Isolation, sensitivity patterns and molecular charactarisation of bacteria isolates from infected diabetic foot ulcers in patients at Kenyatta National Hospital

Abstract

BACKGROUND: The burden of diabetes mellitus is increasing in resource-poor settings and this is associated with a rise in diabetic complications. Diabetic foot ulcers (DFUs) often lead to hospital admissions, amputations and deaths among diabetic patients. In Kenya, there is a lack of up-to-date information on microbial isolates from diabetic foot ulcers and no mention of utilization of molecular techniques. Significance: Identifying bacteria and their sensitivity patterns in infected diabetic foot ulcers could lead to appropriate antibiotic prescriptions, curb antimicrobial drug resistance, and better clinical outcomes thereby reducing health expenditure. OBJECTIVE: To isolate bacteria and determine their antibiotic sensitivity patterns in patients with infected diabetic foot ulcers in a clinical setting using culture-based and molecular techniques while also investigating their risk factors and clinical outcomes. METHODS The study had a cross-sectional design and recruited 84 adult inpatients and outpatients at Kenyatta National Hospital over 12 months with any type of diabetes mellitus and having active foot ulcers. Consecutive sampling was used to enroll participants and informed consent taken. History and physical examinations findings were recorded through a structured questionnaire. The most recent blood tests were also recorded. Samples were collected from the centre of the wound and taken to the laboratory. One part of the sample was used for microbiological tests, and the other for RTPCR was stored at −80 °C. On Day 1, specimens were inoculated on culture media and incubated in aerobic conditions. On Day 2, the most predominant colony was isolated and identified using standard biochemical tests and then VITEK ® 2 machine was utilized for further identification and antibiotic sensitivity. For 51 samples, DNA extraction was performed using kits from Qiagen Hilden, Germany. Staphylococcus aureus DNA, Methicillin-resistant S. aureus (MRSA) DNA and positive control were identified through real-time PCR. Thereafter, clinical outcomes for patients with foot ulcers were retrieved from the patients’ medical records. RESULTS: Majority (68%) were inpatients. The mean age was 60.30 + 12.88 years with 68% living in urban areas and 60% having minimal or no formal education. Eight percent (8%) were newly-diagnosed with diabetes mellitus (DM). The median (IQR) duration of DM was 6.5 (1.25, 12.5) years. A majority (96%) had type II DM. Forty-five percent (45%) were on insulin only, xvi 18% on oral drugs only and 32% on a combination of both. The median (IQR) random blood sugar was 9.60 (5.32, 15.45) mmol/L and glycated haemoglobin was 8.80 (7.17 to 9.92)%. A majority of the patients had good lipid profile, 85% with desirable total cholesterol and 70% having ideal low-density lipoproteins. Eighty-three percent (83%) had one or multiple comorbidities. The median duration of the DFU was 8 (4, 16) weeks. Eighty swabs (94%) were culture-positive; 29% were Gram-positive and 65% were Gram-negative. The organisms isolated were Staphylococcus aureus (16%), Escherichia coli (15%), Proteus mirabilis (11%), Klebsiella pneumonia (7%) and Pseudomonas aeruginosa (7%). The bacterial isolates showed resistance to commonly used antibiotics such as ampicillin, amoxicillin, cefazolin, cefepime, ceftazidime, cefotaxime, cefuroxime, clindamycin, co-trimoxazole (TMPSMX), erythromycin, piperacillin-tazobactam, and tetracycline. More than half (62%) of the S. aureus isolated and 40% of the Gram-negative bacteria were MDROs. MRSA were not identified using culture methods but were identified using PCR. PCR was also more sensitive than culture-based methods to identify S. aureus. There was a high mortality rate (11%) among patients with DFUs. CONCLUSION: There are poor outcomes for patients with DFUs in this setting such as poor wound healing, high recurrence rates, increased lower limb amputations and mortality compared to previous studies. Almost all DFUs were infected, with Gram-negative bacteria being the most common. In order of frequency, S. aureus, E. coli, P. mirabilis, K. pneumonia, P. aeruginosa were the most common isolated species. There was a high prevalence of nosocomial bacteria. In this study, the bacteria isolated showed high resistance to commonly used antibiotics with Gramnegative bacteria showing higher resistance patterns. Recommendations: Patients with DFUs should have microbial swabs for culture and sensitivity performed routinely for correct management and antibiotic stewardship. PCR is an effective way of species identification, and in particular, RT-PCR for S. aureus and MRSA should be considered for patients with adequate healthcare resources. The high prevalence of poor outcomes for patients with DFUs warrants the need to investigate bio-psychosocial risk factors.