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Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5?.

ABSTRACT: A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5? and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5?. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5?. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5?, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5?, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5?.

SUBMITTER: Yassien MA

PROVIDER: S-EPMC4671525 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α.

Yassien M A M MA Elfaky M A MA

Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas 20150908 11

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study ...[more]

PMID: 26375447

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Project description:Typhoid 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.Planktonic 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.It 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

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5?.

Publications

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α.

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