Gastric Mucosa of Ferrets (Mustela putorius furo), and an Emended. Description of ..... This study was supported in part by Public Health Service grant. RR01046 ...
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Oct. 1988, p. 367-370 0020-7713/88/040367-04$02.00/0 Copyright 0 1988, International Union of Microbiological Societies
Vol. 38, No. 4
Campylobacter pylovi subsp. mustelae subsp. nov. Isolated from the Gastric Mucosa of Ferrets (Mustela putorius furo), and an Emended Description of Campylobacter pylovi J. G. FOX,l* N. S. TAYLOR,l PAUL EDMONDS,2 AND DON J. BRENNER3 Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139l; School of Applied Biology, Georgia Institute of Technology, Atlanta, Georgia 303322; and Division of Bacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 303333 The name Cumpylubucter pyluri subsp. mustelue subsp. nov. is proposed for a Campylobacter commonly isolated from normal or inflamed gastric mucosa of ferrets. C. pyluri subsp. mustelue, like C. pyluri, has multiple sheathed flagella, rapidly hydrolyzes urea, is H,S negative on triple sugar iron agar, and has a variable reaction on lead acetate strips. It does not grow in the presence of 3% NaCl, and growth is variable in 0.04% triphenyltetrazolium chloride and 1% glycine. Unlike C. pyluri, this organism reduces nitrate, is susceptible to nalidixic acid, and is resistant to cephalothin. Three strains of C. pylon' subsp. mustelue were highly related (286%) as determined by deoxyribonucleic acid (DNA)-DNA hybridization (hydroxyapatite method, 50 and 65°C). C. pyluri subsp. mustelue was 2 8 5 % related to C. pyluri, whereas the level of relatedness with another seven Campylubucter isolates was 1 2 % at 65°C. The type strain of C. pylon' subsp. mustelue is strain RS5-13-6 (= ATCC 43772), and its DNA has a guanine-plus-cytosinecontent of 38 mol%.
Since the genus Campylobacter was named in 1963 by Sebald and Vkron (20) to accommodate a group of bacteria previously classified as Vibrio, the number of Campylobacter species has grown to 14. One of the most recent additions is Campylobacter pylori, which was first isolated in 1982 and was described by Marshall et al. as "Campylobacter pyloridis" in 1984 (16). The name was later corrected to C. pylori (15). C. pylori is associated with gastritis and peptic ulceration in humans (15, 16). C. pylori was placed in the genus Campylobacter on the basis of overall phenotypic similarity, including similar guanine-plus-cytosine (G+C) content (15, 16). However, it was noted that C. pylori has four polar flagella that are sheathed, a property not found in other Campylobacter species (6, 9). In the original description, C. pylori was incorrectly reported to be unable to hydrolyze urea (16); however, it does, in fact, hydrolyze urea (5, 12, 15). C. pylori differs from other Campylobacter species in other chemotaxonomic characteristics, including the absence of methylated menaquinones (6), a different fatty acid composition (6, lo), and a different sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein banding pattern (21). Morphologically, C. pylori possesses sheathed flagella with terminal bulbs and has a smooth (not rugose) cell wall, features not found in other Campylobacter species (5, 9). Other morphological and subcellular features of C. pylori appear to resemble those of Aquaspirillum rather than those of other Campylobacter species (9). Recent 16s ribosomal ribonucleic acid (rRNA) sequencing data (13, 17, 19) indicate that C. pylori may be more closely related to Wolinella than to Campylobacter and that perhaps it should be reclassified either in Wolinella or in a new genus. In 1985, another gram-negative, Campylobacter-like (CLO) motile rod-shaped organism (3 by 0.5 to 1 km) was isolated from normal and inflamed gastric mucosa of ferrets (Mustela putorius furo) (3). Like C. pylori, this organism is oxidase positive and catalase positive, does not hydrolyze hippurate, and is strongly urease positive. Unlike C. pylori,
it is weakly nitrate positive, susceptible to nalidixic acid, and resistant to cephalothin. An electron microscopic examination indicated the presence of multiple sheathed flagella, both polar and at the side of the organism'(3; J. G. Fox, P. Edmonds, N. S. Taylor, B. Paster, and F. Dewhirst, in Proceedings of the IV International Campylobacter Workshop, Goterna, Sweden, p. 50). However, extensive phenotypic characteristics, G +C content, and deoxyribonucleic acid (DNA) hybridization patterns were not determined. Since the original report (3), this bacterium also has been isolated from gastric mucosa of ferrets residing in England and Canada (18; J. A. Hollingsworth et al., presented at the Conference on C. pylori Infections, Keystone, Colorado, 1987; D. S. Tompkins, A. P. West, P. G. R. Goodwin, J. I. Wyatt, and B. J. Rathbone, Abstr. IV Int. Workshop Campylobacter Infections, abstr. no. 161, 1987). In this paper, we present a further detailed characterization of the CLO isolates from ferrets; we determined phenotypic characteristics and levels of DNA relatedness to C. pylori, to other Campylobacter species, and to Wolinella species. MATERIALS AND METHODS Bacterial strains. A total of 21 CLO isolates from the gastric mucosa of ferrets were studied. Isolates 86-11 and 86-50 were cultured from pet ferrets obtained from Florida veterinary practitioners. The remaining isolates (R85-13-1 through R85-13-14, R86-2-13, 796, 829, 5951, and 7536) were from ferrets purchased from Marshall Farms, Inc., North Rose, N.Y., and used in biochemical research at the Massachusetts Institute of Technology, Cambridge, Mass. The CLO ferret isolates were cultured by previously described methods (3). The isolates were stored at -70°C in brucella broth containing 10% glycerol. For biochemical characterization, strains were grown on brain heart infusion broth or agar (supplemented with 5% sheep blood) in a microaerophilic atmosphere. Cultures were inoculated, placed in vented jars containing an N,-H,-CO, (80:10:10) mixture with a final 0, content of 5%, and incubated for 3 to 5 days. Culture purity was determined by microscopic examination of Gram-
* Corresponding author. 367
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SYST.
BACTERIOL.
TABLE 1. Phenotypic characteristics of C.pylori subsp. mustelae subsp. nov. isolated from the gastric mucosa of ferrets ( M . putorius furo)
Characteristic
Oxidase Catalase Urease Motility Hippurate hydrolysis Yellow pigment Growth at 25°C Growth at 37°C Growth at 42°C Growth on 1.5% NaCl Growth on 2.0% NaCl Growth on 3.0% NaCl Growth on 1.0% glycine Growth on 0.04% triphenyltetrazolium chloride
Reaction of type strain R85-13-6
+ + + +
-
+ +
-
-
-
+
Nitrate reduced to nitrites H2S (triple sugar iron) H2S (Trypticase soy broth, lead acetate strips) Susceptibility to: Nalidixic acid Cephalothin Metronidazole a
% of strains positive
100" 100 100 100 0 0 0 100 100 0 0 0 10 62 100 0
5
100 0 91
Strains giving less common result
R85-13-5, R85-13-9 5951, 7536, R85-13-9, R85-13-5, R86-2-13, R85-13-2, R85-13-1, R85-13-4 R86-2-13
R85-13-10, R85-13-11
n = 21.
stained cells. The type strains of Campylobacter species, Wolinella recta, and Wolinella succinogenes were obtained from the American Type Culture Collection, Rockville, Md. Phenotypic characterization. Tests were performed as described by Gebhart et al. unless otherwise stated (4). Motility was tested by the hanging drop method, using phasecontrast microscopy, and in semisolid medium. Nitrate broth was obtained from GIBCO Laboratories, Grand Island, N. Y. Hippurate hydrolysis was determined by using the method of Hwang and Ederer (8). Urease activity was assessed 5 min after inoculation of a culture onto urea agar slants (GIBCO). All other tests were read after 5 days of incubation. Colony morphology and microscopic observations were done on 72-h-old cultures. The presence of flagella was determined with selected strains grown on 5% sheep blood agar and examined after 72 h of growth. The cells were suspended in sterile water and were negatively stained with 3% phosphotungstic acid. The bacteria were then examined with a transmission electron microscope. DNA studies. Strains were heavily inoculated by streaking onto 30 to 50 Mueller-Hinton agar plates (150 by 15 mm) containing 5% defibrinated sheep blood and were incubated at 35 to 37°C in an atmosphere containing 10% CO,, 10% H,, and 80% N, (oxygen content, 5%) for 5 to 7 days. Cells were harvested and lysed, and DNA was extracted and purified as previously described (1). G+C contents were determined by thermal denaturation in a spectrophotometer (2). DNAs from CLO ferret isolate R85-13-6T (T = type strain) and C. pylori ATCC 43504T were labeled in vitro with [32P]deoxyribose cytidine triphosphate provided in a nick translation reagent kit (catalog no. 8160SB; Bethesda Research Laboratories, Inc., Bethesda, Md.) and were tested for relatedness to unlabeled DNAs from CLO ferret isolates R85-13-10, R85-13-6T, and R85-13-11, from C. pylori ATCC 43504T, and from strains of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter jejuni, Campylobacter laridis, Campylobacter sputorum, "Campylobacter upsaliensis," W . recta, and W . succinogenes.
The hydroxyapatite method was used at 50°C (the temperature for optimal DNA reassociation) and at 65°C (stringent reassociation conditions) (1).The levels of divergence within reassociated (related) DNA heteroduplexes were calculated to the nearest 0.5% on the assumption that a 1%decrease in thermal stability is caused by approximately 1%unpaired bases. RESULTS AND DISCUSSION
The gram-negative, microaerophilic, catalase-positive, thermotolerant, curved rod-shaped bacteria isolated from the gastric mucosa of ferrets phenotypically resemble Campylobacter species. Colonies on agar are 1 mm in diameter, nonpigmented, and translucent. They hydrolyze urea and are motile (Table 1). This combination of characteristics is present only in C. pylori among Campylobacter spp. Although C. pylori flagella are restricted to polar sites, the ferret isolates possess flagella at both lateral and polar locations. In addition to location of flagella and source isolation, the ferret isolates differ from C. pylori strains by the reactions shown in Table 2. The levels of DNA relatedness of three ferret isolates were 96% in 50°C reactions and 88% in 65°C reactions (Table 3). The level of divergence of related sequences was 1.0%. Labeled DNA from ferret isolate R85-13-6T was 100% related to unlabeled DNA from C . pylori ATCC 43504T at 50"C, with 1.0% divergence, and 85% related at 65°C. In reciprocal reactions, labeled C . pylori ATCC 43504T DNA was 92% related to unlabeled ferret isolate R85-13-6T DNA at 50"C, with 1.5% divergence, and 94% related at 65°C. The relatedness values for the ferret isolates and the C . pylori type strain fit the suggested phylogenetic definition of a single species (strains with approximately 70% or greater DNA relatedness with 5% or less divergence) (22). Several investigators have reported phenotypic and chemotaxonomic differences between C. pylori and other Campylobacter species. C . pylori is the only Campylobacter species that hydrolyzes urea (except for Campylobacter
CAMPYLOBACTER PYLORI SUBSP. MUSTELAE SUBSP. NOV.
VOL. 38, 1988
TABLE 2. Biochemical characteristics of gastric campylobacters Characteristic
Oxidase Catalase Glucose oxidation Glucose fermentation Nitrate Urease Alkaline phosphatase, esterase C4, esterase, lipase (CS), acid phosphatase, naphthol-AS-B1phosphohydrolase Leucine arylamidase H,S (lead acetate) Hippurate hydrolysis Susceptible to nalidixic acid Susceptible to cephalothin G1y cine Growth on triphenyltetrazolium chloride
+ +
+-
+ +
-
+
+
t b
+
-b
+I-
+/-
-
-
+
+/+/-
+/+/-
+
-
+, Positive; -, negative; +I-, variable. Data from reference 21. nigrofigilis) and that possesses sheathed flagella (16). C. pylori differs from other Campylobacter species in cellular fatty acid composition (6, lo), in the absence of methylated menaquinones (6), in sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein banding patterns (21), in cell wall structure, and in ultrastructure (6, 9). Von Wulffen reported that C . pylori DNA does not react with DNA from C. jejuni or C . coli in dot blot hybridization assays (21). This author used stringent (65°C) reassociation conditions, at which only faint reactions were observed between C. jejuni and C . coli, which are 30 to 50% related under these conditions (7). Relatedness values as high as 25 or 30% could be missed when these conditions are used, and the possibility of relatedness of C. pylori to species other than C. coli and C. jejuni was not tested. In our study, ferret isolate R85-13-6T was 0 to 2% related under stringent conditions to type or reference strains of seven Campylobacter species, 6% related to the type strain of W . succinogenes, and 4% related to the type strain of W. recta (Table 3). Romaniuk et al. (19) compared 16s rRNA sequences from C. pylori, five Carnpylobacter species, W . succinogenes, and
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Thiovulum species. C . pylori was 10% different from W . succinogenes, 16 to 17% different from Carnpylobacter species, and 20% different from Thiovulurn. Of the Campylubacter species tested, there are close homology with only (other than C. pylori) 2 to 8% differences. Romaniuk et al. concluded that C. pylori is no closer to Carnpylobacter than W . succinogenes and Thiovulum species are and that C. pylori and W . succinogenes probably merit a separate genus. Similar 16s rRNA sequencing results were obtained by Lau et al., who also used five Campylobacter species, C. pylori, and W . succinogenes (13). These results were confirmed and substantially expanded by Paster and Dewhirst, who concluded that on the basis of rRNA sequence data a strong argument can be made that C. pylori should be removed from Carnpylobacter and reclassified in the genus Wolinella (17). These authors did not make a formal proposal to remove C. pylori from Campylobacter pending the availability of additional phenotypic and rRNA-DNA hybridization data (17). Our initial studies indicate that on the basis of 16s rRNA sequencing data the ferret Campylobacter is closely related to C. pylori (Fox et al., IV Int. Campylobacter Workshop). The ferret isolates are phenotypically different from the human isolates of C. pylori and apparently have adapted to a different host. The inclusion of the ferret isolates in the species C. pylori necessitates emending the species description, and their differences from human isolates justify, as was previously done with C. fetus, proposing a separate subspecies as described below. Emended description of Campylobacter pylori (Marshall, Royce, Annear, Goodwin, Pearman, Warren, and Armstrong 1984) Marshall and Goodwin 1987. Fastidious, gram-negative microaerophilic, nonencapsulated, curved rods. Cells are 3 to 5 pm long and 0.5 to 1 pm wide with a spiral periodicity (14). Darting motility by means of multiple sheathed polar flagella or lateral flagella with terminal bulbs or both. Poor growth in most liquid media; grows in 2 to 5 days on brain heart infusion broth or agar, blood agar, and chocolate agar. Colonies are nonpigmented, translucent, and 1 mm in diameter (1416). Optimal growth at 37"C, no growth at 25"C, growth at 30"C, and variable growth at 42°C. No growth in the presence of 3% NaCl. Variable growth in 0.04% triphenyltetrazolium chloride and 1%glycine. Catalase positive. H,S production is negative on triple sugar iron agar and
TABLE 3. DNA relatedness of ferret isolates to each other, to C. pylori, and to representative Campylobacter and Wolinella species 32P0,-labeled DNA from: Source of unlabeled DNA
% Relatedness
at 50°C
Ferret isolate R85-13-6T Ferret isolate R85-13-11 Ferret isolate R85-13-10 C . pylori ATCC 43504T W . succinogenes ATCC 29543T W . succinogenes W602 W . recta ATCC 33238 C. laridis ATCC 35221T C. hyointestinalis ATCC 35217T C . fetus ATCC 27374T C. jejuni ATCC 33560T C. coli ATCC 33559T " C . upsaliensis" NCTC 11541 D, Divergence.
C . pylori ATCC 43504=
Ferret isolate R85-13-6T
100 100 92 100
% D"
0.0 1.0 1.0 1.0
% Relatedness
% Relatedness
100 88 87 85 6 6 4 2 1 0 0 0 0
92
at 65°C
at 50°C
17
%D
1.5
% Relatedness
at 65°C
94
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FOX ET AL.
is variable on lead acetate paper. Urea is rapidly hydrolyzed. Variable reactions for nitrate reduction and hippurate hydrolysis. Variable susceptibility to nalidixic acid, cephalothin, and metronidazole. Susceptible to ampicillin, amoxicillin, erythromycin, gentamicin, kanamycin, penicillin, rifampin, and tetracycline (11, 14). Resistant to vancomycin, lincomycin, and colistin. Isolated from the gastric mucosa of humans and ferrets. In humans, isolates found in cases of gastritis and gastric ulcers, but the role of the organism in these diseases, if any, has not been established. In ferrets, strains are isolated from normal or inflamed gastric mucosa obtained from either laboratory-maintained animals or pets. The G + C content in 36 to 38 mol%. The type strain is Royal Perth Hospital Isolate 13487 (= NCTC 11637 = ATCC 43504). It has a G+C content of 37 mol%. Description of Cumpylobucter pylori subsp. pylori subsp. nov. Cumpylobucter pylori subsp. pylori subsp. nov. (py. lor’i. L. gen. n. pylori, gatekeeper; of the pyloris). Polar flagella. Variable growth at 42°C. Produces H,S in the lead acetate test. Does not reduce nitrates. Resistant to nalidixic acid and susceptible to cephalothin. Exhibits leucine arylanidase activity in enzyme profiles (9). Isolated from the gastric mucosa of humans. The type strain is Royal Perth Hospital Isolate 13487 (= NCTC 11637 = ATCC 43504). Description of Cumpylobucter pylori subsp. mustelae subsp. nov. Cumpylobucter pylori subsp. mustelae subsp. nov. (mus. tel’ ae. L. gen. n. mustelae, of a ferret). Lateral as well as polar flagella (3; Fox et al., IV Int. Carnpylobacter Workshop). Grows at 42°C. Reduces nitrates. Susceptible to nalidixic acid and resistant to cephalothin. Does not exhibit leucine arylanidase activity in enzyme profiles (Tompkins et al., Abstr. IV Int. Workshop Campylobacter Infections). Isolated from the gastric mucosa of ferrets. The type strain is strain R85-13-6 (= ATCC 43772); it has a G+C content of 38 mol%. Characteristics of type strain R85-13-6 are given in Table 1. ACKNOWLEDGMENTS We thank Thomas Ozro MacAdoo, Department of Languages and Literature, Virginia Polytechnic Institute and State University, Blacksburg, for his expert advice on latinization of names. We also thank Charlotte M. Patton, Division of Bacterial Diseases, Centers for Disease Control, Atlanta, Ga., for a critique of the manuscript. This study was supported in part by Public Health Service grant RR01046 from the Division of Research Resources and by Public Health Service grants Pol-CA-28842 and Pol-CA-26731 from the National Cancer Institute. LITERATURE CITED Brenner, D. J. 1982. Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J. Clin. Microbiol. 15133-1140. De Ley, J. 1970. Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J. Bacteriol. 101:738-754. Fox, J. G., B. M. Edrise, E. B. Cabot, C. Beaucage, J. C. Murphy, and K. S. Prostak. 1986. Campylobacter-like organisms isolated from gastric mucosa in ferrets. Am. J . Vet. Res. 47:236-239. Gebhart, C. J., P. Edmonds, E. Ward, H. J. Kurtz, and D. J. Brenner. 1985. Campylobacter hyointestinalis sp. nov.: a new species of Campylohacter found in the intestines of pigs and
INT. J. SYST.BACTERIOL. other animals. J. Clin. Microbiol. 21:715-720. 5. Goodwin, C. S., E. Blincow, J. A. Armstrong, R. K. McCulloch, and D. Collins. 1985. Campylobacter pyloridis is unique: GCLO2 is an ordinary campylobacter. Lancet ii:38-39. 6. Goodwin, C. S., R. K. McCulloch, J. A. Armstrong, and S. H. Wee. 1985. Unusual cellular fatty acids and distinctive ultrastructure in a new spiral bacterium (Campylobacter pyloridis) from the human gastric mucosa. J. Med. Microbiol. 19:257-267. 7. Hebert, G. A., D. G. Hollis, R. E. Weaver, A. G. Steigerwalt, R. M. McKinney, and D. J. Brenner. 1983. Serogroups of Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus defined by direct immunofluorescence. J. Clin. Microbiol. 17:529-53 8. 8. Hwang, M., and G. M. Ederer. 1975. Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. J. Clin. Microbiol. 1:114-115. 9. Jones, D. M., A. Curry, and A. J. Fox. 1985. An ultrastructural study of the gastric campylobacter-like organism “Campylobacter pyloridis.” J. Gen. Microbiol. 131:2335-2341. 10. Lambert, M. A., C. M. Patton, T. J. Barrett, and C. W. Moss. 1987. Differentiation of Campylobacter and Campylobacter-like organism by cellular fatty acid composition. J. Clin. Microbiol. 25706-713. 11. Lambert, T., F. Megraud, G. Gerbaud, and P. Courvalin. 1986. Susceptibility of Campylobacter pyloridis to 20 antimicrobial agents . Antimicrob. Agents Chemother. 30:510-5 11. 12. Langenberg, M. L., G. N. J. Tygat, M. E. I. Schipper, P. J. G. M. Rietta, and H. C. Zanen. 1984. Campylobacter-like organisms in the stomach of patients and healthy individuals. Lancet i:1348. 13. Lau, P. P., B. DeBrunner-Vossbrinck, B. Dunn, K. Miotto, M. T. MacDonnell, D. M. Rollins, C. J. Pillidge, R. B. Hespell, R. R . Colwell, S. L. Mitchell, and G. E. Fox. 1987. Phylogenetic diversity and position of the genus Campylobacter. Syst. Appl. Microbiol. 9:23 1-238. 14. Marshall, B. J., and C. S. Goodwin. 1987. Revised nomenclature of Carnpylobacter pyloridis. Int. J. Syst. Bacteriol. 37:68. 15. Marshall, B. J., D. B. McGechie, P. A. Rogers, and R. J. Glancy. 1985. Pyloric campylobacter infection and gastroduodenal disease. Med. J. Aust. 142:439-444. 16. Marshall, B. J., H. Royce, D. I. Annear, C. S. Goodwin, J. W. Pearman, J. R. Warren, and J. A. Armstrong. 1984. Original isolation of Campylobacter pyloridis from human gastric mucosa. Microbios Lett. 2583-88. 17. Paster, B. J., and F. E. Dewhirst. 1988. Phylogeny of campylobacters, wolinellas, Bacteroides gracilis, and Bacteroides ureolyticus by 16s ribosomal ribonucleic acid sequencing. Int. J. Syst. Bacteriol. 3856-62. 18. Rathbone, B. J., A. P. West, J. I. Wyatt, A. W. Johnson, D. S. Tompkins, and R. V. Heatley. 1986. Campylobacter pyloridis, urease, and gastric ulcers. Lancet i:400-401. 19. Romaniuk, P. J., B. Zoltowaska, T. J. Trust, D. J. Lane, G. J. Olsen, N. R. Pace, and D. A. Stahl. 1987. Campylobacter pylori, the spiral bacterium associated with human gastritis, is not a true Campylobacter sp. J. Bacteriol. 169:2137-2141. 20. Sebald, M., and M. Veron. 1963. Teneur en bases de 1’ ADN et classification des vibrions. Ann. Inst. Pasteur (Paris) 105897910. 21. Von Wulffen, H. 1987. Low degree of relatedness between Campylobacter pyloridis and enteropathogenic Campylobacter species as revealed by DNA-DNA blot hybridization and immunoblot studies. FEMS Microbiol. Lett. 42:129-133. 22. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, 0. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Triiper. 1987. Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Int. J. Syst. Bacteriol. 37:463464.
