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Applied and Environmental Microbiology, December 1998, p. 4789-4795, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Technique To Quantify the Population Size and
Composition of the Biofilm Component in Communities of Bacteria in
the Phyllosphere
Cindy E.
Morris,*
Jean-Michel
Monier,
and
Marie-Agnès
Jacques
INRA, Station de Pathologie
Végétale, Domaine St. Maurice, 84143 Montfavet cedex,
France
Received 9 June 1998/Accepted 30 September 1998
 |
ABSTRACT |
The presence of microbial biofilms in the phyllosphere of
terrestrial plants has recently been demonstrated, but few techniques to study biofilms associated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is assembled in biofilms and to
quantitatively isolate bacteria from the biofilm and nonbiofilm
(solitary) components of phyllosphere microbial communities. This
technique is based on removal of bacteria from leaves by gentle
washing, separation of biofilm and solitary bacteria by filtration, and
disintegration of biofilms by ultrasonication. The filters used for
this technique were evaluated for their nonspecific retention rates of
solitary bacteria and for the efficiency of filtration for different
concentrations of solitary bacteria in the presence of biofilms and
other particles. The lethality and efficiency of disintegration of the
sonication conditions used here were also evaluated. Isolation and
quantification of bacteria by this technique is based on use of culture
media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and
solitary bacteria in the suspensions generated by this technique. Preliminary results from estimates of biofilm abundance in phyllosphere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and
parsley leaves.
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INTRODUCTION |
The presence of microbial biofilms
on leaves of numerous species of terrestrial plants has recently been
demonstrated (12). Biofilms on leaf surfaces are composed of
diverse microorganisms including gram-positive and gram-negative
bacteria as well as yeasts and filamentous fungi. Like biofilms in
other environments, those in the phyllosphere are tens of micrometers
thick, have copious exopolymeric matrices, and, in certain cases, form
extensive networks several millimeters long. These properties are very
similar to those of biofilms in other environments such as the aquatic milieu, where biofilms have been studied extensively (4).
Hence, biofilms in the phyllosphere could have the same significant
impact on resistance to stress, on metabolic and genetic exchange, and on the phenotypic plasticity of epiphytic microorganisms as has been
observed for microorganisms in other environments (3, 4,
18).
To address the role of biofilms in the ecology and physiology of
epiphytic microorganisms, techniques adapted to the study of biofilms
associated with living plant tissues are needed. In our previous paper
(12), we described techniques that allowed us to
characterize the thickness, location, and microbial composition of
individual biofilms on leaf surfaces. Here we report a technique to
quantify the proportion of the total bacterial population on leaves
that is assembled in biofilms.
Techniques to estimate the relative abundance of the biofilm component
of microbial ecosystems have been described previously for aquatic
systems. Unlike the phyllosphere of terrestrial plants, the aquatic
systems studied have several properties that facilitate the estimation
of biofilm abundance. First, biofilms in aquatic systems are attached
to surfaces whereas solitary microorganisms are planktonic. Hence,
aquatic biofilms can be isolated simply by removing a portion of the
substrate from the system (5-7, 11, 14, 16). Furthermore,
where viable plate counts or immunological techniques are used to
assess the population size of the bacteria in biofilms, mechanical
detachment of the biofilms from the substrate also serves to
disintegrate the biofilms (1, 6, 7, 11, 14, 16). Second, in
many of the aquatic systems studied, biofilms are attached to
abiotic
and often inorganicsubstrates. Hence, the size of the
biofilm component in these systems can also be assessed via protein
assays or biomass determination without the need to detach the biofilms
from the substrate (5). Quantitative isolation of biofilm
bacteria from the phyllosphere requires a modified approach adapted to
the particularities of this ecosystem.
We report the combined use of leaf washing, filtration, and
ultrasonication for estimating the sizes of the populations of bacteria
within biofilms on leaf surfaces relative to those not assembled in
biofilms (referred to as solitary). Previous estimates from aquatic
systems have led to the general belief that bacteria in biofilms
dominate aquatic bacterial communities (4). However, no such
information is available for non-water-saturated systems such as leaf
surfaces of terrestrial plants. Here we present preliminary data
describing the relative abundance of the biofilm and solitary components of the total culturable bacterial population of leaf surfaces. We also present estimates for the size of the biofilm and
solitary components in the population of oxidase-positive fluorescent
pseudomonads, a group of bacteria that is found in epiphytic biofilms
(12) and that causes postharvest decay of the plants studied here.
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MATERIALS AND METHODS |
Plant material.
Field-grown plant material was used in our
experiments. Leaves of broad-leaf endive (Cichorium endivia
var. latifolia) were obtained from plants grown at the
Institut National de la Recherche Agronomique Research Center,
Montfavet, France (by using cultivation techniques described in
reference 8) or from plants purchased in the
fresh-produce section of local markets. Leaves of parsley (Petroselinum crispum) were purchased at local markets.
Bacterial suspensions.
The filtration efficiency and the
effect of ultrasonication on bacteria were determined for cultured
single strains of bacteria and for mixtures of microorganisms naturally
occurring on broad-leaf endive or parsley. Strain PF130A of
Pseudomonas fluorescens bv. 5 was isolated from endive. This
spontaneous rifampin-resistant mutant has been described previously
(8). P. putida MC1-100, Corynebacterium
aquaticum MC1-045, MC1-098, and MC1-108, and Hafnia alvei MC1-057 were isolated from a biofilm on endive
(12) and tentatively identified by fatty acid methyl ester
analysis as described by Thompson et al. (15). These
bacteria were cultured on tryptic soy agar (TSA) (1.7 g of tryptone,
0.3 g of Bacto Soytone, 0.25 g of glucose, 0.5 g of
NaCl, 0.5 g of K2HPO4, 15 g of agar, 50 mg of cycloheximide per liter) for 48 h at 25°C and suspended in sterile potassium phosphate buffer (6.75 g of
KH2PO4 per liter, 8.75 g of
K2HPO4 per liter [pH 7]). To obtain mixed
populations of the naturally occurring epiphytic microflora, leaves of
field-grown plants were washed in sterile buffer by gentle agitation of
flasks. Microorganisms were recovered from the washings by filtration through MF-type (mixed cellulose acetate and nitrate) filters (0.22-µm-diameter pores; Millipore, St. Quentin, France). These microorganisms were resuspended in sterile buffer, and the density was
adjusted to the desired concentration.
For some experiments, leaf washings were cultured to obtain microbial
suspensions containing high densities of biofilms. To culture biofilms,
leaf washings were filtered across Isopore polycarbonate filters with
5-µm-diameter pores (Millipore). The filters were then placed
directly into tryptic soy broth (TSA containing neither agar nor
cycloheximide), and loopfuls of 48-h cultures of strains T53
(Pseudomonas fluorescens bv. 3) and MC1-045 were added. The resulting microbial suspension was incubated at room temperature for 3 days without agitation. This culture was then treated to obtain
suspensions containing only solitary bacteria or predominantly biofilm
bacteria as described below.
Filtration efficiency.
The filters compared for their
retention rates of bacteria are described in Table
1. All the filters used in these
experiments were 47 mm in diameter. Filtrations were conducted on
autoclavable plastic filter supports attached to a vacuum aspirator
(SUE 300 Q aspiration pump; draw, 20 liters/min; Heto Lab Equipment,
Allerød, Denmark). The suspensions of microorganisms described above
were prefiltered across SVLP or TMTP filters to obtain homogeneous suspensions of cells that should, theoretically, all pass through the
pores of similar filters. These prefiltered suspensions (PREF suspensions) were divided into aliquots of 50 ml. Each of three aliquots was filtered again across sterile LSWP, SVLP, and TMTP filters, and then the filters were each rinsed with 50 ml of sterile buffer. The bacterial concentration was determined before and after
filtration of the PREF suspensions by dilution plating on TSA. To
determine the number of bacteria remaining on the filter, each filter
was ground for 1 min in 50 ml of sterile buffer with an Ultra-Turrax
(Janke and Kunkel, IKA Labortechnik, Staufen, Germany) at 24,000 rpm.
The number of bacteria in the ground suspension was determined by
dilution plating on TSA. The number of colonies on the plates was
counted after 4 or 5 days of incubation at 25°C.
The effect of microbial aggregates or other particles on filtration
efficiency was studied. Particles were introduced in the
form of crude
leaf washings or mixed biofilm cultures. PREF suspensions
of
P. fluorescens PF130A or the spontaneous rifampin-resistant
P. putida MC1-100,
H. alvei MC1-057, or
C. aquaticum MC1-108 were
mixed at various concentrations with a
range of dilutions of endive
leaf washings or biofilm cultures. The
pure-culture PREF suspensions
and the mixtures were divided into
aliquots of 40 ml. Each of
three or four aliquots was filtered across
TMTP filters, and each
filter was rinsed with 50 ml of sterile buffer.
The concentration
of the rifampin-resistant bacteria in the pure
culture was determined
immediately before and after filtration by
dilution plating on
TSA supplemented with rifampin (50 mg/liter). The
frequency of
naturally occurring rifampin-resistant bacteria in the
leaf washings
and in the biofilm cultures was determined on
TSA-rifampin medium
before preparation of the mixtures. The colonies
were counted
after 4 days of incubation of the plates at 25°C.
Disintegration of biofilms.
Microbial suspensions were
treated with ultrasound for different times and amplitudes with a
Vibracell-72405 100-W ultrasonicator (Bioblock Scientific, Illkirch,
France) with an ultrasonication tip whose diameter was 6 mm. All
sonications were conducted with pulsations (1 s off, 2 s on). To
determine the effect of sonication on bacterial culturability, PREF
suspensions of cultures of mixed populations were prepared to eliminate
biofilms. Aliquots (20 ml) of the PREF suspensions were put in sterile
beakers and kept on ice until analyzed. The density of culturable
bacteria in the suspensions after ultrasonication was determined by
dilution plating on TSA and King's medium B (10)
(containing 50 mg of cycloheximide/liter) (KB) for each of three or
five aliquots for each combination of time and amplitude of
ultrasonication. The samples were processed in time according to a
completely randomized block design. Fluorescent colonies on KB were
counted after 2 and 3 days of incubation at 25°C. Total colonies on
TSA were counted after 5 days of incubation.
The efficiency of ultrasonication for biofilm disintegration was
determined with biofilm cultures. To collect and concentrate
biofilms,
biofilm cultures were filtered across TMTP filters and
the
microorganisms retained on the filter were scraped off with
a sterile
spatula and resuspended in sterile buffer. Aliquots
of the suspension
were prepared and stored as described above.
Three or five aliquots
were ultrasonicated for each combination
of time and amplitude. The
samples were processed in time according
to a completely randomized
block design. After ultrasonication,
each aliquot was again filtered
across a TMTP filter and the filter
was rinsed with two 20-ml aliquots
of sterile buffer. The filter
was then aseptically transferred to a
stomacher bag and stomached
for 2 min in 20 ml of sterile buffer with a
Bagmixer (Interscience,
St. Nom, France). The number of CFU from the
filter was determined
by dilution plating of the washing on TSA and KB.
The plates were
incubated and the colonies were counted as described
above.
The ultrasonication efficiency was also determined by measuring the
particle sizes in the sonicated suspensions. Aliquots
of the sonicated
biofilm suspensions were observed microscopically.
For each combination
of time and sonication amplitude, three separate
aliquots were mounted
on a hemocytometer. For each hemocytometer
preparation, 25 randomly
sampled fields (200 by 250 µm) were observed
under phase-contrast
microscopy at a magnification of ×200 with
a BX 60 microscope (Olympus
Optical Co., Hamburg, Germany) coupled
to a black-and-white video
camera. The lengths of all particles
longer than 5 µm were determined
with Visiolab 1000 software (Biocom,
Lyon,
France).
Estimation of the relative abundance of biofilm and solitary
bacteria on leaves.
To estimate the sizes of solitary and biofilm
bacterial populations, washings of leaves were filtered and sonicated
under the conditions determined in the preceding experiments, as
illustrated in Fig. 1. Plant material
consisted of individual leaves of broad-leaf endive or 10-g bunches of
parsley leaves. The population sizes of culturable bacteria were
determined on TSA and KB.
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FIG. 1.
Steps in the method for quantifying the population sizes
of biofilm and solitary bacteria in the phyllosphere.
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To remove bacteria from the leaves, the endive and parsley leaves were
washed in 150 ml of sterile potassium phosphate buffer
for 3 min by
gentle manual rotation of the flasks (120 rpm). The
leaves were then
removed from the buffer and rinsed in 50 ml of
sterile buffer, and the
rinse and washing suspensions were combined.
To determine the number of
bacteria remaining on the leaves after
the washing step, the leaves
were ground for 2 min in 40 ml of
sterile buffer with an Ultra-Turrax
(24,000 rpm), and the grindings
were plated on media. To determine the
number of solitary and
biofilm bacteria in the leaf washings, the
washings were each
divided into three aliquots of 50 ml. Each aliquot
was filtered
across a sterile TMTP filter, the filters were each rinsed
with
100 ml of buffer, and the concentration of bacteria in the
filtrate
(filtered leaf washing plus rinsing buffer) was determined by
plating on media. To estimate the number of bacteria retained
on the
filters, each filter was aseptically torn into four pieces
to allow
adequate coverage of the filter by the buffer used for
sonication. Each
piece of filter was sonicated for 2 min in 20
ml of sterile buffer at
70% sonicator amplitude with pulsations
(1 s off, 2 s on). Under
these conditions, the energy of ultrasonication
was 0.45 W/ml. The
filter pieces were rinsed by agitation in a
separate 20-ml volume of
buffer, and then the five volumes corresponding
to each filter were
mixed. The bacterial population sizes in these
suspensions were
determined by plating on media. Colonies were
counted after 3 and 5 days of incubation of the plates at 25°C.
The rate of retention of bacteria in PREF suspensions of all leaf
washings was determined as described above. Furthermore,
to determine
the efficiency of sonication for removal of bacteria
from filters, the
four filter pieces were stomached for 2 min
in 20 ml of buffer as
described above. Bacterial populations in
the washing were determined
by plating on media. After being washed,
filter pieces were placed on
TSA and colonies developing on filters
were counted after 3 to 5 days
of incubation at 25°C.
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RESULTS |
Filtration efficiency.
Isopore polycarbonate (TMTP) filters
retained 0.004% or fewer of cells from PREF suspensions of bacteria in
pure culture, whereas the other types of filters tested retained 0.03 to 2% of the bacteria from these suspensions (Table
2). On TMTP filters, the retention rate
of bacteria from leaf washings (0.2 to 4%) was much higher than for
bacteria in pure culture. Furthermore, bacteria recovered in leaf
washings of broad-leaf endive tended to be retained on filters at a
higher frequency than those from parsley (Table
3). The introduction of particles (crude
leaf washings or biofilm cultures) into PREF suspensions had little effect on the passage of cells across the pores of TMTP filters: the
recovery rate of cells from PREF suspensions mixed with other particles
was at least 90% of the recovery rate without the introduction of
particles (Table 4). This recovery rate
was not affected by the concentration of PREF cells in the mixtures
(Table 4).
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TABLE 4.
Filtration efficiency of Isopore polycarbonate filters in
the presence of microbial aggregates and
other particlesa
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Disintegration.
Most ultrasonication treatments caused
insignificant reductions in the number of culturable bacteria in PREF
suspensions of mixed populations (P 
0.05 by Tukey's
honest significant difference test) (Fig.
2). Reductions were significant only for
suspensions sonicated for 4 min at 90% of the maximum energy of the
sonicator. Ultrasonication also resulted in disintegration of biofilms,
as indicated by reductions in the numbers of culturable bacteria from
biofilm cultures that were retained on filters (Fig.
3). These reductions were 50-fold or
greater for total bacteria and 35-fold or greater for fluorescent
bacteria. The number of culturable bacteria retained on filters after
sonication was significantly smaller than that in the control
experiment for all treatments (P < 0.0002 by Tukey's
honestly significant difference test).
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FIG. 2.
Effect of ultrasonication on the culturability of total
(A and B) and fluorescent (C and D) epiphytic bacteria. Bars represent
the mean population densities in three aliquots sonicated for each
combination of time and amplitude. Error bars indicate the standard
error. Panels A and C and panels B and D represent the results of two
independent trials.
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FIG. 3.
Effect of ultrasonication on the number of total (A and
B) and fluorescent (C and D) bacterial CFU retained on Isopore
polycarbonate filters with 5-µm-diameter pores. Bars represent the
mean population densities retained on filters after sonication of three
aliquots for each combination of time and amplitude. Error bars
indicate the standard error. Panels A and C and panels B and D
represent the results of two independent trials.
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Microscopic observations confirmed the effects of sonication on the
disintegration of biofilms. The abundance of aggregates
and the
frequency of large aggregates in microbial suspensions
were
significantly reduced by ultrasonication. Microbial suspensions
sonicated at 70% strength for 2 or 3 min or at 90% strength for
1 min
had significantly (
P 0.05) fewer total aggregates
(i.e.,
all aggregates longer than 5 µm) than did aliquots of the same
suspensions that were not sonicated (Table
5). These sonication
conditions also
significantly reduced the concentration of aggregates
7.3 to 9.9 µm
long and/or 10 to 29 µm long in microbial suspensions
(Table
5).
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TABLE 5.
Effect of ultrasonication on the size of microbial
aggregates in cultures of biofilms as determined by
microscopic observation
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Estimation of the relative abundance of biofilm and solitary
bacteria on leaves.
When leaf washings were analyzed as described
in Fig. 1, between 1 and 38% of the total bacterial and fluorescent
pseudomonad populations on all samples except one of the parsley leaves
(Table 6) were retained on the filter
after the first cycle of filtration (step 2a, Fig. 1). We considered
these values to be estimates of the frequency of biofilm bacteria in
each of these populations. We then compared these values to the
frequencies of bacteria retained on filters from PREF suspensions of
these leaf washings (step 3a-ii, Fig. 1). These latter values, which
represent the nonspecific retention of solitary bacteria on filters,
were low (0.2 to 1% for bacteria from endive; 0.1 to 0.6% for
bacteria from parsley). Therefore, nonspecific retention of solitary
bacteria accounted for only a small fraction of the size of the
bacterial populations that we considered to be the biofilm component
for all but one of these leaves. However, nonspecific retention of
fluorescent bacteria on filters could account for the so-called biofilm
component of the first parsley sample (Table 6). None of these
estimates were affected by bacteria remaining on the filters after
sonication, since over 99% of the bacteria on the filters were removed
by sonication (99.7 to 100% for bacteria from endive; 99.7 to 99.9% for bacteria from parsley).
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TABLE 6.
Population sizes of solitary bacteria and bacteria
aggregated in biofilms on leaves of field-grown broad-leaf endive
and parsley
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Not all bacteria were removed from leaves by washing. In these
experiments, 70 to 93% of the culturable bacteria on endive
leaves and
72 to 87% of those on parsley were removed by gentle
washing. The
efficiency of removal of bacteria will contribute
to errors in
estimates of the frequency of biofilm bacteria if
the proportion of
biofilm bacteria in the populations remaining
on the leaves is not the
same as in those removed from leaves.
With a simple mathematical model,
we calculated the relationship
between the potential error of the
estimate of the frequency of
biofilm bacteria and the efficiency of
leaf washing. We considered
the two extreme cases: (i) that all
bacteria remaining on leaves
after washing were solitary or (ii) that
all remaining bacteria
were in biofilms. Using this model, we
calculated that the potential
value for the mean frequency of biofilm
bacteria in the total
population is in the range of
R ×
nBF to
R ×
nBF + (1
R), where
R is the proportion of the total
bacteria on a leaf that were
removed by washing and
nBF is the estimate of the proportion of
bacteria in biofilms as determined by this technique. For example,
for
the first endive sample (Table
6), 70% of the culturable
bacteria were
removed from this leaf by washing and bacteria in
biofilms constituted
38% of the recovered population. Hence, if
we account for the bacteria
remaining on this leaf after washing,
the potential value for the mean
frequency of biofilm bacteria
in this population is between 27 and
57%. For the second endive
leaf (Table
6), where 93% of the
culturable bacteria were removed
by washing and 32% of these bacteria
were in biofilms, the potential
value for the mean frequency of biofilm
bacteria lies in the range
of 30 to 37%.
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DISCUSSION |
The above technique to estimate the abundance of biofilm bacteria
in epiphytic communities employs methods for recovery and separation of
bacteria that are adapted to the particular conditions of the
phyllosphere ecosystem. In the phyllosphere, solitary cells are not
planktonic and may be as loosely or as firmly attached to the leaf
surface as are biofilms. Hence, bacteria must be removed from leaves
and then biofilms must be separated from solitary cells prior to
disintegration of the biofilms. For the technique described here, we
used gentle agitation of leaves in a buffer, one of several standard
methods for removing bacteria from the phyllosphere (9).
Leaf washing offers advantages and disadvantages for estimation of the
abundance of biofilms in the phyllosphere. First, washing does not
remove all the bacteria from the phyllosphere. However, the efficiency
of removal can be measured. For our technique, we have proposed a
method to evaluate the error in the estimates of mean biofilm abundance
due to washing efficiency. This error should not be confused with the
random errors associated with this estimate. Random errors should be
accounted for by repeated measures from the same sample. Second,
washing could alter biofilm integrity and lead to the liberation of
solitary cells. We suggest that the gentle washing used in the
technique described here leads to very minimal release of solitary
cells from biofilms. For biofilms formed by Pseudomonas
aeruginosa on polymer discs, vigorous shaking for 10 min was
needed to remove loosely attached cells from the biofilms
(16). Furthermore, our study and previous studies clearly show that vigorous mechanical treatments such as high-energy
ultrasonication are needed to significantly alter biofilm integrity
(1, 13). An advantage of leaf washing is that it permits
subsequent dilution plating on culture media and isolation,
characterization, and identification of bacteria in the biofilm and
solitary populations. However, it would also be possible to use
oligonucleotide probes, sera, or epifluorescent indicators of viability
to characterize directly the bacteria in the leaf washing so as to
avoid the use of culture media.
For separation of solitary cells and biofilms in leaf washings, we used
Isopore polycarbonate filters. These filters had the lowest retention
rate of the filters tested. However, because of their relatively low
porosity, they retain a certain proportion of bacteria that are small
enough to pass through the pores. According to the manufacturer of
these filters, a significant increase in their porosity would render
them too fragile to be manipulated. To reduce the retention rates of
bacteria on these filters, we examined numerous filtration conditions
(data not shown). We pretreated the Isopore filters with bovine serum
albumin, proteose peptone, dextrin, skim milk, pectin, or heat-killed
bacterial cells. We also used refrigerated bacterial suspensions and
hydrophobic Isopore filters. None of these treatments caused a
significant reduction in the retention rates relative to those of the
untreated filters. Furthermore, the origin of the phyllosphere bacteria
studied here seemed to affect their ability to stick to these filters.
Hence, when estimating the abundance of biofilm bacteria in
phyllosphere communities, it is necessary to verify that the proportion
of solitary bacteria retained on the filters is lower than the
proportion of biofilm bacteria in the community.
To disintegrate biofilms, chemical and physical approaches are
possible. In preliminary work before performing this study, we examined
physical methods involving glass beads or Ultra-turrax homogenizers as
well as the use of surfactants or pharmaceutical products for
liquefying mucus (acetylcysteine). These techniques were either lethal
or inefficient. Here we have described sonication conditions that do
not significantly reduce the culturability of bacterial cells and that
also effectively lead to the disintegration of biofilms. Sonication has
been used in numerous studies to disintegrate biofilms for enumeration
of cells. In some of these studies, no information or only partial
information concerning the energy and duration of sonication and its
effect on biofilm disintegration and cell viability is presented
(2, 11, 14). Bauer-Kreisel et al. (1) have
described ultrasonication conditions that effectively detach and
disintegrate mixed-culture biofilms from sintered glass beads in a
fluidized-bed reactor but that lead to the destruction of 90% of the
cells. We measured the effectiveness of disintegration by demonstrating
microscopically that large particles were eliminated from biofilm
suspensions and, by using culture techniques, that fewer bacterial CFU
were retained on filters after sonication. Other evidence for the
effectiveness of sonication would be an increase in the number of
solitary bacterial cells after sonication. However, it was not possible
to measure increases in the number of solitary cells in this study
because the initial number of solitary cells was so large that it would
have masked any increases resulting from cells liberated by sonication.
The technique described above will allow quantification of the dynamics
of the population sizes of solitary and biofilm bacteria in the
phyllosphere of diverse plants in response to stimuli from the physical
and biological environment. Such an approach will allow us to address
questions about the role played by biofilm-like structures in the
ecology of epiphytic bacteria; we will be able to measure if the
relative abundance of epiphytic bacteria in biofilm-like structures
increases after various environmental stresses. This technique will
also permit a comparison of the taxonomic composition and the genetic
and physiological profiles of biofilm and solitary bacteria in
phyllosphere communities. The preliminary data that we have obtained by
this technique provides the first measure of biofilm abundance in
epiphytic bacterial communities. For other ecosystems where biofilm
abundance has been estimated, Costerton et al. (4) conclude:
"based on detailed analysis of hundreds of aquatic systems, biofilm
populations predominate in virtually all nutrient-sufficient aquatic
systems independent of system geometry and of the type of ecosystem
involved." Our preliminary data suggests that this generality cannot
be made for epiphytic bacterial populations on terrestrial plants. The tools described above will allow studies of the factors that influence biofilm abundance on leaf surfaces under natural settings. This tool
may also be useful for studies of biofilm abundance in other nonaquatic systems.
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ACKNOWLEDGMENTS |
We thank Catherine Glaux for excellent technical assistance. We
also thank the participants of the biofilms discussion group at
http://www.im.dtu.dk/biofilms for their useful remarks and suggestions.
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FOOTNOTES |
*
Corresponding author. Mailing address: INRA, Station de
Pathologie Végétale, Domaine St. Maurice, B.P. 94, 84143 Montfavet cedex, France. Phone: (33)-490-31-63-84. Fax:
(33)-490-31-63-35. E-mail: morris{at}avignon.inra.fr.
Present address: Department of Environmental Science, Policy and
Management, University of California, Berkeley, CA 94720-3110.
Present address: INRA, Station de Pathologie Végétale,
49071 Beaucouzé cedex, France.
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Applied and Environmental Microbiology, December 1998, p. 4789-4795, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
