Amoxicillin and orange juice

Bacterial concentration was adjusted by measuring optical density at 600 nm to 0.1 for all three bacterial strains.

An aliquot from each bacterial suspension was then serially diluted and plated for checking the colony purity and number of colony forming units, CFUs.

A bacterial mix was prepared in a sterile jar by pooling bacterial suspensions of P . Each well was inoculated with 300 ?l bacterial mix and 1,200 ?l fresh BHI. The amount of bacteria in the inoculum were calculated as: 1.83 ? 10 7 ± 5.79 ? 10 6 CFU/ml F . gingivalis , and 4.43 ? 10 7 ± 1.44 ? 10 7 CFU/ml A .

actinomycetemcomitans (mean and standard deviation).

The plates were incubated anaerobically to allow biofilm formation for 72 hr. The biomass of the biofilm and the viability of bacteria were checked at different time points in preliminary tests in order to develop a mature biofilm for exposure to antibiotics. Bacterial mass in the biofilms was assessed by plate reading by plate reader (Synergy H1 Hybrid reader, BioTek instruments, USA).

The biofilm biomass absorbance was measured at 600 nm immediately after complete growth medium removal.

The viability of bacteria in biofilm was assessed by culture plate counting of CFUs.

First, the growth medium and planktonic cells were removed from the 3?day old biofilms, each biofilm was scrapped off the well by use of a sterile cell scraper (Sarstedt, Newton, USA), suspended in 1 ml fresh BHI, and disrupted by vigorous pipetting. The biofilm suspensions were 10?fold diluted on FAA plates in order to obtain plates containing CFUs in range of 1–500 colonies.

After 5–7 days of incubation in anaerobic conditions, CFUs were enumerated on FAA plates by help of a stereomicroscope, distinguished by the colony morphology. The biofilm development and structure were assessed by confocal laser scanning microscopy (CLSM). Prior to examining under CLSM, the biofilms grown for 3 days on 4?wells chamber slides were stained with FilmTracer ™ LIVE?DEAD Biofilm Viability kit (Molecular Probes BV, Leiden, The Netherlands).

Fully hydrated biofilms were stained for 20–30 min in the dark, in anaerobic conditions, thereafter immediately examined by CLSM (Leica penamox 500 amoxicillin TCS SP5; Leica Microsystems, Germany) equipped with an oil?immersion of 63? or 100? objective (Carl Zeiss, Jena, Germany) with beam path settings at 488 nm and 543 nm, respectively. Fluorescence amoxicillin and tums intensity thresholds were manually set for each fluorescent color when examining a negative control biofilm and not further modified.

At least three different areas on each biofilm were investigated. Image stacks were analyzed with the Leica Confocal Lite ® software (Leica Microsystems).

Antibiotic cocktails to be used in assays were freshly prepared in BHI prior to each experiment. The antibiotic concentrations tested in this study were defined as “high concentration” when combinations used were AMX + MET (1,080 ?g/ml + 2,160 ?g/ml) and PV + MET (1,080 ?g/ml + 2,160 ?g/ml); and “low concentration” when AMX + MET (360 ?g/ml + 720 ?g/ml), respectively, PV + MET (360 ?g/ml + 720 ?g/ml) were used. Growth medium together with planktonic bacteria were carefully removed and antibiotic solutions in BHI at given concentrations were gently placed in each well, in order not to induce biofilm disruption. Biofilms exposed to chlorhexidine (CHX) 0.2% (Corsodyl, GlaxoSmeethKlein, UK) were used as positive control. Fresh BHI alone was used for the negative controls. The challenged biofilms in 24?well plates were incubated anaerobically for 2 hr. Then, the growth medium containing antibiotics or CHX were removed and each biofilm was harvested in 1 ml

fresh

Viable counts were recorded on FAA plates incubated for 5 days.

Paired t test was used to investigate significant differences between the two different combinations of antibiotics. A p value less than .05 was considered

statistically

A mature biofilm with three?dimensional structure with mushroom?like shapes and nutrient channels was formed after 3 days incubation in anaerobic conditions.

Both live and dead bacterial?like structures could be observed by CLSM as shown in Figures 2 and 4a. Exposure of biofilms to CHX for 2 hr resulted in eradication of all three bacterial strains, as no CFU could be detected at plate counting. A shorter exposure time than 2 hr to CHX 0.2% resulted in incomplete elimination of live bacteria from the biofilms; while exposure for 2 hr to CHX 0.2% diluted in 1:2 or 1:4 volumes resulted also in detection of CFU after harvesting of positive?control biofilms (results not shown). gingivalis were completely eliminated from the biofilms by antibiotic treatment, both AMX + MET and PV + MET in high and low concentrations, as presented in Figure 3. actinomycetemcomitans was five logs reduced following exposure of biofilms to high concentration of antibiotics and four logs following low concentration.

There was no statistical difference (paired t test) between the AMX + MET and PV + MET combinations with respect to growth inhibition of A . actinomycetemcomitans neither in the high concentration ( p = .95) nor in the low concentration ( p = .17). The combination PV + MET in high concentration had significantly stronger effect in eliminating A . actinomycetemcomitans than the low concentration ( p = .041).

No significance was achieved when comparing between the effect of low and high concentrations of AMX + MET ( p = .076).

The antibacterial effect of AMX + MET and PV + MET combinations on biofilms was obvious by CLSM imaging, as an increased proportion of red bacterial?like structures were observed throughout the biofilms' three?dimensional structure, in contrast to the few remaining green structures, located mainly at the bottom of the biofilms (Figure 4c,d).

A biofilm consisting of three bacterial strains, one known as intermediate colonizer and two as late colonizers in subgingival plaque, was developed in this study.

The two late colonizers are periodontal pathogens commonly

targeted

actinomycetemcomitans that

cannot

actinomycetemcomitans but incomplete elimination of the other two species, as live CFU of F .

gingivalis could be retrieved from biofilms treated with MET alone in low concentration (results not shown). AMX + MET treatment administrated systemically, in individuals who continue get amoxicillin to experience loss of periodontal attachment following mechanical debridement, is effective particularly in cases where A .

The antibiotic combination eliminates or markedly suppresses the periodontal pathogens that remain after subgingival periodontal instrumentation to a level manageable by the host organism, supporting the host defense in overcoming periodontal infections and reducing the risk for recurrent disease progression (Slots et al., 2004 ). Infections caused by biofilms are difficult to treat as bacteria in biofilms are 100 to 1,000 times more resistant to antimicrobials compared to the same type of bacteria in a planktonic state (Ceri et al., 1999 ; Hoiby et al., 2011 ; Olson, Ceri, Morck, Buret, & Read, 2002 ).

The biofilms thickness increases with the incubation time, containing a higher number of bacteria when reaching the mature state, at 72 hr in our work, as demonstrated in other in vitro studies (Ali Mohammed et al., 2013 ; Sanchez et al., 2011 ). The antibiotics might not penetrate the deeper layers of the biofilm and thus may not reach bacteria protected in this environment.

The

antibiotic

time

actinomycetemcomitans was drastically reduced following AMX + MET treatment, both in high and low antibiotic concentrations, but not completely eliminated as in case of F .

gingivalis , despite the high dose antibiotics used.

This might be explained by the short exposure time to antibiotics that did not fully affect A . actinomycetemcomitans , which is more slowly growing. It is also possible that in our model, the nutrients are quickly consumed by the two other species, more rapidly growing, resulting in stationary phase?like dormant A . It is known that antibiotics affect growing cells, thus they are less efficient on bacteria found in a low metabolic activity located in nutrient?deficient areas in biofilm (Olsen, 2015 ).

Another in vitro study showed that the susceptibility of A .

actinomycetemcomitans in biofilm to six different antibiotics decreases as the biofilm matures (Takahashi, Ishihara, Kato, & Okuda, 2007 ). actinomycetemcomitans resistant to AMX and MET collected from periodontal patients have previously been reported (Eick, Pfister, & Straube, 1999 ; Rams, Degener, & van Winkelhoff, 2014b ). Because bacterial pathogens resistant both to AMX and MET were found in relatively low frequency (Al?Haroni, Skaug, & Al?Hebshi, 2006 ; Rams et al., 2014b ; Rams, Degener, & van Winkelhoff, 2014a ), the combination of MET with AMX was recommended rather than using either one alone in order to slow the incidence of developing resistance to MET (Rams et al., 2014a , 2014b ).

A multitude of studies have demonstrated clinical effect of administration of the AMX + MET in combination (Sgolastra, Gatto, et al., 2012 ; Sgolastra, Petrucci, et al., 2012 ; Zandbergen, Slot, Cobb, & Van der Weijden, 2013 ). Furthermore, synergistic interactions of AMX and MET on an in vitro multispecies biofilm (Soares et al., 2015 ) as well as for both combinations, AMX or PV and MET by Etest (Baumgartner & Xia, 2003 ), have been reported. Pavicic, van Winkelhoff, and de Graaff, 1991 found PG + MET to act synergistically against A . actinomycetemcomitans in vitro using an agar dilution method and checkerboard titrations.

Others found synergy between AMX and MET only on a few of the tested A .