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.     

Molecular Studies on the Ecology of Listeria monocytogenes in the Smoked Fish Processing Industry

We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology of Listeria monocytogenes in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive for L. monocytogenes using a commercial PCR system (BAX for Screening/Listeria monocytogenes), including 57 (27.7%) environmental samples (n = 206), 8 (7.8%) raw material samples (n = 102), 23 (18.1%) samples from fish in various stages of processing(n = 127), and 7 (7.3%) finished product samples (n = 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (P ≤ 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different L. monocytogenes strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.     

Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3⁺ bacteria were present within single-membrane phagosomes that are LAMP1⁺. Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3⁺ bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.     

A nucleic acid sequence-based amplification (NASBA) system for Listeria monocytogenes and simple method for detection of amplimers

A NASBA system amplifying specific sequences of the Listeria monocytogenes hlyA gene and an immunoenzymatic assay for the detection of amplimers was developed. The immunoenzymatic assay utilized a simple microtiter plate format and an anti-RNA:DNA hybrid antibody for the detection of NASBA product (predominantly RNA) hybridized to an immobilized DNA probe. This highly sensitive isothermal amplification and detection system was reactive with genomic DNA from various L. monocytogenes isolates but not with other Listeria or non-Listeria species.     

Isolation and characterization of Listeria monocytogenes from tropical seafood of Kerala, India

Listeria monocytogenes, which is an intracellular pathogen, causes various illnesses in human as well as in animals. The pathogenicity of this organism depends upon the presence of different virulence genes. A total of 324 tropical seafood and fishery environmental samples were screened for L. monocytogenes. The incidence of the human pathogenic species L. monocytogenes was 1.2 % of the samples. Listeria spp. was detected in 32.3, 27.1, and 5 % of fresh, frozen, and dry fish samples, respectively. Listeria innocua was found to be the most prevalent species of Listeria in the tropical seafood and environmental samples of Kerala. Listeria monocytogenes and L. innocua isolates were confirmed by multiplex PCR. L. monocytogenes isolates from the four positive samples showed phosphatidylinositol-specific phospholipase C reaction on Chromocult® Listeria selective agar. Molecular characterization of L. monocytogenes isolates for virulence genes revealed the presence of β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes in all the isolates recovered from the positive samples.     

Rapid and Sensitive Detection of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> by Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) was designed for detection of Listeria monocytogenes, which is an important food-borne kind of pathogenic bacteria causing human and animal disease. The primers set for the hlyA gene consist of six primers targeting eight regions on specific gene. The LAMP assay could be performed within 40 min at 65°C in a water bath. Amplification products were visualized by calcein and manganous ion and agarose gel electrophoresis. Sensitivity of the LAMP assay for detection of L. monocytogenes in pure cultures was 2.0 CFU per reaction. The LAMP assay was 100-fold higher sensitive than that of the conventional PCR assay. Taking this way, 60 chicken samples were investigated for L. monocytogenes. The accuracy of LAMP was shown to be 100% when compared to the “gold standard” culture-biotechnical, while the PCR assay failed to detect L. monocytogenes in two of the positive samples. It is shown that LAMP assay can be used as a sensitive, rapid, and simple detection tool for the detection of L. monocytogenes and will facilitate the surveillance for contamination of L. monocytogenes in food.     

Direct PCR assay for detection ofNeisseria meningitidis in human cerebrospinal fluid

A PCR amplification was performed to detectNeisseria meningitidis insertion sequence1106 (IS-1106) in the humancerebrospinalfluid (CSF) in cases of meningitis. The study included 27 CSF samples from suspected meningitis patients. Although the inflammatory response in most of the samples was slightly increased, the results showed that 7 (26%) and 8 (30%) CSF samples were diagnosed as meningococcal meningitis by Gram staining and by culture, respectively. The primers of theIS-1106 were used for direct diagnosis ofN. meningitidis in the human spinal fluid after a minor treatment of the CSF samples. The sample was diagnosed as meningococcal meningitis, if a DNA band of about 600 bp was detected in the ethidium bromide-stained agarose gel. The 27 CSF samples were analyzed in a random manner. Of these, 18 samples including the Gram staining- and culture-positive samples were also positive in PCR amplification. However, a CSF sample, which was diagnosed to be meningococcal meningitis in culture was negative in both Gram staining and PCR analysis. The specificity of theIS-1106 primers was determined to be 95%, with 100% sensitivity in comparison to Gram staining and culture. The primers were sensitive to 10 pg or more of meningococcal DNA. In addition, the PCR amplification showed high predictive values (89 and 100%) in diagnosing meningitis in patients that were negative and positive responders when tested by culture and by Gram staining. In conclusion, the PCR amplification ofIS-1106 ofN. meningitidis is specific and sensitive to both culture-positive and-negative meningococcal meningitis. Hence, PCR assay is highly recommended for use in a rapid diagnosis of suspected meningitis patients.     

Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5′ nuclease assay

The presence of Listeria monocytogenes as a dairy food contaminant is a lethal threat to dairy industrialists; therefore, products tainted with L. monocytogenes must be quickly detected and removed from production. This fluorogenic PCR-based assay was developed to rapidly detect L. monocytogenes contamination in dairy samples before a final product is distributed. The detection method employed uses a PCR primer pair and a fluorogenic TaqMan probe which bind to a region of a virulence determinant gene specific to L. monocytogenes. As the DNA target is amplified, the 5′ nuclease activity of Taq DNA polymerase hydrolyzes the internal fluorogenic probe creating a change in fluorescence that can be monitored and automatically analyzed with a fluorometer. Sensitivity studies indicated a lower detection limit of under 10 CFU for pure culture extracts and spiked dairy enrichments. A study was performed on 266 dairy product samples obtained from Central California dairy production plants. Eighty-three of these samples were artificially spiked with both high and low concentrations of L. monocytogenes before an overnight enrichment in TSB/LiCl/colostin sulfate/moxalactam media. DNA from enriched samples was obtained using a rapid Chelex extraction specifically designed for dairy sample enrichments and automated analysis. The extraction was followed by the fluorogenic PCR assay and measurement of fluorescence increase. The assay was completed within 24 h, with an observed 95.2% sensitivity, 96.7% specificity, 92.9% positive predictive value, 97.8% negative predictive value, and 96.2% accuracy. According to specificity studies, five other bacterial species cross-reacted with the fluorogenic 5′ nuclease PCR. However, only one of these strains (Listeria grayi) was able to grow in the enrichment medium employed, and was not isolated from any of the 266 dairy product enrichments evaluated in this study. Therefore, this method provides a rapid, sensitive, and automatable analysis alternative to standard culture techniques for the detection of Listeria monocytogenes in dairy samples. Received 4 February 1998/ Accepted in revised form 1 October 1998     

Isolation and Confirmation of <Emphasis Type="Italic">Listeria</Emphasis> Species from Seafood off Goa Region by Polymerase Chain Reaction

Several food borne outbreaks have highlighted the importance of Listeria monocytogenes to the public health and have been recognized as an emerging, important food borne pathogen, and a causative agent of listerioses. A number of genes are involved in the manifestation of Listeria virulence, hlyA is one among them. In the present study, 111 marine fish samples including prawns, finfishes and bivalves were screened for the presence of Listeria species. The isolates were characterized biochemically and further L. monocytogenes were confirmed by polymerase chain reaction (PCR) technique using the hlyA gene as a tool to differentiate between L. monocytogenes and other non-pathogenic Listeria species. Out of 111 samples 5 (4.5%) samples were positive for L. monocytogenes. Among the three different types of samples bivalves were found to have maximum percent (12.5) of L. monocytogenes followed by prawns (3.84) and finfishes (2.9). Among all the 111 samples, 15 (13.51%) samples were positive for other Listeria species. It was observed that Listeria occurrence is more in shellfishes than in fin fishes. All the isolates were sensitive towards five different antibiotics in sequence ciprofloxacin > sulphafurazole > norfloxacin > ampicillin and gentamicin.     

A Quantitative Real-time PCR Assay for Quantification of Viable Listeria Monocytogenes Cells After Bacteriocin Injury in Food-First Insights

Quantitative real-time PCR may be a rapid and automated procedure for detection of bacterial pathogens from food samples. Nevertheless, when testing the effects of antimicrobials on the viability of bacterial pathogens in foods, we found that DNA from dead cells interfered greatly in the detection of viable Listeria monocytogenes after treatment with the broad-spectrum bacteriocin enterocin AS-48. To overcome this problem, a quantitative real-time PCR (qRT-PCR) assay based on bacterial mRNA was adapted to quantify viable L. monocytogenes in food after bacteriocin treatments. The procedure allowed a better and faster estimation of viable cells compared to PALCAM viable cell counts when the threshold level was 2 log units/g of food, while PALCAM viable count allowed detection of one log unit/g. This procedure may be useful to verify the efficacy of bacteriocins against L. monocytogenes in foods.     

Amplified fragment length polymorphism analysis of Listeria monocytogenes by Experion™ automated microfluidic electrophoresis

Fifty Listeria monocytogenes strains were genotyped by sAFLP and PCR products were separated by agarose gel and automated chip-based microfluidic electrophoresis. A high congruency of results was observed comparing the two techniques, although for some cultures a better separation of sAFLP fragments was achieved with microfluidic system, which proved to be a highly reliable and reproducible tool to improve the molecular typing of L. monocytogenes, requiring lower volumes of samples and reducing significantly analysis time.     

Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 10² CFU/cm².     

Development of the SapI/AarI Incision Mediated Plasmid Editing Method

Plasmid engineering and molecular cloning is a virtually ubiquitous tool in biology. Although various methods have been developed for ligating DNA molecules or targeted mutagenesis of plasmids, each has its limitations. Many of the commonly used laboratory strategies are inefficient, while commercially available kits are quite costly and often specialized for highly specific circumstances. Here, we describe the SapI/AarI incision mediated plasmid editing (SIMPLE) method, which allows users to perform site-directed mutagenesis, deletions, and even short insertions into any plasmid in a single PCR reaction, using just one restriction enzyme. In addition, the SIMPLE method can be adapted to insert any sized DNA fragment into a vector using a two-step PCR approach, and can be used to ligate any number of DNA fragments with non-compatible ends in the specific order desired. The SIMPLE method provides researches an efficient and powerful tool with a broad range of applications for molecular cloning.     

Detection and characterization of the most common foodborne pathogens by using multiplex PCR procedure

Food Microbial contamination is one of the most serious problems. A large percentage of food-borne illnesses are caused by food-borne pathogens, and diarrheal agents comprise more than half of the overall prevalence of food-borne illnesses in the globe, and more commonly in developing countries. This study aimed to identify the most-common foodborne organisms from foods in Khartoum state by PCR.A total of 207 food samples (raw milk, fresh cheese, yogurt, fish, sausage, mortadella, and eggs) were collected. DNA was extracted from food samples by guanidine chloride protocol, and then species-specific primers were used to identify Escherichia coli O157: H7, Listeria monocytogenes, Salmonella spp., Vibrio cholerae, V. parahaemolyticus, and Staphylococcus aureus. Out of 207 samples, five (2.41%) were positive for L. monocytogenes, one (0.48%) was positive for S. aureus, and one (0.48%) was positive for both Vibrio cholerae and Vibrio parahaemolyticus. From 91 fresh cheese samples, 2 (2.19%) were positive for L. monocytogenes, and one (1.1%) sample was positive for two different foodborne pathogens (V. cholerae and V. parahaemolyticus). Out of 43 Cow's milk samples, three (7%) samples were positive for L. monocytogenes, and out of 4 sausage samples, one (25 %) was positive for S. aureus. Our study revealed the presence of L. monocytogenes and V. cholera in raw milk and fresh cheese samples. Their presence is considered a potential problem and needs intensive hygiene efforts and standard safety measures before, during, and after food processing operations.     

Ecology and Transmission of Listeria monocytogenes Infecting Ruminants and in the Farm Environment

A case-control study involving 24 case farms with at least one recent case of listeriosis and 28 matched control farms with no listeriosis cases was conducted to probe the transmission and ecology of Listeria monocytogenes on farms. A total of 528 fecal, 516 feed, and 1,012 environmental soil and water samples were cultured for L. monocytogenes. While the overall prevalence of L. monocytogenes in cattle case farms (24.4%) was similar to that in control farms (20.2%), small-ruminant (goat and sheep) farms showed a significantly (P < 0.0001) higher prevalence in case farms (32.9%) than in control farms (5.9%). EcoRI ribotyping of clinical (n = 17) and farm (n = 414) isolates differentiated 51 ribotypes. L. monocytogenes ribotypes isolated from clinical cases and fecal samples were more frequent in environmental than in feed samples, indicating that infected animals may contribute to L. monocytogenes dispersal into the farm environment. Ribotype DUP-1038B was significantly (P < 0.05) associated with fecal samples compared with farm environment and animal feedstuff samples. Ribotype DUP-1045A was significantly (P < 0.05) associated with soil compared to feces and with control farms compared to case farms. Our data indicate that (i) the epidemiology and transmission of L. monocytogenes differ between small-ruminant and cattle farms; (ii) cattle contribute to amplification and dispersal of L. monocytogenes into the farm environment, (iii) the bovine farm ecosystem maintains a high prevalence of L. monocytogenes, including subtypes linked to human listeriosis cases and outbreaks, and (iv) L. monocytogenes subtypes may differ in their abilities to infect animals and to survive in farm environments.     

Simultaneous detection of pathogenic <Emphasis Type="Italic">B. cereus,S. aureus</Emphasis> and <Emphasis Type="Italic">L. monocytogenes</Emphasis> by multiplex PCR

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.     

Anthropogenic impact on the presence of L. monocytogenes in soil, fruits, and vegetables

The aim of this study was to determine the prevalence of Listeria sp. and Listeria monocytogenes in soil samples with reference to type of fertilizers (natural and artificial) and distance from places intensively exploited by men, as well as to determine the relationship between the presence of L. monocytogenes in the soil and in fruits and vegetables. The examined 1,000 soil samples originated from 15 different areas, whilst 140 samples of fruits and 210 samples of vegetables were collected from those areas. L. monocytogenes was isolated only from 5.5 % of all soil samples coming exclusively from meadows intensively grazed by cattle (27.8 %) and areas near food processing plants (25 %) and wild animal forests (24 %). Listeria sp. and L. monocytogenes were not present on artificially fertilized areas and wastelands. L. monocytogenes was detected in 10 % of samples of strawberry, 15 % of potato samples, and 5 % of parsley samples. Our data indicate that Listeria spp. and particularly L. monocytogenes were found in the soil from (1) arable lands fertilized with manure, (2) pasture (the land fertilized with feces of domestic animals), and (3) forests (again, the land fertilized with feces of animals, not domestic but wild). The bacteria were not detected in the soil samples collected at (1) artificially fertilized arable lands and (2) wastelands (the lands that were not fertilized with manure or animal feces). Moreover, a correlation was determined in the presence of L. monocytogenes between soil samples and samples of the examined fruits and vegetables.     

Prevalence and characteristics of Listeria monocytogenes in meat,meat products,food handlers and the environment of the meat processing and the retail facilities of a company in Northern Greece

In this study, we investigated the incidence of Listeria monocytogenes in the receiving meat, the meat products, the personnel and the environment of a vertically integrated company in Northern Greece owing a processing plant and three trading facilities. A total of 303 samples were examined from the receiving raw meat, raw meat preparations, ready-to-eat meat products, processing surfaces and the environment of these facilities as well as the food handlers’ hands and nasal cavities. MALDI-TOF MS was used for Listeria identification; from the 22 (7·26%) positive to Listeria spp. isolates, 12 (3·96%) identified as L. monocytogenes, eight (2·64%) as Listeria innocua and two (0·66%) as Listeria welshimeri. Molecular serotyping of L. monocytogenes isolates by multiplex PCR revealed 11 strains belonging to serogroup IIa (1/2a and 3a) and one to IIc (1/2c and 3c). The assay for the detection of the virulence-associated genes revealed eight isolates carrying all the examined genes (inlA, inlB, inlC, plcA, prfA, actA, hlyA and iap) and four carrying all except the actA gene. Eleven (91·7%) of the isolates showed a strong ability to form biofilm. All isolates were multidrug resistant. The MALDI-TOF Main Spectrum Profile (MSPs), revealed three clusters: one with five isolates (four from environmental samples and one from a food handler), one with five isolates (all from environmental samples) and one with two isolates (both from raw meat products). MALDI-TOF MS seems to be a reliable tool for the identification of niches and contamination routes in processing plants, contributing also to the evaluation and improvement of the applied preventive measures to control L. monocytogenes.     

Multiplex PCR for Simultaneous Detection of Bacteria of the Genus Listeria, Listeria monocytogenes, and Major Serotypes and Epidemic Clones of L. monocytogenes

A multiplex PCR assay which combines detection of bacteria of the genus Listeria, Listeria monocytogenes serotypes 1/2a and 4b, and epidemic clones I, II, and III of L. monocytogenes was developed. The assay provides a rapid, reliable, and inexpensive method for screening and subgrouping this important food-borne pathogen.     

Correlation between the Presence of Virulence-Associated Genes as Determined by PCR and Actual Virulence to Mice in Various Strains of Listeria Spp.

Five chromosomal genes, prfA, plcA, hlyA, mpl and plcB, are implicated in the virulence of Listeria monocytogenes and some of these genes have been used for the identification of bacteria by polymerase chain reaction (PCR). Using 6 strains of L. monocytogenes and 3 L. innocua strains, the relationship was examined between the presence of five virulence-associated genes and actual virulence to mice in terms of 50% lethal dose (LD₅₀), bacterial viability in the organ of infected mice and the intracellular growth in cultured macrophages. None of the five genes could be amplified by PCR in all the L. innocua strains and they were actually avirulent to mice. All L. monocytogenes strains were shown to be virulent and to have intact virulence-associated genes except for the strain ATCC15313. This particular strain was revealed to be avirulent and defective in hlyA and plcA in PCR amplification. It was suggested that PCR detection of genes prfA, mpl, or plcB may not be sufficient to detect virulent strains of L. monocytogenes. It appeared that the ability to produce listeriolysin O (LLO), which is encoded by hlyA, was critical for the expression of virulence regardless of the amount of LLO produced.