Virulence genes, resistome and mobilome of Streptococcus suis strains isolated in France.

Streptococcus suis is a leading cause of infection in pigs, causing extensive economic losses. In addition, it can also infect wild fauna, and can be responsible for severe infections in humans. Increasing antimicrobial resistance (AMR) has been described in S. suis worldwide and most of the AMR genes are carried by mobile genetic elements (MGEs). This contributes to their dissemination by horizontal gene transfer. A collection of 102 strains isolated from humans, pigs and wild boars in France was subjected to whole genome sequencing in order to: (i) study their genetic diversity, (ii) evaluate their content in virulence-associated genes, (iii) decipher the mechanisms responsible for their AMR and their association with MGEs, and (iv) study their ability to acquire extracellular DNA by natural transformation. Analysis by hierarchical clustering on principal components identified a few virulence-associated factors that distinguish invasive CC1 strains from the other strains. A plethora of AMR genes (n=217) was found in the genomes. Apart from the frequently reported erm(B) and tet(O) genes, more recently described AMR genes were identified [vga(F)/sprA, vat(D)]. Modifications in PBPs/MraY and GyrA/ParC were detected in the penicillin- and fluoroquinolone-resistant isolates respectively. New AMR gene-MGE associations were detected. The majority of the strains have the full set of genes required for competence, i.e for the acquisition of extracellular DNA (that could carry AMR genes) by natural transformation. Hence the risk of dissemination of these AMR genes should not be neglected.

Prevalence and molecular epidemiology of mcr-mediated colistin-resistance Escherichia coli from healthy poultry in France after national plan to reduce exposure to colistin in farm.

Introduction: Within the 2007-2014 programme for the surveillance of antimicrobial resistance (AMR) in livestock in France, mcr-1 prevalence average in commensal Escherichia coli was found to be 5.9% in turkeys and 1.8% in broilers, indicating that mobile colistin resistance had spread in farm animals. In 2017, the French national Ecoantibio2 plan was established to tackle AMR in veterinary medicine, with the objective of a 50% reduction in exposure to colistin in farm animals within 5 years (from 2014-2015 to 2020). Our objective was to update data concerning the prevalence and molecular epidemiology of colistin resistance, in consideration of colistin sales in poultry production in France. Methods: Antimicrobial susceptibility of commensal E. coli isolated from broilers and turkeys at slaughterhouse was determined by broth micro-dilution. The mcr genes were screened by polymerase chain reaction (PCR). Whole genome sequencing (WGS) was used to investigate the genetic diversity of colistin-resistant isolates. Transformation experiments enabled identification of the mcr-bearing plasmid replicon types. The correlation between prevalence of colistin resistance and colistin usage data was explored statistically. Results and discussion: In 2020, in France, the resistance prevalence to colistin in poultry production was 3% in turkeys and 1% in broilers, showing a significant highly positive correlation with a -68% decrease of poultry exposure to colistin since 2014. Only the mcr-1 gene was detected among the colistin-resistant E. coli. More than 80% of isolates are multi-drug resistant with 40% of isolates originating from turkeys and 44% originating from broilers co-resistant to the critically important antimicrobial ciprofloxacin. Most of the strains had no clonal relationship. The mcr gene was located in different plasmid types, carrying various other AMR genes. The decrease in colistin resistance among poultry in France can be considered a positive outcome of the national action plans for reduced colistin usage.

Comparative Analysis of Campylobacter jejuni and C. coli Isolated from Livestock Animals to C. jejuni and C. coli Isolated from Surface Water Using DNA Sequencing and MALDI-TOF.

This study evaluated the contribution of cattle, sheep, poultry and pigs to the contamination of surface water from rivers by Campylobacter jejuni and C. coli using MLST, cgMLST and considered MALDI-TOF MS as an alternative technique. The 263 strains isolated from cattle (n = 61), sheep (n = 42), poultry (n = 65), pigs (n = 60) and surface water (n = 35) were distributed across 115 sequence types (STs), 49 for C. jejuni and 66 for C. coli. Considering MLST data, 14.2%, 11.4% and 2.8% of the surface water strains could be attributed to cattle, poultry and sheep, respectively, none to pigs, and 85.7% were non-attributed. Analysis of cg-MLST data with STRUCTURE indicated that C. jejuni strains from water were predominantly attributed to poultry (93.5%), weakly to sheep (<1%) and 6.3% non-attributed, and that conversely, C. coli strains from water were predominantly non-attributed (94.3%) and 5.7% attributed to poultry. Considering the protein profiles with a threshold of 94% and 97% of similarity, respectively, strains from surface water could be attributed to poultry (31.4% and 17.1%), and to cattle (17.1% and 5.7%); 54.1% and 77.1% were non-attributed. This study confirmed these livestock animals might contribute to the contamination of surface water, with a level of contribution depending on the typing technique and the method of analysis. MALDI-TOF could potentially be an alternative approach for source attribution.

First identification of Cryptosporidium parvum virus 1 (CSpV1) in various subtypes of Cryptosporidium parvum from diarrheic calves, lambs and goat kids from France.

Cryptosporidium spp. remain a major cause of waterborne diarrhea and illness in developing countries and represent a significant burden to farmers worldwide. Cryptosporidium parvum virus 1 (CSpV1), of the genus Cryspovirus, was first reported to be present in the cytoplasm of C. parvum in 1997. Full-length genome sequences have been obtained from C. parvum from Iowa (Iowa), Kansas (KSU) and China. We aimed at characterizing the genome of CSpV1 from France and used sequence analysis from Cryptosporidium isolates to explore whether CSpV1 genome diversity varies over time, with geographical sampling location, C. parvum genetic diversity, or ruminant host species. A total of 123 fecal samples of cattle, sheep and goats were collected from 17 different French departments (57 diseased animal fecal samples and 66 healthy animal fecal samples). Subtyping analysis of the C. parvum isolates revealed the presence of two zoonotic subtype families IIa and IId. Sequence analysis of CSpV1 revealed that all CSpV1 from France, regardless of the subtype of C. parvum (IIaA15G2R1, IIaA17G2R1 and IIdA18G1R1) are more closely related to CSpV1 from Turkey, and cluster on a distinct branch from CSpV1 collected from C. parvum subtype IIaA15G2R1 from Asia and North America. We also found that samples collected on a given year or successive years in a given location are more likely to host the same subtype of C. parvum and the same CSpV1 strain. Yet, there is no distinct clustering of CSpV1 per French department or ruminants, probably due to trade, and transmission of C. parvum among host species. Our results point towards (i) a close association between CSpV1 movement and C. parvum movement, (ii) recent migrations of C. parvum among distantly located departments and (iii) incidental transmission of C. parvum between ruminants. All together, these results provide insightful information regarding CSpV1 evolution and suggest the virus might be used as an epidemiological tracer for C. parvum. Future studies need to investigate CSpV1's role in C. parvum virulence and on subtype ability to infect different species.

Circulation of Bluetongue Virus Serotypes 1, 4, 8, 10 and 16 and Epizootic Hemorrhagic Disease Virus in the Sultanate of Oman in 2020-2021.

The circulation of Bluetongue (BT) and Epizootic Hemorrhagic Disease (EHD) in the Middle East has already been reported following serological analyses carried out since the 1980s, mostly on wild ruminants. Thus, an EHD virus (EHDV) strain was isolated in Bahrain in 1983 (serotype 6), and more recently, BT virus (BTV) serotypes 1, 4, 8 and 16 have been isolated in Oman. To our knowledge, no genomic sequence of these different BTV strains have been published. These same BTV or EHDV serotypes have circulated and, for some of them, are still circulating in the Mediterranean basin and/or in Europe. In this study, we used samples from domestic ruminant herds collected in Oman in 2020 and 2021 for suspected foot-and-mouth disease (FMD) to investigate the presence of BTV and EHDV in these herds. Sera and whole blood from goats, sheep and cattle were tested for the presence of viral genomes (by PCR) and antibodies (by ELISA). We were able to confirm the presence of 5 BTV serotypes (1, 4, 8, 10 and 16) and the circulation of EHDV in this territory in 2020 and 2021. The isolation of a BTV-8 strain allowed us to sequence its entire genome and to compare it with another BTV-8 strain isolated in Mayotte and with homologous BTV sequences available on GenBank.

Oronasal or Intramuscular Immunization with a Thermo-Attenuated ASFV Strain Provides Full Clinical Protection against Georgia 2007/1 Challenge.

African swine fever (ASF) is a contagious viral disease of suids that induces high mortality in domestic pigs and wild boars. Given the current spread of ASF, the development of a vaccine is a priority. During an attempt to inactivate the Georgia 2007/1 strain via heat treatment, we fortuitously generated an attenuated strain called ASFV-989. Compared to Georgia, the ASFV-989 strain genome has a deletion of 7458 nucleotides located in the 5'-end encoding region of MGF 505/360, which allowed for developing a DIVA PCR system. In vitro, in porcine alveolar macrophages, the replication kinetics of the ASFV-989 and Georgia strains were identical. In vivo, specific-pathogen-free (SPF) pigs inoculated with the ASFV-989 strain, either intramuscularly or oronasally, exhibited transient hyperthermia and slightly decreased growth performance. Animals immunized with the ASFV-989 strain showed viremia 100 to 1000 times lower than those inoculated with the Georgia strain and developed a rapid antibody and cell-mediated response. In ASFV-989-immunized pigs challenged 2 or 4 weeks later with the Georgia strain, no symptoms were recorded and no viremia for the challenge strain was detected. These results show that the ASFV-989 strain is a promising non-GMO vaccine candidate that is usable either intramuscularly or oronasally.

A three-year evolution and comparison of the blaCTX-M genes in pathogenic and non-pathogenic Escherichia coli isolated from young diarrheic and septicaemic calves in Belgium.

Escherichia coli producing Extended-Spectrum-ß-Lactamases (ESBL) are a major public health hazard worldwide. The most frequent ESBL belong to the CTX-M family. This study follows their prevalence in pathogenic and non-pathogenic ESBL-producing E. coli isolated from young diarrheic and septicaemic calves over three calving seasons. The triplex PCR targeted three main groups: CTX-M-1, CTX-M-2 and CTX-M-9. Of the 394 isolates studied, 388 (98.5%) were positive, with a majority of CTX-M-1 (243, 61.7%), following by CTX-M-9 (74, 18.8%) and CTX-M-2 (64, 16.2%). The progressive decrease of ESBL-resistance of pathogenic E. coli is not linked to any shift in genetic background, blaCTX-M genes still present in 99% of the isolates, or to the proportion of the three CTX-M groups. Moreover, no significant difference was observed in the CTX-M content between pathogenic and non-pathogenic E. coli.

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.

Multiple independent introductions of highly pathogenic avian influenza H5 viruses during the 2020-2021 epizootic in France.

During winter 2020-2021, France and other European countries were severely affected by highly pathogenic avian influenza H5 viruses of the Gs/GD/96 lineage, clade 2.3.4.4b. In total, 519 cases occurred, mainly in domestic waterfowl farms in Southwestern France. Analysis of viral genomic sequences indicated that 3 subtypes of HPAI H5 viruses were detected (H5N1, H5N3, H5N8), but most French viruses belonged to the H5N8 subtype genotype A, as Europe. Phylogenetic analyses of HPAI H5N8 viruses revealed that the French sequences were distributed in 9 genogroups, suggesting 9 independent introductions of H5N8 from wild birds, in addition to the 2 introductions of H5N1 and H5N3.

Concomitant NA and NS deletion on avian Influenza H3N1 virus associated with hen mortality in France in 2019.

An H3N1 avian influenza virus was detected in a laying hens farm in May 2019 which had experienced 25% mortality in Northern France. The complete sequencing of this virus showed that all segment sequences belonged to the Eurasian lineage and were phylogenetically very close to many of the Belgian H3N1 viruses detected in 2019. The French virus presented two genetic particularities with NA and NS deletions that could be related to virus adaptation from wild to domestic birds and could increase virulence, respectively. Molecular data of H3N1 viruses suggest that these two deletions occurred at two different times.

Description and validation of a new set of PCR markers predictive of avian pathogenic Escherichia coli virulence.

Avian colibacillosis is the main bacterial infectious disease in poultry and is caused by avian pathogenic Escherichia coli (APEC). However, E. coli strains are very diverse, and not all are pathogenic for poultry. A straightforward scheme for identifying APEC is crucial to better control avian colibacillosis. In this study, we combined high-throughput PCR and a machine learning procedure to identify relevant genetic markers associated with APEC. Markers related to phylogroup, serotype and 66 virulence factors were tested on a large number of E. coli strains isolated from environmental, faecal or colibacillosis lesion samples in 80 broiler flocks. Nine classification methods and a machine learning procedure were used to differentiate 170 strains presumed non-virulent (obtained from farm environments) from 203 strains presumed virulent (obtained from colibacillosis cases on chicken farms) and to develop a prediction model to evaluate the pathogenicity of isolates. The model was then validated on 14 isolates using a chick embryo lethality assay. The selected and validated model based on the bootstrap aggregating tree method relied on a scheme of 13 positive or negative markers associated with phylogroups (arpA), H4 antigen and virulence markers (aec4, ETT2.2, frzorf4,fyuA, iha, ireA, iroN, iutA1, papA, tsh, and vat). It had a specificity of 84 % and a sensitivity of 85 %, and was implemented as an online tool. Our scheme offers an easy evaluation of the virulence of avian E. coli isolates on the basis of the presence/absence of these 13 genetic markers, allowing for better control of avian colibacillosis.

Impact of mesophilic anaerobic digestion and post-treatment of digestates on the transfer of conjugative antimicrobial resistance plasmids.

Manure is a major source of antimicrobial-resistant bacteria and resistance genes carried by mobile genetic elements such as plasmids. In France, the number of on-farm biogas plants has increased significantly in recent years. Our study investigated the impact of mesophilic anaerobic digestion (AD) and the post-treatment of digestates on the fate of conjugative plasmids, along with their potential transfer of antimicrobial resistance. Samples of raw manure, digestates and post-treated digestates were collected from three on-farm biogas plants. Conjugative plasmids were captured using the Escherichia coli CV601 recipient strain and media supplemented with rifampicin and kanamycin - to which the recipient strain is resistant - and tetracycline, sulfamethoxazole, gentamicin, trimethoprim, amoxicillin, cefotaxime, ciprofloxacin or colistin. Putative transconjugants were identified and characterised by disc diffusion and whole genome sequencing. The results showed that the antimicrobial resistance genes transferred from the different matrices conferred resistance to tetracyclines, sulphonamides, trimethoprim, and/or streptomycin. Transconjugants were obtained from raw manure samples but not from digestates or post-digestates, suggesting that mesophilic AD processes may produce fewer conjugative plasmids potentially able to be transferred to Enterobacterales.

Full-Length Genome Sequence of a Novel European Antigenic Variant Strain of Infectious Bursal Disease Virus.

We report the full-length genome sequence (compared to reference sequences) of a novel European variant strain of infectious bursal disease virus (IBDV), designated 19P009381 (AxB1). This should help to further identify such viruses in Europe.

Efficacy of passive immunization in broiler chicks via an inactivated Escherichia coli autogenous vaccine administered to broiler breeder hens.

Avian pathogenic Escherichia coli (APEC) cause extra-intestinal infections called colibacillosis, which is the dominant bacterial disease in broilers. To date, given the diversity of APEC strains and the need for an acceptable level of protection in day-old chicks, no satisfactory commercial vaccine is available. As part of a French nationwide project, we selected three representative strains among several hundred APEC that cause colibacillosis disease. We first performed experiments to develop colibacillosis in vivo models, using an inoculum of 3 × 107 CFU of each E. coli strain per chick. Two APEC strains (19-381 and 19-383-M1) were found to be highly virulent for day-old chicks, whereas the third strain (19-385-M1) induced no mortality nor morbidity.We then produced an autogenous vaccine using the (Llyod, 1982; MaCQueen, 1967) 19-381 and 19-383-M1 APEC strains and a passive immunization trial was undertaken. Specific-pathogen-free Leghorn hens were vaccinated twice 2 weeks apart, the control group receiving a saline solution. The vaccinated and control hens exhibited no clinical signs, and egg production and fertility of both groups were similar. Fertile eggs were collected for 2 weeks after the second vaccination and chicks were obtained. After challenge with each APEC (19-381 and 19-383-M1), chicks appeared to be partially protected from infection with the 19-383-M1 strain, with 40% mortality compared with 80% for the non-vaccinated chicks. No protection was found when the chicks were challenged with the 19-381 strain. Now, further work is needed to consider some aspects: severity of the pathogen challenge model, persistence of the protection, number of APEC strains in the autogenous vaccine, choice of adjuvants, and heterologous protection by the vaccine made from strain 19-383-M1.RESEARCH HIGHLIGHTS Three APEC strains were characterized and selected to develop in vivo models of colibacillosis.A bivalent autogenous vaccine was produced and a passive immunization trial was carried out.Protection of chicks was demonstrated when challenged with the 19-383-M1 APEC strain (homologous challenge).Further work is needed in particular to evaluate the protection against heterologous challenge.

A World of Viruses Nested within Parasites: Unraveling Viral Diversity within Parasitic Flatworms (Platyhelminthes).

Because parasites have an inextricable relationship with their host, they have the potential to serve as viral reservoirs or facilitate virus host shifts. And yet, little is known about viruses infecting parasitic hosts except for blood-feeding arthropods that are well-known vectors of zoonotic viruses. Herein, we uncovered viruses of flatworms (phylum Platyhelminthes, group Neodermata) that specialize in parasitizing vertebrates and their ancestral free-living relatives. We discovered 115 novel viral sequences, including 1 in Macrostomorpha, 5 in Polycladida, 44 in Tricladida, 1 in Monogenea, 15 in Cestoda, and 49 in Trematoda, through data mining. The majority of newly identified viruses constitute novel families or genera. Phylogenetic analyses show that the virome of flatworms changed dramatically during the transition of neodermatans to a parasitic lifestyle. Most Neodermata viruses seem to codiversify with their host, with the exception of rhabdoviruses, which may switch hosts more often, based on phylogenetic relationships. Neodermata rhabdoviruses also have a position ancestral to vertebrate-associated rhabdo viruses, including lyssaviruses, suggesting that vertebrate-associated rhabdoviruses emerged from a flatworm rhabdovirus in a parasitized host. This study reveals an extensive diversity of viruses in Platyhelminthes and highlights the need to evaluate the role of viral infection in flatworm-associated diseases. IMPORTANCE Little is known about the diversity of parasite-associated viruses and how these viruses may impact parasite fitness, parasite-host interactions, and virus evolution. The discovery of over a hundred viruses associated with a range of free-living and parasitic flatworms, including parasites of economic and clinical relevance, allowed us to compare the viromes of flatworms with contrasting lifestyles. The results suggest that flatworms acquired novel viruses after their transition to a parasitic lifestyle and highlight the possibility that they acquired viruses from their hosts and vice versa. An interesting example is the discovery of flatworm rhabdoviruses that have a position ancestral to rabies viruses and other vertebrate-associated rhabdoviruses, demonstrating that flatworm-associated viruses have emerged in a vertebrate host at least once in history. Therefore, parasitic flatworms may play a role in virus diversity and emergence. The roles that parasite-infecting viruses play in parasite-associated diseases remain to be investigated.

Characterization of a novel picornavirus isolated from moribund gilthead seabream (Sparus aurata) larvae.

Gilthead seabream represents a species of importance in Mediterranean aquaculture. The larval stage is particularly sensitive and frequently impacted in suboptimal environmental or sanitary conditions. In the present study, investigations were carried out in a seabream hatchery following an unusual mortality reaching 70% among 50-day post-hatching. Anorexia, loss of appetite and abnormal swimming behaviour were observed in absence of parasites or pathogenic bacteria. Proliferation of rod-shaped bacteria in the gut lumen was associated with focal degeneration in the intestinal mucosa. Cytopathic effects on an EK-1 cell line after 21 days of culture at 14°C and 20°C in contact with homogenized affected larvae revealed the presence of a viral agent. Molecular characterization by high-throughput sequencing showed a typical picornavirus genome organization with a polyprotein precursor of 2276 amino acids sharing 46.3% identity with that of the Eel Picornavirus-1. A specific real-time PCR confirmed the presence of the viral genome in affected larval homogenate and corresponding cell culture supernatant. We propose the name Potamipivirus daurada for this novel species within the genus Potamipivirus. The etiological role of this virus remains uncertain at this time, and future studies will be necessary to investigate its prevalence in natural and aquaculture-reared populations as well as its ability to cause diseases in gilthead seabream.

Impact of Escherichia coli probiotic strains ED1a and Nissle 1917 on the excretion and gut carriage of extended-spectrum beta-lactamase-producing E. coli in pigs.

We evaluated the impact of the administration of two Escherichia coli probiotic strains (ED1a and Nissle 1917) to pigs on the gut carriage or shedding of extended-spectrum beta-lactamase-producing E. coli. The probiotics were given to four sows from 12 days before farrowing to the weaning day, and to the 23 piglets (infected treated group (IPro)) from birth to the age of 49 days. Four other sows and their 24 piglets (infected non-treated group (INT)) did not receive the probiotics. IPro and INT piglets (n = 47) were orally inoculated with the strain E. coli 17-348F-RifR carrying the bla CTX-M-1 gene and resistant to rifampicin. Cefotaxime-resistant (CTXR) E. coli and rifampicin-resistant (RifR) E. coli were cultured and excretion of probiotics was studied using PCR on individual faecal and post-mortem samples, and from manure collected after the challenge with resistant E. coli. CTXR and RifR E . coli isolates were characterized to detect transfer of the bla CTX-M-1 to other strains.. Overall, there was no significant reduction in faecal excretion of CTXR and RifR E. coli in IPro pigs compared with INT pigs, although the CTXR and RifR E. coli titres were slightly, but significantly lower in the colon, caecum and rectum at post mortem. Excretion of the probiotics decreased with age, but Nissle 1917 was detected in most pigs at post-mortem. No transfer of the bla CTX-M-1 gene to probiotic and other E. coli strains was detected. In conclusion, in our experimental conditions, the used probiotics did not reduce shedding of the challenge strain.

Description of the first isolates of guinea fowl corona and picornaviruses obtained from a case of guinea fowl fulminating enteritis.

Guinea fowl fulminating enteritis has been reported in France since the 1970s. In 2014, a coronavirus was identified and appeared as a possible viral pathogen involved in the disease. In the present study, intestinal content from a guinea fowl involved in a new case of the disease in 2017 was analysed by deep sequencing, revealing the presence of a guinea fowl coronavirus (GfCoV) and a picornavirus (GfPic). Serial passage assays into the intra-amniotic cavity of 13-day-old specific pathogen-free chicken eggs and 20-day-old conventional guinea fowl eggs were attempted. In chicken eggs, isolation assays failed, but in guinea fowl eggs, both viruses were successfully obtained. Furthermore, two GfCoV and two GfPic isolates were obtained from the same bird but from different sections of its intestines. This shows that using eggs of the same species, in which the virus has been detected, can be the key for successful isolation. The consensus sequence of the full-length genomes of both GfCoV isolates was highly similar, and correlated to those previously described in terms of genome organization, ORF length and phylogenetic clustering. According to full-length genome analysis and the structure of the Internal Ribosome Entry Site, both GfPic isolates belong to the Anativirus genus and specifically the species Anativirus B. The availability of the first isolates of GfCoV and GfPic will now provide a means of assessing their pathogenicity in guinea fowl in controlled experimental conditions and to assess whether they are primary viral pathogens of the disease "guinea fowl fulminating enteritis".RESEARCH HIGHLIGHTSFirst isolation of guinea fowl coronaviruses and picornaviruses.Eggs homologous to the infected species are key for isolation.Isolates available to precisely evaluate the virus roles in fulminating enteritis.First full-length genome sequences of guinea fowl picornaviruses.

Selection of a Gentamicin-Resistant Variant Following Polyhexamethylene Biguanide (PHMB) Exposure in Escherichia coli Biofilms.

Antibiotic resistance is one of the most important issues facing modern medicine. Some biocides have demonstrated the potential of selecting resistance to antibiotics in bacteria, but data are still very scarce and it is important to better identify the molecules concerned and the underlying mechanisms. This study aimed to assess the potential of polyhexamethylene biguanide (PHMB), a widely used biocide in a variety of sectors, to select antibiotic resistance in Escherichia coli grown in biofilms. Biofilms were grown on inox coupons and then exposed daily to sublethal concentrations of PHMB over 10 days. Antibiotic-resistant variants were then isolated and characterized phenotypically and genotypically to identify the mechanisms of resistance. Repeated exposure to PHMB led to the selection of an E. coli variant (Ec04m1) with stable resistance to gentamycin (8-fold increase in minimum inhibitory concentration (MIC) compared to the parental strain. This was also associated with a significant decrease in the growth rate in the variant. Sequencing and comparison of the parental strain and Ec04m1 whole genomes revealed a nonsense mutation in the aceE gene in the variant. This gene encodes the pyruvate dehydrogenase E1 component of the pyruvate dehydrogenase (PDH) complex, which catalyzes the conversion of pyruvate to acetyl-CoA and CO2. A growth experiment in the presence of acetate confirmed the role of this mutation in a decreased susceptibility to both PHMB and gentamicin (GEN) in the variant. This work highlights the potential of PHMB to select resistance to antibiotics in bacteria, and that enzymes of central metabolic pathways should be considered as a potential target in adaptation strategies, leading to cross-resistance toward biocides and antibiotics in bacteria.

Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog.

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2'-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens.