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Title
Susceptibility of Actinobacillus
actinomycetemcomitans to six antibiotics decreases
as biofilm matures
Author(s)
AlternativeTakahashi, N; Ishihara, K; Kato, T; Okuda, K
JournalThe Journal of antimicrobial chemotherapy, 59(1):
59-65
URL http://hdl.handle.net/10130/167
Right
This is a pre-copy-editing, author-produced PDF of
an article accepted for publication in The Journal
of antimicrobial chemotherapy following peer
review. The definitive publisher-authenticated
version Takahashi N, Ishihara K, Kato T, Okuda K.
Susceptibility of Actinobacillus
actinomycetemcomitans to six antibiotics decreases
as biofilm matures. J Antimicrob Chemother. 2007
Jan;59(1):59-65. is available online at:
http://jac.oxfordjournals.org/cgi/content/abstract/
59/1/59.
1
Susceptibility of Actinobacillus actinomycetemcomitans to six 2
antibiotics decreases as biofilm matures 3
4
1, 2Naoko Takahashi, 1, 2Kazuyuki Ishihara*, 1Tetsuo Kato, 1 Katsuji Okuda 5
1Department of Microbiology and 2Oral health Science Center 6
Tokyo Dental College 7
1-2-2 Masago, Mihama-ku 8
Chiba 261-8502 9
Japan 10
11
*Corresponding author 12
Kazuyuki Ishihara 13
Tokyo Dental College 14
1-2-2 Masago, Mihama-ku 15
Chiba 261-8502 16
Japan 17
TEL: +81-43-270-3742, Fax: +81-43-270-3744 18
e-mail: [email protected] 19
20
Running title: Antibiotic susceptibility of periodontopathic biofilm 21
Key word: Periodontitis, Bacterial biofilm, Antibiotic resistance, 22
Antibiotic therapy, Actinobacillus actinomycetemcomitans 23
ABSTRACT 24
Objectives: Actinobacillus actinomycetemcomitans is a major causative agent of chronic 25
and aggressive periodontitis. Freshly isolated strains of A. actinomycetemcomitans display 26
rough-type colonies and initiate biofilm formation on glass surfaces. The purpose of this 27
study was to determine the antibiotic susceptibility of A. actinomycetemcomitans biofilm 28
during different phases of maturation. 29
Methods: Using 96-well microtitre plates, we determined the antibiotic susceptibility of 30
rough-type strain 310a to concentrations of from 0.1 to 10 mg/L each of erythromycin, 31
ofloxacin, ampicillin, cefalexin, tetracycline, and minocycline during biofilm formation. 32
Antibiotics were added at the start of the culture (early phase) and at after 24 hours of 33
cultivation (mature phase). 34
Results: Adding 10 mg/L ampicillin, 10 mg/L cefalexin, 0.1 or 1 mg/L tetracycline, or 0.1 35
mg/L minocycline significantly inhibited 310a biofilm formation in the early phase, but 36
not in the mature phase. Although adding 10 mg/L of erythromycin, tetracycline, or 37
minocycline reduced biofilm development in the early phase, it enhanced 310a biofilm 38
development in the mature phase. Ofloxacin exerted a strong inhibitory effect in both the 39
early and mature phases of biofilm formation throughout all experiments. 40
Conclusions: The present study demonstrated that the susceptibility of A. 41
actinomycetemcomitans to many antibiotics decreased after biofilm maturation. 42
INTRODUCTION 43
Biofilms are ubiquitous in natural, industrial, and clinical environments, and 44
participate in many chronic infections, including those involved in infectious kidney 45
stones, bacterial endocarditis, and cystic fibrosis1. In biofilm, the susceptibility of 46
microorganisms to antimicrobial agents differs from that of planktonic cultures of the 47
same bacteria,2 and quorum sensing will lead to alterations in patterns of gene 48
expression.3 These factors are major contributors to the etiology of infectious disease. 49
In the oral cavity, multiple species of microorganisms form biofilms not only on tooth 50
surfaces but also on soft tissue and more than 500 bacterial taxa have been isolated from 51
the oral cavity.4 Dental plaque is a microbial biofilm formed by multiple organisms bound 52
tightly to the tooth surface. An increase in anaerobic Gram-negative rods such as 53
Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis is closely 54
associated with the etiology of periodontitis.5 55
The susceptibility of periodontopathic bacteria to antimicrobial agents changes 56
with formation of biofilm. Wright et al.6 examined the in vitro effects of metronidazole on 57
P. gingivalis, and found that biofilm cells had a 160-times greater MIC than planktonic 58
cells. Larsen7 also investigated the susceptibility of P. gingivalis biofilm cells to 59
amoxicillin, doxycycline, and metronidazole, and found that the MBC for these agents 60
was 2-8 times greater, or in the case of doxycycline, 64 times greater than that for 61
planktonic cultures. 62
A. actinomycetemcomitans is frequently isolated from aggressive periodontitis.8 63
Fresh isolates of this microorganism form rough-type colonies on agar plates, and also 64
form biofilms on the glass surfaces of test tubes by using fimbriae.9, 10 Repeated 65
subculture results in conversion from rough morphological type to smooth type, which 66
results in turbid growth in broth.10 This microorganism has also been reported to invade 67
host epithelial cells.11 Moreover, another study found that A. 68
actinomycetemcomitans-positive sites were at greater risk of further attachment loss,12 69
while absence of this microorganism had a negative predictive value for further 70
attachment loss.13 Periodontal therapies which involve mechanical cleaning such as 71
scaling and root planing and/or open-flap surgery are essential in eliminating biofilm 72
containing periodontopathogens from periodontal lesions. However, mechanical treatment 73
alone cannot reliably eliminate A. actinomycetemcomitans.14 These reports suggest that 74
the eradication of this microorganism requires a combination of mechanical therapy and 75
chemotherapy. The primary objective of this study was to clarify the efficacy of various 76
antibiotics against rough type A. actinomycetemcomitans biofilm formation at different 77
phases of growth and determine which was the most effective in eradicating this 78
microorganism. 79
MATERIALS AND METHODS 80
Bacterial strains and culture conditions. A. actinomycetemcomitans clinical isolates 81
310a (provided by Dr. H. Ohta, Ibaraki University, Japan). Rough- and smooth-type A. 82
actinomycetemcomitans 310a have been described previously.9 Strains were grown on 83
blood agar plates containing Tryptic soy agar (Becton Dickinson Microbiology System, 84
Cockeysville, MD) supplemented with 10% defibrinated horse blood, haemin (5 μg/mL; 85
Sigma Chemical Co., St. Louis, MO) and menadione (0.5 μg/mL; Wako Pure Chemical 86
Industries, Osaka, Japan), or Tryptic soy broth (Becton Dickinson Microbiology System) 87
supplemented with 0.1% Yeast-Extract (Difco Laboratories, Detroit, MI) （TSBYE）in an 88
anaerobic chamber (N2: 80%, H2: 10%, CO2: 10%) at 37oC. Rough-type colony 89
morphology was confirmed by observing the wrinkled colony morphology on the agar 90
plates. Smooth-type 310a was obtained by repeated subculture of the rough types of these 91
strains. 92
Staining of extracellular polysaccharide (EPS) of A. actinomycetemcomitans. The 93
extracellular polysaccharide (EPS) of the bacterial cells was stained with Alcian blue 94
solution (pH 7.0, Nacalai Tesque, INC., Kyoto, Japan), which binds to acidic 95
polysaccharides, according to the method of Sauer et al.15 A. actinomycetemcomitans 310a 96
was precultured for about 40 h. Aliquots of 200 μL of each preculture were inoculated 97
into glass bottom dishes (Matsunami Glass IND., LTD., Kishiwada, Japan) containing 4 98
mL TSBYE. After 48 h incubation, the culture medium was removed, and the 99
microcolonies on the glass surface were washed with phosphate-buffered saline (PBS, pH 100
7.2). Then they were stained with the Alcian blue solution for 30 min at room 101
temperature. After rinsing with distilled water, the specimens were observed under a 102
microscope. The bacteria were also stained with 0.1% crystal violet (Difco) for 15 min 103
and processed for microscopic observation. 104
Antimicrobial agents and MIC determinations. The antibiotics used in this study were 105
ofloxacin (LKT Laboratories, Inc., MI), cefalexin monohydrate (ICN Biomedical, Inc., 106
Aurora, OH), ampicillin sodium (Wako), erythromycin (Wako), tetracycline 107
hydrochloride (Wako), and minocycline hydrochloride (Wako). 108
The MICs for planktonic cells were determined by duplicate tests to compare the 109
susceptibility of the biofilms. Aliquots of 50 μL A. actinomycetemcomitans 310a smooth 110
type were inoculated into 1 mL TSBYE containing each antibiotic and incubated at 37oC 111
in an anaerobic chamber containing 80% N2, 10% H2, and 10% CO2 for 48 h. MIC was 112
determined as the lowest concentration of the antibiotic inhibiting visible growth of the 113
bacteria. 114
Quantification of biofilms. Quantification of biofilms was achieved by staining with 115
crystal violet. Five μL A. actinomycetemcomitans 310a rough type were inoculated into 116
wells of 96-well (flat-bottom) cell culture plates (Sumitomo Bakelite Co, Ltd, Tokyo, 117
Japan) containing 95 μL TSBYE in each well. After the designated incubation time (12, 118
18, 24 and 48 h), the culture medium containing planktonic cells was removed, and the 119
wells were washed with 200 μL distilled water. The adherent bacteria were stained with 120
50 μL of 0.1% crystal violet for 15 min at room temperature. After rinsing twice with 200 121
μL distilled water, the dye bound to the biofilms was extracted with 200 μL of 99% 122
ethanol for 20 min. The extracted dye was then quantified by measuring the absorbance at 123
595 nm with a microplate reader (Model 3550, Bio-Rad Laboratories, Hercules, CA). 124
Effects of antimicrobial agents on biofilm formation by A. actinomycetemcomitans. 125
As described above, 5 μL aliquots of precultured cells were inoculated into the wells of 126
cell culture plates containing 95 μL TSBYE. The plates were incubated under anaerobic 127
conditions at 37oC for 12, 18, 24, and 48 h, and biofilm formation was assayed. The 128
results of the preliminary experiment were used to confirm that the biofilm grew 129
continuously from 0 to 48 h. To determine the relationship between antibiotic resistance 130
and biofilm formation, we evaluated the resistance of these cells to the various antibiotics 131
at two phases of biofilm formation: early phase (the period of microcolony formation) and 132
mature phase (the period after thin biofilm formation). Early phase indicates 0 h to 24 h 133
after inoculation; mature phase indicates 24 h to 48 h after inoculation. 134
To determine the susceptibility of the early-phase biofilm to antibiotics, 135
precultured cells were inoculated into the wells of the plates with TSBYE supplemented 136
with each antibiotic. The plates were incubated under anaerobic conditions at 37oC for 24 137
h and then assayed for quantification of biofilm formation as described above, and for 138
quantification of viability of biofilm as described below. 139
To determine the antibiotic susceptibility of mature-phase biofilm, precultured 140
cells were inoculated into the wells of the plates containing 95 μL TSBYE without 141
antibiotics. After 24 h incubation, 100 μL TSBYE containing a specified concentration of 142
each antibiotic was added to each well. The plates were incubated for a further 24 h and 143
then assayed for biofilm formation and quantification of viability of biofilm as described 144
below. 145
Evaluation of viability of biofilm. To measure the number of viable cells in the biofilms, 146
the bioactivity of A. actinomycetemcomitans was evaluated by an ATP-bioluminescence 147
assay with the Kinshiro KKT-100 (TOYO B-Net CO. LTD., Tokyo, Japan) based on the 148
method of Lundin et al.16. Briefly, grown A. actinomycetemcomitans cells were washed 149
with ATP buffer (50 mM HEPES and 5 mM MgSO4, pH 7.75). Then an ATP extractant 150
solution (TOYO B-Net CO. LTD.) was added, and the mixture was incubated for 10 sec. 151
After addition of a bioluminescent reagent, bioluminescence was measured with a 152
luminometer (Model Lumat LB9507, Berthold Technologies, Bad Wildbad, Germany) for 153
60 sec. The relationship between the adenosine triphosphate (ATP) content and the viable 154
cell count [cfu/mL] in the liquid aliquots was examined before evaluation of cell viability. 155
After determining the relationship, the number of viable cells of A. 156
actinomycetemcomitans 310a rough type on the 96-well plate was then evaluated using 157
bioactivity. The bioactivity of the biofilms after exposure to each antibiotic was 158
calculated and expressed as the relative ATP content. Relative ATP content = ATP levels 159
of antibiotic-treated samples/ATP levels of controls (without antibiotics). 160
Scanning electron microscopic observation of biofilm. Aliquots of 50 μl of precultured 161
A. actinomycetemcomitans 310a rough type were inoculated into 12-well plates 162
(Sumitomo Bakelite Co, Ltd.). Each well contained 1 mL TSBYE and a 12 mm diameter 163
circular glass coverslip (Matsunami Glass IIND, Tokyo, Japan). After 24 h incubation 164
under anaerobic conditions at 37oC, 1 mL TSBYE supplemented with various 165
concentrations of each antibiotic was added to each well, and the plates were incubated 166
again for 24 h. The biofilms formed on the coverslips were fixed in 2% glutaraldehyde in 167
PBS at room temperature for 1 h. After washing with PBS, the cells were dehydrated 168
through a graded series of ethanol, dried at the critical point of t-butyl alcohol, and then 169
coated with osmium. The samples were observed with a scanning electron microscope 170
(SEM, Field Emission Scanning Microscopy, JSM-6340F, JEOL. Ltd., Tokyo, Japan) at 171
an acceleration voltage of 15 kV. 172
Statistics. Each experiment using the 96-well plates was performed more than three 173
times, with each conducted in triplicate. The Mann-Whitney U test was used for 174
quantification of the biofilms, and the ATP-bioluminescence assays to identify 175
statistically significant differences. 176
RESULTS 177
Morphology and growth of A. actinomycetemcomitans biofilm. Rough-type A. 178
actinomycetemcomitans 310a formed microcolonies at the bottoms of the wells in the 179
culture plates. In contrast, smooth-type A. actinomycetemcomitans 310a attached weakly 180
and uniformly (Fig. 1, A and C). A. actinomycetemcomitans 310a rough-type strains 181
stained with Alcian blue, but strain 310a smooth type did not (Fig. 1, B and D). 182
Biofilm growth of rough-type A. actinomycetemcomitans 310a in a 96-well plate 183
is shown in Fig. 2. Growth continued until 48 h of culture. We chose 24 h as the 184
incubation time as that time point occurs well before the plateau of biofilm maturation. 185
MICs for A. actinomycetemcomitans 310a smooth type. The MICs for A. 186
actinomycetemcomitans 310a smooth types are presented in Table 1. Ofloxacin was the 187
most effective, and tetracycline and minocycline were moderately effective among the 188
antibiotics tested. Ampicillin, erythromycin, and cefalexin exhibited a weaker 189
antimicrobial effect against the strains tested. The bacterial strain was not completely 190
resistant to any of the antibiotics used in this study. 191
Effects of antibiotics on biofilm formation by A. actinomycetemcomitans 310a rough 192
type. We investigated the effects of these antibiotics during the early phase of A. 193
actinomycetemcomitans 310a rough-type biofilm formation in 96-well cell culture plates 194
for 24-h (Fig. 3A). When the cells were cultured together with 10 mg/L of any of the 195
antibiotics, they all showed significantly reduced biofilm formation compared to that of 196
the controls (p<0.05). Ofloxacin significantly inhibited biofilm formation at all 197
concentrations used in this study. Tetracycline and minocycline reduced biofilm 198
formation at concentrations of 1-10 mg/L. Erythromycin, ampicillin, and cefalexin 199
reduced biofilm formation only at the highest concentration of 10 mg/L. In contrast, at a 200
low concentration of 0.1 mg/L, erythromycin and ampicillin both increased biofilm 201
formation significantly compared to the controls (p<0.05). 202
The effects of the antibiotics on mature-phase biofilm formation are summarized 203
in Fig. 3B. Ofloxacin completely inhibited biofilm growth to the level where ofloxacin 204
was added. Erythromycin, tetracycline, and minocycline exhibited a moderate inhibitory 205
effect on biofilm growth, even in the mature phase, while cefalexin and ampicillin did 206
not. The mature phase was not affected by addition of 10 mg/L ampicillin, 10 mg/L 207
cefalexin, 0.1 or 1 mg/L tetracycline, or 0.1 mg/L minocycline, all of which showed 208
significant inhibitory effects in the early phase of biofilm formation. Relative mass of 209
mature-phase biofilm at 48 h from 24 h after treatment with 10 mg/L of each antibiotic, 210
apart from ofloxacin and minocycline, showed an increase from 24 h onwards, although a 211
reduction was observed in the 48-h control. 212
Viability of A. actinomycetemcomitans 310a rough-type biofilms. At both 213
quantification points, ofloxacin showed significant inhibitory effects on the early- and 214
mature-phase biofilms, while tetracycline, and minocycline showed significant inhibitory 215
effects on the early-phase biofilms alone (Fig. 3A). However, the assays did not reveal the 216
actual bioactivity of the microorganisms in these biofilms as quantification with crystal 217
violet stained both live and dead cells. It is inherently difficult to evaluate viable cells in 218
biofilms with reproducibility, as releasing and dispersing biofilm cells from hard surfaces 219
is difficult. Therefore, to determine the effects of these antibiotics on the viability of the 220
bacterial cells in the biofilm, we used an ATP-bioluminescence assay to measure of 221
viability (bioactivity). The relationship between viable cell count (cfu) and 222
ATP-bioluminescence is shown in Fig. 4. This result confirms that viable cell number can 223
be quantified by ATP-bioluminescence. 224
The quantity of viable cells in the biofilm was evaluated by 225
ATP-bioluminescence. When 10 mg/L of each antibiotic was added to the early-phase 226
biofilm, the relative ATP content of all the 310a biofilms decreased significantly 227
compared to that of the controls (p<0.05) (Fig. 5A). In contrast, when 10 mg/L of each 228
antibiotic was added at the mature phase of 310a biofilm formation, only ofloxacin 229
showed a significant decrease in relative ATP content (p<0.05). ATP levels in the biofilm 230
increased after addition of ampicillin or cefalexin indicating susceptibility. Unexpectedly, 231
erythromycin, tetracycline, and minocycline showed significantly increased ATP (p<0.05) 232
(Fig. 5B), suggesting that the viable cells existing inside the biofilm were not negatively 233
affected, even if biofilm formation somewhat decreased, as revealed by crystal violet 234
staining. 235
SEM observations. Fig. 6 shows scanning electron micrographs of the A. 236
actinomycetemcomitans 310a rough-type biofilms that formed on the glass coverslips. 237
The most striking feature of the 310a rough type was that it consisted of individual cells 238
bound tightly to each other, forming a biofilm structure resembling a ball (Fig. 6A). This 239
colonial morphology and size were significantly affected by exposure to ofloxacin (Fig. 240
6B). The structure changed into a honeycomb pattern in some parts of the biofilm. In 241
other sections, the cells expanded extraordinarily, until the sizes were about three times 242
larger than normal. Swelling of the cells was observed in cefalexin- and ampcillin-treated 243
biofilm (Fig. 6, D and E). Minocycline and minocycline treatment yielded only a small 244
effect on colony and cell morphology (Fig. 6, E-G). 245
246
DISCUSSION 247
Antibiotic administration in conjunction with mechanical cleansing, such as 248
scaling and root planing, or with periodontal surgery is essential in eradicating A. 249
actinomycetemcomitans from periodontal lesions.12, 13 Biofilm bacteria exhibit a distinct 250
mode of growth which differs from that of planktonic cells.2 However, most evaluations 251
of the susceptibility of A. actinomycetemcomitans to antibiotics have been performed with 252
planktonic organisms, and there are few reports on the susceptibility of rough-type strain 253
to antimicrobial agents.17 The purpose of this study was to characterize the susceptibility 254
of rough type A. actinomycetemcomitans biofilms to various antibiotics. Biofilms consist 255
of cells and EPS, and their accumulation is a net result of planktonic cell attachment, 256
biofilm cell growth, detachment and EPS production.1, 18 The results of Alcian blue 257
staining indicated that A. actinomycetemcomitans 310a rough type produced EPS. This 258
result agrees with the report of Kaplan et al.19 demonstrating that a linear polymer of 259
N-acetyl-D-glucosamine residues in the β (1,6) linkage was a major matrix component of 260
biofilms produced by A. actinomycetemcomitans. 261
In this study, we also demonstrated differences in the susceptibilities of A. 262
actinomycetemcomitans biofilm cells to ofloxacin, cefalexin, ampicillin, erythromycin, 263
tetracycline, and minocycline. These antibiotics were chosen for their different kinetics in 264
drug activity. Some of them are clinically used in the treatment of periodontitis.20 As 265
rough-type strains of A. actinomycetemcomitans form biofilms on glass surfaces of test 266
tubes with no turbidity when cultured in broth,9 we could not use turbidity to define the 267
MICs of the rough type in test tubes. Throughout the study, we demonstrated that the 268
most effective antibiotic against A. actinomycetemcomitans was ofloxacin. This 269
susceptibility to ofloxiacin was similar to that in the reports of earlier investigations using 270
planktonic cells.21 271
We compared the effects of various antibiotics at the early and mature phases of 272
biofilm formation by using crystal violet staining (Fig. 3). Our results suggest that the 273
susceptibility of A. actinomycetemcomitans rough type decreases with maturation of the 274
biofilms. Antibiotic resistance within biofilms has been demonstrated to change in several 275
microorganisms.18, 22 The following reasons for such resistance have been suggested: the 276
antibiotics penetrate slowly or incompletely into a biofilm; microbial nutrients and waste 277
products modify the local environment inside a biofilm; the growth rates of the bacteria 278
decrease inside a biofilm; biofilm/attachment-specific phenotypes develop inside a 279
biofilm. There were two phases in the lifecycle of oral biofilms that we wished to 280
observe: the first occurring after tooth brushing or professional mechanical tooth 281
cleaning, and the second occurring after biofilm has grown over a period of several hours, 282
after which, it reaches maturation. In our simulation, these were designated as “early 283
phase” and “mature phase”. In preliminary tests, we also observed a decrease in the 284
susceptibility of mature phase biofilm using rough-type A. actinomycetemcomitans AB55 285
(data not shown). These results suggest that mechanical removal of biofilm prior to 286
chemotherapy is required to eradicate A. actinomycetemcomitans from the oral cavity. 287
We used SEM to visually investigate the effects of antibiotics on mature-phase A. 288
actinomycetemcomitans 310a biofilms, and found that colonial morphology was 289
distinctively affected depending on the antibiotic. As shown in Fig. 6B, ofloxacin induced 290
the most dramatic morphological changes, indicating that ofloxacin damages A. 291
actinomycetemcomitans in biofilm. Among all the antibiotics used in this study, ofloxacin 292
had the greatest inhibitory effects in both the early and mature phases of biofilm formation 293
through all experiments. Kleinfelder et al. 23 demonstrated that systemic ofloxacin used as 294
an adjunct to open flap surgery was able to suppress A. actinomycetemcomitans to below 295
detectable levels in patients. Our present results agree with those of their report. 296
The ATP-bioluminescence assay has been reported to be a useful tool in 297
evaluating the quantity of bacterial biofilms.24 We used this assay in conjunction with 298
crystal violet staining to evaluate the biofilm formation of A. actinomycetemcomitans. All 299
of the antibiotics used in this study showed an inhibitory effect on the viability of the 300
biofilm cells at the early phase (Fig. 5A). After exposure of mature-phase biofilm to 301
ampicillin or cefalexin, ATP increased significantly. These antibiotics did not affect the 302
growth of A. actinomycetemcomitans. It is possible that this discrepancy between 303
production and consumption of ATP was due to weak inhibition of cell-wall organization 304
by antibiotics under MIC level. Another possibility, however, is that this temporary 305
increase was due to an influx of ATP released from dying microorganisms. Erythromycin, 306
tetracycline, and minocycline enhanced the bioactivity of the 310a cells at the mature 307
phase, in spite of a reduction in biofilm formation revealed by crystal violet staining. 308
They exerted a bacteriostatic effect, but no bactericidal effect. It is possible that the 309
accumulation of ATP in biofilm cells is involved in a survival response to antibiotics . To 310
clarify these issues, further analysis employing other methods of evaluation is required to 311
determine live cell number. Erythromycin showed no effect, either at the early or 312
mature phases of biofilm formation. Several reports have indicated that the macrolide 313
family affects bacterial quorum sensing at sub-MICs, and that this results in an 314
attenuation of biofilm formation.25 It has also been reported that auto-inducer 2 (AI-2) 315
signals in A. actinomycetemcomitans may modulate aspects of virulence, including the 316
uptake of iron, and that they may control cellular adaptation to growth under iron-limiting 317
conditions26. Our results showed that sub-MICs of erythromycin did not affect biofilm 318
formation, which suggests that AI-2 in A. actinomycetemcomitans is not affected by 319
erythromycin, or that the gene induced by AI-2 exerts no effect on susceptibility to 320
antibiotics. Further analyses such as evaluation of AI-2 production are required to clarify 321
this point. 322
The results of our study demonstrated that the susceptibility of rough-type A. 323
actinomycetemcomitans to antibiotics decreases during maturation of the biofilm. These 324
data highlight the difficulty of designing antibiotic therapies for periodontitis. Further 325
study is required to determine the effect of dose in the gingival crevice in chemotherapy. 326
Periodontal pockets harbor a variety of different microbial species with different 327
susceptibilities to different antimicrobials in vivo. Therefore, it is essential to determine 328
the antibiotic susceptibility of biofilms containing different species of subgingival 329
bacteria. 330
ACKNOWLEDGMENTS 331
We wish to thank K. Tadokoro for his assistance with the SEM observations. Part 332
of this work was supported by Grant 16591837 from the Ministry of Education, Science, 333
Sport, and Culture (MEXT) of Japan and Grant HRC7 from the Oral Health Science Center 334
of Tokyo Dental College and a “High-Tech Research Center” Project for Private 335
Universities: matcching fund subsidy from MEXT, 2006-2010.. 336
TRANSPARENCY DECLARATIONS 337
None to declare. 338
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298-308. 408
409
410
Table 1. MICs for A. actinomycetemcomitans 310a and AB55 smooth type. 411
MIC (mg/l)
Strain OFX LEX AMP ERY TET MIN
310a 0.03 6.0 1.0 2.0 0.13 0.25
412
MICs for smooth-type (planktonic-type) A. actinomycetemcomitans performed under 413
same time schedule as early-phase. 50 μL or 200 μL A. actinomycetemcomitans 310a or 414
AB55 smooth type were inoculated into TSBYE medium containing antibiotics and then 415
incubated at 37oC in an anaerobic chamber for 48 h. MICs were determined as lowest 416
concentration of antibiotic inhibiting visible growth of bacteria. Experiments were 417
performed in duplicate. 418
FIGURE LEGENDS 419
Fig. 1. Microscopical observations of A. actinomycetemcomitans 310a biofilm. 420
Microscopy of 48-h cultured A. actinomycetemcomitans 310a (magnification×1000). 421
Pictures: A and B show A. actinomycetemcomitans 310a rough type. Pictures: C and D 422
show A. actinomycetemcomitans 310a smooth type. Pictures: A and C show staining with 423
crystal violet. Pictures: B and D show staining with Alcian blue. 424
425
Fig. 2. Biofilm formation of A. actinomycetemcomitans 310a rough type on 96-well 426
plates. Aliquots of 5 μL cell suspension were inoculated into wells of 96-well plates 427
containing 95 μL medium and incubated for designated times. After incubation, biofilm 428
formation assays were performed. Experiments were performed in triplicate 429
430
Fig. 3. A: Effects of antibiotics on A. actinomycetemcomitans 310a rough-type early 431
phase biofilm formation. Cells were inoculated into wells of 96-well plate and incubated 432
for 24-h with each antibiotic. B: Antibiotic susceptibility in mature phase biofilm. Cells 433
were inoculated into wells of 96-well plate. After 24-h incubation, antibiotics were added, 434
and culture was continued for further 24-h. Polka-dot pattern indicates control without 435
antibiotics incubated for 48 h. Striped-pattern indicates biofilm formation at time 436
antibiotics added (24 h). Experiments were performed more than three times, and each 437
was conducted in triplicate. Effects of antibiotics were statistically analyzed with 438
Mann-Whitney U test, * p<0.05 vs. biofilm level after 48 h incubation [polka-dot pattern]. 439
† p<0.05 vs. biofilm level at time antibiotics added. 440
441
Fig. 4. Relationship between ATP-content and cfu/mL. Aliquots of 200 μL cell 442
suspension of A. actinomycetemcomitans 310a smooth type were inoculated into test 443
tubes containing 2 ml medium and incubated for 24 h. After incubation, ATP content and 444
bacterial viability (cfu/mL) were measured. 445
446
Fig. 5. Effects of antibiotics on A. actinomycetemcomitans 310a rough type bioactivity. 447
A: Effects in early phase of biofilms. Cells were inoculated into wells of 96-well plate 448
and incubated for 24-h with each antibiotic (10 mg/L). B: Effects in mature phase of 449
biofilms. Cells were inoculated into wells of 96-well plate. After 24-h incubation, 450
antibiotics (10 mg/L) were added, and culture was continued for further 24-h. Polka-dot 451
pattern indicates control without antibiotics incubated for 48 h. Striped-pattern indicates 452
biofilm formation at time antibiotics added (24 h). Experiments were performed more 453
than three times, and each was conducted in triplicate. Effects of antibiotics were 454
statistically analyzed with Mann-Whitney U test, * p<0.05 vs. biofilm level after 48 h 455
incubation without antibiotics [polka-dot pattern]. † p<0.05 vs. biofilm level at time 456
antibiotics added. 457
458
Fig. 6. Scanning electron micrographs of biofilms formed by A. actinomycetemcomitans 459
310a rough type on glass coverslips. After 24-h incubation, cells were added to medium 460
supplemented with or without the following antibiotics (10 mg/L each) and incubated 461
further for 24-h; Panels: A, control (without antibiotics); B, ofloxacin; C, cefalexin; D, 462
ampicillin; E, erythromycin; F, tetracycline; and G, minocycline. 463
464
465
466
467
468
469
470
Fig. 3 471
472
473
474
Fig. 5 475
476
477
478
Fig. 1
0
0.2
0.4
0.6
0.8
1
1.2
12 18 24 48
Incubation time (hours)
Abs
orba
nce
(595
nm
)
Fig. 2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 (control) 0.1 1 10
Concentration (mg/L)
Abs
orba
nce
(595
nm
)
OFXLEXAMPERYTETMIN
*
A* *
**
**
*
*
*
****
Fig. 3
0.0
0.5
1.0
1.5
2.0
2.5
0.1 1 10
Concentration (mg/L)
Abs
orba
nce
(595
nm
) OFXLEXAMPERYTETMIN
B
*
†*
048-h 24-h
Control
†*
† ††††
*
†*
†* †*
†*
†*
†*
†*
**
†
Fig. 3
Viable bacteria (×108 cfu/ml)
R2 = 0.86
0
0.5
1
1.5
2
2.5
3
0 1 2 3 4 5
AT
P co
nten
ts (×
10-8
) M
Fig. 4
0
0.5
1
1.5
0 (control) 10
Concentration (mg/L)
OFXLEXAMPERYTETMIN
Rel
ativ
e A
TP
Con
tent
( ant
ibio
tics/
cont
rol )
A
** *
* * *
Fig. 5
0
1
2
3
4
5
6
7
10
Concentration (mg/L)
OFXLEXAMPERYTETMIN
Rel
ativ
e A
TP
Con
tent
( a
ntib
iotic
s/co
ntro
l )B
*
*
*
*
**
Fig. 5
048-h 24-h
Control
† †
†
A B C
D E F
G
Fig. 6
