Molecular diversity of bla genes in Klebsiella pneumoniae and Escherichia coli isolates

Dissertation presented to obtain a Ph.D. degree in Biology, speciality Microbiology, by Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Antimicrobial resistance is a growing problem worldwide, with extended-spectrum β- lactamase (ESBL)-producing organisms remaining an important cause of cephalosporin therapy failure. The main purpose of the work presented in this dissertation was to search for the molecular diversity of Ambler class A β-lactamase encoding genes in clinical Klebsiella pneumoniae and Escherichia coli isolates and its consequences. In a first step, the evaluation of ESBL detection and confirmation methods for K. pneumoniae, using different guidelines, was performed, with nucleotide sequencing allowing the identification of a new ESBL (SHV-55). The enzymatic properties of this new β-lactamase confirmed a higher affinity towards extended-spectrum cephalosporins, which is characteristic of ESBLs, contrasting to the parental enzyme SHV-1. For the new β-lactamase SHV-72, the higher value of 50% inhibitory concentration of clavulanic acid than for SHV-1 correlated with the values of higher afinity towards penicillins,which are characteristic of an inhibitor resistance enzyme. Simulation of molecular dynamics suggested that the Lys234Arg substitution in SHV-72 was responsible for stabilizing an atipical conformation of the Ser130 side chain, which may decrease susceptibility to clavulanic acid by preventing cross-linking between Ser130 and Ser70. Among the collection of K. pneumoniae strains studied, we identified several genes coding for different enzymes belonging to the CTX-M, GES, LEN, OKP, OXA, TEM, and SHV families and, among them, 35 were new β-lactamases. Among ESBL-producing isolates from 1999 and 2006, we detected the presence of CTX-M enzymes only in the latest period. Overall, the complex molecular diversity of the blaSHV genes detected impelled us to propose a classification for this gene family, based on nucleotide synonymous mutations and the presence or absence of the nonsynonymous mutation T92A. Finally, among E. coli isolates colected in Portugal, a predominat multidrug resistant clone, producing TEM, OXA and CTX-M enzymes, was present in different hospital wards and community environments. The high dissemination of the CTX-M enzymes detected could also be associated with horizontal transfer of plasmids or mobile elements, like ISEcp1. In Algeria, we identified the same insertion sequence in clinical E. coli strains producing TEM and either CTX-M-3 or CTX-M-15 β-lactamases. In conclusion, the results from this dissertation extended our knowledge about the most important mechanism of resistance – the β-lactamases production – focusing on the phenotypic characteristics, biochemical properties, structure-function relationships, molecular diversity and dissemination, which are of most importance to the comprehension of the general resistance scenario in Portugal and worldwide.