Listeria monocytogenes: a foodborne pathogen.

Listeriosis, caused by Listeria monocytogenes, appears to be increasing in incidence worldwide. The disease is of great concern to the food industry. A recent outbreak in California was linked to the consumption of Mexican-style soft cheese and involved more than 300 cases, 30% of which were fatal. L. monocytogenes can be found in a variety of dairy products, leafy vegetables, fish and meat products. It can grow in refrigerated foods and is more heat resistant than most vegetative microbes. The epidemiologic features of listeriosis are poorly understood, and the minimum infectious dose is unknown. Those predisposed to listeriosis include immunocompromised people and pregnant women and their fetuses. Meningitis, spontaneous abortion and septicemia are the primary manifestations of the disease. Early recognition is critical for successful treatment, and ampicillin is the preferred drug. Listeriosis should be considered in any febrile patient with neurologic symptoms of unknown origin, as well as in women with unexplained recurrent miscarriages, premature labour or fetal death. A food source should be the prime suspect if any isolated case or outbreak occurs.     

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes

Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures. Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L. monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L. monocytogenes, presumably by maintaining membrane fluidity. The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L. monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region. tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C. However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1. No evidence was seen for a clear lipid phase transition in any sample. We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L. monocytogenes membrane that permits growth at refrigeration temperatures.     

Acanthamoeba release compounds which promote growth of Listeria monocytogenes and other bacteria

Listeria monocytogenes can grow as a saphrophyte in diverse habitats, e.g., soil, rivers, lakes, and on decaying plant material. In these environments, the bacteria are frequently exposed to predatory protozoa such as Acanthamoeba. Although L. monocytogenes is a facultative intracellular pathogen it does not infect or survive intracellular in Acanthamoeba castellanii, unlike several other facultative intracellular bacteria. Instead, motile L. monocytogenes can form large aggregates on amoebal cells and are effectively phagocytosed and eventually digested by Acanthamoeba. Here, we demonstrate that non-motile L. monocytogenes represent a less preferred prey in co-cultures with A. castellanii. Moreover, we found that the presence of Acanthamoeba strongly promotes growth of the bacteria in non-nutrient saline, by releasing nutrients or other growth promoters. Thus, the lack of motility and ability to utilize amoebal metabolites may aid to avoid eradication by amoebal predation in low-nutrient environments.     

Plasmids in Listeria monocytogenes and other Listeria species.

One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance.     

Surface proteins and the pathogenic potential of Listeria monocytogenes

On the basis of the recently determined genome sequence of Listeria monocytogenes, we performed a global analysis of the surface-protein-encoding genes. Only proteins displaying a signal peptide were taken into account. Forty-one genes encoding LPXTG proteins, including the previously known internalin gene family, were detected. Several genes encoding proteins that, like InlB and Ami, possess GW modules that attach them to lipoteichoic acids were also identified. Additionally, the completed genome sequence revealed genes encoding proteins potentially anchored in the cell membrane by a hydrophobic tail as well as genes encoding P60-like proteins and lipoproteins. We describe these families and discuss their putative implications for host-pathogen interactions.     

Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species

We demonstrate here the development of a non-invasive optical forward-scattering system, called 'scatterometer' for rapid identification of bacterial colonies. The system is based on the concept that variations in refractive indices and size, relative to the arrangement of cells in bacterial colonies growing on a semi-solid agar surface will generate different forward-scattering patterns. A 1.2-1.5mm colony size for a 1mm laser beam and brain heart infusion agar as substrate were used as fixed variables. The current study is focused on exploring identification of Listeria monocytogenes and other Listeria species exploiting the known differences in their phenotypic characters. Using diffraction theory, we could model the scattering patterns and explain the appearance of radial spokes and the rings seen in the scattering images of L. monocytogenes. Further, we have also demonstrated development of a suitable software for the extraction of the features (scalar values) calculated from images of the scattering patterns using Zernike moment invariants and principal component analysis and were grouped using K-means clustering. We achieved 91-100% accuracy in detecting different species. It was also observed that substrate variations affect the scattering patterns of Listeria. Finally, a database was constructed based on the scattering patterns from 108 different strains belonging to six species of Listeria. The overall system proved to be simple, non-invasive and virtually reagent-less and has the potential for automated user-friendly application for detection and differentiation of L. monocytogenes and other Listeria species colonies grown on agar plates within 5-10 min analysis time.     

生物传感器应用于食源性致病菌检测研究进展

生物传感器技术是一种由生物、化学、物理、医学、电子技术等多种学科互相渗透形成起来的高新微量分析技术,具有选择性好、灵敏度高、分析速度快、成本低、能在复杂的体系中进行在线连续监测的特点.本文根据生物传感器的分子识别元件将生物传感器分为DNA传感器、免疫传感器、细胞传感器三大类,简要介绍各种生物传感器的原理及其在检测食源性致病菌方面的应用情况,并对未来生物传感器应用于实际检测进行了展望.     

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.     

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.     

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.     

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.     

Application of bacteriocins in the control of foodborne pathogenic and spoilage bacteria

Bacteriocins are antimicrobial peptides or proteins produced by strains of diverse bacterial species. The antimicrobial activity of this group of natural substances against foodborne pathogenic, as well as spoilage bacteria, has raised considerable interest for their application in food preservation. Application of bacteriocins may help reduce the use of chemical preservatives and/or the intensity of heat and other physical treatments, satisfying the demands of consumers for foods that are fresh tasting, ready to eat, and lightly preserved. In recent years, considerable effort has been made to develop food applications for many different bacteriocins and bacteriocinogenic strains. Depending on the raw materials, processing conditions, distribution, and consumption, the different types of foods offer a great variety of scenarios where food poisoning, pathogenic, or spoilage bacteria may proliferate. Therefore, the effectiveness of bacteriocins requires careful testing in the food systems for which they are intended to be applied against the selected target bacteria. This and other issues on application of bacteriocins in foods of dairy, meat, seafood, and vegetable origins are addressed in this review.     

Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria

The realization of rapid, sensitive, and specific methods to detect foodborne pathogenic bacteria is central to implementing effective practice to ensure food safety and security. As a principle of transduction, the impedance technique has been applied in the field of microbiology as a means to detect and/or quantify foodborne pathogenic bacteria. The integration of impedance with biological recognition technology for detection of bacteria has led to the development of impedance biosensors that are finding wide-spread use in the recent years. This paper reviews the progress and applications of impedance microbiology for foodborne pathogenic bacteria detection, particularly the new aspects that have been added to this subject in the past few years, including the use of interdigitated microelectrodes, the development of chip-based impedance microbiology, and the use of equivalent circuits for analysis of the impedance systems. This paper also reviews the significant developments of impedance biosensors for bacteria detection in the past 5 years, focusing on microfabricated microelectrodes-based and microfluidic-based Faradaic electrochemical impedance biosensors, non-Faradaic impedance biosensors, and the integration of impedance biosensors with other techniques such as dielectrophoresis and electropermeabilization.     

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.     

Characterization and pathogenic potential of Listeria monocytogenes isolates from the smoked fish industry

This study was designed to evaluate the hypothesis that some of the Listeria monocytogenes subtypes associated with foods, specifically smoked fish, may have an attenuated ability to cause human disease. We tested this hypothesis by using two different approaches: (i) comparison of molecular subtypes found among 117 isolates from smoked fish, raw materials, fish in process, and processing environments with subtypes found among a collection of 275 human clinical isolates and (ii) the evaluation of the cytopathogenicity of industrial isolates. Ribotyping and PCR-restriction fragment length polymorphism typing of the hlyA and actA genes differentiated 23 subtypes among the industrial isolates and allowed classification of the isolates into three genetic lineages. A significantly higher proportion of human isolates (69.1%) than industrial isolates (36.8%) were classified as lineage I, which contains human sporadic isolates and all epidemic isolates. All other industrial isolates (63.2%) were classified as lineage II, which contains only human sporadic isolates. Lineage I ribotypes DUP-1038B and DUP-1042B represented a significantly higher proportion of the human isolates than industrial isolates (5.1%). Lineage II ribotypes DUP-1039C, DUP-1042C, and DUP-1045, shown previously to persist in the smoked fish processing environment, represented nearly 50% of the industrial isolates, compared to 7.6% of the human isolates. Representatives of each subtype were evaluated with a tissue culture plaque assay. Lineage I isolates formed plaques that were significantly larger than those formed by lineage II isolates. Isolates from the smoked fish industry representing three ribotypes formed no plaques or small plaques, indicating that they had an impaired ability to infect mammalian cells. While L. monocytogenes clonal groups linked to human listeriosis cases and outbreaks were isolated, our data also suggest that at least some L. monocytogenes subtypes present in ready-to-eat foods may have limited human-pathogenic potential.     

Hemin-incorporated nanoflowers as enzyme mimics for colorimetric detection of foodborne pathogenic bacteria

Rapid, sensitive and point-of-care detection of foodborne pathogenic bacteria is essential for food safety. In this study, we found that hemin-concanavalin A hybrid nanoflowers (HCH nanoflowers), as solid mimic peroxidase, could catalyze oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of H₂O₂ to a green-colored product. HCH nanoflowers, integrating the essential functions of both biological recognition and signal amplification, meet the requirements of signal labels for colorimetric immunoassay of bacteria. In view of the excellent peroxidase mimetic catalytic activity of HCH nanoflowers, a colorimetric biosensing platform was newly constructed and applied for sensitive detection of foodborne Escherichia coli O157:H7 (E. coli O157:H7). The corresponding detection limits was as low as 4.1?CFU/mL with wide linear ranges (10¹–10⁶?CFU/mL).     

Effect of cultivation temperature on nucleic acid level in bacteria Listeria monocytogenes and Yersinia pseudotuberculosis

The relationship between the multiplication of bacteria, the content of nucleic acid and the specific rate of their growth during their batch cultivation in nutrient broth and mineral medium at temperatures of 37 degrees C and 4-6 degrees C was studied in the causative agents of saprozoonotic infections with L. monocytogenes and Y. pseudotuberculosis used as typical representatives of such bacteria. The content of DNA was shown to remain practically unchanged after the alteration of cultivation temperature and the conditions of nutrition. The linear relationship between the content of RNA and specific growth rate was registered both at 37 degrees C and 4-6 degrees C. However a higher content of RNA at low temperatures was found to correspond to one and the same specific growth rate, which was linked with the additional synthesis of this nucleic acid.     

Prevalence and level of Listeria monocytogenes and other Listeria sp. in ready-to-eat minimally processed and refrigerated vegetables

Minimally processed and refrigerated vegetables can be contaminated with Listeria species bacteria including Listeria monocytogenes due to extensive handling during processing or by cross contamination from the processing environment. The objective of this study was to examine the microbiological quality of ready-to-eat minimally processed and refrigerated vegetables from supermarkets in Osijek, Croatia. 100 samples of ready-to-eat vegetables collected from different supermarkets in Osijek, Croatia, were analyzed for presence of Listeria species and Listeria monocytogenes. The collected samples were cut iceberg lettuces (24 samples), other leafy vegetables (11 samples), delicatessen salads (23 samples), cabbage salads (19 samples), salads from mixed (17 samples) and root vegetables (6 samples). Listeria species was found in 20 samples (20 %) and Listeria monocytogenes was detected in only 1 sample (1 %) of cut red cabbage (less than 100 CFU/g). According to Croatian and EU microbiological criteria these results are satisfactory. However, the presence of Listeria species and Listeria monocytogenes indicates poor hygiene quality. The study showed that these products are often improperly labeled, since 24 % of analyzed samples lacked information about shelf life, and 60 % of samples lacked information about storage conditions. With regard to these facts, cold chain abruption with extended use after expiration date is a probable scenario. Therefore, the microbiological risk for consumers of ready-to-eat minimally processed and refrigerated vegetables is not completely eliminated.