Dissertation Submitted in Partial Fulfillment of the Regulations

IDENTIFICATION OF GENES CODING FOR CARBAPENEMASES

IN CLINICAL ISOLATES OF CARBAPENEM RESISTANT

KLEBSIELLA SPECIES IN A TERTIARY CARE HOSPITAL

Dissertation submitted in

Partial fulfillment of the Regulations required for the award of

M.D. DEGREE

MICROBIOLOGY– BRANCH IV

The Tamil Nadu

DR. M.G.R. MEDICAL UNIVERSITY

Chennai

MAY 2020.

UNIVERSITY REGISTRATION NO: 201714252

CERTIFICATE

This is to certify that the enclosed work “Identification of Genes

Coding for Carbapenemases in Clinical Isolates of Carbapenem Resistant

Klebsiella Species in a Tertiary Care Hospital” submitted by

Dr.S.Sharon Dorothy to The Tamilnadu Dr. MGR Medical University is based on bonafide cases studied and analysed by the candidate in the

Department of Microbiology, Coimbatore Medical College Hospital during the period from July 2018 to June 2019 under the guidance and supervision of Dr. N.Mythily, MD., Professor & HOD, Department of

Microbiology and the conclusion reached in this study are her own.

Guide

Dr. N.MYTHILY, MD.,

Professor & HOD, Department of Microbiology, Coimbatore Medical College, Coimbatore.

Dr. B. ASOKAN. M.S., M.Ch., Dr., N.MYTHILY, MD.,

Dean, Professor & HOD, Coimbatore Medical College and Hospital, Department of Microbiology, Coimbatore – 14. Coimbatore Medical College, Coimbatore – 14.

DECLARATION

I, Dr.S.Sharon Dorothy solemnly declare that the dissertation entitled

“Identification of Genes Coding for Carbapenemases in Clinical Isolates of

Carbapenem Resistant Klebsiella Species in a Tertiary Care Hospital” was done by me at Coimbatore Medical College Hospital, during the period from July 2018 to June 2019 under the guidance and supervision of Dr. N. Mythily, M.D.,

Professor & HOD, Department of Microbiology, Coimbatore Medical College,

Coimbatore.

This dissertation is submitted to The Tamilnadu Dr. MGR Medical

University towards the partial fulfilment of the requirement for the award of

M.D. Degree (Branch – IV) in Microbiology.

I have not submitted this dissertation on my previous occasion to any

University for the award of any degree.

Place:

Date :

Dr. S.Sharon Dorothy

ACKNOWLEDGEMENT

I express my deep debt of gratitude to our respectful Dean,

Dr.B. Asokan, M.S., M.Ch., for permitting me to do this study.

I wish to place my deep sense of gratitude and sincere thanks to Dr. N. Mythily MD., Professor and Head of the Department of

Microbiology, for the constant encouragement, guidance and timely advice given to me during the course of my post-graduation.

I sincerely place my thanks to Associate Professors

Dr.P.Sankar,M.D., Dr.B.Padmini, M.D., for their support and encouragement.

I express my sincere thanks to my Assistant Professors

Dr.N.Bharathi Santhose M.D., Dr.C.Ashok Kumar MD.,

Dr.R.Radhika,MD., Dr.P.Malini, M.D., and Dr.S.Nirmala Devi, M.D., for their valuable suggestions.

My special thanks to my post graduate colleagues Dr.C.Varsha,

Dr.C.P.Shanthini and Dr.K.Soundarya and other post graduates in the

Department of Microbiology for their co-operation in completing my study.

I would grossly fail in my duty, if I do not mention here of my subjects who have undergone the pain and discomfort of the investigations during this study. I take this opportunity to thank all the technical staffs in the

Department of Microbiology who gave me their kind co-operation throughout my study.

I affectionately thank my family members who are giving their constant support throughout my entire post-graduation course without which this work would not have been successful.

I am thankful to God, who have been with me all throughout my way to reach the destination.

CERTIFICATE - II

This is to certify that this dissertation work titled “Identification of Genes Coding for Carbapenemases in Clinical Isolates of Carbapenem

Resistant Klebsiella Species in a Tertiary Care Hospital” of the candidate

Dr.S.Sharon Dorothy with registration Number 201714252 for the award of Doctor of Medicine in the branch of Microbiology. 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 5% (Five) percentage of plagiarism in the dissertation.

Guide sign with Seal.

CONTENTS

S.NO CONTENTS PAGE NO

1. 01 INTRODUCTION 2. 07 AIMS & OBJECTIVES 3. 08 REVIEW OF LITERATURE 4. 30 MATERIALS & METHODS 5. 54 RESULTS 6. 64 DISCUSSION 7. 78 SUMMARY 8. 81 CONCLUSION 9. BIBLIOGRAPHY 10. ANNEXURE 11. MASTER CHART

LIST OF TABLES

S.NO TABLE 1. SAMPLE WISE DISTRIBUTION OF KLEBSIELLA INFECTIONS 2. TYPES OF KLEBSIELLA SPECIES ISOLATED FROM STUDY POPULATION

3. GENDER WISE DISTRIBUTION OF KLEBSIELLA ISOLATES 4. WARD WISE DISTRIBUTION OF CARBAPENEM RESISTANT KLEBSIELLA SPECIES 5. FACTORS INFLUENCING INCIDENCE OF RESISTANT INFECTIONS IN VARIOUS WARDS 6. AGE WISE DISTRIBUTION OF CARBAPENEM SENSITIVE AND CARBAPENEM RESISTANT ISOLATES OF KLEBSIELLA 7. RISK FACTORS ASSOCIATED WITH INFECTIONS OF KLEBSIELLA SPECIES 8. INVITRO ACTIVITY OF VARIOUS ANTIBIOTICS AGAINST KLEBSIELLA ISOLATES

9. DETECTION OF ESBL AND Amp C β LACTAMASE PRODUCTION BY VARIOUS METHODS 10. VARIOUS TYPES OF β LACTAMASE ENZYME PRODUCERS AMONG ANTIBIOTIC RESISTANT KLEBSIELLA ISOLATES 11. COMPARISON OF VARIOUS PHENOTYPIC METHODS IN THE DETECTION OF CARBAPENEMASES 12. DETECTION OF CARBAPENEMASES BY MOLECULAR METHODS

LIST OF CHARTS

S.NO CHART 1. SAMPLE WISE DISTRIBUTION OF KLEBSIELLA INFECTIONS

2. TYPES OF KLEBSIELLA SPECIES ISOLATED FROM STUDY POPULATION

3. GENDER WISE DISTRIBUTION OF KLEBSIELLA ISOLATES

4. WARD WISE DISTRIBUTION OF CARBAPENEM RESISTANT KLEBSIELLA SPECIES

5. AGE WISE DISTRIBUTION OF CARBAPENEM RESISTANT KLEBSIELLA INFECTIONS

6. RISK FACTORS ASSOCIATED WITH INFECTIONS OF CARBAPENEM RESISTANT KLEBSIELLA INFECTIONS 7. ANTIMICROBIAL RESISTANCE PATTERN OF KLEBSIELLA ISOLATES

8. DETECTION OF β LACTAMASES BY PHENOTYPIC METHODS

9. DETECTION OF CARBAPENEMASES BY VARIOUS PHENOTYPIC METHODS

10. ISOLATES PRODUCING VARIOUS TYPES OF β LACTAMASES DETECTED BY PHENOTYPIC METHODS

11. VARIOUS GENES CODING FOR CARBAPENEMASES DETECTED BY MOLECULAR METHODS

LIST OF COLOUR PLATES

S.NO COLOUR PLATE 1. NUTRIENT AGAR PLATE SHOWING COLONIES OF KLEBSIELLA 2. Mac Conkey AGAR PLATE SHOWING MUCOID LACTOSE FERMENTING COLONIES OF KLEBSIELLA 3. BIOCHEMICAL REACTIONS OF KLEBSIELLA PNEUMONIAE

4. BIOCHEMICAL REACTIONS OF KLEBSIELLA OXYTOCA 5. CARBOHYDRATE FERMENTATION TESTS OF KLEBSIELLA SPECIES 6. AMINO ACID DECARBOXYLATION TESTS OF KLEBSIELLA SPECIES 7. GRAM STAINING PICTURE OF KLEBSIELLA SHOWING SHORT GRAM NEGATIVE BACILLI 8. KLEBSIELLA ISOLATE RESISTANT TO MEROPENEM AND SENSITIVE TO COLISTIN AND TIGECYCLINE 9. E STRIP SHOWING KLEBSIELLA ISOLATE RESISTANT TO MEROPENEM (4 µg/ml) 10. E STRIP SHOWING KLEBSIELLA ISOLATE SENSITIVE TO COLISTIN ( 1 µg/ml) 11. DETECTION OF ESBL BY COMBINED DISC METHOD

12. DETECTION OF ESBL BY E STRIP

13. DETECTION OF Amp C BY COMBINED DISC METHOD 14. DETECTION OF CARBAPENEM RESISTANT ISOLATES BY DISC DIFFUSION METHOD 15.. DETECTION OF CARBAPENEMASES BY Carba NP TEST 16. DETECTION OF CARBAPENEMASE BY mCIM AND eCIM 17. DETECTION OF CARBAPENEMASES BY PCR 18. LOADING SAMPLES IN THERMOCYCLER 19. AGAROSE GEL ELECTROPHORESIS 20 MOLECULAR IMAGER (Gel Doc XR+,Biorad.,USA) 21 DISTRIBUTION OF NDM-1 GENES IN RESISTANT ISOLATES 22 DISTRIBUTION OF OXA -48 like GENES IN RESISTANT ISOLATES

LIST OF ABBREVIATIONS

CDC CENTER FOR DISEASE CONTROL

CLSI CLINICAL AND LABORATORY STANDARDS INSTITUTE

CRE CARBAPENEM RESISTNANT ENTEROBACTERIACEAE

ESBL EXTENDED SPECTRUM β LACTAMASE

PCR POLYMERASE CHAIN REACTION

CRKP CARBAPENEM RESISTANT KLEBSIELLA PNEUMONIAE`

DNA DEOXY RIBONUCLEIC ACID mCIM MODIFIED CARBAPENEM INACTIVATION METHOD eCIM EDTA MODIFIED CARBAPENEM INACTIVATION METHOD

CDT COMBINED DISC TEST

MBL METALLO β LACTAMASE

NDM NEW DELHI METALLO β LACTAMASE

OXA OXACILLINASES

VIM VERONA INTEGRON METALLO β LACTAMASE

IMP IMIPENEMASE

KPC KLEBSIELLA PNEUMONIAE CARBAPENEMASE bla KPC GENE CODING FOR KLEBSIELLA PNEUMONIAE CARBAPENEMASE

TBE TRIS- BORATE- EDT A bp BASE PAIR

EtBr ETHIDIUM BROMIDE

HAI HOSPITAL ACQUIRED NFECTION

TSB TRYPTIC SOY BROTH

SSI SURGICAL SITE INFECTION

INTRODUCTION

Klebsiella belongs to the family Enterobacteriaceae and tribe

Klebsiellae. Klebsiella pneumoniae is considered to be one of the most common Gram negative bacteria. It was` initially named after the scientist

Carl Friedlander as “Friedlander bacillus” and later came to be called as

Klebsiella(1). The genus includes six species- Klebsiella pneumoniae, oxytoca, granulomatis, variicola, singaporensis and alba. Klebsiella pneumoniae is the type genus of this species.

Klebsiella are widely distributed in the gastrointestinal tracts of humans and animals(2). Klebsiella pneumoniae is the most common species frequently recovered from clinical specimens. It causes a classic form of primary pneumonia. It is also found in the oropharynx of normal human beings (1-6%), however hospitalized patients possess a higher prevalence of about 20%(3). Patients with debilitating conditions such as Alcoholism,

Diabetes mellitus and Chronic Obstructive Pulmonary Disease have more chances of developing severe lung diseases. Klebsiella species has evolved to be a frequent cause of nosocomial infections. It is an important cause of outbreaks in hospitals. The management of infections caused by these organisms is further complicated by the increased incidence of hypervirulent strains.

K. pneumoniae is the causative agent of several different healthcare

-associated infections, such as bloodstream infections, wound infections,

1 pneumonia, and meningitis. The various factors that contribute to virulence and pathogenicity of Klebsiella are the capsular serotype, lipopolysaccharide, iron-scavenging systems and adhesions.(4).

Klebsiella accounts for 3% to 7% of all nosocomial bacterial infections, placing them one among the eight important infectious pathogens in medical care facilities. All clinical strains of Klebsiella are intrinsically resistant to Ampicillin, Carbenicillin, and Ticarcillin.

Bacteria have developed multiple antimicrobial resistance mechanisms. One or more similar or different types of resistance mechanisms are found in multi drug resistant pathogens. Transmission of antimicrobial resistance is mainly mediated by plasmids. Klebsiella has a tendency to harbor antibiotic – resistant plasmids thus infections with multiple antibiotic resistant strains can be highly anticipated(5). Many gram negative bacteria are becoming Pan drug resistant (PDR) due to the presence of integron gene cassettes with multiple resistant genes that encode one or more enzymes. When the β lactamase produced by the organism is in higher amounts and the organism also has an increased outer membrane permeability, it becomes resistant to the action of even higher antimicrobials like Carbapenems.

With an alarming increase in the occurrence of ESBL and Amp C mediated β lactamases, Carbapenems known to have a better stability against these enzymes were considered to be the β lactam antimicrobials of choice especially in ICU and high risk wards. Acquired resistance

2 mediated by carbapenem – hydrolyzing enzymes is also a major problem world wide. Many enzymes are being recognized as significant such as serine proteases of Ambler Class A (KPC),Class B – NDM,IMP,VIM and Carbapenem hydrolyzing Class-D Oxacillinases - OXA -48,OXA-181.

These genes are mostly plasmid located and also associated with various genetic structures such as Insertion sequence, Integrons and Transposons which promotes their spread. Carbapenems belong to the class of β lactam antibiotics whose range of activity includes gram positive cocci,

Enterobacteriaeceae, Pseudomonas, Listeria as well as anaerobes like

Bacteroides fragilis and Clostridium difficile. Imipenem is rapidly hydrolysed by renal peptidase. Meropenem on the other hand is not hydrolysed by renal peptidase and is considered as a reserve drug for the treatment of serious nosocomial infections like septicemia , febrile neutropenia, intra-abdominal and pelvic infections caused by cephalosporin resistant bacteria.

Carbapenems have long been used for the treatment of infections caused by microbes belonging to Enterobacteriaceae. The percentage of

Carbapenem-resistant Enterobactericeae (CRE) has been on the rise. The most prominent and recent increase of Multi drug resistance is observed with Klebsiella species. It is important to notice that the patients harboring multi drug resistant Klebsiella species have higher mortality rates. MDR infections are hard-to-treat with limited available choices of antibiotics such as Tigecycline, Colistin and Fosfomycin.

Carbapenem resistance is a major and an on going public health problem globally. The most common and serious pathogens which develop multi drug resistance are named as “ESKAPE” pathogens which stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,

Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species. The occurrence of carbapenem resistance in Klebsiella species is however an evolving global public health problem and calls for urgent action within the medical fraternity.

In India, NDM is the most extensively observed Carbapenemase.

OXA – 181, a variant of OXA – 48 is another carbapenemase reported in India. OXA - 48 is the carbapenemase predominantly observed in

Klebsiella species. Klebsiella species carrying KPC, OXA and NDM enzymes are all clinically important. Klebsiella pneumoniae that produce

K.pneumoniae Carbapenemase (KPC) have spread rapidly across hospitals world wide. KPC producing isolates are typically multi drug resistant(6).

They are resistant to anti microbials like Carbapenems (Ertapenem,

Doripenem, Imipenem, Meropenem) Penicillins, Cephalosporins,

Fluoroquinolones and also Aminoglycosides. Routine methods of antibiotic susceptibility testing may not be helpful in detecting the presence of

CRKP. It is therefore high time to implement efficient infection control measures to control the spread of resistant organisms. Newer phenotypic methods are available for detection of antimicrobial resistance. However,

Molecular methods have a greater speed and accuracy and also provide

4 useful information regarding the underlying mechanisms of antimicrobial resistance .Hence many laboratories therefore have replaced phenotypic methods by molecular methods.

The spread of carbapenem resistance among Klebsiella is a greater threat to the community because the alternatives in treatment available to those affected by these resistant strains is highly limited(7) . This is the reason why early detection of carbapenemase producers is significant.

Hence rapid methods for determining antimicrobial susceptibility are important to ensure adequate and appropriate use of antimicrobial agents and to limit the spread of these bacteria. In order to formulate effective infection control measures and for planning principles in health administrations, a wide understanding of the underlying genetic mechanisms involved in the emergence of resistance is of primary importance.

Global reports documenting the infection burden of common and diverse bacterial pathogens that have developed resistance is increasing in the past decades.(8) This alarming rise of infections has lead to nothing but irrational use of antimicrobials across the globe. Furthermore this has increased the emergence of specific and multidrug resistance among various strains of microorganisms including Klebsiella. Gram-negative bacteria have developed several mechanisms of resistance to currently used antimicrobials. The extensive and irrational use of antimicrobials has paved a way for the development of multidrug resistance in Klebsiella.

Antibiotic efflux pumps represent one of the most important antimicrobial resistance mechanisms used by Klebsiella isolated from clinical specimens.

The efflux pumps pump out the antimicrobial agent which leads to decreased concentration of the drug in the intracellular environment which in turn promotes undisturbed bacterial survival.

Molecular typing and virulence analysis of clinical isolates can be of utmost help in the early diagnosis and efficient management of multidrug resistant (MDR) Klebsiella infections.

AIM & OBJECTIVES

AIMS AND OBJECTIVES

AIM:

To identify the genes coding for carbapenemases in clinical isolates of carbapenem resistant Klebsiella species in Coimbatore Medical College

Hospital.

OBJECTIVES:

 To isolate the organisms in clinical specimens by using conventional

methods.

 To confirm the isolates of Klebsiella by biochemical reactions.

 To evaluate the antimicrobial susceptibility pattern of the organism by

Kirby - bauer disc diffusion method.

 To confirm the resistance pattern by phenotypic methods.

 To detect the presence of genes coding for resistance by molecular

methods.

REVIEW OF LITERATURE

HISTORY:

In 1882: Carl Friedlander first isolated the bacterium from the lungs of patients who died from pneumonia and named it as “Friedlander’s bacillus”.

In 1886: The bacterium was named as Klebsiella after Edwin

Klebs, a German Microbiologist .

In 1929: Resistance of Gram negative organism to β- lactam antibiotics was first discovered by Alexander Fleming.

In 1983 : ESBL K.pneumoniae was first seen in Europe.

In 1989 : ESBL K.pneumoniae was isolated in the United States.

In 1996 : CR-Kp was first reported in India (9).

CLASSIFICATION:

Four major genera are placed under the tribe Klebsiellae.

They are :

Klebsiella

Enterobacter

Hafnia

Serratia.

Genus Klebsiella has six species namely

K.pneumoniae

K.oxytoca

K.granulomatis

K.variicola

K.singaporensis

K.alba.

Genus Species Subspecies

Klebsiella K.pneumoniae aerogenes

ozaenae

pneumoniae

rhinoscleromatis

K.oxytoca

K.granulomatis

K.variicola

K.singaporensis

K.alba

TAXONOMIC CHANGES IN THE GENUS KLEBSIELLA :

Based on the analysis of the 16S rRNA sequence and the rpoB genes of the type strains of Klebsiella species, the species were placed under three phyletic clusters (Drancourt et al.,2001)

Cluster 1 K.pneumoniae subsp pneumoniae

ozaenae

rhinoscleromatis

granulomatis

Cluster 2 Raoultella

Cluster 3 K.Oxytoca – 2 clades – oxy 1 and oxy 2

NATURAL HABITAT:

Klebsiella form a part of the normal microbiota of the nasopharynx and Gastrointestinal tract of humans and are found commonly in human feces and in water . Strains originally isolated from the respiratory tract specimens of humans and animals are usually designated as Klebsiella pneumoniae. K.oxytoca is also encountered occasionally in clinical samples.

Klebsiella granulomatis Causes granuloma inguinale

Klebsiella ozaenae Causes atrophic rhinitis

Klebsiella alba

Klebsiella singaporensis Saprophytic species

Klebsiella variicola

MORPHOLOGY:

Klebsiella are non motile, gram negative rods of about 1-2 μm length, capsulate and abundant gas producing organisms. Klebsiella are aerobic or facultatively anaerobic. They ferment glucose, produce catalase but not oxidase(3) .Greater amounts of capsular material is produced when the organism is cultured on media rich in carbohydrates .The polysaccharides of the different capsular types resemble the K antigens of Escherichia coli.

VIRULENCE FACTORS :

Lipopolysaccharide 7 serotypes

Capsule 77 serotypes

Adhesins Type 1 pili (MSHA)

Type 3 pili (MR/K –HA )

KPF – 28 fimbriae

CF – 29 K (non – fimbrial)

Aggressive Adhesin

Siderophores Enterochelin

Aerobactin

PATHOGENESIS :

CAPSULAR ANTIGENS :

80 capsular antigens have been recognized so far. Strains which possess the antigenic types K1,K2,K3,K5 and K21 are considered important in causing human disease .The capsule prevents phagocytosis by polymorphonuclear granulocytes and protects the bacterium from microbial killing by serum factors.

PILI:

Non flagellar, filamentous projections on the bacterial surface are referred to as the pili. These help the organisms to adhere strongly to the host cells. Two important pili of Klebsiella species are Type 1 and Type

3(10).

LIPOPOLYSACCHARIDES :

The complement cascade plays an important role in bacterial killing.

It is activated by the bacterial Lipopolysaccharide .The activation of the cascade is prevented by two mechanisms . Thick capsule of the bacterium masks the LPS or the O side chains of the LPS projects out of the cell and binds to the complement components thereby preventing their further activation.

SIDEROPHORES :

Siderophores are high –affinity ,low molecular weight iron – chelators that competitively take up iron, bound to the host proteins .

Good iron stores in the host thus lead to enhanced infection by Klebsiella.

CULTURAL CHARACTERISTICS :

Klebsiella species have no complex nutritional requirements and grow readily on Ordinary media like Nutrient agar, MacConkey agar and

Blood agar.

On Nutrient agar plate , Klebsiella is seen as circular ,raised ,dome shaped , mucoid, translucent to opaque colonies of 2-3 mm size.

On MacConkey agar , Klebsiella produces pink mucoid Lactose fermenting colonies.

On Blood agar plate , Klebsiella grow as non hemolytic greyish white mucoid colonies .

BIOCHEMICAL IDENTIFICATION :

Klebsiella produce catalase but not oxidase. Indole is not formed by Klebsiella pneumoniae but K.oxytoca forms indole .Citrate is utilized by all Klebsiellae except K.rhinoscleromatis .Urease is not hydrolysed by

Klebsiella pneumoniae subspecies rhinosceromatis and ozaenae. All others hydolyse urea. Klebsiellae produce abundant gas (Co2) on Triple Sugar iron slant so much so that the butt of the medium is often pushed up

in the tube.

Klebsiellae are methyl red negative and Voges-Proskauer positive

except for K.ozaenae and K.rhinoscleromatis. They ferment all the sugars

namely Glucose, Lactose, Mannitol, Salicin, Adonitol, Inositol, Sorbitol,

Arabinose, Rhamnose , Raffinose, Maltose, Xylose, Trehalose and

Mannose. Klebsiella rhinoscleromatis does not ferment Lactose .Klebsiellae

are Phenylalanine deaminase negative. They are O-nitrophenyl beta D-

galactopyranoside (ONPG) positive except for K.rhinoscleromatis (11)

KEY IDENTIFICATION CHARACTERISTICS :

Tribe:

CIT

KIA H2S IND

LYS

GAS PAD URE

ARG ORN

Klebsiellae MOT

ONPG

K.Pneumoniae A/A ++ _ _ + _ + _ + _ + _ _ +

K.oxytoca A/A ++ _ _ + + + _ + _ + _ _ +

CLINICAL VARIANT OF KLEBSIELLA :

Hypervirulent Klebsiella pneumoniae (hv –kp):

Hypervirulent strains of Klebsiella were first isolated in Taiwan in

the last twientieth century.They were recognized as the etiological agents

of liver abscesses, meningitis , and endopthalmitis in apparently healthy

patients.(12)

The striking features of the Hypervirulent clinical variant of

Klebsiella are:

1. Ability to cause infections in ambulatory and apparently healthy

patients.

2. Causes infection in unusual sites of infection ( liver ,Eye ,CSF)

3. Metastatic spread.

4. Production of extremely mucoviscous colonies on growth media.

The predominant types of capsular polysaccharides in

Hypervirulent Klebsiella pneumoniae are the K1 and K2 serotypes .

Enhanced capsule production is the main factor associated with the high virulence of hv-kp.It is triggered by two genes namely

i. regulator of the mucoid phenotype (rmp A ) gene

ii. mucoviscosity associated gene A (mag A ).(13)

Hypervirulent strains of Klebsiella pneumoniae are confirmed by the string test .

Positive string test :

The string test is considered positive when touching a colony with a bacteriological loop and raising the loop above the rim of the agar plate is able to generate a viscous string of more than 5mm , creating a string of bacteria .

Hypervirulent Klebsiella pneumoniae bacteremia leads to a higher mortality rate of about 27.4 – 37.0% (14).

RISK FACTORS FOR ACQUIRING KLEBSIELLA INFECTION:

i. Alcoholism

ii. Diabetes mellitus

iii. Prolonged hospital stay

iv. Prolonged antibiotic use

v. Indwelling devices

Invasive infections caused by Klebsiella pneumoniae have been associated with co-morbidities such as Cancer , Diabetes Mellitus and previous Organ transplantation(14).

EPIDEMIOLOGY OF KLEBSIELLA INFECTIONS :

Reported carrier rates of Klebsiella in hospitalized patients is 77% in stool,19% in pharynx and 42% in the hands of the patients(10). Since

1970s, Klebsiella were resistant only to Aminoglycoside antibiotics. It was in 1982 that ESBLs evolved (10).Recently in the past two decades,

Klebsiella have become Multi drug resistant treatable only by reserve drugs of higher class such as the Polymyxins.

MECHANISM OF ANTIBIOTIC RESISTANCE IN KLEBSIELLA

SPECIES:

Mobile genes on plasmids are the main reason for the extensive spread of resistance in Gram negative bacteria .(15)

Drug resistance occurs in an organism due to any one of the following mechanisms:

a. Drug inactivation / alteration

b. Modification of drug binding sites / targets

c. Changes in cell permeability

d. Biofilm formation(16)

Drug inactivation / alteration:

This can be brought by the secretion of certain substances like enzymes by the organisms. The β lactamases secreted by the bacterium opens the β lactam ring of the β lactam antimicrobials which leads to the inability of the drug to bind to the cell wall of the organism.

Modification of drug binding sites :

The β lactam antimicrobials bind to specific proteins called

Penicillin Binding Proteins on the cell wall of the bacteria. When these proteins are modified structurally or functionally , the antimicrobials are unable to bind to their active site on the bacterial cell.

Changes in cell permeability :

The changes in cell permeability leads to decreased intracellular accumulation of the drug that leads to ineffective action .This is brought

17 about by the loss of porin channels or formation of efflux pumps .

In Klebsiella pneumoniae , two major outer membrane porins,

OmpK35 and OmpK36 , play roles in both antimicrobial resistance and virulence(17).

BIOFILM FORMATION :

Formation of biofilms increases the adherence of the bacterium to tissues or in-dwelling devices making it difficult to kill them. Type 1 and type 3 fimbriae are found to strongly enhance the formation of biofilms in Klebsiella Type 3 Fimbriae in Klebsiella causing biofilm formation is found to favor the CAUTI due to Klebsiella species (18).

Among the various mechanisms described above , Carbapenem resistance is caused mainly due to the production of different classes of carbapenemases such as A,B or D and others are the production of

ESBLs /Amp C β lactamases with a porin mutation or drug efflux(19).

β LACTAMASES :

These are well characterized enzymes that irreversibly modify and inactivate the β lactam antibiotics .They are highly prevalent and act by hydrolyzing the β lactam ring which is present in all β lactams such as the penicillins, cephalosporins, monobactams and carbapenems .(20)

β lactamases are classified using two classification schemes as below :

a. Ambler scheme (molecular classification).

b. Bush and Jacoby classification

AMBLERS CLASSIFICATION:

CLASS ENZYMES

A Penicillinase

Cephalosporinase

Broad Spectrum β Lactamases

Extended Spectrum β Lactamases

Carbapenemases

B Metallo β Lactamases

C Amp C β lactamases

D Oxacillin hydrolyzing enzymes

CLASS A β lactamases :

The class A β Lactamases consist of the Penicillinases,

Cephalosporinases, Extended Spectrum β Lactamases and Carbapenemases.

This group contains a number of significant enzymes including ESBLs

-TEM , SHV and CTX –M .Most of these enzymes are chromosomally mediated .

CLASS A Carbapenemases :

The Ambler Class A Carbapenemases belong the Subgroup 2f of

Bush and Jacoby classification scheme .These are serine carbapenemases which primarily hydrolyse carbapenems. The main carbapenemase in this class, KPC is plasmid – encoded. (21)

CLASS B –Metallo β lactamases :

They are placed in Group 3 of Bush and Jacoby classification .

Metallo β lactamases are so named because they have zinc ions at their active site.Genes encoding MBLs are found on plasmids .They efficeiently hydrolyse carbapenems but are ineffective in hydrolyzing

Monobactums(3).These enzymes are not inhibited by clavulanic acid or

Tazobactum but are readily inhibited by compounds like EDTA ,

Dipicolinic acid and 1,10 phenanthroline . The most common MBLs are IMP – Imipenemase metallo β lactamases, VIM- Verona Integron

Encoded metallo β lactamases and the newly described New Delhi metallo β lactamase -1 enzymes .(22)

CLASS D – Oxacillinases :

OXA -48 , an important oxacillinase contributing to the carbapenem resistance in Klebsiella species belongs to group 2d of Bush and Jacoby Classification . They are serine carbapenemases that readily hydrolyse carbapenems .

BUSH AND AMBLER ACTIVE ASSOCIATED JACOBY CLASS SITE GENES CLASSIFICATION A 2f Serine KPC VIM B 3 Zinc IMP NDM OXA -48 D 2d Serine OXA -181

β Lactamase inhibitors :

The β lactamase inhibitors are those chemical compounds which can reversibly or irreversibly bind to the β lactamase enzyme secreted by the bacteria thereby allowing the antimicrobial to bind to its active site

.The three important β lactamase inhibitors are :

1. Clavulanic acid

2. Sulbactum

3. Tazobactum

INFECTIONS CAUSED BY KLEBSIELLA:

Magill et al .,2014., stated that Klebsiella species have been identified as the third leading cause of HAI ‘s (9.9%) next only to

Clostridium difficile and Staphylococcus aureus(23).

URINARY TRACT INFECTIONS :

The most common site of infection by Klebsiella pneumoniae is the Urinary tract (24) . Nosocomial UTI are more commonly caused by

Klebsiella .Klebsiella infections are more common in UTI in Diabetic patients .Antibiotic resistance in the organisms isolated from Nosocomial

UTI is greater than that related to community acquired UTI in diabetic patients(25) . Klebsiella pneumoniae also causes CAUTI – Catheter associated Urinary tract infections . It is thought that these are facilitated by the ability to form biofilms and adhere to catheters(18) .

BLOOD STREAM INFECTIONS :

Xu et al ., reported the mortality rate of blood stream infections caused by K pneumoniae varies from 15 to 79% (20).Rebekah et al., in

2018 stated that K pneumoniae is the second leading cause of blood stream infections behind only E.coli(26).

RESPIRATORY INFECTIONS - PNEUMONIA :

Klebsiella causes both Community acquired and Hospital acquired pneumonia Hospital acquired pneumonia is defined as the pneumonia occurring after 48 hrs of hospital admission . Klebsiella has been identified as the third most common organism that causes Hospital acquired pneumonia . K.pneumoniae is the causative agent of approximately 11.8 % of all hospital acquired pneumonia in the world.

Sputum in pneumonia caused by Klebsiella is characteristically referred to as the red currant jelly sputum which is formed due to the extensive

22 inflammation and necrosis of the tissue . It is stated that 8 % to 12% of Ventilator Associated Pneumonia is caused by Klebsiella pneumoniae(27).

MENINGITIS :

The clinical spectrum of K.pneumoniae meningitis can be categorized into 3 distinct forms.

a. Metastatic meningitis particularly from the distant liver abscesses.

b. Post craniotomy meningitis following neurosurgical procedures .

c. Primary or spontaneous meningitis in patients with

immunocompromised conditions(28)

NEONATAL SEPSIS :

Klebsiella pneumoniae is one of the leading causes of neonatal sepsis .It accounts for 4-9% of neonatal sepsis cases in developed countries and 16 -28% in developing countries(29)

LIVER ABSCESS :

Liver abscesses are mainly caused by hypervirulent strains of

Klebsiella in patients with no previous history of hepatobiliary disease.(12)

SURGICAL SITE INFECTIONS:

Klebsiella pneumoniae (26.37%) was the commonest pathogen isolated from wound infections of post surgical cases .(30)

CARBAPENEMS :

β LACTAM ANTIMICROBIALS :

Carbapenems belong to a group of drugs called β Lactam antibiotics(31). Penicillins , Cephalosporins and Monobactams also belong to the same group of drugs. These antimicrobials are so named because they possess a β Lactam ring (32).

CELL WALL OF BACTERIA :

The cell wall of bacteria contain an outer Peptidoglycan layer.

The peptidoglycan layer is thick in gram positive bacteria and comparatively thinner in Gram Negative bacteria. The gram negative bacteria in addition to the peptidoglycan layer possess an additional

Lipopolysaccharide layer which is absent in Gram positive bacteria .The peptidoglycan layer is cross-linked to a greater extent so as to make the cell wall rigid .

MECHANISM OF ACTION OF CARBAPENEMS :

The site of action of all the β Lactam antibiotics is the cell wall of bacteria ; they interfere with the synthesis of cell wall . They bind to specific protein moieties on the cell wall called as the Penicillin Binding proteins and inhibit the transpeptidases responsible for the formation of cross linkages, thereby preventing the formation of a rigid cell wall.This causes the formation of cell wall deficient forms ,which swell and burst and finally lead to bacterial lysis .

β lactamases inhibit the β Lactam antimicrobials by opening the β lactam ring .Carbapenems are the most potent of the β lactam antimicrobials because they are highly resistant to the hydrolysis caused

24 by various β lactamase enzymes .

CARBAPENEMS :

1. Imipenem

2. Meropenem

3. Faropenem

4. Doripenem

5. Ertapenem

6. Biapenem

7. Panipenem

8. Razupenem

IMIPENEM :

Imipenem is one of the most potent and a broad spectrum β

Lactam antibiotic known. It is active against Gram positive, Gram

Negative and anaerobic organisms .

The only disadvantage of Imipenem is that it is rapidly hydrolysed by the dehydropeptidase 1 enzyme produced by the renal tubular cells .

This disadvantage can be surpassed by using it in combination with

Cilastatin. Cilastatin is a reversible inhibitor of dehydropeptidase 1.

It can be used against a wide range of hospital acquired infections even in immunocompromised patients .

MEROPENEM :

Meropenem is considered to be stable than imipenem as it is not hydrolysed by the dehydropeptidase enzyme(33) . It is a reserve drug

25 which can be used in the treatment of serious nosocomial infections caused by cephalosporin resistant bacteria.

FAROPENEM :

It is orally active against many gram positive , gram negative and anaerobic organisms.

MANAGEMENT OF CRKP INFECTIONS:

POLYMYXINS :

Polymyxin B and polymyxin E(Colistin):

The polymyxins are the antimicrobials of choice for treating CRKP infections.They serve as the last resort of treatment for infections caused by Multi drug resistant gram negative pathogens .

Newer antibiotics for CR-KP :

Ceftazidime – avibactum

Plazomicin

Meropenem – varobactum

Imipenem – relabactum

Aztreonam – avibactum

Cefiderocol

These antibiotics are yet to come in the Indian market and in many other countries. .

For CR isolates , Tigecycline and Colistin remain the antimicrobials

26 of choice .In critically ill bacteremic patients with CR-KP, evidence seems to suggest that a combination regimen may be associated with better survival than monotherapy alone . Meropenem in addition to Colistin –

Tigecycline combination may result in a significant increase in survival , even in those patients who are infected with CR isolates, possibly due to potential Colistin – Carbapenem synergy.(34)

EPIDEMIOLOGY OF CARBAPENEM RESISTANCE IN INDIA :

80% of the Indian K.Pneumoniae isolates are resistant to

Cephalosporins and up to 60% resistant to Carbapenem .India has witnessed a rise in Carbapenem resistance rates from 9% in 2008 to 44% in 2010.(35) The first reported CR-Kp was identified in 1996.(9).The New

Delhi metallo –β –Lactamase ,an Ambler class B enzyme , is the main cause of carbapenem resistance in India .This is slowly being accompanied by an increased incidence of the OXA -48 like group ,Ambler Class D enzyme (36).While KPC is the predominant carbapenemase produced in

Europe it has been rarely reported from India.

GLOBAL EPIDEMIOLOGY OF CARBAPENEM RESISTANCE IN

KLEBSIELLA SPECIES :

Reports of Centers for Disease Control show that Approximately

80% of the infections caused by CRE is due to Klebsiella species(37).

Klebsiella species seems to represent a reservoir of resistance, transmittable to other members of the family Enterobacteriaceae .The Klebsiella pneumoniae Carbapenemase (KPC) ,an Ambler Class A β lactamase is

27 the most globally prevalent Carbapenem resistance enzyme is particularly widespread in Europe and North America(36).

Globally , the highest rate of carbapenem resistance has been reported in Greece with 68% resistance followed by India and Eastern

Mediterranean regions with 54% resistance(8).

ENZYMES RESPONSIBLE FOR CARBAPENEM RESISTANCE:

KPC :

Klebsiella pneumoniae Carbapenemase (KPC) was first detected in 2001 in New York(19).In western countries,KPC is the predominant mechanism of carbapenem resistance .In India, KPC in Klebsiella pneumoniae was first reported in 2011.bla KPC, is coded by a gene that is carried on a transposon , a mobile piece of genetic element .This in turn adds to the risk of extensive dissemination of the enzyme coding gene. KPCs are able to hydrolyse a wide range of β lactam antimicrobials.

KPC confers resistance not only to carbapenems but also to

Fluoroquinolones, Aminoglycosides and Trimethoprim-Sulphamethoxazole.

Thus the strains harboring KPC become Multi drug resistant organisms

(MDRO).

NDM :

The New Delhi Metallo β Lactamase was first isolated in a

Swedish patient who had a recent travel history to New Delhi.Hence the enzyme was named as NDM(38) .The gene that codes for the NDM enzyme is labelled as bla NDM.38.5 % of the isolated CRE in India

28 were found to possess the bla NDM gene .

VIM :

Verona –Integron Metallo β lactamase & Imipenemase enzymes are associated with plasmids or transposons and are found embedded in the Class – 1 integrons(39).

The other common enzymes causing Carbapenem resistance ae OXA

-48 and OXA -181.

MATERIALS & METHODS

MATERIALS AND METHODS

Place of study :

This study on the “ Identification of genes responsible for

Carbapenemases in Klebsiella species in a tertiary care hospital “ was done in the Department Of Microbiology, Coimbatore Medical College

Hospital among patients with prolonged hospitalization in intensive care units and various wards.

Study Period :

This study was conducted for a period of one year from July

2018 to June 2019.

Type of Study:

Prospective study

Approval from the ethical committee was obtained prior to the conduct of the study.

Sample:

In the present study, 250 Klebsiella species were isolated from different clinical samples like Pus, Urine, Blood, Wound swabs,

Catheter tip, Endotracheal aspirates and Sputum.

Inclusion Criteria:

1) Patients of all ages and groups.

2) Patients on prolonged hospitalization in the intensive care unit.

3) Patients on ventilator.

4) Patients on indwelling catheters or orthopaedic implants.

5) Patients with suspected sepsis.

6) Patients with wounds of any origin.

Exclusion Criteria:

1) Bacterial isolates other than Klebsiella.

2) Patients admitted for less than 48 hrs .

3) Patients already on antibiotics .

PROCESSING OF SAMPLES :

All the samples were collected under strict aseptic

precautions under standard microbiology procedures and were

processed(40). Two swabs were collected , One for gram’s stain and

the other for bacterial culture.

1) Direct Gram’s Stain :

Microscopic examination for the presence of pus cells and

bacteria.

2) All the collected samples except blood were inoculated onto the

Nutrient agar, MacConkey agar and Blood agar and incubated for 18-

24 hours at 37◦C.

3) Blood and Catheter tip culture:

Blood samples and catheter tips were inoculated initially in BHI

broth and incubated at 37ºC for 48 hours. The broth was regularly

examined for turbidity and subculture was done on Blood Agar plate

and MacConkey Agar plate . Any growth was processed for further

identification of the isolate.

4) Sputum samples were accepted as per Murray and Washington criteria

(41) .

5) Urine Cultures with colony counts > 104cfu/ml were accepted for

identification.

6) BAL specimens cultures with >105cfu/ml colony count were accepted.

7) Mucoid Lactose fermenting colonies on MacConkey agar were

processed for identification(42).

BIOCHEMICAL REACTIONS:

The mucoid lactose fermenting colonies were subjected to the following

tests .

1) Gram Stain

2) Motility

3) Catalase Test

4) Oxidase Test.

Gram negative short and stout bacilli ,non motile , catalase positive

and oxidase negative (11) was subjected to the following biochemical

reactions(43).

S.No Biochemical tests Results

1 Indole test Negative for K.pneumoniae

Positive for K.oxytoca

2 Simmon’s citrate Test Positive

3 Christensen’s Urease Test Positive

4 Triple Sugar Iron Agar Acid /acid with abundant

gas production

5 Methyl red test Negative

6 Nitrate reduction Test Positive

7 Voges – Proskauer test Positive

8 Lysine Decarboxylase Positive

9. Oxidative –Fermentation test Utilizes sugars both

oxidatively and fermentatively

10. Carbohydrate fermentation tests All sugars fermented with

gas formation

CATALASE TEST (TUBE CATALASE METHOD):

Procedure

A sterile gas rod was used to pick up one or two colonies and

introduced into a sterile glass test tube containing 3% hydrogen peroxide.

Catalase production is indicated by the production of prompt effervescence.

Klebsiella species are catalase positive .

OXIDASE TEST (WET FILTER PAPER METHOD ):

Procedure

Oxidase reagent (tetramethyl paraphenylene diamine dihydrochloride) was freshly prepared and poured over a filter paper placed in a petri dish. A colony was then picked up and streaked over the filter paper

.Formation of purple colour within 10 seconds was oxidase positive.

Klebsiella species are oxidase negative.

INDOLE TEST :

Formation of Indole by the splitting of Tryptophan is demonstrated by this test . 0.5ml of Kovac’s reagent was added gently along the sides of a test tube containing 2ml of 24-48 hours broth culture.

Positive test is demonstrated by the fomation of a red colouring at the junction .

Klebsiella pneumoniae - No ring formation -- indole negative

Klebsiella oxytoca - Red colored ring formed at the junction

– Indole positive .

METHYL RED TEST :

The organism to be tested was inoculated in tubes containing 5ml of glucose phosphate broth .The tubes were incubated at 37° C for 48 hours .Methyl red reagent was then added to the broth culture .Formation of red colour indicates a positive reaction .

Klebsiellae are Methyl red negative.

VOGES –PROSKAUER TEST :

The test organism was inoculated in 5ml of glucose phosphate broth and was incubated at 37 ° C for a minimum of 48 hours. To this broth culture, 0.2 ml of 40% KOH and 0.6 ml of 5% α- naphthol is added .Formation of red colour within 5 minutes indicates a positive reaction .

Klebsiella species are Voges Proskauer test positive.

CITRATE UTILIZATION TEST :

The slant of the Simmon’s citrate medium was streaked with the test organism and was incubated at 37◦C for 18 – 24 hrs . Change of green colour of the medium into blue or growth of the organism along the slant of the medium was taken as positive for citrate utilization.

All Klebsiella species are Citrate positive .

UREASE HYDROLYSIS TEST :

Christensens urease medium was used .The slant of the medium was streaked with the organism and was incubated at 37◦C for 18 – 24 hrs. The test was positive when the medium changed its colour from pale to pink.

Klebsiella species are Urease positive .

TRIPLE SUGAR IRON AGAR MEDIUM (TSI):

The triple sugar iron medium contains glucose ,lactose and sucrose.

The medium is poured into tubes such that they have a butt and a slant.

The organism was inoculated into the butt by a stab with a straight wire and was streaked on to the slant. The medium after inoculation was incubated at 37◦C for 18-24 hours .The medium was then examined for the presence of growth and fermentation of sugars.

Klebsiella species ferment all the three sugars and produce abundant amounts of gas . Abundant amounts of gas are produced such that the butt of the medium is pushed up in the tube.

Klebsiella on TSI agar – Acid / Acid (A/A) with gas .

NITRATE REDUCTION TEST :

The bacterium was inoculated in Nitrate broth and was incubated at 37 C for about 5 days.Reagent A ( α- napthylamine ) and Reagent

B ( Sulfanilic acid )was added to the medium .The reduction of nitrates is indicated by the formation of a red diazonium dye, p-sulfobenzene- azo-α- naphthylamine. Absence of formation of red colour gives a negative reaction.

Klebsiella species reduce nitrates to nitrites which is indicated by the formation of red colour.

LYSINE DECARBOXYLATION TESTS:

The enzymatic ability of an organism to decarboxylate an amino acid to form amines is measured by this test .Isolated colonies on the nutrient agar plate were inoculated in 4 tubes with Moeller’s decarboxylase medium containing Lysine ,Arginine , Ornithine and Base. Positive reaction is shown by the formation of purple colour.Yellow colour indicates a negative reaction.

Klebsiella species are Lysine decarboxylase positive .

CARBOHYDRATE FERMENTATION TESTS:

Oxidative utilization of sugars were tested separately . 1% glucose,

1% lactose ,1% sucrose ,1% mannitol and 1% maltose were used .Indicator used was Bromothymol blue .Formation of acid by the bacterium was indicated by the change of the colour of the indicator to yellow and gas production by the formation of gas in the Durhams tube .

Klebsiella species utilize all sugars with formation of gas .

OXIDATIVE FERMENTATION TESTS (HUGH & LEIFSON’S ):

This test is used to determine the ability of the organism to utilize the sugars oxidatively or fermentatively .Isolated colonies of the organism from the primary isolation plate are inoculated in OF medium using a sterile loop. One of the tubes is over laid with 1cm of mineral oil to create an anaerobic environment. The tubes are then incubated at 37° C

37 for 2-3 days and are examined daily. A change in the colour of the medium from green to yellow indicates the utilization of sugars .

Klebsiella utilizes sugars both oxidatively and fermentatively .

ANTIBIOTIC SUSCEPTIBILITY TESTING :

Mueller Hinton agar was prepared as per the manufacturer’s instructions and was poured to a depth of 4mm in 90mm sterile petri dishes.

Preparation of 0.5 McFarland’s standard:

1. 1% w/v sulphuric acid was prepared by adding 1ml of concentrated

sulphuric acid to 99ml of water .The solution was mixed well.

2. 1% w/v of barium chloride solution was prepared by adding

0.5 g of dehydrated barium chloride with 50 ml of distilled

water .

This solution can be stored by refrigeration for upto 6 months .

0.5 Mc Farland’s standard has an optical density equivalent to that of

1.5 x 108 colony forming units/ml .

Disc diffusion method :

Antibiotic susceptibility testing was done by Modified Kirby bauer disc diffusion method on Mueller Hinton agar .A single isolated colony of the bacterium was picked up with a loop and inoculated in peptone water and was incubated (44) .The turbidity was matched with 0.5

Mcfarland turbidity standard .The inoculum was then cultured on Mueller

Hinton agar plate by Lawn culture method using a sterile cotton swab .

Within 15 minutes ,the antibiotic discs were placed at a maximum of 5 on each plate .The plate was then incubated at 37°C for 18 -24 hours.

The zone sizes were interpreted according to CLSI guidelines .

List of Antibiotics used :

According to CLSI guidelines,the antibiotics for the test were selected .

S.no Name of the Strength Sensitive Intermediate Resistant

antibiotic (μg) (mm) (mm) (mm)

1. Amikacin AK 30 μg >17 15 -16 <14

2. Gentamicin G 10 μg >15 13-14 <12

3. Ofloxacin OF 5 μg >16 13-15 <12

4. Cefotaxime CTX 30 μg >26 23-25 <22

5. Ceftriaxone CTR 30 μg >26 23-25 <22

6. Ceftazidime CAZ 30 μg >21 18-20 <17

7. Piperacillin – PIT 100/10 >21 18-20 <17

Tazobactum μg

8. Cefoxitin CX 30 μg >18 15-17 <14

9. Norfloxacin NX 10 μg >17 13-16 <12

10. Trimethoprim- COT 1.25 / >16 11-15 <10

Sulfamethoxazole 23.75

μg

11. Cefepime CPM 30 μg >25 19-24 <18

12. Meropenem MRP 10 μg >23 20-22 <19

13. Ertapenem ETP 10 μg >22 19-21 <18

14. Imipenem IMP 10 μg >23 20-22 <19

The isolates which showed resistance to any one or all of the

Carbapenems , Cephalosporins or Cefoxitin were taken up for further phenotypic and genotypic tests .

Minimum Inhibitory Concentration by Epsilometer ( E test method ):

This test combines the principles of Disc diffusion and MIC. An

E strip comprises of a stable antimicrobial gradient (Meropenem) present on an inert ,thin ,non –porous plastic carrier of 60 mm length and 5mm width .This Meropenem E strip when placed on an inoculated agar medium ,releases the drug immediately and an antimicrobial concentration gradient is established on the medium . The plates are read after an overnight incubation at 37°C for 18-24 hrs .The reading is done by viewing the strip from the top of the plate .A symmetrical inhibition ellipse is produced.The MIC value is indicated by the intersection of the ellipse shaped inhibition zone with the E -strip. The results are interpreted following the CLSI guidelines.

MIC INTERPRETATIVE CRITERIA FOR MEROPENEM:

CATEGORY MIC Value (μg/ml)

SUSCEPTIBLE <1

INTERMEDIATE 2

RESISTANT >4

MIC For COLISTIN:

The E strip method was followed and the MIC value for Colistin was obtained for the isolates which were resistant to one or all of the

Carbapenems .

CATEGORY MIC Value (μg/ml)

SUSCEPTIBLE <2

INTERMEDIATE _

RESISTANT >4

METHODS FOR DETECTION OF ANTIMICROBIAL

RESISTANCE :

Laboratory methods for detection of Extended spectrum β lactamases:

Phenotypic screening test :

An isolate is considered to be an ESBL producer if the organism which produces zone sizes as below for the antibiotics during routine antibiotic susceptibility testing on Mueller hinton agar .

DRUG ZONE SIZE

Cefpodoxime <22mm

Ceftazidime <22mm

Aztreonam <27mm

Cefotaxime <27mm

Cetfriaxone <25mm

ESBL production can be further confirmed by the confirmatory test

procedure according to CLSI guidelines .

Phenotypic confirmatory test :

COMBINED DISC TEST :

This test was done using Cefotaxime (30 μg ) ,Ceftazidime (30

μg), Cefotaxime clavulanic acid(30/10μg ) and Ceftazidime clavulanic acid

(30/10μg ) Himedia antibiotic discs.The organism to be tested was

inoculated in peptone water and was incubated .The turbidity was matched

with McFarlands standards .The inoculum was then lawn cultured on a

Mueller Hinton agar plate . Cefotaxime (30 μg ) ,Ceftazidime (30 μg)

,Cefotaxime clavulanic acid(30/10μg ) and Ceftazidime clavulanic acid

(30/10μg ) Himedia antibiotic discs were placed and the plate was

incubated at 37C for 18- 24 hours(45) .A >5mm increase in the zone

size of the antibiotic used in combination with clavulanate than the

antibiotic used alone is considered to be a positive test for ESBL

production .

Discs used Interpretation Result

Cefotaxime- CTX (30 μg ) >5mm increase in Positive Cefotaxime clavulanic acid (30/10 μg) zone size for CEC (CEC) than CTX Ceftazidime- CAZ (30 μg ) >5mm increase in Positive Ceftazidime clavulanic acid (30/10 μg ) zone size for CAC (CAC) than CAZ

E STRIP Test for ESBL production :

E strip for ESBL production comprises of shorter gradients of two drugs aligned in opposite directions in a single strip. One end generates a stable gradient of one cephalosporin (Cefotaxime ) and the other end generates a combination of a cephalosporin with clavulanate (Cefotaxime

+ Clavulanic acid).A positive result is indicated by a MIC ratio of > 8 between the cephalosporin and the cephalosporin clavulanate drugs .

Laboratory methods for the detection of AmpC β lactamases :

Amp C detection by disc method :

Amp C detection is done by comparing the sizes of the zone of inhibition of Cephamycins (Cefoxitin or Cefotetan ) with that of

Cephamycins with Cloxacillin ( Cefoxitin + Cloxacillin ). Antibiotic discs of Cefoxitin and Cefoxitin + Cloxacillin were placed on an MHA plate inoculated with the test organism and was incubated .A difference in zone size of >5mm around the Cefoxitin + Cloxacillin disc than the cefoxitin disc is considered positive for AmpC production .

Laboratory methods of detection of Metallo β Lactamase :

Imipenem – EDTA Combined disc test:

The antibiotic discs containing Imipenem and Imipenem + EDTA are placed 15 - 20mm apart on an MHA plate inoculated with the test organism .The plate is incubated for 18-24 hrs at 37°C and the zone of

43 inhibition around both the discs are compared .When there is an increase in zone size of >7mm around the Imipenem + EDTA disc than the

Imipenem disc ,the organism is considered to be a Metallo β lactamase producer .

CARBAPENEMASE DETECTION METHODS :

Phenotypic methods:

1. Rapidec Carba NP test

2. Modified Carbapenem Inactivation method

- To confirm carbapenemase detection

3. EDTA Modified Carbapenem Inactivation Method

- To differentiate between metallo β lactamase and serine

carbapenemase .

These tests were performed on organisms that showed a zone of inhibition of < 19mm for Meropenem on routine antibiotic susceptibility testing by disc diffusion method .

Rapidec Carba NP test :

This test described by Nordmann, Poirel and Dortet consists of a ready to use strip for the rapid detection of carbapenemase .

Principle :

The carbapenemase enzyme present in the bacteria hydrolyses the

44 carbapenem drug which leads to a change in pH which further causes a change in the colour of the indicator used (Phenol red) from red to yellow .

The kit was purchased from BioMerieux and the test was performed as per the instructions given by the manufacturer.

The kit contains

1. API suspension medium

2. Ready to use strip with 5 wells

3. Sticks for mixing the contents

4. Black and white chart

The 5 wells in the strip are named as a,b,c,d and e .

The contents of the wells are as below :

WELL CONTENTS

A Phenol red

(Indicator)

B Turbidity control

C Lysis buffer

D Control well without Imipenem

E Reaction well with Imipenem and

Zinc.

Procedure :

1) 100 μl of the API suspension medium (provided in the kit ) was

dispensed into wells a, b and c .

2) The strip was incubated at room temperature for 10 minutes .

3) The contents of the well b (Turbidity control) was mixed

thoroughly with the sticks provided in the kit .

4) Few colonies of the test organism ( Klebsiella ) were picked up

from a Mueller Hinton agar plate (<72 hrs old ) with the stick

provided in the kit and was mixed in the well c (Lysis buffer )

. Lysis buffer lyses the bacterium and leads to the extraction of

the carbapenemase enzyme .

5) Colonies were mixed until the turbidity of the well c was equal

to that of well b.

6) The strip was incubated at room temperature for 30 minutes .

7) 25 μl of the lysate from well c was added to wells d and e .

8) 25 μl from well a (Indicator – Phenol red) was added to wells

d and e .

9) The strip was incubated at 37 C for 30- 40 minutes.

10) The strip was read after incubating for a maximum of two

hours .

11) The colour change in the wells d and e are noted.

Interpretation :

The organism if a Carbapenemase producer hydrolyses the drug in well e and causes a change in pH of the indicator that turns the colour from red to yellow .The contents of the control well d remain red in colour .

If the test well and the control well are both red in colour , the test is considered negative .

CONTROL WELL TEST WELL INTERPRETATION

Red Red Negative

Orange Orange Negative

Red Orange – Yellow Positive

Orange Yellow Positive

Modified Carbapenem Inactivation method (mCIM):

According to the CLSI guidelines, this test is considered to be the phenotypic confirmatory test for carbapenemase production in

Enterobacteriaeceae.

Test Procedure :

1) For each isolate to be tested , a loopful of bacteria from an

overnight blood agar plate is emulsified in tubes containing 2ml

of tryptic soy broth(TSB) .

2) Vortex for 10 – 15 seconds.

3) A Meropenem disc (10 μg) is added to each tube using sterile

forceps .

4) Incubate at 37 °C for 4hrs + 15 minutes.

5) Prepare a 0.5 McFarland suspension of E.coli ATCC 25922 in

nutrient broth just before or on completion of the incubation of

the TSB –Meropenem disc suspension.

6) The E.coli ATCC is lawn cultured on to a Mueller Hinton agar

plate .

7) The meropenem is taken from the TSB using a loop and is

placed on to the plate cultured with E.coli ATCC.

8) The plate is incubated at 37°C for 18 -24 hours .

9) The size of the zone of inhibition are measured after incubation.

Test Interpretation :

Carbapenemase producers hydrolyse the drug when the disc is incubated with the test organism in TSB.So when placed on the plate containing Carbapenem susceptible E.coli ATCC , the drug disc does not cause any zone of inhibition .

Carbapenemase positive :

A zone of inhibition of 6-15 mm or pinpoint colonies within a zone of 16-18mm zone are considered positive .

EDTA Modified Carbapenem Inactivation method (eCIM):

1) eCIM is only done for isolates that are mCIM positive .

2) This test is done to differentiate between the metallo β lactamases

and serine carbapenemases .

Procedure :

1) Add 20μl of 0.5ml EDTA to the 2ml TSB to obtain a final

concentration of 5mM EDTA .

2) The procedure is same as that of mCIM .

3) Both mCIM and eCIM tubes are processed in parallel.

4) The meropenem discs from both the tubes are placed on the same

plate containing E.coli ATCC 25922 indicator strain.

Test Interpretation :

The action of the metallo β lactamase is inhibited by EDTA.

Therefore if the organism is a metallo β lactamase producer , meropenem is not hydrolysed in the tube containing EDTA. An increase in the zone diameter of >5mm for eCIM than that of mCIM is considered positive .

Molecular method:

Polymerase chain reaction :

Conventional PCR was done to detect the presence of genes such as

NDM-1,OXA -48,KPC ,VIM and IMP.DNA was prepared by emulsifying 2-5 colonies in 100μl of molecular grade water (Qiagen,Germany);1 μl of the DNA template was added to 20 μl of the PCR reaction mixture (Invitrogen,USA).

Requirements :

1. Micro Pippetes of variable volume 0.5 -10μl, 10- 100μl and 100-

1000μl.

2. Filtered pippete tips

3. 1.5ml /2ml centrifuge tubes

4. Thermo cycler (Applied Biosystems,Veriti,USA )

5. Apparatus for electrophoresis and power supply unit.

6. Taq polymerase ,PCR buffer ,dNTP mixture and other molecular

reagents.

7. Gel Doc system

8. 0.5% TBE buffer,agarose ,EtBr,100bp 0.5% TBE buffer, agarose

EtBr,100bp marker.

9. Computer for data storage .

Molecular reagents used for Extraction Procedure:

1. 370 μl Molecular grade water

2. 30μl Magnesium chloride buffer

3. 30 μl dNTP

4. 15 μl of Forward primer

5. 15 μl of Reverse primer

The extraction procedure yields purified DNA after lysis of the cell.

The isolated DNA can be used directly for DNA amplification.

PCR amplification steps :

1. Initial DNA release – 94 °C for 5 min

2. Denaturation - 94 °C for 30 s

3. Annealing – 52 °C for 40 s 30 cycles

4. Elongation – 72 °C for 50 s

5. Final elongation – 72° C for 6 min.

Initial denaturation :

First step of amplification is initial denaturation. The temperature

is raised by thermocycler to 94° C for 5 min for Taq enzyme activation.

Denaturation:

The separation of the double stranded template DNA into two

complementary strands occurs in this step. The temperature is elevated

to 94° C for 30 seconds.

Annealing :

Annealing refers to the complementary binding of the two

specific oligonucleotide primers to the DNA template. The temperature

is decreased to 58 °C for 30 seconds in the thermocycler.

Extension:

The DNA polymerase extends the primers when the temperature

is increased to 72° C for 30 seconds .Final extension takes place at 72°C

for 5 minutes . The template DNA is synthesized using deoxynucleotides

(dNTPs)in the reaction mixture .Two single stranded DNA templates and

newly synthesized complementary DNA strands attach together to form

new double stranded DNA copies .Every copy of newly formed DNA

may function as a template for further amplification .The products will

be amplified in an exponential manner in each cycle . At the end of 30

cycles ,the final product will have 2n copies of the template DNA .

Agarose gel preparation:

2% agarose gel was prepared by mixing 2 gm of agarose

in 100ml of TBE buffer and melted using microwave oven.When the

agarose gel temperature reaches around 60°C ,5μl of Ethidium bromide

was added.This warm agarose solution was poured slowly into gel

platform and the gel was kept undisturbed until the gel solidified .TBE

buffer was poured into the gel tank and the solidified gel was placed

carefully into the tank. The buffer level in the tank was maintained 0.5

cm above the level of the gel. PCR samples were loaded after mixing

with gel loading dye along with 10μl of 100bp DNA ladder.Then

electrophoresis was run at a rate of 50 V till the dye reaches three fourth

distance of the gel.Then the gel was visualized by using Molecular

imager (Gel Doc XR+,Bio Rad , USA) .

Primers :

Gene target Primers sequence (5’-3’) Product

size (bp)

NDM – F GGGCAGTCGCTTCCAACGGT 188

NDM –R GTAGTGCTCAGTGTCGGCAT

OXA -48F TTGGTGGCATCGATTATCGG 390

OXA -48 R GAGCACTTCTTTTGTGATGGC

VIM-F GATGGTGTTTGGTCGCATA 743

VIM-R CGAATGCGCAGCACCAG

IMP-F GGAATAGAGTGGCTTAATTCTC 475

IMP-R CCAAACCACTACGTTATCT

KPC F TGTCACTGTATCGCCGTC 1000

KPC R CTCAGTGCTCTACAGAAAACC

Fig 1: Nutrient Agar Plate showing colonies of Klebsiella

FIG 2: Mac Conkey agar plate showing mucoid lactose fermenting colonies of Klebsiella

Fig 3:Biochemical reactions of Klebsiella pneumoniae

Fig 4: Biochemical reactions of Klebsiella oxytoca

Fig 5:Carbohydrate fermentation tests of Klebsiella species

Fig 6: Amino acid decarboxylation tests of Klebsiella species

Fig 7:Gram stain picture of Klebsiella showing short gram negative bacilli

Fig 8: Klebsiella isolate resistant to Meropenem and sensitive to Colistin and Tigecycline

Fig 9: E strip showing Klebsiella isolate resistant to Meropenem (4 µg/ml)

Fig 10 : E strip showing Klebsiella isolate sensitive to Colistin (1 µg/ml)

Fig 11: Detection of ESBL by Combined Disc Method

Fig 12: Detection of ESBL by E strip Method

Fig 13: Detection of Amp C by Combined Disc Method

Fi g 14 :Detection of Carbapenem resistant isolates by disc diffusion method

Fig 15: Detection of Carbapenemases by Carba NP Test

Fig 16: Detection of Carbapenemase by mCIM and eCIM

mCIM - Negative mCIM – Positive

eCIM – Negative

Fig 17 : Detection of Carbapenemases by PCR

Fig 18:Loading samples in Thermocycler

Fig 19:Agarose gel Electrophoresis

Fig 20:Molecular Imager (Gel Doc XR+, Bio Rad,USA)

Fig 21: Distribution of NDM 1 genes in resistant isolates

1 2 3 4 5 6 7 8 9 10 11 12 13 14

DNA Ladder 100bp

Fig 22: Distribution of OXA 48 like genes in resistant isolates

1 2 3 4 5 6 7 8 9 10 11 12 13 14

DNA Ladder 100bp

RESULTS

This study on the“ Identification of genes coding for Carbapenemases In

Klebsiella species in a tertiary care hospital “ included a total of 250 patients in whom Klebsiella caused infections . This study was done at Coimbatore

Medical College for a period of one year from July 2018 to June 2019.Various clinical samples from those patients were processed and were analysed for antimicrobial resistance by phenotypic and genotypic methods.

Table 1: SAMPLE WISE DISTRIBUTION OF KLEBSIELLA

INFECTIONS (n=250)

S.No Sample Carbapenem Carbapenem Total No of sensitive isolates resistant isolates isolates (n=202) (n=48) (n=250) n (%) n (%) n (%) 1. Urine 90 (44.5%) 20(41.6%) 110(44.5%)

2. Pus 76(37.6%) 17(35.4%) 93(37.2%)

3. Sputum 20(9.9%) 6(12.5%) 26(10.4%)

4. Throat swab 10(4.9%) 2(4.1%) 12(4.8%)

5. Blood 4(1.9%) 1(2%) 5(2%)

ET Tube C/S 1(0.4%) 1(2%) 2(0.8%)

6. Bronchial Wash _ 1(2%) 1(0.4%)

7. High Vaginal 1(0.4%) - 1(0.4%) Swabs

Among the 250 Klebsiella isolates taken up for study , 110(44.5% ) isolates were from Urine ,93 (37.2%) were from pus , 26 (10.4%) from

Sputum, 12(4.8%) from Throat swab and 5 (2%) from Blood.2(0.8%) of the isolates were from Endotracheal tube samples and 1(0.4%) were from Bronchial wash and High vaginal swab each.

Among the 48 Carbapenem resistant Klebsiella isolates , 20 (41.6%) were from Urine samples , 35.4% from Pus samples,6(12.5%) were from

Sputum whereas 2(4.1%) were Throat swab samples and 1 (2%) isolate was obtained from Blood, ET tube and Bronchial wash each .

TABLE 2: TYPES OF KLEBSIELLA SPECIES ISOLATED FROM

STUDY POPULATION

Carbapenem Resistant Klebsiella Klebsiella sp S.No species (n=250) (n=48)

Klebsiella pneumoniae 177(70.8%) 32(66.6%)

Klebsiella oxytoca 73(29.2%) 16(33.3%)

Klebsiella pneumoniae and Klebsiella oxytoca were the species isolated of which Klebsiella pneumoniae (70.8%) was the most common species in the study population.

TABLE 3: GENDER WISE DISTRIBUTION OF KLEBSIELLA

ISOLATES (n=250)

Carbapenem Carbapenem Total No of P S.no Gender resistant sensitive isolates value isolates isolates (n=250) 1. MALES 39(81.2%) 114(45.6) 153(61.2%) 0.001 2. FEMALES 9(18.7%) 88(35.2%) 97(38.8%)

Klebsiella infections were more commonly observed in males (61.2%) than females (38.8%). 81.2% of the Carbapenem resistant Klebsiella species were observed in males and 9(18.7%) were observed in females .P value <0.005 is significant.

TABLE 4: WARD WISE DISTRIBUTION OF CARBAPENEM

RESISTANT KLEBSIELLA SPECIES

S.No WARD No of Carbapenem resistant Isolates (n=48) 1. SURGERY 13(27%) 2. ICU 11(22.9%) 3. MEDICINE 10(20.8%) 4. ORTHO 9(18.7%) 5. BURNS 3(6.25%) 6. PAEDIATRICS 1(2%) 7. OG 1(2%)

Table 5: FACTORS INFLUENCING INCIDENCE OF RESISTANT

INFECTIONS IN VARIOUS WARDS

WARDS No of isolates

(n=48)

SURGERY 13(27%)

Elective surgeries 4(30.7%)

Emergency surgeries 9(69.2%)

ICU 11(22.9%)

Mechanical ventilation 6(54.5%)

Urinary Catheterisation 8(72.2%)

MEDICINE 10(20.8%)

Urinary Catheterisation 5(50%)

Prolonged hospitalisation 7(70%)

Previous antibiotic exposure 7(70%)

ORTHO 9(18.7%)

Implant associated 6(66.6%)

Non implant associated 3(33.3%)

Carbapenem resistant Klebsiella isolates were more commonly seen in patients admitted in the Surgical wards (27%) and in Intensive care units

(22.9%).Of the isolates from medical wards 20.8% were Carbapenem resistant.18.7% of the isolates from Orthopaedic wards and 6.25% from burns ward were resistant to Carbapenems. (2%) of the isolates both from Gynaec wards and Pediatric wards each exhibited resistance to Carbapenems.

Table 6: AGE WISE DISTRIBUTION OF CARBAPENEM SENSITIVE

AND CARBAPENEM RESISTANT ISOLATES OF KLEBSIELLA

Carbapenem Carbapenem Total S.No Age resistant sensitive isolates (n=250) isolates 1. 1-10 yrs 35(17.3%) 1(2%) 36(14.4%)

2. 10-20 yrs 17(8.4%) 2(4.1%) 19((7.6%)

3. 20-30 yrs 24(11.8%) 3(6.25%) 27(10.8%)

4. 30-40 yrs 24(11.8%) 9(18.7%) 33(13.2%)

5. 40-50 yrs 26(12.8%) 13(27.0%) 39(15.6%)

6. >50 yrs 76(37.6%) 20(41.6%) 96(38.4%)

Carbapenem resistance was predominantly observed in the age group of more than 50 years (41.6%) ,followed by patients aged 40-50 yrs(27%) and 30-

40 yrs(18.7%).A relatively lower incidence was observed in patients aged 1-10 yrs (2%) and 10-20 yrs(7.6%).

TABLE 7:RISK FACTORS ASSOCIATED WITH INFECTIONS OF

KLEBSIELLA SPECIES

Carbapenem Carbapenem Resistant Sensitive Risk Factor P value Klebsiella species isolates (n=39) (n=250) DIABETES Present 26 119 0.026 Absent 13 131 ICU STAY Present 15 55 0.025 Absent 24 195 Alcoholism / Present 18 74 Chronic liver 0.039 disease Absent 21 176 Invasive Present 22 99 surgical 0.047 procedures / Absent 17 151 Trauma Indwelling Present 19 75 medical devices 0.020 Absent 20 175 Prolonged Present 33 135 hospitalization <0.001 (>7 days ) Absent 6 115

The major risk factors associated with Klebsiella infections were found to prolonged hospitalization more than 1 week and diabetes mellitus .P value

<0.001 is significant. The other risk factors were stay in the Intensive care units

, invasive surgical procedures , alcoholism and indwelling medical devices.

TABLE 8: INVITRO ACTIVITY OF VARIOUS ANTIBIOTICS

AGAINST KLEBSIELLA ISOLATES

Antibiotic Discs S.No Sensitive Resistant (Hi Media ) 1. Amikacin 234(93.6%) 16(6.4%)

2. Gentamicin 160(64%) 90(36.0%)

3. Ciprofloxacin 203(81.2%) 47(18.8%)

4. Norfloxacin (n=110) 90( 81.8%) 20(16.3%)

5. Cefotaxime 157(62.8%) 93(37.2%)

6. Ceftazidime 208(83.2%) 42(16.8%)

7. Cotrimoxazole 171(68.4%) 79(31.6%)

8. Cefepime 173(69.2%) 77(30.8%)

9. Piperacillin - tazobactum 217(86.8%) 33(13.2%)

10. Meropenem 202(80.8%) 48(19.2%)

11. Cefoxitin 239(95.6%) 11(4.4%)

12. Colistin 250(100%)

13. Tigecycline 250(100%)

The sensitivity pattern of the Klebsiella isolates were as follows :The highest sensitivity was for Colistin (100%) and tigecycline (100%) .The sensitivity to all other antibiotics were as follows : Meropenem (80.8%),

Piperacillin- Tazobactum (86.8%), Cefepime (62.4%), Cotrimoxazole (68.4%)

Ceftazidime(83.2%), Cefotaxime (62.8%),Norfloxacin (83.6%), Ciprofloxacin

(81.2%), Gentamicin (64%) and Amikacin (93.6%).

Table 9: DETECTION OF ESBL and Amp C β Lactamase

PRODUCTION BY VARIOUS METHODS

Amp C ESBL detection S.No Test method production (N=250) (n=250)

1. Screening method 135 (54%) 11(4.4%)

2. Combined disc method 115(85.1%) 10(90.9%)

3. E strip 102(75.5%) 9(81.8%)

The isolates which were either Cefotaxime or Ceftazidime resistant were subjected to further phenotypic tests for ESBL detection .135(54%) out of 250 isolates were found to be ESBL producers by screening method .ESBL detection by Combined disc method was about 85.1% and by E strip method was about 75.5%.

Detection Of Amp C Production was done by screening the organism for its sensitivity to Cefoxitin. Klebsiella which were Cefoxitin resistant were further confirmed by Combined disc method .9 of 250 isolates were confirmed as Amp C β lactamase producers by phenotypic methods .

TABLE 10: VARIOUS TYPES OF β LACTAMASE ENZYME

PRODUCERS AMONG ANTIBIOTIC RESISTANT KLEBSIELLA

ISOLATES

S.No Type Of β Lactamase No of Isolates Percentage

1. ESBL 135 54% 2. MBL 43 22.9% 3. AMP C 9 3.6%

Extended spectrum β lactamases were the commonly produced enzyme among the β lactamase producers in our study.135 (54%) isolates of Klebsiella of our study population were found to be ESBL producers .43 (22.9%) isolates were found to produce MBL and 9 (3.6%) isolates were found to produce

Amp C.

Table 11: COMPARISON OF VARIOUS PHENOTYPIC METHODS IN

THE DETECTION OF CARBAPENEMASES

POSITIVE NEGATIVE TEST S.NO ISOLATES ISOLATES TOTAL DONE Number Percentage Number Percentage 1. CARBA 41 85.4% 7 14.5% 48 NP test 2. mCIM 43 89.5% 5 11.6% 48 3. eCIM 16 37.2% 27 62.7% 43

Table 12 : DETECTION OF CARBAPENEMASES BY MOLECULAR

METHODS

NO OF NDM -1 and bla VIM OXA -48 CARBAPENEM NDM-1 OXA -48 like bla IMP like RESISTANT Co producers bla KPC

KLEBSIELLA 22 21 5 ISOLATES Not detected (45.8%) (43.7%) (10.4%) (N=48)

CHARTS

CHART 1: SAMPLE WISE DISTRIBUTION OF KLEBSIELLA INFECTIONS

90 90 76 80 70 60 50 40 20 17 30 20 20 6 10 2 1 1 1 4 10 1 1 0

CARBAPENEM SENSITIVE

CARBAPENEM RESISTANT

CHART 2: TYPES OF KLEBSIELLA SPECIES ISOLATED FROM THE STUDY POPULATION

177 180 160 140 120 100 Carbapenem sensitive 80 73 Carbapenem resistant 60 32 40

20 16 0

Klebsiella pneumoniae Klebsiella oxytoca

CHART 3: GENDER WISE DISTRIBUTION OF KLEBSIELLA ISOLATES

114 120

100 88

80 CARBAPENEM RESISTANT 60 CARBAPENEM SENSITIVE 39 40

20 9

0 MALES FEMALES

CHART 4: WARD WISE DISTRIBUTION OF CARBAPENEM RESISTANT KLEBSIELLA SPECIES

N=48

1 1 3 SURGERY 13 ICU 9 MEDICINE ORTHOPAEDICS BURNS PAEDIATRICS OG 10 11

CHART 5 :AGE WISE DISTRIBUTION OF CARBAPENEM RESISTANT KLEBSIELLA INFECTIONS

20 20

15 13 9 10 CARBAPENEM RESISTANT 5 3 1 2 0 1-10 YRS 10-20 20-30 30-40 40-50 >50 YRS YRS YRS YRS YRS

CHART 6:RISK FACTORS ASSOCIATED WITH CARBAPENEM RESISTANT KLEBSIELLA INFECTIONS

6 35 13 17 30 20 21 24

33 15 ABSENT 26 PRESENT 22 10 19 18 15

CHART 7: ANTIMICROBIAL RESISTANCE PATTERN OF KLEBSIELLA

ISOLATES (N=250)

250 234 217 203 208 202 200 171 173 160 157 150

93 100 90 90 79 77

47 42 48 50 33 16 20

CARBAPENEM RESISTANT CARBAPENEM SENSITIVE

CHART 8:DETECTION OF β LACTAMASES BY PHENOTYPIC METHODS

135 140 120 115 100 102 80 60 11 ESBL 10 40 9 Amp C 20 Amp C 0 ESBL Screening method Combined disc method E strip

CHART 9 : DETECTION OF CARBAPENEMASES BY VARIOUS PHENOTYPIC METHODS

41 CARBA NP mCIM eCIM

CHART 10 : ISOLATES PRODUCING VARIOUS TYPES OF β LACTAMASES DETECTED BY PHENOTYPIC METHODS

ESBL MBL 135 Amp C

CHART 11: VARIOUS GENES CODING FOR CARBAPENEMASES DETECTED BY MOLECULAR METHODS

25 22 21 20

5 5 0

NDM -1 OXA -48 like NDM -1 and OXA 48 like co-producers

DISCUSSION

Klebsiella is one among the Gram negative bacterium which causes a wide array of infections in humans and in the last decade, Antimicrobial resistance among gram negative organisms predominantly in

Enterobacteriaceae has become a dire emergency . CRE was declared a public threat in the United States by the CDC in 2013. Among the infections due to

Carbapenem resistant Enterobacteriaceae , Klebsiella species are responsible for more than 80% of infections(46). Infections due to Carbapenem resistant

Klebsiella species are also being increasingly reported in India(47).

India ranks second only to Greece in reporting Carbapenem resistance with a high resistance rate of 54%(48). Antimicrobial resistance in Klebsiella mainly occurs due to the transfer of genes among the species by Horizontal

Gene Transfer (49).

Carbapenems were considered to be the last resort for treatment of infections caused by Multi drug resistant Klebsiella (50).Carbapenem resistance in Enterobacteriaceae is considered due to the production of Carbapenemases of various classes and hyperproduction of ESBLs and AmpC along with mechanisms that lead to reduced cell wall permeability(5).

In this study , 250 Klebsiella isolates obtained from various clinical samples were evaluated for resistance to Carbapenems .The prevalence of

Carbapenem resistance among Klebsiella species in our study is 19.2%.This is slightly lower than that compared to Suhail et al., who has reported a prevalence of 27.9%(51). Lack of routine screening for Carbapenemases in our laboratory may be the cause for a relatively lower prevalence rate.

SPECIMEN DISTRIBUTION :

Out of the 250 isolates in our study,110 (44.5%) were from Urine samples,93 (37.2%) from pus,26(10.4%) from sputum, 12 (4.8% ) from throat swabs ,5 (2%) from blood and 2 (0.8%) were from Endotracheal tube samples.Isolates from Bronchial wash and High vaginal swabs constituted 0.4% of the total isolates .

This is in accordance with the study conducted by Jadhav et al., who has also reported isolation of Klebsiella to be higher in Urine samples followed by pus samples and sputum samples(52). This is in contrast to the study conducted by Ghanem et al., who has reported highest occurrence of Klebsiella isolates in

Sputum samples(66.6%)(53).

Carbapenem resistant isolates were also found to be more common from

Urine samples( 41.6% ) and from pus (35.4%). Isolates from sputum samples constituted for about 12.5% and isolates from throat swabs constituted for about 4.1% . Blood,Endotracheal tube specimens and Bronchial wash samples contributed to 2% of the isolates each.

Higher incidence of Carbapenem resistance in Urine samples may be attributed to urinary catheterization in a large number of patients admitted to the health care facility(54). Presence of Urinary catheter can raise the resistance of Klebsiella to Carbapenems by about 10%(55). Brizendine et al., in their study state that lowering the Urinary Catheterisation rate in patients may lead to a decrease in the occurrence of infections caused by resistant organisms(54) .

Urinary catheterization in patients admitted in the Intensive care units and Medical wards contributed to a significant number of resistant isolates(54)

. This is in accordance with the study conducted by Akgul et al., who reported prolonged hospitalization,mechanical ventilation and Urinary catheterisation to be risk factors associated with Carbapenem resistant Klebsiella infections (56).

SPECIES DISTRIBUTION :

In our study , Klebsiella pneumoniae 177(70.8%) was the most common species isolated whereas Klebsiella oxytoca [ 73 (29.2%) ] was comparatively lower.

This is in accordance to the study conducted by Acheampong et al., who also reports an occurrence of K.pneumoniae to be 74.4% and K.oxytoca to be 24.1%(57). This is slightly lower when compared to the study conducted by Vasaikar et al., who reported 83.7% of K.pneumoniae and 7.4% of

K.oxytoca (58) .

Our study indicates a need for speciation of Klebsiella species as a routine as other Klebsiella species other than Klebsiella pneumoniae are also on the rise in causing resistant infections .

AGE DISTRIBUTION :

In our study , highest distribution of Klebsiella infections is seen among patients in the age group of more than 50 yrs (38.4%) followed by patients in the age group of 40-50 yrs (15.4%) .

This is in accordance with the study conducted by Suhail et al., who states incidence of Carbapenem resistant Klebsiella to be highest in the age group of more than 60 yrs (51).This is in contrast to the study conducted by

Acheampong at al., who has reported a higher incidence of Klebsiella infections in the age group of 1-9 yrs followed by 30 -39 yrs of age .

Higher incidence of Carbapenem resistant infections in older age groups may be due to existence of co- morbid conditions like Diabetes mellitus which cause immunosuppression. The incidence of resistant infections in the elderly in our study may also be due to the proportionately higher number of samples received from adult wards than paediatric wards.

SEX DISTRIBUTION:

In our study , 153(61.2%) of the Klebsiella isolates were from males and

97(38.8%) were from females. This is in accordance with the study by Ghanem et al., who reported 66% of Klebsiella infections in males and 34% in females(53).

According to our study ,the male to female ratio is 1.5:1.Namratha et al., also have reported a male to female ratio of about 1.7:1 in their study (59).

A male predominance in the occurrence of Carbapenem resistant infections may be due to a higher percentage of male population involved in

Alcoholism and Smoking which predisposes them to a manifold of infections.

WARD DISTRIBUTION:

In our study ,distribution of infections due to Carbapenem resistant

Klebsiella was observed in the wards as follows : Surgical wards

(27%),Intensive care units (22.9%) , Medical wards (20.8%), Orthopaedic wards (18.7%) ,Burns ward (6.25%) Gynaec ward(2%) and Pediatric wards(2%).

A higher incidence of resistant infections in Surgical wards was found in patients who underwent emergency surgeries than in patients with elective surgeries(56) .The time of administration of antibiotics to the patients prior to surgery play an important role in the incidence of infections.

The following measures are to be followed in order to prevent the occurrence of Surgical site infections :

1.Administration of antibiotics 30 minutes before the skin incision is made.

2.Re-administration of antibiotic every 4hours .

3.Discontinuation of the antibiotic only during the intra operative period .(60)

Klebsiella ,being an inhabitant of the Gastro intestinal tract causes most of the SSI in Gastrointestinal tract surgeries (61). Ciprofloxacin, prophylactically administered before surgery is found to be effective in preventing SSI due to Klebsiella species(61).

Indwelling devices due to mechanical ventilation or surgical procedures as in Implant associated infections play a major role in giving rise to resistant organisms as these devices act as a scaffold for Biofilm formation. Biofilm acts as a barrier protecting the bacteria from the host immune response and also the external agents such as antibiotics. Therefore higher doses of antibiotics have to be administered in cases suspected to have biofilm producing strains which further leads to selection pressure on the bacterium and formation of resistant strains(62).

Under developed immune system in the pediatric age group promotes acquisition of infections by resistant organisms.

Moderate number of resistant isolates has been obtained from pus samples of Burns patients. Klebsiella is considered to be the second most common organism causing infections in burns patients(63) . Burns cause a breach in the skin barrier which creates a favourable access for organisms causing infections. Klebsiella therefore can easily colonise a burns victim on account of its saprophytic nature . Prakash et al., states that the organism causing infection in a burns patient and its susceptibility should be known in order to select the appropriate antibiotics so as to prevent the emergence of resistance and its spread(64). Poor infection control practices is found to be an important causation factor for Burn wound infections(65) .

RISK FACTORS :

In our study ,the risk factors associated with acquiring Klebsiella infections were found to be

1. Prolonged hospitalization for more than 7 days

2. History of Diabetes mellitus

3. Undergoing invasive surgical procedures /Trauma

4. Indwelling medical devices

5. History of alcoholism

6. ICU stay .

Prolonged hospitalization( more than 7 days ) in the health care facility plays a major role in the acquisition of resistant infections. It may be due to cross infections from patients and higher rates of carriers among the health care personnel who harbor resistant organisms.

ICU stay has caused 31.2% of Carbapenem resistant Klebsiella infections in our study. ICU stay increases the incidence of infections due to resistant organisms because of the vulnerability of the patients due to illness, exposure of the patients to various procedures like insertion of urinary catheters, intravenous access , mechanical ventilation and administration of various broad spectrum antibiotics. Longer duration of stay in the ICU further complicates the scenario.Li et al., has reported an incidence of 48.1% of Carbapenem resistant

Klebsiella infections in ICU(66) .This is slightly higher when compared to our study and it may be due to the fact that a comparatively lower number of samples were received from Intensive care units than from other wards in our study .

RESISTANCE PATTERN :

Studying the antibiotic susceptibility pattern of this population may yield a considerable information on the usage of antibiotics and to formulate antibiotic policies in a view to prevent further occurrence of drug resistant organisms .

Highest sensitivity was observed for Amikacin (93.6%) followed by

Piperacillin – tazobactum (86.8%) and Ciprofloxacin ( 16.3%).This is in agreement with the study conducted by Sikarwar et al., who also reported highest susceptibility of Klebsiella to the aminoglycoside antibiotic

Amikacin(67). (Refer Table 8)

In our study , resistance to Cefotaxime and Ceftazidime was 37.2% and

16.8% respectively. This is lower than that of the study conducted by

Sarojamma et al., who has reported the resistance to Cefotaxime and

Ceftazidime to be 48% and 56%(68). A lower incidence indicates a need for screening for resistance as our routine.

Two antibiotics that are currently used for the treatment of Carbapenem resistant Klebsiella are Tigecycline and Colistin(69).Recent emergence of

Colistin resistance in Klebsiella has lead to a worsening of the situation of such cases(70).However in our study all the Carbapenem resistant isolates were sensitive to Colistin and Tigecycline .This is similar to the study by Alexander et al,who has reported 90% sensitivity to Colistin and Tigecycline(71) .Higher sensitivity to Colistin and Tigecycline in our study is because of the reduced usage of these drugs in a resource limited setting .

ANTIMICROBIAL RESISTANCE :

ESBL :

ESBL screening was done by using Cefotaxime and Ceftazidime antibiotic discs .Phenotypic confirmation was done using Cefotaxime and

Cefotaxime clavulanic acid Himedia antibiotic discs .135 isolates were found to be ESBL producers by screening method . CDT could detect only 115

(85.1%) and E strip method confirmed only 102(75.5%) to be ESBL producers .

The difference in the detection of ESBL production by various methods can be accounted to the lack of constant sensitivity of the phenotypic tests and the influence of environmental factors on resistance(72).

In our study , ESBL production was found in 135 out of 250 isolates which accounts for about 54%. The percentage of organisms producing ESBL in India ranges from 4 % to 83%(68) .This is in accordance with the study conducted by Krishnamurthy et al ., who has reported ESBL production in

Klebsiella to be 60.4%.

This points towards the need for routine screening of ESBL production.

Increase in ESBL production points to the irrational use of antibiotics which leads to development of resistance in micro organisms.

Amp C β lactamases:

Amp C detection was found by phenotypic methods in 3.6% of the total

Klebsiella isolates. This is slightly lower than the study conducted by

Veeraraghavan et al., who reported 5% of Amp C production in the isolates (48).

More number of isolates detected by screening methods than by confirmatory phenotypic methods can be attributed to the fact that the mechanism of resistance may be others such as porin loss or efflux pump formation(73). Loss of OmpK35 proteins and concurrent formation of Efflux pumps act in combination to pump out the antibiotics and prevent their action.

These mechanisms explain the mechanism of resistance in those isolates which were positive by screening methods but negative by confirmatory phenotypic methods .

DETECTION OF CARBAPENEMASES :

In our study ,isolates that were resistant to Meropenem were subjected to further phenotypic tests for detection of Carbapenemases .Carba NP test,

Modified Carbapenem Inactivation method and EDTA Modified Carbapenem

Inactivation method were the phenotypic tests performed in our study. Modified

Hodge test was not done as it has been removed from CLSI M 100 document in 2018.

Out of 48 ,41 (85.4%) isolates were positive by Carba NP test for

Carbapenemases .This is in agreement with the study conducted by Dutta et al

., who has reported the detection rate of Carbapenemases by Carba NP test to be 86.4%(74). This may be due to a lower sensitivity of the Carba NP test in the detection of Oxacillinases (OXA ).Carba NP test is a comparatively rapid phenotypic test which can yield results on resistance within 4 hours .

43(89.5%) of the isolates were found to be positive for MBL production by mCIM . Metallo β lactamase is the most common type of β lactamase enzyme causing resistance in India .Indian subcontinent is an epidemic region for MBLs of which NDM predominates over other Carbapenemases (75) .

Polymerase chain reaction detected the presence of 22 (45.8%) New

Delhi Metallo β lactamases-1 , 21 (43.7%) OXA -48 like and 5(10.4%) NDM-

1 and OXA -48 like co –producers .This is in accordance with the study conducted by Veeraraghavan et al., who also has detected 19% of NDM , 13% of OXA -48 like and 28% of NDM and OXA -48 like co – producers. Various studies reported the Indian subcontinent to be an important reservoir of the

NDM gene(76) .

New Delhi Metallo β lactamase is the prevalent gene (73%)(77)in a study conducted in North India . The NDM -1 producing bacteria are considered to be the most frequent cause of Urinary tract infections(78).This fact supports

75 our finding of more Carbapenem resistant isolates obtained from Urine samples in this study .They are also found to cause blood stream infections ,wound infections and pneumonia .

OXA -48 Carbapenemase producing clones has lead to a recent increase in Carbapenem resistance.(48) This supports the higher incidence of OXA -48 like Carbapenemases obtained in our study .NDM,OXA-48 like and co- producers of both are the common genes isolated in Carbapenem resistant isolates in a study conducted by Pragasam et al.,(36).

In India ,Carbapenem resistance is predominantly due to NDM and OXA

-48 like enzymes and KPC is hardly to be seen(48,36,79).In our study ,none of the isolates were detected with the presence of KPC,VIM or IMP genes.This is in agreement with the study conducted by Celikbilek et al., who has reported no isolates possessing KPC,VIM or IMP genes(80).

The data thus obtained by our study reiterates the importance of

Klebsiella as an invasive pathogen and is aimed at tracking their occurrence and designing means of reducing their impact .Hence it is high time we realise the threat posed by Carbapenem resistance in Klebsiella species and take measures in controlling the spread of these organisms. An Infection control committee plays a crucial role in monitoring and limiting the spread of infections in a tertiary care hospital. Formulation of an appropriate Antibiotic policy would

76 restrict the usage of antibiotics thereby preventing the spread of resistance.

Strict implementation of infection control practices and judicious use of antibiotics should therefore be adopted before it is out of time.

SUMMARY

This study was conducted at the Department of Microbiology at

Coimbatore Medical College and Hospital, Coimbatore from July

2018 to June 2019 .A total of 250 isolates of Klebsiella were obtained from various clinical samples .The antibiotic susceptibility and resistance pattern of the isolates were evaluated .

 250 Klebsiella isolates were taken up for study of which 202

were sensitive and 48 were found to be resistant to

Carbapenems .

 Among the 202 Carbapenem sensitive isolates, a higher

number of isolates were obtained from Urine samples

followed by from pus samples , sputum, throat swab ,blood ,

Endotracheal tube samples and from Bronchial wash samples.

 Klebsiella pneumoniae (70.8%) were the commonest species

isolated followed by Klebsiella oxytoca(29.2%) .

 Male preponderance was observed in the resistant Klebsiella

isolates .Male to female ratio in our study was observed to be

1.7:1.

 Proportionately higher resistance to Carbapenems was seen in

isolates obtained from patients aged above 50 years .

 Surgical wards and Intensive care units contributed to a

comparatively higher number of resistant isolates .Antibiotic

exposure before surgery ,Mechanical ventilation and Urinary

catheterization in patients were found to play a major role in

giving rise to infections by resistant organisms in Surgical

wards and ICU.

 Factors influencing the risk of acquiring resistant infections

were found to be Prolonged Hospitalisation (more than 7

days),Diabetes mellitus, Invasive surgical procedures ,

Indwelling medical devices, Chronic liver disease

/Alcoholism and ICU stay.

 Highest sensitivity was observed for the aminoglycoside

Amikacin (93.6%) and Piperacillin- tazobacum (83.6%).

 Among the 250 isolates ,187 (74.8%) were found to produce

β lactamases of various types.54% of the isolates were found

to produce ESBL and 4.4% of the isolates were detected with

Amp C β lactamase production.22.9% of the isolates were

found to be positive for Metallo β lactamases by various

phenotypic methods.

 41(85.4%) isolates were positive for Carbapenemase

production by Carba NP test ,43 (89.5%) isolates were

positive by mCIM and 16 (37.2%) isolates were positive for

serine carbapenemases by eCIM.

 PCR detected the presence of NDM-1 gene in 22 (45.8%)

isolates and OXA -48 like gene in 21(43.7%) isolates and

both NDM -1 and OXA -48 like genes in 5(10.4%) isolates .

 Strict Infection control measures and rational use of

antibiotics are the preliminary steps to be implemented at a

war fosting to prevent spread of Antimicrobial resistance .

CONCLUSION

Klebsiella species are one among the commonest Gram negative bacterium causing infections worldwide . Emergence of antimicrobial resistance in Klebsiella leads to life threating infections . Antimicrobial resistance can make previously curable infectious diseases become untreatable.Routine screening of organisms for Antimicrobial resistance in our laboratories may help us to give an early indication on the rational use of antibiotics .

Phenotypic methods are found to be less efficacious than genotypic methods in determining resistance .On the other hand , Genotypic methods have their own limitations like need for costly equipment ,reagents and trained experts which are not always available . Rapid phenotypic methods like Carba

NP test could be used as an alternative method for early reporting of resistance.

However , Molecular assays remain the Gold standard method.

Combination therapy of Colistin or Tigecycline with an aminoglycoside along with a Carbapenem can improve the outcome in patients with

Carbapenem resistance. Antimicrobial stewardship should be made compulsory to curtail the irrational use of antibiotics. Antibiotic policies have to be formulated and followed strictly to avoid further emergence of antimicrobial resistance in microorganisms. Infection control measures like Hand washing, good house keeping and proper disinfection has to be implemented so as to

81 prevent the spread of resistant organisms which in turn leads to a reduction in the incidence of Hospital acquired infections in tertiary care centers .

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ANNEXURES

Statement of consent

I, ______, do hereby volunteer and consent to participate in the study conducted by Dr.S.Sharon Dorothy , I have read and understood the consent form or it has been read and explained to me thoroughly. I am fully aware of the study details as well as aware that I may ask questions to her or withdraw from the study anytime. I am aware that my individual results will be kept confidential and also I consent to publish the analysed report of this study in journals.

ம쏁.毁.ஷாரன் டாரதி அவர்கள், ꏁண்迁யிரியல்鏁றை, ககாறவ ம쏁த்鏁வக்கல்쯂ரி, பரிகசாதறை நடத்த சம்மதம் அளிக்கிகைன். இந்த ஆய்வின் சசய்믁றை மற்쟁ம் இ鏁 சதாடர்பாை அறைத்鏁 விளக்கங்கறள뿁ம் ககட்翁க்சகாண்翁, எை鏁 சந்கதகங்கறள뿁ம் சதளிퟁப翁த்திக்சகாண்கடன் என்பறத뿁ம் சதரிவித்鏁க்சகாள்கிகைன். இந்த ஆய்வில் என் விவரங்கள் பா鏁காக்கப்ப翁வ鏁டன் இதன் 믁羿ퟁகள் ஆய்விதழில் சவளியிடப்ப翁வதில் ஆட்கசபறை இல்றை என்பறத뿁ம் சதரிவித்鏁க்சகாள்கிகைன்.

Signature/left thumb Signature/left thumb

Impression of the subject: Impression of the witness:

Station: Station:

Date: Date: PROFORMA

CASE HISTORY

Name: Age: Sex:

Address:

Occupation:

IP No:

Ward no:

Diagnosis:

Date of admission:

Date of discharge:

Complaints of: Fever : Continuous, intermittent, low grade, high grade Associated with Chills\sweating

Cough : Productive / non-productive , diurnal variation

Sputum : Colour : Purulent/non purulent Foul smelling Stained

Pus : Colour Discharge - watery, purulent, blood stained.

Present history:

Past history : H\O DM, HT, Anaemia, jaundice, Convulsions.

General Examination : H\O Ear discharge Conjunctivitis Trauma Anaemia GeneralisedLymphadenopathy Cyanosis, Clubbing, Icterus, Skin,Hair,Nail changes.

Systemic Examination: CVS: RS: Per abdomen: CNS:

Treatment given previously: Treatment given as per antibiogram:

Lab investigations: Specimen: Lab number: Date: APPENDIX

A) STAINS AND REAGENTS :

Gram staining :

 Methyl violet (2%) – 10 g of Methyl violet in 100ml of Absolute alcohol in 1 litre of Distilled water( Primary stain )  Grams Iodine – 10 g Iodine in 20g KI(fixative)  Acetone – Decolourising agent  1% Carbol fucshin – Counter stain

MEDIA USED :

MacConkey Agar medium Composition Ingredients (gms/ltr)

 Peptone -20 g  Lactose -10 g  NaCl 5 g  Na Deoxycholate 1.0  Neutral red 0.03  Agar 15.0 52 g of dehydrated MacConkey agar medium was suspended in 100ml of Distilled water and boiled to dissolve the medium completely .the solution was then sterilized by Autoclaving 121 C and 15 lbs pressure for 15 minutes . Blood agar medium ( 5% sheep blood agar )

Composition

Ingredients gram/liter  Peptone 10.00  Distilled water 1 ltr.  Sodium chloride 5.00  Agar 15.00

Forty grams of the dehydrated blood agar medium was suspended in 1000 ml cold distilled water in a flask and boiled to dissolve the medium completely. It was then sterilized by autoclaving at 1210C and 15 lbs pressure for 15 minutes. The autoclaved materials were allowed to cool to a temperature of 450C in a water bath. Defibrinated 5-10% sheep blood was then added to the medium aseptically and distributed to sterile petridishes. Sterile media was stored in refrigerator at 40C for future use.

Muller Hinton agar medium Composition Ingredients gram/ltr Beef dehytrated infusion 300 Casein hydrolysate 17.50 Starch agar 1.50 Agar 10.00

Thirty-eight grams of dehydrated Mueller Hinton agar medium was suspended in 1000 ml cold distilled water and boiled to dissolve the medium completely. The solution was then sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes. The autoclaved media was stored at 4°C.pH=7.4

MEDIA REQUIRED FOR BIOCHEMICAL IDENTIFICATION:

1. Catalase test: 3% hydrogen peroxide

Oxidase reagent

Composition Distilled water

Tetramethyl-P- phenylenedimine 0.1 g Indole test Composition Ingredients amount

Peptone 20g Sodium chloride 5g

Distilled water 1 L

After adjustment of the pH to 7.4 , sterilize by autoclaving at 1210C for 15 min.

Kovac.s reagent

Amyl or isoamyl alcohol 150ml p.Dimethyl-aminobenzaldehyde 10g Hydrochloric acid 50ml

Dissolve the aldehyde in the alcohol and slowly add the acid and store in the refrigerator.

Simmon’s Citrate Medium:

 Koser’s medium 1 ltr  Agar 20g  Bromothymol blue 0.2% 40ml  Dispense,Autoclave at 121°for 15 min and allow to set as slopes.

Triple Sugar Iron medium:

 Beef extract 3g  Yeast extract 3g  Peptone 20g  Glucose 1g  Lactose 10g  Sucrose 10g  Ferric citrate 0.3g  Sodium chloride 5g  Sodium thiosulphate 0.3g  Agar 12g  Phenol red 0.2% solution 12 ml  Distilled water 1 ltr

Heat to dissolve the solids,add the indicator solution,mix and tube .Sterilize at 121° for 15 min and cool to form slopes with deep butts.

Methyl Red test/Voges –Proskauer test:

A.MR/VP broth(Glucose broth/phosphate buffer broth)

Polypeptone 7g

Glucose 5g

Dipotassium phosphate 5g

Distilled water 1Ltr

Final pH 6.9

B.Reagents

1. ∝-Napthol,5%(5gm in 100ml of absolute ethyl alcohol)

2. Potassium hydroxide 40%(Potassium hydroxide in 100ml of Distilled water).

Decarboxylase media: Moller decarboxylase broth base: Ingredients gms/ml Peptone 5 Beef extract 5 Bromocresol purple 0.01 Cresol red 0.005 Glucose 0.5 Pyridoxal 0.005 Aminoacid Add 10g of the levo form of the aminoacid for 1000 ml.mix and dispense in sterile tubes.

Hugh-Leifson’s Oxidation-Fermentation test: Peptone 2g Sodium chloride 5g D-glucose 10g Bromothymol blue 0.03g Agar 3g Dipotassium phosphate 0.3g Distilled water 1ltr pH=7.1

Basal medium is autoclaved.1%of sterile sugar solutions is added to the basal medium.Dispense into the sterile test tubes without slant.

EDTA Solution:

0.5M EDTA solution was prepared by adding 186.1 gm of disodium EDTA in 1000 ml of distilled water.pH was 8.Sterilized by autoclaving at 121°C for 15 min.

McFarland Standard 0.5

Composition and preparation 1 % (V/V) solution of chemically pure (0.36N) Sulphuric acid and 1.175 % (W/V) solution of chemically pure (0.048M) barium chloride was prepared in two separate sterile flasks. Then 9.9 ml of sulphuric acid and o.1 ml of barium chloride were added to the clean screw capped test tube and sealed. The barium sulphate suspension corresponds approximately to McFarland standard tube No.1 with corresponding cell density of 3 × 108 organisms/ml. To made the turbidity standard of cell density to one half of the McFarland standard tube No.1 which correspond to cell density of 1.5 × 108 organism/ml for determination of antibiotic sensitivity by Kirby- Bauer inoculated technique 0.5 ml of 1.7 %(W/V) barium chloride (Bacl2 2H2O) was added to 99.5 ml of 1 % (V/V) Sulphuric acid (0.36N), mixed well and 5- 10 ml was distributed in sterile capped test tubes and sealed.

MASTER CHART

S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

1 Umamageshwari 40 F FM1/M1 URINE UTI Y <7 S S S R S S S S S S S S S POS NEG WOUND 2 Lakshmi 65 F OG III PUS Y >7 S S R S S S R S S S S INFECTION S S POS NEG 3 Mohammed niyas 5 mon MCH PAED URINE cystitis N >7 S S S S S S S S S S S S S POS NEG WOUND 4 Anitha 36 F OG PUS N >7 S R S S S S R S S R S INFECTION S S POS NEG 5 Kanimozhi 8 F BMW URINE UTI N >7 S R S R S S S S R S S S S POS NEG 6 Naveen kumar 8 M BMW URINE UTI N >7 S R S R S S S S R S S S S POS NEG 7 Lenin roshan 6 M PICU URINE UTI N >7 S S S R R S R S R S I S S NEG NEG 8 Bairavi 10 F PAED URINE UTI N >7 S S S S S S S R S S S S S POS NEG 9 Sundaravel 9 M PAED URINE UTI N >7 S S S R S S S R R R S S S POS NEG 10 Dhaksha 3 F BMW URINE UTI N >7 S R S S S S R S R S S S S POS NEG WOUND 11 Marathal 25 F OG PUS Y >7 S S S R R S S S R S S INFECTION S S NEG NEG WOUND 12 Thangamal 64 F SD pus Y >7 S S S S S S S S S S INFECTION S S POS NEG 13 Narayanasamy 52 M NEPH URINE UTI Y >7 S R S R S S R S S S I S S POS NEG WOUND 14 Shanthi 40 F FS2 pus Y >7 S S S R S S S S R R INFECTION S S S POS NEG WOUND 15 Lakshmi 37 F PLASTIC WOUND swab Y <7 S S S R S S S R R R DISCHARGE S S S POS NEG 16 Jermakishore 16 M MM2/M3 URINE UTI N <7 S R S R S S R S R S S S S POS NEG 17 Dineshkumar 39 M MM5/M4 URINE UTI Y <7 S S S S S S S S R S I S S POS NEG 18 Ganeshan 53 M MM4/M6 URINE UTI Y >7 S R S S R S S S S S S S S NEG NEG 19 Anguraj 66 M MM3/M1 URINE UTI Y >7 S S S S S S S S R R S S S POS NEG 20 Mahalingan 67 M MM4 URINE UTI Y >7 S S S S S S S S S S I S S POS NEG 21 Dhanushree 3 FCH BMW URINE UTI N >7 S S S S R S S R R S S S S NEG NEG 22 Gomathi 63 F FM2/M6 URINE UTI N >7 S S S R S S S S R R S S S POS NEG 23 Sona 8 FCH BMW URINE UTI N >7 S R S S S S S S R S I S S POS NEG 24 Paramasivam 60 M MM3/M5 URINE UTI Y >7 S S S S S S S S R S S S S POS NEG 25 Gokulsri 8 MCH PAED URINE UTI N >7 S S S S S S R S R S S S S POS NEG WOUND 26 Anitha 36 F OG PUS Y >7 S S S S S S S R S INFECTION R S S S POS NEG 27 Rabiya 60 F FM2/M6 URINE UTI Y >7 S R S S R S S S S S S S S NEG NEG

28 Ilangovan 53 M URO URINE pyelonephritis Y >7 S S S S S R S S S S R S S POS POS WOUND 29 Selval 49 F OG PUS Y >7 S S S S R S R S S S S INFECTION S S NEG NEG S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

WOUND 30 Ajbarnisha 52 F DERM PUS Y >7 S R S S R S S S S S S INFECTION S S NEG NEG 31 Balakrishnan 12 MCH PAED URINE UTI N >7 S S S R S S S R R R S S S POS NEG 32 chitraranjan 54 M NEPHRO URINE UTI Y <7 S S R S R S S S R S S S S NEG NEG WOUND 33 Shanthi 39 F FS2/S1 pus Y <7 S S S S S S R S S S INFECTION R S S POS NEG 34 Ramasamy 60 M imcu URINE UTI Y <7 S R S S R S S S S S S S S NEG NEG 35 Thirumalai 66 M URO URINE UTI Y 7 S S R S R S S S R S S S S NEG NEG 36 Ramakrishnan 47 M NEPHRO URINE UTI Y 7 R S S R R S S S S R S S S NEG NEG

37 Indrani 55 F GYNAEC PUS WOUND INFE Y 7 S R S S R S S S S S R S S NEG NEG 38 Ayeesha 10 FCH PICU blood SEPSIS N 7 S S S S R S R R S S S S S NEG NEG 39 Padmavathi 50 F OG URINE UTI Y 7 S S S S R S S S S S S S S NEG NEG 40 Karthika 21 F IMCU URINE UTI N 7 S R S R R S S S R S S S S NEG NEG 41 Shanthi 33 F URO URINE UTI N 7 S S R S R S S R R R S S S NEG NEG 42 Ponni 47 F SD URINE UTI Y 7 S S R S S S S S S S S S S POS NEG 43 Vedavarshini 15 F URO URINE UTI N 7 S S R R R S S S R S S S S NEG NEG 44 Rabideen 54 M SD URINE UTI Y 7 S R R S S S S S S S S S S POS NEG 45 Rangasamy 65 M SD URINE UTI Y 7 S S R S S S S S S S S S S POS NEG 46 b/o Lakshmi 9m MCH BMW URINE UTI N 7 S S R R S S S S R R S S S POS NEG 47 Shakunthala 55 F NEPHRO URINE UTI Y 7 S S R S S S S S R S S S S POS NEG 48 Kadar mohanthy 55 M SD URINE UTI Y 7 S R S S S S S S S S S S S POS NEG WOUND 49 Keerthana 19 F YZ pus N 7 S S R S S S S R S S S INFECTION S S POS NEG 50 Dhakshan 1yr MCH PAED URINE UTI N 7 S S S S R S S S R R S S S NEG NEG 51 Sankari 30 F OG URINE UTI N 7 S S S R S S R S R S S S S POS NEG IMPLANT 52 Anand muniappan 57 M ORTHO pus Y 7 S R S S S S S S S S S INFECTION S S POS NEG 53 Lakshmi 42 F OG URINE UTI Y 7 S S S S S S S S S S S S S POS NEG 54 BABU 47 M M6 URINE UTI Y 7 S R S S R S S S R S S S S NEG NEG 55 Latha 52 F ICCU URINE UTI Y 7 S S S S S S S R S S I S S POS NEG

56 Duraisamy 60 M NEPHRO URINE pyelonephritis Y 7 S S S S S S S S R S S S S POS NEG 57 Pandi 52 M NEPHRO URINE UTI Y 7 S R S S S S S S S S S S S POS NEG 58 Priya 19 F NEPHRO URINE UTI N 7 S S R S S S S S R S S S S POS NEG 59 meenatchi 6 FCH BMW URINE UTI N 7 R S R R S S S S S S S S S POS NEG 60 B/o saranya 35 d FCH PAED URINE UTI N 7 S R R S S S S R S R S S S POS NEG 61 Arnith 2 MCH PAED URINE UTI N 7 S S R S S S S S R S S S S POS NEG 62 Maran 40 M PLASTIC URINE UTI Y 7 S S R S S S S S R S S S S POS NEG S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

63 Jenifer 7 FCH BMW URINE UTI N 7 S R R R S R S S S S S S S POS NEG 64 MANIKANDAN 35 M UROLOGY URINE UTI Y 7 S S R S R S S S S S S S S NEG NEG 65 Nagarajan 68 M MM4/M6 URINE UTI Y 7 S S R S S S S R R S S S S POS NEG 66 Swatha 64 F OG URINE UTI Y 7 S R R S S S S S R S S S S POS NEG WOUND 67 Ramalingam 36 M MS1/S3 pus Y 7 S S R S S S S S S S S INFECTION S S POS NEG 68 Shanthi 60 F NEPHRO URINE cystitis Y 7 S R R S R S S S S R S S S NEG NEG

69 Subashini 21 F OG THROAT SWAB URTI N 7 S R S S S S S S R S S S S POS NEG 70 b/o maragenam 1m MCH PAED URINE cystitis N 7 S R S S S S R S R S S S S POS NEG 71 Chandrasekar 53 M UROLOGY URINE UTI Y 7 S R S S S S S S S R S S S POS NEG 72 Vijaya 50 M SD URINE UTI Y 7 S R S S R S S R S R S S S NEG NEG 73 MOORTHY 45 M MM3/M3 URINE UTI Y 7 S S S S S S S S R S S S S POS NEG 74 Ganesan 65 M MM2/M2 URINE UTI Y 7 S S S S S S S R R S S S S POS NEG 75 Anand raj 18 M MM4/M4 URINE UTI N 7 S R S S R S S S S S S S S NEG NEG 76 Prreja 25 F FM1/M2 URINE UTI N 7 S S S S R S S S R S S S S NEG NEG 77 Sumathi 73 F FM2/M4 URINE cystitis Y 7 S S S R S S S R S S S S S POS NEG 78 Soundarya 10 FVH PICU URINE UTI N 7 S R S R R S S S R S S S S NEG NEG 79 Gandhi 50 M IMCU URINE UTI Y 7 S S S S S S S S S R S S S POS NEG 80 b/o kamatchi 2m MCH PAED URINE UTI N 7 R S S S S S S S R S S S S POS NEG

81 Arunachalam 77 M SD URINE pyelonephritis Y 7 S R S S R S R S S S S S S NEG NEG 82 Manjula 55 F NEPHRO URINE UTI Y 7 S S S R S S S S S S S S S POS NEG 83 Philomina 70 F FM4/M6 URINE UTI Y 7 S S S S S S S R R S S S S POS NEG WOUND 84 Ragupathy 62 M SKIN pus Y 7 S R S S S S S S R R INFECTION S S S POS NEG WOUND 85 Rajendhran 65 M NEPHRO pus Y 7 S S S R R S S S S S S INFECTION S S NEG NEG

86 Palanisamy 54 M MS1/S2 PUS WOUND INFE Y 7 S S S S S S S S R S S S S POS NEG 87 Raji 63 M FM2/M4 URINE UTI Y 7 S R R S S S S R R S S S S POS NEG WOUND 88 Raja 11 MCH PAED SURG pus N 7 S S S R S S S S S S S INFECTION S S POS NEG WOUND 89 Rithesh 10 M PAED SURG pus N 7 R S R S R S S S S R INFECTION S S S NEG NEG 90 rajendhran 60 M NEPHRO URINE urosepsis Y 7 S S R R S S S R S S S S S POS NEG 91 Saraswathy 65 F SD BLOOD SEPSIS Y 7 S S R R S S S S R S S S S POS NEG 92 Saheena 22 F FM1/M2 URINE UTI N 7 S R R S S S S S S S S S S POS NEG 93 Jothi 36 F SD URINE UTI Y 7 S S R S S R S S S S S S S POS NEG 94 Yohith sharma 7 MCH PAED URINE UTI N 7 S R R S S S R S R R S S S POS NEG 95 Kumar 33 M IMCU URINE UTI N 7 S R R S R S S R S S S S S NEG NEG S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

96 rathnasamy 60 M MM4/M4 URINE UTI Y 7 S S S S R S S S S S S S S NEG NEG 97 Pal pandi 33 M ORTHO URINE UTI N 7 S S R S R S S S R S S S S NEG NEG

98 Renuka 27 F FS2 PUS WOUND INFE N 7 S R R S R S R R S S S S S NEG NEG 99 Rajammal 60 F IMCU URINE UTI Y 7 S R S S R S S S S S S S S NEG NEG 100 Rakkiappan 85 M MM1 URINE UTI Y 7 S S R S R S S S R S S S S NEG NEG 101 Govindharaj 43 M SD URINE UTI Y 7 S R S S S S S R S S S S S POS NEG 102 MOOrthy 45 M ORTHO URINE UTI Y 7 S R S S R S S R S S S S S NEG NEG

103 Vibin 3y MCH BMW THROAT SWAB URTI N 7 S R S S S S R S R S S S S POS NEG 104 ANADHAKUMAR 19 M MM4/M4 THROAT SWAB URTI N 7 S R R S S S S R S S S S S POS NEG

105 Palanathal 65 F FM3/M2 THROAT SWAB LRTI Y 7 S S S S S S S S S S S S S POS NEG

106 Thiruneshwaran 7 MCH BMW THROAT SWAB URTI N 7 S S R S S S S S S S S S S POS NEG PAED pus from 107 Akilesh 7 MCH wound swab N 7 S S S R S S S R R S S SURGERY wound S S POS NEG DISCHARGE 108 Rasathal 56 F FM4/M5 PUS FROM Y 7 R S S S R S R R S S S WOUND S S NEG NEG DISCHARGE 109 Sandhanapriya 3 yrs FCH BMW PUS FROM N 7 S S S S S S S S S S S WOUND S S POS NEG DISCHARGE 110 BAGATH 11m MCH BMW PUS FROM N 7 S R S R S S S S S S S WOUND S S POS NEG WOUND 111 Yuvaraj 29 M UROLOGY PUS N 7 S R S S R S S R S S S INFECTION S S NEG NEG DISCHARGE 112 Dhiyan Yadav 1 yr MCH BMW PUS FROM N 7 S S S R S S R S R S S WOUND S S POS NEG WOUND 113 Sajith Varma 3 MCH PAED PUS N 7 S S R S R S S S S S S INFECTION S S NEG NEG discharge 114 Moorthy 45 M MS2 /S5 wound swab Y 7 S R R S S S S R S S S from wound S S POS NEG DISCHARGE 115 Yasotha 62 F FM4/M5 PUS FROM Y 7 S S S R S S S S S S S WOUND S S POS NEG WOUND 116 manivannnan 73 M MS5/S1 PUS Y 7 S R S S R S S S R S S INFECTION S S NEG NEG DISCHARGE 117 Indira 23 F OG PUS FROM N 7 S S R S R S R R S S S WOUND S S NEG NEG WOUND 118 Alex 11 MCH PAED PUS N 7 S R S R S S S S S S S INFECTION S S POS NEG WOUND 119 Harikandan 11 MCH PAED PUS N 7 S S S S S S S S S S S INFECTION S S POS NEG S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

WOUND 120 Maheshwari 25 F OG PUS N 7 S S S S R S S S R S S INFECTION S S NEG NEG WOUND 121 Thulasi tauif 19 F Fs2 /s4 PUS N 7 S R S S S S S R S S S INFECTION S S POS NEG WOUND 122 Kannan 17 M Plastic PUS N 7 S S R R S S R S S S S INFECTION S S POS NEG WOUND 123 Kaupathaal 37 F Og PUS Y 7 S S R S R S S R S S S INFECTION S S NEG NEG WOUND 124 Kalaivani 23 F Fs2/s4 PUS Y 7 S S R S R S S S R S S INFECTION S S NEG NEG WOUND 125 Subbaiya gounder 72 M Thoracic med PUS Y 7 S R R S R S S S S S S INFECTION S S NEG NEG WOUND 126 Mohammed 21 M Ms4 PUS Y 7 S S R R S S S R S S S INFECTION S S POS NEG OSTEOMYELITI 127 Dharmalingam 52 M ortho PUS Y 7 S S S S S S S S S S S S S S POS NEG WOUND 128 Punitha 42 F Fs2/s4 PUS Y 7 S R S R S S S S R S S INFECTION S S POS NEG

129 Ramya 20 F Og Throat swab pharyngitis N 7 S R S S R S S R S S S S S NEG NEG OSTEOMYELITI 130 Ramachandran 31 M Ortho PUS Y 7 S S S S S S S S S S S S S S POS NEG DISCHARGE 131 Thangaraj 27 M Ortho PUS FROM Y 7 S S R S S S S S S S S WOUND S S POS NEG WOUND 132 Suriya 20 M Trauma PUS Y 7 S S R R S S R S S S S INFECTION S S POS NEG WOUND 133 Vimala 24 f F Fs2/s4 Pus Y 7 S S R S R S S R R S S INFECTION S S NEG NEG WOUND 134 Vimala 24 F Og Pus N 7 S R R S R S S S S S S INFECTION S S NEG NEG WOUND 135 PANDI 35 M FS1/S3 Pus Y 7 S S S S S S S S S S S INFECTION S S POS NEG WOUND 136 Dharmalingam 52 M Ortho Pus Y 7 S S S R S S S s S S S INFECTION S S POS NEG WOUND 137 Velusamy 42 M Plastic Pus Y 7 S S S S R S S S R S S INFECTION S S NEG NEG WOUND 138 Selvaraj 59 M ortho Pus Y 7 S S S S S S R S S S S INFECTION S S POS NEG WOUND 139 Thangamani 50 F Ortho Pus Y 7 S S S S R S S R S S S INFECTION S S NEG NEG WOUND 140 Sasikala 30 F Fs1/s4 Pus Y 7 S S S R S S R S S S S INFECTION S S POS NEG WOUND 141 Sathish 26 M Ortho Pus Y 7 S R S S R S S S R S S INFECTION S S NEG NEG 142 Krishnamoorthi 45 M Diabetology BLOOD SEPSIS Y 7 S S S S R S S R S S S S S NEG NEG WOUND 143 Kumaran 65 M Ms5/s2 Pus Y 7 S S S S R S S S S S S INFECTION S S NEG NEG S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

WOUND 144 Mathesswaran 25 M Thoracic med Pus N 7 S S S S R S S S S S S DISCHARGE S S NEG NEG WOUND 145 Santhamani 65 F Imcu Pus Y 7 S S S S S R R S R S S DISCHARGE S S POS NEG WOUND 146 Thangavel 52 M Ms2 /s6 Pus Y 7 S R S R S S S R S S S INFECTION S S POS NEG WOUND 147 Raj 27 M Ortho Pus Y 7 S S S S R S S S S S S DISCHARGE S S NEG NEG 148 Rajamani 32 F Burns s2 Pus SEPSIS Y 7 S S S S R S S S S S S S S NEG NEG WOUND 149 Sudha 37 F ortho wound swab Y 7 S S S R S S S R S S S INFECTION S S POS NEG WOUND 150 Daniel kumar 61 M Plastic Pus Y 7 S R S S S S S S S S S INFECTION S S POS NEG WOUND 151 Manoj 40 M S4 Pus Y 7 S S S S S S S S S S S INFECTION S S POS NEG WOUND 152 Palaniyammal 46 M Plastic Pus Y 7 S S S R S S S R S S S INFECTION S S POS NEG WOUND 153 Varatharaj 61 M Ms5/s1 Pus Y 7 S S S S S S R S S S S INFECTION S S POS NEG WOUND 154 Velusamy 65 M Ms5/s1 Pus Y 7 S R S R S S S S S S S INFECTION S S POS NEG

155 Alexo 62 M Thoracic med Sputum PNEUMONIA Y 7 S S S R S S S R S S S S S POS NEG 156 Santhi 22 F Og throat swab LRTI N 7 S S S S R S R S S S S S S NEG NEG 157 Sarathal 80 F Og icu Urine urosepsis Y 7 S S S S R S S S S S S S S NEG NEG

158 Karuppaamy 43 M Thoracic med sputum PNEUMONIA Y 7 S R S R S S S S S S S S S POS NEG 159 Baby 75 F Fm2/m6 Urine cystits Y 7 S S S R S S S R S S S S S POS NEG Neuromedicin 160 Rajan 69 M Urine UTI Y 7 S S S R R S R S S S S e S S NEG NEG 161 Nandhini 13 F Fm3/m2 Urine UTI N 7 S S S R S S S S S S S S S POS NEG

162 Suresh kumar 56 M Thoracic med sputum PNEUMONIA Y 7 S R S R S S S S S S S S S POS NEG wound 163 Glote 48 M Ms2/s5 wound swab Y 7 S S S R S S S S S S S discharge S S POS NEG 164 Amasai 71 M Sd Urine UTI Y 7 S S S R S S S S S S S S S POS NEG

165 Selvi 40 F Fs2/s4 PUS WOUND INFE Y 7 S S S R S S S S S S S S S POS NEG 166 Jarnel mohammed 78 M Thoracic sputum PNEUMONIA Y 7 S R S R S S S S S S S S S POS NEG 167 Baby 75 F Taei ward Urine UTI Y 7 S S S R R S R S S S S S S NEG NEG 168 Neelakandan 28 M Mm3/m5 sputum PNEUMONIA N 7 S S S R S S S R S S S S S POS NEG

169 kaliyappan 72 M Thoracic med sputum PNEUMONIA Y 7 S S S R R S S S S S S S S NEG NEG 170 Anandhi 19 F Sd Urine UTI N 7 S R S R S S R S S S S S S POS NEG

171 Saraswathi 50 F Fm4/m6 Urine renal calculi Y 7 S S S R S S S S S S S S S POS NEG S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

172 Rajadurai 21 M Thoracic med sputum PNEUMONIA N 7 S S S R S S S S S S S S S POS NEG

173 Palanisamy 70 M Ms2 PUS WOUND INFE Y 7 S S S R S S S S S S S S S POS NEG pus from 174 Karuppasamy 79 M Plastic Wound swab Y 7 S S S R R S S S S S S wound S S NEG NEG 175 Saranya mohan 21 F Gynaec sputum PNEUMONIA N 7 S R S R R S S S S S S S S NEG NEG

176 Backiyam 50 F Thoracic med throat swab PNEUMONIA Y 7 S S S R R S S S S S S S S NEG NEG 177 Vallaiathal 55 F IMCU Et tube c/s VAP Y 7 S S S R R S S S S S S S S NEG POS 178 Mithunya 1m fch Picu Et tube c/s VAP N 7 S R S R R S S S S S S S S NEG POS WOUND WOUND 179 Ganeshamoorthi 41 M urology Y 7 S S S R R S S S S S S SWAB DISCHARGE S S NEG POS 180 Subramani 45 m Ms2/s1 Urine UTI Y 7 S S S R S S S S S S S S S POS NEG 181 LidiyaR 25 f Og throat swab LRTI N 7 S S S R R S S S S S S S S NEG NEG 182 b/o divya manju 15 d Fch NICU Blood SEPSIS 7 S S S R R S S S S S S S S NEG NEG 183 durai 48 m MM3/M5 Urine UTI 7 S S S R S S S S S S S S S POS NEG 184 SULOCHANA 25 F LW/SD pus SSI 7 S S S R S R S S S S S S S POS POS WOUND 185 ANGATHAL 80 F FS2/S4 pus 7 S S S R S S S S S S S INFECTION S S POS NEG WOUND 186 NATESAN 70 M MM4/M6 pus 7 S S S R S S S S S S S INFECTION S S POS NEG WOUND 187 SUBRAMANI 70 M SICU pus 7 S S S R S S S S S S S INFECTION S S POS NEG 188 RAMYA 20 F OG pus SSI 7 S S S R S S S S S S S S S POS NEG 189 JARNEL MOHAMMED 78 M MM2/M3 Urine urosepsis 7 S S S R R S S S S S S S S NEG NEG THORACIC 190 KALIYAPPAN 72 M Sputum PNEUMONIA 7 S S S R R S S S S S S MED S S NEG NEG 191 ANNADURAI 55 M ORTHO Sputum PNEUMONIA 7 S S S R R S S S S S S S S NEG NEG 192 SUDHA 37 F ORTHO Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 193 VALLIYAMMAL 33 F OG Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 194 SUBRAMANI 36 M MS1/S2 Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 195 RAVICHANDRAN 46 M cts Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 196 GOPAL 65 M EYE Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 197 RAMASAMY 75 M MM3/M5 Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 198 MURUGESAN 47 M MS5/S1 Sputum PNEUMONIA 7 R S S R S S S S S S S S S POS NEG 199 RAMASAMY 60 M ORTHO Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 200 KIRUBA 44 F NS Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 201 KALEESWARI 23 F IMCU Sputum PNEUMONIA 7 S S S R S S S S S S S S S POS NEG 202 Sunil kumar 6 MCH PAED Sputum PNEUMONIA N 7 S R S R S S S S S S S S S NEG NEG 203 John 14 M MM1/M1 Urine UTI N <7 R R S R S S S S S R R S S NEG NEG POS POS POS NEG POS S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

204 Madasamy 19 M MM3/M2 Urine UTI N <7 S R S S S S S S S S R S S NEG NEG POS POS NEG NA POS 205 Arun kumar 26 M MM4/M1 Urine UTI N <7 S R S S S S S S S R R S S POS NEG POS POS POS NEG POS 206 Mani pandi 30 M ORTHO Pus SSI N >7 S R S S S S S S R S R S S NEG NEG POS POS POS NEG POS WOUND 207 Prabhu 32 M Burns s2 pus Y >7 S R S R S S S S R S R POS POS POS NEG POS INFECTION S S NEG NEG WOUND 208 Mani 36 M SURG Pus Y 7 S R S S S S S S S R R POS POS POS POS NEG POS INFECTION S S NEG WOUND 209 rajendhran 39 M Plastic throat swab Y <7 R R S S S S S S R S R POS POS POS NEG POS INFECTION S S NEG NEG 210 Sivamoorthy 34 M MM2/M1 Urine UTI Y <7 S R S S S R S S S S R S S NEG NEG NEG POS POS NEG POS WOUND 211 anupam prakash 35 M MS1 Pus Y >7 S R S S S S S S S R R POS POS POS NEG POS INFECTION S S NEG NEG 212 Nagaraj 32 M MM4/M4 Urine UTI Y <7 S R S S S S S S R S R S S POS NEG POS NEG POS POS POS 213 Kalimuthu 38 M MM2/M2 Urine UTI Y >7 S S S S S S S S S S R S S NEG NEG POS POS POS NEG POS WOUND 214 Vijay 33 M SURG Pus Y <7 R R S S S S S S S S R NEG POS POS NEG POS INFECTION S S NEG NEG 215 Chandran 42 M SURG Urine UTI Y <7 S R S S S S S S R R R S S NEG NEG POS POS NEG NA POS WOUND 216 Ramesh Y 46 M ORTHO Pus Y <7 S R S S S S S S S S R NEG POS POS NEG POS INFECTION S S NEG NEG bronchial 217 Mansoor ali 48 M ICU pneumonia Y <7 S R S S S S S S S S R POS POS POS POS NEG POS wash S S NEG WOUND 218 Arivu 43 M ORTHO Pus Y >7 R R S S S R S S S S R POS POS POS NEG POS INFECTION S S NEG POS WOUND 219 Madhappan 47 M MS2 Pus Y >7 S R S S S S S S R R R POS NEG POS POS POS INFECTION S S NEG NEG acute 220 Shanmugam 45 M MM3/M4 throat swab Y >7 S R S S S S R S S S R POS NEG POS POS POS POS pharyngitis S S NEG WOUND 221 Vinayagam 48 M SURG Pus Y >7 S R S S S S S S S S R POS POS POS NEG POS INFECTION S S NEG NEG WOUND 222 Muthu kumar 49 M ORTHO Pus Y >7 S R S S S S S S R R R POS POS POS NEG POS INFECTION S S NEG NEG 223 Dinesh 42 M ORTHO Urine UTI Y >7 R R S S S S S S S S R S S NEG NEG POS NEG POS POS POS WOUND 224 Velan 46 M ORTHO Pus Y >7 S R S S S S R S S S R POS NEG POS POS POS INFECTION S S NEG NEG 225 Mahesh 52 M ICU Urine UTI Y >7 S R S R S R S S R S R S S POS NEG NEG NEG POS POS POS WOUND 226 Sivakumar 53 M ORTHO Pus Y <7 S R S S S S S S S R R POS POS POS POS POS INFECTION S S NEG NEG 227 Ganesh 56 M ICU Urine UTI Y >7 R R S S S S R S S S R S S NEG NEG POS POS POS NEG POS

228 Subramaniam 55 M nephro Urine pyelonephritis Y >7 S R S S S S S S S S R POS POS POS NEG POS S S NEG NEG 229 Kaliappan 56 M ICU Urine UTI Y <7 S R S S S R S S R R R S S NEG NEG POS POS POS NEG POS 230 kuppusamy 75 M ICU throat swab sepsis Y >7 S R S S R S S S S S R S S NEG NEG POS POS POS NEG POS 231 Veerasamy 65 M surg Blood SEPSIS Y >7 R R S S S S S S R S R S S NEG POS NEG POS POS POS POS 232 Singaravelan 80 M ICU urine urosepsis Y >7 S R S S S S S S S S R S S NEG NEG POS POS POS NEG POS S.NO PATIENTS NAME AGE SEX WARD SAMPLES DIAGNOSIS CMD DOH AK G CIP CTX CPM CX NX CAZ COT P I T MRp T G L C O L ESBL AMPC MBL cARbanp mcim ecim P C R

233 Mani 53 M MS1 URINE UTI N <7 S S S S S S R S R R R S S NEG NEG NEG POS POS NEG POS WOUND 234 Karunakaran 65 M ORTHO Pus Y >7 S S S R R S S S R S R POS POS POS NEG POS INFECTION S S NEG NEG 235 Abdhul salam 78 M ICU Sputum PNEUMONIA Y >7 R S S S S S R S S S R S S NEG NEG NEG POS POS NEG POS 236 Karthikeyan 61 M SURGERY Sputum PNEUMONIA Y >7 S S S S S S S S S R R S S NEG NEG POS POS POS NEG POS 237 Viswanathan 52 M MS2 URINE UTI Y >7 S S S S S S S S S S R S S NEG NEG POS POS POS NEG POS 238 Marimuthu 73 M ICU Urine UTI Y >7 S S S S S S R S R S R S S NEG NEG POS POS POS POS POS 239 Palanisamy 69 M ICU Sputum PNEUMONIA Y <7 S S S S S S S S S R R S S NEG NEG POS NEG NEG NEG POS WOUND 240 Pandiyan 65 M ORTHO Pus Y >7 S S S S S S S S S S R POS POS POS NEG POS INFECTION S S NEG NEG

241 maheshwari 29 F OG Urine UTI N <7 S S S R R S S S S S R S NEG NEG POS POS POS POS POS

242 Eswari 38 F OG vaginal Swab discharge PV N >7 S S S S S S S S R R S S S NEG NEG POS POS POS POS POS

243 baby 46 F Nephro Urine UTI N >7 S S S S S S S S S S R S S NEG NEG POS NEG POS POS POS

244 Ramkumar 42 m SURG Urine UTI Y >7 S S S S R S S R S S R S S NEG NEG NEG POS NEG BEG POS WOUND 245 Poongodi 49 F Burns s pus N >7 S S S S S S S S S S R POS POS POS POS POS INFECTION S S NEG NEG 246 Katha 42 F FM6/M4 Sputum PNEUMONIA N >7 S S S S S S S S R S R S S NEG NEG POS POS POS POS POS WOUND 247 Ayyamal 55 F burns pus Y >7 S S S S R S S R S S R POS POS POS POS POS INFECTION S S NEG NEG 248 Palaniyammal 78 F ICU sputum PNEUMONIA Y >7 S S S S S S R S R S R S S NEG NEG NEG POS POS POS POS 249 Kadeeja 64 F ICU urine urosepsis Y >7 S S S S S S S S S S R S S NEG POS POS POS POS NEG POS DISCHARGE 250 Lakshmi 80 F SURG Pus FROM Y >7 S S S S R S S S R S R POS POS POS NEG POS S S NEG NEG WOUND