Bacteriological Profile and Antimicrobial Susceptibility Patterns of Bacteria Isolated from Pus/Wound Swab Samples from Children Attending a Tertiary Care Hospital in Kathmandu, Nepal

In Nepal, little is known about the microbiological profile of wound infections in children and their antimicrobial susceptibility patterns. Total of 450 pus/wound swab samples collected were cultured using standard microbiological techniques and the colonies grown were identified with the help of biochemical tests. The antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique. Methicillin-resistant Staphylococcus aureus isolates were detected by using cefoxitin disc and confirmed by determining minimum inhibitory concentrations (MIC) of oxacillin. 264 (59%) samples were culture positive. The highest incidence of bacterial infections was noted in the age group of less than 1 year (76%). Out of 264 growth positive samples, Gram-positive bacteria were isolated from 162 (61%) samples and Gram-negative bacteria were found in 102 (39%) samples. Staphylococcus aureus (99%) was the predominant Gram-positive bacteria isolated and Pseudomonas aeruginosa (44%) was predominant Gram-negative bacteria. About 19% of S. aureus isolates were found to be methicillin-resistant MIC of oxacillin ranging from 4 μg/mL to 128 μg/mL. Among the children of Nepal, those of age less than 1 year were at higher risk of wound infections by bacteria. Staphylococcus aureus followed by Pseudomonas aeruginosa were the most common bacteria causing wound infections in children.


Introduction
Injuries among children are major public health problems caused by falls, burns, and road traffic accidents [1]. These various injuries result in development of wounds which drag the patients to the health care facilities. Bacterial infections are serious problems to the successful treatment of the wounds resulting in complications sometimes leading to fatal sepsis. Over 95% of all injury-associated deaths among children occur in low-income and middle-income countries [2].
The common bacterial pathogens responsible for wound infections are Staphylococcus aureus, Pseudomonas aeruginosa, and bacteria belonging to family Enterobacteriaceae [3].
Since the emergence of methicillin-resistant Staphylococcus aureus (MRSA) in 1960, there have been reports of increasing rate of infection by MRSA and this superbug has established itself as the common cause of nosocomial as well as community acquired infections [4]. Further, many reports have shown the increasing rates of infections in children by MRSA [5]. In Nepal, many studies have been conducted to 2 International Journal of Microbiology determine the rates of infections caused by MRSA among adults [6], but only limited data are available regarding the prevalence of MRSA among the strains of S. aureus isolated from children. Further, from literature review, we concluded that no studies have been conducted to determine the bacteriological profile of wound infections among children and their antimicrobial susceptibility patterns. Children have weak immune system, because of that they are prone to serious infections and timely proper treatment is very necessary. In addition, if the local antimicrobial susceptibility data are not available, the chances of haphazard use of antibiotics will be high. As a result, the rate of drug resistance will increase causing a serious problem. So, in this study, we determined the bacteriological profile of wound infections among children and their antimicrobial susceptibility patterns. Further, we also studied the incidence of methicillin-resistant Staphylococcus aureus in causing wound infections among children.

Study Design.
A cross-sectional study was conducted using total of 450 pus/wound swab samples collected from the children attending Kanti Children's Hospital, Kathmandu, Nepal, from September 2012 to May 2013. The samples were collected from the patients who were admitted to the hospital after surgeries or major injuries and had signs of wound infections. The wounds of the injured patients were cleaned with antiseptic solutions before admitting them to the hospital. So, the patients might have got infections in hospital as the signs of infections were seen after the patients had been admitted.

Isolation, Identification, and Antimicrobial Susceptibility
Testing. The samples were subjected to bacteriological culture following standard microbiological techniques [7]. The colonies grown were identified with the help of colony morphology, Gram's staining, and biochemical tests [8]. The antimicrobial susceptibility testing was performed by modified Kirby-Bauer disc diffusion technique following clinical and laboratory standards institute guidelines [9].

Detection of MRSA and Determination of Minimum Inhibitory Concentrations of Oxacillin for Strains of MRSA.
MRSA isolates were detected by using cefoxitin disc (30 g) [9]. Minimum inhibitory concentrations of oxacillin for strains of MRSA (screened by cefoxitin disc diffusion method) were determined by agar dilution method [9, 10].

Data
Analysis. The data were analyzed by using statistical package for the social sciences version 16.00. value < 0.05 was considered as statistically significant.

Minimum Inhibitory Concentrations of Oxacillin for MRSA Screened by Cefoxitin Disc Diffusion Method.
All the 31 MRSA isolates screened by cefoxitin disc diffusion method had minimum inhibitory concentrations of oxacillin ≥ 4 g/mL. The MIC values ranged from 4 g/mL to 128 g/mL.

Discussion
Wound infection is one of the most common and serious complications among the hospital acquired infections [11][12][13]. Wound infection can increase the length of hospital stay and accounts for the mortality rate up to 70-80% [12,13]. The growth positivity reported by Bhatta and Lakhey (60%) was similar to our finding [14]. However, lower rate was reported by Shrestha and Basnet (50%) [15].
The result obtained showed the slightly decreasing trend in rates of wound infections with increase in age. And highest rate of wound infection was among the children below 1 year of age. Our finding was in accordance with the findings by Kai-Yang et al. [16] andÖnen et al. [17]. Due to the weak immune system of the younger children, they are more susceptible to infections.
As in our study, Bhatta and Lakhey [14], Shrestha and Basnet [15], and Garba et al. [18] also reported the S. aureus to be the most prevalent bacteria isolated from the cases of Table 1: Results of biochemical tests of different bacteria isolated in our study.
Yellow colony on mannitol salt agar, Gram-positive cocci, catalase positive, slide and tube coagulase positive, gelatin hydrolysis positive, beta-hemolysis in blood agar, methyl red test and Voges-Proskauer test positive, nitrate reduction positive
Gram-negative bacilli, oxidase negative, catalase positive, lactose fermenting, indole negative, motile, urease differential, hydrogen sulphide positive, gas positive, citrate positive C. freundii Gram-negative bacilli, oxidase negative, catalase positive, lactose non-fermenting, indole positive, motile, urease differential, hydrogen sulphide negative, gas positive, citrate positive C. koseri Gram-negative bacilli, oxidase negative, catalase positive, lactose non-fermenting, indole negative, motile, urease positive, hydrogen sulphide positive, gas positive, citrate positive P. mirabilis  wound infections. In hospital the sources of S. aureus may be the inanimate objects, health care workers, and other patients. Further, due to presence of the S. aureus as normal flora of human body, the endogenous infections are also possible. The relatively higher resistance of the bacteria isolated from the children to the commonly used antibiotics is a matter of great concern. The prevalence of MRSA reported in our study was in accordance with that reported by Subedi and Brahmadathan (15.4%) [19]. However, higher rates were reported by Kshetry [22]. In a recent study from Nepal, Adhikari et al. also reported higher rate of MRSA in comparison to our study [23]. The

Limitations of the Study
Because of resource constraints, we were unable to use molecular level analysis to confirm our results. Further, multicenter study including larger numbers of samples would have generated more significant results.

Conclusions
Among the children of Nepal, those of age less than 1 year were at higher risk of wound infections by bacteria and there was a decreasing trend in rates of wound infections with increase in age. Staphylococcus aureus followed by Pseudomonas aeruginosa were the most common bacteria causing wound infections in children, which are also the predominant pathogens causing wound infections in adults.

Ethical Approval
Before commencing of the study, the research protocol was approved by Kanti Children's Hospital, Kathmandu, Nepal, and Goldengate International College, Kathmandu, Nepal. The research was in compliance with the Helsinki Declaration.

Consent
Informed consent was obtained from all the patient's guardians.