Oxford Nanopore's 2024 sequencing technology for Listeria monocytogenes outbreak detection and source attribution: progress and clone-specific challenges.

Whole genome sequencing is an essential cornerstone of pathogen surveillance and outbreak detection. Established sequencing technologies are currently being challenged by Oxford Nanopore Technologies (ONT), which offers an accessible and cost-effective alternative enabling gap-free assemblies of chromosomes and plasmids. Limited accuracy has hindered its use for investigating pathogen transmission, but recent technology updates have brought significant improvements. To evaluate its readiness for outbreak detection, we selected 78 Listeria monocytogenes isolates from diverse lineages or known epidemiological clusters for sequencing with ONT's V14 Rapid Barcoding Kit and R10.4.1 flow cells. The most accurate of several tested workflows generated assemblies with a median of one error (SNP or indel) per assembly. For 66 isolates, the cgMLST profiles from ONT-only assemblies were identical to those generated from Illumina data. Eight assemblies were of lower quality, with more than 20 erroneous sites each, primarily caused by methylations at the GAAGAC motif (5'-GAAG6mAC-3'/5'-GT4mCTTC-3'). This led to inaccurate clustering, failing to group isolates from a persistence-associated clone that carried the responsible restriction-modification system. Out of 50 methylation motifs detected among the 78 isolates, only the GAAGAC motif was linked to substantially increased error rates. Our study shows that most L. monocytogenes genomes assembled from ONT-only data are suitable for high-resolution genotyping, but further improvements of chemistries or basecallers are required for reliable routine use in outbreak and food safety investigations.

Extended-spectrum ß-lactamase (ESBL)- and Carbapenemase-producing Enterobacterales Isolated from Fresh Herbs and Salads at Retail Level in Switzerland.

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.

Outbreak of Listeriosis Likely Associated with Baker's Yeast Products, Switzerland, 2022-2024.

We traced back a nationwide outbreak of human listeriosis in Switzerland to a persisting production line contamination of a factory producing baker's yeast with Listeria monocytogenes serotype 1/2a sequence type 3141. We used whole-genome sequencing to match clinical isolates to isolates from product samples.

Colonization with resistant bacteria in hospital employees: an epidemiological surveillance and typing study.

The objective of this study was to determine the prevalence, molecular epidemiology, and risk factors for gut colonization with extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), and vancomycin-resistant enterococci (VRE) in healthcare workers (HCWs). In September/October 2022, we performed a cross-sectional study among HCW from 14 institutions in Northeastern Switzerland. HCWs reported risk factors for antimicrobial resistance (covering the last 12-24 months) and provided rectal swabs. Swabs were screened for ESBL-E, CPE, and VRE; whole-genome sequencing (WGS) was performed to assess the genetic relatedness. Logistic regression was used to identify occupational and non-occupational risk factors. Among approximately 22,500 employees, 1,209 participated (median age 46 years, 82% female). Prevalences of ESBL-E (n = 65) and CPE (n = 1) were 5.4% [95% confidence interval (CI) 4.2-6.8] and 0.1% (95% CI 0.0-0.5), respectively; no VREs were detected. In the multivariable analysis, non-European ethnicity [adjusted odds ratio (aOR) 7.0, 95% CI 1.4-27.3], travel to high-risk countries (aOR 4.9, 95% CI 2.5-9.3), systemic antibiotics (aOR 2.1, 95% CI 1.1-3.7), antibiotic eye drops (aOR 4.7, 95% CI 1.7-11.9), and monthly sushi consumption (aOR 2.4, 95% CI 1.4-4.3) were positively associated with ESBL-E colonization, whereas alcohol consumption (aOR 0.5 per glass/week, 95% CI 0.3-0.9) was negatively associated with ESBL-E colonization. Occupational factors showed no association. Among ESBL-Escherichia coli, ST131 (15 of 61, 25%) and blaCTX-M-15 (37/61; 61%) were most common; one isolate co-harbored blaOXA-244. WGS data did not show relevant clustering. Occupational exposure is not associated with ESBL-E colonization in HCW. Given the potential public health and antibiotic stewardship implications, the role of sushi consumption and antibiotic eye drops as risk factors should be further elucidated.

Characteristics and comparative genome analysis of Yersinia enterocolitica and related species associated with human infections in Switzerland 2019-2023.

PURPOSE: We aimed to characterise Yersinia enterocolitica from human clinical specimens in Switzerland using epidemiological, microbiological and whole-genome sequencing (WGS) data. METHODS: Isolates (n = 149) were collected between January 2019 and December 2023. Epidemiological data was noted and strains were characterized by biochemical and serological typing, antimicrobial susceptibility testing (AST), and WGS-based analysis. RESULTS: Most of the isolates (86%) were from stool specimens and 52% were from male patients. The patients' median age was 28 years (range < 1-94 years). Typing assigned the isolates to bioserotype 4/O:3 (44%), biotype 1A (34%), bioserotype 2/O:9 (21%), and bioserotype 3/O:3 (1%). WGS identified Y. enterocolitica (n = 147), Y. alsatica (n = 1) and Y. proxima (n = 1). Seven isolates were multidrug resistant (MDR) and harboured plasmid pAB829 carrying aph(3″)-Ib, aph(6)-Id, and tet(Y) (n = 1), pAC120 carrying aph(6)-Id and tet(A) (n = 2), or a 12.6 kb Tn2670-like transposon containing catA1, aadA12, sul1, and qacEΔ1 (n = 4). Virulence factors (VFs) included ail (n = 99), invB, (n = 145), ystA (n = 99), ystB (n = 48) and pYV-associated VFs (n = 93). MLST and cgMLST analysis showed that BT 1A strains consisted of several STs and were highly diverse, whereas BT 2/O:9 strains were all ST12 and clustered closely, and BT 4/O:3 strains mostly belonged to ST18 but were more diverse. SNP analysis revealed two highly clonal BT 4/O:3 subpopulations with wide spatio-temporal distribution. CONCLUSIONS: Y. enterocolitica BT 1A, BT 2/O:9 and BT 4/O:3 are frequently associated with human yersiniosis in Switzerland. WGS-based subtyping of Y. enterocolitica is a powerful tool to explore the genetic diversity and the pathogenic potential of human isolates.

Disseminated, fatal reactivation of bovine tuberculosis in a patient treated with adalimumab: a case report and review of the literature.

PURPOSE: Tumor necrosis factor inhibitors (TNFi) are known to increase the risk of tuberculosis (TB) reactivation, though cases involving Mycobacterium bovis are rarely reported. CASE PRESENTATION/RESULTS: We describe a case of disseminated TB with M. bovis in a 78-year-old woman with a negative Interferon-Gamma-Release Assay (IGRA), taking adalimumab due to rheumatoid polyarthritis, which resulted in a fatal outcome. The atypical clinical and histopathological features were initially interpreted as sarcoidosis. The case occurred in Switzerland, an officially bovine tuberculosis-free country. The whole genome sequence of the patient's cultured M. bovis isolate was identified as belonging to the animal lineage La1.2, the main genotype in continental Europe, but showed significant genetic distance from previously sequenced Swiss cattle strains. In a literature review, four cases of bovine tuberculosis reactivation under TNFi treatment were identified, with pulmonal, oral and intestinal manifestations. Similar to our patient, two cases presented a negative IGRA before TNFi initiation, which later converted to positive upon symptomatic presentation of M. bovis infection. CONCLUSION: This case highlights the diagnostic challenges of TB in immunosuppressed patients, the limited sensitivity of IGRA, and the importance of considering TB reactivation even in regions declared free of bovine tuberculosis. Detailed patient histories, including potential exposure to unpasteurized dairy products, are essential for guiding preventive TB treatment before TNFi initiation.

Isolation of community-associated methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 30 from house rats (Rattus tanezumi) in Hong Kong.

Methicillin-resistant Staphylococcus aureus (MRSA) is of major public health concern due to its resistance to multiple antibiotics. This resistance has been observed in various settings, including hospitals and communities, and has been detected in both animals and humans. Although peridomestic rat species (Rattus spp.) are well described reservoirs of several human pathogens and antimicrobial resistant bacteria, little is known about their role in MRSA epidemiology. In order to investigate whether Rattus spp. in Hong Kong are potential carriers of MRSA, 221 rats were caught from various ecological areas and nasopharyngeal samples were cultured on MRSA selective media. Genotypic characteristics of MRSA were confirmed by whole genome sequencing. Two clonal sequence type (ST) 30 MRSA isolates, harbouring mecA on staphylococcal chromosome cassette (SCC) mec type IVc, were cultured from two house rats (Rattus tanezumi) caught in two densely populated urban areas. To the best of the authors' knowledge, this is the first detection of community-associated (CA)-MRSA strain ST30 SCCmec IVc in peridomestic rodents in Hong Kong and globally. Our finding indicates that house rats can be carriers of MRSA strains that are widely distributed in the community.

Seroprevalence of Mycobacterium avium subsp. paratuberculosis in Swiss dairy herds and risk factors for a positive herd status and within-herd prevalence.

Introduction: Bovine paratuberculosis (PTB) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). Control of PTB is important given its negative economic consequences and the potential zoonotic role of MAP in Crohn's disease in humans. Methods: To determine the seroprevalence of MAP in Swiss dairy herds and to identify risk factors associated with seropositive herd status and high within-herd seroprevalence, 10,063 serum samples collected from cattle over 12 months of age in 171 Swiss dairy farms were analyzed using a commercial ELISA test. Eight herds were excluded due to non-interpretable ELISA results. Risk factors associated with seropositive herd status and high within-herd seroprevalence were investigated with regression models using results from a questionnaire on management practices possibly associated with the introduction or spread of MAP in the remaining 163 herds. Univariable logistic regression was performed, carrying forward for multivariable regression analysis when p < 0.2. Results: The calculated between-herd true seroprevalence was 3.6% (95% CI, 0.96-8.4%). Due to the low within-herd seroprevalence, it was not possible to calculate the true seroprevalence at animal level; the apparent within-herd seroprevalence ranged from 2.3 to 5.5% with a median of 3.6% in nine positive farms. Herd size (p = 0.037) and the common grazing of lactating cows with cows from other herds (p = 0.014) were associated with seropositive herd status, while heifers sharing alpine pasture with dairy cattle from other herds were associated with a decreased probability of the herd to test seropositive (p = 0.042). Reliable identification of significant risk factors associated with MAP spread and high seroprevalence of PTB within seropositive herds was not possible due to low observed seroprevalence within herds and low sensitivity of the ELISA test. Discussion: These results highlight the limitation of serology for MAP diagnosis in small herds with low infection prevalence.

A Comprehensive Methodology for Microbial Strain Typing Using Fourier-Transform Infrared Spectroscopy.

Timely and accurate detection and characterization of microbial threats is crucial for effective infection and outbreak management. Additionally, in food production, rapid microbe identification is indispensable for maintaining quality control and hygiene standards. Current methods for typing microbial strains often rely on labor-intensive, time-consuming, and expensive DNA- and sera-serotyping techniques, limiting their applicability in rapid-response scenarios. In this context, the IR Biotyper®, utilizing Fourier-transform infrared (FTIR) spectroscopy, offers a novel approach, providing specific spectra for fast strain typing within 3 h. This methodology article serves as a comprehensive resource for researchers and technicians aiming to utilize FTIR spectroscopy for microbial strain typing. It encompasses detailed guidelines on sample preparation, data acquisition, and analysis techniques, ensuring the generation of reliable and reproducible results. We highlight the IR Biotyper®'s rapid and accurate discrimination capabilities, showcasing its potential for real-time pathogen monitoring and source-tracking to enhance public health and food safety. We propose its integration as an early screening method, followed by more detailed analysis with whole-genome sequencing, to optimize detection accuracy and response efficiency in microbial surveillance systems.

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.

Rapid detection of the source of a Listeria monocytogenes outbreak in Switzerland through routine interviewing of patients and whole-genome sequencing.

AIMS OF THE STUDY: Listeriosis is a notifiable disease in Switzerland. In summer 2022, the Swiss Federal Office of Public Health noticed an increase in reports of listeriosis cases, indicating a possible ongoing outbreak. Here we present the approaches applied for rapidly confirming the outbreak, detecting the underlying source of infection and the measures put in place to eliminate it and contain the outbreak. METHODS: For close surveillance and early detection of outbreak situations with their possible sources, listeriosis patients in Switzerland are systematically interviewed about risk behaviours and foods consumed prior to the infection. Listeria monocytogenes isolates derived from patients in medical laboratories are sent to the National Reference Laboratory for Enteropathogenic Bacteria and Listeria, where they routinely undergo whole-genome sequencing. Interview and whole-genome sequencing data are continuously linked for comparison and analysis. RESULTS: In summer 2022, 20 patient-derived L. monocytogenes serotype 4b sequence type 388 strains were found to belong to an outbreak cluster (≤10 different alleles between neighbouring isolates) based on core genome multilocus sequence typing analysis. Geographically, 18 of 20 outbreak cases occurred in northeastern Switzerland. The median age of patients was 77.4 years (range: 58.1-89.7), with both sexes equally affected. Rolling analysis of the interview data revealed smoked trout from a local producer as a suspected infection source, triggering an on-site investigation of the production facility and sampling of the suspected products by the responsible cantonal food inspection team on 15 July 2022. Seven of ten samples tested positive for L. monocytogenes and the respective cantonal authority ordered a ban on production and distribution as well as a product recall. The Federal Food Safety and Veterinary Office released a nationwide public alert covering the smoked fish products concerned. Whole-genome sequencing analysis confirmed the interrelatedness of the L. monocytogenes smoked trout product isolates and the patient-derived isolates. Following the ban on production and distribution and the product recall, reporting of new outbreak-related cases rapidly dropped to zero. CONCLUSIONS: This listeriosis outbreak could be contained within a relatively short time thanks to identification of the source of contamination through the established combined approach of timely interviewing of every listeriosis patient or a representative and continuous molecular analysis of the patient- and food-derived L. monocytogenes isolates. These findings highlight the effectiveness of this well-established, joint approach involving the federal and cantonal authorities and the research institutions mandated to contain listeriosis outbreaks in Switzerland.

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.

Genetic diversity and antimicrobial susceptibility of Streptococcus suis from diseased Swiss pigs collected between 2019 - 2022.

Streptococcus suis is an important pathogen causing severe disease in pigs and humans, giving rise to economic losses in the pig production industry. Out of 65 S. suis isolates collected from diseased pigs in Switzerland between 2019 and 2022, 57 isolates were thoroughly examined by phenotypic and whole genome sequence (WGS) based characterization. The isolates' genomes were sequenced allowing for a comprehensive analysis of their distribution in terms of serovar, sequence type (ST), clonal complex (CC), and classical virulence markers. Antimicrobial resistance (AMR) genes were screened, and phenotypic susceptibility to eight classes of antimicrobial agents was examined. Serovar 6, devoid of any resistance genes, was found to be most prevalent, followed by serovars 1, 3, 1/2, and 9. Thirty STs were identified, with ST1104 being the most prevalent. Serovar 2 and serovar 1/2 were associated with CC1, potentially containing the most virulent variants. Based on single nucleotide polymorphism (SNP) analyses, fifteen isolates belonged to one of seven putative transmission clusters each consisting of two or three isolates. High phenotypic AMR rates were detected for tetracyclines (80%) and macrolides (35%) and associated with the resistance genes tet(O) and erm(B), respectively. In contrast, susceptibility to ß-lactam antibiotics and phenicols was high. Determination of phenotypic AMR profiling, including the minimum inhibitory concentrations (MICs) of the tested antimicrobial agents, sets a baseline for future studies. The study provides valuable insights into the genetic diversity and antimicrobial susceptibility of Swiss S. suis isolates, facilitating the identification of emerging clones relevant to public health concerns.

A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

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.

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.

Development of a Sensitive and Specific Quantitative RT-qPCR Method for the Detection of Hepatitis E Virus Genotype 3 in Porcine Liver and Foodstuff.

As an international and zoonotic cause of hepatitis, hepatitis E virus (HEV) poses a significant risk to public health. However, the frequency of occurrence and the degree of contamination of food of animal origin require further research. The aim of this study was to develop and validate a highly sensitive quantitative RT-qPCR assay for the detection and quantification of HEV contamination in porcine liver and food. The focus was on genotype 3, which is most common as a food contaminant in developed countries and Europe. The selected assay has its target sequence in the open reading frame 1 (ORF1) of the HEV genome and showed good results in inclusivity testing, especially for HEV genotype 3. The developed assay seems to show high efficiency and a low intercept when compared to other assays, while having a comparable limit of detection (LOD). In addition, a standard curve was generated using artificially spiked liver to provide more accurate quantitative results for contamination assessment and tracking in this matrix. Application of the assay to test 67 pig livers from different origins resulted in a positivity rate of 7.5%, which is consistent with the results of numerous other prevalence studies. Quantitative detection of the viral genome in the food chain, particularly in pig livers, is essential for understanding the presence and evolution of HEV contamination and thus ensures consumer safety.

Rombocin, a Short Stable Natural Nisin Variant, Displays Selective Antimicrobial Activity against Listeria monocytogenes and Employs a Dual Mode of Action to Kill Target Bacterial Strains.

Nisin, with its unique mode of action and potent antimicrobial activity, serves as a remarkable inspiration for the design of novel antibiotics. However, peptides possess inherent weaknesses, particularly their susceptibility to proteolytic degradation, such as by trypsin, which limits their broader applications. This led us to speculate that natural variants of nisin produced by underexplored bacterial species can potentially overcome these limitations. We carried out genome mining of two Romboutsia sedimentorum strains, RC001 and RC002, leading to the discovery of rombocin A, which is a 25 amino acid residue short nisin variant that is predicted to have only four macrocycles compared to the known 31-35 amino acids long nisin variants with five macrocycles. Using the nisin-controlled expression system, we heterologously expressed fully modified and functional rombocin A in Lactococcus lactis and demonstrated its selective antimicrobial activity against Listeria monocytogenes. Rombocin A uses a dual mode of action involving lipid II binding activity and dissipation of the membrane potential to kill target bacteria. Stability tests confirmed its high stability at different pH values, temperatures, and in particular, against enzymatic degradation. With its gene-encoded characteristic, rombocin A is amenable to bioengineering to generate novel derivatives. Further mutation studies led to the identification of rombocin K, a mutant with enhanced bioactivity against L. monocytogenes. Our findings suggest that rombocin A and its bioengineered variant, rombocin K, are promising candidates for development as food preservatives or antibiotics against L. monocytogenes.

Cesin, a short natural variant of nisin, displays potent antimicrobial activity against major pathogens despite lacking two C-terminal macrocycles.

Nisin is a widely used lantibiotic owing to its potent antimicrobial activity and its food-grade status. Its mode of action includes cell wall synthesis inhibition and pore formation, which are attributed to the lipid II binding and pore-forming domains, respectively. We discovered cesin, a short natural variant of nisin, produced by the psychrophilic anaerobe Clostridium estertheticum. Unlike other natural nisin variants, cesin lacks the two terminal macrocycles constituting the pore-forming domain. The current study aimed at heterologous expression and characterization of the antimicrobial activity and physicochemical properties of cesin. Following the successful heterologous expression of cesin in Lactococcus lactis, the lantibiotic demonstrated a broad and potent antimicrobial profile comparable to that of nisin. Determination of its mode of action using lipid II and lipoteichoic acid binding assays linked the potent antimicrobial activity to lipid II binding and electrostatic interactions with teichoic acids. Fluorescence microscopy showed that cesin lacks pore-forming ability in its natural form. Stability tests have shown the lantibiotic is highly stable at different pH values and temperature conditions, but that it can be degraded by trypsin. However, a bioengineered analog, cesin R15G, overcame the trypsin degradation, while keeping full antimicrobial activity. This study shows that cesin is a novel (small) nisin variant that efficiently kills target bacteria by inhibiting cell wall synthesis without pore formation. IMPORTANCE The current increase in antibiotic-resistant pathogens necessitates the discovery and application of novel antimicrobials. In this regard, we recently discovered cesin, which is a short natural variant of nisin produced by the psychrophilic Clostridium estertheticum. However, its suitability as an antimicrobial compound was in doubt due to its structural resemblance to nisin(1-22), a bioengineered short variant of nisin with low antimicrobial activity. Here, we show by heterologous expression, purification, and characterization that the potency of cesin is not only much higher than that of nisin(1-22), but that it is even comparable to the full-length nisin, despite lacking two C-terminal rings that are essential for nisin's activity. We show that cesin is a suitable scaffold for bioengineering to improve its applicability, such as resistance to trypsin. This study demonstrates the suitability of cesin for future application in food and/or for health as a potent and stable antimicrobial compound.