In-vitro Antibiotic Susceptibility Tests of Bacterial Isolates from Abdominal Wound Infection in a Nigerian Teaching Hospital

Aims: The trend of increasing antibiotic resistance has been reported from various centres. The aim of this study was to look at the pattern of resistance of bacterial isolates from abdominal wound infections and determine its magnitude in a bid to establish appropriate antibiotic stewardship program in the centre. Study Design: A prospective cross sectional study that looked at pattern of antibiotic susceptibilities in isolated organisms from infected laparotomy wounds. Methods: All adult patients (eighteen years and above) who had either emergency or elective laparotomy for one indication or the other were recruited into this study. Wound inspection was done on days 3, 5, 7; swabs were taken in infected cases under aseptic condition and processed according to microbiological standards. Results: Eighty five (38.1%) patients developed wound infection out of the 223 that met the inclusion criteria. This consists of 157 (70.4%) males and 66 (29.6%) females. Their ages ranged between 18 and 80 years. Males developed wound infection more than females. Dirty wounds had the highest infection rate. The most common isolates were Klebsiella spp (34%), Staphylococcus aureus (30.4%) and Proteus spp (19.6%). Multidrug resistance (>50%) to commonly used antibiotics such as amoxicillin-clavulanate, cotrimoxazole and gentamicin were seen in many isolates. Conclusion: The emergence of multidrug resistant organisms calls for collaborative efforts and judicious use of antimicrobial agents among clinicians.


INTRODUCTION
Surgical site infection (SSI) as recently defined by the Centres for Disease Control and Prevention (CDC) is one occurring after surgery in the part of the body where the surgery took place [1]. The extent of this might range from involvement of layers of the anterior abdominal wall (incision site) to involvement of the deep space/peritoneal cavity or specific organs within the abdominal cavity (organ/space) [1]. Surgical wounds are generally classified based on their degree on microbial contamination into clean, clean contaminated, contaminated and dirty wounds [2]. The susceptibility of a wound to infection is therefore directly related to each class of the wound. For clean wounds, gram positive organisms from the skin flora are usually the cause of infection while in other classes of wounds, polymicrobial aerobic and anaerobic organisms closely resembling the normal endogenous microflora of the affected organ are the usual isolates [3][4][5].
Some of these organisms have developed resistance to the commonly used antimicrobial agents over time. This is as a result of injudicious use of such drugs on the part of the patients and indiscriminate prescription on the part of clinicians [6,7]. This prospective study therefore aimed to look at the pattern of antibiotic resistance by organisms isolated from abdominal wound infection following laparotomies in a Nigerian teaching hospital.

Study Centre and Design
This is a prospective cross sectional study carried out at the Federal Teaching Hospital, a tertiary health care centre which also serves as a referral centre located in Gombe, North -Eastern Nigeria from January 2012 to December 2012.

Recruitment of Study Participants and Sample Collection
The inclusion criteria were all adult patients (eighteen years and above) who had either emergency or elective laparotomy in the General Surgery unit at Federal Medical Centre, Gombe, North eastern, Nigeria and who subsequently developed surgical site infection within thirty days of surgery. [Eighteen years was the cut-off age for pediatric patients in the study centre as at the time of this study]. Informed consent was obtained from all patients recruited into the study and relevant clinical information entered into a proforma designed for the study. Ethical approval was obtained from the Institutional Ethics Review Board.
Patients' wounds were inspected on postoperative days 3, 5 and 7 for local evidence of wound infection. The diagnostic criteria for clinically infected wound were based on the definition provided by the Centres for Disease Control and Prevention (CDC) and the National Healthcare Safety Network (NHSN) [2].
Wound swabs were collected from patients who had suspected or clinically infected wounds. This was done under aseptic procedure, cleansing the wound site with sterile gauze soaked in normal saline; parting the wound edges and dipping the sterile cotton-tipped specimen collection stick to the base of the wound and firmly rotating it while avoiding contact with the wound edges. The specimen were capped and labeled appropriately and thereafter sent to the Medical Microbiology laboratory. Microscopy of the specimen was done using gram straining technique. Each smear was examined at high magnification using an oil immersion (x100) objective lens. Gram positive organisms appeared blue/purple, while Gram negative organisms appeared pinkish red [8].

Culture
The samples collected were inoculated on blood agar, chocolate agar (Oxoid, Basingstoke, UK) and MacConkey agar (Fluka medica) plates using a sterile platinum wire loop. MacConkey and blood agar plates were incubated aerobically at a temperature of 35-37°C for 18-24 hours, while chocolate agar plates were incubated in a candle jar to facilitate the growth of fastidious organisms. Growth on the culture plates were examined macroscopically for colonial morphology. The colonies were subjected to appropriate biochemical tests for identification and classification [9].

Biochemical Confirmation
Biochemical test such as carbohydrate fermentation, oxidase production, catalase utilization, coagulase production, indole production, citrate utilization and ability to produce urease were employed in addition to microscopic findings to identify the organisms [10]. Klebsiella spp was identified as gram negative bacilli, non-motile, lactose fermenting, indole negative with a positive citrate utilization reaction. Staphylococcus aureus was identified as gram positive cocci with positive catalase and coagulase reactions. Escherichia coli was identified as gram negative bacilli, motile, lactose fermenting, positive indole and negative citrate reaction. Proteus spp were identified as gram negative bacilli, non-lactose fermenting with positive urease and negative oxidase reactions, swarming and motile.

DISCUSSION
The pattern of isolated microorganisms from the surgical wounds in this study is similar to the profile that has been observed from other related studies within and outside Nigeria [12][13][14][15][16]. The resistance pattern demonstrated to the tested antibiotics could be seen to vary and a similar scenario has been widely reported [17][18][19]. The development of this resistance pattern could be attributed to injudicious use of antimicrobial agents which is common place in most third world countries as there are no rule prescription of antibiotics.

Fig. 1. Showing the distribution of patients according to different classes of wound
In Nigeria for example, it is a free for all as there has not been any regulations that govern who prescribes what. Antibiotics abound everywhere as over the counter drugs and clinicians most times do not wait before they start antibiotics as an empirical treatment. If they had to wait for culture result, patient may have sepsis. This is same for whole world especially after malignancy operations and complications. This is the common cause of antibiotic resistance [20,21]. The continuous exposure of microbial agents to these drugs over time eventually lead to reduced efficacy borne out of genetic modification of the target receptors on the microorganisms. Some of these organisms which are ubiquitous within the hospital environment have developed resistance to the commonly used antibiotics needed to suppress their proliferation.
The resistance to the commonly used antibiotics occurs through various plasmid-mediated mechanisms. These mechanisms include: decreased intracellular concentration of antibiotics (either increased efflux or reduced influx of the drug); neutralization by inactivating enzymes (β-lactamase); alteration of the target receptor on which the drug is to act and complete elimination of the target on which the drug is to act [22,23].
Bacterial isolates in this study were both monomicrobial and polymicrobial. Monomicrobial isolates were predominant and comprised largely (67%) of gram negative, aerobic organisms while Staphylococcus aureus was the only gram positive pathogen isolated. The gram negative organisms were largely from the intestinal flora as these were seen more in clean contaminated, contaminated and dirty wounds.
Klebsiella spp demonstrated a very good response to the cephalosporins, gentamicin and quinolones. However, there was a poor response to the use of penicillin and amoxicillin/clavulanic acid. The findings from Benue, North central, Nigeria [24] is comparable to ours but at variance with reports from Lagos [25] and Abuja [26] where these organisms showed a high pattern of resistance (>60%) to cephalosporins and gentamicin. Other workers from Ethiopia [27] and India [28] had reported similar resistance profile by Klebsiella spp. This resistance pattern may be due to the increasing development of extended spectrum beta lactamases (ESBLs) in the Klebsiella spp.
Staphylococcus aureus showed a good response to the use of amoxicillin/clavulanic acid, cephalosporins and quinolones. Jido et al. [29] working from Kano, North west, Nigeria had earlier reported a similar profile. Our observations tend to be in agreement with a study from Nepal [30] but inconsistent with that of another Nigerian study from Niger State where a high resistant profile was observed [31]. Although, in an Indian study by Sonawane et al. [28] Staphylococci showed complete (100%) susceptibility to vancomycin. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) and other multidrug resistant patterns in some Nigerian centres is a pointer to the magnitude of the problem in our environment [32,33].   The poor responses of these organisms to the tested antibiotics were borne out of factors that have been identified to be peculiar to our environment. These include injudicious use of such drugs, poor patient compliance, substandard drugs and self medication [35][36][37]. The emergence of multi-drug resistant organisms is a nightmare for clinicians and the patients and the management of such may entail the use of newer generation but expensive antibiotics like meropenem and vancomycin as already reported from different climes [26,28,34]. Other newer agents that have been found useful in resistant cases include the glycopeptides (Dalbavacin, Oritavacin) and quinupristin/dalfopristin combination which have been found particularly useful in cases of vancomycin resistant organisms as well as methicillin resistant staphylococcus aureus (MRSA). Considering the peculiar problem of poverty in our environment, many of our patients might not be able to eventually afford the more potent, newer generation drugs when the need arises as is the case when dealing with multidrug resistant organisms. A change in policy direction and enforcing antibiotic stewardship might be a necessary way of combating this problem.

CONCLUSION
It is evident that there is an emerging problem of multidrug resistant organisms. Collaborative efforts are required among clinicians in order to curtail this trend. Well-structured antibiotic stewardship programmes in our institutions will be of judicious benefit. Government policies should strengthen and restrict the prescription of antibiotics in our hospitals to clinicians at appropriate levels while measures to curb the over the counter sale of antibiotics are put in place.

ACKNOWLEDGEMENT
Special thanks go to of Dr I.A Esin and Dr P.F Adejoh for their supportive role during the course of this research work. The assistance of Mr Kudi Ayuba will not go unappreciated as well.