Caracterización biológica y ecología de balos en una planta de depuración simbiótica de aguas residuales

Abstract

Introduction: Bdellovibrio was discovered by H. Stolp and H. Petzold in the 1960s. They are Gram-negative, vibrio shape, chemoorganotrophic, aerobic, and predatory of other Gram-negative bacteria. Their live cycle is composed of two phases: an attack phase for the prey fixing, and another of growth and division, in which Bdellovibrio grows at the expense of this. Faced with the problem of the increase in multi-resistance to antimicrobials, that constitutes a challenge for antimicrobial therapy, bacteria of the BALOs group (Bdellovibrio And Like Organisms) have raised interest as an alternative resource to the use of antibiotics both in the infectious pathology and agriculture and aquaculture field, due to the capacity that these bacteria present to destroy a wide variety of pathogen bacteria. Objectives: This research aims to verify the genetic diversity among this bacterial group, to establish a methodology that allows an effective recovery of deep-frozen BALOs and another methodology that increase in their detection directly from the medium, and to analyze the BALOs predatory capacity and BALOs specificity against their prey under different Ambiental conditions. Methodology: BALOs were isolated from the Industrial water treatment (E.D.A.R. from the Spanish acronym) located at the University of Murcia, using a procedure called "Double-layer agar technique". For that issue medically bacteria were used as prey, performing the experiment in a variety of temperature and nutritional conditions for the study of different ecologic aspects. Besides, freezing experiments were performed with a variety of glycerol concentrations to find the optimized methodology for the long-term preservation of BALOs and their subsequent reactivation. For the genetic and taxonomic contextualization, two strains of BALOs (MuRa1a y MuRa6a) were sequenced and both bioinformatics techniques and bibliographic searches were used, respectively. Results: Our bibliography review showed us the numerous taxonomic changes suffered by BALOs, until were placed in their current class: Oligoflexia. The results obtained after sequencing the two Bdellovibrio strains reveal a phylogenetic distance between them and corroborate the genetic variation of this bacterial group. The detection of BALOs directly from the aquatic environment was higher when using Escherichia coli strains as prey bacteria after filtration and incubation of the two bacteria types at 30ºC. This detection was increased by using a system that stimulates the self-enrichment of the sample, without the external contribution of nutrients. The freezing experiment resulted in reactivation of BALOs only possible at glycerol concentrations lower than 0.5%, while, in the ecological field, the BALOs showed great predation specificity depending on their original prey bacteria and a greater presence of BALOs was observed in biotopes of the E.D.A.R. where prey abounded regardless of the physico-chemical conditions. Finally, it was possible to observe a transformation of the infective form towards non-mobile compact coccoid forms when BALOs isolated from E.D.A.R. were incubated with their prey during days. Conclusions: BALOs group is a bacterial group with a high genetic and habitat diversity. The decrease in the richness of the culture medium and the self-enrichment of the sample, produces a greater detection of BALOs directly from the medium, also observing that the predatory capacity of the BALO is greater if the prey bacteria of origin are found in the medium. Besides, the use of a low concentration of glycerol during the freezing procedure stimulates the subsequent reactivation of the bacteria under study. Finally, the new coccoid forms observed in the isolated BALO strains seem to appear as a consequence of the impoverishment of the environment.