A STUDY ON ANTIBIOTIC SUSCEPTIBILITY PATTERN OF ESCHERICHIA COLI ISOLATES FROM EXTRA INTESTINAL INFECTIONS IN A TERTIARY CARE HOSPITAL.

plasmids, they also contain other antimicrobial resistant genes.AmpC production in E.coli is through plasmids and mutation in their porin structure. Carbapenems are the drug of choice for ESBL producing Ecoli but recent time development of resistance is increasingly reported due to production of Carbapenemase.The aim of this study is to test the Antimicrobial susceptibility pattern of Extra-intestinal Ecoli isolates. The study was conducted in the department of Microbiology, Stanley Medical College, Chennai during the period October 2018 to May 2019.The institutional ethical committee approval was obtained and clinical samples such as urine, blood, pus, sputum and sterile body fluids were received from 983 patients suspected of bacterial infections. The samples were processed and biochemical test identified 84 Ecoli Isolates. and carbapenemase production were tested. E.coli isolates showed resistance to most of the beta lactam antibiotics such as Ampicillin, CefotaximeandCeftazidime and also to Ciprofloxacin &Cotrimoxazole, The Picture shows the detection of ESBL by Double disc test using Cefotaxime/ Cefotaxime+ Clavulanicacid and Ceftazidime/ Ceftazidime + clavulanic acid. There is an increase in zone diameter of inhibition of more than 5mm The picture shows growth of ESBLs producing magenta coloured EIPEC colonies in high chrome agar. All the 31 ESBL isolates detected by double disk test were found be positive in Hi-chrome agar.

782 nosocomial and community acquired infections 1 .It carries multiple virulence factors that enable to invade and colonize the extra intestinal sites 2 .
The increasing trend of developing antibiotic resistance in ExPEC is of global treat causing morbidity and mortality 3 .As there is no vaccination forExPEC, it is necessary to analyze the antibiotic susceptibility pattern for empirical treatment in emergency situations 4 .The mechanism of development of resistance is hydrolysis of antibiotics by Beta-lactamases enzymes produced by bacteria 5 . Extended spectrum beta lactamases (ESBLs) hydrolyze β-lactam antibiotics of third generation Cephalosporins, Penicillins and Monobactams. Since the ESBL enzyme genes are usually found in large plasmids, they also contain other antimicrobial resistant genes. Thereforemost ESBL producing organisms are also resistant to Aminoglycosides, Fluoroquinolones, chloramphenicol, and sulfonamides and are multi-drug resistant 5 . ESBL E.coli resistant to Cephamyxin is classified as AmpC beta lactamases belong to class C Ambler classification and group1 by Bush-Jacob"s classification 5 . AmpC production in E.coli is through plasmids and mutation in their porin structure 5 .Carbapenems are the drug of choice for ESBL producingEcolibut recent time development of resistance is increasingly reported due to production of Carbapenemase 6 . The genes encoding for carbapenem production commonly detected are KPC, NDM, OXA, VIM, and IMP 6 .
The aim of this study is to test theAntimicrobial susceptibility pattern of Extra-intestinal Ecoli isolates. This study was conducted in the department of Microbiology, Stanley Medical College, Chennai, during the period October 2018 to May 2019.The institutional ethical committee approval was obtained and clinical samples such as urine, blood, pus, sputum and sterile body fluids were received from 983 patients suspected of bacterial infection.

Materials and Methods:-
The study was conducted in the department of Microbiology, Stanley Medical College, Chennaiduring the period October 2018 to May 2019 .The institutional ethical committee approval was obtained and clinical samples such as urine, blood, pus, sputum and sterile body fluids were received from 983 patients suspected of bacterial infection. Processing of the sample including gram staining, motility testing andinoculating into Mac-Conkey agar plate, Blood agar plate and CLED in case of urine and incubating at 37 O C for 18 to 24 hours were done. The culture showed positivity in 362 samples. Lactose fermenting, Motile, Gram negative bacilli, Catalase positive, oxidase negative, Indole test positive, citrate negative, urease negative, TSI: A/A with gas and absent H2S, Fermenting glucose, lactose, mannitol, maltose with gas and not fermenting sucrose, Methyl Red positive, VogesPrausker test negative were identified as Escherichia coli. This included 84Ecoli Isolates and Antimicrobial testing were done modified Kirby-Bauer disk diffusion method as a lawn culture on Mueller-Hinton agar the following drugs:-Ampicillin 10µg, Gentamicin 10µg, Amikacin 30μg, Amoxicillin-clavulanate 20/10 μg, Piperacillin-tazobactam 100/10μg, Cefotaxime 30μg, Ceftazidime 30μg, Cefazolin 30μg (urinary isolates), Aztreonam 30μg, Ciprofloxacin 5μg, Levofloxacin 5μg, Trimethoprim-sulfamethoxazole 1.25/23.75 μg, Fosfomycin 200 μg (urinary isolates) and Nitrofurantoin 300 μg (urinary isolates), Turbidity was compared to a 0.5 MacFarland"s Turbidity. The control was prepared by using E coli ATCC 25922 strains and incubated at 37°C for 18 hours. Inner diameter of the zone of inhibition was measured by using a millimeter scale around each antimicrobial disk was interpreted as sensitive, intermediate or resistant according to the CLSI guidelines of 2018.

ESBL screening
The isolates that were resistant to Ceftazidime (30μg) and Cefotaxime ( 30μg) by disc diffusion method with zones of inhibition of ≤ 22mm for Ceftazidime and ≤ 27mm for Cefotaxime based on the CLSI 2018 guideline were considered as suspected ESBL producers.

Phenotypic Confirmatory Methods for Extended-Spectrum β-Lactamases:
Double disk diffusion method was used to confirm ESBL production by E.colistrains. The test organisms inoculated by lawn culture onto Mueller Hinton agar plate.Ceftazidime (30μg) disc vs. Ceftazidime (30μg)/ Clavulanic acid (30/10μg) disc and Cefotaxime disc vsCefotaxime/ clavulanic acid (30/10μg) disc were placed at least 20 mm apart, and incubated at 37°C for 18 hours. E.coli isolates demonstrating an increase in zone diameters of more than 5 mm either with Ceftazidime / clavulanic or Cefotaximeclavulanic-acid were ESBL producers.

Screening for Amp C beta lactamases:
The isolates were screened for AMPC production by testing their susceptibility to Cefoxitin (30μg) by Kirby Bauer disk diffusion method. All the isolates with an inhibition zone diameter < 18 mm were labeled as Amp C positive.

Confirmatory test for Amp C beta lactamases
Double disk diffusion method using Cefoxitin (30μg) and Cefoxitin (30μg) + Cloxacillin (200μg) combination were placed at a distance of 20mm on a Muller Hinton agar plate inoculated with test isolates. A zone diameter ≥ 4mm around the Cefoxitin+ Cloxacillin than the zone diameter around the Cefoxitin disc alone were considered as AmpC producers 5

Test for Carbapenemase production
The isolates resistant to Carbapenem by disc diffusion method were screened for the production of Carbapenemase. The phenotypic detection of the Carbapenemase production was performed using mCIM and eCIM as per CLSI 2018 guidelines.

Modified Carbapenem inactivation Methods (mCIM)
For each isolate 1-μl loopful of pathogen was emulsified and vortexed for 10-15 sec. To this 10-μg Meropenem disk was added to each tube with a sterile forceps. The entire disk is immersed in the suspension. MRP disk was removed from each trypticase soy broth tube and placed on E. coli ATCC 25922 inoculated MHA and incubated.

EDTA Modified Carbapenem inactivation Methods
The same procedure is repeated after adding 20μl of the 0.5 M, EDTA to the 2-mL Trypticase soy broth tube. The MRP disks from the mCIM and eCIM tubes are placed on the same MHA plate, plated with the Meropenemsusceptible E.coliATCC.
Out of the total 84ExPECisolates, 38 were from urine sample, 29 from pus, 6 from sputum and 11 from blood.

Confirmatory test for ESBL by Double disc test
The Picture shows the detection of ESBL by Double disc test using Cefotaxime/ Cefotaxime+ Clavulanicacid and Ceftazidime/ Ceftazidime + clavulanic acid. There is an increase in zone diameter of inhibition of more than 5mm

ESBL detection by chrome agar
The picture shows growth of ESBLs producing magenta coloured EIPEC colonies in high chrome agar. All the 31 ESBL isolates detected by double disk test were found be positive in Hi-chrome agar.

Confirmatory test for Amp C beta lactamases
The picture shows AmpC confirmatory test using Cefoxitin and Cefoxitin+ Cloxacillin.There is an increase in zone diameter of inhibition of more than 4mm.

Conclusion:-
The early detection ofresistant strains of ExPECplays a vital role in reduction of morbidity and mortality. Judicious use of antibiotics and a good antibiotic policy are needed to limit the emergence and spread of antibiotic resistance in bacteria. The appropriate selection of antibiotics for the treatment depends on the early and prompt antibiotic sensitivity test. Empirical treatment of antibiotics prior to antibiotic sensitivity report should be avoided unless indicated..Good hand hygiene practices, Health education of healthcare personnel, professionals, contact precautions and minimal use of interventional devices would prevent spread of nosocomial resistant strains.