Temporal association of antimicrobial use in livestock with antimicrobial resistance in non-typhoid Salmonella human infections in the Netherlands, 2008-2019.

Background: Antimicrobial use (AMU) in livestock contributes to antimicrobial resistance (AMR) among zoonotic pathogens, such as non-typhoid Salmonella (NTS). Since 2009, the Netherlands has made substantial efforts to reduce AMU in livestock. Objectives: To assess the association between AMU in livestock and AMR in NTS human isolates. Additionally, associations between AMU in broilers/pigs and AMR in NTS broiler/pig isolates, and between AMR in broilers/pigs and in human NTS isolates were assessed. The focus was on Salmonella Enteritidis (SE) and Salmonella Typhimurium including its monophasic variant (ST/STM). Methods: A national population registry-based study was conducted in the Netherlands from 2008 to 2019. Multivariable logistic regression models were used to assess the associations between livestock AMU and NTS resistance proportion in humans and broilers/pigs, overall as well as per class-specific antimicrobials. Correlation analysis was performed to relate AMR proportions between human and broiler/pig NTS isolates. Results: For SE, only a positive association between penicillins use in broilers and resistance to ampicillin among human isolates was significant. For ST/STM, most associations between AMU in livestock and AMR among human isolates were significantly positive, overall and per class-specific antimicrobials, namely for penicillins-ampicillin, tetracyclines-tetracycline and sulfonamides/trimethoprim-sulfamethoxazole/trimethoprim. Significantly positive associations between AMU in broilers/pigs and AMR in broiler/pig ST/STM isolates were also observed, but not between broiler/pig and human AMR levels. Conclusions: Significant associations were generally found between livestock AMU and AMR in human and broiler/pig ST/STM isolates. However, confounding factors, such as imported meat and travel are of concern. To fully comprehend the impact of livestock AMU on resistance in human NTS isolates, it is imperative to enhance AMR surveillance of NTS.

The use of aminopenicillins in animals within the EU, emergence of resistance in bacteria of animal and human origin and its possible impact on animal and human health.

Aminopenicillins have been widely used for decades for the treatment of various infections in animals and humans in European countries. Following this extensive use, acquired resistance has emerged among human and animal pathogens and commensal bacteria. Aminopenicillins are important first-line treatment options in both humans and animals, but are also among limited therapies for infections with enterococci and Listeria spp. in humans in some settings. Therefore, there is a need to assess the impact of the use of these antimicrobials in animals on public and animal health. The most important mechanisms of resistance to aminopenicillins are the ß-lactamase enzymes. Similar resistance genes have been detected in bacteria of human and animal origin, and molecular studies suggest that transmission of resistant bacteria or resistance genes occurs between animals and humans. Due to the complexity of epidemiology and the near ubiquity of many aminopenicillin resistance determinants, the direction of transfer is difficult to ascertain, except for major zoonotic pathogens. It is therefore challenging to estimate to what extent the use of aminopenicillins in animals could create negative health consequences to humans at the population level. Based on the extent of use of aminopenicillins in humans, it seems probable that the major resistance selection pressure in human pathogens in European countries is due to human consumption. It is evident that veterinary use of these antimicrobials increases the selection pressure towards resistance in animals and loss of efficacy will at minimum jeopardize animal health and welfare.

Genomic comparison of mecC-carrying methicillin-resistant Staphylococcus aureus from hedgehogs and humans in the Netherlands.

OBJECTIVES: MRSA carrying the mecC gene (mecC-MRSA) have been found in humans and animals worldwide. A high carriage rate of mecC-MRSA has been described among hedgehogs in different countries. We performed genomic comparison of mecC-MRSA from hedgehogs and humans using next-generation sequencing (NGS) to investigate possible zoonotic transmission in the Netherlands. METHODS: Nasal swabs from hedgehogs (n = 105) were cultured using pre-enrichment and selective plates. Isolates were sequenced using Illumina NGS platforms. These data were compared with sequence data of mecC-MRSA (n = 62) from the Dutch national MRSA surveillance in humans. RESULTS: Fifty hedgehogs were found to be MRSA positive, of which 48 carried mecC. A total of 60 mecC-MRSA isolates derived from 50 hedgehogs were compared with the human isolates. Fifty-nine mecC-MRSA from hedgehogs and all but one isolate from humans belonged to clonal complexes CC130 and CC1943. The mecC gene was located within the SCCmec XI element. Most mecC-MRSA did not carry other resistance genes besides mecC and blaZ. Two human isolates carried erm(C). Isolates differed in the presence of various virulence genes, which were linked to distinct STs and clonal complexes. Some isolates had up to 17 virulence genes, which underlines their pathogenic potential. No genetic clusters of hedgehog and human isolates were found. CONCLUSIONS: mecC-MRSA from hedgehogs and humans mainly belonged to the same two clonal complexes, indicating a common source. No firm evidence for recent zoonotic transmission was found. Further studies are needed to investigate the role of hedgehogs in the occurrence of mecC-MRSA in humans.

Horsing around: Escherichia coli ST1250 of equine origin harbouring epidemic IncHI1/ST9 plasmid with bla CTX-M-1 and an operon for short-chain fructooligosaccharides metabolism.

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.

Species-Specific Patterns of Gut Metabolic Modules in Dutch Individuals with Different Dietary Habits.

Diet is an important determinant of the human gut microbiome. Here, we analyzed fecal metagenomes of Dutch adults following omnivorous, pescatarian, vegan, and vegetarian diets. We compared the taxonomic composition of individuals from our study with publicly available gut metagenomes from westernized and non-westernized societies. We observed that, despite long-term transition to diets rich in plant fibers (vegan or vegetarian), the microbiomes of these were typical of westernized populations, and similar in composition to omnivores. Although there were no major differences in metabolic modules, we identified differences in the species that contributed to particular functions, such as carbohydrate degradation and short-chain fatty acid metabolism. Overall, this study shows functional redundancy of the microbiomes among westernized populations, which is independent of long-term individual dietary habits. IMPORTANCE Diet is an important modulator of the human gut microbiome, which is susceptible to increased consumption of plant fibers in vegan or vegetarian lifestyles. To investigate this, we compared the gut microbiome of Dutch adults following omnivorous, pescatarian, vegan and vegetarian diets. We did not observe major differences in the gut microbiome composition and function between individuals with different dietary habits. However, we observed differences in the species that contribute to the core functions of the gut microbiome. Our study thus emphasizes the need to better understand the species-specific functional changes associated with dietary habits in the human gut microbiome.

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.

Rhodococcus equiU19 strain harbors a nonmobilizable virulence plasmid.

Rhodococcus equiis the causative agent of pyogenic pneumonia in foals, and a virulence-associated protein A (VapA) encoded on the pVAPA virulence plasmid is important for its pathogenicity. In this study, we analyzed the virulence of R. equi strain U19, originally isolated in the Netherlands in 1997 and the genetic characteristics of the pVAPA_U19 plasmid. U19 expressed VapA that was regulated by temperature and pH and underwent significant intracellular proliferation in macrophages. The restriction fragment length polymorphism of pVAPA_U19 digested with EcoRI was similar to that of pREAT701 (85 kb Type I) harbored by R. equi ATCC33701, although the band pattern at 10-20 kb differed. Whole-genome sequencing showed that pVAPA_U19 was 51,684 bp in length and that the vapA pathogenicity island region and the replication/participation were almost identical to those in pREAT701. By contrast, the open reading frames (ORF26-ORF45) genes of pREAT701 (approximately 29,000 bp) were absent from pVAPA_U19. In this lacking region, mobility (MOB) genes, such as relaxase, which allow conjugative DNA processing, and the mating pair formation (MPF) genes, which are a form of the Type IV secretion system and provide the mating channel, were present. Coculture between U19 and five different recipient strains (two plasmid-cured strains and three cryptic plasmid-harboring strains) demonstrated that pVAPA_U19 could not support conjugation. Therefore, pVAPA_U19 does not differ significantly from the previously reported pVAPA in terms of virulence and plasmid replication and maintenance but is a nonmobilizable plasmid unable to cause conjugation because of the absence of genes related to MOB and MPF.

Faecal carriage of Clostridioides difficile is low among veterinary healthcare workers in the Netherlands.

Veterinary healthcare workers are in close contact with many different animals and might be at an increased risk of acquiring Clostridioides difficile. In this cross-sectional study, we assessed the prevalence and risk factors of C. difficile carriage in Dutch veterinary healthcare workers. Participants provided a faecal sample and filled out a questionnaire covering potential risk factors for C. difficile carriage. C. difficile culture positive isolates were polymerase chain reaction (PCR) ribotyped and the presence of toxin genes tcdA, tcdB and cdtA/cdtB was determined. Eleven of 482 [2.3%; 95% confidence interval (CI) 1.3-4.0] veterinary healthcare workers were carriers of C. difficile. Three persons carried C. difficile ribotype 078 (0.6%; 95% CI 0.2-1.8). Risk factors for carriage were health/medication and hygiene related, including poor hand hygiene after patient (animal) contact, and did not include occupational contact with certain animal species. In conclusion, the prevalence of C. difficile carriage in veterinary healthcare workers was low and no indications were found that working in veterinary care is a risk for C. difficile carriage.

Impact of long-term dietary habits on the human gut resistome in the Dutch population.

The human gut microbiome plays a central role in health and disease. Environmental factors, such as lifestyle and diet, are known to shape the gut microbiome as well as the reservoir of resistance genes that these microbes harbour; the resistome. In this study we assessed whether long-term dietary habits within a single geographical region (the Netherlands) impact the human gut resistome. Faecal samples from Dutch omnivores, pescatarians, vegetarians and vegans were analysed by metagenomic shotgun sequencing (MSS) (n = 149) and resistome capture sequencing approach (ResCap) (n = 64). Among all diet groups, 119 and 145 unique antibiotic resistance genes (ARGs) were detected by MSS or ResCap, respectively. Five or fifteen ARGs were shared between all diet groups, based on MSS and ResCap, respectively. The total number of detected ARGs by MSS or ResCap was not significantly different between the groups. MSS also revealed that vegans have a distinct microbiome composition, compared to other diet groups. Vegans had a lower abundance of Streptococcus thermophilus and Lactococcus lactis compared to pescatarians and a lower abundance of S. thermophilus when compared to omnivores. In summary, our study showed that long-term dietary habits are not associated with a specific resistome signature.

ESBL/pAmpC-producing Escherichia coli and Klebsiella pneumoniae carriage among veterinary healthcare workers in the Netherlands.

BACKGROUND: Animals are a reservoir for ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae (ESBL-E/K). We investigated the association between occupational contact with different types of animals and the prevalence of ESBL-E/K carriage among veterinary healthcare workers, assessed molecular characteristics of ESBL-E/K, and followed-up on the ESBL-E/K carriage status of participants and their household members. METHODS: Participants completed a questionnaire about their contact with animals at work and at home, health status, travel behaviour and hygiene, and sent in a faecal sample which was tested for the presence of ESBL-E/K. Resistance genes were typed using PCR and sequencing. ESBL-E/K positive participants and their household members were followed up after 6 months. Risk factors were analysed using multivariable logistic regression methods. RESULTS: The prevalence of ESBL-E/K carriage was 9.8% (47/482; 95%CI 7.4-12.7). The most frequently occurring ESBL genes were blaCTX-M-15, blaCTX-M-14 and blaDHA-1. The predominant sequence type was ST131. None of the occupation related factors, such as contact with specific animal species, were significantly associated with ESBL-E/K carriage, whereas travel to Africa, Asia or Latin America in the past 6 months (OR 4.4), and stomach/bowel complaints in the past 4 weeks (OR 2.2) were. Sixteen of 33 initially ESBL-E/K positive participants (48.5%) tested positive again 6 months later, in 14 persons the same ESBL gene and E. coli ST was found. Four of 23 (17.4%) household members carried ESBL-E/K, in three persons this was the same ESBL gene and E. coli ST as in the veterinary healthcare worker. CONCLUSIONS: Despite the absence of specific occupation related risk factors, ESBL-E/K carriage in veterinary healthcare workers was high compared to the prevalence in the general Dutch population (5%). This indicates that occupational contact with animals is a potential source of ESBL-E/K for the population at large.

The extent of carbapenemase-encoding genes in public genome sequences.

Genome sequences provide information on the genetic elements present in an organism, and currently there are databases containing hundreds of thousands of bacterial genome sequences. These repositories allow for mining patterns concerning antibiotic resistance gene occurrence in both pathogenic and non-pathogenic bacteria in e.g. natural or animal environments, and link these to relevant metadata such as bacterial host species, country and year of isolation, and co-occurrence with other resistance genes. In addition, the advances in the prediction of mobile genetic elements, and discerning chromosomal from plasmid DNA, broadens our view on the mechanism mediating dissemination. In this study we utilize the vast amount of data in the public database PATRIC to investigate the dissemination of carbapenemase-encoding genes (CEGs), the emergence and spread of which is considered a grave public health concern. Based on publicly available genome sequences from PATRIC and manually curated CEG sequences from the beta lactam database, we found 7,964 bacterial genomes, belonging to at least 70 distinct species, that carry in total 9,892 CEGs, amongst which bla NDM, bla OXA, bla VIM, bla IMP and bla KPC. We were able to distinguish between chromosomally located resistance genes (4,137; 42%) and plasmid-located resistance genes (5,753; 58%). We found that a large proportion of the identified CEGs were identical, i.e. displayed 100% nucleotide similarity in multiple bacterial species (8,361 out of 9,892 genes; 85%). For example, the New Delhi metallo-beta-lactamase NDM-1 was found in 42 distinct bacterial species, and present in seven different environments. Our data show the extent of carbapenem-resistance far beyond the canonical species Acetinobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. These types of data complement previous systematic reviews, in which carbapenem-resistant Enterobacteriaceae were found in wildlife, livestock and companion animals. Considering the widespread distribution of CEGs, we see a need for comprehensive surveillance and transmission studies covering more host species and environments, akin to previous extensive surveys that focused on extended spectrum beta-lactamases. This may help to fully appreciate the spread of CEGs and improve the understanding of mechanisms underlying transmission, which could lead to interventions minimizing transmission to humans.

Heterogeneity of antimicrobial susceptibility testing results for sulfamethoxazole/trimethoprim obtained from clinical equine Staphylococcus aureus isolates using different methods.

Based on antimicrobial susceptibility testing (AST), correct classifications as susceptible, intermediate or resistant are challenging for some antimicrobial agent-bacterial species combinations. In this study, we investigated 19 equine Staphylococcus aureus isolates for their susceptibility to the combination sulfamethoxazole/trimethoprim (SXT) by using broth microdilution (BMD), agar disk diffusion (DD) and automated test systems. To elucidate the presence of the corresponding genetic resistance properties among the isolates, whole genome sequence analysis was performed and the genomes were screened for trimethoprim (TMP) resistance genes and mutations in the deduced FolP amino acid (aa) sequences, known to confer sulfonamide resistance. To check for hetero-resistance, zone diameters in DD were screened after 18 and 42 h of incubation. All 19 isolates harboured one of the TMP resistance genes dfrG or dfrS1. Three isolates had an aa exchange in their FolP aa sequence (F17L), which has previously been described to result in sulfonamide resistance. These isolates were classified as SXT-resistant by all methods. The remaining 16 isolates were classified as SXT-susceptible or -intermediate (BMD and/or DD) or SXT-resistant (mainly automated test systems). None of the isolates had relevant aa variations in their FolP aa sequences. All 19 isolates showed slight growth within their SXT inhibition zone by DD, pointing towards hetero-resistance. Overall, automated test systems classified isolates lacking genetic resistance determinants more frequently as SXT-resistant than DD and BMD. Therefore, further studies are needed to define a reliable method for SXT susceptibility testing.

Prolonged carriage of (livestock-associated) MRSA in individuals without professional livestock contact.

OBJECTIVES: To investigate prolonged carriage of MRSA in adults from the general population living in a livestock-dense area, using WGS. METHODS: A cross-sectional study during 2014-15 among 2492 adults without professional livestock contact identified 14 (0.6%) nasal MRSA carriers, 10 of which carried livestock-associated (LA)-MRSA of multiple-locus variable-number tandem repeat analysis (MLVA) complex (MC) 398. Two years later, 12 MRSA-positive and 88 MRSA-negative participants provided a second nasal swab and filled in a short questionnaire. Isolates from persons who were MRSA positive at both timepoints were compared using MLVA and isolates with the same MLVA type were sequenced. The WGS data were used for core-genome MLST (cgMLST) and resistome analysis, including sequenced isolates from the national MRSA surveillance. RESULTS: All MRSA-negative persons tested negative again, while 6 of the 12 initially MRSA-positive persons tested positive again. MLVA revealed that isolate pairs from five individuals had the same MLVA type, of which three were LA-MRSA. cgMLST showed that the distance between these isolate pairs ranged between 3 and 13 genes, while the minimum distance to unrelated isolates from the national MRSA surveillance was 38 genes. Moreover, the resistome present in the five isolate pairs was identical within each pair. None of the prolonged carriers was hospitalized during the 3 months before the sampling moment and none of them with LA-MRSA had contact with livestock in this period. CONCLUSIONS: Prolonged carriage of MRSA, including LA-MRSA, can be demonstrated after more than 30 months in persons without professional livestock contact.

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.

Dynamics of Intestinal Carriage of Extended-Spectrum Beta-lactamase-Producing Enterobacteriaceae in the Dutch General Population, 2014-2016.

BACKGROUND: In the Netherlands, the prevalence of intestinal extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in community-dwelling subjects is ~5%. Little is known about the dynamics of ESBL-E carriage. METHODS: In a nationwide, population-based study (2014-2016) with 4177 community-dwelling subjects, fecal samples from 656 subjects were collected after 1 (time point [T] = 1) and 6 (T = 2) months. The growth of ESBL-E was quantified and a whole-genome sequence analysis was performed. Subjects were categorized as either an incidental, short-term, or long-term carrier or as a noncarrier. Risk factors were determined by random forest models and logistic regression. The transmissibility and duration of ESBL-E carriage was quantified using a transmission model, which also incorporated previous study data. RESULTS: Out of 656 participants, 96 were ESBL-E carriers at T = 0. Of these, 66 (10.1%) subjects were incidental carriers, 22 (3.3%) were short-term carriers, and 38 (5.8%) were long-term carriers; the remaining 530 (80.8%) were noncarriers. The risk factors for long-term carriage were travelling to Asia, swimming in a sea/ocean, and not changing the kitchen towel daily. The log-transformed colony forming units ratio at T = 0 was predictive for ESBL-E carriage at T = 1 (odds ratio [OR], 1.3; 95% confidence interval [CI], 1.2-1.6) and T = 2 (OR, 1.2; 95% CI, 1.1-1.4). Model simulations revealed a median decolonization rate of 2.83/year, an average duration of carriage of 0.35 years, and an acquisition rate of 0.34/year. The trend of the acquisition rate during the study period was close to 0. CONCLUSIONS: The risk factors for long-term ESBL-E carriage were travel- and hygiene-related. The dynamics of ESBL-E carriage in the general Dutch population are characterized by balancing decolonization and acquisition rates.

Attributable sources of community-acquired carriage of Escherichia coli containing ß-lactam antibiotic resistance genes: a population-based modelling study.

BACKGROUND: Extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC), plasmid-mediated AmpC-producing E coli (pAmpC-EC), and other bacteria are resistant to important ß-lactam antibiotics. ESBL-EC and pAmpC-EC are increasingly reported in animals, food, the environment, and community-acquired and health-care-associated human infections. These infections are usually preceded by asymptomatic carriage, for which attributions to animal, food, environmental, and human sources remain unquantified. METHODS: In this population-based modelling study, we collected ESBL and pAmpC gene data on the Netherlands population for 2005-17 from published datasets of gene occurrences in E coli isolates from different sources, and from partners of the ESBL Attribution Consortium and the Dutch National Antimicrobial Surveillance System. Using these data, we applied an established source attribution model based on ESBL-EC and pAmpC-EC prevalence and gene data for humans, including high-risk populations (ie, returning travellers, clinical patients, farmers), farm and companion animals, food, surface freshwater, and wild birds, and human exposure data, to quantify the overall and gene-specific attributable sources of community-acquired ESBL-EC and pAmpC-EC intestinal carriage. We also used a simple transmission model to determine the basic reproduction number (R0) in the open community. FINDINGS: We identified 1220 occurrences of ESBL-EC and pAmpC-EC genes in humans, of which 478 were in clinical patients, 454 were from asymptomatic carriers in the open community, 103 were in poultry and pig farmers, and 185 were in people who had travelled out of the region. We also identified 6275 occurrences in non-human sources, including 479 in companion animals, 4026 in farm animals, 66 in wild birds, 1430 from food products, and 274 from surface freshwater. Most community-acquired ESBL-EC and pAmpC-EC carriage was attributed to human-to-human transmission within or between households in the open community (60·1%, 95% credible interval 40·0-73·5), and to secondary transmission from high-risk groups (6·9%, 4·1-9·2). Food accounted for 18·9% (7·0-38·3) of carriage, companion animals for 7·9% (1·4-19·9), farm animals (non-occupational contact) for 3·6% (0·6-9·9), and swimming in freshwater and wild birds (ie, environmental contact) for 2·6% (0·2-8·7). We derived an R0 of 0·63 (95% CI 0·42-0·77) for intracommunity transmission. INTERPRETATION: Although humans are the main source of community-acquired ESBL-EC and pAmpC-EC carriage, the attributable non-human sources underpin the need for longitudinal studies and continuous monitoring, because intracommunity ESBL-EC and pAmpC-EC spread alone is unlikely to be self-maintaining without transmission to and from non-human sources. FUNDING: 1Health4Food, Dutch Ministry of Economic Affairs, and the EU's Horizon-2020 through One-Health European Joint Programme.

The use of aminoglycosides in animals within the EU: development of resistance in animals and possible impact on human and animal health: a review.

Aminoglycosides (AGs) are important antibacterial agents for the treatment of various infections in humans and animals. Following extensive use of AGs in humans, food-producing animals and companion animals, acquired resistance among human and animal pathogens and commensal bacteria has emerged. Acquired resistance occurs through several mechanisms, but enzymatic inactivation of AGs is the most common one. Resistance genes are often located on mobile genetic elements, facilitating their spread between different bacterial species and between animals and humans. AG resistance has been found in many different bacterial species, including those with zoonotic potential such as Salmonella spp., Campylobacter spp. and livestock-associated MRSA. The highest risk is anticipated from transfer of resistant enterococci or coliforms (Escherichia coli) since infections with these pathogens in humans would potentially be treated with AGs. There is evidence that the use of AGs in human and veterinary medicine is associated with the increased prevalence of resistance. The same resistance genes have been found in isolates from humans and animals. Evaluation of risk factors indicates that the probability of transmission of AG resistance from animals to humans through transfer of zoonotic or commensal foodborne bacteria and/or their mobile genetic elements can be regarded as high, although there are no quantitative data on the actual contribution of animals to AG resistance in human pathogens. Responsible use of AGs is of great importance in order to safeguard their clinical efficacy for human and veterinary medicine.

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.

Correction: Time to acquire and lose carriership of ESBL/pAmpC producing E. coli in humans in the Netherlands.

[This corrects the article DOI: 10.1371/journal.pone.0193834.].