Publications by authors named "Laura Ruiz-Ripa"

38 Publications

Genomic Analysis of of the Lineage CC130, Including -Carrying MRSA and MSSA Isolates Recovered of Animal, Human, and Environmental Origins.

Front Microbiol 2021 25;12:655994. Epub 2021 Mar 25.

Area of Biochemistry and Molecular Biology, OneHealth-UR Research Group, University of La Rioja, Logroño, Spain.

Most methicillin resistant (MRSA) isolates harboring gene belong to clonal complex CC130. This lineage has traditionally been regarded as animal-associated as it lacks the human specific immune evasion cluster (IEC), and has been recovered from a broad range of animal hosts. Nevertheless, sporadic -MRSA human infections have been reported, with evidence of zoonotic transmission in some cases. The objective of this study was to investigate the whole-genome sequences of 18 CC130 isolates [13 methicillin-resistant (-MRSA) and five methicillin-susceptible (MSSA)] from different sequences types, obtained from a variety of host species and origins (human, livestock, wild birds and mammals, and water), and from different geographic locations, in order to identify characteristic markers and genomic features. Antibiotic resistance genes found among MRSA-CC130 were those associated with the SSCXI element. Most MRSA-CC130 strains carried a similar virulence gene profile. Additionally, six MRSA-CC130 possessed and one MSSA-ST130 had . The MSSA-ST700 strains were most divergent in their resistance and virulence genes. The pan-genome analysis showed that 29 genes were present solely in MRSA-CC130 (associated with SCCXI) and 21 among MSSA-CC130 isolates (associated with phages). The SCCXI, PBP3, GdpP, and AcrB were identical at the amino acid level in all strains, but some differences were found in PBP1, PBP2, PBP4, and YjbH proteins. An examination of the host markers showed that the 3' region of the bacteriophage φ3 was nearly identical to the reference sequence. Truncated gene was also found in -negative strains (two of them carrying -type gene). The gene of wild rabbit isolates included novel mutations. The gene was found in the three MSSA-ST700 strains from small ruminants and in one MSSA-ST130 from a red deer; these strains also carried a -type gene, different from the human and equine variants. Finally, a phylogenetic analysis showed that the three MSSA-ST700 strains and the two MSSA-ST130 strains cluster separately from the remaining MRSA-CC130 strains with the gene as marker for the main lineage. The presence of the human IEC cluster in some -MRSA-CC130 strains suggests that these isolates may have had a human origin.
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http://dx.doi.org/10.3389/fmicb.2021.655994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027229PMC
March 2021

Prevalence and Genetic Characteristics of CC398 Isolates From Invasive Infections in Spanish Hospitals, Focusing on the Livestock-Independent CC398-MSSA Clade.

Front Microbiol 2021 9;12:623108. Epub 2021 Feb 9.

Área Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain.

Background: Livestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported.

Objectives: To determine the prevalence of (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates.

Methods: One thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6-12 months in 17 Spanish hospitals (2018-2019) were studied. CC398 lineage identification, detection of -types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing.

Results: Forty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven -types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent -types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin-resistant/(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km) ( = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately -t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD ( < 0.005).

Conclusion: CC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers.
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http://dx.doi.org/10.3389/fmicb.2021.623108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7945039PMC
February 2021

S. pseudintermedius and S. aureus lineages with transmission ability circulate as causative agents of infections in pets for years.

BMC Vet Res 2021 Jan 21;17(1):42. Epub 2021 Jan 21.

Área Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, 26006, Spain.

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], 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 [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.
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http://dx.doi.org/10.1186/s12917-020-02726-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819200PMC
January 2021

Detection of methicillin-resistant coagulase-negative staphylococci and PVL/mecA genes in cefoxitin-susceptible Staphylococcus aureus (t044/ST80) from unpasteurized milk sold in stores in Djelfa, Algeria.

J Dairy Sci 2021 Mar 15;104(3):2684-2692. Epub 2021 Jan 15.

Area Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006 Logroño, Spain. Electronic address:

This study was designed to determine antimicrobial resistance phenotypes and genotypes and virulence factors in Staphylococcus aureus and coagulase-negative staphylococci (CNS) in unpasteurized milk sold in Djelfa, Algeria. Eighty-two unpasteurized cow milk samples were randomly obtained from 82 retail stores in Djelfa and tested to detect staphylococci. Species were identified by biochemical tests and MALDI-TOF. Antimicrobial resistance phenotypes and genotypes were determined by disk diffusion test, PCR, and sequencing. The Staph. aureus isolates were subjected to spa typing, multilocus sequence typing, and detection of virulence genes and the scn gene by PCR and sequencing. Forty-five (54.9%) milk samples were contaminated by staphylococci and 45 isolates were recovered: 10 Staph. aureus (12.2% of total samples) and 35 CNS (42.7%). Resistance to penicillin (blaZ), tetracycline (tetL/tetK), and erythromycin (ermB/msrA/ermC) were the most common phenotypes (genotypes). Three CNS were methicillin-resistant and all were mecA-positive. The Staph. aureus isolates were ascribed to the following lineages [spa type/sequence type/associated clonal complex (number of isolates)]: t267/ST479/CC479 (n = 6), t1510/ST5651/CC45 (n = 1), t359/ST97/CC97/ (n = 1), t346/ST15/CC15 (n = 1), and t044/ST80 (n = 1). The mecA gene was detected in the cefoxitin-susceptible t044/ST80 isolate and co-harbored the lukF/lukS-PV and scn genes. The detection of mecA-PVL-positive Staph. aureus, methicillin-resistant CNS, and multidrug-resistant staphylococcal species indicates a potentially serious health issue and reveals that unpasteurized milk sold in Djelfa city could be a potential vehicle for pathogenic and antimicrobial-resistant staphylococci.
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http://dx.doi.org/10.3168/jds.2020-19270DOI Listing
March 2021

Genetic characterization of extended-spectrum β-lactamase-producing Enterobacteriaceae from a biological industrial wastewater treatment plant in Tunisia with detection of the colistin-resistance mcr-1 gene.

FEMS Microbiol Ecol 2021 03;97(3)

Université de la Manouba, IRESA, École Nationale de Médecine Vétérinaire de Sidi Thabet, Sidi Thabet 2020, Sidi Thabet, Ariana, Tunisia.

This study evaluated the occurrence of extended-spectrum β-lactamases (ESBL) and associated resistance genes, integrons, and plasmid types, as well as the genetic relatedness of enterobacterial isolates in the wastewater treatment plant (WWTP) of La Charguia, Tunis City (Tunisia). A total of 100 water samples were collected at different points of the sewage treatment process during 2017-2019. Antimicrobial susceptibility was conducted by the disc-diffusion method. blaCTX-M, blaTEM and blaSHV genes as well as those encoding non-β-lactam resistance, the plasmid types, occurrence of class1 integrons and phylogenetic groups of Escherichia coli isolates were determined by PCR/sequencing. Genomic relatedness was determined by multi-locus sequence typing (MLST) for selected isolates. In total, 57 ESBL-producer isolates were recovered (47 E. coli, eight Klebsiella pneumoniae, 1 of the Citrobacter freundii complex and 1 of the Enterobacter cloacae complex). The CTX-M-15 enzyme was the most frequently detected ESBL, followed by CTX-M-27, CTX-M-55 and SHV-12. One E. coli isolate harboured the mcr-1 gene. The following phylogroups/sequence types (STs) were identified among ESBL-producing E. coli isolates: B2/ST131 (subclade-C1), A/ST3221, A/ST8900, D/ST69, D/ST2142, D/ST38, B1/ST2460 and B1/ST6448. High numbers of isolates harboured the class 1 integrons with various gene cassette arrays as well as IncP-1 and IncFIB plasmids. Our findings confirm the importance of WWTPs as hotspot collectors of ESBL-producing Enterobacteriaceae with a high likelihood of spread to human and natural environments.
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http://dx.doi.org/10.1093/femsec/fiaa231DOI Listing
March 2021

Human -Carrying MRSA: Clinical Implications and Risk Factors.

Microorganisms 2020 Oct 20;8(10). Epub 2020 Oct 20.

Area of Biochemistry and Molecular Biology, University of La Rioja, 26006 Logroño, Spain.

A new methicillin resistance gene, named , was first described in 2011 in both humans and animals. Since then, this gene has been detected in different production and free-living animals and as an agent causing infections in some humans. The possible impact that these isolates can have in clinical settings remains unknown. The current available information about -carrying methicillin resistant (MRSA) isolates obtained from human samples was analyzed in order to establish its possible clinical implications as well as to determine the infection types associated with this resistance mechanism, the characteristics of these -carrying isolates, their possible relation with animals and the presence of other risk factors. Until now, most human -MRSA infections have been reported in Europe and -MRSA isolates have been identified belonging to a small number of clonal complexes. Although the prevalence of -MRSA human infections is very low and isolates usually contain few resistance (except for beta-lactams) and virulence genes, first isolates harboring important virulence genes or that are resistant to non-beta lactams have already been described. Moreover, severe and even fatal human infection cases have been detected. -carrying MRSA should be taken into consideration in hospital, veterinary and food safety laboratories and in prevention strategies in order to avoid possible emerging health problems.
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http://dx.doi.org/10.3390/microorganisms8101615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589452PMC
October 2020

Frequency and Characterization of Antimicrobial Resistance and Virulence Genes of Coagulase-Negative Staphylococci from Wild Birds in Spain. Detection of -Carrying Isolates.

Microorganisms 2020 Aug 29;8(9). Epub 2020 Aug 29.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain.

The objective of this study was to determine the prevalence and diversity of coagulase-negative staphylococci (CoNS) species from wild birds in Spain, as well as to analyze the antimicrobial resistance phenotype/genotype and the virulence gene content. During 2015-2016, tracheal samples of 242 wild birds were collected in different regions of Spain for staphylococci recovery. The species identification was performed using MALDI-TOF. The antimicrobial resistance phenotype and genotype was investigated by the disk diffusion method and by PCR, respectively. The presence of the virulence genes /-PV, , , , and was investigated by PCR. Moreover, CoNS carrying the gene were subjected to SCC typing. Of the tested animals, 60% were CoNS-carriers, and 173 CoNS isolates were recovered from the 146 positive animals, which belonged to 11 species, with predominance of ( = 118) and ( = 25). A total of 34% of CoNS isolates showed a multidrug resistance phenotype, and 42 -positive methicillin-resistant CoNS (MRCoNS) were detected. The isolates showed resistance to the following antimicrobials (percentage of resistant isolates/antimicrobial resistance genes detected): penicillin (49/ , ), cefoxitin (24/ ), erythromycin and/or clindamycin (92/ (B), (C), (43), (A), (C), (A), (B), (A) and (A)), gentamicin and/or tobramycin (5/ (6')-Ie-(2″)-Ia, (4')-Ia), streptomycin (12/), tetracycline (17/ (K), (L), (M)), ciprofloxacin (4), chloramphenicol (1/ ), fusidic acid (86/ , ) and trimethoprim-sulfamethoxazole (1/ ). None of the isolates harbored the /-PV, , , and genes, but two isolates (1%) carried the gene. Wild birds are frequently colonized by CoNS species, especially . We identified scavenging on intensively produced livestock and feeding on landfills as risk factors for CoNS carriage. High proportions of MRCoNS and multidrug resistant CoNS were detected, which coupled with the presence of important virulence genes is of concern.
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http://dx.doi.org/10.3390/microorganisms8091317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564563PMC
August 2020

Genetic characterization of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from wastewater and river water in Tunisia: predominance of CTX-M-15 and high genetic diversity.

Environ Sci Pollut Res Int 2020 Dec 7;27(35):44368-44377. Epub 2020 Aug 7.

Departamento de Agricultura y Alimentación, Universidad de La Rioja, Madre de Dios, 53, 26006, Logroño, Spain.

Aquatic environments are crucial hotspots for the dissemination of antibiotic resistant microorganisms and resistance genes. Thus, the purpose of this study was to investigate the occurrence and the genetic characterization of cefotaxime-resistant (CTX) Enterobacteriaceae at a Tunisian semi-industrial pilot plant with biological treatment (WWPP) and its receiving river (Rouriche River, downstream from WWPP) located in Tunis City, during 2017-2018. We collected 105 and 15 water samples from the WWPP and the Rouriche River, respectively. Samples were screened to recover ESBL-producing Enterobacteriaceae (ESBL-E) and isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, plasmid types and molecular typing (multilocus sequence typing, MLST). Among 120 water samples, 33 and 4 contained ESBL-producing E. coli and K. pneumoniae isolates, respectively. Most isolates were multidrug resistant and produced CTX-M-15 (28 isolates), CTX-M-1 (4 isolates), CTX-M-55 (2 isolates), CTX-M-27 (one isolate), SHV-12 (one isolate) and VEB beta-lactamases (one isolate). All K. pneumoniae were CTX-M-15-positive. Four colistin-resistant isolates were found (MIC 4-8 μg/ml), but they were negative for the mcr genes tested. Class 1 integrons were detected in 21/25 trimethoprim/sulfamethoxazole-resistant isolates, and nine of them carried the gene cassette arrays: aadA2 + dfrA12 (n = 4), aadA1 + dfrA15 (n = 2), aadA5 + dfrA17 (n = 2) and aadA1/2 (n = 1). The IncP and IncFIB plasmids were found in 30 and 16 isolates, respectively. Genetic lineages detected were as follows: E. coli (ST48-ST10 Cplx, ST2499, ST906, ST2973 and ST2142); K. pneumoniae: (ST1540 and ST661). Our findings show a high rate of CTX-M-15 and high genetic diversity of ESBL-E isolates from WWPP and receiving river water.
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http://dx.doi.org/10.1007/s11356-020-10326-wDOI Listing
December 2020

Mechanisms of Linezolid Resistance Among Enterococci of Clinical Origin in Spain-Detection of - and (D)-Carrying .

Microorganisms 2020 Jul 30;8(8). Epub 2020 Jul 30.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain.

The mechanisms of linezolid resistance among 13 and 6 isolates, recovered from six Spanish hospitals during 2017-2018, were investigated. The presence of acquired linezolid resistance genes and mutations in 23S rDNA and in genes encoding for ribosomal proteins was analyzed by PCR and amplicon sequencing. Moreover, the susceptibility to 18 antimicrobial agents was investigated, and the respective molecular background was elucidated by PCR-amplicon sequencing and whole genome sequencing. The transferability of the linezolid resistance genes was evaluated by filter-mating experiments. The gene was detected in all 13 isolates; and one -positive isolate also carried the recently described (D) gene. Moreover, one isolate displayed the nucleotide mutation G2576T in the 23S rDNA. This mutation was also present in all six isolates. All linezolid-resistant enterococci showed a multiresistance phenotype and harbored several antimicrobial resistance genes, as well as many virulence determinants. The gene was located upstream of the gene in 12 of the isolates. Moreover, an (A)-like gene was located downstream of in two isolates recovered from the same hospital. The gene was transferable in all but one isolates, in all cases along with the gene. The (D) gene was not transferable. The presence of and mutations in the 23S rDNA are the main mechanisms of linezolid resistance among and , respectively. We report the first description of the (D) gene in . The presence of the and (D) genes in Spanish hospitals is a public health concern.
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http://dx.doi.org/10.3390/microorganisms8081155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464793PMC
July 2020

Extended Spectrum β-Lactamase-Producing and from Broiler Liver in the Center of Algeria, with Detection of CTX-M-55 and B2/ST131-CTX-M-15 in .

Microb Drug Resist 2021 Feb 29;27(2):268-276. Epub 2020 Jun 29.

Area Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain.

This study aimed to determine the prevalence and diversity of extended-spectrum β-lactamase (ESBL)-producing and multidrug-resistant (MDR) and isolates from 136 broiler livers randomly purchased in 136 retail markets in Djelfa (Algeria). Isolation was performed on Hektoen agar and bacterial identification was carried out by API20E system and Maldi-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). Antimicrobial susceptibility was tested by the disk diffusion and agar dilution methods. Detection of ESBLs and other resistance and integron genes, phylogenetic grouping, and molecular typing was performed by PCR and sequencing. Seventy-eight isolates (one per positive sample) were recovered: 73 and 5 . Among , 86.3% of isolates were MDR. ESBL activity was revealed in eight and five e isolates (rates of 5.9% and 3.7% in analyzed samples, respectively). ESBL genes detected among were as follows (number of isolates): (3), (3), (1), and (1); all ESBL-producing isolates carried the gene. ESBL-producing isolates were assigned to lineages (phylogroup/sequence type and number of isolates in parenthesis): A/ST48 (1), B1/ST6448 (1), B1/ST5087 (3), B1/ST23 (1), and B2/ST131 (two isolates). isolates were ascribed to sequence types ST2010 and ST3483. Regarding the 65 non-ESBL isolates, the most observed resistance genes were as follows: (A) (75%), (57.1%), and (43.5%). Class1 integrons were revealed in seven non-ESBL isolates (10.7%) and two gene-cassette arrays were identified: 1 and 11. Our study provides evidence that broiler-derived food from Center of Algeria constitutes a source of ESBL and/or MDR-producing Enterobacteriaceae, with detection of relevant ESBL genes and epidemic clones.
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http://dx.doi.org/10.1089/mdr.2020.0024DOI Listing
February 2021

Mechanisms of Linezolid Resistance Among Clinical spp. in Spain: Spread of Methicillin- and Linezolid-Resistant ST2.

Microb Drug Resist 2021 Feb 22;27(2):145-153. Epub 2020 May 22.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain.

This study aimed at determining the mechanisms of linezolid resistance and the molecular characteristics of clinical ( = 2) and coagulase-negative staphylococci ( = 15) isolates obtained from four Spanish hospitals. The detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. The antimicrobial resistance and virulence profile was determined, and the isolates were typed by different molecular techniques. Moreover, the genetic environment of the gene was determined by whole-genome sequencing. The gene was detected in one methicillin-resistant (MRSA) that also displayed the amino acid change Val118Ala in the ribosomal protein L4. The second isolate was methicillin susceptible and showed different alterations in the ribosomal protein L4. All remaining linezolid-resistant ( = 14) and isolates ( = 1) showed the mutation G2576T ( = 14) or C2534T ( = 1) in the 23S rRNA. Moreover, different amino acid changes were detected in the ribosomal proteins L3 and L4 in isolates. All isolates belonged to the multilocus sequence type ST2. Linezolid-resistant staphylococci (LRS) showed a multiresistance phenotype, including methicillin resistance that was detected in all isolates but one, and was mediated by the gene. The gene in the MRSA isolate was located together with the gene on a conjugative 38,864 bp plasmid. Linezolid- and methicillin-resistant ST2 showing mutations in the 23S rRNA and in the ribosomal proteins L3 and L4 are spread among Spanish hospitals, whereas LRS carrying acquired linezolid resistance genes are rarely detected.
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http://dx.doi.org/10.1089/mdr.2020.0122DOI Listing
February 2021

Detection of a cfr-positive MRSA CC398 strain in a pig farmer in Spain.

Enferm Infecc Microbiol Clin 2021 Mar 6;39(3):139-141. Epub 2020 May 6.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain. Electronic address:

Objective: To characterize one linezolid- and methicillin-resistant Staphylococcus aureus (MRSA) isolate recovered from a nasal sample of a pig farmer patient.

Methods: The detection of linezolid resistance mechanisms was performed by PCR and sequencing. The antimicrobial resistance and virulence profile was investigated, and the molecular typing was performed by molecular techniques. The transference of cfr gene was assessed by conjugation experiments and its genetic environment was investigated by specific PCRs.

Results: The linezolid-resistant MRSA isolate was typed as t011-ST398/CC398-SCCmecV-agrI and carried the cfr gene. The isolate was multidrug-resistant but lacked the virulence genes studied. The cfr gene was co-located with the fexA gene on a Tn558 variant and was successfully transferred by conjugation.

Conclusion: We report the first description of LA-MRSA-CC398 carrying the cfr gene in Spain. This finding highlights the importance of surveillance programmes to determine the presence and spread of the cfr gene in the livestock and clinical settings.
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http://dx.doi.org/10.1016/j.eimc.2020.03.006DOI Listing
March 2021

Coagulase-negative staphylococci carrying cfr and PVL genes, and MRSA/MSSA-CC398 in the swine farm environment.

Vet Microbiol 2020 Apr 28;243:108631. Epub 2020 Feb 28.

Department of Food and Agriculture, University of La Rioja, Logroño, Spain. Electronic address:

This work aimed at characterizing four Staphylococcus aureus and 68 coagulase-negative staphylococci (CoNS), recovered from the air and liquid manure tank of two swine farms with intensive- and semi-extensive-production types, for their antimicrobial resistance pheno-/genotypes and their virulence gene content. Molecular typing was performed by spa typing, MLST, agr typing, and SCCmec typing, where applicable. Conjugation experiments were performed to assess the transferability of the linezolid resistance gene cfr, and its genetic environment was determined by Whole-Genome-Sequencing. The four S. aureus (intensive-production farm, IP-farm) were typed as t011-agrI-CC398-ST398, were scn-negative and two of them were methicillin-resistant (MRSA) with the mecA gene (SCCmec-V). Multidrug resistance was seen in 87 % of the CoNS. Statistically significant differences among the antimicrobial resistance rates of CoNS from the two farms were observed for cefoxitin, aminoglycosides, tetracycline, ciprofloxacin and trimethoprim-sulfamethoxazole. Eight methicillin-resistant CoNS, which were recovered from the IP-farm, carried the mecA gene. One S. simulans isolate was PVL-positive and three S. cohnii eta-positive. One S. equorum and one S. arlettae showed linezolid resistance and carried the cfr gene (IP-farm), which was non-transferable by conjugation into S. aureus. The cfr genetic context in both isolates was identical, with the lsa(B) gene located upstream of cfr. The environment of swine farms might contribute to the dissemination of CoNS that show multidrug resistance and harbor important virulence factors.
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http://dx.doi.org/10.1016/j.vetmic.2020.108631DOI Listing
April 2020

Antimicrobial resistance phenotypes and genotypes of methicillin-resistant Staphylococcus aureus CC398 isolates from Spanish hospitals.

Int J Antimicrob Agents 2020 Apr 25;55(4):105907. Epub 2020 Jan 25.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain. Electronic address:

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-CLI (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-CLI 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.
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http://dx.doi.org/10.1016/j.ijantimicag.2020.105907DOI Listing
April 2020

High prevalence of multidrug resistant S. aureus-CC398 and frequent detection of enterotoxin genes among non-CC398 S. aureus from pig-derived food in Spain.

Int J Food Microbiol 2020 May 8;320:108510. Epub 2020 Jan 8.

Departamento de Agricultura y Alimentación, Universidad de La Rioja, Logroño, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2020.108510DOI Listing
May 2020

Penicillin-susceptible Staphylococcus aureus bacteremia: Epidemiological and clinical relevance. Possible therapeutic implications.

Enferm Infecc Microbiol Clin 2020 11 16;38(9):434-437. Epub 2020 Jan 16.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, La Rioja, España.

Introduction: The increase in penicillin susceptibility among Staphylococcus aureus (SA-Pen) might have therapeutic relevance. We aimed to study the current situation in our environment.

Material And Methods: Over a 2.5 years period, all SA isolates from bacteraemia in one hospital were analysed. For all isolates, antimicrobial susceptibility profile, beta-lactam resistance genes (blaZ, mecA) and Panton-Valentine leucocidine encoding-genes were studied. For SA-Pen-blaZ isolates, spa-type, MLST and the presence of other resistance genes were studied.

Results: Among 84 patients with SA bacteraemia (35.7% MRSA and 64.3% MSSA), 77 were analysed; 22.2% of MSSA isolates were Pen and blaZ (Pen-MIC≤0.03μg/ml) corresponding to 14.3% of the total SA. In MSSA-Pen-blaZ isolates, eight spa-types and 7 clonal-complexes were detected.

Conclusion: A high prevalence of MRSA/SA and MSSA-Pen-blaZ/MSSA was detected in blood cultures. Pen-MIC≤0,3μg/ml corresponded to MSSA-Pen-blaZ. This situation raises therapeutic options which should be further evaluated in larger studies and clinical trials.
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http://dx.doi.org/10.1016/j.eimc.2019.12.003DOI Listing
November 2020

Relevance of clonal complex CC398 in bacteremia caused by Staphylococcus aureus in a secondary hospital of Aragon, Spain.

Enferm Infecc Microbiol Clin 2020 10 14;38(8):394-395. Epub 2020 Jan 14.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, La Rioja, España.

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http://dx.doi.org/10.1016/j.eimc.2019.12.007DOI Listing
October 2020

Methicillin-Resistant CC398 in Purulent Lesions of Piglets and Fattening Pigs in Portugal.

Microb Drug Resist 2020 Jul 16;26(7):850-856. Epub 2020 Jan 16.

Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal.

Methicillin-resistant (MRSA) is an important clinical problem. In 2005, a livestock-associated MRSA clone was described, named CC398, being mostly associated with pigs, and causing colonization and infection in pigs and in related humans. The prevalence of these strains in food-producing pigs raised concerns about the possibility of MRSA-CC398 being a foodborne pathogen. The objective of this study was to investigate the presence of and MRSA in 141 carcasses of pigs at three slaughterhouses of Portugal, discarded from the food chain by signs of infection, and to characterize the recovered isolates. isolates were identified by matrix-assisted laser desorption/ionization time-of-flight and they were typed (, CC398-clone, and SCC). The study of antibiotic resistance and virulence genes, and the detection of immune evasion cluster genes and prophages were performed by PCR and sequencing. Twenty-eight were obtained from 141 samples (one/sample, 19.9%), being 22 MRSA and 6 methicillin-susceptible (MSSA). All MRSA strains were typed as CC398 and were ascribed to three types (t011, t108, and t1451). The SCC detected differed according to the types of MRSA isolates (SCCV: t011 and t108; SCCIVa: t1451). The MSSA strains were classified as -t1491-ST1-CC1. All the strains contained a wide range of antimicrobial resistance genes, the resistance to tetracycline being the prevalent one. In contrast, the strains contained only a few virulence genes. Among the 6 integrases of phages tested, three were detected: SΦ1, SΦ2, and SΦ7, with variations between MRSA and MSSA strains. MRSA-CC398 is not only a habitual pig colonizer but also an opportunistic pathogen in these animals, and must be controlled at the level of producers and slaughterhouses because of its impact on public health.
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http://dx.doi.org/10.1089/mdr.2019.0219DOI Listing
July 2020

Detection of poxtA- and optrA-carrying E. faecium isolates in air samples of a Spanish swine farm.

J Glob Antimicrob Resist 2020 09 26;22:28-31. Epub 2019 Dec 26.

Departamento de Agricultura y Alimentación, Universidad de La Rioja, 26006 Logroño, Spain. Electronic address:

Objective: Two linezolid-resistant Enterococcus faecium isolates, C10004 and C10009, were recovered from air samples of a Spanish swine farm and comprehensively characterized.

Methods: Detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. Isolates were characterized by multilocus sequence typing (MLST), antimicrobial susceptibility testing, detection of antimicrobial resistance and virulence genes, and analysis of the genetic environment of the linezolid resistance genes. The characterization of isolate C10009 was performed by Whole-Genome-Sequencing and of isolate C10004 by PCR and amplicon sequencing, where applicable. Conjugation experiments to assess the transferability of the optrA and poxtA genes implicated in linezolid resistance were performed.

Results: The linezolid-resistant E. faecium isolates C10004 and C10009, assigned to ST128 and ST437, respectively, harbored the optrA and poxtA genes. Neither mutations in the 23S rRNA nor in the genes for the ribosomal proteins L3, L4 and L22 were detected. C10004 and C10009 carried fourteen and thirteen antimicrobial resistance genes, respectively. The sequence alignment indicated that the genetic environment of the poxtA gene was identical in both isolates, with a downstream-located fexB gene. The poxtA gene was transferred by conjugation together with the fexB gene, and also with tet(M) and tet(L) in the case of isolate C10004. The optrA gene could not be transferred.

Conclusions: This is the first report of the poxtA gene in Spain. The presence of poxtA- and optrA-carrying E. faecium isolates in air samples represents a public health concern, indicating an involvement of swine farms in the spread of linezolid-resistant bacteria.
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http://dx.doi.org/10.1016/j.jgar.2019.12.012DOI Listing
September 2020

High prevalence of mcr-1 encoding colistin resistance and first identification of bla in ESBL/CMY-2-producing Escherichia coli isolated from chicken faeces and retail meat in Tunisia.

Int J Food Microbiol 2020 Apr 16;318:108478. Epub 2019 Dec 16.

Université la Manouba, IRESA, École Nationale de Médecine Vétérinaire de Sidi Thabet, Sidi Thabet 2020, Sidi Thabet, Ariana, Tunisie.

Avian industries have been reported as an important contributor in the worldwide spread of antibiotic resistance owing to some particular practices especially the overuse of antibiotics. Thus in this study, we aimed to characterize extended-spectrum-beta-lactamase (ESBL) and acquired-AmpC-beta-lactamase (aAmpC)-producing Escherichia coli isolates from chicken faeces and raw meat in Tunisia. During the year 2018, 286 faecal chicken swabs and 47 raw chicken meat samples were collected and processed to recover cefotaxime-resistant E. coli. Antimicrobial susceptibility was performed by disk-diffusion and/or broth-microdilution. bla, bla, bla, and bla genes were investigated by PCR/sequencing. Genes encoding resistance to colistin (mcr-1 to mcr-8), tetracycline (tetA/tetB), sulfonamide (sul1/sul3), and chloramphenicol (cmlA), were analysed by PCR. Class 1 integrons were investigated by PCR/sequencing. Phylogenetic groups of all isolates were determined. PFGE and MLST were performed for representative isolates. PCR-based replicon typing was performed in mcr1-harbouring isolates. Cefotaxime-resistant E. coli was detected in 22.4% (64/286) and 63.8% (30/47) of faeces and meat samples, respectively. Ninety isolates were ESBL-producers and harboured the genes: bla +/- bla (n = 65), bla +/- bla (n = 21), bla (n = 1), and bla (n = 3). The bla gene was detected in four ESBL-negative isolates. Isolates belonged to phylogroups D (50%), A (36.2%), B1 (9.6%), and B2 (4.3%). Fifty-four were colistin-resistant and 52 carried the mcr-1 gene. The tetA, sul1/sul3 and cmlA genes were detected among resistant isolates and 76 harboured class 1 integrons. MLST analysis revealed 13 sequence types (STs). The isolates were classified into 28 PFGE types. The IncP, IncFIB, and IncI1 replicons were detected among mcr-1-positive strains. We report a high frequency of ESBL-producers and colistin-resistant E. coli in chicken and derived food and the detection for the first time of bla in poultry in Tunisia.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2019.108478DOI Listing
April 2020

Genetic characterisation of staphylococci of food-producing animals in Senegal. PVL detection among MSSA.

BMC Vet Res 2019 Nov 4;15(1):391. Epub 2019 Nov 4.

Departamento Agricultura y Alimentación, Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006, Logroño, Spain.

Background: Food-producing animals can be a vehicle for staphylococcal species as well as their virulence and antimicrobial resistance genes. This work aimed to analyse the diversity of staphylococcal species in food-producing animals in Dakar/Senegal, and to determine the antimicrobial resistance phenotype/genotype and virulence factors of recovered isolates. Nasal samples of 149 cows and 199 chickens (348 animals) were collected from one slaughterhouse and a local market respectively, and were inoculated on selective media for staphylococci recovery. For S. aureus isolates, molecular typing (spa-type, MLST) was performed by PCR/sequencing, and the presence of 27 virulence genes (exfoliative and toxic shock toxins, PVL, haemolysins and enterotoxins) as well as the gene scn were analysed by PCR. Susceptibility to twelve antibiotics was studied by disc-diffusion method for all staphylococci; the resistance genes involved were screened by PCR.

Results: Staphylococcus spp. was present in 3 and 26.8% of chicken and cow nasal samples, respectively. Seven S. aureus isolates and forty isolates of other staphylococcal species were identified. S. aureus isolates were recovered from cow (n = 6) and chicken (n = 1) samples, belonging to four genetic lineages: t084/ST15 (n = 1); t10579/ST291 (n = 3); t355, t4690/ST152 (n = 2); and t6618/ST6 (n = 1). All S. aureus were methicillin-susceptible, penicillin-resistant (blaZ), and two of them were also tetracycline-resistant [tet(K)]. All the isolates carried at least one of the virulence genes tested. The PVL genes were detected in three ST15 and ST152 isolates. They all harboured haemolysins encoding genes and lacked the scn gene. The other staphylococci recovered were S. sciuri (n = 16), S. simulans (n = 11), S. hyicus (n = 5), S. haemolyticus (n = 4), S. chromogenes (n = 3), and S. hominis (n = 1); they were all methicillin-susceptible and 27.5% tetracycline-resistant [tet(K) and tet(L)].

Conclusions: A low prevalence of S. aureus was detected among food-producing animals, all susceptible to methicillin. However, the presence of virulence genes (lukF/lukS-PV, eta, tst, sea and see) is worrisome to the extent that they could be transferred to derived food and therefore, to humans.
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http://dx.doi.org/10.1186/s12917-019-2137-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829939PMC
November 2019

Antimicrobial Resistance, Virulence, and Genetic Lineages of Staphylococci from Horses Destined for Human Consumption: High Detection of Isolates of Lineage ST1640 and Those Carrying the Gene.

Animals (Basel) 2019 Nov 1;9(11). Epub 2019 Nov 1.

Departamento de Agricultura y Alimentación, Universidad de La Rioja, 26006 Logroño, Spain.

This work aimed to determine the frequency and diversity of species carriage in horses intended for human consumption, as well as their resistance and virulence determinants. Eighty samples (30 nasal; 50 faecal) were recovered from 73 healthy horses in a Spanish slaughterhouse. The samples were cultured for staphylococci and methicillin-resistant staphylococci (MRS) recovery. The phenotype/genotype of antimicrobial resistance was analysed for all isolates. The -type and sequence-type (ST) were determined in strains; moreover, the presence of virulence and host-adaptation genes (, , , and ) was studied by PCR. species were detected in 27/30 (90%) and 33/50 (66%) of nasal and faecal samples, respectively. Ninety isolates belonging to eight species were recovered, with predominance of ( = 34), ( = 19), and ( = 19). strains were all methicillin-susceptible (MSSA), 28/34 were susceptible to all the antibiotics tested, and the remaining six showed resistance to (gene-detected) streptomycin ( (6)-), penicillin (), and trimetroprim/sulphametoxazole (SXT) (, ). The lineage ST1640/t2559 was predominant ( = 21). The genes and were present in all but the ST1640 isolates. Three isolates were multidrug-resistant. Healthy horses in Spain seem to be a reservoir for virulent MSSA and the lineage ST1640, although the presence of the latter in horses is described for the first time in this study. Moreover, the equine-adapted leukocidin gene is frequent among strains. A large variety of staphylococcal species with low antibiotic resistance rate were also observed.
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http://dx.doi.org/10.3390/ani9110900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912640PMC
November 2019

mcr-1 encoding colistin resistance in CTX-M-1/CTX-M-15- producing Escherichia coli isolates of bovine and caprine origins in Tunisia. First report of CTX-M-15-ST394/D E. coli from goats.

Comp Immunol Microbiol Infect Dis 2019 Dec 4;67:101366. Epub 2019 Oct 4.

Departamento de Agricultura y Alimentación, Universidad de La Rioja, 26006 Logroño, Spain. Electronic address:

The objective of this study was to isolate and characterize ESBL-producing Escherichia coli (ESBL-EC) from raw bovine and caprine milk samples, as well as from bovine faeces in Tunisia. Therefore, 120 bovine faecal samples and 9 caprine raw milk samples were collected from 2 extensive dairy-cow-farms and 5 ovine farms, respectively. In addition, 94 raw bovine milk samples, from containers and holding tanks from 50 small public-markets in the North of Tunisia, were processed for the isolation of cefotaxime-resistant E. coli (CTX). Antimicrobial susceptibility testing was carried out by disc-diffusion/broth-microdilution methods. The presence of genes encoding ESBL, as well as those encoding colistin (mcr-1 to 5 genes)- sulfonamide-, tetracycline-, gentamicin-, quinolone and chloramphenicol-resistance and class 1 integrons were tested by PCR (and sequencing in some cases). ESBL-EC isolates were further characterized by phylogrouping and MLST/PFGE typing. Eight samples (3.6%) contained ESBL-EC isolates (3/2 from raw bovine/goat milk and 3 from cattle faeces) and one isolate/sample was characterized. Four ESBL-EC isolates, all of bovine origin (3 faeces/1 milk), were resistant to colistin (MIC: 8-16 μg/ml), harboured the mcr-1 gene and carried IncP- and IncFIB-type plasmids. The 8 ESBL-EC strains had the following characteristics: a) bovine faeces: mcr-1/CTX-M-1/D-ST1642 (3 strains); b) raw milk: mcr-1/CTX-M-1/A-ST10 (1 strain); CTX-M-15/B1-ST394 (3 strains), and CTX-M-15/A-ST46 (1 strain). Most of bovine ESBL-EC isolates were multidrug-resistant (4/5). Our results showed that ESBL-EC were detected in bovine and caprine samples (CTX-M-1/CTX-M-15 producers), being some of them colistin-resistant (associated with mcr-1 gene), and they belonged to international clonal lineages.
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http://dx.doi.org/10.1016/j.cimid.2019.101366DOI Listing
December 2019

Diversity of staphylococcal species in food producing animals in Spain, with detection of PVL-positive MRSA ST8 (USA300).

Vet Microbiol 2019 Jun 12;233:5-10. Epub 2019 Apr 12.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain. Electronic address:

This work aimed to determine the prevalence, diversity, antibiotic-resistance phenotype/genotype and virulence factors in staphylococci of farm-animals. Nasal samples of 117 farm-animals (calve: 72; lamb: 37; goat: 8) were collected from one slaughterhouse in La Rioja/Spain and cultured for staphylococci and methicillin-resistant Staphylococcus (MRS) recovery. Identification was performed by MALDI-TOF. Antimicrobial resistance phenotype/genotype was determined by susceptibility testing and specific PCRs. Molecular typing (spa-typing, multilocus-sequence-typing, agr-typing, SCCmec), and detection of 12 virulence genes and human Immune-evasive-cluster (IEC) genes were performed by PCR/sequencing in S. aureus. Two marker genes of arginine catabolic mobile element (ACME) were determined by PCR (USA300-MRSA detection). Staphylococci were identified in 50%, 54% and 21% of goat, lamb and calve samples, respectively. Among the 13 S. aureus isolates recovered, 11 were susceptible to all antimicrobials tested, and two were multidrug-resistant-MRSA [beta-lactams (blaZ, mecA), macrolides [(msr(A)/msr(B)] and fluoroquinolones]. The MSSA harboured either tst or enterotoxin genes, while the MRSA harboured the lukF/lukS-PV genes. Five sequence-types were detected. The two MRSA strains (from lamb and goat) were typed as t5173/ST8/agr-I/SCCmec-IVa/ACME-positive, corresponding to USA300 clone, and were IEC-B-positive. Among the 47 coagulase-negative staphylococci (CoNS), six species were identified, predominating S. simulans (n = 25) and S. sciuri (n = 11). Fifty-three percent of CoNS showed resistance to at least one antimicrobial agent (six multidrug-resistant strains), and the following resistance phenotypes/genotypes were detected: streptomycin [27.6%; ant(6)-Ia, str], tetracycline [23.4%; tet(M), tet(L), tet(K)], clindamycin [19.1%; lnu(A), vgaA], erythromycin [10.6%; erm(C), msr(A)/msr(B)], chloramphenicol (8.5%; fexA), tobramycin (6.4%), penicillin-cefoxitin (4.3%; blaZ, mecA), and SXT (2.1%). The detection of the MRSA-USA300 lineage in food animals is worrisome and should be further monitored.
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http://dx.doi.org/10.1016/j.vetmic.2019.04.013DOI Listing
June 2019

Epidemiology of MRSA CC398 in hospitals located in Spanish regions with different pig-farming densities: a multicentre study.

J Antimicrob Chemother 2019 08;74(8):2157-2161

Área Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain.

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.
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http://dx.doi.org/10.1093/jac/dkz180DOI Listing
August 2019

High diversity of coagulase negative staphylococci species in wild boars, with low antimicrobial resistance rates but detection of relevant resistance genes.

Comp Immunol Microbiol Infect Dis 2019 Jun 8;64:125-129. Epub 2019 Mar 8.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain. Electronic address:

This work was focused to determine the prevalence and the species diversity of coagulase-negative staphylococci (CoNS) in wild boars, and to study their antimicrobial resistance phenotype and genotype. Nasal samples of 371 wild boars from six Spanish regions were collected for CoNS recovery. The identification was performed by MALDI-TOF mass-spectrometry. Antimicrobial susceptibility for eight antimicrobial agents was studied by disc-diffusion method and the presence of 31 antimicrobial resistance genes by PCR. CoNS were detected in nasal samples of 136/371 animals tested (36.6%), and 161 isolates were obtained (1-3/animal); a high diversity of species was found (n = 17), with predominance of S. sciuri (n = 64), S. xylosus (n = 21) and S. chromogenes (n = 17). Among CoNS isolates, 22.4% showed resistance to at least one antimicrobial tested. Tetracycline-resistance phenotype was the most frequently detected (10.5%), generally mediated by tet(K) gene [associated or not with tet(L)]. Other relevant resistance genes were identified including unusual ones [mecA, erm(B), erm(F), mphC, erm(43), msr(A)/msr(B), lnu(A), dfrG, fexA, and cat]. This is the first study in which CoNS isolates from wild boars are analysed. The knowledge of antimicrobial phenotype and genotype of CoNS in natural ecosystems is highly important since these staphylococcal species can act as vectors of relevant antimicrobial resistance mechanisms.
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http://dx.doi.org/10.1016/j.cimid.2019.03.006DOI Listing
June 2019

Clonally Diverse Methicillin and Multidrug Resistant Coagulase Negative Staphylococci Are Ubiquitous and Pose Transfer Ability Between Pets and Their Owners.

Front Microbiol 2019 26;10:485. Epub 2019 Mar 26.

Área Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain.

Sixty-eight owners and 66 pets, from 43 unrelated pet-owning households were screened for methicillin-resistant coagulase negative staphylococci (MRCoNS), potential cases of MRCoNS interspecies transmission (IT), and persistence. MRCoNS isolates were identified by microbiological and molecular tests. MLST-based phylogenetic analysis was performed in isolates. Antimicrobial susceptibility was evaluated using phenotypic and molecular methods. SCC type and the presence of biofilm-related locus was PCR-tested. Isolates suspected for MRCoNS IT cases were subjected to -PFGE analysis and individuals from positive households were followed-up for 1 year for carriage dynamics (every 3 months, T0-T4). Nineteen MRCoNS isolates from owners (27.9%) and 12 from pets (16.7%) were detected, coming from 20 households (46.5%). was predominant (90 and 67% of human and animal strains, respectively), showing high phylogenetic diversity (16 STs among 24 strains). Methicillin-resistant (MRSE) strains belonged to CC5 (75%), CC11 (12.5%), singleton S556 (8.3%), and S560 (4.17%). Significant host-associated differences were observed for resistance to aminoglycosides, co-trimoxazole, chloramphenicol (higher in animal isolates) and tetracycline (higher among human strains). Multidrug resistance (MDR) was common (68.4%) and associated with human strains. Great diversity of and complexes were detected, most strains being non-typeable, followed by SCCIV and V. Over one third of isolates (most from owners), carried the locus, all MRSE CC5. Two sporadic IT cases (T0) were identified in owners and dogs from two households (4.7%), with diverse interspecies-exchanged clones detected along the sampling year, especially in dogs. A comparative analysis of all MRCoNS, with all nasal coagulase positive staphylococci (CoPS) recovered from the same individuals at T0, revealed that CoPS alone was predominant in owners and pets, followed by co-carriage of CoPS and MRCoNS in owners but single MRCoNS in pets. Statistical analyses revealed that owners are more prone to co-carriage and that co-existence of IT cases and co-carriage are positively interrelated. MRCoNS from healthy owners and their pets are genetically heterogeneous MDR strains that are spread in the community. Therefore, pets also contribute to the dissemination of successful human clones. Owner-pet inhabitancy increases the risk for staphylococcal temporal concomitance with its subsequent risk for bacterial infection and genetic exchange.
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http://dx.doi.org/10.3389/fmicb.2019.00485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443710PMC
March 2019

Multicenter study of clinical non-β-lactam-antibiotic susceptible MRSA strains: Genetic lineages and Panton-Valentine leukocidin (PVL) production.

Enferm Infecc Microbiol Clin 2019 Oct 14;37(8):509-513. Epub 2019 Mar 14.

Area Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain. Electronic address:

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is considered a major cause of healthcare-associated (HA) and community-acquired (CA) infections. Considering non-β-lactam susceptibility as a potential marker for mecC-MRSA and CA-MRSA, the aim of this study was to determine the frequency and the associated genetic lineages of non-beta-lactam-antibiotic susceptible MRSA (NBLS-MRSA) strains in a multicenter study in Spain.

Methods: A collection of 45 NBLS-MRSA strains recovered in the period from January to June 2016 from 12 Spanish hospitals was analyzed. Molecular typing through spa-type characterization, agr group and multi-locus-sequence typing was performed. Methicillin-resistant genes (mecA and mecC) as well as immune evasion cluster (scn-chp-sak-sea-sep, considering scn gene as the marker of IEC system) and Panton-Valentine leukocidin (PVL) genes were determined with PCR/sequencing.

Results: The NBLS-MRSA phenotype was uncommon in the 12 hospitals analyzed (NBLS-MRSA/MRSA frequency: 0.3%-7.7%). All strains contained the mecA gene (and none contained mecC). Twenty-two different spa-types were detected among NBLS-MRSA strains, with spa-t008/agr-I the most prevalent (27%). The main clonal complexes were (CC/%): CC8/42.2%, CC5/33.3% and CC30/4.4%, with ST8 and ST5 as the main sequence types. The PVL toxin was present in 38% of strains (with spa-types t008, t024, t019, t044, t068, t318 and t3060). The IEC genes were detected in 78% of strains: IEC type-B (n=17), type-F (n=16), type-A (n=1) and type-E (n=1); 10 MRSA isolates were scn-negative.

Conclusion: The NBLS-MRSA phenotype is uncommon in the analyzed hospitals; although no mecC-positive strains were detected, it could be a good marker for MRSA PVL-positive isolates (38%), frequently associated with CA-MRSA infections.
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http://dx.doi.org/10.1016/j.eimc.2019.01.015DOI Listing
October 2019

Detection of MRSA of Lineages CC130-mecC and CC398-mecA and Staphylococcus delphini-lnu(A) in Magpies and Cinereous Vultures in Spain.

Microb Ecol 2019 Aug 29;78(2):409-415. Epub 2019 Jan 29.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006, Logroño, Spain.

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.
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http://dx.doi.org/10.1007/s00248-019-01328-4DOI Listing
August 2019

High frequency of coagulase-positive staphylococci carriage in healthy wild boar with detection of MRSA of lineage ST398-t011.

FEMS Microbiol Lett 2019 02;366(4)

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Madre de Dios 51, 26006 Logroño, Spain.

The objective of this study was to determine the frequency and diversity of coagulase-positive staphylococci (CoPS) in nasal samples of healthy wild boar, to study their resistance phenotypes/genotypes and to check the occurrence of the MRSA-ST398. Nasal samples of 371 wild boars were collected in Spain for staphylococci and MRSA recovery. Staphylococci identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). The susceptibility to 11 antimicrobials was tested by disc-diffusion and the presence of resistance genes by PCR. Molecular typing and virulence factors determination were carried out by PCR and sequencing. The rate of CoPS carriage (Staphylococcus aureus, Staphylococcus hyicus and Staphylococcus pseudintermedius) in wild boar was of 17.8% (13.7%, 2.7% and 1.6%, respectively). Susceptibility to all tested antimicrobials was shown in 74.5% of S. aureus and one strain was MRSA [lineage ST398-t011-agrI, carrying blaZ, mecA, tet(M) and tet(K) genes]. A total of 22 spa-types and 17 STs were detected among S. aureus, including: ST398/CC398 (n = 1), ST2328-ST133/CC133 (n = 20), ST425/CC425 (n = 7), ST5/CC5 (n = 5), ST1/CC1 (n = 3), ST130/CC130 (n = 2) and ST88/CC88 (n = 1). Two spa-types (t02, t15) and four STs (ST455, ST796, ST797, ST798) were detected among the six S. pseudintermedius isolates recovered, and all of them carried the lukF/S-I and siet virulence genes. All S. hyicus isolates were susceptible to antimicrobials tested.
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http://dx.doi.org/10.1093/femsle/fny292DOI Listing
February 2019