Chueca Beatriz, Berdejo Daniel, Gomes-Neto Nelson J, Pagán Rafael, García-Gonzalo Diego
Tecnología de los Alimentos, Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Instituto Agroalimentario de Aragón, Universidad de Zaragoza-CITA Zaragoza, Spain.
Laboratory of Food Microbiology, Department of Nutrition, Health Sciences Center, Federal University of Paraíba João Pessoa, Brazil.
Front Microbiol. 2016 Mar 4;7:273. doi: 10.3389/fmicb.2016.00273. eCollection 2016.
The improvement of food preservation by using essential oils (EOs) and their individual constituents (ICs) is attracting enormous interest worldwide. Until now, researchers considered that treatments with such antimicrobial compounds did not induce bacterial resistance via a phenotypic (i.e., transient) response. Nevertheless, the emergence of genotypic (i.e., stable) resistance after treatment with these compounds had not been previously tested. Our results confirm that growth of Escherichia coli MG1655 in presence of sub-inhibitory concentrations of the ICs carvacrol, citral, and (+)-limonene oxide do not increase resistance to further treatments with either the same IC (direct resistance) or with other preservation treatments (cross-resistance) such as heat or pulsed electric fields (PEF). Bacterial mutation frequency was likewise lower when those IC's were applied; however, after 10 days of re-culturing cells in presence of sub-inhibitory concentrations of the ICs, we were able to isolate several derivative strains (i.e., mutants) displaying an increased minimum inhibitory concentration to those ICs. Furthermore, when compared to the wild type (WT) strain, they also displayed direct resistance and cross-resistance. Derivative strains selected with carvacrol and citral also displayed morphological changes involving filamentation along with cell counts at late-stationary growth phase that were lower than the WT strain. In addition, co-cultures of each derivative strain with the WT strain resulted in a predominance of the original strain in absence of ICs, indicating that mutants would not out-compete WT cells under optimal growth conditions. Nevertheless, growth in the presence of ICs facilitated the selection of these resistant mutants. Thus, as a result, subsequent food preservation treatments of these bacterial cultures might be less effective than expected for WT cultures. In conclusion, this study recommends that treatment with ICs at sub-inhibitory concentrations should be generally avoided, since it could favor the emergence of hyper-resistant strains. To ascertain the true value of EOs and their ICs in the field of food preservation, further research thus needs to be conducted on the induction of increased transient and stable bacterial resistance via such antimicrobial compounds, as revealed in this study.
利用香精油（EOs）及其单一成分（ICs）改善食品保鲜在全球引起了极大的关注。到目前为止，研究人员认为用这类抗菌化合物处理不会通过表型（即短暂性）反应诱导细菌耐药性。然而，此前尚未测试过用这些化合物处理后基因型（即稳定性）耐药性的出现情况。我们的结果证实，在亚抑制浓度的香芹酚、柠檬醛和氧化苎烯等单一成分存在的情况下，大肠杆菌MG1655的生长不会增加对相同单一成分（直接耐药性）或其他保鲜处理（交叉耐药性）（如加热或脉冲电场（PEF））进一步处理的耐药性。当应用这些单一成分时，细菌突变频率同样较低；然而，在亚抑制浓度的单一成分存在下对细胞进行10天的再培养后，我们能够分离出几种对这些单一成分显示出增加的最低抑菌浓度的衍生菌株（即突变体）。此外，与野生型（WT）菌株相比，它们还表现出直接耐药性和交叉耐药性。用香芹酚和柠檬醛筛选出的衍生菌株还表现出形态变化，包括丝状化，并且在生长后期稳定期的细胞计数低于WT菌株。此外，每个衍生菌株与WT菌株的共培养在不存在单一成分的情况下导致原始菌株占优势，这表明在最佳生长条件下突变体不会胜过WT细胞。然而，在单一成分存在下的生长促进了这些耐药突变体的选择。因此，结果是，对这些细菌培养物进行后续的食品保鲜处理可能比对WT培养物预期的效果要差。总之，本研究建议通常应避免用亚抑制浓度的单一成分进行处理，因为这可能有利于超耐药菌株的出现。为了确定香精油及其单一成分在食品保鲜领域的真正价值，因此需要进一步研究此类抗菌化合物诱导细菌短暂性和稳定性耐药性增加的情况，正如本研究中所揭示的那样。
