Select Listeria monocytogenes subtypes commonly found in foods carry distinct nonsense mutations in inlA, leading to expression of truncated and secreted internalin A, and are associated with a reduced invasion phenotype for human intestinal epithelial cells

The surface protein internalin A (InlA) contributes to the invasion of human intestinal epithelial cells by Listeria monocytogenes. Screening of L. monocytogenes strains isolated from human clinical cases (n=46), foods (n=118), and healthy animals (n=58) in the United States revealed mutations in inlA leading to premature stop codons (PMSCs) in L. monocytogenes ribotypes DUP-1052A and DUP-16635A (PMSC mutation type 1), DUP-1025A and DUP-1031A (PMSC mutation type 2), and DUP-1046B and DUP-1062A (PMSC mutation type 3). While all DUP-1046B, DUP-1062A, DUP-16635A, and DUP-1031A isolates (n=76) contained inlA PMSCs, ribotypes DUP-1052A and DUP-1025A (n=72) contained isolates with and without inlA PMSCs. Western immunoblotting showed that all three inlA PMSCs result in the production of truncated and secreted InlA. Searches of the Pathogen Tracker database, which contains subtype and source information for more than 5,000 L. monocytogenes isolates, revealed that the six ribotypes shown to contain isolates with inlA PMSCs were overall more commonly isolated from foods than from human listeriosis cases. L. monocytogenes strains carrying inlA PMSCs also showed significantly (P=0.0004) reduced invasion of Caco-2 cells compared to isolates with homologous 3' inlA sequences without PMSCs. Invasion assays with an isogenic PMSC mutant further supported the observation that inlA PMSCs lead to reduced invasion of Caco-2 cells. Our data show that specific L. monocytogenes subtypes which are common among U.S. food isolates but rare among human listeriosis isolates carry inlA mutations that are associated with, and possibly at least partially responsible for, an attenuated invasion phenotype.     

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.     

Lactic acid bacteria from chicken carcasses with inhibitory activity against Salmonella spp. and Listeria monocytogenes

This study was conducted to isolate psychrotrophic lactic acid bacteria (LAB) from chicken carcasses with inhibitory activity against strains of Salmonella spp. and Listeria monocytogenes. A total of 100 broiler samples were examined for the presence of LAB. Ninety-two LAB isolates that showed antimicrobial effects against Salmonella spp. and L. monocytogenes were further analysed to examine their LAB (Gram-positive, catalase negative, oxidase negative) and psychrotrophic characteristics (ability to grow at 7 °C). Fifty isolates were further selected and identified initially using standard biochemical tests in miniature (Micro-kits API CH 50) and then by sequencing of the 16s-23s rRNA gene boundary region (Intergenic Spacer Region). By molecular identification, these isolates were classified into 5 different LAB species: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Pediococcus acidilactici, and Lactobacillus paralimentarius. None of the isolates produced tyramine or histamine.     

Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion

Abstract Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.     

The relative efficacy of different strain combinations of lactic acid bacteria in the reduction of populations of Salmonella enterica Typhimurium in the livers and spleens of mice

Multispecies probiotics have been reported to be more effective than monostrain probiotics in health promoting for the host. In this study, 12 lactic acid bacteria (LAB) strains were selected based on the level of induction of tumor necrosis factor (TNF)-α in RAW 264.7 macrophage cells. Their adherence to Caco-2 cells and inhibitory effects on Salmonella invasion of Caco-2 cells were compared. Strains with different probiotic properties were then combined and BALB/c mice were fed with LAB strains for 63 days; then the mice were challenged with Salmonella on day 64. For Salmonella-unchallenged mice that received a multistrain combination of LAB strains that have greater TNF-α production in macrophages, greater adherence and inhibit Salmonella invasion of Caco-2 cells to a greater extent, their peritoneal macrophages had greater phagocytic activity. For Salmonella-challenged mice, a significant reduction of Salmonella cells in the livers and spleens of the mice was observed 8 days post challenge. The addition of 12% skim milk powder together with LAB strain combinations significantly enhanced the reduction of Salmonella cells in the mice livers and spleens. In conclusion, we have shown that LAB strain combinations with particular probiotic properties when fed to mice can inhibit Salmonella invasion of the liver and spleen.     

Invasion assay of Listeria monocytogenes using Vero and Caco-2 cells

The invasion ability of Listeria monocytogenes into cultured cells has been used to evaluate its pathogenicity. In this study, invasive ability was investigated using Vero and Caco-2 cell lines. The form of invasion showed no morphological differences between both cell lines inoculated with L. monocytogenes L89-H2 or L96-23C1 strains when double fluorescence stained with rhodamine and FITC or with Giemsa staining. Recovery count and recovery rate of L. monocytogenes from Vero cells was related to the number of inoculated bacteria (2 x 10(5) to 2 x 10(7)/ml) in a bell-shape pattern, though the relationship was unclear in Caco-2 cells. Recovery rate of L. monocytogenes was higher in Vero cells than Caco-2 cells at a multiplicity of infection (MOI) 10, though the rates in both cells showed different stable stages over a considerably wide range of MOI. The recovery rate of all five L. monocytogenes strains from listeriosis patients was 15% at MOI 10 from infected Vero cells, while meat-derived strains showed variable rates regardless of the serovar. These results suggest that the Vero cell line is suitable for an invasion assay and that a recovery rate of 15% may be the critical limit for the expression of pathogenicity in the host.     

Lactobacillus acidophilus LAP5 able to inhibit the Salmonella choleraesuis invasion to the human Caco-2 epithelial cell

The mechanisms for lactic acid bacteria (LAB) to inhibit Salmonella invasion appear to be multifactorial and include the adhesion of LAB to host intestine epithelium, the production of organic acids, or bacteriocin by LAB cells. Previously, we found a strain of Lactobacillus acidophilus isolated from swine, i.e. strain LAP5, was with antagonistic effect against Salmonella typhimurium. This strain LAP5 was also found to meet the requirements for probiotic use. In this study, we evaluate the potential of LAP5 strain to protect the human or swine from infection by Salmonella choleraesuis. We present evidence that the culture of LAP5 was able to inhibit the invasion of S. choleraesuis to human Caco-2 cell line. The LAP5 cell culture showed a higher inhibitory effect on the invasion of S. choleraesuis to Caco-2 cells than the spent culture supernatant (SCS) of LAP5 did. Also, the pH, organic acids or the bacteriocin, which act at low pH conditions, may play the role of antagonistic effect. The addition, adhesion of LAP5 cells to Caco-2 cell line may also play roles to reduce the invasion of S. choleraesuis.     

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.     

Immune effect of heat-killed multistrain of Lactobacillus acidophilus against Salmonella typhimurium invasion to mice

AIMS: This study attempted to determine whether lactic acid bacteria (LAB) could have a better probiotic function when used as a multistrain mixture, i.e. Mix-LAB, than when used as a monostrain. To this end, three strains of Lactobacillus acidophilus, specifically strain LAP5, LAF1 and LAH7, were heat-killed and mixed. This heat-killed Mix-LAB was used to evaluate the effectiveness of multistrain in inhibiting Salmonella invasion into cultured cells and into organs (spleen and liver) of live mice. METHODS AND RESULTS: BALB/c mice were orally administered with heat-killed Mix-LAB or sterile normal saline (control) for seven consecutive days and then challenged with orally administered Salmonella typhimurium on day 8. Results showed that, at day 6 after the challenge, the mice which had received Mix-LAB exhibited lower rates (P < 0.05) of Salmonella invasion into liver and spleen than did the control mice. Also, before the Salmonella challenge, the serum tumour necrosis factor-alpha (TNF-alpha) levels were not significantly different (P > 0.05) between these two groups of mice. After the challenge, however, the serum TNF-alpha level was significantly elevated (P < 0.05) in the control group, but not significantly changed in the Mix-LAB fed mice. To investigate possible factors involved in heat-killed Mix-LABs antagonistic effect on Salmonella invasion of mouse organs, heat-killed single strain and Mix-LAB were evaluated for ability to inhibit Salmonella invasion into cultured human intestinal Int-407 and Caco-2 cells. Results showed that none of the heat-killed strains were able to protect these cultured cells from Salmonella invasion, even though strains of LAP5 and Mix-LAB were adherent to them. However, study of the activation of murine macrophage Raw 264.7 cells showed that heat-killed Mix-LAB stimulated TNF-alpha production, nitric oxide release, and increased phagocytic activity in macrophages. CONCLUSIONS: Our findings suggest that heat-killed Mix-LAB can inhibit Salmonella invasion of mouse organs through the immunomodulating role of activated macrophage. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of heat-killed Mix-LAB to prevent bacterial infection in mice was found to be more significant than that of viable monostrain. This effect may be due to the activation of the immune system rather than to the adherence of LAB to the intestine epithelium.     

Probiotic properties of Lactococcus lactis ssp. lactis HV219, isolated from human vaginal secretions

AIMS: To determine the resistance of Lactococcus lactis ssp. lactis HV219 to acids, bile, antibiotics, inflammatory drugs and spermicides, compare adsorption of the strain to bacteria and Caco-2 cells under stress, and evaluate the antimicrobial activity of bacteriocin HV219. METHODS AND RESULTS: Bacteriocin HV219 activity against Gram-positive and Gram-negative bacteria was confirmed by leakage of DNA and beta-galactosidase, and atomic force microscopy. Adsorption of bacteriocin HV219 to bacteria is influenced by pH, temperature, surfactants and salts. Initially, only 3% of HV219 cells adhered to Caco-2 cells. However, after 2 h, adherence increased to 7%. Strain HV219 and Listeria monocytogenes ScottA did not compete for colonization. Strain HV219 is sensitive to most antibiotics tested, but resistant to amikacin, ceftazidime, nalidixic acid, metronidazole, neomycin, oxacillin, streptomycin, sulphafurazole, sulphamethoxazole, sulphonamides, tetracycline and tobramycin. Ibuprofen, ciprofloxacin, diklofenak and nonoxylol-9 inhibited the growth of strain HV219. CONCLUSION: Strain HV219 is resistant to hostile conditions in the intestinal tract, including therapeutic levels of specific antibiotics and binds to Caco-2 cells, but not in competition with L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain HV219 will only be effective as probiotic if taken with specific antibiotics and not with anti-inflammatory drugs and spermicides.     

Characteristics and frequency of detection of fecal Listeria monocytogenes shed by livestock, wildlife, and humans

Listeria monocytogenes is a facultative intracellular pathogen that can be carried asymptomatically in various animals and can be shed in feces. We investigated the prevalence and characteristics of L. monocytogenes isolated from livestock, wildlife, and human potential sources of contamination in 2 areas in Ontario, Canada. From February 2003 to November 2005, a total of 268 fecal samples were collected from different animals. Listeria monocytogenes was isolated using selective enrichment, isolation, and confirmation procedures, and 15 samples (6%) yielded to the isolation of 84 confirmed strains. Listeria monocytogenes was isolated from livestock (beef and dairy), wildlife (deer, moose, otter, and raccoon), and human (biosolids and septic) fecal sources. Thirty-two isolates were from serovar 1/2a, 34 from serovar 1/2b, 1 from serovar 3a, and 17 from serovar 4b. Listeria monocytogenes populations were resolved into 13 EcoRI ribotypes, and 18 ApaI and 18 AscI pulsotypes, with Simpson indexes of discrimination of 0.878 and 0.907, respectively. A majority (59%) of L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, which was supported by higher entry into Caco-2 cells (9.3%) than isolates producing truncated and secreted internalin A (1.3% of entry). Listeria monocytogenes fecal isolates were on average resistant to 6.4 +/- 2.5 antibiotics out of 17 tested, and potentially virulent isolates exhibited an enhanced resistance to kanamycin, gentamicin, streptomycin, and rifampicin. Livestock, wildlife, and human L. monocytogenes fecal communities exhibited overlapping but distinct populations, and some genotypes and phenotypes were similar to those previously described for surface water isolates in the same area.     

Diverse geno- and phenotypes of persistent Listeria monocytogenes isolates from fermented meat sausage production facilities in Portugal

The persistence of Listeria monocytogenes in food-associated environments represents a key factor in transmission of this pathogen. To identify persistent and transient strains associated with production of fermented meat sausages in northern Portugal, 1,723 L. monocytogenes isolates from raw material and finished products from 11 processors were initially characterized by random amplification of polymorphic DNA (RAPD), PCR-based molecular serotyping, and epidemic clone characterization, as well as cadmium, arsenic, and tetracycline resistance typing. Pulsed-field gel electrophoresis (PFGE) typing of 240 representative isolates provided evidence for persistence of L. monocytogenes for periods of time ranging from 10 to 32 months for all seven processors for which isolates from different production dates were available. Among 50 L. monocytogenes isolates that included one representative for each PFGE pattern obtained from a given sample, 12 isolates showed reduced invasion efficiency in Caco-2 cells, including 8 isolates with premature stop codons in inlA. Among 41 isolates representing sporadic and persistent PFGE types, 22 isolates represented lysogens. Neither strains with reduced invasion nor lysogens were overrepresented among persistent isolates. While the susceptibility of isolates to lysogenic phages also did not correlate with persistence, it appeared to be associated with molecular serotype. Our data show the following. (i) RAPD may not be suitable for analysis of large sets of L. monocytogenes isolates. (ii) While a large diversity of L. monocytogenes subtypes is found in Portuguese fermented meat sausages, persistence of L. monocytogenes in this food chain is common. (iii) Persistent L. monocytogenes strains are diverse and do not appear to be characterized by unique genetic or phenotypic characteristics.     

Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides.

Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria. The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment. However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform. The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod. Mutants of L. mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained. The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.     

Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides.

Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria. The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment. However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform. The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod. Mutants of L. mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained. The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.     

Lactic acid bacteria fermentation of human milk oligosaccharide components, human milk oligosaccharides and galactooligosaccharides

Human milk contains about 7% lactose and 1% human milk oligosaccharides (HMOs) consisting of lactose with linked fucose, N-acetylglucosamine and sialic acid. In infant formula, galactooligosaccharides (GOSs) are added to replace HMOs. This study investigated the ability of six strains of lactic acid bacteria (LAB), Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Streptococcus thermophilus and Leuconostoc mesenteroides subsp. cremoris, to digest HMO components, defined HMOs, and GOSs. All strains grew on lactose and glucose. N-acetylglucosamine utilization varied between strains and was maximal in L. plantarum; fucose utilization was low or absent in all strains. Both hetero- and homofermentative LAB utilized N-acetylglucosamine via the Embden-Meyerhof pathway. Lactobacillus acidophilus and L. plantarum were the most versatile in hydrolysing pNP analogues and the only strains releasing mono- and disaccharides from defined HMOs. Whole cells of all six LAB hydrolysed oNP-galactoside and pNP-galactoside indicating β-galactosidase activity. High β-galactosidase activity of L. reuteri, L. fermentum, S. thermophilus and L. mesenteroides subsp. cremoris whole cells correlated to lactose and GOS hydrolysis. Hydrolysis of lactose and GOSs by heterologously expressed β-galactosidases confirmed that LAB β-galactosidases are involved in GOS digestion. In summary, the strains of LAB used were not capable of utilizing complex HMOs but metabolized HMO components and GOSs.     

16S-ARDRA, a tool for identification of lactic acid bacteria isolated from grape must and wine

Lactic acid bacteria (LAB) are found in a great variety of habitats, including grape must and wines. There is a close relationship between the species of LAB which develop during fermentation and the eventual quality of the wine. For these reasons analytical techniques allowing fast and reliable identification of wine LAB are needed. In this work a simple and accurate protocol for identifying species of LAB isolated from grape must and wine is presented. This protocol is based on the amplification, directly from colony, of 16S rDNA and later digestion with one of the following restriction enzymes BfaI, MseI and AluI. A sequential use of the three enzymes is proposed to simplify LAB wine identification, first MseI, then BfaI and finally, if necessary, AluI digestion. The technique was able to discriminate 32 of the 36 LAB reference species tested and allowed the identification of 342 isolates from musts and wines. The isolates belonged to the species: Lactobacillus brevis, L. collinoides, L. coryniformis, L. bilgardii, L. mali, L. paracasei, Leuconostoc mesenteroides, Oenococcus oeni, Pediococcus parvulus and P. pentosaceus.     

In-vitro assessment of probiotic potential of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WU-P19 isolated from a traditional fermented herb

Samples of fermented herbs were used to isolate lactic acid bacteria (LAB). Of a total of 19 isolates, eight were resistant both to gastric acid and bile salts (glycocholic acid, GCA; taurocholic acid, TCA; glycodeoxycholic acid, GDCA; and taurodeoxycholic acid, TDCA). Most isolates exhibited a pH-dependent surface hydrophobicity: a pH of 4 conferred a greater hydrophobicity compared to a pH of 7. Based on the hydrophobicity characteristics, the LAB isolate WU-P19 from the traditional fermented herb Oroxylum indicum was selected for further study. WU-P19 was identified as Lactobacillus plantarum WU-P19. The presence of bile salts GCA and GDCA in the culture medium induced production of the relevant bile salt hydrolase. Relative to controls, the presence of the bile salts in the culture medium affected the carbon and nitrogen contents of the cells and their hydrophobicity. Cells grown in a medium free of bile salts were morphologically different to cells grown in the presence of GCA and GDCA. WU-P19 was resistant to several antibiotics. It produced β-galactosidase and inhibited growth of the tested pathogenic bacteria at various levels. In vitro, L. plantarum WU-P19 adapted well to conditions typical of the various zones of the human gastrointestinal tract. In view of the promising results, in vivo evaluations are planned for the isolate WU-P19.