Virulence and antimicrobial resistance of Shiga toxin-producing Escherichia coli from dairy goat and sheep farms in The Netherlands.

AIMS: The aim of our study was to investigate the virulence and resistance of STEC from small ruminants farms in The Netherlands. Moreover, the potential transmission of STEC between animals and humans on farms was evaluated. METHODS AND RESULTS: From 182 farms, in total, 287 unique STEC isolates were successfully recovered from animal samples. In addition, STEC was isolated from eight out of 144 human samples. The most detected serotype was O146:H21; however, among other serotypes also O26:H11, O157:H7, and O182:H25 isolates were present. Whole genome sequencing covering all human isolates and 50 of the animal isolates revealed a diversity of stx1, stx2, and eae sub-types and an additional 57 virulence factors. The assessed antimicrobial resistance phenotype, as determined by microdilution, was concordant with the genetic profiles identified by WGS. WGS also showed that three of the human isolates could be linked to an animal isolate from the same farm. CONCLUSIONS: The obtained STEC isolates showed great diversity in serotype, virulence, and resistance factors. Further analysis by WGS allowed for an in-depth assessment of the virulence and resistance factors present and to determine the relatedness of human and animal isolates.

New insights into the epidemiology of Listeria monocytogenes - A cross-sectoral retrospective genomic analysis in the Netherlands (2010-2020).

Introduction: Listeriosis, caused by infection with Listeria monocytogenes (Lm), is a relatively rare but severe disease with one of the highest mortality rates among bacterial foodborne illnesses. A better understanding on the degree of Lm clustering, the temporal distribution of the clusters, and their association with the various food sources is expected to lead to improved source tracing and risk-based sampling. Methods: We investigated the genomic epidemiology of Lm in the Netherlands between 2010 and 2020 by analyzing whole-genome-sequencing (WGS) data of isolates from listerioss patients and food sources from nationwide integrated surveillance and monitoring. WGS data of 756 patient and 770 food/environmental isolates was assessed using core-genome multi-locus sequence typing (cgMLST) with Hamming distance as measure for pairwise distances. Associations of genotype with the epidemiological variables such as patient's age and gender, and systematic use of specific drugs were tested by multinomial logistic regressions. Genetic differentiation of the Lm within and between food categories was calculated based on allele frequencies at the 1701 cgMLST loci in each food category. Results: We confirmed previous results that some clonal complexes (CCs) are overrepresented among clinical isolates but could not identify any epidemiological risk factors. The main findings of this study include the observation of a very weak attribution of Lm types to food categories and a much better attribution to the producer level. In addition, we identified a high degree of temporal persistence of food, patient and mixed clusters, with more than half of the clusters spanning over more than 1 year and up to 10 years. Discussion: Taken together this would indicate that identifying persistent contamination in food production settings, and producers that process a wide variety of raw food produce, could significantly contribute to lowering the Lm disease burden.

One Health surveillance-A cross-sectoral detection, characterization, and notification of foodborne pathogens.

Introduction: Several Proficiency Test (PT) or External Quality Assessment (EQA) schemes are currently available for assessing the ability of laboratories to detect and characterize enteropathogenic bacteria, but they are usually targeting one sector, covering either public health, food safety or animal health. In addition to sector-specific PTs/EQAs for detection, cross-sectoral panels would be useful for assessment of the capacity to detect and characterize foodborne pathogens in a One Health (OH) perspective and further improving food safety and interpretation of cross-sectoral surveillance data. The aims of the study were to assess the cross-sectoral capability of European public health, animal health and food safety laboratories to detect, characterize and notify findings of the foodborne pathogens Campylobacter spp., Salmonella spp. and Yersinia enterocolitica, and to develop recommendations for future cross-sectoral PTs and EQAs within OH. The PT/EQA scheme developed within this study consisted of a test panel of five samples, designed to represent a theoretical outbreak scenario. Methods: A total of 15 laboratories from animal health, public health and food safety sectors were enrolled in eight countries: Denmark, France, Italy, the Netherlands, Poland, Spain, Sweden, and the United Kingdom. The laboratories analyzed the samples according to the methods used in the laboratory and reported the target organisms at species level, and if applicable, serovar for Salmonella and bioserotype for Yersinia. Results: All 15 laboratories analyzed the samples for Salmonella, 13 for Campylobacter and 11 for Yersinia. Analytical errors were predominately false negative results. One sample (S. Stockholm and Y. enterocolitica O:3/BT4) with lower concentrations of target organisms was especially challenging, resulting in six out of seven false negative results. These findings were associated with laboratories using smaller sample sizes and not using enrichment methods. Detection of Salmonella was most commonly mandatory to notify within the three sectors in the eight countries participating in the pilot whereas findings of Campylobacter and Y. enterocolitica were notifiable from human samples, but less commonly from animal and food samples. Discussion: The results of the pilot PT/EQA conducted in this study confirmed the possibility to apply a cross-sectoral approach for assessment of the joint OH capacity to detect and characterize foodborne pathogens.

Prevalence, risk factors and genetic traits of Salmonella Infantis in Dutch broiler flocks.

Salmonella Infantis is a poultry-adapted Salmonella enterica serovar that is increasingly reported in broilers and is also regularly identified among human salmonellosis cases. An emerging S. Infantis mega-plasmid (pESI), carrying fitness, virulence and antimicrobial resistance genes, is also increasingly found. We investigated the prevalence, genetic characteristics and risk factors for (pESI-carrying) S. Infantis in broilers. Faecal samples from 379 broiler flocks (in 198 farms with ≥3000 birds) in the Netherlands were tested. A questionnaire about farm characteristics was also administered. Sampling was performed in July 2018-May 2019, three weeks before slaughter. Fourteen flocks (in 10 farms) were S. Infantis-positive, resulting in a 3.7 % flock-level and 5.1 % farm-level prevalence. Based on multi-locus sequence typing (MLST), all isolates belonged to sequence type 32. All but one isolate carried a pESI-like mega-plasmid. Core-genome MLST showed considerable heterogeneity among the isolates, even within the same farm, with a few small clusters detected. The typical pESI-borne multi-resistance pattern to aminoglycosides, sulphonamide and tetracycline (93 %), as well as trimethoprim (71 %), was found. Additionally, resistance to (fluoro)quinolones based on gyrA gene mutations was detected. S. Infantis was found more often in flocks using salinomycin as coccidiostat, where flock thinning was applied or litter quality was poor, whereas employing external cleaning companies, wheat in feed, and vaccination against infectious bronchitis, were protective. Suggestive evidence for vertical transmission from hatcheries was found. A heterogeneous (pESI-carrying) S. Infantis population has established itself in Dutch broiler flocks, calling for further monitoring of its spread and a comprehensive appraisal of control options.

Genomic Characterization of hlyF-positive Shiga Toxin-Producing Escherichia coli, Italy and the Netherlands, 2000-2019.

Shiga toxin-producing Escherichia coli (STEC) O80:H2 has emerged in Europe as a cause of hemolytic uremic syndrome associated with bacteremia. STEC O80:H2 harbors the mosaic plasmid pR444_A, which combines several virulence genes, including hlyF and antimicrobial resistance genes. pR444_A is found in some extraintestinal pathogenic E. coli (ExPEC) strains. We identified and characterized 53 STEC strains with ExPEC-associated virulence genes isolated in Italy and the Netherlands during 2000-2019. The isolates belong to 2 major populations: 1 belongs to sequence type 301 and harbors diverse stx2 subtypes, the intimin variant eae-ξ, and pO157-like and pR444_A plasmids; 1 consists of strains belonging to various sequence types, some of which lack the pO157 plasmid, the locus of enterocyte effacement, and the antimicrobial resistance-encoding region. Our results showed that STEC strains harboring ExPEC-associated virulence genes can include multiple serotypes and that the pR444_A plasmid can be acquired and mobilized by STEC strains.

Sources and transmission routes of campylobacteriosis: A combined analysis of genome and exposure data.

OBJECTIVES: To determine the contributions of several animal and environmental sources of human campylobacteriosis and identify source-specific risk factors. METHODS: 1417 Campylobacter jejuni/coli isolates from the Netherlands in 2017-2019 were whole-genome sequenced, including isolates from human cases (n = 280), chickens/turkeys (n = 238), laying hens (n = 56), cattle (n = 158), veal calves (n = 49), sheep/goats (n = 111), pigs (n = 110), dogs/cats (n = 100), wild birds (n = 62), and surface water (n = 253). Questionnaire-based exposure data was collected. Source attribution was performed using core-genome multilocus sequence typing. Risk factors were determined on the attribution estimates. RESULTS: Cases were mostly attributed to chickens/turkeys (48.2%), dogs/cats (18.0%), cattle (12.1%), and surface water (8.5%). Of the associations identified, never consuming chicken, as well as frequent chicken consumption, and rarely washing hands after touching raw meat, were risk factors for chicken/turkey-attributable infections. Consuming unpasteurized milk or barbecued beef increased the risk for cattle-attributable infections. Risk factors for infections attributable to environmental sources were open water swimming, contact with dog faeces, and consuming non-chicken/turkey avian meat like game birds. CONCLUSIONS: Poultry and cattle are the main livestock sources of campylobacteriosis, while pets and surface water are important non-livestock sources. Foodborne transmission is only partially consistent with the attributions, as frequency and alternative pathways of exposure are significant.

Genomic epidemiology of emerging ESBL-producing Salmonella Kentucky bla CTX-M-14b in Europe.

Global dissemination of ciprofloxacin-resistant Salmonella Kentucky has been observed over the past decades. In recent years, there have been reports of extended-spectrum ß-lactamase (ESBL) producing S. Kentucky. Routine surveillance at the European Centre for Disease Prevention and Control (ECDC) detected cases with a ciprofloxacin-resistant S. Kentucky with the ESBL-gene bla CTX-M-14b. Ensuing research identified 78 cases in 2013-2018 in eight European countries. Compared to other S. Kentucky and non-typhoidal Salmonella infections, reported to the European Surveillance System, these cases were more likely to be elderly and to present urinary-tract infections. Bayesian time-scaled phylogeny on whole genome sequences of isolates from these cases and supplementary isolates from public sequence databases was used to infer the origin and spread of this clone. We dated the origin of the bla CTX-M-14b clone to approximately 2005 in Northern Africa, most likely in Egypt. The geographic origin predicted by the phylogenetic analysis is consistent with the patients' travel history. Next to multiple introductions of the clone to Europe from Egypt, our analysis suggests that in some parts of Europe the clone might have formed a stable population, from which further spread has occurred. Comparative genomics indicated that the bla CTX-M-14b gene is present on the bacterial chromosome, within the type VI secretion system region. The bla CTX-M-14b gene is integrated downstream of the hcp1 gene, on a 2854 bp plasmid fragment containing also ISEcp1. This is the first report of a chromosomally integrated CTX-M gene in Salmonella spp. in Europe, previous studies having identified similar genes only on plasmids.

Do vegetarians less frequently carry ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae compared with non-vegetarians?

BACKGROUND: ESBL and plasmid-mediated AmpC (pAmpC)-producing Enterobacteriaceae are frequently found on meat products in Dutch retail, especially on poultry. OBJECTIVES: We investigated whether vegetarians are at lower risk of carrying ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae (ESBL-E/K) compared with persons who consume meat. METHODS: Vegetarians, pescatarians (vegetarians who eat fish) and non-vegetarians (persons who eat meat at least three times per week) were asked to send in a faecal sample and a questionnaire. ESBL-E/K were cultured and MLSTs were determined. ESBL/pAmpC genes were analysed using PCR and sequencing. The risk of ESBL-E/K carriage in the three study groups was analysed using multivariable logistic regression. RESULTS: Prevalence of ESBL-E/K carriage was 8.0% in vegetarians (63/785; 95% CI 6.3-10.1), 6.9% in pescatarians (27/392; 95% CI 4.8-9.8) and 3.8% in non-vegetarians (14/365; 95% CI 2.3-6.3). Multivariable analysis showed an OR for ESBL-E/K carriage of 2.2 for vegetarians (95% CI 1.2-4.0) and 1.6 for pescatarians (95% CI 0.8-3.2) compared with non-vegetarians. The predominant MLST was E. coli ST131 and the most common ESBL genes were blaCTX-M-15, blaCTX-M-27, blaCTX-M-14 and blaCTX-M-1 in all diet groups. Independent risk factors for ESBL-E/K carriage were travel to Africa/Latin America/Asia (OR 4.6; 95% CI 2.8-7.7) in the past 6 months and rarely/never washing hands before food preparation (OR 2.5; 95% CI 1.2-5.0). CONCLUSIONS: Vegetarians and pescatarians did not have a lower risk of ESBL-E/K carriage compared with non-vegetarians, indicating that eating meat is not an important risk factor for ESBL-E/K carriage.

Transmission of ESBL-producing Escherichia coli between broilers and humans on broiler farms.

BACKGROUND: ESBL and AmpC ß-lactamases are an increasing concern for public health. Studies suggest that ESBL/pAmpC-producing Escherichia coli and their plasmids carrying antibiotic resistance genes can spread from broilers to humans working or living on broiler farms. These studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these isolates. METHODS: Eleven suspected transmission events among broilers and humans living/working on eight broiler farms were investigated using whole-genome short-read (Illumina) and long-read sequencing (PacBio). Core genome MLST (cgMLST) was performed to investigate the occurrence of strain transmission. Horizontal plasmid and gene transfer were analysed using BLAST. RESULTS: Of eight suspected strain transmission events, six were confirmed. The isolate pairs had identical ESBL/AmpC genes and fewer than eight allelic differences according to the cgMLST, and five had an almost identical plasmid composition. On one of the farms, cgMLST revealed that the isolate pairs belonging to ST10 from a broiler and a household member of the farmer had 475 different alleles, but that the plasmids were identical, indicating horizontal transfer of mobile elements rather than strain transfer. Of three suspected horizontal plasmid transmission events, one was confirmed. In addition, gene transfer between plasmids was found. CONCLUSIONS: The present study confirms transmission of strains as well as horizontal plasmid and gene transfer between broilers and farmers and household members on the same farm. WGS is an important tool to confirm suspected zoonotic strain and resistance gene transmission.

Comparative genomics reveals a lack of evidence for pigeons as a main source of stx2f-carrying Escherichia coli causing disease in humans and the common existence of hybrid Shiga toxin-producing and enteropathogenic E. coli pathotypes.

BACKGROUND: Wild birds, in particular pigeons are considered a natural reservoir for stx2f-carrying E. coli. An extensive comparison of isolates from pigeons and humans from the same region is lacking, which hampers justifiable conclusions on the epidemiology of these pathogens. Over two hundred human and pigeon stx2f-carrying E. coli isolates predominantly from the Netherlands were analysed by whole genome sequencing and comparative genomic analysis including in silico MLST, serotyping, virulence genes typing and whole genome MLST (wgMLST). RESULTS: Serotypes and sequence types of stx2f-carrying E. coli showed a strong non-random distribution among the human and pigeon isolates with O63:H6/ST583, O113:H6/ST121 and O125:H6/ST583 overrepresented among the human isolates and not found among pigeons. Pigeon isolates were characterized by an overrepresentation of O4:H2/ST20 and O45:H2/ST20. Nearly all isolates harboured the locus of enterocyte effacement (LEE) but different eae and tir subtypes were non-randomly distributed among human and pigeon isolates. Phylogenetic core genome comparison demonstrated that the pigeon isolates and clinical isolates largely occurred in separated clusters. In addition, serotypes/STs exclusively found among humans generally were characterized by high level of clonality, smaller genome sizes and lack of several non-LEE-encoded virulence genes. A bundle-forming pilus operon, including bfpA, indicative for typical enteropathogenic E. coli (tEPEC) was demonstrated in 72.0% of the stx2f-carrying serotypes but with distinct operon types between the main pigeon and human isolate clusters. CONCLUSIONS: Comparative genomics revealed that isolates from mild human disease are dominated by serotypes not encountered in the pigeon reservoir. It is therefore unlikely that zoonotic transmission from this reservoir plays an important role in the contribution to the majority of human disease associated with stx2f-producing E. coli in the Netherlands. Unexpectedly, this study identified the common occurrence of STEC2f/tEPEC hybrid pathotype in various serotypes and STs. Further research should focus on the possible role of human-to-human transmission of Stx2f-producing E. coli.

Phylogeographic Analysis Reveals Multiple International transmission Events Have Driven the Global Emergence of Escherichia coli O157:H7.

BACKGROUND: Shiga toxin-producing Escherchia coli (STEC) O157:H7 is a zoonotic pathogen that causes numerous food and waterborne disease outbreaks. It is globally distributed, but its origin and the temporal sequence of its geographical spread are unknown. METHODS: We analyzed whole-genome sequencing data of 757 isolates from 4 continents, and performed a pan-genome analysis to identify the core genome and, from this, extracted single-nucleotide polymorphisms. A timed phylogeographic analysis was performed on a subset of the isolates to investigate its worldwide spread. RESULTS: The common ancestor of this set of isolates occurred around 1890 (1845-1925) and originated from the Netherlands. Phylogeographic analysis identified 34 major transmission events. The earliest were predominantly intercontinental, moving from Europe to Australia around 1937 (1909-1958), to the United States in 1941 (1921-1962), to Canada in 1960 (1943-1979), and from Australia to New Zealand in 1966 (1943-1982). This pre-dates the first reported human case of E. coli O157:H7, which was in 1975 from the United States. CONCLUSIONS: Inter- and intra-continental transmission events have resulted in the current international distribution of E. coli O157:H7, and it is likely that these events were facilitated by animal movements (eg, Holstein Friesian cattle). These findings will inform policy on action that is crucial to reduce the further spread of E. coli O157:H7 and other (emerging) STEC strains globally.

Longitudinal study of ESBL Escherichia coli carriage on an organic broiler farm.

Objectives: To determine the molecular characteristics of ESBL-producing Escherichia coli (ESBL-E) collected during a longitudinal study on an organic broiler farm in order to investigate clonal expansion and horizontal gene transfer. Methods: Isolates were obtained from a longitudinal study performed previously on an organic broiler fattening farm. Samples from individually followed-up broilers, the broiler house, the transport van and persons that took the samples, taken at several timepoints (days 1, 3, 4, 7, 10, 42 and 70) within a production round and during the consecutive one (days 1, 2, 3 and 70), had been investigated for the occurrence of ESBL-E. In the current study, ESBL genes and MLST STs of these ESBL-E were determined. Plasmids were characterized and subtyped. Results: On arrival in round_1, ESBL-E of ST88 predominated, while on days 3, 4, 7 and 10 ST10 was most often found and at slaughter age ST155 and ST1551 prevailed. A shift in STs was also observed in round_2. None of the 35 individually selected broilers followed up in round_1 was positive for the same ESBL-E ST at all sampling times. All isolates carried CTX-M-1 group genes, confirmed as blaCTX-M-1 in 158 isolates. Further analysis of 36 isolates of different STs showed blaCTX-M-1 on IncI1/ST3 plasmids. Conclusions: The rapid dissemination of ESBL-E on this broiler farm was not due to the spread of one specific E. coli clone, but most likely the result of horizontal transfer of an IncI1/ST3 plasmid carrying blaCTX-M-1 resulting in a shift in the predominant ESBL-E population in broilers.

Wild, insectivorous bats might be carriers of Campylobacter spp.

BACKGROUND: The transmission cycles of the foodborne pathogens Campylobacter and Salmonella are not fully elucidated. Knowledge of these cycles may help reduce the transmission of these pathogens to humans. METHODOLOGY/PRINCIPAL FINDINGS: The presence of campylobacters and salmonellas was examined in 631 fresh fecal samples of wild insectivorous bats using a specially developed method for the simultaneous isolation of low numbers of these pathogens in small-sized fecal samples (≤ 0.1 g). Salmonella was not detected in the feces samples, but thermotolerant campylobacters were confirmed in 3% (n = 17) of the bats examined and these pathogens were found in six different bat species, at different sites, in different ecosystems during the whole flying season of bats. Molecular typing of the 17 isolated strains indicated C. jejuni (n = 9), C. coli (n = 7) and C. lari (n = 1), including genotypes also found in humans, wildlife, environmental samples and poultry. Six strains showed unique sequence types. CONCLUSION/SIGNIFICANCE: This study shows that insectivorous bats are not only carriers of viral pathogens, but they can also be relevant for the transmission of bacterial pathogens. Bats should be considered as carriers and potential transmitters of Campylobacter and, where possible, contact between bats (bat feces) and food or feed should be avoided.

Long-term Carriage of Extended-Spectrum ß-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in the General Population in The Netherlands.

Background: This longitudinal study aimed to investigate (risk factors for) persistence of carriage and molecular characteristics of extended-spectrum and plasmid-encoded AmpC ß-lactamase-producing (ESBL/pAmpC) Escherichia coli and Klebsiella pneumoniae (ESBL-E/K) in adults in the Dutch community. Methods: Following a cross-sectional study (ESBL-E/K prevalence, 4.5%), a subset of ESBL-E/K-positive (n = 76) and -negative (n = 249) individuals volunteered to provide 5 monthly fecal samples and questionnaires. ESBL-E/K was cultured using selective enrichment/culture, and multilocus sequence types (MLSTs) were determined. ESBL/pAmpC-genes were analyzed using polymerase chain reaction (PCR) and sequencing. Plasmids were characterized and subtyped by plasmid MLST. Risk factors for persistent carriage were analyzed using logistic regression. Results: Of the initially ESBL-E/K-positive participants, 25 of 76 (32.9%) remained positive in all subsequent samples; 51 of 76 persons (67.1%) tested ESBL-E/K negative at some time point during follow-up, of which 31 (40.8%) stayed negative throughout the longitudinal study. Carriers often carried the same ESBL gene and plasmid, but sometimes in different ESBL-E/K strains, indicative for horizontal transfer of plasmids. Of the 249 initially ESBL-E/K-negative participants, the majority (n = 218 [87.6%]) tested negative during 8 months of follow-up, whereas 31 of 249 (12.4%) participants acquired an ESBL-E/K. Escherichia coli phylogenetic group B2 and D and travel to ESBL high-prevalence countries were associated with prolonged carriage. Conclusions: ESBL-E/K carriage persisted for >8 months in 32.9% of the initially ESBL-positive individuals, while 12.4% of initially negative individuals acquired ESBL-E/K during the study. A single positive test result provides no accurate prediction for prolonged carriage. Acquisition/loss of ESBL-E/K does not seem to be a random process, but differs between bacterial genotypes.

Molecular relatedness of ESBL/AmpC-producing Escherichia coli from humans, animals, food and the environment: a pooled analysis.

Background: In recent years, ESBL/AmpC-producing Escherichia coli (ESBL/AmpC-EC) have been isolated with increasing frequency from animals, food, environmental sources and humans. With incomplete and scattered evidence, the contribution to the human carriage burden from these reservoirs remains unclear. Objectives: To quantify molecular similarities between different reservoirs as a first step towards risk attribution. Methods: Pooled data on ESBL/AmpC-EC isolates were recovered from 35 studies in the Netherlands comprising >27 000 samples, mostly obtained between 2005 and 2015. Frequency distributions of ESBL/AmpC genes from 5808 isolates and replicons of ESBL/AmpC-carrying plasmids from 812 isolates were compared across 22 reservoirs through proportional similarity indices (PSIs) and principal component analyses (PCAs). Results: Predominant ESBL/AmpC genes were identified in each reservoir. PCAs and PSIs revealed close human-animal ESBL/AmpC gene similarity between human farming communities and their animals (broilers and pigs) (PSIs from 0.8 to 0.9). Isolates from people in the general population had higher similarities to those from human clinical settings, surface and sewage water and wild birds (0.7-0.8), while similarities to livestock or food reservoirs were lower (0.3-0.6). Based on rarefaction curves, people in the general population had more diversity in ESBL/AmpC genes and plasmid replicon types than those in other reservoirs. Conclusions: Our 'One Health' approach provides an integrated evaluation of the molecular relatedness of ESBL/AmpC-EC from numerous sources. The analysis showed distinguishable ESBL/AmpC-EC transmission cycles in different hosts and failed to demonstrate a close epidemiological linkage of ESBL/AmpC genes and plasmid replicon types between livestock farms and people in the general population.

Phenotypic Prediction: Linking in vitro Virulence to the Genomics of 59 Salmonella enterica Strains.

The increased availability of whole-genome-sequencing techniques generates a wealth of DNA data on numerous organisms, including foodborne pathogens such as Salmonella. However, how these data can be used to improve microbial risk assessment and understanding of Salmonella epidemiology remains a challenge. The aim of this study was to assess variability in in vitro virulence and genetic characteristics between and within different serovars. The phenotypic behavior of 59 strains of 32 different Salmonella enterica serovars from animal, human and food origin was assessed in an in vitro gastro-intestinal tract (GIT) system and they were analyzed for the presence of 233 putative virulence genes as markers for phenotypic prediction. The probability of in vitro infection, P(inf), defined as the fraction of infectious cells passing from inoculation to host cell invasion at the last stage of the GIT system, was interpreted as the in vitro virulence. Results showed that the (average) P(inf) of Salmonella serovars ranged from 5.3E-05 (S. Kedougou) to 5.2E-01 (S. Typhimurium). In general, a higher P(inf) on serovar level corresponded to higher reported human incidence from epidemiological reporting data. Of the 233 virulence genes investigated, only 101 showed variability in presence/absence among the strains. In vitro P(inf) was found to be positively associated with the presence of specific plasmid related virulence genes (mig-5, pef, rck, and spv). However, not all serovars with a relatively high P(inf), > 1E-02, could be linked with these specific genes. Moreover, some outbreak related strains (S. Heidelberg and S. Thompson) did not reveal this association with P(inf). No clear association with in vitro virulence P(inf) was identified when grouping serovars with the same virulence gene profile (virulence plasmid, Typhoid toxin, peg operon and stk operon). This study shows that the in vitro P(inf) variation among individual strains from the same serovar is larger than that found between serovars. Therefore, ranking P(inf) of S. enterica on serovar level alone, or in combination with a serovar specific virulence gene profile, cannot be recommended. The attribution of single biological phenomena to individual strains or serovars is not sufficient to improve the hazard characterization for S. enterica. Future microbial risk assessments, including virulence gene profiles, require a systematic approach linked to epidemiological studies rather than revealing differences in characteristics on serovar level alone.

Prevalence and types of Campylobacter on poultry farms and in their direct environment.

To study whether broiler and layer farms contribute to the environmental Campylobacter load, environmental matrices at or close to farms, and caecal material from chickens, were examined. Similarity between Campylobacter from poultry and environment was tested based on species identification and Multilocus Sequence Typing. Campylobacter prevalence in caecal samples was 97% at layer farms (n = 5), and 93% at broiler farms with Campylobacter-positive flocks (n = 2/3). Campylobacter prevalence in environmental samples was 24% at layer farms, and 29% at broiler farms with Campylobacter-positive flocks. Campylobacter was detected in soil and surface water, not in dust and flies. Campylobacter prevalence in adjacent and remote surface waters was not significantly (P > 0.1) different. Detected species were C. coli (52%), C. jejuni (40%) and C. lari (7%) in layers, and C. jejuni (100%) in broilers. Identical sequence types (STs) were detected in caecal material and soil. A deviating species distribution in surface water adjacent to farms indicated a high background level of environmental Campylobacter. STs from layer farms were completely deviant from surface water STs. The occasional detection of identical STs in broilers, wastewater at broiler farms and surface water in the farm environment suggested a possible contribution of broiler farms to the aquatic environmental Campylobacter load.

Occurrence and molecular characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses in an equine clinic.

Objectives: To investigate the occurrence and characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses at one equine clinic in the Netherlands. Methods: A total of 91 horses, including residents and patients, were sampled. ESBL/AmpC-producing E. coli were identified by a combination disc diffusion test. Phylogenetic groups and MLST were determined. ESBL/AmpC genes were analysed using PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid MLST. Results: At least one E. coli isolate with a confirmed ESBL/AmpC gene was found in samples from 76 horses (84%). Although phylogenetic group B1 E. coli bla CTX-M-1 predominated, a diverse E. coli population was found, indicating that clonal nosocomial spread was not the only reason for the high occurrence found. MLST analysis revealed the presence of 47 E. coli STs, organized in four clusters of genetically related strains. ST10, ST641, ST1079 and ST1250 were most commonly found. With regard to the genes, bla CTX-M-1 was most prevalent ( n = 91), followed by bla CTX-M-2 ( n = 26). The most frequently found plasmid type was IncHI1, but plasmids belonging to the IncF, IncI1 and IncN groups were also identified. Conclusions: A high occurrence of ESBL-producing E. coli in faecal samples was found among horses in an equine clinic and the variety of STs, ESBL genes and plasmid types suggests nosocomial transmission. ESBL E. coli can cause difficult-to-treat infections in horses and prudent use of antimicrobials is warranted. A further assessment of the risks of transmission to persons in close contact with horses, such as caretakers or veterinarians, is crucial.

Transmission dynamics of extended-spectrum ß-lactamase and AmpC ß-lactamase-producing Escherichia coli in a broiler flock without antibiotic use.

Extended-spectrum and AmpC ß-lactamase-producing Escherichia coli (ESBL/AmpC-EC) are found throughout the broiler production pyramid. Transmission of resistance between broilers and humans could occur at any point, representing a potential public health issue. Insight in farm transmission dynamics could provide a basis for control, leading to fewer contaminated broilers. The aim was quantifying transmission rates and routes of ESBL/AmpC-EC, and specific phylogenetic groups, in an organic broiler flock without antibiotic use. In each of two consecutive production rounds, 80 randomly chosen broilers were followed individually. Cloacal swabs from these, 20 other randomly chosen broilers, and 11 environmental samples were taken at several moments from arrival till slaughter. ESBL/AmpC-EC were isolated by selective pre-enrichment, and ESBL/AmpC-genes and E. coli phylogenetic groups were determined. Transmission parameters (ß) were estimated using a Generalised Linear Model with a susceptible-infectious-susceptible model. Effect of direct broiler contact as compared to contact through the environment and previous carriage c.q. infectious status and their interaction were included as explanatory variables. Multiplying ß by the length of the infectious period gives the reproduction ratio (R). On day 1, prevalence was 28.8% (95%CI 19.2-40.0%) and 0.0% (95%CI 0.0-4.5%) among individually followed broilers, in round 1 and 2 respectively. In round 2, the environment was positive before arrival of day-old chicks. After 3 days, almost 100% of broilers and environmental samples were positive in both rounds. Most samples were positive for CTX-M-1 group genes, and A1 and B1 were predominant phylogenetic groups. From day 3 there was a shift towards more phylogenetic groups. R was 1.70 (95%CI 0.55-5.25) for total ESBL/AmpC-EC. Risk for broilers to become infectious was lower if previously infectious (ßpreviously infectious=0.02 vs. ßnot previously infectious=3.41; P<0.0001). For phylogenetic groups separately, R was 0.88 (95%CI 0.38-2.07), 0.51 (95%CI 0.27-0.98), 0.99 (95%CI 0.65-1.51) for A1, B1 and rest (i.e. A0, B2, D1, D2) groups, respectively. The interaction effect for A1 and B1 was reflected in the fact that when broilers were previous infectious, the environment was relatively more important for transmission of the A1 group, while this was direct contact between broilers for the B1 group. Positive day-old chicks and the environment both play a role in introduction and transmission of ESBL/AmpC-EC in flocks. These results suggest that, even without selective pressure from antibiotics, total ESBL/AmpC-EC persistence, and resulting endemic situation, seem to be caused by shifts in carriage of different phylogenetic groups. It implies that contaminated broilers enter the slaughterhouse.