Antibiotics Sensitivity Pattern of Post-Operative Wound Infections in a Tertiary Care Hospital, Western Nepal

Background: Surgical site infections (SSIs) is one of the most common postoperative complications and cause signicant postoperative morbidity, mortality, prolong hospital stay and increase in hospital cost. The condition is serious in developing countries like Nepal owing to irrational prescriptions of antimicrobial agent. SSIs in those countries rates from 2.5% to 41.9%. This study was performed to nd the common organisms causing surgical site infections and their antibiotics sensitivity pattern in a tertiary care hospital, western Nepal. Materials and methods: Pus or swab samples collected from suspected post- operative wound infections and submitted for culture and sensitivity in the Department of Microbiology were included in this study. Isolation and identication of the organisms was done as recommended by American society of microbiology (ASM). Antibiotic susceptibility test was performed by Kirby Bauer disc diffusion method as recommended by Clinical Laboratory Standard Institute (CLSI) guideline. Results: Out of 152 pus and swab samples processed for culture, (64.5%) showed culture positivity. In total isolates (65.7%) were Gram negative bacteria and (34.3%) Gram positive bacteria. Staphylococcus aureus (23.9%) was the predominant Gram positive isolate and Escherichia coli (18%) was the major Gram negative isolate. S. aureus showed (100%) sensitivity towards Linezolid and (94.4%) towards Vancomycin. Among commonly used antibiotics for Gram positive bacteria Penicillin (94.4%), Erythromycin (80.5%) were highly resistant. Sixty percent of Staphylococcus aureus isolated showed methicillin resistant (MRSA). Gram negative bacteria showed (100%) sensitivity towards the Colistin sulphate and Polymyxin B and were highly resistant towards Ampicillin (98.2%), Cefexime (87.3%), Ceftriaxone (87.3%) and other commonly used antibiotics. Overall multi-drug resistance was found in (89.5%) isolates. Among Gram negative bacterial isolates (23.1%) were MBL producer and (21.7%) were ESBL producer. Conclusion: Culture positivity


Background
Surgical site infections (SSIs) is one of the most common postoperative complications and cause signi cant postoperative morbidity, mortality, prolong hospital stay and increase in hospital cost [1]. The United state centre's for disease control and prevention (CDC) has developed criteria that de ne surgical site infections as infections related to an operative procedure that occur at or near the surgical incision within 30 days or within 1 year if prosthetic material is implanted at surgery. SSIs are a real problem to the surgeons and are considered as major infection control concern across the world [2].
Unrestrained and rapidly spreading anti-microbial resistance among bacterial population become a serious challenge in the management and treatment of patients with post operative wound infections.
Most post operative wound infections are hospital acquired and vary from one hospital to the other hospital, one surgeon to another surgeons, one patient to another patient and are associated with complication of increased morbidity and mortality [3,4]. The emergence of antimicrobial resistance has made the choice of empirical therapy more di cult and expensive [5]. The condition is serious in developing countries due to irrational prescriptions of antimicrobial agent [6]. SSIs in those countries rates from 2.5-41.9% [7]. This study was conducted to assess the current status of bacterial pathogen The methods for the collection, isolation, and identi cation were performed as described by American Society of Microbiology (ASM) and analyzed accordingly [8]. 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 identi cation.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 ful lling the criteria of ASM was also excluded from the study.

Identi cation of Bacterial Isolates
Identi cation 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 [9] .

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 con rmation of ESBL-producing strains [10].

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 [10].
Combination Disk (CD) Method CD methods were used for the con rmation 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 con rmed ESBL [10]. E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as negative controls, respectively.

Screening test
The isolates were subjected for MBL detection when the ZOI for CAZ (30 µg) was < 18 mm [11].

MBL con rmation 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 [10].
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 [10] 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 [10]. 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), cipro oxacin (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 [12]. In this study, if the isolates were resistant to at least three classes of rst-line antimicrobial agents, they were regarded as MDR (multidrug resistance) [13].

Data Processing and analysis
All the data from cases were entered in MS Excel (Microsoft o ce 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 signi cant.

Ethical Consideration
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.

Results
A total of 152 specimens representing surgical site infection from different wards were processed and signi cant bacterial growth was found in 99 (64.5%) specimens. Among 99 specimens with signi cant growth, mixed bacterial growth was observed in 5 (5%) samples and monobacterial growth was found in 94 (95%) samples. Total number of bacteria isolated was 105. Out of 105 bacteria isolated from 99 samples, 69 (65.7%) were Gram negative and 36 (34.3%) were Gram positive bacterial isolates.

Discussion
Out of 152 samples 99 (65.1%) showed culture positivity which is in accordance with the study done by khyati J et al. which showed a culture positivity of (62%) samples [14]. The study done by chaudhary et al. showed culture positivity of (77.6% ) which was higher than our study and another study done by Bastola et al. showed (48.6%) culture positivity which was lower than our study [15,16]. Contamination from the external environment and poor hospital hygiene may be the possible reason for higher rate of surgical site infection [17].
In our study, (95%) of culture positive samples reveled mono-microbial growth and (5%) showed polymicrobial growth. Similarly high percentage (91.6%) of mono-microbial growth was reported by Mama M et al. in the year 2014 [18]. Similarly Acharya J et al. showed (10.5%) mixed bacterial growth [19].In total isolated organisms growth of Gram negative bacteria was (65.7%) which was more than growth of Gram positive bacteria (34.3%), It was similar with study done by Dhakal et al. in the year 2017 [20]. But in contrast Chaudhary et al. showed Gram positive bacteria were more common than Gram negative bactaeria [15]. This variation could be due to the diversity in study population, site of surgery and hospital environment [21].
Although Gram negative bacteria were more than Gram positive bacteria, common causative agent of SSIs in this study was Staphylococcus aureus (23.9%) which was in accordance with the study done by Dhakal et al., Chaudhary et al. and Sawadekar et al. [20,15,22]. E.coli was the most common Gram negative bacteria followed by Acinetobacter species and Klebsiella pneumoniae in our study. Similar study done by Nishanthy M. and Chitralekha et al. showed Klebsiella species was the most common Gram negative bacteria followed by Pseudomonas aeruginosa, Escherichia coli, Proteus species and Acinetobacter [23].The high prevalence of S. aureus infection may be because it is an endogenous source of infections. Infections with this organism may also be due to contamination from the environment e.g.
contamination of surgical instruments. With the disruption of natural skin barrier S. aureus, which is a common bacterium on surfaces, easily nd their way into wounds .Gram negative bacteria were also encountered in high percentage this may be due to contamination of surgical wounds with normal ora of gut [24].
Resistance to the selected antimicrobials was very high. The overall multiple drug resistance of the isolates in this study was (89.5%) in total and (86.9%) among GPC and (91.7%) among GNB, which was in line with study done by Mulu W et al. and Mama M et al [25,18]. But the study done by Pirvanescu H et al. encountered only (27.6%) MDR [26]. High resistance of the isolates to antibiotics may be due to practicing self medication or unavailability of guideline regarding the selection of drugs thereby which lead to inappropriate use of antibiotics [6].  [20].Nepal is a developing country there is no speci c rules for the purchase and use of antibiotics. People can use antibiotics without prescription. Irrational use of antibiotics is a major cause for drug resistant. Our study included indoor patients who get infection with already drug resistant bacteria from hospital source this may be the reason why most routinely used antibiotics were resistant [30,31,32].

Conclusion
In our study (65.1%) samples Showed culture positivity. Staphylococcus aureus was the common causative agent of SSIs. Bacteria showed more than 50% resistance towards commonly used antibiotic like Cefexime, Ceftriaxone, Cipro oxacin which is matter of concern. MDR rate was very high (89.5%) and we encountered (56%) MRSA, (23.7%) MBL and (21.3%) ESBL producer organisms. It is necessary for every medical practitioner to have better knowledge about causative agent of SSIs and their sensitivity pattern to control the wound infection and to minimize the rate of mortality and morbidity due to SSIs. A proper control of antibiotic usage will prevent the emergence of resistant strains of bacteria. Proper sterilization of surgical wards and surgical equipment we can reduce the rate of SSIs.

Recommendation
As Rate of isolation of organism causing SSIs is high, Samples should be collected from every suspected cases to nd out the causative agent. Detection rate of MDR is high so AST should be done regularly for the selection of appropriate antibiotic in the treatment. To minimize SSIs proper sterilization of equipments and wards should be done time to time. Pattern of MDR, MRSA, ESBL and MBL in isolated organisms