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Fresh produce is usually consumed raw or minimally processed, making it a potential vehicle for the transmission of antimicrobial-resistant (AMR) microorganisms to humans. The objective of the study was to assess the occurrence of extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Enterobacterales (ESBL-E and CPE), respectively, in 118 fresh herbs and 101 bagged salads collected at retail level in Switzerland and to characterize the isolates’ phenotypic and genotypic properties using culture-based methods and whole genome sequencing (WGS).Of the fresh herbs, 6/118 contained ESBL-E and 7/118 yielded CPE. Of the salads, 13/101 contained ESBL-E and 1/101 CPE. Antimicrobial susceptibility testing (AST) identified 9/29 isolates as multidrug-resistant (MDR).ESBL-E were Escherichia coli (n = 6), Klebsiella pneumoniae (n = 4) Enterobacter chuandaensis (n = 1), and Kluyvera spp. (n = 1) carrying ß-lactamase (bla) genes belonging to the cefotaximase-München (blaCTX-M)-groups, Proteus spp. (n = 1) containing Hôpital-Universitaire-de-Genève-bla (blahugA), Raoultella ornithinolytica (n = 1) carrying sulfhydryl reagent variable bla (blaSHV), and Serratia fonticola (n = 7) carrying S. fonticula bla (blaFONA) genes. CPE were Enterobacter asburiae (n = 1) E. cloacae (n = 6) and E. vonholyi (n = 1) carrying imipenemase bla (blaIMI) genes.Several K. pneumoniae sequence types (STs) were identified (ST967, ST628, ST219, and ST1823), which have been linked to human disease and nosocomial outbreaks. They carried blaCTX-M-15 on plasmids detected globally in environmental and clinical samples. E. coli (ST10, ST48, ST609, ST2040, ST6215 and ST3580) and enterotoxigenic E. coli (ETEC) ST2040 carrying blaCTX-M-15 were found. E. cloacae (ST820 and ST1516) with blaIMI-1 have been found previously in clinical settings and community outbreaks.The occurrence and consumption of fresh produce containing MDR ESBL-E and CPE pose substantial public health risks and raise significant food safety concerns.

STUDY AIMS: Although non-toxigenic Vibrio cholerae lack the ctxAB genes encoding cholera toxin, they can cause diarrhoeal disease and outbreaks in humans. In Switzerland, V. cholerae is a notifiable pathogen and all clinical isolates are analysed at the National Reference Laboratory for Enteropathogenic Bacteria and Listeria. Up to 20 infections are reported annually. In this study, we investigated the population structure and genetic characteristics of non-toxigenic V. cholerae isolates collected over five years. METHODS: V. cholerae isolates were serotyped and non-toxigenic isolates identified using a ctxA-specific PCR. Following Illumina whole-genome sequencing, genome assemblies were screened for virulence and antibiotic resistance genes. Phylogenetic analyses were performed in the context of 965 publicly available V. cholerae genomes. RESULTS: Out of 33 V. cholerae infections reported between January 2017 and January 2022 in Switzerland, 31 were caused by ctxA-negative isolates. These non-toxigenic isolates originated from gastrointestinal (n = 29) or extraintestinal (n = 2) sites. They were phylogenetically diverse and belonged to 29 distinct sequence types. Two isolates were allocated to the lineage L3b, a ctxAB-negative but tcpA-positive clade previously associated with regional outbreaks. The remaining 29 isolates were placed in lineage L4, which is associated with environmental strains. Genes or mutations associated with reduced susceptibility to the first-line antibiotics fluoroquinolones and tetracyclines were identified in 11 and 3 isolates, respectively. One isolate was predicted to be multidrug resistant. CONCLUSIONS: V. cholerae infections in Switzerland are rare and predominantly caused by lowly virulent ctxAB-negative and tcpA-negative strains. As V. cholerae is not endemic in Switzerland, cases are assumed to be acquired predominantly during travel. This assumption was supported by the phylogenetic diversity of the analysed isolates.

ABSTRACT: 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.

ABSTRACT Chlamydia, comprising several human and zoonotic pathogens, is a genus of the conserved bacterial phylum Chlamydiota. Their obligate intracellular niche serves as a barrier for natural genetic exchange via horizontal gene transfer (HGT), and further limits the development and application of genetic tools. To date, the only example for recent inter-phylum HGT among the Chlamydiota is tetracycline resistance in the potentially zoonotic species Chlamydia suis, a close phylogenetic relative of human C. trachomatis, which causes bacterial sexually transmitted infections and ocular trachoma. Tetracycline resistance in porcine C. suis strains has been described worldwide and is always part of a genomic island dividing invasin (inv), located within a chromosomal region between the rRNA operon (rrn) and the nqrF reductase. Here, we aimed to expand the still modest number of available genetic manipulation systems for Chlamydia by generating allele-replacement and integration vectors for C. suis. These vectors comprised homologous C. suis sequences of the chromosomal region of interest, an E. coli origin of replication (ori) and selection markers but lacked the native chlamydial plasmids or its ori. We first recovered allele-replacement mutants using a vector that targets the tryptophan (trp) operon of C. suis. The vector was further successfully maintained as a free plasmid in C. trachomatis without allele replacement, suggesting complex plasmid dynamics in the absence of a chlamydial ori. Moreover, we showed that the hypervariable rrn-nqrF intergenic region of C. suis is highly susceptible to transformation, resulting in complete vector integration upstream of nqrF without interruption of the targeted inv gene. IMPORTANCE The obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis, the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis, the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration.

Genomic analyses indicate that the ST131 lineage of E. coli, responsible for urinary and bloodstream infections globally, is evolving towards both increased virulence and increased resistance to antimicrobials.

BackgroundLinezolid 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.ObjectiveThis 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.MethodsA 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).ResultsOverall, 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.ConclusionBeef 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.

Paratuberculosis or Johne’s disease is a chronic intestinal disease in domestic and wild ruminants. It affects global dairy economy and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The objective of this study was to analyze strain diversity in MAP-positive fecal samples by using a particular single nucleotide polymorphism (SNP) distinguishing between cattle (C-) and sheep (S-) type MAP and analysis of SNPs within gyrA and gyrB genes differentiating between Types I, II, and III. Moreover, mycobacterial interspersed repetitive unit and variable-number tandem repeat (MIRU-VNTR) analysis using eight established loci was performed. A total of 90 fecal samples from diseased animals presenting diarrhea and/or weight loss, originating from 59 bovine herds across 16 cantons of Switzerland were screened by PCR for the MAP-specific F57 and IS900 genes and were further subtyped. 96.7% and 3.3% of the samples contained C- and S-type MAP, respectively. Ten INRA Nouzilly MIRU-VNTR (INMV) profiles, with a discriminatory index of 0.802, calculated based on 65 epidemiological independent genotypes, were detected: INMV 1 (33.8%), INMV 2 (23.1%), INMV 6 (16.9%), INMV 9 (9.2%), INMV 116 (4.6%), INMV 3 (3.1%), INMV 5 (3.1%) and INMV 72 (1.5%), including two novel INMV profiles, namely INMV 253 (3.1%; S-type III) and INMV 252 (1.5%; C-type). INMV 1, INMV 2, and INMV 6 comprised almost 75% of the F57- and IS900-positive samples. Typing data from 11 herds suggest that there are some herds with intra-herd diversity of genotypes. The results of this study indicate a heterogeneity of MAP in Switzerland.

ABSTRACT: 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.

ABSTRACT Salmonella enterica serovar Typhimurium causes a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. No licensed vaccine is available, but O-antigen-based candidates are in development, as the O-antigen moiety of lipopolysaccharides is the principal target of protective immunity. The vaccines under development are designed based on isolates with O-antigen O-acetylated at position C-2 of abequose, giving the O:5 antigen. Serotyping data on recent Salmonella Typhimurium clinical isolates from the Democratic Republic of the Congo (DRC), however, indicate increasing levels of isolates without O:5. The importance and distribution of this loss of O:5 antigen in the population as well as the genetic mechanism responsible for the loss and chemical characteristics of the O-antigen are poorly understood. In this study, we Illumina whole-genome sequenced 354 Salmonella Typhimurium isolates from the DRC, which were isolated between 2002 and 2017. We used genomics and phylogenetics combined with chemical approaches (1H nuclear magnetic resonance [NMR], high-performance anion-exchange chromatography with pulsed amperometric detection [HPAEC-PAD], high-performance liquid chromatography–PAD [HPLC-PAD], and HPLC-size exclusion chromatography [HPLC-SEC]) to characterize the O-antigen features within the bacterial population. We observed convergent evolution toward the loss of the O:5 epitope predominantly caused by recombination events in a single gene, the O-acetyltransferase gene oafA. In addition, we observe further O-antigen variations, including O-acetylation of the rhamnose residue, different levels of glucosylation, and the absence of O-antigen repeating units. Large recombination events underlying O-antigen variation were resolved using long-read MinION sequencing. Our study suggests evolutionary pressure toward O-antigen variants in a region where invasive disease by Salmonella Typhimurium is highly endemic. This needs to be taken into account when developing O-antigen-based vaccines, as it might impact the breadth of coverage in such regions. IMPORTANCE The bacterium Salmonella Typhimurium forms a devastating burden in sub-Saharan Africa by causing invasive bloodstream infections. Additionally, Salmonella Typhimurium presents high levels of antimicrobial resistance, jeopardizing treatment. No licensed vaccine is available, but candidates are in development, with lipopolysaccharides being the principal target of protective immunity. The vaccines under development are designed based on the O:5 antigen variant of bacterial lipopolysaccharides. Data on recent Salmonella Typhimurium clinical isolates from the Democratic Republic of the Congo (DRC), however, indicate increasing levels of isolates without this O:5 antigen. We studied this loss of O:5 antigen in the population at the genetic and chemical levels. We genome sequenced 354 isolates from the DRC and used advanced bioinformatics and chemical methods to characterize the lipopolysaccharide features within the bacterial population. Our results suggest evolutionary pressure toward O-antigen variants. This needs to be taken into account when developing vaccines, as it might impact vaccine coverage.

Escherichia coli is a major cause of urinary tract infection. Here, Biggel et al. provide a phylogenomic analysis of 907 clinical E. coli isolates and identify the P-fimbriae-encoding locus associated with invasive uropathogenic E. coli isolates.

Bacillus thuringiensis is a microbial insecticide widely used to control agricultural pests. Although generally regarded as safe, B. thuringiensis is phylogenetically intermingled with the foodborne pathogen B. cereus sensu stricto and has been linked to foodborne outbreaks. Limited data on the pathogenicity potential of B. thuringiensis and the occurrence of biopesticide residues in food compromise a robust consumer risk assessment. In this study, we analyzed whole-genome sequences of 33 B. thuringiensis isolates from biopesticides, food, and human fecal samples linked to outbreaks. All food and outbreak-associated isolates genomically matched (≤ 6 wgSNPs; ≤ 2 cgSNPs) with one of six biopesticide strains, suggesting biopesticide products as their source. Long-read sequencing revealed a more diverse virulence gene profile than previously assumed, including a transposase-mediated disruption of the promoter region of the non-hemolytic enterotoxin gene nhe and a bacteriophage-mediated disruption of the sphingomyelinase gene sph in some biopesticide strains. Furthermore, we provide high-quality genome assemblies of seven widely used B. thuringiensis biopesticide strains, which will facilitate improved microbial source tracking and risk assessment of B. thuringiensis-based biopesticides in the future.

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.

Escherichia albertii, a zoonotic pathogen, has sporadically been associated with infectious diarrhea in humans. Poultry and wild birds are considered potential reservoirs. We assessed the occurrence of E. albertii in 280 fecal samples from wild birds (n = 130) and pooled fecal samples collected at slaughterhouse level from poultry flocks (n = 150) in Switzerland. Using an E. albertii-specific PCR targeting the Eacdt gene, 23.8% (31/130) of the samples from wild birds, but not from the pooled poultry fecal samples, tested positive for Eacdt. The positive samples originated from 11 bird species belonging to eight families. Strain isolation was attempted on the PCR-positive samples by subculturing the broth cultures onto xylose–MacConkey plates. Isolation was possible on 12 of the 31 Eacdt-PCR-positive samples. Whole-genome sequencing revealed that the strains belonged to nine distinct sequence types, with ST13420 and ST5967 being represented by two and three isolates, respectively. All strains harbored the eae gene, while two strains were also positive for stx2f. Our study thus shows that E. albertii is present in the Swiss wild bird population, which can potentially act as a source of this pathogen to humans, other animals, and the environment.

Abstract Background The diagnosis of urinary tract infections (UTIs) in institutionalized older adults is often based on vague symptoms and a positive culture. The high prevalence of asymptomatic bacteriuria (ABU), which cannot be easily discriminated from an acute infection in this population, is frequently neglected, leading to a vast over-prescription of antibiotics. This study aimed to identify subpopulations predisposed to transient or long-term ABU. Methods Residents in a long-term care facility were screened for ABU. Mid-stream urine samples were collected during two sampling rounds, separated by 10 weeks, each consisting of an initial and a confirmative follow-up sample. Results ABU occurred in approximately 40% of the participants and was mostly caused by Escherichia coli. Long-term ABU (> 3 months) was found in 30% of the subjects. The frailest women with urinary incontinence and dementia had drastically increased rates of ABU and especially long-term ABU. ABU was best predicted by a scale describing the functional independence of older adults. Conclusions Institutionalized women with incontinence have ABU prevalence rates of about 80% and are often persistent carriers. Such prevalence rates should be considered in clinical decision making as they devalue the meaning of a positive urine culture as a criterion to diagnose UTIs. Diagnostic strategies are urgently needed to avoid antibiotic overuse and to identify patients at risk to develop upper UTI.

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not complete, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters, using cutoffs of 30-50 allele differences. The exceptions are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile.

Background: Companion animal clinics contribute to the spread of antimicrobial resistant microorganisms (ARM) and outbreaks with ARM of public health concern have been described. Methods: As part of a project to assess infection prevention and control (IPC) standards in companion animal clinics in Switzerland, a total of 200 swabs from surfaces and 20 hand swabs from employees were collected during four days in a medium-sized clinic and analyzed for extended spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E), carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant enterococci (VRE), and methicillin-resistant staphylococci (MRS). Results: A total of 22 (11.0%) environmental specimen yielded CPE, 14 (7.0%) ESBL-E, and 7 (3.5%) MRS; MR Staphylococcus aureus were isolated from two (10.0%) hand swabs. The CPE isolates comprised Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Citrobacter braakii, and Serratia marcescens. Whole genome sequencing revealed that all CPE carried closely related blaOXA-48 plasmids, suggesting a plasmidic spread within the clinic. The clinic exhibited major deficits in surface disinfection, hand hygiene infrastructure, and hand hygiene compliance. CPE were present in various areas, including those without patient contact. The study documented plasmidic dissemination of blaOXA-48 in a companion animal clinic with low IPC standards. This poses a worrisome threat to public health and highlights the need to foster IPC standards in veterinary clinics to prevent the spread of ARM into the community.

Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.