Joseph G. Tully

Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris', 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'

Cell-wall-less uncultivated parasitic bacteria that attach to the surface of host erythrocytes currently are classified in the order Rickettsiales, family Anaplasmataceae, in the genera Haemobartonella and Eperythrozoon. Recently 16S rRNA gene sequences have been determined for four of these species: Haemobartonella felis and Haemobartonella muris and Eperythrozoon suis and Eperythrozoon wenyonii. Phylogenetic analysis of these sequence data shows that these haemotrophic bacteria are closely related to species in the genus Mycoplasma (class Mollicutes). These haemotrophic bacteria form a new phylogenetic cluster within the so-called pneumoniae group of Mycoplasma and share properties with one another as well as with other members of the pneumoniae group. These studies clearly indicate that the classification of these taxa should be changed to reflect their phylogenetic affiliation and the following is proposed: (i) that Haemobartonella felis and Haemobartonella muris should be transferred to the genus Mycoplasma as Candidatus Mycoplasma haemofelis and Candidatus Mycoplasma haemomuris and (ii) that Eperythrozoon suis and Eperythrozoon wenyonii should be transferred to the genus Mycoplasma as Candidatus Mycoplasma haemosuis and Candidatus Mycoplasma wenyonii. The former Haemobartonella and Eperythrozoon species described here represent a new group of parasitic mycoplasmas that possess a pathogenic capacity previously unrecognized among the mollicutes. These haemotrophic mycoplasmas have been given the trivial name haemoplasmas. These results call into question the affiliation of the remaining officially named species of Haemobartonella and Eperythrozoon which should be considered species of uncertain affiliation pending the resolution of their phylogenetic status.

Mycoplasma genitalium, a New Species from the Human Urogenital Tract

Two mycoplasmas recovered from human urogenital tracts were similar in their biochemical and serological properties. These organisms possessed a unique terminal structure that appeared to be associated with attachment to tissue cells and erythrocytes. The organisms fermented glucose but did not hydrolyze urea or arginine. Growth occurred at 30 to 37°. Cholesterol was required for growth. Unlike most other mycoplasmas, both strains were susceptible to thallium acetate. These two organisms were serologically distinct from other Mycoplasma species and from a group of unclassified serotypes of mycoplasmas. On the basis of these findings and other morphological, biological, and serological properties of the microorganisms, we propose that mycoplasmas with these characteristics belong to a new species, Mycoplasma genitalium. Strain G-37 (= ATCC 33530) is the type strain.

Mycoplasma penetrans sp. nov., from the urogenital tract of patients with AIDS

An unusual mycoplasma, which was isolated from the urine of a human immunodeficiency virus-positive male homosexual patient, has an elongated flask shape and two unique sharply divided internal compartments. The tiplike compartment is densely packed with fine granules, and the body compartment is loosely filled with coarse granules consistent with ribosomal structures. The organism has properties of adherence, hemadsorption, and cytadsorption and invades many different types of mammalian cells. Adhesion and penetration apparently involve the terminally located tiplike structure. Cholesterol is required for growth, and the mycoplasma ferments glucose and hydrolyzes arginine, but does not hydrolyze urea. The results of DNA homology studies revealed that this organism is not genetically related to previously described mycoplasma species that have the same biochemical properties. The results of serologic studies demonstrated that this organism is antigenically distinct from all previously described mycoplasmas. We propose that this new mollicute species should be named Mycoplasma penetrans sp. nov. The type strain is strain GTU-54-6A1 (= ATCC 55252).

Spiroplasma melliferum, a New Species from the Honeybee (Apis mellifera)

Twenty-eight strains of spiroplasma subgroup I-2 isolated from insects and flower surfaces were similar in their serological properties. Strain BC-3T (T = type strain), which was isolated from the honeybee, was chosen as a representative of this cluster and was characterized according to accepted standards. This strain and other strains of the cluster entered the hemocoel of their insect hosts after per os acquisition, caused pathology in various tissues, and reduced adult longevity. Growth in SM-1 or M1D medium occurred at 20 to 37°, with optimum growth at about 32 to 35°. Cholesterol was required for growth. Glucose, fructose, and other carbohydrates were fermented, and arginine was catabolized. Seventeen strains, including strain BC-3T, reacted with considerable homogeneity in deformation tests and were completely separable from strains of subgroup I-1 (Spiroplasma citri) and subgroup I-3 (corn stunt spiroplasma). A group of five subgroup I-2 strains showed homogeneity upon one-dimensional polyacrylamide gel electrophoresis of cell proteins. Strain BC-3T was also serologically distinct from subgroups I-4 through I-8; from Spiroplasma floricola, Spiroplasma apis, and Spiroplasma mirum; and from representative strains of spiroplasma groups II and VI through XI. Previously published studies on strain BC-3T and related strains demonstrated that (i) these organisms comprise a unique subgroup of the S. citri complex (group I); (ii) deoxyribonucleic acid-deoxyribonucleic acid homologies between strain BC-3T and strains of other group I subgroups do not exceed 70%; (iii) the patterns of protein sharing among group I strains revealed by two-dimensional polyacrylamide gel electrophoresis support molecular genetic indications of partial relatedness; (iv) the EcoRI restriction endonuclease patterns of deoxyribonucleic acids from strain BC-3T and serologically related strains show close relatedness; (v) sequencing of 5S ribosomal ribonucleic acid suggests some degree of relatedness with all organisms now classified in the Mollicutes; (vi) strain BC-3T is capable of viscotactic and chemotactic responses; (vii) strain BC-3T possesses fibrils that may mediate various types of motility; and (viii) a lytic virus (SpV4) isolated from Spiroplasma sp. strain B63 (a representative of subgroup I-2) is morphologically and genomically distinct from other spiroplasma viruses and forms plaques only on lawns of subgroup I-2 spiroplasmas. Previous work on strain AS 576, another member of subgroup I-2, demonstrated (i) a viscotactic response, (ii) moderate sensitivity to osmotic environments, (iii) susceptibility to tetracycline and aminoglycoside antibiotics, (iv) growth in a relatively simple, chemically defined medium, (v) nutritional utilization patterns in defined medium, and (vi) a genome molecular weight of 109. On the basis of our new findings and the previously described properties of strain BC-3T and related subgroup I-2 strains, we propose that spiroplasma strains with the characteristics described here be classified as a new species, Spiroplasma melliferum. Strain BC-3, the type strain, has been deposited in the American Type Culture Collection as strain ATCC 33219.

Spiroplasma poulsonii sp. nov., a new species associated with male-lethality in Drosophila willistoni, a neotropical species of fruit fly.

Progenies from some wild-caught females of Drosophila willistoni and three other sibling species are entirely female. The proclivity for production of unisexual female progeny by these flies was named the sex ratio (SR) trait and was originally thought to be genetic. However, experiments in the laboratory of Donald F. Poulson in the early 1960s demonstrated that this trait was vertically transmitted and infectious, in that it could be artificially transferred by injection from infected females to non-infected females. Motile, helical micro-organisms were observed in females showing the trait. In 1979, the SR organisms were designated as group II in the informal spiroplasma classification system. The organisms proved to be extremely fastidious, but were eventually cultivated in a very complex cell-free medium (H-2) after initial co-cultivation with insect cells. Cultivation in the H-2 medium and the subsequent availability of a triply cloned strain (DW-1T) permitted comparative studies. Cells of strain DW-1T were helical, motile filaments 200-250 nm in diameter and were bound by a single trilaminar membrane. Cells plated on 1.8% Noble agar formed small satellite-free colonies 60-70 microns in diameter with dense centres and uneven edges. The temperature range for growth was 26-30 degrees C; optimum growth occurred at 30 degrees C, with a doubling time in H-2 medium of 15.8 h. The strain passed through filters with 220 nm, but not 100 nm, pores. Reciprocal serological comparisons of strain DW-1T with representatives of other spiroplasma groups showed an extensive pattern of one-way crossing when strain DW-1T was used as antigen. However, variable, usually low-level reciprocal cross-reactions were observed between strain DW-1T and representatives of group I sub-groups. The genome size of strain DW-1T was 2040 kbp, as determined by PFGE. The G + C content was 26 +/- 1 mol%, as determined by buoyant density and melting point methods. The serological and molecular data indicate that strain DW-1T is separated from group I representative strains sufficiently to justify retention of its group status. Continued group designation is also indicated by the ability of SR spiroplasmas to induce male lethality in Drosophila, their vertical transmissibility and their extremely fastidious growth requirements. Group II spiroplasmas, represented by strain DW-1T (ATCC 43153T), are designated Spiroplasma poulsonii.

Revised group classification of the genus Spiroplasma

Significant changes have been made in the systematics of the genus Spiroplasma (class Mollicutes) since it was expanded by revision in 1987 to include 23 groups and eight sub-groups. Since that time, two additional spiroplasmas have been assigned group numbers and species names. More recently, specific epithets have been assigned to nine previously designated groups and three sub-groups. Also, taxonomic descriptions and species names have been published for six previously ungrouped spiroplasmas. These six new organisms are: Spiroplasma alleghenense (strain PLHS-1T) (group XXVI), Spiroplasma lineolae (strain TALS-2T) (group XXVII), Spiroplasma platyhelix (strain PALS-1T) (group XXVIII), Spiroplasma montanense (strain HYOS-1T) (group XXXI), Spiroplasma helicoides (strain TABS-2T) (group XXXII) and Spiroplasma tabanidicola (strain TAUS-1T) (group XXXIII). Also, group XVII, which became vacant when strain DF-1T (Spiroplasma chrysopicola) was transferred to group VIII, has been filled with strain Tab 4c. The discovery of these strains reflects continuing primary search in insect reservoirs, particularly horse flies and deer files (Diptera: Tabanidae). In the current revision, new group designations for 10 spiroplasma strains, including six recently named organisms, are proposed. Three unnamed but newly grouped spiroplasmas are strain TIUS-1 (group XXIX; ATCC 51751) from a typhiid wasp (Hymenoptera: Tiphiidae), strain BIUS-1 (group XXX; ATCC 51750) from floral surfaces of the tickseed sunflower (Bidens sp.) and strain BARC 1901 (group XXXIV; ATCC 700283). Strain BARC 2649 (ATCC 700284) from Tabanus lineola has been proposed as a new sub-group of group VIII. Strains TIUS-1 and BIUS-1 have unusual morphologies, appearing as helices at only certain stages in culture. In this revision, potentially important intergroup serological relationships observed between strain DW-1 (group II) from a neotropical Drosophila species and certain sub-group representatives of group I spiroplasmas are also reported.

Heterogeneity of Genome Sizes within the Genus Spiroplasma

Organisms belonging to the genus Spiroplasma are currently classified into 23 groups, 17 of which have been assigned species epithets. We determined the genome sizes of representatives of 20 groups by using pulsed-field gel electrophoresis. Each genome size was deduced from the mobility of linear nonrestricted DNA, as well as from the sum of the sizes of restriction fragments obtained after digestion with NotI, a restriction endonuclease with a limited number of restriction sites in spiroplasma DNA. The values which we obtained indicated that the genome sizes of members of the genus Spiroplasma range from 940 to 2,220 kbp.

Spiroplasma kunkelii sp. nov.: Characterization of the Etiological Agent of Corn Stunt Disease

Nine strains of spiroplasma subgroup 1-3, which comprise the etiological agent of corn stunt disease, were similar in their serological properties. Strain E275T(T = type strain) was studied by using criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on Taxonomy of Mollicutes for descriptions of new mollicute species. This strain was shown to belong to the class Mollicutes by the ultrastructure of its limiting membrane, its procaryotic organization, its colonial morphology, and its filtration behavior and to the family Spiroplasmataceae by its helical morphology and motility. Although some serological cross-reactions with other group I spiroplasma strains was observed, strain E275Tcould be readily distinguished from representatives of other group I subgroups. Subgroup 1-3 spiroplasmas and other group I strains also differed in their one- and two-dimensional polyacrylamide gel protein patterns, plant and insect host ranges, and pathogenicities. Growth in MIA or MID medium occurred at 20 to 30°C. Cholesterol was required for growth. Glucose was fermented, and arginine was catabolized. Subgroup 1-3 strains, including strain E275T, reacted with considerable homogeneity in deformation tests and were completely separable from strains belonging to subgroup I-1 (Spiroplasma citri) and subgroup I-2 (Spiroplasma melliferum). Strain E275Twas also serologically distinct from subgroups I-4 through I-8, Spiroplasma floricola (group III), Spiroplasma apis (group IV), Spiroplasma mirum (group V), and representative strains of spiroplasma groups II and VI through XI. The deoxyribonucleic acid of strain E275Thybridized with the deoxyribonucleic acid of S. citri at significant levels (33 to 68%, depending on the technique used). These results demonstrate that strain E275Tand similar strains meet the criteria proposed by the International Committee on Systematic Bacteriology Subcommittee for elevation of spiroplasma subgroups to species. We propose that such strains be named Spiroplasma kunkelii. Strain E275Thas been deposited in the American Type Culture Collection as strain ATCC 29320T.

Revised group classification of the genus Spiroplasma (class Mollicutes), with proposed new groups XII to XXIII

Fourteen spiroplasma strains, primarily of insect origin, were analyzed according to criteria previously proposed for description of new serogroups of the genus Spiroplasma. When tested by reciprocal metabolism inhibition, growth inhibition, and deformation serological procedures, 12 of the strains were serologically unrelated to each other and to representative strains previously assigned to groups I to XI and subgroups I-1 to I-8. Examination by dark-field and electron microscopy indicated that each of the 12 strains possessed morphological features typical of spiroplasmas (helicity, motility, lack of a cell wall, and absence of periplasmic fibrils). All strains were resistant to 500 U of penicillin per ml and catabolized glucose but were unable to hydrolyze urea. Ability to hydrolyze arginine varied among strains. The guanine-plus-cytosine contents of the deoxyribonucleic acid of the 12 strains varied from 24 to 29 mol%. Two other strains (MQ-6 and Ar-1357) shared only a partial serological relationship to strain CC-1 (group XVI), suggesting that this group may consist of an assemblage of heterogeneous serovars. On the basis of the unique serological distinctions and other properties reported herein, we propose that the 12 representative strains be assigned new consecutive group designations XII to XXIII.

Spiroplasma mirum, a New Species from the Rabbit Tick (Haemaphysalis leporispalustris)

Three spiroplasma strains recovered from rabbit ticks (Haemaphysalis leporispalustris) in Georgia and Maryland were found to be similar in biochemical, serological, and pathological properties. The organisms grew at temperatures of 20 to 37°C, required cholesterol for growth, fermented glucose, hydrolyzed arginine, and produced a film and spot reaction. The three spiroplasma strains were serologically distinct from the one established species (Spiroplasma citri) in the genus and from all other unclassified spiroplasma serogroups presently known. On the basis of these findings and other morphological, biological, and serological properties of the organism, it is proposed that spiroplasma strains with these characteristics be classified as a new species, Spiroplasma mirum. Strain SMCA (ATCC 29335) is the type strain.