Identification of staphylococcal cassette chromosome mec in Staphylococcus aureus and non-aureus staphylococci from dairy cattle in Belgium: Comparison of multiplex PCR and whole genome sequencing.

The present study compared multiplex PCR (mPCR) and Whole Genome Sequencing (WGS) using the SCCmecFinder database to identify the Staphylococcal Cassette Chromosome (SCC) mec in five Staphylococcus aureus (SA) and nine non-aureus staphylococci (NAS) isolated from dairy cattle. mPCR identified an SCCmecIV in four SA and one NAS, but could not differentiate between SCCmecII and IV in the fifth SA, that all harbored the mecA gene and were phenotypically resistant to cefoxitin. SCCmecFinder confirmed the presence of an SCCmecIVc(2B) in four SA and of the SCCmecIVa(2B) in the fifth SA and the one NAS. Both methods also detected one untypeable SCCmec in another cefoxitin-resistant NAS harboring the mecA gene and a pseudo SCCmec in one cefoxitin-sensitive NAS harboring one mecC-related gene. No SCCmec elements were identified either in one cefoxitin-sensitive NAS harboring the mecA2 gene, or in five NAS (one resistant and four sensitive to cefoxitin) harboring the mecA1 gene. SCCmecFinder could even not identify the presence of any mecA1 gene in these five NAS, whose presence was nevertheless confirmed by ResFinder. The conclusions of this study are: (i) mPCR and WGS sequencing using SCCmecFinder are complementary methodologies to identify SCCmec; (ii) SCCmecFinder and ResFinder to a lesser extent cannot identify all mec gene allotypes; (iii) a specific classification of the SCCmec in NAS would be epidemiologically helpful; (iv) presence of a mecA gene and a complete SCCmec is linked to cefoxitin resistance, whereas presence of other mec genes and of pseudo or no SCCmec is not.

Identification of Five Serotypes of Enteropathogenic Escherichia coli from Diarrheic Calves and Healthy Cattle in Belgium and Comparative Genomics with Shigatoxigenic E. coli.

Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (AE) lesions and cause non-bloody diarrhea in mammals. A minority of bovine EPEC belong to one of the ten classical serotypes of human and bovine AE-STEC. The purpose of this study was to identify five non-classical O serotypes (O123/186, O156, O177, O182, and O183) among bovine EPEC and to characterize their virulence repertoires by whole genome sequencing. Around 40% of the 307 EPEC from 307 diarrheic calves, 368 EPEC from 47 healthy cattle, and 131 EPEC from 36 healthy calves in dairy farms were analyzed. Serotype O177 was the most frequent among EPEC from diarrheic and healthy calves, while the O156 was the most frequent in healthy cattle. The genomic analysis identified different H serotypes, MLSTypes, and/or eae gene subtypes among the O156 and O177 EPEC, while the O182 was homogeneous. The virulence gene profiles of bovine EPEC were closely related to each other and to the profiles of ten bovine and human AE-STEC. These results emphasize the need for additional studies to identify more O:H serotypes of bovine EPEC and to elucidate their origin and evolution of EPEC with regard to AE-STEC belonging to the same O:H serotypes.

Genomic relatedness of a canine Lactococcus garvieae to human, animal and environmental isolates.

Lactococcus (L.) garvieae is a zoonotic fish pathogen that can also cause bacteraemia and endocarditis in humans and has been isolated from healthy or diseased domestic animals. Nevertheless L. garvieae is more an opportunistic, than a primary pathogen since most affected humans have predisposing conditions and comorbidities. L. garvieae is also present in other animal species, most frequently cattle, but also sheep, goats, water buffaloes, and pigs, and much more rarely dogs, cats, horses, camel, turtle, snake and crocodile. The purpose of this study was to genomically (i) confirm the identification by MALDI-TOF MS® of a L. garvieae from the nasal discharge of a dog with chronic respiratory disorders and (ii) compare this canine isolate with human and animal L. garvieae isolates. According to the BLAST analysis after Whole Genome Sequencing, this canine isolate was more than 99% identical to 3 L. garvieae and belonged to a new Multi-Locus Sequence Type (ST45). MLST and whole genomes-based phylogenetic analysis were performed on the canine isolate and the 40 genomes available in Genbank. The canine L. garvieae was most closely related to an Australian camel and an Indian fish L. garvieae and more distantly to human L. garvieae. Twenty-five of the 29 putative virulence-associated genes searched for were detected, but not the 16 capsule-encoding genes. The heterogeneity of the L. garvieae species is reflected by the diversity of the MLSTypes and virulotypes identified and by the phylogenetic analysis.

Whole Genome Sequencing of Staphylococci Isolated From Bovine Milk Samples.

Staphylococci are among the commonly isolated bacteria from intramammary infections in bovines, where Staphylococcus aureus is the most studied species. This species carries a variety of virulence genes, contributing to bacterial survival and spread. Less is known about non-aureus staphylococci (NAS) and their range of virulence genes and mechanisms, but they are the most frequently isolated bacteria from bovine milk. Staphylococci can also carry a range of antimicrobial resistance genes, complicating treatment of the infections they cause. We used Illumina sequencing to whole genome sequence 93 staphylococcal isolates selected from a collection of staphylococcal isolates; 45 S. aureus isolates and 48 NAS isolates from 16 different species, determining their content of antimicrobial resistance genes and virulence genes. Antimicrobial resistance genes were frequently observed in the NAS species as a group compared to S. aureus. However, the lincosamide resistance gene lnuA and penicillin resistance gene blaZ were frequently identified in NAS, as well as a small number of S. aureus. The erm genes conferring macrolide resistance were also identified in several NAS isolates and in a small number of S. aureus isolates. In most S. aureus isolates, no antimicrobial resistance genes were detected, but in five S. aureus isolates three to six resistance genes were identified and all five of these carried the mecA gene. Virulence genes were more frequently identified in S. aureus, which contained on average five times more virulence genes compared to NAS. Among the NAS species there were also differences in content of virulence genes, such as S. chromogenes with a higher average number of virulence genes. By determining the content of a large selection of virulence genes and antimicrobial resistance genes in S. aureus and 16 different NAS species our results contribute with knowledge regarding the genetic basis for virulence and antimicrobial resistance in bovine staphylococci, especially the less studied NAS. The results can create a broader basis for further research into the virulence mechanisms of this important group of bacteria in bovine intramammary infections.

Viral variant visualizer (VVV): A novel bioinformatic tool for rapid and simple visualization of viral genetic diversity.

Here a bioinformatic pipeline VVV has been developed to analyse viral populations in a given sample from Next Generation Sequencing (NGS) data. To date, handling large amounts of data from NGS requires the expertise of bioinformaticians, both for data processing and result analysis. Consequently, VVV was designed to help non-bioinformaticians to perform these tasks. By providing only the NGS data file, the developed pipeline generated consensus sequences and determined the composition of the viral population for an avian Metapneumovirus (AMPV) and three different animal coronaviruses (Porcine Epidemic Diarrhea Virus (PEDV), Turkey Coronavirus (TCoV) and Infectious Bronchitis Virus (IBV)). In all cases, the pipeline produced viral consensus genomes corresponding to known consensus sequence and made it possible to highlight the presence of viral genetic variants through a single graphic representation. The method was validated by comparing the viral populations of an AMPV field sample, and of a copy of this virus produced from a DNA clone. VVV demonstrated that the cloned virus population was homogeneous (as designed) at position 2934 where the wild-type virus demonstrated two variant populations at a ratio of almost 50:50. A total of 18, 10, 3 and 28, viral genetic variants were detected for AMPV, PEDV, TCoV and IBV respectively. The simplicity of this pipeline makes the study of viral genetic variants more accessible to a wide variety of biologists, which should ultimately increase the rate of understanding of the mechanisms of viral genetic evolution.

Molecular characterization of encephalomyocarditis virus strains isolated from an African elephant and rats in a French zoo.

In November 2013, a fatal encephalomyocarditis virus (EMCV) case in a captive African elephant (Loxodonta africana) occurred at the Réserve Africaine de Sigean, a zoo in the south of France. Here we report the molecular characterization of the EMCV strains isolated from samples collected from the dead elephant and from 3 rats (Rattus rattus) captured in the zoo at the same time. The EMCV infection was confirmed by reverse-transcription real-time PCR (RT-rtPCR) and genome sequencing. Complete genome sequencing and sequence alignment indicated that the elephant's EMCV strain was 98.1-99.9% identical to the rat EMCV isolates at the nucleotide sequence level. Phylogenetic analysis of the ORF, P1, VP1, and 3D sequences revealed that the elephant and rat strains clustered into lineage A of the EMCV 1 group. To our knowledge, molecular characterization of EMCV in France and Europe has not been reported previously in a captive elephant. The full genome analyses of EMCV isolated from an elephant and rats in the same outbreak emphasizes the role of rodents in EMCV introduction and circulation in zoos.

DUGMO: tool for the detection of unknown genetically modified organisms with high-throughput sequencing data for pure bacterial samples.

BACKGROUND: The European Community has adopted very restrictive policies regarding the dissemination and use of genetically modified organisms (GMOs). In fact, a maximum threshold of 0.9% of contaminating GMOs is tolerated for a "GMO-free" label. In recent years, imports of undescribed GMOs have been detected. Their sequences are not described and therefore not detectable by conventional approaches, such as PCR. RESULTS: We developed DUGMO, a bioinformatics pipeline for the detection of genetically modified (GM) bacteria, including unknown GM bacteria, based on Illumina paired-end sequencing data. The method is currently focused on the detection of GM bacteria with - possibly partial - transgenes in pure bacterial samples. In the preliminary steps, coding sequences (CDSs) are aligned through two successive BLASTN against the host pangenome with relevant tuned parameters to discriminate CDSs belonging to the wild type genome (wgCDS) from potential GM coding sequences (pgmCDSs). Then, Bray-Curtis distances are calculated between the wgCDS and each pgmCDS, based on the difference of genomic vocabulary. Finally, two machine learning methods, namely the Random Forest and Generalized Linear Model, are carried out to target true GM CDS(s), based on six variables including Bray-Curtis distances and GC content. Tests carried out on a GM Bacillus subtilis showed 25 positive CDSs corresponding to the chloramphenicol resistance gene and CDSs of the inserted plasmids. On a wild type B. subtilis, no false positive sequences were detected. CONCLUSION: DUGMO detects exogenous CDS, truncated, fused or highly mutated wild CDSs in high-throughput sequencing data, and was shown to be efficient at detecting GM sequences, but it might also be employed for the identification of recent horizontal gene transfers.

Genomic polymorphism of Mycoplasma flocculare revealed by a newly developed multilocus sequence typing scheme.

Mycoplasma flocculare is genetically closely related to M. hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, and is frequently isolated with this second species. In this article, we report on the development of the first multilocus sequence typing (MLST) scheme for M. flocculare, based on three genes (adk, rpoB and tpiA). In total, 5022 bp of sequence were analyzed. MLST was used to characterize seven M. flocculare isolates and the reference strain. Eight distinct sequence types were defined, showing the great intraspecies variability of M. flocculare, and the high discriminatory power of the new typing method. The relative contribution of recombinations to the genomic evolution of M. flocculare was revealed by calculating the index of association (IA: 0.0185). This MLST scheme is now available for the acquisition of new knowledge on M. flocculare epidemiology via an online database comprising the DNA sequences of each allele, available at http://pubmlst.org/mflocculare/.

Identification of a Divergent Avian Influenza H3N2 Virus from Domestic Ducks in France.

An avian influenza H3N2 virus was isolated from domestic ducks in France in 2016. Although this French H3N2 virus possesses traits of an avian virus, the genetic distances observed for hemagglutinin (HA) and neuraminidase (NA) show that these two genes most likely evolved independently from other avian influenza sequences.

Characterization of plasmids harboring blaCTX-M genes in Escherichia coli from French pigs.

Resistance to extended-spectrum cephalosporins is prevalent in French pig E. coli isolates. The aim of this study was to characterize the plasmids and genes present in pathogenic and commensal extended-spectrum cephalosporins -resistant isolates. The resistance plasmids of 26 strains were sequenced and then analyzed to identify resistance and virulence genes. Results showed that resistance to extended-spectrum cephalosporins in French pig E. coli isolates is-as in other food animals in France-mainly carried by highly similar blaCTX-M-1 IncI1/ST3 plasmids. These plasmids very often bear other resistance genes such as resistance to sulphonamides (sul2), trimethoprim (dfrA17) and aminoglycosides (aadA5), and occasionally to tetracycline (tet(A)), macrolides (mph(A) and erm genes), phenicols (floR) or streptomycin (strA, strB). Few virulence genes were detected, including colicins, heat-stable enterotoxins, adhesins or temperature-sensitive hemagglutinins. The other cefotaximases detected were blaCTX-M-27 and blaCTX-M-14, the latter being on an IncF plasmid which showed very close identity to a human epidemic plasmid. Importantly, resistance genes for quinolones or polymyxins were never detected on the extended-spectrum cephalosporins resistance plasmids. These results are helpful to evidence the risk of co-selecting cephalosporins -resistance using antibiotics outside this group. They also highlight the occasional presence in pigs of human epidemic plasmids.

Ruminant and chicken: important sources of campylobacteriosis in France despite a variation of source attribution in 2009 and 2015.

Pathogen source attribution studies are a useful tool for identifying reservoirs of human infection. Based on Multilocus Sequence Typing (MLST) data, such studies have identified chicken as a major source of C. jejuni human infection. The use of whole genome sequence-based typing methods offers potential to improve the precision of attribution beyond that which is possible from 7 MLST loci. Using published data and 156 novel C. jejuni genomes sequenced in this study, we performed probabilistic host source attribution of clinical C. jejuni isolates from France using three types of genotype data: comparative genomic fingerprints; MLST genes; 15 host segregating genes previously identified by whole genome sequencing. Consistent with previous studies, chicken was an important source of campylobacteriosis in France (31-63% of clinical isolates assigned). There was also evidence that ruminants are a source (22-55% of clinical isolates assigned), suggesting that further investigation of potential transmission routes from ruminants to human would be useful. Additionally, we found evidence of environmental and pet sources. However, the relative importance as sources varied according to the year of isolation and the genotyping technique used. Annual variations in attribution emphasize the dynamic nature of zoonotic transmission and the need to perform source attribution regularly.

Longitudinal study of Escherichia coli plasmid resistance to extended-spectrum cephalosporins in free-range broilers.

Resistance to extended-spectrum cephalosporins (ESCs) is mostly borne by conjugative plasmids. The aim of the present study was to evaluate the characteristics and diversity of ESC resistance plasmids in Escherichia coli from different free-range broiler flocks in France, and their persistence in flocks during rearing. Two hatcheries were selected. Faecal samples from 11 flocks were collected from before their arrival on the broiler production farm up to their slaughter at the end of the rearing period. A selection of 25 E. coli isolates obtained at different times from different flocks but all harbouring an ESC resistance gene was characterised. The plasmids coding for ESC resistance were sequenced using Mi-seq Illumina technology or the ion proton system (Ion Torrent). Ten IncI1 ST12 plasmids carried the blaCMY-2 gene, and most of them had no other resistance genes. All blaCMY-2 plasmids were obtained from day-old to 7-day-old chicks from four flocks hatched at the same hatchery and sent to three different farms. Sequence comparisons showed identity percentages higher than 99%. Fifteen IncI1 ST3 plasmids were obtained from day-old to 77-day-old broilers from seven flocks on six farms. These plasmids harboured the blaCTX-M-1 gene, and most also had the tet(A) and sul2 genes, with sequence identity higher than 99%. For both types of plasmid, very high identity percentages were also obtained with published sequences of plasmids isolated from broilers in other countries or from other animal species. Thus, unlike the IncI1 ST12 blaCMY-2 plasmids, the epidemic nature of the IncI1 ST3 blaCTX-M-1 plasmids in the French poultry production makes it difficult to determine the origin of a contamination which may persist for weeks in a flock.

Characterization of plasmids harboring blaCTX-M and blaCMY genes in E. coli from French broilers.

Resistance to extended-spectrum cephalosporins (ESC) is a global health issue. The aim of this study was to analyze and compare plasmids coding for resistance to ESC isolated from 16 avian commensal and 17 avian pathogenic Escherichia coli (APEC) strains obtained respectively at slaughterhouse or from diseased broilers in 2010-2012. Plasmid DNA was used to transform E. coli DH5alpha, and the resistances of the transformants were determined. The sequences of the ESC-resistance plasmids prepared from transformants were obtained by Illumina (33 plasmids) or PacBio (1 plasmid). Results showed that 29 of these plasmids contained the blaCTX-M-1 gene and belonged to the IncI1/ST3 type, with 27 and 20 of them carrying the sul2 or tet(A) genes respectively. Despite their diverse origins, several plasmids showed very high percentages of identity. None of the blaCTX-M-1-containing plasmid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the blaCMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The blaCMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a blaCTX-M-1 IncI1/ST3 plasmid. Importantly APEC virulence genes (sitA-D, iucA-D, iutA, hlyF, ompT, etsA-C, iss, iroB-E, iroN, cvaA-C and cvi) were detected on the blaCMY-2 plasmid. In conclusion, our results show the dominance and high similarity of blaCTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a blaCMY-2 plasmid.

Novel Streptococcus suis Sequence Type 834 among Humans, Madagascar.

Two cases of meningitis caused by Streptococcus suis occurred in Madagascar, 1 in 2015 and 1 in 2016. We report the characterization of the novel sequence type, 834, which carried the mrp+/sly+/epf+ virulence marker and a mutation G→T at position 174, leading to a substitution mutS1 to mutS284.

Complete Mitochondrial Genome Sequence of Aethina tumida (Coleoptera: Nitidulidae), a Beekeeping Pest.

We report here the full mitochondrial genome sequence of Aethina tumida, a Nitidulidae species beetle, that is a pest of bee hives. The obtained sequence is 16,576 bp in length and contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNAs.

A single amino acid change in the non-structural NV protein impacts the virulence phenotype of Viral hemorrhagic septicemia virus in trout.

Novirhabdoviruses like the Viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses infecting fish. In the current study, RNA genomes of different VHSV field isolates classified as high, medium or low virulent phenotypes have been sequenced by next-generation sequencing and compared. Various amino acid changes, depending on the VHSV phenotype, have been identified in all the VHSV proteins. As a starting point, we focused our study on the non-virion (NV) non-structural protein in which an arginine residue (R116) is present in all the virulent isolates and replaced by a serine/asparagine residue S/N116 in the attenuated isolates. A recombinant virus derived from a virulent VHSV strain in which the NV R116 residue has been replaced by a serine, rVHSVNVR116S, was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSVNVR116S was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.

Complete Genome Sequence of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Strain from Two Genotype 1 Modified Live Virus Vaccine Strains.

This paper provides information on the complete genome sequence of a porcine reproductive and respiratory syndrome virus (PRRSV) strain isolated on a French pig farm which was identified as a recombinant strain from two commercial modified live virus vaccine strains of genotype 1 (VP-046BIS and DV strains).

Emerging highly pathogenic H5 avian influenza viruses in France during 2015/16: phylogenetic analyses and markers for zoonotic potential.

Several new highly pathogenic (HP) H5 avian influenza virus (AIV) have been detected in poultry farms from south-western France since November 2015, among which an HP H5N1. The zoonotic potential and origin of these AIVs immediately became matters of concern. One virus of each subtype H5N1 (150169a), H5N2 (150233) and H5N9 (150236) was characterised. All proved highly pathogenic for poultry as demonstrated molecularly by the presence of a polybasic cleavage site in their HA protein - with a sequence (HQRRKR/GLF) previously unknown among avian H5 HPAI viruses - or experimentally by the in vivo demonstration of an intravenous pathogenicity index of 2.9 for the H5N1 HP isolate. Phylogenetic analyses based on the full genomes obtained by NGS confirmed that the eight viral segments of the three isolates were all part of avian Eurasian phylogenetic lineage but differed from the Gs/Gd/1/96-like lineage. The study of the genetic characteristics at specific amino acid positions relevant for modulating the adaptation to and the virulence for mammals showed that presently, these viruses possess most molecular features characteristic of AIV and lack some major characteristics required for efficient respiratory transmission to or between humans. The three isolates are therefore predicted to have no significant pandemic potential.

Genome-Wide Identification of Host-Segregating Epidemiological Markers for Source Attribution in Campylobacter jejuni.

Campylobacter is among the most common worldwide causes of bacterial gastroenteritis. This organism is part of the commensal microbiota of numerous host species, including livestock, and these animals constitute potential sources of human infection. Molecular typing approaches, especially multilocus sequence typing (MLST), have been used to attribute the source of human campylobacteriosis by quantifying the relative abundance of alleles at seven MLST loci among isolates from animal reservoirs and human infection, implicating chicken as a major infection source. The increasing availability of bacterial genomes provides data on allelic variation at loci across the genome, providing the potential to improve the discriminatory power of data for source attribution. Here we present a source attribution approach based on the identification of novel epidemiological markers among a reference pan-genome list of 1,810 genes identified by gene-by-gene comparison of 884 genomes of Campylobacter jejuni isolates from animal reservoirs, the environment, and clinical cases. Fifteen loci involved in metabolic activities, protein modification, signal transduction, and stress response or coding for hypothetical proteins were selected as host-segregating markers and used to attribute the source of 42 French and 281 United Kingdom clinical C. jejuni isolates. Consistent with previous studies of British campylobacteriosis, analyses performed using STRUCTURE software attributed 56.8% of British clinical cases to chicken, emphasizing the importance of this host reservoir as an infection source in the United Kingdom. However, among French clinical isolates, approximately equal proportions of isolates were attributed to chicken and ruminant reservoirs, suggesting possible differences in the relative importance of animal host reservoirs and indicating a benefit for further national-scale attribution modeling to account for differences in production, behavior, and food consumption.IMPORTANCE Accurately quantifying the relative contribution of different host reservoirs to human Campylobacter infection is an ongoing challenge. This study, based on the development of a novel source attribution approach, provides the first results of source attribution in Campylobacter jejuni in France. A systematic analysis using gene-by-gene comparison of 884 genomes of C. jejuni isolates, with a pan-genome list of genes, identified 15 novel epidemiological markers for source attribution. The different proportions of French and United Kingdom clinical isolates attributed to each host reservoir illustrate a potential role for local/national variations in C. jejuni transmission dynamics.