High Burden of Antimicrobial Resistance among Bacteria Causing Pyogenic Wound Infections at a Tertiary Care Hospital in Kathmandu, Nepal

Pyogenic wound infections are one of the most common clinical entities caused and aggravated by the invasion of pathogenic organisms. Prompt and aggressive antimicrobial therapy is needed to reduce the burden and complications associated with these infections. In this study, we intended to investigate the common pathogens and their antimicrobial susceptibility patterns from the pyogenic wound infections at a tertiary care hospital in Kathmandu, Nepal. A laboratory based cross-sectional study was carried out among the pyogenic clinical specimens of the patients visiting Manmohan Memorial Teaching Hospital, Kathmandu, Nepal. Processing of clinical specimens and isolation and identification of bacterial pathogens were carried out using standard microbiological methods. Antimicrobial susceptibilities and resistant profiles were determined by following the standard guidelines of Clinical and Laboratory Standards Institute (CLSI). About 65% of the clinical specimens were positive for the bacterial growth and Gram positive bacteria (57.4%) were the leading pathogens among pyogenic wound infections. Staphylococcus aureus (412, 49.28%), Escherichia coli (136, 16.27%), Klebsiella spp. (88, 10.53%), and Pseudomonas spp. (44, 5.26%) were the common pathogens isolated. High level of drug resistance was observed among both Gram positive bacteria (51.9%) and Gram negative bacteria (48.7%). Gram positive isolates were resistant to ampicillin, ciprofloxacin, cotrimoxazole, erythromycin, and cloxacillin. Gram negative isolates were resistant to cephalosporins but were well susceptible to amikacin and imipenem. Pyogenic wound infections are common in our hospital and majority of them were associated with multidrug resistant bacteria. The detailed workup of the prevalent pathogens present in infected wounds and their resistance pattern is clearly pertinent to choosing the adequate treatment.


Background
Infections of the skin and soft tissue due to either trauma, surgery, or burns may result in the generation of exudates composed of dead leucocytes, cellular debris, and necrotic tissues [1]. Pyogenic or pus forming wound infections are characterized by severe local inflammation subsequent to tissue injury leading to generalized clinical disease through the various toxic mechanisms associated with invasion of pyogenic bacteria. Some of the common etiological agents responsible for causing pyogenic infections are bacteria such as Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella spp., Proteus spp., Pseudomonas spp., and Acinetobacter spp. [2,3]. The profusion and diversity of organisms are principally influenced by predisposing conditions, anatomic location of infection including its type, quality, and level of tissue perfusion, and antimicrobial efficacy of the host response [4].
Pyogenic wound infections are significant subgroup of infections encountered by infectious disease physicians in the hospitals worldwide. These infections are associated with higher morbidity and therefore antimicrobial regimens are generally recommended to reduce the burden as well as to prevent associated long term complications [5]. Moreover, surgical drainage is also required in severe closed type wound infections [6]. Despite the advancements in diagnostic techniques, treatment of pyogenic infections in the developing countries is challenging due to the emergence of multidrug resistant (MDR) pathogens. In particular, a large number of methicillin resistant Staphylococcus aureus along with the multidrug resistant Gram negative isolates are increasingly allied with pyogenic infections in recent years [7]. The crisis of antibiotic resistance among pyogenic bacterial infections has been attributed to the inappropriate use of antimicrobial agents particularly in developing country [8].
The antimicrobial resistance has become a global challenge and the resistant pathogen poses a grave threat to the public health worldwide. Different studies are being conducted across the globe to access the bacterial profile in pyogenic wound infection [3]. However, in Nepal, the studies are not consistent enough to reveal the information regarding pyogenic pathogens including their antibiotic susceptibility pattern [9,10]. The appropriate knowledge of the pathogens, their resistant character, and their updated antimicrobial therapy plays a crucial role in the treatment process as well as in infection control measures. Therefore, this study was intended to characterize the bacterial isolates from clinical specimens of pyogenic wound infections and to determine the antibiotic susceptibilities to commonly used therapeutic regimens at a tertiary care hospital of Kathmandu.

Study Design and Samples.
A laboratory based crosssectional study was carried out at the Department of Clinical Microbiology of Manmohan Memorial Teaching Hospital, Kathmandu, Nepal, from April 2016 to March 2017 (over the period of one year). Clinical specimens such as pus, wound aspirate, wound swab, necrotic tissue, and surgical drainage were collected aseptically from suspected patients with pyogenic wound infections and processed in the microbiology laboratory with minimal delay. However, the specimens not fulfilling the criteria of American Society for Microbiology (ASM) [11] and duplicate specimens from same patients were excluded from this study. During the study period, a total of 1,198 specimens representing the pyogenic infections were processed.

Laboratory Methods.
Each aseptically collected specimen was inoculated onto the Blood Agar (BA), Chocolate Agar (CA), and MacConkey Agar (MAC) plates (HiMedia Laboratories, India) by surface streaking method. BA and MAC plates were incubated in aerobic atmosphere and CA plates were incubated in additional 5-10% CO 2 at 37 ∘ C for 24-48 hours. Identification of significant isolates associated with pyogenic infections was carried out following standard microbiological techniques including morphological appearance of the colonies: Gram's staining, catalase test, coagulase test, and oxidase test with other biochemical parameters [11]. Assurance of pure culture inoculum was done by setting purity plate along with the biochemical tests.

Identification of Multidrug Resistant (MDR) Isolates.
Multidrug resistant (MDR) bacterial isolates were identified according to the criteria recommended by international expert committee of the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) [13]. In this study, the isolate resistant to at least one antimicrobial from three different groups of first-line drugs tested was regarded as multidrug resistant (MDR).

Phenotypic Test for Methicillin Resistant (MRSA) and
Inducible Clindamycin Resistant ( ) Staphylococcus aureus. Methicillin resistant Staphylococcus aureus (MRSA) isolates were detected by cefoxitin disk (30 g) method of CLSI. S. aureus isolates were judged as methicillin resistant when the ZOI for cefoxitin was ≤21 mm [12]. Similarly, inducible macrolide-lincosamide streptogramin-B (iMLS B ) resistance was detected in S. aureus by disk approximation using clindamycin (2 g) and erythromycin (15 g) on MHA plates. After overnight incubation, isolates with flattened zone of inhibition adjacent to the erythromycin disk (referred to as a "D" zone) were considered to exhibit inducible clindamycin resistance [12].

Ethical Consideration. Written approval (Ref Number 12/MMIHS/2072) was obtained from Institutional Review
Committee of Manmohan Memorial Institute of Health Sciences (IRC-MMIHS) after submitting and presenting the research proposal. In addition, informed oral consent was taken from every patient for participation in this study.

Data Processing and Analysis.
Data regarding patient demographics, bacterial isolates, antimicrobial susceptibilities, and resistance determinants were entered into a computer program. Data were analyzed using SPSS 20.0 version and interpreted according to frequency distribution, percentage.

Antibiotic Susceptibilities of Gram Negative Bacteria.
Diverse susceptibility pattern was observed among the isolates of Gram negative bacteria (Table 3). Enterobacteriaceae isolates were highly resistant to ampicillin (93%) and cephalosporins (up to 68%). Gentamycin, levofloxacin, piperacillin-tazobactam, and imipenem were the effective antimicrobials for enterobacterial strains. On the other hand, Gram negative nonfermenters, Pseudomonas and Acinetobacter spp., were highly resistant to cephalosporins (up to 66%) but well susceptible to levofloxacin, piperacillintazobactam, and imipenem. Polymixin and colistin sulphate were completely effective against Gram negative bacteria.

Discussion
Every year, millions of people in developing countries like Nepal are experiencing the pyogenic wound infections due to either injury related to trauma, accidents, or burns and their complications with pathogenic microorganisms [2,5,9]. Although pyogenic wound infections are common findings among the patients visiting hospitals of Nepal, there is paucity of documented reports describing the etiological spectrum and antibiotic susceptibility pattern of bacteria causing these infections [9,10]. Moreover, continuous upsurge of antimicrobial resistance among the pathogenic organisms has created a therapeutic challenge for treatment of pyogenic wound infections [14]. Therefore, updated knowledge on the etiology and antimicrobiogram is considered highly valuable to reduce morbidities and associated complications. In this study, overall pyogenic wound infections among study subjects based on the significant bacterial growth in clinical specimens were 64.9%. To the best of our knowledge, this is the highest ever reported rate of growth among the pyogenic clinical specimens from Nepal. Previously, Shrestha and Basnet (50.0%) and Acharya [3,17]. These variations in the growth rates from pyogenic wound specimens might be attributable to the quality of specimens processed, contamination with external microbiota, and standard wound care practices in the healthcare and facilities of bacterial cultivation in the locality [16]. Moreover, we noticed that majority (92.54%) of the clinical specimens were found with monomicrobial  growth and little polymicrobial growth. This is also consistent with the previous reports from Nepal [2,15]. Polymicrobial pyogenic wound infections might be associated with poor wound care, increased microbial survival, and ineffective antimicrobial treatment [17]. Gram positive bacteria have been described as the major cause for pyogenic wound infections in several literatures [2,9,10]. Our findings also supported this fact, as majority of our isolates were Gram positive cocci (57.6%). However, Trojan [7,16,17]. On the other hand, Staphylococcus aureus (49.2%) was the predominant isolate responsible for pyogenic wound infections in this study which is quite similar to several previous studies [2,5,9]. In a recent report from India, Gram negative bacteria, particularly Enterobacteriaceae, were found as major pathogens [7]. Escherichia coli, Klebsiella spp., and Pseudomonas spp. were other common pathogens in our study. It is well known that S. aureus and Gram negative bacterial pathogens produce very potent virulence factors, responsible for maintaining the infection and delaying the process of wound healing [16]. Therefore, our results confirm the usual most prevalent microorganisms found in pyogenic wound infections. Nevertheless, Gram negative bacteria have been described to be associated with nosocomial infections and intra-abdominal surgical procedures [18].
High rates of antimicrobial resistance among the pathogenic bacteria associated with the pyogenic infections are major concerns of this study. The prevalence and pattern of antimicrobial resistance among pyogenic bacterial isolates usually exhibit variability according to the geographic areas, climatic conditions, and endemicity of resistant pathogens in the locality. Of particular concern, among Gram positive bacteria, Staphylococcus aureus in this study was the most resilient organism to develop resistance. Our isolates were highly resistant to ampicillin, ciprofloxacin, cotrimoxazole, erythromycin, cloxacillin, clindamycin, and imipenem. This finding is in agreement with the previous reports of Acharya et al., Rai et al., and Yakha et al. [2,9,10] but higher when compared to the reports by Shrestha and Basnet [15] from nearby hospitals of Kathmandu. However, similar to other previous studies [2,15], the isolates of Streptococcus pyogenes were promisingly susceptible to ampicillin, cotrimoxazole, erythromycin, and cephalosporins. Cotrimoxazole, one of the most widely used antimicrobial agents for treating pyogenic and soft tissue infections, was found susceptible to S. aureus and S. pyogenes [19]. However, isolates of Enterococcus spp. were least susceptible to ampicillin, the drug of choice for enterococcal infections [20]. Remarkable susceptibility of Gram positive bacteria to vancomycin, amikacin, and carbapenems (imipenem) may be the good alternative for pyogenic wound infections in our settings.
Furthermore, almost more than half (52%) of the Gram positive strains in our study were MDR which is comparatively higher than that of previous reports from Nepal [2,10]. Higher rates of MDR strains have been documented in several other studies [17,[21][22][23]. We believe our rate of MDR Gram positive isolates is greatly contributed by the high rates of methicillin resistant Staphylococcus aureus (MRSA) strains [24]. In this study, about 32% of the Staphylococcus aureus isolates were methicillin resistant and were resistant to commonly used antimicrobial agents. The MRSA rate is high when compared to the previous reports of  [14,25]. In addition to this, similar to previous studies [14,26], we found inducible clindamycin resistance (iMLS B ) in 8.73% of the isolates. The possible explanation for variation in the drug susceptibilities might be difference in study population including hospitalized inpatients where more MDR strains are expected.
Alongside, our findings indicate the high incidence of drug resistance among Gram negative isolates too. In Table 3: Antibiotic susceptibilities of gram negative bacterial isolates. Journal of Pathogens this study, Escherichia coli, Klebsiella spp., Citrobacter spp., and Proteus spp. were highly resistant to cephalosporins while Gram negative nonfermenters were resistant to fluoroquinolones, aminoglycosides, and cephalosporins. The findings of the susceptibility pattern of our Gram negative isolates are in agreement with other previous reports from this region [2,9,15]. In recent years, there is an increased concern about Gram negative resistance to commonly used antimicrobials in wound infections [7,27]. In this study, multidrug resistance among Gram negative bacteria was common where Escherichia coli (66.18%), Acinetobacter spp. (60%), Klebsiella spp. (50.0%), and Pseudomonas spp (45.45%) were major MDR strains. This finding is quite high when compared to the previous reports from our country [2,10] but is lower than that of other studies from India [7] and Ethiopia [22]. The high rates of resistance in Gram negative bacteria in our hospital have been previously found as -lactamase producers [28]. In this scenario, non--lactam antibiotics including fluoroquinolones and aminoglycosides would be better therapeutic regimens for pyogenic wound infections in our settings.

Antibiotics
Our findings indicate the existence of high drug resistant bacteria in pyogenic wound infections. The high use oflactam antibiotics and inappropriate infection control procedures in the hospitals might be the cause of rising rates of resistance among these bacteria. Moreover, longer duration of prophylactic antimicrobial exposure in surgical interventions may contribute to organisms for developing resistance.

Limitations
This study was based on characterization of bacterial isolates growing in the aerobic or facultative anaerobic conditions excluding anaerobic bacteria. Furthermore, risk factors for pyogenic wound infections and the treatment outcomes were not measured. Molecular characterization of MDR bacterial isolates would have generated more useful epidemiological results.

Conclusion
Pyogenic wound infections were mainly caused by S. aureus, Escherichia coli, Klebsiella spp., and Pseudomonas spp. High level of drug resistance among both Gram positive and Gram negative bacteria was observed. Continuous surveillance is necessary to update the knowledge of antimicrobial susceptibility profiles of clinical isolates to provide the most appropriate dose regimen and treatment schedule against pyogenic wound infections and to limit the expanding menace of drug resistance.

Consent
Informed consent was taken from every patient or their visitors before participating in this study. Personal information of patients was coded and kept confidential.

Conflicts of Interest
There is nothing to be declared as conflicts of interest.

Authors' Contributions
Basista Prasad Rijal and Narayan Prasad Parajuli designed the study, reviewed the literature, and guided in the laboratory investigations. Deepa Satyal performed the laboratory procedures and participated in data management and analysis. Narayan Prasad Parajuli prepared the manuscript with the help of Basista Prasad Rijal and Deepa Satyal. All authors read the manuscript and approved it for final submission.