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A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility.

ABSTRACT: OBJECTIVES:A whole-genome screen at sub-gene resolution was performed to identify candidate loci that contribute to enhanced or diminished ciprofloxacin susceptibility in Salmonella enterica serovar Typhi. METHODS:A pool of over 1 million transposon insertion mutants of an S. Typhi Ty2 derivative were grown in a sub-MIC concentration of ciprofloxacin, or without ciprofloxacin. Transposon-directed insertion site sequencing (TraDIS) identified relative differences between the mutants that grew following the ciprofloxacin treatment compared with the untreated mutant pool, thereby indicating which mutations contribute to gain or loss of ciprofloxacin susceptibility. RESULTS:Approximately 88% of the S. Typhi strain's 4895 annotated genes were assayed, and at least 116 were identified as contributing to gain or loss of ciprofloxacin susceptibility. Many of the identified genes are known to influence susceptibility to ciprofloxacin, thereby providing method validation. Genes were identified that were not known previously to be involved in susceptibility, and some of these had no previously known phenotype. Susceptibility to ciprofloxacin was enhanced by insertion mutations in genes coding for efflux, other surface-associated functions, DNA repair and expression regulation, including phoP, barA and marA. Insertion mutations that diminished susceptibility were predominantly in genes coding for surface polysaccharide biosynthesis and regulatory genes, including slyA, emrR, envZ and cpxR. CONCLUSIONS:A genomics approach has identified novel contributors to gain or loss of ciprofloxacin susceptibility in S. Typhi, expanding our understanding of the impact of fluoroquinolones on bacteria and of mechanisms that may contribute to resistance. The data also demonstrate the power of the TraDIS technology for antibacterial research.

SUBMITTER: Turner AK

PROVIDER: S-EPMC7443733 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility.

Turner A Keith AK Eckert Sabine E SE Turner Daniel J DJ Yasir Muhammud M Webber Mark A MA Charles Ian G IG Parkhill Julian J Wain John J

The Journal of antimicrobial chemotherapy 20200901 9

<h4>Objectives</h4>A whole-genome screen at sub-gene resolution was performed to identify candidate loci that contribute to enhanced or diminished ciprofloxacin susceptibility in Salmonella enterica serovar Typhi.<h4>Methods</h4>A pool of over 1 million transposon insertion mutants of an S. Typhi Ty2 derivative were grown in a sub-MIC concentration of ciprofloxacin, or without ciprofloxacin. Transposon-directed insertion site sequencing (TraDIS) identified relative differences between the mutant ...[more]

PMID: 32514543

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Project description:BackgroundTyphoid fever is an acute systemic infection of humans caused by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). In chronic carriers, the bacteria survive the harsh environment of the gallbladder by producing biofilm. The phenotype of S. Typhi biofilm cells is significantly different from the free-swimming planktonic cells, and studies have shown that they are associated with antibiotic resistance, immune system evasion, and bacterial persistence. However, the mechanism of this transition and the events leading to biofilm formation are unknown. High throughput sequencing was performed to identify the genes involved in biofilm formation and to postulate the mechanism of action.ResultsPlanktonic S. Typhi cells were cultured using standard nutrient broth whereas biofilm cells were cultured in a stressful environment using high shearing-force and bile to mimic the gallbladder. Sequencing libraries were prepared from S. Typhi planktonic cells and mature biofilm cells using the Illumina HiSeq 2500 platform, and the transcriptome data obtained were processed using Cufflinks bioinformatics suite of programs to investigate differential gene expression between the two phenotypes. A total of 35 up-regulated and 29 down-regulated genes were identified. The identities of the differentially expressed genes were confirmed using NCBI BLAST and their functions were analyzed. The results showed that the genes associated with metabolic processes and biofilm regulations were down-regulated while those associated with the membrane matrix and antibiotic resistance were highly up-regulated.ConclusionsIt is proposed that the biofilm phenotype of S. Typhi allows the bacteria to increase production of the membrane matrix in order to serve as a physical shield and to adhere to surfaces, and enter an energy conservation state in response to the stressful environment. Conversely, the planktonic phenotype allows the bacteria to produce flagella and increase metabolic activity to enable the bacteria to migrate and form new colonies of infection. This data provide a basis for further studies to uncover the mechanism of biofilm formation in S. Typhi and to discover novel genes or pathways associated with the development of the typhoid carrier state.

Dataset Information

A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility.

Publications

A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility.

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