| Summary: | Water is one of the most important habitats for bacteria in the environment. The continuous flux in the urban water cycle carries water through many places, dragging bacteria and numerous chemical contaminants. This makes of water one of the most important vehicles, not only for the dissemination of the chemical substances, but also for the dissemination of organisms and, consequently, the respective resistance genes in the environment. The main goal of this study was to investigate if drinking water production and distribution could represent a hotspot for the proliferation, selection or incoming of antibiotic resistant bacteria, and the likelihood of these organisms to reach the final consumer, via tap water. In order to meet this objective, the study was planned aiming the tracking of bacterial communities and individual isolates from the source to the tap. Firstly, the abundance and diversity of bacteria in raw, treated and final (tap) water was characterized using culture-dependent and culture-independent (16S rRNA-DGGE) approaches. Both approaches showed that the water treatment reduced the bacterial counts, diversity and cultivability, promoting also a shift in the cultivable bacterial community from predominantly Gram-negative to predominately Gram-positive bacteria. Nevertheless, this effect was reverted, and in tap water Gram-negative bacteria became predominant. Moreover, in tap water total and cultivable bacteria counts were higher than in the disinfected water collected from the distribution system. These results suggest the occurrence of bacterial regrowth and/or biofilm formation over the distribution system or at tap level. Although changes in the bacterial community structure over the water circuit were observed, the predominant phylum detected, by 16S rRNA-DGGE, was the same in all the sampling points – Proteobacteria (mainly of classes Alpha, Beta and Gamma). Culture-dependent and culture-independent approaches were compared to assess which groups might be overlooked by cultivation procedures. In order to have a clear evidence of the bacterial groups which could be overlapped using those procedures, culture-dependent and two culture-independent (16S rRNA gene based DGGE and 454 pyrosequencing) methods were compared for their ability to survey the bacterial diversity of a sample. Such a comparison showed that although the different methods detected the same predominant phyla, different bacteria were targeted. Thus, besides the previous expectation that culture-independent methods would detect more bacterial groups than cultivation methods, it was also concluded that both approaches target different bacterial populations. Based on the study of the bacterial diversity, mainly of cultivable bacteria, and in the literature available, two of the most relevant taxonomic groups detected in drinking waters, due to the widespread distribution and/or abundance, were further studied. Thus, Sphingomonadaceae and Pseudomonas spp. isolated from the source to the tap were studied for species diversity, intra-species variability and potential to spread antibiotic resistance. Although members of the same species were detected in different sampled sites, the same genotype was never detected in raw water and in tap water. According to these results, the hypothesis that bacteria detected in tap water had origin in the water source had to be rejected. Other hypotheses, namely the occurrence of regrowth in water pipelines or taps or an external contamination downstream the sampled sites in the distribution system, emerged from this study. Additionally, the analysis of the antibiotic resistance profiles confirmed that both Sphingomonadaceae and Pseudomonas spp. are potential reservoirs of antibiotic resistance. Nevertheless, clear evidences of horizontal gene transfer were not obtained in this study. Indeed, antibiotic resistance patterns were mainly species-, rather than site- or strain-related, suggesting the importance of vertical resistance transmission in water bacteria. Some antibiotic resistance phenotypes were observed in tap water but not upstream. Examples of this situation were the resistance phenotypes to ampicillin-sulbactam, piperacillin plus tazobactam-pyocyanin, imipenem, ceftazidime, cefepime, gentamicin or tobramycin in Sphingomonadaceae, or to streptomycin and rifampicin in Pseudomonas spp. Cultivation-independent methods show invariably that most of the bacteria in a community are unknown, which means that were never cultivated, characterized and integrated in a validly named taxonomic group. Bacterial taxonomy can have a contribution to gradually narrow the tranche corresponding to the unknown bacteria. In this study a new species name Bacillus purgationiresistens sp. nov. was proposed, based in a single isolate recovered from treated water. Drinking water was confirmed as a potential hotspot for the spreading of antibiotic resistant bacteria, with emphasis on the transfer environment-humans.
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