Antibiotics in animal husbandry are used to maintain welfare, but lead to the generation of resistant strains. We analyzed commensal multidrug-resistant Escherichia coli from pigs at the beginning and end of the production cycle in a farm with a farrow-to-finish system in order to investigate whether clonal spread or horizontal gene transfer constitutes the main factor responsible for the prevalence of resistance in this environment. Among 380 isolates, 56 multidrug-resistant E. coli with a similar resistant phenotype were selected for more detailed investigations including a genomic similarity analysis and the detection of mobile elements. Isolates carried blaTEM-1, aadA1, strA/B, tetA, tetB, tetC, dfrA1, dfrA5, dfrA7, dfrA12, sul1, sul2, sul3, and qnrS resistance genes, with the common co-occurrence of genes encoding the same resistance phenotype. A pulse-field gel electrophoresis analysis of the genomic similarity of multidrug-resistant E. coli showed ≤65% similarity of most of the tested strains and did not reveal a dominant clone responsible for the prevalence of resistance. Class 1 and 2 integrons and transposons 7 and 21 were detected among mobile elements; however, some were truncated. Plasmids were represented by 11 different incompatibility groups (K, FIB, I1, FIIA, FIC, FIA, Y, P, HI1, B/O, and T). Genetic resistance traits were unevenly spread in the clonal groups and suggested the major rearrangement of genetic material by horizontal gene transfer. The present results revealed that in commensal E. coli from pigs in a homogeneous farm environment, there was no dominant clone responsible for the spread of resistance and persistence in the population.
Diarrhea is often associated with marked alterations in the intestinal microbiota, termed dysbiosis; however, limited information is currently available on the intestinal microbiota in captive golden snub-nosed monkeys (Rhinopithecus roxellana) with diarrhea. We herein characterized the fecal microbiota in diarrhea and healthy monkeys using the Illumina MiSeq platform. The concentrations of fecal short-chain fatty acids (SCFAs) and copy numbers of virulence factor genes were also assessed using gas chromatography and quantitative PCR (qPCR), respectively. The results obtained showed that diarrhea monkeys harbored a distinctive microbiota from that of healthy monkeys and had 45% fewer Bacteroidetes. Among healthy subjects, old monkeys had the lowest relative abundance of Bacteroidetes. Linear discriminant analysis coupled with the effect size (LEfSe) and canonical correlation analysis (CCA) identified significant differences in microbial taxa between diarrhea and healthy monkeys. A PICRUSt analysis revealed that several pathogenic genes were enriched in diarrhea monkeys, while glycan metabolism genes were overrepresented in healthy monkeys. A positive correlation was observed between the abundance of nutrition metabolism-related genes and the individual digestive capacities of healthy monkeys. Consequently, the abundance of genes encoding heat stable enterotoxin was significantly higher in diarrhea monkeys than in healthy monkeys (P<0.05). In healthy subjects, adult monkeys had significant higher concentrations of butyrate and total SCFAs than old monkeys (P<0.05). In conclusion, the present study demonstrated that diarrhea had a microbial component and changes in the microbial structure were accompanied by altered systemic metabolic states. These results suggest that pathogens and malabsorption are the two main causes of diarrhea, which are closely related to the microbial structure and functions.
Arbuscular mycorrhizal (AM) fungi can improve plant tolerance to heavy metal contamination. This detoxification ability may largely depend on how AM fungi influence the uptake and distribution of metals in host plants. Two experiments were performed in order to gain insights into the mechanisms underlying cadmium (Cd) tolerance in mycorrhizal plants. Stable isotope Cd106 and compartmented pots were adopted to quantify the contribution of the AM fungus, Rhizophagus irregularis, to the uptake of Cd by Lotus japonicus. Moreover, synchrotron radiation μX-ray fluorescence (SR-μXRF) was applied to localize Cd in the mycorrhizal roots at the sub-cellular level. The results obtained indicated that mycorrhizal colonization markedly enhanced Cd immobilization in plant roots. Less Cd was partitioned to plant shoots when only hyphae had access to Cd in the hyphal compartment than when roots also had direct access to the Cd pool. SR-μXRF imaging indicated that Cd absorbed by extraradical hyphae was translocated into intraradical fungal structures, in which arbuscules accumulated large amounts of Cd; however, plant cells without fungal structures and plant cell walls contained negligible amounts of Cd. The present results provide direct evidence for the intraradical immobilization of Cd absorbed by AM fungi, which may largely contribute to the enhanced tolerance of plants to Cd. Therefore, AM fungi may play a role in the phytostabilization of Cd-contaminated soil.
A dissimilatory nitrate reduction to ammonium (DNRA) microbial community was developed under a high organic carbon to nitrate (C/NO3−) ratio in an anoxic semi-continuous sequencing batch reactor (SBR) fed with glucose as the source of carbon and NO3− as the electron acceptor. Activated sludge collected from a municipal wastewater treatment plant with good denitrification efficiency was used as the inoculum to start the system. The aim of this study was to examine the microbial populations in a high C/NO3− ecosystem for potential DNRA microorganisms, which are the microbial group with the ability to reduce NO3− to ammonium (NH4+). A low C/NO3− reactor was operated in parallel for direct comparisons of the microbial communities that developed under different C/NO3− values. The occurrence of DNRA in the high C/NO3− SBR was evidenced by stable isotope-labeled nitrate and nitrite (15NO3− and 15NO2−), which proved the formation of NH4+ from dissimilatory NO3−/NO2− reduction, in which both nitrogen oxides induced DNRA activity in a similar manner. An analysis of sludge samples with Illumina MiSeq 16S rRNA sequencing showed that the predominant microorganisms in the high C/NO3− SBR were related to Sulfurospirillum and the family Lachnospiraceae, which were barely present in the low C/NO3− system. A comparison of the populations and activities of the two reactors indicated that these major taxa play important roles as DNRA microorganisms under the high C/NO3− condition. Additionally, a beta-diversity analysis revealed distinct microbial compositions between the low and high C/NO3− SBRs, which reflected the activities observed in the two systems.
In the model species Streptomyces coelicolor A3(2), the uptake of chitin-degradation byproducts, mainly N,N′- diacetylchitobiose ([GlcNAc]2) and N-acetylglucosamine (GlcNAc), is performed by the ATP-binding cassette (ABC) transporter DasABC-MsiK and the sugar-phosphotransferase system (PTS), respectively. Studies on the S. coelicolor chromosome have suggested the occurrence of additional uptake systems of GlcNAc-related compounds, including the SCO6005–7 cluster, which is orthologous to the ABC transporter NgcEFG of S. olivaceoviridis. However, despite conserved synteny between the clusters in S. coelicolor and S. olivaceoviridis, homology between them is low, with only 35% of residues being identical between NgcE proteins, suggesting different binding specificities. Isothermal titration calorimetry experiments revealed that recombinant NgcESco interacts with GlcNAc and (GlcNAc)2, with Kd values (1.15 and 1.53 μM, respectively) that were higher than those of NgcE of S. olivaceoviridis (8.3 and 29 nM, respectively). The disruption of ngcESco delayed (GlcNAc)2 consumption, but did not affect GlcNAc consumption ability. The ngcESco-dasA double mutation severely decreased the ability to consume (GlcNAc)2 and abolished the induction of chitinase production in the presence of (GlcNAc)2, but did not affect the GlcNAc consumption rate. The results of these biochemical and reverse genetic analyses indicate that NgcESco acts as a (GlcNAc)2- binding protein of the ABC transporter NgcEFGSco-MsiK. Transcriptional and biochemical analyses of gene regulation demonstrated that the ngcESco gene was slightly induced by GlcNAc, (GlcNAc)2, and chitin, but repressed by DasR. Therefore, a model was proposed for the induction of the chitinolytic system and import of (GlcNAc)2, in which (GlcNAc)2 generated from chitin by chitinase produced leakily, is mainly transported via NgcEFG-MsiK and induces the expression of chitinase genes and dasABCD.
Elucidating the soil phosphorus cycle driven by soil microbes is a vital question in soil microbial ecology. The Japanese arable Andisols, occupying half of the Japanese cropland, are known for their high phosphorus sorption capacity. However, limited information is currently available on microbially driven phosphorus mineralization in arable Andisols. We herein report that the phosphorus-mineralizing community in the Japanese arable Andisols showed characteristic distribution and composition patterns, from those in other types of soils. We performed a chemical analysis and microbial community analysis of 43 arable Andisols along the Japanese archipelago. Soil phosphomonoesterase activities measured at pH 11 were approximately 70% of those at pH 6.5, which indicates that alkaline phosphatase contributes to phosphorus cycling, although most soil samples were acidic. Functional gene predictions based on 16S rRNA gene sequencing indicated that the alkaline phosphatase gene phoD was more abundant than other alkaline phosphatase genes and, thus, plays major roles. Hence, amplicon sequencing targeting phoD was performed and the results obtained showed that alphaproteobacterial phoD was dominant. This is in contrast to previously reported phoD compositions in other soils and may be attributed to the nutrient conditions in arable Andisols, which favor copiotrophic Alphaproteobacteria. Furthermore, the composition of phoD correlated with soil pH and bioavailable phosphorus concentrations rather than carbon or nitrogen concentrations. These results were partly different from previous findings, varying in the soil types and geographic ranges of sampling sites. Collectively, the present results indicate that the phosphorus-mineralizing community in the Japanese arable Andisols is regulated differently from those in other soil types.
The present study aimed to reveal the eukaryotic diets of two economically important marine sediment-inhabiting worms, Sipunculus nudus (peanut worm) and Urechis unicinctus (spoon worm), using clone libraries and phylogenetic analyses of 18S rRNA genes. Fungal rDNA was also targeted and analyzed to reveal mycobiomes. Overall, we detected a wide range of eukaryotic phylotypes associated with the larvae of S. nudus and in the gut contents of both worms. These phylotypes included ciliates, diatoms, dinoflagellates, eustigmatophytes, placidids, oomycetes, fungi, nematodes, flatworms, seaweeds, and higher plants. Oomycetes were associated with the planktonic larvae of S. nudus. The composition of eukaryotic diets shifted greatly across the larval, juvenile, and adult stages of S. nudus, and among different gut sections in U. unicinctus, reflecting lifestyle changes during the ontogeny of the peanut worm and progressive digestion in the spoon worm. Malassezia-like fungi were prevalent in mycobiomes. Epicoccum and Trichosporon-related phylotypes dominated mycobiomes associated with larval individuals and in the gut contents of adults, respectively. The gut mycobiome of S. nudus was successively characterized through the midgut, aspiratory intestines, hindgut, and rectum as having a high proportion of Climacodon-Rhizochaete, Ceriporiopsis, Cladosporium-Pseudomicrostroma, and Malassezia-related species in the libraries. These results emphasize the dynamics of diets and gut mycobiomes in marine benthic animals.
Azospirillum sp. B510, a free-living nitrogen-fixing bacterium isolated from the stems of rice (Oryza sativa cv. Nipponbare), was investigated to establish effective conditions for the colonization of rice plants. We analyzed the effects of the nitrogen sources KNO3, NH4Cl, urea (CO[NH2]2), and NH4NO3 at different concentrations (0.01–10 mM) on this colonization. Nitrogen promoted plant growth in a concentration-dependent manner, with minor differences being observed among the different nitrogen sources. Bacterial colonization was markedly suppressed on media containing NH4+ concentrations higher than 1 mM. Since concentrations of up to and including 10 mM NH4+ did not exhibit any antibacterial activity, we analyzed several factors affecting the NH4+-dependent inhibition of endophytic colonization, including the accumulation of the reactive oxygen species H2O2 and the secretion of the chemotactic substrate malic acid. The accumulation of H2O2 was increased in rice roots grown on 1 mM NH4Cl. The amounts of malic acid secreted from NH4-grown rice plants were lower than those secreted from plants grown without nitrogen or with KNO3. Although the bacterium exhibited chemotactic activity, moving towards root exudates from plants grown without nitrogen and KNO3-grown plants, this activity was not observed with root exudates from NH4+-grown plants. NH4+, but not NO3−, caused the acidification of growth media, which inhibited plant bacterial colonization. These NH4+-dependent phenomena were markedly suppressed by the stabilization of medium pH using a buffer. These results demonstrate that the type and concentration of nitrogen fertilizer affects the colonization of rice plants by Azospirillum sp. B510.
Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction−1 and sometimes detected as few as 3 copies reaction−1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3–9.1 log copies 100 mL−1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.
The rapid development of high-throughput techniques and expansion of bacterial databases have accelerated efforts to bring plant microbiomes into cultivation. We introduced plant-only-based culture media as a successful candidate to mimic the nutritional matrices of plant roots. We herein employed a G3 PhyloChip microarray to meticulously characterize the culture-dependent and -independent bacterial communities of the maize root compartments, the endo- and ecto-rhizospheres. An emphasis was placed on the preference of the growth of unculturable candidate divisions/phyla on plant-only-based culture media over standard culture media (nutrient agar). A total of 1,818 different operational taxonomic units (OTUs) were resolved representing 67 bacterial phyla. Plant-only-based culture media displayed particular affinity towards recovering endophytic over ectophytic rhizobacteria. This was shown by the slightly higher recovery of CFUs for endophytes on plant-only-based culture media (26%) than on standard culture media (10%) as well as the higher taxa richness and numbers of exclusive families of unculturable divisions/phyla. Out of 30 bacterial phyla (comprising >95% of the whole population), 13 were of a significantly higher incidence on plant-only-based culture media, 6 phyla of which were not-yet-cultured (Atribacteria, OP9; Dependentiae, TM6; Latescibacteria, WS3; Marinimicrobia, SAR406; Omnitrophica, OP3; BRC1). Furthermore, plant-only-based culture media significantly enriched less abundant and/or hard-to-culture bacterial phyla (Acidobacteria, Gemmatimonadetes, and Tenericutes). These results present conclusive evidence of the ability of plant-only-based culture media to bring the plant-fed in situ microbiome into the status of plant-fed in vitro cultures, and to widen the scope of cultivation of heretofore-unculturable bacterial divisions/phyla.
Nitrite reductase is a key enzyme for denitrification. There are two types of nitrite reductases: copper-containing NirK and cytochrome cd1-containing NirS. Most denitrifiers possess either nirK or nirS, although a few strains been reported to possess both genes. We herein report the presence of nirK and nirS in the soil-denitrifying bacterium Bradyrhizobium sp. strain TSA1T. Both nirK and nirS were identified and actively transcribed under denitrification conditions. Based on physiological, chemotaxonomic, and genomic properties, strain TSA1T (=JCM 18858T=KCTC 62391T) represents a novel species within the genus Bradyrhizobium, for which we propose the name Bradyrhizobium nitroreducens sp. nov.
We investigated biofilm formation on biodegradable plastics in freshwater samples. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) was covered by a biofilm after an incubation in freshwater samples. A next generation sequencing analysis of the bacterial communities of biofilms that formed on PHBH films revealed the dominance of the order Burkholderiales. Furthermore, Acidovorax and Undibacterium were the predominant genera in most biofilms. Twenty-five out of 28 PHBH-degrading isolates were assigned to the genus Acidovorax, while the other three were assigned to the genera Undibacterium and Chitinimonas. These results demonstrated that the order Burkholderiales in biofilms functions as a degrader of PHBH films.
Four salts, SEALIFE (a synthetic sea salt), NaCl, Na2SO4, and NaCl+KCl, were applied to monitor the effects of salinity on “Candidatus Scalindua sp.”, a marine anaerobic ammonium oxidation (anammox) bacterium. The highest ammonium consumption of 10 μmol mg protein−1 d−1 was observed at 88 mmol L−1 of Na in the presence of NaCl. The highest inorganic carbon uptake of 0.6 μmol mg protein−1 d−1 was observed at 117 mmol L−1 of Na and at 16 mmol L−1 of K in the presence of NaCl+KCl. Thus, Na and K are both important for maintaining a high growth rate of “Candidatus Scalindua sp.”
PCR clamping by locked nucleic acid (LNA) oligonucleotides is an effective technique for selectively amplifying the community SSU rRNA genes of plant–associated bacteria. However, the original primer set often shows low amplification efficiency. In order to improve this efficiency, new primers were designed at positions to compete with LNA oligonucleotides. Three new sets displayed higher amplification efficiencies than the original; however, efficiency varied among the primer sets. Two new sets appeared to be available in consideration of bacterial profiles by next-generation sequencing. One new set, KU63f and KU1494r, may be applicable to the selective gene amplification of plant-associated bacteria.