We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

Because a natural entity “species” cannot be recognized as a group of strains that is genetically well separated from its phylogenetic neighbors, a pragmatic approach was taken to define a species by a polyphasic approach (L. G. Wayne, D. J. Brenner, R. R. Colwell, P. A. D. Grimont, O. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Truper, Int. J. Syst. Bacteriol. 37:463-464, 1987), in which a DNA reassociation value of about 70% plays a dominant role. With the establishment of rapid sequence analysis of 16S rRNA and the recognition of its potential to determine the phylogenetic position of any prokaryotic organism, the role of 16S rRNA similarities in the present species definition in bacteriology needs to be clarified. Comparative studies clearly reveal the limitations of the sequence analysis of this conserved gene and gene product in the determination of relationships at the strain level for which DNA-DNA reassociation experiments still constitute the superior method. Since today the primary structure of 16S rRNA is easier to determine than hybridization between DNA strands, the strength of the sequence analysis is to recognize the level at which DNA pairing studies need to be performed, which certainly applies to similarities of 97% and higher.

Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology

The recent advent of DNA sequencing technologies facilitates the use of genome sequencing data that provide means for more informative and precise classification and identification of members of the Bacteria and Archaea. Because the current species definition is based on the comparison of genome sequences between type and other strains in a given species, building a genome database with correct taxonomic information is of paramount need to enhance our efforts in exploring prokaryotic diversity and discovering novel species as well as for routine identifications. Here we introduce an integrated database, called EzBioCloud, that holds the taxonomic hierarchy of the Bacteria and Archaea, which is represented by quality-controlled 16S rRNA gene and genome sequences. Whole-genome assemblies in the NCBI Assembly Database were screened for low quality and subjected to a composite identification bioinformatics pipeline that employs gene-based searches followed by the calculation of average nucleotide identity. As a result, the database is made of 61 700 species/phylotypes, including 13 132 with validly published names, and 62 362 whole-genome assemblies that were identified taxonomically at the genus, species and subspecies levels. Genomic properties, such as genome size and DNA G+C content, and the occurrence in human microbiome data were calculated for each genus or higher taxa. This united database of taxonomy, 16S rRNA gene and genome sequences, with accompanying bioinformatics tools, should accelerate genome-based classification and identification of members of the Bacteria and Archaea. The database and related search tools are available at www.ezbiocloud.net/.

Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.

Diagnostic Criteria of Nontuberculous Mycobacterial Lung Disease Key Laboratory Features of NTM Health Careand Hygiene-associated Disease Prevention Prophylaxis and Treatment of NTM Disease Introduction Methods Taxonomy Epidemiology Pathogenesis Host Defense and Immune Defects Pulmonary Disease Body Morphotype Tumor Necrosis Factor Inhibition Laboratory Procedures Collection, Digestion, Decontamination, and Staining of Specimens Respiratory Specimens Body Fluids, Abscesses, and Tissues Blood Specimen Processing Smear Microscopy Culture Techniques Incubation of NTM Cultures NTM Identification Antimicrobial Susceptibility Testing for NTM Molecular Typing Methods of NTM Clinical Presentations and Diagnostic Criteria Pulmonary Disease Cystic Fibrosis Hypersensitivity-like Disease Transplant Recipients Disseminated Disease Lymphatic Disease Skin, Soft Tissue, and Bone Disease

/pdf/an-official-ats-idsa-statement-diagnosis-treatment-and-1jy41yfwpp.pdf

An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.

Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics

A review of reports on the genetic and phenotypic characteristics of strains of the spirochete which causes Lyme disease revealed that these organisms are representative of a new species of Borrelia. We propose the name Borrelia burgdorferi for this species. The type strain of B. burgdorferi is strain B31 (= ATCC 35210). In two separate studies the guanine-plus-cytosine content of the deoxyribonucleic acid of the type strain was determined to be 29.0 to 30.5 mol% (thermal denaturation method).

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Borrelia burgdorferi sp. nov.: Etiologic Agent of Lyme Disease

The name Escherichia vulneris sp. nov. (formerly called Alma group 1 and Enteric group 1 by the Centers for Disease Control and API group 2 by Analytab Products, Inc.) is proposed for a group of isolates from the United States and Canada, 74% of which were from human wounds. E. vulneris is a gram-negative, oxidase-negative, fermentative, motile rod with the characteristics of the family Enterobacteriaceae. Biochemical reactions characteristic of 61 E. vulneris strains were positive tests for methyl red, malonate, and lysine decarboxylase; a delayed positive test for arginine dihydrolase; acid production from d-mannitol, l-arabinose, raffinose, l-rhamnose, d-xylose, trehalose, cellobiose, and melibiose; negative tests for Voges-Proskauer, indole, urea, H(2)S, citrate, ornithine decarboxylase, phenylalanine deaminase, and DNase; and no acid from dulcitol, adonitol, myo-inositol, and d-sorbitol. Two-thirds of the strains produced yellow pigment. Most strains gave negative or delayed positive reactions in tests for lactose, sucrose, and KCN. The E. vulneris strains tested were resistant to penicillin and clindamycin, were resistant or showed intermediate zones of inhibition to carbenicillin and erythromycin, and were susceptible to 14 other antibiotics. DNA relatedness of 15 E. vulneris strains to the type strain averaged 75% in reactions at 60 degrees C and 69% in reactions at 75 degrees C, indicating that they comprise a separate species. DNA relatedness to other species in the family Enterobacteriaceae was 6 to 39%, an indication that this new species belongs in the family. E. vulneris showed the highest relatedness to species of Escherichia (25 to 39%) and Enterobacter (24 to 35%). On the basis of biochemical similarity, the new species was placed in the genus Escherichia. The type strain of E. vulneris is ATCC 33821 (CDC 875-72).

Escherichia vulneris: a New Species of Enterobacteriaceae Associated with Human Wounds

Twenty-five phenotypically and genotypically similar strains of a fastidious, xylem-limited bacterium were isolated from 10 plant disease sources including Pierce's disease of grapevines, phony disease of peach, periwinkle wilt, and leaf scorches of almond, plum, elm, sycamore, oak, and mulberry. The cells were single (occasionally filamentous), nonmotile, aflagellate straight rods (0.25 to 0.35 by 0.9 to 3.5 μm). They were gram negative, catalase positive, and oxidase negative, utilized hippurate, and produced gelatinase and often beta-lactamase but not beta-galactosidase, coagulase, lipase, amylase, phosphatase, indole, or H2S. The bacteria were strict aerobes with optimum growth at 26 to 28°C and pH 6.5 to 6.9 and had doubling times of 0.45 to 1.98 days in periwinkle wilt broth. Monoclonal antibodies prepared against the Pierce's disease bacterium reacted with all strains. DNA composition was 51 to 53 mol% guanine plus cytosine, and strains were at least 85% related in DNA hybridization. Sequencing of 16S ribosomal ribonucleic acid related them to the xanthomonads. These bacteria form a distinct group, and the name Xylella fastidiosa is proposed, establishing a new genus with one species in the gamma subgroup of the eubacteria. Strain PCE-RR (ATCC 35879) is designated as the type strain.

Xylella fastidiosa gen. nov., sp. nov.: Gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp.

Enterobacter sakazakii is the name proposed for the organism previously known as “yellow-pigmented Enterobacter cloacae.” The type strain (holotype) of this species is ATCC 29544. The proposed change in the classification of this organism is based on differences between E. cloacae and E. sakazakii in deoxyribonucleic acid (DNA)-DNA hybridization, biochemical reactions, pigment production, and antibiotic susceptibility. By DNA hybridization, E. sakazakii was about 50% related to E. cloacae, Citrobacter diversus (“Citrobacter intermedius” biotype b), and “Citrobacter amalonaticus” (“Citrobacter intermedius” biotype a). The new species was placed in Enterobacter rather than Citrobacter because of its closer phenotypic and DNA similarity to E. cloacae, the type species of the genus Enterobacter, and because it was only 41% related by DNA hybridization to Citrobacter freundii, the type species of Citrobacter. E. sakazakii had biochemical reactions very similar to those of E. cloacae but was d-sorbitol negative and positive for extracellular deoxyribonuclease at 2 to 7 days and produced yellow-pigmented colonies. E. sakazakii had larger zones of inhibition around ampicillin and cephalothin antibiotic disks, which also helps to differentiate it from E. cloacae. E. sakazakii grew on the nonselective (but differential) plating media commonly used in enteric bacteriology, but its plating efficiency was reduced on more inhibitory enteric plating media. It has been isolated from human clinical specimens such as sputum, feces, and wounds, where it is probably only a colonizer and not clinically significant. However, it is also a documented, although rare, cause of neonatal meningitis. Other sources have included food, a physician's stethoscope, and an uninoculated blood culture bottle.

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Don J. Brenner

Papers

Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics

Borrelia burgdorferi sp. nov.: Etiologic Agent of Lyme Disease

Escherichia vulneris: a New Species of Enterobacteriaceae Associated with Human Wounds

Xylella fastidiosa gen. nov., sp. nov.: Gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp.

Enterobacter sakazakii: A New Species of “Enterobacteriaceae” Isolated from Clinical Specimens