Lucas Domínguez

Mycobacterium tuberculosis subsp. caprae subsp. nov.: A taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain

Isolates from the Mycobacterium tuberculosis complex cultured from caprine pathological tissue samples were biochemically and genetically characterized. The isolates were negative for nitrate reduction and niacin accumulation, they weakly hydrolysed Tween 80, were sensitive to pyrazinamide (50 micrograms ml-1) and were resistant to 1 and 2 micrograms tiophene-2-carboxylic acid hydrazide ml-1 but not to 5 or 10 micrograms tiophene-2-carboxylic acid hydrazide ml-1. Sequencing of the pncA gene revealed a polymorphism characteristic of M. tuberculosis, whereas oxyR, katG and gyrA sequences were characteristic of Mycobacterium bovis. The fingerprinting patterns obtained with IS6110, direct repeats and polymorphic G+C-rich sequence-associated RFLP and direct variable repeat-spacer oligonucelotide typing (spoligotyping) segregated these isolates from the other members of the complex. The results of this testing, together with the repeated association of this micro-organism with goats, suggest that a new member of this taxonomic complex not matching any of the classical species had been identified. This unusual mycobacterium may play a role in the epidemiology of animal and human tuberculosis in Spain. The name Mycobacterium tuberculosis subsp. caprae subsp. nov. is proposed for these isolates. The type strain of Mycobacterium tuberculosis subsp. caprae subsp. nov. is gM-1T (= CIP 105776T).

Bovine Tuberculosis (Mycobacterium bovis) in Wildlife in Spain

ABSTRACT Mycobacterium bovis infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovis through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (Cervus elaphus), fallow deer (Dama dama), wild boar (Sus scrofa), Iberian lynx (Lynx pardina), hare (Lepus europaeus), and cattle (Bos taurus). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovis strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures.

Detection of CMY-2, CTX-M-14, and SHV-12 β-Lactamases in Escherichia coli Fecal-Sample Isolates from Healthy Chickens

ABSTRACT Genes encoding the CMY-2, CTX-M-14, and SHV-12 β-lactamases were detected in three of five Escherichia coli isolates from fecal samples from healthy chickens which showed resistance or diminished susceptibility to extended-spectrum cephalosporins. A −42 mutation at the promoter region of the ampC gene was detected in the other two isolates.

Comparison of enterococcal populations in animals, humans, and the environment - a European study

The objectives of the present study were to generate knowledge of enterococcal populations in the food chain, by studying the population structure (in measures of abundance and diversity) among enterococci in different geographical regions and in different parts of the food chain, as well as the similarities between different enterococcal populations. Altogether, 2868 samples were collected from humans (healthy and hospitalised individuals and clinical isolates), animals (slaughterhouse carcasses and farm animals), and the environment (pig farms, sewage, and surface water) in four European countries-Sweden, Denmark, UK, and Spain. The samples were characterised with regard to presence and numbers of enterococci, and eight (for faecal samples) or 24 (for environmental samples) isolates per sample were phenotyped and preliminarily identified with the PhP-RF system. In total, more than 20,000 isolates were typed. A majority of the samples (77%) showed the presence of presumed enterococci. The diversities of enterococci in environmental samples were generally high, and also faecal samples normally showed presence of more than one enterococcal strain. The most common species found were Enterococcus faecium (33%), E. faecalis (29%), and E. hirae (24%), but different enterococcal populations differed in their species distribution. Clinical isolates, hospitalised patients, and hospital sewage in Sweden showed a clear dominance of E. faecalis (80%, 57%, and 54%, respectively) whereas healthy individuals and urban sewage contained less E. faecalis (39% and 40%, respectively). The species distribution among isolates from slaughterhouses varied between animal species and also between countries, but E. faecalis seemed to be mainly associated with broiler, and E. hirae with cattle and pigs. The results from the study have indicated a simplified method to study the diversity of bacterial populations. Instead of collecting many samples and analysing one or a few isolates per sample, it is possible to collect fewer samples and analyse several isolates per sample. Both approaches yielded similar information on the diversity of the populations. Another useful information was that since samples from hospital sewage, urban sewage, and manure contained enterococcal populations that reflected those in faecal samples of hospitalised patients, healthy humans, and animals, respectively, such samples may be used as pooled faecal samples and may replace cumbersome samplings from many individuals.

Monitoring and Characterization of Extended-Spectrum β-Lactamases in Escherichia coli Strains from Healthy and Sick Animals in Spain in 2003

ABSTRACT Genes encoding CTX-M-14, CTX-M-9, CTX-M-1, CTX-M-32, SHV-12, TEM-52, or CMY-2 β-lactamases were detected in 21 Escherichia coli strains recovered during 2003 from sick animals (11 of 459 [2.4%] strains) and healthy animals (10 of 158 [6.3%] strains) in Spain. Twelve of these strains harbored blaCTX-M genes and showed unrelated pulsed-field gel electrophoresis patterns.

Occurrence and Relatedness of Vancomycin-Resistant Enterococci in Animals, Humans, and the Environment in Different European Regions

ABSTRACT Vancomycin-resistant enterococcci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 μg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.

Antimicrobial resistance among enterococci from pigs in three European countries

ABSTRACT Enterococci from pigs in Denmark, Spain, and Sweden were examined for susceptibility to antimicrobial agents and copper and the presence of selected resistance genes. The greatest levels of resistance were found among isolates from Spain and Denmark compared to those from Sweden, which corresponds to the amounts of antimicrobial agents used in food animal production in those countries. Similar genes were found to encode resistance in the different countries, but the tet(L) and tet(S) genes were more frequently found among isolates from Spain. A recently identified transferable copper resistance gene was found in all copper-resistant isolates from the different countries.

Multiplex PCR Assay for Detection of Bacterial Pathogens Associated with Warm-Water Streptococcosis in Fish

ABSTRACT A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.

Genotyping of Francisella tularensis Strains by Pulsed-Field Gel Electrophoresis, Amplified Fragment Length Polymorphism Fingerprinting, and 16S rRNA Gene Sequencing

ABSTRACT We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

A microbiological, histopathological and immunohistological study of the intragastric inoculation of Listeria monocytogenes in mice

The course of murine infection after intragastric inoculation of L. monocytogenes was investigated by immunocytochemical, histopathological and microbiological techniques. L. monocytogenes antigen was observed in epithelial cells of intestinal mucosa overlying Peyers patches, but not in mucosa devoid of them. This suggests that penetration of L. monocytogenes into the host organism may take place through epithelium overlying Peyers patches. The efficiency of bacterial penetration appeared to be low, as shown by the small amounts of L. monocytogenes antigen detected and the low counts of bacteria in organs. Gross or histopathological lesions in the intestinal tract were not observed. The presence of L. monocytogenes in spleen, liver and in maxillary and mesenteric lymph nodes, confirmed that the systemic course of infection by this route of inoculation is similar to that of the parenteral routes. The results emphasize the subclinical character of murine listeriosis by the oral route.