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Aims: To perform the molecular characterization of 23 Staphylococcus aureus isolates from pigs with signs of infections recovered in Spanish farms during 2018–2019. Methods and Results: The antimicrobial resistance pattern and virulence profile were determined. The molecular typing was performed by different molecular techniques. The transferability of the cfr gene was assessed by conjugation and its genetic environment was determined by PCR mapping. In all, 21 isolates were methicillin‐resistant S. aureus (MRSA) carrying the mecA gene (SCCmecV or non‐typeable SCCmec), whereas the remaining two were methicillin‐susceptible (MSSA). All but one MRSA isolates (n = 20) belonged to the CC398, being the spa t011 the most prevalent (n = 11). The remaining MRSA and the two MSSA isolates were ascribed to ST9/CC9. The S. aureus isolates exhibited resistance to (number of resistant isolates): β‐lactamics (21), erythromycin and/or clindamycin (20), aminoglycosides (7), tetracycline (22), fluoroquinolones (14), chloramphenicol (5) and linezolid (1). The S. aureus isolates did not carry any of the virulence genes studied. One MRSA belonging to the CC398 showed linezolid resistance mediated by the cfr gene. The cfr gene was co‐located with fexA in the Tn558 variant previously reported in the S. aureus plasmid pSCFS7. Conclusions: Two major livestock‐associated genetic lineages were detected among pigs with signs of infection in Spain. The presence of the cfr gene among LA‐MRSA‐CC398 is of great concern not only for veterinary medicine, but also for humans in close contact. Significance and Impact of the Study: This work describes the molecular characterization of S. aureus isolates recovered from pigs with signs of infection and we report, as far as we know, the first description of MRSA‐CC9 from pigs in Spain. Moreover, the detection of a MRSA‐CC398 isolate carrying the multiresistance cfr gene highlights the need for continuous surveillance and awareness of LA‐MRSA. [ABSTRACT FROM AUTHOR]

The novel erm(T)- cadDX-carrying plasmid pUR3912 has recently been described in the methicillin-susceptible Staphylococcus aureus ST398-t571 strain C3912 from a healthy human in Spain. Structural analysis revealed that pUR3912 belongs to the pC194 replicon family, replicates via a rolling circle mechanism and harbours putative double-strand ( dso) and single-strand ( sso) origins of replication. Besides its plasmid location, a copy of pUR3912 was also found in the chromosomal DNA of strain C3912. Two IS 431 copies, which flank the plasmid, most probably mediated its chromosomal integration. Its ability to not only exist extrachromosomally, but also to integrate into the chromosomal DNA ensures persistence and dissemination of pUR3912. [ABSTRACT FROM AUTHOR]

Skin infection associated with methicillin-resistant Staphylococcus aureus (MRSA)-ST398 was detected in a pig-farmer, and MRSAST398 isolates were also detected in nasal samples of the patient and of 11/12 pigs on his farm. Twelve MRSA isolates were obtained from skin lesions (n = 6) and nasal samples (n = 6) of the patient in two sampling moments and 11 MRSA isolates from nasal samples of pigs. They were typed as t011-SCCmecIVa- agrI and t108-SCCmecV-agrI (patient and pigs) and t588- SCCmecV- agrI (patient). The following resistance genes were detected (number isolates): tet(K) (1), tet(L) (23), tet(M) (13), erm(A) (13), erm(C) (13), msr(A) (11), lnu(A) (21), aph(2¢¢)- acc(6¢) (3), ant(4¢) (13), aph(3¢) (12), dfrS1 (15) and dfrK (22). Seventeen human and animal MRSA-ST398 isolates showed indistinguishable PFGE patterns (A1-spa-t011 or B2-spa-t108) and similar phenotypic-genotypic characteristics, including the presence of the lnu(A) gene, associated with lincomycin resistance. Potential pig-to-human transference of ST398 is suggested in this study. The first detection of the lnu(A) gene in MRSAST398 is reported. [ABSTRACT FROM AUTHOR]

Among the 1233 Salmonella enterica isolates obtained in two Spanish hospitals, five isolates (0.4%) (serovars: Virchow, four; Livingstone, one) had the phenotype of an extended-spectrum β-lactamase (ESBL) producer. The genetic characterization of the ESBL of S. enterica Livingstone revealed a blaSHV-2 gene. The blaCTX-M-10 gene in a phage-related genetic environment was found in one S. enterica Virchow isolate, and the blaCTX-M-9 gene within the In60 integron was found in the three remaining Virchow isolates. These three isolates presented indistinguishable or closely related pulsed-field gel electrophoresis patterns among themselves and also as compared with the two other blaCTX-M-9-containing isolates previously obtained from animals. ESBL production is an emerging mechanism of resistance in S. enterica in the two studied hospitals. [ABSTRACT FROM AUTHOR]

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates were detected in seven of 105 faecal samples from healthy humans, from two Spanish cities, during 2007. In these isolates, five ESBLs were detected, CTX-M-14 ( n = 2), CTX-M-1 ( n = 2), CTX-M-32 ( n = 1), CTX-M-8 ( n = 1) and TEM-52 ( n = 1). Both blaCTX-M-14a (surrounded by IS Ecp1-IS 903) and blaCTX-M-14b variants (included in an integron structure) were identified in this study. This is the first time that the blaCTX-M-8 gene and ESBLs of the CTX-M-8 group have been found in Europe and Spain, respectively. Faecal E. coli of healthy humans therefore constitute a reservoir of blaCTX-M genes with different surrounding genetic elements. [ABSTRACT FROM AUTHOR]

Animal food products are important sources of zoonotic agents, increasing the risk of exposure to antibiotic-resistant bacteria from farm to fork. Therefore, we aimed to detect and fully characterise Extended-Spectrum Beta-Lactamase (ESBL)-producing E. coli from the poultry sector in a One Health approach. From December 2021 to March 2022, 48 chicken meat samples were collected from 16 establishments in La Rioja (Northern Spain). Antibiotic susceptibility testing was assessed by the disk-diffusion method. Forty E. coli isolates were recovered from 33 of the 48 chicken meat samples tested (68.8%) when plated on MacConkey-agar. In addition, six ESBL-E. coli (6/48, 12.5%) were obtained on cefotaxime-supplemented MacConkey-agar, which were Whole-Genome Sequenced. A large diversity of clones and ESBL genes was observed, namely ST1140-E/bla CTX-M-32 (n = 1), ST752-A/bla TEM-52 (n = 1), ST117-B2/bla CTX-M-1 /bla SHV-12 (n = 2), ST10-A/bla SHV-12 (n = 1) and ST223-B1/bla SHV-12 (n = 1). Three IncI1-plasmids (pST3-CC3) were found carrying the bla SHV-12 /bla CTX-M-1 /bla CTX-M-32 genes in two genetic environments: i) IS26-smc-glpR-bla SHV-12 -IS26; and ii) wbuC-bla CTX-M-32 /bla CTX-M-1 -ISEcp1. The bla TEM-52 gene was carried on a P1-like phage-plasmid flanked by an IS4-mediated composite transposon. An IncHI2 plasmid harboured a bla SHV-12 gene flanked by an IS26-mediated composite transposon but also additional genes conferring resistance to aminoglycosides, chloramphenicol, and sulphonamides. To analyse the cross-sectoral relatedness of our ESBL-E. coli isolates, our six genomes were mapped with publicly available genomes (n = 2588) related to the STs detected, revealing that one of our genomes (X3078-ST117) displayed strong similarities (34-40 allelic differences) with few genomes belonging to ST117 from the poultry sector from Germany and USA. This study demonstrated that the proportion of ESBL-E. coli is still high in chicken meat in Spain. In addition, the ST117 clone and the IncI1-bla CTX-M-1-32 /bla SHV-12 plasmids might represent successful clones and plasmids adapted to the chicken host., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZD R ) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZD R enterococci carried plasmid rep genes (2-3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43-86). These findings strongly suggest the transmission of LZD R -E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZD R enterococci in all ecological niches and body parts to direct appropriate control strategies., (© 2024. The Author(s).)

This study determined the nasal staphylococci diversity and characterized their resistome, with a focus on the mobilome of methicillin-susceptible Staphylococcus aureus (MSSA)-CC398 subclade from healthy adults in La Rioja (northern Spain). Nasal staphylococci recovered from 57 healthy individuals (HI) were identified (MALDI-TOF-MS) and their antimicrobial resistance, virulence determinants and genetic lineages were studied. The relatedness of MSSA-CC398 isolates was assessed by core-genome single-nucleotide-polymorphisms (SNPs). One-hundred-forty-three non-repetitive staphylococci were obtained from most HI (98.2%), of which S. epidermidis (87.7%) and S. aureus (36.8%) were the predominant species. About 15% of the 27 S. aureus and 30.1% of the 116 coagulase-negative staphylococci (CoNS) isolates presented a multidrug resistance (MDR) phenotype. All S. aureus isolates were MSSA but 30.2% of CoNS isolates were mecA-positive and carried SCCmec types III, IV, and V. The highest non-beta-lactam resistance (frequency/genes) in S. aureus and CoNS were: erythromycin-clindamycin-inducible (25.9%/ermT, ermC) and mupirocin (30.1%/mupA), respectively. About 85% of S. aureus isolates carried relevant virulence genes. Eight clonal complexes (CCs) of MSSA were identified, of which CC398 was the predominant (33.3%). About 78% of the CC398 isolates harboured rep13-bound ermT gene, however, one carried a rep10-bound ermC gene. Only the ermT-positive MSSA-CC398 isolates were closely related (<50 SNPs) and carried the φSa3. Diverse MDR-S. epidermidis isolates were identified which included the lineages ST59 and ST210. The high rate of toxigenic S. aureus and of MSSA-CC398 subclade highlight the ability of HI to carry and transmit virulent isolates. Moreover, the high frequency of MDR-CoNS, often linked with SCCmec, needs to be monitored for their potential human health implications., Competing Interests: Declaration of Competing Interest None declared by authors., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The molecular ecology of Staphylococcus aureus in migratory birds (such as white storks) is necessary to understand their relevance in the "One Health" ecosystems. This study determined the nasotracheal carriage rates of S. aureus from white storks in Southern Spain and genetically characterized the within-host diversity. A collection of 67 S. aureus strains, previously obtained from 87 white stork nestlings (52 nasal and 85 tracheal samples) fed by their parents with food foraged in natural and landfill habitats, were tested for their antimicrobial resistance (AMR) phenotypes. Moreover, the AMR genotypes, immune evasion cluster (IEC), virulence genes and the detection of CC398 lineage were studied by PCR. The spa types and multilocus-sequencing-typing (MLST) were also determined by PCR and sequencing. Staphylococcus aureus carriage was found in 31% of storks (36.5%/11.9% in nasal/tracheal samples). All isolates were methicillin-susceptible (MSSA) and 8.8% of them were also susceptible to all tested antibiotics. The AMR phenotype/percentage/genes detected were as follows: penicillin/79.1%/blaZ; erythromycin-clindamycin-inducible/19.1%/ermA, ermT; tetracycline/11.9%/tetK; clindamycin/4.5%/lnuA and ciprofloxacin/4.5%. Twenty-one different spa types, including 2 new ones (t7778-ST15-CC15 and t18009-ST26-CC25), were detected and ascribed to 11 clonal complexes (CCs). MSSA-CC398 (8.2%), MSSA-CC15 (7.1%) and MSSA-ST291 (5.9%) were the most prevalent lineages in storks. Moreover, tst-positive (MSSA-CC22-t223 and MSSA-CC30-t1654), eta-positive (MSSA-CC9-t209) and etb-positive strains (MSSA-CC45-t015) were detected in four storks. The 18.5% of storks harboured distinct MSSA strains (with different lineages and/or AMR genes). Nestlings of storks foraging in landfills (10 CCs) had more diverse S. aureus strains than those of parents foraging in natural habitats (3 CCs). Low level of AMR was demonstrated among S. aureus strains. The predominance of MSSA-CC398 (an emergent clade) and toxigenic MSSA strains in stork nestlings highlight the need for continuous surveillance of S. aureus in wild birds., (© 2023. The Author(s).)

Objectives: This study aimed to characterize Escherichia coli isolates from cloacal samples of white stork nestlings, with a special focus on extended-spectrum β-lactamases (ESBLs)-producing E. coli isolates and their plasmid content., Methods: Cloacal samples of 88 animals were seeded on MacConkey-agar and chromogenic-ESBL plates to recover E. coli and ESBL-producing E. coli. Antimicrobial susceptibility was screened using the disc diffusion method, and the genotypic characterization was performed by polymerase chain reaction (PCR) and subsequent sequencing. S1 nuclease Pulsed-Field-Gel-Electrophoresis (PFGE), Southern blotting, and conjugation essays were performed on ESBL-producing E. coli, as well as whole-genome sequencing by short- and long-reads. The four bla ESBL -carrying plasmids were completely sequenced., Results: A total of 113 non-ESBL-producing E. coli isolates were collected on antibiotic-free MacConkey-agar, of which 27 (23.9%) showed a multidrug-resistance (MDR) phenotype, mainly associated with β-lactam-phenicol-sulfonamide resistance (bla TEM /cmlA/floR/sul1/sul2/sul3). Moreover, four white stork nestlings carried ESBL-producing E. coli (4.5%) with the following characteristics: bla SHV-12 /ST38-D, bla SHV-12 /ST58-B1, bla CTX-M-1 /ST162-B1, and bla CTX-M-32 /ST155-B1. Whole-genome sequencing followed by Southern blot hybridizations on S1-PFGE gels in ESBL-positive isolates proved that the bla CTX-M-1 gene and one of the bla SHV-12 genes were carried by IncI1/pST3 plasmids, while the second bla SHV-12 gene and the bla CTX-M-32 gene were located on IncF plasmids. The two bla SHV-12 genes and the two bla CTX-M genes had similar but non-identical close genetic environments, as all four genes were flanked by a variety of insertion sequences., Conclusion: The role played by several genetic platforms in the mobility of ESBL genes allows for interchangeability on a remarkably small scale (gene-plasmid-clones), which may support the spread of ESBL genes., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The antimicrobial resistance (AMR) genes of 268 non-duplicated coagulase-negative staphylococci (CoNS) previously obtained from nasotracheal cavities of nestling storks were characterized. They included S. sciuri isolates (n = 191), and non-sciuri-CoNS isolates (NSc-CoNS, n = 77). All S. sciuri carried the intrinsic salA gene (for clindamycin-resistance) and so, clindamycin was not considered for general analysis in this species. About 71.7%/41.6% of the S. sciuri/NSc-CoNS isolates were susceptible to all antibiotics tested; moreover, 14.1%/16.9% and 3.1%/20.8% of S. sciuri/NSc-CoNS showed single antibiotic resistance and multidrug resistance (MDR) phenotype, respectively. Of the ten mecA-positive CoNS isolates, six were associated with SCCmec types-III, -IV or -V elements. Remarkably was the detection of one MDR-S. lentus isolate carrying both mecA and mecC genes, as well as the SCCmec type-XI element. MDR-CoNS was relatively higher in nestlings of parent storks foraging in landfills (21.3%) than those in natural areas (9.7%) (χ 2 = 3.421, df=1, p = 0.064). AMR phenotypes (and genes detected) include penicillin (blaZ, bla ARL ), erythromycin-clindamycin-constitutive (ermA, ermC, ermT), clindamycin (lnuA, salA, vgaA), erythromycin (msrA, mphC), tetracycline (tetK, tetL, tetM), tobramycin (ant4'), tobramycin-gentamicin (aac6'-aph2″), sulfamethoxazole-trimethoprim (dfrA, dfrG, dfrK), chloramphenicol (fexA, fexB, catPC221), and mupirocin (mupA). Interestingly, one S. epidermidis isolate carried the ermT gene. About 29.9% of nestlings harboured more than one non-duplicated CoNS (with varied 2-5 AMR profiles). This study demonstrated that most of the CoNS isolates were susceptible to all the antibiotics tested (63.1%). However, AMR genes of public health importance were found, including the mecC-mediated methicillin resistance trait., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

This study determined the carriage rates and antimicrobial resistance (AMR) genes of enterococci from nasotracheal samples of three healthy animal species and in-contact humans. Nasal samples were collected from 27 dog-owning households (34 dogs, 41 humans) and 4 pig-farms (40 pigs, 10 pig-farmers), and they were processed for enterococci recovery (MALDI-TOF-MS identification). Also, a collection of 144 enterococci previously recovered of tracheal/nasal samples from 87 white stork nestlings were characterized. The AMR phenotypes were determined in all enterococci and AMR genes were studied by PCR/sequencing. MultiLocus-Sequence-Typing was performed for selected isolates. About 72.5% and 60% of the pigs and pig-farmers, and 29.4% and 4.9%, of healthy dogs and owners were enterococci nasal carriers, respectively. In storks, 43.5% of tracheal and 69.2% of nasal samples had enterococci carriages. Enterococci carrying multidrug-resistance phenotype was identified in 72.5%/40.0%/50.0%/23.5%/1.1% of pigs/pig-farmers/dogs/dogs' owners/storks, respectively. Of special relevance was the detection of linezolid-resistant enterococci (LRE) in (a) 33.3% of pigs (E. faecalis-carrying optrA and/or cfrD of ST59, ST330 or ST474 lineages; E. casseliflavus-carrying optrA and cfrD); (b) 10% of pig farmers (E. faecalis-ST330-carrying optrA); (c) 2.9% of dogs (E. faecalis-ST585-carrying optrA); and (d) 1.7% of storks (E. faecium-ST1736-carrying poxtA). The fexA gene was found in all optrA-positive E. faecalis and E. casseliflavus isolates, while fexB was detected in the poxtA-positive E. faecium isolate. The enterococci diversity and AMR rates from the four hosts reflect differences in antimicrobial selection pressure. The detection of LRE carrying acquired and transferable genes in all the hosts emphasizes the need to monitor LRE using a One-Health approach., (© 2023. The Author(s).)

Migratory storks could be vectors of transmission of bacteria of public health concern mediated by the colonization, persistence and excretion of such bacteria. This study aims to determine genera/species diversity, prevalence, and co-colonization indices of bacteria obtained from tracheal (T) and nasal (N) samples from storks in relation to exposure to point sources through foraging. One-hundred and thirty-six samples from 87 nestlings of colonies of parent white storks with different foraging habits (natural habitat and landfills) were obtained (84 T-samples and 52 N-samples) and processed. Morphologically distinct colonies (up to 12/sample) were randomly selected and identified by MALDI-TOF-MS. About 87.2% of the total 806 isolates recovered were identified: 398 from T-samples (56.6%) and 305 from N-samples (43.4%). Among identified isolates, 17 genera and 46 species of Gram-positive and Gram-negative bacteria were detected, Staphylococcus (58.0%) and Enterococcus (20.5%) being the most prevalent genera. S. sciuri was the most prevalent species from T (36.7%) and N (34.4%) cavities of total isolates, followed by E. faecalis (11.1% each from T and N), and S. aureus [T (6.5%), N (13.4%)]. Of N-samples, E. faecium was significantly associated with nestlings of parent storks foraging in landfills (p = 0.018). S. sciuri (p = 0.0034) and M. caseolyticus (p = 0.032) from T-samples were significantly higher among nestlings of parent storks foraging in natural habitats. More than 80% of bacterial species in the T and N cavities showed 1-10% co-colonization indices with one another, but few had ≥ 40% indices. S. sciuri and E. faecalis were the most frequent species identified in the stork nestlings. Moreover, they were highly colonized by other diverse and potentially pathogenic bacteria. Thus, storks could be sentinels of point sources and vehicles of bacterial transmission across the "One Health" ecosystems., (© 2023. The Author(s).)

Background: Staphylococcus pseudintermedius (SP) and Staphylococcus aureus (SA) are common colonizers of companion animals, but they are also considered opportunistic pathogens, causing diseases of diverse severity. This study focused on the identification and characterization of 33 coagulase-positive staphylococci isolated from diseased pets (28 dogs and five cats) during 2009-2011 in a veterinary hospital in Spain in order to stablish the circulating lineages and their antimicrobial resistance profile., Results: Twenty-eight isolates were identified as SP and five as SA. Nine methicillin-resistant (MR) isolates (27%) carrying the mecA gene were detected (eight MRSP and one MRSA). The 55% of SP and SA isolates were multidrug-resistant (MDR). MRSP strains were typed as ST71-agrIII-SCCmecII/III-(PFGE) A (n=5), ST68-agrIV-SCCmecV-B1/B2 (n=2), and ST258-agrII-SCCmecIV-C (n=1). SP isolates showed resistance to the following antimicrobials [percentage of resistant isolates/resistance genes]: penicillin [82/blaZ], oxacillin [29/mecA] erythromycin/clindamycin [43/erm(B)], aminoglycosides [18-46/aacA-aphD, aphA3, aadE], tetracycline [71/tet(M), tet(K)], ciprofloxacin [29], chloramphenicol [29/cat pC221 ], and trimethoprim-sulfamethoxazole [50/dfrG, dfrK]. The dfrK gene was revealed as part of the radC-integrated Tn559 in two SP isolates. Virulence genes detected among SP isolates were as follow [percentage of isolates]: siet [100], se-int [100], lukS/F-I [100], sec canine [7], and expB [7]. The single MRSA-mecA detected was typed as t011-ST398/CC398-agrI-SCCmecV and was MDR. The methicillin-susceptible SA isolates were typed as t045-ST5/CC5 (n=2), t10576-ST1660 (n=1), and t005-ST22/CC22 (n=1); the t005-ST22 feline isolate was PVL-positive and the two t045-ST45 isolates were ascribed to Immune Evasion Cluster (IEC) type F. Moreover, the t10576-ST1660 isolate, of potential equine origin, harbored the lukPQ and scneq genes. According to animal clinical history and data records, several strains seem to have been acquired from different sources of the hospital environment, while some SA strains appeared to have a human origin., Conclusions: The frequent detection of MR and MDR isolates among clinical SP and SA strains with noticeable virulence traits is of veterinary concern, implying limited treatment options available. This is the first description of MRSA-ST398 and MRSP-ST68 in pets in Spain, as well the first report of the dfrK-carrying Tn559 in SP. This evidences that current transmissible lineages with mobilizable resistomes have been circulating as causative agents of infections among pets for years.

Methicillin-resistant Staphylococcus aureus (MRSA) CC398 is a livestock-associated (LA) lineage, mainly detected in swine. Its dissemination via the food-chain could be a food-safety issue. This work aimed to study the diversity of S. aureus lineages in pork-products, to determine the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) of lineage CC398, and to study the antimicrobial resistance phenotype/genotype and the virulence traits of recovered isolates. One hundred and one samples of pig-derived food were collected in Northern Spain for S. aureus isolation. Antibiotic resistance profile was analysed, and associated resistance genes were screened by PCR. Detection of CC398 lineage, spa-type, multilocus sequence-type, virulence factors, immune evasion cluster (IEC) genes, and phage ΦSa3 integrase was performed by PCR/sequencing. The prevalence of S. aureus and MRSA among pig-derived food was 33.6% and 21.8%, respectively. Thirty-nine S. aureus isolates were recovered and attributed to 19 spa-types and 12 STs, ST398 being the predominant lineage (n = 25; 64%). MRSA-CC398 isolates (n = 23) were mainly spa-t011 (n = 16) and 82.6% were multidrug-resistant (MDR). All MRSA-CC398 were tetracycline-resistant and IEC-negative and four hosted either eta, tst or sea gene. The two MSSA-CC398 isolates detected were spa-t5452, IEC-positive, and were resistant to penicillin (blaZ) and erythromycin/clindamycin (inducible) (ermT with/without ermC + msrA). Among the 14 non-CC398 isolates, only two were MRSA (ST8, PVL-positive, enterotoxin-positive, IEC-negative). The 12 MSSA isolates included two of lineage CC45 and IEC-positive. CC398 lineage is prevalent among S. aureus of pig-derived food (both MRSA and MSSA), LA-MRSA-CC398/t011 being the clone most represented. The presence of the IEC-positive MSSA-CC398 and MSSA-CC45 isolates in food products highlights the potential implication of handlers in transmission of foodborne pathogens. Moreover, given the high frequency of MDR isolates and virulence genes detected, hygienic practices should be improved to limit the dissemination risk of S. aureus via the food chain., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of lineage CC398 is an emerging clone causing human infections but is mostly found in pigs. The aim of this study was to characterize the antimicrobial resistance phenotypes/genotypes of a collection of 137 MRSA CC398 isolates obtained in a previous study from 17 Spanish hospitals, using tetracycline resistance as marker for selection. A multidrug-resistant (MDR) phenotype was present in 79% of analysed isolates, with 17% of them resistant to at least six different antimicrobial families. All tetracycline-resistant isolates (n=137) carried the tetM gene and 75% also carried the tetK gene. Almost 50% of MRSA CC398 isolates showed macrolide and/or lincosamide resistance: a) 39% of isolates were ERY R -CLI R (all with constitutive phenotype), with 87% of them carrying the ermC gene, followed by msrA (25%), ermB (21%), vgaA (17%), ermA (6%), lsaB (4%), linA (2%), linB (2%), and ermT (2%, this isolate with the new spa-type t18071); and b) 9% of MRSA CC398 isolates showed the dissociated ERY S -CLI R phenotype carrying the linA, linB, lsaB and vgaA genes. Other antimicrobial resistance phenotypes in these MRSA CC398 isolates included resistance to ciprofloxacin (67%), aminoglycosides (21%), mupirocin (6%), chloramphenicol (4%) or fusidic acid (2%). The more common resistance genes detected for some of these antimicrobials were: aac(6')-Ie-aph(2'')-Ia (16%) and ant(4')-Ia (12%) for aminoglycosides, and fexA (3%) for chloramphenicol. The high rate of MDR phenotypes with a wide range of antimicrobial resistance genes shown in this study reduce the potential therapeutic options in case of infections., (Copyright © 2020 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Background: Tetracycline resistance (TetR) is a marker of livestock-associated MRSA of lineage CC398., Objectives: To determine the MRSA CC398 prevalence among TetR-MRSA recovered in Spanish hospitals located in regions with different pig-farming densities, and the influence of pig density as a key risk factor for its acquisition., Methods: TetR-MRSA isolates (n = 232) recovered from clinical and epidemiological samples during January-June 2016 in 20 hospitals in 13 regions with different pig-farming densities were analysed. MRSA CC398 identification, detection of spa types, methicillin resistance genes and immune evasion cluster (IEC) genes were performed by PCR/sequencing. Statistical analyses were performed to establish the relationships between MRSA CC398 prevalence and pig density., Results: The global MRSA prevalence was 29.7% (6.9% TetR-MRSA/MRSA), with 137 CC398 isolates recovered, representing 4.1% of total MRSA and 59.1% of TetR-MRSA. Among MRSA CC398, 16 different spa types were recorded (t011: 72.3%), and all but two strains were IEC negative. Higher pig-density regions were associated with significant MRSA CC398 increases in hospitals located in adjacent regions (P < 0.001). Linear regression models explained the relationships between MRSA CC398 and pig density (P < 0.001), with an increase of 6.6 MRSA CC398 cases per 100 MRSA per increase of 100 pigs/km2 in a region., Conclusions: High pig density leads to a significant increase in MRSA CC398 in hospitals in Spain, and its combination with a high human population could help its dissemination. In Spain, the prevalence of the zoonotic CC398 lineage is closely related to pig-farming density; therefore, specific tools could be implemented in order to detect its dissemination., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The aim of this study was to determine the carriage rate of coagulase-positive staphylococci (CoPS) in wild birds and to characterize recovered isolates. Tracheal samples from 324 wild birds, obtained in different Spanish regions during 2015-2016, were screened for CoPS carriage. The antimicrobial resistance profile and the virulence gene content were investigated. Molecular typing was performed by spa, agr, MLST, SCCmec, and S. delphini group classification. CoPS were recovered from 26 samples of wild birds (8.3%), and 27 isolates were further characterized. Two CoPS species were detected: S. aureus (n = 15; eight cinereous vultures and seven magpies) and S. delphini (n = 12; 11 cinereous vultures and one red kite). Thirteen S. aureus were methicillin-resistant (MRSA) and the remaining two strains were methicillin-susceptible (MSSA). Twelve MRSA were mecC-positive, typed as t843-ST1583/ST1945/ST1581/ST1571 (n = 11) and t1535-ST1945 (n = 1) (all of clonal-complex CC130); they were susceptible to the non-β-lactams tested. The remaining MRSA strain carried the mecA gene, was typed as t011-ST398-CC398-agrI-SCCmec-V, and showed a multiresistance phenotype. MSSA isolates were ascribed to lineages ST97-CC97 and ST425-CC425. All S. aureus lacked the studied virulence genes (lukS/F-PV, tst, eta, etb, and etd), and the IEC type E (with scn and sak genes) was detected in four mecC-positive and one MSSA isolates. S. delphini strains were methicillin-susceptible but showed resistance to at least one of the antimicrobials tested, with high penicillin (75%, with blaZ gene) and tetracycline [58%, with tet(K)± tet(L)] resistance rates. All S. delphini isolates presented the virulence genes lukS-I, siet, and se-int, and four carried the clindamycin-resistance lnu(A) gene.

Aims: To determine the Staphylococcus aureus carriage rate in wild mammals in Aragon, northern Spain, to analyse their antimicrobial resistance phenotype/genotype and to characterize the recovered isolates., Methods and Results: Nasal and rectal swabs of 103 mammals were collected in Aragón during the period 2012-2015. Antimicrobial susceptibility, the presence of antimicrobial resistance genes and virulence factors were investigated. Molecular characterization was carried out by spa, MLST, agr and SCCmec. Staphylococcus aureus were recovered from 23 animals (22%). Four of the 23 S. aureus were methicillin-resistant S. aureus (MRSA). Three MRSA were mecC-positive and were isolated from European rabbits and were typed as t843 (ascribed to CC130). The remaining MRSA was a mecA-carrying isolate from European hedgehog, typed as ST1-t386-SCCmecIVa-agrIII and it harboured the blaZ, erm(C), ant(6)-Ia and aph(3´)-IIIa resistance genes. A high diversity of spa-types was detected among the 19 methicillin-susceptible S. aureus isolates, which showed high susceptibility to the antimicrobials tested. The tst gene and different combinations of staphylococcal enterotoxins were found., Conclusions: Staphylococcus aureus were detected in nasal and rectal samples of wild mammals. Wild rabbits could be a reservoir of mecC-MRSA., Significance and Impact of the Study: This work provides information on the presence and characteristics of S. aureus from mammals in a defined geographic region in Spain., (© 2019 The Society for Applied Microbiology.)

Unlabelled: Tetracycline-resistance (Tet(R)) has been postulated as a marker of the livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) lineage CC398., Objectives of the Study: to determine the spa-types and assigned MLST clonal complexes (CCs) among all 98 MRSA-Tet(R) strains recovered during 2011-2012 (from different patients) in a Spanish Hospital, analyzing the possible correlation with livestock-contact of the patients. All 98 strains were assigned to 9 CCs: CC398 (60.2%), CC1 (19.4%), CC5 (12.2%), and other CCs (8.2%). The 98 patients were classified into three groups: (A) contact with livestock-animals (n=25); (B) no-contact with livestock-animals (n=42); (C) no information about animal contact (n=31). A significant higher percentage of CC398 strains was obtained in group A (76%) than in group B (50%) (p<0.05), being the percentage in group C of 61.3%. Most of MRSA-Tet(R)-CC398 strains presented a multi-resistance phenotype, including erythromycin, clindamycin, and ciprofloxacin, and the most prevalent detected genes were tet(M) and erm(C). Three strains presented the phenotype macrolide-susceptibility/lincosamide-resistance and contained the vga(A) gene. MRSA-CC1 strains showed higher percentages of erythromycin/clindamycin resistance (95%/89%) than MRSA-CC398 strains (58%/63%), and this resistance was usually mediated by erm(C) gene. Most of MRSA-CC5 strains showed resistance to ciprofloxacin, tobramycin/kanamycin and erythromycin. None of the strains presented the genes lukF/lukS-PV, tsst-1, eta, etb or etd. All MRSA-CC398 strains lacked the genes of the immune-evasion-cluster, but MRSA-CC1 strains carried these genes (type E). In conclusion, although MRSA CC398 is detected in a significant higher proportion in patients with livestock-contact; its detection in people without this type of contact also indicates its capacity for human-to-human transmission., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Objectives: To determine the rate of Staphylococcus aureus faecal carriage in 101 wild small mammals in Spain and to characterize the isolates obtained., Methods: Faecal samples were seeded on mannitol salt agar and ORSAB plates. The presence of the resistance genes mecA, mecC and blaZ and the new blaZ allotype associated with staphylococcal cassette chromosome mec (SCCmec) XI (blaZ-SCCmecXI) was studied by PCR. S. aureus isolates were characterized by spa typing, agr typing and multilocus sequence typing. The presence of immune evasion cluster (IEC) genes and virulence genes was analysed by PCR., Results: S. aureus was detected in 13/101 studied faecal samples and one isolate per positive sample was further studied. Two S. aureus isolates were methicillin-resistant S. aureus (MRSA) (recovered from wood mice, Apodemus sylvaticus) and 11 were methicillin-susceptible S. aureus (MSSA). Both MRSA isolates harboured the mecC gene and the novel blaZ-SCCmecXI, were typed as spa-t1535/agrIII/ST1945(CC130)/SCCmecXI (where ST stands for sequence type and CC stands for clonal complex), carried the exfoliative toxin etd2 gene and were IEC type E. Eight different spa types were identified among the 11 MSSA isolates (five new) and six different sequence types were identified (two new). All MSSA strains were susceptible to the antibiotics tested except one blaZ-positive penicillin-resistant isolate (spa-t120/agrII/ST15). MSSA isolates were ascribed to the CCs (number of strains) CC5 (1), CC1956 (4) and singleton (6). Nine of 11 MSSA isolates carried the cna virulence gene. Only one MSSA isolate carried IEC genes (type C)., Conclusions: This is the first report of MRSA carrying mecC in faecal samples of wild small mammals in Spain. These resistant isolates carried genes of the IEC system, unusual in S. aureus from animals. Wild small mammals could be a reservoir of the mecC gene with important implications for public health., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The objective of this study was to determine the rate of contamination by Staphylococcus aureus in 100 meat samples obtained during 2011-2012 in La Rioja (Northern Spain), to analyze their content in antimicrobial resistance and virulence genes, as well as in immune evasion cluster (IEC) genes, and to type recovered isolates. Seven of 100 samples (7%) contained S. aureus: 6 samples harbored methicillin-susceptible S. aureus (MSSA) and 1 pork sample harbored methicillin-resistant S. aureus (MRSA). The MRSA isolate corresponded to the ST398 genetic lineage with a multidrug resistance profile and the absence of human IEC genes, which pointed to a typical livestock-associated MRSA profile. MRSA isolate was ascribed to the spa-type t011, agr-type I, and SCCmec-V and showed resistance to erythromycin, clindamycin, tetracycline, and streptomycin, in addition to β-lactams. The remaining six MSSA strains belonged to different sequence types and clonal complexes (three isolates ST45/CC45, one ST617/CC45, one ST5/CC5, and one ST109/CC9), being susceptible to most antibiotics tested but showing a wide virulence gene profile. Five of the six MSSA strains (except ST617/CC45) contained the enterotoxin egc-cluster or egc-like-cluster genes, and strain ST109/CC9 contained eta gene (encoding exfoliatin A). The presence of human IEC genes in MSSA strains (types B and D) points to a possible contamination of meat samples from an undefined human source. The presence of S. aureus with enterotoxin genes and MRSA in food samples might have implications in public health. The IEC system could be a good marker to follow the S. aureus contamination source in meat food products.

Eight coagulase-positive staphylococci from equines with different pathologies obtained between 2005 and 2011 were investigated. Isolates were characterized by different molecular techniques (spa-, agr-, MLST), and clonal relatedness of strains was investigated by ApaI and SmaI PFGE. Anti-microbial resistance and virulence profiles were determined. Six isolates were identified as Staphylococcus aureus, and two as Staphylococcus pseudintermedius. Of these, four isolates were methicillin-resistant S. aureus (MRSA) ST398 and one S. pseudintermedius was mecA positive and typed as ST68. One MRSA ST398 strain was isolated in 2005 and might be one of the earliest MRSA ST398 descriptions in Spain. All 5 mecA-positive strains were multidrug resistant and were isolated from hospitalized equines. Three MRSA ST398 strains carried the recently described transposon Tn559 within the chromosomal radC gene. The mecA-positive S. pseudintermedius ST68 strain was also multidrug resistant and harboured the erm(B)-Tn5405-like element. This ST68 strain presented a clear susceptible phenotype to oxacillin and cefoxitin regardless of the presence of an integral and conserved mecA gene and mecA promoter, which enhances the need for testing the presence of this gene in routine analysis to avoid treatment failures. These data reflect the extended anti-microbial resistance gene acquisition capacities of both bacterial species and evidence their pathogenic properties. The first detection of MRSA ST398 and S. pseudintermedius ST68 in horses in Spain is reported., (© 2013 Blackwell Verlag GmbH.)

Fecal samples of 100 healthy humans were tested for Staphylococcus aureus recovery. Fifteen samples (15 %) contained S. aureus, all methicillin-susceptible (MSSA), being one isolate/sample further studied. These 15 isolates were characterized by spa and agr typing as well as multi-locus sequence typing. High diversity of spa types (n = 11) and sequences types (n = 8) was detected. Two S. aureus of lineages ST398 or ST133 were detected, and six isolates were ascribed to clonal complex 30 (CC30). Strains were susceptible to most of the 17 antimicrobial agents tested with exceptions: erythromycin/clindamycin (three strains, containing erm(C) and/or erm(A) + mph(C) genes) and tobramycin and mupirocin (one strain containing ant(4')-Ia + mup(A) genes). The presence of 18 staphylococcal enterotoxin genes was studied by PCR, and isolates were negative for lukF/lukS-PV genes, although strain ST133 harbored the lukD-lukE + lukM genes. Other virulence genes detected were (number of strains): tsst-1 (6), hla (15), hlb (9), hld (15), hlg (6), hlgv (9), cna (2), aur (14), and egc-like cluster (3). Analysis of immune evasion cluster genes showed six types, highlighting their absence in two strains of lineages ST133 and ST5. A high clonal diversity of MSSA strains was identified in the intestinal microbiota of healthy humans, being CC30 the most frequent one. This is the first report of MSSA ST133 and ST398 isolates in gut microbiota of healthy humans.

A total of 204 methicillin-resistant Staphylococcus aureus (MRSA) isolates were isolated in a Spanish hospital in two different periods (2001 and 2009). The percentages of MRSA isolates detected in 2001 and 2009 were 29 and 27 %, respectively. Genetic lineages, resistance mechanisms, and virulence traits were determined in these isolates. The most frequent detected lineage in both periods was S. aureus protein A (spa)-type t067, assigned to clonal complex (CC) 5 (CC5-t067), being more prevalent in 2001 (93 %) than in 2009 (71 %). The remaining CCs and spa-types detected were (%2001/%2009): CC5-t002 (0/5), CC8-t008 (1/16), CC8-t024 (0/1), CC8-t190 (0/3), CC8-t2849 (0/2), CC22-t032 (0/2), CC30-t012 (1/0), CC228-t109 (1/0), CC228-t1318 (2/0), and CC247-t051 (2/0). Most of the MRSA were isolated from wounds, representing 39 % in 2001 and 63 % in 2009. The emergence of MRSA CC8 isolates, mainly from wounds, seemed to occur in the second period. Resistance to (%2001/%2009) quinolones (99/87), aminoglycosides (98/88), macrolides (32/30), lincosamides (30/17), and tetracycline (2/1) was found in isolates in both periods. Trimethoprim-sulfamethoxazole resistance was detected only in 2001 (1 %), and chloramphenicol (1 %) and mupirocin resistance (11 %) were detected only in 2009. An association between staphylococcal enterotoxin gene profiles and CCs was detected in most of the cases. The egc-cluster was related to CC5, CC22, CC30, and CC228 and most of the CC8 isolates presented the sed, sej, and ser genes. Four tst-1-positive (CC5 and CC30) isolates were detected in 2001 and two lukS/F-PV-positive isolates were detected in 2009. Therefore, there is still a predominance of CC5-t067 in our region, although an increase of lineage CC8 was observed.

Introduction: Linezolid resistance is mainly due to mutations in the 23S rRNA target. The aim of this study was to characterize linezolid and methicillin resistant Staphylococcus epidermidis (SE-LM(R)) and S. haemolyticus (SH-LM(R)) strains detected in a Spanish hospital., Methods: SE-LM(R) and SH-LM(R) strains obtained in the period June 2009-August 2011 in a second level hospital were recorded along with the epidemiological characteristics of the patients. These strains were typed, and their resistance, phenotype, genotype and the factors determining their virulence were analysed., Results: Linezolid resistance was explained by the presence of G2603T mutation (23S rRNA) and aminoacid changes in L3 and L4 ribosomal proteins. The 25 SE-LM(R) strains belonged to sequence type ST2, presented SCCmec typeIII, and two different PFGE patterns. The two SH-LM(R) strains showed non-typeable SCCmec. SE-LM(R) strains harboured the resistance genes aac(6')-aph(2"), and dfrS1. SH-LM(R) strains contained these genes and the gene erm(C). No lincomycin resistance mechanism was identified in SE-LM(R) strains regardless of showing lincomycin resistance and diminished susceptibility to clindamycin., Conclusions: Linezolid resistance is of concern in hospitals, and requires continued vigilance. Several linezolid resistance mechanisms (mutation in 23S RNAr and amino acid changes in L3 and L4) were identified in this study., (Copyright © 2012 Elsevier España, S.L. All rights reserved.)

Objectives: The clonal lineages, resistance mechanisms and virulence traits of tetracycline-resistant (Tet(R)) methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in a Spanish hospital during 2009 and 2010 were investigated., Methods: Fifty-two Tet(R) MRSA strains from unrelated patients were included in this study. Susceptibility to 26 antimicrobial agents was determined and 24 resistance genes were tested for by PCR. The sequences of the genes grlA and gyrA were analysed in all ciprofloxacin-resistant MRSA isolates. For all strains, spa, SCCmec and agr typing was implemented. Multilocus sequence typing was performed for 16 representative strains of the different spa types. The presence of the genes tst, lukF/lukS-PV, eta, etb, etd and cna was investigated by PCR., Results: Fifteen different spa types, four of them new ones, were detected among the 52 strains, being associated with the following clonal complexes (CCs): CC398 (67.3%), CC1 (11.5%), CC5 (11.5%) and CC8 (9.6%). A novel sequence type (ST), ST2077, belonging to CC398 was identified. Most MRSA CC398 strains were typed as SCCmecV-agrI. In addition to β-lactam resistance, isolates showed resistance to (gene/number of strains): tetracycline [tet(K)/36, tet(L)/8 and tet(M)/48], macrolides and lincosamides [erm(B)/6, erm(C)/25, erm(T)/2, msr(A)/msr(B)/4 and mph(C)/4], aminoglycosides [aac(6')-Ie-aph(2')-Ia/8, ant(4')-Ia/13 and aph(3')-IIIa/15], trimethoprim [dfrS1/2 and dfrK/3] and mupirocin (mupA/3). Strains investigated for mutations mediating quinolone resistance revealed an S80F exchange in GrlA and different changes in GyrA. Three strains were Panton-Valentine leucocidin-positive (ST8 and ST94) and 41 strains were cna-positive. All MRSA isolates were negative for the genes tst, eta, etb and etd., Conclusions: Tetracycline resistance could be a good marker for MRSA CC398, although this resistance can also be found in other lineages.

The objective was to identify the methicillin-resistant coagulase-positive staphylococci (MRCoPS) nasal carriage rate of healthy dogs in La Rioja (Spain) and to characterize the recovered isolates by different molecular techniques. Nasal samples from 196 dogs were obtained (98 household-dogs, 98 pound-dogs). Isolates were identified and characterized by spa-, SCCmec- and MLST-typing, SmaI-PFGE, antimicrobial susceptibility, determination of antimicrobial resistance and toxin genes profiling. S. pseudintermedius was the only species recovered. Nine methicillin-resistant S. pseudintermedius (MRSP) were obtained from 9 of 196 sampled dogs (8% pound-dogs, 1% household-dogs). MRSP isolates were typed (MLST/PFGE/spa/SCCmec) as: ST71/A/t02/II-III (7 isolates), ST92/C/t06/V (1 isolate), and ST26/B/non-typable/non-typable (1 isolate). All MRSP were resistant to [resistance gene/number isolates]: β-lactams [mecA+blaZ/9], tetracycline [tet(K)/7, tet(M)/2], macrolides and lincosamides [erm(B)/9], aminoglycosides [aacA-aphD+aadE+aphA-3/9], and co-trimoxazol [dfr(G)/9]. Eight MRSP isolates showed also resistance to fluoroquinolones and amino acid changes in GyrA [Ser84Leu+Glu714Lys, 7 isolates; Ser84Leu, 1 isolate] and GrlA [Ser80Ile, 8 isolates] proteins were detected. The remaining isolate was chloramphenicol resistant and harboured cat(pC221) gene. All MRSP isolates harboured the aadE-sat4-aphA-3 multiresistance-gene-cluster linked to erm(B) gene as well as the siet, si-ent and lukS/F-I toxin genes. MRSP is a moderately common (4.6%) colonizer of healthy dogs in Spain. A major MRSP lineage (ST71) was detected and its future evolution should be tracked., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

S. aureus nasal carriage was investigated in 278 healthy humans, determining the antibiotic resistance mechanisms, virulence traits, and genetic lineages of recovered isolates. Nasal samples were cultured in specific media for S. aureus and methicillin-resistant S. aureus (MRSA) recovery. S. aureus was detected in 53 of 278 nasal samples (19.1%): MRSA was found in one sample (0.4%) and methicillin-susceptible S. aureus (MSSA) in the remaining 52 samples. The MRSA isolate was typed as ST1649-t701-agrI-SCCmec-IVc and only exhibited resistance to beta-lactams. A high diversity of spa types (n=37) was identified among the 52 MSSA, identifying 5 new spa-types. Multi-locus sequence typing (MLST) typing was performed in 30 selected MSSA, detecting 16 different sequence types, 2 of them being new. MSSA strains presented agr types I (30.2%), II (30.2%), III (34%), and IV (5.6%). Eleven strains showed erythromycin resistance and harbored different combinations of erm(A), erm(B), erm(C), erm(T), and msr(A) genes. Two strains exhibited ciprofloxacin resistance, and one of them presented amino acid changes in GyrA and GrlA proteins. The presence of 28 genes encoding staphylococcal toxins was investigated by PCR in all 53 S. aureus isolates. The toxic shock syndrome toxin 1 (TSST-1) gene was detected in 15 MSSA isolates (11 of them typed within the clonal complex CC30) and the gene of exfoliative toxin A in 2 strains. Different combinations of enterotoxin genes were identified among S. aureus strains. None of the S. aureus isolates harbored the Panton-Valentine leukocidin gene. Two MSSA presented the sequence-type ST398 [harboring erm(T) gene], and 2 additional isolates were typed as ST97. Interestingly, MSSA CC398 and CC97 isolates were detected. These clonal complexes are associated with food-producing animals., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Skin lesions by methicillin-resistant Staphylococcus aureus (MRSA) of the lineage ST398-t1451 were detected in a pig-farmer in Spain in 2010. Similar MRSA ST398-t1451 strains were also detected in nasal samples from the patient, his brother, and nine pigs from his farm. All human and animal strains were ascribed to the SCCmec type V and the agr type I, showed tetracycline-erythromycin-clindamycin resistances, and harbored the tetK, tetM, and ermC resistance genes. They were negative for all tested toxin genes (lukS/lukF, tst, eta, etb, and etd). All human and animal strains showed closely related pulsed-field gel electrophoresis-ApaI patterns. Possible MRSA transmission from animals to humans is suggested.

Nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) was evaluated after a mupirocin treatment in a family previously colonized by MRSA sequence type ST398 and ST1, who lived close to a pig farm. Eight nasal samples were swabbed from each of the four family members on different moments after mupirocin treatment. The efficacy of treatment was low in those family members who worked in the farm, and higher in the remaining two family members with sporadic contact with pigs. In addition, nasal and skin swabs from randomly selected pigs of the farm were taken. MRSA were detected in 33% of pigs tested. All MRSA isolates obtained were characterized by Staphylococcal-Cassette-Chromosome mec (SCCmec) determination, Multilocus-Sequence-Typing (MLST), spa- and agr-typing, Pulsed-field-gel-electrophoresis (PFGE), antimicrobial susceptibility, detection of antimicrobial resistance genes, and toxin gene profiling. Spa-types t011, t1255 and t1197 were detected in humans and animals, with indistinguishable PFGE patterns, suggesting animal to human MRSA transmission. Each spa-type was ascribed to a specific pulsotype. Spa-types t127 and t108 were only detected in MRSA isolates obtained from humans, and t012 only in those from animals. MRSA ST1-t127 isolates and some ST398-t011 and ST398-t1197 isolates presented a multiantimicrobial-resistance phenotype. None of them harbored lukF/lukS, tst, eta and etb virulence genes. This study showed that the efficacy of nasal MRSA decolonization in healthy people with very close contact with pigs is especially low., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

An outbreak due to a Klebsiella pneumoniae clone occurred in a neonatal intensive care unit of a Spanish Hospital in which three newborns were infected (all with gestational age ≤29 weeks; two of them died) and seven were colonized (gestational age >32 weeks; none died). One K. pneumoniae strain per patient was further characterized. The 10 strains showed an indistinguishable pulsed-field-gel-electrophoresis pattern, were typed in the phylogenetic group KpI and were ascribed into a new sequence type registered as ST341. All 10 strains presented the same multiple-antibiotic-resistant phenotype, showed extended-spectrum-beta-lactamase production, and harbored the bla(CTX-M-15), bla(SHV-11), bla(OXA-1,)aac(6')-Ib-cr, qnrS1, aac(3)-II, aph(3')-Ia and aadA5 resistance genes. No class 1 or class 2 integrons were detected. The bla(CTX-M-15) gene presented the following genetic environment: ISEcp1-bla(CTX-M-15)-orf477. These strains contained two copies of the aac(6')-Ib-cr gene included in the following new genetic environments: aac(3)-II-IS26-aac(6')-Ib-cr-bla(OXA-1) and aac(3)-II-IS26-ΔcatB3-bla(OXA-1)-aac(6')-Ib-cr (registered at GenBank with accession numbers GQ438247 and GQ438248, respectively). The genetic environment of the qnrS1 gene (IS26-ΔISEcl2-qnrS1) (GenBank accession number GQ438249) was also not described previously. The aac(6')-Ib-cr, qnrS1, bla(CTX-M-15), aac(3)-II, and bla(OXA-1) genes, located in a plasmid of 33.5 kb, could be transferred to Escherichia coli by transformation., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

The objective of this study was to determine the frequency of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in slaughter pigs, to characterize the recovered isolates, and to investigate their genomic relatedness. Nasal swabs were collected from 53 finishing-pigs (F-pigs) and 53 suckling-piglets (S-piglets) at two different abattoirs in La Rioja (Northern Spain) coming from six production holdings. MRSA isolates were characterized by spa−, agr−, SCCmec−, and multilocus sequence typing, pulsed-field gel electrophoresis (PFGE)-ApaI, toxin gene profiling, antimicrobial susceptibility, and determination of antimicrobial resistance genes. MRSA isolates were recovered from 11 F-pigs (14 isolates) and 26 S-piglets (30 isolates). Forty of the 44MRSA presented the spa-types t011, t108, t1197, and t2346, which corresponded to the sequence type ST398 and to the clonal complex CC398. Interestingly, the remaining four isolates from F-pigs presented the spa-type t3992, and they were ascribed to a new sequence type named ST1379 (a single-locus variant of ST97), which was included in clonal complex CC97. Five PFGE-ApaI clusters with up to nine individual patterns detected among our MRSA and low genomic relatedness was observed between F-pig and S-piglet isolates. All MRSA were positive for hla, hld, and hlg hemolysin genes. ST1379 isolates harbored eta, lukE/D, and hlg-2 toxin genes, whereas ST398 isolates were positive for hlb. A great variety of distinct resistance gene patterns were observed, most of them coming from F-pig isolates. MRSA virulence properties seem to be dependent of the isolate clonal lineage. This study showed that slaughter pigs are frequently colonized by MRSA CC398; moreover, the detection of strains belonging to CC97 underlines that other lineages are also able to spread in livestock. Further studies should assess the risk of CC398 and non-CC398 MRSA to enter the food chain as well as the human health implications.

The prevalence and characterisation of integrons and the genetic environment of sulphonamide resistance genes were studied in 135 Escherichia coli isolates recovered from blood cultures in a Spanish hospital during 2007. Class 1 and 2 integrons were identified in 54 isolates (intI1, 52 isolates; intI2, 1 isolate; and intI1+intI2, 1 isolate). Of the 53 intI1-positive isolates, 36 (67.9%) contained the classic class 1 integron including the qacEDelta1-sul1 region, and 11 different gene cassette arrangements were demonstrated in 33 of these isolates. Seventeen intI1-positive isolates lacked the qacEDelta1-sul1 region, and 8 gene cassette arrangements were demonstrated in 12 of these isolates. Seventy-one isolates showed a sulphonamide-resistant phenotype, 63 of which contained sul genes. The sul1 gene was associated with intI1 in 36 of 42 sul1-positive isolates, and the sul3 gene was associated with non-classic class 1 integrons in 5 of 7 sul3-positive isolates. Finally, sul2 was found associated with strA-strB genes in 32 of 35 sul2-positive isolates, identifying 11 genetic structures, 1 of them presenting the IS150 element disrupting the strB gene; this structure was included in GenBank with accession no. FJ705354. Almost one-half of the E. coli isolates from blood cultures contained integrons and sul genes. Moreover, sul genes were detected in different structures, one of them new, and could be important determinants in antibiotic resistance dissemination., (Copyright 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Two-hundred-twenty-nine food samples of animal origin were tested to know the prevalence of vancomycin-resistant enterococci (VRE) after a decade of avoparcin ban as animal growth promoter in Spain. VRE with acquired mechanism of resistance were detected in 9 of these 229 samples (3.9%, obtained from chicken, veal and rabbit), and one VRE per food sample was further characterized. The vanA gene was identified in seven isolates (2 E. faecium, 3 E. durans, and 2 E. hirae), and the vanB2 gene in the remaining 2 isolates (identified as E. faecium). The two vanB2 isolates showed a phenotype of multiresistance that included, in addition to vancomycin, also ampicillin, erythromycin, tetracycline, streptomycin, kanamycin, ciprofloxacin, chloramphenicol and trimethoprim-sulfamethoxazole and contained, among others, erm(B), tet(M), ant(6), and aph(3')-III genes. Most of vanA enterococci showed erythromycin and tetracycline resistance and contained the erm(B) and tet(M) genes. One vanA- and both vanB2-positive E. faecium isolates were classified by MLST analysis into the CC17 clonal complex (ST17 and ST78), and one additional vanA isolate was included in a new sequence type named ST425 (singleton). Co-transference by conjugation of erm(B) and vanA genes was demonstrated in one vanA-positive E. faecium isolate. The inclusion of vanB2 cluster into Tn5382 structure was demonstrated in the two vanB2 isolates, as well as the linkage pbp5-Tn5382, and beta-haemolysis and gelatinase production was identified in one of them. Food sample of animal origin could be a vehicle of transference of VRE of vanA and vanB2 type that could be transferred to humans.

The aim of this study was to characterize the resistance mechanism in four clinical and five intestinal vancomycin-resistant Enterococcus faecium strains with VanB phenotype recovered from unrelated patients confined in two Spanish hospitals and to determine their clonal relationships. MIC values for vancomycin and teicoplanin were 16-32 and 0.5 microg ml-1, respectively. The mechanism of vancomycin resistance, as well as the genetic environment of the implicated gene, was analysed by PCR and sequencing. The vanB2 gene was detected in all nine E. faecium strains and the intergenic vanSB-YB region showed the characteristic mutations of the vanB2 subtype. Two possibly related PFGE patterns, A (seven strains) and B (two strains), were distinguished among these enterococci. The vanXB-ORFC intergenic region was amplified in the nine strains and two amino acid changes were detected in the protein encoded by the vanXB gene in strains of pattern A with respect to those of pattern B. The vanB2 gene cluster was integrated into Tn5382 in all nine strains, being pbp5 gene-linked to this transposon. The ant(6')-Ia, aph(3')-IIIa and erm(B) genes were also detected in all of the strains. Both isolates with PFGE pattern B contained the esp gene. In summary, vanB2-containing E. faecium strains with indistinguishable PFGE patterns were recovered from seven patients from two Spanish hospitals.

Objectives: To characterize the beta-lactamase genes of the expanded-spectrum cephalosporin-resistant Escherichia coli isolates recovered in a Spanish hospital during the March 2002-March 2003 period., Methods: Thirty-four of the 1700 E. coli isolates recovered from unrelated patients in a Spanish hospital showed expanded-spectrum cephalosporin resistance. The presence of genes encoding TEM, SHV, CTX-M, CMY-2-type or FOX beta-lactamases as well as the existence of mutations in the regulatory region of the chromosomal ampC gene were studied by PCR and sequencing in these 34 E. coli isolates., Results: The following extended-spectrum beta-lactamases (ESBLs) or plasmidic class C beta-lactamase genes were detected (number of isolates): bla(CTX-M-14) (14), bla(CTX-M-9) (4), bla(CTX-M-32) (1), bla(TEM-52) (2), bla(SHV-12) (3) and bla(CMY-2) (2). The remaining eight isolates showed a mutation in the promoter/attenuator region of the ampC chromosomal gene at position -42, in combination with mutations at positions -18, -1 and +58. The bla(TEM-1) gene was also detected in 12 of the ESBL-producing isolates, in both CMY-2-producing isolates and in four of the eight isolates that showed a mutation at position -42 of the ampC promoter. Other mutations in the promoter/attenuator region were detected in association with ESBL or CMY-2 genes, such as the combination -18, -1 and +58, -28 and +58, or +22, +26, +27 and +32. No clonal relationship was found among the CTX-M-producing E. coli isolates by PFGE with XbaI enzyme., Conclusions: Approximately 1.5% of the E. coli isolates of our hospital harboured ESBL genes, those of the CTX-M-9 group being the most common ones.

The objective was to analyze the beta-lactamase genes and the clonal relationship in a series of 12 clinical Klebsiella pneumoniae strains resistant to ceftazidime or cefotaxime (MIC >/=16 microg/ml) recovered in the neonatalpediatric intensive care unit (ICU) ward of a Spanish hospital during a 1-year period. TEM, SHV, CTX-M, CMY, or FOX beta-lactamase genes were analyzed by PCR and sequencing. The clonal study was performed by pulsed-field gel electrophoresis (PFGE) using XbaI. All but one of the 12 K. pneumoniae strains harbored the bla (SHV-5) gene, and the bla TEM-1a gene was also detected in one of them. These 11 strains belonged to two different clonal types: A (9 strains) and B (2 strains) and were grouped in the subtypes A(1) (6 strains), A(2), A(3), A(4), B(1), and B(2) (1 strain each). The clonal type A strains were isolated from 9 patients (in five cases from blood) during a 6-month period. The remaining K. pneumoniae strain harbored both the bla (SHV-11) + bla (CTX-M-14) genes and showed the clonal type C. A nosocomial outbreak by a SHV-5-producing multiresistant K. pneumoniae is reported in Spain in a neonatal-pediatric ICU ward. This is the first description of a K. pneumoniae harboring both the bla (SHV-11) and bla (CTX-M-14) genes in Spain.

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. Beta-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different beta-lactams showed by these 20 isolates were as follows (in microg/ml): ampicillin (64-->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8-->128), cefazolin (32-->256), cefoxitin (4-->128), cefotaxime (1-64), ceftazidime (2-->64), ceftriaxone (0.5-64), imipenem (< or = 0.06-0.25), and aztreonam (2-->32). TEM, SHV, CMY, and FOX beta-lactamase genes were analyzed by PCR and sequencing. The beta-lactamase genes detected were the following ones (number of isolates): bla(TEM-1b) (3), bla(TEM-1a) (1), bla(TEM-30f) (2), bla(TEM-1b) + bla(CMY-2) (2), and bla(SHV-12) (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type beta-lactamase in 6 isolates. Besides, a high variety of plasmidic beta-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 beta-lactamase has been detected in E. coli isolates of animal origin.

Fecal samples of healthy animals (66 pigs, 22 pets) recovered during 1998 in La Rioja, Spain, were analyzed for vancomycin-resistant enterococci colonization. Vancomycin resistance mechanisms were analyzed by PCR and sequencing. vanA-containing enterococci were detected in 3 of 66 samples (4.5%) and 5 of 22 samples (22.7%) of the pig and pet samples, respectively. Seven unrelated pulsed-field gel electrophoresis (PFGE) patterns were detected among the 8 vanA isolates (7 Enterococcus faecium, 1 E. faecalis). The tet(M) gene was present in all eight vanA enterococcal isolates, while the erm(B) and aac(6')-Ie-aph(2")-Ia genes were detected in 6 and 3 isolates, respectively. Colonization by vanC-1-containing enterococci (E. gallinarum) was demonstrated in 3% and 4.5% of the pig and pet samples. The aac(6')-Ie-aph(2")-Ia, ant(6)-Ia, aph(3')-IIIa, erm(B) and tet(M) genes were identified in one of the E. gallinarum isolates from a pig fecal sample. One vanB2-containing E. hirae strain was detected in the fecal sample of a healthy pig. In this isolate, the vanB2 gene cluster was integrated into the Tn5382-like element, as demonstrated by specific PCRs and sequencing. The tet(M) and erm(B) genes were also detected in this isolate. This is the first report in which a vanB2-containing enterococci is detected in animals and in E. hirae.

Antibiotic resistance and mechanisms involved were studied in Escherichia coli isolates from fecal samples of healthy children. Fifty fecal samples were analyzed, and one colony per sample was recovered and identified by biochemical and molecular tests. Forty-one E. coli isolates were obtained (82%). MIC testing was performed by agar dilution with 18 antibiotics, and the mechanisms of resistance were analyzed. Ampicillin resistance was detected in 24 isolates (58.5%), and blaTEM, blaSHV, and blaOXA type genes were studied by PCR and sequencing. The following beta-lactamases were detected (number of isolates): TEM (20), SHV-1 (1), and OXA-30 (1). The number of aminoglycoside-resistant isolates detected was as follows: streptomycin (15), tobramycin (1), gentamicin (1), and kanamycin (4). The aac(3)-IV gene was detected in the only gentamicin-resistant isolate. Nine (22%) and 2 (5%) isolates showed nalidixic acid (NALR) and ciprofloxacin resistance (CIPR), respectively. Mutations in GyrA and ParC proteins were shown in both NAL(R)-CIP(R) isolates and were the following: (1) GyrA (S83L + D87N), ParC (S801); and (2) GyrA (S83L + A84P), ParC (S80I + A108V). A single mutation in the S83 codon of the gyrA gene was found in the remaining seven NAL(R)-CIP(S) isolates. Tetracycline resistance was identified in 21 isolates (51%) and the following resistance genes were found (number of isolates): tetA (12), tetB (5), and tetD (1). Chloramphenicol resistance was detected in five isolates (12%). These results show that the intestinal tract of healthy children constitutes a reservoir of resistant bacteria and resistance genes.

Thirty-nine Staphylococcus isolates with different mechanisms of resistance were recovered from the feces of 50 healthy children and tested for their susceptibilities to 17 antibiotics. The percentages of resistance of the staphylococci to some antibiotics were as follows: penicillin, 87%; erythromycin, 64%; tobramycin, 36%; tetracycline, 20.5%; kanamycin, 15%; and gentamicin, 13%. The mecA gene was detected in nine coagulase-negative staphylococci.

Antibiotic resistance was investigated in 474 Escherichia coli isolates recovered from animal faeces (broilers, pigs, pets, bulls and horses), human faeces (patients and healthy volunteers) and food products of animal origin. E. coli isolates (3260) recovered from human significant infectious samples were also included. There was a high frequency of nalidixic acid, ciprofloxacin and gentamicin resistance in E. coli isolates from broilers (88, 38 and 40%, respectively), and from foods (53, 13 and 17%). High levels of resistance to trimethoprim-sulphamethoxazole and tetracycline have been found in E. coli isolates from broilers, pigs and foods. These data raise important questions about the potential impact of antibiotic use in animals and the possible entry of resistant pathogens into the food chain.

Colonization by Campylobacter strains was investigated in human, broiler, and pig fecal samples from 1997-1998, as well as in foods of animal origin, and antibiotic susceptibility testing was carried out for these strains. Campylobacter strains were isolated in the foods of animal origin (55 of 101 samples; 54.4%), intestinal samples from broilers (85 of 105; 81%), and pigs (40 of 45; 88.9%). A total of 641 Campylobacter strains were isolated from 8,636 human fecal samples of clinical origin (7.4%). Campylobacter jejuni was the most frequently isolated species from broilers (81%) and humans (84%), and Campylobacter coli was most frequently isolated from pigs (100%). An extremely high frequency of ciprofloxacin resistance was detected among Campylobacter strains, particularly those isolated from broilers and pigs (99%), with a slightly lower result for humans (72%); cross-resistance with nalidixic acid was almost always observed. A higher frequency of resistance to erythromycin (81.1%), ampicillin (65.7%), gentamicin (22.2%), and amikacin (21.6%) was detected in C. coli strains isolated from pigs compared to those isolated from humans (34.5, 29.3, 8.6, and 0%, respectively). A low frequency of erythromycin resistance was found in C. jejuni or C. coli isolated from broilers. A greater resistance to ampicillin and gentamicin (47.4 and 11.9%, respectively) was detected in C. jejuni isolated from broilers than in human strains (38 and 0.4%, respectively). Beta-lactamase production was found in 81% of the Campylobacter strains tested, although 44% of them were characterized as ampicillin susceptible. The increasing rates of Campylobacter resistance make advisable a more conservative policy for the use of antibiotics in farm animals.