Clinicomicrobiological Profile of Podiatric Infections: A Prospective, Cross-sectional Study

Local infections of the foot, ankle, and lower extremity are known as podiatric infections. Podiatric infections are more frequent in countries where a considerable part of the population walks barefoot.¹ Individuals with diabetes have a lifetime risk of 25% in developing a DFU, thus diabetic foot problems remain a serious public health concern and DFUs continue to be a burden worldwide.² A previous study from India revealed that cellulitis was the most common foot infection among both those without diabetes (52%) and with diabetes (40%).¹ Peripheral vascular disease, peripheral neuropathy, and trauma are some of the risk factors for DFU. DFU accounts for the majority of lower extremity amputation globally.³ Ulcer swab cultures are inferior to deep tissue and/or bone biospy because these specimens are more prone to contamination by superficial normal flora.⁴ Bone biopsy is the most effective way to identify the true pathogen. However, deep tissue samples obtained by debridement of foot ulcers have taken the place of bone biopsy culture.^3,4

There is a paucity of published data on the clinicomicrobiological profile of podiatric infections from parts of southwestern India. A previous study found Staphylococcus aureus to be the most prevalent gram-positive isolate (70.37%) and members of Enterobacterales (67%) and Pseudomonas aeruginosa (33%) to be the most prevalent gram-negative isolates.⁴

Biofilm production by bacteria is the cause of many persistent infections. Detection of biofilm assists in tailoring antibiotic therapy, optimizing treatment duration, and determining the necessity of surgical intervention, ultimately improving infection management and patient outcomes.^4,5 Therefore, the authors of the current study aimed to screen for biofilm production in these common isolates.

The ica A, C, B, and D genes have been associated with cell aggregation and biofilm development. In a study by Elboshra et al,⁵ the ica gene was the most prevalent (73.9%), followed by cna (29.2 %) and hlg (7.6%). In this context, the authors of the current study aimed to screen for the common virulence gene carriage in S aureus for epidemiological purposes. 

Medical professionals and clinical microbiologists in developing countries are equally concerned about the growing threat posed by MDR organisms. Hence, the authors of this study evaluated the microbiological profile, antibiogram, treatment, and clinical outcomes of podiatric infections to formulate a comprehensive treatment plan, especially in patients with diabetes. 

Inclusion and exclusion criteria

This was a prospective, cross-sectional study of patients treated from December 2021 through May 2022 in the Department of Microbiology, Kasturba Medical College, Mangalore, a tertiary care center in South India. Culture-positive isolates from swabs and tissue (deep tissue, bone tissue, or both) from patients with podiatric infections were included. Any specimen from a wound infection other than that of the foot and all other specimens from podiatric infections with no growth in culture were excluded. 

The isolates underwent phenotypic identification using a proprietary microbial identification system (VITEK 2; bioMérieux, Inc) and conventional standard biochemical identification. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method and a proprietary system (VITEK 2 Compact; bioMérieux, Inc) as per the 2021 Clinical and Laboratory Standards Institute performance standards.⁶ The organisms isolated and antibiogram were noted in Excel (Microsoft). Clinical and other laboratory data were collected from medical records. 

Enterobacterales, P aeruginosa, and S aureus were screened for biofilm production by phenotypic method, and all the S aureus isolates were screened for carriage of virulence genes.

Biofilm detection was done using the microtiter plate method.⁷ This assay involves staining the biofilm with gentian violet and measuring the amount of dye bound to the biofilm, which provides an indirect measurement of the amount of biofilm present. A sterile 96 well flat-bottomed polystyrene microtiter plate was used, and 20 µL of overnight bacterial culture broth and 230 µL of trypticase soya broth were added to the wells in triplicate. The well with only broth was used as the negative control. The plates were then incubated aerobically for 24 hours at 35 °C. The wells were emptied of their contents and washed 3 times with 300 µL of sterile distilled water. Methanol 250 µL for 15 minutes was used to fix the bacteria adhering to the wells, and 250 µL of 1% solution of gentian violet for 5 minutes was used to stain the wells. The wells were washed to remove the excess stain and air-dried. The dye bound to the wells was solubilized with 250 µL of 33% (v/v) glacial acetic acid. An enzyme-linked immunosorbent assay auto-reader was used to measure the OD of each well at 490 nm. The average of the results of the tests carried out in triplicate were noted. The cutoff OD was determined as 3 standard deviations above the mean OD of the negative control. Strains were classified as biofilm producer and non-biofilm producer.⁷

Multiplex PCR was done for the detection of genes of virulence (ica, cna, and hlg) in S aureus isolates. DNA was extracted from fresh isolates of S aureus by the boiling method wherein 2 to 3 colonies are suspended in 250 µL nuclease-free water, boiled at 100 °C for 5 minutes, and centrifuged at 10 000 rpm for 8 minutes. Supernatant containing DNA was collected and processed. PCR conditions were as follows: denaturation at 94 °C for 30 seconds, followed by 40 cycles of annealing at 50 °C for 30 seconds, 72 °C for 30 seconds, and 72 °C for 40 seconds, with a final extension step at 72 °C for 5 minutes as recommended by Elboshra et al.⁵ The PCR primer sequence and amplicon size are described in Table 1.

Statistical analysis

The statistical analysis was carried out using SPSS version 25.0 (IBM Corporation). The results were analyzed using crosstabulation and the chi-square test, and a P value of less than .05 was considered statistically significant.

A total of 200 specimens of bone tissue, deep tissue, and swab were collected from 117 patients with suspected podiatric infections. Specimens from 71 patients showed bacterial growth (60.6%), and specimens from 46 patients (39.4%) showed no growth. Of the 71 patients, 61 were male (86%) and 10 were female (14%). Demographic distribution is shown in Table 2. 

A total of 120 bacteria were identified from podiatric patients, of which the majority were from deep tissue (n = 75 [62.5%]), followed by bone tissue (n = 12 [10%]) and swab (n = 33 [27.5%]). The spectrum of organisms was studied. Of the 71 culture-positive cases, 15 (21.1%) were polymicrobial and 56 (78.9%) were monomicrobial. Of the total 120 isolates, 32 (26.7%) were gram-positive bacteria and 88 (73.3%) were gram-negative bacteria. S aureus was the most common bacteria isolated (n = 32 [26.7%]), followed by P aeruginosa (n = 29 [24.2%]), Proteus mirabilis (n = 19 [15.8%]), Klebsiella pneumoniae (n = 12 [10%]), Escherichia coli (n = 8 [6.7%]), Citrobacter sp (n = 7 [5.8%]), Enterobacter cloacae complex (n = 5 [4.2%]), Proteus vulgaris (n = 4 [3.3%]), Morganella sp (n = 3 [2.5%]), and Aeromonas sp (n = 1 [0.83%]).

Antimicrobial resistance pattern was noted, as shown in Table 3. Of the 120 organisms isolated, 29 (24.2%) were MDR strains and 65 (54.2%) isolates were biofilm producers. P aeruginosa was the most prevalent biofilm producer (82.8%), followed by P mirabilis (78.9%), Enterobacter sp (60%), Citrobacter sp (57.1%), and S aureus (53.1%). It was found that 90.9% of the MRSA isolates and 28.6% of the MSSA isolates were biofilm producers. Up to 50% of the K pneumoniae isolates produced biofilms. The other biofilm producers were P vulgaris (50%), E coli (37.5%), and Morganella sp (33.3%). 

Genotypic characterization of virulence genes of S aureus was done using multiplex PCR. None of the isolates of S aureus (n = 32) harbored the virulence genes ica, cna, and hlg. 

There were 62 patients (87.32%) with diabetes, and 9 patients (12.67%) without diabetes. The mean duration of diabetes was 12.85 years ± 8.61 standard deviation. Twenty-six patients with diabetes underwent amputation owing to uncontrolled and progressive podiatric infection–related complications. Other risk factors for podiatric infection noted in this study were hypertension, peripheral vascular disease, steroid therapy, and hemodialysis (Table 4). A statistically significant association was found between biofilm formation and MRSA, MDR, and prior antibiotic therapy with multiple (≥4) antibiotics (P < .05), as shown in Table 5.

Infections of the foot involve complex pathogenesis with varying clinical presentation. Management requires early expert evaluation. An increased level of outdoor physical activity in a hot, humid environment with inadequate and improper foot care among males may contribute to the higher incidence of podiatric infections in males than in females.^1,7 The current study revealed a male preponderance similar to other previous studies.^8,9

In the current study, 21.1% of the podiatric infections were polymicrobial and 78.9% were monomicrobial. This is similar to other studies, which show a predominance of monomicrobial growth over polymicrobial growth in patients with DFU.^9,10 

Of the 120 isolates in the current study, 73.3% (n = 88) were gram-negative bacteria and 26.7% (n = 32) were gram-positive bacteria. These findings are similar to those of Seth et al,⁸ in which 71.4% of isolates were gram-negative bacteria and 28.6% were gram-positive bacteria. Similarly, Banu et al⁷ reported that 24.4% of organisms were gram-positive and 75.6% gram-negative. However, Vidhya Rani and Nithyalakshmi¹¹ showed a higher proportion of gram-positive organisms. Environmental factors such as sanitary habits could contribute to the increased incidence of infections due to gram-negative bacteria, especially in developing countries.¹² 

In this study, S aureus and P aeruginosa were the most common organisms (26.6% and 24.2%, respectively), followed by P mirabilis (15.8%). These findings agree with the results reported by Kaye Bennett et al.¹³ Two studies have reported E coli to be the predominant gram-negative organism, followed by P aeruginosa.^4,7 In the current study, Aeromonas sp was isolated from a single specimen. Case reports by Talan et al¹⁴ and Larka et al¹⁵ show that Aeromonas hydrophila-associated skin and soft tissue infection is rare and may occur owing to exposure of abraded skin to contaminated water or soil.

The rate of MRSA in the current study was 34.4%. According to previously published studies, the prevalence of MRSA in DFU varies from 10.6% to 77.8%.¹⁶ In the current study, the rate of clindamycin resistance was lower in MRSA (60%) compared with MSSA (89.9%). MRSA strains showed a higher rate of resistance to co-trimoxazole (45.4%), gentamicin (36.4%), and linezolid (18.29%) compared with MSSA strains, which showed a lower rate of resistance to fluoroquinolones (47.6%), co-trimoxazole (14.3%), gentamicin (19.04%), and linezolid (0%). This is similar to the study by Mukherjee et al,¹⁶ which showed that MRSA was also resistant to fluoroquinolones, aminoglycosides, erythromycin, clindamycin, and co-trimoxazole. 

The current study reveals that S aureus isolates were sensitive to vancomycin (100%) and linezolid (81.71%). This is similar to previously published studies in which vancomycin and linezolid resistance was not detected in S aureus isolates from patients with DFUs.^4,7,17,18

Most of the Enterobacterales in the current study showed a high level of resistance to commonly used antibiotics: ampicillin (62.7%), cefuroxime (70.7%), and ciprofloxacin (65.1%). These findings are consistent with those of Banu et al.⁷ In the current study, resistance was lower to amikacin (7.92%), gentamicin (19.2%), cefoperazone-sulbactam (25.7%), piperacillin/tazobactam (35.1%), and meropenem (19.9%). Hence, aminoglycosides, cefoperazone-sulbactam, piperacillin/tazobactam, and carbapenems can be used as reserve drugs based on the antibiotic susceptibility reports. This corresponds to the findings of Banu et al⁷ and Otta et al.¹⁷ 

In the current study, P aeruginosa showed varying rates of resistance to the common antipseudomonal drugs: ciprofloxacin (68.97%), cefoperazone-sulbactam (48.28%), piperacillin/tazobactam (44.83%), ceftazidime (41.38%), imipenem (37.9%), gentamicin (34.48%), amikacin (37.93%), cefepime (20.69%), and ticarcillin-clavulanic acid (20.69%). These results are on par with the findings of Bilal Tanvir et al.¹⁸ The varying rate of antimicrobial resistance may be due to extensive use of antibiotics, selective action of the disinfectants and antibiotics, or potential ability of an organism to produce biofilm.^19-21 

Al-Hamead Hefni et al²² found that vancomycin was an effective drug of choice against gram-positive infection, whereas imipenem and amikacin were the most effective against gram-negative infection.

An intact epidermis acts as a barrier against microbial invasion. The protective epidermal barrier is breached by wounds, which allows microbes to invade deeper tissues; microbial invasion into deeper tissue is associated with wound biofilm. These cells then closely bind to one another and produce a matrix that makes it easier for them to overcome dietary intolerance and antimicrobial attacks. Biofilms in deep tissues can best be detected in vitro using biopsy specimens. However, swabs are not recommended because of the risk of contamination by skin flora and lack of anaerobic organisms.^7,23,24 

In the current study, 54.2% of the isolates produced biofilm, compared to the studies of Banu et al (46.3%)⁷ and Shettigar et al (45%).⁴ Increased percentage of biofilm formation may be due to inefficient debridement or a wound persisting for a longer period. P aeruginosa was the predominant biofilm producer in the current study (82.8%). This result corresponds to the findings of Premanath et al,²⁵ who reported that 89% of P aeruginosa isolates were biofilm producers. The other common biofilm producers in the current study were P mirabilis (78.9%), Enterobacter sp (60%), Citrobacter sp (57.1%), and S aureus (53.1%). These findings correspond to those reported by Shettigar et al.⁴ In the current study, up to 90.9% of MRSA isolates were biofilm producers, and there was a statistically significant association between biofilm production and MRSA (P < .05). This supports the importance of biofilm detection in MRSA isolates, because biofilm may be associated with persistent infections. 

Studies have shown that biofilm-producing microorganisms may be 1000 times more resistant to antibiotics compared with free-floating planktonic bacteria.⁷ In the current study, 29 isolates (24.2%) were MDR and up to 93.1% of the MDR isolates were biofilm producers. These findings contrast with those reported by Banu et al,⁷ in which 46.3% of MDR isolates were biofilm producers. Significant association (P < .05) was found between biofilm formation and MDR and also between biofilm formation and prior antibiotic therapy with multiple (≥4) antibiotics (P < .05). This could be attributed to the selective pressure induced by the extensive use of antibiotics, which gives the survival advantage to the pathogenic bacteria.²² 

The common virulence genes of S aureus, that is, ica (intracellular adhesin), cna (collagen-binding protein), and hlg (γ-hemolysin), were not detected in the current study. A similar result was reported by Elboshra et al,⁵ which showed absence of cna gene in MRSA isolates. de Almeida et al²⁶ reported absence of all 3 genes in S aureus isolated from flocks of sheep and concluded there was no prevalence of these genes in S aureus isolates. In the current study, the molecular detection of the virulence gene yielded negative results. The relatively infrequent expression of the cna gene among clinical isolates of S aureus²⁷ and the limited sample size could be contributing factors to the negative results observed. 

DFUs are associated with chronic wound infections by biofilm producers. Diabetic foot is a complication that leads to hospitalization among patients with diabetes.^7,28 In the current study of 71 patients, 62 (87.32%) had diabetes and 9 (12.67%) did not. There is a 15-times greater risk of lower extremity amputation in patients with diabetes compared with those without diabetes.⁷ In the current study, 26 patients with diabetes underwent amputation and there was no significant association between biofilm formation, rate of amputation, and length of hospital stay. Interestingly, a significant association was found between biofilm production and prior antibiotic therapy with multiple (≥4) antibiotics (P < .05). Thus, there is a high likelihood of treatment failure with a single drug regimen in the setting of biofilm producers. 

Apart from diabetes, the other risk factors for podiatric infections noted were hypertension, peripheral vascular disease, steroid therapy, and hemodialysis. Evaluation of these potential confounders is crucial owing to their effect on the ongoing infection and response to therapy, because they might result in significant morbidity and increased duration of hospital stay.

The main limitation of this study is the sample size. The strength of the study findings could have been improved had the study duration been increased to include a greater number of samples and to include periodic follow-up of patients with podiatric infections.

In the current study, the rate of culture positivity of a podiatric specimen was 60.6%, with a predominance of gram-negative (73.3%) and monomicrobial (79.9%) infections. Of the 120 total isolates, S aureus (26.6%) and P aeruginosa (24.2%) were the most common organisms and up to 54.2% of the isolates showed biofilm production. P aeruginosa was the predominant biofilm producer (82.8%). Up to 90.9% of the MRSA isolates and 93.1% of the MDR isolates produced biofilm. There was a significant association between biofilm formation and MRSA, MDR, and prior antibiotic therapy with multiple (≥4) antibiotics. Diabetes was a major risk factor in the podiatric patients. Hence, isolation of MRSA or MDR strain from diabetic foot infections could alert the clinician to the possibility of treatment failure with a single drug regimen due to associated biofilm production. Planning for early debridement and/or cleaning of wounds, special wound care, and effective treatment strategies could prevent the development of persistent infections. Thus, the current study emphasizes the importance of detection of biofilm and evaluation of associated risk factors to minimize the severity and effect of podiatric infections in patients with and without diabetes.