Eleven isolates were identified to species level: B. cereus, B. subtilis, E. cloacae, Micrococcus spp., M. luteus, Pantoea spp., P. oryzihabitans, S. haemolyticus, S. aureus, Group D Streptococcus and B. zoohelcum. To the best of our knowledge, P. oryzihabitans, and B. zoohelcum are not considered to be common colonisers of the skin (27)(50). Interestingly we did not recover any pathogenic fungi which may be due to a variety of factors such as a small sample size, medication or use of a medicated prosthetic material or a good equipment hygiene routine (51)(52).Although some of the isolated organisms could be considered endogenous bacteria, these results suggest that there are a variety of bacteria that are present on the skin and prosthetic devices of prosthesis users, including endogenous skin bacteria, endogenous gastrointestinal bacteria, and exogenous bacteria.
The isolated species have been shown to cause wound or skin and soft tissue infections; the exogenous and endogenous organisms were all opportunistic nosocomial pathogens that primarily affect immunocompromised patients (27)(53)(54)(55), or patients undergoing surgical procedures. such as an amputation (56)(57)(58)(59), excluding B. zoohelcum, which has been isolated from either a cat or dog bite and scratch wounds (60). Interestingly, this organism has also been observed in patients that have had long term contact with cats and dogs, and have been isolated from patients interacting with therapy dogs (61) (62).
Worryingly, we observed resistance to the tested antibiotics from all but two isolates. Eight of the eleven were resistant to two commonly used antibiotics whilst Pseudomonas oryzihabitans was resistant to just one, ceftazidime. Whilst we acknowledge that we employed a single concentration of each antibiotic, it was interesting to observe that for beta lactam antibiotics penicillin (1.5 units) and ampicillin (10 µg), all tested strains were resistant. Two isolates, P. oryzihabitans and B. zoohelcum had observed resistance to the cephalosporin, ceftazidime (10 µg), with B. zoohelcum also demonstrating resistance to the aminoglycoside, gentamicin (10 µg).
None of the participants in the study reported an active known infection or had reported the use of any antimicrobials; however, three participants reported a previous infection which had required antibiotic intervention using floxacillin.
In comparison to the antibiotic treatment, all isolates were sensitive to MH. MH had a bactericidal effect on all isolates with an MBC of 9 % w/v against Micrococcus luteus and Pseudomonas oryzihabitans and 15 % w/v against all other isolates. In comparison, whilst GE was able to inhibit the growth (25-200 µg) of 8 isolates, in only one did we observe a bactericidal effect below 300 µg/mL B. zoohelcum.
To the best of our knowledge there has been no research exploring the use of honey and garlic as antimicrobials against Pantoea spp., P. oryzihabitans, S. haemolyticus and B. zoohelcum and this study highlights the potential for the use of either MH or GE against these microorganisms.
Based on the susceptibility results to the chosen antibiotics, there does not seem to be any explicit link between antibiotic resistance and a higher or lower MIC of MH or GE as concluded in a previous study (63), although this could be further explored using next generation sequencing. From this data, it is reasonable to suggested that GE and MH do not work by binding to the Penicillin Binding Proteins in the same way as β-lactams or bind to the aminoacyl-tRNA recognition site, like aminoglycosides.
This study represents one of the first to explore the microbial diversity of the skin-prosthesis interface and highlights the dangers posed by antimicrobial resistant microorganisms. Whilst the world is running out of antibiotics, the potential application of natural product inspired components, such as those derived from honey or plants should be explored in more detail. Results of this study also suggests there is a requirement for comprehensive bacterial identification and increased antimicrobial susceptibility testing on strains not routinely identified in wound infections; here we isolated the more common microorganisms such as Staphylococcus, alongside those less common such as Bergyella zoohelcum. Indeed, B. zoohelcum is commonly liked to cat and dog bites, with only 5 cases of infection reported worldwide (64), though this may be due to the fastidious growth requirements of the microorganism, leading to systematic underreporting.
Despite being multidrug resistant, the isolate in our study was observed to be the most sensitive in our collection against MH (3 %/ 15% w/v MIC/MBC) and GE (25/200 µg/mL MIC/MBC).
We conclude that antimicrobial resistant microorganisms pose a real threat to the general health and wellbeing of individuals who routinely use lower extremity prostheses. Results of this study identified a small cross section of microorganisms, which will form the basis of a much larger study. We would encourage the increased investigation into the use of natural product extracts, such as Manuka honey and allicin as potential new and innovative interventions to help reduce the prevalence of multidrug resistant microorganisms that are present at the skin-prosthesis interface. In light of these findings, it would be pertinent to explore the use of alternative and novel approaches to ensure that hygiene of an individual’s skin and their prosthetic liners and socket is maintained.