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.

Assessment of Viability of Listeria monocytogenes by Flow Cytometry.

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

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.

Comparing Gene Expression Between Planktonic and Biofilm Cells of Foodborne Bacterial Pathogens Through RT-qPCR.

Like most microorganisms, important foodborne pathogenic bacteria, such as Salmonella enterica, Listeria monocytogenes, and several others as well, can attach to surfaces, of either abiotic or biotic nature, and create biofilms on them, provided the existence of supportive environmental conditions (e.g., permissive growth temperature, adequate humidity, and nutrient presence). Inside those sessile communities, the enclosed bacteria typically present a gene expression profile that differs from the one that would be displayed by the same cells growing planktonically in liquid media (free-swimming cells). This altered gene expression has important consequences on cellular physiology and behavior, including stress tolerance and induction of virulence. In this chapter, the methodology to use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to monitor and comparatively quantify expression changes in preselected genes of bacteria between planktonic and biofilm growth modes is presented.

Identifying Putative Biomarkers of Foodborne Pathogens Using a Metabolomic Approach.

Metabolomics is the study of low molecular weight biochemical molecules (typically <1500 Da) in a defined biological organism or system. In case of food systems, the term "food metabolomics" is often used. Food metabolomics has been widely explored and applied in various fields including food analysis, food intake, food traceability, and food safety. Food safety applications focusing on the identification of pathogen-specific biomarkers have been promising. This chapter describes a nontargeted metabolite profiling workflow using gas chromatography coupled with mass spectrometry (GC-MS) for characterizing three globally important foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica, from selective enrichment liquid culture media. The workflow involves a detailed description of food spiking experiments followed by procedures for the extraction of polar metabolites from media, the analysis of the extracts using GC-MS, and finally chemometric data analysis using univariate and multivariate statistical tools to identify potential pathogen-specific biomarkers.

Long-term persistence of diverse clones shapes the transmission landscape of invasive Listeria monocytogenes.

BACKGROUND: The foodborne bacterium Listeria monocytogenes (Lm) causes a range of diseases, from mild gastroenteritis to invasive infections that have high fatality rate in vulnerable individuals. Understanding the population genomic structure of invasive Lm is critical to informing public health interventions and infection control policies that will be most effective especially in local and regional communities. METHODS: We sequenced the whole draft genomes of 936 Lm isolates from human clinical samples obtained in a two-decade active surveillance program across 58 counties in New York State, USA. Samples came mostly from blood and cerebrospinal fluid. We characterized the phylogenetic relationships, population structure, antimicrobial resistance genes, virulence genes, and mobile genetic elements. RESULTS: The population is genetically heterogenous, consisting of lineages I-IV, 89 clonal complexes, 200 sequence types, and six known serogroups. In addition to intrinsic antimicrobial resistance genes (fosX, lin, norB, and sul), other resistance genes tetM, tetS, ermG, msrD, and mefA were sparsely distributed in the population. Within each lineage, we identified clusters of isolates with ≤ 20 single nucleotide polymorphisms in the core genome alignment. These clusters may represent isolates that share a most recent common ancestor, e.g., they are derived from the same contamination source or demonstrate evidence of transmission or outbreak. We identified 38 epidemiologically linked clusters of isolates, confirming eight previously reported disease outbreaks and the discovery of cryptic outbreaks and undetected chains of transmission, even in the rarely reported Lm lineage III (ST3171). The presence of animal-associated lineages III and IV may suggest a possible spillover of animal-restricted strains to humans. Many transmissible clones persisted over several years and traversed distant sites across the state. CONCLUSIONS: Our findings revealed the bacterial determinants of invasive listeriosis, driven mainly by the diversity of locally circulating lineages, intrinsic and mobile antimicrobial resistance and virulence genes, and persistence across geographical and temporal scales. Our findings will inform public health efforts to reduce the burden of invasive listeriosis, including the design of food safety measures, source traceback, and outbreak detection.

A short-lived peptide signal regulates cell-to-cell communication in Listeria monocytogenes.

Quorum sensing (QS) is a mechanism that regulates group behavior in bacteria, and in Gram-positive bacteria, the communication molecules are often cyclic peptides, called autoinducing peptides (AIPs). We recently showed that pentameric thiolactone-containing AIPs from Listeria monocytogenes, and from other species, spontaneously undergo rapid rearrangement to homodetic cyclopeptides, which hampers our ability to study the activity of these short-lived compounds. Here, we developed chemically modified analogues that closely mimic the native AIPs while remaining structurally intact, by introducing N-methylation or thioester-to-thioether substitutions. The stabilized AIP analogues exhibit strong QS agonism in L. monocytogenes and allow structure-activity relationships to be studied. Our data provide evidence to suggest that the most potent AIP is in fact the very short-lived thiolactone-containing pentamer. Further, we find that the QS system in L. monocytogenes is more promiscuous with respect to the structural diversity allowed for agonistic AIPs than reported for the more extensively studied QS systems in Staphylococcus aureus and Staphylococcus epidermidis. The developed compounds will be important for uncovering the biology of L. monocytogenes, and the design principles should be broadly applicable to the study of AIPs in other species.

Interspecies Interaction of Bacteria Listeria monocytogenes, Yersinia pseudotuberculosis, and Marine Saprotrophs during Long-Term Culturing in Biofilms.

All bacterial strains studied retained the viability and ability to form both mono- and polycultural biofilms under conditions of long-term culturing in artificial seawater at 6°C and without addition of nutrients. Bacillus sp. and Pseudomonas japonica presumably stimulated the growth and reproduction of the pathogenic bacteria Listeria monocytogenes and Yersinia pseudotuberculosis. Preserved cell viability in a monoculture biofilm for a long period without adding a food source can indicate allolysis. At the same time, in a polycultural biofilm, the metabolites secreted by saprotrophic strains can stimulate the growth of L. monocytogenes and Y. pseudotuberculosis.

Wild Vitis Species as Stilbenes Sources: Cane Extracts and Their Antibacterial Activity against Listeria monocytogenes.

Grapevines (Vitis spp.) produce several valuable polyphenol-type secondary metabolites including various stilbenoids. Although the potential application of stilbenes may offer alternative solutions to food safety or health challenges, only little information is available on their antibacterial activity against foodborne pathogens. In this work, high-performance liquid chromatography was used to analyze the stilbenoid profile of various wild Vitis species, including V. amurensis, V. davidii, V. pentagona, and V. romanetii, selected from the gene bank for grapes at the University of Pécs, Hungary. We found that the stilbene profile of cane extracts is strongly genotype-dependent, showing the predominant presence of ε-viniferin with a wide concentration range ≈ 320-3870 µg/g dry weight. A novel yet simple and efficient extraction procedure was developed and applied for the first time on grape canes, resulting in ε-viniferin-rich crude extracts that were tested against Listeria monocytogenes, an important foodborne pathogen. After 24 h exposure, V. pentagona and V. amurensis crude extracts completely eliminated the bacteria at a minimum bactericidal concentration of 42.3 µg/mL and 39.2 µg/mL of ε-viniferin, respectively. On the other hand, V. romanetii extract with 7.8 µg/mL of ε-viniferin resulted in 4 log reduction in the viable bacterial cells, while V. davidii extract with 1.4 µg/mL of ε-viniferin did not show significant antibacterial activity. These findings indicate that the ε-viniferin content was directly responsible for the antibacterial effect of cane extract. However, pure ε-viniferin (purity > 95%) required a higher concentration (188 µg/mL) to eradicate the bacteria under the same conditions, suggesting the presence of other antibacterial compounds in the cane extracts. Investigating the onset time of the bactericidal action was conducted through a kinetic experiment, and results showed that the reduction in living bacterial number started after 2 h; however, the bactericidal action demanded 24 h of exposure. Our results revealed that the canes of V. pentagona and V. amurensis species are a crucial bio-source of an important stilbene with antimicrobial activity and health benefits.

[On two cases of systemic listeriosis without dominant neurological compromise]. / Sobre dos casos de listeriosis sistémica sin compromiso neurológico dominante.

Listeria monocytogenes is a Gram-positive aerobic bacterium; found ubiquitously in nature; which mainly affects newborns, older adults, immunosuppressed patients and pregnant women. However, Listeria disease can occur in the healthy population. Invasive listeriosis has three dominant clinical forms, bacteremia, neurolisteriosis and maternal-neonatal infection. Localized forms are infrequently described. The disease occurs mainly secondary to the consumption of contaminated food, including unpasteurized milk or cheese, and occurs in the form of isolated cases or outbreaks, usually beginning a few days after consumption of the contaminated food; although it has been described up to 2 months after ingesting them. There is also the possibility of direct transmission from animals and vertical transmission. Systemic listeriosis without dominant neurological symptoms is a rare event. Two cases are presented. The first was spondylodiscitis in a normal host and the second was Listeria bacteremia in a febrile immunocompromised patient.

Listeria monocytogenes es una bacteria aeróbica Gram positiva; encontrada enforma ubicua en la naturaleza; que afecta sobre todo a recién nacidos, adultos mayores, pacientes inmunodeprimidos y mujeres embarazadas. Sin embargo, la enfermedad por Listeria puede ocurrir en la población sana. La listeriosis invasiva posee 3 formas clínicas dominantes, bacteriemia, neurolisteriosis e infección materno-neonatal. Las formas localizadas se describen infrecuentemente. La enfermedad se produce fundamentalmente en forma secundaria al consumo de alimentos contaminados, incluidos leche o queso no pasteurizados, y sepresenta en forma de casos aislados o brotes, soliendo comenzar a los pocos días del consumo de éstos; aunque se ha descripto hasta 2 meses después de ingerirlos. También existela posibilidad de transmisión directa desde animales y transmisión vertical. La listeriosis sistémica sin cuadro neurológico dominante es un evento raro. Se presentan dos casos. El primero, una espondilodiscitis en huésped normal y el segundo una bacteriemia por Listeria en un paciente inmunocomprometido febril.

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.

Imprinted membrane-covalent organic framework platform for efficient label-free visual detection of Listeria monocytogenes and Salmonella typhimurium in food samples.

BACKGROUND: Rapid and sensitive detection of foodborne pathogens in food plays a crucial role in controlling outbreaks of foodborne diseases, of which Listeria monocytogenes and Salmonella typhimurium are representative and notable pathogens. Thus, it's of great importance to achieve the effective detection of these pathogens. However, the most common detection methods (culture-based technique, Polymerase Chain Reaction and immunological methods) have disadvantages that cannot be ignored, such as time-consuming, laborious, complex sample preparation process, and the possibility of cross-reaction. Hence, it is essential to develop a facile detection method for the pathogens with high sensitivity and specificity to avoid the above-mentioned disadvantages. RESULTS: We report a label-free visual platform for the simultaneous capture and detection of Listeria monocytogenes and Salmonella typhimurium. For the first time, we have prepared polydimethylsiloxane-Chromotrope 2R membrane which serves as the substrate for bacterial capture and enrichment through the formation of specific recognition sites. The positively charged Pt-covalent organic framework combines with the pathogens through surface charge interaction, thereby the label-free sandwich platform is formed. Remarkable peroxidase activity of Pt-covalent organic framework converts the conversion of bacterial quantity into amplified color signal by catalyzing 3,3',5,5'-Tetramethylbenzidine to oxidized 3,3',5,5'-Tetramethylbenzidine. The platform demonstrates the capability to identify two representative food-borne pathogens within a time frame of 100 min, exhibiting high sensitivity and excellent specificity without the interference from non-target bacteria. The limit of detection of the visual platform toward Listeria monocytogenes and Salmonella typhimurium was 1.61 CFU mL-1 and 1.31 CFU mL-1, respectively. And the limit of quantification toward Listeria monocytogenes and Salmonella typhimurium was 4.94 CFU mL-1 and 2.47 CFU mL-1, respectively. The relative standard derivations of the visual platform for both bacteria were lower than 4.9 %. Furthermore, our proposed platform has obtained reliable and satisfactory results on analyzing diverse food samples. SIGNIFICANCE: This research expands the application of a label-free platform combined with unlabeled nanocomponents in the rapid isolation and detection of diverse of food-borne pathogens. The platform possesses the advantages of simple operation and real-time monitoring, without complicated sample pretreatment process. The whole detection process can realize the simultaneous monitoring of Listeria monocytogenes and Salmonella typhimurium within 100 min. Furthermore, it is also of reference significance for the detection of other common pathogens.

Single-Atom Au-Functionalized Mesoporous SnO2 Nanospheres for Ultrasensitive Detection of Listeria monocytogenes Biomarker at Low Temperatures.

Semiconductor metal oxide gas sensors have been proven to be capable of detecting Listeria monocytogenes, one kind of foodborne bacteria, through monitoring the characteristic gaseous metabolic product 3-hydroxy-2-butanone. However, the detection still faces challenges because the sensors need to work at high temperatures and output limited gas sensing performance. The present study focuses on the design of single-atom Au-functionalized mesoporous SnO2 nanospheres for the sensitive detection of ppb-level 3-hydroxy-2-butanone at low temperatures (50 °C). The fabricated sensors exhibit high sensitivity (291.5 ppm-1), excellent selectivity, short response time (10 s), and ultralow detection limit (10 ppb). The gas sensors exhibit exceptional efficacy in distinguishing L. monocytogenes from other bacterial strains (e.g., Escherichia coli). Additionally, wireless detection of 3-hydroxy-2-butanone vapor is successfully achieved through microelectromechanical systems sensors, enabling real-time monitoring of the biomarker 3-hydroxy-2-butanone. The superior sensing performance is ascribed to the mesoporous framework with accessible active Au-O-Sn sites in the uniform sensing layer consisting of single-atom Au-modified mesoporous SnO2 nanospheres, and such a feature facilitates the gas diffusion, adsorption, and catalytic conversion of 3-hydroxy-2-butanone molecules in the sensing layer, resulting in excellent sensing signal output at relatively low temperature that is favorable for developing low-energy-consumption gas sensors.

Effect of Rheum ribes L. pulp enriched with eugenol or thymol on survival of foodborne pathogens and quality parameters of chicken breast fillets.

The aim of this study was to characterize the pulp of Rheum ribes L. and to determine the effect of the pulp enriched with eugenol (1 %) or thymol (1 %) on the microbiological and physico-chemical quality of chicken breast fillets. Chicken breast fillets, inoculated with Listeria monocytogenes, Salmonella enterica subsp. enterica serovar Typhimurium, and Escherichia coli O157:H7 (~6.0 log10), were marinated for 24 h in a mixture prepared from a combination of Rheum ribes L. pulp with eugenol or thymol. The quality parameters were analyzed for 15 days at +4 °C. The Rheum ribes L. pulp was found to have high antioxidant activity, high total phenolic content and contained 22 different phenolic substances, among which rutin ranked first. The pulp contained high levels of p-xylene and o-xylene as volatile substances and citric acid as an organic acid. The combination of Pulp + Eugenol + Thymol (PET) reduced the number of pathogens in chicken breast fillets by 2.03 to 3.50 log10 on day 0 and by 2.25 to 4.21 log10 on day 15, compared to the control group (P < 0.05). The marinating treatment significantly lowered the pH values of fillet samples on the first day of the study, compared to the control group (P < 0.05). During storage, TVB-N levels showed slower increase in the treatment groups compared to the control group (P < 0.05). In addition, the marinating process led to significant changes in physicochemical parameters such as water holding capacity, color, texture, cooking loss, and drip loss compared to the control group (P < 0.05). In conclusion, the results of this study showed that the pulp of Rheum ribes L., which has a high antioxidant capacity and contains various bioactive compounds. Furthermore, S. Typhimurium, E. coli O157:H7 and L. monocytogenes were inhibited considerably by marinating Rheum ribes L. pulp with a combination of eugenol and thymol.

The Probiotic Enterococcus Lactis SF68 as a Potential Food Fermentation Microorganism for Safe Food Production.

Due to the reports describing virulent and multidrug resistant enterococci, their use has become a topic of controversy despite most of them being safe and commonly used in traditionally fermented foods worldwide. We have characterized Enterococcus lactis SF68, a probiotic strain approved by the European Food Safety Authority (EFSA) for use in food and feed, and find that it has a remarkable potential in food fermentations. Genome analysis revealed the potential of SF68 to metabolize a multitude of carbohydrates, including lactose and sucrose, which was substantiated experimentally. Bacteriocin biosynthesis clusters were identified and SF68 was found to display a strong inhibitory effect against Listeria monocytogenes. Fermentation-wise, E. lactis SF68 was remarkably like Lactococcus lactis and displayed a clear mixed-acid shift on slowly fermented sugars. SF68 could produce the butter aroma compounds, acetoin and diacetyl, the production of which was enhanced under aerated conditions in a strain deficient in lactate dehydrogenase activity. Overall, E. lactis SF68 was found to be versatile, with a broad carbohydrate utilization capacity, a capacity for producing bacteriocins, and an ability to grow at elevated temperatures. This is key to eliminating pathogenic and spoilage microorganisms that are frequently associated with fermented foods.

Colorimetric aptasensor for Listeria monocytogenes detection using dual functional Fe3O4@MIL-100(Fe) with magnetic separation and oxidase-like activities in food samples.

A novel colorimetric aptasensor assay based on the excellent magnetic responsiveness and oxidase-like activity of Fe3O4@MIL-100(Fe) was developed. Fe3O4@MIL-100(Fe) absorbed with aptamer and blocked by BSA served as capture probe for selective isolation and enrichment of Listeria monocytogenes one of the most common and dangerous foodborne pathogenic bacteria. The aptamer absorbed on Fe3O4@MIL-100(Fe) was further used as signal probe that specifically binds with target bacteria conjugation of capture probe for colorimetric detection of Listeria monocytogenes, taking advantages of its oxidase-like activity. The linear range of the detection of Listeria monocytogenes was from 102 to 107 CFU mL-1, with the limit of detection as low as 14 CFU mL-1. The approach also showed good feasibility for detection of Listeria monocytogenes in milk and meat samples. The spiked recoveries were in the range 81-114% with relative standard deviations ranging from 1.28 to 5.19%. Thus, this work provides an efficient, convenient, and practical tool for selective isolation and colorimetric detection of Listeria monocytogenes in food.

A 48-year-old man with fever, nauseous, vomiting, and dizzy: A CARE case report.

RATIONALE: Listeria monocytogenes (LM) is an important foodborne bacterium, and LM meningoencephalitis is rare in clinical practice, with poor prognosis in severe patients. It is prone to misdiagnosis in clinical practice. We first reported a case of severe LM meningoencephalitis with muscle lesions and evaluated the comprehensive condition. PATIENT CONCERNS: A 48-year-old man had a fever and was admitted to the neurology department due to dizziness, nausea, and vomiting for 20 days. DIAGNOSES: LM meningoencephalitis complicated with muscle lesions. INTERVENTIONS: We used moxifloxacin 0.4 g, qd, meropenem 2 g, q8h, and dexamethasone 10 mg, qd to reduce exudation and adhesion. Then due to consideration of side effects, we increased the dose of ampicillin by 2 g, q4h, stopped using meropenem and moxifloxacin, and turned to maintenance treatment with dexamethasone and ampicillin. We comprehensively managed his vital signs and physical organ functions, we also controlled some comorbidities. During the hospitalization period thereafter, we used intravenous anti-infection treatment with moxifloxacin 0.4 g, qd, ampicillin 0.5 g, q4h. OUTCOMES: Half a year later, the reexamination showed only protein elevation in cerebrospinal fluid and hydrocephalus in MRI. Afterward, the symptoms did not recur again. The patient recovered well after discharge. LESSONS: LM meningoencephalitis complicated with lower limb muscle lesions is clinically rare. This report focuses on relevant treatment plans, which provide value for the examination and comprehensive management of patients with LM infection in the future.

Unrevealing the potentialities in food formulations of a low-branched dextran from Leuconostoc mesenteroides.

The search for novel exopolysaccharides (EPS) with targeted functionalities is currently a topic of great interest. This study aimed to investigate the chemical characteristics and technological properties of a novel EPS (named EPS_O) from Leuconostoc mesenteroides. EPS_O was a high-molecular-weight dextran (>6.68 × 105 g/mol) characterized by high water-holding capacity (785 ± 73%) and high water solubility index (about 99%). EPS_O in water (<30 mg/mL) formed viscous solutions, whereas at concentrations >30 mg/mL, it formed weak gels. Notably, lower concentrations (4-5 mg/mL) exhibited antimicrobial activity against various foodborne pathogens, antibiofilm activity against Listeria monocytogenes, and radical-scavenging activity. These properties are significant for maintaining food quality and promoting health. Based on these findings, EPS_O presents itself as a promising food ingredient that could elevate food quality and confer health benefits to consumers.

Formation and recovery of Listeria monocytogenes in viable but nonculturable state under different temperatures combined with low nutrition and high NaCl concentration.

The viable but nonculturable (VBNC) state occurs when bacteria lose their ability to grow and multiply on conventional media when stressed by adverse environmental factors, but they remain active and can revive under certain conditions, posing a food safety risk. In this study, the VBNC state of Listeria monocytogenes was induced with different temperatures combined with low nutrient conditions; the VBNC state of L. monocytogenes was confirmed in conjunction with the housekeeping gene abcZ using a molecular biology assay (PMA-qPCR) to calculate the viable bacterial count; The resuscitation conditions for the VBNC state of L. monocytogenes were investigated utilizing various nutrients in the culture medium and pasteurized milk. Four strains of L. monocytogenes reached the VBNC stage after 14, 21, 21, and 35 days at 20°C with 20% (or 30%) NaCl. Resuscitation studies indicate that Trypticase Soy Broth (TSB) combined with Tween 80 and sodium pyruvate is more effective for resuscitation. The Chinese national standard technology GB 4789.30-2016 was used to inoculate lettuce, chicken, and pasteurized milk with L. monocytogenes ATCC 19115 VBNC state. This research has significant implications for commercial food processing, long-term storage, disinfection, disease prevention, and control.

The combined effect of pH and NaCl on the susceptibility of Listeria monocytogenes to rhamnolipids.

The use of natural and sustainable additives, that are less aggressive to the environment, is a trend in the food industry. Rhamnolipids (RL) biosurfactants have shown potential for controlling food pathogens however, due to the presence of free carboxyl groups, the pH and ionic strength may influence the properties of such surfactants. In this study, we describe the antimicrobial activity of RL under different pH values and NaCl concentrations, towards both planktonic and biofilms of Listeria monocytogenes. RL were effective at pH 5.0 and the addition of 5 % NaCl improved the bactericidal efficacy for planktonic and sessile cells. The effect of NaCl was more pronounced at pH above 6 showing a significant increase in RL antimicrobial activity. At pH 7.0 planktonic population was eradicated by RL only when salt was present whereas biofilm viability was decreased by 5 log with MBIC varying from > 2500.0 mg/L (RL) to 39.0 mg/L (RL + 5 % NaCl). Larger vesicular and lamellar RL self-assembly structures were predominant when NaCl was present, suggesting their association with the antimicrobial activity observed. The pH and ionic strength of the medium are important parameters to be considered for the development of RL-based strategies to control L. monocytogenes.