Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri.

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (K(s)) varied for different substrates. Substrate utilization patterns and K(s) values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.

Proposal that the strains of the Mycoplasma ovine/caprine serogroup 11 be reclassified as Mycoplasma bovigenitalium.

This proposal is our response to the recommendation of the International Committee on Systematics of Prokaryotes (Subcommittee on the taxonomy of Mollicutes) that we 'write a proposal to classify Mycoplasma bovigenitalium and ovine/caprine serogroup 11 as a single species'. Physiological and phylogenetic comparisons between 27 strains of M. bovigenitalium and Mycoplasma serogroup 11 showed that (i) growth and patterns of organic acid substrate use completely overlapped among strains; (ii) all had lipase and phosphatase activities; (iii) the strains were indistinguishable in their SDS-PAGE whole-cell protein profiles, which differed from five other species; (iv) strains were indistinguishable in immunoblotting of cell proteins and cross-reactivity in ELISA, but differed from other Mycoplasma species; (v) DNA-DNA hybridization did not distinguish between the two groups, and (vi) comparison of 16S and 23S rRNA gene sequences of ten strains of Mycoplasma serogroup 11 and six strains of M. bovigenitalium showed that they shared 98-100% similarity across all strains tested, but only 86-95% to other Mycoplasma species. Strains of the Mycoplasma ovine/caprine serogroup 11 must therefore be reassigned as Mycoplasma bovigenitalium.

An evaluation of PCR methods to detect strains of Mycoplasma fermentans.

A panel of 30 putative Mycoplasma fermentans strains, isolated from various sources including human, ovine and cell lines, were tested by a previously described polymerase chain reaction (PCR) to confirm their identity by amplification of a conserved 206 bp region of the insertion sequence IS1550. In addition, the application of another PCR based on the major part of the IS1550 element showed one or two products of different length (1144 and 1341 bp) enabling M. fermentans strains to be divided into two types designated as Type A and Type B. A PCR, which amplifies the macrophage activating lipopeptide gene (malp), supported the identification of all the strains as M. fermentans. Thirteen other species of Mycoplasma from human sources gave negative results in these tests, with the exception of Mycoplasma orale, which was detected by both IS1550-PCRs based on the major part and the conserved 206 bp region of the IS1550 element. This study suggests that all M. fermentans isolates possess both the IS1550 element and the malp gene. In contrast to the IS1550, the malp gene is shown to be species-specific and the use of a malp PCR described here could prove to be a useful adjunct to IS1550 detection as confirmation of the presence of M. fermentans in clinical material.

A rapid chromogenic microtitre assay of arginine aminopeptidase activity in Mycoplasma strains.

Arginine-utilizing strains of Mycoplasma can be screened by assay of their arginine aminopeptidase activity. A standardized chromogenic method is described that enables enzyme detection in small volumes of cell suspension in less than 3 h. Cell suspensions (10 microl) in 96-well microtitre plates are incubated at 37 degrees C, pH 8.0, with 0.1 mM arginyl-beta-naphthylamide (100 microl). This is hydrolysed to release beta-naphthylamine, which gives a coloured product on diazotization with fast garnet. M. alkalescens can be detected in this way with as few as 1.1 x 10(5) viable cells and M. fermentans with 2.3 x 10(6) cells. The method has been shown to enable division of 28 strains into three groups of fermentative and arginine-hydrolysing mycoplasmas. This procedure has potential for routine laboratory use.

Biochemical characterisation of some non fermenting, non arginine hydrolysing mycoplasmas of ruminants.

The pattern and kinetics of substrate oxidation by type and recent field strains of Mycoplasma agalactiae, Mycoplasma bovis, Mycoplasma bovigenitalium and Mycoplasma ovine/caprine serogroup 11 were investigated by measurement of oxygen uptake. Metabolism of a range of organic acids, sugars and alcohols was detected. All the test strains were unable to oxidise sugars, glycerol and the organic acids, fumarate, malate and alpha-ketoglutarate (1 mM). All strains oxidised organic acid l-lactate, 2-oxobutyrate and pyruvate and demonstrated the ability to oxidise alcohols, particularly isopropanol, which was oxidised at a high rate and high affinity (0.5 mol/mol isopropanol). Its oxidation was consistent with acetone formation, which may be of important in relation to pathogenicity. All strains oxidised similar substrates, however differences were observed between strains in terms of the relative rates and kinetic values for some substrates.

Simple method to grow enteric campylobacters in unsupplemented liquid medium without the need for microaerophilic kits.

Campylobacter strains (100 human, animal and environmental isolates) were grown in untreated brain heart infusion broth medium (10 ml in tightly capped 20 ml capacity universal tubes) without using microaerophilic kits. Cells grown in these conditions did not differ in their growth rates, protein profiles or substrate utilisation even after 40 passages compared to cells grown under microaerophilic conditions. Growth in such conditions provides a cost effective, convenient and simple system for growing pure culture of campylobacters and obviates the generation of microaerobic conditions using specialised kits.

Close genetic and phenotypic relatedness between mycoplasma ovine/caprine serogroup 11 and Mycoplasma bovigenitalium.

Strains of Mycoplasma ovine/caprine serogroup 11, isolated from infertile sheep, were compared to the type strain, 2D, and to strains of the cattle pathogen M. bovigenitalium, including the type strain, PG11. Examination of these strains by growth inhibition and immune fluorescence tests showed strong serological cross reactivity between M. serogroup 11 and M. bovigenitalium but not with other ruminant mycoplasmas. Substrate oxidation and growth studies did not show any consistent differences between M. serogroup 11 and M. bovigenitalium strains; all strains assigned to both groups were adapted to the utilisation of a small range of organic acids as energy sources. DNA:DNA hybridisation, carried out between DIG labelled reference strains of M. serogroup 11 and M. bovigenitalium and field isolates of these two mycoplasmas showed a particularly close relationship with hybridisation rates all greater than 70% and, mostly, closer to 90%. Sequencing of the 16S ribosomal RNA gene region of the M. serogroup 11 and M. bovigenitalium strains as well as the respective type strains revealed very high overall homologies of 99.5%. In summary, the results showed a very close phenotypic and genotypic relatedness between these two ruminant mycoplasmas which justifies their classification into a single species.