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Mercury contamination is a global environmental problem. This pollutant is highly toxic and persistent which makes it extremely susceptible to biomagnify, i.e. increase its concentrations as it moves up the food chain, reaching levels that threaten wildlife and, ultimately, ecosystems’ function and structure. Mercury monitoring is thus crucial to determine its potential to damage the environment. In this study, we assessed the temporal trends of the concentrations of Hg in two coastal animal species closely connected by a predator-prey interaction, and evaluated its potential transfer between trophic levels using the δ15N signatures of the two species. For this, we performed a multi-year survey of the concentrations of total Hg and the values of δ15N in the mussel Mytilus galloprovincialis (prey) and the dogwhelk Nucella lapillus (predator) sampled along ∼1500 km of the North Atlantic coast of Spain over a 30-year period (five surveys between 1990 and 2021). Concentrations of Hg decreased significantly between the first and the last survey in the two species studied. Except for the 1990 survey, the concentrations of Hg in mussels were amongst the lowest registered in the literature for the North East Atlantic Ocean (NEAO) and the Mediterranean Sea (MS) between 1985 and 2020. Nonetheless, we detected Hg biomagnification in almost all surveys. Worryingly, trophic magnification factors obtained here for total Hg were high and comparable to the found in the literature for methylmercury, the most toxic and readily biomagnified form of this element. The δ15N values were useful to detect Hg biomagnification under normal circumstances. However, we found that nitrogen pollution of coastal waters differentially affected the δ15N signatures of mussels and dogwhelks limiting the use of this parameter for this purpose. We conclude that Hg biomagnification could constitute an important environmental hazard even when found at very low concentrations in the lower trophic levels. Also, we warn that use of δ15N in biomagnification studies when there is some underlying nitrogen pollution problem might lead to misleading conclusions Authors belong to the Grupo de Referencia Competitiva GRC GI-1252/GPC2020–23 (ED431C 2020/19) which is co-funded by Xunta de Galicia and ERDF (EU) SI

Studying how different plant groups deal with heavy metal exposure is crucial to improve our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used RNA sequencing (RNA-seq) and epigenotyping by sequencing (epiGBS) to assess gene expression and DNA methylation changes respectively in plants from four populations of the metallophyte moss Scopelophila cataractae treated with Cd or Cu in the laboratory. We built RNA-seq and epiGBS sequencing libraries from control and treated samples from each population and sequenced them using Illumina HiSeq 3000 (PE-150 bp) and Illumina HiSeq X-Ten System (PE-150 bp) respectively. For the RNA-seq data, we performed a read quality filter, mapped the reads to the de novo transcriptome created with Trinity, and estimated transcript abundance for each sample. For the epiGBS data, we used a custom pipeline (https://doi.org/10.5281/zenodo.7040291) to map the reads to a de novo reference genome and performed strand-specific nucleotide (single nucleotide polymorphisms, SNPs) and methylation (single cytosine methylation polymorphisms, SMPs) variant calling. We filtered out SNPs and SMPs with low coverage within (positions with10 sequencing reads per sample) and across samples (positions with poor representation on the full set of samples). Finally, we performed pairwise comparisons between control and treated samples from each population and identified differentially expressed genes and differentially methylated cytosines associated to heavy metal exposure. We payed particular attention to the different responses of the more and the less tolerant populations of S. cataractae. These datasets could contribute to future comparative studies of abiotic stress response across plant groups.

The remarkable capacity of bryophytes to tolerate extremely challenging abiotic conditions allows us to enhance our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used next generation sequencing to study DNA methylation and gene expression changes in plants from four populations of the metallophyte moss Scopelophila cataractae experimentally exposed to either Cd or Cu. These populations previously showed differences in tolerance to both metals, so here, we aimed to investigate the molecular basis of this phenotypic differentiation. We found no evidence of genetic differentiation among the populations studied. The epigenetic data, however, showed limited but significant population-specific changes in DNA methylation in response to both metals. Exposure to acute Cu stress in the laboratory led to the downregulation of genes involved in heavy metal tolerance in both the more and the less tolerant populations, but this response was quantitatively higher in the most tolerant. We propose that chronic exposure to varying levels of heavy metals in the field led to potentially non-genetically-based intraspecific differentiation for heavy metal tolerance in S. cataractae. The most tolerant plants invested more in constitutive protection and were more efficient at entering a conservative state when faced with acute Cu stress This research has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 704141-BryOmics. Dr. M. Teresa Boquete has been supported during the development of this project by the Juan de la Cierva-Incorporación program from the Spanish Ministry of Science, Innovation and Universities (IJC2018-035018) and is currently supported by the Maria Zambrano program from the Spanish Ministry of Science, Innovation and Universities SI

Background Chagas disease is endemic in Latin America and, over the last few decades, due to population movements, the disease has spread to other continents. Early diagnosis and treatment are critical in terms of improving outcomes for those living with Chagas disease. However, poor knowledge and awareness is one of barriers that affects access to Chagas disease diagnosis and treatment for the population at risk. Information regarding immigrants’ knowledge concerning Chagas disease control and prevention is insufficient in non-endemic countries and, therefore, this study sought to assess Chagas disease knowledge and awareness within the Bolivian community residing in Madrid. Methods This cross-sectional study was carried out in March–August 2017. A total of 376 Bolivians answered a structured questionnaire. A knowledge index was created based on respondents’ knowledge about transmission, symptoms, diagnosis, and place to seek treatment. Multivariate logistic regressions analyses were performed to assess the factors associated with respondents’ knowledge of Chagas disease. Results A total 159 (42.4%) of Bolivians interviewed about their knowledge of Chagas disease were men and 217 (57.6%) were women. Vinchuca was mentioned as mode of transmission by 71% of the Bolivians surveyed, while only 9% mentioned vertical transmission. Almost half of the Bolivians did not know any symptom of Chagas disease and only 47% knew that a specific blood test is necessary for diagnosis. Most of Bolivians were aware of the severity of Chagas disease, but 45% of Bolivians said that there is no cure for Chagas and 96% did not know any treatment. Based on the index of knowledge generated, only 34% of Bolivians had a good knowledge about Chagas disease transmission, symptoms, diagnosis and treatment. According to the multiple logistic regression analysis, knowledge regarding Chagas disease, diagnosis and treatment was significantly higher amongst older Bolivians who had secondary education at least, as well as amongst those who had already been tested for Chagas disease. Conclusions This study found that most of the Bolivian population living in Spain had poor knowledge about Chagas disease transmission, symptoms, diagnostic methods and treatment. A poor understanding of the disease transmission and management is one of the most important barriers when it comes to searching for early diagnosis and appropriate care.

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ec toparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free-living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced-representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we con ducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identi fied a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontar get effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost-effective way to characterize genome-wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects

The capacity to respond to environmental challenges ultimately relies on phenotypic variation which manifests from complex interactions of genetic and nongenetic mechanisms through development. While we know something about genetic variation and structure of many species of conservation importance, we know very little about the nongenetic contributions to variation. Rhizophora mangle is a foundation species that occurs in coastal estuarine habitats throughout the neotropics where it provides critical ecosystem functions and is potentially threatened by anthropogenic environmental changes. Several studies have documented landscape-level patterns of genetic variation in this species, but we know virtually nothing about the inheritance of nongenetic variation. To assess one type of nongenetic variation, we examined the patterns of DNA sequence and DNA methylation in maternal plants and offspring from natural populations of R. mangle from the Gulf Coast of Florida. We used a reduced representation bisulfite sequencing approach (epi-genotyping by sequencing; epiGBS) to address the following questions: (a) What are the levels of genetic and epigenetic diversity in natural populations of R. mangle? (b) How are genetic and epigenetic variation structured within and among populations? (c) How faithfully is epigenetic variation inherited? We found low genetic diversity but high epigenetic diversity from natural populations of maternal plants in the field. In addition, a large portion (up to ~25%) of epigenetic differences among offspring grown in common garden was explained by maternal family. Therefore, epigenetic variation could be an important source of response to challenging environments in the genetically depauperate populations of this foundation species.

Reduced representation bisulfite sequencing is an emerging methodology for evolutionary and ecological genomics and epigenomics research because it provides a cost-effective, high-resolution tool for exploration and comparative analysis of DNA methylation and genetic variation. Here we describe how digestion of genomic plant DNA with restriction enzymes, subsequent bisulfite conversion of unmethylated cytosines, and final DNA sequencing allow for the examination of genome-wide genetic and epigenetic variation in plants without the need for a reference genome. We explain how the use of several combinations of barcoded adapters for the creation of highly multiplexed libraries allows the inclusion of up to 144 different samples/individuals in only one sequencing lane.

INTRODUCTION: Approximately 120,000 people live with Chagas disease in Europe, 43% of whom are living in Spain. Early diagnosis and treatment are critical to improve outcomes for those living with Chagas, and also for the prevention of ongoing transmission. The decision to be tested for Chagas is affected by a range of factors. Studies have highlighted the need to consider the wider social determinants of healthcare seeking behaviour related to Chagas. In Madrid, 44% of Bolivians undergo Chagas screening, which is a higher rate than other European regions, but studies concerning the factors which determine testing have not been performed. This study aimed to assess, for a first time, the factors associated with screening for Chagas among Bolivians living in Madrid trying to help in developing strategies and health recommendations. METHODS: This was a cross-sectional survey about knowledge of Chagas and practices of Bolivians living in Madrid, Spain. A structured questionnaire was administered to 376 participants regarding Chagas health-seeking behaviour. Determinants were assessed by multiple logistic regressions adjusted by sex. RESULTS: After adjusting for others variables and sex, the factors shown to be associated with Chagas screening were to have between 35 and 54 years of age; coming from a department with high prevalence of Chagas (OR 2.17 95% CI 0.99-4.76); received information about Chagas in Spain (OR 2.44 95% CI 1.32-4.51); and received any advice to do the test, especially if the advice came from a professional. CONCLUSIONS: Health authorities should coordinate and promote strategies addressed to diagnose and treat Chagas taking into account all factors associated with screening. Our study suggests that professional advice appears to be the cornerstone to encourage Bolivians to undergo Chagas screening in Madrid. It is time to change the burden of the decision of being screened from the patient to the doctor. Being diagnosed for Chagas needs to become an institutional strategy. This study was funding by the Insituto de Salud Carlos III (www.isciii.es)PI15CIII/00047 to TBH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Reduced representation bisulfite sequencing is an emerging methodology for evolutionary and ecological genomics and epigenomics research because it provides a cost-effective, high-resolution tool for exploration and comparative analysis of DNA methylation and genetic variation. Here we describe how digestion of genomic plant DNA with restriction enzymes, subsequent bisulfite conversion of unmethylated cytosines, and final DNA sequencing allow for the examination of genome-wide genetic and epigenetic variation in plants without the need for a reference genome. We explain how the use of several combinations of barcoded adapters for the creation of highly multiplexed libraries allows the inclusion of up to 144 different samples/individuals in only one sequencing lane.

Anthropogenic activities have increased exposure to heavy metal pollution in previously uncontaminated ecosystems, threatening plant communities. Considering that phenotypic variation underlies rapid adjustment to challenging environmental conditions in natural populations, the study of variation in traits related to plant response to heavy metal stress provides valuable insight into the likelihood of a population’s survival. This paper investigates patterns of intraspecific phenotypic variation for heavy metal accumulation and tolerance in bryophytes, one of the most resilient and relatively understudied plant taxa. We examined two moss species with contrasting affinities for heavy metals: the heavy metal specialist Scopelophila cataractae, and the facultative metallophyte Ceratodon purpureus. We sampled four populations of S. cataractae in close microhabitats with different contamination levels of Cd and Cu, one population of C. purpureus in an urban area, and separate lab-maintained male and female isolates from one population of C. purpureus growing in axenic conditions. After clonally propagating all populations under control, Cd and Cu treatments, we measured plant fitness, oxidative damage, and Cd and Cu accumulation. Scopelophila cataractae isolates from microhabitats with higher levels of metals in the field (Sc2, Sc3) were more tolerant than those collected in less contaminated microhabitats (Sc1, Sc4). Sc2 and Sc3 accumulated significantly less Cu in the leaves compared to the stem which could limit damage to their main photosynthetic organs and contribute to the observed differences in Cu tolerance. In contrast C. purpureus showed intraspecific differences in tolerance to Cd and Cu, but not in accumulation. These differences arose among isolates that had never been exposed to heavy metals before. We also report the first evidence for sexual dimorphism for Cd tolerance in this species, with females being more tolerant than males. Altogether, our results provide novel insights into the mechanisms used by bryophytes to deal with heavy metal stress, as well as the first evidence for metal-dependent, sex-specific differences in heavy metal tolerance in bryophytes.

In this study, we used amplified fragment length polymorphism analysis to investigate the genetic structure of the terrestrial moss Pseudoscleropodium purum (Hedw.) M. Fleish. naturally exposed to different levels of atmospheric deposition of heavy metals. We also determined the heavy metal concentrations in samples of this moss to evaluate whether there was a relationship between atmospheric pollution and population genetic diversity. A low level of genetic diversity and a limited gene flow among populations were observed which is in accordance to the prevalence of asexual reproduction in this species. In addition, no significant correlation was found between metal content and gene diversity in P. purum, probably because of the common history of the sampled populations and/or to the lack of a drastic reduction of the size of the population; nonetheless, a clear genetic structure was evident in relation to the existing pollution gradient. Thus, based on the results of the principal coordinate analysis and Bayesian analysis of the genotypes, the mixed structure of the second most polluted population would suggest an ongoing differentiation of metal-tolerant genotypes in the most polluted sites of the sampling area.

Background Chagas disease has crossed South America’s borders and in recent years has spread to regions that were not previously affected. Early diagnosis and treatment of Chagas disease improves the clinical prognosis and prevents vertical transmission. Taking into account the lack of evidence of how primary care services manage Chagas disease in a non-endemic country, this study assessed Chagas disease knowledge, attitudes and practices among primary health care professionals. Methods and principal findings Between 2017 and 2019, eight focus groups were formed with 41 family physicians and 40 nurses from healthcare centers in Madrid, Spain, and 70 field notes were collected during non-participant observation. The family physicians and nurses showed a lack of general knowledge about Chagas disease, and they did not identify the country of origin to request the blood test. The family physicians and nurses thought that the population did not talk broadly about Chagas disease because of the stigma or shame. The role of nurses was more focused on vaccination status and chronic disease follow-up, and family physicians assumed a facilitating role to send patients to different hospital facilities. Communication between primary care professionals and the hospital is a barrier frequently experienced by family physicians. Conclusions The diagnosis of CD in non-endemic countries continues being an important challenge for health systems. The results obtained with the study of the knowledge, attitudes and practices at primary care through a qualitative approach allows to obtain evidence that could help to develop strategies for the screening of CD in a protocolized way in order to avoid that the diagnosis depends exclusively on the request of the patient., Author summary Spain is the country with the highest burden of Chagas disease, with an estimated 52,000 cases, 81% of which are reported. Nevertheless, recent publications have shown that only 44% of the Bolivian population living in Madrid had a screening test performed (prevalence = 27.7%). This neglected tropical disease is transmitted through the bite of a kissing bug in endemic regions, while mother-to-child transmission, as well as blood transfusion and organ transplant transmissions occur in endemic and non-endemic countries. Without treatment, Chagas disease could cause severe cardiac and/or gastrointestinal symptoms, and ultimately death. Early diagnosis and treatment of Chagas disease improves the clinical prognosis, prevents vertical transmission and it could also prevent blood transfusion and organ transplant transmissions. Our study is the first study in Spain to use qualitative methods to explore the knowledge, attitudes and practices of primary care professionals about Chagas disease. The results show that is necessary to develop strategies in a protocolized way in order to avoid that the diagnosis depends exclusively on the request of the patient. Our findings have important public health and policy implications, offering different strategies in primary care which could facilitate the early diagnosis of Chagas disease.

BACKGROUND: It is estimated that around 52,000 people live with Chagas in Spain, but only 10% have been diagnosed. Migrants from Bolivia bear the burden of Chagas infection in Spain. However, little is known about their current management of Chagas diagnosis and treatment patterns. This study aimed to assess the Chagas related disease perception and health behaviour of Bolivians living in Madrid. METHODS AND PRINCIPAL FINDINGS: For a first time, a cross-sectional survey about Chagas' knowledges and practices was carried out in Madrid, Spain. A total of 376 Bolivians were interviewed about their Chagas health-seeking behaviour. Differences between men and women were assessed Most of Bolivians living in Madrid have access to the public health services. 44% of Bolivians included in the survey had a Chagas screening test done. However, while women did their test for Chagas mostly at hospital (59.2%), men also used the community campaigns (17.5%) and blood banks (14.3%). The prevalence reported among Bolivians tested was 27.7%. Unfortunately, more than half of those reporting a positive test for Chagas did not begin or completed treatment. Only 45.7% of positives reported having had their children tested for Chagas. CONCLUSIONS: Despite the increase in the number of Chagas diagnoses done in Madrid, the number of Bolivians who tested positive and then started or completed treatment remains very low. The fact that most Bolivians' access to the health system is through the primary healthcare services should be considered for improving management of cases and follow-up of treatment adherence. Local and national protocol establishing guidelines for the screening and treatment of Chagas disease would help improving case detection and management at all levels of the healthcare system. This study was funding by the Insituto de Salud Carlos III (www.isciii.es) PI15CIII/00047 to TBH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Protein expression was assessed in samples of Pseudoscleropodium purum cross-transplanted between one unpolluted (UNP) and two polluted (POLL) sites. Firstly, the level of expression (LE) of 17 proteins differed between native mosses from both types of sites, but differences were only maintained throughout the experiment for 5 of them. The LE of these five proteins changed over time in mosses transplanted from UNP to POLL and vice versa, becoming similar to that in autotransplants. However, these changes occurred slower than changes in the heavy metal concentrations measured in the same samples, and therefore they were not related to atmospheric pollution. Although the proteins identified were associated with moss metabolism, the expected growth reduction in samples autotransplanted within POLL (as a result of the down-regulation of photosynthesis-related proteins), did not occur. This supports the hypothesis that mosses growing in polluted areas adapt to heavy metal pollution and are able to reduce/overcome their toxic effects (i.e., reduced growth). Nevertheless, further specific research must be carried out to identify the proteins involved in this type of response, as lack of information on the bryophyte genome precludes us from reaching further conclusions.

We measured the concentrations of Cd, Cu, Hg, Pb and Zn in samples of the terrestrial moss Pseudoscleropodium purum reciprocally transplanted between an unpolluted and two polluted sampling sites. At the beginning of the experiment, the concentrations of all these elements differed significantly between mosses from the unpolluted site and mosses from the polluted sites. In general, the concentrations of the heavy metals in mosses from both polluted sites transplanted to the unpolluted site decreased until they reached the same levels as in autotransplants at this site (after 480-840 days). However, the concentrations of all heavy metals in mosses transplanted from the unpolluted site to both polluted sites increased to higher levels than in the autotransplants (except for Cu, Hg and Pb at one of the sampling sites). These results led us to conclude that mosses that have been continuously exposed to high atmospheric deposition of heavy metals undergo an adaptive response (probably genotypic) to such conditions. We therefore recommend active rather than passive biomonitoring of air quality in industrial environments because atmospheric deposition could be underestimated, and also recommend further investigation into the mechanisms involved in this response.

Pulsed-field gel electrophoretic analysis of 2,144 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients in Spanish hospitals over a 7-year period revealed 17 predominant profiles. Typing showed the replacement of Iberian clone E1 (ST247-MRSA-I) by two prevalent clones, E7 and E8, that are closely related to each other and have the same genetic background as ST125-MRSA-IV.

In a point-prevalence study performed in 145 Spanish hospitals in 2006, we collected 463 isolates of Staphylococcus aureus in a single day. Of these, 135 (29.2%) were methicillin (meticillin)-resistant S. aureus (MRSA) isolates. Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE) after SmaI digestion, staphylococcal chromosomal cassette mec (SCC mec ) typing, agr typing, spa typing with BURP (based-upon-repeat-pattern) analysis, and multilocus sequence typing (MLST). The 135 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (72.6%), gentamicin (20.0%), erythromycin (66.7%), and clindamycin (39.3%). Among the isolates resistant to erythromycin, 27.4% showed the M phenotype. All of the isolates were susceptible to glycopeptides. Twelve resistance patterns were found, of which four accounted for 65% of the isolates. PFGE revealed 36 different patterns, with 13 major clones (including 2 predominant clones with various antibiotypes that accounted for 52.5% of the MRSA isolates) and 23 sporadic profiles. Two genotypes were observed for the first time in Spain. SCC mec type IV accounted for 6.7% of the isolates (70.1% were type IVa, 23.9% were type IVc, 0.9% were type IVd, and 5.1% were type IVh), and SCC mec type I and SCC mec type II accounted for 7.4% and 5.2% of the isolates, respectively. One isolate was nontypeable. Only one of the isolates produced the Panton-Valentine leukocidin. The isolates presented agr type 2 (82.2%), type 1 (14.8%), and type 3 (3.0%). spa typing revealed 32 different types, the predominant ones being t067 (48.9%) and t002 (14.8%), as well as clonal complex 067 (78%) by BURP analysis. The MRSA clone of sequence type 125 and SCC mec type IV was the most prevalent throughout Spain. In our experience, PFGE, spa typing, SCC mec typing, and MLST presented good correlations for the majority of the MRSA strains; we suggest the use of spa typing and PFGE typing for epidemiological surveillance, since this combination is useful for both long-term and short-term studies.

Since 1986 we have carried out five nationwide point-prevalence studies in Spain analyzing Staphylococcus spp. The 2006 data, corresponding to the sixth study, are presented herein.A total of 145 hospitals from all geographic areas of the country participated in the study. We investigated 866 staphylococcal isolates (463 S. aureus). Antimicrobial susceptibility testing was performed against 16 antimicrobials by an automated microdilution method. Susceptibility to tigecycline was determined by the E-test method.Resistance of S. aureus to oxacillin seemed to have stabilized (31.2% in 2002 vs. 29.2% in 2006), and the same was true for resistance to erythromycin, clindamycin and ciprofloxacin. In 2006, isolates were more susceptible to gentamicin (16.9% resistance in 2002 vs. 8.6% in 2006, P0.001). None of the isolates presented decreased susceptibility to vancomycin, and the resistance to cotrimoxazole (0.9%) and rifampin (0.6%) was minimal. One isolate showed linezolid resistance. Resistance of coagulase negative staphylococci to oxacillin (61.3% in 2002 vs. 66.7% in 2006) and erythromycin (63.0% in 2002 vs. 66.5% in 2006) remained stable, although resistance to gentamicin (27.8% in 2002 vs. 44.2% in 2006, P0.001), ciprofloxacin (44.9% in 2002 vs. 54.3% in 2006, P = 0.010) and clindamycin (33.8% in 2002 vs. 46.2% in 2006, P = 0.001) has increased. Two isolates presented decreased susceptibility to teicoplanin and one was linezolid-resistant. All Staphylococcus spp. were uniformly susceptible to quinupristin-dalfopristin and tigecycline.Resistance of Staphylococcus spp. to oxacillin remains high in Spain, but seems to have stabilized in the last years. Linezolid resistance is emerging.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates producing the Panton-Valentine leukocidin (PVL) have been reported worldwide. We describe the molecular characteristics of PVL-positive CA-MRSA strains isolated in Madrid, Spain, and analyze the clinical features of patients infected with these isolates. From 2004 to 2007, we collected 13 PVL-positive MRSA isolates from patients attending to the emergency department. The isolates were genotyped by pulsed-field gel electrophoresis, SCCmec typing, agr polymorphism, and multilocus sequence typing. Susceptibility to 29 antimicrobials was determined by the broth microdilution and by the E-test methods. The isolates belonged to 3 genotypes: ST8-SCCmec IVc (n = 11), ST5-SCCmec IVa (n = 1), and ST80-SCCmec IVc (n = 1). The corresponding agr types were I, II, and III, respectively. Five isolates were resistant to tetracycline and doxycycline, and 1 was resistant to fusidic acid (ST80). The isolates were from children (n = 9) and adults (n = 4), and were associated with skin and soft tissue infections (n = 9), otitis (n = 1), and bacteremia (n = 1). Nine patients were from South America. Our results indicate the transcontinental importation and recent emergence in Spain of PVL-positive CA-MRSA strains belonging to 3 distinct lineages, including 1 predominant (ST8-SCCmec IVc).

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) isolates in Spain have increased dramatically; in 1986 there were only 1.2% MRSA amongst all nosocomial Staphylococcus aureus (SA) isolates, by 1989 this percentage had risen to 44% in some hospital causing a very serious epidemic situation in the country. We have characterized these isolates by direct, reverse and Fisk phage typing and we have also looked for an additional local set of phages to help us to differentiate these strains. We have been able to differentiate an epidemic strain from other MRSA strains which cause sporadic hospital outbreaks, and we have also distinguished between some variants of the epidemic strain. This research has been supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS), Ministerio de Sanidad y Consumo, Spain; No. 93/0144. Sí