Emergence of Hyper-Resistant Escherichia coli MG1655 Derivative Strains after Applying Sub-Inhibitory Doses of Individual Constituents of Essential Oils

Abstract

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.

A bacterial tragedy of the commons that masks the actual frequency of mutants

“…Bacteria were grown overnight in medium TGP and plated on TGP containing rifampicin (100 μg/ml) and on TG2PP plates (Figure 1c). The frequency of Rif R mutants at the 14 th day was 3.2 × 10 −7 / generation , which is compatible with data reported elsewhere for strain MG1655 [39, 40], but different from others that reported lower frequencies [41, 42, 43]. In contrast, bacteria plated on TG2PP showed the usual pattern of mutant accumulation (first visible colonies at day 3 and a low mutant frequency of 5.8 × 10 −8 /generation, calculated at the 14 th day).…”

Whole-Genome Sequencing and Genetic Analysis Reveal Novel Stress Responses to Individual Constituents of Essential Oils in Escherichia coli

“…Treatment of bacterial cells with these ICs caused the generation of reactive oxygen species (ROS), and bacterial cell death followed the RecA-mediated SOS response pathway (6,7). Although these pathways could lead to increased mutagenesis (10), we observed a lower mutation rate in Escherichia coli cells grown in the presence of sub-MICs of ICs than in cells grown in their absence (11). This protection against mutation was attributed to the potential antioxidant activity of ICs at the low concentrations used (12).…”

“…This protection against mutation was attributed to the potential antioxidant activity of ICs at the low concentrations used (12). Nevertheless, derivative strains showing direct resistance and cross-resistance to multiple antimicrobial treatments (i.e., hyperresistant strains) were isolated after the growth of Escherichia coli MG1655 in the presence of sub-MICs of monoterpene ICs [carvacrol, citral, and (ϩ)-limonene oxide] (11). The stability of the resistant phenotype in these derivative strains after their regrowth in the absence of the ICs supported the notion of genotypic modifications from the original strain.…”

“…Recent studies include the sequencing of methicillin-resistant Staphylococcus aureus isolates from both outbreaks in a regional hospital and intercontinental collections (23)(24)(25), whole-genome sequencing (WGS) of multiresistant Klebsiella (26) or carbapenem-resistant Acinetobacter baumannii (27), and the identification of genes associated with the higher resistance shown by these strains. Therefore, as previously researched in antibiotic-resistant strains (25), WGS of hyperresistant derivative strains previously obtained (11) with ICs would, in principle, permit the identification of single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) in comparison with the WT.…”

“…Here, we performed WGS of WT E. coli MG1655 and the isogenic strains CAR, CIT, and LIM, isolated and characterized in a previous work (11).…”