international Journal of Systematic Bacteriology (1 999), 49, 1695-1 700 Printed in Great Britain

Phylogenetic evidence for reclassification of Calymmatobacterium granulomatis as granulomatis comb. nov.

Jenny 5. Carter,’l2 Francis J. B~wden,~Ivan Ba~tian,~Garry M. Myers,’ K. S. Sriprakash’ and David J. Kemp’

Author for correspondence : David J. Kemp. Tel : + 6 18 8922 84 12. Fax : + 6 18 8927 5 187 e-mail : [email protected]

1 Menzies School of Health By sequencing a total of 2089 bp of the 16s rRNA and phoE genes it was Research, Darwin, demonstratedthat Calymmatobacterium grandomatis (the causative Austra Iia organism of donovanosis) shows a high level of identity with Klebsiella * Centre for Indigenous species pathogenic to humans (Klebsiellapneumoniae, Klebsiella Natural and Cultural Resource Management, rhinoscleromatis). It is proposed that C. grandomatis should be reclassified as Faculty of Aboriginal and Klebsiella granulomatis comb. nov. An emended description of the Torres Strait Islander Klebsiella is given. Studies, Northern Territory University, Darwin,

3 Institute of Medical and Keywords : Calymmatobacteriurn, Klebsiella, sequence data, phylogenetic inferences Veterinary Science, Adelaide, Australia

4 AIDS/STD Unit, Royal Darwin Hospital, Darwin, Australia

Calymmatobacterium granulomatis is the presumed ganism (Richens, 1991) have prevented further char- causative agent of donovanosis, an important cause of acterization of this relationship. genital ulceration that occurs in small endemic foci in all continents except Europe and Antarctica. The name Non-cultivable pathogenic eubacteria have been C. granulomatis was originally given to the pleo- identified by PCR using primers targeting conserved morphic bacterium cultured from donovanosis lesions genes (Fredricks & Relman, 1996). We have shown by Aragiio & Vianna (1913). Although these early through sequencing a 334 bp region of the phosphate cultures are highly dubious and were probably not of porin (phoE) gene that C. granulomatis has a high the organism itself (Richens, 1985), the name C. degree of molecular identity with other Klebsiella granulomatis has retained precedence over others. species in this region (Bastian & Bowden, 1996). We present here an analysis of the almost complete 16s C. granulomatis is known to be an encapsulated, non- rRNA and phoE sequences for C. granulomatis, K. motile, facultatively anaerobic, Gram-negative bac- rh inoscleromat is and . terium (Chandra & Jain, 199 1 ; Davis, 1970; Davis & At least two punch biopsies or swabs were obtained Collins, 1969; Dodson et al., 1974; Kuberski et al., from the lesions of eight patients with clinical dono- 1980). C. granulomatis has been associated with the vanosis. One punch biopsy was fixed and examined for genus Klebsiella because of the above characteristics Donovan bodies by the slow Giemsa technique (Sehgal and common antigenicity (Maddocks et al., 1975; & Jain, 1987) whilst DNA was extracted from the Packer & Goldberg, 1950). Furthermore, two members second punch biopsy (seven samples) (Mullenbach et of the genus Klebsiella that produce clinical disease al., 1989) or swab (one sample) (Beige et al., 1995). and pathologic changes very similar to those of C. DNA was also obtained from type cultures of K. granulomatis are Klebsiella rhinoscleromatis (Levine & pneumoniae [NCTC 9633T (National Collection of Hoyt, 1947; Shaw & Martin, 1961; Welsh et al., 1963), Type Cultures)] and K. rhinoscleromatis (NCTC which is implicated in a granulomatous disease of the 5046T). nose, and Klebsiella ozaenae (Richens, 1985) which is implicated in chronic atrophic rhinitis. Richens (1985) Primer sets used in this study include (5’-ACCTA- goes so far as to place C. granulomatis in the genus CCGCAACACCGACTTCTTCGG-3’ and 5’-TGA- Klebsiella, although difficulties in culturing the or- TCAGAACTGGTAGGTCAT-3’, 604 bp phoE prod-

01071 0 1999lUMS 1695 J. S. Carter and others

Table 7. Strain designations and accession numbers for sequences obtained from the GenBank and EMBL databases, and for sequences novel to this study ......

Abbreviations : NCTC, National Collection of Type Cultures; ATCC, American Type Culture Collection; DSM, DMSZ - Deutsche Sammlung fiir Mikroorganismen und Zellkuturen ; JCM, Japanese Collection of Microorganisms ; PAH, Princess Alexandra Hospital, Brisbane, Australia; T, type Culture.

Organism 16s rRNA phoE

Strain Accession Strain Accession

Calymmatobacteriurn granulomatis * AF009 17 17 * AF00923 17 Klebsiella rhinoscleromatis NCTC 5046T AF009 1697 NCTC 5046T AF0092307 Klebsiella pneumoniae DSM 30104T X87276 NCTC 9633T AF064793T JCM 1665 AB004754 K26 X68022 Klebsiella ornithinolytica JCM 7251 AB004756 Klebsiella plan ticola DSM 3069T X932 15 l A04378 Enterobacter aerogenes JCM 1235 AB004750 ATCC 29935 M5929 1 1 X6802 1 Esc her ic h ia co li ATCC 43895 283205 K12 V003 16 ATCC 13880 M59 160 UOC-5 1 U8 1967 Salmonella typhi Stlll US8545 1 x74595 PAHll472 X68674 * Sequences obtained from clinical specimens. t Sequences novel to this study. 5: Strain not specified in GenBank report.

uct) ; (5’-ATGAAAAAGAGTACTCTGGCA-3’and firming and extending the results obtained previously 5’-CAGACCGAAGTCGAACTGATACTG-3’, (Bastian & Bowden, 1996). 840 bp phoE product) ; (5’-CCTAACACATGCAA- GTCGA-3’ and 5‘-ATCCGACTTGACAGACCG-3’, In order to further investigate this relationship we 538 bp 16s rRNA product); (5’-CGGTCTGTCA- determined the sequence of a coding region spanning AGTCGGAT-3’ and 5’-TGTAGCACGTGTGTA- 95% of the phoE gene (1001/1059 bp) from K. GCC-3’, 653 bp 16s rRNA product). pneumoniae, K. rhinoscleromatis and C. granulomatis isolates, along with sequences for a 1088 bp region of One microlitre of template DNA was included in a the 16s rRNA from C. granulomatis and K. rhino- 25 pl PCR mixture that comprised the following scleromatis. The 16s rRNA sequence of K.pneumoniae solution: 10 mM Tris/HCl pH 8.0, 50mM KCl, [DSM 30104T(DSMZ, Deutsche Sammlung fur Mikro- 2.5 mM MgCl,, 0.01 YOgelatin, 0-2 pM dNTPs, 0.5 pM organismen und Zellkulturen)] was obtained from the primers and 0.5 Units Taq polymerase. Samples were GenBank database. These sequences were aligned with subjected to 30 cycles of denaturation (94 “C, 40 s), the sequences of other Klebsiella sp. and related annealing (55 “C, 40 s forphoE primers; 52 “C, 40 s for available from the GenBank/ 16s primers) and extension (72 “C, 60 s) in a thermal EMBL databases using the program CLUSTAL w cycler (Corbett Research, Sydney). All steps of PCR (Thompson et al., 1994). Due to the limited amount of template preparation, PCR mix and gel analysis were complete phoE sequence data available, Klebsiella conducted in three separate dedicated workrooms. planticola, Klebsiella ornithinolytica and Yersinia enter- Template negative controls were included in each ocolitica were only included in the 16s rRNA align- reaction. Products of the expected size were purified ment. For similar reasons different species of Entero- and sequenced in both directions using either the dye bacter have been compared for the 16s rRNA and terminator method (Applied Biosystems) or a manual phoE genes. T7 sequencing kit (Pharmacia-Biotech). Phylogenetic analyses was performed using the PHYLIP Initially, a 604 bp region of phoE was amplified and software package (Felsenstein, 1993). Programs em- sequenced from 4/4 patients with histologically con- ployed included SEQBOOT: to produce multiple data firmed donovanosis and from 4/4 patients with nega- sets by bootstrap resampling (100 iterations) ;DNADIST : tive histology for whom donovanosis remained the to compute distances between species for all data sets clinical diagnosis. The same Klebsiella-like sequence with the Jukes-Cantor algorithm; NEIGHBOR : to pro- was amplified from all eight patients thereby con- duce 1000 trees (100 data sets x 10 jumbled input

1696 lnternational Journal of Systematic Bacteriology 49 Phylogenetic analysis of C. granulornatis

~~

to this study are provided in Table 1 with the strain Yersinia enterocolitica designations. Phylogenetic trees, including the con- fidence values of branching are shown as cladograms Klebsiella pneumoniae in Fig. 1 for the 16s rRNA andphoE. It is evident from I tree topologies that C. has a close Calymmatobacterium granulomatis granulornatis phylogenetic relationship with members of the Klebsiella rhinoscleromatis Klebsiella genus known to be human pathogens (K. 71 rhinosclerornatis, K. pneurnoniae). Bootstrap analysis ,-- Serratia marcescens data confirmed that this association was highly sig- nificant for both the phoE and the 16s rRNA genes -1 -1 r Salmonellatyphi (bootstrap values 100 YO).Nucleotide similarities be- 54 tween the three isolates ranged from 98.8-99*8% for the 16s rRNA and 99.7-99.8 YOfor phoE. IKlebsiella oxytoca PCR-based identification of non-cultivable microbial pathogens may be confounded by the incidental Enterobacler aerogenes amplification of colonizing non-pathogenic . Citrobacterfreundii Fredricks & Relman (1996) have proposed several '"4 criteria for establishing microbial disease causation by r Klebsiella planticola molecular methods. Their guidelines stipulate that : the sequence-based identification should be reproducible ; L Klebsiella ornithinolytica the nucleic acid sequence should be found at its highest concentration in diseased tissue but be absent, or (b) present at only low copy numbers, in normal tissue; Serratia marcescens the relationship should be biologically plausible with the known phenotypic characteristics of the non- Klebsiella oxytoca cultivable organism ; and ideally the molecular identi- fication should be established at the cellular level by in situ hybridization. 67 991I ,- Klebsiella pneumoniae The studies described in this and our other papers U (Bastian & Bowden, 1996; Carter et al., 1997, 1999) 1001 ICalymmatobacterium granulomatis fulfil these criteria for associating the causative or- ganism of donovanosis with the genus Klebsiella. In IKlebsiella rhinosckromatis this paper, the clinical samples from all eight patients with clinical donovanosis produced PCR products that demonstrated greater than 99 similarity with ,- ,- Enterobacter cloacae Yo K. I pneurnoniae and K. rhinosclerornatis (i.e. the associ- ation is reproducible). We have developed a diagnostic PCR based on the observation that two unique base changes in the phoE gene of C. granulornatis eliminate Citrobacterfreundii HaeIII restriction sites (Carter et al., 1999). All 14 7 clinical donovanosis samples tested with this diag- nostic method gave the restriction digest profile ex- "'L Salmonella typhi pected from sequence data. No products were obtained from patients with unrelated genital conditions (i.e. the Fig. 1. Phylogenetic trees showing the relationship of C. molecular identification is specific). As described grandomatis, members of the genus Klebsiella and some earlier, this molecular association of C. granulornatis related members of Enterobacteriaceae. Bootstrap values, with the Klebsiella genus is biologically plausible based expressed as percentages, are given at branch points. Yersinia enterocolitica and Serratia marcescens were used as the out- on the known phenotypic and antigenic characteristics groups for 165 rRNA and phoE analysis respectively: (a) 165 of the organism. Finally, though in situ hybridization rRNA, comparison of 1088 bp; (b) phof, comparison of 1001 bp. experiments have not been pursued to demonstrate a All GenBanUEMBL accession numbers and strain designations tissue-sequence correlation at the cellular level, we are shown in Table 1. have recently been able to cultivate C. granulornatis in a human epithelial cell line. DNA obtained from the cultured organisms had an identical phoE sequence to orders) with the neighbour-joining method ;CONSENSE : that obtained from clinical specimens (Carter et al., to compute the strict consensus tree by the majority- 1997). rule consensus tree method. We believe that in light of our data it is reasonable to Accession numbers for sequences obtained from the consider reclassifying C. granulomatis as Klebsiella GenBank/EMBL databases and for sequences novel granulornat is.

~ International Journal of Systematic Bacteriology 49 1697 J. S. Carter and others

Emended description of the genus Klebsiella to the cell wall are often seen and may be attached to or detached from the cell wall (Dodson et al., 1974; Klebsiella species are facultatively anaerobic, Gram- Kuberski et al., 1980; Chandra & Jain, 1991). negative, non-motile, generally straight rods arranged singly, in pairs or in short chains and measuring Staining properties. Gram-negative. Well seen with 0.3-1-0 pm in diameter and 0.6-6-0 pm in length Giemsa, Leishman, Wright's or Silver stains. Poorly (Orskov, 1984). K. granulomatis is pleomorphic visualized with haematoxylin and eosin (Richens, (Dienst & Bronwell, 1984), i.e. curved or straight rods, 1991). Periodic acid Schiff-negative (Spagnolo et al., coccoid, diplococcoid, ovoid or elliptical in shape. 1984; Richens, 1991). Most Klebsiella species are capsulated (0rskov, 1984) Cultural and growth conditions. K. granulomntis is though K. granulomatis may be capsulated (mature facultatively intracellular, residing in the cytoplasm of form) or non-capsulated (immature form) (Hart, large mononuclear cells and occasionally within poly- 1997). Most species are cultivable on routine micro- morphonuclear leukocytes (Dodson et al., 1974 ; biological media (0rskov, 1984). K. granulomatis is Kuberski et al., 1980; Spagnolo et al., 1984; Chandra facultatively intracellular, residing within the cyto- & Jain, 1991). In the yolk sac of the developing chick plasm of large mononuclear cells and cannot be embryo a conspicious feature is its residence within cultured on routine microbiological media (Dienst & epithelial cells (Anderson, 1943). Successful cultures Bronwell, 1984). There are currently five recognized have been achieved in vivo in the yolk sac of developing species of Klebsiella : K. pneumoniae, Klebsiella oxy- chick embryos (Anderson, 1943) and in the developing toca, Klebsiella terrigena, Klebsiella planticola and chick embryo brain (Thomison, 195 1). Cultures in Klebsiella ornithinolytica (Orskov, 1984 ; Sakazaki et vitro have been achieved utilizing fresh yolk containing al., 1989) and three recognized subspecies of K. embryonic chick heart (Anderson, 1943) ; chick em- pneumoniae ; K. pneumoniae subsp. pneumoniae, K. bryo amniotic fluid (Anderson et al., 1945) ; semi-solid pneumoniae subsp. ozaenae and K. pneumoniae subsp. medium containing peptone, tryptone, dextrose, sea rhinoscleromatis (Orskov, 1984). In the absence of salt, agar and fresh yolk (Dienst, 1948) ;slants prepared DNA-DNA hybridization studies it is not possible to from beef heart infusion agar and fresh yolk (Dunham determine whether K. granulomatis is a sixth Klebsiella & Rake, 1948) ; Locke-Yolk agar with a Locke solution species or a fourth subspecies of K.pneumoniae. For a overlay (Dulaney et al., 1948) ; thioglycollate broth complete description of the characteristics of the genus with lactalbumin hydrosylate (enzymic digest of albu- Klebsiella see Orskov (1984). min) or Phytone (enzymic digest of soya meal) added (Goldberg, 1959) ; fresh mononuclear cells (Kharsany Description of Klebsiella grandomatis comb. nov. et al., 1996, 1997) and a human epithelial cell line (Carter et al., 1997). The optimal temperature for Kelbsiella granulomatis (gran.u.lo'ma. tis. L. dim. n. growth of K. granulomatis is 37 "C (Anderson, 1943; granulum a small grain; Gr. suff. -oma a swelling or Beveridge, 1946; Dienst & Bronwell, 1984). Two tumour; M.L. n. granuloma a granuloma; M.L. gen. factors present within the yolk sac of developing chick n. granulomatis of a granuloma). embryos have been found to be essential for growth. Cell characteristics. Gram-negative (Dienst & These are a micro-aerophillic environment (Anderson, Bronwell, 1984). Non-sporulating (Richens, 1985). 1943 ; Dienst, 1948 ;Goldberg, 1959) and a polypeptide Non-motile (Dienst & Bronwell, 1984). K. granu- present in the enzymic digests of bovine albumin and lomatis is pleomorphic when observed in a single plane soya meal (Goldberg, 1959). (Dienst & Bronwell, 1984), i.e. curved or straight rods, Storage conditions. K. granulomatis will not remain coccoid, diplococcoid, ovoid or elliptical in shape. viable when stored at 5 or 37 "C (Anderson, 1943). It Mature forms are capsulated, ovoid to elliptical in has been reported that stock egg yolk cultures remain shape and measure 0-5-0-7 pm in diameter and viable for extended periods when stored at 25 "C 1-0-1.5 pm in length (Rajam & Rangiah, 1954; Sehgal (Anderson, 1943) though Dienst (1948) could only & Sharma, 1992; Hart, 1997). Immature forms are maintain viability of cultures in fresh yolk medium for non-capsulated, coccoid, diplococcoid or bacillary in 8-10 d at 25 "C. shape and measure 0-6-1-0pm in length (Rajam & Rangiah, 1954; Sehgal & Sharma, 1992; Hart, 1997). Genetic data. The mol YOG + C content is unavailable Immature, non-capsulated forms may appear like due to the absence of DNA-DNA hybridization closed safety pins with certain stains due to bipolar studies. K. granulomatis shows 99-7-994 YOnucleotide chromatin densities (Rajam & Rangiah, 1954; Sehgal similarity with K.pneumoniae and K. rhinoscleromatis, & Sharma, 1992; Hart, 1997). Division is via in- respectively, in the gene encoding the outer-membrane vagination of the cell wall and cytoplasmic membrane phosphate porin and 98.8-99.8 Yo nucleotide similarity (Anderson et al., 1945; Davis & Collins, 1969; with K. pneumoniae and K. rhinoscleromatis, respect- Spagnolo et al., 1984; Sehgal & Sharma, 1992). ively, in the 16s rRNA gene. Filamentous processes recognized as pili and fimbriae Pathogenicity and habitat. K. granulomatis is not are present on the surface of most organisms (Dodson pathogenic for mice, dogs, chickens, the chorio- et al., 1974; Kuberski et al., 1980; Chandra & Jain, allantoic membrane of chick embryos, rabbits, guinea 1991). Numerous round vesicles or blebs endogenous pigs, Macacus rhesus monkeys, sheep, goats, pigs or

1698 lnterna tional Journal of Systematic Bacteriology 49 Phylogenetic analysis of C. granulomatis cows (Anderson, 1943; Anderson et al., 1945; Davis, C. (1970). . A clinical, histological, Beveridge, 1946; Dienst et al., 1949). With the ex- and ultrastructural study. J Am Med Assoc 211, 632-636. ception of the developing chick embryo (and other Davis, C. & Collins, C. (1969). An ultrastructural study of types of eggs) K. granulomatis is pathogenic only for Calymmatobacterium granulomatis. J Invest Dermatol 53, humans (Dienst & Bronwell, 1984) where infection 3 15-320. results in the chronic genital ulcerative disease known DeMonbreun, W. & Goodpasture, E. (1933). Further studies on as donovanosis. K. granulomatis has been assumed to the etiology of granuloma inguinale. Am J Trop Med 13, be sexually transmitted (Rajam & Rangiah, 1954; 447463. Sengupta, 1981; Spence, 198%; Sehgal & Sharma, Dienst, R. 6. (1948). Laboratory diagnosis of granuloma in- 1992; Hart, 1997) though an enteric habitat has been guinale and studies on the cultivation of the donovan body. Am postulated (DeMonbreun & Goodpasture, 1933 ; J Syph Gonor Vener Dis 32, 301-306. Dunham & Rake, 1948; Goldberg, 1962, 1964). Dienst, R. 6. & Bronwell, G. H. (1984). Genus Calymmato- Goldberg (1962) isolated and cultured K. granulomatis bacterium Aragiio and Vianna 191 3,22 1 -4L. In Bergey’s Manual from the faeces of a patient with donovanosis and of Systematic Bacteriologjj, vol. 1, pp. 585-587. Edited by N. R. DeMonbreun & Goodpasture -(1933) also claim to Krieg. Baltimore, MD: Williams & Wilkins. have achieved successful isolation (though not culture) Dienst, R. B., Chen, C. H. & Greenblatt, R. B. (1949). Experimental of K. granulornatis from the faeces of 2/4 patients with studies on the pathogenicity of Donovania granulomatis. Am J donovanosis. It is currently unknown whether K. Syph Gonor Vener Dis 33, 152-1 57. granulornatis also has a natural environmental habitat. Dodson, R., Fritz, G., Hubler, W., Jr, Rudolf, A., Knox, J. & Chu, L. (1974). Donovanosis: a morphologic study. J Invest Dermatol Type culture. Due to difficulties encountered in the 62, 611. storage of no type culture is currently K. granulomatis, Dulaney, A. D., Guo, K. & Packer, H. (1948). Donovania granu- available. lomatis : cultivation, antigen preparation, and immunological tests. J Imrnunol59, 335-340. Acknowledgements Dunham, W. & Rake, G. (1948). Cultural and serological studies on granuloma inguinale. Am J Syph Gonor Vener Dis 32, This study was funded by Territory Health Services, North- 145-1 49. ern Territory Government. J. S. C. is in receipt of a sholarship Felsenstein, 1. (1 993). PHYLIP (Phylogeny Inference Package) from the National Health and Medical Research Council of version 3.52. Distributed by the author. Department of Gen- Australia. etics, University of Washington, Seattle. Fredricks, D. N. & Relman, D. A. (1996). Sequence-based identi- References fication of microbial pathogens : a reconsideration of Koch’s postulates. Clin Microbiol Rev 9, 18-33. Anderson, K. (1943). The cultivation from granuloma inguinale of a microorganism having the characteristics of Donovan Goldberg, J. (1959). Studies on granuloma inguinale. IV. Growth bodies in the yolk sac of chick embryos. Science 97, 56&561. requirements of Donovania granulomatis and its relationship to the natural habitat of the organism. Br J Vener Dis 35,266-268. Anderson, K., DeMonbreun, W. A., & Goodpasture, E. W. (1945). An etiologic consideration of Donovania granulomatis cul- Goldberg, J. (1962). Studies on granuloma inguinale. V. Isolation tivated from granuloma inguinale (three cases) in embryonic of a bacterium resembling Donovania granulomatis from the yolk. J Exp Med 81, 25-40. faeces of a patient with granuloma inguinale. 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