No induction of antimicrobial resistance in Staphylococcus aureus and Listeria monocytogenes during continuous exposure to eugenol and citral

Размещено на http://www.allbest.ru/

No induction of antimicrobial resistance in Staphylococcus aureus and Listeria monocytogenes during continuous exposure to eugenol and citral

Annotation

Keywords: essential oil, adaptation, resistance, virulence.

Отсутствие противодействия антимикробной устойчивости у моноцитогенов staphylococcus aureus и listeria при непрерывном воздействии евгенола и цитрала

Алрхаиа И. Студент 5 курс, Майкопский государственный технологический университет, факультет «Фармация»

Адаптация к эвгенолу и цитралю осуществлялась путем последовательного воздействия на патогены увеличивающихся концентраций эфирных масел. Стандарт M2-A9 использовали для определения восприимчивости к антибиотикам. Влияние эвгенола и цитраля на адгезионную способность оценивали методом кристаллического фиолетового. Влияние адаптации к эвгенолу на вирулентность оценивали с использованием модели Galleria mellonella. В адаптированных клетках S. aureus, MRSA и L. monocytogenes развития устойчивости к компонентам и антибиотикам не наблюдалось.

Ключевые слова: эфирное масло, адаптация, устойчивость, вирулентность.

Either in clinical or in food-processing environment, the bacterial pathogens encounter a myriad of stresses (to name a few; exposure to antimicrobial agents, acids, bases, different osmotic pressures, different pH and water activity values, heat, freezing and thawing). The action of these stresses compromises the cell viability but at the same time, the cells experience a gene expression alteration directed to protect them against each individual stress or even mount a crossprotection response that not only provide the cell with the means to defeat the individual stress but also a complex of stresses injuries(1,C142-150).The threatening emergence of antibiotic-resistant bacteria is of great concern worldwide, and the possibility of food microbiota being reached by antibiotic resistance is greatly disturbing (2, 1646-1653).

1/ Adaptation to eugenol and citral

The bacterial strains were grown in BHI agar plates during 24 h at 37 or 30 °C. From each plate, a loop was used to inoculate 10 mL of BHI broth, and the culture was incubated overnight. From the overnight, 300 pL culture was centrifuged (2790 x g, 5 min at 4 °C). The pellet was resuspended in 300 pL BHI supplemented with 0.05 mg mL-1 eugenol or citral and used to inoculate 10 mL of BHI (supplemented with the same concentration). Cultures were incubated at 37 °C during 18-24 h. The culture was used to inoculate fresh medium supplemented with eugenol or citral (0.05 mg mL-1). During 4 days, bacteria were transferred sequentially at the same concentration. At each serial passage, the bacterial growth was followed by optical density (OD600 nm). Following the four sequential passages, bacteria were transferred to BHI at higher concentration (increment of 0.02 mg mL-1). The process was repeated until bacteria stop growing. Control cultures (bacteria were grown in medium with no EO component) were maintained in parallel. Three independent replicates were carried out (1,C142-150).

2. Influence of eugenol and citral on adherence

The impact of eugenol and citral on adherence of S. aureus and L. monocytogenes was carried out according to Adriao et al. (2008). Bacterial suspensions (OD600 nm = 0.4-0.5) in BHI supplemented with 0.05 and 0.1 pg mL-1 of eugenol or citral were allowed to adhere for 30 min. Nonadherent cells were removed by washing the wells twice with sterile phosphate-buffered saline (PBS). Wells were air-dried and adherent cells heat-fixed at 80 °C for 30 min and stained with 0.1% crystal violet (3,C1928-1939).

3. Results

Susceptibility to eugenol and citral

The observed MIC values for eugenol and citral determined using the agar dilution and the microdilution methods are indicated in Table1. Differences were observed between the MIC values either of eugenol or of citral, in particular for S. aureus strains with trend to a lower MIC values obtained by the microdilution method; the range of the MIC value for eugenol was 0.10-0.15 mg mL-1 for S. aureus strains determined by the agar dilution method, whereas the MIC value determined by the microdilution method ranged from 0.06 to 0.08 mg mL-1. The susceptibility of S. aureus strains to citral was similar to eugenol (Table 1). In contrast to S. aureus, no differences were observed in the MIC values of eugenol for L. monocytogenes determined by the two methods (Table 1).

Table 1. Minimum inhibitory and minimum bactericidal concentrations of eugenol and citral

eugenol citral oil antibiotic

References

1. Adriao A Vieira M Fernandes I et al. (2008) Marked intra-strain variation in response of Listeria monocytogenes dairy isolates to acid or salt stress and the effect of acid or salt adaptation on adherence to abiotic surfaces. Int J Food Microbiol123: C142-150.

2. Skandamis PN Nychas G-JE (2000) Development and evaluation of a model predicting the survival of Escherichia coli O157:H7 NCTC 12900 in homemade eggplant salad at various temperatures, pHs, and oregano essential oil concentrations. Appl Environ Microbiol66: C1646-1653.

3. Somolinos M., Garda D., Condon S. Mackey B Pagan R (2009) Inactivation of Escherichia coli by citral. J Appl Microbiol108: C1928-1939.

Размещено на Allbest.ru