Monocytogenes Cells Research Articles

Effects of Oleuropein and Peracetic Acid as Sanitizing Agents for Inactivation of Biofilms

The development of more efficient sanitizers for reducing the biofilm produced by pathogenic bacteria is of great importance, especially in the food industry. The aim of this study was to conduct a preliminary evaluation of the efficiency of oleuropein (OLE), extracted from olive leaves, and peracetic acid (PAA), alone or in combination, to inactivate biofilms formed by a strong biofilm-producer strain of Listeria monocytogenes (ATCC 7644). A disk diffusion method was also used to assess the susceptibility of the ATCC 7644 strain cells to OLE and/or PAA. Triplicate biofilm assays were prepared on stainless steel coupons (1x1 cm2) during 48 h without stirring. After incubation, the stainless steel was washed (NaCl 0.85%) and immersed in solutions of OLE (5.0 mg/mL) and/or PAA (2.0%) for 1 min. After 1-min treatment, the disinfectant solutions were removed from the tube. Two mL of sodium thiosulfate 0.1 M was placed in the tube to stop reaction. The inactivation of biofilms was assessed by confocal laser scanning microscopy. L. monocytogenes cells tested in the disk diffusion method was not susceptible to OLE (inhibition zone (IZ) = 6 mm), although its susceptibility was intermediate for PAA (IZ = 8.67 mm) and higher for PAA+OLE (IZ = 14 mm). Compared with PAA, OLE alone in contact with biofilms had also lower bactericidal activity on the biofilms. However, the treatment of OLE in combination with PAA resulted in greater inactivation of L. monocytogenes cells in biofilms. Results indicate a potential application of OLE for enhancing the bactericidal effect of PAA against L. monocytogenes biofilms, although further studies are necessary to understand the mechanisms of action of OLE in combination with commercial chemical sanitizers.

Ralstonia insidiosa induces cell aggregation of Listeria monocytogenes

Biofilm formation is an important strategy for foodborne bacterial pathogens to survive in stressful environments such as fresh produce processing facilities. Bacterial cell aggregation strongly promotes the initiation of microcolonies and the formation of biofilms on abiological surfaces. We previously showed that Ralstonia insidiosa, an environmental bacterial species frequently isolated from fresh produce facilities, may serve as a “bridge bacterium” that strongly enhanced the incorporation of several foodborne bacterial pathogens into dual species biofilms. While the R. insidiosa strain exhibited moderate cell aggregation in liquid culture, co-culturing Listeria monocytogenes with R. insidiosa resulted in significant augmentation of cell aggregation. Electron microscopy indicated that L. monocytogenes cells were initially attracted to the R. insidiosa aggregates and formed large dual species aggregates that were predominately composed of L. monocytogenes cells. The predominant presence of L. monocytogenes in the dual species aggregates was also confirmed by differential plating. These findings suggest that bridge bacteria such as R. insidiosa play critical roles in the survival of foodborne bacterial pathogens, such as L. monocytogenes and Escherichia coli, by promoting multispecies biofilm formation. The implications of such bridge bacteria on food safety need to be further evaluated.

Behavior of Salmonella and Listeria monocytogenes in Raw Yellowfin Tuna during Cold Storage.

Behavior of Salmonella and Listeria monocytogenes in raw yellowfin tuna during refrigeration and frozen storage were studied. Growth of Salmonella was inhibited in tuna during refrigerated storage, while L. monocytogenes was able to multiply significantly during refrigerated storage. Populations of Salmonella in tuna were reduced by 1 to 2 log after 12 days of storage at 5–7 °C, regardless levels of contamination. However, populations of L. monocytogenes Scott A, M0507, and SFL0404 in inoculated tuna (104–105 CFU/g) increased by 3.31, 3.56, and 3.98 log CFU/g, respectively, after 12 days of storage at 5–7 °C. Similar increases of L. monocytogenes cells were observed in tuna meat with a lower inoculation level (102–103 CFU/g). Populations of Salmonella and L. monocytogenes declined gradually in tuna samples over 84 days (12 weeks) of frozen storage at −18 °C with Salmonella Newport 6962 being decreased to undetectable level (<10 CFU/g) from an initial level of 103 log CFU/g after 42 days of frozen storage. These results demonstrate that tuna meat intended for raw consumption must be handled properly from farm to table to reduce the risks of foodborne illness caused by Salmonella and L. monocytogenes.

Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15.

Holins are phage-encoded hydrophobic membrane proteins that spontaneously and non-specifically accumulate and form lesions in the cytoplasmic membrane. The ORF72 gene (also designated HolGH15) derived from the genome of the Staphylococcus aureus phage GH15 was predicted to encode a membrane protein. An analysis indicated that the protein encoded by HolGH15 potentially consisted of two hydrophobic transmembrane helices. This protein exhibited the structural characteristics of class II holins and belonged to the phage_holin_1 superfamily. Expression of HolGH15 in Escherichia coli BL21 cells resulted in growth retardation of the host cells, which was triggered prematurely by the addition of 2,4-dinitrophenol. The expression of HolGH15 caused morphological alterations in engineered E. coli cells, including loss of the cell wall and cytoplasmic membrane integrity and release of intracellular components, which were visualized by transmission electron microscopy. HolGH15 exerted efficient antibacterial activity at 37 °C and pH 5.2. Mutation analysis indicated that the two transmembrane domains of HolGH15 were indispensable for the activity of the full-length protein. HolGH15 showed a broad antibacterial range: it not only inhibited Staphylococcus aureus, but also demonstrated antibacterial activity against other species, including Listeria monocytogenes, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli. At the minimal inhibitory concentration, HolGH15 evoked the release of cellular contents and resulted in the shrinkage and death of Staphylococcus aureus and Listeria monocytogenes cells. To the best of our knowledge, this study is the first report of a Staphylococcus aureus phage holin that exerts antibacterial activity against heterogeneous pathogens.

Phage Inactivation of Listeria monocytogenes on San Daniele Dry-Cured Ham and Elimination of Biofilms from Equipment and Working Environments.

The anti-listerial activity of generally recognized as safe (GRAS) bacteriophage Listex P100 (phage P100) was demonstrated in broths and on the surface of slices of dry-cured ham against 5 strains or serotypes (i.e., Scott A, 1/2a, 1/2b, and 4b) of Listeria monocytogenes. In a broth model system, phage P100 at a concentration equal to or greater than 7 log PFU/mL completely inhibited 2 log CFU/cm2 or 3 log CFU/cm2 of L. monocytogenes growth at 30 °C. The temperature (4, 10, 20 °C) seemed to influence P100 activity; the best results were obtained at 4 °C. On dry-cured ham slices, a P100 concentration ranging from 5 to 8 log PFU/cm2 was required to obtain a significant reduction in L. monocytogenes. At 4, 10, and 20 °C, an inoculum of 8 log PFU/cm2 was required to completely eliminate 2 log L. monocytogenes/cm2 and to reach the absence in 25 g product according to USA food law. Conversely, it was impossible to completely eradicate L. monocytogenes with an inoculum of approximately of 3.0 and 4.0 log CFU/cm2 and with a P100 inoculum ranging from 1 to 7 log PFU/cm2. P100 remained stable on dry-cured ham slices over a 14-day storage period, with only a marginal loss of 0.2 log PFU/cm2 from an initial phage treatment of approximately 8 log PFU/cm2. Moreover, phage P100 eliminated free L. monocytogenes cells and biofilms on the machinery surfaces used for dry-cured ham production. These findings demonstrate that the GRAS bacteriophage Listex P100 at level of 8 log PFU/cm2 is listericidal and useful for reducing the L. monocytogenes concentration or eradicating the bacteria from dry-cured ham.

The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication

Lmof2365_2117 is a Listeria monocytogenes putative cell wall surface anchor protein with a conserved domain found in collagen binding proteins. We constructed a deletion mutation in lmof2365_2117 in serotype 4b strain F2365, evaluated its virulence, and determined its ability to adhere and invade colonic epithelial cells and macrophages. In A/J mice, colonization of liver was significantly higher for F2365 than for F2365Δ2117. The ability of F2365Δ2117 to adhere to Caco-2 cells was significantly lower than F2365. The mutant also showed impaired ability to replicate in intestinal epithelial cell and murine macrophages relative to wild type F2365. Lmof2365_2117 contributed to L. monocytogenes attachment to catfish fillets. Because of its role in adherence to Caco-2 cells, we designated Lmof2365_2117 Listeria adhesion protein B (LapB). The carboxy terminus of LapB is similar to a domain in collagen binding proteins, but our results show that L. monocytogenes does not bind collagen.

The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes.

Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

Influence of temperature on alkali stress adaptation in Listeria monocytogenes

Listeria monocytogenes cells may induce alkali stress adaptation when exposed to sublethal concentrations of alkaline cleaners and sanitizers that may be frequently used in the food processing environment. In the present study, the effect of temperature on the induction and the stability of such alkali stress adaptation in L. monocytogenes were investigated. Our results demonstrate that temperature plays a critical role in the induction of alkali stress adaptation in L. monocytogenes under sublethal alkaline conditions. Pre-exposure to pH 9.0 tryptic soy broth supplemented with 0.6% yeast extract (TSB-YE) at 37 °C induced pronounced alkali stress adaptation whereas sublethal alkaline pre-exposure at 4 °C failed to induce any alkali stress adaptation. Furthermore, this pattern of alkali stress adaptation in L. monocytogenes was not dependent on the length of pre-exposure time, the concentration of sublethal alkali, the types of alkaline agents and the growth phases of cells. In addition, alkali stress adaptation induced at 37 °C was completely reversed in pH 7.2 TSB-YE within 2 h at 37 °C or within 4 h at 22 °C. However, once it was induced at a higher temperature, alkali stress adaptation in L. monocytogenes remained stable at 4 °C for at least 4 h. Our findings suggest that even though cold temperatures do not induce alkali stress adapted phenotypes, but it can maintain the previously acquired alkali stress adaptation much longer in L. monocytogenes.

Quantification of Listeria monocytogenes cells with digital PCR and their biofilm cells with real-time PCR

The purpose of this study was to develop a PCR-based method for quantification of Listeria monocytogenes adhesion in microtitre plates. We optimized isolation of DNA in the microtitre plates using cell lysis, ultrasound treatment, heating, and centrifugation of the lysate. Digital PCR was applied for quantification of L. monocytogenes DNA that was used for construction of the standard curve, and real-time PCR was used for quantification of the attached L. monocytogenes cells. This PCR-based method was applied to quantify different strains of L. monocytogenes at different times of biofilm formation, and to study the anti-adhesive actions of natural bioactive substances (epigallocatechin gallate, (−)-α-pinene). The results show that the PCR-based method developed here can be widely used as a novel approach for adhesion assays and biofilm research.

Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid listeria detection.

A fluorescent DNA aptamer-magnetic bead sandwich assay was developed to detect listeriolysin O (LLO) protein from pathogenic Listeria bacteria using a peroxidase-linked system, Amplex Ultra Red (AUR; derivatized resazurin) substrate, and a custom-designed handheld fluorometer. The assay is highly sensitive with demonstrated limits of detection (LODs) in the range of 4 to 61 L. monocytogenes cells or the equivalent LLO produced by 4 to 61 cells on average in separate titration trials. Total assay processing and analysis time was approximately 30 mins. The assay has demonstrated the ability to detect 6 species of Listeria as desired by the USDA's Food Safety Inspection Service (FSIS). The portable system was designed to be used primarily with surface swab samples from fomites, but it can also be used to assess enrichment cultures. The minimal time to detect a positive enrichment culture in our hands from an initial 10 cell inoculum in 200 ml of broth has been 8 h post-incubation at 37 °C in shaker flask cultures. An optional automated magnetic bead assay processing and wash device capable of simultaneously processing 6 samples with low and consistent fluorescence background for higher volume central laboratories is also described.

Bi-phasic growth of Listeria monocytogenes in chemically defined medium at low temperatures

The present work reports a novel observation regarding the growth of L. monocytogenes in modified Welshimer's broth (MWB) at low temperatures. Specifically, the direct monitoring of the growth of L. monocytogenes Scott A using plate count data revealed that the pathogen displays a bi-phasic growth pattern in MWB at 7°C. This bi-phasic growth pattern is masked (not observed) when optical density (OD) measurements are used to monitor growth due to the inability of OD readings to detect L. monocytogenes population density increases up to 107CFU/mL. This bi-phasic growth phenomenon was further investigated as a function of growth temperature (4°C, 7°C, 10°C, 14°C and 18°C), medium composition (by altering the MWB composition by ten-fold increases in different sets of medium constituents), inoculum level (102, 103, 104, 105, 106, and 107CFU/mL) and L. monocytogenes strain (10 strains). The growth of L. monocytogenes Scott A in MWB at 7°C, 10°C and 14°C was consistently bi-phasic and independent of growth rate; at 18°C, growth was consistently mono-phasic (single-phase, typical sigmoid growth curves), whereas no growth was observed at 4°C. The tested modifications in the composition of MWB did not influence the bi-phasic nature of L. monocytogenes Scott A growth at 7°C, and, overall, we could not point out any strain-, or serotype-specific effects. On the other hand, the initial inoculum level appears to affect the form of the growth curve, as there was a shift towards mono-phasic growth in trials with increasing initial inocula. A mathematical model, based on a stepwise response and described through two sequential sigmoid curves, was used to describe bi-phasic growth and estimate the kinetic parameters of L. monocytogenes growth. An alternative hypothesis, based on the assumption of the existence of two subpopulations, possessing different growth kinetics, materialized under the stress imposed on L. monocytogenes cells due to the combined effect of three factors (defined medium, low temperature and low initial inoculum) was also proposed and formulated.

Reduction of Listeria monocytogenes in cold‐smoked salmon by bacteriophage P100, nisin and lauric arginate, singly or in combinations

SummaryThree GRAS antimicrobials including, lauric arginate (LAE), bacteriophage P100 (phage P100) and bacteriocin nisin, were evaluated either singly or in combinations for the reduction of initial load of Listeria monocytogenes in cold‐smoked salmon (CSS). The stability of phage P100 in the presence of LAE (200 ppm) and nisin (500 ppm) or at 10× and 100× of these concentrations was determined at 4 °C or 30 °C for 24 h in a broth model. Phage P100 was found to be highly stable in the presence of these antimicrobial agents as plaque‐forming units (PFU) did not vary between control and antimicrobial‐treated phage. The survival of L. monocytogenes in the presence of phage P100, nisin and LAE showed remarkable reduction within 24 h both at 4 °C or 30 °C in broth. Treatment of CSS containing 3.5 log CFU cm−2 L. monocytogenes with phage P100 (108 PFU mL−1), nisin (500 ppm) and LAE (200 ppm) showed strong listericidal action and reduced the L. monocytogenes by 2–3 log CFU cm−2 after 24 h. Among the combined treatments, phage P100 + LAE or nisin + LAE exhibited the most listericidal action in which L. monocytogenes cells were reduced to undetectable level within 24 h in CSS.

Selection and characterization of DNA aptamers specific for Listeria species

Single-stranded (ss) DNA aptamers with binding affinity to Listeria spp. were selected using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Listeria monocytogenes cells were grown at 37°C and harvested at mid-log phase or early stationary phase to serve as the targets in SELEX. A total of 10 unique aptamer sequences were identified, six associated with log phase cells and four with stationary phase cells. Binding affinity of the aptamers was determined using flow cytometry and ranged from 10% to 44%. Four candidates having high binding affinity were further studied and found to show genus-specific binding affinity when screened against five different species within the Listeria genus. Using sequential binding assays combined with flow cytometry, it was determined that three of the aptamers (LM6-2, LM12-6, and LM12-13) bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to bind to a different cell surface region. This is the first study in which SELEX targeted bacterial cells at different growth phases. When used together, aptamers that bind to different cell surface moieties could increase the analytical sensitivity of future capture and detection assays.

Comparison of individual-based modeling and population approaches for prediction of foodborne pathogens growth

Individual-based modeling (IBM) approach combined with the microenvironment modeling of vacuum-packed cold-smoked salmon was more effective to describe the variability of the growth of a few Listeria monocytogenes cells contaminating irradiated salmon slices than the traditional population models. The IBM approach was particularly relevant to predict the absence of growth in 25% (5 among 20) of artificially contaminated cold-smoked salmon samples stored at 8 °C. These results confirmed similar observations obtained with smear soft cheese (Ferrier et al., 2013). These two different food models were used to compare the IBM/microscale and population/macroscale modeling approaches in more global exposure and risk assessment frameworks taking into account the variability and/or the uncertainty of the factors influencing the growth of L. monocytogenes. We observed that the traditional population models significantly overestimate exposure and risk estimates in comparison to IBM approach when contamination of foods occurs with a low number of cells (<100 per serving). Moreover, the exposure estimates obtained with the population model were characterized by a great uncertainty. The overestimation was mainly linked to the ability of IBM to predict no growth situations rather than the consideration of microscale environment. On the other hand, when the aim of quantitative risk assessment studies is only to assess the relative impact of changes in control measures affecting the growth of foodborne bacteria, the two modeling approach gave similar results and the simplest population approach was suitable.

Impacts of Iron Oxide Nanoparticles on the Invasion Power of Listeria monocytogenes

Recently, magnetic nanoparticles have been introduced as a novel antimicrobial material. Listeria monocytogenes are the causative agent of listeriosis and stands among the major public health issues. This bacterium can attach to the host cell membrane and trigger molecular responses leading to phagocytosis of the bacterial cell. In the present study, the effect of iron oxide nanoparticles (IONs) on the invasion power of L. monocytogenes was investigated. IONs were synthesized by coprecipitation method and coated with 3-aminopropyltriethoxysilane (APTES). Listeria monocytogenes cells were exposed to the prepared particles and interactions between IONs and bacterial cells were visualized by scanning electron microscopy. Invasion power of listerial cells for penetration HepG-2 cells monolayer was also examined in various concentrations of IONs. IONs have reduced invasion power of L. monocytogenes in a concentration-dependent manner. Almost 4-fold reduction in the invasion power of L. monocytogenes was observed at 20 µg ml-1 IONs concentration. Keywords: Antibacterial, 3-aminopropyltriethoxysilane (APTES), Listeria monocytogenes, magnetic nanoparticles, pathogenesis.

Rapid Detection of Listeria monocytogenes by Real-Time PCR in Processed Meat and Dairy Products

The objectives of this study were to evaluate the detection of Listeria monocytogenes in different ready-to-eat foods using real-time PCR (RT-PCR). Various concentrations (10(0) to 10(5) CFU/ml) of L. monocytogenes ATCC 19115 were inoculated into ham, sausage, ground meat, processed milk, cheese, and infant formula. L. monocytogenes ATCC 19115 in the samples was then enumerated on Oxford agar, and DNA was extracted from the samples before and after incubation at 36°C for 4 h. A set of primers and hybridization probe designed in this study was then used to detect the pathogen. The standard curve was then prepared by plotting cycle threshold values for each dilution versus L. monocytogenes cell counts (log CFU). The specificity of the set of primers and hybridization probe was appropriate. A 4-h incubation at 36°C before DNA extraction produced optimum standard curves in comparison to the results for a 0-h incubation. Thus, a 4-h incubation at 36°C was applied for monitoring L. monocytogenes in collected food samples. To monitor L. monocytogenes in foods, 533 samples (ham, 129; sausage, 226; ground meat, 72; processed cheese, 54; processed milk, 42; and infant formula, 10) were collected from retail markets and from the step before pasteurization in plants. Of all 533 samples, 4 samples (0.8%) showed positive signals in RT-PCR. Two samples from hams (1.6%) and two samples from sausages (0.9%) were determined to be positive for L. monocytogenes at < 100 CFU/g. The results indicate that the RT-PCR detection method with the set of primers and hybridization probe designed in this study should be useful in monitoring for L. monocytogenes in processed meat and milk products.

Effect of citral and carvacrol on the susceptibility of Listeria monocytogenes and Listeria innocua to antibiotics

The aim of this study was to evaluate the antibiotic susceptibility of Listeria innocua (L. innocua) and Listeria monocytogenes (L. monocytogenes) cells in the presence of citral and carvacrol at sublethal concentrations in an agar medium. The presence of terpenes in the L. monocytogenes and L. innocua culture medium provided a reduction in the minimal inhibitory concentration (MIC) of all the antibiotics tested. These effects were dependent on the concentration of terpenes present in the culture medium. The combination of citral and carvacrol potentiated antibiotic activity by reducing the MIC values of bacitracin and colistin from 32.0 and 128.0 μg ml⁻¹ to 1.0 and 2.0 μg ml⁻¹, respectively. Thus, both Listeria species became more susceptible to these drugs. In this way, the colistin and bacitracin resistance of L. monocytogenes and L. innocua was reversed in the presence of terpenes. Results obtained in this study show that the phytochemicals citral and carvacrol potentiate antibiotic activity, reducing the MIC values of cultured L. monocytogenes and L. innocua. Phytochemicals citral and carvacrol potentiate antibiotic activity of erythromycin, bacitracin and colistin by reducing the MIC values of cultured Listeria monocytogenes and Listeria innocua. This effect in reducing the MIC values of the antibiotics tested in both micro-organisms was increased when natural antimicrobials were combined. This finding indicated that the combination among terpenes and antibiotic may contribute in reducing the required dosage of antibiotics due to the possible effect of terpenes on permeation barrier of the micro-organism cell membrane.

Gene Transcription Patterns of pH- and Salt-Stressed Listeria monocytogenes Cells in Simulated Gastric and Pancreatic Conditions

A Listeria monocytogenes subgenomic array, targeting 54 genes involved in the adhesion, adaptation, intracellular life cycle, invasion, and regulation of the infection cycle was used to investigate the gene expression patterns of acid- and salt-stressed Listeria cells after exposure to conditions similar to those in gastric and pancreatic fluids. Three L. monocytogenes strains, one laboratory reference strain (EGDe) and two food isolates (wild strain 12 isolated from milk and wild strain 3 isolated from fermented sausage), were used during the studies. Differences in the expressed genes were observed between the gastric and pancreatic treatments and also between the serotypes. Increased transcripts were observed of the genes belonging to the adaptation and regulation group for serotype 4b (strain 12) and to the invasion and regulation group for serotype 1/2a (strain EGDe). Interestingly, no significantly differentially expressed genes were found for serotype 3c (strain 3) in most cases. The genes related to adaptation (serotype 1/2a) and to intracellular life cycle and invasion (serotype 4b) were down-regulated in order to cope with the hostile environment of the gastric and pancreatic fluids. These findings may provide experimental evidence for the dominance of serotypes 1/2a and 4b in clinical cases of listeriosis and for the sporadic occurrence of serotype 3c.

Listeria monocytogenes virulence factor secretion: don't leave the cell without a chaperone

In Gram-positive bacteria, the secretion of proteins requires translocation of polypeptides across the bacterial membrane into the highly charged environment of the membrane-cell wall interface. Here, proteins must be folded and often further delivered across the matrix of the cell wall. While many aspects of protein secretion have been well studied in Gram-negative bacteria which possess both an inner and outer membrane, generally less attention has been given to the mechanics of protein secretion across the single cell membrane of Gram-positive bacteria. In this review, we focus on the role of a post-translocation secretion chaperone in Listeria monocytogenes known as PrsA2, and compare what is known regarding PrsA2 with PrsA homologs in other Gram-positive bacteria. PrsA2 is a member of a family of membrane-associated lipoproteins that contribute to the folding and stability of secreted proteins as they cross the bacterial membrane. PrsA2 contributes to the integrity of the L. monocytogenes cell wall as well as swimming motility and bacterial resistance to osmotic stress; however its most critical role may be its requirement for L. monocytogenes virulence and viability within host cells. A better understanding of the role of PrsA2 and PrsA-like homologs will provide insight into the dynamics of protein folding and stability in Gram-positive bacteria and may result in new strategies for optimizing protein secretion as well as inhibiting the production of virulence factors.

Impact of acid adaptation on attachment of Listeria monocytogenes to stainless steel during long-term incubation under low or moderate temperature conditions and on subsequent recalcitrance of attached cells to lethal acid treatments

This study aimed to evaluate the possible impact of acid adaptation of Listeria monocytogenes cells on their attachment to stainless steel (SS) during long-term incubation under either low or moderate temperature conditions and on the subsequent recalcitrance of attached cells to lethal acid treatments. Initially, nonadapted or acid-adapted stationary phase L. monocytogenes cells were used to inoculate (ca. 108CFU/ml) brain–heart infusion (BHI) broth in test tubes containing vertically placed SS coupons. Incubation was carried out at either 5 or 30°C for up to 15days, under static conditions. On the 5th, 10th and 15th days of incubation, attached cells were subjected to lethal acid treatments by exposing them, for either 6 or 60min, to pH2, adjusted with either hydrochloric or lactic acid. Following the acid treatments, remaining viable cells were detached (through strong vortexing with glass beads) and enumerated by agar plating, and also indirectly quantified by conductance measurements via their metabolic activity. Results obtained from both quantification techniques, employed here in parallel, revealed that although the numbers of attached cells for nonadapted and acid-adapted ones were similar, the latter were found to present significantly (p<0.05) increased recalcitrance to all the acid treatments for both incubation temperatures and all sampling days. In addition and regardless of acid adaptation, when long (60min) acid treatments were applied, conductance measurements revealed that the weak organic lactic acid exhibited significantly (p<0.05) stronger antilisterial activity compared to the strong inorganic hydrochloric acid (at the same pH value of 2). To conclude, present results show that acid adaptation of L. monocytogenes cells during their planktonic growth is conserved even after 15days of incubation under both low and moderate temperature conditions, and results in the increased recalcitrance of their sessile population to otherwise lethal acid treatments. This “stress hardening” should be severely taken into account when acidic decontamination interventions are used to kill attached to equipment surfaces cells of this important pathogenic bacterium.