This observational and cross-sectional study was conducted at Department of Microbiology in collaboration with Department of Surgery at Universal College of Medical Sciences (UCMS), over a period of six months, March to September 2019. A total of 152 pus or swab samples collected from patients suspected of surgical site infection were studied.
The methods for the collection, isolation, and identification were performed as described by American Society of Microbiology (ASM) and analyzed accordingly . Collected pus or swab samples from suspected infected site were inoculated on Blood agar (BA) and MacConkey agar (MAC) (HiMedia) plates. The plates were incubated at 37 °C for 24 hr. All isolated colonies growing in the BA and MAC agar were processed further for identification.Patients of all age groups with suspected post operative SSIs admitted in different wards with their written consent were enrolled for the study.Patients were excluded from the study if they had wound infection other than postoperative wound, Infection occurring 30 days after operation if there is no implant and after 90 days if implant is in place, Burn injuries and donor sites of its skin grafts. The specimen not fulfilling the criteria of ASM was also excluded from the study.
Identification of Bacterial Isolates
Identification of the isolates were done by the following standard microbiological techniques which involved morphological appearance of the colonies, Gram’s staining reactions, catalase test, oxidase test, and other biochemical properties, for example, Sulphide Indole Motility (SIM) media, Simmons citrate media, Christensen’s urea agar, Triple Sugar Iron agar (TSI), Decarboxylase test media, Hugh and Leifson’s OF (oxidative and fermentative) test media, MR/VP (methyl red/Voges Proskauer) broth, Phenylalanine agar, Nitrate reduction test, and others as required  .
Phenotype Detection for ESBL
The initial screening test for the production of ESBL was performed by using ceftazidime (CAZ) (30 µg) and cefotaxime (CTX) (30 µg) disks (Hi.Media India.). If the zone of inhibition (ZOI) was ≤ 22 mm for ceftazidime and ≤ 27 mm for cefotaxime, the isolate was considered as a potential ESBL producer. The organism was swabbed on to a MHA (Mueller-Hinton agar) plate as done for the screening test in the antibiotic sensitivity test. Then, the combination disk method (CD) was applied for the confirmation of ESBL-producing strains .
Double disk synergy test
Amoxycillin- clavulanic acid (AMC) disk (20/10 µg) was placed at the center and disks containing the 30 µg of CAZ, CTX and CRO were placed separately beside 15 mm distance (edge to edge), away from the central disk, in a horizontal manner. Any enhancement of the zone of inhibition between the disks (either of the cephalosporin disks and clavulanic acid containing disk) indicated the presence of ESBL. Isolates with such pattern were recorded as ESBL producers .
Combination Disk (CD) Method
CD methods were used for the confirmation of ESBL-producing strains in which CAZ and CTX alone and in combination with clavulanic acid (CA) (10 µg) were used. An increased ZOI of ≥ 5 mm for either antimicrobial agent in combination with CA versus its zone when tested alone confirmed ESBL . E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as negative controls, respectively.
Tests for MBL Production
The isolates were subjected for MBL detection when the ZOI for CAZ (30 µg) was < 18 mm .
MBL confirmation by combination disk (CD) method
Two imipenem (IPM) disks (10 µg) were used. In one of them, 10 µL of 0.1 mol/L (292 µg) anhydrous ethylenediaminetetraacetic acid (EDTA) was added. Then the two disks were placed 25 mm apart (center to center). An increase in zone diameter of > 4 mm around the IPM-EDTA disk compared to that of the IPM disk alone was considered positive for an MBL .
Tests for MRSA
30 µg of cefoxitin disk method as recommended by CLSI was put up and agar plates were incubated at 35˚C. The diameter of the zone of inhibition of growth were recorded and interpreted as susceptible or resistant by the criteria of CLSI. S aureus strains ATCC 25923 and ATCC 43300 were used as negative and positive controls respectively. Organisms were considered methicillin resistant when the zone of inhibition was equal or less than 21 mm for S. aureus with cefoxitin disk method 
Antibiotic Susceptibility Testing
The antimicrobial susceptibility tests were performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar (HiMedia, India) as per CLSI recommendations . The antibiotics tested in this study include amoxicillin (10 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefoxitin (30 µg), cefepime (30 µg), aztreonam (30 µg), amoxicillin-clavulanate (30 µg), piperacillin-tazobactam (100/10 µg), gentamicin (10 µg), imipenem (10 µg), ciprofloxacin (5 µg), and cotrimoxazole (25 µg), respectively. All the antibiotics used were purchased from HiMedia Laboratories, Mumbai, India. Interpretation of antibiotic susceptibility results was made according to standard interpretative zone diameters suggested in CLSI guidelines . In this study, if the isolates were resistant to at least three classes of first-line antimicrobial agents, they were regarded as MDR (multidrug resistance) .
Data Processing and analysis
All the data from cases were entered in MS Excel (Microsoft office 2007) and then analyzed by statistical package for social sciences (SPSS) for window version; SPSS 20 Inc, Chicago IL ) All the data were expressed in the term of percentage frequency, mean ± SD and compared by Chi-square test. P-value < 0.05 was considered to be statistically significant.
Study was approved by Institutional Review committee of Universal College of Medical Sciences, UCMS. Written informed consent was obtained from each individual participating in the study.