, and the sequence data generally yield a consensus sequence. Here we describe valuable data that are missing from consensus sequences, variable effects on sequence data generated from nonidentical 16S rRNA amplicons, and the appearance of data displayed by different software programs. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of the Bacillus cereus group, i.e., Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis. These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP) previously reported to be specific to B. anthracis was detected in some B. cereus strains. However, a different SNP, at position 1139, was identified as being specific to B. anthracis, which is a biothreat agent with high mortality rates. Compared with visual analysis of the electropherograms, basecaller software frequently missed gene sequence variations or could not identify variant bases due to overlapping basecalls. Accurate detection of 16S rRNA gene sequences that include intragenomic variations can improve discrimination among closely related species, improve the utility of 16S rRNA databases, and facilitate rapid bacterial identification by targeted DNA sequence analysis or by whole-genome sequencing performed by clinical or reference laboratories. In 1977, Woese and his colleagues introduced the 16S rRNA gene sequence for phylogenetic studies and, based on that sequence, proposed a Tree of Life composed of three domains of living organisms, i.e., Archaea, Bacteria, and Eukarya (1, 2). The domain of bacteria is by far the largest and continues to expand as diverse environments are analyzed (3). Bacterial 16S rRNA genes are located within the rRNA operons, which also contain genes for 23S rRNA, 5S rRNA, tRNA, and associated intergenic spacer regions. Since rRNAs are essential for survival, these operons are expected to be found on the chromosome. However, a recent report by Anda et al. (4) described a clade within the genus Aureimonas for which the sole rRNA operon is located on a small plasmid, which suggests that there is still more to be learned about rRNA in bacterial species. Although the DNA sequences of various rRNA genes and intergenic spacer regions have been used for identification to the genus or species level, the 16S rRNA gene is usually preferred. In addition to being universally distributed among bacteria, this gene contains both highly conserved and hypervariable regions, and there are large and constantly expanding databases of 16S rRNA gene sequences for comparison.Widespread use of DNA sequencing technologies in clinical, public health, and research laboratories has resulted in rapid and accurate molecular diagnostic methods. A bacterial isolate can now be identified more rapidly by 16S rRNA sequence analysis than by conventional methods. In addition to novel or unculturable bacteria, gene sequence analysis has been employed for identification of bacteria with unusual...

Background A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. Methods Bacterial genomic DNA was isolated and digested with a rare‐cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. Results Five different strains of gram‐negative Escherichia coli and six different strains of gram‐positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were ∼100 times faster and ∼200,000 times more sensitive than pulsed‐field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. Conclusions DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)‐based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains. Cytometry 41:203–208, 2000. Published 2000 Wiley‐Liss, Inc.

Burkholderia pseudomallei strain Bp1651, a human isolate, is resistant to all clinically relevant antibiotics. We report here on the finished genome sequence assembly and annotation of the two chromosomes of this strain. This genome sequence may assist in understanding the mechanisms of antimicrobial resistance for this pathogenic species.