Adhesion of the probiotic strains <Emphasis Type="Italic">Enterococcus mundtii</Emphasis> ST4SA and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> 423 to Caco-2 cells under conditions simulating the intestinal tract,and in the presence of antibiotics and anti-inflammatory medicaments

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.     

Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro

Background

Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.

Methodology/Principal Findings

The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp^LAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp^LAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp^LAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp^LAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp^LAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.

Conclusions/Significance

Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp^LAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.     

Lactobacillus casei DN-114 001 Inhibits the Ability of Adherent-Invasive Escherichia coli Isolated from Crohn's Disease Patients To Adhere to and To Invade Intestinal Epithelial Cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

Coexpression of bile salt hydrolase gene and catalase gene remarkably improves oxidative stress and bile salt resistance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

Lactic acid bacteria (LAB) encounter various types of stress during industrial processes and gastrointestinal transit. Catalase (CAT) and bile salt hydrolase (BSH) can protect bacteria from oxidative stress or damage caused by bile salts by decomposing hydrogen peroxide (H₂O₂) or deconjugating the bile salts, respectively. Lactobacillus casei is a valuable probiotic strain and is often deficient in both CAT and BSH. In order to improve the resistance of L. casei to both oxidative and bile salts stress, the catalase gene katA from L. sakei and the bile salt hydrolase gene bsh1 from L. plantarum were coexpressed in L. casei HX01. The enzyme activities of CAT and BSH were 2.41 μmol H₂O₂/min/10⁸ colony-forming units (CFU) and 2.11 μmol glycine/min/ml in the recombinant L. casei CB, respectively. After incubation with 8 mM H₂O₂, survival ratio of L. casei CB was 40-fold higher than that of L. casei CK. Treatment of L. casei CB with various concentrations of sodium glycodeoxycholate (GDCA) showed that ~10⁵ CFU/ml cells survived after incubation with 0.5% GDCA, whereas almost all the L. casei CK cells were killed when treaded with 0.4% GDCA. These results indicate that the coexpression of CAT and BSH confers high-level resistance to both oxidative and bile salts stress conditions in L. casei HX01.     

Nisin‐induced expression of pediocin in dairy lactic acid bacteria

Aims: To test whether a single vector, nisin‐controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. Methods and Results: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0·8‐kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml^?1 nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. Conclusions: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. Significance and Impact of the Study: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.     

Adhesion, Invasion, and Translocation Characteristics of Listeria monocytogenes Serotypes in Caco-2 Cell and Mouse Models

Adhesion is a crucial first step in Listeria monocytogenes pathogenesis. In this study, we examined how the adhesion properties of serotypes correlate with their invasion efficiencies in a cell culture model (Caco-2) and in a mouse model. Adhesion characteristics of all 13 serotypes of L. monocytogenes (25 strains) were analyzed, which yielded three distinct groups (P < 0.05) with high-, medium-, and low-level-adhesion profiles. The efficiency of these strains in invading the Caco-2 cell line was analyzed, which produced two groups; however, the overall correlation (R²) was only 0.1236. In the mouse bioassay, all selected strains, irrespective of their adhesion profiles, translocated to the liver and the spleen with almost equal frequencies that did not show any clear relationship with adhesion profiles. However, the serotypes with increased adhesion showed a slightly increased translocation to the brain (R² = 0.3371). Collectively, these results indicate that an in vitro adhesion profile might not be an accurate assessment of a strain's ability to invade a cultured cell line or organs or tissues in a mouse model.     

Volatiles in perennial ryegrass infected with strains of endophytic fungus: impact on African black beetle host selection

Perennial ryegrass (Lolium perenne) is often infected with the fungal‐endophyte Neotyphodium lolii. In addition to the ‘wild‐type’ strain (E^WT), several ‘selected’ strains of N. lolii are now being marketed as AR1 (E^AR1) and AR37 (E^AR37). Each of these strains impact positively on L. perenne's resistance against many insects, including the African black beetles (Heteronychus arator). The impact of volatile oils produced specifically by each strain in the endophyte–grass association in enhancing the grass's resistance to insects is still largely unknown. Keeping these in view, we determined the volatile oil profiles produced by L. perenne infected with either E^WT or E^AR1 or E^AR37 and determined the impacts of these volatiles on the host‐selection behaviour of H. arator adults. In the absence of endophyte infection (E^–), L. perenne produced 18 different volatile oils. In L. perenne E^WT, quantities of 2‐ethyl‐1‐hexanol acetate (Rt = 14.5 min), (Z)‐2‐octen‐1‐ol (Rt = 22.2 min), and butylated hydroxyl toluene (Rt = 23.2 min) were 24, 16 and 26%, respectively, greater than L. perenne E^–. The strains E^AR1 and E^AR37 affected differently the quantities of the volatile compounds but not their identity. In the four‐choice bioassay, males and females of H. arator were equally attracted to each strain. In Y‐tube olfactometer, compared against E^–, H. arator adults were less attracted to L. perenne E^WT and E^AR1. The attractiveness of E^AR37 was similar in effect to E^– to H. arator. The results indicate that each strain of N. lolii alters the profile of volatile oils in L. perenne differently and that alteration can influence H. arator adult‐host selection.     

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B (σ^B), whose role in protection against acid stress is well established. Here, we investigated the involvement of the stressosome, a sensory hub, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated σ^B and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was solely present retained the ability to induce σ^B activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1-T175A, -T209A, -T241A) and RsbS (rsbS-S56A), or the stressosome kinase RsbT (rsbT-N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1-T209A and rsbR1-T241A mutants displayed constitutive σ^B activity. Mild acid shock upregulates invasion genes inlAB and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of σ^B in L. monocytogenes. Furthermore, they show that RsbR1 can function independently of its paralogues and signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209 but not T175. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.     

Characterization of a fibronectin‐binding protein from Lactobacillus casei BL23

Aims: To characterize the functionality of the Lactobacillus casei BL23 fbpA gene encoding a putative fibronectin‐binding protein. Methods and Results: Adhesion tests showed that L. casei BL23 binds immobilized and soluble fibronectin in a protease‐sensitive manner. A mutant with inactivated fbpA showed a decrease in binding to immobilized fibronectin and a strong reduction in the surface hydrophobicity as reflected by microbial adhesion to solvents test. However, minor effects were seen on adhesion to the human Caco‐2 or HT‐29 cell lines. Purified 6X(His)FbpA bound to immobilized fibronectin in a dose‐dependent manner. Western blot experiments with FbpA‐specific antibodies showed that FbpA could be extracted from the cell surface by LiCl treatment and that protease digestion of the cells reduced the amount of extracted FbpA. Furthermore, surface exposition of FbpA was detected in other L. casei strains by LiCl extraction and whole‐cell ELISA. Conclusions: FbpA can be found at the L. casei BL23 surface and participates in cell attachment to immobilized fibronectin. We showed that FbpA is an important, but not the only, factor contributing to fibronectin binding in BL23 strain. Significance and Impact of the Study: This is the first report showing the involvement of FbpA in fibronectin binding in L. casei BL23 and represents a new contribution to the study of attachment factors in probiotic bacteria.     

Insights into collateral susceptibility and collateral resistance in Acinetobacter baumannii during antimicrobial adaptation

The susceptibility of Acinetobacter baumannii exposed to primary antibiotic can be either increased or decreased when exposed to secondary antibiotic. This study was designed to assess the relative fitness, collateral susceptibility and collateral resistance of polymyxin B- (PMB-) adapted A. baumannii to ciprofloxacin (CIP), meropenem (MER), PMB, tetracycline (TET) and tobramycin (TOB). Strains of wild-type A. baumannii KACC 12454 (AB^KACC), wild-type A. baumannii CCARM 12088 (AB^CCARM), PMB-adapted AB^KACC, PMB-adapted AB^CCARM, stabilized AB^KACC and stabilized AB^CCARM were used in this study. Compared to the wild-type AB^KACC, the MICs of PMB were increased from 2 to 128 μg ml⁻¹ against PMB-adapted AB^KACC, while MICs of CIP, MER, TET and TOB were decreased from 2 to 1 μg ml⁻¹, 16 to 1 μg ml⁻¹, 16 to 2 μg ml⁻¹ and 64 to 16 μg ml⁻¹, respectively. The PMB-adapted AB^CCARM was resistant to CIP (32 μg ml⁻¹) and PMB (64 μg ml⁻¹) compared to the wild-type AB^CCARM. The resistance of stabilized AB^KACC and AB^CCARM to all antibiotics was lost after antibiotic-free culture in the exception of CIP and TET. The susceptibilities of wild-type, PMB-adapted and stabilized AB^KACC and AB^CCARM to CIP, MER, PMB, TET and TOB were increased in the presence of β-lactamase and efflux pump inhibitors. The high levels of relative fitness were observed for stabilized AB^KACC, PMB-adapted AB^CCARM and stabilized AB^CCARM. The stabilized AB^KACC and PMB-adapted AB^CCARM were highly heteroresistance to PMB and TET, respectively. The PMB-adapted AB^KACC and AB^CCARM showed various antibiotic patterns, known as collateral susceptibility and collateral resistance. The results provide useful information for designing effective antibiotic regimens that can enhance the antibiotic activity against A. baumannii infections.     

Effect of Bacteriocins and Conditions that Mimic Food and Digestive Tract on Biofilm Formation, In Vitro Invasion of Eukaryotic Cells and Internalin Gene Expression by Listeria monocytogenes

In this report, Listeria monocytogenes isolates were evaluated for their ability to form biofilms, for adhesion/invasion of eukaryotic cells and for differential expression of internalin A (inl A) gene, which is related to virulence potential. The presence of bacteriocins of lactic acid bacteria and incubation at 5 °C were the main factors that influenced biofilm formation by L. monocytogenes, in comparison with BHI (control). In general, adhesion and invasion of Caco-2 cells were significantly lower in low pH (4.5), in incubation at 5 °C and in the presence of Oxgall 0.3 %. On the other hand, two L. monocytogenes isolates (INCQS 353 and Reg 26c) showed higher invasion rates when cultivated in the presence of NaCl 5 % (P < 0.05). One L. monocytogenes isolate (H-2) showed the strongest ability to form biofilm and to invade Caco-2 cells, under selected conditions, suggesting there is a relationship between biofilm formation and virulence potential. For all isolates, expression of inl A gene was down-regulated by the presence of bacteriocins, Oxgall 0.3 %, pH 4.5 and incubation at 5 °C. Nonetheless, for one L. monocytogenes isolate (HU 471), expression of inl A gene was eight times higher in the presence of sucrose, indicating that food components can increase the infectiveness of L. monocytogenes.     

Recurrent and Sporadic Listeria monocytogenes Contamination in Alheiras Represents Considerable Diversity, Including Virulence-Attenuated Isolates

Microbiological characterization of alheiras, traditional smoked meat sausages produced in northern Portugal, had previously shown that more than 60% of the lots analyzed were contaminated with Listeria monocytogenes at levels higher than 100 CFU/g. In order to better understand L. monocytogenes contamination patterns in alheiras, we characterized 128 L. monocytogenes isolates from alheiras using a variety of subtyping techniques (i.e., molecular serotyping; arsenic, cadmium, and tetracycline resistance typing; and pulsed-field gel electrophoresis [PFGE]). Subtyping of isolates from products collected on two separate dates provided evidence for the persistence of specific L. monocytogenes PFGE types in the production and distribution chains of alheiras from four different processors. A subset of 21 isolates was further characterized using ribotyping and Caco-2 cell invasion assays to evaluate the pathogenic potential of L. monocytogenes present in alheiras. Caco-2 invasion assays revealed seven isolates with invasion efficiencies that were less than 20% of that of the control strain 10403S. All seven isolates had premature stop codons in inlA that represented three distinct mutations, which had previously been observed in isolates from the United States or France. Our findings indicate the need for a comprehensive approach to control L. monocytogenes in alheiras, including strategies to reduce persistence. The presence of considerable diversity in invasion phenotypes among L. monocytogenes strains present in alheiras, including the presence of subtypes likely to be virulence attenuated, may provide an opportunity to initially focus control strategies on the subtypes most likely to cause human disease.     

Antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food

Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10³ CFU ml^?1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10² CFU 25 g^?1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.     

Inflammasome-Mediated Inhibition of Listeria monocytogenes-Stimulated Immunity Is Independent of Myelomonocytic Function

Activation of the Nlrc4 inflammasome results in the secretion of IL-1β and IL-18 through caspase-1 and induction of pyroptosis. L. monocytogenes engineered to activate Nlrc4 by expression of Legionella pneumophilia flagellin (L. monocytogenes L.p.FlaA) are less immunogenic for CD8⁺ T cell responses than wt L. monocytogenes. It is also known that IL-1β orchestrates recruitment of myelomonocytic cells (MMC), which have been shown to interfere with T cell-dendritic cells (DC) interactions in splenic white pulp (WP), limiting T cell priming and protective immunity. We have further analyzed the role of MMCs in the immunogenicity of L. monocytogenes L.p.FlaA. We confirmed that MMCs infiltrate the WP between 24–48 hours in response to wt L. monocytogenes infection and that depletion of MMCs enhances CD8⁺ T cell priming and protective memory. L. monocytogenes L.p.FlaA elicited accelerated recruitment of MMCs into the WP. While MMCs contribute to control of L. monocytogenes L.p.FlaA, MMC depletion did not increase immunogenicity of L.p.FlaA expressing strains. There was a significant decrease in L. monocytogenes L.p.FlaA in CD8α⁺ DCs independent of MMCs. These findings suggest that limiting inflammasome activation is important for bacterial accumulation in CD8α⁺ DCs, which are known to be critical for T cell response to L. monocytogenes.     

Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that causes gastroenteritis, meningitis, encephalitis and maternofetal infections. 20–30% of eubacterial ORFs are predicted to encode membrane proteins. The bacterial cytoplasmic membrane is a macromolecular structure, which plays a key role for the pathogenesis. Despite this, little knowledge exists regarding the function of cytoplasmic membrane proteins of Listeria during infection. Here, we investigated a predicted membrane protein of the pathogen L. monocytogenes, Lmo0412, of unknown function. Lmo0412 is only present in the Listeria genus and low conserved in the non-pathogenic species L. innocua. Bacterial fractionation and western blot analyses showed that Lmo0412 was only detectable in the membrane of L. monocytogenes EGDe during logarithmic growth phase. lmo0412 expression in L. monocytogenes was down-regulated during in vitro infection of JEG-3 epithelial cells. An L. monocytogenes mutant deficient in this membrane protein showed increased invasion of Caco-2 and NRK-49F host cells using in vitro infection models. Moreover, the lack of Lmo0412 in this deletion mutant increased the viable bacteria counts in the spleen and liver of mice compared to the wild type strain. Taken together, these data suggest a selective advantage conferred by the absence of Lmo0412 for the virulence of L. monocytogenes.     

Antagonistic Activity of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> ATCC 4356 on the Growth and Adhesion/Invasion Characteristics of Human <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

The aim of this research was to determine the potential probiotic activity of Lactobacillus acidophilus ATCC 4356 against several human Campylobacter jejuni isolates. The ability to inhibit the pathogen’s growth was evaluated by co-culture experiments as well as by antimicrobial assays with cell-free culture supernatant (CFCS), while interference with adhesion/invasion to intestinal Caco-2 cells was studied by exclusion, competition, and displacement tests. In the co-culture experiments L. acidophilus ATCC 4356 strain reduced the growth of C. jejuni with variable percentages of inhibition related to the contact time. The CFCS showed inhibitory activity against C. jejuni strains, stability to low pH, and thermal treatment and sensitivity to proteinase K and trypsin. L. acidophilus ATCC 4356 was able to reduce the adhesion and invasion to Caco-2 cells by most of the human C. jejuni strains. Displacement and exclusion mechanisms seem to be the preferred modalities, which caused a significant reduction of adhesion/invasion of pathogens to intestinal cells. The observed inhibitory properties of L. acidophilus ATCC 4356 on growth ability and on cells adhesion/invasion of C. jejuni may offer potential use of this strain for the management of Campylobacter infections.     

Protective immunity induced by a LLO‐deficient Listeria monocytogenes

Listeria monocytogenes is a food‐borne pathogen able to cause serious disease in human and animals. Listeriolysin O (LLO), a major virulence factor secreted by this bacterium, is a vacuole‐specific lysin that facilitates bacterial entrance into the host cytosol. Thus, LLO plays a key role in the translocation and intracellular spread of L. monocytogenes. To study the effect of LLO on virulence and immunopotency, a LLO‐deficient L. monocytogenes mutant was constructed using a shuttle vector followed by homologous recombination. The mutant strain had lost hemolytic activity, which resulted in an extremely reduced virulence, 5 logs lower than that of the parent strain, yzuLM4, in BALB/c mice. The number of bacteria detected in the spleens and livers of mice infected with the mutant was greatly reduced, and the bacteria were rapidly eliminated by the host. Kinetics studies in this murine model of infection showed that the invasion ability of the mutant strain was much lower than that of the parent strain. Moreover, immunization with the mutant strain conferred protective immunity against listerial infection. In particular, stimulation with Ag85B_240‐259, strong specific Th1 type cellular immunity was elicited by vaccination C57BL/6 mice with hly deficient strain delivering Mycobacterium tuberculosis fusion antigen Ag85B‐ESAT‐6 via intravenous inoculation. These results clearly show that highly attenuated LLO‐deficient L. monocytogenes is an attractive vaccine carrier for delivering heterologous antigens.     

Inhibitory effect of different chicken-derived lactic acid bacteria isolates on drug resistant Salmonella SE47 isolated from eggs

Lactic Acid Bacteria (LAB) regulate and maintain the stability of healthy microbial flora, inhibit the adhesion of pathogenic bacteria and promote the colonization of beneficial micro-organisms. The drug resistance and pathogenicity of Salmonella enteritis SE47 isolated from retail eggs were investigated. Meanwhile, Enterococcus faecalis L76 and Lactobacillus salivarius LAB35 were isolated from intestine of chicken. With SE47 as indicator bacteria, the diameters of L76 and LAB35 inhibition zones were 12 mm and 8·5 mm, respectively, by agar inhibition circle method, which indicated that both of them had inhibitory effect on Salmonella, and L76 had better antibacterial effect; two chicken-derived lactic acid bacteria isolates and Salmonella SE47 were incubated with Caco-2. The adhesion index of L76 was 17·5%, which was much higher than that of LAB35 (10·21%) and SE47 (4·89%), this experiment shows that the higher the bacteriostatic effect of potential probiotics, the stronger the adhesion ability; then Caco-2 cells were incubated with different bacteria, and the survival of Caco-2 cells was observed by flow cytometry. Compared with Salmonella SE47, the results showed that lactic acid bacteria isolates could effectively protect Caco-2 cells; finally, after different bacteria incubated Caco-2 cells, according to the cytokine detection kit, the RNA of Caco-2 cells was extracted and transcribed into cDNA, then detected by fluorescence quantitative PCR, the results showed that L76 could protect Caco-2 cells from the invasion of Salmonella SE47, with less cell membrane rupture and lower expression of MIF and TNF genes. Therefore, the lactic acid bacteria isolates can effectively inhibit the adhesion of Salmonella and protect the integrity of intestinal barrier.     

Chemiluminescence assay for Listeria monocytogenes based on Cu/Co/Ni ternary nanocatalyst coupled with penicillin as generic capturing agent

Bacterial pathogen control is important in seafood production. In this study, a Cu/Co/Ni ternary nanoalloy (Cu/Co/Ni TNA) was synthesized using the oleylamine reducing method. It was found that Cu/Co/Ni TNA greatly enhanced the chemiluminescence (CL) signal of the hydroxylamine‐O‐sulfonic acid (HOSA)–luminol system. The CL properties of Cu/Co/Ni TNA were investigated systemically. The possible CL mechanism also was intensively investigated. Based on the enhanced CL phenomenon of Cu/Co/Ni TNA, a Cu/Co/Ni TNA, penicillin, and anti‐L. monocytogenes (Listeria monocytogenes) antibody‐based sandwich complex assay for detection of L. monocytogenes was established. In this sandwich CL assay, penicillin was employed to capture and enrich pathogenic bacteria with penicillin‐binding proteins (PBPs) while anti‐L. monocytogenes antibody was adopted as the specific recognition molecule to recognize L. monocytogenes. L. monocytogenes was detected sensitively based on this new Cu/Co/Ni TNA–HOSA–luminol CL system. The CL intensity was proportional to the L. monocytogenes concentration ranging from 2.0 × 10² CFU ml^?1 to 3.0 × 10⁷ CFU ml^?1 and the limit of detection wa 70 CFU ml^?1. The reliability and potential applications of our method was verified by comparison with official methods and recovery tests in environment and food samples.