Background: The aim of the study was to analyze the performance of PCR-DGGE based assay and its applicability as a tool for the identification of bacteria in the middle ear of children with otitis media with effusion ...Background: The aim of the study was to analyze the performance of PCR-DGGE based assay and its applicability as a tool for the identification of bacteria in the middle ear of children with otitis media with effusion (OME). Methods: The middle ear effusions from 20 children with OME were analyzed both by bacterial culture and by 16S rDNA-gene-targeted PCR assay, DGGE fingerprinting and sequencing analysis. Results: In bacterial culture assay, only three middle ear effusions (15%) showed bacterial growth. None of the samples were positive for anaerobic culture. The PCR assay with 16S rDNA-gene-targeted universal primers was positive in 10 (50%) cases. The subsequent DGGE fingerprinting and 16S rDNA sequencing analysis revealed that the most commonly encountered bacteria in the middle ear effusions of children with OME are Haemophilus influenzae, Alloiococcus otitidis and Bacteroides spp. Conclusions: The present study demonstrated the applicability of PCR-DGGE based assay and 16S rDNA sequencing for analyzing of bacterial diversity in the middle ear effusion of children OME. The results of our study may contribute to a better understanding of the etiology of OME.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 [9 with 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, 99 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
基金supported by SF109870 from Estonian Science Foundation.
文摘Background: The aim of the study was to analyze the performance of PCR-DGGE based assay and its applicability as a tool for the identification of bacteria in the middle ear of children with otitis media with effusion (OME). Methods: The middle ear effusions from 20 children with OME were analyzed both by bacterial culture and by 16S rDNA-gene-targeted PCR assay, DGGE fingerprinting and sequencing analysis. Results: In bacterial culture assay, only three middle ear effusions (15%) showed bacterial growth. None of the samples were positive for anaerobic culture. The PCR assay with 16S rDNA-gene-targeted universal primers was positive in 10 (50%) cases. The subsequent DGGE fingerprinting and 16S rDNA sequencing analysis revealed that the most commonly encountered bacteria in the middle ear effusions of children with OME are Haemophilus influenzae, Alloiococcus otitidis and Bacteroides spp. Conclusions: The present study demonstrated the applicability of PCR-DGGE based assay and 16S rDNA sequencing for analyzing of bacterial diversity in the middle ear effusion of children OME. The results of our study may contribute to a better understanding of the etiology of OME.
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 [9 with 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, 99 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.
