Amoxicillin comp

Biofilms exposed to chlorhexidine (CHX) 0.2% (Corsodyl, GlaxoSmeethKlein, UK) were used as positive control.

Fresh

BHI

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 BHI and disrupted as described above.

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 significant.

All three types of bacterial strains were harvested from 3?day old biofilms.

The viable

bacteria

actinomycetemcomitans declined in the biofilm compared to the initial inoculum per well, as depicted in Figure 1.

The bacterial mass of the 3?day old biofilm as measured by plate reading ( n = 7) was 0.568 ± 0.075 AU.

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

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 by systemic antibiotic treatment adjunct to mechanical therapy in periodontal disease (Slots, Research, & Therapy, 2004 ).

In our study, the combination AMX + MET was highly effective against F .

gingivalis , as previously reported in other in vivo and in vitro studies (Astasov?Frauenhoffer et al., 2014 ; Sbordone, Barone, Ramaglia, Ciaglia, & Iacono, 1995 ; Soares et al., 2015 ). These two anaerobic bacteria may be killed by MET treatment only, in contrast to A . actinomycetemcomitans that cannot be completely eliminated by MET alone.

The exposure of our 3 days biofilm to MET alone resulted in a similar reduction of A .

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 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

The antibiotic concentrations used in this study were very high compared to antibiotic concentration that may be detected in

gingival

However, use of the high concentration antibiotics is counterbalanced by the length of the exposure time, which was much shorter than in other in vitro studies (Belibasakis & Thurnheer, 2014 ; Soares et al., 2014 ).

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 . nucleatum strains (Kulik Kunz, Lenkeit, Waltimo, Weiger, & Walter, 2014 ). In our study, PV + MET had similar effect as AMX + MET, by eliminating completely two of the bacterial species from the biofilm and strongly reducing the viable counts of A . The combination PV + MET seemed to be dose?dependent, as exposure of the biofilms to a three times lower antibiotic concentration resulted in one log higher amount of live CFU retrieved.

However, these results should be interpreted with caution, because in our study, the antibiotic concentrations were high, combined with a short exposure time.

It is timely to recall that although even the commensal bacteria in dental biofilm may cause oral pathology, the aim for periodontal treatment is not a complete elimination of resident periodontal species, but to obtain a microbial profile compatible with health by reducing the proportion of pathogenic species (

Marsh

A controlled homeostatic immune?inflammatory state is normal in healthy gingiva, the problems arise when dysbiosis develops with a disturbance in the balance between the host and the microbiota in the biofilm (Lamont & Hajishengallis, 2015 ). In this study, CHX 0.2% was used as a positive control. The plaque inhibiting effect and antibacterial properties of chlorhexidine are well documented in vivo and in vitro (Addy, 1986 ; Schiott et al., 1970 ).

CHX has the ability to penetrate biofilms and having bactericidal effect, acting inside the biofilm (Shapiro, Giertsen, & Guggenheim, 2002 ). We have used this concentration because with regard to

effects

plaque

However, Soares et al., 2014 found that CHX 0.12% decreased the metabolic activity of the biofilms by approximately 95% and suggested it to be appropriate as a positive control.

Regarding clinical effects, most studies do not find statistical

significant

To our knowledge, no studies have

compared

Concluding, in this in vitro biofilm model, the replacement of AMX with PV at the same concentration, in combination with MET, resulted in similar effect on bacterial viability, by eliminating F .

The option of using PV + MET instead of AMX + MET deserves further investigation, as this may contribute to reduce the risk of antibiotic resistance development.

The authors thank Endy Spriet from the MIC platform, University of Bergen, for excellent assistance with confocal microscopy. The work was supported by funding from the University of Bergen.

Azithromycin+Amoxicillin/Clavulanate vs Amoxicillin/Clavulanate in COVID19 Patients With Pneumonia in Non-intensive Unit (AziA) The safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Listing a study does not mean it has been evaluated by the U.S. Know the risks and potential benefits of clinical studies and talk to your health care provider before participating.

Study Details Tabular View No Results Posted Disclaimer How to Read a Study Record. Condition or disease Intervention/treatment Phase COVID-19 Pneumonia Combination Product: Azithromycin with amoxicillin/clavulanate Drug: amoxicillin/clavulanate Phase 3. Layout table for study information Study Type : Interventional (Clinical Trial) Estimated Enrollment : 104 participants Allocation: Randomized Intervention Model: Parallel Assignment Masking: None (Open Label) Primary Purpose: Treatment Official Title: Azithromycin With Amoxicillin/Clavulanate Versus Amoxicillin/Clavulanate Alone in COVID-19 Patients With Pneumonia and Hospitalized in a Non-intensive Care Unit Ward (AziA): a Superiority Open-label Randomized Controlled Trial Estimated Study Start Date : April 30, 2020 Estimated

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