A total of 581 diabetic foot patients hospitalized in the Endocrinology Department of the Third Xiangya Hospital of Central South University from January 1, 2018, to December 31, 2021, were selected as the research subjects, and diabetic foot secretions were collected for microbial culture and drug sensitivity tests. Patients receiving antibiotics 7 days before admission were included. The author should ensure that the work described has been carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.

Patients with DFI should be treated with sterile curette or scalpel before receiving antibiotic treatment, and then clean the wound with sterile saline. The process of wound sample collection was strictly aseptic. For patients with superficial ulcers, sterilized saline cotton swabs were dipped into the secretions or pus at the bottom of the ulcer for collection, and attention was paid to avoid contamination of the skin around the wound. For patients with deep ulcer and foot gangrene, the deep ulcer secretions or pus were collected by probe after debridement. The samples were immediately sent to the laboratory microbiology laboratory for aerobic bacteria, anaerobic bacteria, fungal culture and drug sensitivity tests.

The secretions were inoculated with blood, chocolate, and MacConkey agars plates for aerobic culture. The inoculated medium was incubated in a 5% CO₂ incubator at 35°C for 24-48h. According to the morphology and gram staining characteristics of the colonies, the bacterial identification was carried out by the automatized VITEK 2 Compact system (bioMerieux, France). Kirby-Bauer, VITEK-2 Compact automatic system methods were used to test drug sensitivity. For anaerobic bacteria identification, samples were immediately inoculated on Columbia blood agars plates and Brucella agar plates, and were quickly incubated in an anaerobic incubator at 35°C for 48-72h. The distribution of specific strains was observed, and the strain types were determined with the aid of API20A kit. Drug sensitivity test was performed by using Kirby-Bauer method.

The selection of antimicrobial drugs for various types of bacterial susceptibility testing and the determination of the susceptibility results usually refer to the guidelines of the American Association for Clinical and Laboratory Standardization (CLSI) (16). MDR strains are defined according to the consensus issued by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) in 2012 (17).

The Wagner grade classification is as follows (18): 1) Grade 0: there are risk factors for foot ulceration, but no ulcer; 2) Grade 1: superficial ulcers of the feet without signs of infection; 3) Grade 2: Ulcer extension to the ligament, tendon, joint capsule, or deep fascia without abscess or osteomyelitis; 4) Grade 3: deep infection, bone lesions or abscess; 5) Grade 4:localized gangrene (toe, heel or foot); and 6) Grade 5: fully infected foot. The course of ulcer disease is based on acute and chronic wounds. Chronic ulcers refer to ulcers that have not improved after 4 weeks of treatment or have not been cured within 8 weeks (19).

All data were analyzed using SPSS 21.0 software (SPSS, Inc., Chicago, IL,USA). The measurement data are expressed as the mean ± standard deviation, and count data are expressed as a percentage [n (%)]. The chi-square test (χ2) was used for comparison between groups, and p <0.05 was considered statistically significant.

We included 704 secretion culture results from 581 DFI patients, including 65.06% (378/581) male patients and 34.94% (203/581) female patients. Among these patients, there were 573 (98.62%) patients with type 2 diabetes, 7 (1.20%) patients with type 1 diabetes, and 1 (0.17%) patient with latent autoimmune diabetes in adults (LADA).

The mean age of the patients was 61.29 ± 11.5 years, and 57.48% (334/581) of patients were aged 60 years and older. The mean duration of diabetes was 10.45 ± 6.84 years. Patients with different Wagner grades and wound conditions had different average hospital stays. The duration of hospitalization was similar between acute ulcers and chronic ulcers (χ^{2 =} 0.352, p=0.425). Of the 571 patients, 109 (19.09%) had good glycemic control (HbA1c ≤ 7%), 90 (15.76%) had normal glycemic control (HbA1c 7.1-8), and 372 (65.15%) had poor glycemic control ( Table 1 ).

Before antimicrobial treatment, a total of 534 of the 581 secretion samples (91.57%) cultured pathogens (bacteria/fungi), of which 473 (88.58%) had monomicrobial infections and 61 (11.42%) had polymicrobial infections (number of microorganisms ≥ 2). A total of 20 positive bacteria and 12 negative bacteria were cultured after antibacterial treatment. A total of 656 microorganisms were cultured, including 387 (58.99%) gram-positive organisms (GPO), 235 (35.82%) gram-negative bacteria (GNB), and 21 fungal strains (3.20%). The detection rates of microorganisms were different in different Wager grades (χ² = 9.531, p = 0.049), and multi-pathogen infections mainly occurred in patients with Wager grade 3-4 ulcers. GPO were predominant in Wagner grades 1-3 (grade 1: 96.67%, grade 2: 76.52%, grade 3 62.81%), and the most common was Staphylococcus aureus (grade 1: 31.66%, grade 2: 33.04%, level 3 35.53%). GNB were predominant in grades 4 - 5 (grade 4: 51.46%, grade 5:60%). The most common GNB in Wagner grades 1 -5 was Proteus (100% in grade 1, grade 2: 36.36%, grade 3: 32.43%, grade 4: 27.88%, grade 5: 42.86%), and the most common GPO was Enterococcus (grade 4: 34.48%, grade 5: 25.00%). The detection rate of fungi was 3.36%, mainly distributed in Wagner grade 2, including Candida albicans (33.33%), Candida tropicalis (33.33%), Candida parapsilosis (19.05%) ( Table 2 ).

The culture positivity rates of MDR, extended-spectrum-lactamase (ESBL), and methicillin-resistant Staphylococcus aureus (MRSA) were 41.31% (271/656), 12.16% (27/222), and 37.41% (52/139), respectively. The highest detection rate of MDR at Wagner grade 4 was 38.02%. The highest detection rate of ESBL at Wagner grade 2 was 18.18%. Wagner grade 1 had the highest incidence rates of MRSA at 16.67%.

The positive rate of pathogenic bacteria in the acute ulcer stage of diabetic foot was 89.45%, of which the positive rate of GPO was 59.63% and that of GNB was 23.39%. The positive rate was 93.34% in the chronic ulcer stage, of which the positive rate of GPO was 53.72% and that of GNB was 35.81%; fungi accounted for 3.86%. The most common GPO in the acute and chronic ulcer period were Staphylococcus at 43.20% and 33.85%, followed by enterococci at 11.06% and 13.08%. Proteus was the most common GNB in the acute (8.40%) and chronic ulcer stages (12.82%) ( Figure 1 ).

Staphylococcus was still highly sensitive to vancomycin, linezolid, and tigecycline. Among the 137 strains of Staphylococcus aureus, 2 strains were resistant to vancomycin and 1 strain was intermediary. Two strains were resistant to vancomycin and 3 strains were intermediary in the 37 strains of hemolytic Staphylococcus. Among 33 strains of Staphylococcus epidermidis, 1 strain was intermediary to vancomycin, and 1 strain was intermediary to tigecycline ( Table 3 ). Staphylococcus aureus maintains a high sensitivity to linezolid (100.00%) and tigecycline (100.00%), followed by vancomycin (97.81%), rifampicin (96.35%), sulfamethoxazole (91.24%), and moxifloxacin (83.94%). Other antibacterial drugs were more than 50% sensitive, including clindamycin (51.09%), tetracycline (66.96%), erythromycin (51.09%), ciprofloxacin (71.43%), and levofloxacin (70.80%).

A total of 111 methicillin-resistant Staphylococcus (MRS) strains were cultured, of which 51 strains were MRSA. MRS maintains had the highest sensitivity to linezolid (100.00%), followed by tigecycline (98.99%), vancomycin (92.98%), and rifampicin (87.72%), and had high resistance to clindamycin (35.96%), levofloxacin (30.70%), ciprofloxacin (34.31%), and erythromycin (22.21%) ( Figure 2 ). A total of 79 cases of Enterococcus were detected, of which Enterococcus faecium maintained 100% sensitivity to linezolid, tigecycline, and vancomycin and had high resistance to ampicillin (11.11%), penicillin G (11.11%), high-concentration gentamicin (28.57%), ciprofloxacin (12.50%), and levofloxacin (33.33%). Enterococcus faecalis was highly sensitive to tigecycline (100.00%), followed by linezolid (98.08%), vancomycin (95.52%), ampicillin (95.59%), penicillin G (95.38%), and high-concentration gentamicin (97.14%), and maintained more than 75% sensitivity to quinolones. Among these, two strains of Enterococcus faecalis were resistant to vancomycin and two were intermediary, and one was resistant to linezolid ( Table 3 ).

Most GNB still maintained high sensitivity to meropenem, tigecycline, ertapenem, and amikacin ( Table 4 ). The Proteus genus had the highest detection rate among GNB and the highest sensitivity to amikacin (97.14%), followed by meropenem (92%), ertapenem (80%), ceftazidime (77.27%), piperacillin and tazobactam (73.91%), levofloxacin (72.46%), aztreonam (72.46%), cefepime (71.43%), and gentamicin (71.21%) and was still highly resistant to tigecycline. A total of 26 ESBL-producing bacteria were detected, which remained 100% sensitive to tigecycline and meropenem, followed by amikacin (88.46%), piperacillin and tazobactam (84.62%), cefoxitin (80.77%), while the sensitivity to quinol was below 40% ( Figure 3 ). The sensitivities of Candida to amphotericin B, caspofungin, micafungin, voriconazole, flucytosine, fluconazole, and itraconazole were 100.00%, 95.00%, 95.00%, 95.00%, 94.44%, 80.95%, and 72.22%, respectively ( Figure 4 ).