Eur. J. Epidemiol. 0392-2990 June 1987, p. 187-1:)5
Vol. 3, No. 2
ELECTRON OF HUMAN
MICROSCOPY INTESTINAL
G. DETTORI 1. G. AMALFITANO, R. GRILLO,
STUDIES
SPIROCHETES L. POLONELLI, A. ROSSI, P. PLAISANT
Istituto di Microbiologia, Faco!tit di Medicina e Chirurgia ~,A. Gemelli,,, Universit~ Cattolica del Sacro Cuore, Largo F. Vito, 1 - 00168 Roma (Italy).
Key words: H u m a n
intestinal
spirochetes - Swine intestinal
treponemes
T. h y o d y s e n t e r i a e - T. i n n o c e n s - Ultrastructure.
The ultrastructure of ~wenty human intestinal spirochetes was analyzed using the electron microscope. Negatively stained cells were generally found to be loosely and irregularly waved. The isolates had cell dimensions ranging from 0.18=0.35 um in width and from 3.9-14.2 tzm in length. Twin bundles of flagella were present in the space between the cytoplasmic membrane and the outer membrane. The majority of isolates had five flagella inserted sub.terminally at each cell end. Human intesl~inal spirochetes divide by binary fission. They are morphologically similar to swine intestinal treponemes, both pathogenic (Treponema hyodysenteriae) and non pathogenic (Treponema innocens), and different from Treponema palIidum, Treponema phagedenis and Borrelia burgdorferi. Following treatment with sodium deoxycolate, no bundles of cytoplasmic microtubules were observed in cells obtained from cultures of human and swine intestinal spirochetes or from cells of B. burgdorferi, while these structures were present in similarly treated cells of T. pallidum and T. phagedenis.
INTRODUCTION
ttelical, motile bacteria have been observed in the stools of patients with diarrhea since 1884 (7). H u m a n intestinal spirochetosis was described m o r e recently, in 1967, by H a r l a n d and Lee (10), who microscopically observed spirochetes associated with colonic cells of patients with intestinal disease. Subsequently, intestinal spirochetes have been microscopically found in both patients and healthy subjects (6, 8, 12, 22, 24). In spite of the n u m e r o u s studies p e r f o r m e d on this field, very few reports have described successful in vitro cultivation of h u m a n intestinal spirochetes (4, 15, 17, 19, 30). During the last five years we have isolated
m a n y different anaerobic hemolytic spirochetes f r o m the stools of patients with intestinal disturbances (5, 26). Following in vitro cultivation, studies were planned in our l a b o r a t o r y to obtain i n f o r m a t i o n on morphological, biochemical, antigenic and genetic characteristics of intestinal isolates. Our previous studies have shown that h u m a n intestinal hemolytic spirochetes are, as a whole, a h o m o g e n e o u s group of m i c r o o r g a n i s m s C27). Briefly, each isolate had motile, Gram-negative, helical cells which give rise to hemolytic colonies after two-three days of anaerobic culture on blood agar plates. All intestinal isolates shared m a n y c o m m o n antigens (9). Their genomes were f o u n d to be correlated by DNA-DNA hybridization analysis (1).
1 Corresponding author. 187
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P r e l i m i n a r y studies of the u l t r a s t r u c t u r e of the first intestinal isolate showed five flagella inserted at each cell end (26). This study was planned with a twofold purp o s e : (i) to obtain data on the u l t r a s t r u c t u r e of t w e n t y h u m a n intestinal spirochetes (ii) to c o m p a r e their s t r u c t u r e s with those of selected m e m b e r s belonging to the genera Treponema and BorreIia.
MATERIALS A N D METHODS
Microorganisms. - The h u m a n intestinal hemolytic spirochetes w e r e isolated in o u r l a b o r a t o r y by streaking h u m a n feces onto blood agar plates containing spectinomycin (400 m c g / m l ) and rifampin (30 m c g / m l ) as previously described (26). The h u m a n intestinal isolates w e r e progressively labeled f r o m one to twenty with the following codes: HRM-1, HRM-2, HRM-3 and so on. Two swine intestinal hemolytic t r e p o n e m e s , Treponema hyodysenteriae (11, 18, 21, 28) and Treponema innocens (18), w e r e kindly supplied b y Dr. A. Lysons of the I n s t i t u t e for Research on Animal Diseases, New Compton-near-Berkshire, England. These strains were labeled P18A and M1, respectively. The f o r m e r is e n t e r o p a t h o g e n i c for swine, w h e r e a s M1 is non pathogenic (20). Treponema phagedenis was o b t a i n e d f r o m Dr. S. Kellog, of the Centers for Disease .Control, Atlanta, Georgia, U.S.A.. The strain B31 of Borrelia burgdorferi (2, 25) was o b t a i n e d f r o m Dr. H.W. Wilkinson of the Centers for Disease Control, Atlanta, Georgia, U.S.A. Treponema paUidum Nichols in the strain c u r r e n t l y utilized in our labo r a t o r y as a source of antigen for Fluorescent T r e p o n e m a l Antibody test (FTA-Abs) (3) a n d for Nelson and Mayer immobilization test (23). H u m a n and swine intestinal hemolytic spirochetes and T. phagedenis w e r e g r o w n anaerobically at 37°C in an a t m o s p h e r e containing 85% N2, 10% Ha and 5% CO2. I t e m o l y t i c strains w e r e c u l t u r e d on blood agar plates containing 7% h o r s e blood, as previously described (26). T. phagedenis was grown in Peptone Yeast E x t r a c t b r o t h m e d i u m containing 10% r a b b i t s e r u m (9). B. burgdorferi was p r o p a g a t e d by cultivation at 30°C in air for three to five days in a modified Kelly's m e d i u m (25). T. pallidum Nichols was m a i n t a i n e d by intratesticuJar passage in rabbits. Preparation for negative staini!~g, - After 3-6 days of culture, colonies of intestinal hemolytic spirochetes w e r e suspended in physiological saline and a d j u s t e d to a concentration of 100-150 cells p e r high dry field. T: phagedenis and B. burgdor~eri were h a r v e s t e d b y centrifugation for 10 minu~es at a p p r o x i m a t e l y 7000 x g and r e s u s p e n d e d in physiological saline. T. pal!idum Nichols was
collected f r o m the infected r a b b i t testis following m e t h o d s routinely used in p e r f o r m i n g the Nelson and Mayer test (23). F o r m v a r - c o a t e d reinforced grids (400 m e s h ) were p r e p a r e d for each spirochete. Briefly, a sample of each spirochete was placed over a sheet of a p a r a f i l m containing individual d r o p s of distilled w a t e r and of P h o s p h o t u n g s t i c acid (PTA) 3%, p H 6.3-6.4. After a few seconds, the grids were r e m o v e d f r o m the drop a n d the liquid a d s o r b e d with a piece of paper. After drying, the grids were w a s h e d three times with distilled water, stained with PTA 3% and finally dried. Before staining and drying, some grids were p r e t r e a t e d for 1 m i n u l e with sodium deoxycholate (DOC) 1% in w a t e r or with sodium dodecylsulfate (SDS) 1°,5 in water.
Electron microscopy. - Electron m i c r o s c o p y was p e r f o r m e d using a Philips EM 400 etectron m i c r o s c o p e at p r i m a r y magnifications of x4,600 xl0,000, x22,000 and x60,000. Negatives were obtained on K o d a k EM film 4489 a n d photographically enlarged as desired, on K o d a b r o m e 2RC paper. Over 600 electron m i c r o g r a p h s w e r e analyzed for this study. RESULTS
Human intestinal hemolytic spirochetes. - Negatively stained cells of h u m a n intestinal hemolytic spirochetes w e r e generally found to be loosely and irregularly waved (Fig. 1 A-B). When regular, the waves w e r e p o o r and large. The t w e n t y isolates had dimensions ranging f r o m 0.18-0.35 ~m in w i d t h and f r o m 3.9-14.2 ~tm in length (Table 1). S o m e cells had pointed ends while m a n y others had one b l u n t end and the o t h e r pointed. The m i c r o o r g a n i s m s were covered by an o u t e r m e m b r a n e exterior to the c y t o p l a s m i c m e m b r a n e (Fig. 1C). This layer a p p e a r e d finely folded at the tip of some cell_s. Twin bundles of flagella were p r e s e n t in the space b e t w e e n the c y t o p l a s m i c m e m b r a n e and the o u t e r m e m b r a n e . Starting n e a r the end of the cell, each group of flagella w o u n d a r o u n d and overlaped in the mid region of the c y t o p l a s m i c body. The m a j o r i t y of spirochetes had five flagella inserted subterminally (Table 2). However, cells with four flagella were often o b s e r v e d a m o n g m i c r o o r g a n i s m s of each different isolate. F u r t h e r m o r e , sixteen out of t w e n t y isolates h a d some cells with six flagella (Fig. 1D). E a c h spirochete generally had the same n u m b e r of flagella at b o t h ends of the cell. Spirochetes w e r e t r e a t e d with sodium dodecylsulfate 1% in o r d e r to analyze the s t r u c t u r e of the flagella. Following this t r e a t m e n t , s o m e flagella were still b o u n d to the cell body, b u t others were free of cellular m a t e r i a l (Fig. 2 A-B).
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Morphology of intestinaI spirochetes.
!'.
D
C
Figure 1. - H u m a n intestinal spirochetes. Negatively stained cell of isolate HRM-12: cell is 12.25 ~ m long a n d 0.20 ~ m wide (Fig. 1A). A cell of isolate HRM-7: cell dimensions are 8.13 Ltm in length and 0.25 ~ m in w i d t h (Fig. 1B). Cell end of isolate HRM-7: five flagella (f) r u n a r o u n d the body, between outer m e m b r a n e (om) and cytoplasmic m e m b r a n e . The f o r m e r appears finely folded at the tip of the cell (Fig. 1C). Cell of the isolate HRM-8.with six flagella (f), covered by the outer m e m b r a n e (om) (Fig. 1D). Actual magnification: x 5,600 (Fig. IA-B) Bar, 1 !,~m; X 33,000 (Fig. IC-D) B a r 0.1 l~m.
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TABLE 1. Cell d i m e n s i o n s o f h u m a n ~ n t e s t i n a l slairochetes. Spirochetal isolate
HRM HRM HRM HRM HRM HRM HRM HRM HRM H RM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21
Cell Width (~m) Mean Range 0.30 0.24 0.27 0.24 0.30 0.22 0.21 0.25 0.20 0.25 025 0.23 0.24 0.28 0.26 0.25 0.27 0.26 0.30 0.25
Cell Length (!_tin) Mean Range
0.360.32 0,19-0.33 0.25-0.30 0.22-0.30 0.25-0.35 0.18-0.27 0.2,.0-0.25 0.24-0.26 0.18-0.23 0.20-0.29 0.23-0.25 0.18-0,25 0.20-0.30 0.25-0.31 0.22-0.30 0.20-0.30 0.22-0.30 0,25-0.30 0.25-0.33 0.20-0.30
6.25 7.16 6,48 7.86 9.20 5.55 6.15 5.50 5.23 5,90 7.15 9.66 5.78 7.48 7.94 7.05 8.09 6.20 10.4 7.20
4.9-~8.7 4.2-10.6 4.3-8.4 4.6-12.4 6.7-12.2 5.3 5,9 3.9-8,4 5.0-6,0 4.4-6,1 5.5-6,8 4.4-10.4 6.1-12,2 4.6. 7.7 3.9-10.6 5.7-11.1 6.2-7.9 4.7-10.9 4.9- 8.5 8.0-14.2 5,4-10.0
Number of cells examined 8 20 3 17 5 3 5 2 2 4 7 3 5 7 6 2 8 7 7 8
TABLE 2. Q u a n t i t a t i v e e v a l u a t i o n o f t h e n u m b e r o f flagella p r e s e n t i n cells of h u m a n i n t e s t i n a l s p i r o c h e t e s . *
Spirochetg! Isolate
HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM HRM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21
Number of cells foun~ to have in their ends : 4 Flagella 5 Flagella 6 Flagella 5 20 15 23 7 10 10 4 12 8 8 6 16 10 21 4 7 5 23 2
45 24 35 35 38 33 3l 45 37 43 41 35 31 47 26 45 42 35 25 46
1
2 7 9 I 1 1 9 3 7 4 3 1 10 2 2
Total number of Cells examined 50 45 50 58 47 50 50 50 50 51 50 50 50 64 51 52 50 50 50 50
* T h e s a m e n u m b e r ,of f l a g e l l a ~vas c o n s i s t e n t l y o b s e r v e d i n b o t h e n d s f o r a g i v e n cell. 190
Figure 2. - H u m a n intestinal spirochetes. This cell of isolate HRM-19 (Fig. 2A) has been treated with SDS 1% for 1 minute: five flagella are still b o u n d to the cell body. A flagellum (Fig. 2B) that as been detached f r o m the cell body of the spirochete HRM-18 by t r e a t m e n t with SDS. Their insertion disc (id), collars (c) and hooks (h), are visible. Cells of spirochete HRM-19 (Fig. 2C) engaged in the process of cell division. Actual magnification: x 33,000 (Fig. 2A-13) Bar 0.1 ~m; x 12,000 (Fig. 2C) Bar 0.25 lxm
Vol. 3, i987
Morphology of intestinal spirochetes.
A
B
C
D
Figure 3. - Swine intestinal treponemes. Negatively stained cells (Fig. 3A) of pathogenic swine T. hyodysenteriae (strain P18A): m e a n cell dimension are 7.86 I~m in length and 0.33 !xm in width. Non pathogenic swine T. innocens strain M1 (Fig. 3B) : cell is 7.42 ~m in length and 0.28 u m in width. Blunt end of a cell of T. hyodysenteriae (Fig. 3C): seven flagella (f) are visible. The end of non-pathogenic ~wine treponeme M1 (Fig. 3D): five flagella (f) are visible. Ip indicates their insertion points, om is outer m e m b r a n e . Actual magnification: x 5,600 (Fig. 3A-B) Bar, 1 [xm; x 33,000 (Fig. 3C-D) Bar, 0.1 ~m.
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Each flagellum had an insertion p o r t i o n with a hook, a collar and a basal knob. Intestinal h u m a n spirochetes divided by b i n a r y fission (Fig. 2C). S w i n e intestinal hemolytic trepo/temes. - Negatively stained cells of b o t h swine strains, P18A of T. hyodysenteriae and MI of T. innocens, h a d large irregular "waves with blunt ends (Fig. 3A-B). Strain P18A contained cells 6.7-11.3 ,~m long and 0.30-0.35 Ism wide. Strain M1 of T. innocens was found to have n a r r o w e r cells, 0.2-0.3 ~m in width, while their length was 4.0-11.1 Ixm. E a c h cell end of b o t h swine t r e p o n e m e s had twin bundles of flagella which were i n t e r p o s e d b e t w e e n the o u t e r m e m b r a n e and the c y t o p l a s m i c m e m b r a n e (Fig. 3C-D). Many cells of the e n t e r o p a t h o g e n i c strain P18A h a d eight flagella inserted at each extremity. Some m i c r o o r g a n i s m s w e r e found to possess lower (seven) or greater (nine) n u m b e r s of flagella. The great m a j o r i t y of m i c r o o r g a n i s m s obtained f r o m culture of the non-pathogenic swine strain M1 had four or five flagella at each cell end. Very few cells had six o r seven flagella. Their flagella w e r e equipped with insertion discs with hooks and collars (Fig. 4). Cells of b o t h trepon e m e s divided by binary fission.
A
T. pallidum Nichols, T. phagedenis and B. burgdorferi. - Under the electron microscope, negatively stained cells of T. paUidum Nichols, T. phagedenis and B. burgdorlferi a p p e a r e d regularly w a v e d with pointed or b l u n t ends (Fig. 5A-B-C). Three flagella w e r e found constantly at each cell end of T. palIidum.
Figure 4. - A tuft of flagella released from a cell of swine strain P18A of pathogenic T. hyodysenteriae. Insertion discs (id), hooks (h) and flagella are indicated. Actual magnification: x 33,000 Bar 0.1 ~trn.
192
Figure 5 . - Regularly coiled ceils of T. pallidum, T. phaand B. burgdorferi (Fig. 5A-B-C, respectively). Actual magnification: x 2,500 Bar, 1 [~m.
gedenis
Vol. 3, 1987
MorFhology of intestinal spirochetes.
T. phagedenis possessed celts with five to seven flagella, while B. burgdor[eri had six to eight flagella at each cell end. Cells f r o m all three spirochetes had twin bundles of flagella which w o u n d a r o u n d and overlapped in the middle of Cytoplasmic body. Cytoplasmic microtubules. Samples of all spirochetes were treated with sodi,am deoxycholate while resting on the grids (13). After this
Figure 6. - Flagella and cytoplasmic microtubules of T.
pallidum and of T. phagedenis. Microtubules (m) and flagella (f) are visible in cells of T. pallidum (Fig. 6A) and of T. phagedenis (Fig. 6B) after t r e a t m e n t with DOC 1% for 1 minute. Actual magnification: x 33,000 Bar, 0.1 ~m.
193
treatment, bundles of cytoplasmic microtubules were observed in cells of T. pallidum Nichols and of T. phagedenis (Fig. 6A-B). These structures were absent in similarly treated cells obtained from cultures of h u m a n and swine intestinal spirochetes and f r o m cells of B. burgdorferi. DISCUSSION
This electron m i c r o s c o p y study was undertaken to obtain definite data on the ultrastructures of h u m a n intestinal spirochetes isolated in our laboratory. An i m p o r t a n t aim of our w o r k was also to c o m p a r e their structures with those of swine intestinal treponemes and of selected well k n o w n m e m b e r s belonging to the genera Treponema and Borrelia. At the electron microscope, negatively stained cells obtained f r o m colonies of h u m a n intestinal spirochetes and g r o w n anaerobically on blood agar plates, were found to be loosely and irregularly waved. Their dimensions ranged f r o m 0.18-0.35 ,um in width and f r o m 3.9-14.2 ,am in length. Previous electron m i c r o s c o p y observations, carried out on the first intestinal strain isolated by us (strain D60), showed five flagella inserted at each cell end (26). This was c o n f i r m e d in the present study, since the m a j o r i t y of all spirochetes were seen to have five flagella inserted subterminally at each c e l l end. However, cells with four flagella were also observed a m o n g m i c r o o r g a n i s m s of each isolate. F u r t h e r m o r e ; sixteen of the twenty isolates studied had cells with six flagella. Each flagellum was found to have an insertion p a r t with a hook, a collar and a basal knob. H u m a n intestinal spirochetes divided by binary fission. Their structures were then c o m P a r e d with those of cells of swine intestinal pathogenic (T. hyodysenteriae) and non pathogenic (T. innocens) treponemes, of h u m a n T. pallidum Nichols and of T. phagedenis and of B. burgdorferi. H u m a n and swine intestinal spirochetes appeared to be morphologically similar in that b o t h possessed loosely and irregularly waved cells. This finding c o n t r a s t e d with the regular outlines and waves shared by cells of T. pallidum, T. phagedenis and B. burgdor[eri. The cells of intestinal spirochetes and B. burgdorferi were wider than those of T. paIlidum. Morphological differentiation of .some spirochetes was aided by the analysis of their cell ends. T. pal!idum constantly presented three flagella at each of their pointed cell ends. T. phagedenis was characterized by blunt cell ends h a r b o r i n g five to seven flagella, whereas B. burgdorferi showed pointed cell ends with six to eight flagella.
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T A B L E 3.Microorganism
Selected structural characteristics of spirochetes examined in this study.
Cell Width (!tm) Mean
Cell Length (lain) Mean
Cell Waves
Cell ends
Number of flagella
pointed or blunt blunt
4-6
Not-found
7-9
Not-found
blunt
4-7
Not-found
Human intestinal spirochetes
0.25
7.11
T. hyodysenteriae
0.33
7.86
T. innocens
0.23
6.32
F. pallidum
0.13
8.80
1'. phagedenis
0.26
13.15
loose, irregular !oose, irregular loose, irregular tight, regular regular
B. burgdorferi
0.22
12.00
regular
On the contrary, the twenty h u m a n intestinal isolates studied were indistinguishable f r o m swine T. bmocens by analysis of their cell ends. Due to a greater n u m b e r of flagella, swine T. hyodysenteriae could be differentiated from the other intestinal spirochetes we tested. Analysis of individual flagella detached f r o m the cell body of a given spirochete failed to provide useful data for identification of the organisms. In fact, h u m a n intestinal spirochetes, T. pallidum, T. phao~edenis, B. burgdorferi and swine intestinal treponemes, were all found to have flagella of exactly the same type as those described in Gram-positive bacteria. Hovind-Houghen, who has extensively studied the u l t r a s t r u c t u r e ,of Spirochaetales, reported that all but one t r e p o n e m e she examined were seen to harbour, in their cytoplasm, bundles of microtubules clearly different f r o m flagella (14). Since they were not f o u n d in borrelias, their presence should prove valuable in the c!assification of the treponemes and in differentiating them from borrelias (13). We p e r f o r m e d severa! experiments in order to see if microtubules could be found in h u m a n intestinal spirochetes. Samples of each spirochete were treated with sodium deoxycholate while resting on the grids, a p r o c e d u r e that has been described for this purpose (13). As previously reported by HovindHoughen (14), bundles of microtubules were observed in cells of T. paIlidum Nichols and in cells of T. phagedenis. We did not find these structures in cells obtained from cultures of h u m a n and swine intestinal spirochetes or in B. burgdorferi. It is interesting to note here that cells of Brachispira aalborgi, an intestinal spirochete isolated from h u m a n rectal biopsies, do n o t possess cytoplasmic tubules (15). In conclusion, the results of the present s t u d y indicate that h u m a n and swine intestinal spirochetes are structurally correlated (Table 3).
po,inted
Microtubules
3
Present
blunt
5-7
Present
pointed
6-8
Not-found
It appears from our results that the n u m b e r of flagella in h u m a n intestinal spirochetes is similar to that found in non-pathogenic swine treponemes, while pathogenic strains of T. hyodysenteriae usuallv show a larger n u m b e r of flagella. However, Taylor et al. were able to produce diarrhea and dysentery in pigs with a swine spirochete harboring five flagella in their cell ends (29).
Acknowledgements This work was supported by grants from Ministero della Pubblica Istruzione and the Consiglio Nazionale delle Ricerche - Progetto Finalizzato Controllo Malattie da Infezione - N. 83.00693.32, 84.02040.52, 85.0:0883.52, 86.01629.52.
194
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