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The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs' biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments. [ABSTRACT FROM AUTHOR]

The biosynthesis of polyhydroxyalkanoates (PHAs) from industrial wastes by mixed microbial cultures (MMCs) enriched in PHA-accumulating bacteria is a promising technology to replace petroleum-based plastics. However, the populations' dynamics in the PHA-accumulating MMCs are not well known. Therefore, the main objective of this study was to address the shifts in the size and structure of the bacterial communities in two lab-scale sequencing batch reactors (SBRs) fed with fish-canning effluents and operated under non-saline (SBR-N, 0.5 g NaCl/L) or saline (SBR-S, 10 g NaCl/L) conditions, by using a combination of quantitative PCR and Illumina sequencing of bacterial 16S rRNA genes. A double growth limitation (DGL) strategy, in which nitrogen availability was limited and uncoupled to carbon addition, strongly modulated the relative abundances of the PHA-accumulating bacteria, leading to an increase in the accumulation of PHAs, independently of the saline conditions (average 9.04 wt% and 11.69 wt%, maximum yields 22.03 wt% and 26.33% SBR-N and SBR-S, respectively). On the other hand, no correlations were found among the PHAs accumulation yields and the absolute abundances of total Bacteria, which decreased through time in the SBR-N and did not present statistical differences in the SBR-S. Acinetobacter, Calothrix, Dyella, Flavobacterium, Novosphingobium, Qipengyuania, and Tsukamurella were key PHA-accumulating genera in both SBRs under the DGL strategy, which was revealed as a successful tool to obtain a PHA-enriched MMC using fish-canning effluents. [ABSTRACT FROM AUTHOR]

Background: Marine environments are the widest fonts of biodiversity representing a resource of both unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, exopolysaccharides (EPS) have many physiological functions and practical applications. Since EPS production by many bacteria is too scarce for practical use and only few species are known for their high levels of production, the search of new high EPS producers is of paramount importance. Many marine bacteria, that produce EPS to cope with strong environmental stress, could be potentially exploited at the industrial level. Results: A novel bacterium, strain BM39, previously isolated from sediments collected in the Tyrrhenian Sea, was selected for its production of very high levels of EPS. BM39 was affiliated to Pantoea sp. (Enterobacteriaceae) by 16S rRNA gene sequencing and biochemical tests. According to the phylogenetic tree, this strain, being quite far from the closest known Pantoea species (96% identity with P. agglomerans and P. ananatis) could belong to a new species. EPS production was fast (maximum of ca. 21 g/L in 24 h on glucose medium) and mainly obtained during the exponential growth. Preliminary characterization, carried out by thin layer and gel filtration chromatography, showed that the EPS, being a glucose homopolymer with MW of ca. 830 kDa, appeared to be different from those of other bacteria of same genus. The bacterium showed a typical slightly halophilic behavior growing optimally at NaCl 40% (growing range 0-100%). Flow cytometry studies indicated that good cell survival was maintained for 24 h at 120%. Survival decreased dramatically with the increase of salinity being only 1 h at 280%. The biochemical characterization, carried out with the Biolog system, showed that MB39 had a rather limited metabolic capacity. Its ability, rather lower than that of P. agglomerans, was almost only confined to the metabolization of simple sugars and their derivatives. Few alcohols, organic acids and nitrogen compounds were partially used too. Conclusions: Strain BM39, probably belonging to a new species, due to its remarkable EPS production, comparable to those of known industrial bacterial producers, could be suggested as a new microorganism for industrial applications. [ABSTRACT FROM AUTHOR]

Ninety-five bacterial isolates, obtained in pure cultures from samples collected in various sites of the East sector of the Tyrrhenian Sea, were plate-screened for their ability to produce twelve extracellular enzyme activities in order to find new strains for possible applications. Lipases, DNAase and RNAase were generally present; amilase, phosphatase, pectinase and protease were common. Chitinase and urease were present in a limited number of isolates while glucose oxidase and phenol oxidase were quite rare. Few isolates, producing a limited number of enzymes, could have a low eco-nutritional versatility while most of them, showing a diversified enzymatic competence, are probably more advantaged in the marine environment. However, none of the isolates was able to produce all the tested activities. Few strains (14) showed apparent high level of some extracellular enzyme activities and could be considered as potential high-producers. [ABSTRACT FROM AUTHOR]