B. Orgaz

Effectiveness of Chitosan against Mature Biofilms Formed by Food Related Bacteria

Chitosan has proven antimicrobial properties against planktonic cell growth. Little is known, however, about its effects on already established biofilms. Oriented for application in food industry disinfection, the effectiveness of both medium molecular weight (MMW) chitosan and its enzymatically hydrolyzed product was tested against mature biofilms of four pathogenic strains, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus and Salmonella enterica, and a food spoilage species, Pseudomonas fluorescens. Unexpectedly, log reductions were in some cases higher for biofilm than for planktonic cells. One hour exposure to MMW chitosan (1% w/v) caused a 6 log viable cell reduction on L. monocytogenes monospecies mature biofilms and reduced significantly (3–5 log reductions) the attached population of the other organisms tested, except S. aureus. Pronase-treated chitosan was more effective than MMW chitosan on all tested microorganisms, also with the exception of S. aureus, offering best results (8 log units) against the attached cells of B. cereus. These treatments open a new possibility to fight against mature biofilms in the food industry.

Pathogens protection against the action of disinfectants in multispecies biofilms.

Biofilms constitute the prevalent way of life for microorganisms in both natural and man-made environments. Biofilm-dwelling cells display greater tolerance to antimicrobial agents than those that are free-living, and the mechanisms by which this occurs have been investigated extensively using single-strain axenic models. However, there is growing evidence that interspecies interactions may profoundly alter the response of the community to such toxic exposure. In this paper, we propose an overview of the studies dealing with multispecies biofilms resistance to biocides, with particular reference to the protection of pathogenic species by resident surface flora when subjected to disinfectants treatments. The mechanisms involved in such protection include interspecies signaling, interference between biocides molecules and public goods in the matrix, or the physiology and genetic plasticity associated with a structural spatial arrangement. After describing these different mechanisms, we will discuss the experimental methods available for their analysis in the context of complex multispecies biofilms.

Listeria monocytogenes Impact on Mature or Old Pseudomonas fluorescens Biofilms During Growth at 4 and 20°C

Changes in spatial organization, as observed by confocal laser scanning microscopy (CLSM), viable cell content, biovolume, and substratum surface coverage of the biofilms formed on glass by Pseudomonas fluorescens resulting from co-culture with Listeria monocytogenes, were examined. Two strains of L. monocytogenes, two culture temperatures and two biofilm developmental stages were investigated. Both L. monocytogenes strains, a persistently sampled isolate (collected repeatedly along 3 years from a meat factory) and Scott A, induced shrinkage in matrix volume, both at 20°C and 4°C, in mature or old biofilms, without loss of P. fluorescens cell count per surface unit. The nearly homogeneous pattern of surface coverage shown by mono-species P. fluorescens biofilms, turned into more irregular layouts in co-culture with L. monocytogenes. The upper layer of both mono and dual-species biofilms turned to predominantly consist of matrix, with plenty of viable cells underneath, in old biofilms cultured at 20°C, but not in those grown at 4°C. Between 15 and 56% of the substratum area was covered by biofilm, the extent depending on temperature, time and L. monocytogenes strain. Real biofilms in food-related surfaces may thus be very heterogeneous regarding their superficial components, i.e., those more accessible to disinfectants. It is therefore a hygienic challenge to choose an adequate agent to disrupt them.

Effect of the presence of titania nanoparticles in the development of Pseudomonas fluorescens biofilms on LDPE

In this study the use of TiO2 nanoparticles in the preparation of active packaging film materials is investigated. High energy ball milling was used to uniformly disperse TiO2 nanoparticles within low density polyethylene, LDPE. Differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) were used to characterize the nanocomposites. Growth of Pseudomonas fluorescens and subsequent bio-film formation on the surfaces of LDPE with and without TiO2 nanoparticles were studied with Atomic Force Microscopy (AFM) and viable cell counts. A set of samples placed either face down or face-up in microwell plates were subsequently immersed in P. fluorescens cultures and incubated for up to 48 h at 4 or 30 °C. AFM images showed that the presence of titania nanoparticles affects the growth, size, distribution and arrangement of bacteria on the polymer surfaces. Cell recovery and counting experiments revealed a reduction of at least 1-log (i.e. 90% reduction) in bacterial colony forming units per square centimeter (CFU cm−2) for the TiO2 nanofilled polymer compared to LDPE films, without photoactivation. In the presence of TiO2 nanoparticles, bacterial cells attached to the surfaces formed tight aggregates with apparently minor amounts of “extracellular polymer substances” (EPS) around them.

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.

Patterns of biofilm structure and formation kinetics among Acinetobacter baumannii clinical isolates with different antibiotic resistance profiles

Acinetobacter baumannii is a ubiquitous organism that has been involved in a wide range of nosocomial infections. Its ability to produce biofilms, among other characteristics, allows it to persist in hospitals for prolonged periods. In this study, in order to check the possible relationship between its resistance to different antibiotics and its ability to form biofilms on inert surfaces, the rate of biofilm formation as well as siderophore production and detection of OmpA and CsuE by PCR were investigated for 12 A. baumannii clinical isolates. The biofilms were cultured at 37 °C on steel coupons immersed in BHI broth and the attached viable cells were counted after 5, 24 and 48 h. Confocal Laser Scanning Microscopy (CLSM) images were obtained for some of the strains that were noted to produce a brown pigment. The biofilm volume and substratum coverage were estimated with an image analysis software program. Our data, though preliminary, show that the quicker biofilm formers were strains susceptible to aminoglycosides, whereas the biofilms providing thicker and more uniform surface coverage were produced by carbapenem-resistant strains, producing a brown pigment with a plausible siderophore role. Further investigation into a wider set of isolates could help better understand the relationship between biofilm formation and various clinical findings.

Listeria monocytogenes Colonizes Pseudomonas fluorescens Biofilms and Induces Matrix Over-Production

In food facilities, biofilms or their debris might act as helpers for attracting free floating microorganisms. In this sense, Pseudomonas fluorescens, a dense biofilm producer frequently isolated from food contact surfaces, could be a good candidate for sheltering other microorganisms, such as Listeria monocytogenes. The main objective of this work was to evaluate the ability of L. monocytogenes to colonize pre-established Pseudomonas biofilms. For this, the movement throughout mature Pseudomonas biofilms of a green fluorescent protein (GFP) – tagged strain of L. monocytogenes was tracked for 24 h by confocal laser scanning microscopy (CLSM). Moreover, in order to check the effect of the incorporation of Listeria on the overall matrix production, attached populations of both microorganisms and total biomass (cells + matrix) of the resulting biofilms were measured over time. Planktonic cells of L. monocytogenes efficiently migrated to preformed P. fluorescens biofilms. Moreover, they moved preferentially toward the bottom layers of these structures, suggesting some kind of tropism. When preformed P. fluorescens biofilms were conditioning the surfaces, the L. monocytogenes attached population was on average, 1–2 Log higher than when this organism grew on bare coupons. Furthermore, the arrival of L. monocytogenes to the already established P. fluorescens biofilms led to a matrix over-production. Indeed, biomass values [optical density (OD595 nm)] of the resulting biofilms were double those of the ordinary L. monocytogenes–P. fluorescens mixed biofilms (1.40 vs. 0.6). The fact that L. monocytogenes cells accumulate in the bottom layers of preformed biofilms provides this microorganism an extra protection toward physical–chemical damages. This might partly explain why this microorganism can persist in food industry environments.