Regrowth of Microcosm Biofilms on Titanium Surfaces After Various Antimicrobial Treatments

Abstract

Objectives: Our aim of this work was to investigate the regrowth of implant-related biofilms after various antimicrobial treatments in vitro. Methods: Saliva-derived microcosm biofilms were grown on titanium discs in an active attachment model. Treatments including hydrogen peroxide (HP), citric acid (CA), chlorhexidine (CHX), and distilled water (control), at different concentrations, were applied to 2-day biofilms for 1 or 5 min. The viability, lactic acid production, and composition of the biofilms were followed for 3 days. The biofilm composition was analyzed by 16S rDNA amplicon sequencing. Results: The short treatments of CA, CHX, and HP resulted in a 2-3 log reduction in biofilm viability and lactic acid production immediately. However, both parameters returned to the pre-treatment level within 2 days due to biofilm regrowth. The alpha diversity of the regrown biofilms in antimicrobial-treated groups tended to decrease, whereas the diversity of those in water-treated group increased. The composition of the regrown biofilms altered compared to those before treatments. Streptococcus and Enterobacteriaceae were enriched in the regrown biofilms. Conclusions: Although the antimicrobial treatments were efficient, the multi-species biofilms were indeed able to regrow within 2 days. The regrown biofilms display an altered microbial diversity and composition, which in the oral cavity may lead to an aggressive infection.

Keywords: 16S rDNA; antimicrobials; biofilm regrowth; microcosm; oral microbiome; peri-implantitis.

Figure 1

Experimental design of this study. CA, citric acid; CHX, chlorhexidine; HP, hydrogen peroxide; L, low treatment concentration/short duration; H, high treatment concentration/long duration.

Figure 2

Viable cell counts of biofilms after different decontamination treatments at different time points. Data are presented as mean + SD. * represents statistically significant difference compared with day-3 biofilms of Water control group (Water_day 3) (p < 0.05). For clarity, the Water_day 3 data were repeated at days 4 and 5. CA, citric acid; CHX, chlorhexidine; HP, hydrogen peroxide; L, low treatment concentration/short duration; H, high treatment concentration/long duration.

Figure 3

Lactic acid production of biofilms after different decontamination treatments at different time points. Data are presented as mean + SD. * represents statistically significant difference compared with day-3 biofilms of Water group (Water_day 3) (p < 0.05). For clarity, the Water_day 3 data were repeated at days 4 and 5. The dashed line indicates the detection limit (0.05 mM). CA, citric acid; CHX, chlorhexidine; HP, hydrogen peroxide; L, low treatment concentration/short duration; H, high treatment concentration/long duration.

Figure 4

Relative abundance (average of replicates) of top 15 most abundant bacteria genera or higher taxa (remaining genera are grouped as “other”) in saliva inoculum, day-3 and day-5 biofilms. CA, citric acid; CHX, chlorhexidine; HP, hydrogen peroxide; L, low treatment concentration/short duration; H, high treatment concentration /long duration.

Figure 5

Number of OTUs (A) and Shannon diversity index (B) of biofilms in different groups. Connector with * indicates statistically significant difference between day-3 and day-5 biofilms within the same treatment group (independent samples t test, p < 0.05). CA, citric acid; CHX, chlorhexidine; HP, hydrogen peroxide; L, low treatment concentration/short duration; H, high treatment concentration/long duration.

Figure 6

(A) Principal component analysis (PCA) plots of day-3 (open symbols) and day-5 (filled symbols) biofilms in all groups. Biofilms of day 5 were encircled into two parts to visualize the different shift direction between water control group (red line) and other groups (green line). (B) Operational taxonomic units (OTUs) that were differentially abundant between day-3 (red bars) and day-5 (green bars) biofilms in each group ranked by the effect size in linear discriminant analysis effect size (LEfSe). The species-level taxonomies of the most abundant sequence for the OTUs with the same genus name are: OTU1: Streptococcus salivarius/vestibularis; OTU2: Streptococcus oralis/mitis; OTU3: Veillonella parvula; OTU52: Veillonella atypica/dispar; OTU6: Fusobacterium periodonticum; OTU132: Fusobacterium nucleatum subsp. polymorphum/sp. oral taxon 203. CA, citric acid; CHX, chlorhexidine; HP, hydrogen peroxide; L, low treatment concentration/short duration; H, high treatment concentration/long duration.

Figure 7

The effect of PMA treatment on the microbial composition of the biofilms. (A) PCA plot of non-PMA and PMA treated biofilms. (B) Comparison of Bray-Curtis similarity indices between the non-PMA and PMA treated biofilms per decontamination group. * indicates statistically significant difference between the connected two groups (p < 0.05). CA, citric acid; CHX, chlorhexidine; HP, hydrogen peroxide.