The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens.

Foodborne pathogens continue to be a major health concern worldwide. Culture-dependent methodologies are still considered the gold standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor intensive. The implementation of real-time PCR has allowed to overcome these limitations, and even reduce the cost associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter, a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.

Application of MinION Long-Read Sequencer for Semi-targeted Detection of Foodborne Pathogens.

Foodborne pathogens remain a serious health issue in developed and developing countries. Safeness of food products has been assured for years with culture-based microbiological methods; however, these present several limitations such as turnaround time and extensive hands-on work, which have been typically address taking advantage of DNA-based methods such as real-time PCR (qPCR). These, and other similar techniques, are targeted assays, meaning that they are directed for the specific detection of one specific microbe. Even though reliable, this approach suffers from an important limitation that unless specific assays are design for every single pathogen potentially present, foods may be considered erroneously safe. To address this problem, next-generation sequencing (NGS) can be used as this is a nontargeted method; thus it has the capacity to detect every potential threat present. In this chapter, a protocol for the simultaneous detection and preliminary serotyping of Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 is described.

Maple compounds prevent biofilm formation in Listeria monocytogenes via sortase inhibition.

The Pss exopolysaccharide (EPS) enhances the ability of the foodborne pathogen Listeria monocytogenes to colonize and persist on surfaces of fresh fruits and vegetables. Eradicating listeria within EPS-rich biofilms is challenging due to their increased tolerance to disinfectants, desiccation, and other stressors. Recently, we discovered that extracts of maple wood, including maple sap, are a potent source of antibiofilm agents. Maple lignans, such as nortrachelogenin-8'-O-ß-D-glucopyranoside and lariciresinol, were found to inhibit the formation of, and promote the dispersion of pre-formed L. monocytogenes EPS biofilms. However, the mechanism remained unknown. Here, we report that these lignans do not affect Pss EPS synthesis or degradation. Instead, they promote EPS detachment, likely by interfering with an unidentified lectin that keeps EPS attached to the cell surfaces. Furthermore, the maple lignans inhibit the activity of L. monocytogenes sortase A (SrtA) in vitro. SrtA is a transpeptidase that covalently anchors surface proteins, including the Pss-specific lectin, to the cell wall peptidoglycan. Consistent with this, deletion of the srtA gene results in Pss EPS detachment from listerial cells. We also identified several additional maple compounds, including epicatechin gallate, isoscopoletin, scopoletin, and abscisic acid, which inhibit L. monocytogenes SrtA activity in vitro and prevent biofilm formation. Molecular modelling indicates that, despite their structural diversity, these compounds preferentially bind to the SrtA active site. Since maple products are abundant and safe for consumption, our finding that they prevent biofilm formation in L. monocytogenes offers a viable source for protecting fresh produce from this foodborne pathogen.

Molecular Characterization of Listeria monocytogenes in the Food Chain of the Republic of Kosovo from 2016 to 2022.

The present study describes the genetic characterization of L. monocytogenes strains found in the Republic of Kosovo's food chain. From 2016 to 2022, 995 samples were collected. Overall, 648 samples were from ready-to-eat (RTE) food products, 281 from food products consumed cooked (FPCC), 60 from raw materials, and 6 from environmental samples. Overall, 11.76% (117 out of 995) of the samples were contaminated by L. monocytogenes, comprising 6.33% (41 out of 648) from RTE products, 14.95% (42 out of 281) from FPCC, 55.00% (33 out of 60) from raw materials, and 16.66% (1 out of 6) from environmental samples. All isolates were subjected to molecular serotyping and clonal complex (CC) identification by using real-time PCR, as well as multilocus sequence typing. All isolates were grouped into four molecular serotypes, IIa (34.19%), IIb (3.48%), IIc (32.48%), and IVb (29.91%), as well as Lineage I (33.33%) and Lineage II (66.66%). In total, 14 CCs were identified from 41 RTE isolates; however, CC29 (7), CC2 (6), and CC6 (6) were the most dominant. By contrast, CC9 was by far the most represented CC in both FPCC (21) and RM (14). Moreover, 30 isolates expressed CC1, CC2, CC4, or CC6, which are particularly associated with severe human infections.

Potential Use and Chemical Analysis of Some Natural Plant Extracts for Controlling Listeria spp. Growth In Vitro and in Food.

Listeria are Gram-negative intracellular foodborne pathogens that can cause invasive infections with high mortality rates. In this work, the antibacterial activity of ten essential oils, infusion extracts, and decoction extracts of some medicinal plants was tested against Listeria monocytogenes and listeria ivanovii strains. The effects of different physical conditions including temperature, pH, sodium chloride, and some organic acids were studied. The results showed that the water extracts gave the maximum bacterial inhibition, while ethanolic extract was inactive against the tested Listeria spp. The antibiotic sensitivity of L. monocytogenes LMG10470 and L. ivanovii LMZ11352 was tested against five antibiotics including imipenem, levofloxacin, amikacin, ampicillin, and amoxicillin. Imipenem was the most effective antibiotic, resulting in inhibition zones of 40 mm and 31 mm for L. monocytogenes and L. ivanovii, respectively. When imipenem mixed with Syzygium aromaticum oil, Salvia officinalis oil, Pimpinella anisum infusion, and Mentha piperita infusion each, the water extract of Moringa oleifera leaves and seeds against LMG10470 and LMZ11352 resulted in broader antibacterial activity. The antimicrobial activity of both Pimpinella anisum and Mentha piperita plant extracts is related to a variety of bioactive compounds indicated by gas chromatography-mass spectrometry analysis of these two plant extracts. These two plant extracts seemed to contain many chemical compounds elucidated by gas chromatography-mass spectrometry (GC-MS) and infrared radiation spectra. These compounds could be classified into different chemical groups such as ethers, heterocyclic compounds, aromatic aldehydes, condensed heterocyclic compounds, ketones, alicyclic compounds, aromatics, esters, herbicides, saturated fatty acids, and unsaturated fatty acids. The use of these natural compounds seems to be a useful technological adjuvant for the control of Listeria spp. in foods.

Occurrence, Molecular Serogroups, Antimicrobial Susceptibility and Identification by MALDI-TOF MS of Listeria monocytogenes Isolated from RTE Meat Products in Southern Poland.

L. monocytogenes is considered one of the most dangerous foodborne pathogens. This study aimed to determine the occurrence of L. monocytogenes in RTE meat products from southern Poland, including serogroups and antimicrobial susceptibility, and to assess the usefulness of MALDI-TOF MS as a tool for identifying L. monocytogenes. A total of 848 production batches of RTE meat products were analyzed for L. monocytogenes. All L. monocytogenes isolates were serotyped using the multiplex PCR method, tested for antimicrobial susceptibility using the disk diffusion method and identified using the MALDI-TOF MS method. L. monocytogenes was detected in 52/848 batches of RTE meat products (6.13%). The isolates belonged to four serogroups: 17/52 (33%) isolates to IVb; 15/52 (29%) isolates to IIa; 10/52 (19%) isolates to IIc and 10/52 (19%) isolates to IIb. All isolates (52/52) showed susceptibility to the tested antimicrobials. Using MALDI-TOF MS, 10/52 isolates (19.2%) were identified at the level of secure genus identification, probable species identification; 37/52 isolates (71.2%) were identified at the level of probable genus identification; 3/52 isolates (5.8%) were incorrectly identified as L. innocua; and 2/52 isolates (3.8%) were not identified. The occurrence of L. monocytogenes in RTE meat products was low. Almost half of the analyzed isolates were L. monocytogenes of serogroups, which are most often associated with listeriosis in humans in Poland. All isolates showed susceptibility to five commonly used antimicrobials for treating listeriosis. The use of MALDI-TOF MS as a tool for the identification of L. monocytogenes indicated its limitations related to the insufficient representation of the pathogen in the reference database.

Determination of the combined effect of grape seed extract and cold atmospheric plasma on foodborne pathogens and their environmental stress knockout mutants.

The study aimed to explore the antimicrobial efficacy of grape seed extract (GSE) and cold atmospheric plasma (CAP) individually or in combination against L. monocytogenes and E. coli wild type (WT) and their isogenic mutants in environmental stress genes. More specifically, we examined the effects of 1% (wt/vol) GSE, 4 min of CAP treatment, and their combined effect on L. monocytogenes 10403S WT and its isogenic mutants ΔsigB, ΔgadD1, ΔgadD2, ΔgadD3, as well as E. coli K12 and its isogenic mutants ΔrpoS, ΔoxyR, and ΔdnaK. In addition, the sequence of the combined treatments was tested. A synergistic effect was achieved for all L. monocytogenes strains when exposure to GSE was followed by CAP treatment. However, the same effect was observed against E. coli strains, only for the reversed treatment sequence. Additionally, L. monocytogenes ΔsigB was more sensitive to the individual GSE and the combined GSE/CAP treatment, whereas ΔgadD2 was more sensitive to CAP, as compared to the rest of the mutants under study. Individual GSE exposure was unable to inhibit E. coli strains, and individual CAP treatment resulted in higher inactivation of E. coli in comparison to L. monocytogenes with the strain ΔrpoS appearing the most sensitive among all studied strains. Our findings provide a step toward a better understanding of the mechanisms playing a role in the tolerance/sensitivity of our model Gram-positive and Gram-negative bacteria toward GSE, CAP, and their combination. Therefore, our results contribute to the development of more effective and targeted antimicrobial strategies for sustainable decontamination.IMPORTANCEAlternative approaches to conventional sterilization are gaining interest from the food industry, driven by (i) the consumer demand for minimally processed products and (ii) the need for sustainable, environmentally friendly processing interventions. However, as such alternative approaches are milder than conventional heat sterilization, bacterial pathogens might not be entirely killed by them, which means that they could survive and grow, causing food contamination and health hazards. In this manuscript, we performed a systematic study of the impact of antimicrobials derived from fruit industry waste (grape seed extract) and cold atmospheric plasma on the inactivation/killing as well as the damage of bacterial pathogens and their genetically modified counterparts, for genes linked to the response to environmental stress. Our work provides insights into genes that could be responsible for the bacterial capability to resist/survive those novel treatments, therefore, contributing to the development of more effective and targeted antimicrobial strategies for sustainable decontamination.

Enumeration Agar, Acid Exposure and Sampling Time Are Relevant Factors Accounting for the High-Pressure Inactivation of Vegetative Pathogens in Fruit Puree.

High pressure processing (HPP) is a non-thermal technology with emerging application within the fruit and vegetable sector. The impact of the enumeration agar on the recorded HPP inactivation of L. monocytogenes, Salmonella spp. and E. coli in banana-apple and apple purees was evaluated. Additionally, the HPP inactivation and sublethal injury was quantified in apple puree, considering the impact of acid exposure (24 h before HPP) and sampling time. Inoculated purees were pressurized at 300 MPa for 2 min. Enumeration was performed immediately and 24 h after HPP. HPP inactivation was 0.9-to-4.5-fold higher in apple than banana-apple puree. Compared with nutrient-rich media, selective agar enumeration overestimated the inactivation. HPP inactivation and sublethal injury of L. monocytogenes, Salmonella and E. coli was variable, mainly dependent on the exposure to acid and the sampling time. The 24 h-delayed enumeration slightly increased the inactivation. In apple puree, the CECT5947 strain of E. coli O157:H7 was the most piezo-resistant strain (1.5 log reduction), while L. monocytogenes Scott A was the most piezo-sensitive (6-log reduction when exposed to acid and sampled 24 h after HPP). All the studied factors should be taken into account when designing HPP treatments, performing product-specific validation studies and setting verification procedures.

Growth of Listeria monocytogenes in fresh vegetables and vegetable salad products: An update on influencing intrinsic and extrinsic factors.

The ability of foodborne pathogens to grow in food products increases the associated food safety risks. Listeria monocytogenes (Lm) is a highly adaptable pathogen that can survive and grow under a wide range of environmental circumstances, including otherwise inhibitory conditions, such as restrictive cold temperatures. It can also survive long periods under adverse environmental conditions. This review examines the experimental evidence available for the survival and growth of Lm on fresh vegetables and ready-to-eat vegetable salads. Published data indicate that, depending on certain intrinsic (e.g., nutrient composition) and extrinsic factors (e.g., storage temperature, packaging atmosphere), Lm can survive on and in a wide variety of vegetables and fresh-cut minimally processed vegetable salads. Studies have shown that temperature, modified atmosphere packaging, relative humidity, pH, water activity, background microbiota of vegetables, microbial strain peculiarities, and nutrient type and availability can significantly impact the fate of Lm in vegetables and vegetable salads. The influence of these factors can either promote its growth or decline. For example, some studies have shown that background microbiota inhibit the growth of Lm in vegetables and minimally processed vegetable salads, but others have reported a promoting, neutral, or insignificant effect on the growth of Lm. A review of relevant literature also indicated that the impact of most influencing factors is related to or interacts with other intrinsic or extrinsic factors. This literature synthesis contributes to the body of knowledge on possible strategies for improving food safety measures to minimize the risk of Lm-associated foodborne outbreaks involving vegetables and vegetable salads.

Unveiling the persistent threat: recent insights into Listeria monocytogenes adaptation, biofilm formation, and pathogenicity in foodborne infections.

Listeriosis is a severe disease caused by the foodborne pathogen Listeria monocytogenes, posing a significant risk to vulnerable populations such as the elderly, pregnant women, and newborns. While relatively uncommon, it has a high global mortality rate of 20-30%. Recent research indicates that smaller outbreaks of the more severe, invasive form of the disease occur more frequently than previously thought, despite the overall stable infection rates of L. monocytogenes over the past 10 years. The ability of L. monocytogenes to form biofilm structures on various surfaces in food production environments contributes to its persistence and challenges in eradication, potentially leading to contamination of food and food production facilities. To address these concerns, this review focuses on recent developments in epidemiology, risk evaluations, and molecular mechanisms of L. monocytogenes survival in adverse conditions and environmental adaptation. Additionally, it covers new insights into strain variability, pathogenicity, mutations, and host vulnerability, emphasizing the important events framework that elucidates the biochemical pathways from ingestion to infection. Understanding the adaptation approaches of L. monocytogenes to environmental stress factors is crucial for the development of effective and affordable pathogen control techniques in the food industry, ensuring the safety of food production.

Biofilm Formation, Motility, and Virulence of Listeria monocytogenes Are Reduced by Deletion of the Gene lmo0159, a Novel Listerial LPXTG Surface Protein.

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that causes listeriosis in humans and other animals. Surface proteins with the LPXTG motif have important roles in the virulence of L. monocytogenes. Lmo0159 is one such protein, but little is known about its role in L. monocytogenes virulence, motility, and biofilm formation. Here, we constructed and characterized a deletion mutant of lmo0159 (∆lmo0159). We analyzed not only the capacity of biofilm formation, motility, attachment, and intracellular growth in different cell types but also LD50; bacterial load in mice's liver, spleen, and brain; expression of virulence genes; and survival time of mice after challenge. The results showed that the cross-linking density of the biofilm of ∆lmo0159 strain was lower than that of WT by microscopic examination. The expression of biofilm-formation and virulence genes also decreased in the biofilm state. Subsequently, the growth and motility of ∆lmo0159 in the culture medium were enhanced. Conversely, the growth and motility of L. monocytogenes were attenuated by ∆lmo0159 at both the cellular and mouse levels. At the cellular level, ∆lmo0159 reduced plaque size; accelerated scratch healing; and attenuated the efficiency of adhesion, invasion, and intracellular proliferation in swine intestinal epithelial cells (SIEC), RAW264.7, mouse-brain microvascular endothelial cells (mBMEC), and human-brain microvascular endothelial cells (hCMEC/D3). The expression of virulence genes was also inhibited. At the mouse level, the LD50 of the ∆lmo0159 strain was 100.97 times higher than that of the WT strain. The bacterial load of the ∆lmo0159 strain in the liver and spleen was lower than that of the WT strain. In a mouse model of intraperitoneal infection, the deletion of the lmo0159 gene significantly prolonged the survival time of the mice, suggesting that the lmo0159 deletion mutant also exhibited reduced virulence. Thus, our study identified lmo0159 as a novel virulence factor among L. monocytogenes LPXTG proteins.

Prevalence, antibiogram and molecular characterization of Listeria monocytogenes from ruminants and humans in New Valley and Beheira Governorates, Egypt.

BACKGROUND: Listeriosis is a global health threat to both animals and humans, especially in developing countries. This study was designed to isolate Listeria monocytogenes from faeces; environmental samples; and cow, sheep and goat milk, as well as human stool, to study its molecular characteristics and antibiotic sensitivity in the New Valley and Beheira Governorates, Egypt. The isolation and identification of L. monocytogenes were carried out using traditional culture and biochemical methods, followed by antibiography, genus confirmation of some isolates and detection and sequencing of InlB genes via PCR. RESULTS: Out of 2097 examined samples, the prevalence of L. monocytogenes was 13.4% in animals; the prevalence was 9.2%, 2.4%, 25.4%, 4%, 42.4%, and 6.4% in cattle faeces, cattle milk, sheep faeces, sheep milk, goat faeces, and goat milk, respectively. However, the prevalence of L. monocytogenes was 8.3% in human samples. Both animal and human isolates showed 100% resistance to trimethoprim-sulfamethoxazole, and the isolates showed the highest sensitivity to flumequine (100%), amikacin (99.2%), gentamicin (97.6%), and levofloxacin (94.6%). Multidrug resistance (MDR) was detected in 86.9% of the tested isolates. The 16 S rRNA and inlB genes were detected in 100% of the randomly selected L. monocytogenes isolates. Phylogenetic analysis of three isolates based on the inlB gene showed 100% identity between faecal, milk and human stool isolates. CONCLUSIONS: Faeces and milk are major sources of listeriosis, and the high degree of genetic similarity between animal and human isolates suggests the possibility of zoonotic circulation. The high prevalence of MDR L. monocytogenes in both animal and human samples could negatively impact the success of prevention and treatments for animal and human diseases, thereby imposing serious risks to public health.

Prevalence of Listeria monocytogenes in Milk and Dairy Product Supply Chains: A Global Systematic Review and Meta-analysis.

Listeria monocytogenes, one of the main foodborne pathogens, is commonly found in milk and dairy products. This study aimed to estimate the presence of L. monocytogenes in milk and dairy product supply chains using a meta-analysis based on PubMed, Embase, Web of Science, and Scopus databases. A total of 173 studies were included in this meta-analysis. The pooled prevalence in the supply chain environment was 8.69% (95% confidence interval [CI]: 5.30%-12.78%), which was higher than that in dairy products (4.60%, 95% CI: 1.72%-8.60%) and milk products (2.93%, 95% CI: 2.14%-3.82%). Subgroup analysis showed that L. monocytogenes prevalence in raw milk (3.44%, 95% CI: 2.61%-4.28%) was significantly higher than in pasteurized milk (0.60%, 95% CI: 0.00%-2.06%). The highest prevalence of L. monocytogenes in milk and dairy products was observed in North America (5.27%, 95% CI: 2.19%-8.35%) and South America (13.54%, 95% CI: 3.71%-23.37%). In addition, studies using culture and molecular methods (5.17%, 95% CI: 2.29%-8.06%) had higher prevalence than other detection methods. Serogroup 1/2a and 3a (45.34%, 95% CI: 28.74%-62.37%), serogroup 1/2b and 3b (14.23%, 95% CI: 6.05%-24.24%), and serogroup 4b/4e (13.71%, 95% CI: 6.18%-22.83%) were dominant in these studies. The results of this study provide a better understanding of the prevalence of L. monocytogenes in milk and dairy product supply chains and suggest a potential foodborne pathogen burden.

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens.

Loop-mediated isothermal amplification, LAMP, is nowadays the most popular isothermal nucleic acid amplification technique, and as such, several commercial, ready-to-use master mixes have flourished. Unfortunately, independent studies to determine their performance are limited. The current study performed an independent evaluation of the existing ready-to-use commercial LAMP master mixes WarmStart® LAMP Kit, LavaLAMP™ DNA Master Mix, Saphir Bst Turbo GreenMaster, OptiGene Fast Master Mix ISO-004, and SynLAMP Mix. To reduce bias, three different genes, namely ttr (Salmonella spp.), rfbE (E. coli O157), and hly (Listeria monocytogenes), were targeted. The comparison was based on amplification speed, performance with decreasing DNA concentrations, and the effect of five typical LAMP reaction additives (betaine, DMSO, pullulan, TMAC, and GuHCl). Significant differences were observed among the different master mixes. OptiGene provided the fastest amplification and showed less detrimental effects associated with the supplements evaluated. Out of the chemicals tested, pullulan provided the best results in terms of amplification speed. It is noteworthy that the different additives impacted the master mixes differently. Overall, the current study provides insights into the performance of commercial LAMP master mixes, which can be of value for the scientific community to better select appropriate reagents when developing new methods.

Application of gelatin-based zinc oxide nanoparticles bionanocomposite coatings to control Listeria monocytogenes in Talaga cheese and camel meat during refrigerated storage.

Listeria monocytogenes is a concerning foodborne pathogen incriminated in soft cheese and meat-related outbreaks, highlighting the significance of applying alternative techniques to control its growth in food. In the current study, eco-friendly zinc oxide nanoparticles (ZnO-NPs) were synthesized using Rosmarinus officinalis, Punica granatum, and Origanum marjoram extracts individually. The antimicrobial efficacy of the prepared ZnO-NPs against L. monocytogenes was assessed using the agar well diffusion technique. Data indicated that ZnO-NPs prepared using Origanum marjoram were the most effective; therefore, they were used for the preparation of gelatin-based bionanocomposite coatings. Furthermore, the antimicrobial efficacy of the prepared gelatin-based bionanocomposite coatings containing eco-friendly ZnO-NPs was evaluated against L. monocytogenes in Talaga cheese (an Egyptian soft cheese) and camel meat during refrigerated storage at 4 ± 1 oC. Talaga cheese and camel meat were inoculated with L. monocytogenes, then coated with gelatin (G), gelatin with ZnO-NPs 1% (G/ZnO-NPs 1%), and gelatin with ZnO-NPs 2% (G/ZnO-NPs 2%). Microbiological examination showed that the G/ZnO-NPs 2% coating reduced L. monocytogenes count in the coated Talaga cheese and camel meat by 2.76 ± 0.19 and 2.36 ± 0.51 log CFU/g, respectively, by the end of the storage period. Moreover, G/ZnO-NPs coatings controlled pH changes, reduced water losses, and improved the sensory characteristics of Talaga cheese and camel meat, thereby extending their shelf life. The obtained results from this study indicate that the application of gelatin/ZnO-NPs 2% bionanocomposite coating could be used in the food industry to control L. monocytogenes growth, improve quality, and extend the shelf life of Talaga cheese and camel meat.

New insights into the antibiofilm activity and mechanism of Mannosylerythritol Lipid-A against Listeria monocytogenes EGD-e.

Listeria monocytogenes is one of the leading causative agents of foodborne disease outbreaks worldwide. Herein, the antibiofilm effect and mechanism of Mannosylerythritol Lipid-A against L. monocytogenes EGD-e is reported for the first time. MEL-A effectively attenuated biofilm formation while reducing the viability and motility of bacteria within the biofilm in the early stage, and influenced bacterial adhesion by affecting the secretion of extracellular polysaccharides and eDNA. RT-qPCR revealed that MEL-A significantly suppressed the expression of genes involved in flagellar movement and virulence. Untargeted LC-MS metabolomics indicated that MEL-A affected the fluidity and permeability of cell membranes by significantly upregulating unsaturated fatty acids, lipids and glycoside metabolites, and affected protein biosynthesis, nucleotide metabolism and DNA synthesis and repair by significantly downregulating amino acid metabolism and nucleic acid metabolism. These pathways may constitute the key targets of biofilm formation inhibition by MEL-A. Furthermore, MEL-A showed good removal effects on mature biofilms under different temperatures, different materials and milk. Our data indicated that MEL-A could be used as a novel antibiofilm agent to improve food safety. Our study provides new insights into the possible inhibitory mechanism of MEL-A and the response of L. monocytogenes EGD-e to MEL-A.

The Effectiveness of LISTEXTM P100 in Reducing the Biofilm of Listeria spp. on Steel, Plastic, and Galvanised Surfaces.

BACKGROUND: Eliminating and managing L. monocytogenes, L. welshimeri, and L. ivanovii biofilms is a significant problem for food safety, as listeriosis is among the worst foodborne illnesses. METHOD: The Listex P100 bacteriophage's bactericidal and inhibitory properties have been investigated in relation to varying strains of vegetative cells and biofilms of L. monocytogenes, L. welshimeri, and L. ivanovii. RESULTS: The phage concentrations of 109 and 1010 PFU/ml showed strong antibacterial activity against L. monocytogenes, L. welshimeri, and L. ivanovii at both 10°C and 30°C (P<0.05). In 96- well microplate experiments, bacteriophage treatment inhibited biofilm development and reduced biofilm by up to 57.6% (P ≤ 0.05). When compared to controls, Listex P100 bacteriophage significantly reduced the populations of L. monocytogenes, L. welshimeri, and L. ivanovii biofilms on the surfaces of galvanised, stainless steel, and plastic surfaces where holes were produced and the structure of Listeria spp. was disturbed. CONCLUSION: This study clearly demonstrated that L. monocytogenes, L. welshimeri, and L. ivanovii biofilms on galvanised, stainless steel, and plastic surfaces might be removed by using Listex P100 bacteriophage.

Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

Population dynamics and bidirectional transfer of Listeria monocytogenes and Shiga toxin-producing Escherichia coli during cheese production in wooden vats.

Wooden vats are used in the production of some traditional cheeses as the biofilms on wooden vat surfaces are known to transfer large quantities of microbes to cheese. However, the safety of using wooden vats for cheese production remains controversial as the porous structure of wood provides an irregular surface that may protect any attached pathogen cells from cleaning and sanitation processes. On the other hand, the absence of pathogens in wooden vats has been reported in multiple studies and wooden materials have not been associated with foodborne illness outbreaks. The present study determined the survival of Listeria monocytogenes and Shiga toxin-producing Escherichia coli (STEC) during the production of an uncooked pressed cheese in wooden vats as well as their ability to transfer to the wood and then to milk used in subsequent batches of cheese production in the absence of formal cleaning. Results from the study indicate that pathogens inoculated in milk grew during production of the uncooked cheese, but showed limited ability to colonize the wooden vats and contaminate subsequent batches. These results suggest that the risks of using wooden vats to produce cheese is low if the milk is of high microbiological quality.

Airborne signals of Pseudomonas fluorescens modulate swimming motility and biofilm formation of Listeria monocytogenes in a contactless coculture system.

Bacterial volatile compounds (BVCs) facilitate interspecies communication in socio-microbiology across physical barriers, thereby influencing interactions between diverse species. The impact of BVCs emitted from Pseudomonas on the biofilm formation characteristics of Listeria monocytogenes within the same ecological niche has been scarcely investigated under practical conditions of food processing. The objective of this study was to explore the motility and biofilm formation characteristics of L. monocytogenes under the impact of Pseudomonas BVCs. It was revealed that BVCs of P. fluorescens, P. lundensis, and P. fragi significantly promoted swimming motility of L. monocytogenes (P < 0.05). As evidenced by crystal violet staining, the L. monocytogenes biofilms reached a maximum OD570 value of approximately 3.78 at 4 d, which was 0.65 units markedly higher than that of the control group (P < 0.05). Despite a decrease in adherent cells of L. monocytogenes biofilms among the BVCs groups, there was a remarkable increase in the abundance of extracellular polysaccharides and proteins with 3.58 and 4.90 µg/cm2, respectively (P < 0.05), contributing to more compact matrix architectures, which suggested that the BVCs of P. fluorescens enhanced L. monocytogenes biofilm formation through promoting the secretion of extracellular polymers. Moreover, the prominent up-regulated expression of virulence genes further revealed the positive regulation of L. monocytogenes under the influence of BVCs. Additionally, the presence of BVCs significantly elevated the pH and TVB-N levels in both the swimming medium and biofilm broth, thereby exhibiting a strong positive correlation with increased motility and biofilm formation of L. monocytogenes. It highlighted the crucial signaling regulatory role of BVCs in bacterial interactions, while also emphasizing the potential food safety risk associated with the hitchhiking behavior of L. monocytogenes, thereby shedding light on advancements in control strategies for food processing.