Acid adaptation and survival of Listeria monocytogenes in Italian-style soft cheeses

AIMS: The ability of Listeria monocytogenes to survive and grow at high salt concentrations and low pH makes it a potential hazard after the consumption of milk and dairy products, often implicated in severe outbreaks of listeriosis. This study was designed to evaluate the behaviour of L. monocytogenes in traditional acid and salted Italian-style soft cheeses and to investigate whether Listeria occurrence and growth in these environments may represent a potential increase of hazard. METHODS AND RESULTS: A first approach was addressed to in vitro evaluate survival, acid tolerance response, ability to produce biofilm, and capability to invade intestinal-like cells of a L. monocytogenes strain grown under experimental conditions mimicking environmental features that this pathogen encounters in soft cheeses (such as acid pH and high NaCl content). A second set of experiments was performed to monitor, during the storage at 4 degrees C, the survival of acid-adapted and nonadapted Listeriae in artificially contaminated soft cheeses. Both acid tolerance response and invasion efficiency of acid-adapted bacteria resulted in an increase, even when bacteria were simultaneously pre-exposed to increasing salt stress. The contamination of cheeses with acid-adapted and nonadapted bacteria evidenced in all products a good survival. A significant increased survival, the recovery of bacterial cells highly resistant to lethal pH exposure, and the prevalence of filamentous structures were observed in crescenza cheese during the storage. CONCLUSIONS: The Listeria survival and acid pH tolerance observed during refrigerated storage are probably related to the intrinsic acid and saline features of soft cheeses analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Italian soft cheeses tested may represent a potential hazard for the recovery of acid-adapted L. monocytogenes cells with enhanced ability to adhere to inert surfaces and/or to penetrate host cells.     

Quantification of Listeria monocytogenes in fermented sausages by MPN-PCR method

AIMS: To combine the principles of most-probable-number (MPN) statistics and the conventional PCR technique to enumerate Listeria monocytogenes in fermented sausages. METHODS AND RESULTS: A simple method to enumerate L. monocytogenes in fermented sausages was developed and compared with direct plating in Palcam agar. Species-specific MPN-PCR, but not direct plating, made the enumeration of L. monocytogenes possible in all assayed samples. CONCLUSIONS: MPN-PCR proved to be a rapid and reliable method for enumerating L. monocytogenes in fermented sausages, including low contaminated samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This MPN-PCR technique may facilitate the enumeration of L. monocytogenes for routine analyses in fermented sausages without excessive work.     

Prevalence and lineages of Listeria monocytogenes in Chinese food products

AIMS: This study was undertaken to investigate the prevalence and lineages of Listeria monocytogenes in different kinds of food products in local Chinese markets. METHODS AND RESULTS: A total of 2686 food samples and 645 water samples were collected and L. monocytogenes was isolated from 2.28% (76 of 3331) of all samples. The prevalence of L. monocytogenes (14 of 290, 4.83%) in raw meat products was significantly higher than that in other raw food products (P < 0.05). Among 844 ready-to-eat (RTE) food samples, 21 samples were positive for L. monocytogenes. RTE packaged food products from two supermarkets had a prevalence ranging from 0.00% to 25.00%. The prevalence of L. monocytogenes in meat products of freshly slaughtered hogs was 0.95% (four of 420), significantly lower than that in raw meat products in the retail markets (P < 0.05). Ten isolates were recovered from 645 water samples, which were collected after hands washing by shopkeepers or waiters. A total of 38 isolates were randomly selected for lineage classification based on the nucleotide variation of actA gene. Eighty percentage of isolates from RTE food products belonged to Lineage II while only 20% belonged to Lineage I. CONCLUSIONS: Food products in Chinese markets are contaminated with L. monocytogenes. Raw meat products have the highest contamination rates among all the raw food samples. RTE food products are more likely to be contaminated with Lineage II strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here show the main contamination sources of L. monocytogenes in Chinese food products.     

A novel real-time PCR-based method for the detection of Listeria monocytogenes in food

AIMS: A new real-time PCR-based method was developed for the detection of Listeria monocytogenes in food. METHODS AND RESULTS: A two-step enrichment involving a 24-h incubation in half-Fraser broth followed by a 6-h subculture in Fraser broth was used, followed by cell lysis and real-time PCR with primers and a TaqMan probe previously developed in our laboratory. When the method was evaluated with 144 naturally contaminated food samples, 44 were detected as positive by the PCR-based method and 42 by the standard method EN ISO 11290-1. With 61 food samples artificially contaminated at a level of 10(0) CFU per 25 g, 61 and 58 positive samples were detected by the respective methods. CONCLUSIONS: The developed real-time PCR-based method facilitated the detection of L. monocytogenes in food on the next day after the sample reception, with a reduction of false-positive results because of dead bacterial cells and false-negative results because of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be used for L. monocytogenes detection in food as a faster alternative to current methods.     

A multidrug efflux transporter in Listeria monocytogenes

A chromosomal gene (mdrL) was found in Listeria monocytogenes L028, showing a high degree of similarity with multidrug efflux transporters of the major facilitator superfamily (family 2). An allele-substituted mutant of this gene failed to pump out ethidium bromide and presented lower minimal inhibitory concentrations of macrolides, cefotaxime and heavy metals. This is the first multidrug efflux pump described in Listeria.     

Listeria monocytogenes as a probe of immune function.

For almost half a century, the mouse model of Listeria monocytogenes infection has been used to analyse both innate and adaptive components of immunity and to discover key immune genes. Vast accumulated knowledge about the disease in mice provides a unique framework for identifying and characterising immune molecules using a variety of experimental approaches. To illustrate the range of questions that can be addressed using modern genetics and genomics tools, the authors provide an overview of the analysis of components of immune signalling networks using the mouse model of L. monocytogenes infection.     

Antibodies to listeriolysin O reflect the acquired resistance to Listeria monocytogenes in experimentally infected goats

We induced experimental listeriosis in goats by two sequential oral inoculations of Listeria monocytogenes serovar 1/2a at 8 months' interval. Immunoblot analysis with the goat sera demonstrated listeriolysin O (LLO) as the principal protein antigen of L. monocytogenes. Pre-existing antibodies to LLO were, depending on their initial level, associated with either mild clinical symptoms of short duration or the total absence of clinical symptoms. Similarly, the presence and development of such antibodies corresponded with the disappearance pattern of L. monocytogenes from the gastrointestinal tract. These findings suggest that an association exists between antibodies to LLO and acquired resistance to Listeria infections.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

Identification and disruption of btlA,a locus involved in bile tolerance and general stress resistance in Listeria monocytogenes

A transposon Tn917 mutant of Listeria monocytogenes L028 was isolated on the basis of reduced growth on agar adjusted to pH 5.5. The disrupted gene, designated btlA (bile tolerance locus), encodes a putative secondary transporter of the major facilitator superfamily, which has significant homology to yxiO in Bacillus subtilis (lmo1417 in L. monocytogenes EGDe). The mutant demonstrated decreased growth rates relative to the wild-type when grown in sub-lethal levels of various stressors (acid, salt, ethanol, bile, SDS, ampicillin and phosphomycin). The mutant was also more sensitive to lethal levels of bile. A pORI19 insertion mutant demonstrated similar phenotypes. Murine virulence studies indicated that disruption of btlA does not influence virulence potential. BtlA therefore represents a membrane protein essential for the maintenance of homeostasis under stress conditions, but is not involved in pathogenicity.     

应用乳酸菌生物保护剂控制肉制品中单增李斯特菌的研究进展

肉制品营养丰富,极易被微生物污染,单增李斯特菌是污染肉制品主要病原菌之一。乳酸菌做为生物保护剂已经被广泛应用于食品中控制单增李斯特菌。本文首先分析了我国肉制品中单增李斯特菌的污染状况,总结了乳酸菌应用于肉制品安全控制的概况;然后进一步详细介绍了乳酸菌对单增李斯特菌的抑菌机理,着重探讨了乳酸菌对单增李斯特菌致病能力(生长、抗性和毒性)的影响;文章最后指出了乳酸菌在食品应用中存在的问题,并对未来的研究方向提供了建议,以期为乳酸菌在食品安全控制中的应用提供参考。     

Modification of a virulence-associated phenotype after growth of Listeria monocytogenes on food

AIM: To assess the effect of different foods, which have been implicated or not in cases of listeriosis, on the in vitro virulence-associated phenotype level of different Listeria monocytogenes strains. METHODS AND RESULTS: The virulence-associated phenotype level of L. monocytogenes was studied with the in vitro cell test based on a plaque-forming assay with a human adenocarcinoma cell line (HT-29) monolayer. Three strains of L. monocytogenes were grown in preparations (homogenate, 1-mum filtrate or 0.2-mum filtrate) of different food extracts ['rillettes' (potted minced pork), milk, raw salmon and cold-smoked salmon] or in a control medium, brain heart infusion (BHI). The bacterial suspensions grown in food extracts or in BHI at 37 degrees C were diluted with their growth medium (food extract or BHI) or with minimum essential medium before seeding on confluent HT-29 cell monolayers. Filtration of food extracts had no significant effect on the plaque numbers formed by the bacteria. A significant decrease in the plaque numbers was noted for the three strains when they grew in the rillettes extracts, compared with the other food extracts and BHI. The levels of in vitro virulence-associated phenotype of the strains after growth in the rillettes extract were similar to or lower than that of the hypovirulent internal reference strain L. monocytogenes 442. After growth in milk and cold-smoked salmon, the impact on virulence-associated phenotype depended on the strain. In contrast, plaque-forming assay indicated increased virulence-associated phenotype when the strains were switched from a nutrient-rich medium (food extract or BHI) to a minimum essential medium. CONCLUSIONS: In vitro virulence-associated phenotype level of the studied strains grown in BHI or cold-smoked salmon was the same as the control virulent strain EGD. In contrast, the nutrients present in rillettes may therefore substantially reduce the number of plaques but not the growth of L. monocytogenes. The utilization of minimum essential medium as diluent attenuates changes the effect of the food extract on virulence-associated phenotype in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: In the experimental design of this study, we showed that the nature of the food could affect the in vitro virulence-associated phenotype level of L. monocytogenes.     

Reduction in Listeria monocytogenes and spoilage bacteria on smoked catfish using X-ray treatments

Aims: To determine the efficacy of X‐ray processes in inactivating L. monocytogenes levels in smoked catfish during storage at 5°C and to determine the effects of X‐ray doses on controlling the growth of spoilage bacteria on smoked catfish during storage at 5°C for up to 5 weeks. Methods and Results: Smoked catfish fillets inoculated with L. monocytogenes were treated with 0·0–2·0 kGy X‐ray and stored at 5°C for 5 weeks. The negative controls (uninoculated/untreated) and uninoculated samples treated with the lowest (0·1 kGy) and highest (2·0 kGy) doses were stored at 5°C and tested for psychrotrophs count during the 5 weeks of storage. The initial L. monocytogenes population on smoked catfish was significantly (P < 0·05) reduced to undetectable level by a treatment of 1·0 kGy or higher. The initial psychrotrophs count on smoked catfish was significantly reduced from 4·7 CFU g^?1 to below the detectable level by a treatment with 2·0 kGy. Conclusions: Smoked catfish treated with 2·0 kGy X‐ray had no detectable L. monocytogenes throughout 35 days of storage at 5°C. A treatment with 2·0 kGy X‐ray also kept the levels of psychrotrophs in the smoked catfish within the acceptable level until 35 days. Significance and Impact of the Study: The results of this investigation indicate that X‐ray at 2·0 kGy can eliminate L. monocytogenes and extend the shelf life of smoked catfish stored at refrigeration temperature.     

Evaluation of the pH-dependent,stationary-phase acid tolerance in Listeria monocytogenes and Salmonella Typhimurium DT104 induced by culturing in media with 1% glucose: a comparative study with Escherichia coli O157:H7

AIMS: To comparatively evaluate the adaptive stationary-phase acid tolerance response (ATR) in food-borne pathogens induced by culturing in glucose-containing media, as affected by strain variability and antibiotic resistance, growth temperature, challenge pH and type of acidulant. METHODS AND RESULTS: Antibiotic resistant or sensitive strains of Listeria monocytogenes, Salmonella including S. Typhimurium DT104, and Escherichia coli O157:H7 were cultured (30 degrees C for 24 h; 10 degrees C for up to 14 days) in trypticase soya broth with yeast extract (TSBYE) with 1% or without glucose to induce or prevent acid adaptation, respectively. Cultures were subsequently exposed to pH 3.5 or 3.7 with lactic or acetic acid at 25 degrees C for 120 min. Acid-adapted cultures were more acid tolerant than nonadapted cultures, particularly those of L. monocytogenes and Salmonella. No consistent, positive or negative, influence of antibiotic resistance on the pH-inducible ATR or acid resistance (AR) was observed. Compared with 30 degrees C cultures, growth and acid adaptation of L. monocytogenes and S. Typhimurium DT104 at 10 degrees C markedly reduced their ATR and AR in stationary phase. E. coli O157:H7 had the greatest AR, relying less on acid adaptation. A 0.2 unit difference in challenge pH (3.5-3.7) caused great variations in survival of acid-adapted and nonadapted cells. CONCLUSIONS: Culturing L. monocytogenes and Salmonella to stationary phase in media with 1% glucose induces a pH-dependent ATR and enhances their survival to organic acids; thus, this method is suitable for producing acid-adapted cultures for use in food challenge studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial pathogens may become acid-adapted in foods containing glucose or other fermentable carbohydrates. Low storage temperatures may substantially decrease the stationary-phase ATR of L. monocytogenes and S. Typhimurium DT104, but their effect on ATR of E. coli O157:H7 appears to be far less dramatic.     

Surface Listeria monocytogenes carbohydrate-binding components revealed by agglutination with neoglycoproteins

Carbohydrate-binding components were shown to be present at the surface of Listeria monocytogenes by means of a panel of neoglycoproteins using direct agglutination. These lectin-like components bind on neoglycoproteins bearing d -glucosamine, l -fucosylamine, or para-amino-phenyl-α- d -mannopyrannoside residues. The interactions were inhibited by the carbohydrate moieties specific to the neoglycoproteins. The protein nature of the lectin-like components of L. monocytogenes was ascertained by the loss of carbohydratebinding capacity following protease treatment.     

一种免疫生物传感器对单核细胞增生李斯特菌快速检测方法的初步研究

应用F0F1-ATP酶旋转分子马达和免疫技术相结合,建立免疫生物传感器快速检测技术。首先pH变化敏感荧光物质F1300标记到色素体(chrom atophore)的内表面,然后在F0F1-ATP酶上连接β亚基抗体-生物素-链亲和素-生物素-单核细胞增生李斯特菌多抗复合体,得到可以捕获单核细胞增生李斯特菌的免疫生物传感器。传感器上负载菌量不同,酶活性不同,酶活变化以pH敏感的荧光探针来感应,最后通过荧光扫描仪检测不同菌量负载下的荧光信号。结果表明,该方法对单核细胞增生李斯特菌标准菌株(ATCC 15313)的检测时间为4.5 h,检出浓度为100 CFU/孔。     

Serological response in rabbits to Listeria monocytogenes after oral or intragastric inoculation

Abstract The serological response in rabbits against Listeria monocytogenes after oral or intragastric inoculation was investigated. Both the number of sero-positive and the average serum titres were hgher in animals inoculated by the oral route. This difference was especially marked in rabbits inoculated with the lower dose (1×10³ colony-forming units (cfu)), which developed a strong serological response (average serum titre of 1280 after 4 inoculations) in most of the inoculated animals (80%), without any clinical signs. The implication of these results in the epidemiology of listeriosis is discussed.     

Immunomodulatory properties of Enterococcus faecium JWS 833 isolated from duck intestinal tract and suppression of Listeria monocytogenes infection

The aim of this study was to evaluate the immunomodulatory properties of Enterococcus faecium JWS 833 (JWS 833) isolated from duck intestine and compare them to those of Lactobacillus rhamnosus GG (LGG), a proven immunity-enhancing probiotic. To investigate the immune-enhancing properties of JWS 833, production of nitric oxide (NO) and cytokines was measured in mouse peritoneal macrophages. In addition, a Listeria monocytogenes challenge model was used in the assessment. It was found that heat-killed JWS 833 stimulates mouse peritoneal macrophages to produce NO, interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α) and that oral administration of viable JWS833 enhances NO, IL-1β and TNF-α synthesis upon L. monocytogenes challenge. Moreover, mice fed with JWS 833 were partially protected against lethal challenge with L. monocytogenes. JWS 833 strain has significantly greater immunostimulatory properties than LGG. Moreover, JWS 833 strain partially protects mice against lethal challenge with L. monocytogenes. JWS 833, a novel strain of E. faecium isolated from duck intestine, is potentially a useful feed supplement for controlling pathogens and enhancing host immune responses.     

Bioluminescent Listeria monocytogenes provide a rapid assay for measuring biocide efficacy

Abstract A recombinant derivative of Listeria monocytogenes 23074, engineered to express the luxAB genes of Vibrio fischeri MJ1, has a bioluminescent phenotype that provides a rapid monitor of microbial viability. The antibacterial activity of phenol and chlorhexidine diacetate (Hibitane) was measured using both bioluminescence and viable counts. Concentration exponents were assessed as 7.3 for phenol and 2.63 for chlorhexidine diacetate using plate counts. The rapidity of bioluminescence measurement constitutes a major advantage in biocide assessment.     

Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions

Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure-function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed.     

Listeria monocytogenes is common in wild birds in Helsinki region and genotypes are frequently similar with those found along the food chain

Aims: To evaluate the prevalence and genetic diversity of Listeria monocytogenes in wild birds and to compare the genotypes with isolates previously collected from foods and food processing environments. Methods and Results: Samples of wild birds’ faeces (n = 212) were collected from a municipal landfill site and from urban areas in the Helsinki region and analysed by two‐step enrichment and plating onto L. monocytogenes‐selective agar. The overall prevalence of L. monocytogenes in bird faeces was 36% (95% CI 30–43%), and prevalence on the landfill site was significantly higher. All isolates were analysed with pulsed‐field gel electrophoresis and compared with the L. monocytogenes profiles in an existing collection. Similar pulsotypes were found in birds and in isolates collected along the food chain. Conclusions: Birds commonly carry L. monocytogenes, and strains are frequently similar with those detected in foods and food processing environments. Thus, birds may disseminate L. monocytogenes in nature and may also contaminate foods when entering the food processing environments and outdoor market places. Significance and Impact of the Study: Populations of L. monocytogenes in wild birds and along the food processing chain overlap. Our findings add to the epidemiological data on this significant foodborne pathogen.