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.

Candidatus Methanosphaera massiliense sp. nov., a methanogenic archaeal species found in a human fecal sample and prevalent in pigs and red kangaroos.

Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense (Ca. M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca. M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca. M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca. M. massiliense and possible transfer between hosts.IMPORTANCEMethanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca. M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

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.

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.

Detection of Mycobacterium angelicum in Human Urinary Tract, French Polynesia.

We definitively characterized Mycobacterium angelicum, an aquatic zoonotic opportunistic pathogen of the M. szulgai complex, using a polyphasic approach that included whole-genome sequencing. The sequence was obtained on the island of Tahiti, French Polynesia, from a urine specimen collected from a patient experiencing a urinary tract infection.

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.

Characterization of the Secreted Acid Phosphatase SapS Reveals a Novel Virulence Factor of Staphylococcus aureus That Contributes to Survival and Virulence in Mice.

Staphylococcus aureus possesses a large arsenal of immune-modulating factors, enabling it to bypass the immune system's response. Here, we demonstrate that the acid phosphatase SapS is secreted during macrophage infection and promotes its intracellular survival in this type of immune cell. In animal models, the SA564 sapS mutant demonstrated a significantly lower bacterial burden in liver and renal tissues of mice at four days post infection in comparison to the wild type, along with lower pathogenicity in a zebrafish infection model. The SA564 sapS mutant elicits a lower inflammatory response in mice than the wild-type strain, while S. aureus cells harbouring a functional sapS induce a chemokine response that favours the recruitment of neutrophils to the infection site. Our in vitro and quantitative transcript analysis show that SapS has an effect on S. aureus capacity to adapt to oxidative stress during growth. SapS is also involved in S. aureus biofilm formation. Thus, this study shows for the first time that SapS plays a significant role during infection, most likely through inhibiting a variety of the host's defence mechanisms.

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.

Direct Metagenomic Diagnosis of Community-Acquired Meningitis: State of the Art.

Current routine diagnosis of community-acquired meningitis (CAM) by multiplex real-time polymerase chain reaction (RT-PCR) is limited in the number of tested pathogens and their full characterisation, requiring additional in vitro investigations to disclose genotype and antimicrobial susceptibility. We reviewed 51 studies published through December 2021 reporting metagenomic next generation sequencing (mNGS) directly applied to the cerebrospinal fluid (CSF). This approach, potentially circumventing the above-mentioned limitations, indicated 1,248 investigated patients, and 617 patients dually investigated by routine diagnosis and mNGS, in whom 116 microbes were detected, including 50 by mNGS only, nine by routine methods only, and 57 by both routine methods and mNGS. Of 217 discordant CSF findings, 103 CSF samples were documented by mNGS only, 87 CSF samples by routine methods only, and 27 CSF samples in which the pathogen identified by mNGS was different than that found using routine methods. Overall, mNGS allowed for diagnosis and genomic surveillance of CAM causative pathogens in real-time, with a cost which is competitive with current routine multiplex RT-PCR. mNGS could be implemented at point-of-care (POC) laboratories as a part of routine investigations to improve the diagnosis and molecular epidemiology of CAM, particularly in the event of failure of routine assays.

Emergence of multi-drug-resistant Mycobacterium tuberculosis in Niger: A snapshot based on whole-genome sequencing.

BACKGROUND: Among other West African countries experiencing the high endemicity of deadly tuberculosis, the situation in Niger is poorly evidenced by microbiological investigations. METHODOLOGY/PRINCIPAL FINDINGS: The study of 42 isolates of Mycobacterium tuberculosis from Niger by whole genome sequencing using Illumina iSeq technology yielded four M. tuberculosis lineages: Indo-Oceanic L1 (n = 1) (2.3%), East-Asian (n = 1) (2.3%), East-African Indian L3 (n = 2) (4.7%) and Euro-American L4 (n = 38) (90.4%). The sub-lineage L4.1.3 comprising 18 isolates (47.3%) was predominant, followed by the L4.6.2.2 sub-lineage (Cameroon genotype, n = 13 isolates) (34.2%). Investigating drug resistance profile for 12 antibiotics found 8/42 (19%) pan-susceptible isolates and 34/42 (81%) resistant isolates; with 40/42 (95.2%) isolates being susceptible to clofazimine-bedaquiline. CONCLUSIONS/SIGNIFICANCE: These unprecedented data from Niger highlight the dynamics of tuberculosis transmission and drug resistance in Niger and may assist tuberculosis control in this country which continues to support a high burden of tuberculosis.

Rapid Identification of Mycobacterium tuberculosis Complex Using Mass Spectrometry: A Proof of Concept.

Mycobacteria that form the Mycobacterium tuberculosis complex are responsible for deadly tuberculosis in animals and patients. Identification of these pathogens at the species level is of primary importance for treatment and source tracing and currently relies on DNA analysis, including whole genome sequencing (WGS), which requires a whole day. In this study, we report the unprecedented discrimination of M. tuberculosis complex species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), with WGS as the comparative reference standard. In the first step, optimized peptide extraction applied to 36 isolates otherwise identified in five of the 11 M. tuberculosis complex variants by WGS yielded 139 MALDI-TOF spectra, which were used to identify biomarkers of interest that facilitate differentiation between variants. In a second step, 70/80 (88%) other isolates were correctly classified by an algorithm based on specific peaks. This study is the first to report a MALDI-TOF-MS method for discriminating M. tuberculosis complex mycobacteria that is easily implemented in clinical microbiology laboratories.

A Tetragenococcus halophilus human gut isolate.

Tetragenococcus halophilus (T. halophilus) is a facultative anaerobic, coccus-shaped halophilic lactic acid-producing bacterium previously detected and cultured in various salty foods and credited for beneficial effects on human health. In this study, we investigated the presence of T. halophilus in human samples using a polyphasic approach including scanning electron microscopy, molecular biology methods and microbial culture. This unique investigation yielded the unprecedented presence of T. halophilus in human feces samples, thus enriching the repertoire of halophilic microorganisms colonizing the human gastrointestinal tract with the isolation and culture of T. halophilus for the first time in humans. Using the E-test strips, the MIC was assessed for T. halophilus strain CSURQ6002: rifampicin (MIC at 0.002 µg/mL), benzylpenicillin (MIC at 0.094 µg/mL), amoxicillin (MIC at 0.5 µg/mL), erythromycin (MIC at 2 µg/mL), clindamycin (MIC at 4 µg/mL), and vancomycin (MIC at 8 µg/mL). However, this strain showed a MIC up to 256 µg/mL for ciprofloxacin, fosfomycin, doxycyclin, imipenem, and colistin. In-silico profiling derived from whole genome sequencing (NCBI accession number: PRJNA780809), was confirmed. This discovery suggested that T. halophilus was part of the human digestive microbiota and that its potential role on human health should be considered.

Dynamics of community-acquired meningitis syndrome outbreaks in southern France.

In southern France, cases of community-acquired meningitis syndrome (CAM) are typically clustered as outbreaks with determinants which remain unknown. This 61-month retrospective investigation in Nîmes and Marseille university hospital laboratories, yielded 2,209/20,779 (10.63%) documented CAM cases caused by 62 different micro-organisms, represented by seasonal viral etiologies (78.8%), including Enterovirus, Herpes Simplex Virus (HSV), and Varicella-Zoster Virus (VZV; 1,620/2,209 = 73.4%). Multi correspondence analysis revealed an association of infection with age and sex, with the risk of infection being relatively higher in young men, as confirmed by Fisher's exact test (p < 10-3). Bacterial meningitis accounted for 20% of cases, mostly caused by Streptococcus pneumoniae (27.4% of cases), Neisseria meningitidis (12.5%), and Haemophilus influenzae (9.5%) with bacteria/virus coinfection (0.9%), and only six cases of documented fungal meningitis. In total, 62.6% of cases, of which 88.7% were undocumented, arose from 10 outbreaks. 33.2% of undocumented cases were aged >60 years compared to 19.2% of documented cases (p < 0.001), and viral infection was more common in the summer (87.5%) compared to other seasons (72.3%; p < 0.001). Outbreaks most often started in Nîmes and moved eastward toward Marseille at a speed of ~9 km/day, and these dynamics significantly correlated with atmospheric temperature, especially during summer outbreaks. In particular, the incidence of Enterovirus-driven outbreaks correlated with temperature, revealing correlation coefficients of 0.64 in Nîmes and 0.72 in Marseille, and its occurrence in Marseille lagged that in Nîmes by 1-2 weeks. Tracing the dynamics of CAM outbreak during this retrospective investigation in southern France yielded a speed of displacement that correlated with the variation in temperature between both cities, and these results provide clues for the next occurrence of undocumented outbreaks.

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.

Real-time next-generation sequencing on shell-vial culture to contribute to diagnosis of lymphatic tuberculosis: a case report.

Lymph node tuberculosis is a of limited clinical suspicion form of Mycobacterium tuberculosis infection. After 15 days incubation in a cellular culture and directly from the supernatant, 11 minutes of Oxford Nanopore MinION sequencing provided a preliminary result of an antibiotic-susceptible M. tuberculosis Indo-Oceanic lineage strain. Oxford Nanopore MinION sequencing is a promising tool for optimising the laboratory diagnosis of lymph node tuberculosis.

Direct Diagnosis of Echovirus 12 Meningitis Using Metagenomic Next Generation Sequencing.

The current point-of-care diagnosis of enterovirus meningitis does not identify the viral genotype, which is prognostic. In this case report, more than 81% of an Echovirus 12 genome were detected and identified by metagenomic next-generation sequencing, directly from the cerebrospinal fluid collected in a 6-month-old child with meningeal syndrome and meningitis: introducing Echovirus 12 as an etiological agent of acute meningitis in the pediatric population.

Haemophilus influenzae Meningitis Direct Diagnosis by Metagenomic Next-Generation Sequencing: A Case Report.

Current routine real-time PCR methods used for the point-of-care diagnosis of infectious meningitis do not allow for one-shot genotyping of the pathogen, as in the case of deadly Haemophilus influenzae meningitis. Real-time PCR diagnosed H. influenzae meningitis in a 22-year-old male patient, during his hospitalisation following a more than six-metre fall. Using an Oxford Nanopore Technologies real-time sequencing run in parallel to real-time PCR, we detected the H. influenzae genome directly from the cerebrospinal fluid sample in six hours. Furthermore, BLAST analysis of the sequence encoding for a partial DUF417 domain-containing protein diagnosed a non-b serotype, non-typeable H.influenzae belonging to lineage H. influenzae 22.1-21. The Oxford Nanopore metagenomic next-generation sequencing approach could be considered for the point-of-care diagnosis of infectious meningitis, by direct identification of pathogenic genomes and their genotypes/serotypes.

Direct next-generation sequencing diagnosis of echovirus 9 meningitis, France.

The prognosis of central nervous system infections caused by enteroviruses partially depends on the viral genotype, which is not provided by current point-of-care diagnostic methods. In this study, next-generation sequencing identified an echovirus 9 directly from the cerebrospinal fluid of a patient presenting with meningitis.