The combined effect of chitosan and high hydrostatic pressure on Listeria monocytogenes and Escherichia coli

This study explores the combined effect of different High Hydrostatic Pressures (HHP; 200–300 MPa) with various chitosan concentrations (up to 0.2%) on five Listeria monocytogenes strains and one of Escherichia coli, at temperatures 20 & 35 ◦C. Cells were resuspended in ACES buffer 1 h prior to HHP. A synergistic effect of chitosan and HHP was reported for first time in the above bacterial species tested. Synergistic effect up to 1 log reduction was observed at 300 MPa and chitosan at 20 ◦C against L.monocytogenes LO28 with FBR13 being the most sensitive strain at 250 MPa and 0.1% chitosan. Higher combined effect was found at 35 ◦C compared to 20 ◦C at 200 MPa highlighting for first time the significant role of temperature in the above synergistic action. Pressure and temperature had a greater impact on inactivation and synergism than chitosan concentrations. Synergistic effect (1 log reduction) was also observed in E. coli K12 at 0.1% chitosan and 200 MPa. Industrial relevance: This study presents the significance of combining HHP with natural antimicrobials to control L. monocytogenes and E. coli. Even though the technology is used for 3 decades in the food industry, its cost is still relatively high. Therefore, it is important to investigate novel ways to reduce the pressure intensity resulting in reduced costs, lower energy consumption and a broader product portfolio. This research demonstrates for first time the synergistic action of chitosan and HHP on L. monocytogenes and E. coli and the significant role of temperature that could contribute in the enhancement of the antimicrobial effect and optimization of the processing conditions. This aligns also with the growing demand for more sustainable and natural systems regarding the food production

A novel approach in acidic disinfection through inhibition of acid resistance mechanisms; Maleic acid-mediated inhibition of glutamate decarboxylase activity enhances acid sensitivity of Listeria monocytogenes

Here it is demonstrated a novel approach in disinfection regimes where specific molecular acid resistance systems are inhibited aiming to eliminate microorganisms under acidic conditions. Despite the importance of the Glutamate Decarboxylase (GAD) system for survival of Listeria monocytogenes and other pathogens under acidic conditions its potential inhibition by specific compounds that could lead to its elimination from foods or food preparation premises has not been studied. The effects of maleic acid on the acid resistance of L. monocytogenes were investigated and found that it has a higher antimicrobial activity under acidic conditions than other organic acids, while this could not be explained by its pKa or Ki values. The effects were found to be more pronounced on strains with higher GAD activity. Maleic acid affected the extracellular GABA levels while it did not affect the intracellular. Maleic acid had a major impact mainly on GadD2 activity as also shown in cell lysates. Furthermore, it was demonstrated that maleic acid is able to partly remove biofilms of L. monocytogenes. Maleic acid is able to inhibit the GAD of L. monocytogenes significantly enhancing its sensitivity to acidic conditions and together with its ability to remove biofilms, make a good candidate for disinfection regimes

Investigation into the antimicrobial activity of fumarate against Listeria monocytogenes and its mode of action under acidic conditions

Organic acids such as fumarate are commonly used as antimicrobials in foods. Apart from the classical mechanism of intracellular dissociation, weak acids are active through important additional mechanisms which are not well-defined. Fumarate, based on its low dissociation constants is expected to have a low antimicrobial activity which is not the case, suggesting additional antimicrobial effects. Previously, fumarate has been shown to inhibit the GAD system of E. coli and therefore, we investigated for first time how it affects this system in Listeria monocytogenes. We found that fumarate is highly antimicrobial towards L. monocytogenes under acidic conditions. We also show that in cell lysates and similarly to E. coli, fumarate inhibits the GAD system of L. monocytogenes. However, despite the inhibition and in contrast to E. coli, L. monocytogenes is able to counteract this and achieve a higher extracellular GAD output (measured by GABA export) in the presence of fumarate compared to its absence. The latter is achieved by a dramatic 9.44-fold increase in the transcription of gadD2 which is the main component of the extracellular GAD system. Interestingly, although maleate, the cis-isomer of fumarate results in a more dramatic 48.5-fold gadD2 upregulation than that of fumarate, the final GADe output is lower suggesting that maleate might be a stronger inhibitor of the GAD system. In contrast, the GADe removes more protons in the presence of fumarate than in the presence of HCl at the same pH. All the above suggest that there are additional effects by fumarate which might be associated with the intracellular GAD system (GADi) or other acid resistance systems. We assessed the GADi output by looking at the intracellular GABA pools which were not affected by fumarate. However, there are multiple pathways (e.g. GABA shunt) that can affect GABAi pools and we cannot conclusively suggest that GADi is affected. Furthermore, similarly to maleate, fumarate is able to eliminate L. monocytogenes in biofilms under acidic conditions. Overall, fumarate is a good candidate for L. monocytogenes decontamination and biofilm removal which is not toxic compared to the toxic maleate

Determination of the relative effects of temperature, pH and water activity in food systems: a meta-analysis study

The aim of this study is to use ComBase to determine the relative effects of temperature, pH, and water activity in the inactivation rates of Salmonella enterica in a range of foods. This is performed to determine whether any of the above factors have a dominant effect on survival. The inactivation rates of Salmonella were obtained from original raw data in the ComBase browser and from complete ComBase data for Salmonella. A total of 972 data of different types of food systems and data of individual types of food from ComBase were analysed. Over the range of 0–90°C, the z values calculated for the food data is 14°C. At 0–46°C relevant to intermediate moisture foods (IMF), the z values for the food data was 22°C, indicating a moderate effect of temperature. The z value for inactivation at 47–90°C was 11°C, indicating that temperature has an important effect on survival. This study shows that the effect of temperature is clearer at high temperatures than in the low temperature region. It suggests that the inactivation of Salmonella in food systems is slightly dominated by temperature and that the pH and aw levels appear to be less influential

Biofilm formation of Salmonella enterica and the central role of RpoS sigma factor in stress resistance

Non-typhoidal Salmonella is considered as the leading cause of foodborne illness and it has been associated with highprofile outbreaks in many groups of foods. This work examines the contribution of phenotypic properties related to survival (biofilm formation) and how these are linked with the genetic and functional variability of rpoS gene and RpoS status respectively in Salmonella enterica. The test strains were Salmonella serovars Anatum, Enteritidis (466), Enteritidis (496), Hadar, Heidelberg, Montevideo, Newport and Virchow and two Typhimurium strains previously characterised as either RpoSpositive or RpoS-negative. RpoS status was found to affect biofilm formation. The capability of Salmonella to resist stress and survive under unfavourable conditions can vary between strains. We confirmed that the two strains that were previously sensitive to various stresses harboured significant mutations in the rpoS gene. The rpoS sequencing not only confirmed a link between RpoS and biofilm formation, but it also revealed a link with differences in the utilisation of carbon sources. The RpoS-negative phenotype was linked with an increased growth under different carbon sources suggesting that a functional RpoS is a burden for growth which is in agreement with the SPANC hypothesi

High phenotypic variability among representative strains of common Salmonella enterica serovars with possible implications for food safety

Salmonella is an important foodborne pathogen whose ability to resist stress and survive can vary between strains. This variability is normally not taken into account when predictions are made about survival in foods with negative consequences. Therefore, we examined the contribution of variable phenotypic properties to survival under stress in 10 Salmonella serovars. One strain (S. Typhimurium 10) was intentionally an RpoS-negative however, another one (S. Heidelberg) showed an rpoS mutation rendering it inactive. We assessed an array of characteristics (motility, biofilm formation, bile resistance, acid resistance and colony morphology) showing a major variability between strains associated with a 10- to 19-fold difference between the highest and the lowest strain for most characteristics. The RpoS status of isolates did not affect variability in the characteristics with the exception of resistance to NaCl, acetic acid, lactic acid, and the combination of acetic acid and salt where the variability between the highest and the lowest strain was reduced to 3.1-fold, 1.7-fold, 2-fold and 1.7-fold respectively, showing that variability was significant among RpoS-positive strains. Furthermore, we also found a good correlation between acid resistance and lysine decarboxylase activity showing its importance for acid resistance and demonstrated a possible role of RpoS in the lysine decarboxylase activity in Salmonella

Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels

Anaerobic digestion of steam-exploded wheat straw and co-digestion strategies for enhanced biogas production

Wheat straw (WS) is considered a favourable substrate for biogas production. However, due to its rigid structure and high carbon to nitrogen (C/N ratio), its biodegradability during anaerobic digestion (AD) is usually low. In the present study, the effect of steam explosion pretreatment on WS, combined with C/N adjustment with inorganic nitrogen, on biogas production was evaluated. Additionally, co-digestion of WS with protein-rich agri-industrial by-products [dried distillers’ grains with solubles (DDGS) and rapeseed meal (RM)] was assessed. Steam explosion enhanced biogas production from WS, whereas the addition of NH4Cl was beneficial (p<0.05) for the digestion of steam-exploded wheat straw (SE). Furthermore, mono-digestion of the four different substrates seemed to be efficient in both inoculum to substrate ratios (I/S) tested [3.5 and 1.75 (w/w)]. Finally, during co-digestion of WS and SE with DDGS and RM, an increase in the cumulative methane production was noted when higher amounts of DDGS and RM were co-digested. This study demonstrated that DDGS and RM can be used as an AD supplement to stimulate gas production and improve wheat straw biodegradability, while their addition at 10 % on AD system operating with WS can enhance gas yields, in levels similar to those achieved by steam-exploded straw

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains