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The bacterial secretome comprises polypeptides expressed at the cell surface or released into the extracellular environment as well as the corresponding secretion machineries. Despite their reduced coding capacities, Mycoplasma spp. are able to produce and release several components into their environment, including polypeptides, exopolysaccharides and extracellular vesicles. Technical difficulties in purifying these elements from the complex broth media used to grow mycoplasmas have recently been overcome by optimizing growth conditions and switching to chemically defined culture media. However, the secretion pathways responsible for the release of these structurally varied elements are still poorly described in mycoplasmas. We propose the use of the term 'releasome,' instead of secretome, to refer to molecules released by mycoplasmas into their environment. The aim of this review is to more precisely delineate the elements that should be considered part of the mycoplasmal releasome and their role in the interplay of mycoplasmas with host cells and tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GB is currently organizing a Research Topic with the author FT., (Copyright © 2022 Gaurivaud and Tardy.)

With the appreciation that behavior represents the integration and complexity of the nervous system, neurobehavioral phenotyping and assessment has seen a renaissance over the last couple of decades, resulting in a robust database on rodent performance within various testing paradigms, possible associations with human disorders, and therapeutic interventions. The interchange of data across behavior and other test modalities and multiple model systems has advanced our understanding of fundamental biology and mechanisms associated with normal functions and alterations in the nervous system. While there is a demonstrated value and power of neurobehavioral assessments for examining alterations due to genetic manipulations, maternal factors, early development environment, the applied use of behavior to assess environmental neurotoxicity continues to come under question as to whether behavior represents a sensitive endpoint for assessment. Why is rodent behavior a sensitive tool to the neuroscientist and yet, not when used in pre-clinical or chemical neurotoxicity studies? Applying new paradigms and evidence on the biological basis of behavior to neurobehavioral testing requires expertise and refinement of how such experiments are conducted to minimize variability and maximize information. This review presents relevant issues of methods used to conduct such test, sources of variability, experimental design, data analysis, interpretation, and reporting. It presents beneficial and critical limitations as they translate to the in vivo environment and considers the need to integrate across disciplines for the best value. It proposes that a refinement of behavioral assessments and understanding of subtle pronounced differences will facilitate the integration of data obtained across multiple approaches and to address issues of translation., Competing Interests: Authors SM and MB were employed by company Social & Scientific Systems, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Harry, McBride, Witchey, Mhaouty-Kodja, Trembleau, Bridge and Bencsik.)

Poultry and poultry meat are important contributors to the global antimicrobial burden. Unregulated and illegal use of extended-spectrum cephalosporins (ESC) in this sector has long been identified as a major cause of massive spread of ESC-resistant Escherichia coli , and colistin usage is considered a main driver of plasmid-mediated mcr genes dissemination. In Lebanon, the first mcr-1 -positive E. coli found in poultry dates back to 2015, followed by a few reports of mcr-1 -positive E. coli in poultry, swine, humans, and the environment. On the contrary, a comprehensive picture of the population structure of mcr-1 -positive E. coli and mcr-1 -bearing plasmids carrying the mcr-1 gene using whole-genome analysis is largely lacking. This study reports the prevalence of mcr-1 -positive E. coli in poultry originating from 32 farms across three Lebanese governorates and slaughtered in the same place. We report 27/32 (84.4%) mcr-1 positive farms, leading to a total of 84 non-duplicate E. coli collected, of which 62 presented the mcr-1 gene. Numerous associated resistances were identified, including to ESC through the presence of bla CTX-M or bla CMY genes. The mcr-1 gene was mostly carried by IncX4 ( n = 36) and IncI2 ( n = 24) plasmids, which are both known for their efficient transfer capacities. A high genetic diversity was detected, arguing for the lack of contamination during the slaughter process. ST744 and ST1011 were the most widely identified clones, which have been both regularly associated to mcr-1 -carrying E. coli and to the poultry sector. The wide dissemination of colistin-resistance, coupled to resistances to ESC and numerous other molecules, should urge authorities to implement efficient guidelines for the use of antibiotics in the poultry sector in Lebanon., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Al-Mir, Osman, Drapeau, Hamze, Madec and Haenni.)

After a short background discussing engineered nanomaterials (ENMs) and their physicochemical properties and applications, the present perspective paper highlights the main specific points that need to be considered when examining the question of neurotoxicity of nanomaterials. It underlines the necessity to integrate parameters, specific tools, and tests from multiple sources that make neurotoxicology when applied to nanomaterials particularly complex. Bringing together the knowledge of multiple disciplines e.g., nanotoxicology to neurotoxicology, is necessary to build integrated neurotoxicology for the third decade of the 21st Century. This article focuses on the greatest challenges and opportunities offered by this specific field. It highlights the scientific, methodological, political, regulatory, and educational issues. Scientific and methodological challenges include the determination of ENMs physicochemical parameters, the lack of information about protein corona modes of action, target organs, and cells and dose- response functions of ENMs. The need of standardization of data collection and harmonization of dedicated neurotoxicological protocols are also addressed. This article highlights how to address those challenges through innovative methods and tools, and our work also ventures to sketch the first list of substances that should be urgently prioritized for human modern neurotoxicology. Finally, political support with dedicated funding at the national and international levels must also be used to engage the communities concerned to set up dedicated educational program on this novel field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bencsik and Lestaevel.)

Wildlife has recently been pinpointed as one of the drivers of dissemination of genes conferring resistances to clinically important antimicrobials. The presence of both extended-spectrum beta-lactamase- (ESBL) and carbapenemase-encoding genes has notably been reported in wild birds, that can act as sentinels of antimicrobial resistance (AMR) contamination but also as long-distance spreaders in case of migratory birds. Here, 424 wild birds brought to a rescue center in France were sampled over a 6-month period. These birds encompassed 62 different sedentary or migratory species. A further 16 wild mammals present in the center were also investigated. No carbapenemase-producer was found, but we identified a surprisingly high proportion (24.1%) of ESBL-positive isolates. A total of 144 non-duplicate isolates were collected, including Escherichia coli ( n = 88), Enterobacter cloacae ( n = 51), and Citrobacter freundii ( n = 5), of which 123 carried the bla CTX-M- 9 gene. PFGE, phylogroup, and MLST revealed the presence of a limited number of ESBL-positive clones circulating in these animals, all presenting multiple associated resistances. Next-generation sequencing on a subset of isolates, followed by Southern blot hybridization, showed the wide dissemination of an IncHI2/ST1 plasmid carrying the bla CTX-M- 9 , bla SHV- 12 and mcr-9 genes. In all, our results undoubtedly reflect cross transmissions of ESC-resistance (ESC-R) Enterobacteriaceae within the rescue center - similarly to nosocomial spreads observed at hospital, rather than the true bacterial flora of birds. We also showed that the spread of ESC-R in this rescue center did not only rely on clonal but also on a highly successful plasmidic transmission. Since most animals are intended to get back to nature after a few days or weeks, this is obviously an issue with regard to ESBL dissemination in natural environments., (Copyright © 2020 Haenni, Métayer, Jarry, Drapeau, Puech, Madec and Keck.)

Escherichia coli is a ubiquitous commensal and pathogen that has also been recognized as a multi-sectoral indicator of antimicrobial resistance (AMR). Given that latter focus, such as on resistances to extended-spectrum cephalosporins (ESC) and carbapenems, the reported population structure of E. coli is generally biased toward resistant isolates, with sequence type (ST)131 being widely reported in humans, and ST410 and ST648 being reported in animals. In this study, we characterized 618 non-duplicate E. coli isolates collected throughout France independently of their resistance phenotype. The B2 phylogroup was over-represented (79.6%) and positively associated with the presence of numerous virulence factors (VFs), including those defining the extra-intestinal pathogenic E. coli isolates (presence of ≥2 VFs: papA , sfaS, focG , afaD , iutA , and kpsMTII ) and those more specifically related to uropathogenic E. coli ( cnf1 , hlyD ). The major STs associated with clinical isolates from dogs were by far the dog-associated ST372 (20.7%) and ST73 (20.1%), a lineage that had commonly been considered until now as human-associated. Resistance to ESC was found in 33 isolates (5.3%), along with one carbapenemase-producing isolate, and was mostly restricted to non-B2 isolates. In conclusion, the presence of virulent E. coli lineages may be the issue, rather than the presence of ESC-resistant isolates, and the risk of transmission of such virulent isolates to humans needs to be further studied., (Copyright © 2020 Valat, Drapeau, Beurlet, Bachy, Boulouis, Pin, Cazeau, Madec and Haenni.)

The capacity of Mycoplasmas to engage in horizontal gene transfers has recently been highlighted. Despite their small genome, some of these wall-less bacteria are able to exchange multiple, large portions of their chromosome via a conjugative mechanism that does not conform to canonical Hfr/ oriT models. To understand the exact features underlying mycoplasma chromosomal transfer (MCT), extensive genomic analyses were performed at the nucleotide level, using individual mating progenies derived from our model organism, Mycoplasma agalactiae . Genome reconstruction showed that MCT resulted in the distributive transfer of multiple chromosomal DNA fragments and generated progenies composed of a variety of mosaic genomes, each being unique. Analyses of macro- and micro-events resulting from MCT revealed that the vast majority of the acquired fragments were unrelated and co-transferred independently from the selection marker, these resulted in up to 17% of the genome being exchanged. Housekeeping and accessory genes were equally affected by MCT, with up to 35 CDSs being gained or lost. This efficient HGT process also created a number of chimeric genes and genetic micro-variations that may impact gene regulation and/or expression. Our study unraveled the tremendous plasticity of M. agalactiae genome and point toward MCT as a major player in diversification and adaptation to changing environments, offering a significant advantage to this minimal pathogen., (Copyright © 2019 Dordet-Frisoni, Faucher, Sagné, Baranowski, Tardy, Nouvel and Citti.)

Objectives: To examine the relevance of co-resistance to amoxicillin and tetracycline as an indicator of multidrug resistance (MDR) in animal health., Methods: Escherichia coli isolates collected between 2012 and 2016 by the French surveillance network for antimicrobial resistance in diseased animals (RESAPATH) were analyzed. The proportions of MDR isolates and the proportions of isolates presenting co-resistance to amoxicillin and tetracycline were calculated for seven animal species (cattle, horse, dog, swine, poultry, duck, and turkey). The degree of agreement between these two proportions was estimated by calculating the kappa value., Results: In total, 55,904 isolates were analyzed. MDR proportions were variable among animal species, ranging from 21.9% [20.2; 23.7] in horses to 56.0% [55.4; 56.7] in cattle. A similar situation was observed for proportions of isolates with co-resistance to amoxicillin and tetracycline, with the highest value for cattle 65.0% [64.3; 65.6]. This co-resistance was also most often associated with resistance to other antibiotics, regardless of the animal species considered. Comparative analysis showed substantial agreement between MDR and this co-resistance, with a kappa value of 0.75, all animal species considered., Conclusion: Given the widespread use of penicillins and tetracyclines in animal health, co-resistance to amoxicillin and tetracycline could be an efficient indicator of MDR in E. coli isolates. Based on a specific resistance profile and not an arbitrary number of resistances compared with MDR, this potential indicator is also precise, convenient and suitable for routine use., (Copyright © 2019 Bourély, Cazeau, Jarrige, Jouy, Haenni, Lupo, Madec, Leblond and Gay.)

Extended-Spectrum-Cephalosporin (ESC)-resistant Enterobacteriaceae have widely spread in all settings worldwide. In animals, Extended-Spectrum Beta-Lactamase (ESBL) producers have been frequently identified in veal calves. The objectives of this study were to investigate the trends in the ESBL load and antimicrobial resistance (AMR) proportions, and antimicrobial usages (AMU) in veal calves during the fattening process. Ten fattening farms were selected and 50 animals per farm were sampled. AMR was assessed in bacteria from the dominant flora (collected on non-selective MacConckey agar) and in ESBL/AmpC-carrying bacteria from the subdominant flora (selected on ChromID ESBL selective plates) upon arrival and 5-6 months later before slaughter. The number and types of treatments during fattening were also collected. Rates of ESBL-producing E. coli from the subdominant flora significantly decreased in all farms (arrival: 67.7%; departure: 20.4%) whereas rates of multidrug-resistant E. coli from the dominant flora have significantly increased (arrival: 60.2%; departure: 67.2%; p = 0.025). CTX-M-1 was the most frequently identified ESBL enzyme (arrival: 59.3%; departure: 52.0%). The plasmid-mediated mcr-1 gene was also identified occasionally. In parallel, levels of resistances to non-critically important antimicrobials were already high upon arrival but have still further increased over time until slaughter. Our study also highlighted that if only ESBL-producing isolates were monitored, it might have led to a partial (and partly false) picture of AMR rates globally decreasing during the fattening period. The mean number of antimicrobial treatments per calf (NTPC) was 8.75 but no association between AMU and AMR was evidenced. Most ESBL producers were clonally unrelated suggesting multiple sources and not cross-contaminations among calves during transportation. Feeding milk containing antimicrobial residues to veal calves is hypothesized to explain the high ESBL loads in animals at the entrance on farms.

Bacterial surface polysaccharides play significant roles in fitness and virulence. In Gram-negative bacteria such as Escherichia coli , major surface polysaccharides are lipopolysaccharide (LPS) and capsule, representing O- and K-antigens, respectively. There are multiple combinations of O:K types, many of which are well-characterized and can be related to ecotype or pathotype. In this investigation, we have identified a novel O:K permutation resulting through a process of major genome reorganization in a clade of E. coli . A multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing strain - E. coli 26561 - represented a prototype of strains combining a locus variant of O89 and group 1 capsular polysaccharide. Specifically, the variant O89 locus in this strain was truncated at gnd , flanked by insertion sequences and located between nfsB and ybdK and we apply the term O89m for this variant. The prototype lacked colanic acid and O-antigen loci between yegH and hisI with this tandem polysaccharide locus being replaced with a group 1 capsule (G1C) which, rather than being a recognized E. coli capsule type, this locus matched to Klebsiella K10 capsule type. A genomic survey identified more than 200 E. coli strains which possessed the O89m locus variant with one of a variety of G1C types. Isolates from our collection with the combination of O89m and G1C all displayed a mucoid phenotype and E. coli 26561 was unusual in exhibiting a mucoviscous phenotype more recognized as a characteristic among Klebsiella strains. Despite the locus truncation and novel location, all O89m:G1C strains examined showed a ladder pattern typifying smooth LPS and also showed high molecular weight, alcian blue-staining polysaccharide in cellular and/or extra-cellular fractions. Expression of both O-antigen and capsule biosynthesis loci were confirmed in prototype strain 26561 through quantitative proteome analysis. Further in silico exploration of more than 200 E. coli strains possessing the O89m:G1C combination identified a very high prevalence of multidrug resistance (MDR) - 85% possessed resistance to three or more antibiotic classes and a high proportion (58%) of these carried ESBL and/or carbapenemase. The increasing isolation of O89m:G1C isolates from extra-intestinal infection sites suggests that these represents an emergent clade of invasive, MDR E. coli .

The genus Mycoplasma , a group of free-living, wall-less prokaryotes includes more than 100 species of which dozens are primary pathogens of humans and domesticated animals. Mycoplasma species isolated from wildlife are rarely investigated but could provide a fuller picture of the evolutionary history and diversity of this genus. In 2013 several isolates from wild Caprinae were tentatively assigned to a new species, Mycoplasma ( M.) feriruminatoris sp. nov., characterized by an unusually rapid growth in vitro and close genetic proximity to ruminant pathogenic species. We suspected that atypical isolates recently collected from Alpine ibex in France belonged to this new species. The present study was undertaken to verify this hypothesis and to further characterize the French ibex isolates. Phylogenetic analyses were performed to identify the isolates and position them in trees containing several other mycoplasma species pathogenic to domesticated ruminants. Population diversity was characterized by genomic macrorestriction and by examining the capacity of different strains to produce capsular polysaccharides, a feature now known to vary amongst mycoplasma species pathogenic to ruminants. This is the first report of M. feriruminatoris isolation from Alpine ibex in France. Phylogenetic analyses further suggested that M. feriruminatoris might constitute a 4th species in a genetic cluster that so far contains only important ruminant pathogens, the so-called Mycoplasma mycoides cluster. A PCR assay for specific identification is proposed. These French isolates were not clonal, despite being collected in a restricted region of the Alps, which signifies a considerable diversity of the new species. Strains were able to concomitantly produce two types of capsular polysaccharides, β-(1→6)-galactan and β-(1→6)-glucan, with variation in their respective ratio, a feature never before described in mycoplasmas.