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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 three strains studied, in vitro , the MRSA strain was chosen for the in vivo study.

From an overnight culture of MRSA in a 9 mL tryptic soy broth, aliquots of 100 ? L were transferred to sterile tubes containing 3 mL of TSB. 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 12 h), the patch was placed the night before the beginning of the experiment (induction) and changed every 72 h. 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 was seeded in pure and diluted forms at 10 ?2 , 10 ?4 , and 10 ?6 on Chapman gel. After incubation at 37°C for 48 h, the bacterial load is expressed in CFU per unit mass of bone marrow. Samples of the bone marrow of the positive and negative control rabbits were also made.

The treatment of animals started 72 h after inoculation (day 3), and all five types of treatment were done twice a day, intramuscularly for 4 days. After 4 days of treatment (day 7), bone marrow samples were taken, and the bacterial count was evaluated.

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 about amoxicillin 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 hand, was shown in Tables 3, 4, and 5.

The combination of AMC and gentamicin gave no synergistic effect; however, an additive effect was noted for the MRSA and MSSA 2 strains.

For the combination of antibiotics with 1,8-cineole, a total synergistic effect is noted for the three strains combining AMC with 1,8-cineole, while the combination of gentamicin with 1,8-cineole showed a total synergistic effect for the MRSA strain and a partial synergistic effect for the other two MSSA strains.

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 for the uninfected one.

The temperature was around 38.6°C for all the rabbits at the beginning of the experiment, while it exceeded 40°C on day 2 for the groups of infected animals. 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 experiment. For the groups of infected animals, a weight loss during the three days (day 0–day 3) was noted. However, during the four days of treatment (day 3–day 7), an increase in weight of treated rabbits was observed regardless of the type of treatment, with no significant difference between the groups of treated animals.

At the end of the experiment (day 14), the weights of animals from both the negative control and the AMC + 1,8-cineole group were significantly greater.

Evolution of the Bacterial Load in the Bone Marrow. The evolution of the bacterial load in the bone marrow of all groups of animals is shown in Figure 2 and Table 7. Three days after inoculation, the bacterial load was around 10 7 CFU/g for all groups except the uninfected group.

During the four days of treatment (day3–day7), a decrease in the bacterial load was noted and was very significant for the groups of animals treated with AMC + 1,8-cineole, followed by the group treated with AMC + gentamicin, while a moderate decrease was observed for groups treated by AMC, gentamicin, or 1,8-cineole alone. A slight increase in bacterial load was noted for the group of infected untreated animals. During the second week of the experiment, and despite discontinuation of treatment, the bacterial load continued to decrease slightly in all five treated groups. ? : the efficacy measurement was made by comparing the bacterial load before (day 3 after infection) 236 and after antibacterial therapy (day 7 after infection) (Table 7).

Antibiotic treatment of osteomyelitis remains a clinical challenge [34].

This treatment is confronted with the increasing prevalence of multiresistant bacteria, particularly methicillin-resistant S. Hence, there is an interest in finding alternatives to overcome the growing resistance of S.

The MIC values of AMC and gentamicin obtained are lower than those reported by Entenza et al. This difference is probably due to the use of different techniques; Entenza et al.

used the macrodilution method with a higher inoculum of 10 7 CFU/mL.

Indeed, the bactericidal activity of antibiotics decreases when the inoculum increases, especially for S. The bacterial growth phase is also an important parameter that influences the antibacterial activity of antimicrobial agents [37].

The MIC of gentamicin

obtained

for the MRSA strain (2 ? g/mL) confirms the results of the susceptibility test by the disc diffusion method in which gentamicin resistance was found according to EUCAST [38].

For the AMC, the MICs determined by the microdilution were 1 ? g/mL, 0.5 ? g/mL, and 0.25 ? g/mL for MRSA, MSSA 1 , and MSSA 2 , respectively.

Low MIC (?1 ? g/mL) was obtained by Barry on 4.5% among 397 of cefoxitin-resistant staphylococci strains [39].

Methicillin resistance is mediated by an additional PBP (PBP2a) with low affinity for beta-lactam agents and it confers resistance to methicillin as well as to other beta-lactam antibiotics [40]. However, no clinical breakpoints were available for the AMC [41].

With regard to 1,8-cineole, the MIC values obtained are 16 mg/mL for MSSA 1 and 32 mg/mL for MRSA and use of amoxicillin capsules MSSA 2 . These values are lower than those obtained by Silva et al.

[26] obtained a higher MIC value of 64 mg/mL, using the microdilution method with an inoculum of 5 ? 10 5 CFU/mL.

This could be explained by the fact that the dispersion of EOs

using

either dimethyl sulfoxide (DMSO) or Tween 80 is known to reduce their antimicrobial activity.

Indeed, our laboratory has already demonstrated that

detergents

such as Triton-X100 and Tween 80 or solvents such as ethanol decrease the antimicrobial effect of EOs or MICs [21].



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