Antibacterial Activity of Essential Oils Combinations based on Thymus broussonnetii, and Their Synergism with some Antibiotics.

The present study aimed to evaluate the antibacterial activity of the essential oil (EO) of Moroccan endemic Thymus broussonnetii alone, and in combination with EOs obtained from selected medicinal plants, namely Myrtus communis, Artemisia herba alba, Thymus pallidus, Thymus satureioides, Teucrium polium, and Rosmarinus officinalis. The synergistic interactions between the most effective combinations based on T. broussonnetii EO with two conventional antibiotics (streptomycin and ciprofloxacin) were also investigated. T. broussonnetii EO possessed a higher inhibitory activity against tested pathogenic bacteria with inhibition zone diameter (IZ) ranging from 21.61 ± 0.03 to 40.09 ± 0.02 mm, and MIC values between 0.140 mg/mL and 0.280 mg/mL. M. communis, A. herba alba, T. pallidus, T. satureioides, T. polium, and R. officinalis EOs showed moderate to weak antibacterial activity. Among tested EO mixtures, the highest synergistic antibacterial effect was recorded with the EO combination of T. broussonnetii and T. pallidus against S. aureus, E. coli, and S. enterica (FICI = 0.258). This EO combination was also the most effective mixture to synergistically enhance the antibacterial activity of the two antibiotics with up to a 128-fold increase, particularly against the gram-negative E. coli. These findings suggest that T. broussonnetii EO may be an interesting source of natural antimicrobials, for use in combination therapies with other plant EOs, and with conventional antimicrobial drugs to tackle the emergence of multidrug-resistant bacteria.

New insight into antimicrobial activities of Linaria ventricosa essential oil and its synergetic effect with conventional antibiotics.

The purpose of the present study was to determine for the first time the volatile constituents, the antioxidant and antimicrobial activities of the essential oil (EO) of the endemic Moroccan Linaria ventricosa, alone or in combination with four known antibiotics. The major constituents were 2-methoxy-4-vinylphenol (17.4%), α-terpinene (13.64%) and 3,5-dimethylphenyl isocyanate (12.21%). The EO had moderate antioxidant potency, as measured by DPPH free radical scavenging (1.233 ± 0.031 mg/mL), ferric reducing antioxidant power assay (0.373 ± 0.019 mg/mL) and ß-carotene/linoleic acid (0.922 ± 0.026 mg/mL). EO showed microbicidal activity against all microorganisms tested. The highest effectiveness was recorded against Candida albicans (IZ = 24 mm, MIC = 4.87 mg/mL and MMC = 9.75 mg/mL) and Candida glabrata (IZ = 22 mm, MIC = MMC = 4.87 mg/mL). Gram negative bacteria were the most resistant (MIC = MMC = 39 mg/mL). The combination of EO at sub-inhibitory concentrations with antibiotics showed a significant decrease in their individual MICs from 2 to 128 fold, being the best for ciprofloxacin and fluconazole against E. coli and C. albicans and C. glabrata, respectively.

New Insight into the Chemical Composition, Antimicrobial and Synergistic Effects of the Moroccan Endemic Thymus atlanticus (Ball) Roussine Essential Oil in Combination with Conventional Antibiotics.

This study reported the volatile profile, the antimicrobial activity and the synergistic potential of essential oil (EO) from the Moroccan endemic Thymus atlanticus (Ball) Roussine, in combination with the antibiotics ciprofloxacin and fluconazole for the first time, to the best of our knowledge. The EO chemical composition was determined by gas chromatography coupled to mass spectrometry (GC-MS) analysis and the antimicrobial activity assessed by the disc diffusion method against three Gram positive (Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus) and three Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli and one clinical isolate, Klebsiella pneumonia). The antifungal activity was evaluated in four pathogenic yeasts (Candida albicans, C. glabrata, C. krusei and C. parapsilosis). The minimum inhibition concentration (MIC) and the synergistic effect with ciprofloxacin and fluconazole were determined by the two-fold dilution technique and checkerboard test, respectively. Twenty-one constituents were identified by GC-MS in the EO, including carvacrol (21.62%) and borneol (21.13%) as the major components. The EO exhibited a significant antimicrobial activity with inhibition zones ranging from 0.7 mm to 22 mm for P. aeruginosa and B. subtilis, respectively, and MIC values varying from 0.56 mg/mL to 4.47 mg/mL. The fractional inhibitory concentration index (FICI) values ranged from 0.25 to 0.50 for bacteria and from 0.25 to 0.28 for yeasts. The maximum synergistic effect was observed for K. pneumonia with a 256-fold gain of antibiotic MIC. Our results have suggested that EO from T. atlanticus may be used alone or in association with antibiotics as a new potential alternative to prevent and control the emergence of resistant microbial strains both in the medical field and in the food industry.

Chronic exposure to metam sodium-based pesticide in mice during adulthood elevated anxiety and depression-like behaviors: Involvement of serotoninergic depletion and gut microbiota dysbiosis.

Metam sodium-based pesticide (MS-BP) is widely used in agriculture and public health. We have previously demonstrated that maternal exposure to MS-BP resulted in sensorimotor alterations in mice offspring with long-lasting deficits including anxiety- and depression-like behaviors. Here, we project to verify whether these two neurobehavioral effects occur during adulthood following direct exposure to MS-BP and whether it results in changes in the serotoninergic system and gut microbiota. Our findings showed that chronic exposure to MS-BP increased anxiety- and depression-like behaviors, accompanied by a depletion of serotonin-like neurons within the dorsal raphe nucleus and a reduction in serotoninergic terminals in the infralimbic cortex and the basolateral amygdala. In addition, all MS-BP-exposed animals exhibited a reduced total bacterial number and diversity of gut microbiota. Taken together, our data demonstrated that MS-BP-induced behavioral changes could be related to the impairment of the serotoninergic system and gut microbiota dysbiosis.

Synergistic Anticandidal Effects of Six Essential Oils in Combination with Fluconazole or Amphotericin B against Four Clinically Isolated Candida Strains.

The development of opportunistic pathogenic Candida strains insensitive to several classes of antifungals has emerged as a major health care problem during the last years. Combinational therapy of natural products (e.g., essential oils, EOs) with conventional antifungals has been suggested as a promising alternative to overcome this medical problem. The present study investigates the potential antifungal activity of EOs extracted from some selected medicinal plants, alone and in combination with two common conventional antifungals (fluconazole and amphotericin B) against four clinical Candida isolates. MIC assays indicated that EOs induced strong anticandidal activities with MIC values ranging from 0.162 to 4.950 mg/mL. The combination of amphotericin B with Thymus leptobotrys, Origanum compactum and Artemisia herba alba EOs provided a synergistic effect against C. krusei only, with MIC gain of four-fold, and additive effect against remaining strains (MIC gain = two-fold). Interesting synergistic interactions were observed by combining all studied EOs with fluconazole, with reduction rates of their MICs ranging from 16 to 512-fold. This synergistic effect was very pronounced with the combination of T. leptobotrys EO and fluconazole. These findings indicate that studied EOs can be used as anti-candidals in combination with antifungals, particularly fluconazole, to counteract the emergence of resistant Candida spp.

Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus.

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC3 assay protocol was cuvette - based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

Determination of several potential virulence factors in non-o1 Vibrio cholerae, Pseudomonas aeruginosa, faecal coliforms and streptococci isolated from Marrakesh groundwater.

The dynamic, hemolytic and hemagglutination activities and the antibiotic resistance of non-O1 Vibrio cholerae, Pseudomonas aeruginosa, faecal coliforms (FC) and faecal streptococci (FS), isolated by standard membrane filtration methods from suburban and rural groundwater supplies, were carried out. Detectable non-O1 V. cholerae and P. aeruginosa was present in 81% and 88% of samples. The total occurrence of FC and FS during the period of study was 94%. The annual average densities of non-O1 V. cholerae were 4,903 MPN/100 mL. While, they were 206, 1,891 and 1,246 cfu/100 mL for P. aeruginosa, FC and FS respectively. Non-O1 V. cholerae strains had the highest percentage of hemolytic activities (alpha + beta) (71.29%), whereas 20.71% of FS, 16.88% of FC and 9.13% of P. aeruginosa strains produced hemolysin. Bacterial strains isolated were found to be adhesive, with percentages of 63.09%, 65.09%, 84.06% and 87.98% respectively for non-O1 V. cholerae, FS, FC and P. aeruginosa. As for antibiotic resistance, the overall resistance of non-O1 V. cholerae strains was 79%, whereas it was 100% for the other bacteria. Non-O1 V. cholerae resistance was expressed towards sulfamethoxazole (75%), streptomycin (62%) and cephalothin (60%). Obtained results indicated correlation between bacteriological pollution and their public health implications.

Antibacterial activity of Thymus maroccanus and Thymus broussonetii essential oils against nosocomial infection - bacteria and their synergistic potential with antibiotics.

The aim of this study was to evaluate the antibacterial effect of the association between conventional antibiotics and essential oils (EOs) of endemic Moroccan thyme species, Thymus maroccanus and T. broussonetii, on antibiotic-resistant bacteria involved in nosocomial infections. Synergistic interactions between antibiotics (ciprofloxacin, gentamicin, pristinamycin, and cefixime) and EOs, and between T. maroccanus and T. Broussonetii EOs were determined by the checkerboard test. Serial dilutions of two antimicrobial agents were mixed together so that each row (and column) contained a fixed amount of the first agent and increasing amounts of the second one. The results indicate that the oils had a high inhibitory activity against tested bacteria, except for Pseudomonas aeruginosa. In parallel with the increase of cellular killing, the release of 260nm-absorbing materials from bacterial cells, treated with EOs, increased in response to oil concentration. Out of 80 combinations tested between EOs and antibiotics, 71% showed total synergism, 20% had partial synergistic interaction and 9% showed no effect. Combination with carvacrol, the major constituent of T. maroccanus and T. broussonetii, showed also an interesting synergistic effect in combination with ciprofloxacin. The effect on Gram-positive bacteria was more important than on Gram-negative bacteria. These findings are very promising since the use of these combinations for nosocomial infections treatment is likely to reduce the minimum effective dose of the antibiotics, thus minimizing their possible toxic side effects and treatment cost. However, further investigations are needed to assess the potential for therapeutic application.

Essential oils from Moroccan plants as potential chemosensitisers restoring antibiotic activity in resistant Gram-negative bacteria.

Bacterial drug resistance is a worrying public health problem. Antibiotic efflux is a major non-specific resistance mechanism used by bacteria, and efflux pumps are involved in the low-level susceptibility of various important Gram-negative pathogens. Use of molecules that can block bacterial pumps is an attractive strategy, but several studies report only partial efficacy owing to limits of these molecules (stability, selectivity, bioavailability, toxicity, etc.). The objective of this study was to search for natural sources of molecules able to inhibit efflux pump systems of resistant Gram-negative bacteria (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Salmonella enterica serotype Typhimurium and Pseudomonas aeruginosa). The results indicate that the studied essential oils exhibit interesting activity against the tested bacteria. This activity was significantly enhanced in the presence of an efflux pump inhibitor such as phenylalanine arginyl ß-naphthylamide (PAßN). The role of lipopolysaccharide (LPS) structure in the effect of essential oils was also reported in Salmonella LPS deep-rough mutants. In addition, essential oils of Thymus maroccanus and Thymus broussonetii, used at a low concentration (a fraction of the minimum inhibitory concentration), are able to significantly increase chloramphenicol susceptibility of several resistant isolates. These results demonstrate that these essential oils can alter efflux pump activity and may be attractive candidates to develop new drugs for chemosensitising multidrug-resistant strains to clinically used antibiotics.