Bacterial isolation and antibiotic susceptibility from diabetic foot ulcers in Kenya using microbiological tests and comparison with RT-PCR in detection of S. aureus and MRSA

Abstract

Objectives Diabetic foot ulcers (DFUs) often lead to hospital admissions, amputations and deaths; however, there is no up-to-date information on microbial isolates from DFUs and no mention of utilization of molecular techniques in Sub-Saharan Africa. We conducted a cross-sectional study among 83 adult patients at a tertiary hospital in Kenya over 12 months. The study aimed to isolate, identify bacteria, their antibiotic susceptibility patterns in active DFUs, and to compare standard microbiological methods versus a real-time PCR commercial kit in the detection of Staphylococcus aureus DNA and methicillin-resistant S. aureus (MRSA) DNA.

Results Eighty swabs (94%) were culture-positive; 29% were Gram-positive and 65% were Gram-negative. The main organisms isolated were S. aureus (16%), Escherichia coli (15%), Proteus mirabilis (11%), Klebsiella pneumoniae (7%) and Pseudomonas aeruginosa (7%). The bacterial isolates showed resistance to commonly used antibiotics such as ampicillin, amoxicillin, cefepime, ceftazidime, cefuroxime, clindamycin, erythromycin, piperacillin–tazobactam, tetracycline and trimethoprim–sulphamethoxazole (TMPSMX). Thirty-one percent of the S. aureus isolated and 40% of the Gram-negatives were multi-drug resistant organisms (MDROs). There was a high prevalence of nosocomial bacteria. MRSA were not identified using culture methods but were identified using PCR. PCR was more sensitive but less specific than culture-based methods to identify S. aureus.

Introduction It is estimated that 10–15% of diabetic patients will develop diabetic foot ulcers (DFUs) at some point in their life [1, 2]. In Africa, the overall prevalence of DFUs was found to be 13% in a recent meta-analysis [3]. At presentation, about half of DFUs are clinically infected [4]. Staphylococcus aureus and beta-haemolytic Streptococci are the most common causes of skin infections [5,6,7,8,9,10,11]. In resource-poor countries however, Gram-negatives like Pseudomonas aeruginosa are more prevalent [6, 10]. In Kenya, S. aureus and Escherichia coli were found to be the most common organisms in DFIs [12]. More recently, 73.2% of DFUs were infected while 26.8% were culture-negative [13]. Fungal infections may also cause DFIs [14].

Antimicrobial resistance (AMR) is an emerging problem globally. Methicillin-resistant S. aureus (MRSA) was first observed in the early 1960s and has been associated with increased hospital stay, healthcare costs and mortality [15]. MRSA represented 4.7% of S. aureus isolated in a study in Morocco [16]. In Brazil, 33% cases of MRSA (cefoxitin-resistant) were vancomycin-resistant [17]. Multi-drug resistant organisms (MDROs) are bacteria that are resistant to more than one or more classes of antibiotics. In Tanzania, antibiotic susceptibility tests (AST) of bacterial isolates from DFUs revealed a high AMR [18].

Polymerase chain reaction (PCR) is a molecular method that can be used to identify bacterial species by amplifying the 16S ribosomal RNA (rRNA) gene [4, 10]. Real-time PCR (RT-PCR) allows detection of DNA or RNA through production of fluorescence light during the reaction. In Sub-Saharan Africa, there is a lack of up-to-date information on microbial isolates from diabetic foot ulcers and no mention of utilization of molecular techniques. The only available study from Africa is from Algeria where sequencing target genes identified a high prevalence of Gram-negative bacilli (54.9%) and MDROs (58.5%) [19].

The objective of this present study was therefore to isolate bacteria and determine their antibiotic susceptibility patterns in patients with infected DFUs using culture-based methods and to compare the differences between microbiological methods and RT-PCR in detecting S. aureus and MRSA in a sub-Saharan setting, which is facing an escalating AMR with extensive health, economic and societal implications.