Dataset Information

Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts.

ABSTRACT: The Shigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of Shigella colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence in Shigella flexneri strain 2457T following exposure to bile salts. Typical growth patterns were observed within the physiological range of bile salts; however, growth was inhibited at higher concentrations. Interestingly, extended periods of exposure to bile salts led to biofilm formation, a conserved phenotype that we observed among members of the Enterobacteriaceae Characterization of S. flexneri 2457T biofilms determined that both bile salts and glucose were required for formation, dispersion was dependent upon bile salts depletion, and recovered bacteria displayed induced adherence to HT-29 cells. RNA-sequencing analysis verified an important bile salt transcriptional profile in S. flexneri 2457T, including induced drug resistance and virulence gene expression. Finally, functional mutagenesis identified the importance of the AcrAB efflux pump and lipopolysaccharide O-antigen synthesis for bile salt resistance. Our data demonstrate that S. flexneri 2457T employs multiple mechanisms to survive exposure to bile salts, which may have important implications for multidrug resistance. Furthermore, our work confirms that bile salts are important physiological signals to activate S. flexneri 2457T virulence. This work provides insights into how exposure to bile likely regulates Shigella survival and virulence during host transit and subsequent colonic infection.

SUBMITTER: Nickerson KP

PROVIDER: S-EPMC5442615 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts.

Nickerson Kourtney P KP Chanin Rachael B RB Sistrunk Jeticia R JR Rasko David A DA Fink Peter J PJ Barry Eileen M EM Nataro James P JP Faherty Christina S CS

Infection and immunity 20170523 6

The Shigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of Shigella colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence in Shigella flexneri strain 2457T ...[more]

PMID: 28348056

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Project description:Berberine is a natural isoquinoline alkaloid found in Chinese medicinal herbs which is active against a variety of microbial infections. To examine the potential effects of berberine on Shigella flexneri, a whole-genome DNA microarray was constructed and transcriptome analysis of the cellular responses of S.flexneri when exposed to Berberine Chloride (BC) was performed. When cultures were grown to early exponential growth phase, drug was added. Meanwhile, DMSO solution without BC was added to control cultures to the same final concentration. At 30 min after treatment, samples were collected and washed twice with phosphate buffered saline at 25°C for subsequent RNA isolation. To prepare biological replicates for RNA isolation, each experiment was independently performed three times. After drug treatment, total RNA was extracted using SV RNA Isolation System (Promega). Reverse transcription of 20 ug RNA from each sample was performed with Superscript II system (Invitrogen) to prepare cDNA. According to the manufacturer's instructions, the cDNA was labeled with incorporation of fluorescent dyes (Amersham). Cy3-dCTP monofunctional dye was selected for control cDNA samples and Cy5-dCTP was used for drug-treated samples. Each pair of labeled cDNA was mixed and dried by vacuum centrifugation. The pairs of the labeled cDNA samples were hybridized to the DNA microarrays following the protocol available at http://www.ifr.ac.uk/safety/microarrays/. The slides were scanned by using a GenePix 4100A scanner. Fluorescent spots and local background intensities were quantified using GenePix Pro 6.0 software (Axon). Signal intensities were corrected by subtracting the local background value. The data set was filtered so that spots flagged as "not found," "bad," and "empty" were excluded from further analysis. Moreover, spots either with signal-to-noise ratio below the detection limit of 3 or containing less than 55% of pixels two standard deviations above background in both channels were discarded from the data set. Lowess normalization was performed with the fluorescent signal ratios (Cy5/Cy3) using MIDAS software (http://www.tigr.org/software/tm4) with a 0.33 smooth parameter.

Project description:ObjectiveTo investigate the prevalence and mechanisms of fluoroquinolone resistance in Shigella species isolated in Bangladesh and to compare with similar strains isolated in China.MethodsA total of 3789 Shigella isolates collected from Clinical Microbiology Laboratory of icddr,b, during 2004-2010 were analyzed for antibiotic susceptibility. Analysis of plasmids, plasmid-mediated quinolone-resistance genes, PFGE, and sequencing of genes of the quinolone-resistance-determining regions (QRDR) were conducted in representative strains isolated in Bangladesh and compared with strains isolated in Zhengding, China. In addition, the role of efflux-pump was studied by using the efflux-pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP).ResultsResistance to ciprofloxacin in Shigella species increased from 0% in 2004 to 44% in 2010 and S. flexneri was the predominant species. Of Shigella spp, ciprofloxacin resistant (CipR) strains were mostly found among S. flexneri (8.3%), followed by S. sonnei (1.5%). Within S. flexneri (n = 2181), 14.5% were resistance to ciprofloxacin of which serotype 2a was predominant (96%). MIC of ciprofloxacin, norfloxacin, and ofloxacin were 6-32 mg/L, 8-32 mg/L, and 8-24 mg/L, respectively in S. flexneri 2a isolates. Sequencing of QRDR genes of resistant isolates showed double mutations in gyrA gene (Ser83Leu, Asp87Asn/Gly) and single mutation in parC gene (Ser80Ile). A difference in amino acid substitution at position 87 was found between strains isolated in Bangladesh (Asp87Asn) and China (Asp87Gly) except for one. A novel mutation at position 211 (His→Tyr) in gyrA gene was detected only in the Bangladeshi strains. Susceptibility to ciprofloxacin was increased by the presence of CCCP indicating the involvement of energy dependent active efflux pumps. A single PFGE type was found in isolates from Bangladesh and China suggesting their genetic relatedness.ConclusionsEmergence of fluoroquinolone resistance in Shigella undermines a major challenge in current treatment strategies which needs to be followed up by using empirical therapeutic strategies.

Dataset Information

Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts.

Publications

Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts.

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