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We investigated the pathogenicity, invasiveness, and genetic relatedness of 17 clinical Listeria monocytogenes stains isolated over a period of nine years (2006-2014). All isolates were phenotypically characterised and growth patterns were determined. The antimicrobial susceptibility of L. monocytogenes isolates was determined in E-tests. Invasion assays were performed with epithelial HeLa cells. Finally, L. monocytogenes isolates were subtyped by PFGE and MLST. All isolates had similar phenotypic characteristics (ß-haemolysis and lecithinase activity), and three types of growth curve were observed. Bacterial recovery rates after invasion assays ranged from 0.09% to 7.26% (1.62 ± 0.46). MLST identified 11 sequence types (STs), and 14 PFGE profiles were obtained, indicating a high degree of genetic diversity. Genetic studies unequivocally revealed the occurrence of one outbreak of listeriosis in humans that had not previously been reported. This outbreak occurred in October 2009 and affected three patients from neighbouring towns. In conclusion, the molecular epidemiological analysis clearly revealed a cluster (three human cases, all ST1) of not previously reported listeriosis cases in northwestern Spain. Our findings indicate that molecular subtyping, in combination with epidemiological case analysis, is essential and should be implemented in routine diagnosis, to improve the tracing of the sources of outbreaks.

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We compared the diagnostic performance of two chromogenic media, Brilliance MRSA 2 agar (Thermo Fisher Scientific) and ChromID MRSA agar (bioMérieux), for MRSA confirmation of 239 Staphylococcus aureus isolates from clinical, animal and food samples. Statistically significant differences were not observed between MRSA confirmation by mecA/mecC PCR, and by culture in both chromogenic media. However, a statistically significant difference was observed between the results obtained by both chromogenic media (p = 0.003). Segregated analysis of the results depending on the origin of the isolates (clinical, animal, and food) revealed a significant lower performance in the MRSA confirmation of food-derived isolates by using Brilliance MRSA 2 agar in comparison to PCR confirmation (p = 0.003) or ChromID MRSA agar (p<0.001). Both chromogenic media provided a good diagnostic performance for detection of MRSA isolates of human and animal origin. In conclusion, the use of chromogenic agar plates for MRSA confirmation of S. aureus isolates can provide a good diagnostic performance (sensitivity >92% and specificity >89%) regardless of the type of chromogenic media used or the origin of the S. aureus isolates. However, our results revealed a lower diagnostic performance for MRSA confirmation of S. aureus isolates from food samples by using Brilliance MRSA 2 agar.

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Corynebacterium kroppenstedtii has been associated with infections of the female breast. Genome sequencing of two strains revealed a specific genomic island in the multidrug-resistant isolate CNM633/14 with similarity to the R plasmid pJA144188 of Corynebacterium resistens DSM 45100, being indicative of the horizontal transfer of antibiotic resistance genes to C. kroppenstedtii.

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The illegal entrance of foods to EU through black markets at the EU borders can constitute a neglected route of dissemination of foodborne pathogens, and in particular of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we have assessed the presence of MRSA in foods sold in a black market at an EU border (the southeast part of Romania, on the border with Republic of Moldavia). We performed a search for MRSA among 200 food samples collected from 2012 to 2013. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST) and SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 32 S. aureus isolates were recovered from 16 food samples (8%). One isolate detected in a pork lard sample was MRSA (0.5%). PFGE with the restriction enzyme SmaI revealed 12 genotypes among the 32 S. aureus isolates. The MRSA isolate belonged to sequence type 398, harbored SCCmec type V, tested negative for the presence of the PVL genes and was resistant to ciprofloxacin, tetracycline and cefazolin, besides all ß-lactams. Among 31 methicillin-sensitive S. aureus (MSSA), 29% were resistant to penicillin, 9.7% to tetracycline and 3.2% to ciprofloxacin. In conclusion, in this study we report the presence of livestock-associated MRSA in foods sold in a black market at an EU border: ST398-MRSA-V. These results confirm the potential role of food in the dissemination of MRSA lineages among population, and the potential role of illegally introduced food to EU in the prevalence and evolution of MRSA clones in the community.

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The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in food-producing animals has provoked a great concern in the presence of MRSA in associated foodstuff. In this study, we have assessed for the first time the presence of MRSA in food confiscated from non-EU flights. We performed a search for MRSA among 195 food samples confiscated from passengers on flights from twenty-one non-EU countries in 2012 and 2013. One hundred and seventeen meat samples of diverse animal origin (including antelope, beef, chicken, duck, guinea pig, pork, rodents, and turkey), 75 dairy products (74 cheeses and 1 butter) and 3 eggs were analyzed. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST), SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 66 food samples were positive for S. aureus (33.9%). Six S. aureus strains were MRSA (9.1%), all of them in flights from Bolivia (and 5 from the same passenger). Among methicillin-sensitive S. aureus (MSSA) (60 out of 66S. aureus strains), 44.1% were resistant to penicillin, 10.2% to tetracycline, 8.5% were resistant to aminoglycosides (amikacin and tobramycin) and 3.4% exhibited the M phenotype. MRSA isolates were sensitive to all non-ß-lactam antibiotics tested. SmaI-PFGE analysis provided 40 genotypes among the S. aureus isolates (three genotypes among the six MRSA). Five MRSA isolates belonged to ST8 and harboured SCCmec type IVc as well as PVL genes. One isolate belonged to ST1649, harboured SCCmec type IVc and tested negative for the presence of the PVL genes. In conclusion, in this study, we report for the first time the presence of CA-MRSA in food confiscated from non-EU flights: ST8/ST1649-MRSA-IV. These results confirm the illegal entrance of food as a neglected route of transmission as well as the dissemination of successful CA-MRSA lineages among countries via illegal foods. As a result, illegally imported food could play a role in the prevalence and evolution of MRSA clones in the community.

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Two hundred food samples of animal origin confiscated from passengers arriving on flights from non-European countries at the International Airport of Bilbao (Spain) were tested for the presence of four main bacterial foodborne pathogens (Campylobacter spp., Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp.) during 2012 and 2013. Overall, 20 samples were positive for L. monocytogenes (10%) and 11 for Salmonella spp. (5.5%), whereas Campylobacter spp. and E. coli O157:H7 were not detected in any sample. The positive isolates were widely clustered: 14 and 7 different pulsotypes for L. monocytogenes and Salmonella spp. isolates, respectively. Nine sequence types (ST) were detected for L. monocytogenes: ST2 (45%), ST9 (15% isolates), ST8 and ST87 (10%), and ST308, ST37, ST155 and ST378 (5%). The Salmonella spp. isolates belonged to seven serovars: monophasic serovar 4,12:d:- (3; 27.3%), Rauform (2; 18.2%), Anatum (2; 18.2%), Oranienburg, Enteritidis, Newport and Typhimurium (1; 9.1% each). Antibiotic resistance among L. monocytogenes isolates was high, especially for clindamycin and daptomycin (more than 95% of the isolates). These results indicate that food samples imported by travelers in their personal luggage may harbor the most prevalent L. monocytogenes genotypes and Salmonella spp. serovars responsible for foodborne outbreaks worldwide. Consequently, international travel can play an important role in the prevalence and dissemination of successful clones of foodborne pathogenic bacteria, and continuous monitoring of international movements is of importance to better understand clonal evolution and emergence and dissemination of successful lineages.

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We report for the first time mecC-positive methicillin-resistant Staphylococcus aureus (mecC-MRSA) in livestock in Spain. One isolate (sequence type 130) was found in milk samples among 601 S. aureus isolates obtained from 229 dairy sheep farms. This finding highlights the potential for zoonotic transmission of mecC-positive MRSA and the need for surveillance programs to monitor its presence and clonal evolution.

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Continuous monitoring of methicillin-resistant Staphylococcus aureus (MRSA) is necessary to understand the clonal evolution of successful lineages. In this study, we identified the MRSA clones circulating in a Spanish hospital during a 2-year period, assessed their relationship with antimicrobial resistance profiles, and investigated the presence of the emerging community-associated and livestock-associated MRSA lineages (CA-MRSA, LA-MRSA). CC5-MRSA-IV isolates were the most frequently recovered, which supports the previously reported prevalence of this clone in Spanish hospitals. We observed ST125 isolates that harbored specific cassette chromosome recombinase (ccr) gene elements of the staphylococcal cassette chromosome mec (SCCmec) types IV and VI. That clone, which was first detected only recently, has increased resistance to erythromycin. Furthermore, 94% of the infections were caused by non-multiresistant isolates. Neither CA-MRSA nor LA-MRSA isolates were observed. These findings, along with related events over the last decade, suggest the establishment of a clonal endemic population in the Spanish clinical environment (AU)

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Tularemia outbreaks occurred in northwestern Spain in 1997-1998 and 2007-2008 and affected >1,000 persons. We assessed isolates involved in these outbreaks by using pulsed-field gel electrophoresis with 2 restriction enzymes and multilocus variable number tandem repeat analysis of 16 genomic loci of Francisella tularensis, the cause of this disease. Isolates were divided into 3 pulsotypes by pulsed-field gel electrophoresis and 8 allelic profiles by multilocus variable number tandem repeat analysis. Isolates obtained from the second tularemia outbreak had the same genotypes as isolates obtained from the first outbreak. Both outbreaks were caused by genotypes of genetic subclade B.Br:FTNF002-00, which is widely distributed in countries in central and western Europe. Thus, reemergence of tularemia in Spain was not caused by the reintroduction of exotic strains, but probably by persistence of local reservoirs of infection.

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Continuous monitoring of methicillin-resistant Staphylococcus aureus (MRSA) is necessary to understand the clonal evolution of successful lineages. In this study, we identified the MRSA clones circulating in a Spanish hospital during a 2-year period, assessed their relationship with antimicrobial resistance profiles, and investigated the presence of the emerging community-associated and livestock-associated MRSA lineages (CA-MRSA, LA-MRSA). CC5-MRSA-IV isolates were the most frequently recovered, which supports the previously reported prevalence of this clone in Spanish hospitals. We observed ST125 isolates that harbored specific cassette chromosome recombinase (ccr) gene elements of the staphylococcal cassette chromosome mec (SCCmec) types IV and VI. That clone, which was first detected only recently, has increased resistance to erythromycin. Furthermore, 94% of the infections were caused by non-multiresistant isolates. Neither CA-MRSA nor LA-MRSA isolates were observed. These findings, along with related events over the last decade, suggest the establishment of a clonal endemic population in the Spanish clinical environment.

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Mycoplasma agalactiae isolates from Spain were genetically characterized to investigate their genomic diversity and to better understand their relationship to isolates from other countries. Molecular typing revealed a high genomic homogeneity in Spanish M. agalactiae isolates, which clearly shows the circulation of one endemic clonal population.

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BACKGROUND: Contagious Agalactia (CA) is one of the major animal health problems in small ruminants because of its economic significance. Currently, four Mycoplasma spp. have been associated with this syndrome: M. agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. Their presence has been evaluated in several studies conducted in CA-endemic countries. However, previous Spanish studies have been focused on caprine CA, and there is a knowledge gap regarding which Mycoplasma species are present in sheep flocks from Spain, which has the second highest number of sheep amongst the 27 European Union member states. Consequently, we investigated the presence and geographic distribution of the four CA-causing mycoplasmas in Spanish dairy sheep farms. This is the first time such an investigation has been performed. RESULTS: Three hundred thirty nine out of 922 sheep flocks were positive for M. agalactiae by real time PCR (36.8%) and 85 by microbiological identification (9.2%). Interestingly, all 597 milk samples assessed for the presence of M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens tested negative. To evaluate the intermittent excretion of the pathogen in milk, we sampled 391 additional farms from 2 to 5 times, resulting that in 26.3% of the cases a previously positive farm tested negative in a later sampling. CONCLUSIONS: M. agalactiae was the only Mycoplasma species detected in the study area showing a high frequency of presence and wide distribution. Therefore, the establishment of a permanent surveillance network is advantageous, as well as the implementation of control and prevention measures to hinder the dissemination of M. agalactiae and to prevent the entrance of other Mycoplasma species.

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