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We present the case of a 72-year-old female patient with acute contained rupture of a biological composite graft, 21 months after replacement of the aortic valve and the ascending aorta due to an aortic dissection. Auramine-rhodamine staining of intraoperative biopsies showed acid-fast bacilli, but classical culture and molecular methods failed to identify any organism. Metagenomic analysis indicated infection with Mycobacterium chelonae, which was confirmed by target-specific qPCR. The complexity of the sample required a customized bioinformatics pipeline, including cleaning steps to remove sequences of human, bovine ad pig origin. Our study underlines the importance of multiple testing to increase the likelihood of pathogen identification in highly complex samples.

Background: Oral microbiota has largely escaped attention in Parkinson’s disease (PD), despite its pivotal role in maintaining oral and systemic health. Objective: The aim of our study was to examine the composition of the oral microbiota and the degree of oral inflammation in PD. Methods: Twenty PD patients were compared to 20 healthy controls. Neurological, periodontal and dental examinations were performed as well as dental scaling and gingival crevicular fluid sampling for cytokines measurement (interleukine (IL)-1β, IL-6, IL-1 receptor antagonist (RA), interferon-γ and tumor necrosis factor (TNF)-α). Two months later, oral microbiota was sampled from saliva and subgingival dental plaque. A 16S rRNA gene amplicon sequencing was used to assess bacterial communities. Results: PD patients were in the early and mid-stage phases of their disease (Hoehn & Yahr 2–2.5). Dental and periodontal parameters did not differ between groups. The levels of IL-1β and IL-1RA were significantly increased in patients compared to controls with a trend for an increased level of TNF-α in patients. Both saliva and subgingival dental plaque microbiota differed between patients and controls. Streptococcus mutans, Kingella oralis, Actinomyces AFQC_s, Veillonella AFUJ_s, Scardovia, Lactobacillaceae, Negativicutes and Firmicutes were more abundant in patients, whereas Treponema KE332528_s, Lachnospiraceae AM420052_s, and phylum SR1 were less abundant. Conclusion: Our findings show that the oral microbiome is altered in early and mid-stage PD. Although PD patients had good dental and periodontal status, local inflammation was already present in the oral cavity. The relationship between oral dysbiosis, inflammation and the pathogenesis of PD requires further study.

Background The management of ventilator-associated and hospital-acquired pneumonia requires rapid and accurate quantitative detection of the infecting pathogen(s). To do it, we propose a metagenomics next-generation sequencing (mNGS) assay that includes an internal sample processing control (SPC) for the quantitative detection of 20 relevant bacterial species of interest (SOI) from bronchoalveolar lavage (BAL) samples. Results To avoid very major errors in identification of respiratory pathogens due to “false negative” cases, each sample was spiked with Bacillus subtilis, at a precisely defined concentration, using rehydrated BioBall®. This SPC ensured detection and quantification of the pathogen(s) at defined minimum concentrations. In the presented mNGS workflow, absolute quantification of Staphylococcus aureus was as accurate as quantitative PCR. We defined a metagenomics threshold at 5.3E+3 genome equivalent unit per milliliter of the sample for each SOI, to distinguish colonization from higher amounts of pathogens that may be associated with infection. Complete mNGS process and metrics were assessed on 40 clinical samples, showing 100 % sensitivity compared to microbial culture. However, 19 out of the 29 (66 %) SOI detections above the metagenomics threshold were not associated with bacterial growth above classical culture-based clinical thresholds. Taxonomic classification of 7 (37 %) of these “false positive” detections were confirmed by finding specific 16S/MetaPhlAn2 markers, the 12 other (63 %) “false positive” detections did not yield enough reads to check their taxonomic classification. Conclusions Our SPC design and analytical workflow allowed efficient detection and absolute quantification of pathogens from BAL samples, even when the bacterial DNA quantity was largely below manufacturer’s recommendations for NGS. The frequent "false positive" detection suggested the presence of non culturable cells within the tested BAL samples. Finally, mNGS detected mixed infections including bacterial species that were not reported by routine cultures.

Serine/threonine kinase PknB and its corresponding phosphatase Stp are important regulators of many cell functions in the pathogen S. aureus. Genome-scale gene expression data of S. aureus strain NewHG (sigB+) elucidated their effect on physiological functions. Moreover, metabolic modelling from these data inferred metabolic adaptations. We compared wild-type to deletion strains lacking pknB, stp or both. Ser/Thr phosphorylation of target proteins by PknB switched amino acid catabolism off and gluconeogenesis on to provide the cell with sufficient components. We revealed a significant impact of PknB and Stp on peptidoglycan, nucleotide and aromatic amino acid synthesis, as well as catabolism involving aspartate transaminase. Moreover, pyrimidine synthesis was dramatically impaired by stp deletion but only slightly by functional loss of PknB. In double knockouts, higher activity concerned genes involved in peptidoglycan, purine and aromatic amino acid synthesis from glucose but lower activity of pyrimidine synthesis from glucose compared to the wild type. A second transcriptome dataset from S. aureus NCTC 8325 (sigB−) validated the predictions. For this metabolic adaptation, PknB was found to interact with CdaA and the yvcK/glmR regulon. The involved GlmR structure and the GlmS riboswitch were modelled. Furthermore, PknB phosphorylation lowered the expression of many virulence factors, and the study shed light on S. aureus infection processes.

Capnocytophaga canis is an uncommon cause of septic shock. Only three cases have been previously reported in the literature. In this article, we describe the case of a 70-year-old male admitted to the intensive care unit for septic shock of unknown origin. On day 2, one anaerobic bottle out of the two sets taken at admission turned positive with Gram-negative bacilli. The pathogen was identified by 16S rRNA gene as C. canis. The strain was characterized and compared with other clinical isolates of Capnocytophaga spp.

International audience; Invasive meningococcal disease (IMD), caused by Neisseria meningitidis (Nm) strains, is a life-threatening but vaccine-preventable condition. Bexsero is a four-component vaccine that offers broad protection against Nm of serogroup B (NmB), particularly common in Europe. In Switzerland, Bexsero has not yet been licensed and no information is available concerning the predicted vaccine coverage on isolates of circulating Nm. We performed genotyping of Bexsero antigen loci by whole-genome sequencing (WGS) on 104 NmB collected in Switzerland in the 2010-2015 period. We searched for antigen variants previously defined as predictors of strain coverage and estimated that 50% of IMD NmB strains were potentially covered by the vaccine. Clonal complexes (cc) 32, 41/44 and 269, considered the best covered lineages, were further sub-typed according to Bexsero Antigen Sequence Type (BAST) scheme. We also genotyped by WGS 40 Nm of serogroup W (NmW) collected in the country between 2010 and 2016. NmW cc22 isolates appeared to be covered by the vaccine, which was not the case for cc11 isolates, whose incidence has recently increased in Switzerland and all over Europe. Our work underlines the benefit of using WGS for surveillance of vaccine antigen variant distribution in local Nm population and taking proper measures to prevent the spread of NmB.

We report the selection in a 15-year-old boy of a multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Aeromonas salmonicida after medicinal leech therapy that required an antibiotic prophylaxis based on piperacillin/tazobactam and cotrimoxazole. Whole genome sequencing of the strain indeed revealed 13 antibiotic resistance genes, including the ESBL CTX-M-3 and the unusual β-lactamase SCO-1. Keywords: Aeromonas salmonicida, medicinal leech therapy, multidrug resistance, whole genome sequencing

Staphylococcus aureusis a major human pathogen. Despite high incidence and morbidity, molecular mechanisms occurring during infection remain largely unknown. Under defined conditions, biofilm formation contributes to the severity ofS. aureusrelated infections. Extracellular DNA (eDNA), a component of biofilm matrix released from apoptotic bacteria, is involved in biofilm structure and stability. In many bacterial biofilms, eDNA originates from cell lysis although eDNA can also be actively secreted or exported by bacterial membrane vesicles. By screening the Nebraska transposon library, we identifiedrpiRcas a biofilm regulator involved in eDNA regulation. RpiRc is a transcription factor from the pentose phosphate pathway (PPP) whose product is a polysaccharide intercellular adhesin (PIA) precursor. However,rpiRcmutant strain showed neither susceptibility to DispersinB® (a commercially available enzyme disrupting PIA biofilms) nor alteration oficatranscription (the operon regulating PIA production). Decreased biofilm formation was linked to Sln, an extracellular compound degrading eDNA in an autolysis independent pathway. Biofilm susceptibility to antibiotics in wt and mutant strains was tested using a similar protocol as the Calgary biofilm device. Involvement of RpiRc inS. aureusvirulence was assessedex vivoby internalization experiments into HEK293 cells andin vivoin a mouse model of subcutaneous catheter infection. While minimum inhibitory concentrations (MICs) of planktonic cells were not affected in the mutant strain, we observed increased biofilm susceptibility to almost all tested antibiotics, regardless of their mode of action. More importantly, therpiRcmutant showed reduced virulence in bothex vivoandin vivoexperiments related to decreasedfnbpA-Btranscription and eDNA production. RpiRc is an important regulator involved in eDNA degradation inside the matrix of mature PIA independent biofilms. These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also toS. aureuscell adhesion and virulence during subcutaneous infection.Author summaryBiofilm formation contributes to the severity ofStaphylococcus aureusrelated infections. Biofilm matrix is mainly composed by polysaccharide intercellular adhesion (PIA), proteins and extracellular DNA (eDNA). By screening a mutant library ofS. aureus, RpiRc was identified as a new regulator of eDNA dependent biofilm formation. How RpiRc regulates biofilm and its role in S. aureus virulence was studied in four differentS. aureusstrains. Deletion of RpiRc resulted in a pronounced decreased eDNA dependent biofilm formation, but not PIA dependent biofilm formation. Decreased biofilm formation was not related to increased autolysis, but was linked to extracellular compounds found in the supernatant of mutant biofilms. Sln was identified as one of this compound. RpiRc deletion also decreased biofilm recalcitrance (resistance) to selected antibiotics. Involvement of RpiRc inS. aureuspathogenesis was investigatedex vivoby internalization into HEK293 cells andin vivoin a mouse model of catheter infection. RpiRc deletion resulted in decreased virulence related to decreased expression of surface proteins like the fibronectin binding proteins A and B (FnbpA-B). These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also toS. aureuscell adhesion and virulence during subcutaneous infection.

Objectives Predicting the antibiotic susceptibility phenotype from genomic data is challenging, especially for some specific antibiotics in the order Enterobacterales. Here we aimed to assess the performance of whole genomic sequencing (WGS) for predicting the antibiotic susceptibility in various Enterobacterales species using the detection of antibiotic resistance genes (ARGs), specific mutations and a knowledge-based decision algorithm. Methods We sequenced (Illumina MiSeq, 2×250 bp) 187 clinical isolates from species possessing (n = 98) or not (n = 89) an intrinsic AmpC-type cephalosporinase. Phenotypic antibiotic susceptibility was performed by the disc diffusion method. Reads were assembled by A5-miseq and ARGs were identified from the ResFinder database using Diamond. Mutations on GyrA and ParC topoisomerases were studied. Piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, meropenem, amikacin, gentamicin and ciprofloxacin were considered for prediction. Results A total of 1496 isolate/antibiotic combinations (187 isolates × 8 antibiotics) were considered. In 230 cases (15.4%), no attempt of prediction was made because it could not be supported by current knowledge. Among the 1266 attempts, 1220 (96.4%) were correct (963 for predicting susceptibility and 257 for predicting resistance), 24 (1.9%) were major errors (MEs) and 22 (1.7%) were very major errors (VMEs). Concordance were similar between non-AmpC and AmpC-producing Enterobacterales (754/784 (96.2%) vs 466/482 (96.7%), chi-square test p 0.15), but more VMEs were observed in non-AmpC producing strains than in those producing an AmpC (19/784 (2.4%) vs 3/466 (0.6%), chi-square test p 0.02). The majority of VMEs were putatively due to the overexpression of chromosomal genes. Conclusions In conclusion, the inference of antibiotic susceptibility from genomic data showed good performances for non-AmpC and AmpC-producing Enterobacterales species. However, more knowledge about the mechanisms underlying the derepression of AmpC are needed.

Background Endograft infection is a rare but extremely dangerous complication of aortic repair (25–100% of mortality). We describe here the first case of Listeria monocytogenes abdominal periaortitis associated with a vascular graft. We also discuss the differential diagnosis of periaortitis and provide a literature review of L. monocytogenes infectious aortitis. Case presentation Nine months after endovascular treatment of an abdominal aortic aneurysm (abdominal stent graft), a 76-year-old man was admitted for severe abdominal pain radiating to the back. Laboratory tests were normal apart from elevated C-reactive protein (CRP). Injected abdominal computed tomography (CT) showed infiltration of the fat tissues around the aortic endoprosthesis and aneurysmal sac expansion; positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro- D-glucose integrated with computed tomography (18F-FDG PET/CT) showed a hypermetabolic mass in contact with the endoprosthesis. Blood cultures were negative. At surgical revision, an infra-renal peri-aortic abscess was evident; post-operative antibiotic therapy with ciprofloxacin and doxycycline was started. Cultures of intraoperative samples were positive for L. monocytogenes. Results were further confirmed by a broad-range polymerase chain reaction (PCR) and next-generation sequencing. Antibiotic treatment was switched to intravenous amoxicillin for 6 weeks. Evolution was uneventful with decrease of inflammatory parameters and regression of the abscess. Conclusion An etiologic bacterial diagnosis before starting antibiotic therapy is paramount; nevertheless, culture-independent methods may provide a microbiological diagnosis in those cases where antimicrobials are empirically used and when cultures remain negative.

The risk of acquisition of multidrug-resistant Enterobacteriaceae (MRE) and of occurrence of diarrhea is high when traveling to tropical regions. The relationships between these phenomena and the composition of human gut microbiota have not yet been assessed. Here, we investigated the dynamics of changes of metabolically active microbiota by sequencing total RNA from fecal samples taken before and after travel to tropical regions. We included 43 subjects who could provide fecal samples before and after a travel to tropical regions. When found positive by culturing for any MRE after travel, the subjects sent an additional sample 1 month later. In all, 104 fecal samples were considered (43 before travel, 43 at return, 18 one month after travel). We extracted the whole RNA, performed retrotranscription and sequenced the cDNA (MiSeq 2x300bp). The reads were mapped to the reference operational taxonomic units (OTUs) and species/strains using the 16S Greengenes and 23S SILVA databases. We found that the occurrence of diarrhea during the travel was associated with a higher relative abundance of Prevotella copri before departure and after return. The composition of microbiota, before travel as well as at return, was not correlated with the acquisition of MRE. However, the clearance of MRE one month after return was linked to a specific pattern of bacterial species that was also found before and after return. In conclusion, we found specific OTUs associated to a higher risk of diarrhea during a stay in tropical regions and to a faster clearance of MRE after their acquisition.

To compare bacteria recovered by standard cultures and metataxonomics, particularly with regard to ventilator-associated pneumonia (VAP) pathogens, and to determine if the presence of particular bacteria or microbiota in tracheal and oropharyngeal secretions during the course of intubation was associated with the development of VAP. In this case–control study, oropharyngeal secretions and endotracheal aspirate were collected daily in mechanically ventilated patients. Culture and metataxonomics (16S rRNA gene-based taxonomic profiling of bacterial communities) were performed on serial upper respiratory samples from patients with late-onset definite VAP and their respective controls. Metataxonomic analyses showed that a low relative abundance of Bacilli at the time of intubation in the oropharyngeal secretions was strongly associated with the subsequent development of VAP. On the day of VAP, the quantity of human and bacterial DNA in both tracheal and oropharyngeal secretions was significantly higher in patients with VAP than in matched controls with similar ventilation times. Molecular techniques identified the pathogen(s) of VAP found by culture, but also many more bacteria, classically difficult to culture, such as Mycoplasma spp. and anaerobes. Molecular analyses of respiratory specimens identified markers associated with the development of VAP, as well as important differences in the taxa abundance between VAP and controls. Further prospective trials are needed to test the predictive value of these markers, as well as the relevance of uncultured bacteria in the pathogenesis of VAP.

Predicting the antibiotic susceptibility phenotype from genomic data is challenging, especially for some specific antibiotics in Enterobacteriaceae. Here we aimed to assess the performance of whole genomic sequencing (WGS) for predicting the antibiotic susceptibility in various Enterobacteriaceae species using the detection of antibiotic resistance genes (ARGs), specific mutations and a knowledge-based decision algorithm. We sequenced (Illumina MiSeq 2x250b) 187 clinical isolates from species possessing (n=98) or not (n=89) an intrinsic AmpC-type cephalosporinase. Antibiotic susceptibility was performed by the disc diffusion method. Reads were assembled by A5-miseq and ARGs were identified from the ResFinder database using Diamond. Mutations on GyrA and ParC topoisomerases were studied. We assessed the prediction rates for amoxicillin, co-amoxiclav, piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, meropenem, amikacin, gentamicin and fluoroquinolones. A total of 1,870 isolate/antibiotic combinations were considered. In 295 cases (15.8%) no attempt of prediction was made. Among the 1,575 attempts, 1,537 (97.6%) were correct (1,011 for predicting susceptibility and 526 for predicting resistance), 15 (0.9%) were major errors (MEs) and 23 (1.5%) were very major errors (VMEs). The concordance rates were similar between non-AmpC and AmpC-producing Enterobacteriaceae (907/935 [97.0%] vs 630/640 [98.4%], Chi2 test p=0.07), but more VMEs were observed in non-AmpC producing strains than in those producing an AmpC (20/935 [2.1%] vs 3/640 [0.5%], Chi2 test p=0.007). The majority of VMEs were putatively due to the overexpression of chromosomal genes. In conclusion, the inference of antibiotic susceptibility from genomic data showed good performances for non-AmpC and AmpC-producing Enterobacteriaceae species.

Background: The R-GNOSIS (Resistance in Gram-Negative Organisms: Studying Intervention Strategies) WP3 study was the first multicenter randomized clinical trial systematically investigating fecal microbiota transplantation (FMT) for intestinal decolonization of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) or carbapenemase-producing Enterobacteriaceae (CPE). Here, we characterized the temporal dynamics of fecal microbiota changes in a sub-cohort of the R-GNOSIS WP3 participants before and after antibiotics/FMT using whole metagenome shotgun sequencing. Methods: We sequenced fecal DNA obtained from 16 ESBL-E/CPE carriers having received oral colistin/neomycin followed by FMT and their corresponding seven donors. Ten treatment-na&iuml, ve controls from the same trial were included. Fecal samples were collected at baseline (V0), after antibiotics but before FMT (V2) and three times after FMT (V3, V4 and V5). Results: Antibiotic treatment transiently decreased species richness and diversity and increased the abundance of antibiotic resistance determinants (ARDs). Bifidobacterium species, together with butyrate- and propionate-producing species from Lachnospiraceae and Ruminococcaceae families were significantly enriched in post-FMT microbiota of treated carriers. After FMT, the proportion of Enterobacteriaceae was lower compared to baseline but without statistical significance. Conclusions: Combined antibiotic and FMT treatment resulted in enrichment of species that are likely to limit the gut colonization by ESBL-E/CPE.

Apical periodontitis is an inflammatory disease of the dental periradicular tissues triggered by bacteria colonizing necrotic root canals. Primary apical periodontitis results from the microbial colonization of necrotic pulp tissues. Secondary apical periodontitis results from a persistent infection of incorrectly treated root canals. The aim of this study was to characterize the microbiota present in primary and secondary intraradicular infections associated with apical periodontitis using 16S rRNA gene amplicon sequencing. Teeth exhibiting apical periodontitis with or without root canal treatment were extracted after informed consent. From each tooth, the intraradicular content as well as a dentin sample (control) were collected and subjected to DNA extraction. PCR amplicons of the V3–V4 region of the bacterial 16S rRNA gene were pooled and sequenced (2 × 300) on an Illumina MiSeq instrument. The bioinformatics analysis pipeline included quality filtering, merging of forward and reverse reads, clustering of reads into operational taxonomic units (OTUs), removal of putative contaminant OTUs and assigning taxonomy. The most prevalent and abundant OTU in both dentin and root canal samples was assigned to anaerobic bacterium Fusobacterium nucleatum. Multivariate analysis showed clustering of microbiota by sample type (dentin vs. intraradicular content) and, in root canals, by pathology (primary vs. secondary infection). The proportions of Enterococcus faecalis and F. nucleatum were, respectively, higher and lower when comparing secondary to primary infected root canals. Co-occurrence network analysis provided evidence of microbial interactions specific to the infection type. The identification of bacterial taxa differentially abundant in primary and secondary intraradicular infections may provide the basis for targeted therapeutic approaches aimed at reducing the incidence of apical periodontitis.

The first-in-class lipopeptide antibiotic daptomycin (DAP) is highly active against Gram-positive pathogens including s-lactam and glycopeptide resistant strains. Its molecular mode of action remains enigmatic, since a defined target has not been identified so far and multiple effects, primarily on the cell envelope have been observed. Reduced DAP susceptibility has been described in S. aureus and enterococci after prolonged treatment courses. In line with its pleiotropic antibiotic activities, a unique, defined molecular mechanism of resistance has not emerged, instead non-susceptibility appears often accompanied by alterations in membrane composition and changes in cell wall homeostasis. We compared S. aureus strains HG001 and SG511, which differ primarily in the functionality of the histidine kinase GraS, to evaluate the impact of the GraRS regulatory system on the development of DAP non-susceptibility. After extensive serial passing, both DAPR variants reached a minimal inhibitory concentration of 31 μg/ml and shared some phenotypic characteristics (e.g. thicker cell wall, reduced autolysis). However, based on comprehensive analysis of the underlying genetic, transcriptomic and proteomic changes, we found that both strains took different routes to achieve DAP resistance. Our study highlights the impressive genetic and physiological capacity of S. aureus to counteract pleiotropic activities of cell wall- and membrane-active compounds even when a major cell wall regulatory system is dysfunctional.

The applications of whole-metagenome shotgun sequencing (WMGS) in routine clinical analysis are still limited. A combination of a DNA extraction procedure, sequencing, and bioinformatics tools is essential for the removal of human DNA and for improving bacterial species identification in a timely manner. We tackled these issues with a broncho-alveolar lavage (BAL) sample from an immunocompromised patient who had developed severe chronic pneumonia. We extracted DNA from the BAL sample with protocols based either on sequential lysis of human and bacterial cells or on the mechanical disruption of all cells. Metagenomic libraries were sequenced on Illumina HiSeq platforms. Microbial community composition was determined by k-mer analysis or by mapping to taxonomic markers. Results were compared to those obtained by conventional clinical culture and molecular methods. Compared to mechanical cell disruption, a sequential lysis protocol resulted in a significantly increased proportion of bacterial DNA over human DNA and higher sequence coverage of Mycobacterium abscessus, Corynebacterium jeikeium and Rothia dentocariosa, the bacteria reported by clinical microbiology tests. In addition, we identified anaerobic bacteria not searched for by the clinical laboratory. Our results further support the implementation of WMGS in clinical routine diagnosis for bacterial identification.

International audience; Bone and joint infections (BJI) are severe infections that require a tailored and protracted antibiotic treatment. Yet, the diagnostic based on culturing samples lacks sensitivity, especially for hardly culturable bacteria. Metagenomic sequencing could potentially address those limitations. Here, we assessed the performances of metagenomic sequencing on 24 BJI samples for the identification of pathogens and the prediction of their antibiotic susceptibility. For monomicrobial samples in culture (n = 8), the presence of the pathogen was confirmed by metagenomics in all cases. For polymicrobial samples (n = 16), 32/55 bacteria (58.2%) were found at the species level (and 41/55 [74.5%] at the genus level). Conversely, 273 bacteria not found in culture were identified, 182 being possible pathogens and 91 contaminants. A correct antibiotic susceptibility could be inferred in 94.1% and 76.5% cases for monomicrobial and polymicrobial samples, respectively. Altogether, we found that clinical metagenomics applied to BJI samples is a potential tool to support conventional culture. Context. Bone and joint infections (BJI) are severe infections that affect a growing number of patients 1. Along with the surgical intervention, the microbiological diagnosis is a keystone of the management of BJI in (i) identifying the bacteria causing the infection and (ii) assessing their susceptibility to antibiotics. Currently, this is achieved by culturing surgical samples on various media and conditions, together with a long time of incubation to recover fastidiously-growing bacteria that can be involved in BJI. Still, some bacteria will not grow under these conditions because of extreme oxygen sensitivity, a prior antibiotic intake or metabolic issues (e.g. quiescent bacteria in chronic infections). Consequently, the antibiotic treatment may not span all the bacteria involved in the infection, which can favour the relapse and the need for a new surgery. Clinical metagenomics refers to the concept of sequencing all the DNA (i.e. all the genomes) present in a clinical sample with the purpose of identifying pathogens and inferring their antibiotic susceptibility pattern 2. This new, culture-independent method takes advantages of the thrilling development of next-generation sequenc-ing (NGS) technologies since the mid-2000s. The NGS platforms typically yield thousands to millions of reads (sequences of size ranging from 100 bp to a few kbp), which virtually enables to recover the sequences of all the genes present in the sample, yet in a disorganised fashion. Substantial bio-informatics efforts are thereby needed to reconstruct and reorder the original sequences in genomes, and are referred to as the assembly process. Hence, various information such as the taxonomic identification of the present species, antibiotic resistance determinants (ARDs), mutations (as compared to a reference genome or sequence), single nucleotide variants (SNVs, for clonality assessment) and virulence genes can be determined. Clinical metagenomics is an emerging field in medicine. So far, a few attempts to use metagenomics on clinical samples have been performed (on urines 3, 4 , cerebrospinal fluid or brain biopsy 5, 6 , blood 7 and skin granuloma 8) likely because of the high price of metagenomics and the complexity of the management of sequence data for clinical microbiologists. To the best of our knowledge, metagenomics has never been applied to BJI samples.

International audience; Until recently, Staphylococcus aureus from clonal complex (CC)398 were mostly described as colonizing asymptomatic raised pigs and pig-farmers. Currently, the epidemiology of the CC398 lineage is becoming more complex. CC398 human-adapted isolates are increasingly being identified in bloodstream infections in humans living in animal-free environments. In addition, CC398 isolates are increasingly responsible for invasive infections in various animals. CC398 isolates that colonize asymptomatic pigs and the isolates that infect humans living in animal-free environments (human-adapted isolates) both lack several clinically important S. aureus-associated virulence factors but differ on the basis of their prophage content. Recent findings have provided insight into the influence of a φMR11-like helper prophage on the ability of CC398 isolates to infect humans. To assess the recent spread of the CC398 lineage to various animal species and to investigate the links between the φMR11-like prophage and the emergence of CC398 isolates infecting animals, we studied 277 isolates causing infections in unrelated animals. The prevalence of CC398 isolates increased significantly between 2007 and 2013 (p < 0.001); 31.8% of the animal isolates harbored the φMR11-like prophage. High-density DNA microarray experiments with 37 representative infected-animal isolates positive for φMR11-like DNA established that most infected-animal isolates carried many genetic elements related to antimicrobial resistance and virulence genes, and a φ3 prophage encoding immune-modulating proteins and associated with animal-to-human jumps. Our findings suggest recent clonal expansion and dissemination of a new subpopulation of CC398 isolates, responsible for invasive infections in various animals, with a considerable potential to colonize and infect humans, probably greater than that of human-adapted CC398 isolates, justifying active surveillance.