Combination of mild heat and plant essential oil constituents to inactivate resistant variants of Escherichia coli in buffer and in coconut water.

The growing demand for minimally processed foods with clean labels has stimulated research into mild processing methods and natural antimicrobials to replace intensive heating and conventional preservatives, respectively. However, we have previously demonstrated that repetitive exposure of some bacteria to mild heat or subinhibitory concentrations of essential oil constituents (EOCs) may induce the emergence of mutants with increased resistance to these treatments. Since the combination of mild heat with some EOCs has a synergistic effect on microbial inactivation, we evaluated the potential of such combinations against our resistant E. coli mutants. While citral, carvacrol and t-cinnamaldehyde synergistically increased heat inactivation (53.0 °C, 10 min) of the wild-type MG1655 suspended in buffer, only the combination with carvacrol (200 µl/l) was able to mitigate the increased resistance of all the mutants. Moreover, the combination of heat and carvacrol acted synergistically inactivating heat-resistant variants of E. coli O157:H7 (ATCC 43888). This combined treatment could synergistically achieve more than 5 log10 reductions of the most resistant mutants in coconut water, although the temperature had to be raised to 57.0 °C. Therefore, the combination of mild heat with carvacrol appears to hold promise for mild processing, and it is expected to counteract the development of heat resistance.

Zinc-Induced Transposition of Insertion Sequence Elements Contributes to Increased Adaptability of Cupriavidus metallidurans.

Bacteria can respond to adverse environments by increasing their genomic variability and subsequently facilitating adaptive evolution. To demonstrate this, the contribution of Insertion Sequence (IS) elements to the genetic adaptation of Cupriavidus metallidurans AE126 to toxic zinc concentrations was determined. This derivative of type strain CH34, devoid of its main zinc resistance determinant, is still able to increase its zinc resistance level. Specifically, upon plating on medium supplemented with a toxic zinc concentration, resistant variants arose in which a compromised cnrYX regulatory locus caused derepression of CnrH sigma factor activity and concomitant induction of the corresponding RND-driven cnrCBA efflux system. Late-occurring zinc resistant variants likely arose in response to the selective conditions, as they were enriched in cnrYX disruptions caused by specific IS elements whose transposase expression was found to be zinc-responsive. Interestingly, deletion of cnrH, and consequently the CnrH-dependent adaptation potential, still enabled adaptation by transposition of IS elements (ISRme5 and IS1086) that provided outward-directed promoters driving cnrCBAT transcription. Finally, adaptation to zinc by IS reshuffling can also enhance the adaptation to subsequent environmental challenges. Thus, transposition of IS elements can be induced by stress conditions and play a multifaceted, pivotal role in the adaptation to these and subsequent stress conditions.

Role of 1-acyl-sn-glycerol-3-phosphate acyltransferase in psychrotrophy and stress tolerance of Serratia plymuthica RVH1.

A mutant with a transposon insertion just upstream of the lysophosphatidic acid acyltansferase gene plsC was isolated in a screen for mutants affected in growth at low temperature of the psychrotroph Serratia plymuthica RVH1. This mutant had lost its ability to grow at 4 °C and was severely affected in growth at 10 °C, but showed only slightly reduced growth at 30 °C. Fatty acid analysis of membrane extracts showed that the ratio of C16:1/C18:1 fatty acids was six-to sevenfold reduced in the mutant, although the ratio of unsaturated to saturated fatty acids was unaffected. The homeoviscous adaptation ability of the mutant was also unaffected. Growth and fatty acid composition were mostly restored by overexpressing plsC on a plasmid. Supplementation of C16:1 (palmitoleic acid) into the growth medium partially rescued low temperature growth, indicating that a balanced ratio of the two main unsaturated fatty acids is required for psychrotrophy. The mutant was significantly more strongly inactivated by high pressure treatment at 250 MPa, but not at higher pressures. It also showed reduced growth at low pH, but not at increased NaCl concentrations. This work provides novel information on the role of membrane fatty acid composition in stress tolerance.