Unwrapping the 16sRNA Gene Where Microbes Are Our Passion!

A Look Behind the Curtain of 16s Sequencing

 

 

 

Asaia sp., an unusual spoilage organism of fruit-flavored bottled water.

Moore JE1, McCalmont M, Xu J, Millar BC, Heaney N.

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Abstract

A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>10(6) CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.

PMID: 12147519 PMCID: PMC123994

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Select item 11923384

 

602.

J Clin Microbiol. 2002 Apr;40(4):1511-3.

Molecular evidence of Helicobacter cinaedi organisms in human gastric biopsy specimens.

Peña JA1, McNeil K, Fox JG, Versalovic J.

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Abstract

One hundred twenty-six urease-negative gastric biopsy specimens were evaluated for the presence of Helicobacter genus-specific 16S ribosomal DNA (rDNA) and H. pylori-specific glmM DNA sequences by PCR. The species specificity of the glmM PCR assay was demonstrated, as H. pylori was the only Helicobacter species that yielded the expected glmM amplicon. Most urease-negative specimens (118 of 126 specimens) lacked Helicobacter DNA. However, 8 of 126 urease-negative specimens contained Helicobacter 16S rDNA. In order to identify the Helicobacter species present in urease-negative gastric biopsy specimens, 16S rDNA amplicons were cloned and sequenced. Sequence comparisons were performed by analyses of the sequences in public sequence databases. Two samples contained 16S rDNA that was identified as H. cinaedi with 100% identity and that spanned approximately 400 bp (398 and 398 bp, respectively). In contrast, multiple differences (97% identity; 390 of 398 bp) were observed with H. pylori 16S rDNA in this region. This finding was verified by sequencing an overlapping 537-bp fragment within the 5' portion of 16S rDNA. Although the clinical findings were consistent with H. pylori infection (e.g., duodenal ulcer disease), rapid urease testing and DNA sequence analyses suggested the presence of H. cinaedi organisms and the absence of H. pylori in two human antral biopsy specimens. This study represents the first report of an enteric urease-negative helicobacter in the human stomach. Although these organisms were previously associated with extragastric infections, the roles of these organisms in the pathogenesis of chronic gastritis or peptic ulcer disease remain unclear.

PMID: 11923384 PMCID: PMC140399

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Select item 11923353

 

603.

J Clin Microbiol. 2002 Apr;40(4):1333-8.

rpoB gene sequence-based identification of Staphylococcus species.

Drancourt M1, Raoult D.

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Abstract

The complete sequence of rpoB, the gene encoding the beta subunit of RNA polymerase was determined for Staphylococcus saccharolyticus, Staphylococcus lugdunensis, S taphylococcus caprae, and Staphylococcus intermedius and partial sequences were obtained for an additional 27 Staphylococcus species. The complete rpoB sequences varied in length from 3,452 to 3,845 bp and had a 36.8 to 39.2% GC content. The partial sequences had 71.6 to 93.6% interspecies homology and exhibited a 0.08 to 0.8% intraspecific divergence. With a few exceptions, the phylogenetic relationships inferred from the partial rpoB sequences were in agreement with those previously derived from DNA-DNA hybridization studies and analyses of 16S ribosomal DNA gene sequences and partial HSP60 gene sequences. The staphylococcal rpoB sequence database we established enabled us to develop a molecular method for identifying Staphylococcus isolates by PCR followed by direct sequencing of the 751-bp amplicon. In blind tests, this method correctly identified 10 Staphylococcus isolates, and no positive results were obtained with 10 non-Staphylococcus gram-positive and gram-negative bacterial isolates. We propose partial sequencing of the rpoB gene as a new tool for the accurate identification of Staphylococcus isolates.

PMID: 11923353 PMCID: PMC140360

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Select item 11777011

 

604.

Vet Res. 2001 Nov-Dec;32(6):611-6.

Possible misidentification of Bacteroides sp., probably B. ureolyticus as Taylorella equigenitalis: implications for the laboratory diagnosis of CEM.

Moore JE1, Millar BC, Xu J, Buckley TC.

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Abstract

A wild-type isolate with similar morphological and phenotypic properties to Taylorella equigenitalis, the causative bacterial agent of contagious equine metritis (CEM), was referred for molecular identification by PCR amplification of the 16S rRNA gene. A species-specific PCR failed to yield a product compatible with that of T. equigenitalis. The direct sequencing of the universal 16S rRNA PCR amplicon suggested the presence of a Bacteroides sp., probably Bacteroides ureolyticus, with no consequent effects on the movement and transportation of the animal. Adoption of such a molecular means of identification through sequencing may aid in the identification of the atypical forms of Taylorella equigenitalis, as recently described, as well as differentiating this species from Taylorella asinigenitalis.