Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by Fluorescence In Situ Hybridization

Abstract

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence
in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within
tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens
yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed.
Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt
surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes
in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.

Full text

JOURNAL OF CLINICAL MICROBIOLOGY, May 2009, p. 1393–1401 Vol. 47, No. 5
0095-1137/09/$08.00⫹0 doi:10.1128/JCM.02469-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by
Fluorescence In Situ Hybridization
䌤
Dinah Schmiedel,
1
Hans-Jo¨rg Epple,
2
Christoph Loddenkemper,
3
Ralf Ignatius,
1
Jutta Wagner,
1
Bettina Hammer,
4
Annett Petrich,
1
Harald Stein,
3
UlfB.Go¨bel,
1
Thomas Schneider,
2
and Annette Moter
1
*
Institut fu¨r Mikrobiologie und Hygiene, Charite´—Universita¨tsmedizin Berlin, Charite´ Campus Mitte, Dorotheenstr. 96,
D-10117 Berlin,
1
Medizinische Klinik mit Schwerpunkt Gastroenterologie, Infektiologie, und Rheumatologie
2
and
Institut fu¨r Pathologie/Research Center ImmunoSciences (RCIS),
3
Charite´—Universita¨tsmedizin Berlin,
Charite´ Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, and
MVZ Labor Dr. Switkowski, Wagner, Dr. Bauermann, Karlsruher Str. 7A,
10711 Berlin,
4
Germany
Received 23 December 2008/Returned for modification 2 February 2009/Accepted 28 February 2009
Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of
the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow
growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion
of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the
visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal
biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological
techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates
clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that
of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira
sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the
basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that
detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal
signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of
single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate
technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore
represents a valuable tool for routine diagnosis of HIS.
Human intestinal spirochetosis (HIS) is a histologically de-
fined condition of the human distal intestinal tract character-
ized by helical microorganisms attached at one end to the
surface epithelium of the colonic mucosa. This association
forms a so-called “false brush border” (13). While certain
Brachyspira spp. are recognized as the causative agents of
swine dysentery and porcine intestinal spirochetosis (3, 11),
their clinical significance and pathogenic potential for humans
remain unclear. Various studies have reported on the associ-
ation of these bacteria with intestinal disorders such as chronic
watery diarrhea (8, 12) and on clinical improvement following
antimicrobial therapy (29). In contrast, others have suggested
that intestinal spirochetes are harmless commensals in humans
(7). The prevalence of HIS ranges from 1.2% (23) to ⬎40%
(17, 34), depending on presumable patient risk factors, such as
origin from developing countries, immunodeficiency, or homo-
sexuality. Recently, Peruzzi et al. (30) discovered a prevalence
of 12% in a selected population, indicating that HIS is an
important differential diagnosis for patients with chronic gas-
trointestinal disorders and risk factors.
Two intestinal spirochetes have been identified in humans so
far: Brachyspira aalborgi (15) and Brachyspira pilosicoli (36).
Both species require selective media, and B. aalborgi is an
extremely slow growing, fastidious microorganism that re-
quires anaerobic incubation for as long as 4 weeks (4, 34). For
this reason, HIS is primarily diagnosed histopathologically.
The fuzzy basophilic fringe, 4 to 7 ␮m thick, on the epithelial
layer of the colonic mucosa is visible in hematoxylin-and-eosin
(HE)-stained histological sections and is considered pathogno-
monic for HIS. Tissue morphology usually remains unaltered,
and no inflammatory reaction is observed (20).
However, diagnosis of HIS on the basis of HE staining
requires experienced laboratory personnel and accurate inter-
pretation, and silver staining is often needed to confirm the
diagnosis (10). Therefore, a significant portion of cases may be
missed, especially since B. pilosicoli might also colonize the
epithelium without the characteristic end-on attachment, im-
peding identification by light microscopy at low magnification
(24). Furthermore, histopathology does not provide informa-
tion about the identity of the microorganisms, thereby preclud-
ing epidemiological studies. More importantly, the inability to
identify the organism also hampers accurate therapy, since the
intestinal spirochetes are suspected to differ in virulence, and
therefore some cases of HIS may require antibiotic therapy
more urgently than others (5, 29).
The genus Brachyspira currently comprises seven established
* Corresponding author. Mailing address: Charite´—-Universita¨ts-
medizin Berlin, Institut fu¨r Mikrobiologie und Hygiene, Dorotheenstr.
96, D-10117 Berlin, Germany. Phone: 49 30 450524226. Fax: 49 30
450524902. E-mail: annette.moter@charite.de.
䌤
Published ahead of print on 11 March 2009.
1393

species and several proposed species. Among some Brachyspira
species, the high level of 16S rRNA gene conservation pre-
cludes interspecies differentiation by 16S rRNA gene methods
and necessitates further molecular analyses. However, all
known species isolated from humans can be identified and
differentiated via their 16S rRNA genes. In line with the ge-
netic variation discovered in Brachyspira species, such as
Brachyspira hyodysenteriae (2) and Brachyspira innocens (9),
recent molecular studies have also identified human Brach-
yspira strains genetically distinct from B. aalborgi and B. pilosi-
coli. This heterogeneity was confirmed by sequencing of 16S
rRNA (14, 21) or NADH oxidase (25) genes, fluorescence in
situ hybridization (FISH) (16, 17), or multilocus enzyme elec-
trophoresis (33). Pettersson et al. (31) analyzed biopsy samples
TABLE 1. HIS patients diagnosed in this study by histopathology, FISH and 16S rRNA gene sequencing
Patient Gender
Age at
time of
diagnosis
(yr)
Clinical
feature(s) Treatment, response
Biopsy
specimen
location
Result of:
Results of sequencing of the 16S
rRNA gene
a
Histopathological
diagnosis
FISH with
EUB338 and
BRACHY
HIS1 Female 37 Chronic diarrhea Metronidazole,
partial recovery
Colon Positive Positive B. aalborgi, 99.8% homology with
AF200693 (1,307 bp, 2 MM)
HIS2 Male 84 Bloody stools,
colitis
No treatment Colon Positive Positive B. aalborgi, 100% homology with
AF200693 (1,414 bp, 0 MM)
HIS3 Male 49 Chronic diarrhea,
HIV infection
Metronidazole,
complete recovery
Sigmoid Positive Positive B. pilosicoli-like, 99.4% homology with
U14927 (1,412 bp, 7 MM)
HIS4 Male 38 Chronic diarrhea Metronidazole,
complete recovery
Ileum, colon Positive Positive B. pilosicoli-like, 99.5% homology with
U14927 (1,465 bp, 7 MM)
HIS5 Male 65 Chronic diarrhea,
HIV infection
Metronidazole,
partial recovery
Colon Positive Positive B. pilosicoli-like, 99.4% homology with
U14927 (1,421 bp, 8 MM)
a
Given as the organism identified, the percentage of homology with the closest match in EMBL/GenBank, the accession number of the closest match (the length
of the sequence, the number of mismatches 关MM兴).
TABLE 2. Strain designations, EMBL/GenBank accession numbers, and references of the Brachyspira strains included in this study
Strain/phylotype Brachyspira sp. Origin EMBL/GenBank
accession no.
Reference as given
in GenBank
513A
T
(NCTC 11492
T
)B. aalborgi Human Z22781 Hookey et al. (2003)
W1 B. aalborgi Human AF200693 Kraaz et al. (2000)
719-00 B. aalborgi Human AM039526 Klitgaard et al. (2005)
HIS/ML15/5/02 B. aalborgi Human AB177983 Nakamura et al. (2005)
HISM28/5/02 B. aalborgi Human AB120022 Hirane et al. (2004)
HISM27/5/02 B. aalborgi Human AB120021 Hirane et al. (2004)
HISM26/5/02 B. aalborgi Human AB120020 Hirane et al. (2004)
HISM16/5/02 B. aalborgi Human AB120010 Hirane et al. (2004)
HISM15/5/02 B. aalborgi Human AB120009 Hirane et al. (2004)
W3b B. aalborgi Human AY349949 Raasbaeck et al. (2004)
W2f B. aalborgi Human AY349948 Raasbaeck et al. (2004)
W2b B. aalborgi Human AY349947 Raasbaeck et al. (2004)
Hca20 B. aalborgi Human AF228807 Pettersson et al. (2004)
Tvb03 B. aalborgi Human AF228819 Pettersson et al. (2004)
Tvb40 B. aalborgi Human AF228821 Pettersson et al. (2004)
HIS24/11/99 “B. ibaraki” Human AB079583 Adachi (2003)
OmanN26 B. pilosicoli Human AY187057 Mikosza et al. (2005)
Br1622 B. pilosicoli Porcine AY514024 Fossi et al. (2004)
P43
T
(ATCC 51139
T
)B. pilosicoli Porcine U14927 Fellstroem et al. (2007)
C162 B. pilosicoli Porcine U14928 Fellstroem et al. (2007)
AN916:90 B. pilosicoli Porcine U14929 Fellstroem et al. (2007)
CF8 B. pilosicoli Canine AB120008 Manabe et al. (2004)
CD2S B. pilosicoli Canine AB120007 Manabe et al. (2004)
CD1S B. pilosicoli Canine AB120006 Manabe et al. (2004)
H98-5 B. pilosicoli Canine AY349946 Johansson et al. (2004)
CN 140 B. pilosicoli Canine AY349945 Johansson et al. (2004)
Dog 17 B. pilosicoli Canine AY349944 Johansson et al. (2004)
24072-93a B. pilosicoli Canine AY349943 Johansson et al. (2004)
AN2608/97 B. pilosicoli Canine AF245123 Johansson et al. (2007)
A3077 B. pilosicoli Canine AF245120 Johansson et al. (2007)
B256
T
(ATCC 29790
T
)B. innocens Porcine U14920 Fellstroem et al. (1994)
56–150
T
B. murdochii Porcine AY312492 Johansson et al. (2003)
PSW/A
T
(ATCC 51140
T
)B. intermedia Porcine U23033 Stanton et al. (2001)
B78
T
(ATCC 51933
T
)B. hyodysenteriae Porcine U14930 Harris et al. (1972)
C1
T
(ACTT 51933
T
)B. alvinipulli Avian U23030 Stanton et al. (2006)
A2
T
“B. canis” Canine AY349936 Johansson et al. (2004)
AN3949:2/02 “B. suanatina” Avian AY352290 Jansson et al. (2004)
AN1418:2/01 “B. suanatina” Avian AY352282 Jansson et al. (2004)
HIS1 B. aalborgi Human FM178385 This study
HIS2 B. aalborgi Human FM178386 This study
HIS3 (isolate) B. pilosicoli-like Human FM178387 This study
HIS4 (isolate) B. pilosicoli-like Human FM178388 This study
HIS5 (isolate) B. pilosicoli-like Human FM178389 This study
1394 SCHMIEDEL ET AL. J. CLIN.MICROBIOL.

from two adults by 16S rRNA gene sequencing and conse-
quently proposed to divide the B. aalborgi lineage into three
phylogenetic clusters, including the type strain, B. aalborgi
513A, in the first cluster.
The extent of intraspecies genetic variation in human intes-
tinal spirochetes is unclear and difficult to estimate, because
few complete 16S rRNA gene sequences are available. Further
epidemiologic and phylogenetic investigations are needed to
elucidate spirochete genetic diversity and to facilitate the eval-
uation of the molecular diagnostic tools that are presently
available.
FISH is a microscopic method that allows simultaneous vi-
sualization and identification of microorganisms. Jensen and
colleagues (3, 16, 17) designed several genus- or species-spe-
cific oligonucleotide probes targeting the 16S or 23S rRNA of
Brachyspira spp. and applied them successfully to porcine and
human intestinal biopsy specimens. However, no genus-spe-
cific 16S rRNA-directed probe for diagnostic use targeting all
Brachyspira spp. known so far has been developed.
In the present study, intestinal biopsy specimens from five
patients with possible HIS were analyzed histopathologically
and by culture, FISH, PCR amplification, and 16S rRNA gene
sequencing. Biopsy specimens from a healthy control group
were analyzed retrospectively by histopathology and FISH.
The purpose was (i) to acquire further information about the
phylogenetic structure of the Brachyspira spp. associated with
HIS, (ii) to design a FISH probe covering all Brachyspira spp.
based on the currently available sequence data, and (iii) to
evaluate FISH as a fast and robust diagnostic screening tool
for HIS.
MATERIALS AND METHODS
Patients. Five patients (HIS1 to HIS5), four men and one woman (ages, 37 to
84 years), were included in this study. These patients suffered from chronic
intestinal disorders of unknown etiology. Intestinal biopsies were performed for
further differential diagnosis including HIS (Table 1). The patients had been
admitted to four different German hospitals: HIS1 to the University Hospital
Hamburg-Eppendorf, HIS2 to the Ko¨nigin Elisabeth Herzberge Hospital Berlin,
HIS3 and HIS5 to the Charite´ University Hospital, Campus Benjamin Franklin,
and HIS4 to the Hospital Augsburg. All patients underwent colonoscopies; four
biopsy specimens were obtained from the colon, and one biopsy specimen each
was obtained from the ileum and the sigmoid. Clinical development and response
to treatment were followed up. Additional intestinal biopsy specimens were
collected from patients HIS1, HIS3, and HIS5, who underwent control colonos-
copies after the cessation of treatment. Risk factors for HIS (immunosuppres-
sion, homosexuality, and contact with animals) were assessed for all patients.
To provide negative controls for the biopsy specimens, seven biopsy samples
from healthy patients (four females, 56 to 68 years old, and three males, 63 to 65
FIG. 1. Spirochetosis of the colon. Shown are HE-stained sections of colon biopsy specimens from patient HIS3 (A and B) and control patient
5 (C and D). (A) Low-magnification image showing a “fringed” blue line along the surface epithelium. Bar, 40 ␮m. (B) A high-magnification image
of the area boxed in panel A displays numerous hematoxyphilic organisms (arrow) at the luminal border of the colonic mucosa. Bar, 20 ␮m. (C
and D) Normal tissue from a control patient at low and high magnification, respectively.
VOL. 47, 2009 INTESTINAL SPIROCHETOSIS DIAGNOSED BY FISH 1395

years old) screened for colorectal cancer at Charite´ University Hospital, Campus
Benjamin Franklin, were included in the study. These samples were found to be
normal by histopathology and to be HIS negative by FISH with EUB338 and
BRACHY.
Culture. Biopsy samples were plated onto brain heart infusion agar supple-
mented with 10% bovine blood, 400 ␮g spectinomycin ml
⫺1
,25␮g colistin ml
⫺1
,
and 12.5 ␮g rifampin (rifampicin) ml
⫺1
(4). Plates were incubated at 37°C in
anaerobic jars under an atmosphere of 80% N
2
–10% H
2
–10% CO
2
for 6 weeks.
Histopathological diagnosis. Intestinal biopsy samples were fixed in formalin,
embedded in paraffin wax, sectioned (thickness, 4 ␮m), and stained with HE. The
appearance of the typical hematoxyphilic fringe on the brush border of the
surface epithelium under light microscopy, confirmed by Warthin-Starry silver
staining, was classified as “HIS positive”.
Specimen processing for FISH. Biopsy samples were fixed in 3.7% (vol/vol)
formaldehyde in phosphate-buffered saline (pH 7.4) containing 50% (vol/vol)
ethanol and were stored at 4°C for 24 h. The embedding procedure using cold
polymerizing resin and the sectioning technique were performed as described
elsewhere (26). For FISH, a prewarmed hybridization solution (20 ␮l) containing
0.9 M NaCl, 20 mM Tris HCl (pH 7.3), and 0.01% sodium dodecyl sulfate was
mixed with 20 pmol of the respective oligonucleotide probe and carefully applied
to the tissue sections. Probes were synthesized commercially and 5⬘end labeled
with a fluorochrome, either Cy3 (indocarbocyanine) or Cy5 (indodicarbocya-
nine) (both from Biomers, Ulm, Germany). After incubation in a dark humid
chamber at 50°C for 2 h, slides were rinsed with sterile double-distilled water, air
dried, and mounted with Vectashield mounting medium (Vector Laboratories,
Burlingame, CA) containing DAPI (4⬘,6-diamidino-2-phenylindole). For micros-
copy, an epifluorescence microscope (Axioplan 2; Carl Zeiss, Jena, Germany)
equipped with narrow band filter sets (AHF Analysentechnik, Tu¨bingen, Ger-
many) was used.
Oligonucleotide probes. The 16S rRNA-directed bacterial probe EUB338
(5⬘-GCTGCCTCCCGTAGGAGT-3⬘) (1) and the nonspecific nucleic acid stain
DAPI were used to screen for bacterial colonization. To selectively identify
intestinal spirochetes, we designed a 16S rRNA-directed FISH probe named
BRACHY (5⬘-ATTAGTCCATGTTTCCAT-3⬘; corresponding to Escherichia
coli positions 153 to 170) (6) that is specific for Brachyspira spp. The probe was
evaluated by comparison to all available sequences in the EMBL and GenBank
databases. A clinical isolate of B. pilosicoli and the nearest phylogenetic neigh-
bors at the probe binding site, Enterococcus faecium (ATCC 19434) and Spiro-
chaeta halophila (DSM 10522), with two mismatches each, were used as positive
and negative controls, respectively, and were included throughout the study.
Furthermore, the probe was tested against other cultivable spirochetes, i.e.,
Borrelia garinii (tick isolate; R. Ackermann, University Hospital of Cologne,
Cologne, Germany), the oral treponeme Treponema denticola (ATCC 33521),
and Leptospira biflexa and Leptospira interrogans (provided by V. Sambri, Section
of Microbiology, St. Orsola Hospital, University of Bologna, Bologna, Italy). The
identities of all strains were confirmed by PCR and 16S rRNA gene sequencing.
Bacterial strains for positive and negative controls were fixed as described else-
where (27).
DNA extraction. For DNA extraction, a commercially available respiratory
specimen preparation kit (Amplicor; Roche Molecular Systems Inc., Branch-
burg, NJ) was used on the isolates (for HIS3, HIS4, and HIS5) or on 20 sections
from Technovit-embedded biopsy samples (HIS1 and HIS2) as recommended by
the manufacturer.
PCR amplification and sequencing. The 16S rRNA gene was amplified using
bacterial primers TPU1 (AGA GTT TGA TCM TGG CTC AG; corresponding
to Escherichia coli positions 8 to 27) and RTU8 (AAG GAG GTG ATC CAK
CCR CA; corresponding to E. coli positions 1541 to 1522) (38).
Amplicons were analyzed in an automated capillary DNA sequencer (CEQ
8000, Beckman Coulter, Krefeld, Germany). The sequences obtained were com-
pared with currently available data from the public databases (EMBL and
GenBank) using BLAST and FASTA in the sequence analysis program Husar,
version 4.1. (Deutsches Krebsforschungszentrum, Heidelberg, Germany).
Phylogenetic analysis. The 16S rRNA gene sequences obtained from the
biopsy specimens were aligned and compared with previously published Brach-
yspira sp. sequences available from GenBank by using Husar, version 4.1. The
type strains of B. aalborgi,B. pilosicoli,B. innocens,Brachyspira murdochii,Brach-
yspira intermedia,B. hyodysenteriae,Brachyspira alvinipulli, and “Brachyspira ca-
nis,” as well as 28 other B. aalborgi,B. pilosicoli,“Brachyspira ibaraki,” and
“Brachyspira suanatina” strains (Table 2), were included. A neighbor-joining
phylogenetic tree (32) was constructed using PAUP, version 4.1b, on the basis of
a distance matrix corrected by the two-parameter model of Kimura (19).
Nucleotide sequence accession numbers. Five almost complete 16S rRNA
gene sequences obtained from the tissue isolates were submitted to the EMBL
database with the accession numbers listed in Table 2.
RESULTS
Response to treatment and follow-up. Two patients had no
known risk factors for HIS, whereas patient HIS4 was homo-
sexual, and patients HIS3 and HIS5 were infected with human
immunodeficiency virus (HIV). Four patients were treated
with metronidazole. Upon therapy, the diarrhea of patients
HIS3 and HIS4 resolved, while patients HIS1 and HIS5
FIG. 2. Evaluation of probe BRACHY, specific for Brachyspira
spp. Fixed bacteria from cultures of B. pilosicoli,E. faecium,B. garinii,
T. denticola, and L. biflexa were simultaneously hybridized with
BRACHY
Cy3
(orange) and EUB338
Cy5
(magenta). Left and right pan-
els in each row show identical microscopic fields with filter sets for Cy3
and Cy5, respectively. BRACHY showed specific hybridization, giving
a positive signal solely with B. pilosicoli.
1396 SCHMIEDEL ET AL. J. CLIN.MICROBIOL.

showed partial improvement of symptoms. Patient HIS2 im-
proved without treatment.
Histopathological findings. In all cases, the typical hema-
toxyphilic band on the brush border of the epithelial layer of
the mucosa could be detected in HE-stained sections (Fig. 1).
In addition, the tissue sections of patient HIS2 showed signs of
chronic ulcerative colitis.
For patients HIS1, HIS3, and HIS5, biopsy samples from
control colonoscopies were available for histological follow-up.
In the follow-up biopsy samples of patients HIS1 and HIS3,
minimal residual spirochetosis was suspected on the basis of
the Warthin-Starry silver staining results, whereas patient
HIS5 was completely negative.
Biopsy specimens from the control group were HIS negative
(Fig. 1).
Culture. After 6 weeks of anaerobic incubation, a thin haze
of pinpoint-like colonies was observed on agar plates inocu-
lated with biopsy material from patients HIS3, HIS4, and
HIS5. Single colonies with motile spirochetes as verified by
dark-field microscopy were subcultured. The identities of the
strains were confirmed by PCR and 16S rRNA gene sequenc-
ing, showing the highest homology with the B. pilosicoli type
strain P43 (U14927).
Cultures of the control biopsy samples of patients HIS1 and
HIS5 that were collected after treatment remained negative
for Brachyspira spp. For patients HIS1 and HIS2, no bacterial
isolates could be obtained, because we received only formalin-
fixed specimens.
FISH. The oligonucleotide probe BRACHY, specific for the
genus Brachyspira, was designed and evaluated prior to appli-
cation to tissue sections. Compared to all sequences from
EMBL and GenBank available as of January 2007, probe
BRACHY showed 100% homology solely with the Brachyspira
sp. 16S rRNA gene. Stringent hybridization conditions were
adjusted using B. pilosicoli as a positive control and E. faecium
and S. halophila as negative controls, with two mismatches
each to the probe sequence. All other cultivable spirochetes
investigated yielded no signal with this probe (Fig. 2).
Tissue sections from all five patients showed positive signals
after hybridization with both probes BRACHY and EUB338.
At low magnification (⫻100), this positive reaction could be
easily visualized as a conspicuous bright fringe coating the
surface epithelium (Fig. 3A). Higher magnification (⫻1,000)
revealed densely packed spirochetes attached at one end to the
mucosa (Fig. 3B). In addition to the usual helical shape, mi-
FIG. 3. Visualization and identification of Brachyspira spp. in a colon biopsy specimen by FISH. Sections of colon biopsy specimens from patient
HIS4 (A and B) and control patient 1 (C and D) were hybridized with BRACHY
Cy3
(orange) and stained with DAPI (blue). (A) Overview. An
overlay of the Cy3, fluorescein isothiocyanate, and DAPI filter set results in a bright orange fringe covering the surface epithelium of the crypts
and contrasting with the green background fluorescence of the tissue. (B) A higher magnification of the microscopic field boxed in panel A reveals
the characteristic end-on attachment (arrow) as well as single Brachyspira sp. organisms in the lumen. (C and D) Absence of spirochetes in normal
tissue from a control patient, shown at low and high magnification, respectively.
VOL. 47, 2009 INTESTINAL SPIROCHETOSIS DIAGNOSED BY FISH 1397

croorganisms with different morphologies were detected, yield-
ing positive signals with both EUB338 and BRACHY (Fig. 4).
No spirochetes could be detected in control biopsy samples
collected from patients HIS1, HIS3, and HIS5 after treatment.
The absence of a positive reaction with BRACHY was con-
firmed by the lack of detection of spirochetal morphotypes
with EUB338 or DAPI, both at low and at high magnification.
However, in the samples from patient HIS1, various rods and
cocci in the intestinal lumen stained positive with EUB338 and
DAPI.
No spirochetes were detected in the biopsy specimens from
the control group (Fig. 3C and D).
PCR and analysis of sequence data. For all five HIS cases,
almost complete sequences of the 16S rRNA gene, ranging
from 1,299 to 1,436 bp, were obtained. In comparison with
currently available data from EMBL and GenBank, the se-
quences had the highest homology to previously published
Brachyspira spp. Sequences from patients HIS1 and HIS2
could be identified as B. aalborgi; the HIS1 sequence yielded
99.8% homology with the sequence of accession number
AF200693, and the HIS2 sequence was identical with the
AF200693 sequence (22). Sequences from patients HIS3,
HIS4, and HIS5 showed 99.4%, 99.5%, and 99.4% homology
with the B. pilosicoli type strain, P43 (U14927), differing in
seven, seven, and eight nucleotide positions, respectively. Al-
though these three isolates were from different patients living
in two geographically distant towns in Germany, they were
closely related, exhibiting only three to five nucleotide mis-
matches. At three nucleotide positions at the end of the 16S
rRNA gene, the isolates were identical to each other but dif-
ferent from B. pilosicoli P43
T
.
Accordingly, the HIS1 and HIS2 sequences clustered in the
B. aalborgi group of a phylogenetic tree based on an alignment
comprising 1,264 nucleotide positions (Fig. 5). Sequences
HIS1 and HIS2 clustered together close to the B. aalborgi type
strain, 513A (accession number Z22781), and were included in
cluster 1 of the three phylogenetic clusters defined by Petters-
son et al. (31). The HIS3, HIS4, and HIS5 sequences fell into
the B. pilosicoli lineage. They formed a separate branch within
this group, although a bootstrap percentage of 63% indicated
only moderate stability for the branching of this node.
DISCUSSION
The clinical relevance of intestinal spirochetes for humans is
still unresolved (37). Here we present data from five patients
with possible HIS, suffering from symptoms of chronic intesti-
nal disorders. After confirmation of the diagnosis, four patients
underwent antimicrobial chemotherapy, resulting in clinical
improvement and significant reductions in the numbers of spi-
rochetes in control biopsy samples. The parallel course of
positive FISH signals for symptomatic patients strongly sug-
gests that the intestinal spirochetes were potentially patho-
genic and may have been the causative agent of the intestinal
disorders of our patients. This conclusion conflicts with reports
of healthy patients with histologically diagnosed HIS (28, 31).
FIG. 4. Identification of different Brachyspira morphologies by FISH with BRACHY. A section of a colon biopsy specimen from patient HIS4
was hybridized with BRACHY
Cy3
(orange) and stained with DAPI (blue). In addition to the typical presentation of spirochetes in a helical shape,
attached by one end to the surface epithelium, microorganisms with different morphologies can be visualized. Yielding a positive signal with
BRACHY
Cy3
, these microorganisms are identified as Brachyspira spp., as shown in the black-and-white image (inset) that was taken with the Cy3
filter set only.
1398 SCHMIEDEL ET AL. J. CLIN.MICROBIOL.

The discrepancy might be explained by differences in virulence
between and within Brachyspira spp., as well as by differences
in the immune status of the patients, since HIV infection is a
well-described risk factor for HIS. Brooke et al. (5) have sug-
gested that B. aalborgi may be a commensal human-adapted
species that is able to overgrow and to cause symptoms under
certain conditions, while B. pilosicoli could have greater patho-
genic potential in humans. This discussion reveals the insuffi-
ciency of present diagnostic tools and the need for identifica-
tion of the intestinal spirochetes in addition to visualization. In
order not to miss a case of HIS, we designed a 16S rRNA-
targeting probe specific for all members of the genus Brachyspira,
which yielded a positive signal in all five cases. The positive FISH
signals were obvious even at low magnification and could be easily
detected. Furthermore, FISH permitted the detection and iden-
tification of microorganisms as spirochetes even if they were de-
tached from the surface epithelium and altered in their morphol-
ogy (Fig. 4). Since FISH is simple, rapid, and inexpensive, it can
easily be included in routine diagnostic procedures in addition to
traditional histopathological techniques.
FIG. 5. Neighbor-joining tree showing the phylogenetic relationships among different Brachyspira spp. The evolutionary tree, based on 16S
rRNA gene sequence alignment, comprises 1,264 nucleotide positions. B. hyodysenteriae serves as the outgroup. The scale bar represents 0.001
substitution per nucleotide position. The stability of the branching order is represented by the bootstrap percentages, obtained from 1,000
resamplings of the data and placed at the major nodes. Colors differentiate sequences from human (orange), porcine (red), canine (green), and
avian (blue) strains.
VOL. 47, 2009 INTESTINAL SPIROCHETOSIS DIAGNOSED BY FISH 1399

In contrast to histology using light microscopy, FISH allows
identification of intestinal spirochetes on a genus-specific, spe-
cies-specific, and even intraspecies-specific level (17). How-
ever, this requires a thorough inventory of the species involved.
A challenging aspect of the diagnosis of HIS is the genetic
heterogeneity of the intestinal spirochetes. The sequencing of
the 16S rRNA gene revealed 99.8% and 100% homology with
B. aalborgi for two cases (HIS1 and HIS2). In three cases, the
sequences differed from those of previously published Brach-
yspira spp. and formed a distinct branch in the phylogenetic
tree. Although the HIS3, HIS4, and HIS5 isolates were ob-
tained from three different patients from two geographically
distant German areas, they clustered together in the B. pilosi-
coli group, leading to the speculation that they might be epi-
demiologically and clinically relevant. These sequences, highly
identical with the B. pilosicoli type strain, P43, in the first 800
nucleotides, showed distinct variations at the end of the 16S
rRNA gene. This phenomenon demonstrates the importance
of complete 16S rRNA gene sequencing to avoid underestima-
tion of the variations and to enlarge the databases needed for
critical evaluation and careful optimization of the present di-
agnostic techniques. Therefore, clinicians should consider
sending samples from potential HIS patients to a specialized
center for confirmation of the diagnosis, Brachyspira culture,
and further molecular analysis.
In conclusion, intestinal spirochetes are genetically hetero-
geneous microorganisms with pathogenic potential that can be
reliably visualized and identified by FISH. Since it is inexpen-
sive and rapid, FISH can be included in routine diagnostic
procedures. Furthermore, the use of this method can facilitate
further epidemiological studies to determine the clinical sig-
nificance of Brachyspira spp. and to investigate the extent of
intraspecies genetic variation.
ACKNOWLEDGMENTS
We thank Y. Gra¨ser for access to PAUP, version 4.1b, and help with
the phylogenetic analysis. We are grateful to G. Fiedler, A. Pohlisch,
and J. Imlau for excellent technical assistance. Thanks are due to G.
Jechart for providing biopsy samples from the patient in Augsburg and
to U. Lippert for providing biopsy material from the patient in Ham-
burg. We thank D. Ramsey for critical reading of the manuscript.
This work was supported by the Sonnenfeld-Stiftung, Berlin, Ger-
many; by a grant from Charite´—Universita¨tsmedizin Berlin, Berlin,
Germany, to D.S.; and by a Rahel-Hirsch grant from Charite´—Uni-
versita¨tsmedizin to A.M.
There is no conflict of interest for any of the authors.
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