Fabrice Touzain

Complete Genome Sequence of a Porcine Epidemic Diarrhea S Gene Indel Strain Isolated in France in December 2014

ABSTRACT We report the first and only case of a porcine epidemic diarrhea (PED) outbreak occurring in December 2014 in northern France, and we show using the full-length genome sequence of the French PED virus (PEDV) isolate that it was a PEDV indel strain close to German PEDV strains recently isolated.

Dual Emergence of Usutu Virus in Common Blackbirds, Eastern France, 2015.

References 1. Yokota S, Sato T, Okubo T, Ohkoshi Y, Okabayashi T, Kuwahara O, et al. Prevalence of fluoroquinolone-resistant Escherichia coli O25:H4-ST131 (CTX-M-15-nonproducing) strains isolated in Japan. Chemotherapy. 2012;58:52–9. http://dx.doi.org/10.1159/000336129 2. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing (M100-S25). Wayne (PA): The Institute; 2015. 3. Colpan A, Johnston B, Porter S, Clabots C, Anway R, Thao L, et al.; VICTORY (Veterans Influence of Clonal Types on Resistance: Year 2011) Investigators. Escherichia coli sequence type 131 (ST131) subclone H30 as an emergent multidrug-resistant pathogen among US veterans. Clin Infect Dis. 2013;57:1256–65. http://dx.doi.org/10.1093/cid/cit503 4. Price LB, Johnson JR, Aziz M, Clabots C, Johnston B, Tchesnokova V, et al. The epidemic of extended-spectrumβ-lactamase-producing Escherichia coli ST131 is driven by a single highly pathogenic subclone, H30-Rx. mBio. 2013;4: e00377–13. http://dx.doi.org/10.1128/mBio.00377-13 5. Lau SH, Reddy S, Cheesbrough J, Bolton FJ, Willshaw G, Cheasty T, et al. Major uropathogenic Escherichia coli strain isolated in the northwest of England identified by multilocus sequence typing. J Clin Microbiol. 2008;46:1076–80. http://dx.doi.org/10.1128/JCM.02065-07 6. Peirano G, Schreckenberger PC, Pitout JD. Characteristics of NDM-1-producing Escherichia coli isolates that belong to the successful and virulent clone ST131. Antimicrob Agents Chemother. 2011;55:2986–8. http://dx.doi.org/10.1128/AAC.01763-10 7. Morris D, Boyle F, Ludden C, Condon I, Hale J, O’Connell N, et al. Production of KPC-2 carbapenemase by an Escherichia coli clinical isolate belonging to the international ST131 clone. Antimicrob Agents Chemother. 2011;55:4935–6. http://dx.doi.org/10.1128/AAC.05127-11 8. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16:161–8. http://dx.doi.org/10.1016/S1473-3099(15)00424-7 9. Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, et al. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, 2016. Euro Surveill. 2016;21. http://dx.doi.org/10.2807/1560-7917. ES.2016.21.2730280

Genome-wide identification of host-segregating epidemiological markers for source attribution in Campylobacter jejuni

ABSTRACT 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.

Complete Genome Sequence of Bluetongue Virus Serotype 8, Which Reemerged in France in August 2015

ABSTRACT We announce here the complete genome sequence (coding and noncoding) of the bluetongue virus (BTV) serotype 8, isolated from a ram in Allier department, France, 2015. Sequence analysis confirms the reemergence of the BTV-8 strain that previously circulated in France until 2009 and other European countries until 2010.

Impact of Ceftiofur Injection on Gut Microbiota and Escherichia coli Resistance in Pigs

ABSTRACT Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, blaCTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the blaCTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the blaCTX-M-1 gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.

Emerging highly pathogenic H5 avian influenza viruses in France during winter 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.

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.

Preparation for emergence of an Eastern European porcine reproductive and respiratory syndrome virus (PRRSV) strain in Western Europe: Immunization with modified live virus vaccines or a field strain confers partial protection

The porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses for the swine industry worldwide. In the past several years, highly pathogenic strains that lead to even greater losses have emerged. For the Western European swine industry, one threat is the possible introduction of Eastern European PRRSV strains (example Lena genotype 1.3) which were shown to be more virulent than common Western resident strains under experimental conditions. To prepare for the possible emergence of this strain in Western Europe, we immunized piglets with a Western European PRRSV field strain (Finistere: Fini, genotype 1.1), a new genotype 1 commercial modified live virus (MLV) vaccine (MLV1) or a genotype 2 commercial MLV vaccine (MLV2) to evaluate and compare the level of protection that these strains conferred upon challenge with the Lena strain 4 weeks later. Results show that immunization with Fini, MLV1 or MLV2 strains shortened the Lena-induced hyperthermia. In the Fini group, a positive effect was also demonstrated in growth performance. The level of Lena viremia was reduced for all immunized groups (significantly so for Fini and MLV2). This reduction in Lena viremia was correlated with the level of Lena-specific IFNγ-secreting cells. In conclusion, we showed that a commercial MLV vaccine of genotype 1 or 2, as well as a field strain of genotype 1.1 may provide partial clinical and virological protection upon challenge with the Lena strain. The cross-protection induced by these immunizing strains was not related with the level of genetic similarity to the Lena strain. The slightly higher level of protection established with the field strain is attributed to a better cell-mediated immune response.

Impact of the administration of a third-generation cephalosporin (3GC) to one-day-old chicks on the persistence of 3GC-resistant Escherichia coli in intestinal flora: An in vivo experiment.

The aim of the experiment was to evaluate under controlled conditions the impact on the excretion of 3GC-resistant Escherichia coli of the injection of one-day-old chicks with ceftiofur, a third-generation cephalosporin (3GC). Three isolators containing specific-pathogen-free chicks were used. In the first one, 20 birds were injected with ceftiofur then ten of them were orally inoculated with a weak inoculum of a 3GC-resistant E. coli field isolate containing an IncI1/ST3 plasmid encoding a blaCTX-M-1 beta-lactamase. The other chicks were kept as contact birds. None of the 20 birds in the second isolator were injected with ceftiofur, but ten of them were similarly inoculated with the 3GC-resistant strain and the others kept as contact birds. A third isolator contained ten non-injected, non-inoculated chicks. Fecal samples were collected regularly over one month and the E. coli isolated on non-supplemented media were characterized by antimicrobial agar dilution, detection of selected resistance genes and determination of phylogenetic group by PCR. The titers of 3GC-resistant E. coli in individual fecal samples were evaluated by culturing on 3GC-supplemented media. Results showed that the inoculated strain rapidly and abundantly colonized the inoculated and contact birds. The ceftiofur injection resulted in significantly higher percentages of 3GC-resistant E. coli isolates among the analyzed E. coli. No transfer of the 3GC-encoding plasmid to other isolates could be evidenced. In conclusion, these results highlight the dramatic capacity of 3GC-resistant E. coli to colonize and persist in chicks, and the selecting pressure imposed by the off-label use of ceftiofur.

First complete genome sequence of European turkey coronavirus suggests complex recombination history related with US turkey and guinea fowl coronaviruses

A full-length genome sequence of 27 739 nt was determined for the only known European turkey coronavirus (TCoV) isolate. In general, the order, number and size of ORFs were consistent with other gammacoronaviruses. Three points of recombination were predicted, one towards the end of 1a, a second in 1b just upstream of S and a third in 3b. Phylogenetic analysis of the four regions defined by these three points supported the previous notion that European and American viruses do indeed have different evolutionary pathways. Very close relationships were revealed between the European TCoV and the European guinea fowl coronavirus in all regions except one, and both were shown to be closely related to the European infectious bronchitis virus (IBV) Italy 2005. None of these regions of sequence grouped European and American TCoVs. The region of sequence containing the S gene was unique in grouping all turkey and guinea fowl coronaviruses together, separating them from IBVs. Interestingly the French guinea fowl virus was more closely related to the North American viruses. These data demonstrate that European turkey and guinea fowl coronaviruses share a common genetic backbone (most likely an ancestor of IBV Italy 2005) and suggest that this recombined in two separate events with different, yet related, unknown avian coronaviruses, acquiring their S-3a genes. The data also showed that the North American viruses do not share a common backbone with European turkey and guinea fowl viruses; however, they do share similar S-3a genes with guinea fowl virus.