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.

A prospective multicentre evaluation of BioFire® Joint Infection Panel for the rapid microbiological documentation of acute arthritis.

OBJECTIVES: To assess the performance of the rapid syndromic BioFire® Joint Infection Panel (BF-JIP) to detect bacterial and fungal pathogens, as well as antibiotic resistance genes, directly in synovial fluid specimens collected from patients with acute arthritis. METHODS: The study was conducted in six French bacteriological laboratories. To assess the performances of BF-JIP, results were compared with those of synovial fluid 14-day culture and, in case of discrepancy, with those of complementary molecular methods and intraoperative samples. A total of 308 synovial fluid specimens were tested after collection from 308 adults and children presenting with clinical and biological suspicion of acute arthritis; patients presenting with acute periprosthetic joint infection were included according to the European Bone and Joint Infection Society 2021 criteria. RESULTS: Only one specimen failed (no result). On the basis of the consolidated data, the BF-JIP was concordant with the 14-day culture in 280 (91.2%) of the 307 specimens finally included in the study. The positive percentage agreement was 84.9% (95% CI, 78.8-89.8%) and the negative percentage agreement was 100% (95% CI, 97.2-100%). The positive predictive value was extremely high (100%; 95% CI, 97.6-100%), whereas the negative predictive value was lower (82.6%; 95% CI, 75.7-88.2%), partially explained by the missing target species in the panel. DISCUSSION: The BF-JIP showed high performances to detect pathogens involved in acute arthritis.

Evaluation of the Microbiome Identification of Forensically Relevant Biological Fluids: A Pilot Study.

In forensic sciences, body fluids, or biological traces, are a major source of information, and their identification can play a decisive role in criminal investigations. Currently, the nature of biological fluids is assessed using immunological, physico-chemical, mRNA and epigenetic methods, but these have limits in terms of sensitivity and specificity. The emergence of next-generation sequencing technologies offers new opportunities to identify the nature of body fluids by determining bacterial communities. The aim of this pilot study was to assess whether analysis of the bacterial communities in isolated and mixed biological fluids could reflect the situation observed in real forensics labs. Several samples commonly encountered in forensic sciences were tested from healthy volunteers: saliva, vaginal fluid, blood, semen and skin swabs. These samples were analyzed alone or in combination in a ratio of 1:1. Sequencing was performed on the Ion Gene StudioTM S5 automated sequencer. Fluids tested alone revealed a typical bacterial signature with specific bacterial orders, enabling formal identification of the fluid of interest, despite inter-individual variations. However, in biological fluid mixtures, the predominance of some bacterial microbiomes inhibited interpretation. Oral and vaginal microbiomes were clearly preponderant, and the relative abundance of their bacterial communities and/or the presence of common species between samples made it impossible to detect bacterial orders or genera from other fluids, although they were distinguishable from one another. However, using the beta diversity, salivary fluids were identified and could be distinguished from fluids in combination. While this method of fluid identification is promising, further analyses are required to consolidate the protocol and ensure reliability.

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.

In Vivo-Acquired Resistance to Daptomycin during Methicillin-Resistant Staphylococcus aureus Bacteremia.

Daptomycin (DAP) represents an interesting alternative to treat methicillin-resistant Staphylococcus aureus (MRSA) infections. Different mechanisms of DAP resistance have been described; however, in vivo-acquired resistance is uncharacterized. This study described the phenotypic and genotypic evolution of MRSA strains that became resistant to DAP in two unrelated patients with bacteremia under DAP treatment, in two hospitals in the South of France. DAP MICs were determined using broth microdilution method on the pairs of isogenic (DAP-S/DAP-R) S. aureus isolated from bloodstream cultures. Whole genome sequencing was carried out using Illumina MiSeq Sequencing system. The two cases revealed DAP-R acquisition by MRSA strains within three weeks in patients treated by DAP. The isolates belonged to the widespread ST5 (patient A) and ST8 (patient B) lineages and were of spa-type t777 and t622, respectively. SNP analysis comparing each DAP-S/DAP-R pair confirmed that the isolates were isogenic. The causative mutations were identified in MprF (Multiple peptide resistance Factor) protein: L826F (Patient A) and S295L (Patient B), and in Cls protein: R228H (Patient B). These proteins encoded both proteins of the lipid biosynthetic enzymes. The resistance to DAP is particularly poorly described whereas DAP is highly prescribed to treat MRSA. Our study highlights the non-systematic cross-resistance between DAP and glycopeptides and the importance of monitoring DAP MIC in persistent MRSA bacteremia.

Isolation and Characterization of New Bacteriophages against Staphylococcal Clinical Isolates from Diabetic Foot Ulcers.

Staphylococcus sp. is the most common bacterial genus in infections related to diabetic foot ulcers (DFUs). The emergence of multidrug-resistant bacteria places a serious burden on public health systems. Phage therapy is an alternative treatment to antibiotics, overcoming the issue of antibiotic resistance. In this study, six phages (SAVM01 to SAVM06) were isolated from effluents and were used against a panel of staphylococcal clinical samples isolated from DFUs. A genomic analysis revealed that the phages belonged to the Herelleviridae family, with sequences similar to those of the Kayvirus genus. No lysogeny-associated genes, known virulence or drug resistance genes were identified in the phage genomes. The phages displayed a strong lytic and antibiofilm activity against DFU clinical isolates, as well as against opportunistic pathogenic coagulase-negative staphylococci. The results presented here suggest that these phages could be effective biocontrol agents against staphylococcal clinical isolates from DFUs.

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.

Clinical Features and Outcomes of Enterococcal Bone and Joint Infections and Factors Associated with Treatment Failure over a 13-Year Period in a French Teaching Hospital.

Enterococcal bone and joint infections (BJIs) are reported to have poor outcomes, but there are conflicting results. This study aimed to describe the clinical characteristics and outcomes of patients with enterococcal BJI and to assess the factors associated with treatment failure. We conducted a retrospective cohort study at Nimes University Hospital from January 2007 to December 2020. The factors associated with treatment failure were assessed using a Cox model. We included 90 consecutive adult patients, 11 with native BJIs, 40 with prosthetic joint infections and 39 with orthopedic implant-associated infections. Two-thirds of patients had local signs of infection, but few (9%) had fever. Most BJIs were caused by Enterococcus faecalis (n = 82, 91%) and were polymicrobial (n = 75, 83%). The treatment failure rate was 39%, and treatment failure was associated with coinfection with Staphylococcus epidermidis (adjusted hazard ratio = 3.04, confidence interval at 95% [1.31-7.07], p = 0.01) and with the presence of local signs of inflammation at the time of diagnosis (aHR = 2.39, CI 95% [1.22-4.69], p = 0.01). Our results confirm the poor prognosis of enterococcal BJIs, prompting clinicians to carefully monitor for local signs of infection and to optimize the medical-surgical management in case of coinfections, especially with S. epidermidis.

Peripherally Inserted Central Venous Catheter (PICC) Related Bloodstream Infection in Cancer Patients Treated with Chemotherapy Compared with Noncancer Patients: A Propensity-Score-Matched Analysis.

The use of peripherally inserted central catheters (PICCs) has increased in cancer patients. This study aimed to compare the incidence of PICC-related bloodstream infections (PICCR-BSIs) in cancer patients treated with chemotherapy and in noncancer patients. We performed a secondary analysis from a retrospective, single-center, observational cohort. The PICCR-BSI incidence rates in cancer and noncancer patients were compared after 1:1 propensity-score matching. Then, the factors associated with PICCR-BSI were assessed in a Cox model. Among the 721 PICCs (627 patients) included in the analysis, 240 were placed in cancer patients for chemotherapy and 481 in noncancer patients. After propensity-score matching, the PICCR-BSI incidence rate was 2.6 per 1000 catheter days in cancer patients and 1.0 per 1000 catheter days in noncancer patients (p < 0.05). However, after adjusting for variables resulting in an imbalance between groups after propensity-score matching, only the number of PICC lumens was independently associated with PICCR-BSI (adjusted hazard ratio 1.81, 95% confidence interval: 1.01-3.22; p = 0.04). In conclusion, the incidence rate of PICCR-BSI is higher in cancer patients treated with chemotherapy than in noncancer patients, but our results also highlight the importance of limiting the number of PICC lumens in such patients.

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.

Case Report: Acute Brucellosis Due to Brucella suis in a Brazilian Gold Miner Diagnosed in French Guiana.

A 29-year-old Brazilian illegal gold miner developed intermittent fever. Blood cultures were positive for Gram-negative coccobacilli and, after an initial misidentification by an automated identification system, the diagnosis of brucellosis caused by Brucella suis was confirmed. We hypothesize an association with domestic or wild swine exposure. The patient responded well to standard antibiotic therapy of brucellosis. We report the first case of human brucellosis on the Guiana Shield. This report underlines the importance of considering brucellosis in the presence of a fever of unknown origin, even in the Amazonian rainforest area, where several zoonotic diseases might be considered in the differential diagnosis of unexplained fever.

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 Tocilizumab on Mortality in Patients with SARS-CoV-2 Pneumonia Caused by Delta or Omicron Variants: A Propensity-Matched Analysis in Nimes University Hospital, France.

We aimed to assess the factors associated with mortality in patients treated with tocilizumab for a SARS-CoV-2 pneumonia due to the delta or omicron variants of concern (VOC) and detect an effect of tocilizumab on mortality. We conducted a prospective cohort study in a tertiary hospital from 1 August 2021 to 31 March 2022 including patients with severe COVID-19, treated with tocilizumab. Factors associated with mortality were assessed in a Cox model; then, the 60-day mortality rates of COVID-19 patients treated with standard of care (SoC) +/- tocilizumab were compared after 1:1 propensity score matching. The mortality rate was 22% (N = 26/118) and was similar between delta and omicron cases (p = 0.6). The factors independently associated with mortality were age (HR 1.06; 95% CI (1.02-1.11), p = 0.002), Charlson index (HR 1.33; 95% CI (1.11-1.6), p = 0.002), WHO-CPS (HR 2.56; 95% CI (1.07-6.22) p = 0.03), and tocilizumab infusion within the first 48 h following hospital admission (HR 0.37, 95% CI (0.14-0.97), p = 0.04). No significant differences in mortality between the tocilizumab plus SoC and SoC alone groups (p = 0.5) were highlighted. However, the patients treated with tocilizumab within the 48 h following hospital admission had better survival (p = 0.04). In conclusion, our results suggested a protective effect on mortality of the early administration of tocilizumab in patients with severe COVID-19 regardless of the VOC involved.

Novel Species of Brucella Causing Human Brucellosis, French Guiana.

Human brucellosis is a zoonoses caused by bacteria of the genus Brucella. Infection results in subacute or chronic debilitating disease with nonspecific clinical manifestations and is often associated with consuming unpasteurized dairy products. We report 2 cases of brucellosis in male patients who were hospitalized in distinct towns of French Guiana, an overseas territory of France located on the northeastern shore of South America. Both men were citizens of Brazil working as clandestine goldminers in the deep Amazonian rainforest. Characterization of the 2 bacterial isolates revealed that they represent a potential new species of Brucella. Medical practitioners working in contact with wildlife in this region of the world should be aware of the existence of these pathogens and the potential for human infection.

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.

Clinical factors associated with peripherally inserted central catheters (PICC) related bloodstream infections: a single centre retrospective cohort.

BACKGROUND: Despite their spread in daily practice, few data is available on clinical factors associated with peripherally inserted central catheter (PICC)-related bloodstream infections (PR-BSI). We aimed to assess the PR-BSI incidence, microbiology, and factors associated with PR-BSI with a focus on clinical symptoms. METHODS: We conducted a retrospective cohort study in a French university hospital. We screened all PICC insertions performed from April 1st, 2018, to April 1st, 2019, and included PICC insertions in adult patients. We assessed the PR-BSI incidence, the factors associated with PR-BSI using a Cox model, and negative and positive predictive values (NPVs and PPVs) of each clinical sign for PR-BSI. RESULTS: Of the 901 PICCs inserted in 783 patients (38,320 catheters days), 214 PICCs (24%) presented with a complication. The most prevalent complication was PR-BSI (1.9 per 1000 catheter days; 8.1% of inserted PICCs ). Enterobacterales (N = 27, 37%) and coagulase negative Staphylococci (N = 24, 33%), were the main microorganisms responsible for PR-BSI. Factors independently associated with occurrence of PR-BSI were fever (hazard ratio 13.21, 95% confidence interval 6.00-29.11, p < 0.001) and chills (HR 3.66, 95%CI 1.92-6.99, p < 0.001). All clinical signs and a duration of PICC maintenance ≥ 28 days, had a low PPVs (≤ 67.1%) but high NPVs (≥ 92.5%) for PR-BSI. CONCLUSIONS: Monitoring of clinical signs, especially fever and chills, with caution and limitation of device maintenance duration, could improve PICC management.