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

Abstract

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.

Biofilm-Forming Ability of Microbacterium lacticum and Staphylococcus capitis Considering Physicochemical and Topographical Surface Properties

“…This stress response mainly occurs as nutrients diminish and it enables cells to contribute to the preservation of the biofilm structure as well as to protect the system from energy loss [ 17 , 61 , 62 ]. Moreover, damaged or dead cells usually contribute to a greater extent to outer layers of the biofilm, which is known as a protection mechanism [ 63 ].EFM as an analytical method might over- or underestimate the proportion of live and dead cells, as the method only supports two-dimensional images, while biofilms have complex three-dimensional structures. With flow cytometry as an alternative approach for the viability analysis of biofilms, it is possible to elucidate this error as cell aggregates are dissolved, and single cells inside the biofilm are measurable [ 64 ].…”

Removal of Mixed-Species Biofilms Developed on Food Contact Surfaces with a Mixture of Enzymes and Chemical Agents

“…For example, the EPS matrix of S. Typhimurium is mainly composed of aggregative fimbriae and extracellular polysaccharides (cellulose) [ 29 ]. In contrast, proteins are the main compound in biofilms of Gram-positive bacteria [ 3 ]; however, they also produce polysaccharides as well as dextran [ 30 ]. Therefore, it is recommended to use a mixture of enzymes, because these molecules have specific activity [ 31 ].…”

“…The low removal of biofilms developed in TSB+EY on PP (12.2%) was attributed to the high content of lipids in the egg yolk, which were not decomposed by the enzymes applied [ 32 ]. Ripolles-Avila et al [ 3 ] achieved a removal of ~2.3 log CFU/cm 2 of S . Typhimurium on SS 304 with a mix of enzymes (protease, lipase, and amylase), which is in agreement with the findings in this study.…”

“…Nevertheless, the general composition of EPS includes polysaccharides, proteins, lipids, and extracellular DNA [ 1 , 2 ]. Biofilm development confers advantages to microbial cells, such as physical resistance to refrigeration, heat, desiccation, acidity, and salinity; mechanical resistance to liquid streams in pipelines; and chemical protection against antimicrobials and disinfectants [ 3 , 4 ]. Otherwise, biofilms cause corrosion in equipment, biofouling in water systems, and post-process contamination contributing to food spoilage.…”

Novel Techniques for Microbiological Safety in Meat and Fish Industries

“…Some of the foodborne pathogens that can be suppressed by spoilage microbes are B. cereus, C. jejuni, C. botulinum, E. coli, L. monocytogens and Salmonella spp. Moreover, Pseudomonas and Shewanella putrefaciens and lactic acid bacteria also have the substantial potential to limit the bacterial pathogens' growth activities [17]. Lactic acid bacteria and yeast has a combined effect on Coliforms and Salmonella, while yeast also influences lactic acid bacteria [18].…”