Direct metagenomics investigation of non-surgical hard-to-heal wounds: a review.

BACKGROUND: Non-surgical chronic wounds, including diabetes-related foot diseases (DRFD), pressure injuries (PIs) and venous leg ulcers (VLU), are common hard-to-heal wounds. Wound evolution partly depends on microbial colonisation or infection, which is often confused by clinicians, thereby hampering proper management. Current routine microbiology investigation of these wounds is based on in vitro culture, focusing only on a limited panel of the most frequently isolated bacteria, leaving a large part of the wound microbiome undocumented. METHODS: A literature search was conducted on original studies published through October 2022 reporting metagenomic next generation sequencing (mNGS) of chronic wound samples. Studies were eligible for inclusion if they applied 16 S rRNA metagenomics or shotgun metagenomics for microbiome analysis or diagnosis. Case reports, prospective, or retrospective studies were included. However, review articles, animal studies, in vitro model optimisation, benchmarking, treatment optimisation studies, and non-clinical studies were excluded. Articles were identified in PubMed, Google Scholar, Web of Science, Microsoft Academic, Crossref and Semantic Scholar databases. RESULTS: Of the 3,202 articles found in the initial search, 2,336 articles were removed after deduplication and 834 articles following title and abstract screening. A further 14 were removed after full text reading, with 18 articles finally included. Data were provided for 3,628 patients, including 1,535 DRFDs, 956 VLUs, and 791 PIs, with 164 microbial genera and 116 species identified using mNGS approaches. A high microbial diversity was observed depending on the geographical location and wound evolution. Clinically infected wounds were the most diverse, possibly due to a widespread colonisation by pathogenic bacteria from body and environmental microbiota. mNGS data identified the presence of virus (EBV) and fungi (Candida and Aspergillus species), as well as Staphylococcus and Pseudomonas bacteriophages. CONCLUSION: This study highlighted the benefit of mNGS for time-effective pathogen genome detection. Despite the majority of the included studies investigating only 16 S rDNA, ignoring a part of viral, fungal and parasite colonisation, mNGS detected a large number of bacteria through the included studies. Such technology could be implemented in routine microbiology for hard-to-heal wound microbiota investigation and post-treatment wound colonisation surveillance.

Emergence of multidrug-resistant Staphylococcus haemolyticus in Neonatal Intensive Care Unit in Southern France, a Genomic Study.

An emergence of multidrug-resistant (MDR) Staphylococcus haemolyticus has been observed in the neonatal intensive care unit (NICU) of Nîmes University Hospital in southern France. A case-control analysis was conducted on 96 neonates, to identify risk factors associated with S. haemolyticus infection, focusing on clinical outcomes. Forty-eight MDR S. haemolyticus strains, isolated from neonates between October 2019 and July 2022, were investigated using routine in vitro procedures and whole-genome sequencing. Additionally, five S. haemolyticus isolates from adult patients were sequenced to identify clusters circulating within the hospital environment. The incidence of neonatal S. haemolyticus was significantly associated with low birth weight, lower gestational age, and central catheter use (p<0.001). Sepsis was the most frequent clinical manifestation in this series (20/46, 43.5%) and was associated with five deaths. Based on whole-genome analysis, three S. haemolyticus genotypes were predicted: ST1 (6/53, 11%), ST25 (3/53, 5.7%), and ST29 (44/53, 83%), which included the subcluster II-A, predominantly emerging in the neonatal department. All strains were profiled in silico to be resistant to methicillin, erythromycin, aminoglycosides, and fluoroquinolones, consistent with in vitro antibiotic susceptibility tests. Moreover, in silico prediction of biofilm formation and virulence-encoding genes supported the association of ST29 with severe clinical outcomes, while the persistence in the NICU could be explained by the presence of antiseptic and heavy metal resistance-encoding genes. The clonality of S. haemolyticus ST29 subcluster II-A isolates confirms healthcare transmission causing severe infections. Based on these results, reinforced hygiene measures are necessary to eradicate the nosocomial transmission of MDR strains.

Synergistic effect of tryptamine-urea derivatives to overcome the chromosomally-mediated colistin resistance in Klebsiella pneumoniae.

Colistin is considered as the last-resort antibiotics to treat multi-drug resistant Gram-negative bacterial infections in humans. However, the clinical use of colistin was limited because of the apparition of chromosomal mutations and mobile colistin resistance genes in bacterial isolates. One promising strategy is to combine existing antibiotics with promising non-antibiotics to overcome the widespread emergence of antibiotic-resistant pathogens. Moreover, colistin resistance would be regulated by two component systems PhoP/PhoQ which leads to permanent synthesis of cationic groups compensating for Mg2+ deficiency. In this study, the synthesis of a small library of tryptamine urea derivatives was carried out. In addition, antibiotic susceptibility, antibiotic adjuvant screening and checkerboard assays were used to investigate the antibacterial activity of these synthesized compounds and the potential synergistic activity of their combination with colistin. Conformational analysis of the docked binding modes of the active compound in the predicted binding pocket of bacterial response regulator PhoP were carried out, to see if the active compound inhibits PhoP which is involved in colistin resistance. Finally, hemolytic activity studies have been conducted on the most active compound.

Evolution of the urinary microbiota in spinal cord injury patients with decubitus ulcer: A snapshot study.

Current microbiome investigations of patients with pressure ulcers (PU) are mainly based on wound swabs and/or biopsy sequencing, leaving the colonization scenario unclear. Urinary microbiota has been never studied. As a part of the prospective ESCAFLOR study, we studied urinary microbiota of spinal cord injury (SCI) patients with PU without any urinary tract infection at the inclusion, collected at two times (at admission [D0] and after 28 days [D28]) during the patient's care, investigated by 16S rDNA metagenomics next generation sequencing. Subgroup analyses were carried out between patients with wounds showing improved evolution versus stagnated/worsened wounds at D28. Analysis was done using EPISEQ® 16S and R software. Among the 12 studied patients, the urinary microbiota of patients with improved wound evolution at D28 (n = 6) presented a significant decrease of microbial diversity. This modification was associated with the presence of Proteobacteria phylum and an increase of Escherichia-Shigella (p = 0.005), as well as the presence of probiotic anaerobic bacteria Lactobacillus and Bifidobacterium. In contrast, Proteus abundance was significantly increased in urine of patients with stagnated/worsened wound evolution (n = 6) (p = 0.003). This study proposes urinary microbiota as a complementary factor indirectly associated with the wound evolution and patient cure. It opens new perspectives for further investigations based on multiple body microbiome comparison to describe the complete scenario of the transmission dynamics of wound-colonizing microorganisms.

Antibacterial Activity and Untargeted Metabolomics Profiling of Acalypha arvensis Poepp.

The search for potent antimicrobial compounds is critical in the face of growing antibiotic resistance. This study explores Acalypha arvensis Poepp. (A. arvensis), a Caribbean plant traditionally used for disease treatment. The dried plant powder was subjected to successive extractions using different solvents: hexane (F1), dichloromethane (F2), methanol (F3), a 50:50 mixture of methanol and water (F4), and water (F5). Additionally, a parallel extraction was conducted using a 50:50 mixture of methanol and chloroform (F6). All the fractions were evaluated for their antimicrobial activity, and the F6 fraction was characterized using untargeted metabolomics using SPME-GC×GC-TOFMS. The extracts of A. arvensis F3, F4, and F5 showed antibacterial activity against Staphylococcus aureus ATCC 25923 (5 mg/mL), MRSA BA22038 (5 mg/mL), and Pseudomonas aeruginosa ATCC 27853 (10 mg/mL), and fraction F6 showed antibacterial activity against Staphylococcus aureus ATCC 29213 (2 mg/mL), Escherichia coli ATCC 25922 (20 mg/mL), Pseudomonas aeruginosa ATCC 27853 (10 mg/mL), Enterococcus faecalis ATCC 29212 (10 mg/mL), Staphylococcus aureus 024 (2 mg/mL), and Staphylococcus aureus 003 (2 mg/mL). Metabolomic analysis of F6 revealed 2861 peaks with 58 identified compounds through SPME and 3654 peaks with 29 identified compounds through derivatization. The compounds included methyl ester fatty acids, ethyl ester fatty acids, terpenes, ketones, sugars, amino acids, and fatty acids. This study represents the first exploration of A. arvensis metabolomics and its antimicrobial potential, providing valuable insights for plant classification, phytochemical research, and drug discovery.

Usefulness of dynamic regression time series models for studying the relationship between antimicrobial consumption and bacterial antimicrobial resistance in hospitals: a systematic review.

BACKGROUNG: Antimicrobial resistance (AMR) is on the rise worldwide. Tools such as dynamic regression (DR) models can correlate antimicrobial consumption (AMC) with AMR and predict future trends to help implement antimicrobial stewardship programs (ASPs). MAIN BODY: We carried out a systematic review of the literature up to 2023/05/31, searching in PubMed, ScienceDirect and Web of Science. We screened 641 articles and finally included 28 studies using a DR model to study the correlation between AMC and AMR at a hospital scale, published in English or French. Country, bacterial species, type of sampling, antimicrobials, study duration and correlations between AMC and AMR were collected. The use of ß-lactams was correlated with cephalosporin resistance, especially in Pseudomonas aeruginosa and Enterobacterales. Carbapenem consumption was correlated with carbapenem resistance, particularly in Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Fluoroquinolone use was correlated with fluoroquinolone resistance in Gram-negative bacilli and methicillin resistance in Staphylococcus aureus. Multivariate DR models highlited that AMC explained from 19 to 96% of AMR variation, with a lag time between AMC and AMR variation of 2 to 4 months. Few studies have investigated the predictive capacity of DR models, which appear to be limited. CONCLUSION: Despite their statistical robustness, DR models are not widely used. They confirmed the important role of fluoroquinolones, cephalosporins and carbapenems in the emergence of AMR. However, further studies are needed to assess their predictive capacity and usefulness for ASPs.

The ROSA-Like Prophage Colonizing Staphylococcus aureus Promotes Intracellular Survival, Biofilm Formation, and Virulence in a Chronic Wound Environment.

BACKGROUND: The transition from colonization to invasion is critical in diabetic foot ulcer (DFU). Staphylococcus aureus can colonize DFU, or invade the underlying tissues, causing serious infections. The ROSA-like prophage has previously been implicated in strain colonization characteristics of S aureus isolates in uninfected ulcers. METHODS: In this study, we investigated this prophage in the S aureus-colonizing strain using an in vitro chronic wound medium mimicking the chronic wound environment. RESULTS: Chronic wound medium reduced bacterial growth and increased biofilm formation and virulence in a zebrafish model. CONCLUSIONS: The ROSA-like prophage promoted intracellular survival of S aureus-colonizing strain in macrophages, keratinocytes, and osteoblasts.

Proteomic insights into Helcococcus kunzii in a diabetic foot ulcer-like environment.

PURPOSE: Helcococcus kunzii is a skin commensal, Gram-positive bacterium, mostly isolated from infected chronic wounds. This opportunistic pathogen is usually co-isolated with Staphylococcus aureus. The present dataset explores the production and secretion of H. kunzii bacterial virulence interacting proteins in a growth medium mimicking chronic wounds in exponential and stationary growth phases. EXPERIMENTAL DESIGN: The H. kunzii cellular proteome and exoproteome were assessed by analyzing three biological replicates per condition tested. Samples were analyzed using a Q-Exactive HF mass spectrometer. Comparative and functional analyses were performed to profile the identified protein set. RESULTS: The H. kunzii's cellular proteome encompassed 969 proteins, among which 64 and 53 were specifically identified in the exponential and stationary phase of growth, respectively. Its exoproteome comprised 58 proteins, among which 16 and 14 were characteristic of each growth stage. Metabolic differences between the two phases of growth are discussed. Besides, the production of previously shortlisted and novel putative H. kunzii targets involved in modulating the virulence of S. aureus is investigated. CONCLUSION AND CLINICAL RELEVANCE: This work, pioneering the study of H. kunzii physiology in a chronic wound-like environment, should assist future research on this opportunistic pathogen and the search for innovative approaches for wound management.

Effect of antimicrobial consumption on Escherichia coli resistance: assessment and forecasting using Dynamic Regression models in a French university hospital (2014-2019).

INTRODUCTION: The aim of this study was to determine the correlation between antimicrobial consumption (AMC) and antimicrobial resistance (AMR) in Escherichia coli at a hospital level, and assess the capacity of dynamic regression (DR) models to predict AMR for their use in deployment of antimicrobial stewardship programs (ASPs). METHODS: A retrospective epidemiological study was conducted in a French tertiary hospital between 2014 and 2019. DR models were used to assess the correlation between AMC and AMR from 2014 to 2018. The predictive abilities of the models were estimated by comparing the predicted data with those observed in 2019. RESULTS: Rates of fluoroquinolone and cephalosporin resistance decreased. AMC increased overall but decreased for fluoroquinolone. DR models highlighted that the decrease in use of fluoroquinolone and the increase in use of anti-pseudomonal activity penicillin with beta-lactamase inhibitor (AAPBI) explained 54% of the decrease in fluoroquinolone resistance and 15% of the decrease in cephalosporin resistance. In addition, penicillin/beta-lactamase inhibitor (PBI) consumption explained 53% of PBI resistance, and beta-lactam use explained 36% of penicillin resistance, with both remaining stable over time. DR models had predictive capabilities with margins of error from 8% to 34%. CONCLUSION: Over a six-year period in a French tertiary hospital, decreasing rates of resistance to fluoroquinolones and cephalosporins were correlated with decreasing use of fluoroquinolone and increasing use of AAPBI, whereas rates of resistance to penicillin remained high and stable. The results indicate that DR models should be used with caution for AMR forecasting and ASP implementation.

Effect of Antibiotic Exposure on Staphylococcus epidermidis Responsible for Catheter-Related Bacteremia.

Coagulase-negative staphylococci (CoNS) and especially Staphylococcus epidermidis are responsible for health care infections, notably in the presence of foreign material (e.g., venous or central-line catheters). Catheter-related bacteremia (CRB) increases health care costs and mortality. The aim of our study was to evaluate the impact of 15 days of antibiotic exposure (ceftobiprole, daptomycin, linezolid and vancomycin) at sub-inhibitory concentration on the resistance, fitness and genome evolution of 36 clinical strains of S. epidermidis responsible for CRB. Resistance was evaluated by antibiogram, the ability to adapt metabolism by the Biofilm Ring test® and the in vivo nematode virulence model. The impact of antibiotic exposure was determined by whole-genome sequencing (WGS) and biofilm formation experiments. We observed that S. epidermidis strains presented a wide variety of virulence potential and biofilm formation. After antibiotic exposure, S. epidermidis strains adapted their fitness with an increase in biofilm formation. Antibiotic exposure also affected genes involved in resistance and was responsible for cross-resistance between vancomycin, daptomycin and ceftobiprole. Our data confirmed that antibiotic exposure modified bacterial pathogenicity and the emergence of resistant bacteria.

Antimicrobial activity of antibiotics on biofilm formed by Staphylococcus aureus and Pseudomonas aeruginosa in an open microfluidic model mimicking the diabetic foot environment.

BACKGROUND: Diabetic foot infections (DFIs) represent a public health problem because of their frequency and the severity of their consequences, i.e. amputation and mortality. Polymicrobial biofilms on the skin surface of these ulcers complicate wound healing. Few in vitro models exist to study the antibiotics activity in this context. OBJECTIVES: This study evaluated the in vitro activity of antibiotics against the two main bacteria isolated in DFI, Staphylococcus aureus and Pseudomonas aeruginosa, using a dynamic system (BioFlux™ 200) and a chronic wound-like medium (CWM) that mimic the foot ulcer environment. METHODS: Reference strains and two pairs of clinical S. aureus and P. aeruginosa isolated together from a DFI were cultivated in brain heart infusion and CWM media during 72 h at 37°C, alone and combined in the BioFlux™ 200 system. Antibiotic activity was evaluated after a mechanical debridement. The activities were measured by the reduction of biofilm percentage of bacteria in the microfluidic system using microscopy. RESULTS: Daptomycin for S. aureus and ceftazidime for P. aeruginosa showed excellent activity to reduce biofilm biomass, whereas linezolid action was more mitigated and dalbavancin was ineffective. Ceftazidime + daptomycin presented the most potent in vitro activity on a mixed biofilm. CONCLUSIONS: The combination of CWM and the BioFlux™ microfluidic system represents a valuable tool to screen the potential antimicrobial activity of antibiotics under conditions mimicking those encountered in DFI. It could help clinicians in their management of chronic wounds.

Role of gut microbiota and bacterial translocation in acute intestinal injury and mortality in patients admitted in ICU for septic shock.

Introduction: Sepsis is a life-threatening organ dysfunction with high mortality rate. The gut origin hypothesis of multiple organ dysfunction syndrome relates to loss of gut barrier function and the ensuing bacterial translocation. The aim of this study was to describe the evolution of gut microbiota in a cohort of septic shock patients over seven days and the potential link between gut microbiota and bacterial translocation. Methods: Sixty consecutive adult patients hospitalized for septic shock in intensive care units (ICU) were prospectively enrolled. Non-inclusion criteria included patients with recent or scheduled digestive surgery, having taken laxatives, pre- or probiotic in the previous seven days, a progressive digestive neoplasia, digestive lymphoma, chronic inflammatory bowel disease, moribund patient, and pregnant and lactating patients. The primary objective was to evaluate the evolution of bacterial diversity and richness of gut microbiota during seven days in septic shock. Epidemiological, clinical and biological data were gathered over seven days. Gut microbiota was analyzed through a metagenomic approach. 100 healthy controls were selected among healthy blood donors for reference basal 16S rDNA values. Results: Significantly lower bacterial diversity and richness was observed in gut microbiota of patients at Day 7 compared with Day 0 (p<0.01). SOFA score at Day 0, Acute Gastrointestinal Injury (AGI) local grade, septic shock origin and bacterial translocation had an impact on alpha diversity. A large increase in Enterococcus genus was observed at Day 7 with a decrease in Enterobacterales, Clostridiales, Bifidobacterium and other butyrate-producing bacteria. Discussion: This study shows the importance of bacterial translocation during AGI in septic shock patients. This bacterial translocation decreases during hospitalization in ICUs in parallel to the decrease of microbiota diversity. This work highlights the role of gut microbiota and bacterial translocation during septic shock.

Phenotypic and Genotypic Virulence Characterisation of Staphylococcus pettenkoferi Strains Isolated from Human Bloodstream and Diabetic Foot Infections.

Staphylococcus pettenkoferi is a recently described coagulase-negative Staphylococcus identified in human diseases, especially in infections of foot ulcers in patients living with diabetes mellitus. To date, its pathogenicity remains underexplored. In this study, whole-genome analysis was performed on a collection of 29 S. pettenkoferi clinical strains isolated from bloodstream and diabetic foot infections with regard to their phylogenetic relationships and comprehensive analysis of their resistome and virulome. Their virulence was explored by their ability to form biofilm, their growth kinetics and in an in vivo zebrafish embryo infection model. Our results identified two distinct clades (I and II) and two subclades (I-a and I-b) with notable genomic differences. All strains had a slow bacterial growth. Three profiles of biofilm formation were noted, with 89.7% of isolates able to produce biofilm and harbouring a high content of biofilm-encoding genes. Two virulence profiles were also observed in the zebrafish model irrespective of the strains' origin or biofilm profile. Therefore, this study brings new insights in S. pettenkoferi pathogenicity.

Antibiofilm Properties of Antiseptic Agents Used on Pseudomonas aeruginosa Isolated from Diabetic Foot Ulcers.

In diabetic foot ulcers (DFUs), biofilm formation is a major challenge that promotes wound chronicity and delays healing. Antiseptics have been proposed to combat biofilms in the management of DFUs. However, there is limited evidence on the activity of these agents against biofilms, and there are questions as to which agents have the best efficiency. Here, we evaluated the antibiofilm activity of sodium hypochlorite, polyvinylpyrrolidoneIodine (PVPI), polyhexamethylenebiguanide (PHMB) and octenidine against Pseudomonas aeruginosa strains using static and dynamic systems in a chronic-wound-like medium (CWM) that mimics the chronic wound environment. Using Antibiofilmogram®, a technology assessing the ability of antiseptics to reduce the initial phase of biofilm formation, we observed the significant activity of antiseptics against biofilm formation by P. aeruginosa (at 1:40 to 1:8 dilutions). Moreover, 1:100 to 1:3 dilutions of the different antiseptics reduced mature biofilms formed after 72 h by 10-log, although higher concentrations were needed in CWM (1:40 to 1:2). Finally, in the BioFlux200TM model, after biofilm debridement, sodium hypochlorite and PHMB were the most effective antiseptics. In conclusion, our study showed that among the four antiseptics tested, sodium hypochlorite demonstrated the best antibiofilm activity against P. aeruginosa biofilms and represents an alternative in the management of DFUs.

Isolation and Identification of Flavones Responsible for the Antibacterial Activities of Tillandsia bergeri Extracts.

Plants are an everlasting inspiration source of biologically active compounds. Among these medicinal plants, the biological activity of extracts from some species of the Tillandsia genus has been studied, but the phytochemistry of the hardy species Tillandsia bergeri remains unknown. The aim of the present study was to perform the first phytochemical study of T. bergeri and to identify the compounds responsible for the antibacterial activity of T. bergeri extracts. Soxhlet extraction of predried and grinded leaves was first performed using four increasing polarity solvents. A bio-guided fractionation was performed using agar overlay bioautography as a screening method against 12 Gram-positive, Gram-negative, sensitive, and resistant bacterial strains. The results showed the inhibition of Gram-positive methicillin-sensitive Staphylococcus aureus ATCC 29213 (MSSA), methicillin-resistant S. aureus N-SARM-1 (MRSA), and Staphylococcus caprae ATCC 35538 by the dichloromethane fraction. A phytochemical investigation led to the isolation and identification by high-resolution mass spectrometry and nuclear magnetic resonance of the two flavones penduletin and viscosine, responsible for this antibacterial activity. For viscosine, the minimum inhibitory concentration (MIC) value is equal to 128 µg/mL against MSSA and is equal to 256 µg/mL against MRSA and S. caprae. The combination of these compounds with vancomycin and cloxacillin showed a decrease in MICs of the antibiotics. Penduletin showed synergistic activity when combined with vancomycin against MSSA (FICI < 0.258) and S. caprae (FICI < 0.5). Thus, unexplored Tillandsia species may represent a valuable source for potential antibiotics and adjuvants.

Polymicrobial Biofilm Organization of Staphylococcus aureus and Pseudomonas aeruginosa in a Chronic Wound Environment.

Biofilm on the skin surface of chronic wounds is an important step that involves difficulties in wound healing. The polymicrobial nature inside this pathogenic biofilm is key to understanding the chronicity of the lesion. Few in vitro models have been developed to study bacterial interactions inside this chronic wound. We evaluated the biofilm formation and the evolution of bacteria released from this biofilm on the two main bacteria isolated in this condition, Staphylococcus aureus and Pseudomonas aeruginosa, using a dynamic system (BioFlux™ 200) and a chronic wound-like medium (CWM) that mimics the chronic wound environment. We observed that all species constituted a faster biofilm in the CWM compared to a traditional culture medium (p < 0.01). The percentages of biofilm formation were significantly higher in the mixed biofilm compared to those determined for the bacterial species alone (p < 0.01). Biofilm organization was a non-random structure where S. aureus aggregates were located close to the wound surface, whereas P. aeruginosa was located deeper in the wound bed. Planktonic biofilm-detached bacteria showed decreased growth, overexpression of genes encoding biofilm formation, and an increase in the mature biofilm biomass formed. Our data confirmed the impact of the chronic wound environment on biofilm formation and on bacterial lifecycle inside the biofilm.

Real-time metagenomics-based diagnosis of community-acquired meningitis: A prospective series, southern France.

BACKGROUND: Point-Of-Care (POC) diagnosis of life-threatening community-acquired meningitis currently relies on multiplexed RT-PCR assays, that lack genotyping and antibiotic susceptibility profiling. We assessed the usefulness of real-time metagenomics (RTM) directly applied to the cerebrospinal fluid (CSF) for the identification, typing and susceptibility profiling of pathogens responsible for community-acquired meningitis. METHODS: A series of 52 CSF samples from patients suspected of having community-acquired meningitis, were investigated at POC by direct RTM in parallel to routine real-time multiplex PCR (RT-PCR) and bacterial culture, for the detection of pathogens. RTM-generated sequences were blasted in real-time against an in-house database incorporating the panel of 12 most prevalent pathogens and against NCBI using EPI2ME online software, for pathogen identification. In-silico antibiogram and genotype prediction were determined using the ResFinder bio-tool and MLST online software. FINDINGS: Over eight months, routine multiplex RT-PCR yielded 49/52 positive CSFs, including 21 Streptococcus pneumoniae, nine Neisseria meningitidis, eight Haemophilus influenzae, three Streptococcus agalactiae, three Herpesvirus-1, two Listeria monocytogenes, and one each of Escherichia coli, Staphylococcus aureus and Varicella-Zoster Virus. Parallel RTM agreed with the results of 47/52 CSFs and revealed two discordant multiplex RT-PCR false positives, one H. influenzae and one S. pneumoniae. Both multiplex RT-PCR and RTM agreed on the negativity of three CSFs. While multiplex RT-PCR routinely took 90 min, RTM took 120 min, although the pipeline analysis detected the pathogen genome after 20 min of sequencing in 33 CSF samples; and after two hours in 14 additional CSFs; yielding > 50% genome coverage in 19 CSFs. RTM identified 14 pathogen genotypes, including a majority of H. influenzae b, N. meningitidis B and S. pneumoniae 11A and 3A. In all 16 susceptible cultured bacteria, the in-silico antibiogram agreed with the in-vitro antibiogram in 10 cases, available within 48 h in routine bacteriology. INTERPRETATION: In addition to pathogen detection, RTM applied to CSF samples offered supplementary information on bacterial profiling and genotyping. These data provide the proof-of-concept that RTM could be implemented in a POC laboratory for one-shot diagnostic and genomic surveillance of pathogens responsible for life-threatening meningitis. FUNDING: This work was supported by the French Government under the Investments in the Future programme managed by the National Agency for Research reference: Méditerranée Infection 10-IAHU-03.

Bacterial Interactions in the Context of Chronic Wound Biofilm: A Review.

Chronic wounds, defined by their resistance to care after four weeks, are a major concern, affecting millions of patients every year. They can be divided into three types of lesions: diabetic foot ulcers (DFU), pressure ulcers (PU), and venous/arterial ulcers. Once established, the classical treatment for chronic wounds includes tissue debridement at regular intervals to decrease biofilm mass constituted by microorganisms physiologically colonizing the wound. This particular niche hosts a dynamic bacterial population constituting the bed of interaction between the various microorganisms. The temporal reshuffle of biofilm relies on an organized architecture. Microbial community turnover is mainly associated with debridement (allowing transitioning from one major representant to another), but also with microbial competition and/or collaboration within wounds. This complex network of species and interactions has the potential, through diversity in antagonist and/or synergistic crosstalk, to accelerate, delay, or worsen wound healing. Understanding these interactions between microorganisms encountered in this clinical situation is essential to improve the management of chronic wounds.

Bacteriophage Therapy for Staphylococcus Aureus Infections: A Review of Animal Models, Treatments, and Clinical Trials.

Staphylococcus aureus (S. aureus) is a common and virulent human pathogen causing several serious illnesses including skin abscesses, wound infections, endocarditis, osteomyelitis, pneumonia, and toxic shock syndrome. Antibiotics were first introduced in the 1940s, leading to the belief that bacterial illnesses would be eradicated. However, microorganisms, including S. aureus, began to develop antibiotic resistance from the increased use and abuse of antibiotics. Antibiotic resistance is now one of the most serious threats to global public health. Bacteria like methicillin-resistant Staphylococcus aureus (MRSA) remain a major problem despite several efforts to find new antibiotics. New treatment approaches are required, with bacteriophage treatment, a non-antibiotic strategy to treat bacterial infections, showing particular promise. The ability of S. aureus to resist a wide range of antibiotics makes it an ideal candidate for phage therapy studies. Bacteriophages have a relatively restricted range of action, enabling them to target pathogenic bacteria. Their usage, usually in the form of a cocktail of bacteriophages, allows for more focused treatment while also overcoming the emergence of resistance. However, many obstacles remain, particularly in terms of their effects in vivo, necessitating the development of animal models to assess the bacteriophage efficiency. Here, we provide a review of the animal models, the various clinical case treatments, and clinical trials for S. aureus phage therapy.