Development of a microbial time-temperature indicator for real-time monitoring the quality of Australian vacuum-packed lamb.

A time-temperature indicator (TTI) system based on the pH-dependent colour change caused by the growth of a Carnobacterium maltaromaticum strain was developed to specifically provide a real-time indication of quality and shelf life of Australian vacuum-packed (VP) lamb throughout cold chains. Each component of the developed TTI system was studied to select an optimal concentration of a chemical chromatic indicator (chlorophenol red, CR; between 0.01 % and 0.30 %) and supplementary glucose (between 0 % and 10 %), and an appropriate C. maltaromaticum strain (among four different strains) in a simple BHI medium. BHI medium containing 0.01 % CR and 1 % added glucose, inoculated with C. maltaromaticum strain 1 were required for development of the TTI system to indicate quality and shelf life of VP lamb. Different inoculum levels of C. maltaromaticum strain 1 (103 to 105 CFU/mL) were also examined at 8 °C for their effects on the TTI response. As expected, higher inoculum levels of C. maltaromaticum led to a shorter endpoint of the TTI system but it was found that a 3 log10 higher inoculum level in the TTI than the expected total viable counts of VP lamb was required to accurately predict VP lamb shelf life by the TTI. To further evaluate the applicability of the TTI system, we evaluated its response at two other temperatures (2 °C and 4 °C) relevant to the storage conditions for VP lamb. The data showed a strong agreement between the observed TTI's endpoints and predicted shelf lives of VP lamb. This indicated that the developed TTI has the potential to be developed further for commercial application to provide a real-time, distinct, and accurate indication of Australian VP lamb.

Microbial spoilage mechanisms of vacuum-packed lamb meat: A review.

Lamb meat is an important export commodity, however chilled vacuum-packed (VP) lamb has approximately half the shelf-life of beef under the same storage conditions. This makes the industry more vulnerable to financial losses due to long shipping times and unexpected spoilage. Understanding the spoilage mechanisms of chilled VP lamb in relation to VP beef is important for developing effective strategies to extend the shelf-life of lamb. This review has discussed various key factors (i.e., pH, fat, and presence of bone) that have effects on microbial spoilage of VP lamb contributing to its shorter shelf-life relative to VP beef. A range of bacterial organisms and their metabolisms in relevance to lamb spoilage are also discussed. The data gap in the literature regarding the potential mechanisms of spoilage in VP red meat is highlighted. This review has provided the current understanding of key factors affecting the shelf-life of VP lamb relative to VP beef. It has also identified key areas of research to further understand the spoilage mechanisms of VP lamb. These include investigating the potential influence of fat and bone (including bone marrow) on the shelf-life, as well as assessing changes in the meat metabolome as the spoilage microbial community is developing using an integrated approach. Such new knowledge would aid the development of effective approaches to extend the shelf-life of VP lamb.

Proteome analysis of Campylobacter jejuni poultry strain 2704 survival during 45 min exposure to peracetic acid.

Peracetic acid (PAA) applied to whole poultry carcasses can reduce the number of Campylobacter, a leading cause of human gastroenteritis. However, previous modelling experiments indicated that Campylobacter survived in greater numbers when pre-treated with a thermal stress equivalent to poultry processing scalding prior to chilling with PAA than when subject to chilling with PAA only. To better understand how Campylobacter responds to PAA, proteomes of C. jejuni poultry strain 2704 were measured after exposure to PAA (60 ppm, pH 4.0) for 45 min under laboratory ambient conditions (approximately 23 °C) to establish a foundational map of survival mechanism before combining with other stresses. Analysis of 580 quantified proteins did not indicate a triggered "peroxide shock" response, nor were common heat shock responses detected. Thioredoxin, iron homeostatic, peroxiredoxins and cytochrome c peroxidases became more abundant suggesting that PAA disturbed cytoplasmic redox homeostasis resulting in antioxidant activation and increased prioritisation of iron homeostasis. The PAA treatment led to responses that included an increased priority for oxidative phosphorylation and a simultaneous decrease in central metabolism associated protein abundances. Lon protease was induced suggesting it has a role in maintaining homeostasis during non-thermal stress. Proteins in flagella and chemotaxis became more abundant though whether PAA has a chemorepellent effect requires further investigation. Overall, the proteome data suggests there was a rapid cellular response to applied PAA stress in the first 15 min with the adaptation to the stress completing between 30 and 45 min. The findings will help guide PAA implementation in commercial poultry processing in terms of processing location and length of application.

Spoilage potential of bacterial species from chilled vacuum-packed lamb.

The objective of this study was to establish whether specific organisms play important roles in the spoilage rate of vacuum-packed (VP) lamb at low storage temperatures. The spoilage potential of representative organisms (n = 13) of the spoilage community of VP lamb were investigated through a series of shelf-life challenge trials. Each isolate was individually inoculated onto sterile (irradiated) and non-sterile (i.e., containing natural microbial community) VP lamb meat. Meat quality was assessed over time by measuring sensorial qualities, bacterial growth and pH. Among all test organisms, Clostridium spp. had the highest spoilage potential and had a major effect on the spoilage rate of VP lamb (based on sensory assessment). C. estertheticum caused premature 'blown pack' spoilage; however, the spoilage was delayed in a community setting. C. putrefaciens and C. algidicarnis caused premature spoilage of VP lamb independently and in a community setting. In contrast, all facultative anaerobes and Pseudomonas sp. tested were not capable of spoiling meat independently or within a community, expect for Carnobacterium divergens and Serratia spp., which spoiled meat prematurely when present in a community. Overall, these results highlight that Clostridium could be one of the main taxa driving the faster rate of quality loss of chilled VP lamb compared to beef. This research can help to inform opportunities for shelf-life extension by targeting organisms with 'high' spoilage potential, such as Clostridium.

The effects of glucose on microbial spoilage of vacuum-packed lamb.

Vacuum-packed lamb produced in Australia has a shelf-life of 80-90 days under export conditions (-1 to 0 °C). However, access to some markets could involve >90 days transit time. Studies to understand the potential mechanisms of microbial spoilage of vacuum-packed lamb are, therefore, important to assist the development of shelf-life extension methods. Here, we investigated the effects of glucose on the shelf-life of vacuum-packed lamb. This was done by adding glucose (up to 4.64 mmol/kg) to the surface of meat and conducting a series of shelf-life trials, in which the sensorial qualities, bacterial growth, pH, and residual glucose and lactic acid were measured over time. Based on sensory analysis glucose extended the shelf-life, ranging from 8% to >76% increase relative to the control. Glucose reduced meat pH, potentially affecting the microbial community composition and the accumulation of spoilage metabolites. These results indicate that glucose plays an important role in microbial spoilage of vacuum-packed lamb possibly by pH reduction.

Colonisation dynamics of Listeria monocytogenes strains isolated from food production environments.

Listeria monocytogenes is a ubiquitous bacterium capable of colonising and persisting within food production environments (FPEs) for many years, even decades. This ability to colonise, survive and persist within the FPEs can result in food product cross-contamination, including vulnerable products such as ready to eat food items. Various environmental and genetic elements are purported to be involved, with the ability to form biofilms being an important factor. In this study we examined various mechanisms which can influence colonisation in FPEs. The ability of isolates (n = 52) to attach and grow in biofilm was assessed, distinguishing slower biofilm formers from isolates forming biofilm more rapidly. These isolates were further assessed to determine if growth rate, exopolymeric substance production and/or the agr signalling propeptide influenced these dynamics and could promote persistence in conditions reflective of FPE. Despite no strong association with the above factors to a rapid colonisation phenotype, the global transcriptome suggested transport, energy production and metabolism genes were widely upregulated during the initial colonisation stages under nutrient limited conditions. However, the upregulation of the metabolism systems varied between isolates supporting the idea that L. monocytogenes ability to colonise the FPEs is strain-specific.

Characterisation of Listeria monocytogenes food-associated isolates to assess environmental fitness and virulence potential.

The ability of Listeria monocytogenes isolates to survive within the food production environment (FPE), as well as virulence, varies greatly between strains. There are specific genetic determinants that have been identified which can strongly influence a strains ability to survive in the FPE and/or within human hosts. In this study, we assessed the FPE fitness and virulence potential, including efficacy of selected hygiene or treatment intervention, against 52 L. monocytogenes strains isolated from various food and food environment sources. Phenotypic tests were performed to determine the minimum inhibitory concentration of cadmium chloride and benzalkonium chloride and the sensitivities to five clinically relevant antibiotics. A genomic analysis was also performed to identify resistance genes correlating to the observed phenotypic resistance profiles, along with genetic determinants of interest which may elude to the FPE fitness and virulence potential. A transposon element containing a novel cadmium resistance gene, cadA7, a Tn916 variant insert in the hypervariable Listeria genomic island 1 region and an LGI2 variant were identified. Resistance to cadmium and disinfectants was prevalent among isolates in this study, although no resistance to clinically important antimicrobials was observed. Potential hypervirulent strains containing full length inlA, LIPI-1 and LIPI-3 were also identified in this study. Cumulatively, the results of this study show a vast array of FPE survival and pathogenicity potential among food production-associated isolates, which may be of concern for food processing operators and clinicians regarding L. monocytogenes strains colonising and persisting within the FPE, and subsequently contaminating food products then causing disease in at risk population groups.

Effect of Storage Conditions on Shelf Stability of Undiluted Neutral Electrolyzed Water.

ABSTRACT: Neutral electrolyzed water (NEW) is an oxidizing sanitizer that can be made locally on-site; it is often stored in a ready-to-use format to accumulate the large volumes required for periodic or seasonal use. The shelf stability of NEW sanitizer was, therefore, assessed under various storage conditions to guide the development of protocols for its industrial application. To that end, fresh NEW with an available chlorine concentration (ACC) of 480 mg/L, pH 6.96, and oxidation reduction potential (ORP) of 916 mV was stored under different conditions. These were open or sealed polypropylene bottles, three different surface area-to-volume (SA:V) ratios (0.9, 1.7, and 8.7), and two temperatures (4 and 25°C). NEW stored at 4°C was significantly more stable than NEW stored at 25°C; ACC and pH decreased by 137 mg/L and 0.7, respectively, whereas ORP increased by 23 mV, after 101 days of storage. At 25°C, ACC decreased to <0.01 mg/L after 52 days in bottles with a SA:V ratio of 8.7, with a similar decrease after 101 days in bottles with a SA:V ratio of 1.7. However, pH decreased by up to 3.7 pH units, and ORP increased by up to 208 mV. The antimicrobial efficacy of "aged" electrolyzed oxidizing (EO) water with different ACC and ORP, but the same pH (i.e., 3.4 ± 0.2), was evaluated against Escherichia coli and Listeria innocua to determine any differences in residual antimicrobial activity. EO water with an ACC of ≥7 mg/L and an ORP of 1,094 mV caused a reduction of at least 4.7 log, whereas EO water with nondetectable ACC and considerably high ORP (716 mV) had little antimicrobial effect (<1-log reduction). Results from this study indicate that the efficacy of NEW as a sanitizer for large-scale applications such as horticulture can be maintained for at least 3 months when it is stored in closed containers with low SA:V ratio at low temperatures.

Impact of Poultry Processing Operating Parameters on Bacterial Transmission and Persistence on Chicken Carcasses and Their Shelf Life.

It is important for the poultry industry to maximize product safety and quality by understanding the connection between bacterial diversity on chicken carcasses throughout poultry processing to the end of shelf life and the impact of the local processing environment. Enumeration of total aerobic bacteria, Campylobacter and Pseudomonas, and 16S rRNA gene amplicon sequencing were used to evaluate the processing line by collecting 10 carcasses from five processing steps: prescald, postplucker, pre- and post-immersion chill, and post-air chill. The diversity throughout a 12-day shelf life was also determined by examining 30 packaged carcasses. To identify the sources of possible contamination, scald water tank, immersion chilling water tank, air samples, and wall surfaces in the air-chill room were analyzed. Despite bacterial reductions on carcasses (>5 log10 CFU/ml) throughout the process, each step altered the bacterial diversity. Campylobacter was a minor but persistent component in the bacterial community on carcasses. The combination of scalding, defeathering, and plucking distributed thermophilic spore-forming Anoxybacillus to carcasses, which remained at a high abundance on carcasses throughout subsequent processes. Pseudomonas was not isolated from carcasses after air chilling but was abundant on the wall of the air-chill room and became the predominant taxon at the end of shelf life, suggesting possible contamination through air movement. The results suggest that attention is needed at each processing step, regardless of bacterial reductions on carcasses. Changing scalding water regularly, maintaining good hygiene practices during processing, and thorough disinfection at the end of each processing day are important to minimize bacterial transmission.IMPORTANCE Culture-based and culture-independent approaches were utilized to reveal bacterial community changes on chicken carcasses at different processing steps and potential routes from the local processing environment. Current commercial processing effectively reduced bacterial loads on carcasses. Poultry processes have similar processes across facilities, but various processing arrangements and operating parameters could impact the bacterial transmission and persistence on carcasses differently. This study showed the use of a single tunnel incorporating scalding, defeathering and plucking may undesirably distribute the thermoduric bacteria, e.g., Campylobacter and Anoxybacillus, between the local environment and carcasses, whereas this does not occur when these steps are separated. The length of immersion and air chilling also impacted bacterial diversity on carcasses. Air chilling can transfer Pseudomonas from wall surfaces onto carcasses; this may subsequently influence chicken product shelf life. This study helps poultry processors understand the impact of current commercial processing and improve the chicken product quality and safety.

Application of chlorine dioxide and peroxyacetic acid during spray chilling as a potential antimicrobial intervention for beef carcasses.

Enteric pathogens such as Shiga-toxin producing Escherichia coli (STEC) and Salmonella spp. continue to be a major food safety concern for the beef industry. Currently, no single method is completely effective in controlling these pathogens during carcass processing. Previous research, however, suggested that STEC might become more susceptible to oxidative damage when exposed to carcass chilling (King et al., 2016). We aimed to test that hypothesis by evaluating the antimicrobial effects of an oxidant (chlorine dioxide, ClO2 or peroxyacetic acid, PAA) on beef meat during a simulated spray chilling process (sprayed for 4 s every 15 min for 36 cycles) and/or when applied (sprayed for 144 s) prior to spray chilling with water. In all experiments, the inactivating effects of oxidants were greatest on fat surfaces and much less effective on lean surfaces. ClO2 at 15 ppm, a non-lethal level for E. coli under optimal growth conditions, caused higher log reductions in E. coli numbers (approximately 3-log reduction) when applied during spray chilling than when applied immediately prior to 'normal' spray chilling (approximately 1-log reduction). This confirms the hypothesis that E. coli are more susceptible to oxidative stress during spray chilling. In subsequent studies, both ClO2 and PAA at lethal levels (at ≥20 and ≥ 200 ppm, respectively) applied during spray chilling resulted in pronounced inactivation of both E. coli and Salmonella enterica strains, achieving a ≥4-log reduction at the end of chilling. These results indicate that an oxidant-based application during spray chilling as an antimicrobial intervention could be effective to minimise the problems associated with enteric pathogen contamination on beef meat.

Changes of the bacterial community diversity on chicken carcasses through an Australian poultry processing line.

Understanding the bacterial community profile through poultry processing could help the industry to produce better poultry products. In this study, 10 chicken carcasses were randomly sampled from before and after scalding, before and after immersion chilling, and after air chilling each through a modern commercial processing line, along with the contents of 10 caeca. The sampled processing line effectively reduced the bacterial counts by > 4.6 Log10 CFU/ml for each of Total Viable Counts, Escherichia coli and Campylobacter. However, the metagenomics results suggested that Lactobacillus, Staphylococcus and unclassified Lachnospiraceae persisted at all sampling stages. Pseudomonas, Paeniglutamicibacter, Chryseobacterium and Pseudarthrobacter comprised 47.2% in the bacterial community on samples after air chilling compared to 0.3% on samples after immersion chilling, whereas TVCs were the same. Overall, the current interventions of the investigated poultry processing line were unable to eliminate persistence of certain foodborne pathogens, despite a significant reduction of the overall bacterial counts. Chilling is an important controlling point in contamination/cross-contamination, particularly extended air chilling. Lastly, the large presence of Pseudomonas on chickens after air chilling may lead to downstream spoilage related issues, which needs more investigation to explore quantitatively the effect on the shelf life of poultry products.

Novel Biocontrol Methods for Listeria monocytogenes Biofilms in Food Production Facilities.

High mortality and hospitalization rates have seen Listeria monocytogenes as a foodborne pathogen of public health importance for many years and of particular concern for high-risk population groups. Food manufactures face an ongoing challenge in preventing the entry of L. monocytogenes into food production environments (FPEs) due to its ubiquitous nature. In addition to this, the capacity of L. monocytogenes strains to colonize FPEs can lead to repeated identification of L. monocytogenes in FPE surveillance. The contamination of food products requiring product recall presents large economic burden to industry and is further exacerbated by damage to the brand. Poor equipment design, facility layout, and worn or damaged equipment can result in Listeria hotspots and biofilms where traditional cleaning and disinfecting procedures may be inadequate. Novel biocontrol methods may offer FPEs effective means to help improve control of L. monocytogenes and decrease cross contamination of food. Bacteriophages have been used as a medical treatment for many years for their ability to infect and lyse specific bacteria. Endolysins, the hydrolytic enzymes of bacteriophages responsible for breaking the cell wall of Gram-positive bacteria, are being explored as a biocontrol method for food preservation and in nanotechnology and medical applications. Antibacterial proteins known as bacteriocins have been used as alternatives to antibiotics for biopreservation and food product shelf life extension. Essential oils are natural antimicrobials formed by plants and have been used as food additives and preservatives for many years and more recently as a method to prevent food spoilage by microorganisms. Competitive exclusion occurs naturally among bacteria in the environment. However, intentionally selecting and applying bacteria to effect competitive exclusion of food borne pathogens has potential as a biocontrol application. This review discusses these novel biocontrol methods and their use in food safety and prevention of spoilage, and examines their potential to control L. monocytogenes within biofilms in food production facilities.

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.

Investigation of the Listeria monocytogenes Scott A acid tolerance response and associated physiological and phenotypic features via whole proteome analysis.

The global proteomic responses of the foodborne pathogen Listeria monocytogenes strain Scott A, during active growth and transition to the stationary growth phase under progressively more acidic conditions, created by addition of lactic acid and HCl, were investigated using label-free liquid chromatography/tandem mass spectrometry. Approximately 56% of the Scott A proteome was quantitatively assessable, and the data provides insight into its acquired acid tolerance response (ATR) as well as the relation of the ATR to the growth phase transition. Alterations in protein abundance due to acid stress were focused in proteins belonging to the L. monocytogenes common genome, with few strain-dependent proteins involved. However, one of the two complete prophage genomes appeared to enter lysogeny. During progressive acidification, the growth rate and yield were reduced 55% and 98%, respectively, in comparison to nonacidified control cultures. The maintenance of the growth rate was determined to be connected to activation of cytoplasmic pH homeostatic mechanisms while cellular reproductive-related and cell component turnover proteins were markedly more abundant in acid stressed cultures. Cell biomass accumulation was impeded predominantly due to repression of phosphodonor-linked enzymes involved with sugar phosphotransfer, glycolysis, and cell wall polymer biosynthesis. Acidification caused a shift from heterofermentation to an oxidatively stressed state in which ATP appears to be generated mainly through the pyruvate dehydrogenase/pyruvate oxidase/phosphotransacetylase/acetate kinase and branched chain acid dehydrogenase pathways. Analysis of regulons indicated energy conservation occurs due to repression by the GTP/isoleucine sensor CodY and also the RelA mediated stringent response. Whole proteome analysis proved to be an effective way to highlight proteins involved with the acquisition of the ATR.

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.

Utility of gel-free, label-free shotgun proteomics approaches to investigate microorganisms.

This review will examine the current situation with label-free, quantitative, shotgun-oriented proteomics technology and discuss the advantages and limitations associated with its capability in capturing and quantifying large portions of proteomes of microorganisms. Such an approach allows (1) comparisons between physiological or genetic states of organisms at the protein level, (2) 'painting' of proteomic data onto genome data-based metabolic maps, (3) enhancement of the utility of genomic data and finally (4) surveying of non-genome sequenced microorganisms by taking advantage of available inferred protein data in order to gain new insights into strain-dependent metabolic or physiological capacities. The technology essentially is a powerful addition to systems biology with a capacity to be used to ask hypothesis-driven 'top-down' questions or for more empirical 'bottom-up' exploration.