Genetic Characterization of Antibiotic-Resistant Staphylococcus spp. and Mammaliicoccus sciuri from Healthy Humans and Poultry in Nigeria.

Staphylococcus spp. poses a significant threat to human and animal health due to their capacity to cause a wide range of infections in both. In this study, resistance genes conferring antibiotic resistance in Staphylococcus spp. and Mammaliicoccus sciuri isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 61 Staphylococcus spp. isolates were obtained from urine, wounds, nasal and chicken fecal samples. Species identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was performed using the Kirby-Bauer method for 16 antibiotics. Whole-genome sequencing was used for characterization of the isolates. The 61 investigated isolates included Staphylococcus aureus, S. arlettae, M. sciuri, S. haemolyticus, and S. epidermidis. A total of 47 isolates (77%) belonged to human samples and 14 (23%) isolates were collected from poultry samples. All were phenotypically resistant to at least three antimicrobial(s). Multiple resistance determinants were detected in the human and poultry isolates analyzed. Phylogenetic analysis revealed close relatedness among the isolates within each species for S. arlettae, M. sciuri, and S. haemolyticus, respectively. This study delivered comprehensive genomic insights into antibiotic-resistant Staphylococcus species and M. sciuri isolates from human and poultry sources in Edo state, Nigeria, from a One Health perspective.

Detection of mcr-1-1 Positive Enteropathogenic Escherichia coli Isolates Associated with Post-Weaning Diarrhoea in an Organic Piglet-Producing Farm in Austria.

Postweaning diarrhoea (PWD) is a frequent multifactorial disease occurring in swine stocks worldwide. Since pathogenic Escherichia (E.) coli play a pivotal role in the pathogenesis of PWD and porcine E. coli are often resistant to different antibiotics, colistin is frequently applied to treat piglets with PWD. However, the application of colistin to livestock has been associated with the emergence of colistin resistance. This case report describes the detection of the colistin resistance gene mcr-1-1 in two E. coli isolated from piglets with PWD in an Austrian organic piglet-producing farm, which was managed by two farmers working as nurses in a hospital. Both mcr-1-positive E. coli were further analysed by Illumina short-read-sequencing, including assemblies and gene prediction. Both isolates belonged to the same clonal type and were positive for eaeH and espX5, which are both virulence genes associated with enteropathogenic E. coli (EPEC). Due to the detection of mcr-1-positive EPEC and based on the results of the antimicrobial resistance testing, the veterinarian decided to apply gentamicin for treatment instead of colistin, leading to improved clinical signs. In addition, after replacing faba beans with whey, PWD was solely observed in 2/10 weaned batches in the consecutive months.

Molecular Characterization of Chimeric Staphylococcus aureus Strains from Waterfowl.

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain's genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying "bird-specific" virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA.

Complete genome sequence of Bordetella parapertussis strain 400431-b, isolated from a protracted course of whooping cough in Austria, 2023.

Here, we report the complete genome of Bordetella parapertussis strain 400431-b isolated from a nasopharyngeal swab from a 4-year-old patient presenting with a protracted course of whooping cough, vaccinated with three doses of diphtheria-tetanus-acellular pertussis-hepatitis B-poliomyelitis-Haemophilus influenzae type b vaccine.

Diversity of Staphylococcus aureus isolated from nares of ruminants.

AIMS: To examine the diversity of Staphylococcus aureus isolated from nasal swabs of ruminants in Rwanda. METHODS AND RESULTS: A total of 454 nasal swabs from 203 cows, 170 goats, and 81 sheep were examined for the presence of S. aureus, and 30 S. aureus isolates were detected and characterized pheno- and genotypically. Resistance to penicillin and/or tetracycline was observed. The isolates were assigned to eight different spa types (t21057 (novel), t10103, t18853, t20842, t318, t355, t458, and t9432) belonging to six clonal complexes (CCs) (CC152, CC30, CC3591, CC3666, CC522, and CC97). Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), and the human and bovine variants of the toxic shock syndrome toxin gene tst-1 variants were detected. CONCLUSION: These findings demonstrate that the nares of ruminants in Rwanda are colonized with mastitis-associated S. aureus, including lineages that are also carried by humans, underscoring the zoonotic risk, especially for livestock keepers. These results highlight the crucial importance of hygiene measures when handling livestock.

Characterization of Cronobacter sakazakii and Cronobacter malonaticus Strains Isolated from Powdered Dairy Products Intended for Consumption by Adults and Older Adults.

The objective of this study was to characterize Cronobacter spp. and related organisms isolated from powder dairy products intended for consumption by adults and older adults using whole-genome sequencing (WGS), and to identify genes and traits that encode antibiotic resistance and virulence. Virulence (VGs) and antibiotic resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder, and MOB-suite tools. Susceptibility testing was performed using disk diffusion. Five presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal MLST. Three C. sakazakii strains were of the clinical pathovar ST1, one was ST31, and the remaining isolate was C. malonaticus ST60. In addition, Franconibacter helveticus ST345 was identified. The C. sakazakii ST1 strains were further distinguished using core genome MLST based on 2831 loci. Moreover, 100% of the strains were resistant to cefalotin, 75% to ampicillin, and 50% to amikacin. The C. sakazakii ST1 strains were multiresistant (MDR) to four antibiotics. Additionally, all the strains adhered to the N1E-115 cell line, and two invaded it. Eighteen ARGs mainly involved in antibiotic target alteration and antibiotic efflux were detected. Thirty VGs were detected and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, and genes involved in metabolism and stress. The pESA3, pSP291-1, and pCMA1 plasmids were detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52, and IS26. The isolates of C. sakazakii and C. malonaticus exhibited multiresistance to antibiotics, harbored genes encoding various antibiotic resistance proteins, and various virulence factors. Consequently, these contaminated powdered dairy products pose a risk to the health of hypersensitive adults.

Investigation of 29 Antimicrobial Compounds in Soil Using Newly Developed UHPLC-MS/MS Method.

While the prudent and reasonable use of veterinary antimicrobial agents in food-producing animals is necessary, researchers over the decades have shown that these antimicrobial agents can spread into the environment through livestock manure and wastewater. The analysis of the occurrence of antimicrobial compounds in soil samples is of a great importance to determine potential impacts on human and animal health and the environment. In this study, an affordable, rugged and simple analytical method has been developed for the determination of twenty-nine antimicrobial compounds from five different classes (tetracyclines, fluoro(quinolones), macrolides, sulfonamides and diaminopirimidines). Liquid-liquid extraction (LLE) with extract filtration combined with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was the best strategy for the simultaneous determination of all analytes. The developed method was validated according to the Commission Implementing Regulation (EU) 2021/808. The limit of detections (LODs) ranged from 0.5 to 2.0 µg/kg, while the limit of quantitation (LOQ) was established at 1.0 to 20.0 µg/kg. The developed method was successfully applied for the determination of antimicrobial residues in one hundred and eighteen soil samples obtained from four European countries (Austria, Czech Republic, Estonia and Portugal). Doxycycline in the concentration levels of 9.07 µg/kg-20.6 µg/kg was detected in eight of the analysed samples. Samples were collected from areas where natural fertilizers (swine or cow manure) were applied. Our method can be efficiently used to monitor anti-microbial compounds in soil samples.

Genetic characterization of Escherichia coli and Klebsiella spp. from humans and poultry in Nigeria.

The emergence of antibiotic resistance in livestock, especially food-producing animals, is of major public health importance as a result of the possibility of these bacteria entering the food chain. In this study, the genetic characteristics of antibiotic-resistant Escherichia coli and Klebsiella spp. isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 45 Klebsiella spp. and 46 E. coli isolates were obtained from urine, clinical wounds, nasal and chicken faecal samples. Isolates were recovered and identified as previously described. Species identification was achieved by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and ribosomal multilocus sequence typing. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer method for 12 antibiotics. A double disc synergy test was used to screen for extended-spectrum beta-lactamse (ESBL) production. Whole genome sequencing was performed for strain characterization of the isolates. Thirteen Klebsiella spp. isolates yielded positive results by the ESBL phenotypic test and harboured ESBL genes. Of the 46 E. coli isolates, 21 human and 13 poultry isolates were resistant to at least one of the tested antibiotics. Four human E. coli isolates harboured ESBL genes and revealed positive results when applying ESBL double disc synergy tests. ESBL genes in the Klebsiella spp. and E. coli isolates include bla CTX-M-15 and bla SHV-28. Whole genome-based core gene multilocus sequence typing of the Klebsiella spp. and E. coli isolates revealed a close relatedness among the isolates. An integrated 'One Health' surveillance system is required to monitor transmission of antimicrobial resistance in Nigeria.

Screening of Antibiotic and Virulence Genes from Whole Genome Sequenced Cronobacter sakazakii Isolated from Food and Milk-Producing Environments.

The objective of this study was to use whole-genome sequencing (WGS) to screen for genes encoding for antibiotic resistance, fitness and virulence in Cronobacter sakazakii strains that had been isolated from food and powdered-milk-producing environments. Virulence (VGs) and antibiotic-resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder and PlasmidFinder tools. Susceptibility testing was performed using disk diffusion. Fifteen presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal-MLST. Nine C. sakazakii strains were found in the meningitic pathovar ST4: two were ST83 and one was ST1. The C. sakazakii ST4 strains were further distinguished using core genome MLST based on 3678 loci. Almost all (93%) strains were resistant to cephalotin and 33% were resistant to ampicillin. In addition, 20 ARGs, mainly involved in regulatory and efflux antibiotics, were detected. Ninety-nine VGs were detected that encoded for OmpA, siderophores and genes involved in metabolism and stress. The IncFIB (pCTU3) plasmid was detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52 and ISEhe3. The C. sakazakii isolates analyzed in this study harbored ARGs and VGs, which could have contributed to their persistence in powdered-milk-producing environments, and increase the risk of infection in susceptible population groups.

Mapping the evidence of the effects of environmental factors on the prevalence of antibiotic resistance in the non-built environment: Protocol for a systematic evidence map.

BACKGROUND: Human, animal, and environmental health are increasingly threatened by the emergence and spread of antibiotic resistance. Inappropriate use of antibiotic treatments commonly contributes to this threat, but it is also becoming apparent that multiple, interconnected environmental factors can play a significant role. Thus, a One Health approach is required for a comprehensive understanding of the environmental dimensions of antibiotic resistance and inform science-based decisions and actions. The broad and multidisciplinary nature of the problem poses several open questions drawing upon a wide heterogeneous range of studies. OBJECTIVE: This study seeks to collect and catalogue the evidence of the potential effects of environmental factors on the abundance or detection of antibiotic resistance determinants in the outdoor environment, i.e., antibiotic resistant bacteria and mobile genetic elements carrying antibiotic resistance genes, and the effect on those caused by local environmental conditions of either natural or anthropogenic origin. METHODS: Here, we describe the protocol for a systematic evidence map to address this, which will be performed in adherence to best practice guidelines. We will search the literature from 1990 to present, using the following electronic databases: MEDLINE, Embase, and the Web of Science Core Collection as well as the grey literature. We shall include full-text, scientific articles published in English. Reviewers will work in pairs to screen title, abstract and keywords first and then full-text documents. Data extraction will adhere to a code book purposely designed. Risk of bias assessment will not be conducted as part of this SEM. We will combine tables, graphs, and other suitable visualisation techniques to compile a database i) of studies investigating the factors associated with the prevalence of antibiotic resistance in the environment and ii) map the distribution, network, cross-disciplinarity, impact and trends in the literature.

Draft Genome Sequence of Enterococcus dispar CoE-457-22, Isolated from Traditionally Produced Montenegrin Dry Sausage.

Enterococcus dispar was isolated for the first time from synovial fluid and stool cultures and described as a new species in 1991. Here, we report the genome of E. dispar CoE-457-22, which was obtained from traditionally produced Montenegrin dry sausage (sudzuk).

Are Enterobacteriaceae and Enterococcus Isolated from Powdered Infant Formula a Hazard for Infants? A Genomic Analysis.

Powdered infant formulas (PIF) are the most used dietary substitutes that are used in order to supplement breastfeeding. However, PIF are not sterile and can be contaminated with different microorganisms. The objective of this study was to genomically characterize Enterobacteriaceae (ENT) and Enterococcus strains that were isolated from PIF. Strains were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS). Genomic typing, detection of virulence, and resistance profiles and genes were performed with the Ridom SeqSphere+ software; the comprehensive antibiotic resistance database (CARD) platform; ResFinder and PlasmidFinder tools; and by the disk diffusion method. Nineteen isolates from PIF were analyzed, including ENT such as Kosakonia cowanii, Enterobacter hormaechei, Franconibacter helveticus, Mixta calida, and lactic acid bacteria such as Enterococcus faecium. The strains exhibited resistance to beta-lactams, cephalosporins, and macrolides. Resistance genes such as AcrAB-TolC, marA, msbA, knpEF, oqxAB, fosA, blaACT-7, blaACT-14,qacJ, oqxAB,aac(6')-Ii, and msr(C); and virulence genes such as astA, cheB, cheR, ompA ompX, terC, ironA, acm, and efaAfm, adem were also detected. All the analyzed strains possessed genes that produced heat-shock proteins, such as IbpA and ClpL. In PIF, the presence of ENT and Enterococcus that are multiresistant to antibiotics-together with resistance and virulence genes-pose a health risk for infants consuming these food products.

Draft Genome Sequence of Citrobacter cronae Awk (Sequence Type 880), Associated with Edible Snails Commercially Available in Awka, Nigeria.

We describe the draft genome sequence and annotation of Citrobacter cronae strain Awk (sequence type 880), recovered from fresh edible snails (Achatina achatina) commercially available in Awka Metropolis, Nigeria. The genome contains 4,629 protein-coding genes, 107 RNA-coding genes, and several antimicrobial resistance genes, including blaCMY-98 and qnrB12.

Genome Sequence of OXA-726-Encoding Aeromonas dhakensis Igbk (Sequence Type 1171) from an Edible Snail Traded in Nigeria.

Aeromonas dhakensis is the most virulent Aeromonas species pathogenic to both animals and humans. The degree of its risk to health seems masked by misidentifications. We present the genome sequence of A. dhakensis Igbk (sequence type 1171), associated with snails, harboring the OXA-726 gene in the chromosome.

Outbreak of Cronobacter turicensis in European brown hares (Lepus europaeus).

This is the first report of acute deaths in five European brown hares (Lepus europaeus) attributed to mucoid and necrotizing typhlocolitis caused by genetically different Cronobacter (C.) turicensis strains in northeastern Austria. As this opportunistic pathogen is mainly known for causing disease in immunocompromised humans and neonates, this previously unrecognized potential for a spill over from a wildlife reservoir to humans warrants further attention.

Mycoplasma tauri sp. nov. isolated from the bovine genital tract.

Since 2006, a Mycoplasma species unidentifiable to the species level has been regularly isolated from the semen and prepuce of apparently healthy bulls, and occasionally from cattle displaying inflammatory disease of the genital tract. Seven of these Mycoplasma isolates were subjected to a comprehensive taxonomic study. The strains investigated grew well in modified Hayflick's medium and colonies on agar exhibited typical fried egg morphology and produced 'film and spots'. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas with spherically shaped cells bounded by a bi-layered cell membrane. The strains studied neither produced acid from sugar carbon sources nor did hydrolyse arginine or urea, and genome annotation indicated that organic acids (pyruvate, lactate) are used as energy sources. Phylogenetic analyses of 16S rRNA gene sequences, the 16S-23S intergenic spacer region, and partial rpoB gene and protein sequences placed the strains within the Mycoplasma (M.) bovis cluster of the Hominis group with M. primatum, M. agalactiae, and M. bovis being their closest relatives. Genomic information including whole-genome similarity metrics (ANIb, ANIm, TETRA, dDDH, AAI) and phylogenomics, proteomic features revealed by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry as well as serological reactions and polar lipid profiling strongly indicated that the strains examined were representatives of a hitherto unclassified species of genus Mycoplasma, for which the name Mycoplasma tauri sp. nov. with type strain Zaradi2T (=ATCC BAA-1891T = DSM 22451T) is proposed.

Diversity of West Nile and Usutu virus strains in mosquitoes at an international airport in Austria.

Increased globalization and international transportation have resulted in the inadvertent introduction of exotic mosquitoes and new mosquito-borne diseases. International airports are among the possible points of entry for mosquitoes and their pathogens. We established a mosquito and mosquito-borne diseases monitoring programme at the largest international airport in Austria and report the results for the first two years, 2018 and 2019. This included weekly monitoring and sampling of adult mosquitoes, and screening them for the presence of viral nucleic acids by standard molecular diagnostic techniques. Additionally, we surveyed the avian community at the airport, as birds are potentially amplifying hosts. In 2018, West Nile virus (WNV) was detected in 14 pools and Usutu virus (USUV) was detected in another 14 pools of mosquitoes (minimum infection rate [MIR] of 6.8 for each virus). Of these 28 pools, 26 consisted of female Culex pipiens/torrentium, and two contained male Culex sp. mosquitoes. Cx. pipiens/torrentium mosquitoes were the most frequently captured mosquito species at the airport. The detected WNV strains belonged to five sub-clusters within the sub-lineage 2d-1, and all detected USUV strains were grouped to at least seven sub-clusters among the cluster Europe 2; all strains were previously shown to be endemic in Austria. In 2019, all mosquito pools were negative for any viral nucleic acids tested. Our study suggests that airports may serve as foci of arbovirus activity, particularly during epidemic years, and should be considered when designing mosquito control and arbovirus monitoring programmes.

A core genome multilocus sequence typing scheme for Mycoplasma hyorhinis.

Mycoplasma (M.) hyorhinis is a commensal and pathobiont residing in the upper respiratory tract in swine and with the ability to spread systemically, mainly causing polyserositis and polyarthritis in nursery pigs. Since little is known on the epidemiology of M. hyorhinis infection, whole genome sequences of 73 strains isolated from pigs in Austria (n = 71) and Germany (n = 2), that have been isolated from clinically affected pigs during routine diagnostics, and publicly available genomes of eight M. hyorhinis strains were analyzed in the presented study. For this purpose, a core genome multi locus sequence typing (cgMLST) scheme encompassing 453 target genes was developed using the Ridom© SeqSphere + software. Results were compared to two previously described conventional MLST schemes and to a core genome single nucleotide polymorphism (cgSNP) analysis approach. Core genome MLST showed high diversity among the M. hyorhinis strains studied and while certain isolates from one farm or a single animal formed cgMLST clusters (≤ 8 allele differences), no isolates with identical allele profiles were identified. In addition, cgMLST had superior discriminatory power (Simpson's ID = 0.995) over conventional MLST (Simpson's ID = 0.952 and 0.985), while demonstrating a lack of congruence between conventional MLST and genome-wide relationship. Core genome SNP results were highly congruent with cgMLST results but lacked in resolution when comparing closely related isolates. Thus, cgMLST is the most suitable method for epidemiological investigations such as outbreak analysis, and to gain insights into M. hyorhinis population structure.

The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates.

Escherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC ß-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant ß-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.

Broad-Spectrum Cephalosporin-Resistant Klebsiella spp. Isolated from Diseased Horses in Austria.

The aim of the present study was to investigate the diversity of broad-spectrum cephalosporin-resistant Klebsiella spp. isolated from horses in Austria that originated from diseased horses. A total of seven non-repetitive cefotaxime-resistant Klebsiella sp. isolates were obtained during diagnostic activities from autumn 2012 to October 2019. Antimicrobial susceptibility testing was performed. The isolates were genotyped by whole-genome sequencing (WGS). Four out of seven Klebsiella isolates were identified as K. pneumoniae, two as K. michiganensis and one as K. oxytoca. All isolates displayed a multi-drug resistant phenotype. The detection of resistance genes reflected well the phenotypic resistance profiles of the respective isolates. All but one isolate displayed the extended-spectrum ß-lactamases (ESBL) phenotype and carried CTX-M cefotaximases, whereas one isolate displayed an ESBL and AmpC phenotype and carried cephamycinase (CMY)-2 and sulfhydryl variable (SHV)-type b and Temoniera (TEM) ß-lactamases. Among Klebsiella pneumoniae isolates, for different sequence types (ST) could be detected (ST147, ST307, ST1228, and a new ST4848). Besides resistance genes, a variety of virulence genes, including genes coding for yersiniabactin were detected. Considering the high proximity between horses and humans, our results undoubtedly identified a public health issue. This deserves to be also monitored in the years to come.