Amoxicillin in dentistry

The Antistaphylococcal Activity of Amoxicillin/Clavulanic Acid, Gentamicin, and 1,8-Cineole

Alone

This activity was tested in

vitro

The efficacy of these three antibacterial agents was then tested in vivo by using an experimental model of methicillin-resistant S. This efficacy was assessed after four days of treatment by counting the number of bacteria in the bone marrow.

The obtained results in vitro showed that the combination of the AMC with gentamicin did not induce a synergistic effect, whereas the combination of the two antibiotics with 1,8-cineole did. This effect is stronger when AMC is combined with 1,8-cineole as a total synergistic effect was obtained on the three strains used (FIC ? 0.5). In

vivo

These results demonstrated that 1,8-cineole

showed

Osteomyelitis is a bacterial infection characterized by an acute or chronic inflammatory response that leads to bone loss.

Furthermore, the spread of this infection to surrounding tissues is responsible for significant morbidity and healthcare costs each year [1]. In high-income countries, acute osteomyelitis occurs in about 1of 800,000 children per year [2] but it is considerably more common in low- and middle-income countries [3].

In Morocco, osteomyelitis is more common among children [4].

The most common causative organism of osteomyelitis is S. The antibiotic of choice in the treatment of bone infections is vancomycin. However, the resistance of staphylococci to vancomycin has been reported [7, 8].

New alternatives are becoming essential to overcome the increasing resistance of S. aureus strains

and

Our laboratory, which has extensively worked on essential oils (EOs) and their major compounds, demonstrated the antimicrobial activity of these components in vitro and in vivo [9–12].

The advantage of EOs over other antimicrobial agents is that they offer high antibacterial potency without inducing the production of resistance mechanisms [13–15]. Among the various constituents of EOs, 1,8-cineole has been shown to have pharmacological effects. 1,8-cineole has been used as a percutaneous penetration enhancer, an antibacterial expectorant, and an anti-inflammatory agent [16].

1,8-cineole was reported to induce apoptosis in leukemia

cell

Additionally, the importance of combining EOs with antibiotics to fight resistant bacteria is increasingly recognized [18].

In fact, this combination has shown a synergistic effect against antibiotic-resistant bacteria [19, 20]. The aim of the present research is to evaluate the in vitro antistaphylococcal effect of a major compound of EOs 1,8-cineole and two antibiotics AMC and gentamicin either separately or in combination and to elucidate the in vivo efficacy in a rabbit model of methicillin-resistant S. (i) Amoxicillin/clavulanic acid (AMC): Augmentin® 1 g/200 mg, in powder form, for injectable solution purchased from GlaxoSmithKline (Morocco) was used. It was dissolved in 10 mL (w/v) of sterile distilled water and stirred until totally dispersed. The final concentration of AMC obtained was 100 mg/mL. (ii) Gentamycin: Gentosyl® solution for injection at a concentration of 10 mg/mL, purchased from Laprophan (Morocco), was used in this study. (iii) 1,8-cineole, in liquid form, provided by Sigma-Aldrich (France), was dispersed in a viscous solution of 0.2% (v/v) agar according to the method described by Remmal et al. The stock solution prepared according to this procedure had a concentration of 100 mg/mL. Mueller–Hinton agar (MHA, Biokar®), Mueller–Hinton broth (MHB, Biokar®), tryptic soy agar (TSA, Oxoid®), tryptic soy broth (TSB, Oxoid®), and Chapman agar (Biokar®) were prepared and sterilized according to the manufacturers’ instructions. In this study, the antibacterial activity of each agent and their combination was tested against three bacterial strains: a strain of MRSA and two strains of methicillin-susceptible S.

The methicillin resistance was determined by the cefoxitin disk diffusion test using 30 mg cefoxitin disks on Mueller–Hinton agar, as recommended by CLSI guidelines [22], and confirmed by the detection of mecA gene by PCR assay [23]. They were isolated from the bone marrow of patients suffering from osteomyelitis and were obtained from the Laboratory of Microbiology and Molecular Biology, Faculty of Medicine and Pharmacy of Fez (Morocco).

Stock cultures were kept on a Muller–Hinton agar under refrigeration (4°C). The inoculum suspension was obtained by

taking

the

Ten concentrations of 1,8-cineole and the two antibiotics AMC and gentamycin were prepared in sterile hemolysis tubes by serial dilutions. The concentrations of AMC obtained in the well were between 32 ? g/mL and 0.062 ? g/mL, between 4 ? g/mL and 0.0078 ? g/mL for gentamicin, and between 64 mg/mL and 0.125 mg/mL for 1,8-cineole to determine the MIC values.

Bacterial suspensions were prepared as previously described. These suspensions were diluted in MH broth and plated in 96-well plates at a density of 5 ? 10 5 CFU/well. After the plates were incubated at 37°C for 18 hours, 40 ? L of 0.5% triphenyl-tetrazolium chloride was added to each well.

After two hours of incubation, the MIC corresponds to the lowest concentration that does not produce a red color [24].

The evaluation of the interaction between AMC, gentamicin, and 1,8-cineole was performed according to the method of Mulyaningsih et al. Briefly, eight concentrations of antibiotics and eight concentrations of 1,8-cineole were prepared in sterile hemolysis tubes by successive dilutions 1/2.

For antibiotics, the concentrations were introduced vertically into eight wells in a decreasing manner ranging from MIC ? 2 to MIC/64, while the concentrations of 1,8-cineole were introduced horizontally into eight wells in a decreasing manner ranging from MIC ? 2 to MIC/64.

The combination of AMC

and

The analysis of the combination was obtained by calculating the fractional inhibitory concentration (FIC) index (FICI) using the following formula [27]: FICI = FIC (A) + FIC (B), where FIC (A) = [(MIC (A) in combination/MIC (A) alone)] and FIC (B) = [(MIC (B) in combination/MIC (B) alone)]. The

index

were

Forty-two female New Zealand white rabbits (5–6 weeks old), weighing between 1.2 and 1.8 kg, were used in this study. They were divided into seven groups of six rabbits each. The rabbits were given feed and water ad libitum and were treated in accordance with the National Health and Research Council Ethics Committee guidelines. Adequate ventilation was provided, and the environmental temperature was constantly maintained at 21°C ± 3°C. The photoperiod was adjusted daily to 12 h of light and 12 h of darkness.

For the purpose of acclimatization, the animals of the experiment were kept for a week.

Advice with regard to the surgical procedures was sought from a professional vet and from a surgeon. The animals were randomly divided into seven experimental groups of six rabbits each: Group 1 ( n = 6), positive control group: infected, untreated animals. Group 2 ( n = 6): animals infected and treated with AMC at a dose of 30 mg/kg. Group 3 ( n = 6): animals infected and treated with gentamicin at a dose of 3 mg/kg.

Group 4 ( n = 6): animals infected and treated with 1,8-cineole at a dose of 12 mg/kg.

Group 5 ( n = 6): animals infected and treated with AMC at a dose of 15 mg/kg associated with gentamycin at a

dose