Antimicrobial resistance profile among bacteria isolated from patients presenting with wounds at Kabale Regional Referral Hospital, South western Uganda

Background Bacterial Wound infection and antimicrobial resistance remains a public health challenge. The challenge remains worse due to nosocomial bacterial infection often characterized by multidrug resistance. Infected wounds are often associated with delayed epidermal maturation resulting into prolonged hospitalization. Data on profile of clinical significant bacteria and their respective antibiotic drug resistance in Uganda is still limited. In this study we emphasized on phenotypic characterization of bacteria that cause wound infections at Kabale Regional Hospital (KRRH) and determining the respective antimicrobial susceptibility profiles. Methods Between June 2016 – to June 2017 a total of 276 Pus specimens were collected from patients at KRRH and analyzed for bacterial infection by standard bacterial cultures techniques. Pus specimens were all from wounds (surgical and non-surgical). Antibiotic susceptibility testing was performed and reported based on CLSI guidelines. Results One-hundred and ninety-five specimens were positive following bacterial culture (70.7%). Staphylococcus aureus and Escherichia coli were the most frequently isolated bacteria. Antibiotic drug resistance testing revealed that 68% of S. aureus isolates were Methicillin resistant. For Escherichia coli isolates, 73% were ciprofloxacin and levofloxacin resistant while resistance to Imipenem was common among Klebsiella sp. negative , G positive S. aureus also are high levels of multi antimicrobial resistance among both Gram negative Gram positive

Background Bacterial Wound infection and antimicrobial resistance remains a public health challenge.
The challenge remains worse due to nosocomial bacterial infection often characterized by multidrug resistance. Infected wounds are often associated with delayed epidermal maturation resulting into prolonged hospitalization. Data on profile of clinical significant bacteria and their respective antibiotic drug resistance in Uganda is still limited. In this study we emphasized on phenotypic characterization of bacteria that cause wound infections at Kabale Regional Hospital (KRRH) and determining the respective antimicrobial susceptibility profiles.
Methods Between June 2016 -to June 2017 a total of 276 Pus specimens were collected from patients at KRRH and analyzed for bacterial infection by standard bacterial cultures techniques. Pus specimens were all from wounds (surgical and non-surgical). Antibiotic susceptibility testing was performed and reported based on CLSI guidelines.
Results One-hundred and ninety-five specimens were positive following bacterial culture (70.7%).
Staphylococcus aureus and Escherichia coli were the most frequently isolated bacteria. Antibiotic drug resistance testing revealed that 68% of S. aureus isolates were Methicillin resistant. For Escherichia coli isolates, 73% were ciprofloxacin and levofloxacin resistant while resistance to Imipenem was common among Klebsiella sp.
Conclusions Wound infection is mainly caused by gram negative bacteria particularly, Escherichia coli , Klebsiella spp., Proteus spp., and Pseudomonas spp. G positive cocci particularly S. aureus is also an important pathogen among other implicated gram positive cocci. There are high levels of multiantimicrobial resistance among both Gram negative and Gram positive bacteria.

Background
Antimicrobial resistance remains a public health challenge impacting negatively on the quality of health care delivery [1] . Bacterial drug resistance has demonstrated prospective ability to spread from one individual to another within and outside healthcare setting 2 . Several factors fueling antimicrobial resistance have been described worldwide, with unregulated access to antibiotics and improper stewardship being among the common causes 3 . In addition, overuse and inappropriate use of antimicrobial agents has been well described to pray an important role in emergence and spread of antimicrobial resistance (AMR) 4 . Ability to develop antimicrobial tolerance and resistance with propagation of resistance clones has resulted into therapeutic failure consequently leading to increased mortality and morbidity 5 . Wounds are highly susceptible to colonization and infection, which has posed a major challenge in healing that often results into death of the victims. In hospital or Health care settings, the challenge remains worse due to nosocomial bacterial infection often characterized by multidrug resistance 6 . Infected wounds are often associated with delayed epidermal maturation and deep scar formation that results into prolonged hospitalization and high mortality rate due to sepsis. Bacteria pathogens are the main etiological agents of wound infection in the community and hospital settings 7 . These bacterial agents are increasingly resistant to commonly used antibiotics leading to emergency of cefoTaXime (CTX-M beta-lactamase enzymes) in Klebsiella species and Methicillin Resistant S. aureus (MRSA) infections 8 . The outcome of wounds infected with antimicrobial resistant bacteria is often poor characterized with prolonged debility and increased cost of healthcare to the patients. Therefore, awareness of bacterial pathogens in the wounds, management standards and prevailing susceptibility patterns is a prerequisite for the rational antimicrobials use. Data on profile of clinical significant bacteria and their respective antibiotic drug resistance in Uganda is still limited. In this study we emphasized on phenotypic characterization of bacteria that cause wound infections at Kabale Regional Hospital (KRRH) and determining the drug susceptibility profiles of those agents.

Study design
This was a descriptive cross sectional study and it was conducted on patients presenting with wounds at Kabale Regional Referral Hospital (KRRH).

Sample Collection and Transport
Pus (Swabs and aspirates) were collected from wounds (traumatic wounds and surgical wounds) during routine clinical work from the period of June 2016 -to June 2017. Sterile cotton swabs (ThermoFisher TM ) were used to collect specimens which were immediately taken to the microbiology laboratory for microbiological investigations.

Isolation and Identification of Microorganisms
After accession into Laboratory register, the clinical samples were directly inoculated onto Blood agar (BAP), MacConkey agar (Oxoid TM ) and Chocolate agar (Choc). Both BAP and MacConkey agar were incubated aerobically and while Choc plate was incubated in 5 -10% CO2. All culture plates were incubated under humid condition of 55 -70% at temperature of 35 -37 o C for 24 -72 hours with regular inspection every after 24hours for bacterial growth. Bacterial colonial growth was identified phenotypically through macroscopic appearance, gram staining and biochemical identification using analytical profile index -API (BioMérieux TM ) 9 . On addition to API, API staph was used to confirm staphylococcus species after identification with catalase, coagulase and Staphaurex agglutination test (Remel) 10 .

Antibiotic Susceptibility Testing
Kirby-Bauer technique using Mueller Hinton Agar (Oxoid TM ) was used for antibiotic susceptibility testing (AST) following the Clinical Laboratory Standards Institute (CLSI) 11 . The ASTs results were interpreted in accordance with the antibiotic susceptibility break-points described in the CLSI 2018) guidelines.
Phenotypic screening of Extended Spectrum Beta Lactamase (ESBL) using double disk diffusion testing was done on MHA, using gram negative bacteria that showed phenotypic expression of Extended-Spectrum Beta-lactamases (ESBLs). E. coli, Klebsiella spp. and Proteus mirabilis isolates displaying decreased susceptibility to ceftriaxone (CRO 30 μg) with zone diameter of ≤ 25 mm) and Ceftazidime (CAZ 30 μg) with zone diameter of ≤ 22 mm were selected for ESBL confirmatory testing as per CLSI guidelines 12 . Ceftazidime with clavulanic acid (CAZ/CLA 30/10 μg), Ceftazidime (CAZ 30 μg), cefotaxime (CTX 30 μg), and cefotaxime with clavulanic acid (CTX/CLA 30/10μg) antibiotics were used for this study. Equal or greater than 5 mm (≥ 5mm) diameter zone of inhibition for the CAZ/CLA and CTX/CLA disk versus the corresponding CAZ or CTX disk was considered positive for ESBL 13 .

Quality control
Every new batch of antibiotics disks and culture media used during our study were routinely quality controlled. Media PH was adjusted using 1NNaOH and 1NHCL.  11,14 . Results were documented in the laboratory record book and data extraction forms and reported back to clinical team for patient management. AST results were designated in terms of Sensitive (S), Intermediate (I), and Resistant (R) based on current CLSI -2018 guidelines (9).
Klebsiella pneumoniae ATCC700603 and Escherichia coli ATCC 25922 were used as ESBL-positive and ESBL-negative control strains.

Data Management and Analysis
Data generated were entered into Excel spread sheet, cleaned and processed to give descriptive statistics and presented as a percentage, or frequencies proportions of each isolated bacterium to determine the most prevalent species involved in wound infections and respective antimicrobial resistance patterns.

Bacteria isolated from patients presenting with wounds at KRRH
Two hundred and seventy-six [n=276] wound pus swabs were cultured among which 70.7% (n=195) were culture positive with 50.8% (n=99) having a single pathogen and 49.2% (n=96) having more than one bacterial isolate. 31.79% (n=62) was S. aureus which was the most frequently isolated bacteria followed by 29 figure  1

Discussion
Wound infections is a common occurrence among patients visiting Ugandan hospital and despite the scarcity of documented reports or information describing the profile and antibiotic susceptibility pattern of pathogenic bacteria behind these infections, they are commonly due to either surgical or injury related to trauma, accidents, or burns. Currently, wound infections are often characterized delays in health due to infection with highly virulent and antimicrobial resistant bacterial pathogens that pose a threat to treatment [15 ] . The increase of antimicrobial resistance among pathogenic bacteria among the inpatients and the community threatens the current management of wound infections [16] . who reported Gram negative bacteria as the predominating pathogens in wound infections [17,18,19] . Escherichia coli, Klebsiella spp., Proteus sp. and Pseudomonas spp. were other common gram negative bacteria observed in our study. In a recent report from Ukraine, Gram negative bacteria, particularly Enterobacteriaceae, were found as major pathogens [19] . However, the presence of gram positive bacteria (39%) in the current study is supported by several literatures [20,7,15] .
Staphylococcus aureus (49.2%) was the predominant isolate responsible for bacterial wound infections in this study which is quite similar to several previous studies [21,6,22] . We confirm that the as previously described, these bacteria are traditional most prevalent microorganisms found in wound infections. Though, Gram negative bacteria have been described to be associated with hospital acquired infections and common in abdominal surgical procedures [23,24] . S. aureus and Gram negative bacterial pathogens are implicated to be common bacteria that invades and colonize wounds and are known to produce compelling virulence factors that are highly destructive with consequent delays in wound healing process while sustaining infection [25] .
Of note, we observed presence of more than one bacterial pathogens from one wound source, suggestive of Polymicrobial wound infection. This is always associated with poor wound management coupled with increased microbial survival and antimicrobial resistance. In addition, the complacence towards benefits of wound debridement and wound irrigation without antimicrobials and close wound monitoring are probable cause of multiple bacterial invasion and colonization. Infection control and prevention prays a key role wound management and it has been recommended that antimicrobials should not be prescribed as the initial treatment strategy [26] . Nevertheless, we suggest antimicrobial use as prophylaxis in case of surgical wounds, while the use of systemic antibacterial be indicated when sepsis appears and is spreading to the subcutaneous soft tissues. This can result into bacteremia with serious adverse patient outcome [26,18] . However, longer period of prophylactic antimicrobial exposure in surgical interventions may subsidize to organisms developing resistance fast. Therefore, antimicrobials are required to eliminate microbial colonization and spread 17  Jean-Marie et al. [7,27,21] . However, comparable to other studies in Kinshasa (Democratic republic of Congo -DRC) and India, all the isolates of CONS were identified and highly resistant to Ampicillin, amoxicillin -clavunate, trimethoprim -sulfamethoxazole and chloramphenicol [21,20] . Trimethoprimsulfamethoxazole is one of the most widely used antimicrobial agents for treating pyogenic wound infections, was found not susceptible to all isolates. Pressure due to continuous use of this particularly antibiotics in HIV -AIDS patients as prophylactic agent is the probable cause of resistance. S. pyogenes resistance to ciprofloxacin, trimethoprim -sulfamethoxazole, chloramphenicol and Tetracycline threatens the current treatment choice to this pathogen. Similar trends have been reported elsewhere and probable over use of these antimicrobial agents and poor wound management maybe implicated as the drivers [28] . In addition, S. pyogenes was susceptible to ampicillin, amoxicillin -clavunate, Gentamycin, levofloxacin, Piperacillin -Tazobactam, Penicillin, which are one of the most widely used antimicrobial agents [26] . However, Enterococcus spp., isolates were highly resistant to ampicillin, amoxicillin -clavunate, Gentamycin, levofloxacin, Piperacillin -Tazobactam, Penicillin, ciprofloxacin, trimethoprim -sulfamethoxazole, chloramphenicol and Tetracycline, and remarkable susceptible to vancomycin. It is not a surprise to our study that enterococci sp., are known to be multidrug resistant opportunistic bacteria that become pathogenic when they colonize niches uncommon to them like wounds. They have turn out to be major cause of nosocomial infections especially surgical sites among other sites [29] . Alongside, our findings indicate the high incidence of drug resistance among Gram negative isolates too. In this study, Escherichia coli, Klebsiella spp., Proteus spp., and Citrobacter spp., were highly resistant to cephalosporins and other commonly used antibiotics while Pseudomonas aeruginosa a gram negative non-fermenter was resistant to fluoroquinolones, aminoglycosides, and cephalosporins and Penicillins used in the current study. Such susceptibility pattern of these isolates is in agreement with other previous reports across Africa [30] . Multidrug resistance among Gram negative bacteria resistant to commonly used antimicrobials in wound infections is a serious concern [31,17] . The isolated Gram negative bacteria are known to be normal flora of the gut. To minimize the selection pressure of individual antibacterial on the normal flora, we advise use of narrow-spectrum agents. Empirical treatment should be discouraged rather base on the wound culture results. Acinetobacter sp., isolated was not highly resistant to commonly used antimicrobials. However, this particular pathogen is important nosocomial bacteria often associated with a wide range of beta-lactam resistant and extended spectrum beta lactam resistance with ability to produce biofilms, among other characteristics, allows it to persist in hospitals for prolonged periods [32 33] .

Conclusions
Wound infections were mainly caused by gram negative bacteria particularly, Escherichia coli, Klebsiella spp., Proteus spp., and Pseudomonas spp. As well as gram positive cocci particularly S.
aureus. There were high levels of antimicrobial resistance among both Gram negative and Gram positive bacteria. Continuous surveillance is obligatory to appraise and inform on bacterial and their respective antimicrobial susceptibility profiles in wound isolates. This is essential for provision of suitable antimicrobial agent in the treatment of wound infections and prevention of antimicrobial resistance.

Transparency Declarations
None to declare.

Availability of Data and materials
The datasets used and/or analysed during the present study are available from the corresponding author on reasonable request.