Study site and population
A descriptive cross-sectional study was designed and carried out to determine the bacteriological profile of wound infections. MRSA, MDR and ESBL producing bacteria were identified from the pus samples of patients with wound infection visiting KIST Medical College and Teaching Hospital, Kathmandu, Nepal from November 2014 to August 2015. A total of 182 pus and Fine Needle Aspirate specimens were collected from patients with clinical features of wound infection like patients with pain, complaints of regular discharge, foul smelling and red swelling. During the study, patients of all age groups and both genders from out-patients (39/182) and in-patients (143/182) were included. Patients who were admitted in the hospital for more than 3 days and/or in prior antibiotic treatment and anaerobic wound infections were excluded from this study.
Pus specimens were collected from elective surgery wounds of hospital wards [surgical, post- operative, trauma, orthopedic, ENT (eye-nose-throat), gynecology wards], open and dressed wounds. Sterile cotton swabs and fine needle syringes (FNS) were used to collect pus samples from open wounds then each sample was labeled properly with date/time of sample collection, collection method and the patient’s details. Swabs from open wounds were aseptically collected after cleaned off while pus from dressed wounds were collected after removing the dressing items. The information of each patient was recorded such as site of infection, signs and symptoms, other underlying diseases, and prior antibiotics administration. Before collecting the sample, the area was rinsed with sterile normal saline and then a sterile cotton swab was gently rolled over the surface of the wound. The swab with pus was kept in a sterile test tube with cap where details was labeled properly. For the collection of pus sample from deep wounds, FNS was used. Specimens were collected from wounds of different body parts: leg, hand, back part of body, abdominal part, foot region, breast and chest part, head and neck region. Amies transport medium was used to transport the collected specimens. For Fine Needle Aspiration Cytology (FNAC), the syringe was properly capped, labeled and dispatched to the laboratory immediately.
Processing of samples
Macroscopic examination of samples
Among 182 pus specimens collected, 56 (30.8%) were from the leg region, 43 (23.6%) from hand, 15 (8.2%) from back part of body, 14 (7.8%) from abdominal part, 15 (8.2%) from foot region, 6 (3.3%) from breast and chest part, and 33 (18.1%) were from head and neck region wounds. All the specimens were visually examined for consistency, color, turbidity, presence or absence of blood depending upon the type and site of wound. Additionally, pus swabs were observed whether they were labeled correctly or not.
Microscopic examination of samples
After transportation of specimens to the laboratory, Gram staining of each specimens was performed .
Culture of specimens and identification of isolated bacteria
Pus specimens were inoculated into Chocolate agar, Blood agar, MacConkey agar, Nutrient agar and Potato Dextrose agar plates as per the clinical laboratory guidelines . The preliminary identification of the isolated bacteria was done based on colony form, size, shape, pigmentation, margin, and elevation. The isolated organisms were identified by performing different biochemical tests and Gram staining then antimicrobial susceptibility tests were performed. In case of no growth after 24 h of incubation further incubation was done up to 48 h at 37 ̊C. After proper incubation period, the culture plates were examined for microbial growth. In every case, each plate was carefully observed. Then, biochemical tests were performed in sterile media for the identification of bacterial isolates. Identification of Staphylococci spp. was done by Gram staining, catalase test, slide coagulase and tube coagulase test. Similarly, Gram negative strains were identified based on result of different biochemical tests; Oxidase, Catalase, Methyl Red (MR), Voges Proskauer (VP), Citrate utilization, Urea Hydrolysis, Triple Sugar Iron agar (TSI), Sulfide Motility and Indole test. Colony morphology and microscopic observation were taken in account for identification of Candida spp.
Examination of antimicrobial susceptibility pattern of isolated organism
Antimicrobial susceptibility pattern was performed for isolated and identified bacteria from pus samples following the modified Kirby Bauer disc diffusion technique. A dilution of the identified organism was prepared comparing with the standard 0.5 McFarland turbidity which was used to swab over the Mueller Hinton agar (MHA) medium for the antimicrobial susceptibility test (AST). Discs of antibiotic used for Gram positive bacteria were ampicillin (10 μg), cefotaxime (30 μg), gentamycin (10 μg), ciprofloxacin (5 μg), trimethoprim + sulfamethoxazole (25 μg), cefoxitin (30 μg), amikacin (30 μg) and tetracycline (30 μg) whereas antibiotics used for Gram negative organisms were ampicillin (10 μg), trimethoprim + sulfamethoxazole (25 μg), gentamycin (10 μg), ciprofloxacin (5 μg), cefazolin (30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), amikacin (30 μg), piperacillin (100 μg), tobramycin (10 μg), imipenem (10 μg), and meropenem (10 μg). After 24 h of incubation period at 37 ̊C, the zone of inhibition (ZOI) was measured then the results were analyzed according to the guidelines issued by the Clinical Laboratory Standard Institute (CLSI - M100-S25, 2015) . Isolates resistant to two or more antimicrobial classes were reported as multi drug resistant (MDR) strains. Antimicrobials and their doses were selected based on prescription frequency by physician and availability in the study setting. Minimum inhibitory and bactericidal concentration (MIC and MBC) of used antimicrobials were not determined due to unavailability of all antimicrobials powder at the time of study period.
Screening and confirmation for ESBL producers
Enterobacteriaceae isolates were screened for possible ESBL producing bacteria using antibiotic discs of cefotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg) and aztreonam (30 μg) . According to the guidelines, bacterial isolates showing ceftazidime < 22 mm, and cefotaxime < 27 mm are the possible ESBL producer. The suspected ESBL producer strains were subjected to double disc synergy test (DDST) for the confirmation of ESBL producing Enterobacteriaceae .
All data were examined using iBM SPSS version 21.0. Frequencies were calculated for categorical variables. Chi-square test was calculated to analyze significant difference at 95% of confidence level, p value of < 0.05 was considered significant, unless otherwise noted.
All prepared biochemical and streaking media were checked for their sterility. Strains of E. coli ATCC 25922 and S. aureus ATCC 25923 were used as reference strains for quality control of AST and biochemical tests. The same strain of E. coli was also considered as a negative control during the screening and phenotypic confirmation (DDST) tests of ESBL producing Gram-negative bacilli.