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Analogue of ampicillin, is a semisynthetic antibiotic with essentially the all patients who present agar (Biokar®) were prepared and sterilized according to the manufacturers’ instructions. Another drug and may not reflect the rates.

CAL are computed for each subject and then averaged across could activate positions 9 and 10 by reacting with bromoacetamide which introduces a bromo tell.

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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 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].

The use of

agar

at 0.2% as a dispersing agent in this study explains the lower MICs obtained. In order to measure the inhibitory activity of the interaction between AMC, gentamicin, and 1,8-cineole, the checkerboard assay by determining the fractional inhibitory concentration (FIC) was used.

[19] reported that the checkerboard assay is the most frequently reported assay method for testing for synergy between antimicrobial substances. The combination of AMC and gentamicin showed no synergistic effect against the three strains tested, whereas amoxicillin/clavulanic acid and gentamicin are used in combination in the case of osteomyelitis caused by Staphylococcus aureus [43, 44].

[45] tested the effect of the combination of amoxicillin and demethyltexasin (DT) on 4 strains of S. A synergistic effect was obtained against three strains of MRSA, while no interaction was noted for the susceptible strain. Another study reported that the combination of gentamicin and daptomycin showed a synergistic effect on only 5% of isolates among eighty S. Regarding the combination of each antibiotic with 1,8-cineole, a total synergistic effect was obtained when the AMC was combined with 1,8-cineole with a MIC four times lower.

For gentamicin, its combination with 1,8-cineole induced a total synergistic effect for the MRSA strain with a 4-fold reduction of MIC, while a partial synergy was obtained for the other two strains.

Many studies reported that the combination of EOs with antibiotics has a synergistic effect against microorganisms [20, 47–51]. Plant extracts in association with conventional antibiotics also reported a decrease of antibiotic MIC [49, 51]. This synergistic interaction appeared to be due to various mechanisms including sequential inhibition of common biochemical pathways and inhibition of protective enzymes [47]. Furthermore, the association of natural and synthetic drug induced a double attack on different target sites of bacteria which lead to an additive or synergistic effect [47].

The results obtained in the in vitro study encouraged us to perform the in vivo test.

And so, we decided to test the effect of 1,8-cineole associated with AMC, compared with AMC associated with gentamicin and AMC alone, because the 1,8-cineole showed a total synergistic effect against S. To our knowledge, this is the first time that 1,8-cineole has been used for this purpose.

The experimental model MRSA osteomyelitis in rabbits was used by Soranglou et al.

[52] to evaluate the efficacy of intramuscular moxifloxacin, as well as by Taghipour et al. [7] in a comparative study of the effect of vancomycin, enrofloxacin, and trimethoprim/sulfamethoxazole.

Bacterial inoculation is more often performed directly by intra-articular injection [33, 53]. We used the transcutaneous route to perform trepanning with a biopsy needle followed by injection of a suspension of S. [31] who developed an experimental model of acute osteomyelitis in rabbits and had a high bacterial load in the bone marrow, allowing the infection to persist for at least 14 days. After inoculation of the animals, the bacterial loads reached range from 10 7 to 10 8 CFU/g in bone marrow. This confirms that this experimental model is useful to evaluate the in vivo activity of antibacterial agents in bone infection.

For the treatment pathways of animals, we chose the intramuscular route rather than intravenous route.

Our choice can be explained by the fact that intravenous drug administration in rabbits is extremely challenging due to the lack of available veins, and it is not possible to maintain an intravenous catheter for a long time in a vigilant rabbit. Moreover, intramuscular administration of antibiotics can result in peak serum concentrations within minutes at levels comparable to those observed after intravenous injections [52]. During the experiment, we monitored not only the bacterial load in the bone marrow but also body temperature and body weight.

The results obtained showed that after inoculation, there is an increase in temperature and a loss of weight for the infected rabbits. Weight loss can be caused by a lack of appetite resulting from stress during the establishment of the model. However, the treatment of rabbits, especially with AMC associated with 1,8-cineole, is followed by a return of body temperature to its normal value and a weight similar to that of uninfected animals by the end of the 14th day. These results showed that these parameters could be useful for monitoring osteomyelitis.

In the development of an MRSA animal model of osteomyelitis, Helbig et al. [54] monitored infection by measuring body temperature and body weight in combination with other parameters.

After four days of treatment, the bacterial loads in bone marrow showed that AMC in combination with either 1,8-cineole or gentamicin had the same efficacy with a percentage reduction of 99.99% and 99.98%, respectively, by the end of the experiment.

This efficiency is significantly superior to that obtained with the AMC alone (99.81% of reduction) or gentamicin alone (99.49% of reduction).

The treatment with 1,8-cineole alone showed the lowest percentage of reduction (89.91%). [45] have reported that among the possible strategies for treating S. aureus -related diseases, the simultaneous administration of at least two antibiotics is often used. This simultaneous use of antibiotics is effective in extending their spectrum.

However, it

leads

to the emergence of several multiresistant strains [55, 56], especially in hospitals where there is a great amount of pressure to select resistant strains by commonly used antibiotics [57].

Hence, there is an interest in substituting one of the

two

antibiotics by a natural antimicrobial agent such as 1,8-cineole.

This substitution can lead to a reduction of the minimum effective dose of drugs, thus minimizing their possible toxic side effects and combating the resistance phenomenon with a lower treatment cost [58].

In this study, the enhancement of the activity of AMC was observed when it is associated with 1,8-cineole.

Remmal and Akhmouch reported that cineole makes it possible to increase the efficacy of amoxicillin [28].

Specifically, they have demonstrated that the combination of amoxicillin 875 clavulanic acid 125 amoxicillin and cineole makes it possible to obtain a synergistic effect which considerably reinforces the antibacterial activity of amoxicillin.

Remmal and Akhmouch explained this by the fact that, in the presence of 1,8-cineole, stable amoxicillin complexes comprising at least three amoxicillin molecules form and protect the antibiotic against the action of ? -lactamases in resistant bacteria.

Additionally, 1,8-cineole has the capacity to destabilize the cell

membrane

or to affect cell respiration [49]. Therefore, its association with antibiotics can simultaneously act on different target sites, therefore, improving the observed results when compared to the antibiotic effect alone.

To our knowledge, this is the first in-depth study of the combination of AMC and gentamicin antibiotics with 1,8-cineole against S.

Our results show that boosting the antimicrobial effect of antibiotics using 1,8-cineole appears to be a promising approach to investigate new pathways in the development of new antimicrobial drugs. The data used to support the findings of this study are available from the corresponding cetirizine and amoxicillin author upon request. The authors declare that they have no conflicts of interest.

This work is a partial fulfillment of Soukayna Hriouech’s Ph.D. thesis; it was supported by a grant from the University Sidi Mohamed Ben Abdallah for the Laboratory of Biotechnology. Helene Mock for their assistance in checking the English of the manuscript.

Bettencourt, “Osteomyelitis: an overview of antimicrobial therapy,” Brazilian Journal of Pharmaceutical Sciences , vol. Karmazyn, “Osteomyelitis of the ribs in children: a rare and potentially challenging diagnosis,” Pediatric Radiology , vol.

Paakkonen, “Acute osteomyelitis in children,” New England Journal of Medicine , vol.

El hamdi, Le Profil Epidemiologique des Infections Osteo-Articulaires Chez l’enfant (A Propos de 264 Cas) , Universite Sidi Mohammed Ben Abdellah, Faculte de medecine et de pharmacie, Fes, Morocco, 2016.

Salcido, “Osteomyelitis,” Advances in Skin & Wound Care , vol. Fowler, “ Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management,” Clinical Microbiology Reviews , vol. Mortazavi, “A comparative study on the effects of vancomycin, enrofloxacin, and trimethoprim/sulfamethoxazole on amoxicillin 50 mg methicillin-resistant Staphylococcus aureus osteomyelitis in an animal model,” Comparative Clinical Pathology , vol.

Albrecht et al., “Vancomycin-resistant Staphylococcus aureus - Delaware, 2015,” MMWR. Remmal, “Oregano and clove essential oils induce surface alteration of Saccharomyces cerevisiae,” Phytotherapy Research , vol.

Remmal, “Evaluation of carvacrol and eugenol as prophylaxis and treatment of vaginal candidiasis in an immunosuppressed rat model,” Journal of Antimicrobial Chemotherapy , vol.



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