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Human infectious diseases once treatable with antibiotics are now becoming a serious life-threatening problem due to the emergence of multidrug resistant (MDR) micro-organisms. For this, essential oils (EOs) could constitute a viable source of bioactive compounds capable of inhibiting the mechanisms of this antimicrobial resistance. In this context, our goals were to determine for the first time the chemical composition of EO extracted from aerial parts of Rhus albida, its antibacterial and synergistic interactions with fluoroquinolones and finally to evaluate its ability to inhibit the Staphylococcus aureus NorA efflux pump. The chemical profile and antibacterial activity of R. albida EO were determined using Gas Chromatography coupled to Mass Spectrometry and microdilution assays, respectively. While its NorA efflux-pump-inhibiting activity was assessed using a fluorescent real-time efflux method (based on ethidium bromide (EtBr)) against two S. aureus strains: a NorA overproducing strain (SA1199B) and a wild type strain (SA1199). Results showed that the EO, which is characterized by the abundance sesquiterpenes (δ-cadinene (15.6%), α-copaene (6.3%) and caryophyllene (6.2%)), displayed moderate antimicrobial activities against both SA1199 and SA1199B (MIC = 2048 µg/mL). Nevertheless, its combination with fluoroquinolones contributed to the reduction of their intrinsic MICs only against SA1199B by 4- to 8-fold. Furthermore, the EO at 1/32 MIC, contributed to a large accumulation of EtBr and inhibition of its efflux in SA1199B. Our findings demonstrated the ability of R. albida EO to inhibit the NorA efflux pump. Thus, further studies should be performed in order to make the combination of this natural product with approved antimicrobials a viable option for future administrations in infections with S. aureus strains overproducing NorA EPs.

The aims of the present study were to evaluate for the first time the chemical composition and in vitro antimicrobial potential of essential oil (EO) obtained from endemic Lavandula maroccana (Murb.) singly or in combination with some conventional antimicrobials (ciprofloxacin, vancomycin, fluconazol, and amphotericin B) against a panel of pathogenic microorganisms. The gas chromatography/mass spectrometry (GC/MS) analysis of EO led to the identification of 20 components, representing 98.8% of the total oil. The main constituent was the phenolic monoterpene carvacrol (78.2%), followed by 2-Methoxy-4-vinylphenol (2.5%), and Spathulenol (2.2%). The results of the antimicrobial assays demonstrated that L. maroccana EO displayed moderate activity, with minimal inhibitory concentration (MIC) values of 4.604 mg/mL for yeasts and ranged from 2.302 to 18.416 mg/mL for bacteria. According to FIC index values, the combination between L. maroccana EO and conventional antimicrobials elicited very promising synergistic effects against all tested strains. In fact, at sub-inhibitory concentrations L. maroccana EO contributed to the decrease of fluconazol and amphotericin B MICs of Candida strains by 64- to 128-fold and by 2- to 4-fold, respectively. Regarding bacterial strains, L. maroccana EO has reduced the ciprofloxacin and vancomycin MICs by 4- to 33-fold and by 2- to 4-fold, respectively. From these results, it can be concluded that L. maroccana EO can be considered as a promising source of natural antimicrobials, and constitute a potential candidate to be used in combination antibiotic therapy in order to reduce the overuse of antibiotics, and to combat some human infectious diseases.

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. info:eu-repo/semantics/publishedVersion

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.

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.

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 problem of water scarcity is becoming more pronounced especially in countries with arid and semi-arid climates such as Morocco. Wastewaters discharge into different aquatic ecosystems (groundwater, sea water, river, lake water...), are draining different types of microorganisms and hazardous chemicals. The microbiological risk is not negligible, especially in areas where wastewater or other contaminated water, are reused for irrigation without preliminary treatment or for direct consumption by human and animals.

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.