The increased detection of Shiga toxin-producing Escherichia coli (STEC) O113:H4 among human cases in Belgium questions the importance of this serotype as an emerging pathogen. However, detailed information focusing on serotype O113:H4 from human and non-human sources remains limited. We analysed a collection of 140 STEC O113:H4 isolates and their whole genomes, originating from animal hosts (cattle, deer, goats, and sheep), food, and humans, to determine their genetic relationship and assess key virulence genes. All STEC O113:H4 genomes lacked the locus of enterocyte effacement (LEE) and belonged to Pasteur Sequence Type (pST) 367 complex, dominated by pST367 (ehxA-, stx2d+) and pST1729 (ehxA+, stx2b+). Compared to stx2d+ isolates, stx2b+ isolates carried on median more virulence factors, which might thus contribute to enhanced pathogenicity. Besides, humans appear to be infected with distinct subgroups of STEC O113:H4 carrying distinct stx subtypes and originating from potentially different sources: deer, goats, and sheep for STEC carrying stx2b (alone or in combination with stx1c) and mainly cattle for STEC carrying stx2d. Our results call for improved understanding and continuous surveillance of emerging STEC O113:H4.

Objectives: The role of bacterial communities in the pathophysiology of canine nasal disease is still unclear. How and when to treat dogs with suspected secondary bacterial rhinitis and on which test to rely before making a decision to treat with antimicrobials has not been established. The objective is to compare the results of bacterial identification using agar-plate cultures and 16S rRNA gene amplicon sequencing in dogs with nasal discharge suspected to be of bacterial origin. Materials and Methods: Twenty-nine client-owned dogs presented for investigation of nasal disease were included in the study. Paired swabs were collected from the same affected nasal cavity. One swab was streaked on 4 agar media (Columbia Blood Agar, MacConkey, Chapman and Edward's). The other swab was stored in a sterile cryotube at -80(degrees). Extracted DNA underwent a polymerase chain reaction targeting the V1-V3 region of the 16S rRNA gene. Results: At least one of the species detected by amplicon sequencing with a relative abundance of >10% was also identified by culture in 14 cases (48.3%), in association with marked predominance of one taxon (>80% relative abundance) in six of 14 cases. In 12 dogs (41.4%), the cultured isolates were rare or undetected components of the corresponding sequence libraries. A negative culture in the face of bacterial predominance (>50% relative abundance) of a potentially pathogenic bacteria detected by sequencing occurred in 17% (n=5) of cases; however, the use of other agar media may have decreased this percentage. Clinical Significance: Standard culture does not reliably predict the bacterial profile detected by 16S rRNA gene amplicon sequencing.

La diarrhée est l’une des principales pathologies rencontrées dans les élevages de bovins au Bénin. Les veaux, premiers maillons de la chaîne, en sont les plus atteints. L’objectif de cette étude était d’évaluer la présence des gènes de virulence dans les souches d’Escherichia coli susceptibles de provoquer la diarrhée chez les veaux ainsi que leurs profils de résistance aux antimicrobiens usuels. Pour cela, 106 veaux ont fait objet d’un suivi pendant deux mois après leur naissance dans la commune de Nikki. Au total, 33 échantillons de matières fécales ont été prélevés directement du rectum de 33 veaux atteints de diarrhée et soumis à des analyses bactériologiques. Tous les prélèvements réalisés étaient positifs à E. coli. La caractérisation des souches d’E. coli isolées pour la présence des gènes et facteurs de virulence stx1, stx2, eae, sta, F41 et F5, a révélé la présence du gène stx1 uniquement avec un taux de 63,64 %. La résistance des souches d’E. coli aux antibiotiques les plus utilisés au Bénin a été testée : la doxycycline (taux de résistance de 70 %), l’amoxicilline + acide clavulanique (50 %) et la colistine (50 %). Des études ultérieures sont nécessaires afin de procéder au typage sérologique et au séquençage du génome des souches d’E. coli. Il serait également nécessaire d’étendre l’échantillonnage aux autres régions du Bénin, afin de mieux évaluer le statut des élevages bovins vis-à-vis de ces souches d’E. coli isolées chez les veaux et ainsi identifier un éventuel risque zoonotique.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

The present study compared multiplex PCR (mPCR) and Whole Genome Sequencing (WGS) using the SCCmecFinder database to identify the Staphylococcal Cassette Chromosome (SCC) mec in five Staphylococcus aureus (SA) and nine non-aureus staphylococci (NAS) isolated from dairy cattle. mPCR identified an SCCmecIV in four SA and one NAS, but could not differentiate between SCCmecII and IV in the fifth SA, that all harbored the mecA gene and were phenotypically resistant to cefoxitin. SCCmecFinder confirmed the presence of an SCCmecIVc(2B) in four SA and of the SCCmecIVa(2B) in the fifth SA and the one NAS. Both methods also detected one untypeable SCCmec in another cefoxitin-resistant NAS harboring the mecA gene and a pseudo SCCmec in one cefoxitin-sensitive NAS harboring one mecC-related gene. No SCCmec elements were identified either in one cefoxitin-sensitive NAS harboring the mecA2 gene, or in five NAS (one resistant and four sensitive to cefoxitin) harboring the mecA1 gene. SCCmecFinder could even not identify the presence of any mecA1 gene in these five NAS, whose presence was nevertheless confirmed by ResFinder. The conclusions of this study are: (i) mPCR and WGS sequencing using SCCmecFinder are complementary methodologies to identify SCCmec; (ii) SCCmecFinder and ResFinder to a lesser extent cannot identify all mec gene allotypes; (iii) a specific classification of the SCCmec in NAS would be epidemiologically helpful; (iv) presence of a mecA gene and a complete SCCmec is linked to cefoxitin resistance, whereas presence of other mec genes and of pseudo or no SCCmec is not.

The invasiveness properties of Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) O80:H2 in humans and calves are encoded by genes located on a pS88-like ColV conjugative plasmid. The main objectives of this study in larvae of the Galleria mellonella moth were therefore to compare the virulence of eight bovine STEC and EPEC O80:H2, of two E. coli pS88 plasmid transconjugant and STX2d phage transductant K12 DH10B, of four E. coli O80:non-H2, and of the laboratory E. coli K12 DH10B strains. Thirty larvae per strain were inoculated in the last proleg with 10 μL of tenfold dilutions of each bacterial culture corresponding to 10 to 106 colony-forming units (CFUs). The larvae were kept at 37 °C and their mortality rate was followed daily for four days. The main results were that: (i) not only the STEC and EPEC O80:H2, but also different E. coli O80:non-H2 were lethal for the larvae at high concentrations (from 104 to 106 CFU) with some variation according to the strain; (ii) the Stx2d toxin and partially the pS88 plasmid were responsible for the lethality caused by the E. coli O80:H2; (iii) the virulence factors of E. coli O80:non-H2 were not identified. The general conclusions are that, although the Galleria mellonella larvae represent a useful first-line model to study the virulence of bacterial pathogens, they are more limited in identifying their actual virulence properties.

Three soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against the CMY-2 β-lactamase behaved as inhibitors. The structure of the complex VHH cAb CMY-2 (254)/CMY-2 showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes into the catalytic site.

Enterotoxigenic Escherichia coli (ETEC) causing post-weaning diarrhea (PWD) in piglets have a detrimental impact on animal health and economy in pig production. ETEC strains can adhere to the host’s small intestinal epithelial cells using fimbriae such as F4 and F18. Phage therapy could represent an interesting alternative to antimicrobial resistance against ETEC infections. In this study, four bacteriophages, named vB_EcoS_ULIM2, vB_EcoM_ULIM3, vB_EcoM_ULIM8 and vB_EcoM_ULIM9, were isolated against an O8:F18 E. coli strain (A-I-210) and selected based on their host range. These phages were characterized in vitro, showing a lytic activity over a pH (4–10) and temperature (25–45 °C) range. According to genomic analysis, these bacteriophages belong to the Caudoviricetes class. No gene related to lysogeny was identified. The in vivo Galleria mellonella larvae model suggested the therapeutic potential of one selected phage, vB_EcoS_ULIM2, with a statistically significant increase in survival compared to non-treated larvae. To assess the effect of this phage on the piglet gut microbiota, vB_EcoS_ULIM2 was inoculated in a static model simulating the piglet intestinal microbial ecosystem for 72 h. This study shows that this phage replicates efficiently both in vitro and in vivo in a Galleria mellonella model and reveals the safety of the phage-based treatment on the piglet microbiota.

The origin of human and calf infections by Shigatoxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli O80:H2 is still unknown. The aim of this study was to identify E. coli O80 in healthy cattle with an emphasis on melibiose non-fermenting E. coli O80:H2. Faecal materials collected from 149 bulls at 1 slaughterhouse and 194 cows on 9 farms were tested with O80 antigen-encoding gene PCR after overnight growth in enrichment broths. The 53 O80 PCR-positive broths were streaked on different (semi-)selective agar plates. Five E. coli colonies from 3 bulls and 11 from 2 cows tested positive with the O80 PCR, but no melibiose non-fermenting E. coli was isolated. However, these 16 E. coli O80 were negative with PCR targeting the fliCH2, eae, stx1, stx2 and hlyF genes and were identified by WGS to serotypes and sequence types O80:H6/ST8619 and O80:H45/ST4175. They were phylogenetically related to E. coli O80:H6 and O80:H45 isolated from different animal species in different countries, respectively, but neither to STEC and EPEC O80:H2/ST301, nor to other serotypes of the clonal complex 165. As a conclusion, healthy adult cattle were not identified as a source of contamination of humans and calves by STEC or EPEC O80:H2.

Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (AE) lesions and cause non-bloody diarrhea in mammals. A minority of bovine EPEC belong to one of the ten classical serotypes of human and bovine AE-STEC. The purpose of this study was to identify five non-classical O serotypes (O123/186, O156, O177, O182, and O183) among bovine EPEC and to characterize their virulence repertoires by whole genome sequencing. Around 40% of the 307 EPEC from 307 diarrheic calves, 368 EPEC from 47 healthy cattle, and 131 EPEC from 36 healthy calves in dairy farms were analyzed. Serotype O177 was the most frequent among EPEC from diarrheic and healthy calves, while the O156 was the most frequent in healthy cattle. The genomic analysis identified different H serotypes, MLSTypes, and/or eae gene subtypes among the O156 and O177 EPEC, while the O182 was homogeneous. The virulence gene profiles of bovine EPEC were closely related to each other and to the profiles of ten bovine and human AE-STEC. These results emphasize the need for additional studies to identify more O:H serotypes of bovine EPEC and to elucidate their origin and evolution of EPEC with regard to AE-STEC belonging to the same O:H serotypes.

Avian pathogenic Escherichia coli (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain.

Soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against enzymes may behave as potent inhibitors. The immunization of alpacas with the CMY-2 β-lactamase led to the isolation of three VHHs that specifically recognized and inhibited CMY-2. The structure of the complex VHH cAbCMY-2(254)/CMY-2 was determined by X-ray crystallography. We showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes in the catalytic site. The β-lactamase inhibition was found to follow a mixed profile with a predominant non-competitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binder. Our study identified a binding site that can be targeted by a new class of β-lactamase’s inhibitors designed with the help of a peptidomimetic approach. Furthermore, the use of mono or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enable the development of the first generation of ELISA test for the detection of CMY-2 produced by resistant bacteria.IMPORTANCE The still increasing antimicrobial resistance in human clinic or veterinary medicine is a major threat for modern chemotherapy. Beside the major caution in the use of current antibiotics, it is important to develop new classes of antibiotics. This work was focused on β-lactamases that are the enzymes involved in the hydrolysis of the major class of antibiotics, the β-lactam compounds. We selected camelid antibodies that inhibit CMY-2, a class C β-lactamase produced by bacteria isolated from the veterinary and human settings. We characterized the conformational epitope present in CMY-2 in order to create a new family of inhibitors based on the paratope of the antibody. Finally, we designed a primary version of a detection system based on an ELISA using VHH and polyclonal antibodies.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens that cause human diseases ranging from diarrhea to life-threatening complications including hemolytic–uremic syndrome. Virulence of STEC strains and their ability to cause severe diseases are associated with the activity of prophage-encoded Shiga toxins (Stxs). The first objective of this work was to isolate and characterize the Stx2d phage from STEC O80:H2 and to study the transfer of this phage in non-STEC strains. The second objective was to assess the survival of Galleria mellonella larvae inoculated with these transduced strains. Firstly, one bacteriophage isolated from a STEC O80:H2 strain was used to infect six non-STEC strains, resulting in the conversion of three strains. Then, stability assays were performed, showing that this phage was stable in the new STEC strains after three successive subculturing steps, as confirmed by a combination of short and long read genome sequencing approaches. This phage, vB_EcoS_ULI-O80_Stx2d, is resistant to moderate temperature and pH. It belongs to a currently unclassified genus and family within the Caudoviricetes class, shares 98% identity with Stx2_112808 phage and encodes several proteins involved in the lysogenic cycle. The yecE gene was identified at the insertion site. Finally, G. mellonella experiments showed that the transduced strains caused significantly higher mortality rates than the corresponding non-STEC strains. In conclusion, this study showed that stx2d gene from O80:H2 E. coli can be transferred to non-STEC strains and contributes to their virulence.

Antimicrobial resistance is a major worldwide hazard. Therefore, the World Health Organization has proposed a classification of antimicrobials with respect to their importance for human medicine and advised some restriction of their use in veterinary medicine. In Belgium, this regulation has been implemented by a Royal Decree (RD) in 2016, which prohibits carbapenem use and enforces strict restrictions on the use of third- and fourth-generation cephalosporins (3 GC and 4 GC) for food-producing animals. Acquired resistance to β-lactam antibiotics is most frequently mediated by the production of β-lactamases in Gram-negative bacteria. This study follows the resistance to β-lactam antibiotics in Escherichia coli isolated from young diarrheic or septicaemic calves in Belgium over seven calving seasons in order to measure the impact of the RD. Phenotypic resistance to eight β-lactams was assessed by disk diffusion assay and isolates were assigned to four resistance profiles: narrow-spectrum β-lactamases (NSBL); extended-spectrum β-lactamases (ESBL); cephalosporinases (AmpC); and cephalosporinase-like, NSBL with cefoxitin resistance (AmpC-like). No carbapenemase-mediated resistance was detected. Different resistance rates were observed for each profile over the calving seasons. Following the RD, the number of susceptibility tests has increased, the resistance rate to 3 GC/4 GC has markedly decreased, while the observed resistance profiles have changed, with an increase in NSBL profiles in particular.

Escherichia coli producing Extended-Spectrum-β-Lactamases (ESBL) are a major public health hazard worldwide. The most frequent ESBL belong to the CTX-M family. This study follows their prevalence in pathogenic and non-pathogenic ESBL-producing E. coli isolated from young diarrheic and septicaemic calves over three calving seasons. The triplex PCR targeted three main groups: CTX-M-1, CTX-M-2 and CTX-M-9. Of the 394 isolates studied, 388 (98.5%) were positive, with a majority of CTX-M-1 (243, 61.7%), following by CTX-M-9 (74, 18.8%) and CTX-M-2 (64, 16.2%). The progressive decrease of ESBL-resistance of pathogenic E. coli is not linked to any shift in genetic background, blaCTX-M genes still present in 99% of the isolates, or to the proportion of the three CTX-M groups. Moreover, no significant difference was observed in the CTX-M content between pathogenic and non-pathogenic E. coli.

The bla genes identification present in 94 phenotypically resistant Escherichia coli isolated from feces or intestinal contents of young calves with diarrhea or enteritis in Belgium was performed by microarrays (MA) and whole genome sequencing (WGS). According to their resistance phenotypes to 8 β-lactams at the disk diffusion assay these 94 E. coli produced a narrow-spectrum-β-lactamase (NSBL), an extended-spectrum-β-lactamase (ESBL) or a cephalosporinase (AmpC). All ESBL-encoding genes identified by MA and WGS belonged to the blaCTX-M family, with a majority to the blaCTX-M-1 subfamily. Two different genes encoding an AmpC, blaCMY-2, and blaDHA-1 were detected in isolates with an AmpC phenotype. The blaTEM-1 and the blaOXA-1 were detected alone in isolates with a NSBL phenotype or in combination with ESBL-/AmpC-encoding bla genes. Furthermore, the WGS identified mutations in the ampC gene promoter at nucleotides -42 (C>T) and/or -18 (G>A) that could not be identified by MA, in several isolates with an AmpC-like resistance phenotype. No carbapenemase-encoding gene was detected. To our knowledge this is the first survey on the identification of bla genes in E. coli isolated from young diarrheic or septicemic calves in Belgium.

Extra-intestinal Escherichia coli express several virulence factors that increase their ability to colonize and survive in different localizations. The K1 capsular type is involved in several infections, including meningitis, urinary tract, and bloodstream infections. The aims of this work were to isolate, characterize, and assess the in vivo efficacy of phages targeting avian pathogenic E. coli (APEC) O18:K1, which shares many similarities with the human strains responsible for neonatal meningitis. Eleven phages were isolated against APEC O18:K1, and four of them presenting a narrow spectrum targeting E. coli K1 strains were further studied. The newly isolated phages vB_EcoS_K1-ULINTec2 were similar to the Siphoviridae family, and vB_EcoP_K1-ULINTec4, vB_EcoP_K1-ULINTec6, and vB_EcoP_K1-ULINTec7 to the Autographiviridae family. They are capsular type (K1) dependent and present several advantages characteristic of lytic phages, such as a short adsorption time and latent period. vB_EcoP_K1-ULINTec7 is able to target both K1 and K5 strains. This study shows that these phages replicate efficiently, both in vitro and in vivo in the Galleria mellonella model. Phage treatment increases the larvae survival rates, even though none of the phages were able to eliminate the bacterial load.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.

Lactococcus (L.) garvieae is a zoonotic fish pathogen that can also cause bacteraemia and endocarditis in humans and has been isolated from healthy or diseased domestic animals. Nevertheless L. garvieae is more an opportunistic, than a primary pathogen since most affected humans have predisposing conditions and comorbidities. L. garvieae is also present in other animal species, most frequently cattle, but also sheep, goats, water buffaloes, and pigs, and much more rarely dogs, cats, horses, camel, turtle, snake and crocodile. The purpose of this study was to genomically (i) confirm the identification by MALDI-TOF MS® of a L. garvieae from the nasal discharge of a dog with chronic respiratory disorders and (ii) compare this canine isolate with human and animal L. garvieae isolates. According to the BLAST analysis after Whole Genome Sequencing, this canine isolate was more than 99% identical to 3 L. garvieae and belonged to a new Multi-Locus Sequence Type (ST45). MLST and whole genomes-based phylogenetic analysis were performed on the canine isolate and the 40 genomes available in Genbank. The canine L. garvieae was most closely related to an Australian camel and an Indian fish L. garvieae and more distantly to human L. garvieae. Twenty-five of the 29 putative virulence-associated genes searched for were detected, but not the 16 capsule-encoding genes. The heterogeneity of the L. garvieae species is reflected by the diversity of the MLSTypes and virulotypes identified and by the phylogenetic analysis.

Methicillin-resistant Staphylococcus aureus (MRSA) and non-aureus staphylococci (MRNAS) cause different infections in animals, including mastitis, in livestock and humans. This study aimed to identify and compare the staphylococcal chromosome cassette mec (SCCmec) types of MRSA or MRNAS isolated from several animal species and humans in different countries. Of 1462 S. aureus and non-aureus staphylococci, 68 grew on Chrom MRSA ID® agar, were phenotypically resistant to cefoxitin and tested positive with the PCR for the mecA gene. These 60 MRSA and 8 MRNAS were isolated in Belgium mainly from cows (livestock-associated (LA) MRS) and humans (community-acquired (CA) MRS) and in Japan from dogs and cats. The SCCmec cassettes were identified by multiplex PCR in 52 MRSA and 7 MRNAS and by whole genome sequencing (WGS) in 8 additional MRSA. The SCCmec types IV and V were the most frequent in Belgian LA-MRS and CA-MRS, while the SCCmec type II was identified in four of the five Japanese MRSA. The remaining isolate was a bovine S. haemolyticus in which no SCCmec was identified. These results confirm the high prevalence of the SCCmec types IV and V in LA-MRS and CA-MRS in Belgium, emphasizing the possible public health hazard of the former, and the absence of SCCmec in some MRNAS.