Sciences and Research Centre and has not formed the basis of any Degree ,Diploma, Fellowship titles in this or any other University or other similar Institution of Higher learning.

Signature of the candidate

Place: Dr.S.Sarasa

Date:

Signature of the guide Dr T. S. Vijayalakshmi

Professor & H.O.D Karpaga Vinayaga Institute

of Medical Sciences and Research Centre, Madurantagam.

ACKNOWLEDGEMENT

I express my sincere thanks to Dr. R. Annamalai, Managing Director, Karpaga Vinayaga Institute of Medical Sciences for his kindness in helping with all available resources.

I wish to thank Dr. A.R. Chakravarthy, Dean Karpaga Vinayaga Institute of Medical Sciences for his valuable support and encouragement.

It is beyond words to express my sincere thanks and gratitude to my teacher and guide Dr. T.S. Vijayalakshmi, Professor and Head of Department of Microbiology, KIMS who consistently guided me in each and every step of my thesis work. Her kind support and encouraging words are great pillars of my success.

I wish to proudly thank my teacher and professor Dr. D.Joseph Pushpa Innocent and Dr. T.Sheila Doris for their valuable advice and support.

It also gives immense pleasure in thanking Assistant professors, Dr. K.Balan Dr. A.V.Sangeetha , Dr. J.Abirami Lakshmy and tutor Mrs. E.Vanitha Devi, for helping me in overcoming difficult situations during this thesis work and for their valuable guidance.

I also thank my technical staff Mr Devaraj, Mrs.Amudha, Miss Puspha and Miss Punitha and nontechnical staff of Department of Microbiology for their excellent help in laboratory work.

My completion of thesis would have not been accomplished without the support of my family who are always a pillar of strength in all my endeavours.

Above all I thank the Almighty for His blessings.

TABLE OF CONTENTS

S.

NO. TITLE PAGE

NO.

1. INTRODUCTION 1

2. AIMS AND OBJECTIVES 5

3. REVIEW OF LITERATURE 6

4. MATERIALS AND METHODS 42

5. _RESULTS 49

6. DISCUSSION 68

7. _SUMMARY 77

8. CONCLUSION 79

9. _ANNEXURES

10. BIBLIOGRAPHY

Introduction

1

1. INTRODUCTION

Antimicrobial resistance is really a threat to clinical practice. Bacteria develop various mechanisms of resistance to different group of antibiotics.

Antimicrobial resistance is the resistance of a microorganism to an antimicrobial drug that was originally effective for treatments of infections caused by it, by acquiring genes coding for it.

Antimicrobial resistance is a global concern, as new resistance mechanism evolve, making the treatment of even common infections difficult resulting in disability or death of individual.

β lactamases production is one of the most important mechanisms of microbial resistance to β-lactam antibiotics which consist of four major groups such as ,Penicillins, Cephalosporins, Monobactams, and Carbapenems. Excessive use of antibiotic results in selective pressure, enabling resistant bacteria to thrive, and the beta lactamase gene undergoes continuous mutation.⁽¹⁾Certain acquired mechanism such as plasmids help in exchange of resistant determinants which has become a threat to clinicians as these cause rapid dissemination of genes between species.

The enzymes produced by bacteria are usually distinguished into four types, out of which 3 form major group ,such as extended spectrum β- lactamases (ESBL), class C Cephalosporinases (AmpC)) and β-lactamases with

2

carbapenemase activity in which metallo -β-lactamases (MBL)is of great concern today.⁽²⁾

In the early 1980s, the third-generation Cephalosporins which were very effective were introduced for treating beta lactamase mediated resistance. The advantages of these drugs were decreased nephrotoxic effects when compared to Aminoglycosides and Polymyxin. ⁽³⁾

E.coli and Klebsiella are leading causes of pneumonia, UTI, diarrhea, cholecystitis, meningitis. β lactam antibiotics has been the drug of choice in treating these conditions. However multiple antibacterial resistances in recent decades among Enterobacteriaceae are commonly met and the above conditions and pose a problem in management. ⁽⁴⁾

Over the past few years the occurrence of multidrug-resistant Gram- negative bacteria has increased due to multiple mechanism of resistance, which is a continuous phenomenon. The major concern is the production of AmpC beta-lactamases and/or extended-spectrum beta-lactamase in bacterial strains.

ESBL^’s confer resistance to, Cephalosporins, Oxyimino-cephalosporins (e.g., Ceftriaxone, Cefotaxime, and ceftazidime) Amino- penicillins and AmpC beta- lactamases in addition are resistant to Cephamycin (e.g., Cefoxitin) and Monobactams.⁽⁵⁾

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ESBLs are still a threat to clinicians as they are coded by plasmid and can be easily transmitted between species. ESBL producing organisms are highly effective in inactivating Pencillins, most Cephalosporins and Aztreonam

Amp C beta lactamases are of either chromosomal, or plasmid mediated.

The transfer of chromosomal genes onto plasmids results in Amp C β lactamases called as inducible Amp c lactamases. Hence isolates of Enterobacter aerogenes, Salmonella, Escherichia coli, Klebsiella, and Citrobacter have acquired plasmid mediated Amp C beta lactamases.⁽⁶⁾

Various risk factors are involved for the infection or colonization with the ESBL producing organisms. Prolonged stay in the hospital or ICU, Persons with vascular or urinary catheters, and those undergoing haemo- dialysis or emergency abdominal surgery also fall in this group. The prior antibiotic therapy to any antibiotic such as Aminoglycoside, Quinolones, Trimethoprim- Sulfamethoxazole, and Metronidazole or gut colonization with resistant bacteria also predisposes to acquisition of a resistant microbe.⁽⁷⁾

With the introduction of Carbapenem, there came an end to war against antimicrobial resistance till Carbapenamase enzymes emerged. These were useful drugs as they had broad spectrum of activity to combat infection caused by Pencillin and Cephalosporin resistant bacteria.⁽⁸⁾

Resistance to Carbapenem is predominantly mediated by metallo-beta lactamases, a class B type of beta lactamases that require bivalent metal ions

4

for activation. Hence detection of these β lactamases producing organisms becomes important for effective therapeutic approach and enhanced infection control. This study aims at detecting the prevalence of ESBL, Amp C and carbapenamases production in our hospital.

Aims and Objectives

5

2. AIMS & OBJECTIVES

 2.1 To know the antimicrobial susceptibility profile of the gram negative bacilli included in the study.

 2.2 To study the prevalence of resistance in gram negative clinical isolates.

 2.3 to determine the resistance pattern among the gram negative isolates.

 2.4 To find the prevalence of ESBL, Amp C Carbapenamases production and their combinations {ESBL +Amp C; ESBL +MBL; Amp C +MBL} in the isolates.

Review of Literature

6

3. REVIEW OF LITERATURE

Antibiotics discovery has revolutionized medicine in the 20^th century, as wonder drugs, but regrettably, the concomitant rise of antibiotic resistance in hospitals, communities, and the environment have evolved with their use.

Man's misuse and overuse of antibiotics accelerates the microbes to develop resistance genes, and horizontal gene transmission occurs. Hence for every antibiotic discovered organism tend to develop multiple mechanisms of resistance.⁽⁹⁾

3.1. ANTIBIOTIC HISTORY

Antibiotics are special kind of chemotherapeutic agent obtained from living organism. The antibiotic era began70 years ago with the development of Pencillin. Some observations suggest that even before the extensive use of penicillin, bacteria had mechanism to destroy Pencillin by enzymatic degradation. It was a finding by Abraham and Chain, 1940).⁽¹⁰⁾

The word ‘ANTIBIOTIC’ refers to a metabolic product of one organism that in small amounts kills or inhibits another microorganism ^[12]

Word antibiotic was coined by Washman in 1945⁽¹⁰⁾. In earlier days Europeans used natural quinine from bark of cinchona to treat malaria.

Mercury was used to treat syphilis, until arsenical compound salvarasan known as magic bullet was identified by Paul Erhlich. In 1914 he announced the discovery of neosalvarasan and henceforth he is known as Father of

7

Chemotherapy. Syphilis was the first known disease for which chemotherapeutic agent was used. ⁽¹²⁾

In 1928 Alexander Fleming made accidental discovery of penicillin from the fungus Pencillium notatum, which produces a substance that destroys Staphylococci, this substance was later named penicillin ⁽¹³⁾.

The advent of modern antibiotic era was developed only after Gerhard Domagh found that prontosil [forerunner of Sulphonamides] had dramatic effect on Streptococcal infections in 1935. He exploited the potential for antibiotic production among soil organisms ^(12). In 1940, it was found that Pencillin could be effective therapeutic substance. The discovery of antimicrobials, laid a path for research on chemotherapeutic agents and future drug discovery. This resulted in a number of new antibiotics, some of which made their way for clinical use.^(10)(16)

The next two decades was indeed the golden era, during which the discovery of novel antibiotics occurred, with no new classes further discovered

(10). The golden age of antibiotics will soon come to an end, if the evolution of antibiotic resistance is not hampered, or if replacements for them are not found.

3.2. ANTIBIOTIC RESISTANCE

It is the ability of microorganism to grow in the presence of a chemical (drug) that normally kills or limits its growth. Resistant strains which are capable of inactivating the drug became prevalent, as the antibiotic was widely used and

8

studies were undertaken to modify penicillin molecule so as to prevent the action by Pencillanases.⁽⁹⁾

3.3 SUPERRBUGS AND SUPER RESISTANCE

Microbes with enhanced morbidity and mortality acquired due to multiple mutations are termed as “superbugs”. These endow high levels of resistance to the antibiotic classes which are specifically recommended for their treatment. Thus the choice of treatment for these microbes are decreased and making it more costly. Even the periods of hospital care is extended. Super resistant strains have acquired increased virulence, and enhanced transmissibility in some cases. In reality, antibiotic resistance can be considered a virulence factor for the organism.⁽⁹⁾

A strong correlation between antibiotic use and development of antibiotic resistance in the treatment of the diseases caused by Gram-negative pathogens, such as Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae, has been observed in the past half-century. These organisms cause variety of infections in humans and animals. This is especially observed with the β-lactam class of antibiotics and the β-lactamases which are their related inactivating enzymes,.

3.4CLASSIFICATION OF ANTIBIOTICS

Antibiotics are classified on basis of method of action on bacteria. They act by inhibiting the synthesis of cell wall, damaging the cytoplasmic membrane. The other mechanisms are inhibition of nucleic acid or protein

9

synthesis and inhibition of specific enzyme pathway. ⁽¹⁴⁾β lactam antibiotics fall under the group of cell wall inhibitors.

3.5 CELL WALL SYNTHESIS INHIBITORS

These are agents which exert their effect by acting on the cell wall of bacteria.

3.5.1βlactam antibiotics

Pencillin, Cephalosporins, Methicillin, Carbapenem, Monobactams, come under this group. They are broad class of antibiotics characterized by a beta lactam ring which is as the core structure and the key to the mode of action of this class of antibiotics.

β- Lactam antibiotics act by inhibiting cell wall synthesis of bacteria.

The beta lactam ring binds to PBPs, preventing the cross linking of cell walls.

They target the pencillin binding protein (PBP) {transpeptidases}, which are involved in, rendering them unable to perform their role in cell wall synthesis.⁽¹⁵⁾

3.5.2 Mechanism of Action of β-lactam antibiotics:

Bacteria synthesize UDP – N – Acetyl muramic acid pentapeptide and UDP – N – Acetyl glucosamine pentapeptide residues, which are linked together. UDP is split of leading to final step of cleavage of the terminal D- alanine of the peptide chains by transpeptidases and cross linking between peptide chains of the neighboring strands occurs, β-lactam antibiotic inhibits

10

transpeptidases so that cross linking is not formed and thereby inhibiting the replication of bacteria.

Mechanism of action of beta lactam antibiotics is by inhibition of cell wall synthesis. The antibiotic gets incorporate into the bacterial cell wall and inhibit transpeptidases, which help in cell wall synthesis. Secondly, they attach to the Penicillin binding proteins and lyse the bacterial cell wall by freeing the cell wall hydrolases. Bacteria resist these antimicrobial mechanisms of action, by producing β lactamases or mutated types of penicillin binding proteins. ⁽¹⁸⁾

3.6. β LACTAM ANTIMICROBIAL AGENTS The β lactam antibiotics are classified as follows, ⁽¹⁷⁾

1. Penicillins: Penicillin G, Penicillin

2. Penicillinase resistant Penicillins: Methicillin, Nafcillin, Oxacillin, and Cloxacillin

3. Ureidopenicillins: Mezlocillin, Piperacillin 4. Aminopenicillins: Ampicillin, Amoxicillin 5. Carboxy penicillins: Carbenicillin, Ticarcillin 6. Cephalosporins :

a. First generation: Cefazolin, Cephalothin, Cephalexin

b. Second generation: Cefuroxime, Cefaclor, Cefamandole, Cefamycins (Cefotetan, Cefoxitin)

c. Third generation: Cefotaxime, Ceftriaxone, Cefpodoxime, Ceftizoxime, Cefoperazone, Ceftazidime

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Many genera of gram-negative bacteria possess β-lactamases which is chromosomally mediated. These enzymes are believed to have originated from Penicillin-binding proteins, with which they show some sequence homology.

The selective pressure exerted by β-lactam-producing soil organisms would have led to the development of chromosome mediated beta lactamase.^(20)(24)

3.6 .GENETICS OF RESISTANCE

Organism based resistance can be intrinsic or acquired. Intrinsic resistance resulting from normal structural physiological state of organism.^(14)(16)

Chromosomal resistance occurs as a result of mutation in a locus that controls susceptibility to given antimicrobial drug. The presence of antimicrobial drug serves as selective mechanism to suppress susceptible organism and favors growth of drug resistant mutant. Spontaneous mutation occur with frequency of 10⁷to 10¹² and they are infrequent cause for emergence of clinical drug resistance.⁽¹⁶⁾

Extra chromosomal: The plasmid genes for antimicrobial resistance control the formation of no. of enzymes that are capable of destroying antimicrobial drugs. Transposons and integrons are also involved in transfer of genes of drug resistance.⁽¹⁶⁾

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Difference between two types of drug resistance

Mutational drug resistance Transferable drug resistance Chromosome mediated Plasmid mediated

Resistance to 1 drug Resistance to multiple drugs

Non transferable Transferable

Low degree of resistance High degree of resistance Virulence of organism lowered Virulence not lowered Resistance is by altered metabolic

pathway

Resistance is by production of degrading enzymes

Transfer of drug resistance occurs by various mechanisms like conjugation through the agency of R plasmids [resistance plasmids].They carry 2 or more genes which encode variety of enzymes that degrade or destroy antibiotics. Resistance is also transmitted by transduction via agency of bacteriophage. ⁽¹⁶⁾

Transposons are small pieces of DNA that move from one site of bacterial chromosome to other site of chromosome or to plasmids and transmit resistance to many drugs.

The expression of enzyme activity may be constitutive or inducible and may be expressed as homogenously or heterogeneously.⁽¹³⁾

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3.8. MECHANISM OF ANTIBIOTIC RESISTANCE

There is various mechanism of antibiotic resistance by which microorganisms destroy the antibiotic. ⁽¹⁴⁾

1. Enzymatic inactivation 2. Altered drug target receptors

3. Alteration of antibiotic transport (efflux influx pumps) 4. Alteration of metabolic pathway

The most common mechanism of resistance to β lactam antibiotics is by enzymatic inactivation. Bacterial resistance to β lactams may be mediated by various other mechanism like altering the antibiotic target , decreasing the intracellular uptake of the drug, but β lactamase production contributes as important mechanism of resistance in gram negative pathogens.(15)(4)(18)

In the Gram negative bacteria,` β lactamases remain in the periplasmic space, where they attack the antibiotic before it reaches its receptor site.⁽²⁴⁾In the Gram positive bacteria they are secreted to the outside membrane environment as exoenzymes.

3.9. BETA LACTAMASES PENCILLINASES

These were the first beta lactamases identified. It was isolated by Abraham and Chain in E.coli even before pencillin entered clinical use.⁽¹⁸⁾ β lactamases act by hydrolysing the β lactam ring leading to formation of pencilloic acid and by deacylation the enzyme reverts back to bind more pencillin molecules. ⁽²⁰⁾

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Chromosomally mediated β lactamases are present in most of the genera of gram negative bacteria posses which is a natural phenomenon. This probably assists the bacteria in finding a way of protection when facing competition from other bacteria that naturally produce β-lactams. ⁽²³⁾

In the early 1980s,third generation cephalosporins such as cefotaxime and ceftriaxone was developed and gained importance because of the increasing prevalence of Ampicillin-hydrolysing β-lactamases (TEM-1, TEM-2 and SHV-1) in Enterobacteriaceae, some respiratory pathogens such as Haemophilus influenza and Moraxella catarrhalis, and non-glucose fermenting Gram-negative bacilli.⁽¹⁹⁾

A β-lactamase which hydrolyzed extended spectrum Cephalosporins was documented in strains of Klebsiella pneumonia in 1983,(Germany ),based on genetic and functional characteristics. Same findings were reported quickly from Europe and the US. They are called as extended spectrum β-lactamases because of their spectrum of activity against Oxyimino Cephalosporins.^(23)(30) Currently, over 150 ESBL are known.

In 1965, the first plasmid-mediated β-lactamaseTEM-1 was isolated in E.coli.^(23)(39)The designation “TEM” came from the patient’s name, Temoniera

(20)(39). This occurred in a strain isolated from blood culture, from a patient in Greece. It soon spread to other members of the Enterobacteriaceae family,

18

Pseudomonas aeruginosa, Neisseria gonorrhea, and Haemophilus influenza, because this β lactamase was plasmid borne.

SHV 1 (sulphydryl variable) is the second common plasmid mediated β- lactamase found in K. pneumoniae and E.coli. In K. pneumoniae the SHV1 β- lactamase is chromosomally encoded, but it is usually plasmid mediated in E.

coli.^(24)(39)

The incidence and types of extended spectrum β lactamase enzymes varies with geographical location and time due to extended mutation.⁽²⁴⁾ Detection of these enzymes becomes utmost necessary in clinical practice.

3.10. ESBL

These are group of β-lactamases which confer resistance to third- generation cephalosporins and aztreonam, by hydrolysing the molecule. These are inhibited by clavulanic acids.⁽²⁸⁾

Mutations which alter the amino acid configuration around the active site of these β-lactamases, results in ESBL genes TEM-1, TEM-2, or SHV-1.

ESBL pose a unique challenge to, Clinicians, clinical microbiologists, research scientists and infection control professionals face a challenge with ESBL.

Numerous outbreaks worldwide are caused by Enterobacteriaceae that produce ESBLs. Unfortunately ESBL detection in the laboratory has become complicated by multiresistant bacteria.⁽¹⁹⁾ These resistance is typically caused

19

by acquisition of plasmids containing genes which encode both ESBL, and other resistance genes.

The most common β lactamase in GNB is TEM .TEM -1 production seems to be the common cause of Ampicillin resistance in E. coli I (>90%).Recently140 TEM type enzymes have been described.

3.11. BETA LACTAMASES CLASSIFICATION:-

The basis of functional classification began when cephalosporinases, that had higher rates of hydrolysis for cephalosporins, has to be differentiated from Penicillinase, which –hydrolyze only penicillin. ⁽²⁴⁾

Several classification schemes were followed. It was based on molecular structure substrate profile, and location of genes. The first classification was developed by Richmond and Sykes followed by molecular class of Ambler.

The Sykes classification was based on substrate profile and the location of gene.

This Ambler classification separates beta lactamases to 4 distinct classes A to D based on similarities in amino acid sequence.⁽²⁴⁾

Class B is metallo β lactamases which require zinc atom for its activity.

A C D act by serine based mechanism. Most important is class A which is serine proteases that have either preference for penicillin (or) broad spectrum activities. They are found either on chromosomes (or) plasmids and are easily transferable from one bacterium to another. They may be produced

20

constitutively and may be induced. In this group are the staphylococcus and some Gram negative bacilli.

Class C: Primarily cephalosporinases, either constitutive (or) inducible, found on chromosomes of Gram Negative Bacilli. Class B & D are less important.

The BUSH JACOBY system classifies beta lactamases according to functional similarities [substrate and inhibitor profile]⁽²⁵⁾

 Group 1 Cephalosporinases that are not inhibited by clavulanic acid

 Group2 Pencillanases, Cephalosporinases, and broad spectrum β lactamases that are generally inhibited by beta lactamase inhibitors

 Group 3 Metallobetalactamases that hydrolyses Pencillins, Cephalosporins, and Carbapenems.

ESBLs belong to group 2be which are enzymes derived from 2b, denoting a broad spectrum of action or 2d OXA type with similar activity but differs by being resistance to clavulanic acid.⁽²⁸⁾

More than 890 β lactamases are reported; the largest occurred in class A class D families. To illustrate, there are currently more than 165 TEM β lactamases, 75 ESBL^’ s of TEM family , 36 inhibitor resistant TEM , 126 SHV, OXA 160 enzymes and 91 CTX –M ESBL. Classification table is attached in Annexure-I.

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3.12ESBLs - Epidemiology

Enterobacteriaceae producing ESBL strains is a challenge in treating hospitalized as well as community based patients. Production of ESBL began in Europe, however within few years it spread to United States and Asia. It varies from 6% to 87% from previous studies in India. The uniqueness of ESBL is its specificity. A prospective study on antibiotic co resistance and ESBL in urinary isolates from North East India has noted the ESBL detection rate as 24.56%. A study from New Delhi showed 68.78 per cent of the strains of Gram negative bacteria as ESBL producers.⁽⁷²⁾ Ritu Agarwal et al from Pune in their study on prevalence of ESBL among E.coli and Klebsiella pneumoniae isolates found that 40% of E.coli and 54.5% of Klebsiella pneumoniae were ESBL producers.⁽⁷³⁾ Pushpa D et al from Amristar, Punjab has found ESBL production among 43.83% of their uropathogens. Tankhiwale et al from Nagpur detected 48.3% of uropathogens to be ESBL producers.⁽⁷¹⁾ They also found 90.5% of the ESBL producing isolates were multi drug resistant. ESBL production was detected in about 36% of K. pneumoniae and 47% of E.coli infections in a study from Chennai suburban.⁽⁴⁹⁾ From Mangalore ESBL was reported as 39.5% in a study by Eshwar Singh.⁽³⁹⁾

.

3.13. CTX –M beta lactamases

CTX –M. is the second largest group of ESBL which shows greater activity against Cefotaxime than other oxyimino beta – lactam substrate [Ceftriaxone, Cefepime Ceftazidime].⁽²⁴⁾ they are plasmid acquired genes of beta lactamases which were previously found on the chromosome of kluyvera

22

species. These enzymes are classified to 5 major groups, based on Amino acid sequences. CTX- M1 cluster has been related to chromosomal bla gene present in different Kluyvera cryocrescens CTX- M2 cluster originates from gene KLU A OF Kluyvera ascorbata. The other clusters have genes originated from Kluyvera Georgina.⁽²⁰⁾

3.14. OXA – BETA LACTAMASES

These belong to molecular class D and functional group 2d.Hydrolytic activity to oxacillin and cloxacillin is high but action against clavulanic acid is poor. It acquires the ESBL phenotype by amino acid substitution in OXA enzymes.^[20]

3.15. INHIBITOR RESISTANT β lactamases

Among E. coli beta lactamase that were not resistant to clavulanic acid was discovered in early 1990 “s and were termed as inhibitor resistant β lactamases. Though they are not ESBL^’s, since they are derived from classical TEM SHV enzymes, they are discussed along with ESBL.⁽²⁰⁾There are at least 19 distinct enzymes found mainly in E.coli and few strains of Klebsiella pneumonia .They are susceptible to inhibition to Tazobactam.

3.16. Amp C betalactamases: Class C

Amp C beta-lactamase are Ambler class C or group I cephalosporinases that confer resistance to cephalosporins and cephamycin and are not affected by inhibitors (Tazobactam, Clavulanic acid and Sulbactam).⁽³²⁾

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Their ability to hydrolyse cephamycin as well as other extended- spectrum cephalosporins differentiates them from other extended-spectrum β- lactamases (ESBLs) ⁽³⁰⁾.

These are mostly chromosomal beta lactamases and are produced to greater or lesser extent by almost all GNB. Of the two types of Amp C (plasmid-mediated and chromosomal), the former is encountered in Klebsiella species, Proteus mirabilis and Salmonella species while the latter is seen in organisms such as Morganella morganii, Enterobacter cloacae, Citrobacter freundii, Hafnia alvei and Serratia marcescens. Escherichia coli is unique in that it also expresses chromosomal Amp C at low levels.⁽³²⁾

The widespread distribution of Amp C genes on transmissible plasmids is a continuous challenge for research people. Bacteria lacking chromosomal bla Amp c gene acquired it from transmissible plasmids. Amp C enzymes are inducible in many bacteria and can be expressed at high levels, by mutation.

Boronic acid and cloxacillin inhibit Amp C β lactamases.^(29)(30)

E. coli, Enterobacter cloacae, S. marcescens, Pseudomonas aeruginosa and Citrobacter freundii possess chromosomal sequences which encode β lactamases of the AmpC type. This β-lactamase which is a product of the AmpC gene is broadly active against Cephalosporins but is not inhibited by Clavulanic acid, thus differs from ESBLs.⁽³¹⁾

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Chromosome-encoded AmpC is usually an inducible enzyme that is expressed at low basal levels, but in some species such as E. coli, the enzyme is non inducible.^{(30) (20)}. Beta-lactam antibiotics, such as Cefoxitin, Cefotetan, and Imipenem, induce the expression of AmpC genes which are chromosomally located and mediated by the regulator AmpR. The most common cause of constitutive hyper production of AmpC beta-lactamases is mutation in the repressor gene AmpD. ⁽⁴¹⁾

Numerous pathogens produce plasmid-encoded AmpC β-lactamases, e.g. Salmonella spp, Citrobacter freundii, Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis

They are separated to 4 groups:

 GROUP 1 – plasmid encoded Amp C cephalosporinases consist of those which originated from Amp C of Citrobacter freundii.

 GROUP 2 are related to chromosome Cephalosporinases of E. cloacae.

 GROUP 3 are from Amp C of Pseudomonas

 GROUP 4 CMY beta lactamases

At present there are no CLSI or other approved criteria for detection of Amp C β lactamase. A positive ESBL screening test, and a negative confirmatory test involving increased sensitivity to clavulanic acid is typical of organism producing enough Amp C β lactamase.⁽²⁹⁾

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Treatment of infection with Amp C strains is difficult as most Pencillins and Cephalosporins are ineffective. Cefepime can be used. Imipenem, Ertapenem or Temocilin are found to be active. Fluoroquinolone therapy and Tigecycline is another option for treatment.

Hence in clinical management, recognition of Amp C producing E.coli strain is important as in therapeutic failure occurs often on administration of β lactam drugs.⁽⁵⁰⁾

3.17. CARBAPENAMASES

These are versatile beta lactamases encoded by plasmids which hydrolyze Pencillin, all third generation Cephalosporins, Carbapenems like Imipenem, Meropenem, and Doripenem. ⁽⁴³⁾

They are more stable than extended spectrum β lactamases and Amp C β lactamases. Plasmid mediated MBL genes should be detected at the earliest as they spread rapidly to other gram negative bacteria. This makes necessary for the modification of therapy and initiation of effective infection control.

Recently Metallo- Beta-lactamases (MBLs) has emerged as the most problematic resistance mechanisms because they hydrolyze all beta lactams including carbapenems except Aztreonam.⁽³⁶⁾They are inhibited by EDTA chelators ⁽¹⁹⁾

The first metallo lactamases which were initially studied were chromosomal enzymes. These were detected in Bacillus cereus, Aeromonas

26

spp. and Stenotrophomonas maltophilia, which are environmental and opportunistic pathogenic bacteria. ⁽⁴³⁾

Among Enterobacteriaceae, 1st carbapenamase enzyme (bla) IMP-1was discovered in 1991 in Serratia marscences in Japan , which was followed by reports from Greece and United States on identification of _bla VIM, _blaKPC^(8)(43).

MBL genes seem to have disseminated from Pseudomonas aeruginosa to other members of family Enterobacteriaceae. Worldwide prevalence of MBL is seen with Pseudomonas aeruginosa and also recently among Gram negative bacilli.⁽⁴³⁾As the MBL genes are present in mobile gene cassettes, which are inserted in integrons the resistance may spread rapidly to various species of gram-negative bacilli. Thus there is a need to detect MBL in gram- negative bacilli rapidly in order to prevent dissemination and thereby helping in infection control.⁽³⁷⁾ The infection with these MBL strains still remains a nightmare for clinicians in treating organisms. These MBL strains can lead to increased mortality and morbidity.

There are two types by which these enzymes act, either by serine based mechanism or requiring zinc or metal chelators for their action.

Class A D act by are serine based hydrolytic mechanism SME KPC, IMI NMC SME GES families. Some are chromosomal, (SME, IMI- 1) others are plasmid encoded (GES KPC, IMI-2). All hydrolyze carbapenems and they are partially inhibited by clavulanic acid.

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Class B are Metallobetalactamases [MBL] which require zinc for their activity.( IMP, VIM, SPM, GIM, SIM, NDM -1.)These hydrolyze all beta lactams including Carbapenem, Extended Spectrum Cephalosporins, except Aztreonam. They are inhibited by chelators like EDTA.

KPC - Klebsiella pneumoniae beta lactamases IMP - Imipenamase metallo beta lactamases KPC - Klebsiella pneumonia carbapenamase

VIM - Verona integron encoded metalloβ lactamases

NDM - New Delhi metalloβ lactamases originally discovered from New Delhi in 2009 in a Sweedish patient of Indian origin, who acquired urinary tract infection .This gene is now found in many species of E. Coli and Klebsiella from places in India and Pakistan.⁽³⁷⁾

Class D: These are OXA type β lactamases. The chromosomal gene of Shewanella spp, that moved through plasmid to Enterobacteriaceae, these are not inhibited by Clavulanic acid or EDTA. It was first reported in Turkey.

34 several reports have shown that MBL carrying organism may be susceptible to Carbapenem (in vitro) but actively resistant in vivo (Clare et al 2006, Pelg et al 2005).

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3.18 .LAB DIAGNOSIS OF ESBL

PHENOTYPIC DETECTION METHODS OF ESBL

To detect ESBL several phenotypic tests was developed since 1980s utilizing the two characteristic features of ESBL.⁽³⁵⁾

a) Decreased susceptibility to Cephalosporin b) Inhibition by Clavulanic acid

CLSI (2014M100-SS24) recommends disc diffusion method to screen ESBL among Klebsiella, Escherichia coli and phenotypic confirmatory test for confirmation.

3.18.1: SCREENING TESTS FOE ESBL

Disc diffusion method with indicator Cephalosporin of III generation Ceftriaxone (30 μg/disk): 25 mm, Cefopodoxime (10μg /disk): 17 mm;

Ceftazidime (30 μg/disk): 22 mm; Cefotaxime (30 μg/disk): 27 mm;

Susceptibility testing was performed on Mueller-Hinton agar using McFarland 0.5 standard from overnight cultures, followed by incubation at 35°C for 16 to 18 hours.

Zone diameters and MIC range of various antibiotics Antibiotic(μg) Disc diffusion(resistant) Sensitive

MIC

Resistant MIC

Cefotaxime(30) ≤27mm ≤1μg/ml ≥ 4μg/ml

Ceftazidime(30) ≤22mm ≤4μg/ml ≥16μg/ml

Aztreonam(30) ≤27mm ≤4μg/ml ≥16μg/ml

Ceftriaxone(30) ≤25mm ≤1μg/ml ≥ 4μg/ml

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For Pseudomonas species, the ceftazidime resistance is considered when zone diameter is≤14 mm and sensitive if it is ≥18 mm. while doing MIC, resistance is confirmed when if it is greater than 32 μg⁽¹¹⁾

3.18.2: Confirmatory test:

 Double disc diffusion test

 Combined disc test

 Broth micro dilution test/agar microdilution

 Three dimensional test

 Agar supplemented with clavulanate

 E test

 Automated system

(Vitek test / microscan panel / BD phoenix automated system) CONFIRMATORY TESTS

Double disc diffusion test ⁽¹¹⁾

The test innoculum is spread as lawn culture on Muller Hinton agar plate using sterile cotton swab. Disc of ceftazidime (30μg) and clavulanic acid (10μg) was placed on the surface of MHA, and then the disc of ceftazidime (30μg) was placed at distance of 15 mm from previous disc. The inoculated plates were incubated overnight in the incubator and the zone of inhibition between discs is compared. They are said to be ESBL producers when zone diameter difference is greater than 5mm.

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Double disc synergy test

The innoculum of the organism to be tested is spread on MHA plate using cotton swab which should be sterile. The disc of ceftazidime is placed at distance of 30 mm from Amoxyclavdisc, and incubated overnight 18 – 24 hours, and enhancement of zone of inhibition towards clavulanic acid is suggestive of ESBL production. ⁽²⁸⁾

Implications of positive phenotypic confirmatory tests:

As per CLSI guidelines, isolates tested positive by confirmatory test are reported as resistant to all cephalosporins and Aztreonam, excluding Cefoxitin, Cephamycin and Cefotetan, irrespective of their individual MICs.

MIC determination :It is done by Broth or agar microdilution method

Done by agar dilution method Preparation of media:

Muller Hinton agar was prepared in tubes and autoclaved. It is then allowed to cool in a 50^oC water bath. Serial dilution of the 3^rd Generation Cephalosporins, Ceftazidime and Meropenem is prepared in sterile distilled water to give a final concentration ranging from 2μg – 128μg/ml of agar. After adding the drugs to the medium at 50^o Cit is mixed well and poured into sterile Petri dishes. The media was used immediately otherwise potency of drugs will be affected. We can inoculate upto 20 different organisms in a single plate. A

31

control plate containing the test medium without the antibiotic was prepared for each series of test.

Innoculum Preparation:

Isolates belonging to same morphological type (at least 3-4) were selected from an agar culture plate. These are transferred to a tube containing4- 5ml of broth. The broth culture was incubated at 37^oC till it the required turbidity i.e. 0.5 McFarland’s standard (usually 2-3 hours) is obtained.

Inoculation of test plates:

Plates of various concentrations were divided into required number (15- 20 divisions / plate) 0.003 ml of innoculum was put into the appropriate quadrant and incubated at 37^oC for 16-20 hrs. Minimum inhibitory concentration was the lowest concentration at which no visible growth occurs.

Quality Control used for ESBL detection:

The positive control used was Klebsiella pneumoniae ATCC 700603 and negative control was Escherichia coli ATCC 25922.

Commercially Available Methods for ESBL Detection:

(i)E test for ESBLs

This is a useful test in the diagnostic armamentarium. It consists of antimicrobial agent impregnated plastic strips, produced by AB Biodisc (Solna, Sweden) placed on surface of agar. The principle is expansion of disc diffusion method. Various gradients of ceftazidime are printed linearly on the strip

32

.Elliptical zone of inhibition is taken as positive. The test is done in the presence Clavulanate impregnated on other side along with Ceftazidime. The 8-fold reduction in MICs of Cephalosporin is considered as positive as per manufacture guidelines.

Sensitivity reported for this method is 87 to 100% and the specificity is 95 to 100%.

Automated methods (ii)Vitek ESBL cards:

This test utilizes Cefotaxime and Ceftazidime, alone (at 0.5 μg/ml), and in combination with Clavulanic acid (4 μg/ml). After 4 to 15 hours of incubation once the growth control well has reached a set threshold, the readings are automatically analyzed. The cards are inoculated identical to that performed for regular Vitek cards. Once analysis of all wells is performed, positivity is recorded by comparing the two wells .When there is reduction in the growth of the Cefotaxime or Ceftazidime wells containing Clavulanic acid, compared with that of growth in the well with the cephalosporin alone, it indicates a positive result. Sensitivity and specificity of the test are remarkable.

Molecular ESBL detection techniques:

There are several molecular biological methods to characterize organisms. Detection of specific nucleic acid sequences by hybridization with DNA probes for TEM or SHV enzymes.

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PCR- It is an-vitro technique to replicate specific sequence of DNA. It amplifies a DNA target by repetition of a basic cycle by using specific prime

RPLF- It is based on the detection of base pair changes at restriction sites.

Nucleotide sequencing is the gold standard for determining specific beta lactamase gene.

3.19 .LAB DETECTION OF CARBAPENAMASES

Different studies suggest different methods to detect MBL production as there is no standard set of guidelines. Detection of carbapenamase is complicated because some carbapenamase producing isolates demonstrate elevated but susceptible carbapenem MICs.

3.19.1: Screening tests:

Zone diameter and MIC for Enterobacteriaceae

Antimicrobial agent(μg)

Disc method(mm)

MIC(μg) (sensitive) Resistant Intermediate Sensitive

Ertapenem(10) ≤18 ≤19- 21 ≥22 ≤0.5

Imipenem(10) ≤19 ≤20-22 ≥23 ≤1

Meropenem(10) ≤19 ≤20-22 ≥23 ≤1

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Zone diameter and MIC for Pseudomonas Antimicrobial

agent(μg) Disc method(mm) MIC

sensitive(μg)

Resistant Intermediate Sensitive

Ertapenem (10) ≤15 16-18 ≥19 ≤2

Imipenem(10) ≤15 16-18 ≥19 ≤2

Meropenem(10) ≤15 16-18 ≥19 ≤2

CONFIRMATORY TESTS

Confirmation of carbapenamase production is done by confirmatory tests 1. 19.2 : PHENOTYPIC DETECTION TEST

 DOUBLE DISC SYNERGY TEST

 COMBINED DISC TEST

 MODIFIED HODGE TEST

 MIC

 E TEST

 MOLECULAR METHOD

Confirmatory tests are carried out to detect MBL production in Imipenem resistant cases. This is done by combined disc test with Imipenem and EDTA. This method that is highly sensitive and specific at detecting both carbapenem-susceptible (98.33%) and carbapenem-resistant MBL-carrying isolates among the gram-negative genera. The selection for phenotypic MBL detection is more challenging with the emergence of carbapenem-susceptible

35

MBL-carrying organisms; therefore, screening only carbapenem-resistant organisms, as is mostly performed by clinical laboratories is suboptimal.

[2](74)Hence detection of MBL in all isolates is advocated.

MODIFIED HODGE TEST⁽¹⁰⁾

To detect carbapenamase, E.coli ATCC 25922 lawn culture is done on MHA. Imipenem disc (10μg) is placed in the centre. The strains were streaked from the periphery of the centre disc on all sides. The plate was incubated for 18 hours incubation. Enhancement of growth of indicator strains around Imipenem disc is taken as Modified Hodge test positive. No enhanced growth is taken as negative for carbapenamase production. The appearance of clover leaf pattern is diagnostic. In Remodified Hodge test Imipenem plus zinc disc is used zinc has known to increase metallo beta lactamases activity.

DETECTION OF MBL BASED ON INHIBITION OF CHELATING AGENTS :

This is specific test based on using the chelating agents which has inhibitory effect on MBL. These compounds inactivate MBL that act against beta lactams, depriving them of the hydrolytic essential Zn²⁺cat ion. The DDST and CDT are commonly used forms of MBL detection.

IMIPENEM –EDTA DISC TEST

The MHA plate is inoculated with the test organism by sterile cotton swab, an Imipenem disc (10 μg) is applied over it, another disc of Imipenem and EDTA is placed at distance of 15 cm is placed on the MHA plate . The

36

plate is incubated for 18 -24 hours. A difference in zone diameter of > 7 cm between two discs is indicative of metallo betalactamases production.

Imipenem/Ceftazidime double disc synergy test (DDST):

Test organisms inoculated on to plates with Muller Hinton agar following CLSI recommendations .An imipenem (10 μg) disc placed 20 mm centre to centre from a blank disc containing 10 μL of 0.5 M EDTA (750 μg).

Positive result is interpreted if the area between Imipenem and clavulanate shows enhancement in comparison with the zone of inhibition on the other side of the disc.

MBL E-test:

This is a quantitative method of detecting MIC by using the principles of both dilution and diffusion of antibiotic into the medium. It contains an absorbent strip containing predetermined gradient of IPM (4 to 256 μg/mL) and IPM (1 to 64 μg/ml) in combination with a fixed concentration of EDTA on the other side. It is reported to be the most sensitive test for MBL detection. The Etest is done according to manufacturer's instructions. MIC ratio of IP (Imipenem)/IPI (Imipenem-EDTA) of >8 indicates MBL production. The MIC concentration at which the ellipse intersects at the strip is the MIC value.⁽¹³⁾ MOLECULAR METHODS:

The reference standard for precise identification of carbapenamase is by molecular technique where the gene for resistance is identified. Most of these techniques are based on hybridization techniques. Their cost and the

37

requirement for trained microbiologists limit their routine utility. Several multiplex real time PCR methods and other newer methods are available.

3.20: LAB TESTS FOR AmpC DETECTION

There are no universally approved criteria for AmpC detection even by CLSI. Organisms producing enough AmpC β-lactamase will typically give a positive ESBL screening test but the confirmatory test will be negative and shows sensitivity with clavulanic acid.⁽³²⁾

3.20.1: SCREENING:

Resistance to cefoxitin (zone size < 18 mm) as well as oxyimino cephalosporins suggests Amp C enzyme production.⁽¹¹⁾

CONFIRMATION TESTS Three dimensional tests Cefoxitin agar

Amp C disc test

Inhibitor based tests: using

Ro 48 -1220LN 2 -128, SYN 2190, CLOXACILLIN

Boronic acid, Phenyl boronic acid, 3 – Aminophenylboronic acid THREE DIMENSIONAL TESTS

Standard E. coli strain (ATCC 25922) is streaked on MHA, a circular hole in the agar was cut near about 3mm from the disc cefoxitin. The suspension of test organism is added to the well. Distortion of zone at the point

38

of cut in the agar indicated the presence of enzyme. ⁽¹³⁾It is most sensitive method but technically more demanding to perform.

MODIFICATIONS

A radial slit was employed ,rather than using intact cells ,the test organism were concentrated by centrifugation and the pellet was freeze thawed 7 -8 times to release β lactamases. A heavy innoculum streaked radially from cefoxitin disc on agar surface without using slit.

CEFOXITIN AGAR:

The agar plate containing 4 μg / ml of Cefoxitin is streaked with the sensitive indicator strain. To a well in the plate, freeze thawed cell extract of test organism is added. After incubation presence of growth around the well indicates Cefoxitin hydrolysing enzymes. The advantage of this method is that it is easier to perform, allows multiple samples to be tested per plate and as sensitive as three dimensional test,.⁽²⁹⁾

Amp C disc test.

This is a rapid test for detection of Amp C. This is otherwise known as Tris –EDTA test. Here, E. coli culture (ATCC 25922)was swabbed on MHA plate. A cefoxitin disc was placed .Sterile disks (6 mm) were wetted with sterile saline and inoculated with several colonies of test organism. This disc was then placed beside the cefoxitin disk (almost touching). The plates were incubated overnight at 35°C. A positive test appeared as a flattening or

39

indentation of the cefoxitin inhibition zone in the vicinity of the test disk. A negative test had no distorted zone.⁽⁴²⁾ It is simple, and convenient to perform.

Phenotypic confirmation testing of AmpC

The Etest for AmpC is confirmatory. The test principle comprises a strip impregnated with a concentration gradient of Cefotetan on one half of the strip and Cefotetan with Cloxacillin on the other half of the strip. MICs of Cefotetan alone and Cefotetan with Cloxacillin were determined as recommended by the manufacturer.⁽⁴¹⁾

A 8 fold reduction between the two is taken as positive.

Inhibitor based tests:

The use of inhibitors to detect AmpC is similar to confirmatory test for ESBL using Clavulanic acid. Boronic acid and cloxacillin are usually used.

They inhibit Amp C enzymes and potentiate the action of cephems.⁽⁷⁸⁾ Cefoxitin-cloxacillin double disc synergy test (CC-DDS)

Cefoxitin discs of 30 μg and Cefoxitin plus Cloxacillin (30μg/200μg) were used. The strains were inoculated on Mueller-Hinton agar using McFarland 0.5, and incubated overnight at 35°C. The inhibition zones of Cefoxitin-Cloxacillin and that of Cefoxitin zones are compared. A 4mm difference in zone is indicative for AmpC production.⁽⁴¹⁾

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BORONIC ACID

Various boronic acid derivatives are added to blank βlactam disc which is placed near unmodified beta lactam disc for comparison. Disc potentiation >

5 mm zone of inhibition in boronic acid disk is taken as positive.⁽²⁹⁾

Several other studies evaluated the β-lactams enzyme inhibitors LN-2- 128, Ro 48-1220, and Syn 2190combination with Cefotetan, gives the best results, which was 100% specific and 91% sensitive in AmpC β-lactamase detection. Unfortunately, these inhibitors are not commercially available.^{(39 )}

The inhibitor-based test showed the potential for the detection of organisms producing plasmid mediated Amp C βlactamases by using cefotetan in combination with LN-2-128 and 48-1220.LN-2-128 and 48-1220 are inhibitors of class A -lactamases in addition to AmpC β lactamases. A novel AmpC β-lactamase inhibitor, Syn 2190 does not inhibit class A lactamases.

However LN-2-1220 which is a C-3-substituted cephalosporin-derived inhibitor with a broad spectrum of inhibition and inhibits both class A (e.g., TEM and SHV) and class C (e.g., AmpC) –lactamases.⁽⁴⁰⁾ A positive test for a plasmid mediated Amp C β lactamase is accepted when >4mm increase in zone diameter in present with inhibitor and plain antibiotic alone.

MOLECULAR METHODS:

 PCR : using Multiplex PCR assay six family of genes,

can be identified

41

 REAL TIME PCR: It reduces the reporting time.

 DNA probes Specific for gene family are available

( e.g. MOX, DHA, EBC ,ACC, FOX, , CIT, )

 BETA LACTAMASE INHIBITORS:

These are compounds which resemble β-lactam antibiotics. They can bind to β-lactam antibiotics either reversibly or irreversibly protecting the antibiotics from destruction. They serve as suicide bombers utilizing all available enzymes. These have weak antimicrobial activity but have potent inhibitor effect on many plasmid-encoded and some chromosomal β- lactamases. Three important β-lactamase inhibitors are:

1) Clavulanic acid 2) Sulbactam 3) Tazobactam

Materials and Methods

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MATERIALS AND METHODS

A prospective study was conducted from April 2014 to March 2015 in the department of Microbiology, Karpaga Vinayaga Institute of Medical Sciences and Research Centre, Kancheepuram district. The present study aims to find out the various β lactamases producing gram negative bacteria from the samples received from outpatients and inpatients, who were admitted to different wards in our hospital. 492 Gram negative bacilli (GNB) were isolated from various clinical specimens, from April 2014 to March 2015.

ETHICAL CONSIDERATION:

Ethical committee of Karpaga Vinayaga Institute of Medical Sciences and Research centre gave the Ethical clearance for the study.

METHODOLOGY

Isolates obtained from various clinical samples sent to the microbiology laboratory were identified by standard microbiological techniques. The antimicrobial susceptibility testing was carried out by the disc diffusion method by Kirby Bauer according to CLSI guidelines. Antimicrobial discs [Himedia Mumbai] used were: Amikacin (30ug), Ampicillin (10 µg), Amoxicillin- clavulanic acid (30/10μg), Gentamicin (10ug), Ciprofloxacin (5µg), Cotrimoxazole (1.25/23.75µg) Ceftriaxone (30 µg), Cefotaxime (30µg), Ceftazidime (30µg) and Imipenem (10µg). Quality control was achieved by using E.coli ATCC 25922. The isolates showing resistance to any of the 3GC

43

were stored in stock vials for further processing. Media and disks were tested for quality control with standard strains.

INCLUSION CRITERIA

1. All gram negative clinical isolates from both sexes of all age group were included in the study.

2. Enterobacteriaceae and Nonfermenters showing resistant to any of the third generation cephalosporins were tested for beta lactamase production (ESBL, Amp C and Carbapenamase)

EXCLUSION CRITERIA

1. Non Enterobacteriaceae isolates were excluded.

2. Those samples which showed mixed growth were excluded 3. Isolates sensitive to all antimicrobial disc tested were excluded

The isolates stored in the stock vials were sub cultured at the time of testing. The purity and viability of the isolates was checked. They were subjected to various phenotypic methods of β lactamases detection along with MIC determination. Agar dilution technique was used for MIC was determination.

Preparation of Innoculum: Similar looking 3 to 5 representative colonies were touched with a sterile wire and inoculated in 4-5ml of broth (peptone water) and incubated at 37^oC for 2 – 6 hrs till 0.5 McFarland’s standard is attained. If the broth is more turbid then some more broth was added under

44

sterile condition and to match 0.5 McFarland’s standard. Inoculation of MHA plates was made within 15 minutes by using sterile cotton swab. Excess broth from the swab was removed by pressing firmly sidewall of the tube and Inoculation in Muller Hinton agar plate was done. To ensure even distribution of the innoculum, the procedure was repeated by turning the plate to about 60⁰ angle each time and finally the rim of the agar was streaked.

The standard predetermined set of antimicrobial discs were placed on the agar plates and pressed adequately so that the disc has firm contact with the agar surface. The distance between the antibiotic discs should at least 24 mm from centre to centre of the discs. Inoculated plates were inverted and incubated at 37^oC for 16-18 hrs.

Those specimens which showed resistance to one of III generation cephalosporins was subjected to other tests for detection of ESBL, Amp C, and Carbapenamase production.

Detection of the ESBLs –

Phenotypic tests were done to confirm ESBL i.e. combined disc test which uses Ceftazidime [30μg] and Ceftazidime-clavulanic [30/10μg discs).

Detection of the Amp C β-lactamases:

Cefoxitin disc was used for Amp C detection among the isolates. Those which showed Cefoxitin zone of inhibition ≤18 mm were taken as Amp C positive.

45

Then resistant isolates (Cefoxitin resistance) was subjected to Amp C disc test. The AmpC production is indicated by an indentation or a flattening of the zone of inhibition.⁽⁷⁾

Detection of the Carbapenamases:

The carbapenamase production was detected by the Modified Hodge test and Imipenem– EDTA combined disc test. Clover leaf pattern in Modified Hodge test was taken as positive. The organisms were considered to be MBL producers if the increase in the zone of inhibition between Imipenem and Imipenem–EDTA (10μg/750μg){Himedia} disc was ≥ 7mm in combined disc test.

DETECTION OF ESBL

Combined disc test (Phenotypic Confirmation Test)

Growth of the organism was obtained as lawn culture. A Ceftazidime (30µg) disc alone and its combination with 10mg of clavulanic acid was placed on the plate at distance of 15 mm.The inoculated plates were incubated at 35 ⁰ C in the incubator for 18 – 24 hours. When there was 5mm increase in zone of inhibition between ceftazidime / clavulanic acid (30µg/10µg) and plain Ceftazidime are confirmed as ESBLs producers as per CLSI guidelines.⁽¹¹⁾

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Detection of Amp C Amp C disc test. ⁽⁶⁴⁾

Isolates showing resistant to Cefoxitin (30μg) were subjected to Amp C disc test. Here, ATCC 25922 E coli was used as a lawn culture. The Cefoxitin disc was then placed and beside it a sterile disc, {Whattman No. 1 filter paper (6 mm) size} wetted with sterile saline and inoculated with several colonies of test organism and placed almost touching. The plates were incubated overnight at 35°C. A positive test appeared as a flattening or indentation of the Cefoxitin inhibition zone in the vicinity of the test disc. A negative test had an undistorted zone.

Detection of Carbapenamase production Modified Hodge test ⁽¹¹⁾

Lawn culture of E .coli ATCC 25922 was done on Mueller Hinton agar plate. Imipenem disc (10μg) was placed in centre of the culture plate. Four test strains were inoculated from periphery of the disc on all sides. Following incubation, enhancement of zone of inhibition of growth indicator strains around Imipenem is taken as Modified Hodge test positive.

Combined Disc test

Lawn culture of test isolate was made on Mueller Hinton agar plate. A Imipenem disc (10 μg) was placed on MHA plate, another disc having Imipenem and EDTA(10/750μg) was placed with a of distance of 15 mm from the Imipenem disc . The plates were incubated at 37 ⁰ C in incubator. MBL

47

production was taken as positive if the zone of inhibition is >7mm between the two discs.

PCR for detection of resistance genes:

DNA EXTRACTION:

DNA was extracted from the selective isolates using DNA purification kit (Pure Fast® Bacterial Genomic DNA purification kit). (Helini Biomolecules). The procedure is attached in Annexure II.

The following were the primers used HELINI –TEM gene primer

Forward Primer: 5'-GATAACACTGCGGCCAACTT -3' Reverse Primer: 5'-CTGCAACTTTATCCGCCTCC PCR-3' Product size: 262bp

HELINI -SHV gene primer

Forward Primer: 5'-CGCCGCCATTACCATGAGCGAT -3' Reverse Primer: 5'-ACCCGATCGTCCACCATCCACT PCR-3' Product size: 276bp

HELINI -IMP gene primer

Forward Primer-5'-TTTTGCAGCATTGCTACCGC-3' Reverse primer-5'-CACGCTCCACAAACCAAGTG-3' Product size = 220bp

48

STATISTICAL ANALYSIS:

The observations of the study were recorded and analyzed. The results were compared and discussed by using SSPS software and Chi Square test

Results