Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

Genomic Characterization and Antimicrobial Susceptibility of Dromedary-Associated Staphylococcaceae from the Horn of Africa.

Members of the Staphylococcaceae family, particularly those of the genus Staphylococcus, encompass important human and animal pathogens. We collected and characterized Staphylococcaceae strains from apparently healthy and diseased camels (n = 84) and cattle (n = 7) in Somalia and Kenya. We phenotypically characterized the strains, including their antimicrobial inhibitory concentrations. Then, we sequenced their genomes using long-read sequencing, closed their genomes, and subsequently compared and mapped their virulence- and resistance-associated gene pools. Genome-based phylogenetics revealed 13 known Staphylococcaceae and at least two novel species. East African strains of different species encompassed novel sequence types and phylogenetically distant clades. About one-third of the strains had non-wild-type MICs. They were resistant to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, gentamicin, or streptomycin, encoded by tet(K), blaZ/blaARL, mecA/mecA1, msrA/mphC, salA, dfrG, aacA-aphD, and str, respectively. We identified the first methicillin- and multidrug-resistant camel S. epidermidis strain of sequence type (ST) 1136 in East Africa. The pool of virulence-encoding genes was largest in the S. aureus strains, as expected, although other rather commensal strains contained distinct virulence-encoding genes. We identified toxin-antitoxin (TA) systems such as the hicA/hicB and abiEii/abiEi families, reported here for the first time for certain species of Staphylococcaceae. All strains contained at least one intact prophage sequence, mainly belonging to the Siphoviridae family. We pinpointed potential horizontal gene transfers between camel and cattle strains and also across distinct Staphylococcaceae clades and species. IMPORTANCE Camels are a high value and crucial livestock species in arid and semiarid regions of Africa and gain importance giving the impact of climate change on traditional livestock species. Our current knowledge with respect to Staphylococcaceae infecting camels is very limited compared to that for other livestock species. Better knowledge will foster the development of specific diagnostic assays, guide promising antimicrobial treatment options, and inform about potential zoonotic risks. We characterized 84 Staphylococcaceae strains isolated from camels with respect to their antimicrobial resistance and virulence traits. We detected potentially novel Staphylococcus species, resistances to different classes of antimicrobials, and the first camel multidrug-resistant S. epidermidis strain of sequence type 1136.

Macrococcus armenti sp. nov., a novel bacterium isolated from the skin and nasal cavities of healthy pigs and calves.

Gram-positive coccoid bacteria were isolated from the nasal cavities of pigs and calves as well as from axillar and inguinal skin regions of pigs. Phylogenetic analysis of seven strains based on complete genome, 16S rRNA, hsp60, dnaJ, rpoB and sodA gene sequences and MALDI-TOF MS profiles revealed that they belonged to the genus Macrococcus with the closest relatedness to Macrococcus canis, Macrococcus caseolyticus subsp. caseolyticus and Macrococcus caseolyticus subsp. hominis. DNA relatedness of the type strain JEK37T with the type strains of M. canis, M. caseolyticus subsp. caseolyticus and M. caseolyticus subsp. hominis was 23.4, 23.1 and 23.0 % by digital DNA-DNA hybridization and 80.39, 80.45 and 80.87 % by average nucleotide identity (ANI) calculations, confirming that they do not belong to the same species. The DNA G+C content of JEK37T was 35.65 mol%. The novel strains can be differentiated from M. canis KM 45013T by the ability to fermentate d-ribose and by the absence of DNAase production and haemolysis, from M. caseolyticus subsp. caseolyticus CCUG 15606T by the ability to fermentate sucrose and from both species by the inability to grow in 9 and 12% NaCl. They differ from M. caseolyiticus subsp. hominis by the presence of α-glucosidase. The most common fatty acids of JEK37T were C14 : 0, C18 : 1 ω9c and C18 : 0. Known polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, aminolipid, aminoglycolipid, aminophospholipid, glycolipid and phospholipid. Cell-wall peptidoglycan of JEK37T was of type A3α l-Lys-Gly2-L-Ser-Gly (similar to A11.3) and the respiratory quinolone was menaquinone 6. Based on their genotypic and chemotaxonomic characteristics, these strains represent a novel species of the genus Macrococcus, for which we propose the name Macrococcus armenti sp. nov. The type strain is JEK37T (=DSM 112712T=CCOS 1982T).

Driving Laboratory Standardization of Bacterial Culture and Antimicrobial Susceptibility Testing in Veterinary Clinical Microbiology in Europe and Beyond.

Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for the standardized training of clinical microbiologists and harmonization of diagnostic procedures. This is particularly true for veterinary clinical microbiology, where additional challenges exist when microbiologists are trying to fulfill a professional role very similar to that of their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include (i) harmonization of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); (ii) specific guidelines for interpretation and reporting of AST results for animal pathogens; (iii) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; (iv) standardization of diagnostic procedures for animal clinical specimens; and (v) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues, led by the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), which is bringing together experts in veterinary microbiology, pharmacology, epidemiology, and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners toward standardization and harmonization of laboratory methodologies and optimization of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardization and harmonization of veterinary clinical microbiology methodologies that could then be used as a template for implementation at a wider international level.

The influence of clinical severity and topical antimicrobial treatment on bacteriological culture and the microbiota of equine pastern dermatitis.

BACKGROUND: Equine pastern dermatitis (EPD) is a common dermatological problem in horses, yet its aetiology and pathogenesis are poorly understood. OBJECTIVES: This study aimed to investigate the effects of lesion severity and topical antimicrobial treatment on bacterial flora of EPD-affected skin. ANIMALS: Sixteen horses with EPD were investigated. METHODS AND MATERIALS: An observational study was conducted by assigning a clinical severity score ranging from 0 (macroscopically nonlesional) to 21 (severe), and sampling the most and least severely affected limbs of 16 horses (32 limbs) for bacteriological culture and 16S rRNA sequencing. Topical antimicrobial treatment in the month before sampling was recorded. The limbs were allocated to a nonlesional or mildly affected group (Group A, score 0-3) and a moderate to severely affected group (Group B, score 4-21). RESULTS: The most commonly cultured bacterial species was Staphylococcus aureus (one of 15 Group A versus nine of 17 Group B). Within Group B, S. aureus was found in three of six limbs treated with topical antimicrobials and in six of 11 untreated limbs. ß-haemolytic streptococci (three of 32) and Trueperella pyogenes (two of 32) also were cultured exclusively in the untreated limbs of Group B. Staphylococci and streptococci were found more often by 16S rRNA sequencing than in culture. Limbs with higher lesion severity and topical antimicrobial treatment appeared to have a lower alpha diversity and different beta diversity compared to milder and untreated lesions. CONCLUSIONS AND CLINICAL IMPORTANCE: Observed differences in microbiota of equine skin are likely to be linked to the presence and severity of EPD and topical antimicrobial treatment. Further research is needed to establish causal bacteria.

Methicillin-Resistant Staphylococcus aureus Strains in Swiss Pigs and Their Relation to Isolates from Farmers and Veterinarians.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged over the last few decades as a One Health problem with an increasing prevalence in various animal species. The most notable animals are pigs, as asymptomatic carriers, and horses, where there is often an association with infections. The current study looked at the course of MRSA prevalence in Swiss livestock since 2009, with a special focus on pigs, followed by screening of veterinarians and farmers. Livestock isolates were obtained from the Swiss monitoring program and then characterized by spa typing. Concentrating on the year 2017, we analyzed the prevalence of MRSA in Swiss veterinarians and farmers, followed by whole-genome sequencing of selected human and animal strains. The phylogeny was assessed by applying core-genome multilocus sequence typing (MLST) and single-nucleotide polymorphism (SNP) analyses, followed by screening for resistance genes and virulence factors. The prevalence of MRSA in Swiss pigs showed a dramatic increase from 2% in 2009 to 44% in 2017. Isolates typically belonged to clonal complex 398 (CC398), split between spa t011 and t034. The higher prevalence was mainly due to an increase in t011. spa t034 strains from farmers were found to be closely associated with porcine t034 strains. The same could be shown for spa t011 strains from horses and veterinarians. spa t034 strains had a high number of additional resistance genes, and two strains had acquired the immune evasion cluster. However, all but one of the pig spa t011 strains clustered in a separate group. Thus, the increase in pig spa t011 strains does not directly translate to humans.IMPORTANCE MRSA is an important human pathogen; thus, its increasing prevalence in livestock over the last decade has a potentially large impact on public health. Farmers and veterinarians are especially at risk due to their close contact with animals. Our work demonstrates a dramatic increase in MRSA prevalence in Swiss pigs, from 2% in 2009 to 44% in 2017. Whole-genome sequencing allowed us to show a close association between farmer and pig strains as well as veterinarian and horse strains, indicating that the respective animals are a likely source of human colonization. Furthermore, we could demonstrate that pig spa t011 strains cluster separately and are probably less likely to colonize humans than are pig spa t034 strains. This research may provide a basis for a more substantiated risk assessment and preventive measures.

Typing of mecD Islands in Genetically Diverse Methicillin-Resistant Macrococcus caseolyticus Strains from Cattle.

Macrococcus caseolyticus belongs to the normal bacterial flora of dairy cows and does not usually cause disease. However, methicillin-resistant M. caseolyticus strains were isolated from bovine mastitis milk. These bacteria had acquired a chromosomal island (McRI mecD -1 or McRI mecD -2) carrying the methicillin resistance gene mecD To gain insight into the distribution of McRI mecD types in M. caseolyticus from cattle, 33 mecD-containing strains from Switzerland were characterized using molecular techniques, including multilocus sequence typing, antibiotic resistance gene identification, and PCR-based McRI mecD typing. In addition, the same genetic features were analyzed in 27 mecD-containing M. caseolyticus strains isolated from bovine bulk milk in England/Wales using publicly available whole-genome sequences. The 60 strains belonged to 24 different sequence types (STs), with strains belonging to ST5, ST6, ST21, and ST26 observed in both Switzerland and England/Wales. McRI mecD -1 was found in different STs from Switzerland (n = 19) and England/Wales (n = 4). McRI mecD -2 was only found in 7 strains from Switzerland, all of which belonged to ST6. A novel island, McRI mecD -3, which contains a complete mecD operon (mecD-mecR1m-mecIm [where the subscript m indicates Macrococcus]) combined with the left part of McRI mecD -2 and the right part of McRI mecD -1, was found in heterogeneous STs from both collections (Switzerland, n = 7; England/Wales, n = 21). Two strains from England/Wales carried a truncated McRI mecD -3. Phylogenetic analyses revealed no clustering of strains according to geographical origin or carriage of McRI mecD -1 and McRI mecD -3. Circular excisions were also detected for McRI mecD -1 and McRI mecD -3 by PCR. The analyses indicate that these islands are mobile and may spread by horizontal gene transfer between genetically diverse M. caseolyticus strains.IMPORTANCE Since its first description in 2017, the methicillin resistance gene mecD has been detected in M. caseolyticus strains from different cattle sources and countries. Our study provides new insights into the molecular diversity of mecD-carrying M. caseolyticus strains by using two approaches to characterize mecD elements: (i) multiplex PCR for molecular typing of McRI mecD and (ii) read mapping against reference sequences to identify McRI mecD types in silico In combination with multilocus sequence typing, this approach can be used for molecular characterization and surveillance of M. caseolyticus carrying mecD.

Heterogeneous Genetic Location of mcr-1 in Colistin-Resistant Escherichia coli Isolates from Humans and Retail Chicken Meat in Switzerland: Emergence of mcr-1-Carrying IncK2 Plasmids.

We characterized the genetic environment of mcr-1 in colistin-resistant Escherichia coli strains isolated in Switzerland during 2014 to 2016 from humans (n = 3) and chicken meat (n = 6). Whole-genome and plasmid sequencing identified the mcr-1 gene integrated in IncX4 (of which, one strain carried the mcr-1.2 variant), IncI2, IncHI2, and novel IncK2 plasmids (overall, n = 7), as well as in the bacterial chromosome (n = 2) in single or duplicate copies. Our study supports the easy mobilization of mcr-1 across diverse genetic locations.

European multicenter study on antimicrobial resistance in bacteria isolated from companion animal urinary tract infections.

BACKGROUND: There is a growing concern regarding the increase of antimicrobial resistant bacteria in companion animals. Yet, there are no studies comparing the resistance levels of these organisms in European countries. The aim of this study was to investigate geographical and temporal trends of antimicrobial resistant bacteria causing urinary tract infection (UTI) in companion animals in Europe. The antimicrobial susceptibility of 22 256 bacteria isolated from dogs and cats with UTI was determined. Samples were collected between 2008 and 2013 from 16 laboratories of 14 European countries. The prevalence of antimicrobial resistance of the most common bacteria was determined for each country individually in the years 2012-2013 and temporal trends of bacteria resistance were established by logistic regression. RESULTS: The aetiology of uropathogenic bacteria differed between dogs and cats. For all bacterial species, Southern countries generally presented higher levels of antimicrobial resistance compared to Northern countries. Multidrug-resistant Escherichia coli were found to be more prevalent in Southern countries. During the study period, the level of fluoroquinolone-resistant E. coli isolated in Belgium, Denmark, France and the Netherlands decreased significantly. A temporal increase in resistance to amoxicillin-clavulanate and gentamicin was observed among E. coli isolates from the Netherlands and Switzerland, respectively. Other country-specific temporal increases were observed for fluoroquinolone-resistant Proteus spp. isolated from companion animals from Belgium. CONCLUSIONS: This work brings new insights into the current status of antimicrobial resistance in bacteria isolated from companion animals with UTI in Europe and reinforces the need for strategies aiming to reduce resistance.

Real time detection of pathogenic bacteria in veterinary microbiology using isothermal microcalorimetry - A different approach.

With today's challenges regarding antibiotic resistance and the importance of the implementation of prudent use of antibiotics, fast and reliable diagnostic tools for bacterial infections and subsequent antimicrobial susceptibility testing are of utmost relevance. Isothermal microcalorimetry (IMC) is a broadly applicable method, with which metabolic heat flow in reproducing bacteria can be measured in real time. To the best of the authors' knowledge, this is the first report on examination of 124 urine samples from feline and canine urinary tract infection with an IMC-based prototype instrument. A concentration-dependent time of peak heat flow by dilution series with Escherichia coli and Enterococcus faecalis reference strains demonstrated the general good performance of the prototype for detection of these bacteria. With diagnostic culture being set as a gold standard, the diagnostic sensitivity of IMC compared to bacteriological culture was 80 %, the diagnostic specificity was 97 %. With a Cohens' kappa value (κ) of 0.80, the two methods show good concordance. The results from our study demonstrate that the IMC technology is suitable to allow reliable, but much faster detection of bacteria than conventional culture, especially for Escherichia coli. Thus, implementing IMC technology could markedly speed up the bacteriological diagnostic process in veterinary medicine.

Time-calibrated phylogenetic and chromosomal mobilome analyses of Staphylococcus aureus CC398 reveal geographical and host-related evolution.

An international collection of Staphylococcus aureus of clonal complex (CC) 398 from diverse hosts spanning all continents and a 30 year-period is studied based on whole-genome sequencing (WGS) data. The collection consists of publicly available genomic data from 2994 strains and 134 recently sequenced Swiss methicillin-resistant S. aureus (MRSA) CC398 strains. A time-calibrated phylogeny reveals the presence of distinct phylogroups present in Asia, North and South America and Europe. European MRSA diverged from methicillin-susceptible S. aureus (MSSA) at the beginning of the 1950s. Two major European phylogroups (EP4 and EP5), which diverged approximately 1974, are the main drivers of MRSA CC398 spread in Europe. Within EP5, an emergent MRSA lineage spreading among the European horse population (EP5-Leq) diverged approximately 1996 from the pig lineage (EP5-Lpg), and also contains human-related strains. EP5-Leq is characterized by staphylococcal cassette chromosome mec (SCCmec) IVa and spa type t011 (CC398-IVa-t011), and EP5-Lpg by CC398-SCCmecVc-t011. The lineage-specific antibiotic resistance and virulence gene patterns are mostly mediated by the acquisition of mobile genetic elements like SCCmec, S. aureus Genomic Islands (SaGIs), prophages and transposons. Different combinations of virulence factors are present on S. aureus pathogenicity islands (SaPIs), and novel antimicrobial resistance gene containing elements are associated with certain lineages expanding in Europe. This WGS-based analysis reveals the actual evolutionary trajectory and epidemiological trend of the international MRSA CC398 population considering host, temporal, geographical and molecular factors. It provides a baseline for global WGS-based One-Health studies of adaptive evolution of MRSA CC398 as well as for local outbreak investigations.

ENOVAT: the European Network for Optimization of Veterinary Antimicrobial Treatment.

The global antimicrobial resistance crisis has been the driver of several international strategies on antimicrobial stewardship. For their implementation at the field level, the veterinary sector encounters several specific challenges and in particular: (i) a shortage of experts in key disciplines related to antimicrobial stewardship, (ii) a lack of evidence-based antimicrobial treatment guidelines, and (iii) inferior diagnostic tests available compared to human medicine. The present white paper describes how the COST Action ENOVAT (the European Network for Optimization of Veterinary Antimicrobial Treatment, CA18217), comprising 332 persons from 51 countries, worked towards solutions to these challenges. Initially, surveys were conducted to explore the present state in Europe in terms of existing antimicrobial use guidelines and microbiology practices performed. Concurrently, various research activities were launched to optimize diagnostics, including development of epidemiological cut-offs, clinical breakpoints and matrix-assisted laser desorption ionization time of flight mass spectrometry interpretive criteria. Also, guidelines drafting groups working towards evidence-based antimicrobial treatment guidelines for six conditions in food-producing and companion animals were established. The processes and outcomes, also in terms of capacity building, are summarized in this white paper where emphasis is placed on sustainability of the activities. Although several ENOVAT initiatives and spin-off projects will continue beyond the Action, we recommend that a new European veterinary research agenda is launched focusing on research and funding leading to long-term impacts on veterinary antimicrobial use.

Antimicrobial resistance is an urgent global public health threat that is amplified by over- and misuse of antimicrobials. As a result of antimicrobial resistance, antibiotics and other antimicrobial medicines become ineffective and infections become difficult or impossible to treat. This goes for human infections, but also for infections in animals. In a recently finished European project called ENOVAT we tried to tackle the problem of antimicrobial resistance in animals. We focused on two topics. First we optimized and harmonized diagnostics of bacterial infections in the laboratory, and second we developed evidence-based treatment guidelines to support veterinary practitioners on how and when to use antibiotics in the best way. Improved diagnostics and new treatment guidelines can help veterinary practitioners to a more sensible antibiotic choice and with that less over- and misuse of antimicrobials. This article summarizes the process and progress of the work done in the ENOVAT project. Emphasis is also put on how the project benefitted from a unique consortium encompassing 332 professionals with diverse backgrounds, from 51 countries.

Comparison of genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli on chicken retail meat and at slaughter.

Multilocus sequence typing (MLST) and antibiotic resistance patterns of Campylobacter jejuni and Campylobacter coli from retail chicken meat showed high overlap with isolates collected at slaughterhouses, indicating little selection along the production chain. They also showed significant common sequence types with human clinical isolates, revealing chicken meat as a likely source for human infection.

Surgical site infection after 769 Tibial Plateau Leveling Osteotomies.

Objective: To report surgical site infections (SSI) after Tibial Plateau Leveling Osteotomy (TPLO), treatment course, associated risk factors, bacterial isolates and antimicrobial resistance. Study design: Retrospective clinical cohort study. Study population: Six hundred and twenty seven dogs and 769 TPLO procedures. Methods: Data from electronic medical records of dogs undergoing TPLO between 2005 and 2015 at a single institution have been retrospectively reviewed. A generalized mixed logistic regression was used to determine possible risk factors. The Chi-Square test of independence was used to examine the relationship between the isolation of multidrug-resistant (MDR) bacteria and the development of major infections undergoing additional surgical treatment. To assess the correlation between number of SSI and number MDR isolate per year, Pearson's correlation coefficient was calculated. Results: The overall complication rate was 19.3% (n = 149). SSI was most frequent with 8.5% (n = 65). Major SSI occurred in 6.8% (n = 52) TPLO (80.0% SSI). Staphylococcus (S.) pseudintermedius (n = 37) and S. aureus (n = 10) were most frequently isolated. Multidrug-resistant bacteria were identified in 2.7% (n = 21) TPLO (32.3% SSI) but were not associated with major SSI (p = 0.426). There was a strong positive correlation between number of MDR isolates per year and number of SSI per year [r (9) = 0.79, p = 0.004]. Factors associated with SSI were previous TPLO in the contralateral stifle (p = 0.02, OR = 2.01, 95% CI = 1.11-3.64) and German Shepherd dogs (p = 0.035, OR = 4.41, 95% CI = 1.11-17.54). The use of non-locking implants was found to be protective (p = 0.02, OR = 0.179, 95% CI = 0.18-0.77). Clinical significance: Infection with multidrug-resistant bacteria is an emerging problem in veterinary practice and treatment is challenging. The incidence of major SSI was found to be high but was not associated with the isolation of MDR bacteria.

Pilot testing the EARS-Vet surveillance network for antibiotic resistance in bacterial pathogens from animals in the EU/EEA.

Introduction: As part of the EU Joint Action on Antimicrobial Resistance (AMR) and Healthcare-Associated Infections, an initiative has been launched to build the European AMR Surveillance network in veterinary medicine (EARS-Vet). So far, activities included mapping national systems for AMR surveillance in animal bacterial pathogens, and defining the EARS-Vet objectives, scope, and standards. Drawing on these milestones, this study aimed to pilot test EARS-Vet surveillance, namely to (i) assess available data, (ii) perform cross-country analyses, and (iii) identify potential challenges and develop recommendations to improve future data collection and analysis. Methods: Eleven partners from nine EU/EEA countries participated and shared available data for the period 2016-2020, representing a total of 140,110 bacterial isolates and 1,302,389 entries (isolate-antibiotic agent combinations). Results: Collected data were highly diverse and fragmented. Using a standardized approach and interpretation with epidemiological cut-offs, we were able to jointly analyze AMR trends of 53 combinations of animal host-bacteria-antibiotic categories of interest to EARS-Vet. This work demonstrated substantial variations of resistance levels, both among and within countries (e.g., between animal host species). Discussion: Key issues at this stage include the lack of harmonization of antimicrobial susceptibility testing methods used in European surveillance systems and veterinary diagnostic laboratories, the absence of interpretation criteria for many bacteria-antibiotic combinations of interest, and the lack of data from a lot of EU/EEA countries where little or even surveillance currently exists. Still, this pilot study provides a proof-of-concept of what EARS-Vet can achieve. Results form an important basis to shape future systematic data collection and analysis.

Prevalence and antimicrobial resistance of Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7) in Swiss sheep flocks.

Salmonella (S.) enterica subspecies diarizonae (IIIb) serovar 61:k:1,5,(7) (S. IIIb 61:k:1,5,(7)) is considered to be sheep-associated, as it can be found in the intestine, tonsils and nose of clinically healthy sheep, but it has also been described in separate clinical disorders in sheep. In particular, S. IIIb 61:k:1,5,(7) is described as the causative agent of chronic proliferative rhinitis (CPR) in sheep. In Switzerland, CPR in sheep due to S. IIIb 61:k:1,5,(7) was first described in 2017 in a flock of Texel sheep. Therefore, we assessed the prevalence of S. IIIb 61:k:1,5,(7) within the Swiss sheep population using a representative sampling strategy. From May 2017 to June 2018 a total of 681 nasal swabs from individual clinically healthy sheep of 141 different flocks throughout Switzerland were taken. Swabs were analysed by selective enrichment for the presence of S. IIIb 61:k:1,5,(7). Additionally, antimicrobial resistance of the isolates was determined by broth microdilution. A total of 146 out of 681 nasal swabs tested positive for S. IIIb 61:k:1,5,(7), which corresponds to a prevalence on animal level of 21% (95%CI 18%-25%). In 73 out of 141 flocks tested, at least one sheep tested positive for S. IIIb 61:k:1.5,(7), resulting in a minimal prevalence on flock level of 52% (95%CI 43%-60%). Positive flocks were found in all cantons except the canton of Jura. Adults were significantly more affected than sheep under one year/lambs and positive sheep were found in several breeds. No microbiologically resistant isolates were detected, except for one isolate showing resistance against ampicillin. Because of its widespread occurrence in the Swiss sheep population, further research should focus on the pathogenic impact of S. IIIb 61:k:1,5,(7) on the health status of sheep.

Seroprevalence of Mycoplasma hyopneumoniae in sows fifteen years after implementation of a control programme for enzootic pneumonia in Switzerland.

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), an economically important chronic respiratory disease in pigs. M. hyopneumoniae impacts the mucociliary clearance system by disrupting the cilia and modulates the immune response, resulting in intermittent dry non-productive cough. For progressive control of EP in Switzerland, a corresponding programme was fully implemented in 2004. It is based on total depopulation strategies of affected fattening farms as well as partial depopulation in breeding farms. Surveillance of EP status in Switzerland is mainly based on real-time PCR of nasal swabs from coughing animals or suspicious lungs and thereby sporadic cases are still observed every year. In order to obtain information on the seroprevalence, serum samples of 5021 sows from 968 farms collected in 2018 at eight different slaughterhouses were analyzed for the presence of M. hyopneumoniae-specific antibodies using a commercial ELISA kit. The overall seroprevalence was low with 0.98% of sows testing positive and these seropositive animals could be allocated to 3.92% of farms tested. Most seropositive farms presented weakly positive singleton reactors and only one farm showed several strongly seropositive animals. In conclusion, the serological status mirrors the successful progressive control of M. hyopneumoniae in the Swiss domestic pig population over the years. The current study underlines the added value of serological testing in the surveillance of EP in a country with low prevalence and confirms the sustained benefit of strategic control programmes.

The increase of methicillin-resistant Staphylococcus aureus (MRSA) and the presence of an unusual sequence type ST49 in slaughter pigs in Switzerland.

BACKGROUND: In years past, methicillin-resistant S. aureus (MRSA) has been frequently detected in pigs in Europe, North America and Asia. Recent, yet sporadic studies have revealed a low occurrence of MRSA in Switzerland. In 2009, a monitoring survey of the prevalence and genetic diversity of methicillin-resistant S. aureus (MRSA) in slaughter pigs in Switzerland was conducted using methods recommended by the EU guidelines, and using a sampling strategy evenly distributed throughout the year and representative of the Swiss slaughter pig population. Monitoring should determine if the overall prevalence of MRSA in the entire country is increasing over the years and if specific multi-resistant MRSA clones are spreading over the country. RESULTS: In 2009, the nasal cavities of eight out of 405 randomly selected pigs were positive for MRSA, representing a prevalence of 2.0% (95% CI 0.9-3.9). The following year, 23 out of 392 pigs were positive for MRSA [5.9% prevalence (95% CI 3.8-8.7)]. Three multilocus sequence types (ST), four spa types and two types of staphylococcal cassette chromosome mec (SCCmec) elements were detected. The most frequent genotypes were ST398 (MLST)-(spa)t034-V(SCCmec) (n=18) and ST49-t208-V (n=7), followed by ST398-t011-V (n=4), ST398-t1451-V (n=1), and ST1-t2279-IVc (n=1). The isolates displayed resistance to ß-lactams [mecA, (31/31); blaZ, (19/31)]; tetracycline [tet(M), (31/31); tet(K), (30/31)] (n=31); macrolides and lincosamides [erm(C) (4/31) or erm(A) (18/31)] (n=22); tiamulin [vga(A)v (9/31) or unknown mechanism (18/31)] (n=27); trimethoprim [dfr(G) (18/31); spectinomycin [ant(9)-Ia (19/31) or unknown mechanism (3/31)] (n=22); streptomycin [str (19/31)]; sulphamethoxazole (7/31) and ciprofloxacin (n=1) (mechanisms not determined). CONCLUSIONS: This study is the first to describe the presence of MRSA ST49 in slaughter pigs, and to demonstrate a significant and nearly three-fold increase of MRSA prevalence in pigs within two years. The presence of a specific clonal lineage of MRSA from Switzerland suggests that it has been selected in Swiss pig husbandry. Effective hygiene measures should be enhanced within the entire pig production chain to suppress the spread of these pathogens into the community.

Complete Genome Sequences of the Methicillin-Resistant Strain Staphylococcus aureus 17Gst354 and Its Prophage Staphylococcus Phage vB_StaphS-IVBph354.

We report the complete 2,783,931-bp circular genome sequence of the human methicillin-resistant strain Staphylococcus aureus 17Gst354, isolated from a nasal swab. The strain possessed an additional 4,397-bp plasmid. Moreover, we induced and sequenced its temperate phage Staphylococcus phage vB_StaphS-IVBph354, which has a circular genome of 41,970 bp.

Dynamics of extended-spectrum cephalosporin-resistant Escherichia coli in pig farms: A longitudinal study.

OBJECTIVES: Point prevalence estimates of extended-spectrum cephalosporin-resistant Escherichia coli (ESC-R-Ec) are important surveillance measures but may not uncover the ESC-R-Ec dynamics within pig farms. A longitudinal study was therefore performed by sampling individual pigs, pig farmers and the environment. METHODS: On average, 30 (range 10-46) piglets of 31 Swiss farms were sampled during the suckling, weaning and fattening stages (n= 2437 samples). In addition, stool from pig farmers and environmental samples were obtained and metadata collected by questionnaires. ESC-R-Ec was identified by routine culture, and clonal relationships and resistance genes were derived from whole genome sequencing data. RESULTS: Working on pig farms was not associated with an increased prevalence of ESC-R-Ec in humans. ESC-R-Ec prevalence significantly decreased from 6.2% to 3.9% and 1.8% for the suckling, weaned and fattening pigs, respectively (P < 0.001). Within the 57 ESC-R-positive suckling piglets, persisting carriage was detected in 25 animals at two consecutive time points and one animal at three consecutive time points. Clonal spread (n=7 farms, 22.6%) and horizontal gene transfer (n=1 farm, 3%) within pigs but not between humans and animals was detected. Liquid manure (n=10 samples, 16.7%) was identified as the major environmental reservoir of ESC-R-Ec in the pig farm environment. CONCLUSIONS: Pig farming practices like all-in-all-out systems, but not antimicrobial usage, were associated with reduced risk of ESC-R-Ec at the farm level. As carriage duration is normally short within the individual pigs, the risk of recolonisation and clonal spread of ESC-R-Ec might be reduced by applying appropriate decontamination strategies.