Comparative genomics analysis of two Helcococcus kunzii strains co-isolated with Staphylococcus aureus from diabetic foot ulcers.

Helcococcus kunzii is a commensal Gram-positive bacterial species recovered from the human skin microbiota and considered as an opportunistic pathogen. Although little is known about its clinical significance, its increased abundance has been reported in infected wounds, particularly in foot ulcers in persons with diabetes. This species is usually detected in mixed cultures from human specimens and frequently isolated with Staphylococcus aureus. Modulation of staphylococci virulence by H. kunzii has been shown in an infection model of Caenorhabditis elegans. The aim of this study was to compare the genomes of two H. kunzii strains isolated from foot ulcers -isolate H13 and H10 showing high or low impact on S. aureus virulence, respectively- and the H. kunzii ATCC51366 strain. Whole genome analyses revealed some differences between the two strains: length (2.06 Mb (H13) and 2.05 Mb (H10) bp), GC content (29.3% (H13) and 29.5% (H10)) and gene content (1,884 (H13) and 1,786 (H10) predicted genes). The core-proteome phylogenies within the genus characterised H. kunzii H13 and H10 as genetically similar to their ancestor. The main differences between the strains were mainly in sugar-associated transporters and various hypothetical proteins. Five targets were identified as potentially involved in S. aureus virulence modulation in both genomes: the two-component iron export system and three autoinducer-like proteins. Moreover, H13 strain harbours a prophage inserted in 1,261,110-1,295,549 (attL-attR), which is absent in H10 strain. The prophage PhiCD38_2 was previously reported for its ability to modulate secretion profile, reinforcing the autoinducer-like hypothesis. In the future, transcriptomics or metaproteomics approaches could be performed to better characterize the H13 strain and possibly identify the underlying mechanism for S. aureus virulence modulation.

The Persistence of Staphylococcus aureus in Pressure Ulcers: A Colonising Role.

Decubitus pressure ulcers (PU) are a major complication of immobilised patients. Staphylococcus aureus is one of the most frequently detected microorganisms in PU samples; however, its persistence and role in the evolution of these wounds is unknown. In this study, we analysed S. aureus strains isolated from PU biopsies at inclusion and day 28. Eleven S. aureus (21.1%) were detected in 52 patients at inclusion. Only six PUs (11.5%) continued to harbour this bacterium at day 28. Using a whole genome sequencing approach (Miseq®, Illumina), we confirmed that these six S. aureus samples isolated at D28 were the same strain as that isolated at inclusion, with less than 83 bp difference. Phenotypical studies evaluating the growth profiles (Infinite M Mano, Tecan®) and biofilm formation (Biofilm Ring Test®) did not detect any significant difference in the fitness of the pairs of S. aureus. However, using the Caenorhabditis elegans killing assay, a clear decrease of virulence was observed between strains isolated at D28 compared with those isolated at inclusion, regardless of the clinical evolution of the PU. Moreover, all strains at inclusion were less virulent than a control S. aureus strain, i.e., NSA739. An analysis of polymicrobial communities of PU (by metabarcoding approach), in which S. aureus persisted, demonstrated no impact of Staphylococcus genus on PU evolution. Our study suggested that S. aureus presented a colonising profile on PU with no influence on wound evolution.

Investigating Pathogenicity and Virulence of Staphylococcus pettenkoferi: An Emerging Pathogen.

Staphylococcus pettenkoferi is a coagulase-negative Staphylococcus identified in 2002 that has been implicated in human diseases as an opportunistic pathogenic bacterium. Its multiresistant character is becoming a major health problem, yet the pathogenicity of S. pettenkoferi is poorly characterized. In this study, the pathogenicity of a S. pettenkoferi clinical isolate from diabetic foot osteomyelitis was compared with a Staphylococcus aureus strain in various in vitro and in vivo experiments. Growth kinetics were compared against S. aureus, and bacteria survival was assessed in the RAW 264.7 murine macrophage cell line, the THP-1 human leukemia monocytic cell line, and the HaCaT human keratinocyte cell line. Ex vivo analysis was performed in whole blood survival assays and in vivo assays via the infection model of zebrafish embryos. Moreover, whole-genome analysis was performed. Our results show that S. pettenkoferi was able to survive in human blood, human keratinocytes, murine macrophages, and human macrophages. S. pettenkoferi demonstrated its virulence by causing substantial embryo mortality in the zebrafish model. Genomic analysis revealed virulence factors such as biofilm-encoding genes (e.g., icaABCD; rsbUVW) and regulator-encoding genes (e.g., agr, mgrA, sarA, saeS) well characterized in S. aureus. This study thus advances the knowledge of this under-investigated pathogen and validates the zebrafish infection model for this bacterium.

First Report of CC5-MRSA-IV-SCCfus "Maltese Clone" in Bat Guano.

Methicillin-resistant Staphylococcus aureus (MRSA) is a widespread pathogen that could cause different illnesses in both human and animals. Presence of MRSA in animals raises concerns of their capacity to act as reservoirs, particularly in wild animals. This study aimed to characterize the resistance and virulence patterns of S. aureus strains isolated from bat guano in Algeria. From March to May 2016, 98 bat guano samples from Aokas's cave (Bejaia, Algeria) were collected. Swabs were taken for microbiological studies. Isolates were identified by Vitek® MS system, and antibiotic susceptibility was determined by disk diffusion method. The clonal origin, virulence and antibiotic resistance profiles of S. aureus isolates were characterized by whole genome sequencing. Eleven S. aureus strains were obtained from the 98 guano samples. Seven isolates were sensitive to all antibiotics tested and four (36.3%) were resistant to penicillin G, cefoxitin and fusidic acid. The four MRSA isolates were assigned to the sequence type ST149 and related to spa type t010. These isolates harbored a SCCmecIV element and the fusidic acid resistance element Q6GD50 (fusC). They carried different virulence genes including several enterotoxins (sea, egc enterotoxin locus, sec, sel), and the toxic shock syndrome toxin (tst). Our results highlight that bat guano may constitute an important reservoir of MRSA strains.

Long-Term Intrahost Evolution of Staphylococcus aureus Among Diabetic Patients With Foot Infections.

Staphylococcus aureus is one of the main pathogens isolated from diabetic foot infections (DFI). The purpose of this study was to evaluate the importance of the persistence of S. aureus in this environment and the possible modifications of the bacterial genome content over time. Molecular typing of S. aureus isolates cultured from patients with the same DFI over a 7-year study revealed a 25% rate of persistence of this species in 48 patients, with a short median persistence time of 12weeks (range: 4-52weeks). Non-specific clonal complexes were linked to this persistence. During the follow-up, bla genes were acquired in three cases, whereas some virulence markers were lost in all cases after a long period of colonization (21.5weeks). Only one patient (2%) had a long-term persistence of 48weeks. The genome sequencing of a clonal pair of early/late strains isolated in this patient showed mutations in genes encoding bacterial defence and two-component signal transduction systems. Although, this study suggests that the long-term persistence of S. aureus in DFI is a rare event, genomic evolution is observed, highlighting the low adaptive ability of S. aureus to the specific environment and stressful conditions of diabetic foot ulcers. These results provide the basis for better understanding of S. aureus dynamics during persistent colonization in chronic wounds.

Pressure ulcers microbiota dynamics and wound evolution.

Bacterial species and their role in delaying the healing of pressure ulcers (PU) in spinal cord injury (SCI) patients have not been well described. This pilot study aimed to characterise the evolution of the cutaneous microbiota of PU in SCI cohort. Twenty-four patients with SCI from a French neurological rehabilitation centre were prospectively included. PU tissue biopsies were performed at baseline (D0) and 28 days (D28) and analysed using 16S rRNA gene-based sequencing analysis of the V3-V4 region. At D0, if the overall relative abundance of genus highlighted a large proportion of Staphylococcus, Anaerococcus and Finegoldia had a significantly higher relative abundance in wounds that stagnated or worsened in comparison with those improved at D28 (3.74% vs 0.05%; p = 0.015 and 11.02% versus 0.16%; p = 0.023, respectively). At D28, Proteus and Morganella genera were only present in stagnated or worsened wounds with respectively 0.02% (p = 0.003) and 0.01% (p = 0.02). Moreover, Proteus, Morganella, Anaerococcus and Peptoniphilus were associated within the same cluster, co-isolated from biopsies that had a poor evolution. This pathogroup could be a marker of wound degradation and Proteus could represent a promising target in PU management.

Colistin-Resistant Enterobacterales Isolated from Chicken Meat in Western Algeria.

Aim: In Algeria, colistin is used as a metaphylactic treatment in the poultry industry for the treatment of Gram-negative gastrointestinal infections and also as a feed additive to promote animal growth. The aim of this study was to investigate the importance and genetic characteristics of colistin-resistant Enterobacterales from chicken meat in Western Algeria. Results: A total of 181 samples of chicken meat were collected from three poultry farms across three provinces in Western Algeria. The presence of colistin-resistant Enterobacterales isolates was screened on selective media. Resistance and virulence profiles were characterised by PCR and sequencing. The clonal relatedness of the different mcr positive isolates was studied using repetitive sequence-based PCR (Rep-PCR) and multilocus sequence typing. Transferability and characteristics of plasmids harboring mcr-1 positive gene were performed using conjugation, PCR-based replicon typing, and whole-genome sequencing. A total of 22 isolates with acquired colistin resistance were identified giving an overall prevalence of 12.2% (22/181): 17 Escherichia coli (predominantly ST224 [n = 4, 23.5%]) and 5 Klebsiella pneumoniae (ST17 [n = 2, 40%], ST646 [n = 2, 40%], and ST944 [n = 1, 20%]). mcr-1 gene was exclusively found in 11 E. coli (prevalence of 6.1% [11/181]) and was associated with IncFV (n = 7) and IncFIIK (n = 4) plasmids. All the isolates had a commensal origin (n = 11). One isolate harbored virulence profile, a high colistin resistance (minimum inhibitory concentration = 96 mg/L), with some new mutations in the chromosomic colistin-resistant genes and different pathogenicity islands typically identified in uropathogenic E. coli. Conclusions: This study reports the diffusion of mcr-1 producing Enterobacterales from chicken meat in Western Algeria. This represents a worrisome situation needing continuous monitoring.