Evaluation of housekeeping genes in Listeria monocytogenes as potential internal control references for normalizing mRNA expression levels in stress adaptation models using real-time PCR

Listeria monocytogenes is an important food-borne pathogen that can tolerate a wide range of stress conditions. However, its stress adaptation processes are still poorly understood. Real-time-based quantitative RT-PCR (qRT-PCR) provides a tool to probe gene expression changes underlying stress adaptation. But, a limitation to study mRNA levels by real-time qRT-PCR is that validated reference genes are required for normalization. Such genes are currently lacking for experimental models that may be applied to evaluate stress-related gene expression changes in L. monocytogenes. Therefore, five housekeeping genes (HKG) were studied as potential reference genes. Their expression stability was evaluated across 16 L. monocytogenes strains. Three experimental models designed to assess gene expression changes induced by cold, acid and high NaCl concentration stress adaptation were applied. The 16S rRNA gene was consistently the most stably expressed HKG across the different L. monocytogenes strains under all the experimental conditions. While the expressions of β-glucosidase (bglA), Glyceraldehyde-3P-dehydrogenase (gap), RNA polymerase beta subunit (rpoB) and Ribosomal protein L4 (rplD) was stable amongst the different L. monocytogenes strains, they were prone to significant variations under the different stress adaptation model

16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification

BACKGROUND: E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive [Image: see text] glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated. RESULTS: By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains. CONCLUSION: By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation

Evaluation of three reference genes of Escherichia coli for mRNA expression level normalization in view of salt and organic acid stress exposure in food

Escherichia coli can adapt to various stress conditions encountered in food through induction of stress response genes encoding proteins that counteract the respective stresses. To understand the impact and the induction of these genes under food-associated stresses, changes in the levels of their mRNA expression in response to such stresses can be analysed. Relative quantification of mRNA levels by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) requires normalization to reference genes with stable expression under the experimental conditions being investigated. We examined the validity of three housekeeping genes (cysG, hcaT and rssA) among E. coli strains exposed to salt and organic acid stress. The rssA gene was shown to be the most stably expressed gene under such stress adaptation experimental models. The cysG gene was the least stable, whereas the hcaT gene showed similar interstrain variability as rssA but lower expression stability in the different stress adaptation model

Cold Shock Proteins Promote Nisin Tolerance in Listeria monocytogenes Through Modulation of Cell Envelope Modification Responses

Listeria monocytogenes continues to be a food safety challenge owing to its stress tolerance and virulence traits. Several listeriosis outbreaks have been linked to the consumption of contaminated ready-to-eat food products. Numerous interventions, including nisin application, are presently employed to mitigate against L. monocytogenes risk in food products. In response, L. monocytogenes deploys several defense mechanisms, reducing nisin efficacy, that are not yet fully understood. Cold shock proteins (Csps) are small, highly conserved nucleic acid-binding proteins involved in several gene regulatory processes to mediate various stress responses in bacteria. L. monocytogenes possesses three csp gene paralogs; cspA, cspB, and cspD. Using a panel of single, double, and triple csp gene deletion mutants, the role of Csps in L. monocytogenes nisin tolerance was examined, demonstrating their importance in nisin stress responses of this bacterium. Without csp genes, a L. monocytogenes ΔcspABD mutant displayed severely compromised growth under nisin stress. Characterizing single (ΔcspA, ΔcspB, and ΔcspD) and double (ΔcspBD, ΔcspAD, and ΔcspAB) csp gene deletion mutants revealed a hierarchy (cspD > cspB > cspA) of importance in csp gene contributions toward the L. monocytogenes nisin tolerance phenotype. Individual eliminations of either cspA or cspB improved the nisin stress tolerance phenotype, suggesting that their expression has a curbing effect on the expression of nisin resistance functions through CspD. Gene expression analysis revealed that Csp deficiency altered the expression of DltA, MprF, and penicillin-binding protein-encoding genes. Furthermore, the ΔcspABD mutation induced an overall more electronegative cell surface, enhancing sensitivity to nisin and other cationic antimicrobials as well as the quaternary ammonium compound disinfectant benzalkonium chloride. These observations demonstrate that the molecular functions of Csps regulate systems important for enabling the constitution and maintenance of an optimal composed cell envelope that protects against cell-envelope-targeting stressors, including nisin. Overall, our data show an important contribution of Csps for L. monocytogenes stress protection in food environments where antimicrobial peptides are used. Such knowledge can be harnessed in the development of better L. monocytogenes control strategies. Furthermore, the potential that Csps have in inducing cross-protection must be considered when combining hurdle techniques or using them in a series

Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation

Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB, gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response model

Comparative genomics of dairy-associated Staphylococcus aureus from selected sub-Saharan African regions reveals milk as reservoir for human-and animal-derived strains and identifies a putative animal-related clade with presumptive novel siderophore

Staphylococcus aureus infection is considered to be a neglected tropical disease with huge impact on human and animal health alike. Dairy production in sub-Saharan Africa (SSA) relies heavily on various animals such as cows, goats, and camels, depending on the region. S. aureus causes mastitis and exhibits high prevalence in raw milk. The population structure including genotypic and phenotypic traits of dairy S. aureus in relation to animal and human isolates is, however, unknown for SSA. In this work, 20 S. aureus dairy isolates from East and West Africa were selected for comparative genomics and phenotypic analysis. Comparing their population structure revealed a large diversity of different origins suggesting milk to be a reservoir for human and animal strains alike. Furthermore, a novel putative siderophore was detected in multiple strains in a distinct animal-clade with strains of global origin. This putative siderophore shares a high genetic identity with that from Streptococcus equi suggesting possible horizontal gene transfer. These findings combined with the virulence genes harbored by these dairy-derived strains such as pvl, human evasion factor scn, various enterotoxin, leucocidin and antibiotic resistance genes, stresses the need for an integrative One Health approach to tackle the problem of S. aureus infections in animals and humans in sub-Saharan Africa

Mild NaCl Stress Influences Staphylococcal Enterotoxin C Transcription in a Time-Dependent Manner and Reduces Protein Expression

Enterotoxins (SEs) produced by Staphylococcus aureus are the cause of serious food intoxications. Staphylococcal enterotoxin C (SEC) is one of the main contributors, as it is often highly expressed. S. aureus possesses a competitive growth advantage over accompanying bacterial flora under stress conditions encountered in foods, such as high NaCl concentrations. However, the influence of NaCl as an external stressor on SEC expression is still unclear. We investigated the influence of 4.5% NaCl on sec mRNA and SEC protein levels. A qRT-PCR assay revealed that NaCl stress leads to time-dependently decreased or elevated sec mRNA levels for most strains. SEC protein levels were generally decreased under NaCl stress. Our findings suggest that NaCl stress lowers overall SEC concentration and time-dependently affects sec mRNA levels

Transcriptomic and Phenotypic Analyses of the Sigma B-Dependent Characteristics and the Synergism between Sigma B and Sigma L in Listeria monocytogenes EGD-e

Numerous gene expression and stress adaptation responses in L. monocytogenes are regulated through alternative sigma factors σB and σL. Stress response phenotypes and transcriptomes were compared between L. monocytogenes EGD-e and its ΔsigB and ΔsigBL mutants. Targeted growth phenotypic analysis revealed that the ΔsigB and ΔsigBL mutants are impaired during growth under cold and organic-acid stress conditions. Phenotypic microarrays revealed increased sensitivity in both mutants to various antimicrobial compounds. Genes de-regulated in these two mutants were identified by genome-wide transcriptome analysis during exponential growth in BHI. The ΔsigB and ΔsigBL strains repressed 198 and 254 genes, respectively, compared to the parent EGD-e strain at 3 °C, whereas 86 and 139 genes, respectively, were repressed in these mutants during growth at 37 °C. Genes repressed in these mutants are involved in various cellular functions including transcription regulation, energy metabolism and nutrient transport functions, and viral-associated processes. Exposure to cold stress induced a significant increase in σB and σL co-dependent genes of L. monocytogenes EGD-e since most (62%) of the down-regulated genes uncovered at 3 °C were detected in the ΔsigBL double-deletion mutant but not in ΔsigB or ΔsigL single-deletion mutants. Overall, the current study provides an expanded insight into σB and σL phenotypic roles and functional interactions in L. monocytogenes. Besides previously known σB- and σL-dependent genes, the transcriptomes defined in ΔsigB and ΔsigBL mutants reveal several new genes that are positively regulated by σB alone, as well as those co-regulated through σB- and σL-dependent mechanisms during L. monocytogenes growth under optimal and cold-stress temperature conditions

Potassium Lactate as a Strategy for Sodium Content Reduction without Compromising Salt-Associated Antimicrobial Activity in Salami

Reformulating recipes of ready-to-eat meat products such as salami to reduce salt content can mitigate the negative health impacts of a high salt diet. We evaluated the potential of potassium lactate (KL) as a sodium chloride (NaCl) replacer during salami production. NaCl and KL stress tolerance comparisons showed that four food-derived Listeria innocua isolates were suitable as biologically safe Listeria monocytogenes surrogates. Effects of the high salt (4% NaCl) concentration applied in standard salami recipes and a low salt (2.8% NaCl) plus KL (1.6%) combination on product characteristics and growth of contaminating Listeria and starter culture were compared. Simulated salami-ripening conditions applied in meat simulation broth and beef showed that the low salt plus KL combination retained similar to superior anti-Listeria activity compared to the high salt concentration treatment. Salami challenge tests showed that the low NaCl plus KL combination had comparable anti-Listeria activity as the high NaCl concentration during ripening and storage. No significant differences were detected in starter culture growth profiles and product characteristics between the high NaCl and low NaCl plus KL combination treated salami. In conclusion, KL replacement enabled a 30% NaCl reduction without compromising the product quality and antimicrobial benefits of high NaCl concentration inclusion