In total, 50 diabetic patients with DFU were included in the study between January 21, 2013 and July 20, 2013. The study was approved by the Ethics Committee and Institutional Review Board of the Atta-ur-Rahman School of Applied Biosciences (Islamabad, Pakistan). Patients of all ages and genders with type 2 diabetes with a foot infection or DFU who visited the Diabetic Foot Clinic and Out-Patient Department of Pakistan Institute of Medical Sciences (Islamabad, Pakistan) were included. The exclusion criteria were as follows: Non-diabetic patients with foot infection, diabetic patients that had previously received antibiotic treatment, patients with type 1 diabetes with foot infection, and DFUs with a duration of >3 weeks. Information on the duration of the diabetes and DFUs, glycemic control, and history of previous hospitalization due to the DFU was obtained from all patients. The patients were examined for the presence of sensory neuropathy and peripheral vascular disease by measuring the ankle brachial index. The foot ulcers were classified according to the New University of Texas classification system (28).

Cultures of the specimens were obtained at the time of admission, after the surface of the wound had been washed vigorously with saline, followed by debridement of the superficial tissue from the exudates to avoid the isolation of colonizing flora. Bacteria were isolated through the inoculation of specimens on a set of selective and non-selective media, such as blood agar and MacConkey and chocolate agars (Difco; BD Biosciences, Franklin Lakes, NJ, USA). All inoculated plates were incubated at 37°C for 24–48 h. The bacterial isolates were identified using conventional biochemical tests.

The Kirby-Bauer disk diffusion method (29) was used for antibiotic susceptibility testing. The Clinical and Laboratory Standard Institute (CLSI) guidelines were followed for the selection of media, inoculum turbidity and preparation of media plates along with the application of disks and the interpretation of the zone of inhibition. Suspension was inoculated on the media plate with the assistance of a sterile glass spreader. Oxoid™ antibiotic disks (Thermo Fisher Scientific, Inc., Waltham, MA, USA) were applied using sterile forceps. The zone of inhibition around the tested antibiotics was measured, and interpretations were made using the breakpoints elaborated in the CLSI guidelines (30).

Isolates that showed intermediate resistance to third-generation cephalosporin were screened to detect ESBL production. A double disk synergy test (DDST) was performed for the phenotypic detection of ESBL production (31). A third-generation cephalosporin disk was placed on the plate with another disk containing amoxicillin + clavulanic acid; other combination disks, such as ampicillin + salbactam and piperacillin + tazobactam, were also tested. Plates were incubated at 37°C for 18–24 h and the shape of the zone of inhibition was noted. Isolates that exhibited a distinct potentiation towards the amoxicillin + clavulanic acid disk were considered potential ESBL producers. Escherichia coli (E. coli) ATCC 25922 and Klebsiella pneumoniae (K. pneumoniae) ATCC 700603 were used as negative and positive controls for ESBL, respectively. Phenotypic detection of MBLs was performed using an ethylenediaminetetraacetic acid (EDTA) disk synergy test (32). For this, an imipenem disk was placed on a plate inoculated with bacterial suspension, and 5 µl 0.5 M EDTA was poured on another imipenem disk and placed on the same plate; differences in the size of inhibition zone were then observed.

For the molecular detection of antibiotic resistance genes, the phenol-chloroform method was used to extract DNA from bacterial samples (33). Bacterial cells were grown in Luria Bertani broth overnight at 37°C and suspended in lysis buffer (0.2 mg/ml proteinase K in 1% sodium dodecyl sulfate). The suspension was then incubated for 1 h at 55°C and DNA was extracted twice using phenol-chloroform. Subsequently, sodium acetate (0.3 M) and cold ethanol were added to precipitate DNA. The precipitate was centrifuged (Sigma 1–14; Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) at 14,462 × g for 2 min at room temperature, after which the DNA pellet was suspended in 100 µl Tris-EDTA buffer. mecA, β-lactamase (bla)-SHV, bla-CTX-M, bla-CTX-M-15, bla-TEM and bla-oxacillinase (OXA) genes were detected using polymerase chain reaction (PCR). For PCR, a total reaction mixture volume of 25 µl was prepared containing 2 mM deoxyribonucleotide triphosphates, 10X PCR buffer, 50 mM MgCl2, 50 pM of each primer, 5 µl DNA sample, 1 unit of thermo stable Taq DNA polymerase (Fermentas; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and nuclease-free water to adjust the final volume. The reaction mixture was centrifuged (Sigma 1–14; Sigma Laborzentrifugen GmbH) at 14,462 × g for 30 secs at room temperature for thorough mixing. Subsequently, PCR was conducted using the Swift™ MaxPro thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, USA). The primer sequences are presented in Table I (34–37). The PCR products were separated by 1% agarose gel electrophoresis, in which a 100 bp DNA ladder was used as a reference (Invitrogen; Thermo Fisher Scientific, Inc.).

A total of 50 patients fulfilled the inclusion criteria and were included in the study. Out of those 50 subjects, 29 (58%) were men and 21 (42%) women (age range, 36–80 years; mean age, 58 years). According to the University of Texas Classification, 11 patients had stage-B grade-I ulcer, 16 had stage-B grade-II ulcer, 7 had stage-B grade-III ulcer, 2 had stage-C grade-I ulcer, 1 had stage-C grade-II, 1 had stage-D grade-I ulcer, 5 had stage-D grade-II ulcer and 7 had stage-D grade-III ulcer (Table II). The specimens containing clinically significant pathogens included wound swabs (37/50; 74%), tissue (7/50; 14%), pus (4/50; 8%) and bone (2/50; 4%). Of the 50 patients, 46 patients (92%) had sensory neuropathy while 4 patients (8%) did not (Fig. 1).

A total of 99 bacterial isolates were obtained from the 50 patients with DFUs. In these patients, gram-negative bacilli (59.59%) were isolated more frequently than gram-positive cocci (40.4%). The most common isolate was S. aureus (25%), followed by Pseudomonas aeruginosa (P. aeruginosa; 18.18%) and E. coli (16.16%). The other isolated organisms were Streptococcus spp. (15.15%), Enterococcus spp. (9%), Proteus spp. (15.15%) and K. pneumoniae (3%) (Fig. 2). Single organisms were isolated from 14 samples (28%) and mixed bacterial growths were identified in 36 samples (72%). The details of the organisms isolated from the infected foot lesions are presented in Fig. 2. The mean number of isolates per culture-positive sample was 1.9.

All S. aureus isolates were resistant to penicillin, out of which 90% exhibited resistance against oxacillin, cefoxitin and ceftazidime. Vancomycin showed inhibitory effects for only 20% of the S. aureus isolates. By contrast, 100% of the Streptococcus spp. isolates exhibited resistance to oxicillin, cefotaxime, ceftriaxone, cefepime, cefoxitin, ceftazidime and penicillin, while 40% showed susceptibility to vancomycin. According to the sensitivity data, 70% of the S. aureus and 80% of Streptococcus spp. isolates were multidrug-resistant (Table III). All P. aeruginosa and K. pneumoniae isolates, as well as 83.33% of the Proteus spp. and 75% of the E. coli isolates were found to be resistant to ceftazidime. All gram-negative isolates exhibited high susceptibility to chloramphenicol and meropenem. K. pneumoniae and P. aeruginosa were found to be the most resistant, with >60% of strains exhibiting antibiotic resistance, whereas for Proteus spp. and E. coli, <55% of strains were resistant (Table IV).

The phenotypic prevalence of the mecA gene was found to be 88%. No inhibition zone was observed around the 30-µg cefoxitin disk. Out of 25 S. aureus strains, 21 were found to be positive for the mecA gene PCR, with an overall prevalence of 84%. DDST results showed that 66.66, 33.33, 66.7 and 50% of the Proteus spp., P. aeruginosa, K. pneumoniae and E. coli populations, respectively, were ESBL producers. In the case of the cefotaxime + clavulanic acid/ceftazidime + clavulanic acid combination disk test for ESBL detection, a >5-mm increase was observed in the size of the zone of inhibition around the cefotaxime or ceftazidime disk containing the ESBL inhibitor clavulanic acid, as compared with the simple cefotaxime or ceftazidime disk. According to the cefotaxime + clavulanic acid/ceftazidime + clavulanic acid combination disk test, K. pneumoniae and E. coli had the highest prevalence (100%) of ESBL producers, while the EDTA disk synergy test confirmed P. aeruginosa and K. pneumoniae as having the highest prevalence of MBL producers; E. coli was found to have the lowest prevalence, with only 37% of strains producing MBL (Table V).

PCR showed that all K. pneumoniae and E. coli isolates were positive for bla-CTX-M, bla-CTX-M-15, bla-TEM, bla-OXA and bla-SHV resistance genes (Fig. 3). In addition, 72.2% of the P. aeruginosa strains were positive for bla-SHV and 66.7% for bla-CTX-M, bla-CTX-M-15 and bla-TEM. However, only 33.3% of the P. aeruginosa strains were positive for the bla-OXA gene. In Proteus spp., the resistance gene with the highest prevalence (80%) was bla-SHV (Table VI).