Structural and functional changes in Alicyclobacillus acidoterrestris after the application of several sub-lethal physical or chemical treatments

Abstract

Scientific background: Alicyclobacillus spp. includes spore-forming and thermo-acidophilic microorganisms, usually recovered from soil, acidic drinks, orchards and equipment from juice producers. The genus includes different species and sub-species, but A. acidoterrestris is generally regarded as the most important spoiler for acidic drinks and juices. (i) The investigation of microbial community evolution was performed, since soil species could acquire specific phenotypic traits in relation to available nutrients. (ii) A variety of physical and chemical approaches to control A. acidoterrestris were proposed, and the study of sub-lethal injury related to extend of damage was examined. (iii) In vitro stress treatments were tested in vivo. Open questions: (i) Few data are available on the genotyping and phenotyping traits of A. acidoterrestris; (ii) Few data are available on the effect of innovative approaches to control A. acidoterrestris; and no data are available on the sub-lethal injury in A. acidoterrestris after invasive treatments; (iii) Few data are available on the control of A. acidoterrestris in complex conditions. Aims: (i) Selection and characterization of wild strains of A. acidoterrestris as a contribution to species characterization, (ii) studying the structural and functional changes that may affect alicyclobacilli and Bacillus spp. (used as reference) after the application of several sub-lethal physical or chemical treatments, and (iii) validation in a food to assess if an injury could occur also in a complex system. Planning of the research: In the first part, 25 wild strains of A. acidoterrestris from soil (23 strains) and spoiled pear juice (CB-1 and CB-2) were isolated, identified, genotyped and phenotyped. In the second part, 4 strains of A. acidoterrestris (the type strain, 2 wild strains isolated from soil and a wild strain isolated from spoiled pear juice) and 2 species of Bacillus spp. were studied under in vitro conditions after the application of chemical, physical and combined treatments to assess their susceptibility and the presence of sub-lethal injury. The characterization of damage was also performed. Finally, in the last step the presence of sub-lethal injury in vivo was investigated. Materials and Methods: (i) Genotypic traits were performed through examining of 16S rRNA and RAPD PCR (Random-Amplified-Polymophic-DNA). Phenotypical traits were assessed on the opportune laboratory media. (ii) Injury characterization was evaluated by Leakage of UV-absorbing substances and BSA protein assay. (iii) For in vivo assay, clarified apple juice was used. Results: (i) Data of soil-borne strains pinpointed that they could be divided into three blocks, represented by soil strains and by strains moving from soil to other niches. In this context, phenotyping and genotyping did not group the strains in the same way and many strains phylogenetically different showed the same phenotypic trend, thus suggesting that A. acidoterrestris could exist as a genomovar. In addition, the strain CB-1 was distant from other alicyclobacilli, although it possessed the same traits than the other isolate from juice (CB-2); therefore, it is probably a fast-clock organism or the beginning on an alternative pathway in alicyclobacilli evolution. (ii) Alternative approaches applied to control 4 strains of A. acidoterrestris determined a strain-dependent effect probably related to the isolation source; however, the presence of sub-lethal injury related to released nucleic acids, proteins and DPA by spores pointed out the damage on coat and cortex with loss of barrier properties. (iii) The study performed in apple juice confirmed the results obtained in lab media. Significance and Impact of PhD research: From the earliest times, A. acidotererstris was regarded as an important target in the quality control of acidic beverages. Since soil borne species often contaminate fruit juices and do not need strict extreme requirements of acidity and high temperature for survival, it is a great concern to investigate whether and how soil species could evolve from their ecological niches in microbial community to new environments as fruit juices. The present PhD thesis contributed to species characterization through selection and characterization of wild strains of A. acidoterrestris, and provided essential knowledge to validate the genotyping and phenotyping evolution of some strains of A. acidoterrestris. Moreover, this PhD project was the first attempt to investigate the changes that may affect alicyclobacilli after sub-lethal stress application; the release of proteins, nucleic acids and DPA was strictly strain dependent. Finally, the novelty of this PhD thesis was the study of sub-lethal injury on A. acidoterrestris spores in vivo. Future trends: A future perspective could be a focus on a reassessing of strains nomenclature, considering the evolution of some strains of Alicyclobacillus genus, and the different behavior of the type strain compared to the other targets tested.