Clostridioides difficile in calves, cattle and humans from Dutch dairy farms: Predominance of PCR ribotype 695 (clade 5, sequence type 11) in cattle.

Background: Clostridioides difficile is a leading cause of infectious diarrhea in both humans and livestock. In particular, C. difficile strains belonging to sequence type (ST) 11 are common enteropathogens. The aim of this study was to determine the presence and genetic relatedness of C. difficile types in dairy cattle and calves. Method: Dutch dairy farms were visited between February and December 2021. Feces was collected from adult dairy cattle and calves of two age categories (<4 weeks and 4 weeks-4 months). Fecal samples were also requested from dairy farmers, family members and employees. Fecal samples were cultured in an enrichment medium for 10-15 days and subcultured on solid media for capillary PCR ribotyping and whole genome sequencing. Results: C. difficile was detected on 31 out of 157 (19.8%) dairy farms. The highest prevalence was found in calves <4 weeks (17.5%). None of the 99 human samples collected were positive. Thirty-seven cultured isolates belonged to 11 different PCR ribotypes (RT) of which RT695 (56.8%) and RT078/126 (16.2%) were most abundant. In the database of the Netherlands National Expertise Centre for C. difficile infections (CDI, >10.000 patient isolates), RT695 was found in only two patients with hospital-onset CDI, diagnosed in 2020 and 2021. Sequence analysis of 21C. difficile RT695 from cattle revealed that all isolates belonged to clade 5, ST11 and contained genes encoding toxin A, toxin B and binary toxin. RT695 strains carried antimicrobial resistance genes typically found in clade 5C. difficile. Groups of genetically related RT695 isolates were found between dairy farms, whereas identical strains were only present in individual farms. Conclusions: C. difficile was found in ∼20% of dairy farms with a predominance of the relatively unknown RT695. Isolates of RT695 belonged to the same clade and sequence type as RT078/126, which is recognized as an important zoonotic type.

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.

Implementation of WGS analysis of ESBL-producing Escherichia coli within EU AMR monitoring in livestock and meat.

BACKGROUND: As WGS comes of age, changes in EU legislation implemented in 2021 allow its usage for systematic monitoring of ESBL-producing Escherichia coli from livestock and meat, replacing phenotypic testing. Presently, phenotypic testing correlates well with antimicrobial resistance predicted from WGS data. WGS has added value in the wealth of additional information that is present in the data. OBJECTIVES: In this study we have detected the resistance phenotypes for a panel of antimicrobials while also analysing the molecular epidemiology of ESBL-producing E. coli. METHODS: Susceptibility testing was performed with broth microdilution of selectively isolated E. coli. Short-read WGS was performed in parallel and phenotypes predicted based on the sequence data, which was also used to determine the phylogeny of the isolates. RESULTS: The phenotypically determined resistance and the predicted resistance correlated 90%-100% for the different antimicrobial classes. Furthermore, clonal relationships were detected amongst ESBL-producing E. coli within livestock sectors and the meat produced by this sector. CONCLUSIONS: Further implementation of WGS analysis of ESBL/AmpC-producing E. coli within the AMR monitoring programme of EU member states and global surveillance programmes will contribute to determining the attribution of livestock in the prevalence of ESBL/AmpC-encoding E. coli in humans.

cfr and fexA genes in methicillin-resistant Staphylococcus aureus from humans and livestock in the Netherlands.

Background: Although the Netherlands is a country with a low endemic level of methicillin-resistant Staphylococcus aureus (MRSA), a national MRSA surveillance has been in place since 1989. In 2003 livestock emerged as a major reservoir of MRSA and currently livestock-associated MRSA (clonal complex CC398) make up 25% of all surveillance isolates. To assess possible transfer of resistant strains or resistance genes, MRSA obtained from humans and animals were characterized in detail. Methods: The sequenced genomes of 6327 MRSA surveillance isolates from humans and from 332 CC398 isolates from livestock-related samples were analyzed and resistance genes were identified. Several isolates were subjected to long-read sequencing to reconstruct chromosomes and plasmids. Results: Here we show the presence of the multi-resistance gene cfr in seven CC398 isolates obtained from humans and in one CC398 isolate from a pig-farm dust sample. Cfr induces resistance against five antibiotic classes, which is true for all but two isolates. The isolates are genetically unrelated, and in seven of the isolates cfr are located on distinct plasmids. The fexA gene is found in 3.9% surveillance isolates and in 7.5% of the samples from livestock. There is considerable sequence variation of fexA and geographic origin of the fexA alleles. Conclusions: The rare cfr and fexA resistance genes are found in MRSA from humans and animals in the Netherlands, but there is no evidence for spread of resistant strains or resistance plasmids. The proportion of cfr-positive MRSA is low, but its presence is worrying and should be closely monitored.

Surveillance and characteristics of food-borne outbreaks in the Netherlands, 2006 to 2019.

BackgroundA wide variety of pathogens can cause disease in humans via consumption of contaminated food. Although food-borne outbreaks only account for a small part of the food-borne disease burden, outbreak surveillance can provide insights about the pathogens, food products implied as vehicle, points of contamination, and the settings in which transmission occurs.AimTo describe the characteristics of food-borne outbreaks registered between 2006 and 2019 in the Netherlands.MethodsAll reported outbreaks in which the first case occurred during 2006-19 were analysed. We examined the number of outbreaks, cases and setting by year, aetiology, type of evidence and food commodities.ResultsIn total, 5,657 food-borne outbreaks with 27,711 cases were identified. The contaminated food product could be confirmed in 152 outbreaks (2.7%); in 514 outbreaks (9.1%), a pathogen was detected in cases and/or environmental swabs. Norovirus caused most outbreaks (205/666) and most related cases (4,436/9,532), followed by Salmonella spp. (188 outbreaks; 3,323 cases) and Campylobacter spp. (150 outbreaks; 601 cases). Bacillus cereus was most often found in outbreaks with a confirmed food vehicle (38/152). Additionally, a connection was seen between some pathogens and food commodities. Public eating places were most often mentioned as a setting where the food implicated in the outbreak was prepared.ConclusionLong-term analysis of food-borne outbreaks confirms a persistent occurrence. Control and elimination of food-borne illness is complicated since multiple pathogens can cause illness via a vast array of food products and, in the majority of the outbreaks, the pathogen remains unknown.

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.

Impact of the COVID-19 pandemic on human salmonellosis in the Netherlands.

The public health measures implemented to control coronavirus disease 2019 (COVID-19) may influence also other infectious diseases. Using national laboratory surveillance data, we assessed the impact of the COVID-19 pandemic on human salmonellosis in the Netherlands until March 2021. Salmonellosis incidence decreased significantly after March 2020: in the second, third and fourth quarters of 2020, and in the first quarter of 2021, the incidence decreased by 55%, 57%, 47% and 37%, respectively, compared to the same quarters of 2016-2019. The decrease was strongest among travel-related cases (94%, 84%, 79% and 93% in the aforementioned quarters, respectively). Other significant changes were: increased proportion of cases among older adults and increased proportion of invasive infections, decreased proportion of trimethoprim resistance and increased proportion of serovar Typhimurium monophasic variant vs. Enteritidis. This led to decreased contributions of laying hens and increased contributions of pigs and cattle as sources of human infections. The observed changes probably reflect a combination of reduced exposure to Salmonella due to restrictions on international travels and gatherings, closure of dine-in restaurants, catering and hospitality sectors at large and changes in healthcare-seeking and diagnostic behaviours.

Characterization and whole genome sequencing of closely related multidrug-resistant Salmonella enterica serovar Heidelberg isolates from imported poultry meat in the Netherlands.

Multidrug-resistant Salmonella enterica serovar Heidelberg isolates are frequently recovered in the Netherlands from poultry meat imported from South America. Our aim was to retrospectively assess the characteristics of the antimicrobial determinants, gene content and the clonal relatedness of 122 unique S. Heidelberg isolates from chicken meat from Brazil (n = 119) and Argentina (n = 3) that were imported between 2010 and 2015. These isolates were subjected to antimicrobial susceptibility testing, PCR and Illumina HiSeq2500 whole genome sequencing. Draft genomes were assembled to assess the gene content, and the phylogenetic relationships between isolates were determined using single nucleotide polymorphisms. Ciprofloxacin-resistance was identified in 98.4% of the isolates and 83.7% isolates showed resistance to the extended-spectrum cephalosporins cefotaxime and ceftazidime (83.6% and 82.8% respectively). Of the latter, 97.1% exhibited an AmpC phenotype and contained blaCMY-2, whereas the remaining three isolates contained an extended spectrum beta-lactamase. Of the 99 extended-spectrum cephalosporins-resistant isolates harboring CMY-2 plasmids, 56.6% contained the incompatibility group I1 replicon. Phylogenetic cluster analysis showed that all isolates from Brazil clustered together, with 49% occurring in clusters larger than 5 isolates that revealed intra-cluster similarities based on geographical location and/or resistance profiles. The remaining isolates were classified in smaller clusters or as singletons, highlighting the large diversity of S. Heidelberg in the poultry chain in Brazil that was revealed by this study. Considering the potential public health risk associated with multidrug-resistant S. Heidelberg in imported poultry, collaborative whole genome sequencing-based surveillance is needed to monitor the spread, pathogenic properties and epidemiological distribution of these isolates.

Novel Carbapenemases FLC-1 and IMI-2 Encoded by an Enterobacter cloacae Complex Isolated from Food Products.

Food for human consumption is screened widely for the presence of antibiotic-resistant bacteria to assess the potential for transfer of resistant bacteria to the general population. Here, we describe an Enterobacter cloacae complex isolated from imported seafood that encodes two carbapenemases on two distinct plasmids. Both enzymes belong to Ambler class A ß-lactamases, the previously described IMI-2 and a novel family designated FLC-1. The hydrolytic activity of the novel enzyme against aminopenicillins, cephalosporins, and carbapenems was determined.

Diversity of Plasmids and Genes Encoding Resistance to Extended Spectrum Cephalosporins in Commensal Escherichia coli From Dutch Livestock in 2007-2017.

Extended-spectrum ß-lactamase (ESBL) and plasmid-mediated AmpC ß-lactamase (pAmpC) genes confer resistance to extended spectrum cephalosporin's. The spread of these genes is mostly facilitated by plasmid-mediated horizontal transfer. National surveillance activities to detect ESBL/pAmpC-producers in commensal bacteria from livestock are in place in the Netherlands since several years. This study aimed at reporting gene and plasmid diversity of commensal ESBL/pAmpC-producing Escherichia coli isolated from healthy animals during surveillance activities between 2007 and 2017. A collection of 2304 extended-spectrum cephalosporin-resistant (ESC-R) E. coli isolated from feces of broilers, dairy cattle, slaughter pigs, turkeys, ducks, and veal calves was investigated and ESBL/pAmpC genes were determined. Gene location of a selection of 473 E. coli isolates was determined and typing of plasmids linked to the ESBL/pAmpC genes was performed. Twenty-two different ESBL/pAmpC genes were identified with bla CTX-M-1 being the most prevalent gene in livestock (43.7%), followed by bla CMY -2 and bla SHV -12, independent of the animal source. Prevalence of typically human associated bla CTX-M-15 was highest in cattle. Less than 10% E. coli isolates owed their ESC-R phenotype to promoter mutations of the chromosomal ampC gene. Majority (92%) of ESBL/pAmpC genes analyzed were plasmid located, with IncI1α being the most represented plasmid family in isolates from all animals, followed by IncF (veal calves, dairy cattle and slaughter pigs), IncK (broilers and laying hens), IncX1 in broilers, and emerging IncX3 in broilers and dairy cattle. Prevalence and molecular diversity of ESC-R E. coli isolated from livestock over an 11-year period revealed a composite scenario of gene-plasmid combinations.

A cross sectional study on Dutch layer farms to investigate the prevalence and potential risk factors for different Chlamydia species.

In poultry several Chlamydia species have been detected, but Chlamydia psittaci and Chlamydia gallinacea appear to be most prevalent and important. Chlamydia psittaci is a well-known zoonosis and is considered to be a pathogen of poultry. Chlamydia gallinacea has been described more recently. Its avian pathogenicity and zoonotic potential have to be further elucidated. Within the Netherlands no data were available on the presence of Chlamydia on poultry farms. As part of a surveillance programme for zoonotic pathogens in farm animals, we investigated pooled faecal samples from 151 randomly selected layer farms. On a voluntary base, 69 farmers, family members or farm workers from these 151 farms submitted a throat swab. All samples were tested with a generic 23S Chlamydiaceae PCR followed by a species specific PCR for C. avium, C. gallinacea and C. psittaci. C. avium and psittaci DNA was not detected at any of the farms. At 71 farms the positive result could be confirmed as C. gallinacea. Variables significantly associated with the presence of C. gallinacea in a final multivariable model were 'age of hens,' 'use of bedding material' and 'the presence of horses.' The presence of C. gallinacea was associated with neither clinical signs, varying from respiratory symptoms, nasal and ocular discharges to diarrhoea, nor with a higher mortality rate the day before the visit. All throat swabs from farmers, family members or farm workers tested negative for Chlamydia DNA, giving no further indication for possible bird-to-human (or human-to-bird) transmission.

Extended-Spectrum Cephalosporin-Resistant Salmonella enterica serovar Heidelberg Strains, the Netherlands(1).

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains (JF6X01.0022/XbaI.0251, JF6X01.0326/XbaI.1966, JF6X01.0258/XbaI.1968, and JF6X01.0045/XbaI.1970) have been identified in the United States with pulsed-field gel electrophoresis. Our examination of isolates showed introduction of these strains in the Netherlands and highlight the need for active surveillance and intervention strategies by public health organizations.

Enterobacteriaceae resistant to third-generation cephalosporins and quinolones in fresh culinary herbs imported from Southeast Asia.

Since multidrug resistant bacteria are frequently reported from Southeast Asia, our study focused on the occurrence of ESBL-producing Enterobacteriaceae in fresh imported herbs from Thailand, Vietnam and Malaysia. Samples were collected from fresh culinary herbs imported from Southeast Asia in which ESBL-suspected isolates were obtained by selective culturing. Analysis included identification by MALDI-TOF mass spectrometry, susceptibility testing, XbaI-PFGE, microarray, PCR and sequencing of specific ESBL genes, PCR based replicon typing (PBRT) of plasmids and Southern blot hybridization. In addition, the quinolone resistance genotype was characterized by screening for plasmid mediated quinolone resistance (PMQR) genes and mutations in the quinolone resistance determining region (QRDR) of gyrA and parC. The study encompassed fifty samples of ten batches of culinary herbs (5 samples per batch) comprising nine different herb variants. The herbs originated from Thailand (Water morning glory, Acacia and Betel leaf), Vietnam (Parsley, Asian pennywort, Houttuynia leaf and Mint) and Malaysia (Holy basil and Parsley). By selective culturing 21 cefotaxime resistant Enterobacteriaceae were retrieved. Array analysis revealed 18 isolates with ESBL genes and one isolate with solely non-ESBL beta-lactamase genes. Mutations in the ampC promoter region were determined in two isolates with PCR and sequencing. The isolates were identified as Klebsiella pneumoniae (n=9), Escherichia coli (n=6), Enterobacter cloacae complex (n=5) and Enterobacter spp. (n=1). All isolates tested were multidrug resistant. Variants of CTX-M enzymes were predominantly found followed by SHV enzymes. PMQR genes (including aac(6')-1b-cr, qnrB and qnrS) were also frequently detected. In almost all cases ESBL and quinolone resistance genes were located on the same plasmid. Imported fresh culinary herbs from Southeast Asia are a potential source for contamination of food with multidrug resistant bacteria. Because these herbs are consumed without appropriate heating, transfer to human bacteria cannot be excluded.

Cross-sectional study on prevalence and molecular characteristics of plasmid mediated ESBL/AmpC-producing Escherichia coli isolated from veal calves at slaughter.

OBJECTIVES: The presence of ESBL/AmpC-producing E. coli in cattle has been reported previously, however information on veal calves is limited. This study describes the prevalence and molecular characteristics of E. coli with non-wild type susceptibility to cefotaxime in veal calves at slaughter. METHODS: Faecal samples from 100 herds, 10 individual animals per herd, were screened for E. coli with non-wild type susceptibility for cefotaxime. Molecular characterization of ESBL/AmpC genes and plasmids was performed on one isolate per herd by microarray, PCR and sequence analysis. RESULTS: 66% of the herds were positive for E. coli with non-wild type susceptibility for cefotaxime. Within-herd prevalence varied from zero to 90%. 83% of E. coli producing ESBL/AmpC carried bla(CTX-M) genes, of which bla(CTX-M-1), bla(CTX-M-14) and bla(CTX-M-15) were most prevalent. The dominant plasmids were IncI1 and IncF-type plasmids. CONCLUSIONS: A relatively high prevalence of various bla(CTX-M) producing E. coli was found in veal calves at slaughter. The genes were mainly located on IncI1 and IncF plasmids.