Production and analysis of a Bacillus subtilis biofilm comprised of vegetative cells and spores using a modified colony biofilm model.

Bacillus subtilis is a spore-forming soil bacterium that is capable of producing robust biofilms. Sporulation can occur in B. subtilis biofilms and it is possible that the spores embedded in the protective matrix could present a significant challenge to disinfecting agents or processes. This article describes a method for the growth and quantification of a reproducible B. subtilis ATCC 35021 biofilm comprised of vegetative cells and spores using a modified colony biofilm model. In this method, membranes were inoculated and incubated for a total of 8 days to promote biofilm formation and subsequent sporulation within the biofilm. Representative samples were taken over the course of the incubation period to evaluate the biofilms using enumerative, microscopic, and spectrometric methods. At various time points, the total numbers of cells and spores were quantified. A Congo red agar (CRA) method was utilized to detect the TasA matrix protein, a primary component of the B. subtilis biofilm matrix. The presence of TasA was also confirmed using mass spectrometry. The biofilm morphologies were correlated to the enumeration data with a variety of correlative imaging techniques: confocal microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). At the end of the incubation period, the biofilm contained >7 logs total colony forming units with spores comprising approximately 10% of the biofilm. The biofilm generated using this method allows researchers to use a new, more robust challenge for efficacy testing of chemical and physical antimicrobial treatments such as antibiotics, disinfectants, or heat.

Differential gene expression for investigation of Escherichia coli biofilm inhibition by plant extract ursolic acid.

After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 microg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 microg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 microg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 microg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid).

Detection of Staphylococcus aureus biofilm on tampons and menses components.

Culturing has detected vaginal Staphylococcus aureus in 10%-20% of women. Because growth mode can affect virulence expression, this study examined S. aureus-biofilm occurrence in 44 paired-tampon and vaginal-wash-specimens from 18 prescreened women, using fluorescent in situ hybridization (FISH). All 44 specimens were also analyzed for S. aureus by standard culturing on mannitol salt agar, which produced positive results for 15 of the 44 specimens. FISH detected S. aureus cells in all 44 specimens, and S. aureus biofilm was observed in 37 of the 44 specimens. Independent confirmation of the presence of S. aureus in specimens from all 18 women was also obtained by amplification, via polymerase chain reaction, of an S. aureus-specific nuclease gene. The results of this study demonstrate that S. aureus biofilm can form on tampons and menses components in vivo. Additionally, the prevalence of vaginal S. aureus carriage may be more prevalent than what is currently demonstrated by standard culturing techniques.