Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

D and z Values of Salmonella, Listeria innocua, and Listeria monocytogenes in Fully Cooked Poultry Products

: The D and z values of Salmonella, Listeria innocua, and Listeria monocytogenes were obtained for different ready‐to‐eat poultry products, including chicken, turkey, and duck. The D values of Salmonella, L. innocua, and L. monocytogenes were 151.5 to 0.1 min at 55 to 70°C, and the z values of Salmonella, L. innocua, and L. monocytogenes were 4.9 to 7.0 °C. Significant differences were found for the heat resistance of Salmonella, L. innocua, and L. monocytogenes among turkey, duck, and chicken products, indicating that the kinetic values of a certain pathogen in a specific product should be used for determining process lethality in fully cooked and vacuum‐packaged poultry products during post‐cook heat treatments.     

Irradiation Sensitivity of Planktonic and Biofilm-associated <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">L. innocua</Emphasis> as Influenced by Temperature of Biofilm Formation

The human pathogen Listeria monocytogenes forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation effectively inactivates planktonic Listeria, but no information is available on the relative efficacy of the process against biofilm-associated Listeria. The irradiation sensitivity of planktonic or biofilm cells was determined for L. monocytogenes ATCC 43256 and ATCC 49594 and a commonly used surrogate Listeria innocua ATCC 51742. Biofilms were formed on sterile glass slides incubated for 48 h at 22°C, 28°C, or 37°C. The cultures were gamma irradiated and the irradiation D ₁₀ value was calculated for each combination of isolate/culture/temperature. The effect of temperature of cultivation on the irradiation sensitivity of both planktonic cells and biofilm cells varied for each of the isolates. Depending on isolate and temperature, biofilm cells were equally sensitive or more sensitive (P < 0.05) to irradiation. D ₁₀ values overall tended to increase with temperature of cultivation for L. monocytogenes 49594 and L. innocua 51742, but tended to decrease with increasing temperature for L. monocytogenes 43256. The D ₁₀ values of the various culture/temperature combinations differed significantly among the isolates examined. Irradiation effectively eliminates both planktonic and biofilm-associated cells. The extent to which the biofilm habitat modifies the antimicrobial efficacy of irradiation is dependent on the specific isolate examined and the temperature at which it forms. This study is the first inquiry to show that biofilm Listeria cells are as sensitive or more sensitive to irradiation compared with planktonic cells and that this response is dependent on biofilm formation conditions.     

Effects of Antimicrobial Coatings and Cryogenic Freezing on Survival and Growth of Listeria innocua on Frozen Ready‐to‐Eat Shrimp during Thawing

Foodborne pathogens such as Listeria monocytogenes could pose a health risk on frozen ready‐to‐eat (RTE) shrimp as the pathogen could grow following thawing. In this study, antimicrobial‐coating treatments alone, or in combination with cryogenic freezing, were evaluated for their ability to inhibit the growth of Listeria innocua, a surrogate for L. monocytogenes, on RTE shrimp. Cooked RTE shrimp were inoculated with L. innocua at 3 population levels and treated with coating solutions consisting of chitosan, allyl isothiocyanate (AIT), or lauric arginate ester (LAE). The treated shrimp were then stored at –18 °C for 6 d before being thawed at 4, 10, or 22 °C for either 24 or 48 h. Results revealed that antimicrobial coatings achieved approximately 5.5 to 1 log CFU/g reduction of L. innocua on RTE shrimp after the treatments, depending on the inoculated population levels. The coating‐treated shrimp samples had significantly (P < 0.05) less L. innocua than controls at each thawing temperature and time. Cryogenic freezing in combination with coating treatments did not achieve synergistic effects against L. innocua. Antimicrobial coatings can help to improve product safety by reducing Listeria on RTE shrimp.     

Formation and dispersal of biofilms in dairy substrates

The formation and dispersion of biofilms in the dairy industry is a problem because it increases cross‐contamination, affecting the shelf life of the products and their safety. The objective of this study was to evaluate the influence of different dairy substrates (cows’ milk and whey protein) on the formation and dispersion of Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus in two biofilm systems (mono‐species and multi‐species) on stainless steel at 25 °C. The dominant behaviour of E. faecalis occurred in most of the tests on mono‐species and multi‐species biofilms. A greater dispersion of biofilm cells was observed in skimmed milk.     

Effects of Zataria multiflora boiss essential oil,ultraviolet radiation and their combination on Listeria monocytogenes biofilm in a simulated industrial model

The objective of this study was to investigate the inhibitory effect of Zataria multiflora boiss essential oils (ZEOs), ultraviolet (UV) radiation and their combination against Listeria monocytogenes biofilm in a simulated industrial model (SIM). The effect of minimal inhibitory concentration (MIC) and sub‐MIC concentration of ZEOs, different contact time of UV and their combination was evaluated in a SIM on 6‐ and 12‐day‐old L. monocytogenes biofilm. In a SIM, 0.3% ZEOs were adequate to completely eliminate 6‐ and 12‐day‐old biofilm grown on stainless steel coupons. The population of viable L. monocytogenes biofilm cells under a 15‐ to 45‐min contact time of UV treatment declined significantly at 6‐ and 12‐day‐old biofilm. The combined effect of ZEO and UV showed antagonist effects. These findings indicated that in the single use, ZEO and UV revealed a suitable antilisteria biofilm activity, while combining them is not a promising method to remove listeria biofilms from stainless steel surfaces.     

The probiotic,Leuconostoc mesenteroides,inhibits Listeria monocytogenes biofilm formation

Listeria monocytogenes biofilm formation renders these cells highly resistant to current sanitation methods, and probiotics may be a promising approach to the efficient inhibition of Listeria biofilms. In the present study, three Leuconostoc mesenteroides strains of lactic acid bacteria isolated from kimchi were shown to be effective probiotics for inhibiting Listeria biofilm formation. Biofilms of two L. monocytogenes serotypes, 1/2a (ATCC15313) and 4b (ATCC19115), in dual-species culture with each probiotic strain were decreased by more than 40-fold as compared with single-species Listeria biofilms; for instance, a reduction from 5.4 × 10⁶ colony forming units (CFU)/cm² L. monocytogenes ATCC19115 in single-species biofilms to 1.1 × 10⁵ CFU/cm² in dual-species biofilms. Most likely, one of the Leuconostoc strains, L. mesenteroides W51, led to the highest Listeria biofilm inhibition without affecting the growth of L. monocytogenes. The cell-free supernatant from the L. mesenteroides W51 culture containing large protein molecules (>30 kDa) also inhibited Listeria biofilms. These data indicate that Leuconostoc probiotics can be used to repress L. monocytogenes biofilm contamination on surfaces at food processing facilities.     

Comparison of Two-Stage and Direct Selective Enrichment Techniques for Isolating Listeria spp. from Raw Milk

Isolation of Listeria spp. from 34 milk samples was compared by direct selective enrichment (FDA method) and two-stage enrichment techniques. Nineteen were Listeria-positive; 17 yielding one species only, either L. innocua or L. monocytogenes, and 2 both species. Direct enrichment and two-stage enrichment were equally effective at isolating Listeria spp. The number of isolations of L. monocytogenes was similar by both methods, but direct enrichment for more than 24 hr and use of the KOH step did not enhance the number of isolations.     

Packaging Effects on Growth of Listeria innocua in Shredded Cabbage

Freshly shredded white cabbage was treated with citric acid and sodium erythorbate, inoculated with Listeria innocua (in lieu of Listeria monocytogenes), and packaged in 230g lots in four types of retail bags with oxygen transmission rates (OTRs) of 5.6, 1,500, 4,000, and 6,000 cc O₂/m²/24 hr, then stored 21 days at 11°C. After 14 days, L. Innocua decreased in cabbage stored in three films. After 21 days, Listeria population increased in all packages, but the increase was less (p<0.05), for cabbage packaged in film with the highest OTR (commercial film).     

Reliability of Listeria monocytogenes Identification by Specific PCR Assessed by Phenotypic and Genotypic Techniques

This study evaluates the possibility of using polymerase chain reaction (PCR) for rapid identification of food-borne Listeria monocytogenes as an alternative to API Listeria system and estimates the incidence of API Listeria misidentifications in food-borne Listeria species. A total of 198 strains, 11 L. monocytogenes, 28 other Listeria species, and 159 food isolates were phenotypically and genotypically characterized by API Listeria profiles and randomly amplified polymorphic DNA (RAPD) profiles, respectively. They were also tested for PCR amplification using genus- and species-specific primers. Clustering analysis of phenotypic and genotypic data showed discrepancies in species identification of some isolates by API Listeria profiles. Their identities were confirmed by 16S rDNA sequencing, and thus, it was revealed that 33% of Listeria innocua and 19% of Listeria welshimeri were misidentified as L. monocytogenes by API Listeria profiles. Reliable identification of L. monocytogenes was obtained by LM1–LM2 specific primers which allowed PCR amplification only in reference strains and isolates previously identified as L. monocytogenes by RAPD and 16S rDNA sequence analysis. These results corroborate the suitability of specific PCR as a rapid and accurate test for the identification of L. monocytogenes, avoiding misidentification with other Listeria species commonly found in food products.     

Detection of Listeria monocytogenes in Fresh Produce Using Molecular Beacon—Real‐time PCR Technology

ABSTRACT: The capability of an assay to detect Listeria monocytogenes from artificially inoculated fresh‐cut produce such as cantaloupe and mixed salad was demonstrated. An oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (Molecular Beacon, MB) was used in a real‐time polymerase chain reaction (PCR) assay. As few as 4 to 7 colony‐forming units (CFU) of L. monocytogenes per 25 g of artificially contaminated produce could be detected. A comparison of 2 commercially available kits using MB‐PCR (iQ‐Check, Bio‐Rad Laboratories) and conventional PCR (BAX, Dupont Qualicon) was performed on artificially inoculated produce. The time required to detect L. monocytogenes (from produce to PCR) was considerably shorter for the iQ‐Check protocol (approximately 26 h) compared with the BAX‐PCR (approximately 52 h). The iQ‐Check protocol was also used to confirm the identity of the L. monocytogenes isolates obtained during a microbiological screen of conventional and organic leaf lettuce and alfalfa sprout samples from local supermarkets. The iQ check protocol was successful in differentiating L. monocytogenes isolates from other Listeria spp. such as L. welshimeri, L. innocua, and L. ivanovii. This is the 1st report of the application of the MB probe being used for real‐time detection of L. monocytogenes in whole and fresh‐cut produce.     

Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes

Increase of resistance to the application of benzalkonium chloride (BAC), peracetic acid (PA) and nisin during biofilm formation at 25 °C by three strains of Listeria monocytogenes (CECT 911, CECT 4032, CECT 5873 and BAC-adapted CECT 5873) in different scenarios was compared. For this purpose, resistance after 4 and 11-days of biofilm formation was quantified in terms of lethal dose 90% values (LD₉₀), determined according with a dose-response logistic mathematical model. Microscopic analyses after 4 and 11-days of L. monocytogenes biofilm formation were also carried out. Results demonstrated a relation between the microscopic structure and the resistance to the assayed biocides in matured biofilms. The worst cases being biofilms formed by the strain 4032 (in both stainless steel and polypropylene), which showed a complex “cloud-type” structure that correlates with the highest resistance of this strain against the three biocides during biofilm maturation. However, that increase in resistance and complexity appeared not to be dependent on initial bacterial adherence, thus indicating mature biofilms rather than planctonic cells or early-stage biofilms must be considered when disinfection protocols have to be optimized. PA seemed to be the most effective of the three disinfectants used for biofilms. We hypothesized both its high oxidizing capacity and low molecular size could suppose an advantage for its penetration inside the biofilm. We also demonstrated that organic material counteract with the biocides, thus indicating the importance of improving cleaning protocols. Finally, by comparing strains 5873 and 5873 adapted to BAC, several adaptative cross-responses between BAC and nisin or peracetic acid were identified.     

Susceptibility of Listeria monocytogenes strains to disinfectants and chlorinated alkaline cleaners at cold temperatures

Minimal inhibitory concentration (MIC), suspension and biofilm tests were used in evaluating the disinfecting efficacy of eight commercially available disinfectants and four chlorinated alkaline cleaners against 10 strains of Listeria monocytogenes at refrigerated temperatures. The adaptive response and cross-adaptation of L. monocytogenes to the disinfectants and chlorinated alkaline cleaners were investigated. The bactericidal components in the agents used were chlorine, quaternary ammonium compound (QAC), peracetic acid, ethanol and isopropanol. With some exceptions the disinfectants were efficient against the L. monocytogenes strains. One alkaline hypochlorite containing disinfectant was not efficient in the suspension and MIC tests at the lowest concentration recommended by the manufacturer. The chlorinated alkaline cleaners were effective against L. monocytogenes. A QAC-based disinfectant was found to be the least-effective agent on both glass bead-blasted polyethylene and stainless-steel surfaces. Adaptive and cross-adaptive responses of L. monocytogenes strains were observed towards the QAC-based agent, but over 2-fold increases to other agents were not observed. These results suggest that the adaptive responses of L. monocytogenes to disinfectants or chlorinated alkaline cleaners are of a minor concern.     

Effects of packaging type, gas atmosphere and storage temperature on survival and growth of Listeria spp. in shredded dry coleslaw and its components

The survival and growth of Listeria populations inoculated on to dry coleslaw mix and its components were investigated, focusing on effects of storage temperatures and gas atmospheres within packaging films or storage chambers. There were few significant effects of packaging film at 3 °C, but at 8 °C the elevated CO₂/low O₂ atmospheres generated within orientated polypropylene (OPP) packages and used in controlled atmosphere chambers were inhibitory. Although two strains of Listeria monocytogenes had survival characteristics comparable with Listeria innocua, L. monocytogenes ATCC 19114 survived better at 3 °C and also in the elevated CO₂/low O₂ atmospheres within OPP at 8 °C. The effects of product components on the survival of L. innocua were linked to storage temperature. Shredded carrot reduced initial counts and at 8 °C inhibited survival of L. innocua in comparison with shredded cabbage.     

A study of the occurrence of Listeria species in raw sheep milk

The occurrence of bacteria from the genus Listeria in raw sheep milk and traditional local cheese was studied in three regions of the Karak district of Jordan. Conventional plating methods for the detection of Listeria species were followed to determine the average and the percentage of the contaminated samples. The result shows that there were significant differences between the regions in the study concerning the average and the percentage of positive occurrences of Listeria species in raw sheep milk. The results also showed that mainly L. monocytogenes and, to a lesser degree, L. ivanovii and L. innocua were found in the milk samples, while the occurrence of L. monocytogenes in cheese samples was very low.     

The occurrence of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis

A case of listeriosis was associated with the consumption of a soft cheese produced in England. Goats cheese and other products from the same food manufacturer were examined for the presence of Listeria over the following 11 months. Listeria monocytogenes was isolated from 16 of 25 cheese samples on retail sale, 12 of 24 cheese samples obtained directly from the factory, and from shelving within the plant. Phage-typing of 68 isolates of L. monocytogenes from cheese samples and the factory showed that 66 (97%) were indistinguishable from the strain isolated from the patient's cerebrospinal fluid and stool. L. monocytogenes was not isolated from seven goats milk or two yoghurt samples. Listeria innocua was isolated from 10 cheese samples, two of which contained no other species of Listeria. Levels of L. monocytogenes shortly after production were low (<10/g), but were higher (10⁵–10⁷ cfu/g) in six of the 16 cheese samples obtained from retail outlets. Multiplication of L. monocytogenes was demonstrated in cheeses contaminated at the factory and held at 4°C in the laboratory.     

Cell viability of Listeria monocytogenes biofilms

Several authors have reported biofilm formation by Listeria monocytogenes, and it is suspected that biofilms form a unique niche for extended survival of this foodborne pathogen in food-processing environments. We have evaluated growth of two L. monocytogenes strains (Murray and 7148) in biofilms and analysed the relationship between culturable and viable-but-non-culturable (VBNC) cells. Biofilms were grown on glass slides in static conditions at 37°C for up to 10 days. Culturable cells for L. monocytogenes Murray grew to 10⁵cfu cm⁻²within 2 days, while L. monocytogenes 7148 required 4 days to reach these cell numbers. After 2 days, cell counts of L. monocytogenes Murray decreased, followed by another increase with cell numbers reaching almost 10⁶cfu cm⁻²on day 10. In contrast, cell counts of L. monocytogenes 7148 stayed close to 10⁵cfu cm⁻²until day 10. VBNC cells of L. monocytogenes Murray increased with biofilm age while this was not seen for strain 7148. Also, swabbing removed biofilms of strain Murray more easily than strain 7148. Comparisons of viable counts obtained for swabbed and in situ biofilms indicated that these strain differences are due either to variable composition of extracellular polymeric substances in the two biofilms or to different cell physiology of the two strains.     

Susceptibility of Listeria monocytogenes to high pressure processing: A review

Listeria monocytogenes has been regarded as an emerging food pathogen responsible for listeriosis, a serious disease given its high mortality rate. The need for better food processing methods has led to an increased interest in high pressure processing (HPP), a novel nonthermal method presented as “producer” of safer food products. This review provides an overview of the effects of HPP on Listeria monocytogenes and on L. innocua, with the latter often used as an amenable surrogate for the pathogenic species. The factors that affect the susceptibility of listeriae to HPP, as well as the long-term implications of postprocessing recovery, are discussed in the perspective of the use of HPP to improve the safety of potential food vehicles.     

Recoverability of Listeria monocytogenes after coculture with Tetrahymena pyriformis

Bactivory by protozoa is a major factor that limits the number of bacteria in nature and may control the presence of Listeria monocytogenes. The effectiveness of Tetrahymena pyriformis destruction of L. monocytogenes was measured. Within 1 hr, 35–40 T. pyriformis cells ingested an average of 1,219 CFU of L. monocytogenes. Gentamicin was then added to kill un-ingested Listeria. In 24 hr, the recoverable bacteria were reduced at an exponential rate to undetectable levels (<1 per culture). A genetically diverse set of L. monocytogenes cultures all reduced Listeria recovery by the same degree. In assays without addition of gentamicin, numbers of attached L. monocytogenes cells were lessened from an average of log 6.5 CFU/2 ml culture to log 4.7 CFU/2 ml culture. T. pyriformis was capable of lowering numbers of both free-swimming and attached L. monocytogenes. This technology may have applications to control L. monocytogenes in food processing environments.