| dc.description.abstract | Background and study purpose
Type II diabetes mellitus is highly prevalent worldwide (approximately 6.3-10.5% of the global population), and in the population of the Republic of Ireland (9.5%), and continues to increase in prevalence. Diabetic foot ulcers (DFUs) and DFU infections (DFUIs) are complex and costly co-morbidities, affecting between 15-25% of people with diabetes in their lifetimes, and often leading to lower exteremity amputations. Staphylococcus aureus, a bacterial species highly prevalent in the oro-nasal cavity, is the predominant cause of DFUIs. The oro-nasal prevalence of this S. aureus is higher in people with periodontal disease (PD), an oral condition that is twice as prevalent in people with diabetes. This study sought to investigate the role of the oro-nasal cavity as a microbial reservoir for DFUIs and to determine if the electrochemically activated (ECA) disinfectant solution anolyte has potential as a novel therapeutic agent for treating DFUIs.
Methods
Ethical approval for all aspects of this study was granted by the Tallaght University Hospital (TUH) and St. James?s Hospital (SJH) Joint Research Ethics Committee (REC) in January, 2020. Debrided DFU tissues were collected from consenting participants undergoing routine debridment therapy at the Diabetes Day Centre, St. James? Hospital, Dublin. Collected tissue samples (N=51) were divided into two equal aliquots by wet weight and comparatively subjected to immersion in either sterile saline or 200 parts per million (ppm) anolyte for three minutes. The first 20 samples were treated in 1 ml volumes and the remaining 31 were comparatively treated in 10 ml volumes to determine if higher anolyte volumes further improved disinfection efficacy. Following immersion, the microbial bioburdens of anolyte-treated and saline-treated tissues were compared following culture on Columbia blood agar (CBA) under aerobic and anaerobic conditions and on the staphylococcal-selective chromogenic agar (SaSelectTM) under aerobic conditions. Microbial counts were recorded in colony forming units per gram (CFU/g) of tissue.
To explore the oro-nasal cavity as a potential microbial reservoir for DFUIs, consenting participants with type II diabetes with (N=76) and without (N=76) DFUs, underwent periodontal and DFU (if present) examination and clinical sample collection including saline oral rinse, sampling of two periodontal pockets, and a nasal, finger, toe and, if present, an ulcer swab. Oral and ulcer examinations were undertaken by a calibrated dental hygienist and podiatrist, respectively. Participants also described their oral hygiene routines using a self-reported survey. Staphylococci were recovered and presumptively identified on SaSelectTM, and subsequently definitively identified by Pastorex-plus Staph Plus (Bio-Rad) latex agglutination testing, species- specific PCR, 16S rRNA sequencing and Matrix Assisted Laser Desorption Ionization Time-of-Flight-Mass Spectrometry (MALDI-TOF-MS) analysis. A selection of S. aureus (N=371) and Staphylococcus epidermidis (N=337) isolates chosen as representatives of distinct anatomical sites from participants both with and without DFUs underwent whole genome multilocus sequence typing (wgMLST) to examine the population structure of isolates recovered and to determine if oro-nasal and ulcer isolates were closely-related (?24 wgMLST allelic differences), suggestive of endogenous transmission between these anatomical sites in the same individual.
Results
Significant reductions (P<0.01) were observed in the average bacterial load of 1 ml anolyte-treated tissues following CBA aerobic (227.7-fold [saline: 1.40?106 ? 5.28?105 CFU/g, anolyte: 5.11?104 ? 3.54?104 CFU/g]), CBA anaerobic (74.7-fold [saline: 9.23?105 ? 3.03?105 CFU/g, anolyte: 2.49?104 ? 9.14?103 CFU/g]) and SaSelectTM agar aerobic cultures (1605.6 -fold [saline: 6.29?105 ? 2.38?105 CFU/g, anolyte: 2.42?104 ? 1.48?104 CFU/g]). Further highly significant (P<0.01) fold reductions in average bacterial load (CFU/g) were observed in 10 ml anolyte treated tissues following CBA aerobic (12,243.6-fold [saline: 8.78?105 ? 3.50?105 CFU/g, anolyte: 2.86?104 ? 2.11?104 CFU/g]), CBA anaerobic (2747.2-fold [saline: 9.14?105 ? 4.68?105 CFU/g, anolyte: 4.13?104 ? 3.43?104 CFU/g]) and SaSelectTM agar aerobic cultures (9656.9-fold [saline: 1.67?106 ? 6.79?105 CFU/g, anolyte: 1.98?104 ? 1.33?104 CFU/g]). Staphylococcus aureus was the predominant organism recovered from tissues (30/51 tissue samples, 58.8%), of which 50 isolates representative of the 30 patients underwent wgMLST. The predominant sequence types (STs) identified included ST5 (N=18), ST1 (N=9) and ST15 (N=6). No methiciliin-resistant S. aureus (MRSA) or Panton-Valentine Leukocidin toxin (PVL)-positive isolates were detected in debrided tissue. Highly related isolates were recovered from nine separate patients who attended the same clinic an average of four weeks apart (range 1-16 weeks) between 2020-2023, indicative of transmission. However, as participants attend the DFU clinic weekly and participated in the study only once, definitive epidemiological data to support
transmission indicators was not available. Interestingly, 42/51 tissue samples (82.4%) yielded >105 CFU/g from saline-immersed samples. This is the threshold of microbial bioburden used to define of infection of DFUs and impede wound healing. However, in 39/51 (76.4%) of the patients sampled, there were no DFU clinical signs of infection suggesting that the clinical indicators used are not sufficiently sensitive for DFUI diagnosis.
Staphylococcus aureus was significantly more prevalent across all anatomical sites in patients with DFUs (nares [NS] 36/76 [47.4%], oral cavity [OR] 35/76 [46.1%], periodontal pockets [PPs] 13/54 [24.1%], finger [F] 10/76 [13.2%], toe [T] 11/76 [14.5%], ulcer [U] 38/76 [51.3%]) than participants without DFUs (NS 26/76 [34.2%], OR 25/76 [32.9%], PPs 9/76 [11.8%], F 4/76 [5.3%], T 3/76 [3.9%]). Staphylococcus aureus was identified in both the oro-nasal cavity and DFUs of 26/76 (34.2%) patients. Staphylococcus epidermidis was the most prevalent species identified overall in both participant cohorts. This species was also more prevalent in patients with DFUs (NS 45/76 [59.2%], OR 56/76 [73.6%], PPs 34/76 [62.9%], F 30/76 [39.5%], T 19/76 [25.0%], U 10/76 [13.2%]) than in those without DFUs (NS 39/76 [51.3%], OR 36/76 [47.4%], PPs 27/76 [35.5%], F 17/76 [22.4%], T 16/76 [21.1%]). Staphylococcus epidermidis was identified in both the oro- nasal and DFUs of 10/76 (13.2%) patients.
In total, 371 S. aureus isolates from patients with (N=266) and without (N=105) DFUs underwent wgMLST. The predominant spa types identified were t127 (N=43), t363 (N=19) and t230 (N=18) from patients with DFUs, and t084 (N=22), t908 (N=21), and t349 (N=19) for participants without DFUs. The predominant STs identified in isolates from DFU patients were ST1 (N=46), ST30 (N=33) and ST15 (N=29), and participants without DFUs included ST45 (N=32) and ST199 (N=17), typical of methicillin-susceptible S. aureus populations within Irish hospitals. Only one DFU patient yielded MRSA. PVL-positive isolates (N=35) were recovered from multiple anatomical sites of nine DFU patients. Of the 26 patients from whom S. aureus was recovered from both oro-nasal and DFUs, 19/26 (73.1%) of these paired isolates were indistinguishable or very closely related (median average: two allelic differences), indicative of endogenous transmission between anatomical sites. The majority of the oro-nasal isolates that were indistinguishable or very closely related to DFU isolates from the same patient were recovered from the nares (14/19 [73.6%]) indicative of a natural nasal niche for this species.
In total, 327 isolates of S. epidermidis were investigated by wgMLST from patients with (N=224) and without (N=103) DFUs. The predominant arginine catabolic mobile element (ACME) types identified were II (N=71), I (N=35) and V (N=11) from DFU patients, and III (N=61), II (N=43), I (N=35) from participants without DFUs. The S. epidermidis population was highly diverse. The predominant STs amongst DFU patients were ST5 (N=33), ST2 (N=21), ST87 (N=18) and ST35 (N=14), and participants without DFUs included ST35 (N=22), ST73 (N=10), ST218 (N=6) and ST153 (N=4). Interestingly, ST73 isolates were identified in 13 patients with (N=5) and without (N=8) DFUs. These were predominant in the oro-nasal cavity (19/23 [82.6%] from 13 patients) indicating possible enrichment of this ST in oro-nasal sites. Methicillin- resistant S. epidermidis isolates (N=12) were identified in three DFU patients and SCCmec elements (N=15) were identified in isolates from four DFU patients and one non-DFU patient. Closely related oro-nasal (oral [N=2], nasal [N=2]) and DFU isolates were identified in 4/10 (40.0%) distinct patients from whom S. epidermidis was recovered from both sites.
Conclusions
In conclusion, anolyte (200 ppm) is highly effective at reducing the microbial bioburden of debrided DFU tissues, particularly when treated with 10 ml volumes. These findings indicate that anolyte should be considered for a clinical intervention trial with active DFUs, rather than debrided DFU tissue. The prevalence of S. aureus and S. epidermidis is higher in DFU patients than patients without DFUs across all anatomical sites investigated. Either S. aureus or S. epidermidis were identified in both the oro-nasal cavity and DFUs of 36/76 (47.3%) patients with DFUs. Utilising WGS technology, highly related oro-nasal and DFU isolates were identified in 23/36 (63.9%) distinct patients with DFUs (S. aureus 19/23 [82.6%], S. epidermidis 4/23 [17.4%]), consolidating previous research that the oro-nasal cavity is an endogenous reservoir for DFUIs. | en |
