Fig 1.
Inhibitory effect of MMP1 on E. faecalis biofilms.
Absorption of CV stain at 635 nm (OD635) of MMP1-treated biofilms of E. faecalis FA2-2 and V583 strains over seven days. Experiments with BHI media and protein buffer were used as controls. For inhibition experiments, biofilms were grown in presence of MMP1 from day0 to day7. Typical images of wells in microtiter plates are given at the top of each panel. * indicates p-value: *<0.01, **<0.01, and ***<0.001. Error bars on the data points represent the standard deviations of 3 technical repeats.
Fig 2.
Disruptive effect of MMP1 on E. faecalis biofilms.
Absorption of CV stain at 635 nm (OD635) of MMP1-treated biofilms of FA2-2 and V583 strains over seven days. Experiments with BHI media and protein buffer were used as controls. For disruption experiments, biofilms were first grown in BHI media without MMP1 for desired duration, followed by MMP1 treatment. Typical images of wells in microtiter plates are given at the top of each panel. * indicates p-value: *<0.01, **<0.01, and ***<0.001; ns indicates that the effect is not significant. Error bars on the data points represent the standard deviations of 3 technical repeats.
Fig 3.
SEM micrographs of established E. faecalis biofilms.
Biofilms were first grown for 3 to 7 days and then treated with MMP1. (a) Vancomycin susceptible strain FA2-2 and (b) Vancomycin resistant strain V583. In comparison to the control experiments, active MMP1 led to disruption of biofilms resulting in more empty spaces without any bacteria.
Fig 4.
Confocal microscopy of E. faecalis biofilms.
Biofilms were grown for 3 to 7 days in the presence of MMP1. (a) Vancomycin susceptible strain FA2-2 and (b) Vancomycin resistant strain V583. Orange and green areas indicate the presence of live and dead bacteria respectively. The amount of live and dead bacteria did not show consistent pattern because of the compounding effects of natural cell death, Triton X100, MMP1, and sample preparation.
Fig 5.
Colony forming unit (CFU) assay to quantify viable cells in E. faecalis biofilms.
Quantification (Log 10 CFU) of live bacterial cells in 1, 3 and 7 day old E. faecalis FA2-2 and V583 biofilms treated with and without MMP1 under inhibition (a) and disruption (b) conditions. For inhibition experiments, biofilms were grown in presence of MMP1 from day0 to day7. For disruption experiments, biofilms were first grown in BHI media without MMP1 for desired duration, followed by MMP1 treatment. * indicates p-value: *p<0.01, **p<0.01 and ***p<0.001; ns indicates that the effect is not significant. Error bars on the data points represent the standard deviations of 3 technical repeats.
Fig 6.
Congo red staining assay to quantify effects of MMP1 on E. faecalis biofilms.
Absorption of Congo red stain at 490 nm (OD490) of 1, 3 and 7 day old E. faecalis FA2-2 and V583 biofilms treated with and without MMP1 under inhibition (a) and disruption (b) conditions Experiments with BHI media and protein buffer were used as controls. For inhibition experiments, biofilms were grown in presence of MMP1 from day0 to day7. For disruption experiments, biofilms were first grown in BHI media without MMP1 for desired duration, followed by MMP1 treatment. Typical images of wells in microtiter plates are given at the top of each panel. * indicates p-value: *p<0.01, **p<0.01 and ***p<0.001; ns indicates that the effect is not significant. Error bars on the data points represent the standard deviations of 3 technical repeats.
Fig 7.
Effect of MMP1 on E. faecalis growth.
Growth curves of E. faecalis in presence of active MMP1, BHI media, and protein buffer for (a) FA2-2 and (b) V583 strains respectively. Symbols represent data points. Solid lines are fits to the logistic equation of bacterial growth, y = a/{1+bexp(−kt)}, where k represents the growth rate. Error bars on the data points represent the standard deviations of 6 technical repeats. For FA2-2 strain, the best fit parameters are: a = 0.85±0.01, b = 24.98±4.44, k = 1.38±0.08 (BHI media); a = 0.81±0.01, b = 32.53±7.02, k = 1.49±0.09 (protein buffer); a = 0.80±0.01, b = 36.62±8.88, k = 1.53±0.10 (inactive MMP1); a = 0.71±0.02, b = 8.47±1.22, k = 0.79±0.06 (active MMP1). For V583 strain, the best fit parameters are: a = 0.96±0.01, b = 13.41±1.20, k = 1.05±0.04 (BHI media); a = 0.92±0.01, b = 7.70±0.62, k = 0.85±0.04 (protein buffer); a = 0.92±0.01, b = 7.73±0.65, k = 0.88±0.04 (inactive MMP1); a = 0.75±0.01, b = 10.12±1.21, k = 0.96±0.05 (MMP1). For fit parameters, error bars represent the standard error of the mean.
Fig 8.
Mechanism of MMP1 antibiofilm activity against E. faecalis.
MMP1 degrades cell-associated proteins and biofilm matrix components) leading to poor cell adhesion, bacterial growth inhibition, biofilm inhibition, and disruption (proteins 1–6 are hypothetical biofilm-associated proteins to illustrate the mechanism of MMP1 activity).