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Journal of Clinical Microbiology, October 2000, p. 3555-3560, Vol. 38, No. 10
Department of Pathology,1
Department of Oncology,3 and
Department of Microbiology,4 University
Hospital, S-751 85 Uppsala, Department of Biotechnology, Royal
Institute of Technology, S-100 44 Stockholm,2
and Department of Large Animal Clinical Sciences, Faculty of
Veterinary Medicine, Swedish University of Agricultural Sciences, S-750
07 Uppsala,5 Sweden
Received 17 March 2000/Returned for modification 11 June
2000/Accepted 28 July 2000
In this study we report on the isolation and characterization of
the intestinal spirochete Brachyspira aalborgi using human mucosal biopsy specimens taken from the colon of a young adult male
with intestinal spirochetosis. A selective medium, containing 400 µg
of spectinomycin/ml and 5 µg of polymyxin/ml was used for the
isolation procedure. A high degree of similarity, in terms of
phenotypic properties and 16S ribosomal DNA sequence, was observed between the isolated strain, named W1, and the type strain, 513A, of
B. aalborgi. A similarity of 99.7% in the nucleotide
sequence was found between W1 and 513AT, based on the
almost-complete gene. A short segment of the 16S rRNA gene was
amplified by PCR using genetic material enriched from paraffin-embedded
biopsy specimens, which were taken from the patient on two occasions.
The products showed 16S rRNA gene sequences virtually identical to that
of strain 513AT in the actual region. Immunohistochemistry
was performed on the colonic biopsy specimens with a polyclonal
antibody raised against an intestinal spirochete isolated in a previous
case of human intestinal spirochetosis. The antibody reacted strongly
with the spirochete on the luminal epithelium. No immune reaction was
seen within or below the surface epithelium. Routine histology did not
reveal signs of colitis. Electron microscopy showed spirochetes attached end-on to the colonic mucosal surface. The isolate grew poorly
on a commonly used selective medium for intestinal spirochetes, which
may explain previous failures to isolate B. aalborgi.
A large number of reports of
intestinal spirochetosis (IS) in humans have been presented, but the
clinical importance of human IS is still a controversial issue.
Pathogenicity among human intestinal spirochetes has been indicated in
numerous studies (2, 3, 4, 10, 12, 13, 16, 17, 20, 24, 33)
and questioned in others (11, 18, 23). A constant finding,
originally described by Harland and Lee in 1967 (14), is
end-on attachment by the microbes to the colonic mucosal surface, but
most of the reports do not define the spirochetes involved, and it is
likely that they describe infestations of different microbial species.
In addition to this bacterial infestation, most IS cases show an intact
mucosal surface and no, or only minimal, nonspecific changes in the
lamina propria. However, in some cases inflammatory reactions have been
recorded (1, 13, 17, 24), but the species of the spirochetes
involved were unknown.
Two species of intestinal spirochetes have so far been isolated from
humans, namely, Brachyspira (formerly Serpulina)
pilosicoli and Brachyspira aalborgi. The majority
of the isolates have been referred to as B. pilosicoli
(9, 19, 20, 29, 30). B. pilosicoli is well known
as the agent of porcine IS, a common, worldwide disease in pigs
resulting in mild colitis and diarrhea (31). In addition to
humans and pigs, B. pilosicoli infects other hosts such as
dogs (6) and birds (21). B. aalborgi has been isolated on only one occasion, from a human patient in Aalborg, Denmark (15). Consequently, there is only one
strain available for study (ATCC 43994). However, in a recent study, PCR amplification indicated frequent occurrences of the microbe in
patients with histological evidence of IS (22). Colonic
infections caused by B. aalborgi and B. pilosicoli have also been reported for nonhuman primates
(5). Although somewhat smaller, B. aalborgi shows
a strong phenotypic similarity to B. pilosicoli. Both
organisms are weakly beta-hemolytic anaerobes, regularly waved, with
tapered ends and four flagella inserted subterminally at each end. In 1983, Nielsen et al. claimed that B. aalborgi was
nonpathogenic to humans (23), because they were unable to
relate the presence of the spirochetes to gastrointestinal symptoms.
However, some controversy over the pathological and epidemiological
significance of colonic colonization by B. aalborgi still
exists. For example, two human immunodeficiency virus (HIV)-positive
patients in Washington, D.C., with intestinal B. aalborgi
infection both showed inflammatory infiltrates in the lamina propria.
The infection was considered to be at least a contributing factor to
intestinal symptoms experienced by one of the patients (13,
22).
In this report we describe the case of a young human male with
longstanding intestinal symptoms and with IS diagnosed histologically in routine colonic biopsies. The diagnosis was confirmed by isolation of spirochetes from the biopsy specimens. The isolate was classified as
a strain of B. aalborgi because of a high 16S ribosomal DNA (rDNA) sequence similarity with the type strain of the species. This
isolate of B. aalborgi is the first since 1982 and the
second ever reported.
Case report.
A 23-year-old male with a two-and-a-half-year
history of blood and mucus in the stool was subjected to fiber
endoscopy by his local medical practitioner. His medical history was
unremarkable, except for the intestinal complaints and a mother with a
history of colitis. Physical examination results and laboratory
findings were normal. A biopsy specimen taken from the left colon
showed IS but no evidence of colitis. Because of this finding, the
patient was admitted to the University Hospital, Uppsala, Sweden, for a
total colonoscopy, which showed normal endoscopic findings. Biopsy
specimens were then 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 µg of
spectinomycin per ml, and 5 µg 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 µg of vancomycin HCl solution, 25 µg of colistin
sulfate, and 800 µg 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
(
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Brachyspira aalborgi Infection Diagnosed by Culture
and 16S Ribosomal DNA Sequencing Using Human Colonic Biopsy
Specimens
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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.
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'-GTCTTAAGCATGCAAGTC 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 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.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Light microscopy.
IS was diagnosed histologically in
HE-stained 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.
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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.
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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.
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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 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 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.
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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.| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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, spectinomycin and
polymyxin, was used. A similar medium was used in this study for the
isolation procedure. 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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Large Animal Clinical Sciences, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 7018, S-750 07 Uppsala, Sweden. Phone: 46 18 671473. Fax: 46 18 672919. E-mail: Claes.Fellstrom{at}kirmed.slu.se.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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