Transcriptomic response of Escherichia coli O157 isolates on meat: Comparison between a typical Australian isolate from cattle and a pathogenic clinical isolate.

The majority of foodborne illnesses associated with E. coli O157 are attributed to the consumption of foods of bovine origin. In this study, RNA-Seq experiments were undertaken with E. coli O157 to identify genes that may be associated with growth and survival on meat and the beef carcass at low temperature. In addition, the response of an E. coli O157 isolate representative of the general genetic 'type' found in Australia (E. coli O157:H- strain EC2422) was compared to that of a pathogenic clinical isolate (E. coli O157:H7 strain Sakai) not typically found in Australia. Both strains up-regulated genes involved in the acid stress response, cold shock response, quorum sensing, biofilm formation and Shiga toxin production. Differences were also observed, with E. coli O157:H7 Sakai up-regulating genes playing a critical role in the barrier function of the outer membrane, lipopolysaccharide biosynthesis, extracellular polysaccharide synthesis and curli production. In contrast, E. coli O157:H- EC2422 down-regulated genes involved in peptidoglycan biosynthesis and of the primary envelope stress response Cpx system. The unique gene expression profiles of the strains, indicate that these genotypes may differ in their ability to persist in the meat production environment and therefore also in their ability to cause disease.

Physiological Response of Escherichia coli O157:H7 Sakai to Dynamic Changes in Temperature and Water Activity as Experienced during Carcass Chilling.

Enterohemeorrhagic Escherichia coli is a leading cause of foodborne illness, with the majority of cases linked to foods of bovine origin. Currently, no completely effective method for controlling this pathogen during carcass processing exists. Understanding how this pathogen behaves under those stress conditions experienced on the carcass during chilling in cold air could offer opportunities for development or improvement of effective decontamination processes. Therefore, we studied the growth kinetics and physiological response of exponential phase E. coli O157:H7 Sakai cultures upon an abrupt downshift in temperature and water activity (from 35 °C aw 0.993 to 14 °C aw 0.967). A parallel Biolog study was conducted to follow the phenotypic responses to 190 carbon sources. Exposure of E. coli to combined cold and water activity stresses resulted in a complex pattern of population changes. This pattern could be divided into two main phases, including adaptation and regrowth phases, based on growth kinetics and clustering analyses. The transcriptomic and proteomic studies revealed that E. coli exhibited a "window" of cell susceptibility (i.e. weaknesses) during adaptation phase. This included apparent DNA damage, the downregulation of molecular chaperones and proteins associated with responses to oxidative damage. However, E. coli also displayed a transient induction in the RpoE-controlled envelope stress response and activation of the master stress regulator RpoS and the Rcs phosphorelay system involved in colanic acid biosynthesis. Increased expression was observed for several genes and/or proteins involved in DNA repair, protein and peptide degradation, amino acid biosynthesis, and carbohydrate catabolism and energy generation. Furthermore, the Biolog study revealed reduced carbon source utilization during adaptation phase, indicating the disruption of energy-generating processes. This study provides insight into the physiological response of E. coli during exposure to combined cold and water activity stress, which could be exploited to enhance the microbiological safety of carcasses and related foods.

Global genome response of Escherichia coli O157∶H7 Sakai during dynamic changes in growth kinetics induced by an abrupt temperature downshift.

Escherichia coli O157∶H7 is a mesophilic food-borne pathogen. We investigated the growth kinetics of E. coli O157∶H7 Sakai during an abrupt temperature downshift from 35°C to either 20°C, 17°C, 14°C or 10°C; as well as the molecular mechanisms enabling growth after cold stress upon an abrupt downshift from 35°C to 14°C in an integrated transcriptomic and proteomic analysis. All downshifts caused a lag period of growth before growth resumed at a rate typical of the post-shift temperature. Lag and generation time increased with the magnitude of the shift or with the final temperature, while relative lag time displayed little variation across the test range. Analysis of time-dependent molecular changes revealed, in keeping with a decreased growth rate at lower temperature, repression of genes and proteins involved in DNA replication, protein synthesis and carbohydrate catabolism. Consistent with cold-induced remodelling of the bacterial cell envelope, alterations occurred in the expression of genes and proteins involved in transport and binding. The RpoS regulon exhibited sustained induction confirming its importance in adaptation and growth at 14°C. The RpoE regulon was transiently induced, indicating a potential role for this extracytoplasmic stress response system in the early phase of low temperature adaptation during lag phase. Interestingly, genes previously reported to be amongst the most highly up-regulated under oxidative stress were consistently down-regulated. This comprehensive analysis provides insight into the molecular mechanisms operating during adaptation of E. coli to growth at low temperature and is relevant to its physiological state during chilling in foods, such as carcasses.

Global genome response of Escherichia coli O157∶H7 Sakai during dynamic changes in growth kinetics induced by an abrupt downshift in water activity.

The present study was undertaken to investigate growth kinetics and time-dependent change in global expression of Escherichia coli O157∶H7 Sakai upon an abrupt downshift in water activity (aw). Based on viable count data, shifting E. coli from aw 0.993 to aw 0.985 or less caused an apparent loss, then recovery, of culturability. Exponential growth then resumed at a rate characteristic for the aw imposed. To understand the responses of this pathogen to abrupt osmotic stress, we employed an integrated genomic and proteomic approach to characterize its cellular response during exposure to a rapid downshift but still within the growth range from aw 0.993 to aw 0.967. Of particular interest, genes and proteins with cell envelope-related functions were induced during the initial loss and subsequent recovery of culturability. This implies that cells undergo remodeling of their envelope composition, enabling them to adapt to osmotic stress. Growth at low aw, however, involved up-regulating additional genes and proteins, which are involved in the biosynthesis of specific amino acids, and carbohydrate catabolism and energy generation. This suggests their important role in facilitating growth under such stress. Finally, we highlighted the ability of E. coli to activate multiple stress responses by transiently inducing the RpoE and RpoH regulons to control protein misfolding, while simultaneously activating the master stress regulator RpoS to mediate long-term adaptation to hyperosmolality. This investigation extends our understanding of the potential mechanisms used by pathogenic E. coli to adapt, survive and grow under osmotic stress, which could potentially be exploited to aid the selection and/or development of novel strategies to inactivate this pathogen.

Integrated transcriptomic and proteomic analysis of the physiological response of Escherichia coli O157:H7 Sakai to steady-state conditions of cold and water activity stress.

An integrated transcriptomic and proteomic analysis was undertaken to determine the physiological response of Escherichia coli O157:H7 Sakai to steady-state conditions relevant to low temperature and water activity conditions experienced during meat carcass chilling in cold air. The response of E. coli during exponential growth at 25 °C a(w) 0.985, 14 °C a(w) 0.985, 25 °C a(w) 0.967, and 14 °C a(w) 0.967 was compared with that of a reference culture (35 °C a(w) 0.993). Gene and protein expression profiles of E. coli were more strongly affected by low water activity (a(w) 0.967) than by low temperature (14 °C). Predefined group enrichment analysis revealed that a universal response of E. coli to all test conditions included activation of the master stress response regulator RpoS and the Rcs phosphorelay system involved in the biosynthesis of the exopolysaccharide colanic acid, as well as down-regulation of elements involved in chemotaxis and motility. However, colanic acid-deficient mutants were shown to achieve comparable growth rates to their wild-type parents under all conditions, indicating that colanic acid is not required for growth. In contrast to the transcriptomic data, the proteomic data revealed that several processes involved in protein synthesis were down-regulated in overall expression at 14 °C a(w) 0.985, 25 °C a(w) 0.967, and 14 °C a(w) 0.967. This result suggests that during growth under these conditions, E. coli, although able to transcribe the required mRNA, may lack the cellular resources required for translation. Elucidating the global adaptive response of E. coli O157:H7 during exposure to chilling and water activity stress has provided a baseline of knowledge of the physiology of this pathogen.

Transcriptomic analysis of Escherichia coli O157:H7 and K-12 cultures exposed to inorganic and organic acids in stationary phase reveals acidulant- and strain-specific acid tolerance responses.

The food-borne pathogen Escherichia coli O157:H7 is commonly exposed to organic acid in processed and preserved foods, allowing adaptation and the development of tolerance to pH levels otherwise lethal. Since little is known about the molecular basis of adaptation of E. coli to organic acids, we studied K-12 MG1655 and O157:H7 Sakai during exposure to acetic, lactic, and hydrochloric acid at pH 5.5. This is the first analysis of the pH-dependent transcriptomic response of stationary-phase E. coli. Thirty-four genes and three intergenic regions were upregulated by both strains during exposure to all acids. This universal acid response included genes involved in oxidative, envelope, and cold stress resistance and iron and manganese uptake, as well as 10 genes of unknown function. Acidulant- and strain-specific responses were also revealed. The acidulant-specific response reflects differences in the modes of microbial inactivation, even between weak organic acids. The two strains exhibited similar responses to lactic and hydrochloric acid, while the response to acetic acid was distinct. Acidulant-dependent differences between the strains involved induction of genes involved in the heat shock response, osmoregulation, inorganic ion and nucleotide transport and metabolism, translation, and energy production. E. coli O157:H7-specific acid-inducible genes were identified, suggesting that the enterohemorrhagic E. coli strain possesses additional molecular mechanisms contributing to acid resistance that are absent in K-12. While E. coli K-12 was most resistant to lactic and hydrochloric acid, O157:H7 may have a greater ability to survive in more complex acidic environments, such as those encountered in the host and during food processing.

Comparative evaluation of methods commonly used to determine antimicrobial susceptibility to plant extracts and phenolic compounds.

A comparison was made to evaluate the ability of the most commonly used qualitative agar diffusion methods and a quantitative broth dilution assay to determine the antimicrobial activity of a plant extract and a variety of phenolic compounds. A disc and well diffusion technique and a microtiter broth microdilution (MBM) assay were used as antimicrobial susceptibility tests of a plant extract and several phenolic compounds against 7 bacterial species. In both the well and disc diffusion assays, the level of reproducibility was poor and a linear or logarithmic relationship did not exist between inhibition zone size and the concentration of the agents. The MBM method produced the most consistent results and allowed the determination of the relative sensitivities of each species and the relative antimicrobial activities of each agent. This study demonstrated that when a diffusion method is used, multiple concentrations of the agent must be assayed to ensure that a relationship exists between the concentration of the agent and inhibition zone size. When a relationship does not exist, antimicrobial activity should be determined by a quantitative dilution technique.

Genotype-by-environment interactions influencing the emergence of rpoS mutations in Escherichia coli populations.

Polymorphisms in rpoS are common in Escherichia coli. rpoS status influences a trade-off between nutrition and stress resistance and hence fitness across different environments. To analyze the selective pressures acting on rpoS, measurement of glucose transport rates in rpoS+ and rpoS bacteria was used to estimate the role of F(nc), the fitness gain due to improved nutrient uptake, in the emergence of rpoS mutations in nutrient-limited chemostat cultures. Chemostats with set atmospheres, temperatures, pH's, antibiotics, and levels of osmotic stress were followed. F(nc) was reduced under anaerobiosis, high osmolarity, and with chloramphenicol, consistent with a reduced rate of rpoS enrichment in these conditions. F(nc) remained high, however, with alkaline pH and low temperature but rpoS sweeps were diminished. Under these conditions, F(sp), the fitness reduction due to lowered stress protection, became significant. We also estimated whether the fitness need for the gene was related to its regulation. No consistent pattern emerged between the level of RpoS and the loss of rpoS function in particular environments. This dissection allows an unprecedented view of the genotype-by-environment interactions controlling a mutational sweep and shows that both F(nc) and F(sp) are influenced by individual stresses and that additional factors contribute to selection pressure in some environments.

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions.

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.

A regulatory trade-off as a source of strain variation in the species Escherichia coli.

There are few existing indications that strain variation in prokaryotic gene regulation is common or has evolutionary advantage. In this study, we report on isolates of Escherichia coli with distinct ratios of sigma factors (RpoD, sigmaD, or sigma70 and RpoS or sigmaS) that affect transcription initiated by RNA polymerase. Both laboratory E. coli K-12 lineages and nondomesticated isolates exhibit strain-specific endogenous levels of RpoS protein. We demonstrate that variation in genome usage underpins intraspecific variability in transcription patterns, resistance to external stresses, and the choice of beneficial mutations under nutrient limitation. Most unexpectedly, RpoS also controlled strain variation with respect to the metabolic capability of bacteria with more than a dozen carbon sources. Strains with higher sigmaS levels were more resistant to external stress but metabolized fewer substrates and poorly competed for low concentrations of nutrients. On the other hand, strains with lower sigmaS levels had broader nutritional capabilities and better competitive ability with low nutrient concentrations but low resistance to external stress. In other words, RpoS influenced both r and K strategist functions of bacteria simultaneously. The evolutionary principle driving strain variation is proposed to be a conceptually novel trade-off that we term SPANC (for "self-preservation and nutritional competence"). The availability of multiple SPANC settings potentially broadens the niche occupied by a species consisting of individuals with narrow specialization and reveals an evolutionary advantage offered by polymorphic regulation. Regulatory diversity is likely to be a significant contributor to complexity in a bacterial world in which multiple sigma factors are a universal feature.

The effect of growth atmosphere on the ability of Listeria monocytogenes to survive exposure to acid, proteolytic enzymes and bile salts.

Four isolates of Listeria monocytogenes from food, human and environmental sources were grown separately in broth (pH 6.0 at 8 degrees C) under atmospheres of air, 100% N(2), 40% CO(2):60% N(2) or 100% CO(2). Exponential and stationary phase cells were harvested to determine if growth atmosphere and growth phase influenced this pathogen's ability to survive exposure to an acid environment coupled with proteolytic enzymes, and the activity of bile salts. In general, isolates were more resistant to the acid environment than the bile salts environment and stationary phase cells were significantly more resistant to both environments than exponential phase cells. Irrespective of prior growth atmosphere, none of the isolates when in exponential phase remained detectable following full exposure to the acid environment (110 min at 37 degrees C) or the bile environment (3 h at 37 degrees C). With the exception of one isolate grown under the atmosphere of 40% CO(2):60% N(2), all isolates when in stationary phase were detectable following full exposure to the acid environment but death rates varied significantly. Stationary phase cells of all isolates grown under 40% CO(2):60% N(2) and 100% CO(2) were highly susceptible to the bile salts environment: cells were not detectable after a 2-min exposure whereas stationary phase cells grown under air or 100% N(2) were recovered following full exposure to the bile environment. Survival curves were characterised by a population decline of at least 3 log(10)/ml (from an initial level of 7 log(10) CFU/ml) in the first 15 min; thereafter a constant population number of approximately 4 log(10)/ml was maintained over the remaining exposure period. No survival was observed when stationary phase cells of L. monocytogenes FRRB 2538 grown in air and 100% N(2) were subjected to the acid environment followed by immediate exposure to the bile salts environment. The results showed that growth atmosphere and growth phase could influence survival of this pathogen against conditions that imitate the extremes of the most important nonspecific defence mechanisms against microbial infection: the acid environment of the stomach coupled with the activity of proteolytic enzymes, and the activity of bile salts in the small intestine.

rpoS mutations and loss of general stress resistance in Escherichia coli populations as a consequence of conflict between competing stress responses.

The general stress resistance of Escherichia coli is controlled by the RpoS sigma factor (phi(S)), but mutations in rpoS are surprisingly common in natural and laboratory populations. Evidence for the selective advantage of losing rpoS was obtained from experiments with nutrient-limited bacteria at different growth rates. Wild-type bacteria were rapidly displaced by rpoS mutants in both glucose- and nitrogen-limited chemostat populations. Nutrient limitation led to selection and sweeps of rpoS null mutations and loss of general stress resistance. The rate of takeover by rpoS mutants was most rapid (within 10 generations of culture) in slower-growing populations that initially express higher phi(S) levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on phi(70) and involved in the hunger response to nutrient limitation. Indeed, the mutation of rpoS led to significantly higher expression of genes contributing to the high-affinity glucose scavenging system required for the hunger response. Hence, rpoS polymorphism in E. coli populations may be viewed as the result of competition between the hunger response, which requires sigma factors other than phi(S) for expression, and the maintenance of the ability to withstand external stresses. The extent of external stress significantly influences the spread of rpoS mutations. When acid stress was simultaneously applied to glucose-limited cultures, both the phenotype and frequency of rpoS mutations were attenuated in line with the level of stress. The conflict between the hunger response and maintenance of stress resistance is a potential weakness in bacterial regulation.