A rationale for the high limits of quantification of antibiotic resistance genes in soil.

The determination of values of abundance of antibiotic resistance genes (ARGs) per mass of soil is extremely useful to assess the potential impacts of relevant sources of antibiotic resistance, such as irrigation with treated wastewater or manure application. Culture-independent methods and, in particular, quantitative PCR (qPCR), have been regarded as suitable approaches for such a purpose. However, it is arguable if these methods are sensitive enough to measure ARGs abundance at levels that may represent a risk for environmental and human health. This study aimed at demonstrating the range of values of ARGs quantification that can be expected based on currently used procedures of DNA extraction and qPCR analyses. The demonstration was based on the use of soil samples spiked with known amounts of wastewater antibiotic resistant bacteria (ARB) (Enterococcus faecalis, Escherichia coli, Acinetobacter johnsonii, or Pseudomonas aeruginosa), harbouring known ARGs, and also on the calculation of expected values determined based on qPCR. The limits of quantification (LOQ) of the ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) were observed to be approximately 4 log-units per gram of soil dry weight, irrespective of the type of soil tested. These values were close to the theoretical LOQ values calculated based on currently used DNA extraction methods and qPCR procedures. The observed LOQ values can be considered extremely high to perform an accurate assessment of the impacts of ARGs discharges in soils. A key message is that ARGs accumulation will be noticeable only at very high doses. The assessment of the impacts of ARGs discharges in soils, of associated risks of propagation and potential transmission to humans, must take into consideration this type of evidence, and avoid the simplistic assumption that no detection corresponds to risk absence.

Characterization of bacterial communities from Masseiras, a unique Portuguese greenhouse agricultural system.

"Masseiras" is an ancient Portuguese agriculture system, where soil was developed from sand dunes enriched with seaweeds over more than a century. Due to the importance for the local economy, this system evolved for greenhouse structures. In this study we compared the bacterial community composition and structure of "Masseiras" soil, aiming at assessing the potential impact of different agricultural practices. The bulk soil of two greenhouses (following or not the recommended agriculture good practices, FGP and NFGP, respectively) was compared based on their physicochemical properties and bacterial community. In both FGP and NFGP, Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Gemmatimonadetes were in a proportion of 5:1:1:1:1:1. However, the bacterial community of soil FGP was richer and more diverse than that of soil NFGP. Members of the classes Bacilli and Gemm-1, with higher relative abundance in NFGP and FGP, respectively, were those contributing most for distinguishing the bacterial communities of both soils. The differences in the structure of the bacterial communities correlated (Mantel test) with some soil physicochemical properties, such as electrical conductivity and nitrate and Zn contents, which were significantly higher in soil NFGP than in soil FGP.

Ozonation and UV254nm radiation for the removal of microorganisms and antibiotic resistance genes from urban wastewater.

Conventional wastewater treatment has a limited capacity to reduce antibiotic resistant bacteria and genes (ARB&ARG). Tertiary treatment processes are promising solutions, although the transitory inactivation of bacteria may select ARB&ARG. This study aimed at assessing the potential of ozonation and UV254nm radiation to inactivate cultivable fungal and bacterial populations, and the selected genes 16S rRNA (common to all bacteria), intI1 (common in Gram-negative bacteria) and the ARG vanA, blaTEM, sul1 and qnrS. The abundance of the different microbiological parameters per volume of wastewater was reduced by ∼2 log units for cultivable fungi and 16S rRNA and intI1 genes, by∼3-4 log units, for total heterotrophs, enterobacteria and enterococci, and to values close or below the limits of quantification for ARG, for both processes, after a contact time of 30min. Yet, most of the cultivable populations, the 16S rRNA and intI1 genes as well as the ARG, except qnrS after ozonation, reached pre-treatment levels after 3days storage, suggesting a transitory rather than permanent microbial inactivation. Noticeably, normalization per 16S rRNA gene evidenced an increase of the ARG and intI1 prevalence, mainly after UV254nm treatment. The results suggest that these tertiary treatments may be selecting for ARB&ARG populations.

Proteobacteria become predominant during regrowth after water disinfection.

Disinfection processes aim at reducing the number of viable cells through the generation of damages in different cellular structures and molecules. Since disinfection involves unspecific mechanisms, some microbial populations may be selected due to resilience to treatment and/or to high post-treatment fitness. In this study, the bacterial community composition of secondarily treated urban wastewater and of surface water collected in the intake area of a drinking water treatment plant was compared before and 3-days after disinfection with ultraviolet radiation, ozonation or photocatalytic ozonation. The aim was to assess the dynamics of the bacterial communities during regrowth after disinfection. In all the freshly collected samples, Proteobacteria and Bacteroidetes were the predominant phyla (40-50% and 20-30% of the reads, respectively). Surface water differed from wastewater mainly in the relative abundance of Actinobacteria (17% and <5% of the reads, respectively). After 3-days storage at light and room temperature, disinfected samples presented a shift of Gammaproteobacteria (from 8 to 10% to 33-65% of the reads) and Betaproteobacteria (from 14 to 20% to 31-37% of the reads), irrespective of the type of water and disinfection process used. Genera such as Pseudomonas, Acinetobacter or Rheinheimera presented a selective advantage after water disinfection. These variations were not observed in the non-disinfected controls. Given the ubiquity and genome plasticity of these bacteria, the results obtained suggest that disinfection processes may have implications on the microbiological quality of the disinfected water.

Assessment of copper and zinc salts as selectors of antibiotic resistance in Gram-negative bacteria.

Some metals are nowadays considered environmental pollutants. Although some, like Cu and Zn, are essential for microorganisms, at high concentrations they can be toxic or exert selective pressures on bacteria. This study aimed to assess the potential of Cu or Zn as selectors of specific bacterial populations thriving in wastewater. Populations of Escherichia coli recovered on metal-free and metal-supplemented culture medium were compared based on antibiotic resistance phenotype and other traits. In addition, the bacterial groups enriched after successive transfers in metal-supplemented culture medium were identified. At a concentration of 1mM, Zn produced a stronger inhibitory effect than Cu on the culturability of Enterobacteriaceae. It was suggested that Zn selected populations with increased resistance prevalence to sulfamethoxazole or ciprofloxacin. In non-selective culture media, Zn or Cu selected for mono-species populations of ubiquitous Betaproteobacteria and Flavobacteriia, such as Ralstonia pickettii or Elizabethkingia anophelis, yielding multidrug resistance profiles including resistance against carbapenems and third generation cephalosporins, confirming the potential of Cu or Zn as selectors of antibiotic resistant bacteria.

Wastewater reuse in irrigation: a microbiological perspective on implications in soil fertility and human and environmental health.

The reuse of treated wastewater, in particular for irrigation, is an increasingly common practice, encouraged by governments and official entities worldwide. Irrigation with wastewater may have implications at two different levels: alter the physicochemical and microbiological properties of the soil and/or introduce and contribute to the accumulation of chemical and biological contaminants in soil. The first may affect soil productivity and fertility; the second may pose serious risks to the human and environmental health. The sustainable wastewater reuse in agriculture should prevent both types of effects, requiring a holistic and integrated risk assessment. In this article we critically review possible effects of irrigation with treated wastewater, with special emphasis on soil microbiota. The maintenance of a rich and diversified autochthonous soil microbiota and the use of treated wastewater with minimal levels of potential soil contaminants are proposed as sine qua non conditions to achieve a sustainable wastewater reuse for irrigation.

Bacterially induced weathering of ultramafic rock and its implications for phytoextraction.

The bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of two Arthrobacter strains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO4 solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulator Alyssum serpyllifolium subsp. malacitanum was grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.

Phytoremediation of hexachlorocyclohexane (HCH)-contaminated soils using Cytisus striatus and bacterial inoculants in soils with distinct organic matter content.

The performance of Cytisus striatus in association with different microbial inoculant treatments on the dissipation of the insecticide hexachlorocyclohexane (HCH) was studied. Two soils with different organic matter (A and B soil) content were spiked with 0 or 65 mg HCH kg(-1). Plants were either not inoculated (NI), or inoculated with the endophyte Rhodococcus erythropolis ET54b and the HCH-degrader Sphingomonas sp. D4 separately or in combination (ET, D4 and ETD4). Unplanted pots were also established. HCH phytotoxicity was more pronounced in the B soil. Soil HCH concentrations in unplanted pots were similar to initial concentrations, whereas concentrations were reduced after plant growth: by 20% and 8% in A and B soil, respectively. Microbial inoculants also modified HCH dissipation, although effects were soil-dependent. Inoculation with the combination of strains (ETD4) led to a significant enhancement in HCH dissipation: up to 53% in the A soil and 43% in the B soil.