Publications by authors named "Myriam Zarazaga"

94 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

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

Simultaneous Nasal Carriage by Methicillin-Resistant and Methicillin Susceptible of Lineage ST398 in a Live Pig Transporter.

Pathogens 2020 May 21;9(5). Epub 2020 May 21.

Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK.

Methicillin-resistant (MRSA) sequence type (ST)398 is a livestock associated (LA) lineage with zoonotic potential, especially in humans with live pig contact. The objective of this study was to characterize two strains of lineage ST398 (one methicillin-resistant (MRSA), one methicillin-susceptible (MSSA)) isolated from the same nasal sample of a patient admitted in the Intensive-Care Unit of a Spanish Hospital, and with previous occupational exposure to live pigs, by whole-genome-sequencing (WGS). The sample was obtained during routine surveillance for MRSA colonization. Purified genomic DNA was sequenced using Illumina HiSeq 2000 and processed using conventional bioinformatics software. The two isolates recovered were both t011/ST398 and showed similar resistance-phenotypes, other than methicillin susceptibility. The possession of antibiotic resistance genes was the same, except for the A-gene located in SCCV in the MRSA isolate. The MSSA isolate harbored remnants of a SCC following the deletion of 17342bp from a recombination between two putative primases. Both isolates belonged to the livestock-associated clade as defined by three canonical single-nucleotide-polymorphisms, and neither possessed the human immune evasion cluster genes, , or The core genome alignment showed a similarity of 99.6%, and both isolates harbored the same mobile genetic elements. The two nasal ST398 isolates recovered from the patient with previous occupational exposure to pigs appeared to have a livestock origin and could represent different evolutionary steps of animal-human interface lineage. The MSSA strain was formed as a result of the loss of the A gene from the livestock-associated-MRSA lineage.
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http://dx.doi.org/10.3390/pathogens9050401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281718PMC
May 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

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

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

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

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

Production and Antimicrobial Activity of Nisin Under Enological Conditions.

Front Microbiol 2018 5;9:1918. Epub 2018 Sep 5.

Instituto de Ciencias de la Vid y del Vino (Universidad de La Rioja, CSIC, Gobierno de La Rioja), Logroño, Spain.

Lactic acid bacteria (LAB) are responsible for the malolactic fermentation of wines, and, therefore, controlling the growth of these bacteria is a key factor for elaborating premium wines. Sulfur dioxide has been traditionally used as an efficient antimicrobial and antioxidant agent, however, nowadays consumers' demand tends toward a reduction of sulfur dioxide levels in wine and other fermented foods. A previous study of our research group had demonstrated the effectiveness of the bacteriocin nisin to inhibit the growth of enological LAB, and its activity had been tested in culture broths. The aim of this study was to investigate the possibility of controlling the growth of bacteria in wine by the use of nisin in combination with sulfur dioxide, and to study nisin production by the natural producer LM29 under enological conditions. Our results showed that LM29 produced nisin in the presence of 2 and 4% ethanol (v/v), while higher concentrations of ethanol fully inhibited the production of nisin. We obtained a nisin enriched active extract (NAE) from the cell-free supernatant of a culture of LM29 in MRS broth containing 60% (v/v) sterile grape juice, and the extract was fully active in inhibiting the growth of the enological LAB tested by the microtiter method. Moreover, the nisin concentration of the obtained NAE could actually prevent the formation of an undesirable biofilm of LAB strains. Finally, our results of wine ageing under winery conditions showed that the use of 50 mg/L nisin decreased fourfold the concentration of sulfur dioxide required to prevent LAB growth in the wines.
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http://dx.doi.org/10.3389/fmicb.2018.01918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134021PMC
September 2018

Staphylococcus pseudintermedius Human Infection Cases in Spain: Dog-to-Human Transmission.

Vector Borne Zoonotic Dis 2017 04 11;17(4):268-270. Epub 2017 Jan 11.

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

Staphylococcus pseudintermedius is an opportunistic pathogen that has been identified as infectious agent or colonizer mainly in dogs. S. pseudintermedius has been also detected in humans and more specifically in people in contact with dogs. In this study, the possible S. pseudintermedius pet-to-human transmission was analyzed in four clinical human cases. Two patients were dog owners and S. pseudintermedius was also detected as colonizer in these healthy animals. S. pseudintermedius isolates from patients and dogs of the same household showed identical pulsed-field gel electrophoresis patterns, sequence types (STs), and antimicrobial resistance phenotypes and genotypes, and were methicillin susceptible. Resistance to erythromycin, clindamycin, tetracycline, trimetoprim-sulfamethoxazole, and/or ciprofloxacin was identified among S. pseudintermedius strains. The lineages ST241 and the new ST521 were detected in the strains of the two dog-owner patients, respectively. The strains from the other two patients presented two new STs, ST719 and ST720. To our knowledge, this is the first description of human infections caused by S. pseudintermedius in Spain.
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http://dx.doi.org/10.1089/vbz.2016.2048DOI Listing
April 2017

Diversity of species and antimicrobial resistance determinants of staphylococci in superficial waters in Spain.

FEMS Microbiol Ecol 2017 01 8;93(1). Epub 2016 Oct 8.

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

The objectives were to determine the presence and diversity of staphylococcal species in surface waters in La Rioja region (Spain), and to characterize recovered isolates. Staphylococci were detected in 42 of 47 evaluable samples, and 72 isolates were obtained, of which 13 were coagulase-positive (CoPS) and 59 were coagulase-negative (CoNS). Twelve CoPS were identified as S. aureus and typed as follows (number of strains): t002/t502/ST5 (four), t10668/ST425 (one), t10712//ST1643 (one), t843/ST130 (one), t10855/ST2461 (one), t3369/ST2657 (one), t1166/ST133 (one), t8083/ST2049 (one) and t045/ST2460 (one); and one as S. pseudintermedius ST147. Virulence genes tst, cna and lukS/F-I were detected, and one strain showed the immune evasion cluster type F. Regarding CoNS, 12 different species were recovered (number of strains): S. epidermidis (11), S. vitulinus (10), S. sciuri (nine), S. fleurettii (seven), S. lentus (six), S. simulans (five), S. xylosus (four), S. chromogenes (two), S. hominis (two), and S. equorum, S. succinus and S. warneri (one each). Fourteen CoNS isolates presented a multidrug resistance phenotype, with the following resistance genes: blaZ, mecA, fusB, fusC, erm(C), mph(C), erm(A), msr(A)/(B), mph(C), ant(4')-Ia, tet(K), tet(L), cat and str The high diversity of staphylococcal species, as well as multiple resistance and virulence genes, highlights the importance of surface waters as a temporary reservoir and source of transmission.
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http://dx.doi.org/10.1093/femsec/fiw208DOI Listing
January 2017

Evidence for Human Adaptation and Foodborne Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus.

Clin Infect Dis 2016 Nov 20;63(10):1349-1352. Epub 2016 Sep 20.

National Institute for Public Health and the Environment, Bilthoven, The Netherlands.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.
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http://dx.doi.org/10.1093/cid/ciw532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091345PMC
November 2016

Staphylococcus aureus in Animals and Food: Methicillin Resistance, Prevalence and Population Structure. A Review in the African Continent.

Microorganisms 2016 Feb 4;4(1). Epub 2016 Feb 4.

Area of Biochemistry and Molecular Biology, University of La Rioja, Madre de Dios 53, Logroño 26006, Spain.

The interest about Staphylococcus aureus (S. aureus) and methicillin resistant S. aureus (MRSA) in livestock, and domestic and wild animals has significantly increased. The spread of different clonal complexes related to livestock animals, mainly CC398, and the recent description of the new mecC gene, make it necessary to know more about the epidemiology and population structure of this microorganism all over the world. Nowadays, there are several descriptions about the presence of S. aureus and/or MRSA in different animal species (dogs, sheep, donkeys, bats, pigs, and monkeys), and in food of animal origin in African countries. In this continent, there is a high diversity of ethnicities, cultures or religions, as well as a high number of wild animal species and close contact between humans and animals, which can have a relevant impact in the epidemiology of this microorganism. This review shows that some clonal lineages associated with humans (CC1, CC15, CC72, CC80, CC101, and CC152) and animals (CC398, CC130 and CC133) are present in this continent in animal isolates, although the mecC gene has not been detected yet. However, available studies are limited to a few countries, very often with incomplete information, and many more studies are necessary to cover a larger number of African countries.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029517PMC
http://dx.doi.org/10.3390/microorganisms4010012DOI Listing
February 2016

Characterization of staphylococci in urban wastewater treatment plants in Spain, with detection of methicillin resistant Staphylococcus aureus ST398.

Environ Pollut 2016 May 1;212:71-76. Epub 2016 Feb 1.

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

The objective of this study was to determine the prevalence of Staphylococcus in urban wastewater treatment plants (UWTP) of La Rioja (Spain), and to characterize de obtained isolates. 16 wastewater samples (8 influent, 8 effluent) of six UWTPs were seeded on mannitol-salt-agar and oxacillin-resistance-screening-agar-base for staphylococci and methicillin-resistant Staphylococcus aureus recovery. Antimicrobial susceptibility profile was determined for 16 antibiotics and the presence of 35 antimicrobial resistance genes and 14 virulence genes by PCR. S. aureus was typed by spa, agr, and multilocus-sequence-typing, and the presence of immune-evasion-genes cluster was analyzed. Staphylococcus spp. were detected in 13 of 16 tested wastewater samples (81%), although the number of CFU/mL decreased after treatment. 40 staphylococci were recovered (1-5/sample), and 8 of them were identified as S. aureus being typed as (number of strains): spa-t011/agr-II/ST398 (1), spa-t002/agr-II/ST5 (2), spa-t3262/agr-II/ST5 (1), spa-t605/agr-II/ST126 (3), and spa-t878/agr-III/ST2849 (1). S. aureus ST398 strain was methicillin-resistant and showed a multidrug resistance phenotype. Virulence genes tst, etd, sea, sec, seg, sei, sem, sen, seo, and seu, were detected among S. aureus and only ST5 strains showed genes of immune evasion cluster. Thirty-two coagulase-negative Staphylococcus of 12 different species were recovered (number of strains): Staphylococcus equorum (7), Staphylococcus vitulinus (4), Staphylococcus lentus (4), Staphylococcus sciuri (4), Staphylococcus fleurettii (2), Staphylococcus haemolyticus (2), Staphylococcus hominis (2), Staphylococcus saprophyticus (2), Staphylococcus succinus (2), Staphylococcus capitis (1), Staphylococcus cohnii (1), and Staphylococcus epidermidis (1). Five presented a multidrug resistance phenotype. The following resistance and virulence genes were found: mecA, lnu(A), vga(A), tet(K), erm(C), msr(A)/(B), mph(C), tst, and sem. We found that Staphylococcus spp. are normal contaminants of urban wastewater, including different lineages of S. aureus and a high diversity of coagulase-negative species. The presence of multiple resistance and virulence genes, including mecA, in staphylococci of wastewater can be a concern for the public health.
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http://dx.doi.org/10.1016/j.envpol.2016.01.038DOI Listing
May 2016

Detection of MRSA ST3061-t843-mecC and ST398-t011-mecA in white stork nestlings exposed to human residues.

J Antimicrob Chemother 2016 Jan 21;71(1):53-7. Epub 2015 Oct 21.

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

Objectives: The objective of this study was to analyse the prevalence of tracheal carriage of Staphylococcus aureus/MRSA in storks and to study the resistance and virulence genes in the obtained isolates.

Methods: Tracheal samples from 92 stork nestlings of two landfill-associated and two natural-habitat colonies were inoculated in specific media for S. aureus and MRSA recovery. Antimicrobial susceptibility was tested, and the presence of resistance, virulence and immune evasion cluster (IEC) genes was analysed by PCR. S. aureus isolates were characterized by spa and agr typing. Staphylococcal cassette chromosome (SCC) mec type was determined for mecC-positive isolates, and MLST was performed for 17 selected S. aureus isolates.

Results: S. aureus isolates were identified in 32/92 samples (34.8%), and 38 isolates were recovered. The prevalence of S. aureus was higher in nestlings from landfills (24/43, 55.8%) than in those from natural habitats (8/49, 16.3%). Three birds from landfill-associated colonies carried MRSA, two with mecA-positive strains [clonal complex (CC) 5-spa-t002 and CC398-spa-t011] and one with a mecC-positive strain [sequence type (ST) 3061-CC130-spa-t843-agr-III-SCCmecXI). None of the MRSA isolates presented IEC genes. Thirty-five MSSA isolates, which showed 18 different spa types (ascribed to CC5, CC7, CC22, CC30, CC45, CC59, CC133 and CC398), were obtained. The agr types detected were I (63%), II (29%) and III (8%). Resistance and virulence genes identified in MSSA were blaZ (n = 25), erm(T) (n = 9), erm(A) (n = 1), tet(M) (n = 2), fexA (n = 3), str (n = 2), tst (n = 2), eta (n = 1) and cna (n = 15). The IEC types B, C, D and G were found in MSSA isolates, and two new STs were identified (ST3060 and ST3061).

Conclusions: White storks are frequently tracheal carriers of S. aureus, including ST398 isolates. MRSA isolates of lineages CC398-mecA and CC130-mecC were detected in storks from landfill-associated colonies exposed to human residues.
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http://dx.doi.org/10.1093/jac/dkv314DOI Listing
January 2016

GelJ--a tool for analyzing DNA fingerprint gel images.

BMC Bioinformatics 2015 Aug 26;16:270. Epub 2015 Aug 26.

Biochemistry and Molecular Biology Area, University of La Rioja, Ed. Científico Tecnológico-CCT. C/ Madre de Dios, 53, Logroño, 26006, Spain.

Background: DNA fingerprinting is a technique for comparing DNA patterns that has applications in a wide variety of contexts. Several commercial and freely-available tools can be used to analyze DNA fingerprint gel images; however, commercial tools are expensive and usually difficult to use; and, free tools support the basic functionality for DNA fingerprint analysis, but lack some instrumental features to obtain accurate results.

Results: In this paper, we present GelJ, a feather-weight, user-friendly, platform-independent, open-source and free tool for analyzing DNA fingerprint gel images. Some of the outstanding features of GelJ are mechanisms for accurate lane- and band-detection, several options for computing migration models, a number of band- and curve-based similarity methods, different techniques for generating dendrograms, comparison of banding patterns from different experiments, and database support.

Conclusions: GelJ is an easy to use tool for analyzing DNA fingerprint gel images. It combines the best characteristics of both free and commercial tools: GelJ is light and simple to use (as free programs), but it also includes the necessary features to obtain precise results (as commercial programs). In addition, GelJ incorporates new functionality that is not supported by any other tool.
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http://dx.doi.org/10.1186/s12859-015-0703-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549892PMC
August 2015

Characterization of Staphylococcus aureus from Raw Meat Samples in Tunisia: Detection of Clonal Lineage ST398 from the African Continent.

Foodborne Pathog Dis 2015 Aug 18;12(8):686-92. Epub 2015 Jun 18.

1 Laboratoire de Microorganismes et Biomolécules Actives, Département de Biologie, Faculté de Sciences de Tunis, Campus Universitaire , Tunis, Tunisia .

Livestock-associated Staphylococcus aureus isolates, and especially those belonging to ST398, have been increasingly described in colonized and infected animals and humans, and also in food samples in several countries. The purpose of this study was to determine the frequency of S. aureus and methicillin-resistant S. aureus (MRSA) isolates in raw meat samples destined for food consumption in Tunisia, and to characterize the recovered isolates. One hundred sixty-nine food samples of animal origin were collected. Samples were inoculated onto selective mediums for S. aureus and MRSA recovery. Different molecular typing methods were implemented (pulsed-field gel electrophoresis [PFGE], multilocus sequence typing, spa-, agr-, and SCCmec typing). MRSA was detected in 2 of these 169 samples (1.2%), both of which were of chicken origin. The two MRSA isolates (one/sample) were typed as ST30-CC30-t012-agrIII-SCCmecV and ST398-CC398-t4358-agrI-SCCmecIVa. The MRSA ST398 strain presented resistance, in addition to β-lactams, to tetracycline (tet[M]) and erythromycin (erm[C]) and harbored the sen, hla, hlg, and hlgv virulence genes. Methicillin-susceptible S. aureus (MSSA) isolates were recovered from 42 of the 169 tested samples (24.8%). A high diversity of spa types (n=21) and SmaI-PFGE patterns (27 different profiles; 4 nontypeable) were detected among MSSA isolates. Four MSSA isolates were typed as ST398/CC398. The percentage of antimicrobial resistance and detected genes in MSSA isolates were as follows: tetracycline (28.6% tet[K] and tet[L]), kanamycin (9.5%, aph[3']-IIIa), tobramycin (2.4%, ant[4']-Ia), erythromycin (14.3%, erm[A], erm[C], msr[A]), and penicillin (95%). The genes lukS-lukF were detected in two MSSA isolates (4.5%), the gene tst in one isolate, and the gene eta in five isolates. To our knowledge, this is the first detection of MRSA and MSSA ST398 in food in an African country. The risk of transmission of S. aureus and MRSA carrying different antimicrobial resistance and virulence genes through the food chain cannot be ignored.
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http://dx.doi.org/10.1089/fpd.2015.1958DOI Listing
August 2015

Molecular characterization of Staphylococcus aureus isolated from humans related to a livestock farm in Spain, with detection of MRSA-CC130 carrying mecC gene: A zoonotic case?

Enferm Infecc Microbiol Clin 2016 May 11;34(5):280-5. Epub 2015 May 11.

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

Objectives: To conduct a study of Staphylococcus aureus carriage in members of a livestock-farmer's family with different degrees of animal contact, and to characterize the recovered isolates.

Methods: Nasal samples from 11 members of the family were taken in three sampling periods (every six months) (n=31), and 9 skin samples from superficial lesions were also obtained in 5 of them. Samples were analyzed for S. aureus susceptible (MSSA) and resistant to methicillin (MRSA). S. aureus isolates were tested for antibiotic-resistance phenotype and genotype and for the detection of virulence and IEC-system genes. Molecular typing of isolates was also performed (spa- and multilocus-sequence typing).

Results: Eighteen S. aureus isolates were recovered (1 MRSA and 17 MSSA) in the 40 samples analyzed. S. aureus was detected in nasal and skin samples of 7/11 and 4/5 of tested humans, respectively. The MRSA strain was detected in the skin lesion of a farmer with high animal contact, and carried the mecC gene, and was typed as ST130-CC130-t843. The 17 MSSA isolates were ascribed to 9 different spa-types and sequence types included in the clonal complexes CC22, CC30, CC45, CC121, and in the livestock-associated lineages CC9 and CC133. Six strains harbored eta or tsst-1 genes. Three of 18 strains lacked the immune-evasion-cluster (IEC) genes (MRSA-ST130, MSSA-ST1333, and MSSA-ST133), and the remaining isolates were ascribed to IEC type-A or -B.

Conclusions: Animal-associated S. aureus lineages were detected in samples of the farmer's family, highlighting the detection of MSSA-CC133 and mecC-MRSA-ST130.
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http://dx.doi.org/10.1016/j.eimc.2015.03.008DOI Listing
May 2016

High prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene in a semi-extensive red deer (Cervus elaphus hispanicus) farm in Southern Spain.

Vet Microbiol 2015 Jun 14;177(3-4):326-31. Epub 2015 Apr 14.

Area of Biochemistry and Molecular Biology, University of La Rioja, Logroño, Spain. Electronic address:

The objective was to determine the prevalence of Staphylococcus aureus nasal carriage in red deer of a semi-extensive farm and in humans in contact with the estate animals, and to characterize obtained isolates. Nasal swabs of 65 deer and 15 humans were seeded on mannitol-salt-agar and oxacillin-resistance-screening-agar-base. Isolates were identified by microbiological and molecular methods. Antimicrobial susceptibility profile was determined for 16 antibiotics by disk-diffusion and the presence of eight antibiotic resistance genes, seven virulence genes and genes of immune-evasion-cluster (IEC) was analyzed by PCR. S. aureus was typed by PFGE-SmaI, spa, agr, SCCmec and MLST. Isolates were detected in 16 deer (24.6%). Eleven S. aureus isolates were methicillin-resistant (MRSA), and five were methicillin-susceptible (MSSA). All MRSA harbored mecC gene and were agr-III/SCCmecXI/ST1945 (four spa-t843 and seven spa-t1535). All mecC-MRSA carried blaZ-SCCmecXI and etd2, were IEC-type-E, and belonged to the same PFGE pattern. The five MSSA were typed as spa-t2420/agr-I/ST133. Regarding humans, S. aureus was recovered from six samples (40%). The isolates were MSSA and were typed as spa-t002/agr-II, spa-t012/agr-III or spa-t822/agr-III and showed different IEC types (A, B, D and F). blaZ and erm(A) genes were detected, as well as cna and tst genes. As conclusion, red deer analyzed in this study are frequent carriers of mecC-MRSA CC130 (16.9%), they are characterized by few resistance and virulence determinants, and by the presence of IEC type-E. Deer could be a source of mecC-MRSA which could potentially be transmitted to other animals, or even to humans.
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http://dx.doi.org/10.1016/j.vetmic.2015.03.029DOI Listing
June 2015

A survey of tools for analysing DNA fingerprints.

Brief Bioinform 2016 11 29;17(6):903-911. Epub 2015 Mar 29.

DNA fingerprinting is a genetic typing technique that allows the analysis of the genomic relatedness between samples, and the comparison of DNA patterns. This technique has multiple applications in different fields (medical diagnosis, forensic science, parentage testing, food industry, agriculture and many others). An important task in molecular epidemiology of infectious diseases is the analysis and comparison of pulsed-field gel electrophoresis (PFGE) patterns. This is applied to determine the clonal diversity of bacteria in the follow-up of outbreaks or for tracking specific clones of special relevance. The resulting images produced by DNA fingerprinting are sometimes difficult to interpret, and multiple tools have been developed to simplify this task. In this article, we present a survey of tools for analysing DNA fingerprints. In particular, we compare 33 tools using a set of predefined criteria. The comparison was carried out by hands-on experiences-whenever possible-and inspecting the documentation of the tools. As no system is preferred in all the possible scenarios, we have created a spreadsheet that can be customized by researchers to determine the best system for their needs.
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http://dx.doi.org/10.1093/bib/bbv016DOI Listing
November 2016

Molecular characterization of Staphylococcus aureus from nasal samples of healthy farm animals and pets in Tunisia.

Vector Borne Zoonotic Dis 2015 Feb;15(2):109-15

1 Laboratoire des Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Université Tunis-El Manar , Tunis, Tunisia .

A total of 261 healthy farm and pet animals (75 cattle, 52 goats, 100 dogs, and 34 cats) from different regions of Tunisia were screened for Staphylococcus aureus nasal carriage. Molecular typing of isolates (by spa- and multilocus sequence-typing) was performed, and their antimicrobial resistance and virulence genotypes were determined by PCR and sequencing. S. aureus isolates were detected in 17 of 261 tested samples (6.5%). All S. aureus isolates recovered were methicillin-susceptible (MSSA), and one isolate/sample was further studied. Eight different spa types were detected (t189, t279, t582, t701, t1166, t1268, t1534, and t1773), and eight different sequence types were identified (ST6, ST15, ST45, ST133, ST188, ST700 [clonal complex CC130], ST2057, and a new ST2121). MSSA from pets (six isolates) showed resistance to (number of isolates, resistance gene): penicillin (six, blaZ), tetracycline (one, tet[M]), erythromycin one, erm[A]), streptomycin (one, ant[6]-Ia), and ciprofloxacin (one). All isolates from farm animals showed susceptibility to the tested antimicrobials, except for two penicillin-resistant isolates. Five S. aureus isolates from goats and cats harbored the lukF/lukS-PV genes, encoding the Panton-Valentine leukocidin, and six isolates from goats harbored the tst virulence gene. In addition, diverse combinations of enterotoxin genes were detected, including two variants of the egc cluster. Goats and cats could represent a reservoir of important toxin genes, with potential implications in animal and human health.
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http://dx.doi.org/10.1089/vbz.2014.1655DOI Listing
February 2015

Characterization of tetracycline and methicillin resistant Staphylococcus aureus strains in a Spanish hospital: is livestock-contact a risk factor in infections caused by MRSA CC398?

Int J Med Microbiol 2014 Nov 26;304(8):1226-32. Epub 2014 Sep 26.

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

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.
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http://dx.doi.org/10.1016/j.ijmm.2014.09.004DOI Listing
November 2014

Methicillin-resistant coagulase-negative staphylococci from healthy dogs in Nsukka, Nigeria.

Braz J Microbiol 2014 18;45(1):215-20. Epub 2014 Apr 18.

Biochemistry and Molecular Biology University of La Rioja Logroño Spain.

The occurrence, resistance phenotype and molecular mechanisms of resistance of methicillin-resistant staphylococci from groin swabs of 109 clinically healthy dogs in Nsukka, Nigeria were investigated. The groin swab samples were cultured on mannitol salt agar supplemented with 10 μg of cloxacillin. Sixteen methicillin-resistant coagulase negative staphylococci (MRCoNS), all harbouring the mecA gene were isolated from 14 (12.8%) of the 109 dogs studied. The MRCoNS isolated were: S. sciuri subspecies rodentium, S. lentus, S. haemolyticus, and S. simulans with S. sciuri subspecies rodentium (62.5%) being the predominant species. Thirteen (81.3%) of the MRCoNS were resistant to tetracycline while 12 (75%) and 10 (62.5%) were resistant to kanamycin and trimthoprim-sulphamethoxazole respectively. None of the isolates was resistant to fusidic acid, linezolid and vancomycin. Thirteen (81.3%) of the MRCoNS were multi-drug resistance (MDR). Other antimicrobial genes detected were: blaZ, tet(K), tet(M), tet(L), erm(B), lnu(A), aacA-aphD, aphA3, str, dfr(G), cat pC221 , and cat pC223 . Methicillin-resistant staphylococci are common colonizers of healthy dogs in Nigeria with a major species detected being S. sciuri subsp. rodentium.
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http://dx.doi.org/10.1590/S1517-83822014005000034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4059299PMC
February 2015

Detection of methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene in wild small mammals in Spain.

J Antimicrob Chemother 2014 Aug 7;69(8):2061-4. Epub 2014 Apr 7.

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

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.
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http://dx.doi.org/10.1093/jac/dku100DOI Listing
August 2014

Genetic lineages, antimicrobial resistance, and virulence in Staphylococcus aureus of meat samples in Spain: analysis of immune evasion cluster (IEC) genes.

Foodborne Pathog Dis 2014 May 30;11(5):354-6. Epub 2014 Jan 30.

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

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.
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http://dx.doi.org/10.1089/fpd.2013.1689DOI Listing
May 2014

A novel fexA variant from a canine Staphylococcus pseudintermedius isolate that does not confer florfenicol resistance.

Antimicrob Agents Chemother 2013 Nov 26;57(11):5763-6. Epub 2013 Aug 26.

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

Transposon Tn558 integrated in the chromosomal radC gene was detected for the first time in Staphylococus pseudintermedius. It carried a novel fexA variant (fexAv) that confers only chloramphenicol resistance. The exporter FexAv exhibited two amino acid substitutions, Gly33Ala and Ala37Val, both of which seem to be important for substrate recognition. Site-directed mutagenesis that reverted the mutated base pairs to those present in the original fexA gene restored the chloramphenicol-plus-florfenicol resistance phenotype.
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http://dx.doi.org/10.1128/AAC.00948-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811270PMC
November 2013