High inter-species and low intra-species variation in 16S-23S rDNA spacer sequences of pathogenic avian mycoplasmas offers potential use as a diagnostic tool.

In order to investigate its value for phylogenetic analysis, species characterisation and diagnosis, the 16S-23S rDNA intergenic spacer regions (ISRs) of the type strain of 23 avian Mycoplasma species were amplified and the sequences determined. Also sequenced were the reference strains of Mycoplasma iowae serotypes J, K, N, Q and R and a number of field strains of Mycoplasma synoviae, Mycoplasma gallisepticum, Mycoplasma meleagridis and M. iowae. The ISRs demonstrated a high level of size variation (178-2488bp) between species and did not include tRNA genes. Phylogenetic analysis performed using the information conflicted with that based on the 16S rDNA and was therefore not helpful for phylogenetic studies. However, the ISR did appear to be of value for determining species since there was high inter-species variation between all 23 avian Mycoplasma species, and in addition there was low intra-species variation, at least in the four pathogenic species. It could also be very useful as additional information in the description of a new species and as a target for species-specific PCRs.

Mycoplasma host specificity: fact or fiction?

Bacteria of the genus Mycoplasma are the smallest organisms known to be capable of self-replication. They only occur in association with animal host cells on which they are dependant for many pre-formed nutrients since they lack many of the metabolic pathways associated with energy production and the synthesis of cell components found in other species of bacteria. It is generally thought that most species of Mycoplasma are very host specific but there are many reports of mycoplasmas in hosts that are not perceived as their normal habitat. Sometimes these "crossings" may have a pathological impact particularly where there may be predisposing conditions such as immunodeficiency. These are often reported in humans but may also occur in animals whose immune or physiological status is not known. This review brings together some of these reported incidents and speculates on their potential impact for laboratory diagnosis.

Mycoplasma amphoriforme sp. nov., isolated from a patient with chronic bronchopneumonia.

A mycoplasma was isolated from the sputum of an immunodeficient patient with recurrent bronchitis. The isolate designated strain A39T was very fastidious and atypical for a mycoplasma in its colonial appearance. Classical biochemical tests for mycoplasma speciation could not differentiate the isolate from the pathogens Mycoplasma pneumoniae and Mycoplasma genitalium and serological identification as a recognized Mycoplasma species was lacking. Specific PCR detection for these two species was negative. Subsequently, other strains were isolated from human patients that appeared to be similar to strain A39T in their physiological and genetic characteristics. Analysis of the 16S rRNA gene placed strain A39T and other isolates in the pneumoniae group of mycoplasmas, with the highest sequence similarity to Mycoplasma testudinis (96.8 %), but with only 93.0 % similarity to M. pneumoniae and M. genitalium. Examination of the 16S-23S rRNA internally transcribed spacer sequence, protein electrophoresis profile, genome size and serological reactions indicated that this organism represents a novel species, for which the name Mycoplasma amphoriforme sp. nov. is proposed, with strain A39T (=NCTC 11740T=ATCC BAA-992T) as the type strain.

'Candidatus Odyssella thessalonicensis' gen. nov., sp. nov., an obligate intracellular parasite of Acanthamoeba species.

An intracellular bacterium, strain L13, was observed infecting an environmental isolate of an Acanthamoeba species. The bacterium could not be recovered on axenic medium but was recovered and cultivated in vitro using cultures of Acanthamoeba polyphaga. The 16S rRNA gene sequence of L13 was found to be new, sharing less than 84% similarity with other sequences in the GenBank/EMBL database. L13 was found to be a member of the alpha-Proteobacteria, sharing an evolutionary line of descent with a group of uniquely obligate intracellular organisms comprised of Caedibacter and Holospora species and the NHP bacterium. Viable bacteria appeared to be highly motile within amoebae. Ultrastructural analysis of the bacterium demonstrated that it is rod-shaped and possesses a typical Gram-negative cell wall, but has no other outstanding features except small vesicle-like structures often associated with the outer surface of each bacterium. The host range of L13 was found to be limited to the genus Acanthamoeba. In A. polyphaga, L13 infection was slow to manifest when cultures were incubated below 30 degrees C, but at higher temperatures bacteria multiplied prolifically and induced host cell lysis. The protein profile of the bacterium purified from the amoebae was assessed by SDS-PAGE and its G+C content was estimated to be 41 mol%. Although these results support the proposal of L13 as a new species, its obligate intracellular nature prevented isolation of a definitive type strain. L13 is therefore proposed as 'Candidatus Odyssella thessalonicensis' gen. nov., sp. nov.

Characterization of aerobic non-lipophilic coryneforms from human feet.

Aerobic coryneform bacteria from human feet have been studied by analysis of the cell-wall sugars, lipids and diamino acids and by phenotypic tests. Although many isolates studied fall into the established genus Brevibacterium at least two previously unreported taxa of coryneform have been identified bringing the number of aerobic, non-lipophilic taxa known on skin to four. Simple tests can be used to distinguish between these taxa. Studies on the human skin flora continue to reveal the diversity of organisms present.

Phylogenetic analysis of some LL-diaminopimelic acid-containing coryneform bacteria from human skin: description of Propionibacterium innocuum sp. nov.

A new species, Propionibacterium innocuum, is proposed to accommodate strains of coryneform bacteria from human skin with phenotypic characters similar to those of the classical propionibacteria but differing in exhibiting primarily aerobic respiration and possessing a unique cell wall composition in which LL-diaminopimelic acid and arabinose occur together. The partial 16S rRNA sequence confirms an affinity with the genus Pro-pionibacterium and indicates that the species represents a distinct line within the genus. The type strain of Propionibacterium innocuum is NCTC 11082.

A numerical analysis of ribosomal RNA gene patterns for typing clinical isolates of Corynebacterium group D2.

Restriction digest fragments of DNA from 46 clinical isolates identified as Corynebacterium group D2, were separated by electrophoresis, Southern blotted onto nylon membranes and hybridized to a ribosomal RNA gene probe. The resulting band patterns were subjected to unweighed pair-group cluster analysis. Representative strains from the main clusters were compared with similarly prepared band patterns from type strains of human Corynebacterium species. The results indicate that strains identified as Corynebacterium group D2 represent a unique taxon and that computer-assisted analysis of rRNA gene restriction fragment polymorphism (ribotyping) could be a useful technique in epidemiological studies of these bacteria.

Identification of Brevibacterium from clinical sources.

Coryneform bacteria of the genus Brevibacterium occur on the normal skin surface, but reports of human infection with this genus are lacking. A number of cultures of coryneform bacteria sent to the National Collection of Type Cultures for identification have been identified as Brevibacterium spp on the basis of their cell wall composition and ability to produce methane-thiol from L-methionine. We describe a rapid method for the detection of methane-thiol and confirmatory tests which differentiate Brevibacterium from morphologically similar genera.

Rapid identification of cell wall components as a guide to the classification of aerobic coryneform bacteria from human skin.

In a survey of over 1000 isolates of aerobic skin coryneforms from a wide variety of sources, chromatographic methods were used to identify the major cell-wall components in whole-cell hydrolysates. Most of the skin isolates-like members of the genus Corynebacterium--possessed meso-diaminopimelic acid and arabinose. However, substantial numbers of coryneforms apparently resident on the skin did not have this pattern; the sites from which they were isolated suggested that some were derived from the environment whilst others (possessing meso-DAP and galactose but not arabinose as the major wall components) were members of the resident skin flora.