Setting and patients. The study was conducted in the Departments of Microbiology and General Surgery associated with a tertiary care university hospital in North India. All patients with a DFU who were enrolled at the hospital’s wound clinic/completed the registration form and treated were eligible to participate if they met the following inclusion criteria: age 18 to 80 years, clinical diagnosis of infection at the time of presentation, DFUs of >6 weeks’ duration (suggesting chronic nature), and not on antibiotics for at least 2 weeks (antibiotics significantly alter and modify bacterial flora). Clinical suspicion of infection was determined by the presence of 1 or more factors: exudate, redness, discolored ulcer edges, pain, local edema, and foul odor. Patients who were pregnant and patients diagnosed with malignancy or osteomyelitis, sinus leading to bone, an ulcer involving the toe or gangrenous area, and systemic sepsis relating to conditions with severity or risk of increased infection were excluded. Written informed consent was obtained from all participants and 1 family member. The study was approved by the institution’s ethics committee.
Data collection and clinical assessment of the ulcers. Patient demographic data (including age, gender) and clinical history (chief complaints, past medical history, current medication) of diabetes were collected from every participant based on a pretested, predesigned questionnaire. Details such as name and contact information were avoided to maintain patient anonymity; data were recorded using codes. Information regarding the ulcers included anatomical location, surface area calculated by multiplying the greatest length with the perpendicular greatest width as measured by ruler, ulcer duration, and duration of diabetes; digital images were taken at the first visit for assessment and documentation. Relevant laboratory investigations to ascertain glycemic control, anemia, liver function (measurement of levels of serum glutamic-oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatise, and serum bilirubin), and renal function (serum urea, creatinine and uric acid) were noted. The data were accessible only to the research team of this study.
Sample collection and wound culture. Samples were collected both by Levine’s technique11 and needle aspiration. Although tissue biopsy could have provided both qualitative and quantitative data, this invasive technique was not applied due to the added risk for wound infection. Instead, the swab technique was employed, which has been reported in a systematic review12 to provide results comparable to biopsy without being invasive.
Before obtaining the sample, the wound was cleansed with sterile gauze moistened with sterile normal saline to remove surface contamination. Subsequently, using Levine’s technique, a sterile cotton swab was rotated over a 1 cm2 area for 5 seconds with sufficient pressure to cause tissue fluid to be expressed. The tip of the swab was broken off in a sterile transport media (phosphate-buffered saline [PBS]) and transported immediately to the hospital’s microbiology laboratory. Tissue fluid also was collected by sterile syringe in sterile vials and sent for culture. The samples were divided into 2 parts — one for quantitative aerobic bacterial culture and the other for detection of common anaerobes by polymerase chain reaction (PCR).
The laboratory processed the samples per protocol. All samples were gram stained for direct examination. For culture, cotton swabs in PBS were thoroughly vortexed before quantitative culture and then serially diluted in normal saline and plated on MacConkey’s agar media and blood agar media (Hi Media Pvt Labs, Mumbai, India). Tissue fluid was processed in the same manner. After overnight incubation, the plates were visually inspected and colonies of each type of bacteria were counted. Plates with 50 to 500 colonies were considered readable and the exact number of colonies was counted.13 CFUs then were calculated to determine the total bacterial count on each plate using the formula:
CFU/mL = (number of colonies x dilution factor)/volume of culture plate.
CFU >1 x 105 was considered critical colonization/infection and counts below were considered benign colonization.11
Bacterial colonies that grew aerobically were identified and reported using standard biochemical tests.14 Gram stain findings and swab culture of tissue fluids were corroborated for uniformity.
PCR for detection of anaerobes. PCRs were performed to detect common anaerobes (ie, Bacteroides, Clostridium, Peptostreptococcus, Peptococcus, and Ruminococcus) because an anaerobic culture facility was not available. These anaerobes were selected for study based on their predominance in DFUs from previous research.4,6,7,10 DNA was isolated from the tissue fluid samples using QIAamp DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The quality of the DNA was measured by NanoDrop analysis (Thermo Fisher Scientific, Waltham, MA). PCR amplifications were performed by using primer sets from a previous study with slight modifications.15 The primer sequences and the reaction conditions are described in Table 1.14,15
16SrRNA gene sequencing. Samples that did not yield/demonstrate any aerobic or anaerobic flora were subjected to 16S ribosomal RNA (16SrRNA) PCR and sequencing16 to confirm whether they were bacteriologically sterile (see Table 1).14,15 The sequences from 16SrRNA PCR positive samples were analyzed using the mothur package version 1.23.17 Based on instructions, the sequences were screened and aligned to the SILVA reference set of NAST aligner of mothur for identification of the bacteria. Bacterial identification was done based on designated operational taxonomic units of the sequences.
Antibiotic susceptibility testing. The modified Kirby-Bauer disc diffusion method was used to test antibiotic susceptibility of the aerobic isolates per the 2017 Clinical and Laboratory Standards Institute guidelines.18 The antibiotics used in this study are listed in Table 2. Isolates showing resistance to ≥1 antimicrobial agent in ≥3 antimicrobial categories were considered multidrug resistant (MDR), and resistance to ≥1 antimicrobial agent in all but ≤2 antimicrobial categories were included as extensively drug resistant (XDR).19
Data and statistical analysis. All patients with clinical signs of infections and positive for microorganisms were divided into 2 groups based on culture and PCR results. One group comprised cases with 1 type of microflora (monomicrobial) and the other group comprised of cases with more than 1 type of microflora (polymicrobial). Quantitative variables were denoted by mean ± standard deviation, and qualitative variables were shown as percentages. The clinical parameters of the DFU were correlated with the monomicrobial and polymicrobial culture results by chi-squared test using SPSS, version 19 (IBM Corporation, Armonk, NY). Multiple logistic regressions were used to identify independent predictors of monomicrobial and polymicrobial infections. Comorbid factors (eg, anemia and abnormalities in liver and renal function) were not considered for analysis. The critical value of P, indicating the probability of significant difference, was taken as <.05 for comparison.