Transcriptome analysis of alkali shock and alkali adaptation in Listeria monocytogenes 10403S.

Alkali stress is an important means of inactivating undesirable pathogens in a wide range of situations. Unfortunately, Listeria monocytogenes can launch an alkaline tolerance response, significantly increasing persistence of the pathogen in such environments. This study compared transcriptome patterns of alkali and non-alkali-stressed L. monocytogenes 10403S cells, to elucidate the mechanisms by which Listeria adapts and/or grows during short- or long-term alkali stress. Transcription profiles associated with alkali shock (AS) were obtained by DNA microarray analysis of midexponential cells suspended in pH 9 media for 15, 30, or 60 min. Transcription profiles associated with alkali adaptation (AA) were obtained similarly from cells grown to midexponential phase at pH 9. Comparison of AS and AA transcription profiles with control cell profiles identified a high number of differentially regulated open-reading frames in all tested conditions. Rapid (15 min) changes in expression included upregulation of genes encoding for multiple metabolic pathways (including those associated with Na+/H+ antiporters), ATP-binding cassette transporters of functional compatible solutes, motility, and virulence-associated genes as well as the σ(B) controlled stress resistance network. Slower (30 min and more) responses to AS and adaptation during growth in alkaline conditions (AA) involved a different pattern of changes in mRNA concentrations, and genes involved in proton export.

Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress.

Many of the considerable abilities of Listeria monocytogenes to persist and grow in a wide range of adverse environmental conditions are thought to be at least partly under the control of the alternative sigma factor (sigmaB), encoded by the sigB gene. However, little is known about the role of this master regulon in the impressive ability of Listeria to persist and grow under conditions of alkaline pH. In this study, Northern blot analysis of parent Listeria mRNA revealed that alkali adaptation (pH 9.5 for 1 h) significantly increased the expression of sigB-derived mRNA. The study included a comparison of the relative survival of mid-exponential populations of adapted and nonadapted parent type (sigmaB expressing) and mutant (not sigmaB expressing, deltasigB) Listeria strains during subsequent alkaline (pH 12.0), osmotic (25% NaCl, wt/vol), or ethanol (16.5%) stress. Alkali-adapted parent strains were more resistant to pH 12.0 than were adapted deltasigB type strains, but both alkali-adapted parent and deltasigB strains were more resistant to pH 12.0 than were nonadapted strains. Alkali-adapted parent strains were more resistant to osmotic stress than were adapted deltasigB type strains. No significant differences in viability were observed between alkali-adapted parent and deltasigB strains after ethanol stress, suggesting that cross-protection against osmotic stress is mediated by sigmaB whereas cross-protection against ethanol is sigmaB independent. Overall, alkali-induced cross-protection against osmotic and ethanol challenges may have serious implications for food safety and human health because such stress conditions are routinely used as part of food preservation and surface cleaning processes.

Genomic and proteomic analysis of the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes 10403S.

BACKGROUND: Information regarding the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes is very limited. Treatment of alkali-adapted cells with the protein synthesis inhibitor chloramphenicol has revealed that the AlTR is at least partially protein-dependent. In order to gain a more comprehensive perspective on the physiology and regulation of the AlTR, we compared differential gene expression and protein content of cells adapted at pH 9.5 and un-adapted cells (pH 7.0) using complementary DNA (cDNA) microarray and two-dimensional (2D) gel electrophoresis, (combined with mass spectrometry) respectively. RESULTS: In this study, L. monocytogenes was shown to exhibit a significant AlTR following a 1-h exposure to mild alkali (pH 9.5), which is capable of protecting cells from subsequent lethal alkali stress (pH 12.0). Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. The observed variability between results of cDNA arrays and 2D gel electrophoresis may be accounted for by posttranslational modifications. Interestingly, several alkali induced genes/proteins can provide a cross protective overlap to other types of stresses. CONCLUSION: Alkali pH provides therefore L. monocytogenes with nonspecific multiple-stress resistance that may be vital for survival in the human gastrointestinal tract as well as within food processing systems where alkali conditions prevail. This study showed strong evidence that the AlTR in L. monocytogenes functions as to minimize excess alkalisation and energy expenditures while mobilizing available carbon sources.

Recovery and transfer of Salmonella typhimurium from four different domestic food contact surfaces.

Domestic food contact surfaces can play an important role in the transmission of foodborne disease, yet debate continues as to which surface materials pose the greatest risk to consumer health in terms of cross-contamination during food preparation. Salmonella Typhimurium was inoculated onto stainless steel, Formica, polypropylene, or wooden surfaces (25 cm2) in the presence or absence of protein (tryptic soy broth supplemented with 5% horse serum) and held at room temperature. The pathogen was recovered from the test surfaces immediately after inoculation (T=0) and every hour for up to 6 h, by a conventional microbiological sampling technique and by direct transfer onto a model ready-to-eat food (cucumber slices). On all surfaces, pathogen numbers declined during the 6-h holding period, with the most rapid reductions occurring within the first hour. The presence of protein significantly increased (P < 0.05) the number of bacteria recovered from all surface types. However, regardless of application medium or holding time, the number of bacteria recovered from Formica (in all cases) and stainless steel (in most cases) was significantly higher than were the numbers on polypropylene or wood. Similarly, regardless of application medium or holding time, significantly higher bacterial numbers were transferred to the model food from Formica or stainless steel than from polypropylene or wooden surfaces. These differences were greater when the bacteria were applied in a protein-rich medium and the test surfaces held for 1 h or more. The results of this study emphasize that differences, both in recoverability and in the number of bacteria transferred to the model food rather than simply reflecting differences in pathogen survival, may also reflect differences in the ability of the test bacteria to remobilize from the different surface types. However, the results also demonstrate a fundamental problem when choosing food contact surfaces, i.e., that those characteristics that make a surface "easy to clean" may also render it more likely to release contaminating pathogens during common food preparation practices.

Morphological changes in Listeria monocytogenes subjected to sublethal alkaline stress.

Scanning electron microscopy (SEM) studies revealed that exposure to 4lethal alkaline stress induced statistically significant (P<0.05) changes in mean cell length, radius and volume in Listeria monocytogenes and a derived sigma(B) deficient mutant. Bacterial morphology was altered at pH values above 9.0, to include single filamentous or elongated chain forms. Such filamentation and chain formation was observed in the parent strain and in the sigma(B) deficient strain, and in buffered and non-buffered media. Giemsa staining revealed that the filaments were multi-nucleate, with nucleoids spaced along the length of the atypical cells. In buffered media, longer alkaline exposure was associated with increases in the frequency and length of filamentation. In non-buffered medium, longer exposure was associated with gradual decline in length and the frequency of observation of filaments. Transfer of alkaline treated cells to neutral conditions was associated with the formation of septa within filaments, cell division, and a rapid return to normal morphology, i.e. within 3 h. The observed effects, and their reversibility, may be important in increasing the alkaline tolerance of this pathogen during phagocytosis within the innate human immune system response, and in adaptation/survival in food environments treated with alkali detergents and/or sanitisers. Such atypical cells may be associated with increased survival of L. monocytogenes in adverse environments and may also contribute to qualitative and quantitative underestimation of this important pathogen in food processing environments, with potential implications in public health.

Environmental stress and antibiotic resistance in food-related pathogens.

This study investigated the possibility that sublethal food preservation stresses (high or low temperature and osmotic and pH stress) can lead to changes in the nature and scale of antibiotic resistance (ABR) expressed by three food-related pathogens (Escherichia coli, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus). The study found that some sublethal stresses significantly altered antibiotic resistance. Incubation at sublethal high temperature (45 degrees C) decreased ABR. Incubation under increased salt (>4.5%) or reduced pH (<5.0) conditions increased ABR. Some of the pathogens continued to express higher levels of ABR after removal of stress, suggesting that in some cases the applied sublethal stress had induced stable increases in ABR. These results indicate that increased use of bacteriostatic (sublethal), rather than bactericidal (lethal), food preservation systems may be contributing to the development and dissemination of ABR among important food-borne pathogens.

Habituation to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia) is associated with reduced susceptibility to antibiotics in human pathogens.

OBJECTIVES: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic susceptibility profiles of significant human pathogens and commensals. METHODS: The study compared the antibiotic susceptibility (Etest) patterns of Escherichia coli, Staphylococcus aureus/methicillin-resistant S. aureus (MRSA) and Salmonella spp. after broth culture for 72 h in the presence or absence of sub-lethal concentrations of TTO (0.25%, 0.25% and 0.1%). RESULTS: All habituated cultures (exposed to sub-lethal concentrations of TTO) displayed reduced susceptibility to a range of clinically relevant antibiotics compared with non-habituated (control) cultures. CONCLUSIONS: Although TTO may be an effective antimicrobial agent when appropriately used at bactericidal concentrations, its application at sub-lethal concentrations may contribute to the development of antibiotic resistance in human pathogens.

Role of branched-chain fatty acids in pH stress tolerance in Listeria monocytogenes.

In alkaline conditions, Listeria monocytogenes cells develop higher proportions of branched-chain fatty acids (FAs), including more anteiso forms. In acid conditions, the opposite occurs. Reduced growth of pH-sensitive mutants at adverse pH (5.0/9.0) was alleviated by the addition of 2-methylbutyrate (an anteiso-FA precursor), suggesting that anteiso-FAs are important in adaptation to adverse pH. The balance between anteiso- and iso-FAs may be more important than changes in the amounts and/or degrees of saturation of FAs in pH adaptation.

Survival and recovery of Salmonella enterica serovar Typhimurium DT104 at low temperature and water activity in a broth system.

This study investigated the survival of Salmonella enterica serovar Typhimurium DT104 in a broth system under conditions of low temperature (4 degrees C) and low water activity (aw, 0.92 to 0.96). Incubation under these conditions resulted in significant reductions in the viability of stationary phase cells, determined by direct plating on selective XLD medium. Reductions in viable numbers were related to injury associated with initial osmotic shock (hyperosmosis) and further injury associated with longer-term storage under the above conditions. Such injured cells were, however, capable of recovering on a nonselective medium (TSA) and contributing to overall viable cell numbers in nonselective post-storage conditions. Storage at more extreme conditions, at lower aw values, led to cell death at rates influenced by storage temperature. Finally, the data obtained are considered in relation to pathogen survival on the surfaces of beef carcasses during chilling.

Prevalence of Campylobacter Species on Fresh Retail Chicken Wings in Northern Ireland.

Campylobacter contamination was surveyed in 153 chicken wing samples purchased from retail outlets in Northern Ireland over a 10-week sampling period; 64.7% of samples were found to be positive for Campylobacter jejuni/coli using the API Campy: Identification System for Campylobacter. These results support the assertion that retail chicken produets are commonly contaminated with Campylobacter spp. and pose a potential risk to consumers if hygiene and cooking practices are not adequate to prevent cross-contamination and facilitate destruction.