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

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

Removal of Listeria monocytogenes biofilms on stainless steel surfaces through conventional and alternative cleaning solutions.

Conventional treatments are not effective enough to completely remove Listeria monocytogenes biofilms from surfaces, thus implying the presence of certain persistent bacterial forms. In this study, eleven treatments (i.e. two enzymatic agents applied at two different temperatures and concentrations, two alkaline cleaners and one acid detergent) were used to remove mature L. monocytogenes S2-bac biofilms. A combined treatment was then selected for its application to four different L. monocytogenes strains (i.e. CECT 5672, CECT 935, S2-bac, EDG-e). Effectivity of the treatments was evaluated quantitatively using TEMPO and qualitatively by direct epifluorescent microscopy (DEM). Bacterial detachment obtained after the application of acid, alkaline and chlorinated alkaline treatments were 6.03, 6.24 and 4.76 Log CFU/cm2, respectively. Enzymatic treatments applied at 50 °C obtained the greatest detachment and biocidal activity. The results derived from the observation of the remaining biofilm structure by DEM proved that conventional treatments were unable to completely remove conformed structures with the potential risk this entails. Last, the application of a combined treatment using a chlorinated alkaline cleaner followed by an enzymatic treatment enhanced the dispersal of the bacterial cells from surfaces, thus consolidating this as a good option to recommend for the 5-step cleaning procedure.

Dual-species biofilms formation between dominant microbiota isolated from a meat processing industry with Listeria monocytogenes and Salmonella enterica: Unraveling their ecological interactions.

Alternatives to combat the persistence of pathogens need to consider the microbiota established on industrial surfaces as they can influence the protection or replacement (i.e. reduction/inhibition) of pathogens. The objective of the present study was to determine the ecological interactions established in dual-species biofilms between Listeria monocytogenes and Salmonella enterica as target pathogens, and isolates recovered from a meat processing facility (i.e.Pseudomonas fluorescens, Pseudomonas fragi, Bacillus safensis, Bacillus megaterium, and Candida zeylanoides). Results showed different ecological relations in biofilms depending on the species evaluated. Pseudomonas spp. did not influence the growth of either pathogen, although tested species tended to protect the pathogens in the structures generated. B. megaterium and C. zeylanoides affected the two pathogens differently, demonstrating a reduction of L. monocytogenes adhered cells within the formed biofilm. B. safensis reduced or presented non-influence on S. enterica depending on the incubation conditions. Contrarily, B. safensis was the microorganism that demonstrated the highest replacement capacity for L. monocytogenes, reducing its growth by up to 4 log CFU/cm2. The in vitro study of bispecies biofilms is important for the food industry, helping to understand how they behave and to find an effective way to eliminate them.

New approach for the removal of mature biofilms formed by wild strains of Listeria monocytogenes isolated from food contact surfaces in an Iberian pig processing plant.

One of the main objectives of the food industry is to guarantee food safety by providing innocuous food products. Therefore, this sector must consider all the possible biotic or abiotic contamination routes from the entry of raw materials to the release of the final product. Currently, one important problem in this regard is the presence of biofilms on food contact surfaces which can transmit pathogens such as L. monocytogenes. In industrial conditions biofilms are found in a mature state, so it is essential that when carrying out removal effectiveness studies in vitro the tests are realized with models that produce these structures in a similarly mature state. The main objective of this study was to evaluate the effectiveness of an alternative treatment (i.e. enzymatic detergent that include natural antimicrobial agents) and a conventional treatment (i.e. chlorinated alkaline) for the elimination of mature L. monocytogenes biofilms. The results showed a cell detachment from the formed mature biofilms with an effectivity of between 74.75%-97.73% and 53.94%-94.02% for the enzymatic treatment and the chlorinated alkaline detergent, respectively. On a qualitative level, it was observed that the dispersion in the structure was much higher for the enzymatic treatment than for the chlorinated alkaline, which continued to show obvious structure integrity. All this leads to the conclusion that treatments with an enzymatic detergent have a significantly greater impact on the removal of mature L. monocytogenes biofilms, although a further disinfection process would be needed, enhancing even more the treatment effectivity. This may imply that the industrial approach to addressing this problem should be modified to include new perspectives that are more effective than traditional ones.

Detection of Salmonella Typhimurium and Listeria monocytogenes biofilm cells exposed to different drying and pre-enrichment times using conventional and rapid methods.

The capacity of real-time PCR (RT-PCR), the VIDAS immunoassay system, and the conventional count method for detecting Salmonella enterica serovar Typhimurium and Listeria monocytogenes biofilm cells was evaluated in this study. After biofilm formation, tests were performed under different drying times (0, 6, 12, 24, and 72 h) and pre-enrichment times (0, 6, 18, and 25 h). The direct epifluorescence microscopic results demonstrated that Salmonella Typhimurium and L. monocytogenes biofilm cells can remain viable for 72 h under drying conditions. Pre-enrichment time and type of medium played an essential role in the detection of both microorganisms after drying. Furthermore, RT-PCR was more sensitive than VIDAS and the conventional method for detecting Salmonella Typhimurium and L. monocytogenes cells at different drying times and without pre-enrichment (0 h), with a detection range between 102 and 107 CFU/mL. TSBYE-T80 used as a pre-enrichment medium was effective for detecting both bacteria and was more effective than Demi Fraser-T80 medium for detecting L. monocytogenes. Therefore, pre-enrichment is recommended to avoid false positives and false negatives due to the presence of dead cells or a very low initial concentration of cells after drying.

Quantification of mature Listeria monocytogenes biofilm cells formed by an in vitro model: A comparison of different methods.

The presence of biofilms in food industrial environments is one of the main causes associated with food product contamination by L. monocytogenes. Biofilm control in the food industry is very relevant to public health and finding reliable and realistic quantification methods is essential. The aim of this study is to compare five L. monocytogenes biofilm quantification methods - conventional plate count, TEMPO, DEM, VIDAS and qPCR - and to examine a biodetector to visually detect biofilms in industrial settings. Results show that depending on the biofilm matrix production, the recovery of cells that conform the biofilm can be low and therefore, if it is an indirect method, microbial counts can be underestimated. At a species level, the methods that did not present significant differences were plate count, TEMPO (P = 0.998), DEM and qPCR (P = 0.508), so correlation studies were performed which established high correlation for plate count and TEMPO, but not for DEM and qPCR. The VIDAS method was adjusted so that it could quantify the biofilms, but the standard curve only allowed counts from 7 Log CFU cm-2. Results also revealed that the different strains of L. monocytogenes possess different biofilm-forming abilities, although it was not possible to correlate the capacity to produce these structures with the distinct serotypes. Last, visually detecting biofilms on stainless steel coupons proved that in industrial environments nowadays they can be rapidly and qualitatively detected so that relevant decisions can immediately be taken.