Multicenter Study of the Burden of Multidrug-Resistant Bacteria in the Etiology of Infected Diabetic Foot Ulcers

Background Infected diabetic foot ulcer (IDFU) is a public health issue and a leading cause of non-traumatic limb amputation. Very few published data on IDFU is available in most West African countries. The objective of this study was to investigate the etiological agents of IDFU and the challenge of antibacterial drug resistance in the management of infections. Methods This was a prospective cross-sectional hospital-based study involving three tertiary healthcare facilities. Consecutive eligible patients presenting in the facilities were recruited. Tissue biopsies and/or aspirates were collected and cultured on a set of selective and non-selective media and incubated in appropriate atmospheric conditions for 24 to 72 hours. Isolates were identified by established standard methods. Antibiotic susceptibility testing was performed using modified Kirby-Bauer disc diffusion method. Specific resistance determinants were investigated by polymerase chain reaction-based protocols. Data analysis was done with SPSS version 20. Results Ninety patients with clinical diagnosis of DFI were studied between July 2016 and April 2017. A total of 218 microorganisms were isolated, comprising 129 (59.2%) Gram-negative bacilli (GNB), 59 (27.1%) Gram-positive cocci (GPC) and 29 (13.2%) anaerobic bacteria. The top five facultative/aerobic bacteria encountered were: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Citrobacter spp. representing 41 (18.8%), 23 (10.5%), 20 (9.2%), 19 (8.7%) and 19 (8.7%) isolates, in that order, respectively. The commonest anaerobes were Bacteroides spp., and Peptostreptococcus anaerobius which accounted for 7 (24.1%) and 6 (20.7%), respectively. Of the 93 IDFU cases, 74 (80%) were infected by multidrug-resistant (MDR) bacteria predominantly methicillin-resistant S. aureus, extended-spectrum β-lactamase-producing GNB, mainly of the CTX-M variety. Only 4 (3.1%) GNB were carbapenemase-producers encoded by blaVIM. Factors associated with presence of MDR bacteria were peripheral neuropathy (r= 4.05, P= 0.042) and duration of foot infection >1 month(r= 7.63, P= 0.015). Conclusions MDR facultative/aerobic bacteria are overrepresented amongst agents causing IDFU. A relatively low proportion of the etiological agents were anaerobic bacteria. This finding should help formulate empirical therapeutic options for managing IDFU. Furthermore, drastic reduction in inappropriate use of cocktail of antibiotics for IDFUs is advocated to combat infection by MDR bacteria in these patients.


Introduction:
Infected diabetic foot ulcer (IDFU) is associated with inflammation or purulence occurring in a site below the ankle in a person with diabetes mellitus (DM). 1 It is a major global public health with substantial medical, socio-economic and psychological burden. IDFU is one of the most common diabetes-related infections in clinical practice, and a common indication for hospital admission. 2 At 7.2% (95%CI: 5.1-9.3%) and higher than the global prevalence of 6.3% (95%CI: 5.4-7.3%), Africa has the second highest global prevalence of diabetic foot ulcer, a precursor of IDFU. 3 Paradoxically, foot infections are most common and lethal in Africa than elsewhere globally. 4 Between 25-60% of diabetic patients with background foot ulcer will develop IDFU which remains a major reason for non-traumatic amputation of the lower limbs. 5 Wide varieties of organisms, including anaerobic bacteria, have been implicated in the etiology of IDFU depending on severity of infection and time from onset to presentation at the healthcare facility. Advanced IDFUs with features of sepsis at admission usually harbor anaerobic pathogens. 2 Emergence and current global threat of antimicrobial resistance in the face of dwindling antibiotics in the pipeline has added a new twist to the burden of IDFU. 6 Increasing involvement of multi-drug resistant organisms in diabetic patients with infected foot ulcers has significantly reduced antibiotic treatment options, thus posing a serious challenge in resource-constrained low-and middle-income countries where access to antimicrobial drugs is of grave concern. 7 A recent systematic review and meta-analysis on the global burden of diabetic foot ulceration in Cameroon, a West African country, has concluded that paucity of data is a bane on the strategy for treatment and prevention of foot infections in diabetic patients. 3 Thus, our study was designed to determine the prevalent bacteria involved in IDFUs, assess the burden of MDR bacteria among the isolates and evaluate the associated risk factors.

Patients:
This prospective cross-sectional hospital-based multicenter study was carried out at the

Samples collection and bacterial identification:
Aspirates were obtained from deep-seated abscesses while tissue samples were collected after washing the wound vigorously with sterile saline and debridement of the slough to exclude mere colonizers. Necrotic tissues were curetted into Anaerobic Basal Broth (Oxoid, Basingstoke, Hants, UK) for anaerobic culture. The samples were immediately transported to the laboratories and processed within 2 hours of sample collection by inoculating them onto a set of selective and non-selective media which were: 5% (v/v) sheep blood agar (BA: Oxoid,

Antibiotic susceptibility test:
Antibiotic susceptibility testing for aerobic and facultative anaerobes was performed by with sterile cotton swab. The swabbed MHA were allowed to dry at room temperature and a set of six antibiotics discs were placed evenly on each of them. After 18 -24 hours of incubation, the diameter of the zone of inhibition around each antibiotic disc was measured, recorded and interpreted as "sensitive", "intermediate" or "resistant" in accordance with CLSI guidelines. 8 Isolates with intermediate sensitivity were regarded as "resistant".
Extended-spectrum β-lactamase production was determined among Enterobacteriaceae and other GNB that have shown reduced susceptibility to at least one third generation cephalosporin or aztreonam by combination disc method according to CLSI guidelines. 8 Gram-negative bacilli with intermediate sensitivity or resistance to one or more carbapenems were tested for production of carbapenemases by the Modified Hodge test (MHT) and interpreted by CLSI guidelines. 8 Methicillin-resistant S. aureus (MRSA) was detected by disc diffusion test, using cefoxitin disc (30 μg) on Mueller-Hinton agar according to CLSI guidelines. 8 Organisms that were phenotypically multidrug-resistant (MDR), including ESBL-producing GNB, carbapenem-resistant GNB and MRSA, were further tested for resistance determining genes using PCR-based protocols with specific oligonucleotide primers (Table 1) and template DNA of the bacteria extracted by boiling method. 9 Electrophoresis of each PCR product (5μl) was carried out in 1.5% (w/v) Agarose gel (Biomatik, Ontario, Canada) in 1X Tris-Acetate-EDTA (TAE) buffer for 45 minutes. The size of amplified products was estimated using 100bp molecular weight marker (100 -1200bp).

Statistical analysis:
Data analysis was performed with Statistical Package for Social Sciences (SPSS) version 20.Comparison of mean values was done using the Student's t test for continuous and chisquare test for categorical variables. Risk factors for infection of diabetic foot by MDR organisms among were identified by logistic regression analysis. A p-value of 0.05 was considered to be statistically significant.  Peptostreptococcus anaerobius (6; 2.3%) as shown in Table 3.
Analysis of antibacterial resistance profiles of the organisms showed that of the188 aerobic isolates, 121 (64.4%) were multidrug-resistant (MDR), being resistant to one or more agents in at least three antibiotic classes (Table 4). Further analysis of specific MDR phenotypes showed that 13 (31.7%) of S. aureus were methicillin-resistant (MRSA), while 43 (33.3%) and 10 (7.8%) of Gram-negative bacteria were ESBL-producing and carbapenem-resistant respectively (       Antibiotic resistance remains a huge problem among diabetic foot ulcer infections; it worsens prognosis and makes treatment outcomes poor. 16 Seventy four (80%) of the 93 IDFU cases in this series harbour one or more MDR bacteria, largely attributable to inappropriate antibiotics use and unrestricted access to antimicrobial drugs in many low-and middle-income countries. 17 This contrasts with several studies in high-income countries including France with low prevalence of MDR bacteria among patients with IDFU. 16,18 A wide spectrum of aerobic and facultative anaerobic bacteria are found to be multidrug-resistant in this study, comparable to findings elsewhere in Africa and Asia. 19,20 A third of the S. aureus isolates were methicillin-resistant (MRSA). Though prevalence of MRSA appears to be rising in Africa, most of the countries have rates lower than 50%. 21 This study also revealed that mecA was detected in 77% of the MRSA and this is similar to the observation of Chaudhry et al. who detected the gene in 20 (84%) of the 25 phenotypically confirmed MRSA isolates. 22 That there are MRSA that lack mecA gene may be on account of mecC, a variant of mecA discovered in 2011 as well as other mutations of penicillin-binding proteins as alternate mechanisms of penicillin resistance. 23 Extended-spectrum β-lactamases were produced by 33.3% of all Gram-negative bacilli isolated and all the organisms except two were members of the family Enterobacteriaceae. The leading bacterial hosts producing ESBLs were E. coli, Klebsiella and Citrobacter species. Published rates of ESBL-producing bacteria vary widely across countries in Africa; as high as 96% in Mali to 0.3% in South Africa. 24 have all been reported. The most prevalent ESBL type was the CTX-M which has been reported as the most predominant variant worldwide. 25 In this study, only 10 (7.8%) of the Gram-negative bacteria were resistant to the carbapenems. Carbapenem resistance determining genes were present in Acinetobacter baumannii, Hafnia alvei and Morganella morganii. Carbapenems are drugs of last resort in the treatment of resistant Gram-negative bacilli infections and with variable and increasing rates of resistance being reported. 26,27 Independent risk factors for acquisition of MDR bacteria found in our study are peripheral sensory neuropathy and foot infection duration > 1 month. This is similar to reports among IDFU cases from India. 28,29 Other authors also documented prolonged duration of wound infection as a predictor of infection of diabetic foot ulcers with MDR bacteria. 30,31 Contrary findings have however been documented from other studies in China, Iran and Portugal. 18,20,32 Our findings is also discordant with the report of Noor et al. which established that ulcer size is a risk factor for infection by multidrug-resistant organisms. 31 This study also observed a significant association between presence of multidrug-resistant bacteria in IDFU and long duration of hospitalization (>1 months) similar to previously documented reports by another author in Turkey. 33 We did not find any socio-demographic factors that were significantly associated with occurrence of MDR IDFU in our study as with other reports. [28][29][30] However, on the contrary to our finding, Trivedi et al. in the United States noted smoking as an independent risk factor for multidrug-resistant foot wound infection. 34

Conclusion:
The spectrum of agents causing IDFU is wide and includes numerous species of aerobic and anaerobic bacteria. There is a high prevalence of MDR aerobic bacteria among them which poses a great limitation to effective treatment of cases. Improved health seeking attitude and timely antibiotic coverage for MDR bacteria will therefore mitigate IDFU in our environment.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.