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Objectives To investigate the optrA-carrying genetic elements and their transferability in two linezolid-resistant Streptococcus dysgalactiae subsp. equisimilis (SDSE) strains of swine origin. Methods SDSE strains (V220 and V1524) were phenotypically and genotypically characterized. Transferability of oxazolidinone resistance genes (filter mating), genetic elements and relatedness between isolates (WGS) were analysed. Excision of the genetic elements was assayed by inverse PCR. Results SDSE isolates were resistant to chloramphenicol, florfenicol and linezolid, but susceptible to tedizolid and both carried the optrA gene. In SDSE V220 optrA was located on a 72.9-kb ICESdyV220 inserted in the 3′ end of the chromosomal rum gene. It was 94%–96% identical (coverage, from 31% to 61%) to other optrA-carrying ICEs. In-depth ICESdyV220 sequence analysis revealed that optrA was carried by an IMESdyV220 (17.9 kb), also containing the tet(O/W/32/O) gene. Inverse PCR assays excluded the ICESdyV220 mobility. In SDSE V1524, optrA was carried by the ΦSdyV1524 prophage, integrated near the 5′ end of the chromosomal had gene, showing a genetic organization similar to that of other streptococcal phage. Conjugation and transduction assays failed to demonstrate the optrA transferability to streptococcal recipients. V220 and V1524 belonged to two novel sequence types (ST704 and ST634, respectively). Conclusions To the best of our knowledge, this is the first identification of the optrA gene on a prophage and an ICE in SDSE isolates from swine brain. These findings are consistent with the current belief in the key role of bacteriophages and ICEs in the streptococcal evolution and adaptation.

This study aims to give an overview of the prevalence of Listeria monocytogenes and Salmonella spp. in 9727 samples (2996 for L. monocytogenes and 6731 for Salmonella spp.) from different categories of ready-to-eat (RTE) foods, collected over 2 years from 28 large retailers and 148 canteens in the regions of northern Italy. The RTE samples were classified into two groups according to the preparation methods: (i) multi-ingredient preparations consisting of fully cooked food ready for immediate consumption, or with minimal further handling before consumption (Group A), and (ii) multi-ingredient preparations consisting of cooked and uncooked food, or preparations consisting of only raw ingredients (Group B). L. monocytogenes and Salmonella spp. were investigated in both of these categories. The overall prevalence of L. monocytogenes and Salmonella spp. was 0.13% and 0.07%, respectively. More specifically, L. monocytogenes was found in 0.04% of 2442 analysed RTE food samples belonging to group A and in 0.54% of 554 samples belonging to group B. Furthermore, 0.03% of 5367 RTE food samples from group A and 0.21% of 1364 samples from group B tested positive for Salmonella spp. In conclusion, the results obtained in this study can provide a significant contribution to L. monocytogenes and Salmonella spp. risk analysis in RTE foods.

International audience; Enterotoxigenic Escherichia coli (ETEC) is the aetiological agent of postweaning diarrhoea (PWD) in piglets. The SNPs located on the Mucine 4 (MUC4) and Fucosyltransferase 1 (FUT1) genes have been associated with the susceptibility to ETEC F4 and ETEC F18, respectively. The interplay between the MUC4 and FUT1 genotypes to ETEC infection and the use of amoxicillin in modifying the intestinal microbiota during a natural infection by multiresistant ETEC strains have never been investigated. The aim of this study was to evaluate the effects of the MUC4 and FUT1 genotypes and the administration of amoxicillin through different routes on the presence of diarrhoea and the faecal microbiota composition in piglets naturally infected with ETEC. Seventy-one piglets were divided into three groups: two groups differing by amoxicillin administration routes-parenteral (P) or oral (O) and a control group without antibiotics (C). Faecal scores, body weight, presence of ETEC F4 and F18 were investigated 4 days after the arrival in the facility (T0), at the end of the amoxicillin administration (T1) and after the withdrawal period (T2). The faecal bacteria composition was assessed by sequencing the 16S rRNA gene. We described that MUC4 and FUT1 genotypes were associated with the presence of ETEC F4 and ETEC F18. The faecal microbiota was influenced by the MUC4 genotypes at T0. We found the oral administration to be associated with the presence of diarrhoea at T1 and T2. Furthermore, the exposure to amoxicillin resulted in significant alterations of the faecal microbiota. Overall, MUC4 and FUT1 were confirmed as genetic markers for the susceptibility to ETEC infections in pigs. Moreover, our data highlight that group amoxicillin treatment may produce adverse outcomes on pig health in course of multiresistant ETEC infection. Therefore, alternative control measures able to maintain a healthy faecal microbiota in weaners are recommended.

The transmission of antimicrobial resistance bacteria from animals to humans has become an important concern. The extended-spectrum beta-lactamase (ESBL) -AmpC- producing Escherichia coli (ESBL-AmpC EC) and quinolones resistant E. coli are of particular interest. The present study aimed to evaluate the load and prevalence of antibiotic-resistant commensal E. coli along the goose production cycle on 2 free-range farms in central Italy. On A farm, oxytetracycline was administered, while the B farm did not use antibiotics during the geese productive cycle. One hundred geese of 1-day-old from the same batch were divided into the two farms. At hatching, the animals showed an average of E. coli loads was 6.83 ± 0.48 log CFU/g, and 0.28 ± 0.28, 0, 5.12 ± 0.54 log CFU/g for E. coli resistant to nalidixic acid (E. colinal), to cefotaxime (E. colicef) and to tetracyclines (E. colitet), respectively. The loads of E. coli, E. colinal, E. colicef and E. colitet on 224 environmental faecal pools were determined at 8 time points. Antimicrobial susceptibility and molecular characterization of E. colicef isolates were performed. The ANOVA was used to assess the difference in bacterial loads between the two farms. We described more than 50% of resistances for tetracyclines in both farms, and sulphonamides and cephazolin in the A farm. The loads of E. coli and E. colinal in faeces were estimated at approximately 6–7 log (CFU/g) and 5–6 log (CFU/g) in the two farms, respectively. The average load of extended-spectrum beta-lactamase Escherichia coli (ESBL EC) in goose faeces varied broadly along the production cycle: in the first weeks, a sharp increase was observed in both farms, while later on A farm, the burden of ESBL EC remained steady until the end of the production cycle and on B farm the load dramatically decreased from 6 wk of age onward. An increase in the proportion of E. colinal was observed on A farm shortly after the antibiotic administration. Our study shows that the dynamics of antibiotic-resistant E. coli in farmed geese are similar to the ones observed in broilers. However, the risk of the emergence of antibiotic-resistant commensal E. coli, might be mitigated by the adoption of good management practices, including prudent use of antibiotics.

The serotypes of Pasteurella multocida were predicted based on whole genomic sequences (WGS) with specific genes of the capsular and liposaccharide (LPS) outer core polysaccharide regions as targets. A total of 56 strains were whole genomic sequenced and in addition all assembled genomes from NCBI were included for comparison. BIGSdb (Bacterial Isolate Genome Sequence Database) was installed on a Linux server and targets for capsular types A, B, D, E and F were defined as gene sequences of hyaD, bcbD, dcbF, ecbJ and fcbD, respectively and targets for LPS groups 1, 2, 3, 4, 5, 6, 7 and 8 were defined as gene sequences of pcgB, nctA, gatF, latB, rmlA, nctB, ppgB and natG, respectively. The serotypes of P. multocida were predicted from WGS by designating the capsular type and LPS group as well as subtype alleles to isolates. Comparisons between WGS predictions of capsular types and classical phenotypic typing showed correspondence in 87 % of cases whereas comparisons of WGS predictions of LPS groups to phenotypic typing corresponded for 82 % of the strains. In total 93 % and 94 % of the strains available with WGS could be capsular and LPS group typed, respectively. The server is free to access from https://ivsmlst.sund.ku.dk.

Simple Summary Clostridioides difficile is a leading cause of nosocomial and community-acquired diarrhoea in men. The infection most commonly occurs in people who have recently been treated with antibiotics. Indistinguishable C. difficile strains have been isolated from livestock and humans, which has shed light on a possible zoonotic origin of this infection. This study aimed to assess the prevalence and risk factors of C. difficile in calves bred in dairy and beef cattle farms of the Umbria, central Italy. We estimated a 19.8% prevalence of farms positive for C. difficile. The C. difficile isolates from calves were potentially toxigenic and resistant to antibiotics, including lincosamides, quinolones, vancomycin and linezolid. Isolates belonging to ribotype RT-126, which is also commonly reported in humans, showed the highest number of resistance to the antimicrobials tested. Furthermore, we observed an almost sixfold increased risk for C. difficile on farms where penicillins had been prescribed. This, together with the detection of toxigenic and antibiotic-resistant isolates, strongly suggests the need for a reduction of antibiotic use in cattle. Abstract The emergence of Clostridioides difficile as the main agent of antibiotic-associated diarrhoea has raised concerns about its potential zoonotic role in different animal species. The use of antimicrobials is a major risk factor for C. difficile infection. Here, we provide data on C. difficile infection in dairy and beef calves in Umbria, a region in central Italy. This cross-sectional study focuses on prevalence, risk factors, ribotypes, toxinotypes and antimicrobial resistance profiles of circulating ribotypes. A prevalence of 19.8% (CI95%, 12–27.6%) positive farms was estimated, and the prescription of penicillins on the farms was associated with C. difficile detection (OR = 5.58). Eleven different ribotypes were found, including the ST11 sublineages RT-126 and -078, which are also commonly reported in humans. Thirteen isolates out of 17 showed resistance to at least one of clindamycin, moxifloxacin, linezolid and vancomycin. Among them, multiple-drug resistance was observed in two isolates, belonging to RT-126. Furthermore, RT-126 isolates were positive for tetracycline resistance determinants, confirming that tetracycline resistance is widespread among ST11 isolates from cattle. The administration of penicillins increased the risk of C. difficile in calves: this, together with the recovery of multi-resistant strains, strongly suggests the need for minimising antibiotic misuse on cattle farms.

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments, however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm.

Simple Summary Salmonella is one of the most important pathogens responsible for food poisoning in humans and pork is recognized as one of the major sources for human salmonellosis. Pigs can become infected with Salmonella on the farm or in the very last phases of the production chain that include transport, lairage, and slaughter. The transport of animals from the farm to the slaughterhouse plays an important role in the transmission of this pathogen from pigs to pigs. The aim of our study was to investigate if the transport from the farm to the slaughterhouse increases the load of Salmonella in feces and to determine a modification of the fecal microbiota in pigs. Our study showed that the load of Salmonella increases after transport, confirming that this phase of the production is a critical point for the control of Salmonella contamination. The fecal microbiota composition was modified in Salmonella-positive animals after transport, in accordance with what is already published in scientific literature. In this stage, a natural Salmonella infection causes a severe modification of the fecal microbiota which is similar to the one observed in studies carried out in experimental facilities. Abstract Contaminated pork is a significant source of foodborne Salmonellosis. Pork is contaminated at the slaughterhouse and the intestinal content is the predominant source of Salmonella for carcass contamination. The prevalence of Salmonella-positive pigs increases significantly when the time of transport to the slaughterhouse is longer than two hours. The hypothesis behind this study is that transport to the slaughterhouse increases the load of Salmonella in feces and determines a shift of the fecal microbiota in finishing pigs. Fecal samples were collected in a pig herd positive for Salmonella spp., the day before the transport and at the slaughterhouse. Salmonella loads were estimated by the most probable number (MPN) technique, according to the ISO/TS 6579-2:2012/A1. Moreover, the fecal bacteria composition was assessed by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Our study showed that the load of Salmonella increases after transport, confirming that this phase of the production chain is a critical point for the control of Salmonella contamination. A lower richness and an increased beta-diversity characterized the fecal microbiota composition of Salmonella-positive animals after transport. In this stage, a natural Salmonella infection causes a disruption of the fecal microbiota as observed in challenge studies.

Ancienne clé UT "early access" : 000486708300001; International audience; Understanding gut microbiota similarities and differences across breeds in horses has the potential to advance approaches aimed at personalized microbial modifications, particularly those involved in improving sport athletic performance. Here, we explore whether faecal microbiota composition based on faecal 16S ribosomal RNA gene sequencing varies across six different sport breeds at two time points 8 months apart within a cohort of 189 healthy horses cared for under similar conditions. Lusitano horses presented the smallest and Hanoverians the greatest bacterial diversity. We found subtle but significant differences in beta-diversity between Lusitano, Anglo Arabian and the central European breeds, and we reproduced these results across the two time points. Repeat sampling of subjects showed community to be temporally more stable in Lusitano and Anglo Arabian breeds. Additionally, we found that 27 genera significantly varied in abundance across breeds. Overall, 33% of these taxa overlapped with previously identified taxa that were associated with genetic variation in humans or other species. However, a non-significant correlation was observed between microbial composition and the host pedigree-based kinship. Despite a notable variation in the diversity and composition of the faecal microbiota, breed exerted limited effects on the equine faecal microbiota.

Even though SARS-CoV-2's primary transmission pathway is person-to-person, the role played by surfaces and food contact materials in carrying viral RNA should be further explored. For this purpose, the study aimed to investigate the persistence of SARS-CoV-2 using the strain ATCC® VR-1986HK™ on flow pack polyethylene (FPP) and polystyrene food trays (PFT). Samples of FPP and PFT were contaminated with heat-inactivated SARS-CoV-2 and were incubated at a temperature of 24 ± 1 °C and at controlled relative humidity (RH 65%). The experimental design included analyses at the time 0, 3, 6, 12, 24, 36, 48 and after every 24 h until the viral RNA was no longer detectable. The results showed a significant decrease (P

In poultry production, probiotics have shown promise to limit campylobacteriosis at the farm level, the most commonly reported zoonosis in Europe. The aim of this trial was to evaluate the effects of Saccharomyces supplementation in Campylobacter jejuni challenged chickens on performance and intestinal ecosystem. A total of 156 day old male Ross 308 chicks were assigned to a basal control diet (C) or to a Saccharomyces cerevisiae boulardii CNCM I-1079 supplemented diet (S). All the birds were orally challenged with C. jejuni on day (d) 21. Live weight and growth performance were evaluated on days 1, 21, 28 and 40. The histology of intestinal mucosa was analyzed and the gut microbiota composition was assessed by 16S rRNA. Performance throughout the trial as well as villi length and crypt depth were positively influenced by yeast supplementation. A higher abundance of operational taxonomic units (OTUs) annotated as Lactobacillus reuteri and Faecalibacterium prausnitzii and a lower abundance of Campylobacter in fecal samples from S compared to the C group were reported. Supplementation with Saccharomyces cerevisiae boulardii can effectively modulate the intestinal ecosystem, leading to a higher abundance of beneficial microorganisms and modifying the intestinal mucosa architecture, with a subsequent improvement of the broilers&rsquo, growth performance.

In poultry production, probiotics have shown promise to limit campylobacteriosis at the farm level, the most commonly reported zoonosis in Europe. The aim of this trial was to evaluate the effects of Saccharomyces supplementation in Campylobacter jejuni challenged chickens on performance and intestinal ecosystem. A total of 156 day old male Ross 308 chicks were assigned to a basal control diet (C) or to a Saccharomyces cerevisiae boulardii CNCM I-1079 supplemented diet (S). All the birds were orally challenged with C. jejuni on day (d) 21. Live weight and growth performance were evaluated on days 1, 21, 28 and 40. The histology of intestinal mucosa was analyzed and the gut microbiota composition was assessed by 16S rRNA. Performance throughout the trial as well as villi length and crypt depth were positively influenced by yeast supplementation. A higher abundance of operational taxonomic units (OTUs) annotated as Lactobacillus reuteri and Faecalibacterium prausnitzii and a lower abundance of Campylobacter in fecal samples from S compared to the C group were reported. Supplementation with Saccharomyces cerevisiae boulardii can effectively modulate the intestinal ecosystem, leading to a higher abundance of beneficial microorganisms and modifying the intestinal mucosa architecture, with a subsequent improvement of the broilers’ growth performance.

Poultry production is the fastest growing meat sector worldwide. In the last five years, growing concerns have been expressed by international health agencies and consumers about the transmission of antibiotic-resistant bacteria from poultry meat to human. Consequently, poultry producers have adopted alternative production systems based on reduced antibiotic usage, including organic and antibiotic-free (AF) production. However, the effect of these production systems on the antibiotic resistance of the gut flora in slaughtered poultry has been poorly investigated. We hypothesized that organic and AF production systems reduce the risk of antibiotic resistance in the commensal Escherichia coli of broilers at slaughter compared with conventional production. Cecal content from broilers raised in conventional (292), AF (291), or organic (272) flocks (855 broilers in total) belonging to the same company was sampled. E. coli loads [colony-forming units (CFU/g)] and numbers of E. coli resistant to nalidixic acid (E. colinal) were determined for each sample. Antibiotic susceptibility of one isolate per sample was evaluated using the disc diffusion method; colistin resistance was determined by using the broth microdilution method. The differences in bacterial loads from the three production types were evaluated using one-way ANOVA. Differences in the proportion of resistant isolates in the three production lines were evaluated using Pearson's χ2 or Fisher's test. The strength of the association was evaluated by using odds ratio (OR), with the conventional production type as a reference (OR = 1). Overall, the analysis revealed a high level of resistance (50% or higher) to ampicillin, cefazolin, sulfonamides, nalidixic acid, and tetracycline, independently of the production type. High proportion of ciprofloxacin resistance (52%) was observed, with 4.5% isolates resistant to cefotaxime and 1.8% resistant to colistin. The average loads (log CFU/g cecal content) of E. colinal were determined as 6.84 for AF, 6.38 for organic type, and 7.27 for conventional type. The difference was significant (p AF > organic) was confirmed by qualitative data. However, the magnitude of the effect, measured as a reduced risk of resistance, varied broadly for the antibiotics tested. These findings suggest that poultry production systems alternative to the conventional broiler production are associated with reduced frequency of antibiotic-resistant E. coli among the commensal gut flora, posing a lower risk to the environment and the consumer.

Pasteurella multocida is a widespread pathogen associated with major animal diseases of economic significance. Despite this, little is known about the capsular types, virulence gene pattern, and antimicrobial susceptibility of isolates from hosts affected by different diseases, and no data are available in Italy. One hundred eighty six isolates of P. multocida, were taken from different species in different states of health in several Italian regions, and were tested for genes encoding for capsular types (cap) and major virulence factors (tbpA, toxA, hgbB and pfhA). Antimicrobial susceptibility was investigated with the agar diffusion test. The majority of isolates was capA+. However, the distribution differed according to species and disease of origin, with a greater heterogeneity in isolates from rabbits; capE was never found, while capB was detected once. Only capA+ and capF+ strains tested positive for pfhA. Conversely, almost all capD+ isolates were hgbB+. In bovine respiratory disease, pfhA+/tbpA+/capA+ isolates predominated, while tbpA+/toxA+/capD+ isolates predominated in sheep. Overall, low levels of resistance were found, with full susceptibility to ceftiofur and florfenicol. Lower susceptibility to older antimicrobials was recorded, since only approximately 1/3 of the isolates showed susceptibility to tylosin and erythromycin, and resistance to tetracycline (7.5%), and trimethoprim - sulphametoxazole (4.8%) was also observed.

In rabbit, P. multocida is considered a predominant pathogenic agent; despite this, few data on the molecular epidemiology are available so far. The aim of this work was to characterize P. multocida isolates from rabbit affected by various diseases in Italy. Comparison was made to reference strains from other countries. Thirty-nine isolates were tested using PCRs to detect the genes coding capsular antigens, virulence factors and lipopolysaccharide structures (LPS). Multilocus sequence typing (MLST) was performed and 19 STs registered that belonged to 9 clonal complexes. Italian isolates were all related to P. multocida subsp. P. multocida. Three sequence types dominated (ST9, ST50 and ST74). The isolates were assigned to capsular types A (20/39), D (9/39) and F (10/39), to virulence genes pfhA (13/39), hgbB (21/39) and pfhA+hgbB (4/39) (one without virulence factors) and the isolates either belonged to the LPS genotypes 3 (22/39) or 6 (17/39). The clonal relationships of the Italian strains from rabbit had similarity to previously reported rabbit isolates that belonged to ST9, ST74, ST204 and ST206, however, they differed from other rabbit references strains that belonged to six other STs. In particular, ST9 with capsular type F has been previously reported from diseased rabbit in Czech Republic and ST74 has been observed for older rabbit isolates. ST50 has probably been reported from Spain. ST9 and ST50 have previously also been reported from birds and pig, respectively, whereas ST74 has exclusively been reported from pig. It remains to be investigated if the isolates obtained from diseased rabbit in Italy represent introductions from other host or they are primarily of rabbit origin.

Contaminated pork is a significant source of foodborne Salmonella infections. Pork is contaminated at the slaughterhouse; however, the mechanisms driving Salmonella contamination of carcasses are still poorly understood. The aim of this study was to investigate whether the amount of Salmonella carried by slaughtered pigs in their guts has an influence on carcass contamination. On that account, we tested whether the number of carcasses contaminated during a slaughter day was associated with the prevalence of highly contaminated pigs (HCP: Salmonella caecal loads ≥ 3log/g), or with the prevalence of pigs that simply carry Salmonella spp. in their guts. Three hundred and six pigs were sampled in a slaughterhouse from Central Italy. Salmonella loads in the caecum and on the carcass of each pig were estimated by the most probable number (MPN) technique. The overall prevalence of Salmonella was 34.64% and 7.19% for the caeca and carcasses, respectively. S . Derby and Salmonella enterica 4,[5],12:i:– were the most frequently isolated serovars. The prevalence of HCP was 11.44%. We found a higher number of contaminated carcasses on days of high prevalence of HCP than on days of low prevalence of HCP ( p = 0.0011). Conversely, carcass contamination did not vary with the prevalence of pigs that simply carried Salmonella spp. in their guts ( p = 0.7970). Therefore, the prevalence of HCP, but not the prevalence of pigs carrying Salmonella spp., was related to carcass contamination. Taken together, these findings suggest that reduction of Salmonella loads in the guts of slaughtered pigs would result in fewer contaminated carcasses, and consequently, help to minimise the risk of human infection due to the consumption of contaminated pork.