Journal of Applied Bacteriology 1992,72, 214-219

Prevalence of coaggregation reactions among chicken

lactobacilli
L. Vandevoorde, H. Christiaens and W. Verstraete
Laboratory of Microbial Ecology, Faculty of Agricultural Sciences, State University of Gent, Belgium
3686/06/91:accepted 2 September 1991

L. VANDEVOORDE, H. CHRISTIAENS A N D w. VERSTRAETE. 1992. Interbacterial adherence was

frequently encountered among chicken lactobacilli. Fourteen of 45 combinations involving
nine adhering strains were shown to be coaggregative. T h e coadherence mechanism was
mediated by complementary heat- a n d sonication-sensitive cell surface structures. It was
shown that intrageneric adherence enabled lactobacilli to maintain higher numbers in
fed-batch reactors simulating t h e gastrointestinal tract. The mechanism of coaggregation can
substantially increase the colonization potential of lactobacilli in environments with short
residence times.

INTRODUCTION exhibit certain coaggregative interactions. In this study, the

occurrence of coaggregation as well as the nature of the
Bacterial coaggregation was first demonstrated as highly
surface components involved was investigated. Moreover,
specific partnerships between streptococci and actino-
the ecological significance of coaggregation was considered.
mycetes in the oral cavity (Gibbons & Nygaard 1970).
Since that time it has been noted that various species of
oral bacteria are involved in coaggregation, including strep-
tococci with actinomycetes (Ellen & Balcerzak-Raczkowski MATERIALS AND METHODS
1977; McIntire et al. 1978; Cizar et al. 1979; Kolenbrander
& Williams 1981, 1983), Fusobacterium (Kelstrup & Bacterial strains
Funder-Nielson 1974), Veillonella (Weerkamp & McBride
Lactobacillus strains used are listed in Table 1. All strains
1981) and Bacteroides (Kolenbrander et al. 1985; Kolen-
were isolated from the intestinal tract of chickens, except
brander & Andersen 1988). From an ecological viewpoint,
strain Lp80 which is a silage strain. All strains were identi-
cells with the ability to coaggregate with bacteria of the
fied with API 50 CH.
dental plaque have a great advantage over non-
coaggregating cells which are removed from the oral
environment by the salivary flow. Isolation of antibiotic-resistant mutants
As opposed to the prevalence of coaggregative reactions
Spontaneously-occuring antibiotic-resistant mutants of
among oral bacteria, evidence of interbacterial adherence in
LD5 and LP80 were isolated on MRS Agar (Difco) supple-
other environments has hardly been presented. Recently, it
has been found that the competitive exclusion of uro-
pathogens by lactobacilli in the urogenital tract coincided Table 1 Lactobarillus strains used in this study
with their ability to interact closely with these undesired
organisms (Reid et al. 1988). T h e latter phenomenon was Strain code Identification Source
~ ~

suspected to account for the defence mechanism attributed Lactobacillus

to the lactobacilli. LC2 fermentum Crop
Considering the highly specific coaggregation accounting LC3 lactis Crop
for the development of oral microbial communities, the lac- LC5 salivarius Crop
tobacilli, colonizing the transient environments of the crop LC9 fermentum Crop
and the gastrointestinal tract of chicken, could possibly also LClO fermentum Crop
LDS crispat us Duodenum
LCA9 salivarius Caeca
Correspondence to :Dr W . Verstraete, Laboratory of Microbral Ecology,
LCA1156 salivarius Caeca
Faculty of .-lgricultural Sciences, State University of Gent, Coupure L., 653,
Lp80 plantarum Grass silage
8-9000 Gent, Belgium.

mented with rifampicin (10 mg/l) and fusidic acid (25
mg/l). Resistant strains were nominated respectively LD5R
and Lp8OR.
Culture media
Strains were grown overnight in M R S Broth (Difco) at
37°C and harvested in the stationary phase.
Coaggregatlon assay
Bacterial cultures were harvested by centrifugation at
10000 g for 10 min and washed twice with phosphate-
buffered saline (PBS) containing (g/l): NaC1, 8; KH2P04,
0.34; K2HP04, 1.21. They were resuspended in the same
buffer to give a final optical density of 0.60 _+ 0.02 at 600
nm with a double beam spectrophotometer (Shimadzu UV
190). Volumes (4 ml) of the washed cell suspension of both
strains were treated similarly and used as a control. After
mixing, 4 ml volumes of the suspension were transferred to
cuvettes and the optical density was monitored for 4 h at
room temperature. T h e coaggregation was expressed as
follows (Handley et al. 1987)
coaggregation ( O h )
- (O.D.l + 0.D.2) - 2 x 0.D.12
x 100
(O.D.l + 0.D.2)
where O.D.l = optical density of strain 1; 0.D.2 = optical
density of strain 2; 0.D.12 = optical density of mixed
strain 1 and strain 2.
Treatment of bacteria
Cultures were harvested by centrifugation at 10 000 g for
10 min, washed twice with distilled water and resuspended
in the appropriate buffer. Cell suspensions were subjected
to heat, lipase, pepsin and sodium periodate. The heat
treatment (30 min at 70°C or 10 min at 95°C) and lipase
treatment (0.5 mg/ml, E C 3.1.1.3; Sigma) were performed
in PBS, pH 7.2 and 7.7, respectively. T h e effect of pepsin
(0.5 mg/ml, EC 3.4.23.1; Sigma) was assayed in 0.1 mol/l
citrate/phosphate buffer p H 2.8 and sodium periodate (10
mg/ml) in 0.1 mol/l acetate buffer p H 4.5. Subsequently,
bacterial cell resuspensions were centrifuged and washed
twice with PBS prior to the coaggregation assay. Washed
bacterial cells were also subjected to a sonication treatment
at 4 f 1 pm amplitude. T h e sonicated cells and sonication
supernatant fluid were respectively examined for coaggre-
gation inhibition. All tests were performed in duplicate.
C O A G G R E G A T I O N OF L A C T O B A C I L L I 215
Electrophoretic mobility
Electrophoretic mobility was measured by laser Doppler
velocitimetry with a Zetasizer (Malvern Instruments). T w o
ml of the appropriate bacterial cell suspensions were
injected in the electrophoresis cell, where bacteria were
subjected to an electrical field. The concomitant frequency
shift of the diffracted light by the bacterial movement was
recorded. T h e mobility, directly related to the bacterial
charge, was expressed as lo-' m2/V.s. The electrokinetic
or zeta potential can be calculated from the electrophoretic
mobility, provided the bacterial conductance is known.
Colonization assay
Batch experiment
Strain LC3 was grown in 100 ml vessels containing 80 ml
MRS modified medium (without Tween 80). For the col-
onization assay, M R S medium was prepared without
Tween 80, as it was shown to discourage the biofilm forma-
tion on the glass wall. After growth of LC3 and coverage of
the glass wall with adherent cells, the medium was dis-
carded and 80 ml of PBS-suspended cells of LD5X and
Lp80R were added. Controls in vessels without LC3 adher-
ing cells were included in the experiment. After shaking for
1 h at 150 rev/min, the vessels were emptied and rinsed
three times with 80 mi PBS and were sonicated at 4 1
pm for 1 min. Viable counts of LDSR and Lp80R cells that
resisted the rinsing procedure were determined by serial
dilution in physiological solution and surface inoculation on
Rogosa Agar (Oxoid) supplemented with 10 mg/l rifampi-
cin and 25 mg/l fusidic acid to prevent growth of the
remaining LC3 cells.
Fed-batch experiment
T o simulate the chicken tract, a fed-batch experiment was
set up as shown in Fig. 1. Two glass tubes, incubated at
37°C and shaken at 150 rev/min, were fed with MRS modi-
fied medium as described above. One tube was inoculated
with the LC3 strain, allowing biofilm formation. Subse-
quently, both reactors were inoculated with LD5R and
were maintained for a residence time of respectively 1 and
3 h. T h e number of viable LD5R cells was monitored by
regular sampling and surface inoculation after serial dilu-
tion on Rogosa agar supplemented with 10 mg/l rifarnpicin
and 25 mg/l fusidic acid, allowing growth only of LD5R.
RESULTS
In Table 2, an overview is presented of the coaggregation
results of paired strains of chicken lactobacilli. Bacterial
216 L. V A N D E V O O R D E E T A L

Fig. 1 Scheme of a fed-batch reactor

set-up. 1, Influent vessel; 2, buffer vessel
(to prevent growth in the influent); 3,
pumps; 4, waterbath; 5, glass tubes; 6,
sampling; 7, effluent vessel

suspensions of nine strains were combined and visually
evaluated for coaggregation. I n 27% of the mixtures, flocs
were formed within 30 min (at room temperature). I n par-
ticular, strains LC3 and LCA9 were frequently involved in
coaggregation reactions, whereas LC2 did not physically
interact with any of the organisms tested.
In order to quantify the interbacterial adherence, a coag-
gregation assay was developed. Bacterial cultures were
paired and the optical density was monitored spectrophoto-
metrically. Coaggregation was expressed as the mean rela-
tive optical density reduction compared with the unpaired
strains. I n Fig. 2, the effect of shaking on paired and single
bacterial cultures is shown. Clearly, shaking markedly
increased the coaggregation percentage. T h e coaggregation
procedure was standardized by mixing the cultures for 30
min and allowing the flocs to settle for 1 h in 4 ml cuvettes
before measuring the optical density.
To study the influence of environmental conditions, bac-
terial cells were examined for coaggregation at different p H
and electrolyte concentrations. In Fig. 3, the coaggregation
of LC3 and LD5 cells was measured at p H values ranging
from 4 to 7. Decreasing the p H provoked an increase of
coaggregative activity. T h e higher interbacterial affinity
may result from a lowered bacterial charge and inherent
reduced repulsion. Indeed, the electrophoretic mobility of
LC3 and LD5, an indicative value of the bacterial surface
charge, was reduced at lower p H values as can be observed
in Fig. 3, most likely due to the neutralization of ionized
functional groups.
A similar effect was noticed with LC3 and LD5 sus-
pended cells at different electrolyte concentrations (Fig. 4).
Lowering the electrolyte strength below 3 mmol/l reduced,
and even completely inhibited coaggregation. The latter
phenomenon may be attributed to an increased double layer
thickness of the bacterial cell surface. Indeed, Coulomb
repulsion forces become dominant and therefore prevent
bacterial cells coming into close proximity, not allowing
bacterial surface structures to interact.
T o characterize the surface structures that promote bac-
terial coaggregation, cells were treated with lipase, pepsin,
periodate and by heating. Only lipase was shown to be inef-
fective in reducing coaggregation (Fig. 5). Heat, pepsin and
periodate markedly inhibited coaggregation of LC3 and
LD5. Although periodate has been shown to act mainly on
carbohydrates, C-C bonds of some polar amino acids are
also sensitive to oxidative cleavage (Dyer 1956). T h e latter
results thus suggest that the determinants for interbacterial
aggregation are of proteinic nature.
LC3 and L D 5 were sonicated in an attempt to remove
surface structures and to examine its effect on coaggrega-
tion. Beside sonicated cells, the supernatant fluid of the
treated samples was also tested for its ability to prevent

Table 2 Visible coaggregation of chicken

Strain* LC2 LC3 LC4 LC5 LC9 LClO LD5 LCA9 LCA1156 lactobacilli
LC2 - - - - - - - - -
LC3 - - + - - + + + -
LC4 - + - - + - - + -
LC5 - - - - - - - + -
LC9 - - + - - + - + -
LClO - + - - + - - + -
LD5 - + - - - - - + -
LCA9 - + + + + + + - +-
LCA11556 - - - - - - - +
* L, Lactobacillus; C, crop; D, duodenum; CA, caecum.
 COAGGREGATION OF L A C T O B A C I L L I 217

- I2

-$ 0.5 -- -----------:>.
0 0.4 -
0 -
-d 0.3 -
0 -
-
0.2 m

u A

'A
0
0.1 -
20
I I I I I

0
.'
0~0001 0.00 I
/'
0.m
A ' I

0.I
Electrolyte Concentration (rnol/C)

Fig. 4 Effect of electrolyte concentration on 0 ,the coaggregation

I

of LC3 and LD5 and A,the theoretical double layer thickness

-8 - t0.4

2 0.3 was applied to LC3 and LD5 for short time intervals, only
outer cell surface structures were released. T h e outcome of
the sonication experiment of LC3 and LD5 cells is present-
ed in Table 3. Sonicated LC3 cells completely failed to
adhere to LD5 cells. Also, the addition of the supernatant
0 I 2 3 4 5 fluid of sonicated LC3 cells totally prevented the inter-
Time ( h )
action of LC3 lactobacilli and LD5 cells. T h e inability to
Fig. 2 Optical density (O.D.) reduction of paired and unpaired coaggregate most probably results from the release of
strains (a) without and (b) with shaking. 0 ,LC3; A,LD5; surface structures, binding to the receptor molecules of
I, LC3 + LD5
LD5 and consequently blocking normal coaggregation.
Heating the supernatant fluid completely abolished the
coaggregation by blockage of the bacterial receptors. T o inhibitory effect and restored the interbacterial adherence.
determine whether the cells were not killed and disrupted The heat sensitivity of the blocking agent is in accordance
during the procedure, the viability of LC3 and LD5 was
examined during the treatment. Both cells readily survived
the ultrasonication treatment for as long as 4 min (results 120-
not shown). It can thus be assumed that when sonication
- 100-
$?
v
90 - -0
0,
-(0.2)
-; c
2
._ 80-
a5 -
\
' \

.
\
\ \ a
r
\ \
E
.-C
-
0."
- 4 . \I~ - (0.4)7
0 c 60-
-3 ao- >\>

4
I

+
0
._
-
0 -
x ,

, \
~ (0.6)
.-
0
z
0
0
40-
f
80 75-
'A
,>\ "
- (0.8) -
-2

?
0
0
0
0 . 0
- ( I )
20 -
70
-
A
%; -
65 I 1 I I I 1 I .(1.4)
3.5 4 4.5 5 5.5 6 6.5 7 7.5 95OC Periodai
Fig. 5 Effect of treatments on coaggregation of LC3 and LD5.
m,(LC3) + LD5; [7, LC + (LD5); m,(LC3) (LD5). +
Parentheses indicate a treated strain. *No inhibition
218 L. VANDEVOORDE € T A L .

Table 3 Effect of sonication on coaggregation of Lactobacillus

strains LC3 and LD5

Treatment Coaggregation (%)

Control 61 + 4
LC3 sonicated 13 f 3
LD5 sonicated +
61 2
Addition of LC3 sonication 2+2
supernatant fluid
Addition of LC3 sonication 58 f 3
supernatant fluid Time ( h )
(treated for 10 min at 95°C)

with the hypothesis that proteins account for the coaggrega-

tion phenomenon.
To investigate the ecological significance of coaggrega-
tion, LC3 was inoculated in 100 ml vessels, containing 80
ml Tween 80-depleted M R S broth and grown overnight to
allow glass wall adherence. After removal of the broth and
repeated washing, LDSR lactobacilli were tested for their
ability to interact with the adherent LC3 cells. Compared t
50 5 10 15 20 25 30

with the control vessels and the non-aggregative reference Time ( h )

strain Lp80R, the number of LDSR remaining after the
washing procedure were markedly higher (Table 4). T h e
results of this experiment suggest that these bacteria can
make use of their coaggregative forces to withstand
removal. In order to corroborate the latter hypothesis, an
experiment was conducted in glass tubes, fed with Tween
80-depleted MRS at regular time intervals to simulate the
chicken crop and tract. T h e specific growth rate of LC3
and LDSR in Tween 80-depleted MRS broth amounted to
0.75 and 0-20/h, respectively. A test was performed to
verify the effect of the presence of a coaggregative strain
LC3 on the colonizing capacity of LDSR (Fig. 6). At short
residence times (1 h), the number of viable LDSR cells was
markedly higher in the reactor previously inoculated with
LC3. At higher residence times (3 h), the effect was less
pronounced. This may be attributed to the p H drop ( < 5)
in the reactor inoculated with LC3, possibly decreasing the
growth rate of LDSR. I n addition, the higher residence
Table 4 Effect of the presence of adhering partner cells on the
establishment of coaggregating LD5R cells and non-aggregating
Lp80R cells
Presence of
Tested strains LC3 biofilm cfu/ml
LD5R (9.6 f 6.1)104
LD5R (2.2 f 1.0)106
Lp80R (4.7 f 1.9)104
Lp80R (10.4 f 5.6)104
Fig. 6 The colonization of a fed-batch reactor by the coaggregation
strain LD5R in 0, the presence or 0 ,the absence of the adhering
strain LC3 at two different residence times: (a) 1 and (b) 3 h
time reduced the wash-out rate of LDSR in the control
reactor. Still, these results indicate the importance of coag-
gregation in continuous flow ecosystems.
DISCUSSION
Gibbons & Nygaard (1970) were the pioneers in examining
specific adhesion between different bacterial species. In
fact, ever since the initial demonstration of coaggregative
reactions between the oral micro-organisms, Actinomyes
viscosus and Act. naeslundii with Streptococcus sanguas, this
subject has received increasing attention, especially with
regard to its role in the establishment of the dental plaque
microbial community.
In this study the Lactobacillus population of chickens was
shown to be equipped with outer cell surface structures
enabling intrageneric interactions. Indeed, by combining
Lactobacillus strains, it became obvious that these
organisms, capable of adhering to the crop epithelium as
previously shown, also exhibited coaggregative properties.
T h e coaggregation reactions were prevalent among chicken
lactobacilli, yet the pairing ability with potential partner
cells appeared to be strain-specific. Environmental condi-
 C O A G G R E G A T I O N OF L A C T O B A C I L L I 219

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