Betaproteobacteria are ubiquitous in the urban water cycle, with multiple opportunities for interaction with humans. The combination of the physiological and ecological properties of some Betaproteobacteria explains their ability to survive in the environment, persist after disinfection of drinking water and sometimes colonize animals, including humans. Metabolic versatility, ability to form biofilm and tolerance to antimicrobial agents and metals, are examples of characteristics underlying the aforementioned ubiquity. However, this information is not clearly available in the scientific literature, so one of the objectives of this work was to investigate and systematize information on the occurrence of Betaproteobacteria in drinking water. Three bacterial genera of Betaproteobacteria, whose presence in water exemplifies the mentioned ubiquity are Achromobacter, Burkholderia and Ralstonia, sometimes related to outbreaks of infection associated with drinking water. Ralstonia spp. aroused particular attention since it was recently isolated from a group of sources as diverse as mineral water or hospital sewage. Ralstonia spp., mainly the species Ralstonia pickettii and R. mannitolilytica, have recently been associated with cases of infection and this ubiquity has aroused interest. Members of these species may tolerate metals, antibiotics and disinfectants, which may contribute to their ubiquity and eventual severity as infectious agents. In this study it was sought to understand if there was an interrelation between resistance to metals and antibiotics and how this could influence the behavior of the strains. The experimental work involved the characterization of strains of the R. pickettii and R. mannitolilytica species, isolated from hospital sewage, tap water and bottled mineral water, and aimed to identify possible associations of resistance phenotypes to antibiotics and metals and other types of stress, as well as their genetic basis. Specifically, in a first trial, water isolates of R. pickettii and R. mannitolilytica from hospital effluent (n = 2), tap water (n = 2), mineral water (n = 1) were studied, and resistance phenotypes to antibiotics and metals as well as disinfection response and some parameters of growth kinetics were evaluated. To achieve these objectives, the disc diffusion and microdilution method was used to determine minimum inhibitory concentrations (MICs), growth curves were made in Mueller-Hinton broth culture medium supplemented with the antimicrobial agents of interest, in order to study the parameters of growth kinetics, the biofilm formation capacity was evaluated and the behavior was tested in the presence of ultraviolet radiation, sodium hypochlorite and hydrogen peroxide. This study suggested an association between resistance to gentamicin (MIC > 256 mg/L) and increased tolerance to arsenite (MIC = 1.4 mmol/L). The cross-resistance hypothesis was ruled out since sub-inhibitory concentrations of gentamicin or arsenite significantly decreased the growth rate and yield of Ralstonia spp., although only the arsenite caused a significant increase in the lag phase. In addition, in strains resistant to gentamicin, biofilm formation was stimulated in the presence of aminoglycosides or arsenite. Ultraviolet or hypochlorite disinfection exhibited similar inactivation rates in resistant and susceptible gentamicin strains. In contrast, the same dose of hydrogen peroxide caused faster inactivation in strains sensitive to gentamicin than in resistant strains. The association between resistance to gentamicin and arsenite was explored in a larger group of R. pickettii. A group of 37 strains, 14 from mineral water, 17 from tap water and 6 from sewage hospital were included. The antibiotic and arsenite resistance profiles were screened as described above and selected resistance genetic determinants, including those relating to Integrative and Conjugative Elements (ICEs), plasmids, and genes associated with efflux and arsenic resistance were analyzed. Most of the isolates (32/37) were resistant to gentamicin, beta-lactam and colistin. However, not all isolates were resistant to gentamicin and the association of this phenotype with increased arsenite tolerance was confirmed. This division of susceptible vs. resistant strains to gentamicin and arsenite was consistent with the separation of two phylogenetic groups, based on analysis of the 16S rRNA gene. Moreover, isolates resistant to gentamicin and arsenite showed ICEs and the arsH and acr3 genes, related to the resistance to arsenite. Most of the R. pickettii isolates (36) had one or two plasmids with sizes between 77 and 260 kbp. The deduced amino acid sequences of the efflux pump, whose gene amplified in all strains, differed in resistant and susceptible isolates. Although genotypes of resistant strains susceptible to gentamicin differed in a manner consistent with the phenotypes and with the phylogenetic distinction of both groups, it was not possible to find a genetic explanation for the observed phenotype nor for the association of arsenite and gentamicin resistance. However, for all the analyzed genetic elements there was a clear division between both groups, suggesting that approaches of comparative genomics could contribute to elucidate the genetic bases of the phenotypes under analysis - resistance vs. susceptibility to gentamicin and arsenite. In this sense, we compared the genomes of one resistant and one susceptible strain, and with special focus on the functional categories "Membrane transport", "Stress and response" and "Virulence, disease and defense" were analyzed the genes present only in the resistant strain or mutations detected between both. This analysis has shown some functions that may explain either the ability to acquire or develop new resistance mechanisms. This type of study allows elucidating the processes of development or acquisition of resistance in ubiquitous species characterized by mechanisms of intrinsic resistance, since they are common to the majority of members of a species.
|Date of Award||2019|
|Supervisor||Célia Manaia (Supervisor)|