Complete genome sequence of the linezolid-resistant clinical Enterococcus faecalis N23-3408 linked to a livestock lineage in Switzerland.

Here, we report the complete genome of human clinical linezolid-resistant Enterococcus faecalis N23-3408. N23-3408 harbored a 59.5 kb plasmid with antimicrobial resistance genes cat, erm(B), fexA, optrA, tet(L), and tet(M). Closely related E. faecalis harboring this plasmid was previously obtained from livestock animals and pet food in Switzerland.

Sucrose-fermenting Salmonella Typhimurium N23-2364: a challenge for the diagnostic laboratory.

We describe a case of Salmonella infection caused by a sucrose-fermenting Salmonella enterica Typhimurium sequence type 12 which acquired transposon CTnscr94 carrying the sucrose operon scrKYABR. Sucrose-fermenting Salmonella are particularly challenging for culture-based detection and may lead to failure to detect Salmonella in clinical samples.

Antimicrobial resistance and phylogenetic relatedness of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in peridomestic rats (Rattus norvegicus and Rattus tanezumi) linked to city areas and animal farms in Hong Kong.

Extended-spectrum ß-lactamase-producing Escherichia (E.) coli (ESBL-EC) in the clinical setting have emerged as a major threat to public and animal health. Wildlife, including Rattus spp. may serve as reservoirs and spreaders of ESBL-EC in the environment. Peridomestic rats are well adapted to living in proximity to humans and animals in a variety of urban and agricultural environments and may serve as sentinels to identify variations of ESBL-EC within their different habitats. In this study, a set of 221 rats (Rattus norvegicus, R. tanezumi, R. andamanensis, and Niviventer huang) consisting of 104 rats from city areas, 44 from chicken farms, 52 from pig farms, and 21 from stables of horse-riding schools were screened for ESBL-EC. Overall, a total of 134 ESBL-EC were isolated from the caecal samples of 130 (59%) rats. The predominant blaESBL genes were blaCTX-M-14, blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65. Phylogenetic analysis revealed a total of 62 sequence types (STs) and 17 SNP clusters. E. coli ST10 and ST155 were common to ESBL-EC from city areas and chicken farms, and ST44 were found among ESBL-EC from city areas and pig farms. Extra-intestinal pathogenic E. coli (ExPEC) ST69, ST131 and ST1193 were found exclusively among rats from city areas, and avian pathogenic E. coli (APEC) ST177 was restricted to ESBL-EC originating from chicken farms. Phylogenetic analysis showed that the populations of rodent ESBL-EC from city areas, chicken farms and pig farms were genetically different, suggesting a certain degree of partitioning between the human and animal locations. This study contributes to current understanding of ESBL-EC occurring in rats in ecologically diverse locations.

Whole genome sequence-based characterization of Campylobacter isolated from broiler carcasses over a three-year period in a big poultry slaughterhouse reveals high genetic diversity and a recurring genomic lineage of Campylobacter jejuni.

Campylobacter is among the most frequent agents of bacterial gastroenteritis in Europe and is primarily linked to the consumption of contaminated food. The aim of this study was to assess genomic diversity and to identify antimicrobial resistance and virulence genes of 155 Campylobacter isolated from broiler carcasses (neck skin samples) in a large-scale Swiss poultry abattoir over a three-year period. Samples originated from broilers from three different types of farming systems (particularly animal-friendly stabling (PAFS), free-range farms, and organic farms). Campylobacter jejuni (n = 127) and Campylobacter coli (n = 28) were analysed using a whole genome sequencing (WGS) approach (MiniSeq; Illumina). Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into complex types (CTs) using the cgMLST SeqSphere+ scheme. Antimicrobial resistance genes were identified using the Resistance Gene Identifier (RGI), and virulence genes were identified using the virulence factor database (VFDB). A high degree of genetic diversity was observed. Many sequence types (C. jejuni ST19, ST21, ST48, ST50, ST122, ST262 and C. coli ST827) occurred more than once and were distributed throughout the study period, irrespective of the year of isolation and of the broiler farming type. Antimicrobial resistance determinants included blaOXA and tet(O) genes, as well as the T86I substitution within GyrA. Virulence genes known to play a role in human Campylobacter infection were identified such as the wlaN, cstIII, neuA1, neuB1, and neuC1. Subtyping of the Campylobacter isolates identified the occurrence of a highly clonal population of C. jejuni ST21 that was isolated throughout the three-year study period from carcasses from farms with geographically different locations and different farming systems. The high rate of genetic diversity observed among broiler carcass isolates is consistent with previous studies. The identification of a persisting highly clonal C. jejuni ST21 subtype suggests that the slaughterhouse may represent an environment in which C. jejuni ST21 may survive, however, the ecological reservoir potentially maintaining this clone remains unknown.

Oxazolidinone resistance genes in florfenicol-resistant enterococci from beef cattle and veal calves at slaughter.

Background: Linezolid is a critically important oxazolidinone antibiotic used in human medicine. Although linezolid is not licensed for use in food-producing animals, the use of florfenicol in veterinary medicine co-selects for oxazolidinone resistance genes. Objective: This study aimed to assess the occurrence of cfr, optrA, and poxtA in florfenicol-resistant isolates from beef cattle and veal calves from different herds in Switzerland. Methods: A total of 618 cecal samples taken from beef cattle and veal calves at slaughter originating from 199 herds were cultured after an enrichment step on a selective medium containing 10 mg/L florfenicol. Isolates were screened by PCR for cfr, optrA, and poxtA which are genes known to confer resistance to oxazolidinones and phenicols. One isolate per PCR-positive species and herd was selected for antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS). Results: Overall, 105 florfenicol-resistant isolates were obtained from 99 (16%) of the samples, corresponding to 4% of the beef cattle herds and 24% of the veal calf herds. Screening by PCR revealed the presence of optrA in 95 (90%) and poxtA in 22 (21%) of the isolates. None of the isolates contained cfr. Isolates included for AST and WGS analysis were Enterococcus (E.) faecalis (n = 14), E. faecium (n = 12), E. dispar (n = 1), E. durans (n = 2), E. gallinarum (n = 1), Vagococcus (V.) lutrae (n = 2), Aerococcus (A.) urinaeequi (n = 1), and Companilactobacillus (C.) farciminis (n = 1). Thirteen isolates exhibited phenotypic linezolid resistance. Three novel OptrA variants were identified. Multilocus sequence typing identified four E. faecium ST18 belonging to hospital-associated clade A1. There was a difference in the replicon profile among optrA- and poxtA-harboring plasmids, with rep9 (RepA_N) plasmids dominating in optrA-harboring E. faecalis and rep2 (Inc18) and rep29 (Rep_3) plasmids in poxtA-carrying E. faecium. Conclusion: Beef cattle and veal calves are reservoirs for enterococci with acquired linezolid resistance genes optrA and poxtA. The presence of E. faecium ST18 highlights the zoonotic potential of some bovine isolates. The dispersal of clinically relevant oxazolidinone resistance genes throughout a wide variety of species including Enterococcus spp., V. lutrae, A. urinaeequi, and the probiotic C. farciminis in food-producing animals is a public health concern.

Serotypes, Antimicrobial Resistance Profiles, and Virulence Factors of Salmonella Isolates in Chinese Edible Frogs (Hoplobatrachus rugulosus) Collected from Wet Markets in Hong Kong.

Salmonella is an important agent of gastrointestinal disease in humans. While livestock, such as cattle, poultry, and pigs, are well-recognised animal reservoirs of Salmonella, there is a lack of data on Salmonella in edible frogs, even though frog meat is a popular food worldwide. In this study, 103 live edible Chinese frogs (Hoplobatrachus rugulosus) were collected from wet markets throughout Hong Kong. After euthanasia, faeces or cloacal swabs were examined for Salmonella. Overall, Salmonella spp. were isolated from 67 (65%, CI: 0.554-0.736) of the samples. The serotypes included S. Saintpaul (33%), S. Newport (24%), S. Bareilly (7%), S. Braenderup (4%), S. Hvittingfoss (4%), S. Stanley (10%), and S. Wandsworth (16%). Many isolates were phylogenetically related. A high number of genes encoding for resistance to clinically relevant antimicrobials, and a high number of virulence determinants, were identified. Antimicrobial susceptibility testing (AST) identified multidrug resistance (MDR) in 21% of the isolates. Resistance to ampicillin, ciprofloxacin, nalidixic acid, and tetracycline was common. These results demonstrate that a high percentage of live frogs sold for human consumption in wet markets are carriers of multidrug-resistant Salmonella. Public health recommendations for handling edible frogs should be considered, to mitigate the risk of Salmonella transmission to humans.

Emergence of blaSHV-12 and qnrS1 encoded on IncX3 plasmids: Changing epidemiology of extended-spectrum ß-lactamases among Enterobacterales isolated from broilers.

OBJECTIVES: The occurrence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in broilers represents a risk to public health because of the possibility of transmission of ESBL producers and/or blaESBL genes via the food chain or within settings where human-animal interfaces exist. METHODS: This study assessed the occurrence of ESBL producers among faecal samples of broilers at slaughter. Isolates were characterised by multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing. RESULTS: The flock prevalence, determined by sampling crates of 100 poultry flocks, was 21%. The predominant blaESBL gene was blaSHV-12, identified in 92% of the isolates. A variety of Escherichia coli and Klebsiella pneumoniae sequence types (STs) were identified, including extraintestinal pathogenic E. coli ST38, avian pathogenic E. coli ST10, ST93, ST117, and ST155, and nosocomial outbreak clone K. pneumoniae ST20. Whole-genome sequencing was used to characterise a subset of 15 isolates, including 6 E. coli, 4 K. pneumoniae, 1 Klebsiella grimontii, 1 Klebsiella michiganensis, 1 Klebsiella variicola, and 1 Atlantibacter subterranea. Fourteen isolates carried identical or closely related 46338-54929 bp IncX3 plasmids encoding blaSHV-12 and qnrS1. One E. coli isolate carried a 46338 bp IncX3 plasmid, which was integrated chromosomally into ydbD. CONCLUSIONS: The blaSHV-12 gene has replaced the previously predominant blaCTX-M-1 in ESBL-producing Enterobacterales from broilers in Switzerland. Broilers may play a role in the dissemination of blaSHV-12 and qnrS1 associated with epidemic IncX3 plasmids, representing a risk to human and animal health.

Whole genome sequence-based characterisation of Shiga toxin-producing Escherichia coli isolated from game meat originating from several European countries.

Game meat is becoming increasingly popular but may be contaminated with pathogenic bacteria such as Shiga toxin-producing Escherichia coli (STEC). STEC cause gastrointestinal illnesses including diarrhoea, haemorrhagic colitis (HC), and the haemolytic uremic syndrome (HUS). The aim of this study was to assess the occurrence of STEC in 92 meat samples from chamois (n = 2), red deer (n = 27), roe deer (n = 38), and wild boar (n = 25), from Switzerland and other European countries. After enrichment, Shiga-toxin encoding genes (stx) were detected by PCR in 78 (84%) of the samples and STEC were isolated from 23 (25%) of the same samples. Nine different serotypes and eight different sequence types (STs) were found, with O146:H28 ST738 (n = 10) and O110:H31 ST812 (n = 5) predominating. None of the STEC belonged to the so-called top-five serogroups O26, O103, O111, O145, and O157. Subtyping of stx identified stx1c (n = 9), stx2a (n = 1), stx2b (n = 19), stx2e (n = 2), and stx2g (n = 1). Additional virulence factors (VFs) comprised ehx (n = 12), iha (n = 21), sta1 (n = 1), and subAB (n = 19). None of the isolates contained the eae gene. Twenty-one STEC contained VFs associated with extra-intestinal pathogenic E. coli (ExPEC). Overall, the pathogenic potential of STEC in game meat is moderate, though the isolation of one STEC strain carrying stx2a, and of STEC/ExPEC hybrids suggests a role of game meat as a potential source of STEC infections in humans. Therefore, detailed knowledge of the safe handling and preparation of game meat is needed to prevent foodborne infections.

High occurrence of Enterococcus faecalis, Enterococcus faecium, and Vagococcus lutrae harbouring oxazolidinone resistance genes in raw meat-based diets for companion animals - a public health issue, Switzerland, September 2018 to May 2020.

IntroductionEnterococci harbouring genes encoding resistance to florfenicol and the oxazolidinone antimicrobial linezolid have emerged among food-producing animals and meat thereof, but few studies have analysed their occurrence in raw meat-based diets (RMBDs) for pets.AimWe aimed to examine how far RMBDs may represent a source of bacteria with oxazolidinone resistance genes.MethodsFifty-nine samples of different types of RMBDs from 10 suppliers (three based in Germany, seven in Switzerland) were screened for florfenicol-resistant Gram-positive bacteria using a selective culture medium. Isolates were phenotypically and genotypically characterised.ResultsA total of 27 Enterococcus faecalis, Enterococcus faecium, and Vagococcus lutrae isolates were obtained from 24 of the 59 samples. The optrA, poxtA, and cfr genes were identified in 24/27, 6/27 and 5/27 isolates, respectively. Chloramphenicol and linezolid minimum inhibitory concentrations (MICs) ranged from 24.0 mg/L-256.0 mg/L, and 1.5 mg/L-8.0 mg/L, respectively. According to the Clinical and Laboratory Standards Institute (CLSI) breakpoints, 26 of 27 isolates were resistant to chloramphenicol (MICs ≥ 32 mg/L), and two were resistant to linezolid (MICs ≥ 8 mg/L). Multilocus sequence typing analysis of the 17 E. faecalis isolates identified 10 different sequence types (ST)s, with ST593 (n = 4 isolates) and ST207 (n = 2 isolates) occurring more than once, and two novel STs (n = 2 isolates). E. faecium isolates belonged to four different STs (168, 264, 822, and 1846).ConclusionThe high occurrence in our sample of Gram-positive bacteria harbouring genes encoding resistance to the critical antimicrobial linezolid is of concern since such bacteria may spread from companion animals to humans upon close contact between pets and their owners.

Epidemiological links and antimicrobial resistance of clinical Salmonella enterica ST198 isolates: a nationwide microbial population genomic study in Switzerland.

Salmonella is a leading cause of foodborne outbreaks and systemic infections worldwide. Emerging multi-drug resistant Salmonella lineages such as a ciprofloxacin-resistant subclade (CIPR) within Salmonella enterica serovar Kentucky ST198 threaten the effective prevention and treatment of infections. To understand the genomic diversity and antimicrobial resistance gene content associated with S. Kentucky in Switzerland, we whole-genome sequenced 70 human clinical isolates obtained between 2010 and 2020. Most isolates belonged to ST198-CIPR. High- and low-level ciprofloxacin resistance among CIPR isolates was associated with variable mutations in ramR and acrB in combination with stable mutations in quinolone-resistance determining regions (QRDRs). Analysis of isolates from patients with prolonged ST198 colonization indicated subclonal adaptions with the ramR locus as a mutational hotspot. SNP analyses identified multiple clusters of near-identical isolates, which were often associated with travel but included spatiotemporally linked isolates from Switzerland. The largest SNP cluster was associated with travellers returning from Indonesia, and investigation of global data linked >60 additional ST198 salmonellosis isolates to this cluster. Our results emphasize the urgent need for implementing whole-genome sequencing as a routine tool for Salmonella surveillance and outbreak detection.

Faecal carriage of enterococci harbouring oxazolidinone resistance genes among healthy humans in the community in Switzerland.

OBJECTIVES: This study aimed to investigate the faecal carriage of enterococci harbouring oxazolidinone resistance genes among healthy humans in Switzerland and to genetically characterize the isolates. METHODS: A total of 399 stool samples from healthy individuals employed in different food-processing plants were cultured on a selective medium containing 10 mg/L florfenicol. Resulting enterococci were screened by PCR for the presence of cfr, optrA and poxtA. A hybrid approach combining short-read and long-read WGS was used to analyse the genetic context of the cfr, optrA and poxtA genes. RESULTS: Enterococcus faecalis (n = 6), Enterococcus faecium (n = 6), Enterococcus gallinarum (n = 1) and Enterococcus hirae (n = 2) were detected in 15/399 (3.8%) of the faecal samples. They carried cfr + poxtA, optrA, optrA + poxtA or poxtA. Four E. faecalis harbouring optrA and one E. faecium carrying poxtA were resistant to linezolid (8 mg/L). In most optrA-positive isolates, the genetic environments of optrA were highly variable, but often resembled previously described platforms. In most poxtA-positive isolates, the poxtA gene was flanked on both sides by IS1216E elements and located on medium-sized plasmids. CONCLUSIONS: Faecal carriage of Enterococcus spp. harbouring cfr, optrA and poxtA in healthy humans associated with the food-production industry demonstrates the possibility of spread of oxazolidinone resistance genes into the community. Given the importance of linezolid as a last-resort antibiotic for the treatment of serious infections caused by Gram-positive pathogens, the detection of the oxazolidinone resistance determinants in enterococci from healthy humans is of concern for public health.

Antimicrobial resistance profiles of Escherichia coli and prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae in calves from organic and conventional dairy farms in Switzerland.

This study compared the antimicrobial resistance (AMR) among commensal Escherichia coli in the fecal microbiota of young calves raised on organic and on conventional dairy farms in Switzerland. Further, fecal carriage of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae was assessed for calves from both farming systems. Where possible, data on antimicrobial usage (AMU) were obtained. Antimicrobial susceptibility testing was performed on a total of 71 isolates using the disk diffusion method. ESBL producers were characterized by polymerase chain reaction-based multilocus sequence typing and sequencing of the blaESBL genes. Organically raised calves were significantly more likely to harbor E. coli that showed AMR to ampicillin (odds ratio [OR]: 2.78, 95% confidence interval [CI]: 1.02-7.61, p = 0.046), streptomycin (OR: 3.22, 95% CI: 1.17-8.92, p = 0.046), kanamycin (OR: 11.3, 95% CI: 2.94-43.50, p < 0.001), and tetracycline (OR: 3.25, 95% CI: 1.13-9.31, p = 0.028). Calves with reported AMU were significantly more likely to harbor E. coli with resistance to ampicillin (OR: 3.91, 95% CI: 1.03-14.85, p = 0.045), streptomycin (OR: 4.35, 95% CI: 1.13-16.7, p = 0.045), and kanamycin (OR: 8.69, 95% CI: 2.01-37.7, p = 0.004). ESBL-producing Enterobacteriaceae (18 E. coli and 3 Citrobacter braakii) were detected exclusively among samples from conventionally farmed calves (OR: infinity [∞], 95% CI: 2.3-∞, p < 0.0013). The observations from this study suggest that AMR is highly prevalent among commensal E. coli in young dairy calves, irrespective of the farm management system, with proportions of certain resistance phenotypes higher among organic calves. By contrast, the occurrence of ESBL producers among young dairy calves may be linked to factors associated with conventional farming.

Fattening Pigs Are a Reservoir of Florfenicol-Resistant Enterococci Harboring Oxazolidinone Resistance Genes.

ABSTRACT: The use of florfenicol in farm animals may select enterococci that carry resistance genes that confer resistance to linezolid, a critically important oxazolidinone antibiotic used in human medicine. This cross-sectional study aimed to assess the occurrence of oxazolidinone resistance genes in florfenicol-resistant enterococci from fattening pigs in Switzerland and to characterize a subset of the isolates using whole genome sequencing. A total of 31 florfenicol-resistant enterococcal isolates were obtained from 27 (5%) of 565 cecal samples of fattening pigs from seven (11%) of 62 farms. Screening by PCR revealed the presence of cfr-poxtA in 1 of 31, optrA in 15 of 31, and poxtA in 15 of 31 enterococcal isolates. One randomly selected isolate per PCR-positive Enterococcus species and positive farm was selected for further analysis (n = 10). In nine of the 10 isolates, the presence of oxazolidinone resistance genes did not result in phenotypic resistance. Whole genome sequencing analysis showed the presence of E. faecalis (n = 1), E. faecium (n = 1), and E. hirae (n = 1), harboring optrA18, optrA7, and a new optrA allele, respectively. E. durans (n = 1), E. faecium (n = 4), and E. hirae (n = 1) carried the wild-type poxtA, and E. faecalis (n = 1) coharbored cfr(D) and poxtA2. Except for optrA7, all oxazolidinone resistance genes were found on plasmids. Multilocus sequence typing analysis identified E. faecalis ST19 and ST376, E. faecium ST80 belonging to hospital-adapted clade A1, and E. faecium ST21, ST55, ST269, and ST416 belonging to clade A2, which represents human commensals and animal strains. The occurrence of cfr(D), optrA, and poxtA in various porcine Enterococcus spp. demonstrates the spread of oxazolidinone resistance genes among enterococci from fattening pigs in Switzerland. The presence in one sample of poxtA-carrying E. faecium ST80 emphasizes the potential risk to human health through dissemination of strains carrying oxazolidinone resistance genes into the food chain.

Finding of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in wild game meat originating from several European countries: predominance of Moellerella wisconsensis producing CTX-M-1, November 2021.

IntroductionMeat can be a vehicle for food-borne transmission of antimicrobial resistant bacteria and antimicrobial resistance genes. The occurrence of extended-spectrum beta-lactamase (ESBL) producing Enterobacterales has been observed in meat from livestock production but has not been well studied in meat from wild game.AimWe aimed to investigate, particularly in central Europe, to what extent ESBL-producing Enterobacterales may be present in wild game meat.MethodsA total of 111 samples of different types of game meat supplied by butchers, hunters, retail stores and a large game-processing establishment in Europe were screened for ESBL-producing Enterobacterales using a selective culture medium. Isolates were genotypically and phenotypically characterised.ResultsThirty-nine samples (35% of the total) yielded ESBL-producing Enterobacterales, with most (35/39) supplied by the game-processing establishment. Isolates included 32 Moellerella wisconsensis, 18 Escherichia coli and one Escherichia marmotae. PCR screening identified bla CTX-M-1 (n = 31), bla CTX-M-32 (n = 8), bla CTX-M-65 (n = 4), bla CTX-M-15 (n = 3), bla CTX-M-8 (n = 1), bla CTX-M-14 (n = 1), bla CTX-M-55 (n = 1), and bla SHV-12 (n = 2). Most E. coli belonged to phylogenetic group A (n = 7) or B1 (n = 9), but several isolates belonged to extraintestinal pathogenic E. coli (ExPEC) sequence types (ST)58 (n = 4), ST68 (n = 1) and ST540 (n = 1). Whole genome sequencing of six selected isolates localised bla CTX-M-1 on megaplasmids in four M. wisconsensis and bla CTX-M-32 on IncN_1 plasmids in one M. wisconsensis and one E. marmotae. Forty-eight isolates (94%) exhibited a multidrug-resistance phenotype.ConclusionWe found a high occurrence of ESBL-producing Enterobacterales in wild game meat, suggesting wildlife habitat pollution and possible microbial contamination events occurring during skinning or cutting carcasses.

Distribution of virulence factors, antimicrobial resistance genes and phylogenetic relatedness among Shiga toxin-producing Escherichia coli serogroup O91 from human infections.

Shiga toxin-producing Escherichia coli (STEC) belonging to the serogroup O91 are among the most common non-O157 STEC serogroups associated with human illness in Europe. This study aimed to analyse the virulence factors, antimicrobial resistance genes and phylogenetic relatedness among 48 clinical STEC O91 isolates collected during 2003-2019 in Switzerland. The isolates were subjected to whole genome sequencing using short-read sequencing technologies and a subset of isolates additionally to long-read sequencing. They belonged to O91:H10 (n=6), O91:H14 (n=40), and O91:H21 (n=2). Multilocus sequence typing showed that the O91:H10 isolates all belonged to sequence type (ST)641, while the O91:H14 isolates were assigned to ST33, ST9700, or were non-typeable. Both O91:H21 isolates belonged to ST442. Shiga toxin gene stx1a was the most common Shiga toxin gene subtype among the isolates, followed by stx2b, stx2d and stx2a. All isolates were LEE-negative and carried one or two copies of the IrgA adhesin gene iha. In a subset of long-read sequenced isolates, modules of the Locus of Adhesion and Autoaggregation pathogenicity island (LAA-PAI) carrying iha and other genes such as hes, lesP or agn43 were identified. A large proportion of STEC O91:H14 carried the subtilase cytotoxin gene subA, colicin genes (cba, cea, cib and cma) or microcin genes (mcmA, mchB, mchC and mchF). STEC O91:H14 were further distinguished from STEC O91:H10/H21 by one or more virulence factors found in extraintestinal pathogenic E. coli (ExPEC), including hlyF, iucC/iutA, kpsE and traT. The hlyF gene was identified on a novel mosaic plasmid that was unrelated to hlyF+ plasmids described previously in STEC. Core genome phylogenetic analysis revealed that STEC O91:H10 and STEC O91:H21 were clonally conserved, whereas STEC O91:H14 were clonally diverse. Among three STEC O91:H14 isolates, a number of resistance genes were identified, including genes that mediate resistance to aminoglycosides (aadA, aadA2, aadA9, aadA23, aph(3'')-Ib and aph(6)-Id), chloramphenicol (cmlA), sulphonamides (sul2 and sul3), and trimethoprim (drfA12). Our data contribute to understanding the genetic diversity and differing levels of virulence potential within the STEC O91 serogroup.

Colistin resistance in Gram-negative bacteria analysed by five phenotypic assays and inference of the underlying genomic mechanisms.

BACKGROUND: Colistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation. RESULTS: Of 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L. CONCLUSIONS: The Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance.

Seroprevalence of Toxoplasma gondii and Salmonella in Hunted Wild Boars from Two Different Regions in Switzerland.

Toxoplasma gondii and Salmonella are zoonotic foodborne pathogens that may be transmitted to humans through the consumption of raw or undercooked meat, including game. The aim of this study was to determine the seroprevalence of T. gondii and Salmonella antibodies in wild boars in two different regions in Switzerland. During the hunting season of 2020, a total of 126 diaphragm muscle samples of hunted wild boars were collected and the meat juice of these samples was analysed for pathogen-specific IgG antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits. The overall seroprevalences were 35% for T. gondii and 17% for Salmonella, respectively. In general, seropositivity increased with the age of the animals. Seroprevalences of T. gondii were similar for animals from the northern region (29%) to those from the southern region (36.8%), indicating that T. gondii is widespread in the sylvestrian environment. By contrast, Salmonella seropositivity was remarkably higher in wild boars from the north (52%) compared with those from the south (5.3%). The high occurrence of Salmonella may represent a risk of transmission to compatriot domestic animals such free-range farmed pigs as well as to humans. Further, meat of hunted wild boars may present a source of human toxoplasmosis or salmonellosis.

Spread of vancomycin-resistant Enterococcus faecium ST133 in the aquatic environment in Switzerland.

OBJECTIVES: The global dissemination of vancomycin-resistant enterococci (VRE) has become a serious public-health concern. Although outbreaks are typically caused by nosocomial transmission, contaminated food and water may contribute to the spread of VRE. The aim of this study was to assess the presence of VRE in flowing surface water bodies in Switzerland and to characterise the isolates. METHODS: Surface water was sampled from rivers, streams and canals throughout Switzerland and was screened for the presence of VRE. Whole-genome sequencing was used to identify antimicrobial resistance genes and the phylogenetic similarity of the obtained isolates. RESULTS: VRE were detected in 6 (3.1%) of 191 water samples. The six VRE-containing samples were all collected near treated wastewater discharge sites. The six isolates were identified as Enterococcus faecium sequence type 133 (ST133) and harboured the vancomycin resistance-conferring vanA gene cluster on transposon Tn1546. They showed a close phylogenetic relationship to ST133 swine faecal isolates obtained during a previously reported screening in Switzerland. CONCLUSION: Our results suggest that surface water contributes to the environmental dissemination of VRE. Repeated identification of ST133 clones in geographically distinct water sampling sites and swine faecal samples collected in slaughterhouses may indicate a local dominance of this VRE lineage in Switzerland.

High Occurrence of Shiga Toxin-Producing Escherichia coli in Raw Meat-Based Diets for Companion Animals-A Public Health Issue.

Feeding pets raw meat-based diets (RMBDs) is becoming increasingly popular but comes with a risk of pathogenic bacteria, including Shiga toxin-producing Escherichia coli (STEC). In humans, STEC may cause gastrointestinal illnesses, including diarrhea, hemorrhagic colitis (HC), and the hemolytic uremic syndrome (HUS). The aim of this study was to evaluate commercially available RMBDs with regard to the occurrence of STEC. Of 59 RMBD samples, 59% tested positive by real-time PCR for the presence of Shiga toxin genes stx1 and/or stx2. STECs were recovered from 41% of the 59 samples, and strains were subjected to serotyping and virulence gene profiling, using whole genome sequencing (WGS)-based methods. Of 28 strains, 29% carried stx2a or stx2d, which are linked to STEC with high pathogenic potential. Twenty different serotypes were identified, including STEC O26:H11, O91:H10, O91:H14, O145:H28, O146:H21, and O146:H28, which are within the most common non-O157 serogroups associated with human STEC-related illnesses worldwide. Considering the low infectious dose and potential severity of disease manifestations, the high occurrence of STEC in RMBDs poses an important health risk for persons handling raw pet food and persons with close contact to pets fed on RMBDs, and is of concern in the field of public health.

Genetic Context of optrA and poxtA in Florfenicol-Resistant Enterococci Isolated from Flowing Surface Water in Switzerland.

Linezolid is an important last-resort antibiotic for the treatment of multidrug-resistant enterococci. The aim of this study was to further characterize the genetic context of optrA and poxtA in 10 florfenicol-resistant enterococci isolated from flowing surface water. In most genomes, optrA and poxtA were embedded in transposition units integrated into plasmids or into the chromosomal radC. For the first time, a chromosomally integrated optrA in an Enterococcus raffinosus isolate is described.