Sensitivity of bacterial biofilms and planktonic cells to a new antimicrobial agent, Oxsil 320N.

The effective concentrations of disinfectants were determined for planktonic bacteria using the norms EN 1040 and NF T 72-150. This concentration corresponds to biocide efficacy after 5 min of contact, followed by neutralization. However, micro-organisms often colonize a substratum and form microcolonies or biofilms where they are enclosed in exopolymer matrices. Biofilms are commonly resistant to a broad range of antimicrobial agents, and resistance mechanisms involve exopolymer matrices, changes in gene expression and metabolic alterations. Due to these different resistance mechanisms, it is difficult to select and titrate antimicrobial agents to be effective against biofilms. In this context, SODIFRA developed a new disinfectant, Oxsil 320N (French patent 94 15 193). Oxsil 320N is an association of three active principles: hydrogen peroxide, acetic acid/peracetic acid and silver. This biocide was tested on planktonic bacteria and on 24-h biofilms formed on AISI 304 stainless steel surfaces. The effective concentration of Oxsil 320N was also determined on biofilms using SODIFRA recommendations (without neutralization of the biocide). Data showed that the antimicrobial efficacy measured on planktonic bacteria is not a reliable indicator of performance when biofilm is present. When biofilms were exposed to Oxsil 320N, the concentration needed to achieve a 10(5)-fold decrease in concentration was 10 times higher than that for bacterial suspensions (0.313% Oxsil 320N). An effective concentration of Oxsil 320N of 3.13% was required.

[Compared viability of planctonic bacteria and bacteria in biofilms by flow cytometry]. / Comparaison de la vitalité des bactéries libres et des bactéries en biofilm par cytométrie en flux.

Study of biofilms adhering to various surfaces shows the presence of several layers of cells. A small percentage can grow immediately. Are the others viable? These cells are in a resting phase and exhibit a metabolic gradient through the depth of the biofilm. In order to assess the health hazard of active recovery of these resting cells, we studied biofilms with two fluorochromes: a fluorescein diacetate derivative, CDF, which detects esterase activity and gives a green fluorescence to cells, and propidium iodide, IP, which enters cells with injured membranes and gives a red fluorescence to nucleic acids. Confocal microscopy can be used to localized red and green cells at various depths in biofilms but flow cytometry is necessary for a semi-quantitative analysis. We focused on parameters required to obtain valid flow cytometry results. After elimination of bacterial and glycocalyx autofluoresce and choosing non-toxic marker concentrations flow cytometry revealed a large proportion of double-marked cells, i.e. cells exhibiting esterase synthesis and membrane injuries. The question is whether these cells are a public health hazard. Can these cells repair their injuries and recover viability when they leave the biofilm? Do they recover metabolic activity, virulence, and sensitivity to antibacterials? Flow cytometry enabled the identification of four levels of viability in oral streptococci biofilms.

Evaluation of biohazards in dehydrated biofilms on foodstuff packaging.

Plastic materials used for food packaging are clean but not sterile when the food is just packaged. Accidental wet contamination may occur at every moment between packaging and opening by the consumer: on polyethylene (PET), bacteria may adhere strongly and constitute a biofilm in less than 24 h. By rolling on themselves, PET sheets may contaminate food. We tried to show that contact with salted foodstuffs favoured microbial recovery. Four strains were chosen to perform biofilms on PET: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli. Biofilms were dried up 24 h. Biofilm bacteria were stressed by adhesion, by starvation and by dehydration. However, they were capable of recovery in salted solutions or media, probably because one (or more) stress protected them against another stress. Stress was demonstrated by stress protein production, by mean of electrophoresis, and membrane lesions by mean of flow cytometry. Stress recovery was performed in aqueous salted solutions or salted brain-heart infusion with NaCl 9, 15, 20 and 30 g/l. Staphylococci were more sensitive to these stresses and recovery was a function of salt concentration. Gram-negative bacteria were little affected by stresses; salt effects were less important. If all these biofilms were capable of recovery from stresses in salted media, flexible PET could possibly lead to a health hazard when it is used for wet salt meats, e.g.

[Prevention of cariogenic dental plaque. Study of the structures implicated in the adhesion and coaggregation in Streptococcus mutans and Streptococcus sobrinus]. / Prévention de la plaque dentaire cariogène étude des structures impliquées dans l'adhésion et la coagrégation chez Streptococcus mutans et Streptococcus sobrinus.

Cariogenic dental plaque may be assimilated to a biofilm resulting from the adhesion of S. mutans, then from the coaggregation of other streptococci, or other genus. We used a static monospecific biofilm model. Supports or bacteria were treated with inhibitors before adhesion in order to clarify the nature of adhesins responsible for the primary adhesion of S. mutans and S. sobrinus on Tygon. To determine the bindings of coaggregation, inhibitors were applied on one-day-old biofilms. Analysis of effects were performed by automatic inoculator Spiral (Interscience) for microbiological methods, and by SEM JEOL 5400 LV for microscopic methods. In the aim of preventing adhesion and coaggregation, different traps were assayed:sugars, chemical inhibitors such as F- and EDTA salts. Of these, only the latter showed efficiency. This confirmed the role of bivalent mineral ions and electrostatic attraction forces in the adhesion and coaggregation of streptococci.