Antibiotics amoxicillin

Bacteria were precultured for 24 hr in liquid growth medium.

Then an aliquot of the bacterial suspension was inoculated in fresh BHI and further incubated overnight and allowed to reach late log?phase. All incubations were done in anaerobic conditions (5% CO 2 , 10% H 2 , and 85% N 2 ) at 37°C (Anoxomat System, MART Microbiology, Lichtenvoorde, The Netherlands).

Biofilms were prepared in 24?well plates (Corning Inc., NY, USA) by adapting a previously described protocol (Sanchez et al., 2011 ).

Briefly, bacterial suspensions were

prepared

cultures

aliquot

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

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

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 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 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

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 in biofilms were mainly represented by F .

gingivalis, which showed a nearly two?log increase in numbers during

the

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% amoxicillin mk 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 amoxicillin in 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 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 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

The antibiotics might not penetrate the deeper layers of the biofilm and thus may not reach bacteria protected in this environment.

The antibiotic concentrations used in this study were very high compared to antibiotic concentration that may be detected in gingival crevicular fluid (Giedrys?Leeper, Selipsky, & Williams, 1985 ; Shaddox & Walker, 2009 ; Tenenbaum, Jehl, Gallion, & Dahan, 1997 ). 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

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, Head, & Devine, 2015 ).

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

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 our knowledge, no studies have compared the two CHX concentrations and evaluated the probing pocket depth and/or the periodontal attachment level. 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, amoxicillin angina 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.

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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 Primary Completion Date : July 30, 2020 Estimated Study Completion Date : July 30, 2020.

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For general information, Learn About Clinical Studies. Layout table for eligibility information Ages Eligible for Study: 18 Years to 99 Years (Adult, Older Adult) Sexes Eligible for Study: All Accepts Healthy Volunteers: No.

Adult ? 18 ans, Patient with positive SARS-CoV-2 RT-PCR on nasopharyngeal sample at randomization or within the previous 48 hours, Patient with pneumonia diagnosed by thorax CT-scan or echography, Patient able to take per os medication, Written and signed consent of the patient, Patients affiliated with or benefitting from a social security scheme.