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This study explored the assembly mechanisms and physicochemical dynamics of microbial communities within atmospheric bioaerosols, focusing on the influence of different aerial trajectories. Over two years, samples near Seoul were classified into 'North', 'Southwest', and 'Others' categories based on their aerial trajectories. Physicochemical analysis of the PM2.5 particles revealed distinct ion compositions for each cluster, reflecting diverse environmental influences. Microbial community analysis revealed that shared dominant bacterial phyla were present in all clusters. However, distinct taxonomic profiles and biomarkers were also evident, such as coastal bacteria in the 'Southwest' cluster correlating with wind speed, and arid soil-originated bacteria in the 'North' cluster correlating with cations. These findings demonstrate that biomarkers in each cluster are representative of the distinct environments associated with their aerial trajectories. Notably, cluster 'Southwest' the highest microbial diversity and a strong alignment with the neutral community model, suggesting a large influence of passive dispersal from marine environments. Contrarily, 'North' and 'Others' were more influenced by niche-dependent factors. This study highlights the complex interplay between environmental factors and microbial dynamics in bioaerosols and provides important insights for environmental monitoring and public health risk assessment.

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Recognizing that microbial community composition within the human microbiome is associated with the physiological state of the host has sparked a large number of human microbiome association studies (HMAS). With the increasing size of publicly available HMAS data, the privacy risk is also increasing because HMAS metadata could contain sensitive private information. I demonstrate that a simple test statistic based on the taxonomic profiles of an individual's microbiome along with summary statistics of HMAS data can reveal the membership of the individual's microbiome in an HMAS sample. In particular, species-level taxonomic data obtained from small-scale HMAS can be highly vulnerable to privacy risk. Minimal guidelines for HMAS data privacy are suggested, and an assessment of HMAS privacy risk using the simulation method proposed is recommended at the time of study design.

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[This corrects the article DOI: 10.1371/journal.pone.0096197.].

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The genetic diversity and population structure of Vibrio vulnificus isolates from Korea and Taiwan were investigated using PCR-based assays targeting putative virulence-related genes and multilocus sequence typing (MLST). BOX-PCR genomic fingerprinting identified 52 unique genotypes in 84 environmental and clinical V. vulnificus isolates. The majority (> 50%) of strains had pathogenic genotypes for all loci tested; moreover, many environmental strains had pathogenic genotypes. Although significant (p < 0.05) inter-relationships among the genotypes were observed, the association between genotype and strain source (environmental or clinical) was not significant, indicating that genotypic characteristics alone are not sufficient to predict the isolation source or the virulence of a given V. vulnificus strain and vice versa. MLST revealed 23-35 allelic types per locus analyzed, resulting in a total of 44 unique sequence types (STs). Two major monophyletic groups (lineages A and B) corresponding to the two known lineages of V. vulnificus were observed; lineage A had six STs that were exclusively environmental, whereas lineage B had STs from both environmental and clinical sources. Pathogenic and nonpathogenic genotypes predominated in MLST lineages B and A, respectively. In addition, V. vulnificus was shown to be in linkage disequilibrium (p < 0.05), although two different recombination tests (PHI and Sawyer's tests) detected significant evidence of recombination. Tajima's D test also indicated that V. vulnificus might be comprised of recently sub-divided lineages. These results suggested that the two lineages revealed by MLST correspond to two distinct ecotypes of V. vulnificus.

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To find environmental variables (EVs) shaping the ecological niche of the archaeal phylum Thaumarchaeota in terrestrial environments, we determined the abundance of Thaumarchaeota in various soil samples using real-time PCR targeting thaumarchaeotal 16S rRNA gene sequences. We employed our previously developed primer, THAUM-494, which had greater coverage for Thaumarchaeota and lower tolerance to nonthaumarchaeotal taxa than previous Thaumarchaeota-directed primers. The relative abundance estimates (RVs) of Thaumarchaeota (RTHAUM), Archaea (RARCH), and Bacteria (RBACT) were subjected to a series of statistical analyses. Redundancy analysis (RDA) showed a significant (p < 0.05) canonical relationship between RVs and EVs. Negative causal relationships between RTHAUM and nutrient level-related EVs were observed in an RDA biplot. These negative relationships were further confirmed by correlation and regression analyses. Total nitrogen content (TN) appeared to be the EV that affected RTHAUM most strongly, and total carbon content (TC), which reflected the content of organic matter (OM), appeared to be the EV that affected it least. However, in the path analysis, a path model indicated that TN might be a mediator EV that could be controlled directly by the OM. Additionally, another path model implied that water content (WC) might also indirectly affect RTHAUM by controlling ammonium nitrogen (NH4+-N) level through ammonification. Thus, although most directly affected by NH4+-N, RTHAUM could be ultimately determined by OM content, suggesting that Thaumarchaeota could prefer low-OM or low-WC conditions, because either of these EVs could subsequently result in low levels of NH4+-N in soil.

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Based on comparative phylogenetic analysis of 16S rRNA gene sequences deposited in an RDP database, we constructed a local database of thaumarchaeotal 16S rRNA gene sequences and developed a novel PCR primer specific for the archaeal phylum Thaumarchaeota. Among 9,727 quality-filtered (chimeral-checked, size >1.2 kb) archaeal sequences downloaded from the RDP database, 1,549 thaumarchaeotal sequences were identified and included in our local database. In our study, Thaumarchaeota included archaeal groups MG-I, SAGMCG-I, SCG, FSCG, RC, and HWCG-III, forming a monophyletic group in the phylogenetic tree. Cluster analysis revealed 114 phylotypes for Thaumarchaeota. The majority of the phylotypes (66.7%) belonged to the MG-I and SCG, which together contained most (93.9%) of the thaumarchaeotal sequences in our local database. A phylum-directed primer was designed from a consensus sequence of the phylotype sequences, and the primer's specificity was evaluated for coverage and tolerance both in silico and empirically. The phylum-directed primer, designated THAUM-494, showed >90% coverage for Thaumarchaeota and <1% tolerance to non-target taxa, indicating high specificity. To validate this result experimentally, PCRs were performed with THAUM-494 in combination with a universal archaeal primer (ARC917R or 1017FAR) and DNAs from five environmental samples to construct clone libraries. THAUM-494 showed a satisfactory specificity in empirical studies, as expected from the in silico results. Phylogenetic analysis of 859 cloned sequences obtained from 10 clone libraries revealed that >95% of the amplified sequences belonged to Thaumarchaeota. The most frequently sampled thaumarchaeotal subgroups in our samples were SCG, MG-I, and SAGMCG-I. To our knowledge, THAUM-494 is the first phylum-level primer for Thaumarchaeota. Furthermore, the high coverage and low tolerance of THAUM-494 will make it a potentially valuable tool in understanding the phylogenetic diversity and ecological niche of Thaumarchaeota.

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Various enteric viruses including norovirus, rotavirus, adenovirus, and astrovirus are the major etiological agents of food-borne and water-borne disease outbreaks and frequently cause non-bacterial gastroenteritis worldwide. Sensitive and high-throughput detection methods for these viral pathogens are compulsory for diagnosing viral pathogens and subsequently improving public health. Hence, we developed a sensitive, specific, and high-throughput analytical assay to detect most major enteric viral pathogens using "Combimatrix" platform oligonucleotide probes. In order to detect four different enteric viral pathogens in a sensitive and simultaneous manner, we first developed a multiplex RT-PCR assay targeting partial gene sequences of these viruses with fluorescent labeling for the subsequent microarray. Then, five olignonucleotides specific to each of the four major enteric viruses were selected for the microarray from the oligonulceotide pools targeting the specific genes obtained by multiplex PCR of these viruses. The oligonucleotide microarray was evaluated against stool specimens containing single or mixed viral species. As a result, we demonstrated that the multiplex RT-PCR assay specifically amplified partial sequences of four enteric viruses and the subsequent microarray assay was capable of sensitive and simultaneous detection of those viruses. The developed method could be useful for diagnosing enteric viruses in both clinical and environmental specimens.

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The bacterial diversity of the continental shelf sediment in the Yellow Sea was investigated by the cloning and sequencing of PCR-amplified 16S rRNA genes. The majority of the cloned sequences were distinct phylotypes that were novel at the species level. The richness estimator indicated that the sediment sample might harbor up to 32 phylum-level taxa. A large number of low-abundance, phylum-level taxa accounted for most of the observed phylogenetic diversity at our study site, suggesting that these low-abundance taxa might play crucial roles in the shelf sediment ecosystem.

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We investigated the phylogenetic diversity of ammonia-oxidizing bacteria (AOB) in Yellow Sea continental shelf sediment by the cloning and sequencing of PCR-amplified amoA and 16S rRNA genes. Phylogenetic analysis of the amoA-related clones revealed that the diversity of AOB was extremely low at the study site. The majority (92.7%) of amoA clones obtained belonged to a single cluster, environmental amoA cluster-3, the taxonomic position of which was previously unknown. Phylogenetic analysis on AOB-specific 16S rRNA gene sequences also demonstrated a very low diversity. All of the cloned 16S rRNA gene sequences comprised a single phylotype that belonged to the members of uncultured Nitrosospira cluster-1, suggesting that AOB belonging to the uncultured Nitrosospira cluster- 1 could carry amoA sequences of environmental amoA cluster-3.

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We performed a comprehensive phylogenetic analysis of the phylum Acidobacteria and developed novel, group-specific PCR primers for Acidobacteria and its class-level subgroups. Acidobacterial 16S rRNA gene sequences deposited in the RDP database were used to construct a local database then subsequently analyzed. A total of 556 phylotypes were observed and the majority of the phylotypes belonged to five major subgroups (subgroups 1, 2, 3, 4, and 6), which comprised >80% of the acidobacterial sequences in the RDP database. Phylum-specific and subgroup-specific primers were designed from the consensus sequences of the phylotype sequences, and the specificities of the designed primers were evaluated both in silico and empirically for coverage and tolerance. The phylum-specific primer ACIDO, which was designed in this study, showed increased coverage for Acidobacteria, as compared to the previous phylum-specific primer 31F. However, the tolerance of the primer ACIDO for non-target sequences was slightly higher than that of the primer 31F. We also developed subgroup-specific PCR primers for the major subgroups of Acidobacteria, except for subgroup 4. Subgroup-specific primers S1, S2, and S3, which targeted subgroups 1, 2, and 3, respectively, showed high coverage for their target subgroups and low tolerance for non-target sequences. However, the primer S6 targeting subgroup 6 showed a lower specificity in its empirical evaluation than expected from the in silico results. The subgroup-specific primers, as well as the phylum-specific primer designed in this study, will be valuable tools in understanding the phylogenetic diversity and ecological niche of the phylum Acidobacteria and its subgroups.

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The distribution pattern of the phylum Acidobacteria, a previously uncultured bacterial group, was investigated by molecular ecological analyses of global soil samples collected from pristine ecosystems across five continents. Acidobacterial 16S rDNAs were observed in almost all soil samples, and members of acidobacterial primer group A were detected in all samples that harbored the phylum Acidobacteria. Other primer groups, Y, G, and O, showed limited distribution patterns. We further divided the primer groups into acidobacterial subdivisions (class-level). Subdivisional distribution patterns were determined by comparing the observed T-RFs with theoretical T-RFs predicted by in silico digestion of acidobacterial 16S rDNAs. Consistent with the PCR results obtained with subgroup-specific primers, T-RFLP analyses showed that acidobacterial subdivision 1 belonging to primer group A was present in the majority of the soil samples. This study revealed that the phylum Acidobacteria could be globally distributed. At the subdivisional level, acidobacterial subdivision 1 might be the most widely distributed group in this phylum, indicating that members of subdivision 1 might be adapted to various soil environments, and members belonging to other subdivisions might be restricted to certain geographic regions or habitats.

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A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed for the rapid and sensitive detection of L. monocytogenes. PCR primers generating a 132-bp amplicon and a capture probe able to hybridize to the PCR amplicon were designed based on the L. monocytogenes-specific hly gene encoding listeriolysin. The detection limit of PCR-ELISA for L. monocytogenes was determined to be as low as 10 cells per PCR reaction, and this level of detection was achieved within 5 h. These results indicate that the PCR-ELISA provides a valuable tool for the rapid and sensitive detection of L. monocytogenes for the ready-to-eat food industry.

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A culture-independent survey was performed to search for 16S rRNA gene sequences representing dominant and metabolically active bacteria in rhizosphere soil. PCR- and reverse transcription-PCR-derived clone libraries were constructed from DNA and RNA directly extracted from the soil sample. Acidobacteria-related sequences occupied an unusually large proportion (>50%) of both rDNA- and rRNA-derived clone libraries. This study suggested that the bacteria belonging to the phylum Acidobacteria might be numerically dominant as well as metabolically active in the soil sample, implying that the phylum Acidobacteria might be highly involved in the biogeochemical cycles of the rhizosphere soil.

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Molecular and cultivation techniques were used to characterize the bacterial communities of biobead reactor biofilms in a sewage treatment plant to which an Aerated Up-Flow Biobead process was applied. With this biobead process, the monthly average values of various chemical parameters in the effluent were generally kept under the regulation limits of the effluent quality of the sewage treatment plant during the operation period. Most probable number (MPN) analysis revealed that the population of denitrifying bacteria was abundant in the biobead #1 reactor, denitrifying and nitrifying bacteria coexisted in the biobead #2 reactor, and nitrifying bacteria prevailed over denitrifying bacteria in the biobead #3 reactor. The results of the MPN test suggested that the biobead #2 reactor was a transition zone leading to acclimated nitrifying biofilms in the biobead #3 reactor. Phylogenetic analysis of 16S rDNA sequences cloned from biofilms showed that the biobead #1 reactor, which received a high organic loading rate, had much diverse microorganisms, whereas the biobead #2 and #3 reactors were dominated by the members of Proteobacteria. DGGE analysis with the ammonia monooxygenase (amoA) gene supported the observation from the MPN test that the biofilms of September were fully developed and specialized for nitrification in the biobead reactor #3. All of the DNA sequences of the amoA DGGE bands were very similar to the sequence of the amoA gene of Nitrosomonas species, the presence of which is typical in the biological aerated filters. The results of this study showed that organic and inorganic nutrients were efficiently removed by both denitrifying microbial populations in the anaerobic tank and heterotrophic and nitrifying bacterial biofilms well-formed in the three functional biobead reactors in the Aerated Up-Flow Biobead process.

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To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.

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