Biofilms in Focus : A Threat to Foods

The world of food microbiology, and by extension that of food safety, has evolved significantly at the turn of the century. In fact, from carrying out controls on the presence or absence of pathogens in finished products, we have gone on to try to detect, not only where the pathogens are found, to apply the appropriate corrective measures to the problem, but also to look beyond. At this point, the number of publications related to biofilms, considered as forms of resistance of microorganisms in food facilities, has increased significantly. Thus, according to PubMed, since 2010, 1494 articles have been published on biofilms and food safety. Therefore, the study of biofilms is being considered as an important element to improve food safety conditions nowadays

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

Altres ajuts: acords transformatius de la UABThe 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

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

Altres ajuts: acord transformatiu CRUE- CSICAntimicrobial 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 straindependent trend. Moreover, species-dependent differences were observed in the conformation of the extracellular matrix

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

Altres ajuts: acord transformatiu CRUE-CSICConventional 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/cm, 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

Microbial ecology evaluation of an iberian pig processing plant through implementing sch sensors and the influence of the resident microbiota on Listeria monocytogenes

This study was supported by Research Project grants RTI2018-098267-R-C32 from the Spanish Ministerio de Ciencia, Innovaci?n y Universidades. Acknowledgments: The authors thank Dolors Busquets Soler for her technical assistance in the laboratory and Sarah Davies for the English grammar review.There is a whole community of microorganisms capable of surviving the cleaning and disinfection processes in the food industry. These persistent microorganisms can enhance or inhibit biofilm formation and the proliferation of foodborne pathogens. Cleaning and disinfection protocols will never reduce the contamination load to 0; however, it is crucial to know which resident species are present and the risk they represent to pathogens, such as Listeria monocytogenes, as they can be further used as a complementary control strategy. The aim of this study was to evaluate the resident surface microbiota in an Iberian pig processing plant after carrying out the cleaning and disinfection processes. To do so, surface sensors were implemented, sampled, and evaluated by culture plate count. Further, isolated microorganisms were identified through biochemical tests. The results show that the surfaces are dominated by Bacillus spp., Pseudomonas spp., different enterobacteria, Mannheimia haemolytica, Rhizobium radiobacter, Staphylococcus spp., Aeromonas spp., lactic acid bacteria, and yeasts and molds. Moreover, their probable relationship with the presence of L. monocytogenes in three areas of the plant is also explained. Further studies of the resident microbiota and their interaction with pathogens such as L. monocytogenes are required. New control strategies that promote the most advantageous profile of microorganisms in the resident microbiota could be a possible alternative for pathogen control in the food industry. To this end, the understanding of the resident microbiota on the surfaces of the food industry and its relation with pathogen presence is crucial

Pathogenic mono-species biofilm formation on stainless steel surfaces : quantitative, qualitative, and compositional study

Acord transformatiu CRUE-CSICThe aims of this study were, first, to determine Listeria monocytogenes, Salmonella enterica, ser. Typhimurium, and Staphylococcus aureus biofilm counts, structure, and their composition in macromolecules by direct epifluorescence microscopy (DEM); and second, to evaluate the distribution of the components of the produced biofilms through 3D representations obtained by confocal laser scanning microscopy (CLSM). Results showed that all assessed strains have a high capacity to generate biofilms, with counts greater than 7 log CFU cm⁻ in all cases. The highest and lowest survival percentages were obtained by S. aureus with 94.13 ± 1.77% and S. Typhimurium with 60.31 ± 4.45%, respectively. Biofilm matrix composition in macromolecules was as follows: (a) L. monocytogenes: proteins 83.35 ± 5.81%, polysaccharides 10.98 ± 5.77%, and extracellular DNA 5.67 ± 2.5%; (b) S. Typhimurium: 83.37 ± 3.35%, 0.68 ± 0.77%, and 15.95 ± 3.39%, respectively; and (c) S. aureus: 82.55 ± 2.39%, 8.48 ± 2.07%, and 8.97 ± 2.27%, respectively. The qualitative analysis of the 3D representations of the biofilms formed by the pathogens modelized showed no homogeneity and/or ordered distribution of their components within the biofilm architecture. These findings could lead to the development of addressed cleaning and disinfection alternatives

In Vitro Preformed Biofilms of Bacillus safensis Inhibit the Adhesion and Subsequent Development of Listeria monocytogenes on Stainless-Steel Surfaces

Listeria monocytogenes continues to be one of the most important public health challenges for the meat sector. Many attempts have been made to establish the most efficient cleaning and disinfection protocols, but there is still the need for the sector to develop plans with different lines of action. In this regard, an interesting strategy could be based on the control of this type of foodborne pathogen through the resident microbiota naturally established on the surfaces. A potential inhibitor, Bacillus safensis, was found in a previous study that screened the interaction between the resident microbiota and L. monocytogenes in an Iberian pig processing plant. The aim of the present study was to evaluate the effect of preformed biofilms of Bacillus safensis on the adhesion and implantation of 22 strains of L. monocytogenes. Mature preformed B. safensis biofilms can inhibit adhesion and the biofilm formation of multiple L. monocytogenes strains, eliminating the pathogen by a currently unidentified mechanism. Due to the non-enterotoxigenic properties of B. safensis, its presence on certain meat industry surfaces should be favored and it could represent a new way to fight against the persistence of L. monocytogenes in accordance with other bacterial inhibitors and hygiene operations

Development of a dairy fouling model to assess the efficacy of cleaning procedures using alkaline and enzymatic products

Dairy fouling is defined as the accumulation of thermally insulating materials or deposits from process fluids which are especially formed on heat transfer surfaces. The selection of suitable cleaning strategies to remove dairy fouling requires the understanding of its composition and the relationships with the surfaces where it is formed. For the industry, the development of novel strategies to test cleaning products, as well reducing water and energy consumption during the dairy processing operations is of enormous interest. The results showed the development of a laboratory-milk fouling model (MFM) with an average content of 52.8 mg/cm2 of fouling in the test coupons. Seven different cleaners were tested with a fouling removal effectiveness of between 55% and 97%. Additionally, for evaluating the cleaning process of the model, the turbidity of the cleaning solutions was assessed. We presented an enzymatic alternative to the use of traditional cleaning products, with a similar efficacy against the dairy fouling. 78% of fouling removal after the use of enzymatic solution, in comparison to the 72% of fouling removal after the use of alkaline cleaning products. A reduction in water (−33.3%) and temperature (−28.5%), as well as shorter cleaning times (−33%) than its chemical alternative, was observed.info:eu-repo/semantics/acceptedVersio

Quantitative and Compositional Study of Monospecies Biofilms of Spoilage Microorganisms in the Meat Industry and Their Interaction in the Development of Multispecies Biofilms

Food spoilage is a serious problem in the food industry, since it leads to significant economic losses. One of its main causes is the cross-contamination of food products from industrial surfaces. Three spoilage bacterial species which are highly present in meat and the gastrointestinal tract of chickens were selected: Pseudomonas fragi, Leuconostoc gasicomitatum, and Lactobacillus reuteri. The dual aim was to determine their ability to form monospecies biofilms and to examine how they interact when they coexist together. To do so, mature monospecies biofilms were produced statically for seven days at a temperature of 30 ◦C. L. gasicomitatum was also used to investigate the behavior of P. fragi and L. reuteri in the formation of multispecies biofilms. The structure and composition of the monospecies biofilms were evaluated by direct epifluorescence microscopy, and the multispecies biofilms were evaluated by plate counting. Both L. gasicomitatum and L. reuteri were able to form biofilms, with counts of approximately 7 Log CFU/cm2 and a defined structure. However, P. fragi obtained counts to the order of 4 Log CFU/cm2 , which is significantly different from the previous species (P < 0.05), and it had no network of cell conglomerates. The content of the L. gasicomitatum and L. reuteri biofilm matrices were 70-80% protein, unlike P. fragi, which presented a higher polysaccharide content (P < 0.05). In the multispecies biofilms, the presence of P. fragi did not affect the growth of L. gasicomitatum, which remained at between 5.76 to 6.1 Log CFU/cm2 . However, L. reuteri was able to displace L. gasicomitatum growth after 24 h of coexisting in a mixed biofilm, presenting differences in counts of approximately 2 Log CFU/cm2 . The study of the biofilms constructed by food industry resident microbiota can help to understand the ecological relations that exist between species, characterize them, and propose strategies to eliminate them. The name of genes and species should be written in italic