Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR.

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.

Selenized non-Saccharomyces yeasts and their potential use in fish feed.

Selenium (Se) is a vital trace element, essential for growth and other biological functions in fish. Its significance lies in its role as a fundamental component of selenoproteins, which are crucial for optimal functioning of the organism. The inclusion of Se in the diets of farmed animals, including fish, has proved invaluable in mitigating the challenges arising from elemental deficiencies experienced in captivity conditions due to limitations in the content of fishmeal. Supplementing diets with Se enhances physiological responses, particularly mitigates the effects of the continuous presence of environmental stress factors. Organic Se has been shown to have higher absorption rates and a greater impact on bioavailability and overall health than inorganic forms. A characteristic feature of yeasts is their rapid proliferation and growth, marked by efficient mineral assimilation. Most of the selenized yeasts currently available in the market, and used predominantly in animal production and aquaculture, are based on Saccharomyces cerevisiae, which contains selenomethionine (Se-Met). The object of this review is to highlight the importance of selenized yeasts. In addition, it presents metabolic and productive aspects of other yeast genera that are important potential sources of organic selenium. Some yeast strains discussed produce metabolites of interest such as lipids, pigments, and amino acids, which could have applications in aquaculture and further enrich their usefulness.

Promising bioprocesses for the efficient removal of antibiotics and antibiotic-resistance genes from urban and hospital wastewaters: Potentialities of aerobic granular systems.

The use, overuse, and improper use of antibiotics have resulted in higher levels of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs), which have profoundly disturbed the equilibrium of the environment. Furthermore, once antibiotic agents are excreted in urine and feces, these substances often can reach wastewater treatment plants (WWTPs), in which improper treatments have been highlighted as the main reason for stronger dissemination of antibiotics, ARB, and ARGs to the receiving bodies. Hence, achieving better antibiotic removal capacities in WWTPs is proposed as an adequate approach to limit the spread of antibiotics, ARB, and ARGs into the environment. In this review, we highlight hospital wastewater (WW) as a critical hotspot for the dissemination of antibiotic resistance due to its high level of antibiotics and pathogens. Hence, monitoring the composition and structure of the bacterial communities related to hospital WW is a key factor in controlling the spread of ARGs. In addition, we discuss the advantages and drawbacks of the current biological WW treatments regarding the antibiotic-resistance phenomenon. Widely used conventional activated sludge technology has proved to be ineffective in mitigating the dissemination of ARB and ARGs to the environment. However, aerobic granular sludge (AGS) technology is a promising technology-with broad adaptability and excellent performance-that could successfully reduce antibiotics, ARB, and ARGs in the generated effluents. We also outline the main operational parameters involved in mitigating antibiotics, ARB, and ARGs in WWTPs. In this regard, WW operation under long hydraulic and solid retention times allows better removal of antibiotics, ARB, and ARGs independently of the WW technology employed. Finally, we address the current knowledge of the adsorption and degradation of antibiotics and their importance in removing ARB and ARGs. Notably, AGS can enhance the removal of antibiotics, ARB, and ARGs due to the complex microbial metabolism within the granular biomass.

Deciphering the Role of WWTPs in Cold Environments as Hotspots for the Dissemination of Antibiotic Resistance Genes.

Cold environments are the most widespread extreme habitats in the world. However, the role of wastewater treatment plants (WWTPs) in the cryosphere as hotspots in antibiotic resistance dissemination has not been well established. Hence, a snapshot of the resistomes of WWTPs in cold environments, below 5 °C, was provided to elucidate their role in disseminating antibiotic resistance genes (ARGs) to the receiving waterbodies. The resistomes of two natural environments from the cold biosphere were also determined. Quantitative PCR analysis of the aadA, aadB, ampC, blaSHV, blaTEM, dfrA1, ermB, fosA, mecA, qnrS, and tetA(A) genes indicated strong prevalences of these genetic determinants in the selected environments, except for the mecA gene, which was not found in any of the samples. Notably, high abundances of the aadA, ermB, and tetA(A) genes were found in the influents and activated sludge, highlighting that WWTPs of the cryosphere are critical hotspots for disseminating ARGs, potentially worsening the resistance of bacteria to some of the most commonly prescribed antibiotics. Besides, the samples from non-disturbed cold environments had large quantities of ARGs, although their ARG profiles were highly dissimilar. Hence, the high prevalences of ARGs lend support to the fact that antibiotic resistance is a common issue worldwide, including environmentally fragile cold ecosystems.

Structure of fungal communities in sequencing batch reactors operated at different salinities for the selection of triacylglyceride-producers from a fish-canning lipid-rich waste stream.

Oleaginous fungi natively accumulate large amounts of triacylglycerides (TAG), widely used as precursors for sustainable biodiesel production. However, little attention has been paid to the diversity and roles of fungal mixed microbial cultures (MMCs) in sequencing batch reactors (SBR). In this study, a lipid-rich stream produced in the fish-canning industry was used as a substrate in two laboratory-scale SBRs operated under the feast/famine (F/F) regime to enrich microorganisms with high TAG-storage ability, under two different concentrations of NaCl (SBR-N: 0.5 g/L; SBR-S: 10 g/L). The size of the fungal community in the enriched activated sludge (EAS) was analyzed using 18S rRNA-based qPCR, and the fungal community structure was determined by Illumina sequencing. The different selective pressures (feeding strategy and control of pH) implemented in the enrichment SBRs throughout operation increased the abundance of total fungi. In general, there was an enrichment of genera previously identified as TAG-accumulating fungi (Apiotrichum, Candida, Cutaneotrichosporon, Geotrichum, Haglerozyma, Metarhizium, Mortierella, Saccharomycopsis, and Yarrowia) in both SBRs. However, the observed increase of their relative abundances throughout operation was not significantly linked to a higher TAG accumulation.

Simplified engineering design towards a competitive lipid-rich effluents valorization.

Medium- and long-chain fatty acids and glycerol contained in the oily fraction of many food-industry effluents are excellent candidates to produce biobased high-value triacylglycerides (TAGs) and polyhydroxyalkanoates (PHAs). The typical process configuration for TAGs recovery from lipid-rich streams always includes two steps (culture enrichment plus storage compounds accumulation) whereas, for PHAs production, an additional pretreatment of the substrate for the obtainment of soluble volatile fatty acids (VFAs) is required. To simplify the process, substrate hydrolysis, culture enrichment, and accumulation (TAG and PHA storage) were coupled here in a single sequencing batch reactor (SBR) operated under the double growth limitation strategy (DGL) and fed in pulses with industrial waste fish oil during the whole feast phase. When the SBR was operated in 12 h cycles, it was reached up to 51 wt % biopolymers after only 6 h of feast (TAG:PHA ratio of 50:51; 0.423 CmmolBIOP/CmmolS). Daily storage compound production was observed to be over 25% higher than the reached when enrichment and accumulation stages were carried in separate operational units. Increasing the feast phase length from 6 to 12 h (18 h cycle) negatively affected the DGL strategy performance and hence system storage capacity, which was recovered after also extending the famine phase in the same proportion (24 h cycle). Besides, the carbon influx during the feast phase was identified as a key operational parameter controlling storage compounds production and, together with the C/N ratio, culture selection. The different cycle configurations tested clearly modulated the total fungal abundances without no significant differences in the size of the bacterial populations. Several PHA and TAG producers were found in the mixed culture although the PHA and TAG productions were poorly associated with the increased relative abundances (RAs) of specific operational taxonomic units (OTUs).

Effects of sulphur amino acids on the size and structure of microbial communities of aerobic granular sludge bioreactors.

Granular activated sludge has been described as a promising tool in treating wastewater. However, the effect of high concentrations of sulphur amino acids, cysteine and methionine, in the evolution, development and stability of AGS-SBRs (aerobic granular sludge in sequential batch reactors) and their microbial communities is not well-established. Therefore, this study aimed to evaluate microbial communities' size, structure and dynamics in two AGS-SBRs fed with two different concentrations of amino acids (50 and 100 mg L-1 of both amino acids). In addition, the impact of the higher level of amino acids was also determined under an acclimatization or shock strategy. While N removal efficiency decreased with amino acids, the removal of the organic matter was generally satisfactory. Moreover, the abrupt presence of both amino acids reduced even further the removal performance of N, whereas under progressive adaptation, the removal yield was higher. Besides, excellent removal rates of cysteine and methionine elimination were found, in all stages below 80% of the influent values. Generally considered, the addition of amino acids weakly impacts the microbial communities' total abundances. On the contrary, the presence of amino acids sharply modulated the dominant bacterial structures. Furthermore, the highest amino acid concentration under the shock strategy resulted in a severe change in the structure of the microbial community. Acidovorax, Flavobacterium, Methylophilus, Stenotrophomonas and Thauera stood out as the prominent bacteria to cope with the high presence of cysteine and methionine. Hence, the AGS-SBR technology is valuable for treating influents enriched in sulphur Aa inclusively when a shock strategy was used.

Dynamics of PHA-Accumulating Bacterial Communities Fed with Lipid-Rich Liquid Effluents from Fish-Canning Industries.

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.

Valorization of lipid-rich wastewaters: A theoretical analysis to tackle the competition between polyhydroxyalkanoate and triacylglyceride-storing populations.

The lipid fraction of the effluents generated in several food-processing activities can be transformed into polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs), through open culture biotechnologies. Although competition between storing and non-storing populations in mixed microbial cultures (MMCs) has been widely studied, the right selective environment allowing for the robust enrichment of a community when different types of accumulators coexist is still not clear. In this research, comprehensive metabolic analyses of PHA and TAG synthesis and degradation, and concomitant respiration of external carbon, were used to understand and explain the changes observed in a laboratory-scale bioreactor fed with the lipid-rich fraction (mainly oleic acid) of a wastewater stream produced in the fish-canning industry. It was concluded that the mode of oxygen, carbon, and nitrogen supply determines the enrichment of the culture in specific populations, and hence the type of intracellular compounds preferentially accumulated. Coupled carbon and nitrogen feeding regime mainly selects for TAG producers whereas uncoupled feeding leads to PHA or TAG production function of the rate of carbon supply under specific aeration rates and feast and famine phases lengths.

Agrobacterium leguminum sp. nov., isolated from nodules of Phaseolus vulgaris in Spain.

Two endophytic strains, coded MOVP5T and MOPV6, were isolated from nodules of Phaseolus vulgaris plants grown on agricultural soil in Southeastern Spain, and were characterized through a polyphasic taxonomy approach. Their 16S rRNA gene sequences showed 99.3 and 99.4 %, 98.9 and 99.6 %, and 99.0 and 98.7% similarity to 'A. deltaense' YIC 4121T, A. radiobacter LGM 140T, and A. pusense NRCPB10T, respectively. Multilocus sequence analysis based on sequences of recA and atpD genes suggested that these two strains could represent a new Agrobacterium species with less than 96.5 % similarity to their closest relatives. PCR amplification of the telA gene, involved in synthesis of protelomerase, confirmed the affiliation of strains MOPV5T and MOPV6 to the genus Agrobacterium. Whole genome average nucleotide identity and digital DNA-DNA hybridization average values were less than 95.1 and 66.7 %, respectively, with respect to its closest related species. Major fatty acids in strain MOPV5T were C18 : 1 ω7c/C18 : 1 ω6c in summed feature 8, C19 : 0 cyclo ω8c, C16 : 0 and C16 : 0 3-OH. Colonies were small to medium, pearl-white coloured on YMA at 28 °C and growth was observed at 10-42 °C, pH 5.0-10.0 and with 0.0-0.5 % (w/v) NaCl. The DNA G+C content was 59.9 mol%. These two strains differ from all other genomovars of Agrobacterium found so far, including those that have not yet given a Latin name. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strain MOPV5T as representing a novel species of Agrobacterium, for which the name Agrobacterium leguminum sp. nov. is proposed. The type strain is MOPV5T (=CECT 30096T=LMG 31779T).

Salinity is the major driver of the global eukaryotic community structure in fish-canning wastewater treatment plants.

Fish-canning wastewater is characterized frequently by a high content of salt (NaCl), making its treatment particularly difficult; however, the knowledge of the effect of NaCl on eukaryotic communities is very limited. In the present study, the global diversity of eukaryotes in activated sludges (AS) from 4 different wastewater treatment plants (WWTPs) treating fish-canning effluents varying in salinity (0.47, 1.36, 1.72 and 12.76 g NaCl/L) was determined by sequencing partial 18S rRNA genes using Illumina MiSeq. A greater diversity than previously reported was observed in the AS community, which comprised 37 and 330 phylum-like and genera-like groups, respectively. In this sense, the more abundant genus-like groups (average relative abundance (RA) > 5%) were Adineta (6.80%), Lecane (16.80%), Dictyostelium (7.36%), Unclassified_Fungi7 (6.94%), Procryptobia (5.13) and Oocystis (5.07%). The eukaryotic communities shared a common core of 25 phylum-like clades (95% of total sequences); therefore, a narrow selection of the eukaryotic populations was found, despite the differences in the abiotic characteristics of fish-canning effluents and reactor operational conditions inflicted. The differences in NaCl concentration were the main factor that influenced the structure of the eukaryotic community, modulating the RAs of the different phylum-like clades of the common core. Higher levels of salt increased the RAs of Ascomycota, Chlorophyta, Choanoflagellata, Cryptophyta, Mollusca, Nematoda, Other Protists and Unclassified Fungi. Among the different eukaryotic genera here found, the RA of Oocystis (Chlorophyta) was intimately correlated to increasing NaCl concentrations and it is proposed as a bioindicator of the global eukaryotic community of fish-canning WWTPs.

A Novel OmpR-Type Response Regulator Controls Multiple Stages of the Rhizobium etli - Phaseolus vulgaris N2-Fixing Symbiosis.

OmpR, is one of the best characterized response regulators families, which includes transcriptional regulators with a variety of physiological roles including the control of symbiotic nitrogen fixation (SNF). The Rhizobium etli CE3 genome encodes 18 OmpR-type regulators; the function of the majority of these regulators during the SNF in common bean, remains elusive. In this work, we demonstrated that a R. etli mutant strain lacking the OmpR-type regulator RetPC57 (ΔRetPC57), formed less nodules when used as inoculum for common bean. Furthermore, we observed reduced expression level of bacterial genes involved in Nod Factors production (nodA and nodB) and of plant early-nodulation genes (NSP2, NIN, NF-YA and ENOD40), in plants inoculated with ΔRetPC57. RetPC57 also contributes to the appropriate expression of genes which products are part of the multidrug efflux pumps family (MDR). Interestingly, nodules elicited by ΔRetPC57 showed increased expression of genes relevant for Carbon/Nitrogen nodule metabolism (PEPC and GOGAT) and ΔRetPC57 bacteroids showed higher nitrogen fixation activity as well as increased expression of key genes directly involved in SNF (hfixL, fixKf, fnrN, fixN, nifA and nifH). Taken together, our data show that the previously uncharacterized regulator RetPC57 is a key player in the development of the R. etli - P. vulgaris symbiosis.

Maize Endophytic Bacterial Diversity as Affected by Soil Cultivation History.

The bacterial endophytic communities residing within roots of maize (Zea mays L.) plants cultivated by a sustainable management in soils from the Quechua maize belt (Peruvian Andes) were examined using tags pyrosequencing spanning the V4 and V5 hypervariable regions of the 16S rRNA. Across four replicate libraries, two corresponding to sequences of endophytic bacteria from long time maize-cultivated soils and the other two obtained from fallow soils, 793 bacterial sequences were found that grouped into 188 bacterial operational taxonomic units (OTUs, 97% genetic similarity). The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from fallow soils. A mean of 30 genera were found in the fallow soil libraries and 47 were in those from the maize-cultivated soils. Both alpha and beta diversity indexes showed clear differences between bacterial endophytic populations from plants with different soil cultivation history and that the soils cultivated for long time requires a higher diversity of endophytes. The number of sequences corresponding to main genera Sphingomonas, Herbaspirillum, Bradyrhizobium and Methylophilus in the maize-cultivated libraries were statistically more abundant than those from the fallow soils. Sequences of genera Dyella and Sreptococcus were significantly more abundant in the libraries from the fallow soils. Relative abundance of genera Burkholderia, candidatus Glomeribacter, Staphylococcus, Variovorax, Bacillus and Chitinophaga were similar among libraries. A canonical correspondence analysis of the relative abundance of the main genera showed that the four libraries distributed in two clearly separated groups. Our results suggest that cultivation history is an important driver of endophytic colonization of maize and that after a long time of cultivation of the soil the maize plants need to increase the richness of the bacterial endophytes communities.

Denitrification and Biodiversity of Denitrifiers in a High-Mountain Mediterranean Lake.

Wet deposition of reactive nitrogen (Nr) species is considered a main factor contributing to N inputs, of which nitrate ([Formula: see text]) is usually the major component in high-mountain lakes. The microbial group of denitrifiers are largely responsible for reduction of nitrate to molecular dinitrogen (N2) in terrestrial and aquatic ecosystems, but the role of denitrification in removal of contaminant nitrates in high-mountain lakes is not well understood. We have used the oligotrophic, high-altitude La Caldera lake in the Sierra Nevada range (Spain) as a model to study the role of denitrification in nitrate removal. Dissolved inorganic Nr concentration in the water column of la Caldera, mainly nitrate, decreased over the ice-free season which was not associated with growth of microbial plankton or variations in the ultraviolet radiation. Denitrification activity, estimated as nitrous oxide (N2O) production, was measured in the water column and in sediments of the lake, and had maximal values in the month of August. Relative abundance of denitrifying bacteria in sediments was studied by quantitative polymerase chain reaction of the 16S rRNA and the two phylogenetically distinct clades nosZI and nosZII genes encoding nitrous oxide reductases. Diversity of denitrifiers in sediments was assessed using a culture-dependent approach and after the construction of clone libraries employing the nosZI gene as a molecular marker. In addition to genera Polymorphum, Paracoccus, Azospirillum, Pseudomonas, Hyphomicrobium, Thauera, and Methylophaga, which were present in the clone libraries, Arthrobacter, Burkholderia, and Rhizobium were also detected in culture media that were not found in the clone libraries. Analysis of biological activities involved in the C, N, P, and S cycles from sediments revealed that nitrate was not a limiting nutrient in the lake, allowed N2O production and determined denitrifiers' community structure. All these results indicate that denitrification could be a major biochemical process responsible for the N losses that occur in La Caldera lake.

Spatio-Temporal Variations in the Abundance and Structure of Denitrifier Communities in Sediments Differing in Nitrate Content.

Spatial and temporal variations related to hydric seasonality in abundance and diversity of denitrifier communities were examined in sediments taken from two sites differing in nitrate concentration along a stream Doñana National Park during a 3-year study. We found a positive relationship between the relative abundance of denitrifiers, determined as narG, napA, nirK, nirS and nosZ denitrification genes, and sediment nitrate content, with similar spatial and seasonal variations. However, we did not find association between denitrification activity and the community structure of denitrifiers. Because nosZ showed the strongest correlation with the content of nitrate in sediments, we used this gene as a molecular marker to construct eight genomic libraries. Analysis of these genomic libraries revealed that diversity of the nosZ-bearing communities was higher in the site with higher nitrate content. Regardless of nitrate concentration in the sediments, the Bradyrhizobiaceae and Rhodocyclaceae were the most abundant families. On the contrary, Rhizobiaceae was exclusively present in sediments with higher nitrate content. Results showed that differences in sediment nitrate concentration affect the composition and diversityof nosZ-bearing communities.

Evolution of bacterial diversity during two-phase olive mill waste ("alperujo") composting by 16S rRNA gene pyrosequencing.

Microorganisms are the main contributing factor responsible for organic matter degradation during composting. In this research, the 454-pyrosequencing of the 16S rRNA gene was used to elucidate evolution of bacterial diversity during mesophilic, thermophilic and maturation composting stages of the two-phase olive mill waste ("alperujo"), the main by-product of the Spanish olive oil industry. Two similar piles were performance composting AL with sheep manure as bulking agent. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the main phyla found in genomic libraries from each composting phase. Shannon and Chao1 biodiversity indices showed a clear difference between the mesophilic/thermophilic and maturation phases, which was mainly due to detection of new genera. PCA analysis of the relative number of sequences confirmed maturation affected bacterial population structure, and Pearson correlation coefficients between physicochemical composting parameters and relative number of genera sequences suggest that Planomicrobium and Ohtaekwangia could be considered as biomarkers for AL composting maturation.

Bacterial Communities in the Rhizosphere of Amilaceous Maize (Zea mays L.) as Assessed by Pyrosequencing.

Maize (Zea mays L.) is the staple diet of the native peasants in the Quechua region of the Peruvian Andes who continue growing it in small plots called chacras following ancestral traditions. The abundance and structure of bacterial communities associated with the roots of amilaceous maize has not been studied in Andean chacras. Accordingly, the main objective of this study was to describe the rhizospheric bacterial diversity of amilaceous maize grown either in the presence or the absence of bur clover cultivated in soils from the Quechua maize belt. Three 16S rRNA gene libraries, one corresponding to sequences of bacteria from bulk soil of a chacra maintained under fallow conditions, the second from the rhizosphere of maize-cultivated soils, and the third prepared from rhizospheric soil of maize cultivated in intercropping with bur clover were examined using pyrosequencing tags spanning the V4 and V5 hypervariable regions of the gene. A total of 26031 sequences were found that grouped into 5955 distinct operational taxonomic units which distributed in 309 genera. The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from bulk soil. One hundred ninety seven genera were found in the bulk soil library and 234 and 203 were in those from the maize and maize/bur clover-cultivated soils. Sixteen out of the 309 genera had a relative abundance higher than 0.5% and the were (in decreasing order of abundance) Gp4, Gp6, Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Gemmatimonas, Dechloromonas, Ohtaekwangia, Rhodoferax, Gaiella, Opitutus, Gp7, Spartobacteria genera incertae sedis, Terrimonas, Gp5, Steroidobacter and Parcubacteria genera incertae sedis. Genera Gp4 and Gp6 of the Acidobacteria, Gemmatimonas and Rhodoferax were the most abundant in bulk soil, whereas Flavobacterium, Dechloromonas and Ohtaekwangia were the main genera in the rhizosphere of maize intercropped with bur clover, and Gp4, Subdivision3 genera incertae sedis of phylum Verrucomicrobia, Gp6 and Rhodoferax were the main genera in the rhizosphere of maize plants. Taken together, our results suggest that bur clover produces specific changes in rhizospheric bacterial diversity of amilaceous maize plants.

Isolation of N2 -fixing rhizobacteria from Lolium perenne and evaluating their plant growth promoting traits.

Twenty one dinitrogen (N2 )-fixing bacteria were isolated from the rhizosphere of Lolium perenne grown for more than 10 years without N-fertilization. The nearly complete sequence of the 16S rRNA gene of each strain and pairwise alignments among globally aligned sequences of the 16S rRNA genes clustered them into nine different groups. Out of the 21 strains, 11 were members of genus Bacillus, 3 belonged to each one of genera Paenibacillus and Pseudoxanthomonas, and the remaining 2 strains to each one of genera Burkholderia and Staphylococcus, respectively. A representative strain from each group contained the nifH gene and fixed atmospheric N2 as determined by the acetylene-dependent ethylene production assay (acetylene reduction activity, ARA). The nine selected strains were also examined to behave as plant growth promoting bacteria (PGPRs) including their ability to act as a biocontrol agent. The nine representative strains produced indol acetic acid (IAA) and solubilized calcium triphosphate, five of them, strains C2, C3, C12, C15, and C16, had ACC deaminase activity, and strains C2, C3, C4, C12, C16, and C17 produced siderophores. Strains C13, C16, and C17 had the capability to control growth of the pathogen Fusarium oxysporum mycelial growth in vitro. PCA analysis of determined PGPR properties showed that ARA, ACC deaminase activity, and siderophore production were the most valuable as they had the maximal contribution to the total variance.

Diversity, distribution and quantification of antibiotic resistance genes in goat and lamb slaughterhouse surfaces and meat products.

The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated 'hot spots.' The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination.

Spatial distribution of N-cycling microbial communities showed complex patterns in constructed wetland sediments.

Constructed wetlands are used for biological treatment of wastewater from agricultural lands carrying pollutants such as nitrates. Nitrogen removal in wetlands occurs from direct assimilation by plants and through microbial nitrification and denitrification. We investigated the spatial distribution of N-cycling microbial communities and genes involved in nitrification and denitrification in constructed wetland sediments receiving irrigation water. We used quantitative real-time PCR (qPCR) to characterize microbial communities. Geostatistical variance analysis was used to relate them with vegetation cover and biogeochemical sediment properties. The spatial distribution of the N-cycling microbial communities of sediments was heterogeneous and complex. Total communities of bacteria and crenarchaea showed different spatial distributions. Analysis of autocorrelation patterns through semivariance indicated a tendency towards a patchy distribution over scales around 10 m for genes involved in the nitrification and denitrification processes. In contrast, biogeochemical sediment properties showed diverse spatial distributions. While almost no patchiness was found for pH and moisture, patchiness at scales between 8 and 10 m was detected for carbon, nitrate and ammonia. Denitrification variables showed spatial autocorrelation at scales comparable to genes. However, denitrifying enzyme activity and potential N(2)O production showed a common spatial pattern, different from that of the N(2)O/(N(2)O + N(2)).