TSST-1 production in biofilms in tampon sacs.

<p><i>Staphylococcus aureus</i> MN8 (10⁷/0.1 ml) were placed inside dialysis tubes that had been tied off on one end, and then tampons were inserted. The tampon sacs were immersed beneath Todd Hewitt 0.8% agar such that the open end of the dialysis tubing remained above the surface. The agar was allowed to solidify, and the tampons sacs were incubated for indicated times. TSST-1 was measured within the dialysis tube by knowing the pre- and post-weight of the tampons, eluting TSST-1 with distilled water, and quantifying TSST-1 by Western immunoblot analysis.</p

Community-associated <i>Staphylococcus aureus</i> 128 growing as biofilms on tampon fibers and cellulose acetate dialysis tubing.

<p><i>Staphylococcus aureus</i> 128, a TSST-1⁺ organism at 10⁷/0.1 ml, was inoculated onto the inside of pre-wetted dialysis tubing that had been tied off on one end. A tampon was then inserted into the dialysis tubing and the tubing immersed under Todd Hewitt broth containing 0.8% agar. The open end of the dialysis tubing remained above the agar surface such that the only source of nutrients for the growing microbes was media absorbed across the dialysis tubing. Magnification of A was 6000x and B was 9000x. Both frames show organisms embedded in a complex matrix of extracellular material.</p

Effect of GML on non-typable <i>Haemophilus influenzae</i> cultured in biofilms.

<p><i>Haemophilus influenzae</i> was cultured for 24 and 48 hours in a 96 well microtiter plate. In one set of three wells for the microbe, the wells were agitated 3 times by pipetting up and down and then supernates removed for plate counting to determine CFUs/ml. * indicates significant mean reduction compared to starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size. Subsequent to removing bacterial cells and washing three times with phosphate-buffered saline, the wells were treated with crystal violet for 30 min. The wells were then washed three times with phosphate-buffered saline to remove unbound crystal violet. Finally, the wells were treated with ethanol to solubilize biofilm-associated crystal violet. Absorbances at 595 nm were determined by an ELISA reader. # indicates significant mean reduction in absorbance at 595 nm wavelength between no GML and GML-treated wells at p<0.01.</p

Antibacterial activity of non-aqueous gel alone and in combination with glycerol monolaurate against <i>Staphylococcus aureus</i>.

<p>Non-aqueous gel (v/v) was incubated with 1 x 10⁶ CFU/ml <i>Staphylococcus aureus</i> for 24 h at 37°C at high aeration (shaking, 225 revolutions/min). Plate counts were used to determine CFU/ml, and error bars represent standard deviation of three replicates (A). Sub-growth-inhibitory concentration of non-aqueous gel was also delivered with glycerol monolaurate and bacteria, and CFU/ml was likewise determined (B).</p

Effect of pH on GML activity against <i>Escherichia coli</i> Watson strain.

<p>Todd Hewitt broth was buffered to pH 7.0, 6.0, and 5.0. Susceptibility of <i>Escherichia coli</i> to GML at the indicated pH was measured in 24 hour assays. CFUs/ml were determined by plate counts. * indicates means of No GML samples and GML samples differ significantly at p<0.001. Dashed line indicates the inoculum size.</p

Ability of pretreatment of GML with <i>Staphylococcus aureus</i> MN8 spent culture fluids to reduce antimicrobial activity.

<p><i>Staphylococcus aureus</i> MN8 or <i>Streptococcus pyogenes</i> T25₃curedT12 sterile stationary phase culture fluids were incubated with GML (1000 μg) for 24 hours. Subsequently, treated GML was evaluated for antibacterial activity against <i>Staphylococcus aureus</i> MN8 in a 24 hour assay, in comparison to untreated GML. * indicates mean significantly reduced from starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size.</p

Effect of a nonaqueous (NA) gel on GML antibacterial activity against <i>Staphylococcus aureus</i> MN8.

<p>Non-aqueous gel was diluted to 25% and 10% with Todd Hewitt broth. GML and <i>Staphylococcus aureus</i> MN8 were added, and cultures were incubated for 24 hours. CFU/ml were determined by plate counts. Dashed line indicates the inoculum size. * indicates mean differs significantly from the GML alone control at p<0.001.</p

Anti-biofilm activity of glycerol monolaurate against <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Acinetobacter baumannii</i>.

<p>Stationary biofilms were cultured in 96-well microtiter plates and treated with sub-growth-inhibitory concentrations of glycerol monolaurate. Twenty-four hours post-exposure, media was removed, wells were washed 3 times with PBS, biofilms were fixed with ethanol (<i>Staphylococcus aureus</i> biofilms only), and wells were treated with crystal violet. Wells were then washed to remove unbound crystal violet. Remaining crystal violet was solubilized with 33% acetic acid (<i>Staphylococcus aureus</i>) or 95% ethanol (<i>Pseudomonas aeruginosa</i> or <i>Acinetobacter baumannii</i>) and absorbance was determined by Tecan reader. Pre-formed and forming <i>Staphylococcus aureus</i> biofilms treated with sub-growth-inhibitory concentration of glycerol monolaurate and assessed for effect on biomass removal at 24 h post-treatment (A) and across time (B). Pre-formed <i>Pseudomonas aeruginosa</i> and <i>Acinetobacter baumannii</i> were treated with glycerol monolaurate and assessed for biomass remaining at 24 h post-treatment (C,D respectively). Bars represent standard deviation of at least three independent replicates.</p

Ability of GML to remove pre-formed biofilms.

<p><i>Staphylococcus aureus</i> MN8 and nontypable <i>Haemophilus influenzae</i> were grown for 48 hours in 96 well microtiter plates to allow biofilm formation. CFUs/ml were determined after suspension of organisms (A). Subsequently, the wells were treated or not treated with GML (500 μg/ml) for 60 min, and then CFUs/ml determined again (A). * indicates significant mean reduction compared to starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size. Wells were stained with crystal violet after removal of non-adherent bacteria or after removal of non-adherent bacteria and treatment with GML, followed by removal of unbound crystal violet and determination of absorbance at 595 nm (B). # indicates significant mean reduction in absorbance at 595 nm wavelength between no GML and GML-treated wells at p<0.01.</p

Ability of R versus S and 1/3 versus 2 forms of GML to inhibit growth of <i>Streptococcus pyogenes</i> 594.

<p>R form and mixture of R and S forms of GML and 2 position lauric acid versus mixture of 1/3 and 2 position lauric of GML were incubated with <i>Streptococcus pyogenes</i> 594, a highly susceptible organism to GML, for 24 hours. Plate counts were used to determine GML killing. * indicates the mean for the 2 form of GML is significantly different from the mixture at p<0.001. Dashed line indicates starting inoculum size.</p