Browsing by MeSH term "DNA, Bacterial"
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Publication An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene(Oxford University Press, 2016) Ferrandiz-Avellano, Maria-Jose; Martin-Galiano, Antonio Javier; Arnanz, Cristina; Camacho-Soguero, Isabel; Tirado-Velez, JM; de la Campa, Adela G; Ministerio de Economía y Competitividad (España)We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 μM) and 72.9% (8 μM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 μM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7-31.4 kb in length and included 9-22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria.Publication Analysis of the genome content of Lactococcus garvieae by genomic interspecies microarray hybridization(BioMed Central (BMC), 2010-03-16) Aguado-Urda, Mónica; López-Campos, Guillermo H; Fernández-Garayzábal, José F; Martin-Sanchez, Fernando; Gibello, Alicia; Domínguez, Lucas; Blanco, María M; Ministerio de Ciencia e Innovación (España)BACKGROUND: Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study, the genomic content of L. garvieae CECT 4531 was analysed using bioinformatics tools and microarray-based comparative genomic hybridization (CGH) experiments. Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 were used as reference microorganisms. RESULTS: The combination and integration of in silico analyses and in vitro CGH experiments, performed in comparison with the reference microorganisms, allowed establishment of an inter-species hybridization framework with a detection threshold based on a sequence similarity of >or= 70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of the genes are related to ribosomal, sugar metabolism or energy conversion systems. Some of the identified genes, such as als and mycA, could be involved in the pathogenesis of L. garvieae infections. CONCLUSIONS: In this study, we identified 267 genes that were potentially present in L. garvieae CECT 4531. Some of the identified genes could be involved in the pathogenesis of L. garvieae infections. These results provide the first insight into the genome content of L. garvieae.Publication Arthropod-Borne Bacteria Cause Nonmalarial Fever in Rural Ethiopia: A Cross-Sectional Study in 394 Patients(Mary Ann Liebert, 2019-11) Ramos, Jose M; Pérez-Tanoira, Ramón; Martín-Martín, Inés; Prieto-Pérez, Laura; Tefasmariam, Abraham; Tiziano, Gebre; Escudero, Raquel; Gil-Zamorano, Judit; Gil-Gil, Horacio; Górgolas, Miguel; Jado, Isabel; Autonomous University of Madrid (España)Bacterial arthropod-borne pathogens are a common cause of fever in Africa, but their precise impact is unknown and usually underdiagnosed in the basic rural laboratories of low-resourced African countries. Our aim was to determine the prevalence of arthropod-borne bacterial diseases causing fever among malaria smear-negative patients in a rural hospital located in Ethiopia. The study population included patients aged 2 years or older; referred to Gambo Rural General Hospital (West Arsi, Ethiopia), between July and November 2013, for fever or report of fever in the previous 48 h; attending the outpatient department; and testing negative for malaria by Giemsa-stained thin blood smears. We extracted DNA from 394 whole blood samples, using reverse line blot assays of amplicons to look for bacteria from the genera: Anaplasma, Bartonella, Borrelia, Coxiella, Ehrlichia, Francisella, and Rickettsia. Thirteen patients showed presence of DNA for these pathogens: three each by Borrelia spp., the Francisella group (F. tularensis tularensis, F. tularensis holartica, and F. novicia), Rickettsia bellii, and Rickettsia Felis, and one by Bartonella rochalimae. Thus, in this rural area of Africa, febrile symptoms could be due to bacteria transmitted by arthropods. Further studies are needed to evaluate the pathogenic role of R. bellii.Publication Bacterial DNA translocation and liver disease severity among HIV-infected patients with chronic hepatitis C(Lippincott Williams & Wilkins (LWW), 2012-12-15) Garcia-Alvarez, Monica; Berenguer, Juan; Guzman-Fulgencio, Maria; Alvarez, Emilio; Cosín, Jaime; Micheloud, Dariela; Jimenez-Sousa, Maria Angeles; Fernandez-Rodriguez, Amanda; Aldámiz-Echevarría, Teresa; Carrero, Ana; Miralles, Pilar; Resino, Salvador; Instituto de Salud Carlos III; Redes Temáticas de Investigación Cooperativa en Salud (RETICS) (España); Fundación para la Investigación y la Prevención del Sida en EspañaWe carried out a cross-sectional study to explore whether bacterial 16S ribosomal DNA (bactDNA) shows association with severity of liver disease among human immunodeficiency virus/hepatitis C virus coinfected patients. Patients with advanced fibrosis (F3/F4), moderate activity grade (A2/A3), and high fibrosis progression rate (FPR > 0.15) had higher values of plasma bactDNA levels than did patients without these markers of liver disease (P < 0.05). The chance of having a fibrosis stage or activity grade increased was 1.20 [95% confidence interval (CI) = 1.0 to 1.44, P = 0.045] and 1.22 (95% CI = 1.1 to 1.45, P = 0.029) times greater for every 100 copies per microliter of plasma bactDNA. Likewise, the odds of having values of FPR > 0.15 was 1.18 (95% CI = 0.98 to 1.42, P = 0.089). In addition, patients with high bactDNA levels (≥175 copies per microliter) had the highest odds of having high values of Metavir score and FPR (P < 0.05). Our data show that bacterial translocation is associated with severe liver disease among human immunodeficiency virus-infected patients with chronic hepatitis C.Publication Epidemiological tracing of Salmonella enterica serotype Abortusovis from Spanish ovine flocks by PFGE fingerprinting(Cambridge University Press, 2007-05) Valdezate, Sylvia; Astorga, R; Herrera-León, Silvia; Perea, A; Usera, Miguel A.; Huerta, B; Echeita, Aurora; Instituto de Salud Carlos III; Regional Government of Andalusia (España)Salmonella enterica serotype Abortusovis, an ovine host-specific serotype, rare in most countries, is responsible for epidemic abortion episodes in Spain. With the aim of surveillance and detection of the spread of specific clones, 55 Abortusovis isolates collected during 1996-2001 from flocks in 11 provinces, were studied using XbaI-PFGE. Despite the fact that the strains were geographically and spatially related, PFGE demonstrated an epidemiologically acceptable discriminating power, identifying 20 clones (similarity, 52-96%). Clones Sabv6, 1, 5 were disseminated in seven, five and two areas respectively, while another 17 clones appeared in single places. Clones from nearby geographic regions showed a high relatedness (one band of difference in the PFGE profile) Sabv1-2-3, Sabv5-6, Sabv7-8, and Sabv13-14, suggesting a common ancestor. Co-isolation in the same flock (Sabv5-6, Sabv1-3, Sabv1-6) was detected. PFGE surveillance detected the predominance and widespread distribution of clone Sabv6 in 21 out of the 55 Abortusovis serotype episodes studied in Spain.Publication Fluoroquinolone Resistance in Penicillin-resistantStreptococcus pneumoniaeClones, Spain(Centers for Disease Control and Prevention (CDC), 2004-10) de la Campa, Adela G; Balsalobre-Arenas, Maria Luz; Ardanuy, Carmen; Fenoll, Asuncion; Pérez-Trallero, Emilio; Liñares, Josefina; Instituto de Salud Carlos III; Ministerio de Ciencia y Tecnología (España)Among 2,882 Streptococcus pneumoniae sent to the Spanish Reference Laboratory during 2002, 75 (2.6%) were ciprofloxacin-resistant. Resistance was associated with older patients (3.9% in adults and 7.2% in patients > or =65 years of age), with isolation from noninvasive sites (4.3% vs. 1.0%), and with penicillin and macrolide resistance. Among 14 low-level resistant (MIC 4-8 microg/mL) strains, 1 had a fluoroquinolone efflux phenotype, and 13 showed single ParC changes. The 61 high-level ciprofloxacin-resistant (MIC > or =16 microg/mL) strains showed either two or three changes at ParC, ParE, and GyrA. Resistance was acquired either by point mutation (70 strains) or by recombination with viridans streptococci (4 strains) at the topoisomerase II genes. Although 36 pulsed-field gel electrophoresis patterns were observed, 5 international multiresistant clones (Spain23F-1, Spain6B-2, Spain9V-3, Spain14-5 and Sweden15A-25) accounted for 35 (46.7%) of the ciprofloxacin-resistant strains. Continuous surveillance is needed to prevent the dissemination of these clones.Publication Impact of human-associated Escherichia coli clonal groups in Antarctic pinnipeds: presence of ST73, ST95, ST141 and ST131(Nature Publishing Group, 2018) Mora, Azucena; García-Peña, Francisco Javier; Alonso, María Pilar; Pedraza-Diaz, Susana; Ortega-Mora, Luis Miguel; Garcia-Parraga, Daniel; López, Cecilia; Viso, Susana; Dahbi, Ghizlane; Marzoa, Juan; Sergeant, Martin J; García, Vanesa; Blanco, Jorge; Ministerio de Ciencia e Innovación (España); Ministerio de Economía y Competitividad (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Agencia Estatal de Investigación (España); Instituto de Salud Carlos III; Xunta de Galicia (España)There is growing concern about the spreading of human microorganisms in relatively untouched ecosystems such as the Antarctic region. For this reason, three pinniped species (Leptonychotes weddellii, Mirounga leonina and Arctocephalus gazella) from the west coast of the Antartic Peninsula were analysed for the presence of Escherichia spp. with the recovery of 158 E. coli and three E. albertii isolates. From those, 23 harboured different eae variants (α1, β1, β2, ε1, θ1, κ, ο), including a bfpA-positive isolate (O49:H10-A-ST206, eae-k) classified as typical enteropathogenic E. coli. Noteworthy, 62 of the 158 E. coli isolates (39.2%) exhibited the ExPEC status and 27 (17.1%) belonged to sequence types (ST) frequently occurring among urinary/bacteremia ExPEC clones: ST12, ST73, ST95, ST131 and ST141. We found similarities >85% within the PFGE-macrorrestriction profiles of pinniped and human clinic O2:H6-B2-ST141 and O16:H5/O25b:H4-B2-ST131 isolates. The in silico analysis of ST131 Cplx genomes from the three pinnipeds (five O25:H4-ST131/PST43-fimH22-virotype D; one O16:H5-ST131/PST506-fimH41; one O25:H4-ST6252/PST9-fimH22-virotype D1) identified IncF and IncI1 plasmids and revealed high core-genome similarities between pinniped and human isolates (H22 and H41 subclones). This is the first study to demonstrate the worrisome presence of human-associated E. coli clonal groups, including ST131, in Antarctic pinnipeds.Publication Inactivation of the Thymidylate Synthase thyA in Non-typeable Haemophilus influenzae Modulates Antibiotic Resistance and Has a Strong Impact on Its Interplay with the Host Airways(Frontiers Media, 2017-06) Rodríguez-Arce, Irene; Martí, Sara; Euba, Begoña; Fernández-Calvet, Ariadna; Moleres, Javier; López-López, Nahikari; Barberán, Montserrat; Ramos-Vivas, José; Tubau, Fe; Losa, Carmen; Ardanuy, Carmen; Leiva, José; Yuste, Jose Enrique; Garmendia, Junkal; Ministerio de Economía y Competitividad (España); Comunidad Foral de Navarra (España); Sociedad Española de Neumología y Cirugía TorácicaAntibacterial treatment with cotrimoxazol (TxS), a combination of trimethoprim and sulfamethoxazole, generates resistance by, among others, acquisition of thymidine auxotrophy associated with mutations in the thymidylate synthase gene thyA, which can modify the biology of infection. The opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently encountered in the lower airways of chronic obstructive pulmonary disease (COPD) patients, and associated with acute exacerbation of COPD symptoms. Increasing resistance of NTHi to TxS limits its suitability as initial antibacterial against COPD exacerbation, although its relationship with thymidine auxotrophy is unknown. In this study, the analysis of 2,542 NTHi isolates recovered at Bellvitge University Hospital (Spain) in the period 2010-2014 revealed 119 strains forming slow-growing colonies on the thymidine low concentration medium Mueller Hinton Fastidious, including one strain isolated from a COPD patient undergoing TxS therapy that was a reversible thymidine auxotroph. To assess the impact of thymidine auxotrophy in the NTHi-host interplay during respiratory infection, thyA mutants were generated in both the clinical isolate NTHi375 and the reference strain RdKW20. Inactivation of the thyA gene increased TxS resistance, but also promoted morphological changes consistent with elongation and impaired bacterial division, which altered H. influenzae self-aggregation, phosphorylcholine level, C3b deposition, and airway epithelial infection patterns. Availability of external thymidine contributed to overcome such auxotrophy and TxS effect, potentially facilitated by the nucleoside transporter nupC. Although, thyA inactivation resulted in bacterial attenuation in a lung infection mouse model, it also rendered a lower clearance upon a TxS challenge in vivo. Thus, our results show that thymidine auxotrophy modulates both the NTHi host airway interplay and antibiotic resistance, which should be considered at the clinical setting for the consequences of TxS administration.Publication Long-range dispersal moved Francisella tularensis into Western Europe from the East(Microbiology Society, 2016) Dwibedi, Chinmay; Birdsell, Dawn; Lärkeryd, Adrian; Myrtennäs, Kerstin; Öhrman, Caroline; Nilsson, Elin; Karlsson, Edvin; Hochhalter, Christian; Rivera, Andrew; Maltinsky, Sara; Bayer, Brittany; Keim, Paul; Scholz, Holger C; Tomaso, Herbert; Wittwer, Matthias; Beuret, Christian; Schuerch, Nadia; Pilo, Paola; Hernández Pérez, Marta; Rodríguez-Lázaro, David; Escudero, Raquel; Anda, Pedro; Forsman, Mats; Wagner, David M; Larsson, Pär; Johansson, Anders; Ministerio de Economía y Competitividad (España)For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species.Publication Mefloquine and new related compounds target the F(0) complex of the F(0)F(1) H(+)-ATPase of Streptococcus pneumoniae(American Society for Microbiology (ASM), 2002-06) Martín-Galiano, Antonio Javier; Gorgojo, Begoña; Kunin, Calvin M; de la Campa, Adela G; Comunidad de Madrid (España); Instituto de Salud Carlos IIIThe activities of mefloquine (MFL) and related compounds against previously characterized Streptococcus pneumoniae strains carrying defined amino acid substitutions in the c subunit of the F(0)F(1) H(+)-ATPase were studied. In addition, a series of MFL-resistant (Mfl(r)) strains were isolated and characterized. A good correlation was observed between inhibition of growth and inhibition of the membrane-associated F(0)F(1) H(+)-ATPase activity. MFL was about 10-fold more active than optochin and about 200-fold more active than quinine in inhibiting both the growth and the ATPase activities of laboratory pneumococcal strain R6. Mutant strains were inhibited by the different compounds to different degrees, depending on their specific mutations in the c subunit. The resistant strains studied had point mutations that changed amino acid residues in either the c subunit or the a subunit of the F(0) complex. Changes in the c subunit were located in one of the two transmembrane alpha helices: residues M13, G14, G20, M23, and N24 of helix 1 and residues M44, G47, V48, A49, and V57 of helix 2. Changes in the a subunit were also found in either of the transmembrane alpha helices, helix 5 or 6: residue L186 of helix 5 and residues W206, F209, and S214 of helix 6. These results suggest that the transmembrane helices of the c and a subunits interact and that the mutated residues are important for the structure of the F(0) complex and proton translocation.Publication Microbial Signature in Adipose Tissue of Crohn's Disease Patients(Multidisciplinary Digital Publishing Institute (MDPI), 2020-07-31) Serena, Carolina; Queipo-Ortuño, Maribel; Millan, Monica; Sanchez-Alcoholado, Lidia; Caro, Aleidis; Espina, Beatriz; Menacho, Margarita; Bautista, Michelle; Monfort-Ferré, Diandra; Terrón-Puig, Margarida; Núñez-Roa, Catalina; Maymó-Masip, Elsa; Rodriguez, M. Mar; Tinahones, Francisco J.; Espin, Eloy; Martí, Marc; Fernández-Veledo, Sonia; Vendrell, Joan; [Serena,C; Monfort-Ferré,D; Terrón-Puig,M; Núñez-Roa,C; Maymó-Masip,E; Rodriguez,MM; Fernández-Veledo,S; Vendrell,J] Hospital Universitari de Tarragona Joan XXIII, Institut d’Investigació Sanitària Pere Virgili Universitat Rovira i Virgili, Tarragona, Spain. [Serena,C; Terrón-Puig,M; Núñez-Roa,C; Maymó-Masip,E; Rodriguez,MM; Fernández-Veledo,S; Vendrell,J] CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain. [Queipo-Ortuño,M; Sanchez-Alcoholado,L] CIBER de Obesidad y Nutrición (CIBERObN), Instituto de Salud Carlos III, Madrid, Spain. [Queipo-Ortuño,M; Sanchez-Alcoholado,L] Unidad de Gestión Clínica Intercentros de Oncología Medica, Hospitales Universitarios Regional y Virgen de la Victoria de Málaga, Instituto de Investigación Biomédica de Málaga (IBIMA)-CIMES-UMA, Málaga, Spain. [Millan,M; Caro,A; Espina,B] Colorectal Surgery Unit, Hospital Universitari Joan XXIII, Tarragona, Spain. [Millan,M] Colorectal Surgery Unit, Hospital Universitari La Fe, Valencia, Spain. [Menacho,M; Bautista,M] Digestive Unit, University Hospital Joan XXIII, IISPV, Tarragona, Spain. [Tinahones,FJ] Endocrinology and Nutrition Department, Biomedical Research Institute from Malaga (IBIMA), University Hospital Virgen de la Victoria of Malaga, Malaga University, Málaga, Spain. [Espin,E; Martí,M] Colorectal Surgery Unit, General Surgery Service, Valle Hebron Hospital, Autonomous University of Barcelona, Barcelona, Spain. [Vendrell,J] Medicine and Surgery Department, School of Medicine, Universitat Rovira i Virgili, Tarragona, Spain.Crohn's disease (CD) is characterized by compromised immune tolerance to the intestinal commensal microbiota, intestinal barrier inflammation, and hyperplasia of creeping fat (CF) and mesenteric adipose tissue (AT), which seems to be directly related to disease activity. Gut microbiota dysbiosis might be a determining factor in CD etiology, manifesting as a low microbial diversity and a high abundance of potentially pathogenic bacteria. We tested the hypothesis that CF is a reservoir of bacteria through 16S-rRNA sequencing of several AT depots of patients with active and inactive disease and controls. We found a microbiome signature within CF and mesenteric AT from patients, but not in subcutaneous fat. We failed to detect bacterial DNA in any fat depot of controls. Proteobacteria was the most abundant phylum in both CF and mesenteric AT, and positively correlated with fecal calprotectin/C-reactive protein. Notably, the clinical status of patients seemed to be related to the microbiome signature, as those with the inactive disease showed a reduction in the abundance of pathogenic bacteria. Predictive functional profiling revealed many metabolic pathways including lipopolysaccharide biosynthesis and sulfur metabolism overrepresented in active CD relative to that in inactive CD. Our findings demonstrate that microbiota dysbiosis associated with CD pathophysiology is reflected in AT and might contribute to disease severity.Publication Molecular Epidemiology and Antibiotic Susceptibility of Vibrio cholerae Associated with a Large Cholera Outbreak in Ghana in 2014(Public Library of Science (PLOS), 2016-05-27) Eibach, Daniel; Herrera-León, Silvia; Gil, Horacio; Hogan, Benedikt; Ehlkes, Lutz; Adjabeng, Michael; Kreuels, Benno; Nagel, Michael; Opare, David; Fobil, Julius N; May, Jürgen; German Center for Infection Research (Alemania)BACKGROUND: Ghana is affected by regular cholera epidemics and an annual average of 3,066 cases since 2000. In 2014, Ghana experienced one of its largest cholera outbreaks within a decade with more than 20,000 notified infections. In order to attribute this rise in cases to a newly emerging strain or to multiple simultaneous outbreaks involving multi-clonal strains, outbreak isolates were characterized, subtyped and compared to previous epidemics in 2011 and 2012. METHODOLOGY/PRINCIPAL FINDINGS: Serotypes, biotypes, antibiotic susceptibilities were determined for 92 Vibrio cholerae isolates collected in 2011, 2012 and 2014 from Southern Ghana. For a subgroup of 45 isolates pulsed-field gel electrophoresis, multilocus sequence typing and multilocus-variable tandem repeat analysis (MLVA) were performed. Eighty-nine isolates (97%) were identified as ctxB (classical type) positive V. cholerae O1 biotype El Tor and three (3%) isolates were cholera toxin negative non-O1/non-O139 V. cholerae. Among the selected isolates only sulfamethoxazole/trimethoprim resistance was detectable in 2011, while 95% of all 2014 isolates showed resistance towards sulfamethoxazole/trimethoprim, ampicillin and reduced susceptibility to ciprofloxacin. MLVA achieved the highest subtype discrimination, revealing 22 genotypes with one major outbreak cluster in each of the three outbreak years. Apart from those clusters genetically distant genotypes circulate during each annual epidemic. CONCLUSIONS/SIGNIFICANCE: This analysis suggests different endemic reservoirs of V. cholerae in Ghana with distinct annual outbreak clusters accompanied by the occurrence of genetically distant genotypes. Preventive measures for cholera transmission should focus on aquatic reservoirs. Rapidly emerging multidrug resistance must be monitored closely.Publication Molecular epidemiology, antimicrobial susceptibilities and resistance mechanisms of Streptococcus pyogenes isolates resistant to erythromycin and tetracycline in Spain (1994-2006)(BioMed Central (BMC), 2012-09-21) Rubio-Lopez, Virginia; Valdezate, Sylvia; Álvarez, David; Villalon-Panzano, Pilar; Medina-Pascual, Maria Jose; Salcedo, Celia; Saez, Juan Antonio; Instituto de Salud Carlos III; Ministerio de Ciencia e Innovación (España)BACKGROUND: Group A Streptococcus (GAS) causes human diseases ranging in severity from uncomplicated pharyngitis to life-threatening necrotizing fasciitis and shows high rates of macrolide resistance in several countries. Our goal is to identify antimicrobial resistance in Spanish GAS isolates collected between 1994 and 2006 and to determine the molecular epidemiology (emm/T typing and PFGE) and resistance mechanisms of those resistant to erythromycin and tetracycline. RESULTS: Two hundred ninety-five out of 898 isolates (32.8%) were erythromycin resistant, with the predominance of emm4T4, emm75T25, and emm28T28, accounting the 67.1% of the 21 emm/T types. Spread of emm4T4, emm75T25 and emm28T28 resistant clones caused high rates of macrolide resistance. The distribution of the phenotypes was M (76.9%), cMLSB (20.3%), iMLSB (2.7%) with the involvement of the erythromycin resistance genes mef(A) (89.5%), msr(D) (81.7%), erm(B) (37.3%) and erm(A) (35.9%).Sixty-one isolates were tetracycline resistant, with the main representation of the emm77T28 among 20 emm/T types. To note, the combination of tet(M) and tet(O) tetracycline resistance genes were similar to tet(M) alone reaching values close to 40%. Resistance to both antibiotics was detected in 19 isolates of 7 emm/T types, being emm11T11 and the cMLSB phenotype the most frequent ones. erm(B) and tet(M) were present in almost all the strains, while erm(A), mef(A), msr(D) and tet(O) appeared in less than half of them. CONCLUSIONS: Spanish GAS were highly resistant to macrolides meanwhile showed minor resistance rate to tetracycline. A remarkable correlation between antimicrobial resistance and emm/T type was noticed. Clonal spread of emm4T4, emm75T25 and emm28T28 was the main responsable for macrolide resistance where as that emm77T28 clones were it to tetraclycline resistance. A wide variety of macrolide resistance genes were responsible for three macrolide resistance phenotypes.Publication Nosocomial transmission of multidrug-resistant Mycobacterium tuberculosis in Spain(Centers for Disease Control and Prevention (CDC), 1996-04) Rullán, John V; Herrera Guibert, Dionisio; Cano-Portero, Rosa; Moreno, Victoria; Godoy, Pere; Peiró, Enrique F; Castell, Juan; Ibañez, Consuelo; Ortega, Arturo; Sánchez Agudo, Leopoldo; Pozo, Francisco; Instituto de Salud Carlos IIIBefore 1990, outbreaks of multidrug-resistanttuberculosis (MDRTB) were uncommon (1); sincethen, more than 10 outbreaks have been reported,all in hospitals and prisons in the eastern UnitedStates (2-7). Persons traditionally considered atrisk for MDRTB (foreign-born TB patients andthose inadequately treated for TB) have not beenassociated with these outbreaks. Instead, the pres-ence of patients with active TB near immunocom-promised patients in HIV-dedicated wards has ledto MDRTB-infected HIV patients whose TB casesoften go unrecognized. The patients receive inade-quate treatment in facilities without effective pro-cedures for isolating acid-fast bacilli; thesecircumstances favor nosocomial transmission.Health officials in other geographic areas whereHIV and TB are major public health threats havebeen alerted to this emerging problem, and sur-veillance systems have been designed (8).Publication Rapid and Highly Sensitive DNA Flow Technology Platform to Detect Tick-Borne Bacterial Pathogens in Clinical Samples(Mary Ann Liebert, 2020-02) Jado, Isabel; Escudero, Raquel; Espigares, Belén; Lara, Ester; Rodriguez-Vargas, Manuela; Garcia-Amil, Cristina; Lobo, Bruno; Rodriguez-Moreno, Isabel; Toledo, AlvaroZoonotic diseases represent a significant public health concern worldwide due to the emergence/re-emergence of vector-borne diseases in the last decade. Ticks are the most important vectors in the northern hemisphere and can transmit diseases such as Lyme disease, human granulocytic anaplasmosis, and spotted fever rickettsioses, among others. Therefore, there is a growing need to develop better and faster diagnostic tools that can detect zoonotic human pathogens in clinical samples. In this study, we present the results for a new kit tick-borne bacteria flow chip (TBFC), which allows the simultaneous screening of seven different bacterial pathogens in human samples using a DNA flow technology platform (hybriSpot system). The analytical sensitivity and specificity of the TBFC were calculated spiking bacterial DNA in human DNA samples, and the results were compared with an in-house single PCR-reverse line blot (RLB) routinely used for diagnosis at the National Center for Microbiology in Spain. The analytical sensitivity and specificity of the TBFC were almost identical to the PCR-RLBs used in diagnosis. In addition, samples from patients (n = 212) with a wide range of clinical signs/symptoms consistent with multisystem disorders suggestive of a tick-borne infection were tested using the TBFC, and the results were compared with those obtained by PCR-RLB. The concordance of both methods using patient samples was 97.2%. The TBFC kit is a rapid new and cost-efficient diagnostic molecular tool capable of detecting tick-borne pathogens in clinical samples.Publication Role of global and local topology in the regulation of gene expression in Streptococcus pneumoniae(Public Library of Science (PLOS), 2014-07-14) Ferrandiz-Avellano, Maria-Jose; Arnanz, Cristina; Martin-Galiano, Antonio Javier; Rodriguez-Martin, Carlos; de la Campa, Adela G; Ministerio de Ciencia e Innovación (España); Instituto de Salud Carlos IIIThe most basic level of transcription regulation in Streptococcus pneumoniae is the organization of its chromosome in topological domains. In response to drugs that caused DNA-relaxation, a global transcriptional response was observed. Several chromosomal domains were identified based on the transcriptional response of their genes: up-regulated (U), down-regulated (D), non-regulated (N), and flanking (F). We show that these distinct domains have different expression and conservation characteristics. Microarray fluorescence units under non-relaxation conditions were used as a measure of gene transcriptional level. Fluorescence units were significantly lower in F genes than in the other domains with a similar AT content. The transcriptional level of the domains categorized them was D>U>F. In addition, a comparison of 12 S. pneumoniae genome sequences showed a conservation of gene composition within U and D domains, and an extensive gene interchange in F domains. We tested the organization of chromosomal domains by measuring the relaxation-mediated transcription of eight insertions of a heterologous Ptccat cassette, two in each type of domain, showing that transcription depended on their chromosomal location. Moreover, transcription from the four promoters directing the five genes involved in supercoiling homeostasis, located either in U (gyrB), D (topA), or N (gyrA and parEC) domains was analyzed both in their chromosomal locations and in a replicating plasmid. Although expression from the chromosomal PgyrB and PtopA showed the expected domain regulation, their expression was down-regulated in the plasmid, which behaved as a D domain. However, both PparE and PgyrA carried their own regulatory signals, their topology-dependent expression being equivalent in the plasmid or in the chromosome. In PgyrA a DNA bend acted as a DNA supercoiling sensor. These results revealed that DNA topology functions as a general transcriptional regulator, superimposed upon other more specific regulatory mechanisms.Publication Spread of epidemic MRSA-ST5-IV clone encoding PVL as a major cause of community onset staphylococcal infections in Argentinean children(Public Library of Science (PLOS), 2012-01-23) Sola, Claudia; Paganini, Hugo; Egea, Ana L; Moyano, Alejandro J; Garnero, Analia; Kevric, Ines; Culasso, Catalina; Vindel, Ana; Study Group of CA-MRSA in Children, Argentina-2007; Lopardo, Horacio; Bocco, José L; Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Agencia Nacional de Promoción Científica y Tecnológica (Argentina); National University of Córdoba (Argentina)BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus-(CA-MRSA) strains have emerged in Argentina. We investigated the clinical and molecular evolution of community-onset MRSA infections (CO-MRSA) in children of Córdoba, Argentina, 2005-2008. Additionally, data from 2007 were compared with the epidemiology of these infections in other regions of the country. METHODOLOGY/PRINCIPAL FINDINGS: Two datasets were used: i) lab-based prospective surveillance of CA-MRSA isolates from 3 Córdoba pediatric hospitals-(CBAH1-H3) in 2007-2008 (compared to previously published data of 2005) and ii) a sampling of CO-MRSA from a study involving both, healthcare-associated community-onset-(HACO) infections in children with risk-factors for healthcare-associated infections-(HRFs), and CA-MRSA infections in patients without HRFs detected in multiple centers of Argentina in 2007. Molecular typing was performed on the CA-MRSA-(n: 99) isolates from the CBAH1-H3-dataset and on the HACO-MRSA-(n: 51) and CA-MRSA-(n: 213) isolates from other regions. Between 2005-2008, the annual proportion of CA-MRSA/CA-S. aureus in Córdoba hospitals increased from 25% to 49%, P<0.01. Total CA-MRSA infections increased 3.6 fold-(5.1 to 18.6 cases/100,000 annual-visits, P<0.0001), associated with an important increase of invasive CA-MRSA infections-(8.5 fold). In all regions analyzed, a single genotype prevailed in both CA-MRSA (82%) and HACO-MRSA(57%), which showed pulsed-field-gel electrophoresis-(PFGE)-type-"I", sequence-type-5-(ST5), SCCmec-type-IVa, spa-t311, and was positive for PVL. The second clone, pulsotype-N/ST30/CC30/SCCmecIVc/t019/PVL(+), accounted for 11.5% of total CA-MRSA infections. Importantly, the first 4 isolates of Argentina belonging to South American-USA300 clone-(USA300/ST8/CC8/SCCmecIVc/t008/PVL(+)/ACME(-)) were detected. We also demonstrated that a HA-MRSA clone-(pulsotype-C/ST100/CC5) caused 2% and 10% of CA-MRSA and HACO-MRSA infections respectively and was associated with a SCCmec type closely related to SCCmecIV(2B&5). CONCLUSIONS/SIGNIFICANCE: The dissemination of epidemic MRSA clone, ST5-IV-PVL(+) was the main cause of increasing staphylococcal community-onset infections in Argentinean children (2003-2008), conversely to other countries. The predominance of this clone, which has capacity to express the h-VISA phenotype, in healthcare-associated community-onset cases suggests that it has infiltrated into hospital-settings.Publication StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae(Multidisciplinary Digital Publishing Institute (MDPI), 2023-03-22) de Vasconcelos Júnior, Antônio Alexandre; Tirado-Velez, JM; Martin-Galiano, Antonio Javier; Megías, Diego; Ferrandiz-Avellano, Maria-Jose; Hernández, Pablo; Amblar, Monica; de la Campa, Adela G; Agencia Estatal de Investigación (España); Ministerio de Economía e Innovación (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.Publication Starkeya nomas sp. nov., a prosthecate and budding bacterium isolated from an immunocompromized patient(Microbiology Society, 2023-08) Sahuquillo-Arce, José Miguel; Reyes-Prieto, Mariana; Hernández-Cabezas, Alicia; Molina-Moreno, José Miguel; Saez-Nieto, Juan Antonio; Marín, María Del Pilar; Alcoriza-Balaguer, María Isabel; Lahoz, Agustín; Sanz, Jaime; González-Tarancón, Lola; Ferrús, María Loreto; Martínez-Priego, Llúcia; Magraner-Martínez, Adolfo; López-Hontangas, José LuisStrain HF14-78462T is an environmental bacterium found in clinical samples from an immunocompromized patient in 2014 at Hospital Universitari i Politècnic La Fe (Valencia, Spain). Phenotypically, strain HF14-78462T cells were Gram-stain-negative, aerobic, non-spore forming and non-motile small rods which formed mucous and whitish-translucent colonies when incubated at 20-36 °C. Phylogenetic analyses based on the 16S rRNA genes and the whole genomes of closest sequenced relatives confirmed that strain HF14-78462T is affiliated with the genus Starkeya. The strain was oxidase, catalase and urease positive; but indole, lysine decarboxylase, ornithine decarboxylase and DNase negative, did not produce H2S and was able to utilize a wide variety of carbon sources including acetamide, adonitol, amygdalin, l-arabinose, citric acid, glucose, mannitol and melibiose. Unlike Starkeya novella and Starkeya koreensis, strain HF14-78462T failed to grow in thiosulphate-oxidizing media and had a narrower temperature growth range. Its genome was characterized by a size of 4.83 Mbp and a C+G content of 67.75 mol%. Major fatty acids were C18:1 ω7c, cyclo C19 : 0 and C16 : 0, its polar acids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and an aminophospholipid; while the ubiquinones were Q9 (1.8 %) and Q10 (98.2 %). Digital DNA-DNA hybridization values were 41 and 41.4 against S. novella and S. koreensis, respectively, while average nucleotide identity values were around 84 %. Phenotypic, average nucleotide identity and phylogenomic comparative studies suggest that strain HF14-78462T is a new representative of the genus Starkeya and the name Starkeya nomas sp. nov. is proposed. The type strain is HF14-78462T (=CECT 30124T=LMG 31874T).Publication The genome of Streptococcus pneumoniae is organized in topology-reacting gene clusters(Oxford University Press, 2010-06) Ferrandiz-Avellano, Maria-Jose; Martin-Galiano, Antonio Javier; Schvartzman, Jorge B; de la Campa, Adela G; Ministerio de Ciencia e Innovación (España); Comunidad de Madrid (España); Centro de Investigación Biomedica en Red - CIBERThe transcriptional response of Streptococcus pneumoniae was examined after exposure to the GyrB-inhibitor novobiocin. Topoisomer distributions of an internal plasmid confirmed DNA relaxation and recovery of the native level of supercoiling at low novobiocin concentrations. This was due to the up-regulation of DNA gyrase and the down-regulation of topoisomerases I and IV. In addition, >13% of the genome exhibited relaxation-dependent transcription. The majority of the responsive genes (>68%) fell into 15 physical clusters (14.6-85.6 kb) that underwent coordinated regulation, independently of operon organization. These genomic clusters correlated with AT content and codon composition, showing the chromosome to be organized into topology-reacting gene clusters that respond to DNA supercoiling. In particular, down-regulated clusters were flanked by 11-40 kb AT-rich zones that might have a putative structural function. This is the first case where genes responding to changes in the level of supercoiling in a coordinated manner were found organized as functional clusters. Such an organization revealed DNA supercoiling as a general feature that controls gene expression superimposed on other kinds of more specific regulatory mechanisms.