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Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7.

ABSTRACT: Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.

SUBMITTER: Lisle JT

PROVIDER: S-EPMC90905 | biostudies-literature | 1998 Dec

REPOSITORIES: biostudies-literature

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Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7.

Lisle J T JT Broadaway S C SC Prescott A M AM Pyle B H BH Fricker C C McFeters G A GA

Applied and environmental microbiology 19981201 12

Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assay ...[more]

PMID: 9835545

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Project description:Treatments of wastewater and fresh produce commonly employ chlorine as an antimicrobial. However, there are increasing levels of concerns regarding the safety and antimicrobial efficacy of chlorine treatments. Numerous studies have reported the antimicrobial properties of chlorine dioxide (ClO2) treatment in a variety of applications but information regarding how ClO2 affects bacteria is limited. In the present study, a mixed-method approach utilizing both quantitative and qualitative methodologies was used to observe Escherichia coli O157:H7 membrane damage after exposure to ClO2 (2.5, 5, or 10 mg/L) for 5, 10, or 15 min. For comparison, controls of 0.1% peptone, 70% isopropanol, and 10 mg/L NaOCl were applied for 15 min. After treatment, cells were enumerated on selective media overlaid with non-selective media and simultaneously analyzed for damage using the following fluorescent probes (1) Bis-(1,3-Dibutylbarbituric Acid) trimethine oxonol (DiBAC4(3)) for membrane polarization, (2) SYTO 9/propidium iodide (LIVE/DEAD) for membrane permeability, (3) 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) for active glucose uptake, and (4) lipid peroxidation through accumulation of malondialdehyde (MDA). Bacterial log reductions after ClO2 treatment ranged from 0.2 to 5.5 and changes in relative fluorescence units after membrane permeability and glucose uptake assays were not consistent with viability, indicating membrane permeability and metabolism were not substantially altered. Depolarization was observed after NaOCl treatment, however, the polarity of cells treated with ClO2 were like those treated with water (P < 0.05). Accumulation of MDA was detected only after 10 mg/L ClO2 treatments, indicating that membrane peroxidation occurred at higher concentrations. Transmission electron microscopy imaging revealed that separation of the cell wall from the cytosol occurred after the 10 mg/L ClO2 treatment, but the cell wall itself appeared to be unbroken. These data suggest that ClO2 damage to E. coli O157:H7 is not primarily located at the cell wall and harms cells significantly different than NaOCl at comparable concentrations.

Project description:In open environments such as water, enterohemorrhagic Escherichia coli O157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. This activates the phosphate-specific transport (Pst) system that contains a high-affinity Pi transporter. In the Δpst mutant, PhoB is constitutively activated and regulates the expression of genes in the Pho regulon. Here, we show that Pi starvation and deletion of the pst system enhance E. coli O157:H7 biofilm formation. Among differentially expressed genes of EDL933 grown under Pi starvation conditions and in the Δpst mutant, we have found that a member of the PhoB regulon, waaH, predicted to encode a glycosyltransferase, was highly expressed. Interestingly, WaaH contributed to biofilm formation of E. coli O157:H7 during both Pi starvation and in the Δpst mutant. In the Δpst mutant, the presence of waaH was associated with lipopolysaccharide (LPS) R3 core type modifications, whereas in E. coli O157:H7, waaH overexpression had no effect on LPS structure during Pi starvation. Therefore, waaH participates in E. coli O157:H7 biofilm formation during Pi starvation, but its biochemical role remains to be clarified. This study highlights the importance of the Pi starvation stress response to biofilm formation, which may contribute to the persistence of E. coli O157:H7 in the environment.IMPORTANCE Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen that causes bloody diarrhea that can result in renal failure. Outside of mammalian hosts, E. coli O157:H7 survives for extended periods of time in nutrient-poor environments, likely as part of biofilms. In E. coli K-12, the levels of free extracellular Pi affect biofilm formation; however, it was unknown whether Pi influences biofilm formation by E. coli O157:H7. Our results show that upon Pi starvation, PhoB activates waaH expression, which favors biofilm formation by E. coli O157:H7. These findings suggest that WaaH is a target for controlling biofilm formation. Altogether, our work demonstrates how adaptation to Pi starvation allows E. coli O157:H7 to occupy different ecological niches.

Dataset Information

Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7.

Publications

Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7.

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