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The aim of this study was to determine the chemical profile of the essential oils (EOs) of three Moroccan lavender species (Lavandula pedunculata, LP; Lavandula angustifolia, LA; and Lavandula maroccana, LM) and to investigate, for the first time, the synergistic effect of the optimal mixture of the EOs with conventional antibiotic ciprofloxacin against three pathogenic foodborne bacteria. Gas chromatography/mass spectrometry analysis showed that eucalyptol (39.05%), camphor (24.21%) and borneol (8.29%) were the dominant compounds of LA-EO. LP-EO was characterized by the abundance of camphor (74.51%) and fenchone (27.06%), whereas carvacrol (42.08%), camphor (17.95%) and fenchone (12.05%) were the main constituents of LM-EO. EOs alone or combined showed a remarkable antimicrobial activity against the tested bacteria with minimum inhibitory concentrations (MICs) ranging from 3.53 mg/mL to 15.96 mg/mL. The optimal mixture, calculated using a mixture design, corresponded to 19% LA, 38% LP and 43% LM. All combination of the EOs and the best EO mixture with ciprofloxacin exhibited a total synergism with fractional inhibitory concentration index (FICI) values ranging from 0.27 to 0.37. The best EO mixture showed the highest gain of 128 fold, especially against Salmonella spp., more than that found testing the EOs separately. These findings should be taken into consideration for a possible application in the pharmaceutical and food industries.

Ficus carica is one of the oldest and earliest cultivated fruits in the world. To our knowledge, little information is available on the antimicrobial and antioxidant activities of F. carica...

Over the past years, the emergence of multidrug-resistant pathogens has become a pressing public health problem. It is an important cause of morbidity and mortality worldwide and puts a considerably financial burden on the health care system. The development of new antimicrobials is becoming increasingly difficult, time-consuming and expensive. Hence, new approaches are urgently needed to replenish our arsenal of antimicrobial agents. Plant tissues contain a wild variety of antimicrobial constituents discovered and used to reverse drugs resistance and restore their efficiency. For this, essential oil (EO) extracted from aerial parts of Senecio anteuphorbium (L) Haw, a Moroccan endemic plant, was studied for the first time for its: (i) Qualitative and quantitative chemical profile, (ii) antimicrobial activities, (iii) and synergistic interactions with two conventional antifungals and ciprofloxacin. The data of the chemical composition showed that S. anteuphorbium EO was characterized by the abundance of γ-selinene (27.2%), cyperene (21.7%), γ-cadinene (11.4%) and α-cyperone (8.1%). Regarding antimicrobial assays, results demonstrated that S. anteuphorbium EO displayed moderate antimicrobial activities with MIC values in the range of 0.51 mg/mL and 1.02 mg/mL for yeasts and between 2.3 mg/mL and 4.6 mg/mL for bacteria. However, the combination of the studied EO with fluconazole and ciprofloxacin showed a significant decrease in their individual MICs. In fact, at sub-inhibitory concentrations S. anteuophorbium EO contributed to the decrease of fluconazole MICs of Candida strains by 4- to 64- fold and by 2- to 16- fold regarding ciprofloxacin MICs. The overall data suggest that the topical or systemic use of S. anteuphorbium EO in combinational treatments may be a promising strategy to combat infectious diseases and further studies should be performed in order to well determine the main mechanism of these synergistic associations.

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 β-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.

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.

Introduction Combinations of plant essential oils (EOs) provide a novel approach to increasing effectiveness against resistant bacteria. In this context, the combined antibacterial effects of EOs obtained from Lavandula maroccana (Murb.), Thymus pallidus Batt., and Rosmarinus officinalis L. against three multi-drug resistant Gram-negative bacteria were evaluated. Methods The antibacterial effect of each EO alone was evaluated by agar diffusion and broth microdilution methods, while the effects of EO binary combinations were evaluated through the determination of the fractional inhibitory concentration index (FICI) using the checkerboard method. Results GC-MS analysis of EOs showed that T. pallidus was dominated by γ-terpinene, thymol and p-cymene, while the main constituents of R. officinalis were 1,8-cineole, camphor, and α-pinene. L. maroccana EO was characterized by a high percentage of carvacrol. EOs tested individually demonstrated moderate antibacterial activity with MIC and MBC values from 1.3 mg/mL to72 mg/mL, whereas the FICI of their binary combinations revealed interesting interactions. The combination of EOs obtained from T. pallidus and L. maroccanas showed the highest number of effective combinations, with synergistic effects against P. aeruginosa and K. pneumoniae, and an additive effect against E. coli. The mixture of L. maroccana and R. officinalis EOs showed synergistic effects against E. coli and P. aeruginosa with an additive effect against K. pneumoniae. The combination of R. officinalis and T. pallidus EOs showed a synergistic effect against K. pneumoniae only. Conclusion These findings suggest that combining L. maroccana with T. pallidus and/or R. officinalis EOs may constitute a promising option for controlling the emergence of multidrug-resistant bacteria.

Inhibition of bacterial efflux mechanisms has been investigated as a promising target to combat the bacterial resistance to antibiotics. The present work investigated the ability of Artemisia herba-alba and Cymbopogon citratus essential oils (EOs) to inhibit efflux pumps of some selected antibiotic-resistant Gram-negative bacteria. The chemical profiles of tested EOs were evaluated by GC–MS. The major constituents of the oils were chrysanthenone (47.0%), camphor (24.0%), and verbenone (7.2%) in A. herba-alba EO and geranial (46.3%), neral (35.2%), and geraniol (5.4%) in C. citratus EO. Antibacterial tests showed that these EOs had high antibacterial activity. This activity was significantly enhanced in the presence of an efflux pump inhibitor, phenylalanine arginyl s-naphthylamide (PAsN). When combined with PAsN, the MIC values against EA27 and AG102 (strains overexpressing efflux pumps) were decreased 32-fold and >32-fold, respectively, for A. herba-alba , and 512-fold and 1024-fold, respectively, for C. citratus EO. The involvement of a lipopolysaccharide (LPS) structure in the resistance to the active compounds of the studied EOs was also demonstrated in Salmonella LPS deep-rough mutants. Moreover, the combination of A. herba-alba and C. citratus EOs, at a low concentration, reduced significantly chloramphenicol MIC 2–4-fold, 4-fold and 8–16-fold, respectively, for EAEP294 (deleted of AcrAB), EA27 and AG102. For Salmonella strains, all tested combinations recorded a gain greater than or equal to 64-fold. It was concluded that these EOs can alter efflux pump activity and may be good candidates for the development of new chemosensitizer drugs able to restore antibiotic activity of some drug-resistant Gram-negative bacteria.

The in vitro antibacterial activity of Thymus riatarum essential oil was evaluated in the present study using different assays toward human pathogenic isolates collected during serious infections. The phytochemical analysis of T. riatarum essential oil (EO) indicated the presence of 20 components, including mainly borneol (41.67%), terpinen-4-ol (8.65%), and trans -caryophyllene (7.59%). Determination of antibacterial activity showed that the EO possesses a noticeable potential of inhibiting the growth of tested strains with Minimum Inhibitory Concentrations (MICs) ranging from 3.57 mL/L to 7.5 mL/L. However, Pseudomonas aeruginosa showed the least sensitivity and was only inhibited with high concentrations (MIC > 7.5 mL/L). The release of cytoplasmic material absorbing at 260 nm, measured in the presence of T. riatarum EO, increased in response to oil concentration. The ability of T. riatarum EO to block efflux pumps systems of resistant Gram-negative bacteria was also studied. The results indicated that the EO activity was significantly enhanced in the presence of an efflux pump inhibitor such as phenylalanine arginyl s-naphthylamide (PAsN). In addition, the tested EO, used at MIC/4, was able to increase chloramphenicol susceptibility of several resistant isolates. These results demonstrated that T. riatarum EO could permeabilize bacterial membrane impairing efflux pump activity and may be used in combination with conventional antibiotics against multidrug-resistant bacteria. To our knowledge, this is the first report about the antibacterial activity and the mode of action of T. riatarum EO.

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.

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 β-naphthylamide (PAβN). 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.

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 (α + β) (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.

One hundred twenty-two different actinomycete strains were isolated from sample collected from several depths of the Marrakech wastewater infiltration-percolation system. To evaluate the antimicrobial effect of the different actinomycetes recovered, eleven wastewater-associated micro-organisms known as human potential pathogens were used. Results showed that 44 isolates had an in vitro inhibitory effect toward at least seven of the indicator microorganisms while only five active strains inhibited all these pathogens. All five selected active isolates belonged to the genus Streptomyces. Three were identified as Streptomyces violaceorubidus. These isolates showed the broad activity spectrum against a wastewater-associated pathogenic yeast (Candida albicans), Gram-negative (Salmonella sp. CCMM B17) and Gram-positive (Staphylococcus aureus CCMM B3). These findings indicate the potential involvement of antagonistic actinomycetes in the removal of wastewater-associated pathogens.

The dynamic and antibiotic resistance of faecal coliforms, faecal streptococci and Pseudomonas aeruginosa , isolated by standard membrane filtration methods from suburban and rural untreated groundwater supplies, were scrutinized in this study. The annual average densities of these bacteria were respectively 1891 colony forming units (c.f.u.)/100 mL, 1246 c.f.u./100 mL and 206 c.f.u./100 mL. The total occurrence of faecal coliforms and faecal streptococci during the period of study was 94%. Detectable P. aeruginosa was present in 88% of samples. A total of 320 faecal coliforms, 338 faecal streptococci and 208 P. aeruginosa strains, were examined for their resistance to eighteen antibiotics. The overall resistance (resistance to at least one antibiotic) of these bacteria was 100%. The multiresistance (resistance to at least two antibiotics) of faecal coliforms and faecal streptococci was 94%, whereas 100% of P. aeruginosa strains were multiresistant. The monoresistance (resistance to one antibiotic) of fae...

In Morocco, the occurrence of toxic cyanobacteria blooms is confirmed in some water bodies used for recreational and/or as drinking water reservoirs. According to WHO recommendations, the establishment of a monitoring program for microcystins is a necessity. This paper presents toxicological studies of 19 toxic cyanobacteria strains of Microcystis, Synechocystis, Pseudanabaena, and Oscillatoria. These strains were isolated from various water bodies including natural lakes, reservoirs, and ponds located in central regions of Morocco. The isolation, culture, and biomass production of these strains was made on Z8 or BG13 media under laboratory controlled conditions. The hepatotoxicity of cyanobacterial lyophilized material was confirmed by mouse bioassays. The amount of microcystins produced by each strain was determined by the enzyme-linked immunosorbent assay (ELISA). The detection and identification of microcystin variants was performed by high performance liquid chromatography (HPLC) with photodiode array detection. Almost all strains showed medium to high toxicity, the estimated LD50 i.p. mice bioassay ranged between 28 to 350 mg/kg body weight. The concentrations of microcystins varied between 2.16 to 944 micrograms/g and 26.8 to 1884 micrograms/g dry weight determined by ELISA and HPLC, respectively. The screening of bloom-forming and microcystin producer cyanobacteria strains in these fresh water bodies leads us to propose the need for the establishment of a survey of cyanobacteria and a cyanotoxin-monitoring program.

In waste stabilisation pond systems the interactions between algae and bacteria have an important ecological effect and appeared to play a key role in the self-purification process. The aim of the present study is to evaluate the interactions between two axenic cyanobacteria: Synechocystis sp. and Pseudanabaena sp. with heterotrophic bacteria and some pathogenic bacteria: E. coli, Salmonella sp. and non-O1 V. cholerae. The results obtained showed that Synechocystis sp. (planktonic cyanobacterium) and Pseudanabaena sp. (benthic cyanobacterium) stimulated the growth and the survival of heterotrophic bacteria and non-O1 V. cholerae and reduced the survival of E. coli and Salmonella. Blooms of these cyanobacteria during hot periods which are a particularity in stabilisation ponds of Marrakech, could explain the dynamics of bacteria studied in this sewage treatment process. The presence of heterotrophic bacteria, showing relative high densities during hot periods, could be considered as an important bioticfactor which led to the cyanobacteria blooms in the Marrakech ponds which function under an arid Mediterranean climate.

Experimental study of the effects of sunlight and Synechocystis sp. (picocyanobacterium) on the incidence of antibiotic resistance in Salmonella sp. and faecal coliforms (FC), was carried out in wastewaters of Marrakesh stabilization ponds (Morocco). The effect of sunlight was found to be important on the incidence of antibiotic resistance of these enteric bacterial populations. In microcosms seeded with the mixed inoculum (40 different strains) of Salmonella or FC and exposed to sunlight, the overall resistance of these bacteria (72.5 and 57.5% respectively) was significantly higher than that noted in the dark (45 and 32.5% respectively). In microcosms seeded with a monobacterial inoculum, some FC isolates became resistant to the antibiotics cephalothin, amoxicillin or sulphamethoxazole, after their incubation in sunlight. Synechocystis appeared to have no significant effect on the incidence of bacterial antibiotic resistance. The ecological significance of sunlight and Synechocystis sp. in reducing densities and increasing antibiotic resistance of pathogenic bacteria through wastewater stabilization ponds is discussed.

UNLABELLED Several mechanisms are involved in the bacterial resistance towards antimicrobial agents. The membrane-associated mechanisms of resistance were studied in Escherichia coli strains after incubation with Thymus maroccanus essential oil, its major components (carvacrol and thymol) or with certain antibiotics. The minimum inhibitory concentration (MIC) and the expression of membrane proteins, porins and efflux pumps were determined in wild type and derivative strains. Derivative strains adapted to different compounds displayed a high level of resistance to all tested antibiotics. The MIC increase is associated with an overexpression of an efflux pump immunorelated to AcrAB-TolC in various variants. Interestingly, the expression of outer membrane proteins slightly decreases in these strains. We demonstrate that the increase in antibiotic resistance correlates with membrane changes observed in the variants. This type of bacterial adaptation to natural compounds can occur in vivo providing the emergence/selection of bacteria less susceptible to clinically used antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY Thymus maroccanus essential oil and some major components are able to select variants that modify the expression of transporters involved in the influx (porins) and in the efflux (AcrAB family) of various drugs. Importantly, these membrane proteins are involved in the transport of natural compounds and several antibiotic families. This special 'membrane adaptation' can explain the persistence of less susceptible/tolerant bacteria in the environment where natural compounds are present and the continuous stimulation of efflux systems in these bacteria.

The abundance and antibiotic resistance of non-O1 Vibrio cholerae strains were studied in wastewater before and after treatment in stabilization ponds in an arid Mediterranean climate. The seasonal abundance of non-O1 Vibrio cholerae was the inverse of those of fecal coliforms, with high densities in hot periods and low densities in cold periods. Although the stabilization pond presents a good efficiency in removing fecal coliforms (97.97%), this treatment system did not produce any significant reduction in non-O1 V. cholerae abundances between the inflow and outflow stations. Among the 240 non-O1 V. cholerae strains isolated before and after treatment in the stabilization ponds, 89 (37.1%) isolates were resistant to at least one of 14 tested antibiotics. The levels of antibiotic resistance at the inflow and outflow points of the system were respectively 40 and 34%. High ampicillin, amoxicillin and mezlocillin resistance was observed at all sampling points, followed by resistance to cefalexin, cefoperazone and amikacin. Antibiotic resistance can be transferred from non-O1 V. cholerae to other members of the Enterobacteriaceae family such as Escherichia coli K12. Transfer frequencies in nutrient broth and filtered wastewater were 3 × 10−5 and 2 × 10−8, respectively.

The discovery of antifungal drugs had eradicated some infections that ravaged the humankind. But their indiscriminate use has led to the development of multidrug resistant pathogens. One strategy employed to overcome these resistance mechanisms is the use of combination of the essential oils (EOs) of medicinal plants and conventional drugs. In this study, we investigated a possible synergistic effect of the EOs of two Moroccan endemic thymes (Thymus broussonetii and T. maroccanus) with amphotericin B (Amp B) and fluconazol against Candida albicans. The fractional inhibitory concentration indices (FICI) of T. maroccanus and T. broussonetii EOs combined with Amp B and fluconazol, calculated from the checkerboard titer assay, were 0.49, 0.27, 0.37 and 0.3, respectively. Also, our results indicate that the synergistic effect of EOs with fluconazol was stronger than the combination with Amp B. All these data highlight that the EOs tested potentiate the antifungal action of Amp B and fluconazol, suggesting a possible utilization of these EOs in addition to antifungal drugs for the treatment of some candidiasis due to C. albicans. The use of these combinations is likely to reduce the minimum effective dose of the drugs, thus minimizing their toxic side effects and the treatment cost.

The stabilization pond is one of the more important biological wastewater treatment systems, applied in many countries. An experiment treating wastewater by stabilization ponds under the arid climate of Marrakesh (Morocco) has been underway since 1985. The experimental installation, made from two lined stabilization ponds, received domestic sewage which carried not only the organic load but also a significant bacterial load and other microorganisms. In this new habitat, the cells' bacterial behaviour was affected by various physico-chemical and biological factors. It appears that in such treatment system, known for excessive algal production, the microalgae has evidently an influence on bacterial growth. In this paper, we proposed to appreciate how microalgae essentially: Chlorella (Chlorophyta), Synechococcus andSynechocystis (Cyanobacteria), can affect the behaviour, survival and temporal evolution of Escherichia coli and Vibrio cholerae. In wastewater stabilization ponds of Marrakesh high levels of V. cholerae and low concentrations of coliform bacteria were noted during summer periods. This period coincided with a bloom of picocyanobacteria associated with a weak relative abundance of Chlorella. Some interactions tests were carried out with these bacteria and these algae, using a treated wastewater batch culture. Results show that the green algae reduces V. cholerae (pathogenic bacteria) abundances more than E. coli (fecal contamination bacteria) where as better survival of this pathogenic bacteria was noted in presence of Cyanobacteria. The die-off of E. coli appears to be more reduced in presence of Cyanobacteria than Chlorella. Furthermore, the alkaline pH seems to present a more bactericidal effect on E. coli than on V. cholerae. Thus, the Cyanobacteria blooms, associated with a weak percentage of Chlorella abundance, occurring periodically during summer in sewage stabilization ponds of Marrakesh, will be considered as one of the major factors leading to high levels of V. cholerae and low abundances of fecal coliform bacteria during the hot period.