George Golovko

Metagenomic Analysis of the Airborne Environment in Urban Spaces

The organisms in aerosol microenvironments, especially densely populated urban areas, are relevant to maintenance of public health and detection of potential epidemic or biothreat agents. To examine aerosolized microorganisms in this environment, we performed sequencing on the material from an urban aerosol surveillance program. Whole metagenome sequencing was applied to DNA extracted from air filters obtained during periods from each of the four seasons. The composition of bacteria, plants, fungi, invertebrates, and viruses demonstrated distinct temporal shifts. Bacillus thuringiensis serovar kurstaki was detected in samples known to be exposed to aerosolized spores, illustrating the potential utility of this approach for identification of intentionally introduced microbial agents. Together, these data demonstrate the temporally dependent metagenomic complexity of urban aerosols and the potential of genomic analytical techniques for biosurveillance and monitoring of threats to public health.

GPR43 mediates microbiota metabolite SCFA regulation of antimicrobial peptide expression in intestinal epithelial cells via activation of mTOR and STAT3

The antimicrobial peptides (AMP) produced by intestinal epithelial cells (IEC) play crucial roles in the regulation of intestinal homeostasis by controlling microbiota. Gut microbiota has been shown to promote IEC expression of RegIIIγ and certain defensins. However, the mechanisms involved are still not completely understood. In this report, we found that IEC expression levels of RegIIIγ and β-defensins 1, 3, and 4 were lower in G protein-coupled receptor (GPR)43−/− mice compared to that of wild-type (WT) mice. Oral feeding with short-chain fatty acids (SCFA) promoted IEC production of RegIIIγ and defensins in mice. Furthermore, SCFA induced RegIIIγ and β-defensins in intestinal epithelial enteroids generated from WT but not GPR43−/− mice. Mechanistically, SCFA activated mTOR and STAT3 in IEC, and knockdown of mTOR and STAT3 impaired SCFA induction of AMP production. Our studies thus demonstrated that microbiota metabolites SCFA promoted IEC RegIIIγ and β-defensins in a GPR43-dependent manner. The data thereby provide a novel pathway by which microbiota regulates IEC expression of AMP and intestinal homeostasis.

Assessing the utility of metabarcoding for diet analyses of the omnivorous wild pig (Sus scrofa)

Abstract Wild pigs (Sus scrofa) are an invasive species descended from both domestic swine and Eurasian wild boar that was introduced to North America during the early 1500s. Wild pigs have since become the most abundant free‐ranging exotic ungulate in the United States. Large and ever‐increasing populations of wild pigs negatively impact agriculture, sport hunting, and native ecosystems with costs estimated to exceed

Microbiome interaction networks and community structure from laboratory-reared and field-collected Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus mosquito vectors

1.5 billion/year within the United States. Wild pigs are recognized as generalist feeders, able to exploit a broad array of locally available food resources, yet their feeding behaviors remain poorly understood as partially digested material is often unidentifiable through traditional stomach content analyses. To overcome the limitation of stomach content analyses, we developed a DNA sequencing‐based protocol to describe the plant and animal diet composition of wild pigs. Additionally, we developed and evaluated blocking primers to reduce the amplification and sequencing of host DNA, thus providing greater returns of sequences from diet items. We demonstrate that the use of blocking primers produces significantly more sequencing reads per sample from diet items, which increases the robustness of ascertaining animal diet composition with molecular tools. Further, we show that the overall plant and animal diet composition is significantly different between the three areas sampled, demonstrating this approach is suitable for describing differences in diet composition among the locations.

The ability of human nuclear DNA to cause false positive low-abundance heteroplasmy calls varies across the mitochondrial genome

Microbial interactions are an underappreciated force in shaping insect microbiome communities. Although pairwise patterns of symbiont interactions have been identified, we have a poor understanding regarding the scale and the nature of co-occurrence and co-exclusion interactions within the microbiome. To characterize these patterns in mosquitoes, we sequenced the bacterial microbiome of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus caught in the field or reared in the laboratory and used these data to generate interaction networks. For collections, we used traps that attracted host-seeking or ovipositing female mosquitoes to determine how physiological state affects the microbiome under field conditions. Interestingly, we saw few differences in species richness or microbiome community structure in mosquitoes caught in either trap. Co-occurrence and co-exclusion analysis identified 116 pairwise interactions substantially increasing the list of bacterial interactions observed in mosquitoes. Networks generated from the microbiome of Ae. aegypti often included highly interconnected hub bacteria. There were several instances where co-occurring bacteria co-excluded a third taxa, suggesting the existence of tripartite relationships. Several associations were observed in multiple species or in field and laboratory-reared mosquitoes indicating these associations are robust and not influenced by environmental or host factors. To demonstrate that microbial interactions can influence colonization of the host, we administered symbionts to Ae. aegypti larvae that either possessed or lacked their resident microbiota. We found that the presence of resident microbiota can inhibit colonization of particular bacterial taxa. Our results highlight that microbial interactions in mosquitoes are complex and influence microbiome composition.

Using High Throughput DNA Sequencing to Evaluate the Accuracy of Serial Dilution Based Tests of Microbial Activities in Oil Pipelines

BackgroundLow-abundance mutations in mitochondrial populations (mutations with minor allele frequency ≤ 1%), are associated with cancer, aging, and neurodegenerative disorders. While recent progress in high-throughput sequencing technology has significantly improved the heteroplasmy identification process, the ability of this technology to detect low-abundance mutations can be affected by the presence of similar sequences originating from nuclear DNA (nDNA). To determine to what extent nDNA can cause false positive low-abundance heteroplasmy calls, we have identified mitochondrial locations of all subsequences that are common or similar (one mismatch allowed) between nDNA and mitochondrial DNA (mtDNA).ResultsPerformed analysis revealed up to a 25-fold variation in the lengths of longest common and longest similar (one mismatch allowed) subsequences across the mitochondrial genome. The size of the longest subsequences shared between nDNA and mtDNA in several regions of the mitochondrial genome were found to be as low as 11 bases, which not only allows using these regions to design new, very specific PCR primers, but also supports the hypothesis of the non-random introduction of mtDNA into the human nuclear DNA.ConclusionAnalysis of the mitochondrial locations of the subsequences shared between nDNA and mtDNA suggested that even very short (36 bases) single-end sequencing reads can be used to identify low-abundance variation in 20.4% of the mitochondrial genome. For longer (76 and 150 bases) reads, the proportion of the mitochondrial genome where nDNA presence will not interfere found to be 44.5 and 67.9%, when low-abundance mutations at 100% of locations can be identified using 417 bases long single reads. This observation suggests that the analysis of low-abundance variations in mitochondria population can be extended to a variety of large data collections such as NCBI Sequence Read Archive, European Nucleotide Archive, The Cancer Genome Atlas, and International Cancer Genome Consortium.

Microbiota dysbiosis and its pathophysiological significance in bowel obstruction

Microbial activities have detrimental effects on industrial infrastructure. If not controlled, microbial presence can result in corrosion, biofilm formation, and product degradation. Serial dilution tests are routinely used for evaluating presence and abundance of microorganisms by diluting samples and culturing microbes in specific media designed to support microorganisms with particular properties, such as sulfate reduction. A high-throughput sequencing approach was used to evaluate changes in microbial composition during four standard serial dilution tests. Analysis of 159 isolates revealed significant differences in the microbial compositions of sequential serial dilution titers and identified several cases where: (a) bacteria known to have a detrimental metabolic function (such as acid production) were lost in the serial dilution medium designed to test for this function; (b) bacteria virtually absent in the original sample became dominant in the serial dilution medium. These observations raise concerns regarding the accuracy and overall usefulness of serial dilution tests.

Broom: application for non-redundant storage of high throughput sequencing data

Bowel obstruction (OB) causes local and systemic dysfunctions. Here we investigated whether obstruction leads to alterations in microbiota community composition and total abundance, and if so whether these changes contribute to dysfunctions in OB. Partial colon obstruction was maintained in rats for 7 days. The mid colon and its intraluminal feces - proximal to the obstruction - were studied. OB did not cause bacterial overgrowth or mucosa inflammation, but induced profound changes in fecal microbiota composition and diversity. At the phylum level, the 16S rRNA sequencing showed a significant decrease in the relative abundance of Firmicutes with corresponding increases in Proteobacteria and Bacteroidetes in OB compared with sham controls. Daily treatment using broad spectrum antibiotics dramatically reduced total bacterial abundance, but increased the relative presence of Proteobacteria. Antibiotics eliminated viable bacteria in the spleen and liver, but not in the mesentery lymph node in OB. Although antibiotic treatment decreased muscle contractility in sham rats, it had little effect on OB-associated suppression of muscle contractility or inflammatory changes in the muscle layer. In conclusion, obstruction leads to marked dysbiosis in the colon. Antibiotic eradication of microbiota had limited effects on obstruction-associated changes in inflammation, motility, or bacterial translocation.

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

Motivation: The data generation capabilities of high throughput sequencing (HTS) instruments have exponentially increased over the last few years, while the cost of sequencing has dramatically decreased allowing this technology to become widely used in biomedical studies. For small labs and individual researchers, however, storage and transfer of large amounts of HTS data present a significant challenge. The recent trends in increased sequencing quality and genome coverage can be used to reconsider HTS data storage strategies. Results: We present Broom, a stand‐alone application designed to select and store only high‐quality sequencing reads at extremely high compression rates. Written in C++, the application accepts single and paired‐end reads in FASTQ and FASTA formats and decompresses data in FASTA format. Availability and implementation: C++ code available at https://scsb.utmb.edu/labgroups/fofanov/broom.asp. Supplementary information: Supplementary data are available at Bioinformatics online.