br Discussion In the context of managing diabetes foot infec

Discussion In the context of managing diabetes foot infections from an infectious disease viewpoint, current guidelines based on culture-dependent data, are now subject to the scrutiny of molecular DNA based approaches. Furthermore, studies employing amplification and sequence analysis of the 16S rRNA gene to characterize the microorganisms involved in DFI, none to date have sampled participants with overt clinical signs of infection. Instead the available data report on chronic, new or recurrent DFUs that are clinically non-infected (Dowd et al., 2008a,b; Gardner et al., 2013; Price et al., 2009; Smith et al., 2016; Wolcott et al., 2015). Given the increasing utilisation of genomic analysis from both a clinical and research domain, it is essential to understand the microbiota of clinically infective DFUs and if current anti-infective practices can be improved through the translation of complex bioinformatics arising from the DNA analysis of microbial communities. We analysed robust microbiota datasets from Infections of the feet in people with diabetes, and detailed their clinical outcomes, relating this back to current anti-infective practices. We found that the duration of a DFU prior to presenting with a new clinical infection may help clinicians decide on the antimicrobial regimen of choice. We identify Staphylococci spp. as the most commonly sequenced 5-fluorocytosine bacteria in approximately one third of DFUs in this study, followed closely by Corynebacterium spp. In a recent review by our group on the bacteriology of DFUs from both a molecular and culture based approach (Malone et al., 2016), the predominant pathogen/s of infection for DFI was S. aureus. Additionally, Corynebacterium spp., Streptococcus spp., and obligate anaerobes belonging to Clostridiales family XI all identified as major players in this study were similarly identified in studies of chronic non-infected wounds. Based on our molecular data and that of previous molecular and culture based publications, current guidelines that promote the use of antimicrobials targeting Gram-positive aerobic cocci as a first line treatment are appropriate. Corynebacterium spp. has provided a continuing source of debate regarding its role as a non-pathogenic skin commensal (Citron et al., 2007), or as a pathogen of infection in the presence of an in immunocompromised patient (Dowd et al., 2008a,b; Uçkay et al., 2015). In this study, we seldom identified the presence Corynebacterium spp. as a sole pathogen (High frequency taxa), with it almost exclusively occurring in combination with other known pathogens of DFI. This suggests that there may be a role for Corynebacterium spp. as part of a polymicrobial infection. Given that many first line antimicrobials of choice for DFI are active against this microorganism, there may not be a requirement to target this sole microorganism unless a mono-microbial culture is identified. Community structure is essentially the composition of a community, including the number of species in that community, their relative numbers (Richness) and their complexity (Diversity). We identify that the duration of DFU is a major driver behind the microbiome, with longer duration DFUs typically having greater species richness and diversity. This correlated to increased relative abundances of Gram-negative proteobacteria and reduced relative abundances of firmicutes in a pattern previously described by Gardener et al. on neuropathic non-infected DFUs (Gardner et al., 2013). Proteobacteria are commonly identified in wounds (Dowd et al., 2008a; Wolcott et al., 2015) and largely belong to the Pseudomonadaceae and Enterobacteriaceae families. It is unclear from our data if these microorganisms require special attention, for example P. aeruginosa was present as minor taxa in only one quarter of samples (eight DFUs), thus supporting the general consensus (Lipsky et al., 2012) that P. aeruginosa is not a typical pathogen of infection in DFI (excluding southern hemisphere locations) (Sivanmaliappan and Sevanan, 2011), and may not require additional tailored therapy should it be identified through cultivation based methods.