Predominance of multi-resistant gram-negative bacteria colonizing chronic lower limb ulcers (CLLUs) at Bugando Medical Center

Background Infections, trauma, malignances and poorly controlled diabetes are common causes of chronic lower limb ulcerations in developing countries. Infected wound with multi-drug resistant bacteria usually are associated with increased morbidity and mortality. We report the distribution of bacteria pathogens colonizing the chronic lower limb ulcers and their drug susceptibility pattern from Bugando Medical Centre (BMC) a tertiary hospital in Tanzania. Findings Three hundred non-repetitive wound swabs were aseptically collected from 300 patients with chronic lower limb ulcers using sterile swabs and processed following standard operative procedures. Isolates were identified using in house biochemical testing and in case of ambiguous confirmation was done using API 20NE and API 20E. Susceptibility was determined using disc diffusion test following clinical laboratory standard Institute guidelines (CLSI). Of 300 swabs from patients with chronic lower limbs ulcers, 201 (67.7%) had positive aerobic culture within 48 hours of incubation. Of 201 isolates, 180(89.6%) were gram-negative bacteria. Out of 180 gram negative bacteria, resistance was detected for ampicillin (95%, n = 171), amoxicillin/clavulanate (83.9%, n = 151), trimethoprim-sulphamethoxazole (78.9%, n = 142), ceftriaxone (46.7%, n = 84), ceftazidime (45.6%, n = 82), gentamicin (39.4%, n = 71), ciprofloxacin (17.8%, n = 32) and meropenem 28(15.6%, n = 25). A total of 41 (35%) of enterobacteriaceae were found to be extended spectrum beta-lactamases (ESBL) producers while of 18 Staphylococcus aureus, 8(44.4%) were found to be methicillin resistant Staphylococcus aureus (MRSA). Conclusion There is high prevalence of ESBL and MRSA isolates in surgical wards at BMC. We recommend infection control and antibiotic stewardship programs in these wards to minimize spread of multi-resistant organisms.


Background
In developing countries infections, trauma, malignances and poorly controlled diabetes are the most common causes of chronic lower limb ulcerations [1,2]. An infected wound complicates the postoperative course and results in prolonged hospital stay and delayed recovery [3]. Prolonged hospital stay usually exposes the patient to health care associated infections (HCAs) [4], and more risk to infection due multi-drug resistant bacteria like extended spectrum beta lactamase (ESBL) producers and methicillin resistant Staphylococcus aureus (MRSA) [5][6][7][8]. In Tanzania about 50% of Klebsiella pneumonia and 25 -45% of Escherichia coli isolated from HCAIs are reported to be ESBL producers [9][10][11]. Also in Tanzania S. aureus has been reported to be the commonest cause of surgical site infections of which 18.8% are reported to be MRSA [3].
Clinical experience and outcome of patients regarding chronic lower limb ulcers in this centre has been described in previous publication [12]. This article is building from the same study but focusing on distribution of bacteria pathogens, susceptibility pattern of gram negative and gram positive isolates from chronic lower limb ulcers. In addition more information regarding the susceptibility pattern that predicts ESBL phenotype is presented.
The predominance of multi-drug resistant gram negative bacteria colonizing and infecting lower limbs necessitates the scaling up of infection control practices and the introduction of antibiotic stewardship in surgical wards in developing countries.

Study population
A cross sectional study involving all patients with chronic lower limbs ulcers was conducted between November 2011 and February 2012 in surgical wards of Bugando Medical Centre (BMC), a 1000 bed capacity tertiary hospital in the northwestern part of Tanzania as previously described [12].

Laboratory procedures
A total of 300 non-repetitive wound swabs were collected from 300 patients with chronic lower limb ulcers using sterile swabs. All swabs were processed as previously described [13]. Briefly specimens were inoculated on MacConkey agar and 7% sheep blood agar (HIME-DIA, INDIA) and incubated at 35°C aerobically for 24-48 hrs. Identification of bacteria was done using colonies characteristics, hemolysis on blood agar, lactose fermentation on differential media and in house biochemical tests using colonies from pure cultures; in case of ambiguous results confirmation was done using API 20NE and API 20E [11].
The study was approved by Bugando Medical Centre/ Catholic University of Health and Allied Sciences ethics committee and the informed consent were obtained from all patients.

Discussion
The microbiological profile of chronic ulcers of the lower limbs is very important in the provision of appropriate management of ulcers as well as institution-specific antibiotic policy in the surgical wards [19]. As reported previously [3], Pseudomonas aeruginosa was the most frequent gram negative bacteria isolated while Staphylococcus aureus was the commonest gram positive bacteria. Most of these isolates were multiply-resistant to commonly used antibiotics. This is due to the fact that most of these patients were hospitalized for more than 72 hrs signifying health care associated infections [3].
Compared to previous data in the surgical wards [3] the ESBL rates among Klebsiella spp and E. coli colonizing CLLU were lower than those involved in surgical site infections. Also this study confirmed the previous observation [10,11] that the resistance to trimethoprim-sulphamethoxazole, gentamicin, and ciprofloxacin is a predictor of ESBL phenotype. Most of ESBL producers in this study are multi-drug resistance, due to the fact that most of ESBL conjugative plasmids observed previously in this settings [20,21] carry resistance markers for other antibiotic classes such as tetracycline, gentamicin and trimethoprim-sulphamethoxazole. Similar to other studies [11,22], majority of gram negative and gram positive isolates were sensitive to meropenem and vancomycin respectively. Increased trend was observed for MRSA at BMC whereby in 2009 and 2011 about 16.3% and 18.8% of S. aureus were found to be MRSA respectively [3,17] while in the current study about 44% of Staphylococcus aureus were found to be MRSA.
Despite the importance of these data some limitations were failure to perform; anaerobic culture, molecular characterization and PCR confirmation of ESBL and MRSA phenotype.
control and antibiotic stewardship programs in these wards to minimize spread of multi-resistant organisms. Coordinated surveillance of multi drug resistant isolates in Tanzania and other developing countries is highly needed so that this worldwide public health problem is controlled.