scholarly journals Active Ammonia Oxidizers in an Acidic Soil Are Phylogenetically Closely Related to Neutrophilic Archaeon

2013 ◽  
Vol 80 (5) ◽  
pp. 1684-1691 ◽  
Author(s):  
Baozhan Wang ◽  
Yan Zheng ◽  
Rong Huang ◽  
Xue Zhou ◽  
Dongmei Wang ◽  
...  
Keyword(s):  
16S Rrna ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Acidic Soil ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Evidence ◽  
Ammonia Oxidizing Bacteria ◽  
Content Type ◽  
Metabolic Versatility

ABSTRACTAll cultivated ammonia-oxidizing archaea (AOA) within theNitrososphaeracluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence ofNitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth ofNitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis ofamoAgenes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the “heavy” DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that13CO2assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both13C-labeledamoAand 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strainsNitrososphaera viennensisEN76 and JG1 within theNitrososphaeracluster. Our results provide strong evidence for the adaptive growth ofNitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

scholarly journals Phylogenetically Distinct Phylotypes Modulate Nitrification in a Paddy Soil

2015 ◽  
Vol 81 (9) ◽  
pp. 3218-3227 ◽  
Author(s):  
Jun Zhao ◽  
Baozhan Wang ◽  
Zhongjun Jia
Keyword(s):  
High Throughput ◽  
Paddy Soil ◽  
Stable Isotope Probing ◽  
Paddy Fields ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Evidence ◽  
Ammonia Oxidizing Bacteria ◽  
Soil Nitrification ◽  
Content Type

ABSTRACTPaddy fields represent a unique ecosystem in which regular flooding occurs, allowing for rice cultivation. However, the taxonomic identity of the microbial functional guilds that catalyze soil nitrification remains poorly understood. In this study, we provide molecular evidence for distinctly different phylotypes of nitrifying communities in a neutral paddy soil using high-throughput pyrosequencing and DNA-based stable isotope probing (SIP). Following urea addition, the levels of soil nitrate increased significantly, accompanied by an increase in the abundance of the bacterial and archaealamoAgene in microcosms subjected to SIP (SIP microcosms) during a 56-day incubation period. High-throughput fingerprints of the total 16S rRNA genes in SIP microcosms indicated that nitrification activity positively correlated with the abundance ofNitrosospira-like ammonia-oxidizing bacteria (AOB), soil group 1.1b-like ammonia-oxidizing archaea (AOA), andNitrospira-like nitrite-oxidizing bacteria (NOB). Pyrosequencing of13C-labeled DNA further revealed that13CO2was assimilated by these functional groups to a much greater extent than by marine group 1.1a-associated AOA andNitrobacter-like NOB. Phylogenetic analysis demonstrated that active AOB communities were closely affiliated withNitrosospirasp. strain L115 and theNitrosospira multiformislineage and that the13C-labeled AOA were related to phylogenetically distinct groups, including the moderately thermophilic “CandidatusNitrososphaera gargensis,” uncultured fosmid 29i4, and acidophilic “CandidatusNitrosotalea devanaterra” lineages. These results suggest that a wide variety of microorganisms were involved in soil nitrification, implying physiological diversification of soil nitrifying communities that are constantly exposed to environmental fluctuations in paddy fields.

scholarly journals Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

2015 ◽  
Vol 81 (14) ◽  
pp. 4607-4615 ◽  
Author(s):  
Xiaoqing Wang ◽  
Christine E. Sharp ◽  
Gareth M. Jones ◽  
Stephen E. Grasby ◽  
Allyson L. Brady ◽  
...  
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Soil Bacteria ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Degrading Bacteria ◽  
Aerobic Soil

ABSTRACTThe exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced byGluconacetobacter xylinusor the EPS produced byBeijerinckia indica. The latter is a heteropolysaccharide comprised primarily ofl-guluronic acid,d-glucose, andd-glycero-d-mannoheptose.13C-labeled EPS and13C-labeled cellulose were purified from bacterial cultures grown on [13C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from13C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However,B. indicaEPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylumPlanctomycetes. In one incubation, members of thePlanctomycetesmade up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance ofPlanctomycetessuggested that they were primary degraders of EPS. Other bacteria assimilatingB. indicaEPS included members of theVerrucomicrobia, candidate division OD1, and theArmatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

scholarly journals RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

2014 ◽  
Vol 80 (7) ◽  
pp. 2240-2247 ◽  
Author(s):  
Gerald W. Tannock ◽  
Blair Lawley ◽  
Karen Munro ◽  
Ian M. Sims ◽  
Julian Lee ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Bacterial Species ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Cells ◽  
Bacterial Populations ◽  
Content Type ◽  
And Function

ABSTRACTKnowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed thatBacteroides uniformis,Blautia glucerasea,Clostridium indolis, andBifidobacterium animaliswere the main users of the13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified.B. uniformisutilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.

scholarly journals Competing Formate- and Carbon Dioxide-Utilizing Prokaryotes in an Anoxic Methane-Emitting Fen Soil

2011 ◽  
Vol 77 (11) ◽  
pp. 3773-3785 ◽  
Author(s):  
Sindy Hunger ◽  
Oliver Schmidt ◽  
Maik Hilgarth ◽  
Marcus A. Horn ◽  
Steffen Kolb ◽  
...  
Keyword(s):  
16S Rrna ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Soil Microcosms ◽  
Content Type ◽  
Formyltetrahydrofolate Synthetase ◽  
Low Concentrations ◽  
Family Level ◽  
Methyl Coenzyme M Reductase

ABSTRACTMethanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and acetogenesis in anoxic microcosms of soil from the fen Schlöppnerbrunnen. Formate dissimilation also yields CO2as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO2-utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [13C]formate or13CO2(i.e., [13C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes,mcrA(encoding the alpha-subunit of methyl-coenzyme M reductase), andfhs(encoding formyltetrahydrofolate synthetase). Methanogens, acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated withMethanocellaceae,Methanobacteriaceae,Acetobacteraceae, andRhodospirillaceaewere13C enriched (i.e., labeled) in [13C]formate treatments, whereas genes affiliated withMethanosarcinaceae,Conexibacteraceae, andSolirubrobacteraceaewere labeled in13CO2treatments. [13C]acetate was enriched in [13C]formate treatments, but labeling of known acetogenic taxa was not detected. However, several phylotypes were affiliated with acetogen-containing taxa (e.g.,Sporomusa).Methanosaetaceae-affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives.Crenarchaeotaconstituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schlöppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate thatMethanocellaceae,Methanobacteriaceae,Methanosarcinaceae, andMethanosaetaceaewere linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.

scholarly journals Shifts in Identity and Activity of Methanotrophs in Arctic Lake Sediments in Response to Temperature Changes

2012 ◽  
Vol 78 (13) ◽  
pp. 4715-4723 ◽  
Author(s):  
Ruo He ◽  
Matthew J. Wooller ◽  
John W. Pohlman ◽  
John Quensen ◽  
James M. Tiedje ◽  
...  
Keyword(s):  
16S Rrna ◽  
Lake Sediments ◽  
Incubation Temperature ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Type I ◽  
Content Type ◽  
Arctic Lake ◽  
Methanotrophic Communities

ABSTRACTMethane (CH4) flux to the atmosphere is mitigated via microbial CH4oxidation in sediments and water. As arctic temperatures increase, understanding the effects of temperature on the activity and identity of methanotrophs in arctic lake sediments is important to predicting future CH4emissions. We used DNA-based stable-isotope probing (SIP), quantitative PCR (Q-PCR), and pyrosequencing analyses to identify and characterize methanotrophic communities active at a range of temperatures (4°C, 10°C, and 21°C) in sediments (to a depth of 25 cm) sampled from Lake Qalluuraq on the North Slope of Alaska. CH4oxidation activity was measured in microcosm incubations containing sediments at all temperatures, with the highest CH4oxidation potential of 37.5 μmol g−1day−1in the uppermost (depth, 0 to 1 cm) sediment at 21°C after 2 to 5 days of incubation. Q-PCR ofpmoAand of the 16S rRNA genes of type I and type II methanotrophs, and pyrosequencing of 16S rRNA genes in13C-labeled DNA obtained by SIP demonstrated that the type I methanotrophsMethylobacter,Methylomonas, andMethylosomadominated carbon acquisition from CH4in the sediments. The identity and relative abundance of active methanotrophs differed with the incubation temperature. Methylotrophs were also abundant in the microbial community that derived carbon from CH4, especially in the deeper sediments (depth, 15 to 20 cm) at low temperatures (4°C and 10°C), and showed a good linear relationship (R= 0.82) with the relative abundances of methanotrophs in pyrosequencing reads. This study describes for the first time how methanotrophic communities in arctic lake sediments respond to temperature variations.

scholarly journals Responses of Active Ammonia Oxidizers and Nitrification Activity in Eutrophic Lake Sediments to Nitrogen and Temperature

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Ling Wu ◽  
Cheng Han ◽  
Guangwei Zhu ◽  
Wenhui Zhong
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Ammonia Oxidizing Bacteria ◽  
Ammonia Oxidizers ◽  
Nitrogen Input ◽  
Nitrification Activity ◽  
Ammonia Oxidizing Archaea ◽  
Nitrogen Inputs

ABSTRACTAmmonium concentrations and temperature drive the activities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), but their effects on these microbes in eutrophic freshwater sediments are unclear. In this study, surface sediments collected from areas of Taihu Lake (China) with different degrees of eutrophication were incubated under three levels of nitrogen input and temperature, and the autotrophic growth of ammonia oxidizers was assessed using13C-labeled DNA-based stable-isotope probing (SIP), while communities were characterized using MiSeq sequencing and phylogenetic analysis of 16S rRNA genes. Nitrification rates in sediment microcosms were positively correlated with nitrogen inputs, but there was no marked association with temperature. Incubation of SIP microcosms indicated that AOA and AOBamoAgenes were labeled by13C at 20°C and 30°C in the slightly eutrophic sediment, and AOBamoAgenes were labeled to a much greater extent than AOAamoAgenes in the moderately eutrophic sediment after 56 days. Phylogenetic analysis of13C-labeled 16S rRNA genes revealed that the active AOA were mainly affiliated with theNitrosopumiluscluster, with theNitrososphaeracluster dominating in the slightly eutrophic sediment at 30°C with low ammonium input (1 mM). Active AOB communities were more sensitive to nitrogen input and temperature than were AOA communities, and they were exclusively dominated by theNitrosomonascluster, which tended to be associated withNitrosomonadaceae-like lineages.Nitrosomonassp. strain Is79A3 tended to dominate the moderately eutrophic sediment at 10°C with greater ammonium input (2.86 mM). The relative abundance responses of the major active communities to nitrogen input and temperature gradients varied, indicating niche differentiation and differences in the physiological metabolism of ammonia oxidizers that are yet to be described.IMPORTANCEBoth archaea and bacteria contribute to ammonia oxidation, which plays a central role in the global cycling of nitrogen and is important for reducing eutrophication in freshwater environments. The abundance and activities of ammonia-oxidizing archaea and bacteria in eutrophic limnic sediments vary with different ammonium concentrations or with seasonal shifts, and how the two factors affect nitrification activity, microbial roles, and active groups in different eutrophic sediments is unclear. The significance of our research is in identifying the archaeal and bacterial responses to anthropogenic activity and climate change, which will greatly enhance our understanding of the physiological metabolic differences of ammonia oxidizers.

scholarly journals Temporal Variability of Coastal Planctomycetes Clades at Kabeltonne Station, North Sea

2011 ◽  
Vol 77 (14) ◽  
pp. 5009-5017 ◽  
Author(s):  
Ilaria Pizzetti ◽  
Bernhard M. Fuchs ◽  
Gunnar Gerdts ◽  
Antje Wichels ◽  
Karen H. Wiltshire ◽  
...  
Keyword(s):  
16S Rrna ◽  
North Sea ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Group A ◽  
Related Group ◽  
Group B ◽  
Card Fish ◽  
Group D

ABSTRACTMembers of the bacterial phylumPlanctomycetesare reported in marine water samples worldwide, but quantitative information is scarce. Here we investigated the phylogenetic diversity, abundance, and distribution ofPlanctomycetesin surface waters off the German North Sea island Helgoland during different seasons by 16S rRNA gene analysis and catalyzed reporter deposition fluorescencein situhybridization (CARD-FISH). GenerallyPlanctomycetesare more abundant in samples collected in summer and autumn than in samples collected in winter and spring. Statistical analysis revealed thatPlanctomycetesabundance was correlated to theCentralesdiatom bloom in spring 2007. The analysis of size-fractionated seawater samples and of macroaggregates showed that ∼90% of thePlanctomycetesreside in the >3-μm size fraction. Comparative sequence analysis of 184 almost full-length 16S rRNA genes revealed three dominant clades. The clades, namedPlanctomyces-related group A, unculturedPlanctomycetesgroup B, andPirellula-related group D, were monitored by CARD-FISH using newly developed oligonucleotide probes. All three clades showed recurrent abundance patterns during two annual sampling campaigns. UnculturedPlanctomycetesgroup B was most abundant in autumn samples, whilePlanctomyces-related group A was present in high numbers only during late autumn and winter. The levels ofPirellula-related group D were more constant throughout the year, with elevated counts in summer. Our analyses suggest that the seasonal succession of thePlanctomycetesis correlated with algal blooms. We hypothesize that the niche partitioning of the different clades might be caused by their algal substrates.

scholarly journals Quantitative Assessment of Ammonia-Oxidizing Bacterial Communities in the Epiphyton of Submerged Macrophytes in Shallow Lakes

2010 ◽  
Vol 76 (6) ◽  
pp. 1813-1821 ◽  
Author(s):  
M. Coci ◽  
G. W. Nicol ◽  
G. N. Pilloni ◽  
M. Schmid ◽  
M. P. Kamst-van Agterveld ◽  
...  
Keyword(s):  
16S Rrna ◽  
Shallow Lakes ◽  
Submerged Macrophytes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gene Copy ◽  
Potamogeton Pectinatus ◽  
Rrna Gene ◽  
Ammonia Oxidizing Bacteria ◽  
Potential Nitrification

ABSTRACT In addition to the benthic and pelagic habitats, the epiphytic compartment of submerged macrophytes in shallow freshwater lakes offers a niche to bacterial ammonia-oxidizing communities. However, the diversity, numbers, and activity of epiphytic ammonia-oxidizing bacteria have long been overlooked. In the present study, we analyzed quantitatively the epiphytic communities of three shallow lakes by a potential nitrification assay and by quantitative PCR of 16S rRNA genes. On the basis of the m2 of the lake surface, the gene copy numbers of epiphytic ammonia oxidizers were not significantly different from those in the benthic and pelagic compartments. The potential ammonia-oxidizing activities measured in the epiphytic compartment were also not significantly different from the activities determined in the benthic compartment. No potential ammonia-oxidizing activities were observed in the pelagic compartment. No activity was detected in the epiphyton of Chara aspera, the dominant submerged macrophyte in Lake Nuldernauw in The Netherlands. The presence of ammonia-oxidizing bacterial cells in the epiphyton of Potamogeton pectinatus was also demonstrated by fluorescent in situ hybridization microscopy images. By comparing the community composition as assessed by the 16S rRNA gene PCR-denaturing gradient gel electrophoresis approach, it was concluded that the epiphytic ammonia-oxidizing communities consisted of cells that were also present in the benthic and pelagic compartments. Of the environmental parameters examined, only the water retention time, the Kjeldahl nitrogen content, and the total phosphorus content correlated with potential ammonia-oxidizing activities. None of these parameters correlated with the numbers of gene copies related to ammonia-oxidizing betaproteobacteria.

scholarly journals Mucilaginibacter koreensis sp. nov., isolated from leaf mould

2014 ◽  
Vol 64 (Pt_7) ◽  
pp. 2274-2279 ◽  
Author(s):  
Cheol Su Park ◽  
Kyudong Han ◽  
Tae-Young Ahn
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
Sequence Similarity ◽  
Optimal Growth ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Strictly Aerobic ◽  
Content Type ◽  
Link Type

A Gram-staining-negative, strictly aerobic, rod-shaped, pale-pink pigmented bacterial strain, designated TF8T, was isolated from leaf mould in Cheonan, Republic of Korea. Its taxonomic position was determined through a polyphasic approach. Optimal growth occurred on R2A agar without NaCl supplementation, at 25–28 °C and at pH 6.0–7.0. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain TF8T belongs to the genus Mucilaginibacter in the family Sphingobacteriaceae . The sequence similarity between 16S rRNA genes of strain TF8T and the type strains of other species of the genus Mucilaginibacter ranged from 92.1 to 94.7 %. The closest relatives of strain TF8T were Mucilaginibacter lutimaris BR-3T (94.7 %), M. soli R9-65T (94.5 %), M. litoreus BR-18T (94.5 %), M. rigui WPCB133T (94.0 %) and M. daejeonensis Jip 10T (93.8 %). The major isoprenoid quinone was MK-7 and the major cellular fatty acids were iso-C15 : 0 (33.0 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 24.8 %) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 13.0 %). The major polar lipids of TF8T were phosphatidylethanolamine and three unidentified aminophospholipids. The G+C content of the genomic DNA was 46.2 mol%. On the basis of the data presented here, strain TF8T is considered to represent a novel species of the genus Mucilaginibacter , for which the name Mucilaginibacter koreensis sp. nov. is proposed. The type strain is TF8T ( = KACC 17468T = JCM 19323T).

scholarly journals Wastewater Irrigation Increases the Abundance of Potentially Harmful Gammaproteobacteria in Soils in Mezquital Valley, Mexico

2014 ◽  
Vol 80 (17) ◽  
pp. 5282-5291 ◽  
Author(s):  
Melanie Broszat ◽  
Heiko Nacke ◽  
Ronja Blasi ◽  
Christina Siebe ◽  
Johannes Huebner ◽  
...  
Keyword(s):  
16S Rrna ◽  
Rainy Season ◽  
Soil Microbial Communities ◽  
Wastewater Irrigation ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Resistance Determinants ◽  
Oxacillin Resistance ◽  
Irrigated Soils

ABSTRACTWastewater contains large amounts of pharmaceuticals, pathogens, and antimicrobial resistance determinants. Only a little is known about the dissemination of resistance determinants and changes in soil microbial communities affected by wastewater irrigation. Community DNAs from Mezquital Valley soils under irrigation with untreated wastewater for 0 to 100 years were analyzed by quantitative real-time PCR for the presence ofsulgenes, encoding resistance to sulfonamides. Amplicon sequencing of bacterial 16S rRNA genes from community DNAs from soils irrigated for 0, 8, 10, 85, and 100 years was performed and revealed a 14% increase of the relative abundance ofProteobacteriain rainy season soils and a 26.7% increase in dry season soils for soils irrigated for 100 years with wastewater. In particular,Gammaproteobacteria, including potential pathogens, such asPseudomonas,Stenotrophomonas, andAcinetobacterspp., were found in wastewater-irrigated fields. 16S rRNA gene sequencing of 96 isolates from soils irrigated with wastewater for 100 years (48 from dry and 48 from rainy season soils) revealed that 46% were affiliated with theGammaproteobacteria(mainly potentially pathogenicStenotrophomonasstrains) and 50% with theBacilli, whereas all 96 isolates from rain-fed soils (48 from dry and 48 from rainy season soils) were affiliated with theBacilli. Up to six types of antibiotic resistance were found in isolates from wastewater-irrigated soils; sulfamethoxazole resistance was the most abundant (33.3% of the isolates), followed by oxacillin resistance (21.9% of the isolates). In summary, we detected an increase of potentially harmful bacteria and a larger incidence of resistance determinants in wastewater-irrigated soils, which might result in health risks for farm workers and consumers of wastewater-irrigated crops.

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