Listeria Monocytogenes Biofilm Research Articles

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.

The combined application of ultrasound and UVC have the potential to control mature Listeria monocytogenes and Bacillus subtilis biofilms

The present study aims to determine the bactericidal and disintegrating effect of ultraviolet type C treatments combined with ultrasound against biofilms of Listeria monocytogenes and Bacillus subtilis formed at different incubation periods (i.e. 24, 48, and 72 h). Treatments applied are UVC doses ranging from 60 to 360 mJ/cm2 (5–30 s) and US doses of 5–15 min, depending on the maturity of biofilms and the microorganism. Initially, we investigated the effects of UVC on planktonic cells, revealing a significantly higher lethality in L. monocytogenes compared to B. subtilis (P < 0.05). Moreover, B. subtilis demonstrated greater resistance in both its spore and vegetative forms (P < 0.05). The application of UVC on biofilms demonstrates that, as the more structured and robust the biofilm is, the more difficult it is for UVC to penetrate the biofilm structure and reach the cells that conform them, which is why higher lethality is observed in the shortest formation period (i.e. 24 h). In addition, as the UVC dose increases, microorganisms show a higher resistance to the treatment, thus showing a greater reduction in the microbial population at lower doses. The single application of ultrasound (US) manages to reduce the cell load between 1 and 4 log CFU/cm2, depending on the microorganism and exposure time. The results obtained after the combination of both technologies achieve higher lethality, 5.2 log reductions at the lowest doses (i.e. applying 5 min of US and 5 s of UVC). Furthermore, the maximum reduction possible for these experiments (≥5.6 log) is achieved in any treatment applying a 15-s UVC exposure time. Significant differences are observed (P < 0.05) as the maturity stage increases ([+Let] 24a > 48b > 72c hours). The combination of these two technologies demonstrates highly promising outcomes across all examined bacterial structures, including spores and vegetative biofilms.

Synergistic and antibiofilm activity of DNase I and glucose oxidase loaded chitosan nanoparticles against dual-species biofilms of Listeria monocytogenes and Salmonella

Salmonella and Listeria monocytogenes are two of the most common foodborne pathogens in the food industry. They form dual-species biofilms, which have a higher sensitivity to antimicrobial treatment and a greater microbial adhesion. In this experiment, we loaded DNase I and glucose oxidase (GOX) on chitosan nanoparticles (CSNPs) to explore their inhibitory effects on and disruption of dual-species biofilms of Salmonella enterica and L. monocytogenes. Transmission electron microscopy (TEM) showed that CSNP-DNase-GOX and CSNPs were spherical in shape. CSNP-DNase-GOX was shifted and altered compared to the infrared peaks of CSNPs. CSNPs loaded with DNase I and GOX showed an increase in the particle size and an alteration in the polydispersity index (PDI) and the zeta potential. Compared to free DNase I or GOX, DNase I and GOX loaded on CSNPs had higher stability at different temperatures. CSNP-DNase-GOX was more effective in inhibiting dual-species biofilms than CSNP-GOX. Scanning electron microscopy (SEM) and fluorescence microscopy were used to observe the structure of the biofilm, which further illustrated that CSNP-DNase-GOX disrupted the dual-species biofilms of S. enterica and L. monocytogenes.

Inhibition and eradication of Listeria monocytogenes biofilm using the combined treatment with nisin and sesamol

Listeria monocytogenes can form biofilms, which enables it to persist in the food industry and poses a significant threat to food safety. The combination of nisin (NS) and sesamol (SE) has been found to inhibit the proliferation of L. monocytogenes. The objective of this study is to evaluate the effects of the combined use of NS and SE on the biofilm formation of L. monocytogenes and investigate the potential underlying mechanisms. The results showed that the combination of NS and SE completely inhibited the biofilm formation of both strains on the stainless steel sheet within 48 h. The existing L.monocytogenes biofilm on the stainless steel sheet was completely removed within 4 h after the combined treatment of NS and SE. Compared with individual treatment, the combined use of NS and SE resulted in a more significant downregulation in gene expression of fbpA, which is associated with L. monocytogenes biofilm formation. The findings suggest that the combined strategy of NS and SE in food processing has greater potential to control the formation of L. monocytogenes biofilms and eliminate existing ones, compared with using NS or SE alone.

Preparation and characterization of nisin-loaded chitosan nanoparticles functionalized with DNase I for the removal of Listeria monocytogenes biofilms.

Listeria monocytogenes biofilms represent a continuous source of contamination, leading to serious food safety concerns and economic losses. This study aims to develop novel nisin-loaded chitosan nanoparticles (CSNPs) functionalized with DNase I and evaluate its antibiofilm activity against L. monocytogenes on food contact surfaces. Nisin-loaded CSNPs (CS-N) were first prepared by ionic cross-linking, and DNase I was covalently grafted on the surface (DNase-CS-N). The NPs were subsequently characterized by Zetasizer Nano, transmission electron microscopy, Fourier transform infrared (FT-IR), and X-ray diffraction (XRD). The antibiofilm activity of NPs was evaluated against L. monocytogenes on polyurethane (PU). The DNase-CS-N was fabricated and characterized with quality attributes (particle size-427.0±15.1nm, polydispersity [PDI]-0.114±0.034, zeta potential-+52.5±0.2mV, encapsulation efficiency-46.5%±3.6%, DNase conjugate rate-70.4%±0.2). FT-IR and XRD verified the loading of nisin and binding of DNase I with chitosan. The DNase-CS-N caused a 3 logcolony-forming unit (CFU)/cm2 reduction of L. monocytogenes biofilm cells, significantly higher than those in CSNPs (1.4log), CS-N (1.8log), and CS-N in combination with DNase I (2.2log) treatment groups. In conclusion, nisin-loaded CSNPs functionalized with DNase I were successfully prepared and characterized with smooth surface and nearly spherical shape, high surface positive charge, and good stability, which is effective to eradicate L. monocytogenes biofilm cells on food contact surfaces, exhibiting great potential as antibiofilm agents in food industry. PRACTICAL APPLICATION: Listeria monocytogenes biofilms are a common safety hazard in food processing. In this study, novel nanoparticles were successfully constructed and are expected to be a promising antibiofilm agent in the food industry.

Surface topography and chemistry of food contact substances, and microbial nutrition affect pathogen persistence and symbiosis in cocktail Listeria monocytogenes biofilms

The foodborne pathogen Listeria monocytogenes (L. monocytogenes) can form very persistent biofilms on food contact substances, either in monospecies, or in conjunction with background microflora. Although some studies have investigated how bacterial symbiosis facilitates L. monocytogenes survival in complex cocktail biofilms, very little is known about how this core symbiotic relationship is affected by environmental factors. Here, we investigate how L. monocytogenes persistence is affected by symbiosis with Escherichia coli O157:H7, Pseudomonas fluorescens, and Ralstonia insidiosa, and how these relationships are impacted by environmental factors, including food contact surface topography and chemistry, and microbial nutrition. We find that symbiosis can significantly enhance L. monocytogenes persistence in four-species cocktail biofilms (5.71 ± 0.03 Log CFU/cm2) by up to 1.9 Log CFU/cm2 compared to monospecies biofilms (3.81 ± 0.08 Log CFU/cm2). We also report that surface topography and chemistry, microbial nutrition, and symbiont species significantly impact the symbiotic relationships involving L. monocytogenes, revealing the adaptability of bacterial symbiosis to changing environmental conditions and the complex nature of cocktail pathogen biofilms. Antagonistic and synergistic interactions involving pathogens in cocktail biofilms, and factors affecting those interactions, are elucidated and can establish a foundation to study symbiosis and its role in mitigating pathogen persistence in food systems, and helping identify unanticipated increased food safety risks of pathogens due to enhanced persistence.

Degradation of Listeria monocytogenes biofilm by phages belonging to the genus Pecentumvirus.

Listeria monocytogenes is a pathogenic bacterium that is especially dangerous for children, the elderly, pregnant women, and immune-compromised people. Because of this, the food industry takes its presence in their plants seriously. Food recalls due to L. monocytogenes are common with a high associated economic cost. In food-processing plants, Listeria spp. typically reside in biofilms, which are structures produced by bacteria that shield them from environmental stressors and are often attached to surfaces. The significance of our work is that we show a bacteriophage-a virus-infecting bacteria-can reduce Listeria counts by two orders of magnitude when the bacterial biofilms were grown under simulated dairy processing conditions. This work provides insights into how phages may be tested and used to develop biosanitizers that are effective but are not harmful to the environment or human health.

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 10years. 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.

Microscopic and transcriptomic characterization of Listeria monocytogenes aggregation and biofilm formation in cantaloupe juice

Plant exudate is likely the major nutrient source supporting the survival and biofilm formation of microorganisms in fresh produce processing environments. This study compared the impact of cantaloupe juice on the physiology and gene expression of Listeria monocytogenes (Lm) using microscopic and transcriptomic tools.Lm showed a strong autoaggregation and formed significantly more biofilms on stainless steel (SS) coupons when grown in cantaloupe juice in comparison to in TSB. SEM images revealed a different attachment profile of Lm on SS coupons. In TSB, Lm cells were mainly found in scratches and grooves of the metal surface, whereas, in cantaloupe juice they attached to the smooth surface as well. Lm planktonic and biofilm cells in cantaloupe juice exhibited stress-induced phenotypes, including strong cell elongation and increased proportion of cells with membrane permeability.Lm growth in cantaloupe juice resulted in distinct transcriptional profiles of both biofilm and planktonic cells in comparison to that in TSB. Functional annotation indicated that the significantly differentially expressed genes mainly participated in metabolism, signaling and stress response. Notably, certain pathways downregulated for planktonic cells were significantly upregulated for biofilm cells in cantaloupe juice compared to TSB, including ABC transporters, two-component system, quorum sensing, chemotaxis, and flagellar assembly.

Eugenol nanoemulsion reduces Listeria monocytogenes biofilm by modulating motility, quorum sensing, and biofilm architecture

Listeria monocytogenes is a major foodborne pathogen in the United States that is capable of forming sanitizer-tolerant biofilms on diverse food contact surfaces and under varying temperature conditions. A plethora of research in the last decade has explored the potential of phytochemicals as antibiofilm agents. However, the low solubility of phytochemicals is a significant challenge that needs to be addressed to develop plant-based disinfectants that can be applied in the industry for controlling L. monocytogenes biofilms and improving food safety. This study investigated the efficacy of eugenol nanoemulsion (EGNE) in inhibiting biofilm formation in two strains of L. monocytogenes (Scott A and AT19115) on stainless steel surfaces at two temperatures (25 or 10°C). In addition, the effect of EGNE on pathogen motility, extracellular polymeric substances (EPS) production, eDNA production, and quorum sensing activity during biofilm formation was studied using standard bioassays. Moreover, the efficacy of EGNE in killing mature L. monocytogenes biofilm was also investigated against both the strains and temperature combinations. All experiments had a completely randomized design with duplicate samples and were repeated at least three times. EGNE had a particle size of ~75 nm, a polydispersity index of 0.25, and a high negative surface charge. EGNE 700 mg/L inhibited L. monocytogenes biofilm formation significantly by ~1.89 log in 72 h at 25°C and ~1.25 log on day 16 at 10°C, when compared to control (p &amp;lt; 0.05). EGNE at 2,750 mg/L concentration completely inactivated (~7 log CFU/coupon reduction as compared to control) L. monocytogenes biofilm cells developed at 25 or 10°C as early as 1 min of treatment time (p &amp;lt; 0.05). In addition, EGNE was able to significantly reduce the motility, EPS, eDNA production, and quorum sensing activity which plays a major role in biofilm formation. Both L. monocytogenes Scott A and AT19115 strains exhibited similar sensitivity to EGNE treatments. The results suggest that EGNE could potentially be used as a natural sanitizer to effectively control L. monocytogenes biofilms in food processing environments.

Inactivation of dried cells and biofilms of Listeria monocytogenes by exposure to blue light at different wavelengths and the influence of surface materials.

Antimicrobial blue light (aBL) in the 400-470 nm wavelength range has been reported to kill multiple bacteria. This study assessed its potential for mitigating an important foodborne pathogen, Listeria monocytogenes (Lm), focusing on surface decontamination. Three wavelengths were tested, with gallic acid as a photosensitizing agent (Ps), against dried cells obtained from bacterial suspensions, and biofilms on stainless-steel (SS) coupons. Following aBL exposure, standard microbiological analysis of inoculated coupons was conducted to measure viability. Statistical analysis of variance was performed. Confocal laser scanning microscopy was used to observe the biofilm structures. Within 16 h of exposure at 405 nm, viable Lm dried cells and biofilms were reduced by approx. 3 log CFU/cm2 with doses of 2,672 J/cm2. Application of Ps resulted in an additional 1 log CFU/cm2 at 668 J/cm2, but its effect was not consistent. The highest dose (960 J/cm2) at 420 nm reduced viable counts on the biofilms by 1.9 log CFU/cm2. At 460 nm, after 800 J/cm2, biofilm counts were reduced by 1.6 log CFU/cm2. The effect of material composition on Lm viability was also investigated. Irradiation at 405 nm (668 J/cm2) of cells dried on polystyrene resulted in one of the largest viability reductions (4.0 log CFU/cm2), followed by high-density polyethylene (3.5 log CFU/cm2). Increasing the dose to 4,008 J/cm2 from 405 nm (24 h), improved its efficacy only on SS and polyvinyl chloride. Biofilm micrographs displayed a decrease in biofilm biomass due to the removal of biofilm portions from the surface and a shift from live to dead cells suggesting damage to biofilm cell membranes. These results suggest that aBL is a potential intervention to treat Lm contamination on typical material surfaces used in food production. IMPORTANCE Current cleaning and sanitation programs are often not capable of controlling pathogen biofilms on equipment surfaces, which transmit the bacteria to ready-to-eat foods. The presence of native plant microbiota and organic matter can protect pathogenic bacteria by reducing the efficacy of sanitizers as well as promoting biofilm formation. Post-operation washing and sanitizing of produce contact surfaces might not be adequate in eliminating the presence of pathogens and commensal bacteria. The use of a dynamic and harmless light technology during downtime and close of operation could serve as a useful tool in preventing biofilm formation and persistence. Antimicrobial blue light (aBL) technology has been explored for hospital disinfection with very promising results, but its application to control foodborne pathogens remains relatively limited. The use of aBL could be a complementary strategy to inactivate surfaces in restaurant or supermarket deli settings.

Combined effects of cold and acid on dual-species biofilms of Pseudomonas fluorescens and Listeria monocytogenes under simulated chilled beef processing conditions

Interactions across bacterial species boundaries are usually influenced by environmental stresses, yet little has been evaluated regarding multifactorial stresses on the fate of dual-species biofilm formation in food industry. In this study, the processing conditions of chilled beef were established as a combination of cold and acid stresses (4 °C and pH 5.4), with pH 7.0 or 25 °C serving as the controls, to investigate the interaction of dual-species biofilm between Pseudomonas fluorescens and Listeria monocytogenes. Dual-species biofilms significantly increased biofilm formation at 72 h under the condition of 25°C-pH7.0 and 25°C-pH5.4 (P < 0.05). Compared with mono-species biofilms, the cell numbers of L. monocytogenes in dual-species biofilms were lower at 25 °C (P < 0.05), however, the adherent cells of L. monocytogenes was higher in dual-species biofilms at 4 °C (P < 0.05). Furthermore, the amount of extracellular polysaccharides and proteins secreted by single P. fluorescens biofilms at 4 °C was more than three times than those at 25 °C. The surface-enhanced Raman spectroscopy further profiled the variability of extracellular polymeric substances (EPS) composition. Additionally, RT-qPCR results revealed an upregulation of biofilm-related and genes in co-culture species. It provides valuable insights into the strategies for removing mixed biofilms under diverse stressful conditions in practical food processing.

Mechanism of acid and alkali electrolyzed water on the elimination of Listeria monocytogenes biofilm based on proteomic analysis

Acidic electrolyzed water is a relatively mature bactericide, which has a certain inhibitory effect on a variety of microorganisms, and is widely used in the field of food processing for cleaning, sterilization and disinfection. This study investigated the deactivation mechanisms of Listeria monocytogenes by Tandem Mass Tags quantitative proteomics analysis. Samples were treated through A1S4 (Alkaline electrolytic water treatment for 1 min and Acid electrolytic water treatment for 4 min), S3A1S1 (Acid electrolyzed water treatment 3 min, Alkaline electrolyzed water treatment 1 min and Acid electrolyzed water treatment 1 min), S5 (Acid electrolytic water treatment for 5 min). Proteomic analysis showed that the mechanism of acid alkaline electrolyzed water treatment to eliminate the inactivation of the biofilm of L. monocytogenes was related to protein transcription and extension, RNA processing and synthesis, gene regulation, sugar and amino acid transport and metabolism, signal transduction and ATP binding. The study on the influence mechanism and action mechanism of the combination of acidic and alkaline electrolyzed water to remove L. monocytogenes biofilm is helpful to understand the development of the process of removing biofilm by electrolyzed water, and provides theoretical support for the treatment of other microbial contamination problems in food processing by electrolyzed water.

Effects of pH, sodium chloride, and temperature on the growth of Listeria monocytogenes biofilms

AbstractListeria monocytogenes is able to form biofilms on food contact surfaces. Effectiveness of salt concentration, pH, and temperature on the formation of L. monocytogenes biofilms was evaluated individually and in combinations using microtiter plate assay by measuring the optical density. The tested strains differed in their biofilm formation (low, moderate, and strong) ability. At 37 °C, decreasing amounts of biofilms was observed in almost all L. monocytogenes strains when the NaCl concentration increased from 0.05 to 15%, but all strains were able to form biofilm even at 1 °C. There was no significant difference in biofilm formation between pH 4, 5, and 6, except for some strains. When stress conditions were tested in combination, the addition of 15% NaCl significantly inhibited the growth of L. monocytogenes at 1 °C and 4 °C, and the weak biofilm-forming strains were less sensitive to the temperature and to NaCl treatments than the strong biofilm-forming strains. These results enhance our knowledge of the application of NaCl, temperature, and pH stresses in the food industry and provide basis to develop new strategies for control of biofilm formation of this pathogen.

Activity of rosemary (Rosmarinus officinalis - Family: Lamiaceae) essential oil compared to peracetic acid in Listeria monocytogenes biofilms

The objective of this study was to evaluate the activity of Rosmarinus officinalis essential oil (EO) compared to peracetic acid (PA) regarding formation and elimination of Listeria monocytogenes biofilms on polystyrene surface. The minimum inhibitory concentration (MIC) was determined according to standard protocol. Isolates were inoculated according to MIC standards polystyrene plate wells, which were then incubated at 37°C/96 hours for evaluation of biofilm formation. Regarding the evaluation of biofilm elimination, the biofilms were treated under MIC for 10 minutes. The MIC obtained were 2.0 and 3.0 mg mL-1 for EO and 0,015% for PA. Therefore, the results showed a reduction in the formation of biofilm with the presence of EO and PA, EO being more efficient (p &lt; 0.05). Both compounds had a good capacity of eliminating biofilms, however the EO reduced the biofilm formation when compared to PA, highlighting its potential as an antibacterial agent and antibiofilm.

Liposomes-Based Drug Delivery Systems of Anti-Biofilm Agents to Combat Bacterial Biofilm Formation.

All currently approved antibiotics are being met by some degree of resistance by the bacteria they target. Biofilm formation is one of the crucial enablers of bacterial resistance, making it an important bacterial process to target for overcoming antibiotic resistance. Accordingly, several drug delivery systems that target biofilm formation have been developed. One of these systems is based on lipid-based nanocarriers (liposomes), which have shown strong efficacy against biofilms of bacterial pathogens. Liposomes come in various types, namely conventional (charged or neutral), stimuli-responsive, deformable, targeted, and stealth. This paper reviews studies employing liposomal formulations against biofilms of medically salient gram-negative and gram-positive bacterial species reported recently. When it comes to gram-negative species, liposomal formulations of various types were reported to be efficacious against Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and members of the genera Klebsiella, Salmonella, Aeromonas, Serratia, Porphyromonas, and Prevotella. A range of liposomal formulations were also effective against gram-positive biofilms, including mostly biofilms of Staphylococcal strains, namely Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus subspecies bovis, followed by Streptococcal strains (pneumonia, oralis, and mutans), Cutibacterium acnes, Bacillus subtilis, Mycobacterium avium, Mycobacterium avium subsp. hominissuis, Mycobacterium abscessus, and Listeria monocytogenes biofilms. This review outlines the benefits and limitations of using liposomal formulations as means to combat different multidrug-resistant bacteria, urging the investigation of the effects of bacterial gram-stain on liposomal efficiency and the inclusion of pathogenic bacterial strains previously unstudied.

Impact of gas ultrafine bubbles on the efficacy of antimicrobials for eliminating fresh and aged Listeria monocytogenes biofilms on dairy processing surfaces

AbstractUltrafine bubbles (UFB) are a novel concept that has the potential to enhance the potency of antimicrobials to eliminate biofilms. This study investigated the impact of incorporating gas (air, CO2, and N2) UFB on the potency of chlorine (Cl2; 50, 100, and 200 ppm) and peracetic acid (PAA; 20, 40, and 80 ppm) antimicrobial (AM) solutions against fresh (3 days) and aged (30 days) Listeria monocytogenes biofilms on polypropylene, silicone, and stainless steel surfaces. Listeria monocytogenes biofilms were statically grown on polypropylene, silicone, and stainless steel coupons (7.62 × 2.54 cm) at 25°C for 3 or 30 days, by immersing in a three‐strain cocktail of L. monocytogenes in brain heart infusion (BHI) broth. The coupons were treated by submerging in AM solutions with or without UFB for 1 min, then swabbed into Dey‐Engley neutralizing broth and enumerated on BHI agar. Incorporation of air, CO2, and N2 UFB in AM solutions resulted in significantly increased log reductions (0.4–1.5 logs) of fresh and aged L. monocytogenes biofilms on polypropylene and stainless steel surfaces, whereas incorporation of CO2 UFB in AM solutions resulted in ~1 log greater reductions of fresh and aged L. monocytogenes biofilms on silicone surfaces compared with AM solutions without UFB. This study also demonstrated that 200 ppm Cl2 was most effective against fresh and aged L. monocytogenes biofilms on polypropylene, silicone, and stainless steel surfaces compared with 50 ppm Cl2, 20 ppm PAA, and 40 ppm PAA.

Combined Effects of Sanitizers and UV-C Light on Listeria monocytogenes Biofilm Growth and Survivability on Produce-Harvesting Materials Used in the Tree Fruit Production Industry

Listeria monocytogenes is an aggressive biofilm former that can establish and persist in food processing environments. Commonly associated with ready-to-eat and dairy products, this pathogenic bacterium has recently been increasingly linked to fresh produce outbreaks. Equipment used during harvesting and handling of produce can provide a niche environment for biofilm growth and persistence. Based on a survey conducted among stakeholders in the tree fruit production industry, three favored materials for storing and harvesting produce were identified: nylon, wood, and plastic. The purpose of this study was to investigate the application of the generally recognized as safe sanitizers lactic acid, thymol, and silver citric acid (SDC) and UV-C light alone or in combination for 2 or 5 min on different food-contact surfaces used during tree fruit harvesting and storing. Multistrain L. monocytogenes biofilms were grown in a Centers for Disease Control and Prevention biofilm reactor for 96 h on wood, nylon, and polycarbonate coupons at 20 ± 2°C. After each treatment, coupons were neutralized and the remaining cells were enumerated. Results showed that the most effective treatment was the simultaneous use of UV-C light and SDC (4-log reduction) and that the least effective treatment was UV-C light alone (P &lt; 0.05). The type of material was found to play a significant role in the efficacy of the sanitizers (P &lt; 0.05). This study demonstrates the ability of L. monocytogenes to grow and form biofilms on different surfaces and contributes to an understanding of the response of this food safety threat against antimicrobial intervention strategies.

Proposal and Verification of the Theory of Layer-by-Layer Elimination of Biofilm in Listeria monocytogenes

Biofilms are microbial communities that represent a high abundance of microbial life forms on Earth. Within biofilms, structural changes during clearance processes occur in three spatial and temporal dimensions; therefore, microscopy and quantitative image analysis are essential in elucidating their function. Here, we present confocal laser scanning microscopy (CLSM) in conjunction with ISA-2 software analysis for the automated and high-throughput quantification, analysis, and visualisation of biofilm interiors and overall biofilm properties in three spatial and temporal dimensions. This paper discusses the removal process of Listeria monocytogenes (LM) biofilms using slightly acidic electrolytic water, non-electrolytic hypochlorite water, and alternating the use of strongly acidic and strongly alkaline electrolytic water. The results show that the biofilm gradually thins and gutters from the initial viscous dense and thick morphology under the action of either biocide. This process is consistent with first-level kinetics. After CLSM filming to observe the biofilm structure, analysis software was used to process and quantify the biovolume, average biofilm thickness, biofilm roughness and other indicators; fluorescence enzyme markers were used to verify the remaining amount of extracellular nucleic acid. In this study, we proposed and validated the theory of layer-by-layer elimination of LM biofilm.

Biogenic nanosilver bearing antimicrobial and antibiofilm activities and its potential for application in agriculture and industry.

Due to the increasing resistance of bacteria and fungi to antimicrobials, it is necessary to search for effective alternatives to prevent and treat pathogens causing diseases in humans, animals, and plants. In this context, the mycosynthesized silver nanoparticles (AgNPs) are considered as a potential tool to combat such pathogenic microorganisms. AgNPs were synthesized from Fusarium culmorum strain JTW1 and characterized by Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy, Nanoparticle Tracking Analysis (NTA), Dynamic Light Scattering (DLS) and Zeta potential measurement. The minimum inhibitory (MIC) and biocidal concentrations (MBC) were determined against 13 bacterial strains. Moreover, the combined effect of AgNPs with antibiotics (streptomycin, kanamycin, ampicillin, tetracycline) was also studied by determining the Fractional Inhibitory Concentration (FIC) index. The anti-biofilm activity was examined by crystal violet and fluorescein diacetate (FDA) assays. Furthermore, antifungal activity of AgNPs was evaluated against a panel of phytopathogenic fungi viz., Botrytis, Colletotrichum, Fusarium, Phoma, Sclerotinia, and an oomycete pathogen Phytophthora by agar well-diffusion and micro-broth dilution method to evaluate the minimal AgNPs concentrations that inhibit fungal spore germination. Fungi-mediated synthesis resulted in the formation of small (15.56 ± 9.22 nm), spherical and stable (zeta potential of - 38.43 mV) AgNPs with good crystallinity. The results of FTIR spectroscopy indicated the presence of various functional groups, namely hydroxyl, amino, and carboxyl ones, from the biomolecules on the surface of AgNPs. The AgNPs showed antimicrobial and antibiofilm formation activities against Gram-positive and Gram-negative bacteria. The values of MIC and MBC ranged between 16-64 and 32-512 μg mL-1, respectively. The enhanced effect of AgNPs in combination with antibiotics was confirmed against human pathogens. The highest synergistic effect (FIC = 0.0625) was demonstrated by the combination of AgNPs with streptomycin against two strains of Escherichia coli (ATCC 25922 and ATCC 8739), followed by Klebsiella pneumoniae and Pseudomonas aeruginosa (FIC = 0.125). Enhanced effects of AgNPs with ampicillin were also shown against Staphylococcus aureus ATCC 25923 (FIC = 0.125) and P. aeruginosa (FIC = 0.25), as well as kanamycin against S. aureus ATCC 6538 (FIC = 0.25). The crystal violet assay revealed that the lowest concentration of AgNPs (0.125 μg mL-1) reduced the development of biofilms of Listeria monocytogenes and Salmonella enterica, while the maximum resistance was shown by Salmonella infantis, its biofilm was reduced after exposure to a concentration of 512 μg mL-1. A high inhibitory effect on the activity of bacterial hydrolases was observed by the FDA assay. AgNPs at a concentration of 0.125 μg mL-1 reduced the hydrolytic activity of all biofilms formed by the tested pathogens, except E. coli ATCC 25922, P. aeruginosa, and Pectobacterium carotovorum (efficient concentration was 2-fold higher, at 0.25 μg mL-1), while the hydrolytic activity of E. coli ATCC 8739, Salmonella infantis and S. aureus ATCC 6538 was suppressed after treatment with AgNPs at concentrations of 0.5, 2 and 8 μg mL-1, respectively. Moreover, AgNPs inhibited fungal growth and spore germination of Botrytis cinerea, Phoma lingam, and Sclerotinia sclerotiorum. MIC and MFC values of AgNPs against spores of these fungal strains were determined at 64, 256, and 32 μg mL-1, and zones of growth inhibition were 4.93, 9.54, and 3.41 mm, respectively. Fusarium culmorum strain JTW1 was found to be an eco-friendly biological system for an easy, efficient and inexpensive synthesis of AgNPs. In our study, the mycosynthesised AgNPs demonstrated remarkable antimicrobial (antibacterial and antifungal) and antibiofilm activities against a wide range of human and plant pathogenic bacteria and fungi singly and in combination with antibiotics. These AgNPs could be applied in medicine, agriculture, and food industry to control such pathogens that cause numerous human diseases and crop losses. However, before using them extensive animal studies are required to evaluate the toxicity, if any.