Unravelling the complex interplay between antibiotic consumption and adaptive changes in methicillin-resistant Staphylococcus aureus.

OBJECTIVES: This study aims to elucidate the genomic dynamics driving the emergence of antimicrobial resistance (AMR), with a specific focus on the interplay between AMR and antimicrobial usage. METHODS: We conducted a comprehensive analysis using a ST239 methicillin-resistant Staphylococcus aureus (MRSA) dataset over a continuous 12-year period from a single hospital. Genomic analyses were performed tracking the changes in MRSA populations, particularly the emergence of reduced vancomycin susceptibility, and assessing the impact of glycopeptide use on these emergence events. RESULTS: Our findings reveal a significant correlation between hospital glycopeptide usage and the selection of MRSA strains with reduced vancomycin susceptibility. Genomic analyses provided insights into the molecular mechanisms driving resistance emergence, including the slowing of the molecular clock rate in response to heightened antimicrobial consumption. CONCLUSIONS: In conclusion, this study the highlights the complex dynamics between AMR and antimicrobial use at the hospital level. The observed correlation between antimicrobial consumption and the development of less susceptible MRSA strains underscores the importance of antimicrobial stewardship programmes and the establishment of optimal consumption thresholds for mitigating AMR effectively.

In vitro and in vivo evaluation of the antimicrobial effectiveness of non-medicated hydrophobic wound dressings.

There is an increasing use of non-medicated wound dressing with claims of irreversible bacterial binding. Most of the data are from in vitro models which lack clinical relevance. This study employed a range of in vitro experiments to address this gap and we complemented our experimental designs with in vivo observations using dressings obtained from patients with diabetes-related foot ulcers. A hydrophobic wound dressing was compared with a control silicone dressing in vitro. Test dressings were placed on top of a Pseudomonas aeruginosa challenge suspension with increasing concentrations of suspension inoculum in addition to supplementation with phosphate buffered saline (PBS) or increased protein content (IPC). Next, we used the challenge suspensions obtained at the end of the first experiment, where bacterial loads from the suspensions were enumerated following test dressing exposure. Further, the time-dependent bacterial attachment was investigated over 1 and 24 h. Lastly, test dressings were exposed to a challenge suspension with IPC, with or without the addition of the bacteriostatic agent Deferiprone to assess the impacts of limiting bacterial growth in the experimental design. Lastly, two different wound dressings with claims of bacterial binding were obtained from patients with chronic diabetes-related foot ulcers after 72 h of application and observed using scanning electron microscope (SEM). Bacteria were enumerated from each dressing after a 1-h exposure time. There was no statistical difference in bacterial attachment between both test dressings when using different suspension inoculum concentrations or test mediums. Bacterial attachment to the two test dressings was significantly lower (p < 0.0001) when IPC was used instead of PBS. In the challenge suspension with PBS, only the hydrophobic dressing achieved a statistically significant reduction in bacterial loads (0.5 ± 0.05 log colony forming units; p = 0.001). In the presence of IPC, there was no significant reduction in bacterial loads for either test dressing. When bacterial growth was arrested, attachment to the test dressings did not increase over time, suggesting that the number of bacteria on the test dressings increases over time due to bacterial growth. SEM identified widespread adsorption of host fouling across the test dressings which occurred prior to microbial binding. Therein, microbial attachment occurred predominantly to host fouling and not directly to the dressings. Bacterial binding is not unique to dialkylcarbamoyl chloride (DACC) dressings and under clinically relevant in vitro conditions and in vivo observations, we demonstrate (in addition to previously published work) that the bacterial binding capabilities are not effective at reducing the number of bacteria in laboratory models or human wounds.

pSK41/pGO1-family conjugative plasmids of Staphylococcus aureus encode a cryptic repressor of replication.

The majority of large multiresistance plasmids of Staphylococcus aureus utilise a RepA_N-type replication initiation protein, the expression of which is regulated by a small antisense RNA (RNAI) that overlaps the rep mRNA leader. The pSK41/pGO1-family of conjugative plasmids additionally possess a small (86 codon) divergently transcribed ORF (orf86) located upstream of the rep locus. The product of pSK41 orf86 was predicted to have a helix-turn-helix motif suggestive of a likely function in transcriptional repression. In this study, we investigated the effect of Orf86 on transcription of thirteen pSK41 backbone promoters. We found that Orf86 only repressed transcription from the rep promoter, and hence now redesignate the product as Cop. Over-expression of Cop in trans reduced the copy number of pSK41 mini-replicons, both in the presence and absence of rnaI. in vitro protein-DNA binding experiments with purified 6 × His-Cop demonstrated specific DNA binding, adjacent to, and partially overlapping the -35 hexamer of the rep promoter. The crystal structure of Cop revealed a dimeric structure similar to other known transcriptional regulators. Cop mRNA was found to result from "read-through" transcription from the strong RNAI promoter that escapes the rnaI terminator. Thus, PrnaI is responsible for transcription of two distinct negative regulators of plasmid copy number; the antisense RNAI that primarily represses Rep translation, and Cop protein that can repress rep transcription. Deletion of cop in a native plasmid did not appear to impact copy number, indicating a cryptic auxiliary role.

Neuroinflammation in Alzheimer's Disease: A Potential Role of Nose-Picking in Pathogen Entry via the Olfactory System?

Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by progressive cognitive decline and memory impairment. Many possible factors might contribute to the development of AD, including amyloid peptide and tau deposition, but more recent evidence suggests that neuroinflammation may also play an-at least partial-role in its pathogenesis. In recent years, emerging research has explored the possible involvement of external, invading pathogens in starting or accelerating the neuroinflammatory processes in AD. In this narrative review, we advance the hypothesis that neuroinflammation in AD might be partially caused by viral, bacterial, and fungal pathogens entering the brain through the nose and the olfactory system. The olfactory system represents a plausible route for pathogen entry, given its direct anatomical connection to the brain and its involvement in the early stages of AD. We discuss the potential mechanisms through which pathogens may exploit the olfactory pathway to initiate neuroinflammation, one of them being accidental exposure of the olfactory mucosa to hands contaminated with soil and feces when picking one's nose.

Human dermal fibroblast subpopulations and epithelial mesenchymal transition signals in hidradenitis suppurativa tunnels are normalized by spleen tyrosine kinase antagonism in vivo.

Hidradenitis Suppurativa is a chronic inflammatory disease of which the pathogenesis is incompletely understood. Dermal fibroblasts have been previously identified as a major source of inflammatory cytokines, however information pertaining to the characteristics of subpopulations of fibroblasts in HS remains unexplored. Using in silico-deconvolution of whole-tissue RNAseq, Nanostring gene expression panels and confirmatory immunohistochemistry we identified fibroblast subpopulations in HS tissue and their relationship to disease severity and lesion morphology. Gene signatures of SFRP2+ fibroblast subsets were increased in lesional tissue, with gene signatures of SFRP1+ fibroblast subsets decreased. SFRP2+ and CXCL12+ fibroblast numbers, measured by IHC, were increased in HS tissue, with greater numbers associated with epithelialized tunnels and Hurley Stage 3 disease. Pro-inflammatory CXCL12+ fibroblasts were also increased, with reductions in SFRP1+ fibroblasts compared to healthy controls. Evidence of Epithelial Mesenchymal Transition was seen via altered gene expression of SNAI2 and altered protein expression of ZEB1, TWIST1, Snail/Slug, E-Cadherin and N-Cadherin in HS lesional tissue. The greatest dysregulation of EMT associated proteins was seen in biopsies containing epithelialized tunnels. The use of the oral Spleen tyrosine Kinase inhibitor Fostamatinib significantly reduced expression of genes associated with chronic inflammation, fibroblast proliferation and migration suggesting a potential role for targeting fibroblast activity in HS.

Dry eye disease in astronauts: a narrative review.

Long-duration spaceflight can have adverse effects on human health. One of the most common ocular conditions experienced by astronauts is dry eye disease (DED). Symptoms of DED include feelings of eye irritation, eye strain, foreign body sensation and blurred vision. Over 30% of International Space Station expedition crew members reported irritation and foreign body sensation. We reviewed the current literature on the prevalence and mechanisms of DED in astronauts and its potential implications for long-duration spaceflight, including the influence of environmental factors, such as microgravity and fluid shift on tear film physiology in space. DED has negative effects on astronaut performance, which is why there is a need for further research into the pathophysiology and countermeasures. As an in-flight countermeasure, neurostimulation seems to be among the most promising options.

In vivo observations of biofilm adhering to a dialkylcarbamoyl chloride-coated mesh dressing when applied to diabetes-related foot ulcers: A proof of concept study.

In this proof-of-concept study of twenty participants, we sought to determine if a DACC (Dialkylcarbamoyl chloride)-coated mesh dressing demonstrates an ability to adhere biofilm when placed on Diabetes Related Foot Ulcers (DRFUs) with chronic infection. The study also sought to determine if removal of the DACC-coated mesh dressings contributes to reducing the total number of bacteria in DRFUs, by exploring the total microbial loads, microbial community composition, and diversity. Standard of care was provided in addition to the application of DACC or DACC hydrogel every three days for a total of two weeks. Wound swabs, tissue curettage, and soiled dressings were collected pre and post-treatment. Tissue specimens obtained pre-treatment were analysed with scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridisation (PNA-FISH) with confocal laser scanning microscopy and confirmed the presence of biofilm in all DRFUs. SEM confirmed the presence of biofilms readily adhered to soiled DACC-coated mesh dressings pre- and post-treatment in all participants. Real-time quantitative polymerase chain reaction (qPCR) demonstrated the mean total microbial load of DRFUs in 20 participants did not change after two weeks of therapy (pre-treatment = 4.31 Log10 16 S copies (±0.8) versus end of treatment = 4.32 Log10 16 S copies (±0.9), P = .96, 95% CI -0.56 to 0.5). 16 S sequencing has shown the microbial composition of DACC dressings and wound swabs pre- and post-treatment remained similar (DACC; R = -.047, P = .98, Swab; R = -.04, P = .86), indicating the microbial communities originate from the ulcer. Biofilms adhere to DACC-coated mesh dressings; however, this may not reduce the total microbial load present within DRFU tissue. Wound dressings for use in hard-to-heal wounds should be used as an adjunct to a good standard of care which includes debridement and wound bed preparation.

Metatranscriptome sequencing identifies Escherichia are major contributors to pathogenic functions and biofilm formation in diabetes related foot osteomyelitis.

Osteomyelitis in the feet of persons with diabetes is clinically challenging and is associated with high rates of amputation. In this study RNA-sequencing was employed to explore microbial metatranscriptomes with a view to understand the relative activity and functions of the pathogen/s responsible for diabetes foot osteomyelitis (DFO). We obtained 25 intraoperative bone specimens from persons with confirmed DFO, observing that Escherichia spp. (7%), Streptomyces spp. (7%), Staphylococcus spp. (6%), Klebsiella spp. (5%) and Proteus spp. (5%) are the most active taxa on average. Data was then subset to examine functions associated with pathogenesis (virulence and toxins), biofilm formation and antimicrobial/multi-drug resistance. Analysis revealed Escherichia spp. are the most active taxa relative to pathogenic functions with K06218 (mRNA interferase relE), K03699 (membrane damaging toxin tlyC) and K03980 (putative peptidoglycan lipid II flippase murJ), K01114 (membrane damaging toxin plc) and K19168 (toxin cptA) being the most prevalent pathogenic associated transcripts. The most abundant transcripts associated with biofilm pathways included components of the biofilm EPS matrix including glycogen synthesis, cellulose synthesis, colonic acid synthesis and flagella synthesis. We further observed enrichment of a key enzyme involved in the biosynthesis of L-rhamnose (K01710 -dTDP-glucose 4,6-dehydratase rfbB, rmlB, rffG) which was present in all but four patients with DFO.

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids.

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.

Culture-Free Phylogenetic Analysis of Legionella pneumophila Using Targeted CRISPR/Cas9 Next-Generation Sequencing.

Currently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method's utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms. IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires' disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.

A metatranscriptomic approach to explore longitudinal tissue specimens from non-healing diabetes related foot ulcers.

Cellular mechanisms and/or microbiological interactions which contribute to chronic diabetes related foot ulcers (DRFUs) were explored using serially collected tissue specimens from chronic DRFUs and control healthy foot skin. Total RNA was isolated for next-generation sequencing. We found differentially expressed genes (DEGs) and enriched hallmark gene ontology biological processes upregulated in chronic DRFUs which primarily functioned in the host immune response including: (i) Inflammatory response; (ii) TNF signalling via NFKB; (iii) IL6 JAK-STAT3 signalling; (iv) IL2 STAT5 signalling and (v) Reactive oxygen species. A temporal analysis identified RN7SL1 signal recognition protein and IGHG4 immunoglobulin protein coding genes as being the most upregulated genes after the onset of treatment. Testing relative temporal changes between healing and non-healing DRFUs identified progressive upregulation in healed wounds of CXCR5 and MS4A1 (CD20), both canonical markers of lymphocytes (follicular B cells/follicular T helper cells and B cells, respectively). Collectively, our RNA-seq data provides insights into chronic DRFU pathogenesis.

Host-microbe metatranscriptome reveals differences between acute and chronic infections in diabetes-related foot ulcers.

Virtually all diabetes-related foot ulcers (DRFUs) will become colonized by microorganisms that may increase the risk of developing an infection. The reasons why some ulcerations develop acute clinical infections (AI-DRFUs) whilst others develop chronic infection (CI-DRFUs) and the preceding host-microbe interactions in vivo remain largely unknown. Establishing that acute and chronic infections are distinct processes requires demonstrating that these are two different strategies employed by microbes when interacting with a host. In this study, dual-RNA seq was employed to differentiate the host-microbe metatranscriptome between DRFUs that had localized chronic infection or acute clinical infection. Comparison of the host metatranscriptome in AI-DRFUs relative to CI-DRFUs identified upregulated differentially expressed genes (DEGs) that functioned as regulators of vascular lymphatic inflammatory responses, T-cell signalling and olfactory receptors. Conversely, CI-DRFUs upregulated DEGs responsible for cellular homeostasis. Gene set enrichment analysis using Hallmark annotations revealed enrichment of immune and inflammatory profiles in CI-DRFUs relative to AI-DRFUs. Analysis of the microbial metatranscriptome identified the DEGs being enriched within AI-DRFUs relative to CI-DRFUs included several toxins, two-component systems, bacterial motility, secretion systems and genes encoding for energy metabolism. Functions relevant to DRFU pathology were further explored, including biofilm and bacterial pathogenesis. This identified that the expression of biofilm-associated genes was higher within CI-DRFUs compared to that of AI-DRFUs, with mucR being the most highly expressed gene. Collectively, these data provide insights into the host-microbe function in two clinically-distinct infective phenotypes that affect DRFUs. The data reveal that bacteria in acutely infected DRFUs prioritize motility over biofilm and demonstrate greater pathogenicity and mechanisms, which likely subvert host cellular and immune pathways to establish infection. Upregulation of genes for key vascular inflammatory mediators in acutely infected ulcers may contribute, in part, to the clinical picture of a red, hot, swollen foot, which differentiates an acutely infected ulcer from that of a chronic infection.

Efficacy of a Topical Wound Agent Methanesulfonic Acid and Dimethylsulfoxide on In Vitro Biofilms.

A topical desiccating wound agent containing methanesulfonic acid, dimethylsulfoxide and amorphous silica was evaluated in three in vitro models for its efficacy against biofilms produced by Pseudomonas aeruginosa (ATCC-15442) and Staphylococcus aureus (ATCC-6538). The in vitro biofilm models used were; the MBEC Assay®, Centre for Disease Control (CDC) Biofilm Reactor® and a Semi-solid biofilm model. A 30-s exposure of a topical wound desiccating agent was used in each model. A complete eradication of viable cells was demonstrated in all models for both strains (p < 0.0001). Imaging with scanning electron microscopy (SEM) was performed where possible. All three models demonstrated complete eradication of viable cells with a 30 s application of a topical wound desiccating agent.

The Resistome and Mobilome of Multidrug-Resistant Staphylococcus sciuri C2865 Unveil a Transferable Trimethoprim Resistance Gene, Designated dfrE, Spread Unnoticed.

Methicillin-resistant Staphylococcus sciuri (MRSS) strain C2865 from a stranded dog in Nigeria was trimethoprim (TMP) resistant but lacked formerly described staphylococcal TMP-resistant dihydrofolate reductase genes (dfr). Whole-genome sequencing, comparative genomics, and pan-genome analyses were pursued to unveil the molecular bases for TMP resistance via resistome and mobilome profiling. MRSS C2865 comprised a species subcluster and positioned just above the intraspecies boundary. Lack of species host tropism was observed. S. sciuri exhibited an open pan-genome, while MRSS C2865 harbored the highest number of unique genes (75% associated with mobilome). Within this fraction, we discovered a transferable TMP resistance gene, named dfrE, which confers high-level TMP resistance in Staphylococcus aureus and Escherichia coli. dfrE was located in a novel multidrug resistance mosaic plasmid (pUR2865-34) encompassing adaptive, mobilization, and segregational stability traits. dfrE was formerly denoted as dfr_like in Exiguobacterium spp. from fish farm sediment in China but escaped identification in one macrococcal and diverse staphylococcal genomes in different Asian countries. dfrE shares the highest identity with dfr of soil-related Paenibacillus anaericanus (68%). Data analysis discloses that dfrE has emerged from a single ancestor and places S. sciuri as a plausible donor. C2865 unique fraction additionally enclosed novel chromosomal mobile islands, including a multidrug-resistant pseudo-SCCmec cassette, three apparently functional prophages (Siphoviridae), and an SaPI4-related staphylococcal pathogenicity island. Since dfrE seems not yet common in staphylococcal clinical specimens, our data promote early surveillance and enable molecular diagnosis. We evidence the genome plasticity of S. sciuri and highlight its role as a resourceful reservoir for adaptive traits. IMPORTANCE The discovery and surveillance of antimicrobial resistance genes (AMRG) and their mobilization platforms are critical to understand the evolution of bacterial resistance and to restrain further expansion. Limited genomic data are available on Staphylococcus sciuri; regardless, it is considered a reservoir for critical AMRG and mobile elements. We uncover a transferable staphylococcal TMP resistance gene, named dfrE, in a novel mosaic plasmid harboring additional resistance, adaptive, and self-stabilization features. dfrE is present but evaded detection in diverse species from varied sources geographically distant. Our analyses evidence that the dfrE-carrying element has emerged from a single ancestor and position S. sciuri as the donor species for dfrE spread. We also identify novel mobilizable chromosomal islands encompassing AMRG and three unrelated prophages. We prove high intraspecies heterogenicity and genome plasticity for S. sciuri. This work highlights the importance of genome-wide ecological studies to facilitate identification, characterization, and evolution routes of bacteria adaptive features.

A multiomics approach to identify host-microbe alterations associated with infection severity in diabetic foot infections: a pilot study.

Diabetic foot infections (DFIs) are a major cause of hospitalization and can lead to lower extremity amputation. In this pilot study, we used a multiomics approach to explore the host-microbe complex within DFIs. We observed minimal differences in the overall microbial composition between PEDIS infection severities, however Staphylococcus aureus and Streptococcus genera were abundant and highly active in most mild to moderate DFIs. Further, we identified the significant enrichment of several virulence factors associated with infection pathogenicity belonging to both Staphylococcus aureus and Streptococcus. In severe DFIs, patients demonstrated a greater microbial diversity and differential gene expression demonstrated the enrichment of multispecies virulence genes suggestive of a complex polymicrobial infection. The host response in patients with severe DFIs was also significantly different as compared to mild to moderate DFIs. This was attributed to the enrichment of host genes associated with inflammation, acute phase response, cell stress and broad immune-related responses, while those associated with wound healing and myogenesis were significantly depleted.

Efficacy of a topical concentrated surfactant gel on microbial communities in non-healing diabetic foot ulcers with chronic biofilm infections: A proof-of-concept study.

This proof-of-concept study sought to determine the effects of standard of care (SOC) and a topically applied concentrated surfactant gel (SG) on the total microbial load, community composition, and community diversity in non-healing diabetic foot ulcers (DFUs) with chronic biofilm infections. SOC was provided in addition to a topical concentrated SG, applied every 2 days for 6 weeks. Wound swabs were obtained from the base of ulcers at baseline (week 0), week 1, mid-point (week 3), and end of treatment (week 6). DNA sequencing and real-time quantitative polymerase chain reaction (qPCR) were employed to determine the total microbial load, community composition, and diversity of patient samples. Tissue specimens were obtained at baseline and scanning electron microscopy and peptide nucleic acid fluorescent in situ hybridisation with confocal laser scanning microscopy were used to confirm the presence of biofilm in all 10 DFUs with suspected chronic biofilm infections. The application of SG resulted in 7 of 10 samples achieving a reduction in mean log10 total microbial load from baseline to end of treatment (0.8 Log10 16S copies, ±0.6), and 3 of 10 samples demonstrated an increase in mean Log10 total microbial load (0.6 log10 16S copies, ±0.8) from baseline to end of treatment. Composition changes in microbial communities were driven by changes to the most dominant bacteria. Corynebacterium sp. and Streptococcus sp. frequently reduced in relative abundance in patient samples from week 0 to week 6 but did not disappear. In contrast, Staphylococcus sp., Finegoldia sp., and Fusobacterium sp., relative abundances frequently increased in patient samples from week 0 to week 6. The application of a concentrated SG resulted in varying shifts to diversity (increase or decrease) between week 0 and week 6 samples at the individual patient level. Any shifts in community diversity were independent to changes in the total microbial loads. SOC and a topical concentrated SG directly affect the microbial loads and community composition of DFUs with chronic biofilm infections.

Effect on total microbial load and community composition with two vs six-week topical Cadexomer Iodine for treating chronic biofilm infections in diabetic foot ulcers.

This study compares two vs six weeks of topical antimicrobial therapy with Cadexomer Iodine in patients with diabetic foot ulcers (DFUs) complicated by chronic biofilm infections. Patients with non-healing DFUs with suspected chronic biofilm infections were eligible for enrolment. Patients were randomised to receive either two or six weeks of treatment with topical Cadexomer Iodine. Tissue biopsies from the ulcers were obtained pre-and-post treatment and underwent DNA sequencing and real-time quantitative polymerase chain reaction (PCR) to determine the total microbial load, community composition, and diversity of bacteria. Scanning electron microscopy confirmed biofilm in all 18 ulcers with suspected chronic biofilm infections. Cadexomer Iodine resulted in 14 of 18 (78%) samples achieving a mean 0.5 log10 reduction in microbial load. Regardless of treatment duration, there was no statistical difference in the reduction of total microbial loads. No difference in the rate of wound healing in the two groups was seen at 6 weeks. Cadexomer Iodine reduces the total microbial load in DFUs with chronic biofilm infections and affects microbial community composition and diversity. All ulcers in both groups showed an initial reduction in wound size with application of Cadexomer Iodine, which might reflect its effect on biofilms.

Analysis of proximal bone margins in diabetic foot osteomyelitis by conventional culture, DNA sequencing and microscopy.

Multiple approaches were employed to detect pathogens from bone margins associated with Diabetic Foot Osteomyelitis (DFO). Intra-operative bone specimens of 14 consecutive subjects with suspected DFO were collected over a six-month study period from Liverpool Hospital. Infected bone and a proximal bone margins presumed to be 'clean/non-infected' were collected. Bone material was subjected to conventional culture, DNA sequencing and microscopy. In total, eight of 14 (57%) proximal bone margins had no growth by conventional culture but were identified in all proximal bone specimens by DNA sequencing. Proximal margins had lower median total microbial counts than infected specimens, but these differences were not statistically significant. Pathogens identified by sequencing in infected specimens were identified in proximal margins and the microbiomes were similar (ANOSIM = 0.02, p = 0.59). Using a combination of SEM and/or PNA-FISH, we visualized the presence of microorganisms in infected bone specimens and their corresponding proximal margins of seven patients (50%) with DFO. We identify that bacteria can still reside in what seems to be proximal 'clean' margins. The significance and implications of clinical outcomes requires further analysis from a larger sample size that incorporates differences in surgical and post-operative approaches, correlating any outcomes back to culture-sequence findings.

Remodeling of pSK1 Family Plasmids and Enhanced Chlorhexidine Tolerance in a Dominant Hospital Lineage of Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is a significant human pathogen whose evolution and adaptation have been shaped in part by mobile genetic elements (MGEs), facilitating the global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs, in particular, how changes in the structure of multidrug resistance (MDR) plasmids may influence important staphylococcal phenotypes, is incomplete. Here, we undertook a population and functional genomics study of 212 methicillin-resistant S. aureus (MRSA) sequence type 239 (ST239) isolates collected over 32 years to explore the evolution of the pSK1 family of MDR plasmids, illustrating how these plasmids have coevolved with and contributed to the successful adaptation of this persistent MRSA lineage. Using complete genomes and temporal phylogenomics, we reconstructed the evolution of the pSK1 family lineage from its emergence in the late 1970s and found that multiple structural variants have arisen. Plasmid maintenance and stability were linked to IS256- and IS257-mediated chromosomal integration and disruption of the plasmid replication machinery. Overlaying genomic comparisons with phenotypic susceptibility data for gentamicin, trimethoprim, and chlorhexidine, it appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA isolates through two mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing the rates of gentamicin resistance and reduced chlorhexidine susceptibility and (ii) changes in the plasmid configuration linked with further enhancement of chlorhexidine tolerance. While the exact mechanism of enhanced tolerance remains elusive, this research has uncovered a potential evolutionary response of ST239 MRSA to biocides, one of which may contribute to the ongoing persistence and adaptation of this lineage within health care institutions.