Microbiol. Immunol. Vol. 36 (6), 563-573, 1992
Ultrastructural Study on the Adherence of Branhamella catarrhalis to Oropharyngeal Epithelial Cell Kamruddin
AHMED, * Naoto and
RIKITOMI,
Keizo
Tsuyoshi
NAGATAKE,
MATSUMOTO
Departmentof Internal Medicine, Institute of Tropical Medicine, Nagasaki University,Nagasaki, Nagasaki 852, Japan (Accepted for publication, March 9, 1992) Abstract
In
the present
to the microplicae
study,
it was
of the oropharyngeal
observed
that
epithelial
cells.
Branhamella
catarrhalis
adhere
Both long and short
micro-
plicae patterns are present on the surface of oropharyngeal epithelial cells and the adherence ability of fimbriated Branhamella catarrhalis also varies according to the microplicae
pattern.
one surface
of the
receptors attach red
are more to the
staining
It was
found
epithelial
on one surface
mucus
layer
specimen
ruthenium
red positive
component,
external
that
cell than but
layer to the
than directly
showed
that
Branhamella
to the
catarrhalis suggesting
on the other. to the
membrane
attached that
Branhamella
epithelial
Branhamella
on the microplicae unit
other,
the
catarrhalis
cell surface.
catarrhalis
attached
epithelial
cell
to
did
of not
Ruthenium to a granular
and also to a ruthenium of the
more presence
red positive
membrane.
During the last several years, we have experienced many patients with chronic respiratory diseases infected by Branhamella catarrhalis (B. catarrhalis) (10, 13). The occurrence of B. catarrhalis infection in patients with chronic respiratory diseases was correlated with the adherence of this bacteria to the oropharyngeal epithelial cell (12). Evidence has shown that respiratory pathogens first attach to the pharyngeal cells, then according to its virulence, tissue tropism and host resistance, causes different types of respiratory injections. Several important events of adherence of B. catarrhalis have been reported from our department (1, 12, 15, 16) with the aim to identifying the adhesin and receptor. There are many complicated events which occur during the attachment process and an electron microscope (EM) is useful to observe the details of the interaction between the bacteria and the host cell (4, 6, 7, 14, 17, 18). EM can reveal the events more precisely than the light microscope (LM). Furthermore, only EM has been used to understand the adherence of many bacteria (3, 8, 11, 21). Therefore, in the present experiment, we have done morphological studies on the adherence of B. catarrhalis to oropharyngeal cells by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). MATERIALS
Bacteria.
Strain
B-88-152,
AND
a fimbriated 563
METHODS
strain,
and
strain
F, a nonfimbriated
564
K.
strain,
were
Bacteria
used
were
incubator. (PBS)
in
this
cultured The
scraping
the
bacteria
1/15 times
at
for
30
done
with
min.
To by
Transmission staining, suspension uranyl
a
paper
filter
on
in
was
present
a
in
with
a in
(JEOL
resin.
of
washed
of
pharyngeal
equal
37
volume,
C in
five
an
incu-
washings
fimbriation One
grid
uranyl
drop
and acetate
then
for
England). were
operated
30
were
by
negative
of
bacterial
then
stained
with
was
removed
with
at
cut
( JEOL
Ltd.,
After
and
dried
sputter-coating with
Postfixation
a scanning
were by
with electron
0.075%
Ultra
(9). and
Cut
E
examined
operated
at cells
80
kV. fixed
0.1
%
through
point 15
drying
nm
microscope
and with
were
and
dehydrated a critical
(RR)
solutions
were
assay,
was red
of
Reichert
specimens Tokyo)
solution
Ruthenium
paraformaldehyde
Specimens
acetate,
a
osmium
washing
ethanol
2%
buffer 1%
hr.
1 hr.
adherence
2%
in
with 1
graded
All
of
cacodylate
a concentration
with
citrate.
After
10
for
solution
done
times for
a
in was
two
at
in
CaC12
buffer
through
were
min.
fixed
%
washed
solution
(SEM).
examined
0.01
buffer
glutaraldehyde,
isoamyl
specimens
were
lead
microscope
microscopy
were
cacodylate
fixation
and
Watford,
Ltd.)
and
at
total
Postfixation
dehydrated
Sections
2%
buffer,
solutions,
the
by tubes
were
in
kept
saline.
assay,
cacodylate
and were
electron
containing
(Polaron, dium,
7.2
adult into
cells
mixed
identify
copper excess
for 1 hr. 1 hr. cells
in
acetate
electron
cacodylate
ethanol
CO2
min.
To
and
7.2), for
in
washing
epoxy
100CX
solution
a
normal
adherence
staining,
specimens
uranyl
Scanning
pH buffer
tetroxide
both
fixed
JEM
a pH
healthy
cfu/ml,
were and
10
carbon-coated
glutaraldehyde
osmium
in
stained
(2).
in
twirling
The
1 •~ 108
cells/ml
for
in
a
then
(PB).
bacteria,
x g
The
from
temperature
paraformaldehyde
red
2%
1%
embedded
7.2
C
saline,
and
of
104
(TEM).
sec.
after
cacodylate, cacodylate
in
The
a
30
cells,
ruthenium
done
80
37
temperature.
unattached
suspended
for
2%
(0.1 M sodium tetroxide in
fixed
room
before
at
tip.
glutaraldehyde,
For
at
microscopy
applied acetate
2.5 •~
at
was
Oropharyngeal
and
min
pH
room
concentration of
centrifuging
catarrhalis was
0.75%
a
separate
electron
B.
at
10
collected swab,
of
at
described Agar
phosphate-buffered
were
buffer 80 •~ g,
been
Infusion
in
cells
phosphate
g for
have
Heart
cotton-tipped
a concentration
750 •~
PBS
a
at
strains
Brain
Mg2+. epithelial
Bacteria,
at
on
ET AL
suspended
with
assay.
centrifuged bator
and
centrifuging
cells,
Both hr
were
mmol
by
Adherence epithelial
18
oropharynx
containing three
study.
for
containing Ca2+ Cells. Oropharyngeal
AHMED
in
CaC12 graded
apparatus
platinum-pallaJSM-840A
kV.
RESULTS
TransmissionElectron Microscopy Peritrichously distributed fimbriae were observed in the strain B-88-152 (Fig. 1A) and were absent in strain F (2). B. catarrhalis was observed to be attached to the microplicae of epithelial cells (Fig. 1B). Many bacteria were observed to be
ULTRASTRUCTURAI,
ADHERENCE
OF
B. CATARRHALLS
565
A
B
Fig. 1. Transmission electron microscopy of strain B-88-152. (A) After negative 0.75% uranyl acetate staining. Bar, 300 nm. (B) Bacteria attached to the microplicae of the epithelial cell. Bar, 400 nm.
K.
566
Fig.
2. are other
Adherence attached surface.
of strain to
one
Bar,
surface
B-88-152 of
the
AHMED
to
ET AL
oropharyngeal
epithelial
cell
epithelial while
a few
cells. bacteria
Most are
of
the
attached
bacteria on
the
2 ƒÊm.
attached to one surface of the epithelial cell, though the other surface of the epithelial cell contained microplicae but with attachment of few (Fig. 2) or no bacteria. Morphological change was not observed inside the epithelial cell at the site of bacterial adherence as compared to the site where bacteria were not attached. Specimen treated with RR was also observed by TEM (Fig. 3). The entire epithelial cells were observed to be covered with a granular RR positive layer. In some cases, thickness of the RR positive layer is greater at the site of bacterial adherence than at other areas of the epithelial cell. In the usual TEM preparation, some unattached bacteria were observed to be very close to the epithelial cell. However, with RR staining, bacteria very near to the epithelal cells were observed to be attached with a granular RR positive structure emerging from the epithelial cell surface (Fig. 3). ScanningElectron Microscopy B. catarrhalis adhere to certain areas but not to others on the same surface of epithelial cells (Fig. 4). Bacteria, attached in agglutinated form, were observed in a few epithelial cells (Fig. 5). These agglutinations were composed of few bacteria. On some epithelial cells, B. catarrhalis were attached on both surfaces (Fig. 6). Both strains utilized two microplicae on the cell surface for attachment. On the surface of pharyngeal epithelial cells many different patterns and length
ULTRASTRUCTURAL
Fig.
3.
Ruthenium
red-stained
scope.
Arrowhead
indicates
positive are
also
structure
emerging
seen
to
be
ADHERENCE
preparation that
strain
from
the
attached
to
one
OF
observed B-88-152 surface
is of
surface
under
of the
transmission
attached
the
567
B. CATARRHALIS
to
epithelial
cell.
epithelial
electron
a granular
cell.
In
this
Bar,
micro-
ruthenium figure,
red bacteria
1 ƒÊm.
of microplicae were observed. In cells with short microplicae (Fig. 7A, upper cell) the length of the microplicae was equal to the width, therefore the short microplicae had a dot-like appearance on the cell surface. Moreover, the term short microplicae were restricted to those microplicae not more than 2.5 pm in length. Other than this, microplicae were designated as long microplicae (Fig. 7A, lower cell and Fig. 7B). On repeated observation, it appears that B. catarrhalis were attached in higher incidence on those cells with short microplicae than epithelial cells with long microplicae (Fig. 7). An amorphous material, suggested to be mucus (14), was seen on the surface of some of the epithelial cells (Figs. 4A and 7B), though B. catarrhalis were not attached to these amorphous materials. DISCUSSION
In
this
adherence very
of
paper, of
B.
useful
for
In
study
this
epithelial
the
cells.
we
could
catarrhalis
to
provide
understanding it was It
found
some
morphological
oropharyngeal of that
is interesting
the
epithelial adherence
B. catarrhalis that
in
TEM
observations cells,
which
on could
the
prove
process. were some
attached bacteria
to the were
microplicae found
very
568
K.
AHMED
ET AL
A
B
Fig. 4. Adherence of Branhamella catarrhalis to oropharyngeal epithelial cell , observed under the scanning electron microscope. (A) Adherence of B-88-152 , 'V' indicates amorphous material. (B) Adherence of strain F.
ULTRASTRUCTURAL
Fig.
5.
epithelial
Scanning cells.
electron Agglutinated
ADHERENCE
microscopy form
of of
the
bacteria,
OF
adherence as indicated
569
B. CATARRHALIS
of
B-88-152 by
with
arrowhead,
oropharyngeal is seen
on
the
cell.
close to the epithelial cell but were not attached. However, when RR was used for TEM observation, it was found that bacteria attached to an RR positive structure emerging from the surface of the epithelial cell. A similar phenomenon was also observed in EM studies of the Escherichia coli (E. coli) attachment process (8). Since RR specifically stains polysaccharide, RR positive layer covering the cell surface of epithelial cell might be polysaccharide in nature. As this B. catarrhalis was observed to be attached to these polysaccharide substances, therefore the receptor of B. catarrhalis on pharyngeal epithelial cell is possibly polysaccharide in nature. In SEM, the surface of the nonfimbriated B. catarrhalis appeared more smooth compared to the fimbriated B. catarrhalis. Due to the presence of fimbriae, platinum-palladium coating made the surface rough in fimbriated B. catarrhalis. Both strains utilized two microplicae on the cell surface for attachment. Bacteria bridge the furrow formed by two adjacent microplicae. This may help the bacteria in many ways for colonization. On the epithelial cells, the length of the microplicae varies greatly. We do not know whether there is any correlation between the expression of microplicae and the maturation of epithelial cells, but small microplicae seem to be more attractive for the fimbriated B. catarrhalis. Further investigation is, however, necessary to elucidate this interesting phenomenon to understand whether the expression of the receptor for B. catarrhalis depends upon the
570
K. AHMED
Fig.
6.
electron
Adherence microscope.
of B-88-152 Bacteria
ET AL
to oropharyngeal are
attached
epithelial to
both
cell
surfaces
observed of the
under epithelial
the scanning cell.
character of the microplicae of oropharyngeal epithelial cells. Rikitomi et al (15) reported that B. catarrhalis attached more with cells from patients with chronic respiratory diseases than cells from normal persons. Therefore the question arises whether there are any morphological changes on the oropharyngeal epithelial cells from patients with chronic respiratory diseases which would lead to easy colonization by increased attachment of B. catarrhalis and subsequently cause infections. It is proved that some species of Streptococcus attach more to keratinized than to nonkeratinized human oral epithelial cells (19). It has also been proved that Pseudomonas aeruginosa is more adherent to nasal and tracheal cells than to buccal cells; nasal and tracheal cells have a loose network of surface microplicae while buccal cells have densely packed microplicae (14). It is therefore necessary to find out whether there is any correlation between the cell surface microplicae and the adherence of B. catarrhalis. B. catarrhalis can attach to both surfaces of the epithelial cell, though the number of attached bacteria is much greater on one surface than the other. E. coli, however, attach exclusively to the basal surface of the superficial uroepithelial cell (20). It may be concluded that the receptors for B. catarrhalis are present on both surfaces of the oropharyngeal epithelial cell. The number of receptors, however, might be fewer on one surface than the other of oropharyngeal cells. Experiments are being conducted to identify the luminal and basal surface of the oropharyngeal epithelial cell. B. catarrhalis were found to adhere to certain areas but not to others on the
ULTRASTRUCTURAL
ADHERENCE
OF
571
B. CATARRHALIS
A
B
Fig. 7. Scanning electron microscopic photograph of the adherence of 13-88-152 with oropharyngeal epithelial cells. (A) Numerous bacteria are attached to the cell having smaller microplicae. No bacteria is attached to the adjacent epithelial cell with longer microplicae. (B) Few bacteria are attached to the epithelial cell with longer microplicae.
572
K.
AHMED
ET AL
same surface of the epithelial cells. This observation suggests that receptors for B. catarrhalis may have a patchy distribution in the host cell membrane. The receptor of Streptococcus pyogenesis also found to have a patchy distribution (5). In SEM, an amorphous material, suggestive of mucus (14), was seen on the surface of some epithelial cells. B. catarrhalis were found to be attached directly to the cell surface. We observed that adherent bacteria varied from cell to cell. This can be explained by the fact that different cells may have a different amount of receptors. By LM observation, proof can be found to support this idea. Adherence of B. catarrhalis in agglutinated form was observed in a few cells. The agglutinations were composed of very few bacteria. This strain is not a spontaneously agglutinating strain. This agglutinated form is due to some nonspecific phenomenon which may occur during the preparation of specimens. The results presented in this paper provide a new view on the adherence of B. catarrhalis to oropharyngeal epithelial cells. We
gratefully
acknowledge
the
assistance
of Mr.
Akitoyo
Ichinose
for
the
electron
microscopy.
REFERENCES
1)
2) 3) 4) 5) 6)
7)
8)
9) 10) 11) 12)
13)
Ahmed, K., Matsumoto, K., Rikitomi, N., Nagatake, T., Yoshida, Y., and Watanabe, K. 1990. Effect of ampicillin, cefmetazole and minocycline on the adherence of Branhamella catarrhalis to pharyngeal epithelial cells. Tohoku J. Exp. Med. 161 : 1-7. Ahmed, K., Rikitomi, N., Nagatake, T., and Matsumoto, K. 1990. Electron microscopic observation of Branhamella catarrhalis. Microbiol. Immunol. 34: 967-975. Araake, M., Yayoshi, M., and Yoshioka, M. 1984. Electron microscopic studies on the attachment of Mycoplasmapulmonis to mouse synovial cells cultured in vitro. Microbiol. Immunol. 28 : 379-384. Beachey, E.H., and Ofek, I. 1976. Epithelial cell binding of group A Streptococciby lipoteicoic acid on fimbriae denuded of M protein. J. Exp. Med. 143: 759-771. Beachey, E.H. 1981. Bacterial adherence: adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143: 325-345. Craven, D.E., Peppler, M.S., Frasch, C.E., Mocca, L.F., McGrath, P.P., and Washington, G. 1980. Adherence of isolates of Neisseria meningitidisfrom patients and carriers to human buccal epithelial cells. J. Infect. Dis. 142: 556-568. Fletcher, M., and Floodgate, G.D. 1973. An electron microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J. Gen. Microbiol. 74: 325-334. Knutton, S., Williams, P.H., Lloyd, D.R., Candy, D.C.A., and McNeish, A.S. 1984. Ultrastructural study of adherence to and penetration of cultured cells by two invasive Escherichia coli strains isolated from infants with enteritis. Infect. Immun. 44: 599-608. Luft, J.H. 1971. Ruthenium red and violet. I. Chemistry, purification, methods to use for electron microscopy and mechanism of action. Anat. Rec. 171: 347-368. Matsumoto, K. 1981. Branhamella catarrhalis infections in chronic respiratory infections. Jpn. Med. J. 2961: 31-40 (in Japanese). Matsuyama, T., and Takino, T. 1980. Scanning electronmicroscopic studies of Bordetella bronchoseptica on the rabbit tracheal mucosa. J. Med. Microbiol. 13: 159-161. Mbaki, N., Rikitomi, N., Nagatake, T., and Matsumoto, K. 1987. Correlation between Branhamella catarrhobis adherence to oropharyngeal cells and seasonal jnthdence of lower respiratory tract infections. Tohoku J. Exp. Med. 153: 111-121. Nagatake, T. 1985. Clinical significance of respiratory infection caused by Branhamella catarrhalis
ULTRASTRUCTURAL
ADHERENCE
OF
B. C.ATARRHALIS
573
with special references to B-lactamase producing strains. Tohoku J. Exp. Med. 147: 1-13. 14) Niederman, M.S., Rafferty, T.D., Sasaki, C.T., Merrill, W.W., Matthay, R.A., and Reynolds, H.Y. 1983. Comparison of bacterial adherence to ciliated and squamous epithelial cells obtained from the human respiratory tract. Am. Rev. Respir. Dis. 127: 85-90. 15) Rikitomi, N., Mbaki, N., Nagatake, T., and Matsumoto, K. 1986. Adherence of Branhamella catarrhalis to human pharyngeal cells: relationship between adherence in vitro and chronic respiratory infections. Jpn. J. Thorac. Dis. 24: 633-638 (in Japanese with English abstract). 16) Rikitomi, N., Andersson, B., Matsumoto, K., Lindstedt, R., and Svanborg, C. 1991. Mechanism of adherence of Moraxella (Branhamella) catarrhalis. Scand. J. Infect. Dis. 23: 559-569. 17) Rumphal, R., Small, P.M., Shands, J.W., Fischlschweiger, W., and Small, P.A. 1980. Adherence of Pseudomonasaeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation. Infect. Immun. 27: 614-619. 18) Rumphal, R., and Pyle, M. 1983. Adherence of mucoid and nonmucoid Pseudomonasaeruginosa to acid-injured tracheal epithelium. Infect. Immun. 41: 345-351. 19) Sklavounou, A., and Germaine, G.R. 1980. Adherence of oral streptococcito keratinized and nonkeratinized human oral epithelial cells. Infect. Immun. 27: 686-689. 20) Tomochika, K., Jie, L., Kanemasa, Y., Kumon, H., and Ohmori, H. 1990. Studies on experimental cystitis caused by uropathogenic Escherichia coli 738. Bacterial Adherence Kenkyukai 4: 60-68 (in Japanese with English abstract). 21) Wilson, M.H., and Collier, A.M. 1976. Ultrastructural study of Mycoplasmapneumoniaein organ culture. J. Bacteriol. 125: 332-339. (Received for publication, October 14, 1991; in revised form, February
17, 1992)
Ultrastructural Study on the Adherence of Branhamella catarrhalis to Oropharyngeal Epithelial Cell Kamruddin
AHMED, * Naoto and
RIKITOMI,
Keizo
Tsuyoshi
NAGATAKE,
MATSUMOTO
Departmentof Internal Medicine, Institute of Tropical Medicine, Nagasaki University,Nagasaki, Nagasaki 852, Japan (Accepted for publication, March 9, 1992) Abstract
In
the present
to the microplicae
study,
it was
of the oropharyngeal
observed
that
epithelial
cells.
Branhamella
catarrhalis
adhere
Both long and short
micro-
plicae patterns are present on the surface of oropharyngeal epithelial cells and the adherence ability of fimbriated Branhamella catarrhalis also varies according to the microplicae
pattern.
one surface
of the
receptors attach red
are more to the
staining
It was
found
epithelial
on one surface
mucus
layer
specimen
ruthenium
red positive
component,
external
that
cell than but
layer to the
than directly
showed
that
Branhamella
to the
catarrhalis suggesting
on the other. to the
membrane
attached that
Branhamella
epithelial
Branhamella
on the microplicae unit
other,
the
catarrhalis
cell surface.
catarrhalis
attached
epithelial
cell
to
did
of not
Ruthenium to a granular
and also to a ruthenium of the
more presence
red positive
membrane.
During the last several years, we have experienced many patients with chronic respiratory diseases infected by Branhamella catarrhalis (B. catarrhalis) (10, 13). The occurrence of B. catarrhalis infection in patients with chronic respiratory diseases was correlated with the adherence of this bacteria to the oropharyngeal epithelial cell (12). Evidence has shown that respiratory pathogens first attach to the pharyngeal cells, then according to its virulence, tissue tropism and host resistance, causes different types of respiratory injections. Several important events of adherence of B. catarrhalis have been reported from our department (1, 12, 15, 16) with the aim to identifying the adhesin and receptor. There are many complicated events which occur during the attachment process and an electron microscope (EM) is useful to observe the details of the interaction between the bacteria and the host cell (4, 6, 7, 14, 17, 18). EM can reveal the events more precisely than the light microscope (LM). Furthermore, only EM has been used to understand the adherence of many bacteria (3, 8, 11, 21). Therefore, in the present experiment, we have done morphological studies on the adherence of B. catarrhalis to oropharyngeal cells by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). MATERIALS
Bacteria.
Strain
B-88-152,
AND
a fimbriated 563
METHODS
strain,
and
strain
F, a nonfimbriated
564
K.
strain,
were
Bacteria
used
were
incubator. (PBS)
in
this
cultured The
scraping
the
bacteria
1/15 times
at
for
30
done
with
min.
To by
Transmission staining, suspension uranyl
a
paper
filter
on
in
was
present
a
in
with
a in
(JEOL
resin.
of
washed
of
pharyngeal
equal
37
volume,
C in
five
an
incu-
washings
fimbriation One
grid
uranyl
drop
and acetate
then
for
England). were
operated
30
were
by
negative
of
bacterial
then
stained
with
was
removed
with
at
cut
( JEOL
Ltd.,
After
and
dried
sputter-coating with
Postfixation
a scanning
were by
with electron
0.075%
Ultra
(9). and
Cut
E
examined
operated
at cells
80
kV. fixed
0.1
%
through
point 15
drying
nm
microscope
and with
were
and
dehydrated a critical
(RR)
solutions
were
assay,
was red
of
Reichert
specimens Tokyo)
solution
Ruthenium
paraformaldehyde
Specimens
acetate,
a
osmium
washing
ethanol
2%
buffer 1%
hr.
1 hr.
adherence
2%
in
with 1
graded
All
of
cacodylate
a concentration
with
citrate.
After
10
for
solution
done
times for
a
in was
two
at
in
CaC12
buffer
through
were
min.
fixed
%
washed
solution
(SEM).
examined
0.01
buffer
glutaraldehyde,
isoamyl
specimens
were
lead
microscope
microscopy
were
cacodylate
fixation
and
Watford,
Ltd.)
and
at
total
Postfixation
dehydrated
Sections
2%
buffer,
solutions,
the
by tubes
were
in
kept
saline.
assay,
cacodylate
and were
electron
containing
(Polaron, dium,
7.2
adult into
cells
mixed
identify
copper excess
for 1 hr. 1 hr. cells
in
acetate
electron
cacodylate
ethanol
CO2
min.
To
and
7.2), for
in
washing
epoxy
100CX
solution
a
normal
adherence
staining,
specimens
uranyl
Scanning
pH buffer
tetroxide
both
fixed
JEM
a pH
healthy
cfu/ml,
were and
10
carbon-coated
glutaraldehyde
osmium
in
stained
(2).
in
twirling
The
1 •~ 108
cells/ml
for
in
a
then
(PB).
bacteria,
x g
The
from
temperature
paraformaldehyde
red
2%
1%
embedded
7.2
C
saline,
and
of
104
(TEM).
sec.
after
cacodylate, cacodylate
in
The
a
30
cells,
ruthenium
done
80
37
temperature.
unattached
suspended
for
2%
(0.1 M sodium tetroxide in
fixed
room
before
at
tip.
glutaraldehyde,
For
at
microscopy
applied acetate
2.5 •~
at
was
Oropharyngeal
and
min
pH
room
concentration of
centrifuging
catarrhalis was
0.75%
a
separate
electron
B.
at
10
collected swab,
of
at
described Agar
phosphate-buffered
were
buffer 80 •~ g,
been
Infusion
in
cells
phosphate
g for
have
Heart
cotton-tipped
a concentration
750 •~
PBS
a
at
strains
Brain
Mg2+. epithelial
Bacteria,
at
on
ET AL
suspended
with
assay.
centrifuged bator
and
centrifuging
cells,
Both hr
were
mmol
by
Adherence epithelial
18
oropharynx
containing three
study.
for
containing Ca2+ Cells. Oropharyngeal
AHMED
in
CaC12 graded
apparatus
platinum-pallaJSM-840A
kV.
RESULTS
TransmissionElectron Microscopy Peritrichously distributed fimbriae were observed in the strain B-88-152 (Fig. 1A) and were absent in strain F (2). B. catarrhalis was observed to be attached to the microplicae of epithelial cells (Fig. 1B). Many bacteria were observed to be
ULTRASTRUCTURAI,
ADHERENCE
OF
B. CATARRHALLS
565
A
B
Fig. 1. Transmission electron microscopy of strain B-88-152. (A) After negative 0.75% uranyl acetate staining. Bar, 300 nm. (B) Bacteria attached to the microplicae of the epithelial cell. Bar, 400 nm.
K.
566
Fig.
2. are other
Adherence attached surface.
of strain to
one
Bar,
surface
B-88-152 of
the
AHMED
to
ET AL
oropharyngeal
epithelial
cell
epithelial while
a few
cells. bacteria
Most are
of
the
attached
bacteria on
the
2 ƒÊm.
attached to one surface of the epithelial cell, though the other surface of the epithelial cell contained microplicae but with attachment of few (Fig. 2) or no bacteria. Morphological change was not observed inside the epithelial cell at the site of bacterial adherence as compared to the site where bacteria were not attached. Specimen treated with RR was also observed by TEM (Fig. 3). The entire epithelial cells were observed to be covered with a granular RR positive layer. In some cases, thickness of the RR positive layer is greater at the site of bacterial adherence than at other areas of the epithelial cell. In the usual TEM preparation, some unattached bacteria were observed to be very close to the epithelial cell. However, with RR staining, bacteria very near to the epithelal cells were observed to be attached with a granular RR positive structure emerging from the epithelial cell surface (Fig. 3). ScanningElectron Microscopy B. catarrhalis adhere to certain areas but not to others on the same surface of epithelial cells (Fig. 4). Bacteria, attached in agglutinated form, were observed in a few epithelial cells (Fig. 5). These agglutinations were composed of few bacteria. On some epithelial cells, B. catarrhalis were attached on both surfaces (Fig. 6). Both strains utilized two microplicae on the cell surface for attachment. On the surface of pharyngeal epithelial cells many different patterns and length
ULTRASTRUCTURAL
Fig.
3.
Ruthenium
red-stained
scope.
Arrowhead
indicates
positive are
also
structure
emerging
seen
to
be
ADHERENCE
preparation that
strain
from
the
attached
to
one
OF
observed B-88-152 surface
is of
surface
under
of the
transmission
attached
the
567
B. CATARRHALIS
to
epithelial
cell.
epithelial
electron
a granular
cell.
In
this
Bar,
micro-
ruthenium figure,
red bacteria
1 ƒÊm.
of microplicae were observed. In cells with short microplicae (Fig. 7A, upper cell) the length of the microplicae was equal to the width, therefore the short microplicae had a dot-like appearance on the cell surface. Moreover, the term short microplicae were restricted to those microplicae not more than 2.5 pm in length. Other than this, microplicae were designated as long microplicae (Fig. 7A, lower cell and Fig. 7B). On repeated observation, it appears that B. catarrhalis were attached in higher incidence on those cells with short microplicae than epithelial cells with long microplicae (Fig. 7). An amorphous material, suggested to be mucus (14), was seen on the surface of some of the epithelial cells (Figs. 4A and 7B), though B. catarrhalis were not attached to these amorphous materials. DISCUSSION
In
this
adherence very
of
paper, of
B.
useful
for
In
study
this
epithelial
the
cells.
we
could
catarrhalis
to
provide
understanding it was It
found
some
morphological
oropharyngeal of that
is interesting
the
epithelial adherence
B. catarrhalis that
in
TEM
observations cells,
which
on could
the
prove
process. were some
attached bacteria
to the were
microplicae found
very
568
K.
AHMED
ET AL
A
B
Fig. 4. Adherence of Branhamella catarrhalis to oropharyngeal epithelial cell , observed under the scanning electron microscope. (A) Adherence of B-88-152 , 'V' indicates amorphous material. (B) Adherence of strain F.
ULTRASTRUCTURAL
Fig.
5.
epithelial
Scanning cells.
electron Agglutinated
ADHERENCE
microscopy form
of of
the
bacteria,
OF
adherence as indicated
569
B. CATARRHALIS
of
B-88-152 by
with
arrowhead,
oropharyngeal is seen
on
the
cell.
close to the epithelial cell but were not attached. However, when RR was used for TEM observation, it was found that bacteria attached to an RR positive structure emerging from the surface of the epithelial cell. A similar phenomenon was also observed in EM studies of the Escherichia coli (E. coli) attachment process (8). Since RR specifically stains polysaccharide, RR positive layer covering the cell surface of epithelial cell might be polysaccharide in nature. As this B. catarrhalis was observed to be attached to these polysaccharide substances, therefore the receptor of B. catarrhalis on pharyngeal epithelial cell is possibly polysaccharide in nature. In SEM, the surface of the nonfimbriated B. catarrhalis appeared more smooth compared to the fimbriated B. catarrhalis. Due to the presence of fimbriae, platinum-palladium coating made the surface rough in fimbriated B. catarrhalis. Both strains utilized two microplicae on the cell surface for attachment. Bacteria bridge the furrow formed by two adjacent microplicae. This may help the bacteria in many ways for colonization. On the epithelial cells, the length of the microplicae varies greatly. We do not know whether there is any correlation between the expression of microplicae and the maturation of epithelial cells, but small microplicae seem to be more attractive for the fimbriated B. catarrhalis. Further investigation is, however, necessary to elucidate this interesting phenomenon to understand whether the expression of the receptor for B. catarrhalis depends upon the
570
K. AHMED
Fig.
6.
electron
Adherence microscope.
of B-88-152 Bacteria
ET AL
to oropharyngeal are
attached
epithelial to
both
cell
surfaces
observed of the
under epithelial
the scanning cell.
character of the microplicae of oropharyngeal epithelial cells. Rikitomi et al (15) reported that B. catarrhalis attached more with cells from patients with chronic respiratory diseases than cells from normal persons. Therefore the question arises whether there are any morphological changes on the oropharyngeal epithelial cells from patients with chronic respiratory diseases which would lead to easy colonization by increased attachment of B. catarrhalis and subsequently cause infections. It is proved that some species of Streptococcus attach more to keratinized than to nonkeratinized human oral epithelial cells (19). It has also been proved that Pseudomonas aeruginosa is more adherent to nasal and tracheal cells than to buccal cells; nasal and tracheal cells have a loose network of surface microplicae while buccal cells have densely packed microplicae (14). It is therefore necessary to find out whether there is any correlation between the cell surface microplicae and the adherence of B. catarrhalis. B. catarrhalis can attach to both surfaces of the epithelial cell, though the number of attached bacteria is much greater on one surface than the other. E. coli, however, attach exclusively to the basal surface of the superficial uroepithelial cell (20). It may be concluded that the receptors for B. catarrhalis are present on both surfaces of the oropharyngeal epithelial cell. The number of receptors, however, might be fewer on one surface than the other of oropharyngeal cells. Experiments are being conducted to identify the luminal and basal surface of the oropharyngeal epithelial cell. B. catarrhalis were found to adhere to certain areas but not to others on the
ULTRASTRUCTURAL
ADHERENCE
OF
571
B. CATARRHALIS
A
B
Fig. 7. Scanning electron microscopic photograph of the adherence of 13-88-152 with oropharyngeal epithelial cells. (A) Numerous bacteria are attached to the cell having smaller microplicae. No bacteria is attached to the adjacent epithelial cell with longer microplicae. (B) Few bacteria are attached to the epithelial cell with longer microplicae.
572
K.
AHMED
ET AL
same surface of the epithelial cells. This observation suggests that receptors for B. catarrhalis may have a patchy distribution in the host cell membrane. The receptor of Streptococcus pyogenesis also found to have a patchy distribution (5). In SEM, an amorphous material, suggestive of mucus (14), was seen on the surface of some epithelial cells. B. catarrhalis were found to be attached directly to the cell surface. We observed that adherent bacteria varied from cell to cell. This can be explained by the fact that different cells may have a different amount of receptors. By LM observation, proof can be found to support this idea. Adherence of B. catarrhalis in agglutinated form was observed in a few cells. The agglutinations were composed of very few bacteria. This strain is not a spontaneously agglutinating strain. This agglutinated form is due to some nonspecific phenomenon which may occur during the preparation of specimens. The results presented in this paper provide a new view on the adherence of B. catarrhalis to oropharyngeal epithelial cells. We
gratefully
acknowledge
the
assistance
of Mr.
Akitoyo
Ichinose
for
the
electron
microscopy.
REFERENCES
1)
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13)
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ULTRASTRUCTURAL
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573
with special references to B-lactamase producing strains. Tohoku J. Exp. Med. 147: 1-13. 14) Niederman, M.S., Rafferty, T.D., Sasaki, C.T., Merrill, W.W., Matthay, R.A., and Reynolds, H.Y. 1983. Comparison of bacterial adherence to ciliated and squamous epithelial cells obtained from the human respiratory tract. Am. Rev. Respir. Dis. 127: 85-90. 15) Rikitomi, N., Mbaki, N., Nagatake, T., and Matsumoto, K. 1986. Adherence of Branhamella catarrhalis to human pharyngeal cells: relationship between adherence in vitro and chronic respiratory infections. Jpn. J. Thorac. Dis. 24: 633-638 (in Japanese with English abstract). 16) Rikitomi, N., Andersson, B., Matsumoto, K., Lindstedt, R., and Svanborg, C. 1991. Mechanism of adherence of Moraxella (Branhamella) catarrhalis. Scand. J. Infect. Dis. 23: 559-569. 17) Rumphal, R., Small, P.M., Shands, J.W., Fischlschweiger, W., and Small, P.A. 1980. Adherence of Pseudomonasaeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation. Infect. Immun. 27: 614-619. 18) Rumphal, R., and Pyle, M. 1983. Adherence of mucoid and nonmucoid Pseudomonasaeruginosa to acid-injured tracheal epithelium. Infect. Immun. 41: 345-351. 19) Sklavounou, A., and Germaine, G.R. 1980. Adherence of oral streptococcito keratinized and nonkeratinized human oral epithelial cells. Infect. Immun. 27: 686-689. 20) Tomochika, K., Jie, L., Kanemasa, Y., Kumon, H., and Ohmori, H. 1990. Studies on experimental cystitis caused by uropathogenic Escherichia coli 738. Bacterial Adherence Kenkyukai 4: 60-68 (in Japanese with English abstract). 21) Wilson, M.H., and Collier, A.M. 1976. Ultrastructural study of Mycoplasmapneumoniaein organ culture. J. Bacteriol. 125: 332-339. (Received for publication, October 14, 1991; in revised form, February
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