P. Rodríguez-López

Numerical spatio-temporal characterization of Listeria monocytogenes biofilms

As the structure of biofilms plays a key role in their resistance and persistence, this work presents for the first time the numerical characterization of the temporal evolution of biofilm structures formed by three Listeria monocytogenes strains on two types of stainless-steel supports, AISI 304 SS No. 2B and AISI 316 SS No. 2R. Counting methods, motility tests, fluorescence microscopy and image analysis were combined to study the dynamic evolution of biofilm formation and structure. Image analysis was performed with several well-known parameters as well as a newly defined parameter to quantify spatio-temporal distribution. The results confirm the interstrain variability of L. monocytogenes species regarding biofilm structure and structure evolution. Two types of biofilm were observed: homogeneous or flat and heterogeneous or clustered. Differences in clusters and in attachment and detachment processes were due mainly to the topography and composition of the two surfaces although an effect due to motility was also found.

Removal of Listeria monocytogenes dual-species biofilms using combined enzyme-benzalkonium chloride treatments

Abstract The effects of pronase (PRN), cellulase (CEL) or DNaseI alone or combined with benzalkonium chloride (BAC) against Listeria monocytogenes-carrying biofilms were assayed. The best removal activity against L. monocytogenes–Escherichia coli biofilms was obtained using DNaseI followed by PRN and CEL. Subsequently, a modified logistic model was used to quantify the combined effects of PRN or DNaseI with BAC. A better BAC performance after PRN compared to DNaseI eradicating L. monocytogenes was observed. In E. coli the effects were the opposite. Finally, effects of DNaseI and DNaseI–BAC treatments were compared against two different L. monocytogenes-carrying biofilms. DNaseI–BAC was more effective against L. monocytogenes when co-cultured with E. coli. Nonetheless, comparing the removal effects after BAC addition, these were higher in mixed-biofilms with Pseudomonas fluorescens. However, a high number of released viable cells was observed after combined treatments. These results open new perspectives of enzymes as an anti-biofilm strategy for environmental pathogen control.

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes–Escherichia coli dual-species biofilms

Abstract This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes–Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r2 = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.

Biofilm Formation of Staphylococcus aureus

Abstract Biofilm is considered as part of the normal life cycle of Staphylococcus aureus in the environment, in which planktonic cells present attach to solid surfaces, proliferating and accumulating in multilayer cell clusters embedded in an organic polymer matrix. The structural components of this extracellular matrix, mainly proteins and polysaccharides, allow the accumulation of biomass, as well as the cohesion and stabilization of the biofilm. In addition, the presence of extracellular DNA into the biofilm matrix can promote the horizontal gene transfer and, thus, the spreading of antimicrobial resistances between embedded cells. Biofilm protects the bacterial community from environmental stresses, from the host immune system, and from antimicrobial attacks, as opposed to the situation for exposed planktonic cells. Moreover, the dispersal of biofilm cells in clumps may provide a sufficient number of cells for an infective dose that is not typically found in bulk fluid, enabling an enhanced transmission and infection of S. aureus.

Current Knowledge on Listeria monocytogenes Biofilms in Food-Related Environments: Incidence, Resistance to Biocides, Ecology and Biocontrol

Although many efforts have been made to control Listeria monocytogenes in the food industry, growing pervasiveness amongst the population over the last decades has made this bacterium considered to be one of the most hazardous foodborne pathogens. Its outstanding biocide tolerance capacity and ability to promiscuously associate with other bacterial species forming multispecies communities have permitted this microorganism to survive and persist within the industrial environment. This review is designed to give the reader an overall picture of the current state-of-the-art in L. monocytogenes sessile communities in terms of food safety and legislation, ecological aspects and biocontrol strategies.