AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using ...AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.展开更多
AIM: To identify the bacterial flora in conditions such as Barrett's esophagus and reflux esophagitis to determine if they are similar to normal esophageal flora.METHODS: Using broad-range 16S rDNA PCR,esophageal ...AIM: To identify the bacterial flora in conditions such as Barrett's esophagus and reflux esophagitis to determine if they are similar to normal esophageal flora.METHODS: Using broad-range 16S rDNA PCR,esophageal biopsies were examined from 24 patients [9with normal esophageal mucosa, 12 with gastroesophageal reflux disease (GERD), and 3 with Barrett's esophagus].Two separate broad-range PCR reactions were performed for each patient, and the resulting products were cloned.In one patient with Barrett's esophagus, g9 PCR clones were analyzed.RESULTS: Two separate clones were recovered from each patient (total = 48), representing 24 different species, with 14 species homologous to known bacteria,5 homologous to unidentified bacteria, and 5 were not homologous (<97% identity) to any known bacterial 16S rDNA sequences. Seventeen species were found in the reflux esophagitis patients, 5 in the Barrett's esophagus patients, and 10 in normal esophagus patients.Further analysis concentrating on a single biopsy from an individual with Barrett's esophagus revealed the presence of 21. distinct bacterial species. Members of four phyla were represented, including Bacteroidetes,Firmicutes, Proteobacteria, and Actinobacteria.Microscopic examination of each biopsy demonstrated bacteria in intimate association with the distal esophageal epithelium, suggesting that the presence of these bacteria is not transitory.CONCLUSION: These findings provide evidence for a complex, residential bacterial population in esophageal reflux-related disorders. While much of this biota is present in the normal esophagus, more detailed comparisons may help identify potential disease associations.展开更多
Our relationship with the colonic bacterial flora has long been viewed as benign, but recent studies suggest that this symbiosis has risks as well as benefits. This relationship requires that the host not only provide...Our relationship with the colonic bacterial flora has long been viewed as benign, but recent studies suggest that this symbiosis has risks as well as benefits. This relationship requires that the host not only provide a supportive environment for the symbiotic bacteria, but also actively maintain intact mechanisms for properly managing the physiologic stresses that are closely associated with the symbiont’s essential survival functions. Failure to do so breaches the host- symbiont contract, and can result in serious effects on the health of the host. Recent investigations that employ several knockout mouse models reveal the consequences of genetic deficiency in the host regarding these mechanisms, and the latent, pro-inflammatory, tumorigenic nature of normal bacterial flora. Further study of the interactions between normal bacterial flora and hosts could shed light on the etiologies and pathogenesis of inflammatory diseases and related cancers, with implications for human health.展开更多
基金Supported by(in part)Grants UH2CA140233 from the Human Microbiome Project of the NIH Roadmap Initiative and National Cancer InstituteR01AI063477 from the National Institute of Allergy and Infectious Diseases+1 种基金DE-11443 from the National Institute of Dental and Craniofacial ResearchU19DE018385 from the National Institute of Dental & Craniofacial Research
文摘AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.
基金Supported by R01CA97946, R21DK57941, R01GM63270,R01 DK58587, and R01CA77955, and by the General Clinical Research Center core grant to New York University School of Medicine (NIH/NCRR M01 RR00096) from the National Institutes of Health, by the Medical Research Service of the Department of Veterans Affairs, and by the Ellison Medical Foundation
文摘AIM: To identify the bacterial flora in conditions such as Barrett's esophagus and reflux esophagitis to determine if they are similar to normal esophageal flora.METHODS: Using broad-range 16S rDNA PCR,esophageal biopsies were examined from 24 patients [9with normal esophageal mucosa, 12 with gastroesophageal reflux disease (GERD), and 3 with Barrett's esophagus].Two separate broad-range PCR reactions were performed for each patient, and the resulting products were cloned.In one patient with Barrett's esophagus, g9 PCR clones were analyzed.RESULTS: Two separate clones were recovered from each patient (total = 48), representing 24 different species, with 14 species homologous to known bacteria,5 homologous to unidentified bacteria, and 5 were not homologous (<97% identity) to any known bacterial 16S rDNA sequences. Seventeen species were found in the reflux esophagitis patients, 5 in the Barrett's esophagus patients, and 10 in normal esophagus patients.Further analysis concentrating on a single biopsy from an individual with Barrett's esophagus revealed the presence of 21. distinct bacterial species. Members of four phyla were represented, including Bacteroidetes,Firmicutes, Proteobacteria, and Actinobacteria.Microscopic examination of each biopsy demonstrated bacteria in intimate association with the distal esophageal epithelium, suggesting that the presence of these bacteria is not transitory.CONCLUSION: These findings provide evidence for a complex, residential bacterial population in esophageal reflux-related disorders. While much of this biota is present in the normal esophagus, more detailed comparisons may help identify potential disease associations.
基金Supported by US Public Health Service Grants, R01CA97946 and R01AI063477 and the Medical Research Service of the Department of Veterans Affairs, United States
文摘Our relationship with the colonic bacterial flora has long been viewed as benign, but recent studies suggest that this symbiosis has risks as well as benefits. This relationship requires that the host not only provide a supportive environment for the symbiotic bacteria, but also actively maintain intact mechanisms for properly managing the physiologic stresses that are closely associated with the symbiont’s essential survival functions. Failure to do so breaches the host- symbiont contract, and can result in serious effects on the health of the host. Recent investigations that employ several knockout mouse models reveal the consequences of genetic deficiency in the host regarding these mechanisms, and the latent, pro-inflammatory, tumorigenic nature of normal bacterial flora. Further study of the interactions between normal bacterial flora and hosts could shed light on the etiologies and pathogenesis of inflammatory diseases and related cancers, with implications for human health.