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Introduction: Fecal microbiota transplantation (FMT) is recommended as safe and effective treatment for recurrent Clostridioides difficile infections. Freezing the FMT preparation simplifies the process, allowing a single stool sample to be used for multiple receivers and over an extended period of time. We aimed to assess the effect of long-term frozen storage on bacterial taxonomic profiles of a stool suspension prepared for FMT.Methods: DNA was extracted from a stool suspension before freezing and sequentially during the 18-month storage period at −80°C. Two different protocols were used for DNA extraction. The first relied on a classical mechanical and chemical cell disruption to extract both intra- and extracellular DNA; the second included specific pre-treatments aimed at removing free DNA and DNA from human and damaged bacterial cells. Taxonomic profiling of bacterial communities was performed by sequencing of V3–V4 16S rRNA gene amplicons.Results: Microbiota profiles obtained by whole DNA extraction procedure remained relatively stable during frozen storage. When DNA extraction procedure included specific pre-treatments, microbiota similarity between fresh and frozen samples progressively decreased with longer frozen storage times; notably, the abundance of Bacteroidetes decreased in a storage duration-dependent manner. The abundance of Firmicutes, the main butyrate producers in the colon, were not much affected by frozen storage for up to 1 year.Conclusion: Our data show that metataxonomic analysis of frozen stool suspensions subjected to specific pre-treatments prior to DNA extractions might provide an interesting indication of bacterial resistance to stress conditions and thus of chances of survival in FMT recipients.

Primary and revision arthroplasties are increasing worldwide, as are periprosthetic joint infections (PJI). The management of PJI requires surgery, the strategy of which is dictated by the acute or chronic nature of the infection, with an exchange of the implant in the event of a chronic PJI or in the case of recurrence with the same pathogen. We report the case of a 63-year-old man with two episodes of Streptococcus dysgalactiae subsp. equisimilis PJI within 9 months. Based on clinical suspicion of an haematogenous PJI, the patient was treated by DAIR (debridement, antibiotics, implant retention), while genomic sequencing revealed two different strains, confirming our hypothesis that no additional surgery was needed. Hence, we report a case where genomic analysis was decisive for the decision of the best therapeutic strategy.

Diagnosis of culture-negative infective endocarditis usually implies indirect pathogen identification by serologic or molecular techniques. Clinical metagenomics, relying on next-generation sequencing (NGS) is an emerging approach that allows pathogen identification in challenging situations, as evidenced by a clinical case. We sequenced the DNA extracted from the surgically-removed frozen valve tissue from a patient with suspected infective endocarditis with negative blood and valve cultures. Mapping of the sequence reads against reference genomic sequences, a 16S rRNA gene database and clade-specific marker genes suggested an infection caused by Cardiobacterium hominis.

The most common causes of infective endocarditis (IE) are Staphylococcus, Streptococcus, Enterococcus, and HACEK-related organisms. In 15–30% of the IE cases, standard blood cultures remain sterile. We aimed at identifying the causative agent of a blood-culture-negative IE by whole metagenome shotgun sequencing (WMGS). A 54-year old woman diagnosed with community-onset pneumonia by a general practitioner, was admitted with dyspnea, cough and fever. The patient's blood cultures were repeatedly negative. The transesophageal echocardiography and transthoracic echocardiography showed an echo density on the left coronary leaflet of the aortic valve and signs suggestive of a ruptured abscess of the mitro-aortic junction. The patient underwent a semi-urgent aortic valve replacement by a mechanical prosthetic valve. We extracted DNA from the surgically-removed fresh valve tissue. The extraction procedure included bacterial/fungal DNA enrichment procedure. Nextera XT library prepared from the valve DNA extract was sequenced (2 × 250) on an Illumina MiSeq instrument. Sequence reads were mapped against bacterial genomic sequences, 16S rRNA genes and clade-specific taxonomic markers. Most of the 103,136 sequencing reads classified as bacterial were assigned to Neisseria meningitidis. In line with these data, mapping of reads against clade-specific and 16S rRNA gene markers revealed N. meningitidis as the most represented species. Assembled metagenomic fragments had the best average nucleotide identity (ANI) with N. meningitidis. Comparison of assembled contigs to reference alleles showed that this strain belongs to the ST-41/44 complex. N. meningitidis is commonly associated with meningitis and/or septicemia but should not be neglected as a causative agent of IE, which became exceedingly rare with the introduction of antibiotics. Our data show that WMGS may be used as a diagnostic procedure to strengthen the diagnosis of IE and to obtain draft genomic sequence of the pathogen and typing information.

Until 2007, Staphylococcus aureus from clonal complex 398 (CC398) was exclusively associated with livestock species and companion animals. Recently, several studies described the emergence of S. aureus CC398 as etiologies of severe infections in humans living in an animal-free environment. Recent sequencing efforts showed that the mobile genetic elements found in CC398 isolates were specific for each population and enabled differentiation of strains responsible for asymptomatic colonization from strains involved in bloodstream infections. We mobilized prophages from a human CC398 isolate and introduced them into two naïve ancestral isolates devoid of prophages that exclusively colonize animals. These lysogenized ancestral CC398 isolates acquired features related to virulence, such as an increased capacity to adhere to human extracellular matrix proteins and the ability to invade and survive within non-phagocytic cells. Pathogenicity of several clinical isolates from the CC398 lineage as well as ancestral and in vitro lysogenized ancestral counterparts was assessed in a model of infectious endocarditis in rats. Natural and artificial lysogens were not only more invasive than their prophage-free parent but also showed an increased capacity to multiply within aortic vegetations. This study identified prophages as mediators of bacterial virulence in a model of infectious endocarditis, probably through promotion of interaction with extracellular matrix components. Further studies are needed to identify mechanisms leading to promotion of intrinsic virulence.

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.

Here, we sequenced DNA extracted from a necrotic hepatic lesion from a patient with suspected chronic hepatic brucelloma but negative culture results. Although most of the taxonomically classified sequencing reads corresponded to human genome sequences, our data suggest that whole-metagenome shotgun sequencing may be used together with other tests to strengthen the diagnosis of hepatic brucelloma.