Introduction: Wound infection is the one of the most important causes of morbidity and mortality worldwide and antibiotics resistant bacteria are the great part of complications on treatment of the infection.

Objectives: The present study was conducted to isolate and identify the etiological agents of wound infection and assess the antimicrobial susceptibility pattern of the isolates.

Methods: The study was carried out on wound infection suspected patients visiting B and B Hospital for six months duration. The collected pus specimen were first observed macroscopically then streaked on MacConkey agar and blood agar, incubated at 370C for 24 hours. The isolated bacteria were identified by macroscopic and microscopic observations and biochemical reactions. Antibiotic susceptibility pattern of the isolates was assessed by Modified Kirby Baur disc diffusion technique.

Results: A total of 503 pus samples were collected; of which 43.7% showed bacterial growth. Out of a total 220 bacterial isolates; 158 (71.82%) were Gram negative and 62 (28.18%) were Gram positive bacteria. Pseudomonas spp. (34.55%) was most common followed by Staphylococcus aureus (21.36%), Escherichia coli (11.82%), Acinetobacter baumannii (11.36%), Enterobacter aerogens (8.18%), Coagulase negative Staphylococci (CoNS) (5.45%), Klebsiella pneumoniae (4.55%), Citrobacter freundii (1.36%), Streptococcus spp. (0.91%) and Enterococcus spp. (0.46%). Most of Gram negative bacteria (63.9%) and Gram positive bacteria (93.5%) were susceptible to amikacin and chloramphenicol respectively. Among 220 isolates, 138 (62.73%) were found to be multi drug resistant (MDR). All the isolates of A. baumannii were isolated from in-patients and almost 96.4% (24/25) were MDR.

Conclusion: In this study, significant number of MDR strains was found as the causative agent of wound infection. So, routine microbiological analysis of the wound specimen and their antibiotic susceptibility testing are recommended that will guide medical practitioners for empirical treatment of wound infection, so as to reduce the spread of resistant bacteria.

Keywords: wound infection, antibiotic susceptibility, MDR

The exposure of subcutaneous tissue following a loss of skin integrity (i.e. a wound) provides a moist warm and nutritive environment that is conducive to microbial colonization and proliferation. The progression of a wound to an infected state is likely to involve a multitude of microbial and host factors.¹ Wound can be infected by a variety of microorganisms ranging from bacteria to fungi and parasites as well as virus.² The most common organisms are S. aureus, P. aeruginosa, E. coli, Klebsiella spp. and Acinetobacter spp.^3,4

Antibiotics, although, have been of great value in treatment and in prophylaxis to prevent infections, the timing of administration, choice of antimicrobial agent, durations of administration have clearly defined the value of antibiotics in reducing wound infections.⁵ Advance in control of infection have not completely eradicated the problem of the wound infection because of development of drug resistance.⁶

Wound infection results in sepsis, limb loss, long hospital stays, higher costs and is responsible for significant human mortality and morbidity worldwide.⁷ It is one of the most common hospital acquired infections.⁸ It remains an ongoing problem which although, cannot be completely eradicated however, by taking prompt control measures against the most commonly isolated organism and proper care of wound may lead to the minimum of wound infection.¹ Hence, the present study was carried out to identify the causative agent of wound infection and antibiotic susceptibility pattern of the isolates, which will be beneficial as guidance for medical practitioners to select empirical antimicrobial therapy and on the implementation of infection control measures that plays an important role in minimizing the emergence rate of antimicrobial resistance (MDR).

A cross-sectional study was carried out in a total of 503 aseptically collected pus samples from wound infection suspected patients visiting B and B hospital, Lalitpur from October 2013 to April 2014. The pus specimen were macroscopically analyzed for their color and odor then were streaked on MacConkey agar and Blood agar plates and incubated at 370C for 24 to 48 hours. All the isolates were identified by colony morphology, staining reaction and the biochemical properties.⁹ The antimicrobial susceptibility test of isolates was performed by Modified Kirby Bauer disc diffusion method using the standard guidelines of the¹⁰ and MDR strain was identified as resistant to ≥3 antimicrobial classes.¹¹ The computer data were studied using the statistical software SPSS version 17. Chi-square test was used to calculate probabilities and determine significance. A p-value of less than or equal to 0.05 was considered to be statistically significant p<0.05).

Out of 503 pus samples from wound infection suspected patients, 220 (43.7%) showed bacterial growth whereas 283 (56.3%) were growth negative (Table 1). Out of 302 samples from in-patients, 160 (53.0%) samples and among 201 samples from out patients, 60 (29.9%) samples showed bacterial growth. The higher percentage of culture positivity seen among in-patients was found to be statistically significant (P<0.001) (Table 2).

Among 370 (73.56%) male patients and 133 (26.44%) female patients, 172 (46.50%) and 48 (36.1%) were found growth positive respectively. The result was statistically significant (P=0.038) (Table 3). Among 220 positive cases the highest positive cases 67 (30.45%) was found in the patients of age group 21-30 years (Table 4).

Among 220 bacterial isolates, Gram negative bacteria were predominant with 158 (71.82%) isolates, while Gram positive bacteria contributed 62 (28.18%) of total isolates. Altogether 10 different bacterial species were isolated, among which Pseudomonas spp. (34.55%) were predominant followed by S. aureus (21.36%) (Table 5). Out of 220 isolates, most of the isolates were isolated from in-patient (72.73%) than out-patient (27.27%). All the A. baumannii were from in-patients (Table 6).

Vancomycin was used only for the isolates resistant to cloxacillin. Most of the Gram positive bacterial isolates (more than 90%) were found to be sensitive to chloramphenicol. Penicillin G was ineffective (100%) to all the Gram positive isolates followed by amoxycillin (Table 7). Most of the Gram negative bacterial isolates (more than 48%) were found to be sensitive to amikacin, gentamicin and cefoperazone/ sulbactum whereas 73.4% of them were resistant to ciprofloxacin (Table 8).

Out of 47 isolates of S. aureus, 95.7% showed sensitivity towards chloramphenical, clindamycin, and cloxacillin. Penicillin G was ineffective (100%), followed by amoxycillin (89.4%). Among the S. aureus isolates, two isolates were found to be methicillin resistant S. aureus (MRSA), sensitive to vancomycin (Table 9).

The effective antibiotics against Pseudomonas spp. were amikacin with (68.4%) sensitivity and gentamicin with (63.2%) respectively. The most ineffective antibiotic was ceftazidime (94.7%) (Table 10).

The most effective antibiotics against A. baumannii isolates was colistin with 100% sensitivity, the opposite was ceftriaxone with 100% respectively. Out of the isolates, more than 92% were resistant to ciprofloxacin, ofloxacin, gentamicin, cefoperazone/ sulbactum and piperacillin/ tazobactam (Table 11).

Out of 220 bacterial isolates, 138 were found to be the MDR isolates. The higher MDR pattern was found in Gram negative isolates 106 (48.18%) than in Gram positive isolates 32 (14.55%). The highest percentage (96%) of MDR pattern was shown by A. baumannii followed by K. pneumoniae and the least (21.92%) by E. coli (Table 12).

Among the 160 isolates from in-patients, 96 (60%) were MDR strains and out of 60 out-patient, 42 (70%) were found to be MDR strains, were statistically insignificant (P=0.172) (Table 13). Out of 172 isolates from male 103 (59.88%) were MDR strains and among 48 isolates from female patients, 35 (72.92%) were MDR strains, were statistically insignificant (P=0.99) (Table 14).

Out of 503 samples, 220 (43.7%) showed bacterial growth. In similar studies by Maharjan.¹² showed similar result 50.95% growth but by Puyal.¹³ showed 71.84% growth. The lesser percentage of growth positive cases may be due to the collection of samples from patients taking antibiotics.

The occurrence of higher number of male patients 370 (73.56%) than female patients for the collection of pus samples may be due to the higher involvement of males in physical outdoor works for earning livelihood as compared to females and more chances of accidents during the activities. The higher growth positive cases in male patients (78.18%) than in female (21.82%) was observed in this study and was supported by the similar studies carried out by KC et al.⁷ & Shrestha.¹⁴

The higher number of bacterial growth was observed in in-patients 160 (72.73%) than in out-patients 60 (27.27%). Similarly, Acharya¹⁵ & Bhattrai¹⁶ also reported the higher number of growth positive cases in in-patients. This may be due to the factors associated with acquisition of nosocomial pathogens in in-patients and immunologically weak health status facilitates the wound infection in them.

The highest positive cases 30.45% (67/226) were observed in the age group of 21-30 years might be due to the active participation of this age group of people in different physical and mechanical works and during they may get injured.

Among the total 220 bacterial isolates, 71.82% were Gram negative and 28.18% were Gram positive bacteria. In similar study conducted by Yakha et al.¹⁷ & Acharya¹⁵ Gram negative bacteria were found predominant. Isolation of Gram negative bacteria, during this study was higher, as they are more prevalent aerobes and facultative anaerobes in abscesses and skin wound, these bacteria have well recognized property for abscess formation in open as well as in visceral infection, which increases their incidence in both open and closed types of wound. Isolation of Gram negative bacteria also increases in the cases of Hospital acquired infections (HAIs). A study conducted by Banjara¹⁸ at TUTH showed the high rate of Gram negative bacteria in HAI.

Altogether, 10 different bacterial species were isolated with Pseudomonas spp. being the predominant one (34.55%) followed by S. aureus (21.36%), E. coli (11.82%), A. baumannii (11.36%), E. aerogens (8.18%) Coagulase negative Staphylococci (CoNS) (5.45%), K. pneumoniae (4.55%), C. freundii (1.36%), Streptococcus spp (0.91%) and Enterococcus spp (0.46%). Similar studies carried out by Hani and Adnan¹⁹ & Ranjan et al.²⁰ also showed Pseudomonas spp. was the most prevalent bacteria among the total cases with 27.8% and 29.6% respectively. But, the study conducted by Zafar et al.²¹ showed Pseudomonas spp. the second commonest bacteria with 18.35% and S. aureus as the most predominant isolates with 41.28% of the total cases. Pseudomonas spp was predominant (36.25%) among in-patients followed by A. baumannii (15.63%) but S. aureus was the most prevalent (40%) among out-patients. All the isolates of A. baumannii isolated from in-patients.

Regarding antibiotic susceptibility testing, chloramphenicol (93.5%) and amikacin (63.9%) were the most effective antibiotics, in general to the Gram positive and Gram negative bacteria respectively. Among 47 S. aureus, two were found to be methicillin resistant (MRSA). 89.4% of S. aureus were resistant to amoxycillin. The resistance of S. aureus to β- lactam antibiotics is due to the production of penicillinas that hydrolyzed the β-lactam ring. MRSA resistance to β-lactam antibiotics is conferred primarily by the production of a bacterial cell wall penicillin binding protein, PBP2a. This has low binding to β-lactams which allows the organism to survive and multiply. PBP2a production is mecA gene encode.⁹ 51.06% (24/47) S. aureus isolates were MDR. The studies by Puyal,¹³ Acharya¹⁵ and Bhattarai¹⁶ support this result.

In present study, out of 76 Pseudomonas spp. isolates, 52 (68.42%) were found to be MDR isolates. Colistin was absolutely effective antibiotic against Pseudomonas spp. followed by imipenem (91.67%), amikacin (68.4%) and gentamicin (63.2%) respectively. ceftazidime was found to be the most ineffective (94.7%) antibiotics. Similar study by Acharya¹⁵ found 62.16% MDR among Pseudomonas spp. isolates and amikacin was found to be 84.61% effective antibiotics. The study by Hani and Adnan¹⁹ showed amikacin and gentamicin effectiveness, 78% and 72% respectively against Pseudomonas spp. But the study carried out by Falagas et al.²² isolated the organism from ICU patients which was almost resistant to amikacin, gentamicin and other anti-pseudomonal antibiotics. The study conducted by Li et al.²³ showed that active efflux played role in the resistance to various non-β-lactam agents by Pseudomonas spp. strains and de-energization by the addition of a proton conductor increased the accumulation level to that expected for equilibration across the cytoplasmic membrane.

 In this study, A. baumannii constituted 25 (15.82%) of the Gram negative isolates and 11.82% among the total bacterial isolates. Among the isolated A. baumannii 24 (96%) were found to be MDR strains. In the study conducted by Kelper et al.,²⁴ out of 237 samples 38 (16.03%) were found to be Acinetobacter spp. out of which 29 (76.31%) isolates were found to be MDR. Almost all the antibiotics used in this study were found to be resistant to the organism. However, the imipenem and meropenem were effective 24% and 12% respectively against the A. baumannii. MDR A. baumannii is an important nosocomial pathogen. It has the capacity to survive in dry environments, which increases the risk for nosocomial transmission. A. baumannii can cause infection in any organ system, including bacteremia, pneumonia, endocarditis, meningitis, urinary tract infection, intra-abdominal abscess, osteomyelitis, soft tissue infection, surgical site infections.²⁵

The prevalence of MDR isolates was found independent of gender and types of patients. Imipenem was found to be the most effective antibiotic for Gram negative bacteria from in-patients. P. aeruginosa and S. aureus were found as the most frequent etiological agents from pus samples. The prevalence of MDR was found to be 62.7% (138/220) among the total bacterial isolates. All the A. baumannii were isolated from in-patients and almost 96% (24/25) were found to be MDR. The result obtained from this study will be helpful for policy makers in evaluating the infection control measures in hospitals. The routine antimicrobial susceptibility testing before antibiotic administration is highly recommended since it can play key role to limit the wound infections to minimum as the emergence of drug resistant bacteria is less likely when there is empirical drug therapy, fasten the process of wound healing hence the appropriate use of drug decreases the cost of wound infection treatment.

There is no conflict of interest.

None.

None.

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