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

It was demonstrated previously that abrupt transfer of vigorously aerated cultures of Mycobacterium tuberculosis to anaerobic conditions resulted in their rapid death, but gradual depletion of available O2 permitted expression of increased tolerance to anaerobiosis. Those studies used a model based on adaptation of unagitated bacilli as they settled through a self-generated O2 gradient, but the model did not permit examination of homogeneous populations of bacilli during discrete stages in that adaptation. The present report describes a model based on culture of tubercle bacilli in deep liquid medium with very gentle stirring that keeps them in uniform dispersion while controlling the rate at which O2 is depleted. In this model, at least two stages of nonreplicating persistence were seen. The shift into first stage, designated NRP stage 1, occurred abruptly at a point when the declining dissolved O2 level approached 1% saturation. This microaerophilic stage was characterized by a slow rate of increase in turbidity without a corresponding increase in numbers of CFU or synthesis of DNA. However, a high rate of production of glycine dehydrogenase was initiated and sustained while the bacilli were in this state, and a steady ATP concentration was maintained. When the dissolved O2 content of the culture dropped below about 0.06% saturation, the bacilli shifted down abruptly to an anaerobic stage, designated NRP stage 2, in which no further increase in turbidity was seen and the concentration of glycine dehydrogenase declined markedly. The ability of bacilli in NRP stage 2 to survive anaerobically was dependent in part on having spent sufficient transit time in NRP stage 1. The effects of four antimicrobial agents on the bacilli depended on which of the different physiologic stages the bacilli occupied at a given time and reflected the recognized modes of action of these agents. It is suggested that the ability to shift down into one or both of the two nonreplicating stages, corresponding to microaerophilic and anaerobic persistence, is responsible for the ability of tubercle bacilli to lie dormant in the host for long periods of time, with the capacity to revive and activate disease at a later time. The model described here holds promise as a tool to help clarify events at the molecular level that permit the bacilli to persist under adverse conditions and to resume growth when conditions become favorable. The culture model presented here is also useful for screening drugs for the ability to kill tubercle bacilli in their different stages of nonreplicating persistence.

https://iai.asm.org/content/iai/64/6/2062.full.pdf

An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence.

There is ample clinical evidence, as well as evidence from animal experiments, that Mycobacterium tuberculosis can persist in tissues for months to decades without replicating, yet with the ability to resume growth and activate disease. Our knowledge of both macrophage physiology and the nature of tuberculous lesions in man and animals suggests that hypoxia is a major factor in inducing nonreplicating persistence (NRP) of tubercle bacilli. In vitro models reinforce this conclusion and provide insights into mechanisms that make NRP possible. There is evidence from in vitro models that the strategies employed by the bacilli to permit hypoxic NRP include restriction of biosynthetic activity to conserve energy, induction of alternative energy pathways, and stabilization of essential cell components to lessen the need for repair or replacement.

Nonreplicating persistence of mycobacterium tuberculosis.

Very abrupt exposure to anaerobic conditions has a lethal effect on actively growing cultures of Mycobacterium tuberculosis. However, incubation under conditions in which oxygen is depleted gradually causes M. tuberculosis to shift down from active replication to dormancy. The dormant bacilli are resistant to the bactericidal effects of anaerobiosis and also exhibit partial or complete resistance to the bactericidal effects of isoniazid and rifampin. On the other hand, metronidazole, a drug specific for anaerobes, kills dormant tubercle bacilli under anaerobic conditions, but it has no effect on actively growing aerobic cultures. The lethal effect of metronidazole under anaerobic conditions is enhanced by rifampin. The possible implications of these findings on the phenomenon of latency in tuberculosis are discussed.

Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis.

A specific assay for free pyrazinoic acid has been applied to a study of pyrazinamidase activity of mycobacteria. Evidence is presented for differences in rates of appearance of enzymatic activity, which helps to explain variability in test results often seen in mycobacterial identification. A simple technique is described for pyrazinamidase testing that can be applied to identification of mycobacteria in a routine diagnostic laboratory. An evaluation of a simple urease test is also presented.

Lawrence G. Wayne

Papers

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

An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence.

Nonreplicating persistence of mycobacterium tuberculosis.

Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis.

Simple pyrazinamidase and urease tests for routine identification of mycobacteria.