Genome-wide assessment of antimicrobial tolerance in Yersinia pseudotuberculosis under ciprofloxacin stress.

Samuel Willcocks,Kristin K Huse,Andrew Scott,Richard Stabler,Helen S Atkins,Petra C F Oyston,Brendan W Wren

doi:10.1099/mgen.0.000304

Abstract

Yersinia pseudotuberculosis is a Gram-negative bacterium capable of causing gastrointestinal infection and is closely related to the highly virulent plague bacillus Yersinia pestis . Infections by both species are currently treatable with antibiotics such as ciprofloxacin, a quinolone-class drug of major clinical importance in the treatment of many other infections. Our current understanding of the mechanism of action of ciprofloxacin is that it inhibits DNA replication by targeting DNA gyrase, and that resistance is primarily due to mutation of this target site, along with generic efflux and detoxification strategies. We utilized transposon-directed insertion site sequencing (TraDIS or TnSeq) to identify the non-essential chromosomal genes in Y. pseudotuberculosis that are required to tolerate sub-lethal concentrations of ciprofloxacin in vitro. As well as highlighting recognized antibiotic resistance genes, we provide evidence that multiple genes involved in regulating DNA replication and repair are central in enabling Y. pseudotuberculosis to tolerate the antibiotic, including DksA (yptb0734), a regulator of RNA polymerase, and Hda (yptb2792), an inhibitor of DNA replication initiation. We furthermore demonstrate that even at sub-lethal concentrations, ciprofloxacin causes severe cell-wall stress, requiring lipopolysaccharide lipid A, O-antigen and core biosynthesis genes to resist the sub-lethal effects of the antibiotic. It is evident that coping with the consequence(s) of antibiotic-induced stress requires the contribution of scores of genes that are not exclusively engaged in drug resistance.

Highlights

Yersinia pseudotuberculosis is a facultatively anaerobic, Gram- negative pathogen of humans and animals, capable of causing fever and acute gastrointestinal infection, yersiniosis, when transmitted by the faecal–oral route [1]
Most cases of enteric disease caused by Y. pseudotuberculosis are self-limiting and do not require treatment, there is some evidence that antibiotics can reduce faecal shedding, potentially reducing the risk of transmission [2, 3]
By sequencing a transposon library in Yersinia pseudotuberculosis subjected to sub-lethal concentrations of ciprofloxacin, we have identified all the genes required by the pathogen to tolerate this stress, as without functional versions of the gene(s), the mutants cannot survive

Summary

Introduction

Yersinia pseudotuberculosis is a facultatively anaerobic, Gram- negative pathogen of humans and animals, capable of causing fever and acute gastrointestinal infection, yersiniosis, when transmitted by the faecal–oral route [1]. Infection can result in septicaemia, which carries an associated risk of mortality [1]. Most cases of enteric disease caused by Y. pseudotuberculosis are self-limiting and do not require treatment, there is some evidence that antibiotics can reduce faecal shedding, potentially reducing the risk of transmission [2, 3]. There are other rare immunological complications associated with enteric yersiniosis [4], which is why there is a requirement for diagnosis of what is usually a self- limiting gastric infection. In cases with more serious clinical presentation, such as scarlet-like fever or sepsis, antibiotic therapy is required, usually with cefotaxime, ceftriaxone or ciprofloxacin

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