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International audience; Objectives - Daptomycin has become a first-line therapeutic option for vancomycin-resistant Enterococcus faecium infective endocarditis (IE). Although high doses (≥8 mg/kg) are often recommended, optimal doses, particularly for strains with MICs close to the susceptibility breakpoint (4 mg/L), are still debated.Methods - Daptomycin efficacy at doses equivalent to 8 mg/kg daptomycin (DAP8) and 12 mg/kg daptomycin (DAP12) in humans was evaluated in a rabbit model of aortic valve IE induced by 108 cfu of E. faecium reference strain Aus0004 (daptomycin MIC = 2 mg/L) or its in vitro mutant strain Mut4 (daptomycin MIC = 4 mg/L). Treatment began 48 h post-inoculation and lasted 5 days.Results - With Aus0004, the median log10 cfu/g of vegetations was significantly lower after DAP8 and DAP12 versus controls [6.05 (n = 12) and 2.15 (n = 10) versus 9.14 (n = 11), respectively; P Conclusions - DAP12 was the most successful strategy against IE due to a WT E. faecium strain (daptomycin MIC = 2 mg/L). To treat IE strains with MIC = 4 mg/L, DAP8 or DAP12 monotherapy was poorly effective with the risk of resistant mutant emergence. Reassessment of the daptomycin susceptibility breakpoint for enterococci seems necessary.

Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6"-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6"-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection. Fil: Sauvageot, Nicolas. Universite de Caen Basse Normandie; Francia Fil: Mokhtari, Abdelhamid. Université Paris-Saclay; Francia. Institut National de la Recherche Agronomique; Francia. 8 May 1945 University of Guelma; Argelia Fil: Joyet, Philippe. Université Paris-Saclay; Francia. Institut National de la Recherche Agronomique; Francia Fil: Budin Verneuil, Aurélie. Universite de Caen Basse Normandie; Francia Fil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Repizo, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Henry, Céline. Université Paris-Saclay; Francia. Institut National de la Recherche Agronomique; Francia Fil: Pikis, Andreas. National Institutes of Health; Estados Unidos. Center for Drug Evaluation and Research, Food and Drug Administration; Estados Unidos Fil: Thompson, John. National Institutes of Health; Estados Unidos Fil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Hartke, Axel. Universite de Caen Basse Normandie; Francia Fil: Deutscher, Josef. Institut National de la Recherche Agronomique; Francia. Université Paris-Saclay; Francia. Centre National de la Recherche Scientifique; Francia. Université Paris Diderot - Paris 7; Francia

The objective of this study was to perform an inventory of the extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae isolates responsible for infections in French hospitals and to assess the mechanisms associated with ESBL diffusion. A total of 200 nonredundant ESBL-producing Enterobacteriaceae strains isolated from clinical samples were collected during a multicenter study performed in 18 representative French hospitals. Antibiotic resistance genes were identified by PCR and sequencing experiments. The clonal relatedness between isolates was investigated by the use of the DiversiLab system. ESBL-encoding plasmids were compared by PCR-based replicon typing and plasmid multilocus sequence typing. CTX-M-15, CTX-M-1, CTX-M-14, and SHV-12 were the most prevalent ESBLs (8% to 46.5%). The three CTX-M-type EBSLs were significantly observed in Escherichia coli (37.1%, 24.2%, and 21.8%, respectively), and CTX-M-15 was the predominant ESBL in Klebsiella pneumoniae (81.1%). SHV-12 was associated with ESBL-encoding Enterobacter cloacae strains (37.9%). qnrB , aac(6 ′ )-Ib-cr , and aac(3)-II genes were the main plasmid-mediated resistance genes, with prevalences ranging between 19.5% and 45% according to the ESBL results. Molecular typing did not identify wide clonal diffusion. Plasmid analysis suggested the diffusion of low numbers of ESBL-encoding plasmids, especially in K. pneumoniae and E. cloacae . However, the ESBL-encoding genes were observed in different plasmid replicons according to the bacterial species. The prevalences of ESBL subtypes differ according to the Enterobacteriaceae species. Plasmid spread is a key determinant of this epidemiology, and the link observed between the ESBL-encoding plasmids and the bacterial host explains the differences observed in the Enterobacteriaceae species.

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O -acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d -alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O -acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N -acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.

International audience; Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen-host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming the identification of secreted proteins that are differently expressed at 30 degrees C versus 37 degrees C and at mid-exponential versus early-stationary growth phase and antigenic proteins from R. equi ATCC 33701. A total of 48 proteins was identified regardless of growth conditions. The cholesterol oxidase ChoE appears to be the major secretory protein. Moreover, four proteins revealed high homologies with the mycolyl transferases of the Ag85 complex from Mycobacterium tuberculosis. The sequence analysis predicted that 24 proteins are transported by a signal peptide-dependent pathway. Moreover, five antigenic proteins of R. equi were identified by immunoblot, including a novel strongly immunoreactive protein of unknown function. In conclusion, the elucidation of the secretome of R. equi identified several proteins with different biological functions and a new candidate for developing vaccines against R. equi infection in horse.

International audience; Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptides are rare and exhibit highly sought-after activities. In an effort to expand the repertoire of such molecules, we heterologously expressed, in Streptomyces coelicolor, the gene cluster encoding sviceucin, a type I lasso peptide with two disulfide bridges originating from Streptomyces sviceus, which allowed it to be fully characterized. Sviceucin and its reduced forms were characterized by mass spectrometry and peptidase digestion. The three-dimensional structure of sviceucin was determined using NMR. Sviceucin displayed antimicrobial activity selectively against Gram-positive bacteria and inhibition of fsr quorum sensing in Enterococcus faecalis. This study adds sviceucin to the type I lasso peptide family as a new representative. Moreover, new clusters encoding disulfide-bond containing lasso peptides from Actinobacteria were identified by genome mining. Genetic and functional analyses revealed that the formation of disulfide bonds in sviceucin does not require a pathway-encoded thiol-disulfide oxidoreductase. Most importantly, we demonstrated the functional exchangeability of the sviceucin and microcin J25 (a non-disulfide-bridged lasso peptide) macrolactam synthetases in vitro, highlighting the potential of hybrid lasso synthetases in lasso peptide engineering.

International audience; Enterococcus faecalis is a Gram-positive commensal bacterium inhabiting the gastrointestinal tracts of human and other mammals, but is also increasingly recognized as an opportunistic human pathogen. Oxidative stress is one of the major challenges encountered by enterococci, both in their natural environment and during infection. In this paper, we evaluated the transcriptomic response of E. faecalis to oxidative stress, and showed that transcript abundance was reduced for 93 genes and increased for 39 genes during growth in medium containing 1.75 mM H2O2. The presence of hydrogen peroxide affected several metabolic pathways, including a large decrease in ethanolamine utilization and methylglyoxal metabolism, and an increase in transcript abundance for several transport systems. In particular, four operons encoding iron transporters appeared highly induced. By contrast, in our experimental conditions, the expression of most of the genes known to be involved in the enterococcal response to oxidative stress, did not appear significantly altered.

International audience; During the 2011 measles outbreak in Paris (France), patients with clinical suspicion of measles were tested for virological confirmation of measles virus (MV) infection. To assess the practical value of molecular diagnosis in an epidemic setting, 171 oral fluid samples and 235 serum samples collected from 270 patients were tested prospectively for MV-RNA using a novel one-step real-time RT-PCR assay including an internal control. Serum samples were also tested for MV-specific IgG and IgM antibodies. MV infection was confirmed by detection of MV-RNA and/or MV-IgM for 229 of the 270 patients. The results for the 102 cases with both serum and oral fluid samples available were used to compare the techniques. The detection rate of MV-RNA by RT-PCR was 98% (100/102) for oral fluid and 95% (97/102) for serum samples. The detection rate of MV-IgM was 85% (87/102). Negative MV-IgM results were observed mostly for serum samples collected early after the onset of the rash. A MV-RNA standard of known concentration obtained by in vitro transcription was used to quantify MV-RNA in samples. MV-RNA copy numbers were significantly higher in oral fluid than in serum samples, but did not correlate with time of sampling (within 1 week after the onset of the rash), patient age, or vaccination status. During the early stage of infection, the MV-RNA viral load in serum was lower in patients positive than in those negative for MV-IgG. In conclusion, the one-step real-time RT-PCR assay is a simple and sensitive tool suitable for MV diagnosis within hours. J. Med. Virol. © 2013 Wiley Periodicals, Inc.

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6-phospho-α-glucosidase, which in B. subtilis hydrolyses maltose 6'-P into glucose and glucose 6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6-P into glucose 1-P and glucose 6-P. However, purified MalP phosphorolyses maltose but not maltose 6'-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6'-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6'-P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6'-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalysed metabolism. Dephosphorylation assays with a wide variety of phospho-substrates revealed that MapP preferably dephosphorylates disaccharides containing an O-α-glycosyl linkage.

Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.

International audience; To cope with medium acidity, Lactococcus lactis has evolved a number of inducible mechanisms commonly referred as acid stress response. To better understand the molecular basis of this response, several mutants constitutively tolerant to acidity were previously obtained by insertional random mutagenesis of L. lactis MG1363. Mutants in which the GMP synthase gene (i.e. guaA), the (p)ppGpp synthase gene (i.e. relA*) or the high affinity phosphate transport system (i.e. pstS) are inactivated are further characterized in this study. 2-DE was performed and showed that 42, 26, and 35 protein spots are positively deregulated in the guaA, relA*, and pstS mutants, respectively, as compared to the wild-type strain. Most of these proteins were identified by MS. Proteomes comparison of the mutants guaA, relA*, and pstS as well as the acid adaptation proteome of the wild-type strain revealed (i) the presence of numerous overlaps and (ii) that only five proteins were overexpressed in the four conditions, suggesting that these proteins play a crucial role in the constitutive acid stress tolerance of the mutants and in the acid tolerance response of the wild-type strain.

International audience; Exponentially growing cells of Lactococcus lactis MG1363 are able to develop an Acid Tolerance Response (ATR) when incubated at pH 5, in both rich (M17)--and chemically defined (SA)--culture media. Physiological and proteomic characterization of this adaptive response indicated that L. lactis reorganizes its metabolism in response to acid stress to a great extent and quite differently in the two media. The development of ATR was fully dependent on protein de novo synthesis in SA and only partly dependent in M17. 2D gel electrophoresis revealed a total of 90 spots induced by acidity, 80 of which were identified by mass spectrometry. Only 10 proteins (BglA, PycA, GlmS, HasC, ArgS, GatA, AtpA, ArcB, Cfa, and SodA) were overproduced in the two media. A transcriptional analysis of the corresponding genes suggested that for half of them the mode of regulation may differ in the two media. Among the protein spots upregulated during the ATR in SA but not in M17, 13 already displayed an elevated rate of synthesis in M17 at neutral pH. These proteins could play an important role in the development of the protein de novo synthesis-independent ATR observed in M17.

International audience; Cyclopropane fatty acid synthase (cfa) catalyses the transfer of a methyl group from S-adenosylmethionine (SAM) to unsaturated fatty acids. Northern blot experiments demonstrated that the Lactococcus lactis MG1363 cfa gene is mainly expressed as a bicistronic transcript together with metK, the gene encoding SAM synthetase, and is highly induced by acidity. The cfa promoter was characterized by 5'-RACE PCR, and fused to beta-galactosidase by cloning into the pAK80 plasmid. This transcriptional fusion was highly induced by acidity (23-fold at pH 5) as well as during entry into the stationary phase (8-fold) in L. lactis. Interestingly, the cfa promoter expression is repressed in a L. lactis relA* mutant which accumulates (p)ppGpp, whereas its induction by acidity appeared independent of (p)ppGpp in L. lactis and in Escherichia coli.

Five strains of Pseudoalteromonas, isolated from oyster haemolymph, have exhibited antibacterial activity against several Gram-negative bacteria. Bioactive compounds have been identified in their cell-free supernatant and characterised as alterins, which are cyclolipopeptides comprising a heptapeptidic ring connected to a fatty acid chain. Using ultra-performance liquid chromatography-high-resolution mass spectrometry, this paper describes 37 structural analogues differing from each other by one or more amino acid residue, the length of the fatty acid chain, its hydroxylation and the presence of unsaturation.

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. Genome sequences reveal that most P. aeruginosa strains contain a significant number of accessory genes gathered in genomic islands. Those genes are essential for P. aeruginosa to invade new ecological niches with high levels of antibiotic usage, like hospitals, or to survive during host infection by providing pathogenicity determinants. P. aeruginosa pathogenicity island 1 (PAPI-1), one of the largest genomic islands, encodes several putative virulence factors, including toxins, biofilm genes and antibiotic-resistance traits. The integrative and conjugative element (ICE) PAPI-1 is horizontally transferable by conjugation via a specialized GI-T4SS, but the mechanism regulating this transfer is currently unknown. Here, we show that this GI-T4SS conjugative machinery is directly induced by TprA, a regulator encoded within PAPI-1. Our data indicate that the nucleotide associated protein NdpA2 acts in synergy with TprA, removing a repressive mechanism exerted by MvaT. In addition, using a transcriptomic approach, we unravelled the regulon controlled by Ndpa2/TprA and showed that they act as major regulators on the genes belonging to PAPI-1. Moreover, TprA and NdpA2 trigger an atypical biofilm structure and enhance ICE PAPI-1 transfer.

International audience; Background: The prevalence of strangles, one of the most frequently diagnosed infectious diseases of horses worldwide, remains underestimated in France, despite causing economic losses to the equine industry. Streptococcus equi subsp equi (S. equi) is the causative agent and its persistence in guttural pouches of sub-clinical carriers plays an important role in the spread of the disease.Objectives: The aim of this study was to use whole genome sequencing to understand the genetic relationships of S. equi isolates recovered in France from 2016 to 2019 and compare them with international data.Study design: Longitudinal field study.Methods: The French Network for epidemio-surveillance of equine diseases (RESPE), collected 55 isolates from 23 horses implicated in 13 different outbreaks. DNAs from 18 of these strains were extracted, purified and whole genome sequenced according to Illumina recommendations. Genomes were analysed using Pathogenwatch and visualised in Microreact.Results: All isolates were classified as ST-179, recognised as “classic” S. equi. Genomes clustered into BAPS2 (Bayesian Analysis of Population Structure) but fell into two groups. Sequences in the first group, from different outbreaks (two in 2016 and one in 2017), differed from only some SNPs across the core genome and their closest relatives were previously recovered from horses in the United Arab Emirates. The second group were most closely related to other strains from France, Belgium, Spain, Sweden and the United Kingdom.Main limitations: Not all available isolates have been sequenced to date and the history of the horses from which these isolates were derived requires further investigation.Conclusions: Genomic analyses are undergoing to understand the differences between isolates from horses with acute strangles and sub-clinical carriers, by highlighting differences in genes involved in virulence, resistance, persistence or evasion of the immune system.

International audience; Background: With more than 100 species colonizing animal hosts, bacteria of the genus Mycoplasma include important livestock pathogens. They are wall-less and considered as the smallest organisms capable of autonomous growth in vitro. Because their isolation can be laborious, they often lagged behind other bacteria in terms of general knowledge and diagnosis. In horses, three species, namely Mycoplasma (M.) equirhinis, M. pulmonis and M. felis, have been reported from respiratory tract samples, with an unknown pathogenic role but a potential implication on poor-performance [1-2]. In our laboratory, amongst equine respiratory samples submitted routinely for diagnosis, 15% per year turned out to be positive by real-time PCR (rt-PCR) for Mycoplasma spp. but the corresponding species have not been explored yet. Objectives: The aim of this study was to evaluate the prevalence of different species in respiratory clinical samples and characterize their intra species diversity. Study Design: 687 samples (68% tracheal wash, 26% bronchoalveolar lavage and 6% others) collected in 2020 were analysed. Methods: Samples were analysed by culture, rt-PCR and identified by 16S rRNA sequencing. Isolates were subtyped when necessary. Results: To date, 126 samples (18.3%) were found positive by rtPCR and 30 strains have been isolated (still ongoing). A majority of M. equirhinis (42/126 samples and 24/30 strains) was identified, while M. pulmonis and M. felis have been detected only sporadically so far. Main limitation: A gold standard method for Mycoplasma spp. detection and identification in equine samples would be helpful. Conclusions: A Multi Locus Sequence Typing scheme is being developed to analyse the genetic heterogeneity of M. equirhinis isolates. Subtypes will be examined it in the light of their clinical history to have a first insight into the potential pathogenic role of M. equirhinis.1. Allam and Lemcke. doi: 10.1017/s00221724000469082. Antal et al. doi.org/10.1111/j.1439-0450.1988.tb00496.x

International audience; Background: Didecyldimethylammonium chloride (DDAC) is a quaternary ammonium compound used in several disinfection products in veterinary and hospital environments. In Human medicine, strains of Pseudomonas aeruginosa (PSAE) have been showed to present a decreased susceptibility to DDAC according to the NFEN-13727+A2 referential by incompletely understood mechanisms. Decreased susceptibility to antibiotics and disinfectant agents is a major concern to public health as it allows pathogens to persist in their environment. Objectives: The purpose of this study was to evaluate the existence of PSAE strains with DDAC decreased susceptibility among animals in Normandy, France. Study design: Based on a “One health” approach, our global project aims at a better understanding of the resistance mechanisms to disinfectants and antibiotics for PSAE. Methods: Between 1996 and 2020, 183 strains isolated from animal samples (including 146 equines) were provided by our reference laboratory and routine diagnostic service. Minimum inhibitory concentrations (MICs) of DDAC were determined using broth microdilution method. Decreased susceptibility was defined as MIC≥64mg/L, corresponding to the concentration of DDAC in a disinfectant solution according to the manufacturer’s instructions. Results: During the 1996–2015 period all strain tested were sensitive to DDAC, whereas for years 2017, 2018, 2019 and 2020 respectively 20%; 26.3%; 6.5% and 12% of strains presented decrease susceptibility. Over all 10.4% of the strains presented this phenotype and 84.21% of them were equine. Finally, equine respiratory and genital samples are those which have shown the most decrease in sensitivity, representing respectively 42.11% and 36.84% of all strains. Main limitation: From 1996 to 2015, only 23 strains have been isolated; for years 2017 and 2018, only strains showing antibimicrobial resistances phenotype were selected. Conclusion: Decreased susceptibility to DDAC is also present in the veterinary industry. Genomic analyses are underway to determine the relatedness of theses strains and to understand the mechanisms involved.Short title: Adaptation to a major disinfectant-componentSources of funding: Conseil Régional de Normandie and Ministère de l’Agriculture (EcoAntibio) support this study

International audience; Background: Streptococcus equi subspecies equi (S.equi) is the cause of the highly contagious equine respiratory disease ‘strangles’. Approximately 10% of recovered animals can persistently carry the bacteria and transmit it to naïve animals. The global movement of horses is an ideal mechanism for widespread transmission to geographically distant locations.Objectives: Utilise whole-genome sequence data to disentangle the transmission of S. equi and subsequent outbreaks of strangles.Study design: In vitro analysis of micro-organisms.Methods: Isolates (n = 670) of S. equi were recovered from clinical samples submitted to multiple collaborating clinics and institutions globally. Following species confirmation, isolates underwent whole-genome sequencing using Illumina technology. Sequence reads passing quality control measures were assembled and uploaded to Pathogenwatch, which assigned a phylogeny based upon sequences of core genome alleles. Population structure was inferred using the population mixture analysis in BAPS.Results: BAPS clustered the isolates into six different clusters (BAPS 1-6) and showed dominant lineages in different geographical areas but also global transmission within the clusters. Sub-groups within the clusters highlighted multiple outbreaks at local, national and international scales and highlighted population structures and transmission dynamics within single locations. For example, four different strains collected over just seven months were identified in a single location. Sequence data also identified a statistically significant decline in BAPS-5 since 2010.Main limitations: Pathogenwatch has shown its utility in investigating S. equi transmission and population structure. However, it is based upon a curated set of 1286 core genome loci. Further investigations will need to be conducted using the full spectrum of data available from whole-genome sequencing.Conclusions: Pathogenwatch was used as a tool to rapidly identify and visualise the whole-genome sequence data of a large S. equi dataset. The data demonstrate widespread transmission of multiple S. equi lineages and provide strong evidence that asymptomatic carrier horses are perpetuating this dissemination.

BackgroundEnterococci intrinsically resistant to cephalosporins represent a major cause of healthcare-associated infections, and the emergence of MDR makes therapeutic approaches particularly challenging.ObjectivesTeichoic acids are cell wall glycopolymers present in Gram-positive bacteria. Teichoic acids can be modified by d-alanylation, which requires four proteins encoded by the dltABCD operon. Our objective was to evaluate the Dlt system as a druggable target to treat enterococcal infections.MethodsThe susceptibility of a d-alanylation-deficient strain of Enterococcus faecalis to β-lactam antibiotics individually and/or in combination was analysed. Moreover, a DltA inhibitor was synthesized to test pharmacological inhibition of d-alanylation in vivo and in host using the animal model Galleria mellonella with different clinical isolates of E. faecalis and Enterococcus faecium.ResultsMost cephalosporins used as mono treatment had no impact on survival of the parental strain, but were slightly lethal for the dltA mutant of E. faecalis. Addition of a very low concentration of amoxicillin significantly increased killing of the dltA mutant under these conditions. The most spectacular effect was obtained with a combination of cefotaxime (1 mg/L) and amoxicillin (0.03 mg/L). In the presence of the inhibitor, the WT strain was as susceptible to this combination treatment as the dltA mutant. This molecule associated with the antibiotics was also effective in killing other E. faecalis clinical isolates and successfully prevented death of Galleria infected with either E. faecalis or E. faecium.ConclusionsThe combined results support the potential usefulness of the Dlt system as a target to potentiate antibiotic combination therapies for the treatment of drug-resistant enterococci.

International audience; The Enterobacter cloacae complex (ECC) consists of closely-related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare-associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram-negative outer membrane. Many Enterobacteriaceae fortify their outer membrane with cationic amine-containing moieties to prevent CAMP binding, which can lead to cell lysis. The PmrAB two-component system (TCS) directly activates 4-amino-4-deoxy-l-arabinose (l-Ara4N) biosynthesis to result in cationic amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB is dispensable for CAMP resistance in E. cloacae. Interestingly, some ECC clusters exhibit colistin heteroresistance, where a subpopulation of cells exhibit clinically significant resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with l-Ara4N to induce CAMP heteroresistance and the regulatory mechanism is independent of the PmrAB TCS. Instead, PhoP binds to the arnB promoter to induce l-Ara4N biosynthesis and PmrAB-independent addition to the lipid A disaccharolipid. Therefore, PhoPQ contributes to regulation of CAMP heteroresistance in some ECC clusters.

International audience; Francisella tularensis is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using F. tularensis live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain F. tularensis subsp. tularensis SCHU S4 deleted for the homologous FupA protein exhibited even higher FQ resistance. In addition to an increased FQ minimal inhibitory concentration, LVS Delta fupA/B displayed tolerance toward bactericidal compounds including ciprofloxacin and gentamicin. Interestingly, the FupA/B deletion was found to promote increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of vesicles from LVS and LVS Delta fupA/B identified 801 proteins, including a subset of 23 proteins exhibiting differential abundance between both strains which may therefore contribute to the reduced antibiotic susceptibility of the FupA/B-deleted strain. We also demonstrated that OMVs are key structural elements of LVS Delta fupA/B biofilms providing protection against FQ. These results provide a new basis for understanding and tackling antibiotic resistance and/or persistence of Francisella and other pathogenic members of the Thiotrichales class.

National audience; Avec plus de 100 espèces colonisant diverses espèces animales, les bactéries du genre Mycoplasma sont responsables de maladies ayant des conséquences économiques et sanitaires majeures dans différentes filières de production. Les mycoplasmes sont dénués de paroi cellulaire et sont considérés comme les plus petits organismes capables de multiplication autonome in vitro. Diverses caractéristiques dont une croissance fastidieuse in vitro font que, de façon générale, la caractérisation biologique et le diagnostic des mycoplasmes sont en retrait en comparaison d’autres germes bactériens. Des données anciennes font état de trois espèces isolées au niveau du tractus respiratoire équin, Mycoplasma (M.) equirhinis, M. pulmonis et M. felis, sans que leur réel pouvoir pathogène ou rôle dans d’éventuelles contre-performances sportives soient clairement établis. A LABÉO, sur l’ensemble des prélèvements respiratoires équins analysés en routine, 15% sont positifs par PCR temps-réel pour Mycoplasma spp. chaque année. Toutefois, les espèces associées n’ont pas encore été déterminées.Le but de mon doctorat est de préciser la prévalence et la diversité des espèces mycoplasmiques impliquées dans les troubles respiratoires du cheval et d’analyser leur profil de résistance aux antibiotiques.Des outils de caractérisation des mycoplasmes équins (détection, culture, isolement, identification et conservation) ont été mis au point sur les 687 échantillons respiratoires collectés à LABÉO en 2020. Les premiers résultats montrent une prédominance de l’espèce M. equirhinis, dont la diversité sera caractérisée à la lumière des contextes cliniques des prélèvements.L’exploration d’une population équine asymptomatique selon les mêmes modalités expérimentales permettra de comparer les prévalences des différentes espèces en conditions pathologiques versus de portage et donc de préciser leur rôle dans les troubles respiratoires observés. Enfin le profil de résistance des souches isolées et dont la pertinence clinique aura été établie ainsi que les mécanismes moléculaires sous-jacents seront étudiés afin d’orienter le vétérinaire vers un traitement adapté.A terme, ce projet éclairera la filière équine sur la pertinence de prendre en considération les mycoplasmes dans le diagnostic différentiel des troubles respiratoires équins.

Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections, which is largely used as a model organism to study antibiotic resistance and pathogenesis. As other species of the genus, its wide metabolic versatility appears to be attractive to study biotechnological applications. However, its natural resistance to antibiotics and its capacity to produce a wide range of virulence factors argue against its biotechnological potential. By reducing the genome of the reference strain PAO1, we explored the development of four hypovirulent and hypersusceptible recombinant DNA hosts (rDNA hosts). Despite deleting up to 0.8% of the core genome, any of the developed strains presented alterations of fitness when cultured under standard laboratory conditions. Other features such as antibiotic susceptibility, cytotoxicity, in vivo pathogenesis, and expression of heterologous peptides were also explored to highlight the potential applications of these models. This work stands as the first stage of the development of a safe-platform strain of Pseudomonas aeruginosa that will be further optimized for biotechnological applications.

National audience; Souvent considérée comme banale et associée à un tableau clinique respiratoire, les professionnels de la filière équine oublient parfois que la Gourme, hautement contagieuse, peut provoquer des abcès purulents et que, dans les formes les plus graves, elle peut être mortelle. La bactérie responsable de cette infection est Streptococcus equisubsp equi(S. equi), elle se transmet par contact direct ou indirect. LABÉO a mené, entre 2016 et 2019, une étude de terrain afin de déterminer,pour cette infection, le test diagnostique le plus performant (bactériologie, sérologie et/ou amplification génique par PCR) à associer au bon prélèvement (écouvillon naso-pharyngé oulavage despoches gutturales). Les résultats montrent que le lavage est le meilleur prélèvement pour isoler des souches de chevaux malades (M) ou porteurs asymptomatiques(PA), et que la PCR est le test diagnostic à réaliser en première intention. La caractérisation plus fine des souches isolées n’a pas mis en évidence de nouveau variant circulant en France. Toutefois, une différence de profil entre souches isoléesde M et PA dans leur capacité à produire du biofilm a été observée tendant à démontrer que la formation de chondroïdes dans les poches gutturales de PApourrait ne pas être la seule forme de persistance de S. equi

National audience; Background: Didecyldimethylammonium chloride (DDAC) is a quaternary ammonium compound used in several disinfection products in veterinary and hospital environments. In Human medicine, strains of Pseudomonas aeruginosa (PSAE) have been showed to present a decreased susceptibility to DDAC according to the NFEN-13727+A2 referential by incompletely understood mechanisms. Decreased susceptibility to antibiotics and disinfectant agents is a major concern to public health as it allows pathogens to persist in their environment. Objectives: The purpose of this study was to evaluate the existence of PSAE strains with DDAC decreased susceptibility among animals in Normandy, France. Study design: Based on a “One health” approach, our global project aims at a better understanding of the resistance mechanisms to disinfectants and antibiotics for PSAE. Methods: Between 1996 and 2020, 183 strains (Table I) isolated from animal samples (including 146 equines) were provided by our reference laboratory and routine diagnostic service. Minimum inhibitory concentrations (MICs) of DDAC were determined using broth microdilution method. Decreased susceptibility was defined as MIC≥64 mg/L, corresponding to the concentration of DDAC in a disinfectant solution according to the manufacturer’s instructions. Results: During the 1996–2015 period all strain tested were sensitive to DDAC , whereas for years 2017, 2018, 2019 and 2020 respectively 20%; 26.3%; 6.5% and 12% of strains presented decrease susceptibility (Figure I). Over all 10.4% of the strains presented this phenotype and 84.21% of them were equine (Figure II). Finally, equine respiratory and genital samples are those which have shown the most decrease in sensitivity, representing respectively 42.11% and 36.84% of all strains. Main limitation: From 1996 to 2015, only 23 strains have been isolated; for years 2017 and 2018, only strains showing antibimicrobial resistances phenotype were selected. Conclusion: Decreased susceptibility to DDAC is also present in the veterinary industry. Genomic analyses are underway to determine the relatedness of theses strains and to understand the mechanisms involved.Short title: Adaptation to a major disinfectant-componentSources of funding: Conseil Régional de Normandie and Ministère de l’Agriculture (EcoAntibio) support this study

Background Staphylococcus lugdunensis is a coagulase-negative Staphylococcus part of the commensal skin flora but emerge as an important opportunistic pathogen. Because iron limitation is a crucial stress during infectious process, we performed phenotypic study and compared proteomic profiles of this species incubated in absence and in presence of the iron chelator 2,2′-dipyridyl (DIP). Results No modification of cell morphology nor cell wall thickness were observed in presence of DIP. However iron-limitation condition promoted biofilm formation and reduced the ability to cope with oxidative stress (1 mM H2O2). In addition, S. lugdunensis N920143 cultured with DIP was significantly less virulent in the larvae of Galleria mellonella model of infection than that grown under standard conditions. We verified that these phenotypes were due to an iron limitation by complementation experiments with FeSO4. By mass spectrometry after trypsin digestion, we characterized the first iron-limitation stress proteome in S. lugdunensis. Among 1426 proteins identified, 349 polypeptides were differentially expressed. 222 were more and 127 less abundant in S. lugdunensis incubated in iron-limitation condition, and by RT-qPCR, some of the corresponding genes have been shown to be transcriptionally regulated. Our data revealed that proteins involved in iron metabolism and carriers were over-expressed, as well as several ABC transporters and polypeptides linked to cell wall metabolism. Conversely, enzymes playing a role in the oxidative stress response (especially catalase) were repressed. Conclusions This phenotypic and global proteomic study allowed characterization of the response of S. lugdunensis to iron-limitation. We showed that iron-limitation promoted biofilm formation, but decrease the oxidative stress resistance that may, at least in part, explained the reduced virulence of S. lugdunensis observed under low iron condition.

Disponible uniquement sous abonnement; National audience; Biofilm is a community lifestyle that allows bacteria to survive in a hostile environment through greater resistance to host immune response, antimicrobial drug and disinfectants compared to bacteria in their free form. The biofilm is a complex heterogeneous structure consisting of one or more bacterial populations, embedded in a self-produced extracellular matrix, which has the ability to attach itself to a biotic or abiotic surface. First described in human medicine, biofilms are also involved in bacterial infections in animals and equine in particular. A better knowledge of these biofilms (training mechanism and composition) makes it possible today to consider new therapeutic strategies.; Le biofilm est un mode de vie communautaire qui permet aux bactéries de survivre dans un environnement hostile grâce à une plus grande résistance à la réponse immunitaire de l’hôte, aux antibiotiques et aux désinfectants en comparaison des bactéries sous leur forme libre. Le biofilm est une structure hétérogène complexe constituée d’une ou de plusieurs populations bactériennes, enchâssées dans une matrice extracellulaire autoproduite, qui a la capacité de se fixer sur une surface biotique ou abiotique. D’abord décrits en médecine humaine, les biofilms sont également impliqués dans les infections bactériennes animales, équines notamment. Une meilleure connaissance de ces biofilms (mécanisme de formation et composition) permet aujourd’hui d’envisager de nouvelles stratégies thérapeutiques.

International audience; Objectives: The aim of the study was to characterize phenotypically and genotypically an uncommon mechanism of resistance to macrolides, lincosamides, and streptogramins (MLS) in a Streptococcus milleri group clinical isolate.Materials and Methods: The isolate UCN96 was recovered from an osteoradionecrosis wound, and was identified using the matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and the partial sequencing of the sodA gene. Antimicrobial susceptibility testing were carried out by the disk diffusion method and minimal inhibitory concentrations (MICs) were determined by the broth microdilution technique. PCR screening was performed for MLS resistance genes described in Gram-positive bacteria. Specific mutations in the ribosomal proteins L3-, L4-, and L22-encoding genes were also screened and those in domain V of the 23S rRNA gene (rrl). The number of mutated copies of the rrl gene was determined using amplification-refractory mutation system quantitative-polymerase chain reaction (qPCR) analysis.Results: The clinical isolate UCN96 was unambiguously identified as Streptococcus constellatus. It was susceptible to all macrolides and lincosamides (ML) antibiotics except spiramycin (MIC >256 mg/L) while it was also resistant to streptogramins. Screening for all acquired resistance genes was negative and no mutation was found in genes coding for L3, L4, and L22 ribosomal proteins. Of interest, a single mutation, A2062C (according to Escherichia coli numbering), was detected in the domain V of 23S rRNA.Conclusion: Mutations at the position 2062 of 23S rRNA have been detected once in Streptococcus pneumoniae, and not yet in other Streptococcus spp. This mechanism is very likely uncommon in Gram-positive bacteria because different copies of 23S rRNA operons should be mutated for development of such a resistance pattern.

International audience; Enterococcus faecium has become a major opportunistic pathogen with the emergence of vancomycin-resistant enterococci (VRE). As part of the gut microbiota, they have to cope with numerous stresses including effects of antibiotics and other xenobiotics, especially in patients hospitalized in intensive care units (ICUs) who receive many medications. The aim of this study was to investigate the impact of the most prescribed xenobiotics for ICU patients on fitness, pathogenicity and antimicrobial resistance of the vanB-positive E. faecium Aus0004 reference strain. Several phenotypic analyses were carried out and we observed that caspofungin, an antifungal agent belonging to the echinocandins family, had an important effect on E. faecium growth in vitro. We confirmed this effect by electron microscopy and peptidoglycan analysis and showed that, even at a subinhibitory concentration (¼ × MIC, 8 mg/L), caspofungin had an impact on cell wall organization especially in abundance of some muropeptide precursors. By RNA-seq, it was also shown that around 20% of the transcriptome were altered in the presence of caspofungin with 321 and 259 significantly upregulated and downregulated genes, respectively. Since the fungal target of caspofungin (i.e., beta-(1,3)-glucan synthase) was absent in bacteria, the mechanistic pathway of caspofungin activity was investigated. The repression of genes involved in the pyruvate metabolism seemed to have a drastic impact on bacterial cell viability while decrease of glycerol metabolism could explain the conformational modifications of peptidoglycan. This is the first report of caspofungin antibacterial activity against E. faecium, highlighting the potential impact of non-antibiotic xenobiotics against bacterial pathogens.

The increasing spread of antibiotic resistant bacteria is a major human health concern. The challenging development of new effective antibiotics has led to focus on seeking synergistic antibiotic combinations. Vancomycin (VAN) is a glycopeptide antibiotic used to treat Staphylococcus aureus and enterococci infections. It is targeting D-Alanyl-D-Alanine dimers during peptidoglycan biosynthesis. D-cycloserine (DCS) is a D-Alanine analogue that targets peptidoglycan biosynthesis by inhibiting D-Alanine:D-Alanine ligase (Ddl). The VAN-DCS combination was found to be synergistic in VAN resistant S. aureus strains lacking van genes cluster. We hypothesize that this combination leads to opposite effects in S. aureus and enterococci strains harboring van genes cluster where VAN resistance is conferred by the synthesis of modified peptidoglycan precursors ending in D-Alanyl-D-Lactate. The calculated Fractional Inhibitory Concentration of VAN-DCS combination in a van- vancomycin-intermediate, VanA type, and VanB type strains were 0.5, 5 and 3, respectively. As a result, VAN-DCS combination leads to synergism in van-lacking strains, and to antagonism in strains harboring van genes cluster. The VAN-DCS antagonism is due to a mechanism that we named van-mediated Ddl inhibition bypass. Our results show that antibiotic combinations can lead to opposite effects depending on the genetic backgrounds.

International audience; Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.

Species of the Enterobacter cloacae complex (ECC) represent an increasing cause of hospital-acquired infections and commonly exhibit multiple antibiotic resistances. In order to identify genes that may play a role in its ability to colonize the host, we used the transposon-sequencing (Tn-seq) approach. To this end, a high-density random transposon insertion library was obtained from E. cloacae subsp. cloacae ATCC 13047, which was used to analyze the fitness of ca. 300,000 mutants in Galleria mellonella colonization model. Following massively parallel sequencing, we identified 624 genes that seemed essential for the optimal growth and/or the fitness within the host. Moreover, 63 genes where mutations resulted in positive selection were found, while 576 genes potentially involved in the in vivo fitness were observed. These findings pointed out the role of some transcriptional regulators, type VI secretion system, and surface-associated proteins in the in vivo fitness of E. cloacae ATCC 13047. We then selected eight genes based on their high positive or negative fold changes (FCs) and tested the corresponding deletion mutants for their virulence and ability to cope with stresses. Thereby, we showed that ECL_02247 (encoding the NAD-dependent epimerase/dehydratase) and ECL_04444 (coding for a surface antigen-like protein) may correspond to new virulence factors, and that the regulator ECL_00056 was involved in in vivo fitness. In addition, bacterial cells lacking the flagellum-specific ATP synthase FliI (ECL_03223) and the hypothetical protein ECL_01421 were affected for mobility and resistance to H2O2, respectively. All these results yield valuable information regarding genes important for infection process and stress response of E. cloacae ATCC 13047 and participate to a better understanding of the opportunistic traits in this bacterial pathogen.

Fosfomycin resistance in Escherichia coli results from chromosomal mutations or acquisition of plasmid-mediated genes. Because these mechanisms may be absent in some resistant isolates, we aimed at decipher the genetic basis of fosfomycin resistance in E. coli. Different groups of isolates were studied: fosfomycin-resistant mutants selected in vitro from E. coli CFT073 (MIC = 1 mg/L) and two groups (wildtype and non-wildtype) of E. coli clinical isolates. Single-nucleotide allelic replacement was performed to confirm the implication of novel mutations into resistance. Induction of uhpT expression by glucose-6-phosphate (G6P) was assessed by RT-qPCR. The genome of all clinical isolates was sequenced by MiSeq (Illumina). Two first-step mutants were obtained in vitro from CFT073 (MICs, 128 mg/L) with single mutations: G469R in uhpB (M3); F384L in uhpC (M4). Second-step mutants (MICs, 256 mg/L) presented additional mutations: R282V in galU (M7 from M3); Q558∗ in lon (M8 from M4). Introduction of uhpB or uhpC mutations by site-directed mutagenesis conferred a 128-fold increase in fosfomycin MICs, whereas single mutations in galU or lon were only responsible for a 2-fold increase. Also, these mutations abolished the induction of uhpT expression by G6P. All 14 fosfomycin-susceptible clinical isolates (MICs, 0.5–8 mg/L) were devoid of any mutation. At least one genetic change was detected in all but one fosfomycin-resistant clinical isolates (MICs, 32 – >256 mg/L) including 8, 17, 18, 5, and 8 in uhpA, uhpB, uhpC, uhpT, and glpT genes, respectively. In conclusion, novel mutations in uhpB and uhpC are associated with fosfomycin resistance in E. coli clinical isolates.

National audience

International audience; The commercial Rapid Polymyxin NP test was evaluated to detect colistin-resistant Enterobacteriaceae. A total of 223 enterobacterial isolates corresponding to 136 resistant (including 38 MCR-like producers), 19 heteroresistant, and 78 colistin-susceptible isolates were tested. The test was performed according to the manufacturer's instruction, and the color of the wells was read after 2 and 3 hours of incubation. The results were compared with those of the homemade Rapid Polymyxin NP test, and manual broth microdilution according to EUCAST guidelines was used as the reference method to determine the performance of the test. Excellent performance of the commercial Rapid Polymyxin NP test was found with a very major error rate, a major error rate, a sensitivity, and a specificity of 1.9%, 5.1%, 98.1%, and 94.9%, respectively. The performance of the homemade Polymyxin NP test was similar, with a slightly better value for the very major error (1.2 %).

Microbial endocrinology has demonstrated for more than two decades, that eukaryotic substances (hormones, neurotransmitters, molecules of the immune system) can modulate the physiological behavior of bacteria. Among them, the hormones/neurotransmitters, epinephrine (Epi) and norepinephrine (NE), released in case of stress, physical effort or used in medical treatment, were shown to be able to modify biofilm formation in various bacterial species. In the present study, we have evaluated the effect of Epi on motility, adhesion, biofilm formation and virulence of Pseudomonas aeruginosa, a bacterium linked to many hospital-acquired infections, and responsible for chronic infection in immunocompromised patients including persons suffering from cystic fibrosis. The results showed that Epi increased adhesion and biofilm formation of P. aeruginosa, as well as its virulence towards the Galleria mellonella larvae in vivo model. Deciphering the sensor of this molecule in P. aeruginosa and the molecular mechanisms involved may help to find new strategies of treatment to fight against this bacterium.

International audience; Objectives: This study aimed to analyse antimicrobial susceptibility evolution of equine pathogens isolated from clinical samples from 2006-2016.Methods: A collection of 25 813 bacterial isolates was studied, clustered according to their origins (respiratory tract, cutaneous, genital and other), and analysed for their antimicrobial susceptibility using the disk diffusion method.Results: The most frequently isolated pathogens were group C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%), Pseudomonas aeruginosa (4.0%), Enterobacter spp. (3.4%), Klebsiella pneumoniae (2.4%), and Rhodococcus equi (1.8%). Of the isolates, 9512 were from respiratory samples (36.8%), 7689 from genital origin (29.8%), and 4083 from cutaneous samples (15.8%). Over the 11-year period, the frequency of multidrug-resistant (MDR) strains fluctuated between 6.4-20.4% for group C Streptococci and 17-37.7% for Klebsiella pneumoniae. From 2006-2009, 24.5-43.0% of Staphylococcus aureus isolates were MDR; after 2009 the level did not exceeded 27.6%. For Escherichia coli and Enterobacter spp., these levels were mostly >30.0% until 2012, but significantly decreased thereafter (22.5-26.3%).Conclusions: This study is the first large-scale analysis of equine pathogens, by the number of samples and duration of study. The results showed high levels of MDR strains and the need to support veterinary antimicrobial stewardship to encourage proper use of antibiotics.

Background Respiratory syncytial virus (RSV) is responsible for severe respiratory infections and higher costs in medical care. The two aims of this work were to assess the performances of SOFIA®RSV tests in “real-life-laboratory” conditions (study 1) and implemented at point-of-care testing in a pediatric emergency department (ED, study 2), during two consecutive winter seasons. Methods In study 1, fresh nasopharyngeal swabs from patients of all ages were sampled in 1.5 ml of Universal virological Transport Medium (UTM) and prospectively tested using SOFIA®RSV tests. In study 2, conducted in a pediatric ED, nasopharyngeal swabs were placed in 3 ml of UTM. All SOFIA®RSV tests were confirmed by molecular testing, considered as reference method. The epidemiological and clinical features of tested patients, as well as the care of these patients after obtaining quick results were evaluated. Results The sensitivities of SOFIA®RSV in infants (aged under 24 months) performed in the laboratory and in the pediatric ED were respectively 95% (95% CI: 86.8–98.1) and 74.8% (95% CI: 68.0–80.9) compared to PCR. In study 1, the sensitivity among children (from 2 to 15 years old) and adults (above 15 years old) dropped to 45% (95% CI: 23.1–68.5) and 59% (95% CI: 32.9–81.6), respectively. In study 2, there were some differences in bed-management of SOFIA®RSV positive compared to SOFIA®RSV negative infants. Conclusions SOFIA®RSV tests performed in the laboratory and in the pediatric ED show high and satisfactory sensitivities among young children under 24 months, which supports its robustness and reliability. However, the impact of these tests on patient care at point-of-care cannot be clearly assessed when considering the limits of the study 2 design. Electronic supplementary material The online version of this article (doi:10.1186/s12879-017-2557-8) contains supplementary material, which is available to authorized users.

A rare CTX-M gene bla CTX-M-12 used to be carried on plasmids was detected in the chromosome of three clinical E. kobei strains. They shared a new sequence type assigned as ST591, and were differed by 57 single-nucleotide polymorphisms. An intra-clonal diversification on the capacity of biofilm formation was detected.

International audience; The reality and intensity of antibiotic resistance in pathogenic bacteria calls for the rapid development of new antimicrobial drugs. In bacteria, trans-translation is the primary quality control mechanism for rescuing ribo-somes arrested during translation. Because trans-translation is absent in eukaryotes but necessary to avoid ri-bosomal stalling and therefore essential for bacterial survival, it is a promising target either for novel antibiotics or for improving the activities of the protein synthesis inhibitors already in use. Oxadiazole derivatives display strong bactericidal activity against a large number of bacteria, but their effects on trans-translation were recently questioned. In this work, a series of new 1,3,4-oxadiazole derivatives and analogs were synthesized and assessed for their efficiency as antimicrobial agents against a wide range of gram-positive and gram-negative pathogenic strains. Despite the strong antimicrobial activity observed in these molecules, it turns out that they do not target trans-translation in vivo, but they definitely act on other cellular pathways.

Enterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions. Fil: Grand, Maxime. Universite de Caen Basse Normandie; Francia Fil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Espariz, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Deutscher, Josef. Université Paris-Saclay; Francia. Universite de Paris; Francia Fil: Pikis, Andreas. Center For Drug Evaluation And Research; Estados Unidos Fil: Hartke, Axel. Universite de Caen Basse Normandie; Francia Fil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Sauvageot, Nicolas. Universite de Caen Basse Normandie; Francia

National audience

Summary: Whereas the primary actions of β-lactams are well characterized, their downstream effects are less well understood. Although their targets are extracellular, β-lactams stimulate respiration in Escherichia coli leading to increased intracellular accumulation of reactive oxygen species (ROS). Here, we show that β-lactams over a large concentration range trigger a strong increase in ROS production in Enterococcus faecalis under aerobic, but not anaerobic, conditions. Both amoxicillin, to which the bacterium is susceptible, and cefotaxime, to which E. faecalis is resistant, triggers this response. This stimulation of ROS formation depends mainly on demethylmenaquinone (DMK), a component of the E. faecalis respiratory chain, but in contrast to E. coli is observed only in the absence of respiration. Our results suggest that in E. faecalis, β-lactams increase electron flux through the respiratory chain, thereby stimulating the auto-oxidation of reduced DMK in the absence of respiration, which triggers increased extracellular ROS production. : Léger et al. show that β-lactams treatment enhances superoxide and hydrogen peroxide production in Enterococcus faecalis in a mainly demethylmenaquinone (DMK)-dependent process. In the absence of respiration, the antibiotics trigger the accumulation of the reduced form of DMK, a respiratory chain component, which increases the adventitious reactivity with oxygen. Keywords: Enterococcus, β-lactams, oxidative stress, ROS, respiratory chain

International audience; Staphylococcus lugdunensis is a commensal bacterium of human skin that has emerged as a virulent Coagulase-Negative Staphylococcus in both community-acquired and healthcare associated infections. Genotyping methods have shown a clonal population structure of this pathogen but failed to identify hypervirulent lineages. Here, complete genomes of three pathogenic and three carriage S. lugdunensis strains were obtained by Single-Molecule sequencing (PacBio) and compared to 15 complete genomes available in GenBank database. The aim was to identify (i) genetic determinants specific to pathogenic or carriage strains or specific to clonal complexes (CCs) defined by MultiLocus Sequence Typing, and (ii) antibiotic resistance genes and new putative virulence factors encoded or not by mobile genetic elements (MGE). Comparative genomic analysis did not show a strict correlation between gene content and the ability of the six strains to cause infections in humans and in a Galleria mellonella infection model. However, this study identified new MGEs (five prophages, two genomic islands and one plasmid) and genetic variations of some putative virulence-associated loci, especially in CC3 strains. For a clonal population, high variability and eight CC-dependent genetic organizations were observed for the ess locus, which encodes a putative type VII secretion system (T7SS) homologous to that of S. aureus. Further phenotypic and functional studies are needed to characterize this particular CC3 and to evaluate the role of T7SS in the virulence of S. lugdunensis.

International audience; Species of Enterobacter cloacae complex (ECC) are widely distributed in the environment and are part of the gut microbiota of both animals and humans. Anolis marmoratus, an endemic and anthropophilic lizard of Guadeloupe, is a natural carrier of ECC and in particular of strains resistant to third generation cephalosporins (3GCR). Bacteria belonging to this complex are also major opportunistic pathogens which draw attention by their involvement in nosocomial infections and the emergence of multidrug resistant clones. Using hsp60 genotyping, ECC is divided into 12 genetic clusters (cluster I-XII) and a loosely knit group (cluster XIII). Strains belonging to clusters III, VI and VIII are prominent in human infections.We collected ECC strains (n=155) isolated from different sources (anoles, environment and clinical samples). We characterized these strains according to their antibiotic resistance phenotypes. Nearly 28% of strains sampled from environment and anoles were resistant to 3GC. This specific resistance was related to the overexpression of the AmpC cephalosporinase. We identified the cluster for each strain by using hsp60 genotyping. Cluster VIII was predominant in A. marmoratus feces (n=37, 38.9%) and in water catchment (n=10, 45.6%). We sequenced 58 strains of C-VIII (33 from clinical isolates: 26 from France and 7 from Guadeloupe, and 25 from anoles). A Maximum likelihood analysis based on core genome alignment revealed a clear split of isolates into two main clusters depending on sources: the first one clustering most of clinical samples together (from France and Guadeloupe, n=29) but also 5 strains isolated from anoles. The second cluster was mainly constituted by strains from anoles (n=20) and by 4 clinical samples. These results suggest first that bacteria resistant to third generation cephalosporins exist in environmental reservoirs in Guadeloupe. Moreover, exchange of ECC C-VIII strains may happen between human and anoles within a One Health appro

International audience; Enterococcus faecalis has to cope with major stress conditions during colonization. To understand the effects of stress encountered during infection, the present study assessed the transcriptomic response of the bacteria facing exposure to serum, urine, bile salts, acid pH, or oxidative stress. Compared to non-stressed culture, 30% of the E. faecalis genes were differentially expressed. The transcriptome analysis reveals common but also specific responses, depending on stresses encountered: thus, urine exposure has the most important impact, and the highest number of genes with modified expression is involved in transport and metabolism. The results also pinpoint many stress-related sRNA or intergenic regions not yet characterized. This study identified the general stress stimulon related to infection: when the commensal bacterium initiates its response to stress related to infection, it increases its ability to survive to rough conditions for colonization, rather than promoting expression of virulence factors, and becomes this opportunistic pathogen that thrives in hospital settings.

National audience; Introduction et objectifs :Les souches de Pseudomonas aeruginosa (Pyo) proche de la toto-résistance aux antibiotiques sont en augmentation. Pyo tend également à acquérir des résistances aux ammoniums quaternaires (CDMA) utilisés dans les détergents/désinfectants usuels. Cette problématique est autant retrouvée en médecine vétérinaire et humaine. Les objectifs sont ici, sur 10 souches de Pyo hospitalière (5) et équine (5), de déterminer leur niveau de résistance aux antibiotiques et au CDMA, d’étudier leur formation de biofilm et de déterminer leurs supports génétiques associés.Matériels et méthodes :Les profils de résistance à 6 familles d’antibiotiques et les concentrations minimales inhibitrices (CMI) vis-à-vis du CDMA ont été déterminés selon les recommandations du CA-SFM. Le biofilm formé a été caractérisé en « statique » par impédancemétrie couplée à la microscopie 3D et en « dynamique » par microfluidique. Enfin, le séquençage complet des génomes, une approche transcriptomique et des essais de conjugaison ont été réalisés pour tenter d’identifier les supports génétiques de la résistance au CDMA.Résultats, discussion et conclusion :Les profils de résistances aux antibiotiques sont variés. Les CMI au CDMA les plus élevées (128mg/L) retrouvées pour la moitié des souches (2 cliniques et 3 équines) ne sont pas corrélées à un profil type de résistance aux antibiotiques. Le résistome obtenu explique la résistance à toutes les classes antibiotiques retrouvées. Le gène de la carbapénémase de type VIM-2, de support plasmidique, est retrouvé exclusivement sur les souches cliniques. La pompe RND (Résistance-Nodulation-Division) MexA/B-OprM est surexprimée pour les souches résistantes au CDMA, et plusieurs de ses régulateurs présentent des altérations chromosomiques (substitutions ou délétions sur rocS2 et mexR). Concernant la formation de biofilm associée aux souches étudiées, les mesures d’impédancemétrie et en microfluidique ont mis en évidence plusieurs profils de production distincts. Cependant, ces profils semblent ne pas être corrélés aux profils de résistance aux antibiotiques et/ou au CDMA. La résistance aux biocides et antibiotiques s’accumule chez Pyo. Ces molécules très utilisées tant en filière animale qu’en clinique humaine aboutissent à des impasses qui nécessitent une meilleure connaissance des mécanismes et l’évaluation d’autres biocides.Mots clés : P. aeruginosa – antibiorésistance - ammonium quaternaire – biofilm - pompe RND.

International audience; The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-beta-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of beta-1,6- rather than beta-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6 '-P with a Kd of 0.04 mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6 '-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.

National audience; INTRODUCTION/OBJECTIF DU TRAVAILUn nombre de 65 cas cliniques à Pseudomonas aeruginosa producteurs de carbapènémases de type VIM-2 (pyoVIM2) ont été notifiées dans notre établissement entre 2011 et 2018, et ce, malgré les précautions prises (dépistage des patients, isolement contact et procédure spécifique d’entretien des locaux). Cela nous a amené à évaluer l’activité bactéricide des détergents-désinfectants (DD) utilisés dans notre établissement sur des souches Pseudomonas de notre écologie hospitalière.MATÉRIEL & MÉTHODESLa détermination de l’activité bactéricide de sept DD de surfaces a été effectuée sur 12 souches de Pseudomonas :- deux souches de référence (PAO1 et ATCC15442)- trois souches de pyoVIM2 cliniques ou environnementales en 2018- Six souches de Pseudomonas autre que aeruginosa et productrices de VIM-2 (putida, oleovorans, nitroreducens, monteilii, pleicoglossicida)- Deux transconjugants (TC) obtenus par conjugaison, en utilisant une souche donneuse de plasmide VIM-2 et la souche receveuse PAO1. - L’activité bactéricide in vitro a été évaluée selon le protocole de filtration sur membrane en conditions de propreté (NF EN 13727 + A2). Par ailleurs, 5 souches parmi elles ont été séquencées en génome entier afin d’étudier le contenu en gènes de résistance aux biocides (antibiotiques et ammonium quaternaire).RÉSULTATSSix DD sur sept présentent une activité bactéricide aux dilutions d’usage préconisées par le fabricant sur ces souches. Un seul DD ayant la particularité de ne contenir qu’un seul ammonium quaternaire ne retrouve son activité bactéricide qu’à double dose sur les souches pyoVIM2 (souches cliniques, environnementales et TC). Après analyse des génomes, il a été mis en évidence la co-localisation plasmidique de gènes de résistance à de nombreuses classes antibiotiques (béta-lactamines incluant les carbapénèmes, les aminosides et les fluoroquinolones) ainsi que du gène qacEdelta connu pour conférer la résistance aux ammoniums quaternaires.CONCLUSIONL’utilisation de ce protocole a permis de révéler une sensibilité diminuée à un DD largement utilisé pour l’entretien des locaux dans les établissements de santé en France. Ce constat nous a fait préconiser l’utilisation de ce DD à double dose pour l’entretien des surfaces dans les services où circulait le pyoVIM-2. L’acquisition de cette résistance semble liée à celle du plasmide VIM-2. Nous allons poursuivre la compréhension du mécanisme de la résistance aux biocides et étendre l’étude de sensibilité à d’autres bactéries multi-résistantes aux antibiotiques.