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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| Lenguaje: | spa |
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Universidad de La Rioja (España)
2013
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| Acceso en línea: | https://dialnet.unirioja.es/servlet/oaites?codigo=40435 |
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| Sumario: | 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. |
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