Listeria Monocytogenes Cells Research Articles

Effect of exposure to sub-inhibitory concentrations of biocides on the susceptibility to antibiotics of sessile cells of Listeria monocytogenes and Salmonellaenterica

The effect of sub-inhibitory concentrations of several disinfectants on the antibiotic resistance of sessile cells of Listeria monocytogenes and Salmonella enterica was investigated. Biofilms were formed in the presence of biocides at MIC/2. Five compounds were used: peracetic acid (PAA), benzalkonium chloride (BZK), sodium hypochlorite (SHY), polyvinyl pyrrolidone iodine (PVI) and bis(3-aminopropyl)amine (BAPA). Two strains of L. monocytogenes (LM30 and LM28) and S. enterica (S. Typhimurium -ST- and S. Enteritidis -SE-) were used. Mono-species biofilms (MSB) and mixed biofilms (MB, LM30 + ST and LM28 + SE) were tested. LM30 went from susceptible to resistant to tetracycline and erythromycin after exposure to BAPA in MB. LM28 went from susceptible to resistant to tetracycline in the presence of PVI in MSB, from susceptible to intermediate to chloramphenicol after exposure to PAA in both MSB and MB, from susceptible to resistant to erythromycin after exposure to BAPA in MB, and from susceptible to resistant to ampicillin after exposure to SHY and BAPA in MB. ST went from intermediate to resistant to gentamicin after exposure to SHY, PVI and BAPA (MSB), or to PVI and BAPA (MB). This strain went from susceptible to intermediate to cefoxitin after exposure to BZK and PVI (MSB) or to BZK, PVI and BAPA (MB). For fosfomycin, ST went from susceptible to intermediate after exposure to PAA, PVI and BAPA (MSB) or to SHY, BZK, PVI and BAPA (MB). In the case of cephalothin, ST moved from intermediate to resistant category in MSB exposed to BAPA. SE moved from susceptible to intermediate to cefoxitin after contact with PAA in both MSB and MB. By contrast, in several occasions, strains increased their susceptibility to antibiotics after exposure to biocides. In some additional cases, contact with disinfectants caused significative modifications in the MIC of various antibiotics without the strains changing from one category to another. These findings show that contact with low concentrations of biocides can modify the antibiotic resistance of sessile cells of L. monocytogenes and S. enterica. The need to use adequate doses of disinfectants in food facilities in order to avoid changes in bacterial resistance is highlighted.

Reaction kinetics and mechanism of UVC-LED265–285nm and UVC-LED265–285nm/hydrogen peroxide toward foodborne pathogenic Listeria monocytogenes cells and its chromosomally encoded virulent hly genes

To investigate the principle-based inactivation kinetics and mechanisms of the foodborne pathogen Listeria monocytogenes (Lm), along with its representative virulence gene (hly) encoded in both intracellular and extracellular genomic DNA, we applied disinfection treatments using UVC-LED265–285nm and UVC-LED265–285nm combined with hydrogen peroxide (10 mg/L). These treatments were conducted under controlled model reaction matrices (phosphate-buffered solutions at pH 6.2 and 7.9) and/or actual agricultural water, depending on filtration pre-treatment. Comprehensive analytical methods, such as plate count, flow cytometry, and quantitative real-time PCR, were adopted for the qualitative/quantitative determination of bacterial or gene inactivation parameters. According to the experimentally determined fluence-based first-order rate constants (kUVC-LED and kUVC-LED/H2O2), the k value of Lm viability and hly genes was 1.14–2.19 cm2/mJ and 2.18 × 10−2–2.42 × 10−1 cm2/mJ, respectively. Damage to the longer amplicon or the extracellular form of the hly gene was higher than that to the shorter amplicon or the intracellular form by a factor of 2∼10 fold. Increasing pH in reaction matrices were marginally affected to the rates of inactivation kinetics. Dose-dependent inactivation levels tested in agriculture water matrices were comparable with the kinetics from the PB solutions of both UVC-LED265–285nm and UVC-LED265–285nm/hydrogen peroxide owing to OH radical scavenging by the water matrix components. These results will be valuable for optimizing UVC-LED and UVC-LED-based advanced oxidation process disinfection systems, specifically tailored to the unique challenges of bacterial or gene inactivation in the (waste)water at food processing and agricultural sectors.

Disinfectant efficacy of glabridin against dried and biofilm cells of Listeria monocytogenes and the impact of residual organic matter

Glabridin is an antimicrobial compound which can be extracted from plants, such as liquorice (Glycyrrhiza glabra) roots. Although its activity against foodborne pathogens and spoilage microorganisms has already been reported, the investigation of potential applications as a surface disinfectant is still largely unexplored. Hence, this study evaluated the disinfectant efficacy of glabridin against Listeria monocytogenes. The activity of glabridin was first tested in vitro in a nutrient-rich medium against eight strains of L. monocytogenes, including food isolates and the model strain EGDe. The tested strains showed similar susceptibility with minimal inhibitory and bactericidal concentrations of 12.5 µg/mL and 25 µg/mL, respectively. Subsequently, L. monocytogenes L6, FBR17 and EGDe were selected to assess the efficacy of glabridin against dried cells (according to the European standard EN 13697:2015 + A1:2019) and biofilm cells on stainless steel surfaces. Moreover, the impact of food residual organic matter was investigated using skim milk, cantaloupe and smoked salmon solution as soiling components. Our results showed that applying 200 µg/mL of glabridin resulted in a substantial reduction (>3 log10) of dried and biofilm cells of L. monocytogenes in standard conditions (i.e. low level of residual organic matter). Cantaloupe soiling components slightly reduced the activity of glabridin, while the efficacy of glabridin when tested with salmon and skim milk residuals was substantially affected. Comparative analysis using standardized protein contents provided evidence that the type of food matrices and type of proteins may impact the activity of glabridin as a disinfectant. Overall, this study showed low strain variability for the activity of glabridin against L. monocytogenes and shed light on the possible application of this natural antimicrobial compound as a surface disinfectant.

Flow cytometry: Unravelling the real antimicrobial and antibiofilm efficacy of natural bioactive compounds

Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring system for preventing the spread risk of foodborne disease. The present work used a combined culture-based and FCM approach to assess the in vitro efficacy of essential oils (EOs) from condiment plants commonly used in Mediterranean Europe (i.e., thyme EO, oregano EO, basil EO, and lemon EO) against planktonic and sessile cells of food-pathogenic Listeria monocytogenes 56 LY, and contaminant and alterative species Escherichia coli ATCC 25922 and Pseudomonas fluorescens ATCC 13525. Evaluation of the bacterial response to the increasing concentrations of natural compounds posed FCM as a crucial technique for the quantification of the live/dead, and viable but non-culturable (VBNC) cells when antimicrobial agents exert no real bactericidal action. Furthermore, the FCM results displayed higher numbers of viable bacteria expressed as Active Fluorescent Units (AFUs) with a greater level of repeatability compared with outcomes of the plate-count method. Overall, accurate counting of viable microbial cells is a critically important parameter in food microbiology, and flow cytometry provides an innovative approach with high-throughput potential for applications in the food industry as “flow microbiology”.

Drastic Microbial Count Reduction in Soy Milk Using Continuous Short-Wave Ultraviolet Treatments in a Tubular Annular Thin Film UV-C Reactor.

Vegetative cells of Listeria monocytogenes and Escherichia coli and spores of Bacillus subtilis and Aspergillus niger were inoculated in soy milk at an initial concentration of ≈5 log CFU/mL. Inoculated and control (non-inoculated) soy milk samples were submitted to three types of treatments using a tubular annular thin film short-wave ultraviolet (UV-C) reactor with 1 mm of layer thickness. Treatments applied depended on the flow rate and the number of entries to the reactor, with UV-C doses ranging from 20 to 160 J/mL. The number of entries into the reactor tube (NET) was established as the most determining parameter for the efficiency of the UV-C treatments. Conidiospores of A. niger were reported as the most resistant, followed by B. subtilis spores, while vegetative cells were the most sensible to UV-C, with Listeria monocytogenes being more sensible than Escherichia coli. Treatments of just 80 J/mL were needed to achieve a 5 log CFU/mL reduction of L. monocytogenes while 160 J/mL was necessary to achieve a similar reduction for A. niger spores.

Fate of osmotically adapted and biofilm Listeria monocytogenes cells after exposure to salt, heat, and liquid smoke, mimicking the stresses induced during the processing of hot smoked fish

The study aimed to investigate the response of osmotically adapted and detached biofilm Listeria monocytogenes cells following sequential stresses that occur during the processing of hot smoking, such as heating and smoke application. Thermal resistance of L. monocytogenes was significantly affected by previous osmotic adaptation of the cells. D60oC-values of osmotically adapted L. monocytogenes cells were significantly higher than control cells. The osmotically adapted and subsequently heat-injured cells were more resistant to PALCAM and less resistant to TSAYE with 5.00% NaCl (TSAYE/NaCl) than control cells. Detached biofilm cells were more thermotolerant and less resistant to PALCAM and TSAYE/NaCl than control cells. The sequential effect of smoking against heat-treated (60 °C, 20 min) and osmotically adapted or detached L. monocytogenes biofilm cells was investigated using two liquid smoke extracts (L9 and G6). L9 led to significantly higher reductions (>3.00-Log CFU) compared to G6. The heat-treated, detached biofilm cells revealed resistance to L9, presumably due to metabolic downregulation and physical protection by the extracellular polymeric substances (EPS). These data highlight the potential of the food industry to make informed decisions for using safe heat treatments during hot smoking to effectively inactivate L. monocytogenes and maintain rigorous environmental sanitation practices to control biofilm cells.

Effect of wet steam on the survival of Salmonella and Listeria monocytogenes cells attached to Hass whole avocado

The effect of wet steam treatment on the attached cells of Salmonella and Listeria monocytogenes on Hass avocados surface, according to the nozzle position of the steam generator and application time, was evaluated. Furthermore, the transfer of pathogenic bacteria from the wet steam-treated avocado epicarp to the pulp was investigated. Avocado epicarp was inoculated with Salmonella or L. monocytogenes and stored at 25 °C for 72 h. Later, avocados were washed with water and steamed from the top, upper front, and bottom front parts for 30, 45, and 60s. Both pathogens' loosely (LA) and strongly (SA) attached cells on the avocado surface were enumerated. Pulp samples treated avocados with steam from the top for 60 s were extracted to enumerate both pathogens. Salmonella was more susceptible than L. monocytogenes at steam treatments, whose efficacy varied according to the nozzle position and exposure time. For both pathogens, the maximum effectiveness of steam was obtained from the top for 60 s. Pathogens count in the pulp of avocados steamed from the top for 60 s was lower than in the pulp obtained from untreated avocados. These results indicate that steaming Hass avocados enhances the safety of rind and pulp.

Estimation by flow cytometry of percentages of survival of Listeria monocytogenes cells treated with tetracycline, with or without prior exposure to several biocides.

In certain circumstances, disinfectants are used at sublethal concentrations. The aim of this research work was to determine whether contact of Listeria monocytogenes NCTC 11994 with subinhibitory concentrations of three disinfectants widely used in food processing environments and in the health-care system, benzalkonium chloride (BZK), sodium hypochlorite (SHY) and peracetic acid (PAA), can cause the adaptation of the strain to the biocides and increase its resistance to tetracycline (TE). The minimum inhibitory concentrations (MIC; ppm) were 2.0 (BZK), 3500.0 (SHY) and 1050.0 (PAA). On exposure to increasing subinhibitory concentrations of the biocides, the maximum concentrations (ppm) of the compounds that allowed the strain to grow were (ppm) 8.5 (BZK), 3935.5 (SHY) and 1125.0 (PAA). Both the control cells (non-exposed) and the cells that had been in contact with low doses of biocides were treated with different concentrations of TE (0 ppm, 250 ppm, 500 ppm, 750 ppm, 1000 ppm and 1250 ppm) for 24, 48 and 72 h, and the survival percentages determined using flow cytometry, following dying with SYTO 9 and propidium iodide. The cells previously exposed to PAA presented higher survival percentages (P < 0.05) than the rest of the cells for most of the concentrations of TE and treatment times trialled. These results are worrying because TE is sometimes used to treat listeriosis, highlighting the importance of avoiding the use of disinfectant at subinhibitory doses. Furthermore, the findings suggest that flow cytometry is a fast and simple technique to obtain quantitative data on bacterial resistance to antibiotics.

Fate of Listeria monocytogenes, Salmonella spp., and Shiga Toxin-Producing Escherichia coli on Slices of an All-Beef Soppressata during Storage.

Cells of Listeria monocytogenes, Salmonella spp., or Shiga toxin-producing Escherichia coli (STEC) were inoculated (ca. 4.0 log CFU/slice) onto slices (ca. 4 g each slice) of an all-beef soppressata (ca. pH 5.05 and aw 0.85). The storage of vacuum-sealed slices of inoculated soppressata at 4 °C or 20 °C for 90 days resulted in reductions of all three pathogens by ca. 2.2 to 3.1 or ca. ≥3.3 log CFU/slice, respectively. When pathogen levels decreased to below detection (≤1.18 log CFU/slice) by direct plating, it was possible to recover each of the target pathogens by enrichment, albeit more frequently from slices stored at 4 °C (p < 0.05) compared to 20 °C. In summary, the slices of the commercially produced beef soppressata selected for this study did not provide a favorable environment for either survival or outgrowth of surface-inoculated cells of L. monocytogenes, Salmonella spp., or STEC during storage.

Oil-Based Sanitization in Low-Moisture Environments: Delivery of Acetic Acid with Water-in-Oil Emulsions.

Contamination with Salmonella spp. and Listeria monocytogenes is concerning across low-moisture food (LMF)-processing environments due to the pronounced survival of these organisms under dry conditions. This study treated desiccated bacteria with acetic acid delivered by oil with and without water-in-oil (W/O) emulsion. The influences of cellular desiccation, emulsion water concentration, water activity (aw), and treatment temperature were investigated. Acetic acid dissolved in oil (i.e., acidified oil) showed low levels of antimicrobial efficacy. After treatment with acidified oil (200 mM acetic acid at 22°C for 30 min), Salmonella enterica serovar Enteritidis phage type 30 cells desiccated to 75% equilibrium relative humidity (ERH) and 33% ERH were reduced by 0.69 and 0.05 log CFU/coupon, respectively. The dispersion of a low level of water (≥0.3%, vol/vol) within the acidified oil with the surfactant (i.e., acidified W/O emulsion) significantly enhanced the antimicrobial efficacy. After treatment with the acidified W/O emulsion (200 mM acetic acid at 22°C for 20 min), desiccated Salmonella (4-strain cocktail) and L. monocytogenes (3-strain cocktail) cells were reduced by >6.52 log most probable number (MPN)/coupon, regardless of the desiccation levels. Increased efficacy was observed with temperature elevation. Reduced efficacy was observed when glycerol was added to the aqueous phase of the emulsion to decrease the solution aw, indicating that the enhanced efficacy of the acidified W/O emulsion was associated with differential osmotic pressure. The antimicrobial mechanism may be due to the membrane disruption induced by acetic acid, in combination with the hypoosmotic stress provided by W/O emulsion, creating cellular lysis, as illustrated by electron micrographs. IMPORTANCE Aqueous-based cleaning and sanitation are undesirable in processing facilities that manufacture low-moisture foods such as peanut butter and chocolate. Alcohol-based sanitization is advantageous because it leaves no residue on the contact surface but requires the processing facility to close temporarily due to flammability. At >6.52 log kill of desiccated Salmonella and Listeria monocytogenes cells, the developed oil-based formulation has the potential to be an effective dry sanitation method.

Formation of Listeria monocytogenes persister cells in the produce-processing environment

Persisters are a subpopulation of growth-arrested cells that possess transient tolerance to lethal doses of antibiotics and can revert to an active state under the right conditions. Persister cells are considered as a public health concern. This study examined the formation of persisters by Listeria monocytogenes (LM) in an environment simulating a processing plant for leafy green production. Three LM strains isolated from California produce-processing plants and packinghouses with the strongest adherence abilities were used for this study. The impact of the cells' physiological status, density, and nutrient availability on the formation of persisters was also determined. Gentamicin at a dose of 100 mg/L was used for the isolation and screening of LM persisters. Results showed that the physiological status differences brought by culture preparation methods (plate-grown vs. broth-grown) did not impact persister formation (P > 0.05). Instead, higher persister ratios were found when cell density increased (P < 0.05). The formation of LM persister cells under simulated packinghouse conditions was tested by artificially inoculating stainless steel coupons with LM suspending in media with decreasing nutrient levels: brain heart infusion broth (1366 mg/L O2), produce-washing water with various organic loads (1332 mg/L O2 and 652 mg/L O2, respectively), as well as sterile Milli-Q water. LM survived in all suspensions at 4 °C with 85 % relative humidity for 7 days, with strain 483 producing the most persister cells (4.36 ± 0.294 Log CFU/coupon) on average. Although persister cell levels of LM 480 and 485 were reasonably steady in nutrient-rich media (i.e., BHI and HCOD), they declined in nutrient-poor media (i.e., LCOD and sterile Milli-Q water) over time. Persister populations decreased along with total viable cells, demonstrating the impact of available nutrients on the formation of persisters. The chlorine sensitivity of LM persister cells was evaluated and compared with regular LM cells. Results showed that, despite their increased tolerance to the antibiotic gentamicin, LM persisters were more susceptible to chlorine treatments (100 mg/L for 2 min) than regular cells.

Viability of Listeria monocytogenes and Salmonella spp. on Slices of a German-Style Bologna Containing Blends of Organic Acid Salts During Storage at 4 or 12°C

Viability of cells of Listeria monocytogenes or Salmonella spp. was quantified on slices of a German-style bologna manufactured by a local butcher to contain no added antimicrobials or to include 0.9% or 1.3% of a blend of potassium acetate and sodium diacetate (K-Ace) or 2.5% of a blend of potassium lactate and sodium diacetate (K-Lac) as ingredients. After slicing (ca. 7.1 cm L by 6.7 cm W, ca. 0.5 cm thick, ca. 22.4 g each), a single slice of bologna was placed into a nylon–polyethylene bag and surface inoculated with 250 µL per side of a five-strain mixture of either cells of L. monocytogenes or Salmonella spp. to achieve an initial level of ca. 3.5–4.0 log CFU/slice. The packages were vacuum-sealed and then stored at 4 or 12°C for 90 and 30 days, respectively. Without antimicrobials added to the formulation, L. monocytogenes numbers increased by ca. 5.4 and 6.0 log CFU/slice at both 4 and 12°C during the entire 90- and 30-day storage period, respectively. Likewise, levels of Salmonella also increased by ca. 6.0 log CFU/slice at 12°C in the absence of added antimicrobials; however, levels of this pathogen decreased by ca. 1.7 log CFU/slice after 90 days at 4°C. With the inclusion of 0.9% or 1.3% K-Ace or 2.5% K-Lac in the bologna formulation, levels of L. monocytogenes decreased by ca. ≤0.7 log CFU/slice after 90 days at 4°C, whereas levels of Salmonella decreased by ca. 1.6–2.3 log CFU/slice. After 30 days at 12°C, levels of L. monocytogenes increased by ca. ≤3.4 log CFU/slice on product containing 0.9% K-Ace or 2.5% K-Lac but remained relatively unchanged on slices formulated with 1.3% K-Ace. For Salmonella, in the presence of 0.9% or 1.3% K-Ace or 2.5% K-Lac, pathogen levels decreased by ca. ≤0.7 log CFU/slice at 12°C after 30 days. Our data validate that the inclusion of K-Ace (0.9% or 1.3%) or K-Lac (2.5%) as ingredients is effective for controlling L. monocytogenes and Salmonella on slices of bologna during refrigerated storage.

Pathogen-Imprinted Polymer Film Integrated probe/Ti3C2Tx MXenes Electrochemical Sensor for Highly Sensitive Determination of Listeria Monocytogenes

As one of the most hazardous and deadliest pathogens, Listeria monocytogenes (LM) posed various serious diseases to the human being, thus designing effective strategy for its detection is of great significance. In this work, by preparing Ti3C2Tx MXenes nanoribbon (Ti3C2TxR) as carrier and selecting thionine (Th) acted simultaneously as signal probe and functional monomer, a LM pathogen-imprinted polymers (PIP) integrated probe electrochemical sensor was design to monitor LM for the first time, that was carried out through the electropolymerization of Th on the Ti3C2TxR/GCE surface in the existence of LM. Upon eluting the templates from the LM imprinted cavities, the fabricated PIP/Ti3C2TxR/GCE sensor can rebound LM cells effectively. By recording the peak current of Th as the response signal, it can be weakened when LM cell was re-bound to the LM imprinted cavity on PIP/Ti3C2TxR/GCE, and the absolute values of peak current change increase with the increasement of LM concentrations. After optimizing three key parameters, a considerable low analytical limit (2 CFU mL−1) and wide linearity (10–108 CFU mL−1) for LM were achieved. In addition, the experiments demonstrated that the PIP/Ti3C2TxR sensor offers satisfactory selectivity, reproducibility and stability.

Repeated sub-inhibitory doses of cassia essential oil do not increase the tolerance pattern in Listeria monocytogenes cells

Antimicrobial resistance in Listeria monocytogenes biofilms is considered a risk. When using disinfectants, the minimum inhibitory concentration (MIC) must be contemplated to avoid resistance. The objective of the present study was to determine the MIC of four essential oils in four L. monocytogenes strains. Once the MICs were obtained, the effect of subinhibitory doses of the most effective oil was determined. Strains were subsequently subjected to increasing doses of cassia and the MICs were determined again to evaluate differences. The effect of subinhibitory doses for biofilm formation was evaluated using quantitative and observational methodologies. Last, it was studied whether the strains were more sensitive to antibiotics after being in contact with the oil. After continuous exposure to 1/2 MIC, a decrease in the initial MIC (P = 0.013) was observed, specifically for strains belonging to serotype 1/2a (P = 0.041). In contrast, the formation of biofilms did not show differences between the control and exposed groups (P > 0.05). The qualitative study showed that there were no differences in the structure of the biofilms before and after contact with cassia, except for the CECT 935 strain, indicating a strain-dependent trend. Moreover, species-dependent differences were observed in the conformation of the extracellular matrix.

The applicability of predictive microbiology tools for analysing Listeria monocytogenes contamination in butter produced by the traditional batch churning method

The study was carried out on the assumption that raw materials for butter production by the traditional batch churning method can be contaminated with Listeria monocytogenes cells. Raw milk and cream used in butter making were experimentally contaminated with the pathogen, and two contamination scenarios were considered. In the first variant, it was assumed that cream for butter making is contaminated with L. monocytogenes. The second scenario relied on the assumption that butter is contaminated during packaging. The experimental material was divided into samples and stored at a temperature of 3, 6, 9, 12 or 21 °C. The study demonstrated that L. monocytogenes cells proliferated twice as fast in butter produced from contaminated cream as in butter that was contaminated during packaging. Results of microbiological tests conducted on raw materials, products, and rinses during batch butter production can predict the direction and rate of L. monocytogenes cell growth/inactivation.

An antimicrobial peptide specifically active against Listeria monocytogenes is secreted by Bacillus pumilus SF214

BackgroundMembers of the Bacillus genus produce a large variety of antimicrobial peptides including linear or cyclic lipopeptides and thiopeptides, that often have a broad spectrum of action against Gram-positive and Gram-negative bacteria. We have recently reported that SF214, a marine isolated strain of Bacillus pumilus, produces two different antimicrobials specifically active against either Staphylococcus aureus or Listeria monocytogenes. The anti-Staphylococcus molecule has been previously characterized as a pumilacidin, a nonribosomally synthesized lipopetide composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length.ResultsOur analysis on the anti-Listeria molecule of B. pumilus SF214 indicated that it is a peptide slightly smaller than 10 kDa, produced during the exponential phase of growth, stable at a wide range of pH conditions and resistant to various chemical treatments. The peptide showed a lytic activity against growing but not resting cells of Listeria monocytogenes and appeared extremely specific being inactive also against L. innocua, a close relative of L. monocytogenes.ConclusionsThese findings indicate that the B. pumilus peptide is unusual with respect to other antimicrobials both for its time of synthesis and secretion and for its strict specificity against L. monocytogenes. Such specificity, together with its stability, propose this new antimicrobial as a tool for potential biotechnological applications in the fight against the dangerous food-borne pathogen L. monocytogenes.

Combining loop-mediated isothermal amplification and nanozyme-strip for ultrasensitive and rapid detection of viable Listeria monocytogenes cells and biofilms

Foodborne diseases caused by Listeria monocytogenes (L. monocytogenes) occur frequently. The rapid, ultrasensitive, and visual methods are urgently needed for the detection of viable L. monocytogenes. We developed a novel lateral flow strip for visual and quantitative detection of viable L. monocytogenes cells and biofilms based on propidium monoazide-loop-mediated isothermal amplification (PMA-LAMP) and nanozyme. PMA-LAMP was used to identify viable L. monocytogenes, targeting hlyA gene by FITC- and BIO-modified primers in LAMP process. The optimized Fe3O4 nanoparticles with small size and good dispersibility combined with LAMP products of L. monocytogenes, which were captured by anti-FITC antibody modified on nanozyme strip under a suitable condition. This strip had features of stability, specificity, and visualization, which can also quantify L. monocytogenes with the detection limit of 10 CFU mL−1. Furthermore, the practicality of nanozyme strip had been proved in detecting biofilm formed by L. monocytogenes on stainless steel and lettuce surfaces.

Morphological Alterations and Virulence Gene Expression of Listeria Monocytogenes Cells in Response to Gamma Irradiation Treatments

Gamma irradiation is one of the most popular ray treatments in food industry. It's used to control micro-organisms proliferation in a wide range of products however the response of bacteria to low doses is still unknown. In this study we mainly focus on morphological alteration and virulence gene expression in Listeria monocytogenes after gamma irradiation treatments. The atomic force micrographs (AFM) showed that 0.5 kGy dose has no effect on the membrane morphology of L. monocytogenes. However, after 0.7 kGy treatment, the cells lost their typical shape and smooth membrane and 1 kGy dose was totally destructive. Moreover, membrane fatty acid composition was analyzed by the chromatographic method after different gamma doses. Significant modifications on fatty acids composition were detected in the irradiated strain showing a novo synthesis of membrane lipids: C12:0; C14:0; C15:0; C16:0 and C18:0. In addition, we reported an increase of the saturated fatty acid, essential for membrane adaptation under stress conditions. The expression levels of three virulence genes (hlyA, fri and prfA) were studied in the same conditions using real-time PCR technique. The analysis revealed that both prfA and fri genes were up-regulated after gamma treatment. The induction of prfA, which is a regulator gene, may affect the expression other genes controlling the adaptive form in the treated strain. This study open prospects for further researches to explain the regulatory mechanisms of the adaptive response in Listeria monocytogenes when exposed to sublethal irradiation-stress.

Cold atmospheric pressure plasma inactivation of dairy associated planktonic cells of Listeria monocytogenes and Staphylococcus aureus

The effect of cold atmospheric pressure plasma (CAPP) on three strains of Staphylococcus aureus and one strain of Listeria monocytogenes on gel discs was studied. A plasma jet operating in argon was used and exposure times were from 5 to 120 s. Bacteria had been isolated from dairy environments. In comparison, peracetic acid (PAA, 0.5% v/v) applied on cell suspensions at the same treatment times was studied. The cell viability was measured by flow cytometry and the culturable cells were determined by plate count technique. PAA reduced nearly 7 log cycles of both microorganisms after a short time (10 s). Concurrently, membrane permeabilization, RNA and DNA damage, and lack of esterase activity could be demonstrated. CAPP showed a mild biocidal effect with a maximum reduction of 1–2 log cycle after 120 s, and no significant impact on membrane permeabilization, RNA and DNA damage, or esterase activity. Therefore, for the selected processing parameters, PAA remains as more effective treatment in terms of inactivating pathogenic bacteria in dairy environments, reaching ~7 log cycles reductions. Further studies should determine the effect of higher CAPP exposure times on S. aureus and L. monocytogenes strains.

Properties of the Extracellular Polymeric Substance Layer from Minimally Grown Planktonic Cells of Listeria monocytogenes.

The bacterium Listeria monocytogenes is a serious concern to food processing facilities because of its persistence. When liquid cultures of L. monocytogenes were prepared in defined media, it was noted that planktonic cells rapidly dropped out of suspension. Zeta potential and hydrophobicity assays found that the cells were more negatively charged (−22, −18, −10 mV in defined media D10, MCDB 202 and brain heart infusion [BHI] media, respectively) and were also more hydrophobic. A SEM analysis detected a capsular-like structure on the surface of cells grown in D10 media. A crude extract of the extracellular polymeric substance (EPS) was found to contain cell-associated proteins. The proteins were removed with pronase treatment. The remaining non-proteinaceous component was not stained by Coomassie blue dye and a further chemical analysis of the EPS did not detect significant amounts of sugars, DNA, polyglutamic acid or any other specific amino acid. When the purified EPS was subjected to attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, the spectra obtained did not match the profile of any of the 12 reference compounds used. An x-ray diffraction (XRD) analysis showed that the EPS was amorphous and a nuclear magnetic resonance (NMR) analysis detected the presence of glycerol. An elemental energy dispersive x-ray (EDX) analysis showed traces of phosphorous as a major component. In conclusion, it is proposed that the non-proteinaceous component may be phospholipid in nature, possibly derived from the cell wall lipoteichoic acid.