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•The mcr-1gene was identified for the first time on an IncI1 plasmid.•The plasmid was present in a commensal Escherichia colifrom livestock.•The IncI1 plasmid was genetically distinct from resistance-encoding IncI1 plasmids previously isolated in the Netherlands.•mcr-1was flanked by two copies of ISApl1, which are likely responsible for its insertion.•The plasmid was conjugative between E. colistrains under laboratory conditions.

Broilers are an important reservoir of extended spectrum beta-lactamase and AmpC beta-lactamase (ESBL/pAmpC)-producing bacteria. In previous studies, a single supply of a competitive exclusion (CE) product before challenge with a high dose of ESBL/pAmpC-producing Escherichia coliled to reduced colonization, excretion, and transmission, but could not prevent colonization. The hypothesized mechanism is competition; therefore, in this study the effect of a prolonged supply of CE products on colonization, excretion, and transmission of ESBL-producing E. coliafter challenge with a low dose at day 0 or day 5 was investigated. Day-old broilers (Ross 308) (n = 220) were housed in isolators. Two CE products, containing unselected fermented intestinal bacteria (CEP) or a selection of pre- and probiotics (SYN), were supplied in drinking water from day 0 to 14. At day 0 or 5, broilers were challenged with 0.5 mL with 101or 102 cfu/mL E. coliencoding the beta-lactamase gene blaCTX-M-1on an IncI plasmid (CTX-M-1-E. coli). Presence and concentration of CTX-M-1-E. coliwere determined using cloacal swabs (days 0–14, 16, 19, and 21) and cecal content (day 21). Cox proportional hazard model and a mixed linear regression model were used to determine the effect of the intervention on colonization and excretion (log10 cfu/g). When challenged on the day of hatch, no effect of CEP was observed. When challenged at day 5, both CEP and SYN led to a prevention of colonization with CTX-M-1-E. coliin some isolators. In the remaining isolators, we observed reduced time until colonization (hazard ratio between 3.71 × 10−3and 3.11), excretion (up to −1.60 log10 cfu/g), and cecal content (up to −2.80 log10 cfu/g), and a 1.5 to 3-fold reduction in transmission rate. Colonization after a low-dose challenge with ESBL-producing E. colican be prevented by CE products. However, if at least 1 bird is colonized it spreads through the whole flock. Prolonged supply of CE products, provided shortly after hatch, may be applicable as an intervention to reduce the prevalence of ESBL/pAmpC-producing bacteria in the broiler production chain.

Extended-spectrum β-lactamase-producing Escherichia coli(ESBL-EC), plasmid-mediated AmpC-producing E coli(pAmpC-EC), and other bacteria are resistant to important β-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.

ABSTRACTExtended-spectrum-cephalosporin-resistant Enterobacteriaceaeare a public health concern due to limited treatment options. Here, we report on the occurrence and the molecular characteristics of extended-spectrum-cephalosporin-resistant Enterobacteriaceaerecovered from wild birds (kelp gulls). Our results revealed kelp gulls as a reservoir of various extended-spectrum cephalosporinase genes associated with different genetic platforms. In addition, we report for the first time the presence of a known epidemic clone of Salmonella entericaserotype Heidelberg (JF6X01.0326/XbaI.1966) among wild birds.

ABSTRACTThe aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coliand Salmonella entericaisolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation in traYand excAgenes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

AbstractThe combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella entericasubsp. entericastrains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonellaserovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n= 9) or two (n= 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonellato adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

Multidrug-resistant Salmonellaserovar Concord infections have been reported from children adopted from Ethiopia. We interviewed patients, characterized the isolates, and gathered information about adoptions from Ethiopia to assess public health implications.

The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00–2.49 log CFU/g of manured soil, ranging between 10–5and 10–4transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillusand Nocardioides) and manure (Comamonasand Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.

The purpose of this work was to study the genetic determinants responsible for extended-spectrum beta-lactamase (ESBL) resistance of Salmonella isolated from Dutch poultry, poultry meat and hospitalized humans.

The presence and character of class 1 integrons in multidrug-resistant Escherichia coli from slaughter animals and meat was determined by integrase-specific PCR and conserved segment PCR-restriction fragment length polymorphism (RFLP). At least five different class 1 integron types were found and three types were shared between hospitalized patients, humans in the community, meat, and slaughter animals. Common integron types indicate that antibiotic resistance genes are exchanged via the food chain between different reservoirs of both human and animal origin.

ABSTRACTWe report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546derivatives. Differences between the Tn1546types included point mutations in orf1, vanS,vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element inorf1, of IS1251in the vanS-vanHintergenic region, and of IS1216Vin thevanX-vanYintergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546to VRE isolates causing infections in hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546combined with epidemiological data suggest that the DNA polymorphism among Tn1546variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.

ABSTRACTMarA and its homologue, RamA, have been implicated in multidrug resistance (MDR). RamAoverexpression in Salmonella entericaserovar Typhimurium and Escherichia coliconferred MDR independently of marA. Inactivation of ramAdid not affect the antibiotic susceptibilities of wild-type S. entericaserovar Typhimurium or 15 unrelated clinical MDR isolates. Thus, ramAoverexpression is not a common MDR mechanism in Salmonella.

The objective of this study was to elucidate the genetic and evolutionary relatedness of blaCMY-2- and blaSHV-12-carrying IncI1-Iγ plasmids. Phylogenomic analysis based on core genome alignments and gene presence/absence was performed for different IncI1-Iγ sequence types (STs).

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 cloacaecomplex isolated from imported seafood that encodes two carbapenemases on two distinct plasmids.

ABSTRACTIncK plasmids are some of the main carriers of blaCTX-M-14and blaCMY-2genes and show high similarity to other plasmids belonging to the I complex, including IncB/O plasmids. Here, we studied the phylogenetic relationship of 37 newly sequenced IncK and IncB/O plasmids. We show that IncK plasmids can be divided into two compatible lineages named IncK1 and IncK2.

ABSTRACTCarbapenems are considered last-resort antibiotics in health care. Increasing reports of carbapenemase-producing bacteria in food-producing animals and in the environment indicate the importance of this phenomenon in public health. Surveillance for carbapenemase genes and carbapenemase-producing bacteria in Dutch food-producing animals, environmental freshwater, and imported ornamental fish revealed several chromosome-based blaOXA-48-like variants in Shewanellaspp., including two new alleles, blaOXA-514and blaOXA-515. Carbapenemase genes were not associated with mobile genetic elements or Enterobacteriaceae.

Background The purpose of this study was to determine the prevalence and risk factors for colonization with extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli in daycare center (DCC)-attending children. Methods This is a prospective cohort study including 44 DCCs in the Netherlands, combining DCC characteristics and monthly collected stool samples from their attendees, and was performed in 2010–2012. During a 22-month study period, 852 stool samples were collected and screened for ESC-R E coli. Risk factors were studied using logistic regression analysis. Results In DCC-attending children (<4 years old), the overall prevalence of ESC-R E coli was 4.5%, and it was 8% in <1-year-old attendees. Among the 38 children carrying ESC-R E coli, the most common types were blaCMY-2 (26%), blaCTX-M-1 (16%), and chromosomal AmpC type 3 promoter mutants (13%). Extended-spectrum cephalosporin-resistant E coli was less common in DCCs where stricter hygiene protocols were enforced, eg, not allowing ill children to enter the DCC (odds ratio [OR], 0.34; 95% confidence interval [CI], 0.14–0.84), performing extra checks on handwashing of ill children (OR, 0.42; 95% CI, 0.20–0.87), and reporting suspected outbreaks to local health authorities (OR, 0.27; 95% CI, 0.11–0.69). Conclusions The distribution of ESC-R E coli types in DCCs differs from that of the general population. Extended-spectrum cephalosporin-resistant E coli carriage in DCC-attending children is associated with the hygiene policies enforced in the DCC. Although our results are not conclusive enough to change current DCC practice beyond ensuring compliance with standing policies, they generated hypotheses and defined the degree of ESC resistance among DCC attendees, which may influence empiric antibiotic therapy choices, and tracked the increasing trend in ESC resistance.

Background The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II) was funded by the European Union (FAIR5-QLK2-2002-01146) for the period 2003–2005, with the aim to establish a continuous monitoring of antimicrobial susceptibility among veterinary laboratories in European countries based on validated and harmonised methodologies. Available summary data of the susceptibility testing of the bacterial pathogens from the different laboratories were collected.Method Antimicrobial susceptibility data for several bovine pathogens were obtained over a three year period (2002–2004). Each year the participating laboratories were requested to fill in excel-file templates with national summary data on the occurrence of antimicrobial resistance from different bacterial species.Results Data from 25,241 isolates were collected from 13 European countries. For Staphylococcus aureus from bovine mastitis major differences were apparent in the occurrence of resistance between countries and between the different antimicrobial agents tested. The highest frequency of resistance was observed for penicillin. For Mannheimia haemolytica resistance to ampicillin, tetracycline and trimethoprim/sulphonamide were observed in France, the Netherlands and Portugal. All isolates of Pasteurella multocida isolated in Finland and most of those from Denmark, England (and Wales), Italy and Sweden were susceptible to the majority of the antimicrobials. Streptococcus dysgalactiae and Streptococcus uberis isolates from Sweden were fully susceptible. For the other countries some resistance was observed to tetracycline, gentamicin and erythromycin. More resistance and variation of the resistance levels between countries were observed for Escherichia coli compared to the other bacterial species investigated.Conclusion In general, isolates from Denmark, England (and Wales), the Netherlands, Norway, Sweden and Switzerland showed low frequencies of resistance, whereas many isolates from Belgium, France, Italy, Latvia and Spain were resistant to most antimicrobials tested. In the future, data on the prevalence of resistance should be used to develop guidelines for appropriate antimicrobial use in veterinary medicine.

Background The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II) was funded by the European Union (FAIR5-QLK2-2002-01146) for the period 2003–05. The aim of this project was to establish a program for the continuous monitoring of antimicrobial susceptibility of pathogenic and indicator bacteria from food animals using validated and harmonised methodologies. In this report the first data on the occurrence of antimicrobial resistance among bacteria causing infections in pigs are reported.Methods Susceptibility data from 17,642 isolates of pathogens and indicator bacteria including Actinobacillus pleuropneumoniae, Streptococcus suis and Escherichia coli isolated from pigs were collected from fifteen European countries in 2002–2004.Results Data for A. pleuropneumoniae from infected pigs were submitted from five countries. Most of the isolates from Denmark were susceptible to all drugs tested with the exceptions of a low frequency of resistance to tetracycline and trimethoprim – sulphonamide.Conclusion Bacterial resistance to some antimicrobials was frequent with different levels of resistance being observed to several antimicrobial agents in different countries. The occurrence of resistance varied distinctly between isolates from healthy and diseased pigs, with the isolates from healthy pigs generally showing a lower level of resistance than those from diseased pigs.

&lt;it>Objective&lt;/it>: To determine the within-host population dynamics of &lt;it>Campylobacter jejuni&lt;/it> and &lt;it>Escherichia coli&lt;/it> in chickens during and after treatment with fluoroquinolones. &lt;it>Materials and methods&lt;/it>: Total and resistant faecal counts were determined from cloacal swabs during and after treatment with enrofloxacin. Chickens were housed individually to avoid confounding as a result of interaction between animals, and to be able to focus solely on the within-host dynamics. To determine the molecular basis of resistance, a number of isolates were checked for mutations in &lt;it>gyrA&lt;/it>. &lt;it>Results&lt;/it>: Treatment with enrofloxacin at doses routinely prescribed (50 ppm) rapidly reduced the faecal counts of &lt;it>E. coli&lt;/it> below the detection limit and did not induce resistance. In &lt;it>C. jejuni&lt;/it>, on the other hand, treatment with enrofloxacin quickly selected for high frequencies of fluoroquinolone-resistant strains. In all phenotypically resistant isolates, resistance was traced to mutations in the &lt;it>gyrA&lt;/it> gene. &lt;it>Conclusions&lt;/it>: (1) A licensed dosage (50 ppm) of enrofloxacin in drinking water of chickens is effective (i.e. markedly reduced faecal counts) and is safe on a short time scale in &lt;it>E. coli&lt;/it> (i.e. did not rapidly select for resistance), but is neither safe nor effective in &lt;it>C. jejuni&lt;/it>. (2) The rapid emergence of resistance to quinolones in &lt;it>C. jejuni&lt;/it> does not necessarily result from horizontal transmission of resistant strains among chickens, but could solely be the result of &lt;it>de novo&lt;/it> selection of resistance in individual chickens.