Caracterización del entorno genético de genes blaBLEE y plásmidos asociados en cepas circulantes de Escherichia coli y Klebsiella pneumoniae en España.

Student thesis: Doctoral thesis

Abstract

Extended-spectrum beta-lactamases (ESBL), described frequently in Escherichia coli, Klebsiella pneumoniae confer resistance to extended spectrum cephalosporins and monobactams. ESBL are a heterogeneous group of enzymes that were initially described in hospital outbreaks of K. pneumoniae, and derivates of the SHV-1 and TEM-1 were the most common. In recent years, detection of the the CTX-M type has become more frequent. The spread of ESBL in bacteria of the same or different species has been associated with genetic elements such as insertion sequences, transposons and plasmids. The increase in the prevalence of ESBL-producing microorganisms has been reported by several authors, both in Spain and internationally. For this reason, the Spanish Network for Research on Infectious Pathology (REIPI) recognized the need for a multicenter study. Based on the findings, it was possible to identify and update the progress and the type of ESBL in E. coli and K. pneumoniae in several Spanish hospitals. Furthermore, molecular studies ruled out the possible clonal spread of ESBL-producing microorganisms. The present study was conducted as Project No. 3 (Clinical epidemiology and molecular characterization of ESBL-producing enterobacteria) of the REIPI. It involved four centers: Hospital Ramon y Cajal, Hospital Universitario Son Dureta, National Center for Microbiology and Hospital de la Santa Creu i Sant Pau. A total of 92 strains of E. coli and 32 K. pneumoniae isolated from 11 Spanish hospitals in 2004 were analyzed. The clonal relationship between strains was ruled out by genomic studies comparing the XbaI-PFGE patterns. The ESBL were characterized by isoelectric focusing, PCR and sequencing. E. coli showed a high clonal diversity in the restriction patterns obtained by PFGE, and a predominance of the enzymes CTX-M-14 (45. 7%), CTX-M-9 (20. 6%) and SHV-12 (21. 7%), whereas K. pneumoniae had a lower clonal diversity with a predominance of the enzymes CTX-M type (62. 5%) involving CTX-M-1, CTX-M-9, CTXM- 14 and CTX-M-15. We then studied the genetic environments and plasmid profiles of 58 strains of E. coli and K. pneumoniae and determined the incompatibility group of plasmids by rep typing, S1 enzyme digestion, pulsed field gel electrophoresis, southern-blot and hybridization with specific probes for each incompatibility group. As a result there was a large variability of plasmids containing blaESBL genes associated with different genetic environments. The gene with the greatest plasmid variability was blaCTX-M-9, associated to Inc groups I1, HI2, FIB and K. The blaSHV-12 gene was found in plasmids of groups Inc I1, FII, K and HI2. However, blaCTX-M-14 was found only on IncK plasmids. In a third phase of the study, the National Microbiology Center in Spain performed the Multi Locus Sequence Typing (MLST) and determined the phylogenetic groups of strains of E. coli to determine the possible association between ST, phylogenetic groups and ESBL. A wide variety of MLST were detected, and the most common patterns were ST131 and ST10 and ST23 complexes. In addition, strains of E. coli ST131 carried a variety of betalactamase (CTX-M-15, CTX-M-9, CTX-M-10, CTX-M-14, SHV-12 and CTX-M-1). Regarding the phylogenetic groups, we found that ST10 and ST23 complexes were linked to phylogenetic group A, whereas the pattern ST131 was related to the virulent extraintestinal group B2. In conclusion, this study identified a greater variety of ESBL than observed in the multicenter study of 2000. These ESBL were transported by genetic elements such as plasmids and insertion sequences which were very diverse within the group of strains studied. We did not detect an association between the different types of MLST, the filogenetic groups and the ESBL-producing E. coli.
Date of Award29 Oct 2010
Original languageSpanish
SupervisorFerran Navarro Risueño (Director)

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