Detección y bases genéticas de beta-lactamasas AmpC y carbapenemasas en aislados clínicos y comensales de enterobacterias stars

Abstract

Microorganisms of the Enterobacteriaceae family are opportunistic pathogens that normally are part of the human and other mammal intestinal microbiota. Some enterobacteria species possess intrinsic AmpC beta-lactamase genes in their chromosome, and mutations in those genes can cause the increase in their spectrum of action. On the other hand, the enterobacteria are able to acquire resistance mechanisms to different antibiotic families. In the last years, concerns do exist about the spread of carbapenemase genes due to the difficulty to treat infections caused by carbapenemase expressing bacteria. In this thesis, the detection and characterization of AmpC beta-lactamase and carbapenemase genes have been carried out in enterobacteria isolates; as well as the development of new carbapenemase detection methods. The first objective was to isolate and to characterize enterobacteria from healthy human fecal samples. The resistance phenotype and the presence of AmpC and Extended spectrum beta-lactamases (ESBL) were analyzed in the 70 obtained islates. Resistance to ampicillin was detected in 57% of the isolates, 22 with AmpC phenotype and 3 with ESBL phenotype. Among the 22 isolates with AmpC phenotype, the species-specific ampC gene was detected in 21 isolates (13 Citrobacter freundii complex, 8 Enterobacter cloacae). Moreover, at least two copies of the blaCMY-2 gene were localized in an IncK conjugative plasmid of 78 Kb in an Escherichia coli ST405 isolate. The prevalence of enterobacteria with plasmidic ampC genes in healthy humans was of 1,4 %. The second objective was the characterization of enterobacteria from clinical origin. Eighty-one isolates suspicious of harbouring AmpC beta-lactamases or belonging to species that carry cAmpC, were studied. ampC genes were detected in 57 of these isolates, 8 of them of plasmid location (E. coli, Citrobacter, Proteus and Klebsiella) and with chromosomal location in the 49 remaining isolates (24 Citrobacter, 16 Enterobacter, 7 Morganella and 2 Proteus). The presence of other resistance genes was studied in all the isolates from both origins. The ESBL-positive isolates recovered from fecal samples of healthy humans carried blaSHV-12 (2 isolates) and blaCTX-M-15+blaOXA-1 (1) genes. A wide diversity of beta-lactam resistance genes was detected, as well as the presence of resistance genes to aminoglycosides, sulfamides, chloramphenicol, tetracyclines, rifampicin and quinolones. Class 1 integrons were detected in 20 isolates, with diverse gene cassette arrays; class 2 integrons were detected in 3 isolates. The third objective was to analyze the polymorphism in ampC and qnrB genes among the Citrobacter, Enterobacter and Morganella genera, as well as to study the clonal diversity of isolates of both origins. A high degree of polymorphism of ampC genes was detected, as well as in the ampR gene and in the ampR/ampC intergenic regions. A total of 29 CMY variants, 15 ACT, 4 DHA and 2 MIR were detected. Among the detected variants, 43 had not been previously described, for this reason they were included in GenBank and www.lahey.org/Studies. The isolates of these genera, not only showed a high varieties ty in these genes, but also diversity of clones among the isolates. Moreover, 12 qnrB variants were detected among the 18 positive-qnrB-Citrobacter isolates (5/13 of fecal samples/clinical samples, respectively). The qnrB50 variant has been described for the first time in this thesis. The fourth objective was to carry out the biochemical characterization of ESAC betalactamases, as well as to study the avibactam effect on isolates with ESAC phenotype. The study determined that Asn298 deletion was the responsible of ESAC phenotype. This deletion did not affect the beta-lactamase inhibition by avibactam. The changes in aminoacid sequence in the studied ESAC beta-lactamases did not modify the inhibition effect of avibactam. The fifth objective was to characterize the carbapenem resistance mechanisms among carbapenem resistant enterobacteria including two clinical cases of special relevance. The first clinical case was due to an infection by a multiresistant E. coli ST448/B1 that had 6 betalactamase encoding genes (blaVIM-1, blaKPC-3, blaSHV-12, blaOXA-9, blaTEM-1a and blaCMY-2), as well as other 11 resistance genes and the virulence factor fimA gene. This isolate harbored the new class 1 integron In916, non previously reported.The second case was related to a coinfection in a patient by carbapenem resistant C. freundii and P. aeruginosa isolates. Both isolates carried the blaVIM-2 gene into class 1 integrons, but with different structures and promoters among them. This study is the first description of a blaVIM-2-carrying C. freundii isolate in Spain. Finally, the sixth objective was the generation of a phenotypic carbapenemase detection protocol. In this study, a new algorithm for the phenotypic carbapenemase detection is proposed. This algorithm includes two new phenotypic methods that are based on the use of inhibitors and that allows the differentiation of the diverse carbapenemases types, including OXA-48.