Faculty Bibliography

AIM: To identify the bacterial flora in conditions such as Barrettos 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 Barrettos esophagus]. Two separate broad-range PCR reactions were performed for each patient, and the resulting products were cloned. In one patient with Barrettos 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 Barrettos esophagus patients, and 10 in normal esophagus patients. Further analysis concentrating on a single biopsy from an individual with Barrettos 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

Bacillus anthracis is a Gram-positive, spore-forming rod that causes anthrax. Culture-based methods are the gold standard for the identification of virulent B. anthracis strains but these require days for completion. The experience from the anthrax attacks in September and October of 2001 revealed the urgent need for methods that can rapidly detect this pathogen with high reliability. Because of the extensive homology among non-anthrax Bacillus sp. at the chromosomal level, rapid detection of virulent B. anthracis strains depends oil markers associated with the two plasmids, pXO1 and pXO2, responsible for its virulence. Genes encoding toxins and capsules have been used as markers for pXO1 and pXO2, respectively, in methods that are designed for rapid and sensitive detection of B. anthracis DNA, Such as real-time polymerase chain reaction, direct liquid phase hybridization, and DNA microarrays. A variety of platforms can be modified to suit the needs for rapid detection of B. anthracis antigens, but little is known about plasmid-encoded antigens expressed in spores. Future studies should be aimed at detecting markers for pXO1 and pXO2in viable spores. (c) 2005 Lippincott Williams & Wilkins

Nucleic acid-based tests are rapidly expanding in the field of diagnostic microbiology, due to their unique high sensitivity and specificity as well as rapid assay turnaround time. However, the potential of false positives and false negatives can hinder the wide application of these novel techniques. This mini-review article summarizes common causes and potential solutions for false-positives and false-negatives encountered in the field of diagnostic molecular microbiology

The esophagus, like other luminal organs of the digestive system, provides a potential environment for bacterial colonization, but little is known about the presence of a bacterial biota or its nature. By using broad-range 16S rDNA PCR, biopsies were examined from the normal esophagus of four human adults. The 900 PCR products cloned represented 833 unique sequences belonging to 41 genera, or 95 species-level operational taxonomic units (SLOTU); 59 SLOTU were homologous with culture-defined bacterial species, 34 with 16S rDNA clones, and two were not homologous with any known bacterial 16S rDNA. Members of six phyla, Firmicutes, Bacteroides, Actinobacteria, Proteobacteria, Fusobacteria, and TM7, were represented. A large majority of clones belong to 13 of the 41 genera (783/900, 87%), or 14 SLOTU (574/900, 64%) that were shared by all four persons. Streptococcus (39%), Prevotella (17%), and Veilonella (14%) were most prevalent. The present study identified approximately 56-79% of SLOTU in this bacterial ecosystem. Most SLOTU of esophageal biota are similar or identical to residents of the upstream oral biota, but the major distinction is that a large majority (82%) of the esophageal bacteria are known and cultivable. These findings provide evidence for a complex but conserved bacterial population in the normal distal esophagus

Pneumocystis produces respiratory infection in immunocompromised individuals of several species of mammals, including humans. Each mammalian species has its own specific Pneumocystis species, which does not cross-infect other mammals. The species infecting humans has now been renamed P. jerovici, since P. carinii is reserved for one of two species infecting rats. Long believed to be a protozoan, Pneumocystis is now classified as an Archiascomycetous fungus. This is based on new molecular taxonomic techniques using DNA sequence analysis of srRNA genes. Only two of about 140 copies of the gene that exist in Pneumocystis were used for sequencing, so the evidence is not conclusive; however, it is supported by morphological evidence such as fungus-specific nucleus-associated organelles for cell division. There is also ultrastructural evidence of meiotic division and sexual conjugation. Clinically, several lines of evidence suggest the improbability of latent infection. Adult infections appear to be new infections, a fact that invites a new perspective on prevention

We performed polymerase chain reaction analysis, for Mycobacterium species 16S rRNA, rpoB, and IS6110 sequences, on 25 tissue specimens from patients with sarcoidosis and on 25 control tissue specimens consisting of mediastinal or cervical lymph nodes and lung biopsies. Mycobacterium species 16S rRNA sequences were amplified from 12 (48%) rpoB sequences and from 6 (24%) of the sarcoidosis specimens. In total, 16S rRNA or rpoB sequences were amplified from 15 sarcoidosis specimens (60%) but were not detected in any of the control tissues (p=0.00002, chi square). In three specimens, the sequences resembled Mycobacterium species other than M. tuberculosis. All specimens with sequences consistent with M. tuberculosis were negative for IS6110. We provide evidence that one of a variety of Mycobacterium species, especially organisms resembling M. tuberculosis, is found in most patients with sarcoidosis

Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays in C. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model

The 29-kDa protein PEB4, a major antigen of Campylobacter jejuni, is present in all C. jejuni strains tested and elicits an antibody response in infected patients. By screening a lambda gt11 library of chromosomal DNA fragments of C. jejuni strain 81-176 in Escherichia coli Y1090 cells with antibody raised against purified PEB4, a recombinant phage with a 2-kb insert expressing an immunoreactive protein of 29 kDa was isolated. DNA sequence analysis revealed that the insert contains two complete open reading frames ORF-A and ORF-B. ORF-A (peb4A) encodes a 273-residue protein with a calculated molecular mass of 30,460 daltons. The deduced amino acid sequence, composition and pl of the recombinant mature protein are similar to those determined for purified PEB4. The first 21 residues resemble a signal peptide. Gene bank searches indicated 33.7% identity with protein export protein PrsA of Bacillus subtilis and 23.8% identity with protease maturation protein precursor PrtM of Lactococcus lactis. PCR experiments indicate that peb4A is highly conserved among C. jejuni strains. ORF-B begins 2 bp after the last codon of peb4A and encodes a putative protein of 353 residues with 63.4% identity with E. coli fructose 1,6-biphosphate aldolase. The sequence arrangement suggests that these two genes form an operon