Chemical composition and synergistic effect of three Moroccan lavender EOs with ciprofloxacin against foodborne bacteria: a promising approach to modulate antimicrobial resistance.

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 to 15·96 mg ml-1 . 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 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.

Natural extracts stimulate membrane-associated mechanisms of resistance in Gram-negative bacteria.

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.

Thymus maroccanus essential oil, a membranotropic compound active on Gram-negative bacteria and resistant isolates.

AIMS: The effects of Thymus maroccanus essential oil (EO) on the integrity of the cell membranes and the permeability of the outer membrane (OM) and inner membrane (IM) of Escherichia coli, Enterobacter aerogenes and Salmonella enterica Typhimurium were investigated. METHODS AND RESULTS: The bacterial release of intracellular proteins, cytoplasmic ß-galactosidase and periplasmic ß-lactamase induced by T. maroccanus EO was compared to the membranotropic activity of polymyxin B (PB) known as an effective permeabilizer of the membrane of Gram-negative bacteria. Results showed that T. maroccanus EO increased the permeability of the OM and IM of studied bacteria and induced the release of intracellular proteins into the external medium. CONCLUSIONS: The effect of T. maroccanus EO on the outer membrane was comparable to that of PB, and both T. maroccanus EO and PB induce similar levels of ß-lactamase release. In addition, it also promoted the release of the cytoplasmic ß-galactosidase. Moreover, the lipopolysaccharide molecules and the overexpression of efflux pumps seem to play a crucial role in the level of susceptibility of studied bacteria to the permeabilizing effect of T. maroccanus EO. SIGNIFICANCE AND IMPACT OF STUDY: These results demonstrate that T. maroccanus EO can restore antibiotic activity by targeting the two bacterial membranes and would be attractive candidates for developing new adjuvants for combating resistant Gram-negative bacteria.

Anticandidal activity of the essential oils of Thymus maroccanus and Thymus broussonetii and their synergism with amphotericin B and fluconazol.

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.

Environmental pollutions impacts on the bacteriological and physicochemical quality of suburban and rural groundwater supplies in Marrakesh area (Morocco).

This study scrutinized bacteriological and chemical quality of groundwater supplies of Marrakesh (Morocco) within a year. It assessed the influence of some chemical factors on fecal and opportunistic pathogenic bacterial communities. The annual average densities of fecal coliforms, fecal streptococci and Pseudomonas aeruginosa were respectively: 1891 colony forming units (CFU)/100 mL, 1246 CFU/100 mL and 206 CFU/100 mL. The total occurrence of these bacteria during the period of study was 94%. Detectable non-O1 Vibrio cholerae was present in 81% of samples and the mean abundances ranged from 0 to 11100 MPN/100 mL. Significant correlations between fecal coliforms and streptococci and between fecal coliforms and non-O1 V. cholerae (p < 0.01) were found. Fecal coliforms can be used to detect the presence of non-O1 V. cholerae in this groundwater. These well waters were greatly mineralized, hard, salt with high concentrations of nitrogenous ions and major elements (Ca(2+), Na(+), Cl(-), SO(2-)(4) and K(+)). Most of chemical parameters were relatively steady with time except for calcium, ammonium, nitrites and organic matter which were seasonally influenced. The degree of correlation between chemical parameters and the abundances of isolated bacteria is heterogeneous. The concentrations of calcium and nitrites favored (p < 0.01) the abundance of fecal coliforms and streptococci, but reduced P. aeruginosa abundances. Calcium, salinity, chlorides, nitrites and nitrates tests can be used for screening in situ when the laboratory facilities are limited. This work is expected to assist local authorities in developing plans and actions to reduce the pollution to acceptable levels.

Bioactive compounds from Pseudanabaena species (Cyanobacteria).

Bioactive products of the multicellular filamentous benthic cyanobacterium, Pseudanabaena species, isolated from wastewater stabilization ponds at Marrakech, were tested against some pathogenic micro-organisms. Extracellular and intracellular products released by this blue-green alga in the stationary, growth phase, reduced the survival of Escherichia coli, Salmonella sp., Staphylococcus aureus and Candida albicans, and stimulated the growth of non-O1 V. cholerae, whereas no significant effect was noted on the growth of Candida tropicalis. Intracellular substances (methanolic extract) had a stronger inhibitory effect on these micro-organisms than that produced by extracellular substances. These antibacterial and antifungal substances may have a pharmacological value. They may have an important ecological effect on the composition of the bacterial community in Marrakech stabilization ponds when blooms of Pseudanabaena species occurred in this sewage treatment ecosystem during hot periods.

Dynamics of non-O1 Vibrio cholerae and fecal coliforms in experimental stabilization ponds in the arid region of Marrakesh, Morocco, and the effect of pH, temperature, and sunlight on their experimental survival.

We studied the spatial-temporal dynamics of non-O1 Vibrio cholerae numbers at a stabilization pond treatment plant. This bacterium's seasonal dynamics were the inverse of those of fecal coliforms, with high levels in hot periods and low levels in cold periods. Stabilization pond treatment did not significantly reduce non-O1 V. cholerae numbers between the system's inflow and outflow points. In contrast, fecal coliforms were reduced by 98.95% in hot periods and by 94.91% in cold periods. Significant rho coefficient values for the Spearman correlation between numbers of non-O1 V. cholerae and temperature and pH of 0.91 and 0.76 respectively, were found at the system's outflow point. An experimental study of the effects of pH, temperature, and sunlight on the survival of non-O1 V. cholerae and fecal coliforms confirmed the inverse behaviour of the two bacterial groups noted in the stabilization ponds. Alkaline pH values of 8 and 8.8 promoted the survival of non-O1 V. cholerae and inhibited that of Escherichia coli. Low temperatures (8 degrees C) prolonged E. coli survival (k = 0.002/h), while a temperature of 23 degrees C reduced it markedly (k = 0.022/h). Non-O1 V. cholerae did not survive as well as E. coli at 8 degrees C (k = 0.009/h). The effect of temperature on non-O1 V. cholerae appeared to be closely linked to nutrient levels. Non-O1 V. cholerae appeared to be less sensitive to sunlight than E. coli whose survival was markedly reduced particularly during summer periods. Non-O1 V. cholerae and E. coli did not behave in the same way in water subjected to stabilization pond treatment. The use of fecal coliforms as an indicator of the potential health hazard of the effluent may not be adequate for this treatment procedure.

Drug resistance in Salmonella strains isolated from domestic wastewater before and after treatment in stabilization ponds in an arid region (Marrakech, Morocco).

Some 118 Salmonella strains isolated before and after treatment in stabilization ponds were tested for antimicrobial resistance. In the treatment plant, which decreases the abundance of Salmonella by 99%, a significantly lower level of antibiotic resistance (P<0.01) was identified at the system's inflow point (19%) than at its outflow (29%). The serotypes most frequently identified as having multiple antibiotic resistance were Salmonella paratyphi B and S. typhimurium. High tetracycline resistance was observed at all sampling points, followed by resistance to ampicillin and streptomycin. Antibiotic resistance can be transferred from Salmonella to other members of the Enterobacteriaceae family, such as Escherichia coli K12; transfer frequencies in nutrient broth and filtered sewage water were 4.5×10(-4) and 7×10(-7), respectively.