Cell-based internal standard for qPCR determinations of antibiotic resistance indicators in environmental water samples

Quantitative PCR (qPCR) has been used to quantify antibiotic resistance genes (ARGs) in water, wastewater, soil, sediment and tissue samples. Concerns regarding the comparability of data obtained in different laboratories has been a major bottleneck to incentivize the compilation of publicly available of ARGs quantifications gathered from different reports. In this study, the influence of the DNA extraction kits (NZY Tissue gDNA Isolation kit or DNeasy PowerWater kit) and of the operator on the DNA extraction yield and on qPCR genes quantification was assessed. Since in wastewater and water samples the matrix effect can affect the DNA recovery and, therefore, gene quantification, an internal standard, consisting in a cloned gene not found in environmental samples, was tested. The aim was to assess how qPCR determinations in wastewater and water samples can be affected by the matrix effect. The results show that the DNA extraction operator did not significantly influence DNA yield. The use of distinct kits resulted in qPCR gene quantifications that did not differ in more than 1 log-unit mL−1. The matrix effect, assessed based on the use of an internal standard, was associated with an underestimation that ranged 0.1–0.9 log gene copy number mL−1 of sample, irrespective of the water type. The reliability on the use of a DNA extraction kit that costs about 3 times less than the most commonly used can be an incentive for the use of DNA based analyses of ARGs in environmental waters. Moreover, the fact that both the DNA extraction operator and the reduced matrix effect have little influence on the final results, are good news, encouraging the compilation of data produced in distinct laboratories. Nevertheless, harmonization efforts are still necessary to minimize bias that may be due associated with other conditions, such as equipment.info:eu-repo/semantics/publishedVersio

Factors influencing antibiotic resistance burden in municipal wastewater treatment plants

Municipal wastewater treatment plants are recognized reservoirs of antibiotic-resistant bacteria. Three municipal wastewater treatment plants differing on the dimensions and bio-treatment processes were compared for the loads of amoxicillin-, tetracycline-, and ciprofloxacinresistant heterotrophic bacteria, enterobacteria, and enterococci in the raw inflow and in the treated effluents. The sewage received by each plant, in average, corresponded to 85,000 inhabitant equivalents (IE), including pretreated industrial effluents (≤30%) in plant activated sludge, 105,000 IE, including pretreated hospital effluents (≤15%) in plant trickling filter, and 2,000 IE, exclusively of domestic sewage, in plant submerged aerated filter. The presence of pretreated industrial effluents or of pretreated hospital sewage in the raw inflow did not imply significantly higher densities (per milliliter or per IE) of antibiotic-resistant bacteria in the raw wastewater. Longer hydraulic residence periods (24 h) corresponded to higher bacterial removal rates than shorter periods (12 and 9 h), although such efficiency did not imply significant average decreases in the antibiotic resistance prevalence of the treated effluent. The bacterial loads in the treated effluent could be ranked according to the treatment efficiency, suggesting that the characteristics of the raw inflow may have less relevance on the quality of the treated wastewater than other aspects, such as the inflow volume, the type of biological treatment, or the hydraulic residence time.info:eu-repo/semantics/publishedVersio

Development of an internal standard for quantitative PCR analyses of antibiotic resistance genes

info:eu-repo/semantics/publishedVersio

Differential patterns of antimicrobial resistance in population subsets of Escherichia coli isolated from waste- and surface waters

The species Escherichia coli comprises different subgroups with distinct phylogeny, physiology and ecology and, thus, presumably, with different roles in antimicrobial resistance dissemination. E. coli strains isolated from raw and treated municipal wastewater and from urban water streams were characterized in terms of phylogenetic groups, antimicrobial resistance patterns and the presence of class 1 and class 2 integrons. Our main objective was to investigate the contribution of the different phylo-groups in antimicrobial resistance dissemination in urban waters. Groups A and B1 were predominant in all types of water, evidencing, respectively, the lowest and the highest resistance prevalence. Municipal wastewater treatment was accompanied by significant increases of ciprofloxacin and streptomycin resistance (pb0.01). Antimicrobial resistance prevalence differed significantly between the different phylo-groups and within the same group, mainly in group A. Such differences contributed to explain the higher ciprofloxacin and streptomycin resistance rates observed in treated effluent in comparison with the raw wastewater. We conclude that the dynamics of the bacterial populations has a major role on the dissemination of antimicrobial resistance in the environment.info:eu-repo/semantics/acceptedVersio

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

Caenibacterium thermophilum gen. nov., sp. nov., isolated from a thermophilic aerobic digester of municipal sludge

A bacterial strain, N2-680T (=DSM 15264T=LMG 21760T), isolated from a thermophilic aerobic digester of municipal sludge, was characterized with respect to its morphology, physiology and taxonomy. Phenotypically, the isolate was a Gram-negative rod with a polar flagellum, catalase- and oxidase-positive, containing cytoplasmic inclusions of poly-b-hydroxybutyrate and had an optimal growth temperature of about 47 6C. Strain N2-680T was unable to reduce nitrate and could use organic acids, amino acids and carbohydrates as single carbon sources. Chemotaxonomic analysis revealed that ubiquinone 8 was the major respiratory quinone of this organism and that phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids. At 50 6C, the major components in fatty acid methyl ester analysis were C16 : 0 and cyclo-C17 : 0. The highest 16S rDNA sequence identity of isolate N2-680T was to Leptothrix mobilis and Ideonella dechloratans (95?7%) and to Rubrivivax gelatinosus and Aquabacterium commune (95?6 %). 16S rDNA sequence similarities to species of two related thermophilic genera, Caldimonas manganoxidans and Tepidimonas ignava, were lower (93?6 and 94?7 %). On the basis of phylogenetic analyses and physiological and chemotaxonomic characteristics, it is proposed that isolate N2-680T represents a new genus and species, for which the name Caenibacterium thermophilum gen. nov., sp. nov. is propose

Tepidiphilus margaritifer gen. nov., sp. nov., isolated from a thermophilic aerobic digester

A moderately thermophilic bacterium is described, strain N2-214T, that was isolated from an enrichment culture, growing on caprolactone, obtained from a sample from a water-treatment sludge aerobic digester operating at temperatures around 60 °C. The organism was aerobic, Gram-negative, oxidase- and catalase-positive, with a polar flagellum, and capable of growth at temperatures as high as 61 °C. The major fatty acids of strain N2-214T were C16 : 0, C18 : 1 and cyclo-C19 : 0. The phylogenetic relationships of the strain, derived from 16S rRNA gene sequence comparisons, demonstrated it to be a member of the {beta}-subclass of the Proteobacteria. The highest 16S rDNA sequence similarity of isolate N2-214T was to Azoarcus buckelii (91·9 %), Thauera aromatica (92 %) and Hydrogenophilus thermoluteolus (92·7 %). On the basis of phylogenetic analyses and physiological and chemotaxonomic characteristics, it is proposed that isolate N2-214T (=DSM 15129T=LMG 21637T) represents a new genus and species, Tepidiphilus margaritifer gen. nov., sp. nov

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

The balance between treatment efficiency and receptor quality determines wastewater impacts on the dissemination of antibiotic resistance

This study investigated the balance between treatment efficiency and impact caused by urban wastewater treatment plants (UWTPs) on the dissemination of antibiotic resistance. Four full-scale UWTPs (PT1-PT4) and the receiving river were sampled over four campaigns. The 16 S rRNA gene, two mobile genetic elements (MGEs), eight antibiotic resistance genes (ARGs), and culturable bacteria were monitored over different treatment stages and in hospital effluent. The bacterial and antibiotic resistance load was not significantly different in the inflow of the four UWTPs (p > 0.01). Biological treatment promoted ARGs reduction values up to 2.5 log-units/mL, while UV (PT1, PT2) or sand filtration/ozonation (PT3) led to removal values < 0.6 log-units/mL. The final effluent of PT3, with the highest removal rates and significantly lower ARGs abundance, was not significantly different from the receiving water body. Emerging ARGs (e.g., blaVIM, blaOXA-48, and blaKPC) were sporadically detected in the river, although more frequent downstream. Hospital effluent might contribute for the occurrence of some, but not all these ARGs in the river. A major conclusion was that the impact of the UWTPs on the river was not only determined by treatment efficiency and final effluent quality, but also by the background contamination of the river and/or dilution rate.info:eu-repo/semantics/publishedVersio

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

info:eu-repo/semantics/publishedVersio