Global diversity of enterococci and description of 18 previously unknown species.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in broilers.

OBJECTIVES: Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. METHODS: The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. RESULTS: Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. CONCLUSIONS: Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.

Azole Resistance in Veterinary Clinical Aspergillus fumigatus Isolates in the Netherlands.

Aspergillus fumigatus is a saprophytic fungal pathogen that causes opportunistic infections in animals and humans. Azole resistance has been reported globally in human A. fumigatus isolates, but the prevalence of resistance in isolates from animals is largely unknown. A retrospective resistance surveillance study was performed using a collection of clinical A. fumigatus isolates from various animal species collected between 2015 and 2020. Agar-based azole resistance screening of all isolates was followed by in vitro antifungal susceptibility testing and cyp51A gene sequencing of the azole-resistant isolates. Over the 5 year period 16 (11.3%) of 142 A. fumigatus culture-positive animals harbored an azole-resistant isolate. Resistant isolates were found in birds (15%; 2/13), cats (21%; 6/28), dogs (8%; 6/75) and free-ranging harbor porpoise (33%; 2/6). Azole-resistance was cyp51A mediated in all isolates: 81.3% (T-67G/)TR34/L98H, 12.5% TR46/Y121F/T289A. In one azole-resistant A. fumigatus isolate a combination of C(-70)T/F46Y/C(intron7)T/C(intron66)T/M172V/E427K single-nucleotide polymorphisms in the cyp51A gene was found. Of the animals with an azole-resistant isolate and known azole exposure status 71.4% (10/14) were azole naive. Azole resistance in A. fumigatus isolates from animals in the Netherlands is present and predominantly cyp51A TR-mediated, supporting an environmental route of resistance selection. Our data supports the need to include veterinary isolates in resistance surveillance programs. Veterinarians should consider azole resistance as a reason for therapy failure when treating aspergillosis and consider resistance testing of relevant isolates.

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.

Antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) abundance in animal faeces, meat, production environments and human faeces in Europe.

BACKGROUND: Real-time quantitative PCR (qPCR) is an affordable method to quantify antimicrobial resistance gene (ARG) targets, allowing comparisons of ARG abundance along animal production chains. OBJECTIVES: We present a comparison of ARG abundance across various animal species, production environments and humans in Europe. AMR variation sources were quantified. The correlation of ARG abundance between qPCR data and previously published metagenomic data was assessed. METHODS: A cross-sectional study was conducted in nine European countries, comprising 9572 samples. qPCR was used to quantify abundance of ARGs [aph(3')-III, erm(B), sul2, tet(W)] and 16S rRNA. Variance component analysis was conducted to explore AMR variation sources. Spearman's rank correlation of ARG abundance values was evaluated between pooled qPCR data and earlier published pooled metagenomic data. RESULTS: ARG abundance varied strongly among animal species, environments and humans. This variation was dominated by between-farm variation (pigs) or within-farm variation (broilers, veal calves and turkeys). A decrease in ARG abundance along pig and broiler production chains ('farm to fork') was observed. ARG abundance was higher in farmers than in slaughterhouse workers, and lowest in control subjects. ARG abundance showed a high correlation (Spearman's ρ > 0.7) between qPCR data and metagenomic data of pooled samples. CONCLUSIONS: qPCR analysis is a valuable tool to assess ARG abundance in a large collection of livestock-associated samples. The between-country and between-farm variation of ARG abundance could partially be explained by antimicrobial use and farm biosecurity levels. ARG abundance in human faeces was related to livestock antimicrobial resistance exposure.

Risk factors for the abundance of antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) in pig and broiler faeces in nine European countries.

OBJECTIVES: The occurrence and zoonotic potential of antimicrobial resistance (AMR) in pigs and broilers has been studied intensively in past decades. Here, we describe AMR levels of European pig and broiler farms and determine the potential risk factors. METHODS: We collected faeces from 181 pig farms and 181 broiler farms in nine European countries. Real-time quantitative PCR (qPCR) was used to quantify the relative abundance of four antimicrobial resistance genes (ARGs) [aph(3')-III, erm(B), sul2 and tet(W)] in these faeces samples. Information on antimicrobial use (AMU) and other farm characteristics was collected through a questionnaire. A mixed model using country and farm as random effects was performed to evaluate the relationship of AMR with AMU and other farm characteristics. The correlation between individual qPCR data and previously published pooled metagenomic data was evaluated. Variance component analysis was conducted to assess the variance contribution of all factors. RESULTS: The highest abundance of ARG was for tet(W) in pig faeces and erm(B) in broiler faeces. In addition to the significant positive association between corresponding ARG and AMU levels, we also found on-farm biosecurity measures were associated with relative ARG abundance in both pigs and broilers. Between-country and between-farm variation can partially be explained by AMU. Different ARG targets may have different sample size requirements to represent the overall farm level precisely. CONCLUSIONS: qPCR is an efficient tool for targeted assessment of AMR in livestock-related samples. The AMR variation between samples was mainly contributed to by between-country, between-farm and within-farm differences, and then by on-farm AMU.

Variation in haplotypes in single cysts of assemblages C and D, but not of assemblage E of Giardia duodenalis.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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. Presence of more than 1 haplotype per cyst in assemblage C and D suggests differences between the non-daughter nuclei, in contrast to the one haplotype in assemblage E.

Biosecurity and antimicrobial use in broiler farms across nine European countries: toward identifying farm-specific options for reducing antimicrobial usage.

Broiler chickens are among the main livestock sectors worldwide. With individual treatments being inapplicable, contrary to many other animal species, the need for antimicrobial use (AMU) is relatively high. AMU in animals is known to drive the emergence and spread of antimicrobial resistance (AMR). High farm biosecurity is a cornerstone for animal health and welfare, as well as food safety, as it protects animals from the introduction and spread of pathogens and therefore the need for AMU. The goal of this study was to identify the main biosecurity practices associated with AMU in broiler farms and to develop a statistical model that produces customised recommendations as to which biosecurity measures could be implemented on a farm to reduce its AMU, including a cost-effectiveness analysis of the recommended measures. AMU and biosecurity data were obtained cross-sectionally in 2014 from 181 broiler farms across nine European countries (Belgium, Bulgaria, Denmark, France, Germany, Italy, the Netherlands, Poland and Spain). Using mixed-effects random forest analysis (Mix-RF), recursive feature elimination was implemented to determine the biosecurity measures that best predicted AMU at the farm level. Subsequently, an algorithm was developed to generate AMU reduction scenarios based on the implementation of these measures. In the final Mix-RF model, 21 factors were present: 10 about internal biosecurity, 8 about external biosecurity and 3 about farm size and productivity, with the latter showing the largest (Gini) importance. Other AMU predictors, in order of importance, were the number of depopulation steps, compliance with a vaccination protocol for non-officially controlled diseases, and requiring visitors to check in before entering the farm. K-means clustering on the proximity matrix of the final Mix-RF model revealed that several measures interacted with each other, indicating that high AMU levels can arise for various reasons depending on the situation. The algorithm utilised the AMU predictive power of biosecurity measures while accounting also for their interactions, representing a first step toward aiding the decision-making process of veterinarians and farmers who are in need of implementing on-farm biosecurity measures to reduce their AMU.

Determinants for antimicrobial resistance genes in farm dust on 333 poultry and pig farms in nine European countries.

Livestock feces with antimicrobial resistant bacteria reaches the farm floor, manure pit, farm land and wider environment by run off and aerosolization. Little research has been done on the role of dust in the spread of antimicrobial resistance (AMR) in farms. Concentrations and potential determinants of antimicrobial resistance genes (ARGs) in farm dust are at present not known. Therefore in this study absolute ARG levels, representing the levels people and animals might be exposed to, and relative abundances of ARGs, representing the levels in the bacterial population, were quantified in airborne farm dust using qPCR. Four ARGs were determined in 947 freshly settled farm dust samples, captured with electrostatic dustfall collectors (EDCs), from 174 poultry (broiler) and 159 pig farms across nine European countries. By using linear mixed modeling, associations with fecal ARG levels, antimicrobial use (AMU) and farm and animal related parameters were determined. Results show similar relative abundances in farm dust as in feces and a significant positive association (ranging between 0.21 and 0.82) between the two reservoirs. AMU in pigs was positively associated with ARG abundances in dust from the same stable. Higher biosecurity standards were associated with lower relative ARG abundances in poultry and higher relative ARG abundances in pigs. Lower absolute ARG levels in dust were driven by, among others, summer season and certain bedding materials for poultry, and lower animal density and summer season for pigs. This study indicates different pathways that contribute to shaping the dust resistome in livestock farms, related to dust generation, or affecting the bacterial microbiome. Farm dust is a large reservoir of ARGs from which transmission to bacteria in other reservoirs can possibly occur. The identified determinants of ARG abundances in farm dust can guide future research and potentially farm management policy.

Serologic Screening of Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Cats and Dogs during First Coronavirus Disease Wave, the Netherlands.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many animal species, including minks, cats, and dogs. To gain insights into SARS-CoV-2 infections in cats and dogs, we developed and validated a set of serologic assays, including ELISA and virus neutralization. Evaluation of samples from animals before they acquired coronavirus disease and samples from cats roaming SARS-CoV-2-positive mink farms confirmed the suitability of these assays for specific antibody detection. Furthermore, our findings exclude SARS-CoV-2 nucleocapsid protein as an antigen for serologic screening of cat and dog samples. We analyzed 500 serum samples from domestic cats and dogs in the Netherlands during April-May 2020. We showed 0.4% of cats and 0.2% of dogs were seropositive. Although seroprevalence in cats and dogs that had unknown SARS-CoV-2 exposure was low during the first coronavirus disease wave, our data stress the need for development of continuous serosurveillance for SARS-CoV-2 in these 2 animal species.

Transboundary Spread of Brucella canis through Import of Infected Dogs, the Netherlands, November 2016-December 2018.

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.

Duplicated TLR5 of zebrafish functions as a heterodimeric receptor.

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.

Antimicrobial Resistance in Salmonella enterica Serovar Paratyphi B Variant Java in Poultry from Europe and Latin America.

Salmonella enterica serovar Paratyphi B variant Java sequence type 28 is prevalent in poultry and poultry meat. We investigated the evolutionary relatedness between sequence type 28 strains from Europe and Latin America using time-resolved phylogeny and principal component analysis. We sequenced isolates from Colombia, Guatemala, Costa Rica, and the Netherlands and complemented them with publicly available genomes from Europe, Africa, and the Middle East. Phylogenetic time trees and effective population sizes (Ne) showed separate clustering of strains from Latin America and Europe. The separation is estimated to have occurred during the 1980s. Ne of strains increased sharply in Europe around 1995 and in Latin America around 2005. Principal component analysis on noncore genes showed a clear distinction between strains from Europe and Latin America, whereas the plasmid gene content was similar. Regardless of the evolutionary separation, similar features of resistance to ß-lactams and quinolones/fluoroquinolones indicated parallel evolution of antimicrobial resistance in both regions.

Specific staphylococcal cassette chromosome mec (SCCmec) types and clonal complexes are associated with low-level amoxicillin/clavulanic acid and cefalotin resistance in methicillin-resistant Staphylococcus pseudintermedius.

BACKGROUND: Staphylococcus pseudintermedius is a common pathogen in dogs and methicillin resistance has emerged over recent decades. According to the current guidelines, S. pseudintermedius displaying oxacillin resistance should be reported as resistant to all ß-lactams. OBJECTIVES: To identify possible associations between ß-lactam resistance levels and clonal complexes (CCs) and/or staphylococcal cassette chromosome mec (SCCmec) types in methicillin-resistant S. pseudintermedius (MRSP). METHODS: MICs of oxacillin, penicillin, ampicillin, amoxicillin/clavulanic acid and cefalotin were determined by broth microdilution for 86 clinical canine MRSP isolates from Denmark and the Netherlands. PCR and sequencing were used for SCCmec typing and MLST. RESULTS: Isolates belonged to CC71 (n = 36), CC258 (n = 33), CC45 (n = 11), CC68 (n = 1) and five singleton STs. SCCmecII-III was exclusively found in CC71 and SCCmecIV was significantly associated with CC258. SCCmecV and non-typeable SCCmec types occurred in 4 and 14 isolates, respectively. SCCmecIV was associated with lower MICs of oxacillin (<2 mg/L), ampicillin (<8 mg/L) and amoxicillin/clavulanic acid (<4 mg/L) and with susceptibility to cefalotin (<4 mg/L). All isolates harbouring SCCmecV were susceptible to cefalotin as well. CONCLUSIONS: SCCmec types were associated with different CCs and with either high- or low-level resistance to different ß-lactams. The finding of amoxicillin/clavulanic acid (20%) and cefalotin (70%) in vitro susceptibility across all CCs might have clinical implications, since amoxicillin/clavulanic acid and first-generation cephalosporins are first-choice antibiotics for treatment of S. pseudintermedius infections. Pharmacokinetic/pharmacodynamic and clinical outcome studies are warranted to evaluate the in vivo efficacy of these ß-lactams for treatment of MRSP infections.

Fecal Carriage of Extended-Spectrum-ß-Lactamase/AmpC-Producing Escherichia coli in Horses.

A nationwide study on the occurrence of extended-spectrum ß-lactamase (ESBL)/AmpC in nonhospitalized horses in the Netherlands was performed. Molecular characterization was done, and questionnaires were analyzed to identify factors associated with carriage. In total, 796 horse owners were approached; 281 of these submitted a fecal sample from their horse(s), resulting in 362 samples. All samples were cultured qualitatively in Luria-Bertani (LB) broth and subsequently on MacConkey agar, both supplemented with 1 mg/liter cefotaxime (LB+ and MC+). Positive samples were subsequently cultured quantitatively on MC+. Initial extended-spectrum-ß-lactamase (ESBL)/AmpC screening was performed by PCR, followed by whole-genome sequencing on selected strains. Associations between ESBL/AmpC carriage and questionnaire items were analyzed using a univariate generalized estimating equation (GEE) regression analysis, followed by a multiple GEE model for relevant factors. In total, 39 of 362 samples (11%) were determined to be positive for ESBL/AmpC. blaCTX-M-1-carrying isolates were obtained from 77% of positive samples (n = 30). Other ESBL/AmpC genes observed included blaCTX-M-2, blaCTX-M-14, blaCTX-M-15, blaCTX-M-32, blaSHV-12, blaCMY-2, and blaACT-10 A high association between the presence of blaCTX-M-1 and IncHI1 plasmids was observed (46% of samples; n = 18). Based on core genome analysis (n = 48 isolates), six Escherichia coli clusters were identified, three of which represented 80% of the isolates. A negative association between ESBL/AmpC carriage and horses being in contact with other horses at a different site was observed. The presence of a dog on the premises and housing in a more densely human-populated region were positively associated.IMPORTANCE Extended-spectrum ß-lactamases (ESBLs) are widespread in human and animal populations and in the environment. Many different ESBL variants exist. The dissemination of ESBLs within and between populations and the environment is also largely influenced by genetic mobile elements (e.g., plasmids) that facilitate spread of these ESBLs. In order to identify potential attributable ESBL sources for, e.g., the human population, it is important to identify the different ESBL variants, the bacteria carrying them, and the potential risk factors for ESBL carriage from other potential sources. This nationwide study focuses on ESBL carriage in the open horse population and investigated the molecular characteristics, geographical distribution throughout the Netherlands, and potential risk factors for fecal ESBL carriage in horses. These data can be used for future attribution studies in order to reduce potential transmission of ESBL-producing bacteria between sources.

Incompatibility and phylogenetic relationship of I-complex plasmids.

Plasmid incompatibility is the inability of two plasmids to be stably maintained in one cell, resulting in loss of one of the plasmids in daughter cells. Dislodgement is a phenotypically distinct form of incompatibility, described as an imperfect reproduction, manifesting in rapid exclusion of a resident plasmid after superinfection. The relationship between plasmids of the phenotypic incompatibility groups IncB/O and IncZ is unclear. Their inability to co-exist was initially referred to as dislodgement while other research reached the conclusion that IncB/O and IncZ plasmids are incompatible. In this manuscript we re-evaluated the relationship between IncB/O and IncZ plasmids to settle these conflicting conclusions. We performed dislodgement testing of R16Δ (IncB/O) and pSFE-059 (IncZ) plasmids by electroporation in a bacterial cell and checked their stability. Stability tests of the obtained plasmid pair showed that the IncB/O plasmid was exclusively and almost completely lost from the heteroplasmid Escherichia coli population. Other IncB/O - IncZ pairs could not form a heteroplasmid population, using conjugation or electroporation. Our data supports the previous suggestion that IncB/O and IncZ plasmids may be considered phenotypically incompatible.

Leptospira infection and shedding in dogs in Thailand.

BACKGROUND: Leptospirosis is a widespread zoonosis and has been recognized as a re-emerging infectious disease in humans and dogs, but prevalence of Leptospira shedding in dogs in Thailand is unknown. The aim of this study was to determine urinary shedding of Leptospira in dogs in Thailand, to evaluate antibody prevalence by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA), and to assess risk factors for Leptospira infection. In Northern, Northeastern, and Central Thailand, 273 stray (n = 119) or client-owned (n = 154) dogs from rural (n = 139) or urban (n = 134) areas were randomly included. Dogs that had received antibiotics within 4 weeks prior to sampling were excluded. No dog had received vaccination against Leptospira. Urine was evaluated by real-time polymerase chain reaction (PCR) specific for lipL32 gene of pathogenic Leptospira. Additionally, urine was cultured for 6 months in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. Antibodies were measured by ELISA and MAT against 24 serovars belonging to 15 serogroups and 1 undesignated serogroup. Risk factor analysis was performed with backwards stepwise selection based on Wald. RESULTS: Twelve of 273 (4.4%; 95% confidence interval (CI): 2.0-6.8%) urine samples were PCR-positive. In 1/273 dogs (0.4%; 95% CI: 0.01-1.1%) Leptospira could be cultured from urine. MAT detected antibodies in 33/273 dogs (12.1%; 95% CI: 8.2-16.0%) against 19 different serovars (Anhoa, Australis, Ballum, Bataviae, Bratislava, Broomi, Canicola, Copenhageni, Coxi, Grippotyphosa, Haemolytica, Icterohaemorrhagiae, Khorat, Paidjan, Patoc, Pyrogenes, Rachmati, Saxkoebing, Sejroe). In 111/252 dogs (44.0%; 95% CI: 37.9-50.2%) immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies were found by ELISA. Female dogs had a significantly higher risk for Leptospira infection (p = 0.023). CONCLUSIONS: Leptospira shedding occurs in randomly sampled dogs in Thailand, with infection rates comparable to those of Europe and the USA. Therefore, the potential zoonotic risk should not be underestimated and use of Leptospira vaccines are recommended.

Campylobacter pinnipediorum subsp. caledonicus and C. pinnipediorum subsp. pinnipediorum recovered from abscesses in pinnipeds.

Campylobacter pinnipediorum was described recently for isolates recovered from pinnipeds. The novel species was further split into 2 subspecies based on host and geography, with C. pinnipediorum subsp. pinnipediorum recovered from otariid seals in California (USA) and C. pinnipediorum subsp. caledonicus recovered from phocid seals in Scotland. We report details of the infections of 7 pinnipeds from which C. pinnipediorum was isolated: C. pinnipediorum subsp. caledonicus was isolated from 2 harbour seals Phoca vitulina and a single grey seal Halichoerus grypus, and C. pinnipediorum subsp. pinnipediorum was isolated from California sea lions Zalophus californianus. Six of the isolates were recovered from samples collected at post-mortem investigation. In 2 of the Scottish seals and in 3 of the California seals, C. pinnipediorum was the sole bacterial isolate recovered from abscesses present and suggests they may have resulted from conspecific or intraspecific bite wounds.

Zoonotic Endocarditis in a Man, the Netherlands.

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.

Phylogenomic Investigation of IncI1-Iγ Plasmids Harboring blaCMY-2 and blaSHV-12 in Salmonella enterica and Escherichia coli in Multiple Countries.

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). Most IncI1-Iγ/ST12 and IncI1-Iγ/ST231 plasmids had near-identical core genomes. The data suggest that widely occurring blaCMY-2-carrying IncI1-Iγ/ST12 plasmids originate from a common ancestor. In contrast, blaSHV-12 was inserted independently into different IncI1-Iγ/ST231-related plasmids.