Faculty Bibliography

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 typical polymerase 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.

Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 +/- 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% +/- 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2 degrees structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in "Candidatus Protochlamydia amoebophila." Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.

We report the first case of adult meningitis confirmed to be due to Streptococcus gallolyticus subsp. pasteurianus. Phenotypically reported as Streptococcus bovis biotype II/2, 16S rRNA sequencing revealed S. gallolyticus subsp. pasteurianus. Because of taxonomic uncertainties, S. gallolyticus subsp. pasteurianus may be an underrecognized agent of systemic infections

Objective: To report a rare case of an adenomatoid tumor (AT) of the adrenal gland. Methods: We present a case report, including clinical, radiologic, and pathologic findings, and review the relevant literature. Results: We describe a 60-year-old man in whom an AT of the adrenal gland was discovered incidentally, and review the literature. Fewer than 20 cases of these tumors are reported. The tumors range in size from 0.5 to 11 cm, are more common in men, and are more frequently located in the left adrenal gland. A specific imaging phenotype has not been characterized. Histologic features, including infiltration into the surrounding tissue, and presence of signet ring cells, may raise concern of a more aggressive neoplasm. Immunologic staining for mesothelial and endothelial markers may be necessary to establish a definitive diagnosis. Conclusion: ATs of the adrenal gland are rare benign lesions. They are difficult to differentiate from more common adrenal tumors by computerized tamography (CT) or magnetic resonance imaging (MRI). Pathologic findings also can present challenges in distinguishing these lesions from more aggressive neoplasms. ATs should be considered in the differential diagnosis of adrenal masses, whether discovered as incidental radiologic, surgical, or pathologic findings, or at autopsy, so that the potential consequences of misdiagnosis may be avoided

BACKGROUND & AIMS: Gastroesophageal reflux causes inflammation and intestinal metaplasia and its downstream sequelum adenocarcinoma in the distal esophagus. The incidence of esophageal adenocarcinoma has increased approximately 6-fold in the United States since the 1970s, accompanied with a significant increase in the prevalence of gastroesophageal reflux disease (GERD). Despite extensive epidemiologic study, the cause for GERD and the unexpected increases remain unexplainable. Microbes are among the environmental factors that may contribute to the etiology of GERD, but very little research has been done on the esophageal microbiome, particularly in its relation to GERD. This is the first comprehensive reported correlation between a change in the esophageal microbiome and esophageal diseases. METHODS: Biopsy samples of the distal esophagus were collected from 34 patients. Host phenotypes were histologically defined as normal, esophagitis, or Barrett's esophagus (intestinal metaplasia). Microbiomes from the biopsy samples were analyzed by bacterial 16S ribosomal RNA gene survey and classified into types using unsupervised cluster analysis and phenotype-guided analyses. Independence between host phenotypes and microbiome types were analyzed by Fisher exact test. RESULTS: Esophageal microbiomes can be classified into 2 types. The type I microbiome was dominated by the genus Streptococcus and concentrated in the phenotypically normal esophagus. Conversely, the type II microbiome contained a greater proportion of gram-negative anaerobes/microaerophiles and primarily correlated with esophagitis (odds ratio, 15.4) and Barrett's esophagus (odds ratio, 16.5). CONCLUSIONS: In the human distal esophagus, inflammation and intestinal metaplasia are associated with global alteration of the microbiome. These findings raise the issue of a possible role for dysbiosis in the pathogenesis of reflux-related disorders

The preponderance of pancreatic tumors is adenocarcinoma of the ductal type; carcinomas with multiple lineage differentiation are extremely rare. We report an unusual case of pancreatic carcinoma with combined acinar and neuroendocrine differentiation and minor ductal component with concurrent acinar-ductal metaplasia (ADM), an early lesion implicated in ductal carcinogenesis. The patient is a 56-year-old man with vague complaints of dull left upper quadrant pain with radiation across the mid-portion of his abdomen. A computer tomography scan revealed an irregular enlargement of the distal 3.2 cm of the pancreatic body. A distal pancreatectomy was then performed. Histologic examination revealed a pancreatic carcinoma with cellular features of eosinophilic granular cytoplasm and salt-pepper nuclei. The acinar differentiation of the carcinoma was confirmed by positivity on periodic acid-Schiff stain resistant to diastase digestion (dPAS), positivity for antitrypsin on immunohistochemistry (IHC), and presence of zymogen granules on electron microscopy (EM). The neuroendocrine differentiation was evident by positive synaptophysin and chromogranin stain on IHC and neuroendocrine granules on EM. The ductal component was only visible by PAS stain and immunostains for CEA and CK19A and accompanied by a number of the acinar-ductal metaplasia lesions adjacent to the main tumor. Thus, the histological, histochemical, immunohistochemical and electron-microscopic evidence all suggested that the pancreatic carcinoma underwent trilineage differentiation

BACKGROUND: The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons. METHODOLOGY/PRINCIPAL FINDINGS: Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4%) genomes (mean 0.40%, range 0.01%-4.04%). Significant (1.17%-4.04%) intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition). In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS), ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes. CONCLUSIONS/SIGNIFICANCE: These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy

The hoatzin is unique among known avian species because of the fermentative function of its enlarged crop. A small-bodied flying foregut fermenter is a paradox, and this bird provides an interesting model to examine how diet selection and the gut microbiota contribute to maximizing digestive efficiency. Therefore, we characterized the bacterial population in the crop of six adult hoatzins captured from the wild. A total of 1,235 16S rRNA gene sequences were grouped into 580 phylotypes (67% of the pooled species richness sampled, based on Good's coverage estimator, with C(ACE) and Chao1 estimates of 1,709 and 1,795 species-level [99% identity] operational taxonomic units, respectively). Members of 9 of the approximately 75 known phyla in Bacteria were identified in this gut habitat; the Firmicutes were dominant (67% of sequences, belonging to the classes Clostridia, Mollicutes, and Bacilli), followed by the Bacteroidetes (30%, mostly in the order Bacteroidales), Proteobacteria (1.8%), and Lentisphaerae, Verrucomicrobia, TM7, Spirochaetes, Actinobacteria, and Aminanaerobia (all <0.1%). The novelty in this ecosystem is great; 94% of the phylotypes were unclassified at the 'species' level and thus likely include novel cellulolytic lineages

For psoriasis, an idiopathic inflammatory disorder of the skin, the microbial biota has not been defined using cultivation-independent methods. We used broad-range 16S rDNA PCR for archaea and bacteria to examine the microbiota of normal and psoriatic skin. From 6 patients, 19 cutaneous samples (13 from diseased skin and 6 from normal skin) were obtained. Extracted DNA was subjected to the broad range PCR, and 1,925 cloned products were compared with 2,038 products previously reported from healthy persons. Using 98% sequence identity as a species boundary, 1,841 (95.6%) clones were similar to known bacterial 16S rDNA, representing 6 phyla, 86 genera, or 189 species-level operational taxonomic unit (SLOTU); 84 (4.4%) clones with <98% identity probably represented novel species. The most abundant and diverse phylum populating the psoriatic lesions was Firmicutes (46.2%), significantly (P<0.001) overrepresented, compared to the samples from uninvolved skin of the patients (39.0%) and healthy persons (24.4%). In contrast, Actinobacteria, the most prevalent and diverse phylum in normal skin samples from both healthy persons (47.6%) and the patients (47.8%), was significantly (P<0.01) underrepresented in the psoriatic lesion samples (37.3%). Representation of Propionibacterium species were lower in the psoriatic lesions (2.9+/-5.5%) than from normal persons (21.1+/-18.2%; P<0.001), whereas normal skin from the psoriatic patients showed intermediate levels (12.3+/-21.6%). We conclude that psoriasis is associated with substantial alteration in the composition and representation of the cutaneous bacterial biota

CONTEXT: Leptin and ghrelin, hormones involved in human energy homeostasis, are both produced in the stomach. OBJECTIVE: We sought to determine whether the presence of Helicobacter pylori affects gastric and systemic levels of leptin and ghrelin. DESIGN, SETTING, AND PATIENTS: We consecutively enrolled 256 patients referred for upper endoscopy at a Veterans Affairs outpatient endoscopy center. OUTCOMES: We obtained fasting serum, fundic and antral biopsies, and gastric juice. Based on histological, biochemical, and serological assays, patients were categorized as H. pylori+ or H. pylori-. Leptin and total ghrelin levels in serum, gastric biopsies, and gastric juice were determined by specific ELISAs. RESULTS: Of the 256 subjects, 120 were H. pylori+ and 96 were H. pylori-; 40 patients of indeterminate status were excluded. Serum and fundic leptin levels correlated with body mass index in the H. pylori+ (r = 0.35; P < 0.0001 and r = 0.35; P < 0.0001, respectively) and H. pylori- (r = 0.65; P < 0.0001 and r = 0.41; P < 0.0001, respectively) groups, but H. pylori+ subjects had significantly lower serum leptin levels [median 2.2 ng/ml (interquartile range 0.9-4.6) vs. 4.0 ng/ml (1.7-7.2); P = 0.0003]. Serum ghrelin levels were similar in the H. pylori+ and H. pylori- groups [median 1651 pg/ml (interquartile range 845-2247) vs. 1629 pg/ml (992-2886); P = 0.23]. H. pylori status did not significantly affect gastric biopsy leptin and ghrelin levels. Ghrelin levels in gastric juice varied over 4 log(10) (<80-776,000 pg/ml) and correlated with gastric juice pH in the H. pylori+ group (r = 0.68; P < 0.0001). CONCLUSIONS: These findings provide evidence that H. pylori status affects leptin and ghrelin homeostasis, presumably via intragastric interactions.