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Background: There is limited clinical or epidemiological knowledge regarding Bartonella infection in cats, and no serological studies have compared the presence of antibodies against different Bartonella species. Moreover, there are limited feline Bartonella studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate Bartonella spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors., Methods: We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quintana-, and Bartonella koehlerae-specific antibodies using endpoint in-house immunofluorescence antibody assays. Bartonella real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms (Anaplasma, Babesia, Cytauxzoon, Ehrlichia, Hepatozoon, hemotropic Mycoplasma, and Theileria spp.)., Results: From 135 cats studied, 80.7% were seroreactive against at least one Bartonella species. Bartonella quintana, B. koehlerae, and B. henselae seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three Bartonella species. Colony cats were more likely to be Bartonella spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be Bartonella spp.-seroreactive. Bartonella spp. DNA was detected in the blood of 11.9% (n = 16) of cats. Cats were infected with B. henselae (n = 12), B. clarridgeiae (n = 3), and B. koehlerae (n = 1). Mycoplasma spp. DNA was amplified from 14% (n = 19) of cat blood specimens. Cats were infected with Mycoplasma haemofelis (n = 8), Candidatus M. haemominutum (n = 6), Candidatus Mycoplasma turicensis (n = 4), and Mycoplasma wenyonii (n = 1). Anaplasma, Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria spp. DNA was not amplified from any blood sample. Of the 16 Bartonella spp.-infected cats based on PCR results, six (37%) were co-infected with Mycoplasma spp., Conclusions: Bartonella spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with Anaplasma spp., Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria infections were not detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowledge, this study is the first to document M. wenyonii is infection in cats., (© 2021. The Author(s).)

The purpose of this study was to increase our knowledge about Mycobacterium africanum and report the incidence and characteristics of tuberculosis (TB) due to their lineages in Aragon, Spain, over the period 2003-2019. The study includes all the cases in our region, where all the M. tuberculosis complex isolates are systematically characterised. We detected 31 cases of M. africanum among 2598 cases of TB in the period studied. TB caused by M. africanum is rare (1.19%) in our population, and it affects mainly men of economically productive age coming from West African countries. Among the isolates, Lineage (L) 6 was more frequent than L5. The genotyping of these strains identified five clusters and 13 strains with a unique pattern. The isolates' characterisation identified a copy of IS6110 within the moaX gene, which turned out to be specific for L6. It will allow the differentiation of this lineage from the rest of MTBC with a simple PCR reaction. It remains to be established whether this polymorphism may limit M. africanum transmission. Furthermore, a mutation in the mutT2 promoter was found as specific for L6 strains, which could be related to the high variability found for L6 compared to L5.

Introduction: Borrelia miyamotoi is a tick-borne pathogen belonging to the relapsing fever group. It had not been reported from Spain, but its wide distribution and the presence of the tick-vector (Ixodes ricinus) made us suspect its circulation. The aim of this study was to investigate the presence of Borrelia spp. in I. ricinus in Spain., Methods: A total of 652 I. ricinus nymphs collected in northern Spain were processed. The DNA was extracted using incubations with ammonium hydroxide. Borrelia spp. DNA was amplified using Borrelia-specific PCR assays (glpQ, 16S rRNA and flagellin genes)., Results: B. miyamotoi was amplified in 4 specimens, and Borrelia burgdorferi sensu lato in 27 (8 Borrelia afzelii, 7 Borrelia garinii, 8 Borrelia lusitaniae, 3 Borrelia valaisiana and 1 B. burgdorferi sensu stricto)., Conclusion: B. miyamotoi should be considered in the differential diagnoses of patients with confirmed or suspected tick-bite in Spanish endemic areas for Lyme disease., (Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

The solar salterns located in the Odiel marshlands, in southwest Spain, are an excellent example of a hypersaline environment inhabited by microbial populations specialized in thriving under conditions of high salinity, which remains poorly explored. Traditional culture-dependent taxonomic studies have usually under-estimated the biodiversity in saline environments due to the difficulties that many of these species have to grow at laboratory conditions. Here we compare two molecular methods to profile the microbial population present in the Odiel saltern hypersaline water ponds (33% salinity). On the one hand, the construction and characterization of two clone PCR amplified-16S rRNA libraries, and on the other, a high throughput 16S rRNA sequencing approach based on the Illumina MiSeq platform. The results reveal that both methods are comparable for the estimation of major genera, although massive sequencing provides more information about the less abundant ones. The obtained data indicate that Salinibacter ruber is the most abundant genus, followed by the archaea genera, Halorubrum and Haloquadratum . However, more than 100 additional species can be detected by Next Generation Sequencing (NGS). In addition, a preliminary study to test the biotechnological applications of this microbial population, based on its ability to produce and excrete haloenzymes, is shown.

Purpose: Acanthamoeba keratitis (AK) is a painful and potentially blinding corneal infection caused by Acanthamoeba spp. In Madrid, environmental studies have demonstrated a high presence of these free-living amoebae in tap water. Since most of AK cases occur in contact lenses (CL) wearers with inadequate hygiene habits, the presence of Acanthamoeba in discarded CL has been studied and compared with other common etiological agents of keratitis, such as Pseudomonas aeruginosa and Staphylococcus aureus., Methods: One hundred and seventy-seven healthy individuals from Madrid contributed their discarded CL and answered a questionnaire on hygiene habits. DNA was extracted from the CL solution and analyzed by real-time PCR for Acanthamoeba, Pseudomonas aeruginosa and Staphylococcus aureus. These CL and their solutions were also cultured on non-nutrient agar to isolate Acanthamoeba., Results: Among the 177 samples, Acanthamoeba DNA was detected in 87 (49.2%), P. aeruginosa DNA in 14 (7.9%) and S. aureus DNA in 19 (10.7%). Cultivable amoebae, however, were observed in only one sample (0.6%). This isolate was genotyped as T4. The habits reported by this CL owner included some recognized risk factors for AK, but in this study only the practice of "not cleaning the CL case" presented some statistical significant association with Acanthamoeba DNA presence. Detection of the investigated bacterial DNA did not demonstrate statistical significant association with the studied practices, but the presence of P. aeruginosa revealed a possible inhibition of Acanthamoeba in these samples., Conclusions: The PCR results suggest a high presence of Acanthamoeba spp. in healthy CL wearers from Madrid, but we can assume that CL solutions are properly disinfecting the CL since only 1.1% of the positive PCR samples correspond to viable amoebae and, after four years, only one participant reported stronger ocular problems. Nevertheless, more studies are necessary to corroborate this hypothesis.

Two gram-negative, facultatively anaerobic, motile and rod-shaped bacteria, strains P2G1(T) and P1A11, were isolated from the Ter River in Ripoll, Spain. Phylogenetic analysis based on 16S rRNA gene sequences showed that both strains are closely related to each other and that their closest relatives were Aeromonas media ATCC 33907(T) (99.4%) and Aeromonas hydrophila ATCC 7966(T) (99.3%). Multilocus sequence analysis (MLSA) based on the partial sequences of gyrA, gyrB, rpoD, recA, and dnaJ genes suggested that these two strains represent a novel species that clustered with A. media ATCC 33907(T). This was further supported by DNA-DNA hybridization analysis between P2G1(T) and A. media LMG 9073(T). Phenotypic features also allowed their differentiation from closely related species. These two strains should, therefore, be considered to represent a novel species within the genus Aeromonas, for which the name Aeromonas rivipollensis sp. nov. is proposed., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An automated nucleic acid amplification assay that simultaneously identifies Mycobacterium tuberculosis and rifampicin resistance, the Xpert® MTB/RIF test, has undergone extensive evaluation in sputum samples. Our aim was to define its diagnostic accuracy when performed on pleural fluid specimens. In 67 patients with pleural effusions, of whom half had tuberculous pleuritis, Xpert yielded 15% sensitivity and 100% specificity in the detection of tuberculosis (TB). Positive Xpert results tended to be more common in patients with microbiologically confirmed TB. Due to its low sensitivity, Xpert testing of pleural fluids has a limited role in the work-up of pleural effusions.

Background: Presence of intestinal microbes is a prerequisite for the development of ulcerative colitis (UC), although deviation of the normal intestinal microbiota composition, dysbiosis, is presumably implicated in the etiology of UC., Methods: The fecal microbiota of 30 UC samples obtained from 15 patients who were sampled twice and from 15 healthy control subjects originating from 2 geographic locations was analyzed using highly reproducible phylogenetic microarray that has the capacity for detection and quantification of more than 1000 intestinal bacteria in a wide dynamic range., Results: The fecal microbiota composition is not significantly influenced by geographic location, age, or gender, but it differs significantly between the patients with UC and the control subjects (P = 0.0004). UC-associated microbiota is stable during remission and similar among all patients with UC. Significant reduction of bacterial diversity of members of the Clostridium cluster IV and significant reduction in the abundance of bacteria involved in butyrate and propionate metabolism, including Ruminococcus bromii et rel. Eubacterium rectale et rel., Roseburia sp., and Akkermansia sp. are markers of dysbiosis in UC. Increased abundance of (opportunistic) pathogens including Fusobacterium sp., Peptostreptococcus sp., Helicobacter sp., and Campylobacter sp. as well as Clostridium difficile were found to be associated with UC., Conclusions: Dysbiosis in UC is stable in time and shared between patients from different geographic locations. The microbial alterations offer a mechanistic insight into the pathogenesis of the disease.

Benthic microbial biofilms attached to rocks (epilithic) are major sites of carbon cycling and can dominate ecosystem primary production in oligotrophic lakes. We studied the bacterial community composition of littoral epilithic biofilms in five connected oligotrophic high mountain lakes located at different altitudes by genetic fingerprinting and clone libraries of the 16S rRNA gene. Different intra-lake samples were analyzed, and consistent changes in community structure (chlorophyll a and organic matter contents, and bacterial community composition) were observed along the altitudinal gradient, particularly related with the location of the lake above or below the treeline. Epilithic biofilm genetic fingerprints were both more diverse among lakes than within lakes and significantly different between montane (below the tree line) and alpine lakes (above the tree line). The genetic richness in the epilithic biofilm was much higher than in the plankton of the same lacustrine area studied in previous works, with significantly idiosyncratic phylogenetic composition (specifically distinct from lake plankton or mountain soils). Data suggest the coexistence of aerobic, anaerobic, phototrophic, and chemotrophic microorganisms in the biofilm, Bacteroidetes and Cyanobacteria being the most important bacterial taxa, followed by Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Chlorobi, Planctomycetes, and Verrucomicrobia. The degree of novelty was especially high for epilithic Bacteroidetes, and up to 50 % of the sequences formed monophyletic clusters distantly related to any previously reported sequence. More than 35 % of the total sequences matched at <95 % identity to any previously reported 16S rRNA gene, indicating that alpine epilithic biofilms are unexplored habitats that contain a substantial degree of novelty within a short geographical distance. Further research is needed to determine whether these communities are involved in more biogeochemical pathways than previously thought.

Aim: To determine whether intestinal Staphylococcus spp. and their pathogenic features differed between coeliac disease (CD) patients and healthy controls., Methods: 60 children, including active CD (n=20) and non-active CD (n=20) patients and healthy controls (n=20), were studied. Staphylococci were isolated from faeces and identified by PCR and DNA sequencing. The carriage of virulent genes, including adhesion (atlE and fbe), cell aggregation (icaD), global regulatory (agr and sar) and methicillin-resistant (mecA) genes, was analysed by PCR., Results: Staphylococcus epidermidis was more abundant in the microbiota of active and non-active CD patients than in controls. Staphylococcus haemolyticus was more abundant in active CD patients than in control subjects. Staphylococcus aureus was less abundant in active CD patients than in the other child groups. Staphylococcus spp. diversity was higher in active CD patients than in non-active CD patients and controls. The presence of the mecA gene and the simultaneous presence of both the mecA and atlE genes were higher in S. epidermidis clones isolated from CD patients, with active and non-active disease, than in those from control subjects. The individual presence of the other virulent genes was lower in S. epidermidis from active CD patients than in those from the other -child- groups., Conclusions: Increased abundance of S. epidermidis carrying the mecA gene, in active and non-active CD patients, most likely reflects increased exposure of these subjects to opportunistic pathogens and antimicrobials.

In order to study the microbial caecal ecosystem of wild and domestic rabbits through the fermentation characteristics and concentration and diversity of bacterial and archaeal communities, caecal samples from sixteen wild rabbits (WR) were contrasted with two groups (n = 4) of farm rabbits receiving low (LSF) or high (HSF) soluble fibre diets from 28 (weaning) to 51 days of age. DNA was extracted for quantifying bacteria and Archaea by qPCR and for biodiversity analysis of microbial communities by DGGE. Samples from WR had lower caecal pH and ammonia and higher volatile fatty acids concentration than farm animals. Lower acetate and higher butyrate proportions were detected in WR. Bacterial and archaeal DGGE profiles were clearly different between wild and farm rabbits, and diet-affected population of farm rabbits. Similarity index of bacteria was lower than 0.40 among WR, and 0.52 among farm rabbits. In conclusion, caecal fermentation characteristics differ between wild and farm rabbits, which harbour clearly different bacterial and archaeal communities. In farm rabbits, diversity is influenced by the dietary level of soluble fibre., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) continues to be an important public health problem. Currently, 2,100 million people--one third of the world population--are infected by Mycobacterium tuberculosis, with an estimated annual rate of 9.4 million new cases, and 440,000 cases of multidrug-resistant (MDR) TB in 2008; furthermore, cases of extensively-resistant (XDR) TB have been detected in 57 countries. While TB cases are constantly declining in industrialized countries, the rates and mortality due to this infection in developing countries remain alarming and will continue to be so in the future. Although the priorities in these countries are at present simpler, methods allowing rapid diagnosis of TB and of resistant strains will obviously contribute to better control of the disease. Nucleic acid amplification techniques allow M. tuberculosis detection in clinical samples in a few hours, while liquid media cultures may yield positive results in only 2 to 4 weeks, half the time that is usually required for growth in conventional solid media, which also allows more rapid determination of drug susceptibilities. Similarly, based on molecular biology, several approaches may rapidly identify gene mutations associated with resistance to antituberculosis drugs in clinical samples. Finally, the main obstacle to treatment adherence among patients--its length--could be minimized in the future if the new combinations of drugs currently under investigation, and some promising new vaccines, confirm similar rates of efficacy to those used at present., (Copyright © 2011 Sociedad Española de Neumología y Cirugía Torácica. Published by Elsevier Espana. All rights reserved.)

Study Design: Case-control study for assessing a diagnostic test., Objective: The aim of this study was to analyze the diagnostic yield of a multiplex real-time polymerase chain reaction (PCR) assay in the differential diagnosis of tuberculous vertebral osteomyelitis (TVO) and brucellar vertebral osteomyelitis (BVO)., Summary of Background Data: Vertebral osteomyelitis (VO) is one of commonest osteoarticular complications of tuberculosis and brucellosis. However, the very similar clinical, radiologic, and histologic characteristics of these entities mean that diagnosis requires etiological confirmation, but conventional microbiologic methods have important limitations., Methods: Fifteen vertebral samples from patients with TVO or BVO and 9 from pyogenic and nontuberculous mycobacteria VO were studied by multiplex PCR and conventional microbiologic techniques. To identify Brucella DNA, we used a fragment of 207 bp from the conserved region of the gene coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and for Mycobacterium tuberculosis complex, a fragment of 164 bp from the intergenic region SenX3-RegX3., Results: The histopathologic findings were inconclusive in 4 of 14 cases (28.6%) with TVO or BVO and cultures were positive in 11 of 15 cases (73.3%). Multiplex PCR correctly identified 14 of the 15 samples from patients with TVO and BVO and was negative in all the control samples. Thus, the overall sensitivity and specificity of the multiplex PCR were 93.3% and 90%, respectively, with an accuracy of 92% (95% CI, 81.4%-100%)., Conclusion: These results suggest that multiplex real-time PCR is far more sensitive than conventional cultures, and this, together with its speed, makes this technique a very practical approach for the differential diagnosis between TVO and BVO.

A sample of 279 European wild rabbits, Oryctolagus cuniculus (141 males, 138 females), captured alive in Andalusia (Spain) and belonging to the two haplotype classes previously described for this species (230 and 49 corresponding with haplotypes A and B, respectively), were tested for the presence of Bartonella alsatica DNA. Two species-specific nested polymerase chain reaction assays targeting for 16S-23S rRNA intergenic spacer region and RNA polymerase β subunit genes have been developed. Forty-eight (17.20%) rabbits were infected with B. alsatica. Two-way contingency table analyses and the calculation of Cramer's V statistic showed no differences in infection rate, considering haplotype lineage or sex. The risk of infection of human population, especially for hunters in close contact with this demonstrated human pathogen, should be considered.

Limited information on the presence of bacterial and hematozoan infections in parasitic arthropods from Spain is available. In an attempt to address this issue, the prevalence of Theileria, Babesia, Hepatozoon, and Rickettsia species was investigated by polymerase chain reaction plus sequencing. In a survey for zoonotic pathogens in ectoparasites, 42 wild animals (which included rodents, carnivores, Sciuridae, and Cervidae) were captured in Burgos (Spain). A total of 256 arthropods (including 107 ticks, 76 fleas, and 73 mites) were collected from these mammals. Molecular diagnostic results showed that (i) Rickettsia felis was found in fleas (two Ctenocephalides felis), (ii) Hepatozoon sp. infected some fleas (two Ctenophtalmus sp. and a DNA pool of Ceratophyllus sciurorum) and Acari (one Neotrombicula sp.), and (iii) Theileria annae was found in Ixodes ricinus and I. hexagonus (each a single infected specimen). All microorganisms and parasites were genetically identical to pathogens already described in Spain or elsewhere. Infected arthropods were recovered from beech marten, bank vole, squirrel, wood mouse, and red fox. Our findings emphasize the potential risk for transmission of rickettsias to humans (namely, R. felis) in Burgos, since C. felis is capable to seek out humans for feeding. No hemoprotozoa with proven significance as human pathogens were found in the survey. However, finding T. annae in ticks recovered from wild canids suggests possible links of sylvatic and domestic cycles for some Piroplasmida.

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes was used to characterize bacterial populations in the surface soil of a commercial pear orchard consisting of different pear cultivars during two consecutive growing seasons. Pyrus communis L. cvs Blanquilla, Conference, and Williams are among the most widely cultivated cultivars in Europe and account for the majority of pear production in Northeastern Spain. To assess the heterogeneity of the community structure in response to environmental variables and tree phenology, bacterial populations were examined using PCR-denaturing gradient gel electrophoresis (DGGE) followed by cluster analysis of the 16S ribosomal DNA profiles by means of the unweighted pair group method with arithmetic means. Similarity analysis of the band patterns failed to identify characteristic fingerprints associated with the pear cultivars. Both environmentally and biologically based principal-component analyses showed that the microbial communities changed significantly throughout the year depending on temperature and, to a lesser extent, on tree phenology and rainfall. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant bacterial populations. Most DGGE band sequences were related to bacterial phyla, such as Bacteroidetes, Cyanobacteria, Acidobacteria, Proteobacteria, Nitrospirae, and Gemmatimonadetes, previously associated with typical agronomic crop environments.

There are evidences that wild boar and domestic pig populations share the vulnerability to certain pathogens and, in consequence, the risk of pathogen transmission. Based on serological evidences, it is known that Mycoplasma hyopneumoniae (Mhyo) is able to infect the wild boar. However, if this infection causes lesions and disease in this species is still unknown. The objectives of the present study were to assess the seroprevalence to Mhyo, as well as the rates of detection of Mhyo (by nested polymerase chain reaction, nPCR) in upper and lower respiratory tract of the wild boar. Moreover, presence of enzootic pneumonia (EP)-like gross and microscopic lesions in these animals was also assessed. Antibodies against Mhyo were detected in 92 out of 428 (21%) serum samples tested. Moreover, Mhyo DNA was detected by nPCR in 17 out of 85 (20%) nasal swabs and in 12 out of 156 (8%) lung samples. No gross EP-like lesions were observed in any of the studied pigs. Besides, presence of EP-like microscopic lung lesions was observed in 18 out of 63 (29%) animals. Mhyo DNA was detected at lung level in 2 out of these 18 (11%) animals. Presence of EP-like microscopic lesions was significantly related (p<0.05) with Mhyo detection at nasal swab swab but not (p>0.05) with its detection in lung samples; such lesions were also linked (p<0.05) with presence of Metastrongylus spp.-like parasite structures. Results of the present study confirm that Mhyo is able to infect and might be able to cause EP-like microscopic lesions in wild boar but, apparently, with a subclinical impact., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDSPAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

The conservation of paleolithic paintings such as those in Altamira Cave (Spain) is a primary objective. Recent molecular studies have shown the existence of unknown microbial communities in this cave including anaerobic microorganisms on cave walls. Herein, we analyzed an anaerobic microbial group, the sulfate-reducing bacteria (SRB), from Altamira Cave with potential negative effects on painting conservation. In the present work, the communities of bacteria and SRB were studied through PCR-DGGE analysis. Data suggest that SRB communities represent a significant, highly diverse bacterial group in Altamira Cave. These findings represent a first report on this physiological group on caves with paleolithic paintings and their potential biodegradation consequences. Expanding our knowledge on microbial communities in Altamira Cave is a priority to design appropriate conservation strategies.

A total of 691 questing adult ixodid ticks of the genera Ixodes, Haemaphysalis, Dermacentor, and Rhipicephalus were tested by polymerase chain reaction (PCR) and reverse line blot (RLB) for the presence of Anaplasma phagocytophilum, Coxiella burnetii, Borrelia spp., and spotted fever group (SFG) rickettsiae. Ticks were collected by blanket dragging during 2 sampling years (2003-2005) in 10 recreational areas in the Basque Country (Northern Spain). Adult ticks were collected every month of the year and eight different species were identified among which Ixodes ricinus was the most abundant and widespread. Three pathogens for humans, Borrelia burgdorferi, A. phagocytophilum, and C. burnetii, as well as rickettsiae of unknown pathogenicity were detected. The latter were identified as Rickettsia sp. RpA4/DnS14 by sequencing of the citrate synthase (gltA) gene. The infection rates varied from 0.1%-6.9%. DNA of A. phagocytophilum was detected mainly in I. ricinus, but also in Haemaphysalis punctata, H. concinna, and Rhipicephalus bursa. Coxiella burnetii was detected in only one specimen of H. punctata, and Borrelia spp. in eight ticks. Furthermore, PCR-RLB analysis specific for B. burgdorferi sensu lato detected one H. punctata with positive hybridization with the B. burgdorferi sensu stricto probe, and two I. ricinus positive for B. afzelii and B. garinii. SFG rickettsiae were the pathogens most frequently found, present in 48 of 97 D. reticulatus analyzed. Mixed infections were not found in any of the analyzed ticks. These results are compared and discussed with data obtained in previous studies carried out in the same and other regions.

In an attempt to know the potential risk of human disease after exposure to ticks in La Rioja (North of Spain), the objective of our study was to investigate the presence of Rickettsia species in Haemaphysalis ticks collected in our area. A total of 177 Haemaphysalis spp. belonging to three species (H. punctata, H. sulcata, and H. inermis) were subjected to DNA extraction and tested by polymerase chain reaction (PCR) targeting three rickettsial genes: gltA, ompB, and ompA. Six (3 H. inermis, 2 H. punctata, and 1 H. sulcata) of the 177 specimens were found to be infected (3.4%). The rickettsiae in H. inermis ticks (n = 3) were identified as Rickettsia aeschlimannii by sequencing of the genes coding for gltA, ompB, and ompA. Nucleotide sequences from H. punctata and H. sulcata samples that yielded PCR products (n = 3), showed >99% similarity with sequences of Rickettsia endosymbiont of H. sulcata and 'Candidatus Rickettsia hoogstraalii' for gltA and ompB genes, respectively. Attempts to amplify ompA from these two H. punctata and one H. sulcata failed. This study suggests that H. inermis could be a vector for tick-borne spotted fever caused by R. aeschlimannii in the north of Spain. Further studies on characterization and culture of rickettsial endosymbionts found in Haemaphysalis spp. should be performed.

Rodents (87 Mus spretus and 10 Rattus norvegicus) collected in a suburban area of Seville (Andalusia, Spain) between September 2003 and April 2004 were tested by polymerase chain reaction for the presence of Bartonella DNA using primers amplifying a fragment of the 16S/23S rRNA internal transcribed spacer (ITS). Sequence analyses of the ITS of these Bartonella demonstrated that rodents in this region harbored at least three strains: two Bartonella genotypes detected in M. spretus, which clustered separately, and one genotype corresponding with B. tribocorum in R. norvegicus. The finding of Bartonella species in a high proportion of the rodent samples suggests the need to investigate whether these agents might be responsible for cases of febrile illnesses of unknown etiology in Southern Spain.

The aim of the present study was to characterize and identify vibrios isolated from cultured clams in Galicia (NW Spain). A total of 759 isolates were obtained, phenotypically characterized, grouped and assigned to the genus Vibrio. Subsequently, the genomic diversity of 145 representative strains was analyzed by means of amplified fragment length polymorphism (AFLP), which revealed a high genetic diversity amongst these isolates. Only 57 out of 145 strains could be identified to the species level, and they were distributed in 13 AFLP clusters. V. cyclitrophicus, V. splendidus and V. alginolyticus were the most abundantly represented species. Eighty-eight isolates remained unidentified, 59 were distributed over 16 clusters, while 29 were unclustered. Sequencing of the 16S rRNA and two house-keeping genes (rpoA and recA) from representative strains belonging to eight unidentified clusters with the highest number of isolates confirmed their assignation to the Vibrionaceae family, and some of these probably represent new species within the genus. The present study confirmed that the phenotypic characterization of vibrios is not sufficient to identify them at the species level. A wide diversity of vibrios was found in cultured clams from all four geographic locations analyzed. In total, more than 12 Vibrio species and at least three potential new species in this genus were identified.

Objective: To describe the epidemiologic and clinical aspects of patients with tick-borne lymphadenopathy (TIBOLA)., Patients and Methods: The study included all patients treated in our hospital who presented characteristic clinical signs of TIBOLA consisting in the presence of an inoculation lesion after a tick bite with regional lymphadenopathies. The study was carried out at the "Corporació Parc Taulí" hospital in the city of Sabadell, Barcelona, Spain. The inclusion period was from April 2000 to April 2006. Serology tests were performed for Rickettsia conorii, R. slovaca, and Borrelia burgdorferi. The presence of Rickettsia was assessed by means of polymerase chain reaction (PCR)-restriction fragment length polymorphism (PCR-RFLP) and linear-after-the exponential-PCR in blood, skin biopsy, and ticks from the patients., Results: Thirty-six patients fulfilled the inclusion criteria. Twenty-eight patients were children. In most cases, infection occurred between the months of October and April. In 34 patients the tick bite was on the scalp. A necrotic eschar surrounded by a perilesional erythematous halo was observed in 29 cases. All patients had painful regional lymphadenopathies. Serology for R. conorii was positive in 8 cases and for R. slovaca in 2. PCR was negative. The tick, identified as Dermacentor marginatus, was studied in 7 patients. Four of the 7 D. marginatus were positive for sequences compatible with R. slovoca as determined by RFLP or DNA sequencing. Thirty-three patients received antibiotic treatment. Progress was satisfactory in all cases., Conclusions: TIBOLA is present in Catalonia (Northeastern Spain) mainly in children. Although clinical and epidemiologic manifestations are very specific microbiologic confirmation is difficult.

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.

Volcanic eruptions originated the Canary Islands (Spain), and today, different landscapes clearly show that active volcanism. The microbial communities present in these volcanic environments, both at high (above 70 degrees C) and moderate (25-35 degrees C) temperatures, were analyzed. Rock samples were processed using molecular techniques based on both DNA and RNA and complemented with the culture of microorganisms at a wide range of temperatures. Microorganisms detected through RNA-based molecular surveys were mostly related to mesophilic microorganisms, while representatives of hyperthermophiles were not detected. RNA decay curves at 80 degrees C resulted in half-life estimates of less than 2 min for mesophilic microorganisms, supporting a recent and continuous arrival of microorganisms to the high temperature sampled sites. This study constitutes a unique experimental approach showing a great natural potential of microorganisms to spread out to different natural environments. The implications are of great interest for the biogeography, dispersal, and potential distribution of microorganisms, and their genetic information, in natural environments.

The prokaryotic diversity of macroscopic filaments located at the water surface in an extreme acidic environment, Río Tinto (SW, Spain), has been analysed through denaturing gradient gel electrophoresis (DGGE), cloning of 16S rRNA genes and fluorescence in situ hybridization (FISH). The dominant species present in the macrofilaments were Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans and Acidiphilium spp., which represented the three main bacterial genera found in the water column of the river. However, our results also showed the presence of other microorganisms not previously detected in the Río Tinto. Within the Gammaproteobacteria class, sequences closely related to the iron-oxidizing bacteria WJ2 and DSM 2392 were found, as well as sequences related to the non-acidophilic genera Aeromonas and Acinetobacter. In addition, two other new phylotypes related to Gram-positive species from the genera Desulfosporosinus, Clostridium and Mycobacterium were identified. The presence of these anaerobic microorganisms strongly suggests that the filaments could originate in the deeper parts of the river as a typical acid streamer attached to the rocks or the sediments and be pulled up towards the surface when they reach certain buoyancy.

Background: Streptococcus agalactiae remains susceptible to penicillin; nevertheless, an increase in the resistance to other antimicrobial families, such as macrolides and more rarely fluoroquinolones, has been described., Methods and Results: From 2003 to 2004, two fluoroquinolone-resistant S. agalactiae strains were detected. The strains presented one mutation in parC (Ser79 --> Phe) and an additional mutation in gyrA (Glu85 --> Ala or Glu85 --> Lys)., Conclusion: This study describes the first S. agalactiae strains highly resistant to fluoroquinolones in Spain.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes several pathogens such as A. marginale and A. phagocytophilum that have an impact on veterinary and human health. In this study, we characterized A. marginale and A. phagocytophilum infections in humans, wild and domestic animals, and ticks in southern Europe (particularly in south-central Spain and in Sicily) by means of serologic study, PCR, and sequence analysis of major surface proteins (msp) 1alpha and 4 and 16S rDNA. The results suggest that A. marginale infections in this region are maintained in cattle and deer, with ticks and tabanids serving as biological and mechanical vectors of the pathogen, respectively. Infections with A. phagocytophilum may occur in humans and are maintained in cattle, donkeys, deer, and birds and are most likely transmitted by several tick species with as yet an unknown role as reservoir hosts for other wild and domesticated mammals. The presence of concurrent infections in cattle and deer suggests that these pathogens may multiply in the same reservoir host and illustrates the complexity of the epidemiology of bovine and human anaplasmosis in this region.

Strain F-32T, which produces exopolysaccharides and contains polyunsaturated fatty acids, was isolated from a hypersaline water sample collected from Fuente de Piedra (southern Spain). Phylogenetic analyses indicated conclusively that the strain in question belonged to the genus Alteromonas. Phenotypic tests showed that it could be assigned to the genus Alteromonas although it had a number of distinctive characteristics: it is moderately halophilic, growing best with 7.5-10 % w/v NaCl; it grows at 4 degrees C and produces H2S; it does not grow with d-cellobiose, d-fructose, d-galactose, d-glucose or lactose as sole sources of carbon and energy; and its fatty-acid profile is typical of Alteromonas but it also contains a large amount of an unusual acid with three double bonds [18 : 3omega6c (6, 9, 12); 5.01 %, w/v]. The major isoprenoid quinone is Q8. The DNA G+C content is 46.3 mol%. The phylogenetic, phenotypic and genetic properties of strain F-32T place it within a novel species, for which the name Alteromonas hispanica sp. nov. is proposed. The type strain is F-32T (=CECT 7067T=LMG 22958T).

Two Gram-positive, rod-shaped, endospore-forming bacteria (strains CR-119(T) and CR-95(T)) were isolated from brackish sediments in the mouth of the river Velez in Malaga, southern Spain, and subjected to a polyphasic taxonomic study. Phenotypic tests showed that these strains were related to other Bacillus species at a similarity level of less than 87.6 %. Both strains are halotolerant, aerobic, chemoheterotrophic, motile with peritrichous flagella and biosurfactant producers. Their endospores are oval, subterminal and non-deforming structures. The predominant menaquinone in both strains is MK-7. The fatty-acid profiles of both strains contain large quantities of branched and saturated fatty acids. The major fatty acids (%) are 15 : 0 anteiso (32.4), 15 : 0 iso (16.8), 17 : 0 iso (13.4), 16 : 0 (11.5) and 17 : 0 anteiso (10.2) in strain CR-119(T) and 15 : 0 anteiso (37.5), 17 : 0 iso (16.0) and 17 : 0 anteiso (15.8) in strain CR-95(T). The G + C contents of strains CR-119(T) and CR-95(T) are 41.0 and 42.5 mol%, respectively. RAPD analysis confirmed the low degree of similarity between the two strains and also amongst other Bacillus species. 16S rRNA gene analysis of strain CR-119(T) showed the highest sequence similarity to be 97.4 %, with Bacillus mojavensis and Bacillus subtilis subsp. spizizenii. In the case of strain CR-95(T), the maximum similarity value was 99.5 %, with B. mojavensis. DNA-DNA hybridization of strains CR-119(T) and CR-95(T) with the above species produced values lower than 46.9 %. Therefore, on the basis of phenotypic characteristics, phylogenetic data and genomic distinctiveness, we conclude that these Bacillus strains merit classification as novel species, for which we propose the names Bacillus axarquiensis sp. nov. (type strain CR-119(T) = CECT 5688(T) = LMG 22476(T)) and Bacillus malacitensis sp. nov. (type strain CR-95(T) = CECT 5687(T) = LMG 22477(T)).

The aim of the present research was to isolate and identify bacteria from spent nuclear fuel pools of a Spanish nuclear power plant. Water samples were collected and inoculated onto different culture media to isolate the highest number of species. 16S rDNA fragments from colonies growing on solid media were amplified and analysed by denaturing gradient gel electrophoresis. Sequencing revealed the presence of 21 different bacteria belonging to several phylogenetic groups (alpha, beta, and gamma-Proteobacteria, Actinomycetales, Flavobacterium, and the Bacillus/Staphylococcus group). The isolation of these microorganisms in this particular environment (oligotrophic and radioactive) is highly interesting because of the possibility of their being used for the bioremediation of radionuclide-contaminated waters.

Two xylan-degrading bacterial strains were isolated from a decayed Ulmus nigra tree in Spain. The isolates were Gram-positive, non-motile, aerobic and formed substrate mycelium which fragmented into irregular rods. 16S rRNA gene sequence analysis indicated that the isolates form a separate branch within the genus Agromyces phylogenetic cluster, with Agromyces mediolanus DSM 20152(T) being their closest relative (97.7 and 97.6 % sequence similarity). Catalase, nitrate reduction and urease tests differentiated these strains from A. mediolanus. Cell-wall peptidoglycan composition, major menaquinone, predominant fatty acids and phospholipid pattern were typical of the genus Agromyces. The DNA G+C content determined for the type strain XIL01(T) was 72 mol%. Based on the data presented, a novel species Agromyces ulmi sp. nov. is proposed. The type strain is XIL01(T) (=LMG 21954(T)=DSM 15747(T)).

A new Vibrio species, Vibrio ponticus, is proposed to accommodate four marine bacteria isolated from sea water, mussels and diseased sea bream (Sparus aurata), at the Mediterranean coast of Spain. Strains are Gram negative, slightly halophilic bacteria that require Na+ ion for growth, oxidase and catalase positive, negative for arginine dihydrolase and ornithine decarboxylase but positive for lysine decarboxylase and indole, and utilize beta-hydroxybutyrate as a sole carbon source. Phylogenetic analysis locate these marine bacteria in the vicinity of the V. fluvialis-V. furnissii clade, sharing with these two species 16S rDNA sequence similarities slightly above 97% (97.1 and 97.3%, respectively). DNA-DNA hybridisation values confirm that the four strains form a genospecies and represent a new species in the genus Vibrio. We propose strain 369T (CECT 5869T, DSM 16217T) as the type strain.

A group of moderately halophilic bacteria able to degrade aromatic organic compounds contaminating hypersaline habitats in southern Spain have been isolated and characterized. The taxonomic position of these strains was determined using phenotypic, phylogenetic and genotypic methods. The G + C content of their DNA ranged from 61.0 to 62.9 mol%. DNA-DNA hybridization studies showed that they constitute a genospecies, having DNA-DNA hybridization values of 90-100 %. Analysis of the complete 16S rRNA gene sequence revealed a high level of similarity with members of the genus Halomonas, sharing 98 % 16S rRNA gene sequence similarity with the type strains of Halomonas salina and Halomonas halophila. However, phenotypic differences and the low level of DNA-DNA hybridization suggest the placement of these strains as a novel species within the genus Halomonas. The name Halomonas organivorans sp. nov. is proposed, with strain G-16.1T (= CECT 5995T = CCM 7142T) as the type strain. This novel species of Halomonas is characterized by its ability to use a wide range of organic compounds (benzoic acid, p-hydroxybenzoic acid, cinnamic acid, salicylic acid, phenylacetic acid, phenylpropionic acid, phenol, p-coumaric acid, ferulic acid and p-aminosalicylic acid), and it could be useful for the decontamination of polluted saline habitats.

Two bacterial strains, F23T and R22T, have been isolated from hypersaline habitats in Málaga (S. Spain) and Murcia (E. Spain). The novel strains, similar to previously described Idiomarina species, are slightly curved rods, Gram-negative, chemo-organotrophic, strictly aerobic and motile by a single polar flagellum. Both strains produce catalase and oxidase. They hydrolyse aesculin, gelatin, casein, Tween 20, Tween 80 and DNA but not starch or tyrosine. The strains differ from the hitherto described Idiomarina species in their capacity to produce extracellular polysaccharides and their different patterns of carbon sources and antimicrobial susceptibility. They are moderate halophiles capable of growing in NaCl concentrations of 0.5 to 25 % w/v, the optimum being 3-5 % w/v. Cellular fatty acids are predominantly iso-branched. The main fatty acids in strain FP23T are 15 : 0 iso (26.75 %), 16 : 1omega7c (11.33 %) and 16 : 0 (11.73 %) whilst 15 : 0 iso (24.69 %), 17 : 0 iso (12.92 %) and 17 : 1omega9c (11.03 %) are predominant in strain R22T. The DNA G + C composition is 46.0 mol% in strain FP23T and 48.7 mol% in strain R22T. Phylogenetic analyses indicate conclusively that the two strains belong to the genus Idiomarina. DNA-DNA hybridization revealed that they represent novel species. In the light of the polyphasic evidence accumulated in this study, it is proposed that they be classified as novel species of the genus Idiomarina, with the names Idiomarina fontislapidosi sp. nov. (type strain F23T = CECT 5859T = LMG 22169T) and Idiomarina ramblicola sp. nov. (type strain R22T = CECT 5858T = LMG 22170T).

A novel rapidly growing, non-pigmented mycobacterium was isolated from blood samples obtained from 17 patients with febrile syndrome. Bacterial growth occurred at 30 and 37 degrees C on Löwenstein-Jensen medium and also on MacConkey agar without crystal violet. Strains contained alpha- and alpha'-mycolates in their cell wall. Sequence analysis of the hsp65 and 16S rRNA genes identified the isolates as rapidly growing mycobacteria. Sequences of both genes were unique within the mycobacteria. DNA-DNA hybridization showed that the isolates had less than 15 % reassociation with 13 other recognized rapidly growing mycobacteria. The name Mycobacterium canariasense sp. nov. is proposed for this novel opportunistic pathogen, which is most closely related to Mycobacterium diernhoferi. The type strain is 502329T (= CIP 107998T = CCUG 47953T).

We report the detection and characterization of the first vancomycin-resistant VanB-type Enterococcus faecium to be isolated in a Spanish hospital. Sequence analysis of the vanB gene showed that this isolate belonged to subtype vanB2. Moreover, PCR amplification analysis indicated that the vanB gene cluster was linked to a Tn5382-like transposon.

Mastitis is a serious problem for sheep and rabbit farms, Staphylococcus aureus being the main causal agent. Fifty strains of S. aureus isolated from sheep and rabbits from farms located in diverse geographical regions of Spain were studied. Their resistance pattern and plasmid profile was related to the pulsotypes obtained by pulsed-field gel electrophoresis (PFGE). The results showed great heterogeneity in staphylococci isolated from sheep, both in pulse-type and plasmid profile. We found in addition, antibiotic-resistant strains and aminoglycoside-modifying enzyme (AGMEs) producer strains. The genotypes corresponding to staphylococci isolated from rabbits were less heterogeneous, although they also could be subdivided by plasmid profile and resistance patterns. Resistance to antibiotics such as methicillin or AGMEs production could indicate possible human origin of the strains or a possible source of resistant strains for human beings.

Tito Bustillo cave (Ribadesella, Spain) contains valuable Palaeolithic paintings, which date back 15 000-20 000 years. Since 1969, the cave has been open to the public. Rock wall surfaces, spelaeothems and soils are covered by apparent biofilms of phototrophic microorganisms, which develop under artificial lighting. In addition, rock surfaces present conspicuous bacterial growth in the form of round colonies of different colours and about 1-2 mm in diameter. Even the famous Paintings Panel shows some evident microbial growth. In the present study, bacterial communities on the paintings and on the rock surfaces near the paintings were analysed by culture-independent techniques, including polymerase chain reaction (PCR) amplification of bacterial 16S rRNA genes (16S rDNA), phylogenetic sequence analyses and genetic community fingerprinting by denaturing gradient gel electrophoresis (DGGE). DGGE fingerprints showed complex bacterial community patterns. Forty-one clones matching DGGE bands of the community fingerprints were sequenced, representing about 39% of DNA fragments in the DGGE patterns. Phylogenetic sequence analyses revealed a high number of phylogenetically novel 16S rDNA sequence types and a high diversity of putatively chemotrophic and heterotrophic bacteria. Sequences were phylogenetically most closely related to the Proteobacteria (20 clones), green non-sulphur bacteria (three clones), Planctomycetales order (one clone), Cytophaga-Flexibacter- Bacteroides division (one clone) and the Actinobacteria (four clones). Furthermore, we report the presence of members of the Acidobacterium division (12 clones) in a karstic hypogean environment. Members of this phylum have not so far been detected in these particular environments.

Introduction: Chlamydia pneumoniae has relationship with artherosclerosis of carotid artery by seroepidemiological studies and by demonstration of the bacteria in atheromata. We made a case-control study to know the seroprevalence of chronic infection of C. pneumoniae and the presence of the bacteria in arterial biopsies., Patients and Methods: The cases group was constituted by 26 patients undergoing carotid surgery. In the control group there were 50 patients without atherosclerosis and who underwent removal of their varicose veins. There were matched for sex, age and smoking. We obtained serum samples to determinate IgG antibodies against MOMP by MIF and ELISA. In the cases group, we got the arterial biopsies from carotid artery, and from pudendal arteries in control group. We determined chlamydial DNA on the biopsies by heminested PCR (primers: HL-1, HM-1, HR-1). We measured fibrinogen in both groups., Results: By MIF technique, the seroprevalence was (IgG > 1:32) 69.23% cases versus 24% controls (OR: 7.12, CI: 2.47-20.48). The ELISA showed 76.92% of seropositivity in cases versus 16% in controls (OR: 17.5, CI: 95%: 5.35-57.23). The DNA of C. pneumoniae was found in 18 cases and 6 controls, (p < 0.0001, chi 2). We did not find any relationship between fibrinogen levels and groups., Conclusion: We think that there is a relationship between chronic infection with C. pneumoniae and carotid atherosclerosis.

Two rickettsial strains, 16B (previously isolated) and FB1, were isolated from blood from patients with Mediterranean spotted fever in Catalonia, Spain. These are the only 2 human rickettsial isolates of the spotted fever group obtained so far in Spain. These strains were identified by the polymerase chain reaction and sequence analysis of a fragment of the outer membrane protein A (ompA) gene. The partial ompA sequence was found to be 100% identical with that of Rickettsia conorii (Malish 7 strain) for both strains. These results confirm the presence of R. conorii in Catalonia, despite the fact that in a previous study, no R. conorii were isolated, but a new rickettsial strain of the spotted fever group (Bar29) was isolated from dog ticks (Rhipicephalus sanguineus) in Catalonia. Further studies are necessary to get a better knowledge of the epidemiology of rickettsiae in Catalonia.

We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 microg, thereby avoiding false-negative results.

A total of 129 Mycobacterium bovis strains from 5 different Spanish locations were fingerprinted using the IS6110 repetitive element. We demonstrated the presence of multiple copies (from 2 to 13) of IS6110 in a large proportion (47.4%) of the M.bovis strains isolated from cattle and we showed that these strains can be successfully differentiated by means of the RFLP with IS6110. All of the M. bovis strains isolated from goats had multiple copies of IS6110 and 4 bands of 2, 1.7, 1.4 and 1.3 kb were common in all the caprine RFLP patterns. The caprine strains formed a clearly separate cluster from the bovine strains.

Restriction endonuclease analysis (REA) and immunoblotting were used to compare field strains of Mycoplasma mycoides subsp. mycoides biotype Small Colony from different parts of Europe and Africa. Of the 27 strains tested by REA, the European ones (n = 15), except 3 from the Iberian peninsula, differed strikingly from the African ones (n = 10). Vaccinal strains T1-SR and KH3J had a unique REA pattern. Polyclonal immunoblot profiles of 46 strains appeared fairly similar. However most of the Italian strains (22 out of 24) shared a characteristic, in the 100 kDa area, unlike that of the other European strains. Immunoprofiles of the reference strain PG1 and vaccinal strain T1-SR were different from the others. These results allowed the determination of possible links between different geographic region of enzootic contagious bovine pleuropneumonia.

Forty-two methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during 1992-1995 at a hospital in the north of Portugal were characterized by a variety of genomic fingerprints. Hybridization of ClaI and SmaI restriction digests with the mecA- and Tn554-specific DNA probes was used to identify polymorphs and determine their localization in chromosomal DNA preparations, and pulsed-field gel electrophoretic analysis of SmaI digests was used to determine chromosomal backgrounds. A major clone (and its variants) carrying the mecA polymorph I, Tn554 type E in the PFGE background of pattern A, accounted for 85% of all MRSA tested in 1992-1993 and 66% in 1994-1995. This clone is closely related to the epidemic Iberian clone that was associated with outbreaks in Spain during 1989-1993 and was endemic in 1992-1993 in two hospitals in Lisbon (Portugal).