Abstract
BackgroundNalidixic acid resistance among Salmonella Typhimurium clinical isolates has steadily increased, whereas the level of ciprofloxacin resistance remains low. The main objective of this study was to characterize the fluoroquinolone resistance mechanisms acquired in a S. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability.Methodology/Principal FindingsThree different amino acid substitutions were detected in the quinolone target proteins of the resistant mutant (MIC of ciprofloxacin, 64 µg/ml): D87G and G81C in GyrA, and a novel mutation, E470K, in ParE. A protein analysis revealed an increased expression of AcrAB/TolC and decreased expression of OmpC. Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis. A decreased percentage of invasion ability was detected when compared with the susceptible clinical isolate in a gentamicin protection assay. The microarray results revealed a decreased expression of genes which play a role during the invasion process, such as hilA, invF and the flhDC operon. Of note was the impaired growth detected in the resistant strain. A strain with a reverted phenotype (mainly concerning the resistance phenotype) was obtained from the resistant mutant.Conclusions/SignificanceIn conclusion, a possible link between fluoroquinolone resistance and decreased cell invasion ability may exist explaining the low prevalence of fluoroquinolone-resistant S. Typhimurium clinical isolates. The impaired growth may appear as a consequence of fluoroquinolone resistance acquisition and down-regulate the expression of the invasion genes.
Highlights
Salmonella enterica is a Gram-negative facultative intracellular anaerobe of worldwide importance causing gastroenteritis in humans after ingestion of contaminated food or water
Salmonella pathogenicity islands (SPIs)-1 [5] and SPI-2 [6] have been reported to encode the specific machinery that delivers the effectors into the cytoplasm of the eukaryotic cells; these are the socalled type 3 secretion systems (T3SS) which play a central role in the Salmonella-host interaction [7]
In order to study the whole process of high-level fluoroquinolone resistance acquisition, intermediate mutants (50-0.007, 50-0.015, 50-0.03, 50-0.6, 50-0.25, 50-2 and 50-16) of this stepwise selection procedure were included
Summary
Salmonella enterica is a Gram-negative facultative intracellular anaerobe of worldwide importance causing gastroenteritis in humans after ingestion of contaminated food or water. Serovars Enteritidis and Typhimurium are the most frequently isolated among the more than 2500 serovars characterized in Salmonella enterica [1,2]. Upon colonization of the intestine by virulent strains, bacteria localize to the apical epithelium and induce invasionassociated virulence machinery [2]. Most of these virulence genes are organized within particular regions of the genome, termed pathogenicity islands, which are regulated by complex regulatory networks: the delicate balance of expression of many genes acting at the correct time in the correct location [3,4]. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability
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