Bacterial diversity and antibiotic resistance from the water source to the tap

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.A água é um dos habitats mais importantes para as bactérias no ambiente. O fluxo contínuo da água, nomeadamente ao longo do seu ciclo urbano, faz com que chegue a muitos locais, arrastando microrganismos e inúmeros contaminantes químicos. Isto faz da água um dos veículos mais importantes para a disseminação no ambiente, não só de substâncias químicas, mas também de bactérias e, consequentemente, dos respetivos genes de resistência. O principal objetivo deste estudo foi investigar se a produção e distribuição de água de consumo poderá representar um ponto-chave para a proliferação, seleção e entrada de bactérias resistentes a antibióticos, bem como a probabilidade de estes organismos chegarem até ao consumidor final, através da água da torneira. O estudo foi planeado para atingir esse objectivo, através do rastreio de comunidades bacterianas e de isolados individuais desde a captação até à torneira. Inicialmente, a abundância e diversidade bacteriana em água não-tratada, tratada e final (torneira) foi caracterizada através do uso de abordagens dependentes e independentes (16S rRNA-DGGE) de cultivo. Ambas as abordagens mostraram que o tratamento da água reduziu as contagens, a cultivabilidade e a diversidade bacteriana, promovendo também a alteração da comunidade bacteriana cultivável de predominantemente bactérias Gram-negativas para predominantemente Gram-positivas. No entanto, este efeito foi revertido, e na água de torneira as bactérias Gram-negativas voltaram a ser predominantes. Adicionalmente, na água de torneira as contagens de microrganismos totais e de bactérias cultiváveis foram mais elevadas do que para a água tratada recolhida no sistema de distribuição. Estes resultados sugerem a ocorrência de reactivação e crescimento bacteriano e/ou a formação de biofilme ao longo do sistema de distribuição e ao nível das torneiras. Apesar de se terem observado alterações na estrutura da comunidade bacteriana ao longo do circuito da água, o filo detetado como predominante, por 16S rRNA-DGGE, foi o mesmo em todos os pontos de amostragem – Proteobacteria (principalmente das classes Alpha, Beta e Gamma). Abordagens dependentes e independentes de cultivo foram comparadas para avaliar quais os grupos que poderão ser ignorados quando se caracterizam comunidades bacterianas usando métodos de cultivo. De forma a ter uma evidência mais clara dos grupos bacterianos que se sobrepõem usando as duas abordagens, o método dependente e dois independentes de cultivo (DGGE e pirosequenciação 454 com base no gene 16S rRNA) foram comparados quanto à sua capacidade para detectar a diversidade bacteriana de uma amostra de água. Esta comparação mostrou que apesar de os diferentes métodos identificarem o mesmo filo como sendo predominante, as bactérias detetadas eram diferentes. Assim, além da expectativa anterior de que os métodos independentes de cultivo detetassem mais grupos bacterianos do que os dependentes de cultivo, concluiu-se também que as duas abordagens incidem sobre diferentes populações bacterianas. Com base no estudo da diversidade bacteriana, principalmente das bactérias cultiváveis, e no que se encontra disponível na literatura, dois grupos taxonómicos de grande relevância em água de consumo, devido à sua ampla distribuição e/ou abundância, foram estudados. Assim, Sphingomonadaceae e Pseudomonas spp., isoladas desde a captação até à torneira foram caracterizadas para a diversidade de espécies, a variabilidade intra-espécie e o potencial para propagar resistências a antibióticos. Apesar de membros da mesma espécie terem sido identificados em diferentes locais, o mesmo genótipo nunca foi detetado na captação ou sistema de distribuição e em água de torneira. De acordo com estes resultados, a hipótese de que as bactérias detetadas em água de torneira teriam origem na água da captação teve de ser rejeitada. Contudo, este estudo conduz a outras hipóteses, nomeadamente a ocorrência de reactivação e crescimento microbiano nas canalizações ou torneiras, ou de uma contaminação externa, a jusante dos pontos amostrados no sistema de distribuição. Adicionalmente, a análise dos perfis de resistência a antibióticos confirmaram que tanto Sphingomonadaceae como Pseudomonas spp. são potenciais reservatórios de resistência a antibióticos. No entanto, este estudo não permitiu obter evidências claras da ocorrência de transferência horizontal de genes. Na verdade, os padrões de resistência a antibióticos relacionaram-se principalmente com a espécie e não com o local ou estirpe, sugerindo a importância da transmissão vertical de resistências em bactérias da água. Alguns fenótipos de resistência a antibióticos detectados em água de torneira não foram detetados a montante. São exemplos os fenótipos de resistência a ampicilina-sulbactame, piperacilina e tazobactam-piocianina, imipenemo, ceftazidima, gentamicina ou tobramicina nas Sphingomonadaceae, ou a estreptomicina e rifampicina nas Pseudomonas spp. Os métodos independentes de cultivo mostram invariavelmente que a maioria das bactérias de uma comunidade são desconhecidas, o que significa que nunca foram cultivadas, caracterizadas e integradas num grupo taxonómico validamente descrito. A taxonomia bacteriana pode ter um importante contributo para gradualmente se reduzir a parcela correspondente às bactérias desconhecidas. Neste estudo o novo nome Bacillus purgationiresistens sp. nov. foi proposto, com base num único isolado recuperado de água tratada. A água de consumo foi confirmada como potencial ponto-chave para a disseminação de bactérias resistentes a antibióticos, com destaque para a transferência ambiente-humanos

Persistence of wastewater antibiotic resistant bacteria and their genes in human fecal material

Domestic wastewater is a recognized source of antibiotic resistant bacteria and antibiotic resistance genes (ARB&ARGs), whose risk of transmission to humans cannot be ignored. The fitness of wastewater ARB in the complex fecal microbiota of a healthy human was investigated in feces-based microcosm assays (FMAs). FMAs were inoculated with two wastewater isolates, Escherichia coli strain A2FCC14 (MLST ST131) and Enterococcus faecium strain H1EV10 (MLST ST78), harboring the ARGs blaTEM, blaCTX, blaOXA-A and vanA, respectively. The FMAs, incubated in the presence or absence of oxygen or in the presence or absence of the antibiotics cefotaxime or vancomycin, were monitored based on cultivation, ARGs quantification and bacterial community analysis. The fecal bacterial community was dominated by members of the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia. The ARGs harbored by the wastewater isolates could be quantified after one week, in FMAs incubated under both aerobic and anaerobic conditions. These observations were not significantly different in FMAs incubated anaerobically, supplemented with sub-inhibitory concentrations of cefotaxime or vancomycin. The observation that ARGs of wastewater ARB persisted in presence of the human fecal microbiota for at least one week supports the hypothesis of a potential transmission to humans, a topic that deserves further investigation.info:eu-repo/semantics/submittedVersio

Association between gentamicin resistance and stress tolerance in water isolates of Ralstonia pickettii and R. mannitolilytica

Members of the species Ralstonia pickettii and R. mannitolilytica, although ubiquitous and lacking major virulence factors, have been associated with nosocomial outbreaks. Tolerance to metals, antibiotics, and disinfectants may represent an advantage for their ubiquity and opportunistic pathogenic potential. In this study, we compared five strains that differed on the origin (hospital effluent, tap water, mineral water) and in the susceptibility to aminoglycosides, regarding their tolerance to metals and disinfection. The growth kinetics and biofilm formation capacity were tested in four R. pickettii strains and one R. mannitolilytica at sub-inhibitory concentrations of aminoglycosides or arsenite. The survival to UV radiation, chlorine, or hydrogen peroxide was also compared in aminoglycoside resistant and susceptible strains. Aminoglycoside-resistant strains presented a higher tolerance to arsenite than the susceptible ones and either aminoglycosides or arsenite was observed to stimulate the biofilm formation. Sub-inhibitory concentrations of the aminoglycoside gentamicin or arsenite significantly decreased the growth rate and yield, but only arsenite caused a significant increase of the lag phase. Hydrogen peroxide presented higher disinfection effectiveness against aminoglycoside susceptible than against resistant strains, an effect that was not observed for UV or chlorine. Although this conclusion needs validation based on a larger number of isolates, including clinical, the results suggest that aminoglycoside resistance may be associated with traits that influence Ralstonia spp. fitness in the environment.info:eu-repo/semantics/acceptedVersio

Neighbor urban wastewater treatment plants display distinct profiles of bacterial community and antibiotic resistance genes

Urban wastewater treatment plants (UWTPs) are among the major recipients of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues in urban environments. Although during treatment, bacteria of human and animal origin are removed, some are able to survive, persisting in the final effluent. The occurrence of these bacteria, especially those harboring ARGs, may have a direct impact on the quality of the treated wastewater that is returned to the environment. In this study, we aimed to assess if the final effluent bacterial communities of three UWTPs (PT1, PT2, and PT3) located next to each other were distinct and if such differences were related with the antibiotic resistance profiles. It was observed that the bacterial community (16S rRNA gene Illumina sequencing) and load of selected ARGs of final effluent differed among the three UWTPs, irrespective of sampling time. Members of the families Aeromonadaceae, Campylobacteraceae, Veillonellaceae, [Weeksellaceae], and Porphyromonadaceae were observed to be positively correlated with some ARGs (bla(CTX-M), bla(OXA-A), bla(SHV)) and intI1 (p < 0.05), while Intrasporangiaceae were observed to be negatively correlated. While Aeromonadaceae are recognized relevant ARG harbors, the other bacterial families may represent bacteria that co-exist with the ARG hosts, which may belong to minor bacterial groups omitted in the analyses. These findings suggest the importance of bacterial dynamics during treatment to the ARB&ARGs removal, a rationale that may contribute to design new strategies to apply in the UWTPs to prevent the spread of antibiotic resistance.info:eu-repo/semantics/acceptedVersio

Microbial community and antibiotic resistance genes distribution in different urban wastewater treatment plants effluents

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Betaproteobacteria are predominant in drinking water: are there reasons for concern?

Betaproteobacteria include some of the most abundant and ubiquitous bacterial genera that can be found in drinking water, including mineral water. The combination of physiology and ecology traits place some Betaproteobacteria in the list of potential, yet sometimes neglected, opportunistic pathogens that can be transmitted by water or aqueous solutions. Indeed, some drinking water Betaproteobacteria with intrinsic and sometimes acquired antibiotic resistance, harbouring virulence factors and often found in biofilm structures, can persist after water disinfection and reach the consumer. This literature review summarises and discusses the current knowledge about the occurrence and implications of Betaproteobacteria in drinking water. Although the sparse knowledge on the ecology and physiology of Betaproteobacteria thriving in tap or bottled natural mineral/spring drinking water (DW) is an evidence of this review, it is demonstrated that DW holds a high diversity of Betaproteobacteria, whose presence may not be innocuous. Frequently belonging to genera also found in humans, DW Betaproteobacteria are ubiquitous in different habitats, have the potential to resist antibiotics either due to intrinsic or acquired mechanisms, and hold different virulence factors. The combination of these factors places DW Betaproteobacteria in the list of candidates of emerging opportunistic pathogens. Improved bacterial identification of clinical isolates associated with opportunistic infections and additional genomic and physiological studies may contribute to elucidate the potential impact of these bacteria.info:eu-repo/semantics/aceptedVersio

Fate of pseudomonas aeruginosa and blaVIM in soil under selective pressure by copper and zinc

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Schlegelella

Schle.gel.el'la. L. fem. dim. ending ‐ella; N.L. fem. n. Schlegelella named in honor of H. G. Schlegel, a pioneer in PHA research. Proteobacteria / Betaproteobacteria / Burkholderiales / Comamonadaceae / SchlegelellaShort rods 0.8–2.8 μm long and 0.4–0.6 μm wide. Motile. Gram‐negative. Nonsporulating. Aerobe. Moderately thermophilic or mesophilic, with optimal growth occurring at 45–50°C or 30–37°C, respectively. Chemoorganotroph. Accumulates polyhydroxyalkanoates (PHA). Catalase and cytochrome c oxidase variable. The major fatty acids are C16:0 and cyclo‐C17:0 or C16:1 ω7c and/or C16:1 ω6c, depending on the species. The type species is Schlegelella thermodepolymerans. Schlegelella aquatica and Schlegelella brevitalea are two other species with validly published names. DNA G + C content (mol%): 69.2–70.0 (HPLC) or 67.5–70.3 (genome analysis, GenBank). Type species: Schlegelella thermodepolymerans Elbanna et al. 2003VP.info:eu-repo/semantics/acceptedVersio

Melaminivora

Melaminivora [Me.la.mi.ni.vo'ra. N.L. neut. n. melaminum melamine; L. v. voro to eat, to devour; N.L. fem. n. Melaminivora melamine eating]. Proteobacteria / Betaproteobacteria / Burkholderiales / Comamonadaceae / Melaminivora Rods 2.0–3.5‐μm long and 0.5–0.9‐μm wide. Motile by a single unipolar flagellum. Gram‐negative. Nonsporulating. Aerobe. Nitrate is reduced to nitrite. Mesophilic, with the ability to grow between 15 and 50°C, pH 6–9.5, and at NaCl concentrations up to 7%. Chemoorganotroph. Catalase‐ and cytochrome c oxidase‐positive. The respiratory quinone is ubiquinone 8. The major fatty acids are summed feature 3 (C16:1 ω7c/iso‐C15:0 2‐OH), C16:0, and C18:1 ω7c. C10:0 3‐OH is also present. Predominant polar lipids are phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. DNA G + C content (mol%): 69.5–69.6 (HPLC). Type species: Melaminivora alkalimesophila Wang et al. 2014bVP.info:eu-repo/semantics/acceptedVersio

Bacterial diversity and antibiotic resistance in water habitats: searching the links with the human microbiome

Water is one of the most important bacterial habitats on Earth. As such, water represents also a major way of dissemination of bacteria between different environmental compartments. Human activities led to the creation of the so-called urban water cycle, comprising different sectors (waste, surface, drinking water), among which bacteria can hypothetically be exchanged. Therefore, bacteria can be mobilized between unclean water habitats (e.g. wastewater) and clean or pristine water environments (e.g. disinfected and spring drinking water) and eventually reach humans. In addition, bacteria can also transfer mobile genetic elements between different water types, other environments (e.g. soil) and humans. These processes may involve antibiotic resistant bacteria and antibiotic resistance genes. In this review, the hypothesis that some bacteria may share different water compartments and be also hosted by humans is discussed based on the comparison of the bacterial diversity in different types of water and with the human-associated microbiome. The role of such bacteria as potential disseminators of antibiotic resistance and the inference that currently only a small fraction of the clinically relevant antibiotic resistome may be known is discussed.info:eu-repo/semantics/publishedVersio