Genomic characteristics of clinical non-toxigenic Vibrio cholerae isolates in Switzerland: a cross-sectional study.

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.

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.

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.

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.

Serotype Diversity and Antimicrobial Resistance Profile of Salmonella enterica Isolates From Freshwater Turtles Sold for Human Consumption in Wet Markets in Hong Kong.

Chelonians are recognized as a source of human salmonellosis through direct contact or consumption of their meat. Freshwater turtles sold for food are widely available in wet markets in Asia. In this pilot study, 50 turtles belonging to three species were randomly sampled from wet markets throughout Hong Kong. The turtles were humanely euthanised and their feces or the colon were sampled for Salmonella culture. The Salmonella isolates obtained were serotyped and examined for phenotypic antimicrobial resistance and the presence of antimicrobial resistance genes. The study reports a high prevalence (42%, 95% CI: 29.4-55.8) and considerable serotype diversity of Salmonella among turtles sold in wet markets. The most common among the 11 serotypes isolated were S. Oranienburg and S. Thompson, which have been reported in turtles previously. The serotype S. Manhattan is reported in chelonians for the first time. Resistance to streptomycin and chloramphenicol was common, despite the latter being banned from aquaculture in mainland China since 2002. Resistance against fluoroquinolones and third-generation cephalosporins which represent first-line treatment options for salmonellosis was also observed. The multidrug-resistance gene cfr is identified for the first time in Salmonella. This is a worrying finding as it indicates an expansion of the cfr reservoir and potential horizontal spread to other bacteria. The results of this study emphasize the need for close surveillance of Salmonella from turtles sold as food and better regulation of turtle farming to safeguard public health and improve animal welfare.

Feedborne Salmonella enterica Serovar Jerusalem Outbreak in Different Organic Poultry Flocks in Switzerland and Italy Linked to Soya Expeller.

Poultry feed is a leading source of Salmonella infection in poultry. In Switzerland, heat-treated feed is used to reduce Salmonella incursions into flocks in conventional poultry production. By contrast, organic feed is only treated with organic acids. In 2019, the Swiss National Reference Center for Enteropathogenic Bacteria identified the rare serovar S. Jerusalem from samples of organic soya feed. Further, in July 2020, the European Union's Rapid Alert System for Food and Feed published a notification of the detection of S. Jerusalem in soya expeller from Italy. During 2020, seven S. Jerusalem isolates from seven different poultry productions distributed over six cantons in Switzerland were reported, providing further evidence of a possible outbreak. Using whole-genome sequencing (WGS), S. Jerusalem isolates from feed and from animals in Switzerland were further characterized and compared to S. Jerusalem from organic poultry farm environments in Italy. WGS results showed that feed isolates and isolates from Swiss and Italian poultry flocks belonged to the sequence type (ST)1028, grouped in a very tight cluster, and were closely related. This outbreak highlights the risk of spreading Salmonella by feed and emphasizes the need for a heat-treatment process for feed, also in organic poultry production.

Global Transcriptional Response of Three Highly Acid-Tolerant Field Strains of Listeria monocytogenes to HCl Stress.

Tolerance to acid is of dual importance for the food-borne pathogen Listeria monocytogenes: acids are used as a preservative, and gastric acid is one of the first defenses within the host. There are considerable differences in the acid tolerance of strains. Here we present the transcriptomic response of acid-tolerant field strains of L. monocytogenes to HCl at pH 3.0. RNAseq revealed significant differential expression of genes involved in phosphotransferase systems, oxidative phosphorylation, cell morphology, motility, and biofilm formation. Genes in the acetoin biosynthesis pathway were upregulated, suggesting that L. monocytogenes shifts to metabolizing pyruvate to acetoin under organic acid stress. We also identified the formation of cell aggregates in microcolonies as a potential relief strategy. A motif search within the first 150 bp upstream of differentially expressed genes identified a novel potential regulatory sequence that may have a function in the regulation of virulence gene expression. Our data support a model where an excess of intracellular H+ ions is counteracted by pumping H+ out of the cytosol via cytochrome C under reduced activity of the ATP synthase. The observed morphological changes suggest that acid stress may cause cells to aggregate in biofilm microcolonies to create a more favorable microenvironment. Additionally, HCl stress in the host stomach may serve as (i) a signal to downregulate highly immunogenic flagella, and (ii) as an indicator for the imminent contact with host cells which triggers early stage virulence genes.

Whole-Genome Sequences of Six Listeria monocytogenes Strains Isolated from Food.

Here, we report the whole-genome sequences of six Listeria monocytogenes strains isolated from meat and milk products in Switzerland. All of these strains carry premature stop codons or amino acid deletions in inlA.

Surviving host - and food relevant stresses: phenotype of L. monocytogenes strains isolated from food and clinical sources.

The aim of this study was to compare the phenotype of 40 strains of L. monocytogenes under food and host relevant stress conditions. The strains were chosen to represent food and clinical isolates and to be equally distributed between the most relevant clonal complexes for clinical and food isolates (CC1 and CC6 vs CC121 and CC9), plus one group of eight strains of rare clonal complexes. Human-associated CC1 had a faster maximal growth rate than the other major complexes, and the lag time of CC1 and CC6 was significantly less affected by the addition of 4% NaCl to the medium. Food-associated CC9 strains were hypohemolytic compared to other clonal complexes, and all strains found to be resistant to increased concentrations of benzalkonium chloride belonged to CC121 and were positive for Tn6188 carrying the qacH gene. Lactic acid affected the survival of L. monocytogenes more than HCl, and there was a distinct, strain specific pattern of acid tolerant and sensitive strains. Strains from CC6 and human clinical isolates are less resilient under acid stress than those from other complexes and from food. One strain isolated from a human patient exhibited significant growth defects across all conditions.

Whole-Genome Shotgun Sequencing of Three Listeria monocytogenes Strains Isolated from a Ready-to-Eat Salad-Producing Facility in Switzerland.

Ready-to-eat (RTE) raw foods harbor the risk of transmitting Listeria monocytogenes from the environment to the consumer. We isolated three strains from a facility producing RTE salad. These strains were used to perform challenge tests on different RTE salad products. Here, we present the shotgun genome sequences of all three of these strains.