Resistencia a beta-lactámicos y fluoroquinolonas en Salmonella enterica. Mecanismos moleculares y elementos de movilización génica
Salmonella enterica is an important zoonotic pathogen frequently implicated in human foodborne infections. The emergency of clinical isolates resistant to antibiotics involves serious limits for their treatments. The first objective of this thesis was to study the resistance phenotype and its relati...
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Universidad de La Rioja (España)
2013
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Salmonella enterica is an important zoonotic pathogen frequently implicated in human
foodborne infections. The emergency of clinical isolates resistant to antibiotics involves serious
limits for their treatments. The first objective of this thesis was to study the resistance
phenotype and its relation with the serotype in 280 S. enterica isolates obtained from Hosp. San Pedro in Logrono (2007-2009) and Hosp. Clinico Universitario Lozano Blesa in Zaragoza (2009-2010). The main serotypes were Typhimurium (52%) and Enteritidis (33%), being S.Typhimurium highly associated with multi-resistance phenotypes, and in particular with resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline
(ACSSuT, 29.5%). Low resistance percentages of resistance to ciprofloxacin or third generation
cephalosporins were observed.
The second objective was to characterize the mechanisms of resistance to betalactams
(and to other antibiotics) in 203 ampicillin-resistant (AMPR) S. enterica isolates
obtained from hospitals of five regional communities (including both previously mentioned).
Susceptibility to amoxicillin-clavulanic acid (AMCS) was detected in 79 of these isolates, and
reduced susceptibility or resistance (AMCI/R) in 124 additional isolates. The blaTEM-1 gene was
basically identified among AMPR-AMCS isolates and the blaOXA-1 or blaPSE-1 genes among AMPR-
AMCI/R ones. Class 1 integrons were detected in the 59% of AMPR isolates, showing 12 different
structures, and the aadA2/blaPSE-1 (55%) and blaOXA-1-aadA1 (32%) being the main ones. Seven
class 1 integrons, four of them lacking the 3'-conserved region, harbored the trimethoprim
resistance dfr genes. The estX+psp+aadA2+cmlA1+aadA1+qacH+IS440+sul3+orf1+mef(B)?IS26
integron was detected in a S. Typhimurium isolate, and the In37 integron carrying the
aac(6')-Ib-cr+blaOXA-1+catB3+arr3 structure and the unusual PcWTGN-10 promoter was also
detected in a S. Thompson isolate. The ACSSuT penta-resistance phenotype was associated
with the major genotypes blaPSE-1-floR-aadA2-sul1-tet(G) or blaOXA-1-catA-aadA1/strA-strB-sultet (B).
The third objective was focused on the characterization of 65 blaPSE-1-positive isolates,
all of them S. Typhimurium, obtained in the previous part. The Salmonella Genomic Island type 1 (SGI1), carrying blaPSE-1, floR, aadA2, sul1 and tet(G) genes, was identified in all the strains, and a new variant was detected (GenBank JF775513). All the blaPSE-1-positive strains displayed indistinguishable or closely related pulsotypes (PFGE, XbaI, SpeI enzymes), and were assigned to the sequence type ST19 (Clonal Complex CC1). The detection of virulence genes grouped the strains in three virulotypes. An 89% of them showed the same profile and included the genes located in pathogenicity islands (SPI 1-5), prophage related genes and the virulence plasmid.
The fourth objective was to study 11 isolates that showed an extended-spectrum betalactamase (ESBL) or AmpC phenotype. The associated genes were the following ones: blaCTX-M-9 (serotype Virchow, 2 isolates), blaCTX-M-10 (Virchow, 2), blaCTX-M-14a (Enteritidis, 1), blaCTX-M-15 (Gnesta, 1, and S. enterica group C, 1), blaSHV-2 (Livingstone, 1), blaSHV-12 (Enteritidis, 1) and blaCMY-2 (Bredeney, 2). The IncI1 or IncA/C plasmids carried the blaCTX-M-14a, blaCTX-M-15, blaSHV-2, blaSHV-12 or blaCMY-2 genes. Whereas the blaCTX-M-9 gene, included in the In60 complex integron, and the blaCTX-M-10 gene, located in a phage related environment, were found in non-typeable plasmids. The conjugative transfer of ESBL/AmpC genes was successful in 8 of the 11 strains, co-transferring in most of the cases other additional resistance genes. The stability of the ESBL/AmpC phenotype was evaluated after 100 daily passages in the absence of antibiotic selection pressure. Five of the analyzed strains, carrying the blaCTX-M-14a, blaCTX M-15, blaSHV-2, blaSHV-12 and blaCMY-2 genes, lost the plasmidic copy of the beta-lactamase gene. In two of these, the complete loss of the IncI1 plasmid harboring the blaCMY-2 or blaSHV-12 genes was observed.
Other resistance genes, such as tet(A), tet(B), and the dfrA12- and dfrA16-positive integrons, were lost in two additional strains.
The fifth objective was to characterize in detail the single ciprofloxacin resistant isolate
(S. Typhimurium Se20) found in this thesis. It belonged to an in vivo selection of
fluoroquinolones and aminoglycosides resistance case report, associated with the
aac(6')-Ib-cr4 gene acquisition, after a ciprofloxacin treatment during 7 days. After checking by
PFGE and MLST that both pre- and post-treatment strains belonged to the same clone, the
characterization of the molecular resistance mechanisms involved was performed. The genetic
location of the resistance determinants, the plasmids and the in vitro conjugative transference were assessed. This work is the first description of in vivo selection of resistance to fluoroquinolones and aminoglycosides in a qnrS1-positive S. Typhimurium strain mediated by the acquisition of the aac(6')-Ib-cr4 gene and a substitution in the GyrA protein. Finally, the small acc(6')-Ib-cr4-carrying plasmid, named as pMdT1 (GenBank JX457478) was fully characterized. |
| author2 |
Torres Manrique, Carmen (Universidad de La Rioja) |
| author_facet |
Torres Manrique, Carmen (Universidad de La Rioja) Toro Hernando, María de |
| format |
text (thesis) |
| author |
Toro Hernando, María de |
| spellingShingle |
Toro Hernando, María de Resistencia a beta-lactámicos y fluoroquinolonas en Salmonella enterica. Mecanismos moleculares y elementos de movilización génica |
| author_sort |
Toro Hernando, María de |
| title |
Resistencia a beta-lactámicos y fluoroquinolonas en Salmonella enterica. Mecanismos moleculares y elementos de movilización génica |
| title_short |
Resistencia a beta-lactámicos y fluoroquinolonas en Salmonella enterica. Mecanismos moleculares y elementos de movilización génica |
| title_full |
Resistencia a beta-lactámicos y fluoroquinolonas en Salmonella enterica. Mecanismos moleculares y elementos de movilización génica |
| title_fullStr |
Resistencia a beta-lactámicos y fluoroquinolonas en Salmonella enterica. Mecanismos moleculares y elementos de movilización génica |
| title_full_unstemmed |
Resistencia a beta-lactámicos y fluoroquinolonas en Salmonella enterica. Mecanismos moleculares y elementos de movilización génica |
| title_sort |
resistencia a beta-lactámicos y fluoroquinolonas en salmonella enterica. mecanismos moleculares y elementos de movilización génica |
| publisher |
Universidad de La Rioja (España) |
| publishDate |
2013 |
| url |
https://dialnet.unirioja.es/servlet/oaites?codigo=40435 |
| work_keys_str_mv |
AT torohernandomariade resistenciaabetalactamicosyfluoroquinolonasensalmonellaentericamecanismosmolecularesyelementosdemovilizaciongenica |
| _version_ |
1718346611978928128 |
| spelling |
oai-TES00000056892019-06-21Resistencia a beta-lactámicos y fluoroquinolonas en Salmonella enterica. Mecanismos moleculares y elementos de movilización génicaToro Hernando, María deSalmonella enterica is an important zoonotic pathogen frequently implicated in human foodborne infections. The emergency of clinical isolates resistant to antibiotics involves serious limits for their treatments. The first objective of this thesis was to study the resistance phenotype and its relation with the serotype in 280 S. enterica isolates obtained from Hosp. San Pedro in Logrono (2007-2009) and Hosp. Clinico Universitario Lozano Blesa in Zaragoza (2009-2010). The main serotypes were Typhimurium (52%) and Enteritidis (33%), being S.Typhimurium highly associated with multi-resistance phenotypes, and in particular with resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline (ACSSuT, 29.5%). Low resistance percentages of resistance to ciprofloxacin or third generation cephalosporins were observed. The second objective was to characterize the mechanisms of resistance to betalactams (and to other antibiotics) in 203 ampicillin-resistant (AMPR) S. enterica isolates obtained from hospitals of five regional communities (including both previously mentioned). Susceptibility to amoxicillin-clavulanic acid (AMCS) was detected in 79 of these isolates, and reduced susceptibility or resistance (AMCI/R) in 124 additional isolates. The blaTEM-1 gene was basically identified among AMPR-AMCS isolates and the blaOXA-1 or blaPSE-1 genes among AMPR- AMCI/R ones. Class 1 integrons were detected in the 59% of AMPR isolates, showing 12 different structures, and the aadA2/blaPSE-1 (55%) and blaOXA-1-aadA1 (32%) being the main ones. Seven class 1 integrons, four of them lacking the 3'-conserved region, harbored the trimethoprim resistance dfr genes. The estX+psp+aadA2+cmlA1+aadA1+qacH+IS440+sul3+orf1+mef(B)?IS26 integron was detected in a S. Typhimurium isolate, and the In37 integron carrying the aac(6')-Ib-cr+blaOXA-1+catB3+arr3 structure and the unusual PcWTGN-10 promoter was also detected in a S. Thompson isolate. The ACSSuT penta-resistance phenotype was associated with the major genotypes blaPSE-1-floR-aadA2-sul1-tet(G) or blaOXA-1-catA-aadA1/strA-strB-sultet (B). The third objective was focused on the characterization of 65 blaPSE-1-positive isolates, all of them S. Typhimurium, obtained in the previous part. The Salmonella Genomic Island type 1 (SGI1), carrying blaPSE-1, floR, aadA2, sul1 and tet(G) genes, was identified in all the strains, and a new variant was detected (GenBank JF775513). All the blaPSE-1-positive strains displayed indistinguishable or closely related pulsotypes (PFGE, XbaI, SpeI enzymes), and were assigned to the sequence type ST19 (Clonal Complex CC1). The detection of virulence genes grouped the strains in three virulotypes. An 89% of them showed the same profile and included the genes located in pathogenicity islands (SPI 1-5), prophage related genes and the virulence plasmid. The fourth objective was to study 11 isolates that showed an extended-spectrum betalactamase (ESBL) or AmpC phenotype. The associated genes were the following ones: blaCTX-M-9 (serotype Virchow, 2 isolates), blaCTX-M-10 (Virchow, 2), blaCTX-M-14a (Enteritidis, 1), blaCTX-M-15 (Gnesta, 1, and S. enterica group C, 1), blaSHV-2 (Livingstone, 1), blaSHV-12 (Enteritidis, 1) and blaCMY-2 (Bredeney, 2). The IncI1 or IncA/C plasmids carried the blaCTX-M-14a, blaCTX-M-15, blaSHV-2, blaSHV-12 or blaCMY-2 genes. Whereas the blaCTX-M-9 gene, included in the In60 complex integron, and the blaCTX-M-10 gene, located in a phage related environment, were found in non-typeable plasmids. The conjugative transfer of ESBL/AmpC genes was successful in 8 of the 11 strains, co-transferring in most of the cases other additional resistance genes. The stability of the ESBL/AmpC phenotype was evaluated after 100 daily passages in the absence of antibiotic selection pressure. Five of the analyzed strains, carrying the blaCTX-M-14a, blaCTX M-15, blaSHV-2, blaSHV-12 and blaCMY-2 genes, lost the plasmidic copy of the beta-lactamase gene. In two of these, the complete loss of the IncI1 plasmid harboring the blaCMY-2 or blaSHV-12 genes was observed. Other resistance genes, such as tet(A), tet(B), and the dfrA12- and dfrA16-positive integrons, were lost in two additional strains. The fifth objective was to characterize in detail the single ciprofloxacin resistant isolate (S. Typhimurium Se20) found in this thesis. It belonged to an in vivo selection of fluoroquinolones and aminoglycosides resistance case report, associated with the aac(6')-Ib-cr4 gene acquisition, after a ciprofloxacin treatment during 7 days. After checking by PFGE and MLST that both pre- and post-treatment strains belonged to the same clone, the characterization of the molecular resistance mechanisms involved was performed. The genetic location of the resistance determinants, the plasmids and the in vitro conjugative transference were assessed. This work is the first description of in vivo selection of resistance to fluoroquinolones and aminoglycosides in a qnrS1-positive S. Typhimurium strain mediated by the acquisition of the aac(6')-Ib-cr4 gene and a substitution in the GyrA protein. Finally, the small acc(6')-Ib-cr4-carrying plasmid, named as pMdT1 (GenBank JX457478) was fully characterized.Salmonella enterica es un importante patogeno zoonotico frecuentemente implicado en toxiinfecciones alimentarias y la emergencia de aislados clinicos resistentes a los antibioticos supone graves limitaciones para su tratamiento. Por ello, el primer objetivo de esta tesis fue estudiar el fenotipo de resistencia a antibioticos y su relacion con el serotipo en los 280 aislados de S. enterica recogidos en el Hosp. San Pedro de Logrono (2007-2009) y Hosp. Clinico Universitario Lozano Blesa de Zaragoza (2009-2010). Los serotipos mayoritarios fueron Typhimurium (52%) y Enteritidis (33%), estando S. Typhimurium altamente asociado con fenotipos de multirresistencia y especificamente con la resistencia a ampicilina, cloranfenicol, estreptomicina, sulfamidas y tetraciclina (ACSSuT, 29,5%). Se detectaron bajos porcentajes de resistencia a ciprofloxacina o cefalosporinas de tercera generacion. El segundo objetivo fue caracterizar los mecanismos de resistencia a beta-lactamicos (y a otros antibioticos) en los 203 aislados de S. enterica resistentes a ampicilina (AMPR) obtenidos en hospitales de cinco comunidades autonomas (incluidos los dos anteriormente indicados). Se detecto sensibilidad a amoxicilina-acido clavulanico (AMCS) en 79 de estos aislados y sensibilidad intermedia o resistencia (AMCI/R) en 124 aislados. El gen blaTEM-1 se identifico fundamentalmente en los aislados con fenotipo AMPR-AMCS y los genes blaOXA-1 o blaPSE-1 en los aislados AMPR-AMCI/R. En el 59% de los aislados AMPR se detectaron integrones de clase 1 con 12 estructuras distintas, siendo mayoritarias las estructuras aadA2/blaPSE-1 (55%) y blaOXA-1-aadA1 (32%). Siete integrones de clase 1, cuatro de ellos carentes de la region 3'- conservada, albergaban genes dfr de resistencia a trimetoprim. El integron estX+psp+aadA2+cmlA1+aadA1+qacH+IS440+sul3+orf1+mef(B)?IS26 se detecto en un aislado S. Typhimurium y el integron In37 de estructura aac(6')-Ib-cr+blaOXA-1+catB3+arr3 con el promotor PcWTGN-10 inusual en un aislado de S. Thompson. Se observo el fenotipo de pentarresistencia ACSSuT, asociado a los genotipos mayoritarios blaPSE-1-floR-aadA2-sul1-tet(G) o blaOXA-1-catA-aadA1/strA-strB-sul-tet(B). El tercer objetivo se centro en caracterizar los 65 aislados blaPSE-1-positivos, todos ellos S. Typhimurium, obtenidos en el objetivo anterior. La Isla Genomica de Salmonella de tipo 1 (SGI1), portadora de los genes blaPSE-1, floR, aadA2, sul1 y tet(G), se detecto en todos los aislados, identificandose una nueva variante de SGI1 (GenBank JF775513). Todas las cepas blaPSE-1-positivas presentaron pulsotipos indistinguibles o altamente relacionados (PFGE, enzimas XbaI, SpeI) y fueron adscritas a la secuencia tipo ST19 (Complejo Clonal CC1). La deteccion de genes de virulencia agrupo a las cepas en tres virulotipos; detectandose el mayoritario en el 89% de las mismas e incluyendo genes localizados en islas de patogenicidad, en el plasmido de virulencia o relacionados con profagos. El cuarto objetivo fue estudiar los 11 aislados que presentaban un fenotipo de betalactamasa de espectro extendido (BLEE) o AmpC y que se asociaron a los genes blaCTX-M-9 (serotipo Virchow, 2 aislados), blaCTX-M-10 (Virchow, 2), blaCTX-M-14a (Enteritidis, 1), blaCTX-M-15 (Gnesta, 1, y S. enterica grupo C, 1), blaSHV-2 (Livingstone, 1), blaSHV-12 (Enteritidis, 1) y blaCMY-2 (Bredeney, 2). Plasmidos de tipo IncI1 o IncA/C portaban los genes blaCTX-M-14a, blaCTX-M-15, blaSHV-2, blaSHV-12 o blaCMY-2; mientras que el gen blaCTX-M-9, incluido en un integron complejo In60, y el gen blaCTX-M-10, presente en un entorno relacionado con fagos, se encontraron en plasmidos no tipables. La transferencia por conjugacion de los genes BLEE/AmpC resulto positiva en 8 de las 11 cepas estudiadas, cotransfiriendo otros genes de resistencia adicionales en la mayoria de los casos. Se realizaron experimentos de estabilidad del fenotipo BLEE/AmpC tras 100 pases consecutivos en ausencia de presion selectiva antibiotica. Cinco de las cepas analizadas, portadoras de los genes de resistencia blaCTX-M-14a, blaCTX-M-15, blaSHV-2, blaSHV-12 y blaCMY-2, perdieron la copia plasmidica del gen. En dos de estos casos se evidencio la perdida completa del plasmido IncI1 portador del gen blaCMY-2 o blaSHV-12. Otros genes de resistencia, tales como tet(A), tet(B), y los integrones portadores de los genes dfrA12 y dfrA16, se perdieron en dos cepas adicionales. El quinto objetivo fue la caracterizacion en profundidad del unico aislado (S. Typhimurium Se20) resistente a ciprofloxacina obtenido en esta tesis. Correspondio a un caso clinico de seleccion in vivo de resistencia a fluoroquinolonas y aminoglucosidos, asociado a la adquisicion gen aac(6')-Ib-cr4, tras 7 dias de tratamiento con ciprofloxacina. Tras comprobar mediante PFGE y MLST que las cepas pre- y post-tratamiento pertenecian al mismo clon, se realizo la caracterizacion de los mecanismos moleculares de resistencia implicados. Se determino la localizacion genetica de los determinantes de la resistencia, detectando los plasmidos portadores, y se comprobo mediante experimentos in vitro la transferencia por conjugacion de los mismos. Este trabajo supone la primera evidencia de seleccion in vivo de resistencia a fluoroquinolonas y aminoglucosidos en S. Typhimurium portadora del gen qnrS1 y aac(6')-Ib-cr4 y con mutaciones en la proteina GyrA. Por ultimo, se caracterizo el plasmido de pequeno tamano portador del gen aac(6')-Ib-cr4, que se denomino pMdT1 (GenBank JX457478).Universidad de La Rioja (España)Torres Manrique, Carmen (Universidad de La Rioja)Sáenz Domínguez, Yolanda (Universidad de La Rioja)2013text (thesis)application/pdfhttps://dialnet.unirioja.es/servlet/oaites?codigo=40435spaLICENCIA DE USO: Los documentos a texto completo incluidos en Dialnet son de acceso libre y propiedad de sus autores y/o editores. Por tanto, cualquier acto de reproducción, distribución, comunicación pública y/o transformación total o parcial requiere el consentimiento expreso y escrito de aquéllos. Cualquier enlace al texto completo de estos documentos deberá hacerse a través de la URL oficial de éstos en Dialnet. Más información: https://dialnet.unirioja.es/info/derechosOAI | INTELLECTUAL PROPERTY RIGHTS STATEMENT: Full text documents hosted by Dialnet are protected by copyright and/or related rights. 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