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.

The ARIMA model approach for the biofilm-forming capacity prediction of Listeria monocytogenes recovered from carcasses.

The present study aimed to predict the biofilm-formation ability of L. monocytogenes isolates obtained from cattle carcasses via the ARIMA model at different temperature parameters. The identification of L. monocytogenes obtained from carcass samples collected from slaughterhouses was determined by PCR. The biofilm-forming abilities of isolates were phenotypically determined by calculating the OD value and categorizing the ability via the microplate test. The presence of some virulence genes related to biofilm was revealed by QPCR to support the biofilm profile genotypically. Biofilm-formation of the isolates was evaluated at different temperature parameters (37 °C, 22 °C, 4 °C and - 20 °C). Estimated OD values were obtained with the ARIMA model by dividing them into eight different estimation groups. The prediction performance was determined by performance measurement metrics (ME, MAE, MSE, RMSE, MPE and MAPE). One week of incubation showed all isolates strongly formed biofilm at all controlled temperatures except - 20 °C. In terms of the metrics examined, the 3 days to 7 days forecast group has a reasonable prediction accuracy based on OD values occurring at 37 °C, 22 °C, and 4 °C. It was concluded that measurements at 22 °C had lower prediction accuracy compared to predictions from other temperatures. Overall, the best OD prediction accuracy belonged to the data obtained from biofilm formation at -20 °C. For all temperatures studied, especially after the 3 days to 7 days forecast group, there was a significant decrease in the error metrics and the forecast accuracy increased. When evaluating the best prediction group, the lowest RMSE at 37 °C (0.055), 22 °C (0.027) and 4 °C (0.024) belonged to the 15 days to 21 days group. For the OD predictions obtained at -20 °C, the 15 days to 21 days prediction group had also good performance (0.011) and the lowest RMSE belongs to the 7 days to 15 days group (0.007). In conclusion, this study will guide in using indicator parameters to evaluate biofilm forming ability to predict optimum temperature-time. The ARIMA models integrated with this study can be useful tools for industrial application and risk assessment studies using different parameters such as pH, NaCl concentration, and especially temperature applied during food processing and storage on the biofilm-formation ability of L. monocytogenes.

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.

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.

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.

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.

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.

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.

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.

DRG2 in macrophages is crucial for initial inflammatory response and protection against Listeria monocytogenes infection.

Innate immune response is critical for the control of Listeria monocytogenes infection. Here, we identified developmentally regulated GTP-binding protein 2 (DRG2) in macrophages as a major regulator of the innate immune response against L. monocytogenes infection. Both whole-body DRG2 knockout (KO) mice and macrophage-specific DRG2 KO mice had low levels of IL-6 during early infection and increased susceptibility to L. monocytogenes infection. Following an initial impaired inflammatory response of macrophages upon i.p. L. monocytogenes infection, DRG2-/- mice showed delayed recruitment of neutrophils and monocytes into the peritoneal cavity, which led to elevated bacterial burden, inflammatory cytokine production at a late infection time point, and liver micro-abscesses. DRG2 deficiency decreased the transcriptional activity of NF-κB and impaired the inflammatory response of both bone marrow-derived and peritoneal macrophages upon L. monocytogenes stimulation. Our findings reveal that DRG2 in macrophages is critical for the initial inflammatory response and protection against L. monocytogenes infection.

Characterization of the glmS Ribozymes from Listeria Monocytogenes and Clostridium Difficile.

The glmS ribozyme regulates the expression of the essential GlmS enzyme being involved in cell wall biosynthesis. While >450 variants of the glmS ribozyme were identified by in silico approaches and homology searches, only a few have yet been experimentally investigated. Herein, we validate and characterize the glmS ribozymes of the human pathogens Clostridium difficile and Listeria monocytogenes. Both ribozymes, as their previous characterized homologs rely on glucosamine-6-phosphate as co-factor and the presence of divalent cations for exerting the cleavage reaction. The observed EC50 values in turn were found to be in the submicromolar range, at least an order of magnitude lower than observed for glmS ribozymes from other bacteria. The glmS ribozyme of L. monocytogenes was further shown to bear unique properties. It discriminates between co-factors very stringently and other than the glmS ribozyme of C. difficile retains activity at low temperatures. This finding illustrates that albeit being highly conserved, glmS ribozymes have unique characteristics.

Comprehensive metagenomic analysis of stress-resistant and -sensitive Listeria monocytogenes.

Listeria monocytogenes is a pathogenic bacterium which can live in adverse environments (low pH, high salinity, and low temperature). Even though there are various whole genome sequencing (WGS) data on L. monocytogenes, investigations on genetic differences between stress-resistant and -sensitive L. monocytogenes grown under stress environments have been not fully examined. This study aims to investigate and compare genetic characteristics between stress-resistant and -sensitive L. monocytogenes using whole genome sequencing (WGS). A total of 47 L. monocytogenes strains (43 stress-resistant and 4 stress-sensitive) were selected based on the stress-resistance tests under pH 3, 5% salt concentration, and 1 °C. The sequencing library for WGS was prepared and sequenced using an Illumina MiSeq. Genetic characteristics of two different L. monocytogenes groups were examined to analyze the pangenome, functionality, virulence, antibiotic resistance, core, and unique genes. The functionality of unique genes in the stress-resistant L. monocytogenes was distinct compared to the stress-sensitive L. monocytogenes, such as carbohydrate and nucleotide transport and metabolism. The lisR virulence gene was detected more in the stress-resistant L. monocytogenes than in the stress-sensitive group. Five stress-resistant L. monocytogenes strains possessed tet(M) antibiotic resistance gene. This is the first study suggesting that deep genomic characteristics of L. monocytogenes may have different resistance level under stress conditions. This new insight will aid in understanding the genetic relationship between stress-resistant and -sensitive L. monocytogenes strains isolated from diverse resources. KEY POINTS: • Whole genomes of L. monocytogenes isolated from three different sources were analyzed. • Differences in two L. monocytogenes groups were identified in functionality, virulence, and antibiotic resistance genes. • This study first examines the association between resistances and whole genomes of stress-resistant and -sensitive L. monocytogenes.

Growth models for Salmonella, E. coli O157:H7 and L. monocytogenes give different predictions for pathogen growth in cut leafy greens transportation, but are consistent in identifying higher risk conditions.

Leafy greens are frequently implicated in foodborne disease outbreaks and cut-leafy greens are a food that requires time and temperature control for safety. Predictive microbiology uses mathematical models to predict the growth of bacteria based on environmental conditions. The objective of our study was to compare published square root growth models for Salmonella (n = 6), pathogenic E. coli (n = 6) and Listeria monocytogenes (n = 4) using real world transport temperature data. Data from trucks transporting fresh-cut leafy greens during cross-country shipments were used as temperature inputs to the models. Bacterial growth was computed using the temperatures from each probe in every truck over the duration of transit, which resulted in 12-18 growth predictions per truck for each model. Each model generally gave significantly different predictions than other models for the same organism. The exception was for the two Salmonella models predicting the least growth and the two Salmonella models predicting the most growth which gave predictions that were not significantly different. Although different models tended to give different predictions, their ability to rank risk by truck was generally consistent across models. While absolute risk might be dependent upon choice of model, relative risk is independent of model choice.

Cross-contamination of mature Listeria monocytogenes biofilms from stainless steel surfaces to chicken broth before and after the application of chlorinated alkaline and enzymatic detergents.

The objectives of this study were, firstly, to compare a conventional (i.e., chlorinated alkaline) versus an alternative (chlorinated alkaline plus enzymatic) treatment effectivity for the elimination of biofilms from different L. monocytogenes strains (CECT 5672, CECT 935, S2-bac and EDG-e). Secondly, to evaluate the cross-contamination to chicken broth from non-treated and treated biofilms formed on stainless steel surfaces. Results showed that all L. monocytogenes strains were able to adhere and develop biofilms at approximately the same growth levels (≈5.82 log CFU/cm2). When non-treated biofilms were put into contact with the model food, obtained an average transference rate of potential global cross-contamination of 20.4%. Biofilms treated with the chlorinated alkaline detergent obtained transference rates similar to non-treated biofilms as a high number of residual cells (i.e., around 4 to 5 Log CFU/cm2) were present on the surface, except for EDG-e strain on which transference rate diminished to 0.45%, which was related to the protective matrix. Contrarily, the alternative treatment was shown to not produce cross-contamination to the chicken broth due to its high effectivity for biofilm control (<0.50% of transference) except for CECT 935 strain that had a different behavior. Therefore, changing to more intense cleaning treatments in the processing environments can reduce risk of cross-contamination.

[Epidemiological study and serotyping by multiple PCR of Listeria monocytogenes isolated from food matrices in Argentina]. / Estudio epidemiológico y serotipificación por PCR múltiple de Listeria monocytogenes aislada de matrices alimentarias en Argentina.

Listeria monocytogenes is an opportunistic foodborne pathogen. It can resist stress conditions by adapting through the production of biofilms, which represents a serious problem for the food industry. It is classified into 14 serotypes, although only four (1/2a, 1/2b, 1/2c, and 4b) account for 89.0-98.0% of listeriosis cases worldwide. The objective of this study was to detect and serotype L.monocytogenes isolated from different food matrices from processing plants in Argentina. In the period 2016-2021, 1832 samples (meat, ready-to-eat foods, ice cream, dairy foods, and frozen vegetables) were analyzed, of which 226 (12.34%) isolates compatible with L.monocytogenes were detected. At the same time, environmental and surface samplings were performed in processing plants for ready-to-eat foods, sausages and dairy products, where environmental contamination with L.monocytogenes was detected in numerous critical points of the process, yielding a positivity rate of 22.7%. The molecular analysis of serogroups was performed, where it was observed that serogroup IIb was the most frequent with 66.5% (n=107), and in descending order IIc with 22.3% (n=36), and IIa (n=9) and IVb (n=9) with 5.6%. The serogroup mostly isolated in environmental monitoring was IIb. This work highlights the importance of the detection and serotyping of L.monocytogenes for taking actionable measures and identifying outbreaks, and is the first study in Argentina to describe an extensive study in food matrices.

Role of horizontally transferred copper resistance genes in Staphylococcus aureus and Listeria monocytogenes.

Bacteria have evolved mechanisms which enable them to control intracellular concentrations of metals. In the case of transition metals, such as copper, iron and zinc, bacteria must ensure enough is available as a cofactor for enzymes whilst at the same time preventing the accumulation of excess concentrations, which can be toxic. Interestingly, metal homeostasis and resistance systems have been found to play important roles in virulence. This review will discuss the copper homeostasis and resistance systems in Staphylococcus aureus and Listeria monocytogenes and the implications that acquisition of additional copper resistance genes may have in these pathogens.

The efficiency of technologies used for epidemiological characterization of Listeria monocytogenes isolates: an update.

The characterization of pathogenic bacteria by providing information regarding the identification and source-tracking of the causes of outbreaks is vital for the epidemiological investigations of foodborne diseases. The knowledge of transmission of Listeria monocytogenes (L. monocytogenes) strains from the environment, directly or indirectly (through food processing facilities) to the final food products, due to the complexity of evaluating numerous, affecting parameters is quite limited. The food trade globalization also adds difficulties in tracking the association between the infection occurrence and causative pathogens, aiming to prevent their spread. The occurrence of listeriosis, a notifiable disease throughout the world, can either be sporadic or outbreak-related. Due to the importance of foodborne outbreaks from a public health aspect and its correspondence enormous economic losses, cross-linked surveillance studies regarding the contamination of foods by L. monocytogenes, besides identifying clusters and tracing the sources of infections on an international-scale to prevent and control L. monocytogenes outbreaks sounds very crucial. Contrary to the conventional typing methods, molecular-based techniques, such as whole-genome sequencing, owing to the capacity to discriminate L. monocytogenes strains down to single nucleotide differences, provide an accurate characterization of strains and tracking the causes of outbreaks. However, routinely using molecular-based methods depends on the required improvements in the affordability, proper timing, and preparing reliable, standardized bioinformatics facilities. This work was conducted to critically review the practical potential of diverse typing methods have been used for the characterization of L. monocytogenes and discuss how they might change the future of efforts for control of listeriosis.

Isolation and characterization of competitive exclusion microorganisms from animal wastes-based composts against Listeria monocytogenes.

AIM: To isolate the slow-growing or viable but non-culturable competitive exclusion (CE) microorganisms from composts and then verify the anti-Listeria monocytogenes activities of those CE isolates in compost. METHODS AND RESULTS: CE strains were isolated from composts using double- or triple-layer agar methods, purified, and then characterized. Both compost extracts and solid compost samples were spiked with a cocktail of 3 L. monocytogenes strains which were co-inoculated with or without CE strain cocktail and incubated at both 22 and 35°C for 168 h. Results indicated that the addition of resuscitation promoting factor (Rpf) promoted the growth of slow-growing species from composts. About 50% of the isolated CE strains (n = 40) were identified as Bacillus spp., 17 strains can inhibit more than 10 tested L. monocytogenes strains, and nine strains were motile and competitive biofilm formers. In compost extracts, the growth potentials of L. monocytogenes were reduced up to 2.2 logs when co-culturing with CE strains. In compost samples, the addition of CE strains reduced L. monocytogenes population by ca. 1.3 log CFU/g at 22°C after 24-168 h incubation. CONCLUSION: Our modified double/triple-layer agar procedure with Rpf as growth supplement coupled with spot-on-lawn testing can be a quick and efficient method for isolating CE candidates from composts. The efficacy of CE strains against L. monocytogenes in compost extracts and compost samples was affected by compost type, nutrient level, and incubation temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Compost is a rich source of CE microorganisms, and compost-adapted CE microorganisms have the potential as a biological agent to control L. monocytogenes in agricultural environments.

Oxidative lesions and post-treatment viability attenuation of listeria monocytogenes triggered by atmospheric non-thermal plasma.

AIMS: The aim of the current study was to investigate the effect of plasma-mediated oxidative stress on the post-treatment viability of Listeria monocytogenes at the physiological and molecular levels. METHODS AND RESULTS: 107  CFU/ml L. monocytogenes in 10 ml phosphate-buffered saline (PBS) was treated with atmospheric non-thermal plasma for 0, 30, 60, 90 and 120 s respectively. Optical diagnostics using optical emission spectroscopy (OES) confirmed that dielectric barrier discharge (DBD) plasma was a significant source of ample exogenous reactive oxygen and nitrogen species (RONS). The development of extracellular main long-lived species was associated with plasma exposure time, accompanied by a massive accumulation of intracellular ROS in L. monocytogenes (p < 0.01). With the exception of virulence genes (hly), most oxidation resistance genes (e.g. sigB, perR, lmo2344, lmo2770 and trxA) and DNA repair gene (recA) were upregulated significantly (p < 0.05). A visible fragmentation in genomic DNA and a decline in the secretion of extracellular proteins and haemolytic activity (p < 0.01) were noticed. The quantitate oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) confirmed the viability attenuation from the aspect of energy metabolism. Survival assay in a real food system (raw milk) further suggested not only the viability attenuation, but also the resuscitation potential and safety risk of mild plasma-treated cells during post-treatment storage. CONCLUSION: DBD plasma had the potential to inactivate and attenuate the virulence of L. monocytogenes, and it was recommended that plasma exposure time longer than 120 s was more suitable for attenuating viability and avoiding the recovery possibility of L. monocytogenes in raw milk within 7 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The current results presented a strategy to inactivate and attenuate the viability of L. monocytogenes, which could serve as a theoretical basis for better application of non-thermal plasma in food in an effort to effectively combat foodborne pathogens.