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.

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.

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.

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.

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.

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.

Chlamydia suis displays high transformation capacity with complete cloning vector integration into the chromosomal rrn-nqrF plasticity zone.

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.

Completely assembled genome sequence of the florfenicol-resistant Enterococcus faecalis strain 90_2023 isolated from a raw sausage imported from Italy to Switzerland.

Here we report the genome sequence of the florfenicol-resistant Enterococcus faecalis strain 90_2023 isolated from a raw-meat sausage (Finocchiona) imported from Italy to Switzerland. It has a genome of 2.75 Mbp and harbors 16 antimicrobial resistance genes, including catA8, fexA, and a truncated optrA gene on a RepA_N plasmid.

Dissemination of ESBL-producing E. coli ST131 through wastewater and environmental water in Switzerland.

The E. coli lineage ST131 is a major cause of multidrug-resistant urinary tract and bloodstream infections worldwide. Recently emerged ST131 sublineages spread globally within few years, but their dissemination routes are incompletely understood. In this study, we investigate the potential role of wastewater and surface water in the spread of extended-spectrum ß-lactamase (ESBL)-producing ST131. Streams, lakes, and two wastewater treatment plants (WWTPs) in the canton of Zug, Switzerland, were consecutively sampled over 1.5 years. ST131 was detected in 38% of the samples taken downstream (1-5 km) of WWTP discharge sites, but usually absent in water bodies distant from urban areas or WWTP discharge. Specific strains were repeatedly isolated (≤5 pairwise cgSNP distance) from wastewater or river sites downstream of effluent discharge, indicating their repeated entry or persistence in WWTPs in large concentrations. Genetic characterization of the ESBL-producing water isolates revealed a predominance of clades A and C1 and an emerging ciprofloxacin-resistant sublineage with mutations in quinolone resistance determining regions (QRDR) within clade A. Multiple isolates belonged to internationally circulating sublineages, including C1-M27 and papGII + sublineages with chromosomally encoded ESBLs. This study demonstrates that the clinically relevant E. coli lineage ST131 pollutes river ecosystems, representing a significant challenge to public health and to technologies to minimize their entry into the water environment.

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.

Strain diversity in Mycobacterium avium subsp. paratuberculosis-positive bovine fecal samples collected in Switzerland.

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.

Complete Genome Sequence of the Extensively Drug-Resistant Extended-Spectrum ß-Lactamase-Producing Proteus mirabilis Isolate HK294, Obtained from Poultry Feces in Hong Kong.

Here, we report the complete genome sequence of Proteus mirabilis isolate HK294, recovered from pooled poultry feces in Hong Kong in 2022. The chromosome contained 32 antimicrobial resistance genes, including the extended-spectrum ß-lactamases blaCTX-M-65 and blaCTX-M-3. Almost all resistance genes were part of either an integrative conjugative element or a Tn7-like transposon.

Draft Genome Sequence of Pseudomonas carnis Strain 23-145, Causing Blue Discolorations on Rabbit Carcasses.

Here, we report the genome sequence of Pseudomonas carnis strain 23-145, which was recovered from a rabbit carcass with blue discolorations. The strain harbored two trpABCDF loci involved in tryptophan biosynthesis, which is characteristic of blue-pigment-producing Pseudomonas strains.

Draft Genome Sequences of Four Escherichia albertii Isolates from Hunted Wild Boars in Switzerland.

Here, we report the genome sequences of four Escherichia albertii isolates that were recovered from hunted wild boars in Switzerland in 2022 and 2023. The genome sizes of KBWS15i, KBWS35i, KBWS50i, and KBSW171i were 4.4 Mbp, 4.5 Mbp, 4.5 Mbp, and 4.7 Mbp, respectively.

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.

Draft Genome Sequences of Two Escherichia albertii Isolates Collected from Healthy Pets in Switzerland.

Here, we report the genome sequences of two Escherichia albertii isolates recovered from a healthy dog (KBD171i) and cat (KBK128i) in Switzerland in 2022. The genome sizes of KBK128i and KBD171i were 4.7 Mbp and 4.9 Mbp, respectively.

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.

Whole-genome-based characterization of Campylobacter jejuni from human patients with gastroenteritis collected over an 18 year period reveals increasing prevalence of antimicrobial resistance.

Campylobacteriosis is the most common cause of acute gastrointestinal bacterial infection in Europe, with most infections linked to the consumption of contaminated food. While previous studies found an increasing rate of antimicrobial resistance (AMR) in Campylobacter spp. over the past decades, the investigation of additional clinical isolates is likely to provide novel insights into the population structure and mechanisms of virulence and drug resistance of this important human pathogen. Therefore, we combined whole-genome sequencing and antimicrobial-susceptibility testing of 340 randomly selected Campylobacter jejuni isolates from humans with gastroenteritis, collected in Switzerland over an 18 year period. In our collection, the most common multilocus sequence types (STs) were ST-257 (n=44), ST-21 (n=36) and ST-50 (n=35); the most common clonal complexes (CCs) were CC-21 (n=102), CC-257 (n=49) and CC-48 (n=33). High heterogeneity was observed among STs, with the most abundant STs recurring over the entire study period, while others were observed only sporadically. Source attribution based on ST assigned more than half of the strains to the 'generalist' category (n=188), 25  % as 'poultry specialist' (n=83), and only a few to 'ruminant specialist' (n=11) or 'wild bird' origin (n=9). The isolates displayed an increased frequency of AMR from 2003 to 2020, with the highest rates of resistance observed for ciprofloxacin and nalidixic acid (49.8 %), followed by tetracycline (36.9 %). Quinolone-resistant isolates carried chromosomal gyrA mutations T86I (99.4 %) and T86A (0.6 %), whereas tetracycline-resistant isolates carried tet(O) (79.8 %) or mosaic tetO/32/O (20.2 %) genes. A novel chromosomal cassette carrying several resistance genes, including aph(3')-III, satA and aad(6), and flanked by insertion sequence elements was detected in one isolate. Collectively, our data revealed an increasing prevalence of resistance to quinolones and tetracycline in C. jejuni isolates from Swiss patients over time, linked to clonal expansion of gyrA mutants and acquisition of the tet(O) gene. Investigation of source attribution suggests that infections are most likely related to isolates from poultry or generalist backgrounds. These findings are relevant to guide future infection prevention and control strategies.

PorinPredict: In Silico Identification of OprD Loss from WGS Data for Improved Genotype-Phenotype Predictions of P. aeruginosa Carbapenem Resistance.

The increasing integration of genomics into routine clinical diagnostics requires reliable computational tools to identify determinants of antimicrobial resistance (AMR) from whole-genome sequencing data. Here, we developed PorinPredict, a bioinformatic tool that predicts defects of the Pseudomonas aeruginosa outer membrane porin OprD, which are strongly associated with reduced carbapenem susceptibility. PorinPredict relies on a database of intact OprD variants and reports inactivating mutations in the coding or promoter region. PorinPredict was validated against 987 carbapenemase-negative P. aeruginosa genomes, of which OprD loss was predicted for 454 out of 522 (87.0%) meropenem-nonsusceptible and 46 out of 465 (9.9%) meropenem-susceptible isolates. OprD loss was also found to be common among carbapenemase-producing isolates, resulting in even further increased MICs. Chromosomal mutations in quinolone resistance-determining regions and OprD loss commonly co-occurred, likely reflecting the restricted use of carbapenems for multidrug-resistant infections as recommended in antimicrobial stewardship programs. In combination with available AMR gene detection tools, PorinPredict provides a robust and standardized approach to link P. aeruginosa phenotypes to genotypes. IMPORTANCE Pseudomonas aeruginosa is a major cause of multidrug-resistant nosocomial infections. The emergence and spread of clones exhibiting resistance to carbapenems, a class of critical last-line antibiotics, is therefore closely monitored. Carbapenem resistance is frequently mediated by chromosomal mutations that lead to a defective outer membrane porin OprD. Here, we determined the genetic diversity of OprD variants across the P. aeruginosa population and developed PorinPredict, a bioinformatic tool that enables the prediction of OprD loss from whole-genome sequencing data. We show a high correlation between predicted OprD loss and meropenem nonsusceptibility irrespective of the presence of carbapenemases, which are a second widespread determinant of carbapenem resistance. Isolates with resistance determinants to other antibiotics were disproportionally affected by OprD loss, possibly due to an increased exposure to carbapenems. Integration of PorinPredict into genomic surveillance platforms will facilitate a better understanding of the clinical impact of OprD modifications and transmission dynamics of resistant clones.