Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.

Cepas com altas taxas de mutação (mutadoras) foram detectadas em diversos gêneros bacterianos. A alta taxa de mutação está relacionada a defeitos em sistemas de reparo de DNA. Uma alta incidência de isolados clínicos de Proteus mirabilis com altas frequências de mutação foi descrita anteriormente. O fenômeno foi induzido em Escherichia coli, quando transformada com um plasmídeo de P. mirabilis. Com coleção de 77 isolados clínicos de P. mirabilis, medimos a frequência de mutantes espontâneos e verificamos a presença do elemento conjugativo ICE SXT/R391, para desvendar possível relação entre a presença do ICE e a frequência de mutação. 9 isolados clínicos apresentam o ICE. A frequência de mutantes mostrou que não existem mutadores verdadeiros, mas 11 isolados apresentam uma alta frequência de mutantes FosR. Considerando o alto índice de infecções por P. mirabilis, é importante entender a resistência à fosfomicina, já que esta é usada na clínica. Não existe relação entre uma frequência de mutantes espontânea e a presença de ICE SXT/R391 em isolados de P. mirabilis. / Strains with high mutation rates (mutators) were detected in several bacterial genera. The increased mutation rate is related to defects in DNA repair systems. A high incidence of Proteus mirabilis clinical isolates with high mutation frequencies were described previously. The phenomenon was induced in Escherichia coli, when transformed with a plasmid of P. mirabilis. 77 P. mirabilis clinical isolates were tested for the frequency of spontaneous mutants and the presence of a conjugative element found in this species, ICEs SXT/R391, to verify if there is a relation between the element and the mutation frequencies. 9 isolates carry the ICE SXT/R391. The frequency of mutants showed no true mutators among the isolates. 11 isolates show a high frequency of FosR mutants. Considering the high rate of infections by P. mirabilis, it is important to understand the fosfomycin phenomenon, since it is currently used to treat urinary infections. We have seen no relation between a high spontaneous mutation frequency and the presence of ICE SXT/R391 in isolates of P. mirabilis.