Amoxicillin clav 875 mg

(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

Stock cultures were kept on a Muller–Hinton agar under refrigeration (4°C). The inoculum suspension was obtained by taking colonies from 24 h cultures on tryptic soy agar.

These colonies were suspended in sterile saline (0.9% NaCl) and shacked for 15 seconds. The density was adjusted to the turbidity of a 0.5 McFarland Standard (equivalent to 1.5 ? 10 8 CFU/mL) [24]. Minimal Inhibitory Concentration (MIC) The MICs of AMC, gentamicin, and 1,8-cineole were determined by microdilution assays in 96-well plates according to the standards of the CLSI [25].

Ten concentrations of 1,8-cineole and the two antibiotics AMC and gentamycin were prepared in sterile hemolysis tubes by amoxicillin 125 mg price 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 gentamicin was performed in the same way.

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 values of the fractional inhibitory concentrations were interpreted as follows: FICI ? 0.5 means synergy; 0.5 4 means antagonism.

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

For the purpose of acclimatization, the animals of the experiment were kept for a week. Advice with regard to the surgical

procedures

Group 2 ( n = 6): animals infected and treated with AMC at a dose of 30

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 of 1.5 mg/kg.

Group 6 ( n = 6): animals infected and treated with AMC at a dose of 15 mg/kg combined with 1,8-cineole at a dose of 6 mg/kg. Group 7 ( n = 6), negative control group: neither infected nor treated animals.

The doses administered were calculated according to the weight by imitating the recommended human dose for each drug; 12 mg/Kg given twice daily for 1,8-cineole [28], 31.83 mg/kg/twice daily AMC [29], and 6 mg/kg once daily [30]. Among

the

These tubes were incubated for 3 h at 37°C to obtain log-phase growth [31].

After incubation, the tubes were centrifuged for 10 min at 1000 g, the supernatant was decanted, and the remaining pellet was washed twice with phosphate-buffered saline (PBS). Under spectrophotometric control (McFarland score), the bacterial sediment was added to the PBS. A fentanyl patch (Durogesic®) was used for the management of pain during the study.

Due to the delay in action (

about

On the first day of the study, which was considered to be day zero (day 0); the rabbits were anesthetized by injection of a mixture of xylazine at 1 mg/kg and ketamine at 20 mg/kg into the marginal vein of the ear, then the right knee of the animal was shaved, and the skin was disinfected with povidone-iodine (Betadine®).

We used a percutaneously transarticular route to perform a femoral trepanation using a Jamshidi bone marrow biopsy needle (8 Ga).

The Jamshidi needle was inserted between the two femoral condyles and through the epiphysis, physis, and metaphysis to reach the medullary canal.

Then, a 1 mL suspension containing 10 9 CFU/mL of MRSA was injected into the tibia.

The procedures used in this experimental model are described by Gaudin et al. The infection was allowed to develop for three days.

On the third day, in order to quantify the infection, the rabbits were anesthetized as before, and bone marrow samples were taken using 8 Ga syringes, weighed, and mixed with 200 ? L of physiological serum, and the resulting

solution

On the 14 th day, the animals were euthanized by intravenous injection of a lethal dose of 100 mg thiopental under the marginal vein of the ear [33], the proximal half of the tibia was dissected into aseptic conditions, and bone marrow samples were taken.

The bacterial load was then evaluated in the same way as on the third day and the seventh

day

Rectal temperature was taken on days 0, 3, 7, and 14 using a digital thermometer. The individual weighing was carried out on days 0, 3, 7, and 14 using a digital scale. The results were expressed as mean values ± SEM (standard error of the mean).

A statistical analysis of the data was performed with a one-way analysis of variance followed by Tukey?s Multiple Comparison Test (ANOVA followed by Tukey?s test) (Graph Pad Prism, version 5.03).

The AMC, gentamicin, and 1,8-cineole MIC values were shown in Table 2. Effect of the Combination of Antibiotics and 1,8-Cineole.

The effect of the two antibiotics tested against three strains of S.

aureus by combining the two antibiotics, on the one hand, and combining each one of them with 1,8-cineole, on the

other

with

synergistic

Figure 1 shows the evolution of the rabbit’s body temperature of different groups on days 0, 3, 7, and 14.

It illustrates that, after inoculation, an increase in the body temperature of all groups of rabbits was observed

except

During four days of treatment, the body temperature of treated rabbits decreased gradually reaching almost the normal temperature. In contrast, the body temperature of infected untreated rabbits remained above 40°C.

Table 6 shows the evolution of the rabbit’s weight during

the

the