Person: Domínguez Rodríguez, Lucas José
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First Name
Lucas José
Last Name
Domínguez Rodríguez
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
Department
Sanidad Animal
Area
Sanidad Animal
Identifiers
15 results
Search Results
Now showing 1 - 10 of 15
Item Molecular epidemiology of Types I/III strains of Mycobacterium avium subspecies paratuberculosis isolated from goats and cattle(Veterinary Microbiology, 2006) Juan Ferré, Lucía De; Álvarez Sánchez, Julio; Aranaz Martín, Alicia; Rodríguez Bertos, Antonio Manuel; Romero Martínez, Beatriz; Bezos Garrido, Javier; Mateos García, Ana Isabel; Domínguez Rodríguez, Lucas JoséMolecular characterization of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolates classifies them into three groups: cattle or Type II, sheep or Type I, and intermediate or Type III. To avoid problems associated with characterization of extremely slow growth strains, PCR-based techniques that divide the M. a. paratuberculosis strains in two main groups (cattle or Type II, and sheep or Types I/III) can be performed. The objectives of this study were to characterize the M. a. paratuberculosis isolates identified by different PCR-based tests (IS1311-PCR and restriction endonuclease analysis, PCR test based on a DNA sequence difference, and a PCR aimed at three Type I-specific loci), and to determine the clinical and epidemiological implications of Types I/III M. a. paratuberculosis strains in livestock. One hundred and fifty-eight M. a. paratuberculosis strains from domestic ruminants were analyzed. One hundred and six M. a. paratuberculosis isolates (61 from goats and 45 from cattle) were classified as Type II strains; and 52 (29 from cows, 20 from goats, and three from sheep) were included in the Types I/III. The Types I/III M. a. paratuberculosis strains were associated to Spanish native breeds. The majority of these animals had not been in direct or indirect contact with sheep flocks infected with M. a. paratuberculosis. This fact should be taken into account when implementing paratuberculosis control programs.Item First characterization of fluoroquinolone resistance in Streptococcus suis(Antimicrobial Agents and Chemotherapy, 2007) San Millán, Álvaro; Catalán, Ana; González de la Campa, Adela; Rivero, Estefanía; Lopez, Gema; Escudero García-Calderón, José Antonio; Domínguez Rodríguez, Lucas José; Moreno Romo, Miguel Ángel; González Zorn, BrunoWe have identified and sequenced the genes encoding the quinolone-resistance determining region (QRDR) of ParC and GyrA in fluoroquinolone-susceptible and -resistant Streptococcus suis clinical isolates. Resistance is the consequence of single point mutations in the QRDRs of ParC and GyrA and is not due to clonal spread of resistant strains or horizontal gene transfer with other bacteria.Item Genetic diversity of Mycobacterium avium isolates recovered from clinical samples and from the environment: molecular characterization for diagnostic purposes(Journal of clinical microbiology, 2008) Álvarez Sánchez, Julio; Gómez García, Ignacio; Aranaz Martín, Alicia; Bezos Garrido, Javier; Romero Martínez, Beatriz; Juan Ferré, Lucía De; Mateos García, Ana Isabel; Gómez Mampaso, Enrique; Domínguez Rodríguez, Lucas JoséIsolation of Mycobacterium avium complex (MAC) organisms from clinical samples may occur in patients without clinical disease, making the interpretation of results difficult. The clinical relevance of MAC isolates from different types of clinical samples (n = 47) from 39 patients in different sections of a hospital was assessed by comparison with environmental isolates (n = 17) from the hospital. Various methods for identification and typing (commercial probes, phenotypic characteristics, PCR for detection of IS1245 and IS901, sequencing of the hsp65 gene, and pulsed-field gel electrophoresis) were evaluated. The same strain was found in all the environmental isolates, 21 out of 23 (91.3%) of the isolates cultured from urine samples, and 5 out of 19 (26.3%) isolates from respiratory specimens. This strain did not cause disease in the patients. Testing best characterized the strain as M. avium subsp. hominissuis, with the unusual feature that 81.4% of these isolates lacked the IS1245 element. Contamination of certain clinical samples with an environmental strain was the most likely event; therefore, characterization of the environmental mycobacteria present in health care facilities should be performed to discard false-positive isolations in nonsterile samples, mainly urine samples. Molecular techniques applied in this study demonstrated their usefulness for this purpose.Item Spoligotyping Profile Change Caused by Deletion of a Direct Variable Repeat in a Mycobacterium tuberculosis Isogenic Laboratory Strain(Journal of Clinical Microbiology, 2004) Aranaz Martín, Alicia; Romero Martínez, Beatriz; Montero Serra, Natalia; Álvarez Sánchez, Julio; Bezos Garrido, Javier; Juan Ferré, Lucía De; Mateos García, Ana Isabel; Domínguez Rodríguez, Lucas JoséSpoligotyping is a major tool for molecular typing of Mycobacterium tuberculosis complex organisms. For epidemiological purposes, strains are considered clonal only when their spoligotyping patterns are identical. We report a change in the spoligotyping profiles of truly isogenic strains (a clinical isolate and a subculture derived in the laboratory) caused by deletion of a direct variable repeat. Without the information about the relationship between them, a link between these strains would have gone unnoticed. Evolutionary events should be taken into account in the interpretation of spoligotyping results and in the design of databases.Item Single-nucleotide polymorphism in two representative multidrug-resistant Mycobacterium bovis isolates collected from patients in a Spanish hospital harboring a human infection outbreak(Journal of clinical microbiology, 2008) Romero Martínez, Beatriz; Aranaz Martín, Alicia; Bezos Garrido, Javier; Álvarez Sánchez, Julio; Juan Ferré, Lucía De; Mateos García, Ana Isabel; Gómez Mampaso, Enrique; Domínguez Rodríguez, Lucas JoséMycobacterium bovis is the etiological agent of tuberculosis in domestic and wild animals. Its involvement as a human pathogen has been highlighted again with the recent descriptions of transmission through dairy products (18), reactivation or primary infection in human immunodeficiency virus-infected patients (5), and association with meat industry workers, animal keepers, or hunters (3). Strains resistant to antituberculous drugs (M. bovis is naturally resistant to pyrazinamide) pose an additional risk (2). Several studies have demonstrated that mutations in target genes are associated with resistance to antituberculous drugs (4, 7, 10, 11, 16). However, most of them have been developed in Mycobacterium tuberculosis strains and limited data are available regarding M. bovis isolates. The aim of this study was to characterize by sequencing the main genes involved in antibiotic resistance in two multidrug-resistant (MDR) M. bovis isolates in a human outbreak detected in a hospital in Madrid that subsequently spread to several countries (5, 6, 15). The isolates were resistant to 11 drugs, but only their rpoB and katG genes have been analyzed so far (1, 14). We studied the first (93/R1) and last (95/R4) M. bovis isolates of this nosocomial outbreak, characterized by spoligotyping as SB0426 (hexacode 63-5F-5E-7F-FF-60 in the database at www.mbovis.org) (1, 13). Several genes involved in resistance to isoniazid (katG, ahpC, inhA, and the oxyR-ahpC intergenic region), rifampin (rpoB), streptomycin (rrs, rpsL), ethambutol (embB), and quinolones (gyrA) were studied. These genes, or fragments of genes, were amplified and sequenced as previously described (12). The sequence analysis revealed polymorphisms in five (ahpC, rpoB, rpsL, embB, and gyrA) out of nine analyzed genes (Table 1). Nucleotide substitutions in four genes cause a change in the encoded amino acid. Two additional synonymous mutations in ahpC and rpsL differentiated the first and last isolates from the outbreak.Item Assessment of diagnostic tools for eradication of bovine tuberculosis in cattle co-infected with Mycobacterium bovis and M. avium subsp. paratuberculosis(Veterinary Research, 2006) Aranaz Martín, Alicia; Juan Ferré, Lucía De; Bezos Garrido, Javier; Álvarez Sánchez, Julio; Romero Martínez, Beatriz; Lozano, Francisco; Paramio, José L.; López-Sánchez, Jesús; Mateos García, Ana Isabel; Domínguez Rodríguez, Lucas JoséThe intradermal tuberculin (IDTB) test and the interferon-gamma (IFN-$\gamma$) assay are used worldwide for detection of bovine tuberculosis in cattle, but little is known about the effect of co-infecting agents on the performance of these diagnostic tests. This report describes a field trial conducted in a cattle herd with dual infection (bovine tuberculosis and paratuberculosis) during 3.5 years. It has been based on a strategic approach encompassing serial parallel testing (comparative IDTB test, the IFN-$\gamma$ assay and serology of paratuberculosis) that was repeated 8 times over the period, and segregation of animals into two herds. The IDTB test detected 65.2% and the IFN-$\gamma$ test detected 69.6% of the Mycobacterium bovis culture-positive cattle. However, the IDTB test performed better during the first part of the trial, while the IFN-$\gamma$ test was the only method that detected infected animals during the following three samplings. The number of false positive reactors with the IDTB and/or the IFN-$\gamma$ tests was remarkably high compared to other reports, and could be caused by cross-reactivity with M. avium subsp. paratuberculosis. Also, the M. bovis isolates from cattle and wildlife from the same property were characterised using molecular techniques to disclose an epidemiological link. The IDTB test may not be appropriate to eradicate bovine tuberculosis in herds with dual mycobacterial infections. This report highlights the need to use several diagnostic techniques for the accurate detection of M. bovis infected animals in these herds.Item Bovine Tuberculosis (Mycobacterium bovis) in Wildlife in Spain(Journal of Clinical Microbiology, 2004) Aranaz Martín, Alicia; Juan Ferré, Lucía De; Montero Serra, Natalia; Sánchez Ramos, Celia; Galka, Margarita; Delso, Consuelo; Álvarez Sánchez, Julio; Romero Martínez, Beatriz; Bezos Garrido, Javier; Vela Alonso, Ana Isabel; Briones Dieste, Víctor; Mateos García, Ana Isabel; Domínguez Rodríguez, Lucas JoséMycobacterium 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.Item Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp. paratuberculosis strains isolated from cattle and goats(Applied and Environmental Microbiology, 2006) Juan Ferré, Lucía De; Álvarez Sánchez, Julio; Romero Martínez, Beatriz; Bezos Garrido, Javier; Castellanos, Elena; Aranaz Martín, Alicia; Mateos García, Ana Isabel; Domínguez Rodríguez, Lucas JoséCulture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and Löwenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.Item Interference of paratuberculosis with the diagnosis of tuberculosis in a goat flock with a natural mixed infection(Veterinary Microbiology, 2008) Álvarez Sánchez, Julio; Juan Ferré, Lucía De; Bezos Garrido, Javier; Romero Martínez, Beatriz; Sáez, Jose Luis; Reviriego Gordejo, F.J.; Briones Dieste, Víctor; Moreno Romo, Miguel Ángel; Mateos García, Ana Isabel; Domínguez Rodríguez, Lucas José; Aranaz Martín, AliciaDetection of infected animals is a key step in eradication programs of tuberculosis. Paratuberculosis infection has been demonstrated to compromise the specificity of the diagnostic tests. However, its effect on their sensitivity has not been clarified. In the present study, skin tests and the interferon-gamma (IFN-γ) assay were evaluated in a goat flock (n = 177) with a mixed tuberculosis–paratuberculosis infection in order to assess the possible effect of paratuberculosis on their sensitivity. Culture of mycobacteria was performed as the gold standard to determine the true infection status. All techniques showed lower sensitivities than previously described; the single intradermal tuberculin (SIT) test and the IFN-γ assay detected 71% (62.4–78.6, 95% C.I.) of the infected animals; the single intradermal cervical comparative tuberculin (SICCT) test detected only 42.7% (34.1–51.7, 95% C.I.) of infected animals. The highest level of sensitivity was obtained when SIT test and IFN-γ assay were combined in parallel (90.8%, 84.5–95.2, 95% C.I.). Sensitivities of the tests were also assessed by comparing animals suffering tuberculosis and animals with a mixed infection; tests were found to be more effective in the former group. Paratuberculosis seems to have a major effect in the sensitivity of the diagnostic tests under study, and therefore must be taken into account; in particular, the use of the SICCT test should be questioned when both tuberculosis and paratuberculosis are present.Item Multiresistance in Pasteurella multocida is mediated by coexistence of small plasmids(Antimicrobial agents and chemotherapy, 2009) San Millán, Álvaro; Gutiérrez, Belén; Hidalgo, Laura; Llagostera, Montserrat; González Zorn, Bruno; Domínguez Rodríguez, Lucas José; García Benzaquén, Nerea; Escudero García-Calderón, José AntonioIn most gram-negative bacteria, acquired multiresistance is conferred by large plasmids compiling numerous antimicrobial resistance genes. Here, we show an evolutionary alternative strategy used by Pasteurella multocida to become resistant to multiple clinically relevant antibiotics. Thirteen beta-lactam-resistant clinical isolates, concomitantly resistant to tetracyclines and/or streptomycin as well as to sulfonamides, were studied. Pulsed-field gel electrophoresis analysis revealed different profiles among the isolates, showing that clonal dissemination was not the sole event responsible for the spread of multiresistance. Each P. multocida strain carried two or three small plasmids between 4 and 6 kb in size. A direct association between resistance profile and plasmid content was found. Complete nucleotide sequencing of all plasmids revealed seven different replicons, six of them belonging to the ColE1 superfamily. All plasmids carried one, or a maximum of two, antimicrobial resistance determinants. Plasmids pB1000 and pB1002 bore bla(ROB-1), pB1001 carried tet(B), pB1003 and pB1005 carried sul2 and strA, pB1006 harbored tet(O), and p9956 bore the tet(H) gene. All plasmids except pB1002 and pB1006 were successfully transformed into Escherichia coli. pB1000, also involved in beta-lactam resistance in Haemophilus parasuis (A. San Millan et al., Antimicrob. Agents Chemother. 51:2260-2264, 2007), was mobilized in E. coli using the conjugation machinery of an IncP plasmid. Stability experiments proved that pB1000 was stable in P. multocida but highly unstable in E. coli. In conclusion, bla(ROB-1) is responsible for beta-lactam resistance in P. multocida in Spain. Coexistence and the spread of small plasmids are used by P. multocida to become multiresistant.