International Journal of Systematic and Evolutionary Microbiology (2002), 52, 587–597 DOI: 10.1099/ijs.0.01965-0

Proposal of Ureaplasma parvum sp. nov. and emended description of (Shepard et al. 1974) Robertson et al. 2001

1,2 Department of Medical Janet A. Robertson,1 Gerald W. Stemke,2 John W. Davis, Jr,3 Microbiology and 4 5 6 7 Immunology1 and Ryo# Harasawa, David Thirkell, Fanrong Kong, Maurice C. Shepard † Department of Biological and Denys K. Ford8‡ Sciences2 , University of Alberta, Edmonton, Alberta, Canada Author for correspondence: Janet A. Robertson. Tel: j1 780 492 2335. Fax: j1 780 492 7521. 3 Department of Biology e-mail: janet.robertson!ualberta.ca and Medical Laboratory Technology, Bronx Community College – City The phenotypic and genotypic properties of Ureaplasma urealyticum (family University of New York, , order Mycoplasmatales, class ) are reviewed Bronx, NY, USA here. The 14 recognized serovar standard strains found in humans exhibit no 4 Animal Centre for serological cross-reactivity with ureaplasmas from other hosts and uniquely Biomedical Research, The University of Tokyo, express human immuoglobulin A1 protease activity. However, they exhibit Tokyo, Japan many characteristics which place them in two distinct clusters known as the T 5 School of Biology, parvo biovar (or biovar 1 or B) and the T960 biovar (or biovar 2 or A). University of St Andrews, Established phenotypic markers of the biovars include clustering of antigenic St Andrews, UK types, polypeptide patterns of whole-cell preparations, differential inhibition 6 Centre for Infectious by manganese, and polymorphism among their ureases, pyrophosphatases and Diseases and diaphorases. Established genotypic markers of the biovars are DNA–DNA Microbiology, Institute of Clinical Pathology and hybridization of 60% between biovars, and distinctive RFLP patterns and Medical Research, genome sizes. Divergent nucleotide sequences of several highly conserved Westmead, NSW, genes attest to the phylogenetic distinctiveness of the two biovars. PCRs Australia founded upon the sequences for 16S rRNA, the 16S–23S rRNA intergenic 7 Camp Lejeune, NC, USA regions, the genus-defining urease, the serovar-defining, multiple-banded 8 Department of Medicine, antigen genes or randomly amplified polymorphic DNA tests differentiate the University of British biovars unambiguously. With the availability of rapid, reliable and economical Columbia, Vancouver, BC, Canada tests for biovar determination, it is now appropriate to propose that the taxonomic status of U. urealyticum be emended. Serovar standard strains exhibiting traits of biovar parvo (serovars 1, 3, 6 and 14) will be designated as a separate species, Ureaplasma parvum sp. nov., as befits its smaller genome size. The serovar 3 standard (strain 27T) will be the type strain of U. parvum and is represented by ATCC 27815T and NCTC 11736T. Serovar standard strains exhibiting traits of biovar T960T (2, 4, 5, 7, 8T, 9, 10, 11, 12 and 13) will retain the U. urealyticum designation and type strain, the serovar 8 standard (strain T960T), represented by ATCC 27618T and NCTC 10177T.

Keywords: Ureaplasma parvum, Ureaplasma urealyticum, biovars, phenotyping, genotyping

...... † Present address: 1008 River Street, Jacksonville, NC 28540-5703, USA. ‡ Present address: 4380 Locarno Crescent, Vancouver, BC, Canada V6R 1G3. A zymogram is available as supplementary data in IJSEM Online (http://ijs.sgmjournals.org).

01965 # 2002 IUMS Printed in Great Britain 587 J. A. Robertson and others

INTRODUCTION within the Mycoplasma pneumoniae clade of the Mollicutes (Weisburg et al., 1989). Strains of Urea- Organisms of the class Mollicutes are exceptionally plasma from humans are distinct from Ureaplasma small prokaryotic cells which lack a cell wall. The strains from other mammalian and avian hosts and Mycoplasmataceae, Family I of the order Myco- comprise two clusters (Fig. 1). One cluster or biovar is plasmatales, are non-helical, sterol-requiring (and thus composed of strains of an emended Ureaplasma digitonin-sensitive) strains which are not obligate urealyticum species and the other of the proposed anaerobes. They comprise two genera: Mycoplasma species Ureaplasma parvum (Robertson et al., 1994; and Ureaplasma. The of the genus Urea- Harasawa et al., 1996; Stemke & Robertson, 1996). plasma was last described by Taylor-Robinson & Gourlay (1984). Phylogenetically, on the basis of 16S The tiny (‘T’) strain mycoplasmas were discovered in rRNA gene homology, the genus Ureaplasma falls human urogenital tract samples by Shepard (1954, 1956). Their status as self-replicating biological entities was confirmed by Ford et al. (1962). The most notable features of these isolates were their tiny (‘T’) colonies, which were significantly smaller than those of other members of the Mycoplasmataceae, themselves gen- erally undetectable by the naked eye, and the unusual pH growth optimum of 6n0–6n5 (Shepard & Lunceford, 1965). Unique among the Mollicutes, the ‘T’ strains were shown to hydrolyse urea (Purcell et al., 1966; Shepard, 1966; Ford & McDonald, 1967; Shepard & Lunceford, 1967) but not glucose (Black, 1973), other carbohydrates (Robertson & Howard, 1988) or argi- nine (Woodson et al., 1965). Urease activity was shown to be associated with, and, later, essential to the generation of ATP by a chemiosmotic mechanism (Romano et al., 1980; Smith et al., 1993). The genus and the first species, U. urealyticum, were named by Shepard et al. (1974). Although ureaplasmas from humans are serologically distinct from members of the genus isolated sub- sequently from other mammals or avian species, they also are the only isolates which have been shown to produce human immunoglobulin A1 protease activity (Robertson et al., 1984; Kilian et al., 1984; Stemke et al., 1984; Kapatais-Zoumbos et al., 1985). Ureaplasma colonies usually develop after 1–3 d in- ...... cubation. Depending upon the strain and cultural Fig. 1. Phylogenetic tree, prepared using the DNAMAN program, conditions, ureaplasmas sometimes produce tiny based upon 16S rRNA gene sequences and bootstrapped with ‘fried-egg’ colonies but more often produce only part 300 replications. It indicates the relationships between the type of such a typical Mollicutes growth pattern (Fig. 2a). strains of the emended species Ureaplasma urealyticum strain T960T (l ATCC 27618T l NCTC 10177T) and sp. nov. Ureaplasma Although ureaplasma colony sizes can be enhanced by parvum strain 27T (l ATCC 27815T l NCTC 11736T) proposed incorporating a suitable buffer into the medium herein and the type species of the other named species of the (Manchee & Taylor-Robinson, 1969), they in- genus Ureaplasma. The latter are as follows: the avian isolate T T T frequently reach diameters similar to those of the so- Ureaplasma gallorale D6-1 (l ATCC 43346 l NCTC 11707 ) called ‘large colony mycoplasmas’ from which they (Koshimizu et al., 1987); the feline isolates Ureaplasma cati FT2- BT (l ATCC 49228T l NCTC 11710T) and Ureaplasma felinum F2T can be differentiated by positive tests for urease activity (l ATCC 49229T l NCTC 11709T) (Harasawa et al., 1990); the (Fig. 2b). Cultures in broth can reach populations of T ) −" canine isolate Ureaplasma canigenitalium D6P-C (l ATCC approximately 10 organisms ml , but, because of the 51252T)) (Harasawa et al., 1993); and the bovine isolate small cellular mass, they do not cause detectable Ureaplasma diversum A417T (l ATCC 43321T l NCTC 10182T) (Howard & Gourlay, 1982). The most recently deposited turbidity (Shepard et al. 1974). GenBank DNA sequences for Ureaplasma were used; these The spherical or coccobacillary-shaped cells typical of were, respectively, AF073450, AF073456, U62937, D78649, D78651, D78648 and D78650 (Harasawa et al., 1996; Stemke & the genus Ureaplasma have been reported to have Robertson, 1996; Kong et al., 1999). The rRNA gene sequence diameters of between 0n1 and 1n0 µm. Extremely small included as a basis for comparison was that of Mycoplasma or large cells or filamentous cells may result from pneumoniae of the same Mollicutes clade, as represented by adverse cultural conditions or as artefacts of prep- the type strain, FHT (l ATCC 15531T l NCTC 10119T) (Somerson et al., 1963) (GenBank accession no. M29061; Weisburg et al., aration for microscopy (Shepard & Masover, 1979). 1989), not the sequence of strain M129 (GenBank accession no. By morphometry, using electron microscopy, cellular U00089; Himmelreich et al., 1996). diameters of exponential phase cells were determined

588 International Journal of Systematic and Evolutionary Microbiology 52 Ureaplasmas of humans

(a) (b) (c) fe c c

p

...... Fig. 2. Mycoplasma colonies shown in (a) and (b) were cultivated on a genital mycoplasma agar which had been incubated at 37 mC for 48 h in 5% CO2 in N2. (a) Three colonial types of the serovar 3 standard and proposed type strain of U. parvum described variously as a typical ‘fried egg’ (fe) colony, a partial ‘fried egg’ (p) colony and the ‘cauliflower head’ or core (c) colony, which lacks any peripheral, surface growth. Bar, 50 µm. (J. A. Robertson, unpublished photo- micrograph.) The demonstration of urease activity differentiates the genus Ureaplasma from all other members of the Mollicutes. The arrowhead in (b) indicates a darkening U. parvum colony seconds after exposure to the urease spot test (Shepard & Howard, 1970). U. parvum sometimes produces colonies in the size range of many Mycoplasma species. The three other, non-reactive colonies are Mycoplasma hominis. Bar, 100 µm. (J. A. Robertson, unpublished photo- micrograph). (c) A cell from exponential growth of U. parvum cells infecting a HeLa 229 cell culture (top). The culture was fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate and then embedded in Araldite; thin sections were stained with uranyl acetate and lead citrate. The six ovoid sections of U. parvum demonstrate the main structural features of exponential phase cells, i.e. the lack of a cell wall, and the single membrane enclosing a cytoplasm that is dense with ribosomes. Bar, 0n5 µm. (Alfa, 1986; M. J. Alfa, , M. C. Chen and J. A. Robertson, unpublished electron micrograph).

to be "0n5 µm (Robertson et al., 1983). Typical clinical isolates confounded interpretation, this ap- ultrastructural features are shown in Fig. 2(c). proach was not recommended when large numbers of Although members of Mollicutes are of Gram-positive strains were involved (Stemke & Robertson, 1985). lineage, in the absence of a bacterial cell wall they cannot hold the crystal violet of the Gram stain but The phenotypic and genotypic evidence that has take on the colour of the counter-stain. The minute accumulated over the last two decades has confirmed cells are, however, rarely discernable in Gram-stained fundamental differences among human ureaplasmas preparations examined at i1000 magnification. which were consistent with two species. The recent, independent confirmation of several species-defining Under the early conventions which were applied to the criteria, the development of several practical tests for taxonomy of other members of Mollicutes, each strain unambiguously differentiating them, as well as the with a distinct antigenic specificity was accorded considerable interest in their roles in human disease, separate species status. However, when updating the make this taxonomic adjustment timely. minimal standards for the taxonomy of the class Mollicutes, the International Committee on Systematic Bacteriology’s Subcommittee on the Taxonomy of Mollicutes (ICSB\STM, 1979) echoed the opinion of METHODS Shepard et al. (1974) in stating that ‘The utility of the Strains. The sources and designations of the serovar standard growth inhibition and immunofluorescence serological strains of the two biovars of the ureaplasmas associated with reactions [serotyping tests] as criteria for species humans are provided in Table 1, along with culture- distinction, does not apply within the genus Urea- collection accession numbers. Serological identification of plasma and is of untested validity in other genera’. the serovar standard strains has been made by using The criteria to be applied to the taxonomy of Urea- metabolic inhibition tests (Purcell et al., 1966; Robertson & plasma species were to include serology, PAGE and Stemke, 1979), the mycoplasmacidal test (Lin et al., 1972) or DNA–DNA hybridization (ICSB\STM, 1984, 1995). an indirect colony epifluorescence test (Stemke & Robertson, Although serological tests differentiated ureaplasmas 1981). from various hosts, intraspecies serological hetero- Media. The serovar standard strains were cultivated at geneity was the first means used to distinguish in- 35–37 mC in media supplemented with animal serum as dividual isolates within the species U. urealyticum source of sterols (Edward, 1947) but modified by the addition (Ford, 1967; Black, 1970). Fourteen distinct speci- of exogenous urea and various other growth factors. ficities have been identified (Robertson & Stemke, Incubation was at 35–37 mC. Cultures in liquid medium were 1982). However, because the serotyping methods were usually incubated in air, whilst those on solid medium were demanding to perform and the multiple serovars of usually incubated under conditions of reduced oxygen http://ijs.sgmjournals.org 589 J. A. Robertson and others

Table 1. Sources and identification of the serovar standard strains of the two biovars of the present species Ureaplasma urealyticum ...... Strains shown in bold are the representative strains for each biovar or species.

Name* Serovar no.† Culture collection number(s)‡

Biovar described as 1, B or Parvo§ and proposed as Ureaplasma parvum sp. nov. 27T 3(III) ATCC 27815T ; NCTC 11736T 7 1 (I) ATCC 27813 Pi 6 (VI) ATCC 27818 U26 14 ATCC 33697 Biovar described as 2, A or T960T§ proposed as the Ureaplasma urealyticum emend. T960T-(CX8) 8 (VIII) ATCC 27618T ; NCTC 10177T 23 2 (II) ATCC 27814 58 4 (IV) ATCC 27816 354 5 (V) ATCC 27817 Co 7 (VII) ATCC 27819 Vancouver 9 ATCC 33175 Western 10 ATCC 33699 K2 11 ATCC 33695 U24 12 ATCC 33696 U38 13 ATCC 33698

* The sources of the strains are listed in Robertson & Stemke (1982). † Although Black (1970) had introduced Roman numeral designations (shown here in parentheses) for the first eight antigenic specificities, when expanding the serotyping scheme Robertson & Stemke (1982) introduced Arabic numerals to reduce the possibility of labelling errors. ‡ ATCC, American Type Culture Collection, Manassas, VA, USA; NCTC, National Culture Type Collection, Colindale, UK. § The clusters of strains having similar traits were first referred to as biotypes 1 and 2 (Robertson, 1978). Effect of manganese on the growth of Ureaplasma urealyticum (T-strain mycoplasma). In Abstracts of the American Society for Microbiology, p. 74) and were later referred to as Group B and Group A, respectively (Mouches et al., 1981). To avoid confusion with the numbered serovars, Robertson et al. (1990) introduced the biovar descriptors T-960 and parvo (Latin for small); the latter referred to the smaller genome size of the members of the parvo biovar. The biovars of all serotype standard strains have been confirmed by PCRs based upon differences in the 16S rRNA sequences (Robertson et al., 1993); for confirmation of biovar or a newly defined species, see Table 4. concentration. Cells were harvested by ultracentrifugation ability of the serovar 3 standard strain to haemadsorb at approximately 15000 g at 4 mC. (Manchee & Taylor-Robinson, 1969), these strains Determination of phenotypic traits. References to the de- appeared homogeneous. The first genomic studies, tailed, study-specific methods used for the preparation of showing a GjC composition of between 27 and 28 cells, PAGE, immunoblotting, and determination of the mol% (Bak & Black, 1968; Black et al., 1972; Howard #+ et al., 1978) and genome sizes between 4 1 and effect of Mn on growth are provided in Table 2. ) n 4 8 10 Da (701 and 735 kpb) (Bak et al., 1969; Black Determination of genotypic traits. References to the detailed, n i et al., 1972), reinforced this view. The DNA of the study-specific methods used for preparation of cells, DNA T isolation, hybridization, endonuclease digestion, agarose recently sequenced genome of strain 27 (serovar 3) gel electrophoresis, nucleotide sequencing, PCR, and other had a GjC content of 26 mol% (Glass et al., 2000). molecular biological techniques are provided in Table 3. Newer methodology has resulted in revised genome sizes (see Genome size, below). RESULTS AND DISCUSSION Strain clustering to biovar Initial characterization Upon further investigation into non-serologic pheno- Initial characterization of the first set of (eight) typy (Table 2) and genotypy (Table 3), the hetero- serovars of the initial U. urealyticum included over 30 geneity of U. urealyticum was revealed. However, by criteria (Black, 1973). Apart from already recognized then, ureaplasmas had been found in hosts other than antigenic variation (Ford, 1967; Table 1) and the humans. Concern was centred not upon the taxonomic

590 International Journal of Systematic and Evolutionary Microbiology 52 Ureaplasmas of humans

Table 2. Phenotypic traits that distinguish the two biovars of ureaplasmas from humans

Trait Reference(s)

Electrophoretic patterns of cell proteins Isoelectric focusing and SDS-PAGE Sayed & Kenny (1980) One-dimensional SDS-PAGE Howard et al. (1981) Two-dimensional SDS-PAGE Mouches et al. (1981) Swenson et al. (1983) # Differential growth response to 1 mM Mn +* Robertson & Chen (1984) Immunoblots of cell proteins Polypeptides recognized by polyclonal antisera Horowitz et al. (1986) Membrane proteins recognized by polyclonal antisera Lee & Kenny (1987) Thirkell et al. (1989) 72 kDa urease subunit recognized by monoclonal antibodies Thirkell et al. (1990) MacKenzie et al. (1996) Enzyme polymorphism Davis et al. (1987); Davis & Villaneuva (1990) Urease Pyrophosphatase† Diaphorase

* Growth of the parvo and T960T biovars was inhibited temporarily and permanently, respectively. Serovar 13 gave an anomalous response (Stemler et al., 1987). † Zymogram available in IJSEM Online (http:\\ijs.sgmjournals.org).

Table 3. Genotypic traits that distinguish the two biovars of ureaplasmas from humans

Trait Reference(s)

DNA–DNA hybridization (%) Christiansen et al. (1981); Harasawa et al. (1991) RFLP patterns of DNA Genomic DNA Razin et al. (1983), Cocks & Finch (1987) Genomic DNA probed with rRNA genes Harasawa et al. (1991) 5h Regions of multiple-banded antigen genes Teng et al. (1994) Urease genes Neyrolles et al. (1996)* Genome size determined by PFGE Robertson et al. (1990) Gene heterogeneity Urease gene subunits and adjoining upstream regions Blanchard (1990)*, Neyrolles et al. (1996), Kong et al. (1999) 16S rRNA genes Robertson et al. (1993, 1994), Kong et al. (1999) 16S–23S rRNA intergenic spacer regions Harasawa & Kanamoto (1999), Kong et al. (1999) 5h Regions of multiple-banded antigen genes Kong et al. (1999) Biovar-specific PCR amplification With primers based upon 16S rRNA Robertson et al. (1993) With arbitrarily primed PCRs Grattard et al. (1995a)

* Although Blanchard (1990) identified the first biovar-specific PCR, two misidentified strains resulted in an anomalous pattern of partition for the serovar standard strains, and its usefulness was not immediately recognized. The correction was reported by Teng et al. (1994) and independently confirmed by Kong et al. (1999). status of strains from the same (human) host species hindsight was it clearly recognized that the intra- but on whether isolates from another host (bovine) species, serological cross-reactions in ureaplasmas warranted separate species status (e.g. Howard & from humans were biovar-restricted (e.g. Robertson Gourlay, 1982). Until now, of the six named Urea- et al., 1993). plasma species (Fig. 1), only the serologically dis- tinguishable Ureaplasma cati and Ureaplasma felinum Exceptional traits (Harasawa et al., 1990) are associated with the same host (the cat). Most interestingly, when the serovar Traits which do not partition the strains to one or standard strains of ureaplasmas from humans showed other biovar are few. For example, the ability of differing traits, usually they were partitioned to the colonies of the serovar 3 standard strain to adsorb same two clusters or biovars (Table 1). Only with erythrocytes (Manchee & Taylor-Robinson, 1969) http://ijs.sgmjournals.org 591 J. A. Robertson and others pertained to that strain but not to all isolates carrying of 70% [DNA–DNA homology] does not take into the serovar 3 determinant or classified within the parvo account is the possibility that the tempo and mode of biovar (Robertson & Sherburne, 1991). The distri- changes differ in different prokaryotic strains.’ Thus, bution of certain other phenotypic traits among the concept of the relatively fast evolution of the the serovar standards, e.g. extramembranous layers Mollicutes proposed by Maniloff (1992) would merely (Robertson & Smook, 1976), phospholipase A",A# enhance the already convincing hybridization data. and C activities (De Silva & Quinn, 1986, 1999), or antimicrobial-susceptibility patterns has not been es- tablished. Genome sizes In 1990, a decade after the first DNA–DNA hybrid- Role in disease ization studies had shown that ureaplasmas of humans fell into two biovars, genome size (as determined by The ureaplasmas are mucosal parasites which, in then new PFGE methodology) revealed a second humans, may be found as genito-urinary commensals genomic criterion that supported separate species but also as agents of urethritis; moreover, in immuno- status. Unlike the previously reported estimates (701– compromised patients they may cause abscesses and 735 kbp), based upon buoyant-density determinations pyogenic arthritis. Although generally considered (Black et al., 1972), PFGE data showed a broader opportunistic pathogens, the extent of ureaplasmal range of genome sizes (760–1140 kpb). Furthermore, pathogenicity is poorly understood (Taylor-Robinson, there was a discontinuity of " 70 kbp between 1996). Indeed, it is the hope of clinical microbiologists genomes of the parvo biovar (769 kbp) and the T960T that taxonomic recognition of these two distinct biovar (840–1140 kbp) (Robertson et al., 1990) which species will serve to clarify their role in human had been defined earlier by both the partition of a infections. In view of such uncertainty, pathogenicity number of phenotypic traits (Table 2) and the first as a phenotypic trait was excluded from this assess- DNA–DNA hybridizations (Table 3). By PFGE, the ment. genome size of strain 27T, the serovar 3 standard and proposed type strain of U. parvum, had been estimated DNA–DNA hybridization as " 760 kbp (Robertson et al., 1990). Recently, its genome was determined to contain 751719 bp (Glass Although we have adopted a polyphasic approach, et al., 2000), within 1% of that estimate. Differences genomic traits have been particularly helpful in re- in genome sizes were the first, clearly recognizable solving the taxonomic status of ureaplasmas of macromolecular difference between the two biovars, humans. In the last edition of Bergey’s Manual, and led to the introduction of the parvo and T960T Johnson (1984) indicated that, although taxonomic descriptors of the biovars (see Table 1, footnote §). decisions were indeed arbitrary, experience had shown that strains showing " 70% DNA homology (intra- group) invariably were the same species, whilst those Conserved gene sequences showing ! 60% homology (intergroup) were not. Clear evidence of the evolutionary divergence between Two decades ago, in 1980, the first DNA–DNA the biovars accompanied the discovery of the third hybridization studies, using a direct binding method genotypic parameter of ureaplasma biovar-differ- with membrane filters, showed parvo biovar strain entiation, the variable sequences of highly conserved hybridizations of 90–102% within that cluster but T gene sequences. The phylogenetic division indicated by 38–60% with strains of the T960 biovar. The data the 16S rRNA gene data (Robertson et al., 1993, 1994) were 69–100% and 49–52%, respectively, for the T has been confirmed by the sequences of the urease T960 biovar. Only one of the 36 hybridizations was in (Blanchard, 1990; Teng et al., 1994) and the multiple- the 60–70% transition zone (Christiansen et al., 1981). banded antigen genes (Teng et al., 1994) as well as by Later, when the hybridization patterns were confirmed the sequence patterns of the 16S–23S rRNA intergenic using a hydroxy apatite column method (Harasawa spacer regions (Harasawa et al., 1996; Harasawa & et al., 1991) and respecting the maximum 5 mC ∆Tm Kanamoto, 1999). All have been substantiated (Kong requirement (Wayne et al., 1987), none of the 14 et al., 1999). We note that although Kamla et al. (1996) serovars showed homologies within the transition have advanced the argument that phylogeny based zone. Thus, the results of both studies were consistent upon the sequences of elongation factor Tu better with U. urealyticum comprising two distinct taxa. In delineated relationships among the Mollicutes than did the determination of bacterial species, the superiority the 16S rRNA tree, at present these sequences are of DNA–DNA hybridization data over more recently available only for serovars 3 (Glass et al., 2000) and 14 available 16S rRNA gene sequences has yet to be (Kamla et al., 1996). Both are serovars of the parvo successfully challenged. Stackebrandt & Goebel (1994) biovar; they differ by a single adenosine in a string of have allowed that the more conveniently determined adenosines. 16S rRNA gene sequences may be substituted for hybridization determinations unless heterogeneity is Although both the DNA–DNA hybridization and % 3% when their resolution is limited. They have genome size analyses provided dependable parameters emphasized, however, that ‘What the threshold value for discriminating between biovars, like many of the

592 International Journal of Systematic and Evolutionary Microbiology 52 Ureaplasmas of humans phenotypic traits, they were impractical for application separate taxa, a distinction which will better serve the in clinical settings. However, knowledge of the 16S needs of the scientific community. rRNA sequences of the biovar-representative strains brought the benefit of PCRs which identified, to biovar Table 4 provides a protocol for the identification of the level, both laboratory-adapted strains and wild-type ureaplasmas from humans for clinical and for taxo- isolates (Robertson et al., 1993). When the 16S rRNA nomic purposes. Descriptions of the proposed new sequences for five of the 14 serovars were compared, species, U. parvum, and the emended U. urealyticum the variance was % 0n3% among serovars within follow. either biovar but & 1n1% between the two bio- vars (Robertson et al., 1994). Temporarily, it was questioned whether " 1% heterogeneity between 16S Description of Ureaplasma parvum Robertson et al. rRNA gene sequences was compatible with separate 2001 species identity. Fox et al. (1992) upheld the superiority of hybridization data to determine species status and Ureaplasma parvum (parhvum. L. neut. adj. parvum persuasively argued that 16S rRNA sequences were small, referring to the fact that its genome is sig- ‘more appropriate for determining inter- and intra- nificantly smaller than that of U. urealyticum). genic relationships than taxonomic decisions’. How- Phenotypic characteristics. Growth inhibition of cells by ever, the ICSB\STM, while acknowledging the justi- (or colony reaction with) polyclonal antisera to one or fication for a second Ureaplasma species for strains more of the specific antigenic determinants associated from humans, advised awaiting further evidence of the with the present parvo biovar of the human urea- efficacy of molecular differentiation methods (ICSB\ plasmas (serovars 1, 3, 6 or 14) but not to any antigenic STM, 1993) before agreeing, in principle, to the determinants associated with the emended U. urea- separation of the initial U. urealyticum into two species lyticum (T960T biovar of the human ureaplasmas or its (ICSB\STM, 1997). Kong et al. (1999) have shown the serovars 2, 4, 5, 7, 8, 9, 10, 11, 12 or 13). Parvo biovar- 16S rRNA genes to be the most highly conserved of the specific polypeptide patterns obtained by PAGE: these four (previously identified) variable regions of these may be enhanced through reaction with polyclonal genomes. On the basis of their data, the maximum antiserum or monoclonal antibodies to the antigenic variance in the 16S rRNA gene sequences for all specificities associated with the species. Growth is # 14 serovars is 0n97%, as opposed to the 4n5% value for inhibited temporarily in the presence of 1 mM Mn +. the 16S–23S rRNA spacer regions, 6n2–24n4% for the urease gene subunits and adjoining spacer regions, and Genotypic characteristics. Genomes of " 760 kbp dem- 26n0% for the 5h-end of the multiple-banded antigen onstrate " 70% hybridization patterns with the DNA genes and 41n0% upstream of the multiple-banded of other strains of the present parvo biovar of antigen genes. The multiple-banded antigen genes code ureaplasmas. RFLP patterns which are similar to for putative virulence proteins on the surfaces of those characteristic of other serovars of the parvo ureaplasma membranes (Watson et al., 1990) and biovar but differ from those characteristic of the exhibit serovar-related heterogeneity (Teng et al., emended U. urealyticum; amplification by PCRs for 1994; Kong et al., 1999). parvo biovar-specific nucleotide sequences for the urease-associated genes, the 16S rRNA genes, the 16S–23S rRNA intergenic regions, and the 5h end of Conclusions the genes of the multiple-banded antigens, and the parvo biovar-specific RAPD PCR. The type strain of Altogether, we have identified at least 14 biovar- U. parvum will be the serovar 3 standard (strain 27T) defining parameters. With a single exception (Thirkell represented by ATCC 27815T and NCTC 11736T. The et al., 1990), all studies cited here (Tables 2 and 3) have GjC content of the DNA is 26 mol%; its genome size examined both proposed type strains and multiple, is 751719 kbp. The most recent GenBank accession representative strains of each biovar, if not the com- numbers for the sequences of its 16S rRNA and plete set of serovar standards. Eight of the 14 par- 16S–23S rRNA intergenic spacer region are AF073456 ameters have been confirmed independently. These and AF059323, respectively. The genome sequence are the numerous examples of biovar-defining, carries US patent number 5728522 (the annotated protein\polypeptide patterns, including the specific sequence may be found at http:\\genome.microbio. urease polymorphism (Table 2), and the DNA–DNA uab.edu\uu\uugen.htm). hybridization and RFLP patterns and the hetero- geneity of the four genes which have been sequenced (Table 3). The comprehensive and consistent data Emended description of Ureaplasma urealyticum presented in Tables 1, 2 and 3 both fulfil and exceed the (Shepard et al. 1974) Robertson et al. 2001 present minimal standards criteria established by the ICSB\STM (1995) to guide the taxonomy of Mol- Phenotypic characteristics. Growth inhibition of cells by licutes. They represent a compelling argument that (or colony reaction with) polyclonal antisera to one or ureaplasmas of the parvo biovar differ sufficiently more of the specific antigenic determinants associated from the T960T biovar of U. urealyticum to justify with the present T960T biovar of the human urea- http://ijs.sgmjournals.org 593 J. A. Robertson and others

Table 4. Practical protocol for identifying and discriminating between clinical isolates of Ureaplasma species isolated from humans

1 Identification to genus level (a and b): a Presence of tiny, mycoplasma-like colonies* on agar b Demonstration of the ability of these colonies to degrade urea, using either an endogenous† or exogenous urease test‡ or a urea-utilization test in liquid indicator medium 2 Identification to species level§ Amplification of species-specific DNA sequences by PCRR

* Colonies are usually between 15 and 60 µm in diameter (Shepard et al., 1974; Robertson, 1982) although larger colonies may develop when small numbers of them are present or cultivation is under unusual circumstances. See Fig. 2 (a, b) for variation in colony types. For details of current isolation and identification practices, see Waites et al. (2001). † Inclusion of indicator CaCl# in agar medium (Shepard, 1983). ‡ Addition of the urease spot test to selected colonies on agar (Shepard & Howard, 1970). § For routine clinical identification, the reporting of the isolation of Ureaplasma species or ‘ureaplasmas’ should both suffice and be taxonomically acceptable. The identification of ureaplasmas of humans to the species level is, at present, largely a research tool. For taxonomic purposes, a 16S rRNA-based PCR and a second PCR, based upon another gene region conserved among prokaryotes (e.g. the 16S–23S rRNA intergenic regions), should be considered. R The usefulness of biovar-specific PCRs for biotyping wild-type isolates has been demonstrated for serotyped isolates by using a pair of PCRs based upon 16S rRNA genes (Robertson et al., 1993) and multiple-banded antigen genes (Pitcher et al., 2001), and for non- serotyped isolates by using a number of different PCRs. These include single-test PCRs based upon the following: 16S rRNA genes (Jacobs et al., 1994; Abele-Horn et al., 1997); urease gene regions (Blanchard et al., 1993) by those workers and by Povlsen et al., 1998; multiple-banded antigen genes (PCRs; Teng et al., 1995) by Nelson et al. (1998) and Kong et al. (2000); and by an arbitrarily primed PCR (Grattard et al., 1995a, b).

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