To quantify associations between antimicrobial use and acquired resistance in indicator Escherichia coli over a period of time which involved sector-wide antimicrobial use reductions in broilers and ...pigs (years 2004-14), veal calves (2007-14) and dairy cattle (2005-14). Prevalence estimates of resistance were predicted for a hypothetical further decrease in antimicrobial use.
Data reported annually for the resistance surveillance programme in the Netherlands were retrieved. Two multivariate random-effects logistic models per animal sector were used to relate total and class-specific antimicrobial use (as defined daily dosages per animal per year, DDDA/Y) with the probability of E. coli resistance to a panel of 10 antimicrobial agents.
Positive dose-response relationships (ORs) were obtained from all models. Specific resistance phenotypes were more often associated with total antimicrobial use than with class-specific use. The most robust associations were found in pigs and veal calves. Resistance to historically widely used antimicrobials (e.g. penicillins, tetracyclines) was, in relative terms, less influenced by drug use changes over time than resistance to newer or less prescribed antimicrobials (e.g. third-/fourth-generation cephalosporins, fluoroquinolones). In pigs and veal calves, prevalence estimates for the most common resistance phenotypes were projected to decline ∼5%-25% during 2014-16 if total antimicrobial use reduction reached 80%; projections for poultry and dairy cows were more modest.
Epidemiological evidence indicated that drug use history and co-selection of resistance are key elements for perpetuation of resistance. Data suggest that recent Dutch policies aimed at reducing total use of antimicrobials have decreased E. coli resistance in the pig and veal calf production sectors while the impact on the dairy cattle and poultry sectors is less clear.
Toll-like receptor 5 (TLR5) of mammals, birds, and reptiles detects bacterial flagellin and signals as a homodimeric complex. Structural studies using truncated TLR5b of zebrafish confirm the ...homodimeric TLR5–flagellin interaction. Here we provide evidence that zebrafish (Danio rerio) TLR5 unexpectedly signals as a heterodimer composed of the duplicated gene products drTLR5b and drTLR5a. Flagellin-induced signaling by the zebrafish TLR5 heterodimer increased in the presence of the TLR trafficking chaperone UNC93B1. Targeted exchange of drTLR5b and drTLR5a regions revealed that TLR5 activation needs a heterodimeric configuration of the receptor ectodomain and cytoplasmic domain, consistent with ligand-induced changes in receptor conformation. Structure-guided substitution of the presumed principal flagellin-binding site in human TLR5 with corresponding zebrafish TLR5 residues abrogated human TLR5 activation, indicating a species-specific TLR5–flagellin interaction. Our findings indicate that the duplicated TLR5 of zebrafish underwent subfunctionalization through concerted coevolution to form a unique heterodimeric flagellin receptor that operates fundamentally differently from TLR5 of other species.
Brucella canis had not been isolated in the Netherlands until November 2016, when it was isolated from a dog imported from Romania. Including this case, 16 suspected cases were notified to the ...authorities during the following 25 months. Of these 16 dogs, 10 were seropositive; tracking investigations found another 8 seropositive littermates. All seropositive animals were rescue dogs imported from Eastern Europe. B. canis was cultured from urine, blood, and other specimens collected from the dogs. Genotyping of isolates revealed clustering by litter and country. Isolating B. canis in urine indicates that shedding should be considered when assessing the risk for zoonotic transmission. This case series proves introduction of B. canis into a country to which it is not endemic through import of infected dogs from B. canis–endemic areas, posing a threat to the naive autochthonous dog population and humans.
Abstract
Objectives
To determine associations between farm- and flock-level antimicrobial usage (AMU), farm biosecurity status and the abundance of faecal antimicrobial resistance genes (ARGs) on ...broiler farms.
Methods
In the cross-sectional pan-European EFFORT study, conventional broiler farms were visited and faeces, AMU information and biosecurity records were collected. The resistomes of pooled faecal samples were determined by metagenomic analysis for 176 farms. A meta-analysis approach was used to relate total and class-specific ARGs (expressed as fragments per kb reference per million bacterial fragments, FPKM) to AMU (treatment incidence per DDD, TIDDDvet) per country and subsequently across all countries. In a similar way, the association between biosecurity status (Biocheck.UGent) and the resistome was explored.
Results
Sixty-six (38%) flocks did not report group treatments but showed a similar resistome composition and roughly similar ARG levels to antimicrobial-treated flocks. Nevertheless, we found significant positive associations between β-lactam, tetracycline, macrolide and lincosamide, trimethoprim and aminoglycoside antimicrobial flock treatments and ARG clusters conferring resistance to the same class. Similar associations were found with purchased products. In gene-level analysis for β-lactams and macrolides, lincosamides and streptogramins, a significant positive association was found with the most abundant gene clusters blaTEM and erm(B). Little evidence was found for associations with biosecurity.
Conclusions
The faecal microbiome in European broilers contains a high diversity of ARGs, even in the absence of current antimicrobial selection pressure. Despite this, the relative abundance of genes and the composition of the resistome is positively related to AMU in European broiler farms for several antimicrobial classes.
Classifications of the Campylobacter fetus subspecies fetus and venerealis were first described in 1959 and were based on the source of isolation (intestinal versus genital) and the ability of the ...strains to proliferate in the genital tract of cows. Two phenotypic assays (1% glycine tolerance and H2S production) were described to differentiate the subspecies. Multiple molecular assays have been applied to differentiate the C. fetus subspecies, but none of these tests is consistent with the phenotypic identification methods. In this study, we defined the core genome and accessory genes of C. fetus, which are based on the closed genomes of five C. fetus strains. Phylogenetic analysis of the core genomes of 23 C. fetus strains of the two subspecies showed a division into two clusters. The phylogenetic core genome clusters were not consistent with the phenotypic classifications of the C. fetus subspecies. However, they were consistent with the molecular characteristics of the strains, which were determined by multilocus sequence typing, sap typing, and the presence/absence of insertion sequences and a type I restriction modification system. The similarity of the genome characteristics of three of the phenotypically defined C. fetus subsp. fetus strains to C. fetus subsp. venerealis strains, when considering the core genome and accessory genes, requires a critical evaluation of the clinical relevance of C. fetus subspecies identification by phenotypic assays.
Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of ...antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74) and non-pseudintermedius genomes (n = 138). Six sequences specific for S. pseudintermedius were identified (sequence length between 300-500 nt). One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54), and eight other staphylococcal species (n = 43). In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.
The spread of an infectious agent in a population can be reduced by interfering in the infectiousness or susceptibility of individuals, and/or in their contact structure. The aim of this study was to ...quantify the effect of prevention of direct contact between infectious and susceptible pigs on the transmission of Streptococcus suis (S. suis). In three replicate experiments, S. suis-free pigs were housed in boxes either in pairs (25 pairs) or alone (15 pigs). The distance between the boxes was ±1 m. At 7 weeks of age, one pig of each pair was inoculated intranasally with S. suis serotype 9; the other pigs were exposed to S. suis by either direct (pairs) or indirect contact (individually housed pigs). Tonsillar brush and saliva swab samples from all pigs were collected regularly for 4 weeks post inoculation to monitor colonization with S. suis. All inoculated pigs became infected, and their pen mates became colonized within 2 days. Thirteen indirectly exposed pigs became positive within 7-25 days after exposure. The rate of direct transmission βdir was estimated to be 3.58 per pig per day (95% CI: 2.29-5.60). The rate of indirect transmission increased in time, depending on the cumulative number of days pigs tested positive for the presence of S. suis. The estimate β'ind was 0.001 (95% CI: 0.0006-0.0017) new infections per pig per day for each day that an infected pig was tested positive for S. suis. We conclude that prevention of direct contact reduces the rate at which susceptible pigs become colonized. Simulation studies using these parameters showed, however, that such intervention measure would not limit S. suis serotype 9 spread in a commercial pig farm to a relevant extent, implying that spatial separation of groups op pigs within a compartment would not be effective on a farm.
Objectives The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal ...distribution in Europe and North America. Methods A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR–restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. Results Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II–III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n = 2), IV (n = 6), V (n = 14) or VII-241 (n = 4) or were non-typeable (n = 2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II–III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated β-lactam resistance, isolates showed resistance to trimethoprim dfr(G) (90.3%), gentamicin/kanamycin aac(6′)-Ie–aph(2′)-Ia (88.3%), kanamycin aph(3′)-III (90.3%), streptomycin ant(6′)-Ia (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides erm(B), lnu(A) (89.3%), fluoroquinolones (87.4%), tetracycline tet(M) and/or tet(K) (69.9%), chloramphenicol (catpC221) (57.3%) and rifampicin (1.9%). Conclusions Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II–III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.
Giardia duodenalis, a single-celled intestinal parasite, is divided into eight assemblages (A-H), with differences in host specificity. Giardia duodenalis reproduces asexually and cycles between the ...binucleated trophozoite (4 N) and the infectious cyst with four nuclei (16 N). Interaction between the nuclei is limited. Therefore, genetic drift causes differences in genetic make-up between the non-daughter nuclei; the allelic sequence heterozygosity (ASH). The ASH is low (0.01%--0.0023%) for the related assemblages A and E, higher (0.43-0.53) for assemblage B and much higher (0.74% -0.89%) for the assemblage C and D at the root of the phylogenetic tree. The heterozygosity in assemblage F, in the same clade as assemblage A and E, was unknown. The heterozygosity in the sequences of the gdh and dis3 genes was used as proxy for the ASH and whole genome amplification of single cysts followed by cloning and Sanger sequencing of dis3 fragment could reveal the genetic variation within the cyst. The aim of the study was to determine the level of heterozygosity within pooled and single cysts of different assemblages. The heterozygosity in gdh and dis3 was determined in pooled cysts of the assemblages A to F. Heterozygosity in the isolates of the assemblages C (n = 2) and D (n = 1) ranged from 0.41% to 0.82% for gdh and dis3 and no heterozygosity was found in the isolates of the assemblages A (n = 4), E (n = 3) and F (n = 3). The heterozygosity in assemblage B (n = 7) was intermediate (0% to 0.62%). Next, the number of haplotypes of dis3 was determined for single cysts of assemblages C, D and E. In the assemblages C and D, two to four haplotypes were found per cyst, while in assemblage E only one haplotype was identified. Having high heterozygosity is characteristic for the assemblages C and D, while having a low heterozygosity is characteristic for the clade with the assemblages A, E and F.
In 2017, endocarditis caused by Streptococcus equi subspecies zooepidemicus was diagnosed in a man in the Netherlands who had daily contact with horses. Whole-genome sequencing of isolates from the ...man and his horses confirmed the same clone, indicating horse-to-human transmission. Systematic reporting of all zoonotic cases would help with risk assessment.
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