(2) 3556. KRAAZ ET AL.. J. CLIN. MICROBIOL.. taken from the terminal ileum, cecum, ascending colon, transverse colon, descending colon, colon sigmoideum, and rectum. Corresponding biopsy specimens were used as inocula for the culture of spirochetes and taken for both routine histology and immunostaining with an antispirochete polyclonal antibody. Histopathology. The biopsy specimens were processed and paraffin embedded for histological examination by standard techniques. The paraffin sections were stained with hematoxylin-eosin (HE) and examined at ⫻400 magnification. Immunohistochemistry. A polyclonal rabbit antiserum was produced by intravenously immunizing rabbits with suspensions of an “uncharacterized” intestinal spirochete. The spirochete had been isolated from a colonic mucosal biopsy specimen in a previous case of human IS at the University Hospital, Uppsala. The specificity of the antibody was determined by indirect immunofluorescence (27) with the type strains of five species: human B. aalborgi and porcine Brachyspira hyodysenteriae, Brachyspira innocens, Brachyspira murdochii, and B. pilosicoli. A strain of Escherichia coli was used as a negative control. The immunohistochemical examination was carried out on paraffin sections using the avidin-biotin complex (ABC) method (34). Electron microscopy. For transmission electron microscopy (TEM), the tissue was cut from one paraffin block in small cubes measuring about 2 mm3. The tissue was deparaffinized, postfixed in 1% osmium tetroxide (OsO4) in phosphate buffer (pH 7.4), embedded in Epon (agar 100 resin), stained with uranyl acetate and lead citrate, and thereafter mounted on copper grids. The grids were analyzed with a Philips 420 electron microscope, operating at 60 kV. Isolation and culture of spirochetes. Isolation was attempted on seven specimens of biopsy material obtained during colonoscopy from sites covering the ileum-colon-rectum of the patient. The biopsy specimens were streaked onto agar plates within 1 h after sampling. A selective medium was used, consisting of tryptose soy agar (TSA) to which was added 10% bovine blood, 400 g of spectinomycin per ml, and 5 g of polymyxin per ml (15). The same medium, but without antibiotics, was used to determine the intensity of beta-hemolysis and for maintenance of the isolates. Spirochetes were also cultured on FA agar (Fastidious Anaerobe agar; Lab M code LAB 90 BaktDia; National Veterinary Institute, Uppsala, Sweden) and in two liquid media, brain heart infusion (BHI) broth with 5% calf blood and a Borrelia medium, BSK-H (Sigma). Finally, an attempt was made to culture the spirochetes on Serpulina agar plates (blood agar base. number 2 [Oxoid code CM 271], supplemented with 5% citrated sheep blood, 1% sodium ribonucleat, 25 g of vancomycin HCl solution, 25 g of colistin sulfate, and 800 g of spectinomycin per ml). All plates were incubated in anaerobic jars at 37°C for 5 to 28 days under an atmosphere of 90 to 95% H2 and 5 to 10% CO2. Plates were also incubated in a cabinet (model 1024/1028; Forma Scientific) under an atmosphere of N, CO, and H (80:10:10). Growth of spirochetes was confirmed, and the organisms were studied, by phase-contrast microscopy (1,000⫻). After examination and biochemical testing, cultures were frozen and stored in liquid nitrogen (⫺196°C). The storage medium used was beef broth with 10% horse serum and 15% glycerol. Plates containing the spirochetes were also stored anaerobically at room temperature for as long as 3 months. Type strains of six species of intestinal spirochetes, B. hyodysenteriae B78, Brachyspira intermedia PWS/A, B. innocens B256, B. murdochii 155-20, B. pilosicoli P43, and B. aalborgi 513A (NCTC 11,492), originating from a strain collection at the National Veterinary Institute, were cultured as previously described (7). Biochemical testing. The enzymatic reactions of isolated human spirochetes and the type strains were tested with the API-ZYM system, as described by the manufacturer (API, Marcy-I’Etoile, France). A spot test (28) was used to determine indole production by smearing the growth from a culture onto a filter paper saturated with the indole reagent (1% p-dimethylaminocinnamaldehyde in 10% hydrochloric acid). The method of Rübsamen and Rübsamen (26) was used to test for hippurate hydrolysis. PCR amplification, sequencing, and sequence analysis of the 16S rDNA gene. A pair of primers to specifically detect the presence of the 16S rDNA gene of spirochetes of the genus Brachyspira (Serpulina) was designed from sequences obtained from the GenBank sequence database. Primers 5⬘-GTCTTAAGCAT GCAAGTC and 3⬘-AACAGGCTAATAGGCCG, generating a 207-bp fragment, were used. The PCR assay was carried out on two biopsy specimens taken during colonoscopy on two subsequent occasions from the patient in this study. The PCR amplicons were used directly for sequencing (U. Thunberg, unpublished data). The virtually complete 16S rDNA sequence of the isolated spirochete was determined by direct solid-phase DNA sequencing with primers and protocols as. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. FIG. 1. HE-stained section shows a hematoxyphilic fringe on the brush border of the colonic surface epithelium. The fringe consists of microorganisms attached end-on to the epithelial surface..
(3) DIAGNOSIS OF BRACHYSPIRA AALBORGI. VOL. 38, 2000. 3557. described previously (25). This sequence and the partial 16S rDNA sequences were aligned with that of B. aalborgi strain 513AT (accession number Z22781). Nucleotide sequence accession number. The virtually complete 16S rDNA sequence of the isolated spirochete was deposited in GenBank under accession number AF200693.. RESULTS Light microscopy. IS was diagnosed histologically in HEstained sections by the presence of a hematoxyphilic “fuzzy coat” on the brush border of the surface epithelium. Spirochete attachment to epithelial cells was present in all sections of the large intestine, with decreasing intensity, however, from the cecum to the rectum. No bacteria were seen in the crypts. At ⫻400 magnification this coat seemed to consist of a forest of thin sinusoidal microorganisms attached end-on to the cell membrane (Fig. 1). The mucosal crypts showed normal architecture and undamaged goblet cells. There was no evidence of colitis. Immunohistochemistry. The antiserum reacted strongly with the spirochetal antigen in the paraffin sections, producing a marked contrast of the fuzzy coat against the epithelial surface with a preserved spiral shape of the microbes (Fig. 2). No further antigen deposits were detected below the surface epithelium, either in the crypts or in the lamina propria. The polyclonal antibody reacted with all the tested type strains of the intestinal spirochetes, but not with the E. coli strain, in the indirect immunofluorescence test.. Electron microscopy. Numerous spiral-shaped microorganisms attached to the luminal surface of the epithelium were observed (Fig. 3). Except for effacement of the microvilli, the epithelial cells appeared unaffected. The spirochetes were always attached end-on to the cell membrane, between and parallel to the microvilli. Condensation of uncharacterized electron-dense material was observed along the apical cytoplasm of colonized epithelial cells. Culture of human spirochetes. After 1 to 2 weeks of incubation, a thin haze of small, pinpoint-like colonies of bacterial growth in a mixed flora could be seen on all seven agar plates inoculated with biopsy material. Phase-contrast microscopy revealed spirochetes and unclassified cocci. Spirochetes were routinely transferred to fresh TSA and FA agar plates every 2 weeks to obtain pure cultures for further analyses. With the exception of the Serpulina plates, where only poor growth was achieved, the types of solid medium used did not appear to affect either the number of organisms or the type of growth. The spirochetes grew well in both of the liquid media tested, BSK-H (Sigma) and BHI broth with 5% calf blood. Viable spirochetes could be recovered from plates which had been kept in an anaerobic jar for more than 3 months at room temperature. Enhanced microbial growth was achieved after subculturing. The first appearance of bacterial growth was then seen after 5 to 7 days of incubation as pinpoint-like, transparent colonies with weak hemolytic activity. After 2 to 4 weeks, the spirochete colonies fused, forming opaque, grayish carpets. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. FIG. 2. Upon immunostaining the microorganisms are seen to react with the spirochete antiserum, producing a marked contrast with the fringe detected over the colonic mucosal surface. Antigen deposits cannot be detected in the lamina propria..
(4) 3558. KRAAZ ET AL.. J. CLIN. MICROBIOL.. at the sites of inoculation. Spirochetes from one plate were selected for further studies. This isolate was designated W1. Biochemical testing. The enzymatic and biochemical reactions of the isolated human spirochete W1 and the six type strains of intestinal spirochetes are presented in Table 1. The reaction pattern of W1 was similar to that of the type strain of B. aalborgi, 513A. Minor differences were noted with regard to the alkaline and esterase activities. Furthermore, a resemblance between the biochemical reaction pattern of W1 and that of the B. pilosicoli type strain, P43, was noted. W1 shared. DISCUSSION This is the first reported isolation of B. aalborgi by culture since 1982 and the second ever reported. We found that B. aalborgi grew poorly on a selective medium which included three antibiotics; vancomycin, colistin, and spectinomycin. Similar media are commonly used in routine diagnostics of intestinal spirochetes. In the original description of B. aalborgi (15), a selective medium which included only two antibiotics,. TABLE 1. Biochemical reactions of B. aalborgi strain W1 and six Brachyspira type strains Strain. B. B. B. B. B. B. B. a. aalborgi W1 aalborgi 513AT hyodysenteriae B78T intermedia PWS/AT innocens B256T murdochii 155-20T pilosicoli P43T. Reactiona with enzymeb: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 0 0 0 0 0 0 0. 0 1 3 2 3 3 3. 1 0 1 1 2 3 3. 1 1 1 1 1 1 2. 0 0 0 0 0 0 0. 0 0 0 0 0 0 0. 0 0 0 0 0 0 0. 0 0 0 0 0 0 0. 0 0 0 0 0 0 0. 0 0 0 0 0 0 0. 2 2 3 2 4 3 2. 2 2 2 2 2 2 2. 0 0 0 0 5 0 2. 5 5 5 5 5 5 5. 0 0 0 0 1 0 0. 0 0 3 2 3 3 0. 0 0 4 3 5 4 0. 0 0 0 0 0 0 0. 0 0 0 0 0 0 0. 0 0 0 0 0 0 0. Hc. Id. Hee. w w 0 0 0 0 s. 0 0 ⫹ ⫹ 0 0 0. w w s w w w w. Numerals indicate levels of reaction; 0, negative reaction; 1, weak positive reaction; 5, strong positive reaction. Enzyme numbers 1 to 20 refer to the following enzymes: 1, control; 2, alkaline phosphatase; 3, esterase; 4, esterase-lipase; 5, lipase; 6, leucine arylamidase; 7, valine arylamidase; 8, cysteine arylamidase; 9, trypsin; 10, chymotrypsin; 11, acid phosphatase; 12, naphthol-AS-BI-phosphohydrolase; 13, ␣-galactosidase; 14, ␤-galactosidase; 15, ␤-glucuronidase; 16, ␣-glucosidase; 17, ␤-glucosidase; 18, N-acetyl-␤-glucosidase; 19, ␣-mannosidase; 20, ␣-fucosidase. c H, hippurate cleavage reaction; w, weak reaction; s, strong reaction. d I, indole reaction; ⫹, positive reaction. e He, hemolysis. b. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. FIG. 3. Transmission electron micrograph of organisms attached end-on to the colonic mucosa, showing a “false brush border.” The photomicrograph shows effacement of the microvilli and condensation of uncharacterized electron-dense material along the apical cytoplasm of colonized epithelial cells. Magnification, ⫻11,000.. a negative ␤-glucosidase reaction with P43. A negative ␤-glucosidase reaction of intestinal spirochetes has been described only for B. aalborgi (15) and B. pilosicoli (8). Strain W1 differed from P43T by showing negative ␣-galactosidase activity. Finally, W1 showed weaker hippurate hydrolysis and alkaline phosphatase, esterase, and esterase-lipase reactions than P43. A strong hippurate cleavage reaction is a feature commonly used for identification of B. pilosicoli. Morphology of spirochetes in phase-contrast microscopy. On primary plates, the appearance of the organisms was very similar to the original description of B. aalborgi (15). The spirochetes were short and thin (Fig. 1). Some cells were comma-shaped, and others were helical with one or two complete turns. Notably, and in agreement with the original description, many of the microbes were attached to the cover glass by one end, around which they rapidly gyrated. After several freeze-thaw steps, microbes of strain W1 looked larger, more motile, and similar to intestinal spirochetes of other species, e.g., B. hyodysenteriae and B. innocens. PCR amplification and sequence analysis of the 16S rDNA gene. PCR products of the expected size, ⬃207 bp, were obtained from both of the biopsy specimens, investigated. The partial 16S rRNA sequences were identical with the corresponding sequence of the type strain of B. aalborgi, 513A. Nucleotide sequence comparison using almost-complete primary structures from strain W1 and B. aalborgi 513AT revealed a nucleotide similarity of 99.7%. Differences in nucleotide composition were as follows (positions given according to E. coli numbering). Strain W1 was found to have guanosine residues in positions 38, 1089, 1094, and 1388, where B. aalborgi 513AT lacks nucleotide information. Furthermore, strain W1 had G, G, C, and G in positions 630, 1099, 1246, and 1475, while B. aalborgi 513AT has A, C, T, and T, respectively. Both its phenotypic properties and its high 16S rDNA similarity to B. aalborgi 513AT justify the classification of strain W1 as belonging to the species B. aalborgi..
(5) DIAGNOSIS OF BRACHYSPIRA AALBORGI. VOL. 38, 2000. ACKNOWLEDGMENTS This work was supported by grants from the Ivar and Elsa Sandbergs Foundation. We acknowledge the skillful technical assistance of Mia Thorsélius, Zhongmin Guo, Tapio Nikkilä, and Ulla Zimmerman.. REFERENCES 1. Antonakopoulos, G., J. Newman, and M. Wilkinson. 1981. Intestinal spirochaetosis: an electron microscopic study of an unusual case. Histopathology 6:477–488. 2. Cotton, D. W. K., N. Kirkham, and D. A. Hicks. 1984. Rectal spirochaetosis. Br. J. Vener. Dis. 60:106–109. 3. Crusioli, V., and A. Busuttil. 1981. Human intestinal spirochaetosis. Scand. J. Infect. Dis. 16:177–179. 4. Douglas, J. G., and V. Crusioli. 1981. Spirochaetosis: a remediable cause of diarrhoea and rectal bleeding. Br. Med. J. 283:1362. 5. Duhamel, G. E., R. O. Elder, N. Muniappa, M. R. Mathiesen, W. J. Wong, and R. P. Tarara. 1997. 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As the antibacterial resistance pattern of B. aalborgi is unknown, the inclusion of antibacterials different from spectinomycin and polymyxin in selective media may very well explain previous failures to isolate B. aalborgi. Another reason for the rarity of B. aalborgi isolates may be the slow growth in vitro compared to other known species of intestinal spirochetes (15). B. aalborgi resembles the frequently isolated intestinal spirochete B. pilosicoli in many respects, e.g., morphology and the end-on attachment to the epithelial surface. Furthermore, our results indicate that commonly used biochemical tests cannot provide reliable differentiation between the two species. Although some differences were noted between the B. aalborgi strains and the type strain of B. pilosicoli (e.g., B. aalborgi strains showed a negative ␣-galactosidase reaction and no evident hippurate cleavage capacity), B. pilosicoli isolates with divergent biochemical reactions have been reported (8). The slower growth rate of B. aalborgi after primary isolation (⬎1 week) compared to B. pilosicoli (2 to 4 days), as well as the lack of ␣-galactosidase and evident hippurate cleavage capacity, may be used to indicate the presence of B. aalborgi. However, more strains have to be isolated and tested. A more reliable diagnosis might be achieved by using a B. aalborgi-specific PCR assay. Such assays, based on 16S rDNA sequences, have been described previously (5, 22). A problem with the PCR assays is that, due to the lack of available B. aalborgi strains, the primer pairs used have been designed without knowledge of the intraspecies 16S rRNA nucleotide variation. Therefore, it is not known if the primer pairs designed detect all strains of B. aalborgi. B. aalborgi has been identified only in humans and nonhuman primates. It is not known if other hosts exist that could act as sources of infection for humans. We found that the microbes could survive for more than 3 months if they were stored anaerobically at room temperature. Therefore it is likely that fecal environmental contamination constitutes a longstanding potential risk of infection with B. aalborgi. In this study we described a patient whose symptoms were characterized by blood and mucus in the stool, but with normal endoscopic and laboratory findings. The only abnormal feature was the presence of large numbers of B. aalborgi organisms attached to the colonic surface epithelium. No histological signs of microbial invasion and/or inflammatory reactions were found. A literature survey did not reveal whether infection by B. aalborgi is always harmless, or whether the spirochetes may be pathogenic under certain conditions. The blockage of passive absorption by large numbers of spirochetes on the colonic epithelium has been suggested as a possible pathogenic mechanism in IS (10). Such a blockage could probably result in diarrhea, but this was not a prominent symptom in this patient. Furthermore, the spirochetes might irritate the mucin-secreting cells of the mucosa, thus leading to increased mucin production. The pathogenicity of the organism may therefore be dependent on the extent and degree of infestation (10). A third explanation for the intestinal complaints may be the organism’s interference with neural transmission, causing altered colonic motility. In the latter case, the symptoms may persist even after the resolution of the infection (24). However, in conclusion, the capacity of B. aalborgi to cause disease in humans still requires further assessment.. 3559.
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