ResFinderFG v2.0: a database of antibiotic resistance genes obtained by functional metagenomics.

Metagenomics can be used to monitor the spread of antibiotic resistance genes (ARGs). ARGs found in databases such as ResFinder and CARD primarily originate from culturable and pathogenic bacteria, while ARGs from non-culturable and non-pathogenic bacteria remain understudied. Functional metagenomics is based on phenotypic gene selection and can identify ARGs from non-culturable bacteria with a potentially low identity shared with known ARGs. In 2016, the ResFinderFG v1.0 database was created to collect ARGs from functional metagenomics studies. Here, we present the second version of the database, ResFinderFG v2.0, which is available on the Center of Genomic Epidemiology web server (https://cge.food.dtu.dk/services/ResFinderFG/). It comprises 3913 ARGs identified by functional metagenomics from 50 carefully curated datasets. We assessed its potential to detect ARGs in comparison to other popular databases in gut, soil and water (marine + freshwater) Global Microbial Gene Catalogues (https://gmgc.embl.de). ResFinderFG v2.0 allowed for the detection of ARGs that were not detected using other databases. These included ARGs conferring resistance to beta-lactams, cycline, phenicol, glycopeptide/cycloserine and trimethoprim/sulfonamide. Thus, ResFinderFG v2.0 can be used to identify ARGs differing from those found in conventional databases and therefore improve the description of resistomes.

IncC plasmid genome rearrangements influence the vertical and horizontal transmission tradeoff in Escherichia coli.

It has been shown that an evolutionary tradeoff between vertical (host growth rate) and horizontal (plasmid conjugation) transmissions contributes to global plasmid fitness. As conjugative IncC plasmids are important for the spread of multidrug resistance (MDR), in a broad range of bacterial hosts, we investigated vertical and horizontal transmissions of two multidrug-resistant IncC plasmids according to their backbones and MDR-region rearrangements, upon plasmid entry into a new host. We observed plasmid genome deletions after conjugation in three diverse natural Escherichia coli clinical strains, varying from null to high number depending on the plasmid, all occurring in the MDR region. The plasmid burden on bacterial fitness depended more on the strain background than on the structure of the MDR region, with deletions appearing to have no impact. Besides, we observed an increase in plasmid transfer rate, from ancestral host to new clinical recipient strains, when the IncC plasmid was rearranged. Finally, using a second set of conjugation experiments, we investigated the evolutionary tradeoff of the IncC plasmid during the critical period of plasmid establishment in E. coli K-12, by correlating the transfer rates of deleted or non-deleted IncC plasmids and their costs on the recipient strain. Plasmid deletions strongly improved conjugation efficiency with no negative growth effect. Our findings indicate that the flexibility of the MDR-region of the IncC plasmids can promote their dissemination, and provide diverse opportunities to capture new resistance genes. In a broader view, they suggest that the vertical-horizontal transmission tradeoff can be manipulated by the plasmid to improve its fitness.

Inter-phylum circulation of a beta-lactamase-encoding gene: a rare but observable event.

Beta-lactamase-mediated degradation of beta-lactams is the most common mechanism of beta-lactam resistance in Gram-negative bacteria. Beta-lactamase-encoding genes can be transferred between closely related bacteria, but spontaneous inter-phylum transfers (between distantly related bacteria) have never been reported. Here, we describe an extended-spectrum beta-lactamase (ESBL)-encoding gene (blaMUN-1) shared between the Pseudomonadota and Bacteroidota phyla. An Escherichia coli strain was isolated from a patient in Münster (Germany). Its genome was sequenced. The ESBL-encoding gene (named blaMUN-1) was cloned, and the corresponding enzyme was characterized. The distribution of the gene among bacteria was investigated using the RefSeq Genomes database. The frequency and relative abundance of its closest homolog in the global microbial gene catalog (GMGC) were analyzed. The E. coli strain exhibited two distinct morphotypes. Each morphotype possessed two chromosomal copies of the blaMUN-1 gene, with one morphotype having two additional copies located on a phage-plasmid p0111. Each copy was located within a 7.6-kb genomic island associated with mobility. blaMUN-1 encoded for an extended-spectrum Ambler subclass A2 beta-lactamase with 43.0% amino acid identity to TLA-1. blaMUN-1 was found in species among the Bacteroidales order and in Sutterella wadsworthensis (Pseudomonadota). Its closest homolog in GMGC was detected frequently in human fecal samples. This is, to our knowledge, the first reported instance of inter-phylum transfer of an ESBL-encoding gene, between the Bacteroidota and Pseudomonadota phyla. Although the gene was frequently detected in the human gut, inter-phylum transfer was rare, indicating that inter-phylum barriers are effective in impeding the spread of ESBL-encoding genes, but not entirely impenetrable.

Oropharyngeal and intestinal concentrations of opportunistic pathogens are independently associated with death of SARS-CoV-2 critically ill adults.

BACKGROUND: The composition of the digestive microbiota may be associated with outcome and infections in patients admitted to the intensive care unit (ICU). The dominance by opportunistic pathogens (such as Enterococcus) has been associated with death. However, whether this association remains all throughout the hospitalization are lacking. METHODS: We performed a single-center observational prospective cohort study in critically ill patients admitted with severe SARS-CoV-2 infection. Oropharyngeal and rectal swabs were collected at admission and then twice weekly until discharge or death. Quantitative cultures for opportunistic pathogens were performed on oropharyngeal and rectal swabs. The composition of the intestinal microbiota was assessed by 16S rDNA sequencing. Oropharyngeal and intestinal concentrations of opportunistic pathogens, intestinal richness and diversity were entered into a multivariable Cox model as time-dependent covariates. The primary outcome was death at day 90. RESULTS: From March to September 2020, 95 patients (765 samples) were included. The Simplified Acute Physiology Score 2 (SAPS 2) at admission was 33 [24; 50] and a Sequential Organ Failure Assessment score (SOFA score) at 6 [4; 8]. Day 90 all-cause mortality was 44.2% (42/95). We observed that the oropharyngeal and rectal concentrations of Enterococcus spp., Staphylococcus aureus and Candida spp. were associated with a higher risk of death. This association remained significant after adjustment for prognostic covariates (age, chronic disease, daily antimicrobial agent use and daily SOFA score). A one-log increase in Enterococcus spp., S. aureus and Candida spp. in oropharyngeal or rectal swabs was associated with a 17% or greater increase in the risk of death. CONCLUSION: We found that elevated oropharyngeal/intestinal Enterococcus spp. S. aureus and Candida spp. concentrations, assessed by culture, are associated with mortality, independent of age, organ failure, and antibiotic therapy, opening prospects for simple and inexpensive microbiota-based markers for the prognosis of critically ill SARS-CoV-2 patients.

Towards monitoring of antimicrobial resistance in the environment: For what reasons, how to implement it, and what are the data needs?

Antimicrobial resistance (AMR) is a global threat to human and animal health and well-being. To understand AMR dynamics, it is important to monitor resistant bacteria and resistance genes in all relevant settings. However, while monitoring of AMR has been implemented in clinical and veterinary settings, comprehensive monitoring of AMR in the environment is almost completely lacking. Yet, the environmental dimension of AMR is critical for understanding the dissemination routes and selection of resistant microorganisms, as well as the human health risks related to environmental AMR. Here, we outline important knowledge gaps that impede implementation of environmental AMR monitoring. These include lack of knowledge of the 'normal' background levels of environmental AMR, definition of high-risk environments for transmission, and a poor understanding of the concentrations of antibiotics and other chemical agents that promote resistance selection. Furthermore, there is a lack of methods to detect resistance genes that are not already circulating among pathogens. We conclude that these knowledge gaps need to be addressed before routine monitoring for AMR in the environment can be implemented on a large scale. Yet, AMR monitoring data bridging different sectors is needed in order to fill these knowledge gaps, which means that some level of national, regional and global AMR surveillance in the environment must happen even without all scientific questions answered. With the possibilities opened up by rapidly advancing technologies, it is time to fill these knowledge gaps. Doing so will allow for specific actions against environmental AMR development and spread to pathogens and thereby safeguard the health and wellbeing of humans and animals.

Evaluation of SARS-CoV-2 in semen, seminal plasma, and spermatozoa pellet of COVID-19 patients in the acute stage of infection.

To date, there is limited information about the presence of SARS-CoV-2 in semen especially in the acute phase of the infection. While available data from cohort studies including a total of 342 patients in the acute or recovery phase of the infection are reassuring, one study mentioned detecting virus in the semen of 6/38 COVID-19 patients. Here we assessed SARS-CoV-2 presence in the semen of COVID-19 positive patients in the acute stage of infection, within 24 hours of the positive nasopharyngeal swabs. Semen, seminal plasma and spermatozoa pellet were screened for SARS-CoV-2 and manual or airborne contamination during semen sampling. Among the 32 COVID-19 volunteers, the median interval from the onset of symptoms to semen collection was 4 days [IQR: 0-8]. Only one presented positive SARS-CoV-2 PCR in semen and seminal plasma fractions, although the spermatozoa pellet was negative. Viral cultures were all negative. We observed slightly higher concentrations of bacterial DNA in the SARS-CoV-2 positive specimen than in all negative samples. The bacteria identified neither confirm nor rule out contamination by oropharyngeal secretions during collection. SARS-CoV-2 was rarely present in semen during the acute phase of the disease. This very rare situation could be connected to oral or manual contamination during semen collection. The possible presence of SARS-CoV-2 in semen calls for nasopharyngeal viral testing and strict hygiene protocols during semen collection before assisted reproductive attempts.

Endocarditis caused by Thalassospira sp.

We report a case of an infective endocarditis caused by a Thalassospira sp. in a 53-year-old man with pre-existing valvular lesions and living in French Polynesia as a fisherman. The strain was identified with DNA-sequecing methods while it was not by mass spectrometry.

Evidence for contamination as the origin for bacteria found in human placenta rather than a microbiota.

Until recently the in utero environment of pregnant women was considered sterile. Recent high-sensitivity molecular techniques and high-throughput sequencing lead to some evidence for a low-biomass microbiome associated with the healthy placenta. Other studies failed to reveal evidence for a consistent presence of bacteria using either culture or molecular based techniques. Comparing conflicting "placental microbiome" studies is complicated by the use of varied and inconsistent protocols. Given this situation, we undertook an evaluation of the in utero environment sterility using several controlled methods, in the same study, to evaluate the presence or absence of bacteria and to explain contradictions present in the literature. Healthy pregnant women (n = 38) were recruited in three maternity wards. Placenta were collected after cesarean section with or without Alexis® and vaginal delivery births. For this study we sampled fetal membranes, umbilical cord and chorionic villi. Bacterial presence was analyzed using bacterial culture and qPCR on 34 fetal membranes, umbilical cord and chorionic villi samples. Shotgun metagenomics was performed on seven chorionic villi samples. We showed that the isolation of meaningful quantities of viable bacteria or bacterial DNA was possible only outside the placenta (fetal membranes and umbilical cords) highlighting the importance of sampling methods in studying the in utero environment. Bacterial communities described by metagenomics analysis were similar in chorionic villi samples and in negative controls and were dependent on the database chosen for the analysis. We conclude that the placenta does not harbor a specific, consistent and functional microbiota.

[Microbiota establishment: an in utero colonization decisive for future health?] / Établissement du microbiote - Une colonisation in utero déterminante pour la santé future ?

Some diseases seem to have a developmental origin. Today, the microbiota is recognized as a determinant in health and diseases and one important step is its establishment in the neonate. Some variations in its composition including an imbalance (also called dysbiosis) have been associated to several pathologies. Recent studies suggest a bacterial colonization in the non-pregnant uterus, in the amniotic fluid and in the placenta, which were previously thought sterile. So, during deve-lopmental phases, the fetus could have encounter bacteria in utero. These bacteria could contribute to its microbiota establishment before parturition and therefore before the encounter with all microorganisms from vaginal, fecal and cutaneous microbiotas according to the delivery mode. However, studies stating the existence of such in utero microbiota, characterized by a low biomass, are somewhat disputed.