IRUCAA@TDC : Susceptibility of Actinobacillus actinomycetemcomitans to six antibiotics decreases as biofilm matures

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(1)Title. Author(s) Alternative Journal. Susceptibility of Actinobacillus actinomycetemcomitans to six antibiotics decreases as biofilm matures Takahashi, N; Ishihara, K; Kato, T; Okuda, K The 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.. Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/.

(2) 1 2. 3. Susceptibility of Actinobacillus actinomycetemcomitans to six antibiotics decreases as biofilm matures. 4 5. 1, 2. Naoko Takahashi, 1, 2Kazuyuki Ishihara*, 1Tetsuo Kato, 1 Katsuji Okuda. Department of Microbiology and 2Oral health Science Center. 6. 1. 7. Tokyo Dental College. 8. 1-2-2 Masago, Mihama-ku. 9. Chiba 261-8502. 10. Japan. 11 12. *Corresponding author. 13. Kazuyuki Ishihara. 14. Tokyo Dental College. 15. 1-2-2 Masago, Mihama-ku. 16. Chiba 261-8502. 17. Japan. 18. TEL: +81-43-270-3742, Fax: +81-43-270-3744. 19. e-mail: [email protected]. 20 21. Running title: Antibiotic susceptibility of periodontopathic biofilm. 22. Key word: Periodontitis, Bacterial biofilm, Antibiotic resistance,. 23. Antibiotic therapy, Actinobacillus actinomycetemcomitans.

(3) 24. ABSTRACT. 25. Objectives: Actinobacillus actinomycetemcomitans is a major causative agent of chronic. 26. and aggressive periodontitis. Freshly isolated strains of A. actinomycetemcomitans display. 27. rough-type colonies and initiate biofilm formation on glass surfaces. The purpose of this. 28. study was to determine the antibiotic susceptibility of A. actinomycetemcomitans biofilm. 29. during different phases of maturation.. 30. Methods: Using 96-well microtitre plates, we determined the antibiotic susceptibility of. 31. rough-type strain 310a to concentrations of from 0.1 to 10 mg/L each of erythromycin,. 32. ofloxacin, ampicillin, cefalexin, tetracycline, and minocycline during biofilm formation.. 33. Antibiotics were added at the start of the culture (early phase) and at after 24 hours of. 34. cultivation (mature phase).. 35. Results: Adding 10 mg/L ampicillin, 10 mg/L cefalexin, 0.1 or 1 mg/L tetracycline, or 0.1. 36. mg/L minocycline significantly inhibited 310a biofilm formation in the early phase, but. 37. not in the mature phase. Although adding 10 mg/L of erythromycin, tetracycline, or. 38. minocycline reduced biofilm development in the early phase, it enhanced 310a biofilm. 39. development in the mature phase. Ofloxacin exerted a strong inhibitory effect in both the. 40. early and mature phases of biofilm formation throughout all experiments.. 41. Conclusions:. 42. actinomycetemcomitans to many antibiotics decreased after biofilm maturation.. The. present. study. demonstrated. that. the. susceptibility. of. A..

(4) 43. INTRODUCTION. 44. Biofilms are ubiquitous in natural, industrial, and clinical environments, and. 45. participate in many chronic infections, including those involved in infectious kidney. 46. stones, bacterial endocarditis, and cystic fibrosis1.. 47. microorganisms to antimicrobial agents differs from that of planktonic cultures of the. 48. same bacteria,2 and quorum sensing will lead to alterations in patterns of gene. 49. expression.3. 50. In the oral cavity, multiple species of microorganisms form biofilms not only on tooth. 51. surfaces but also on soft tissue and more than 500 bacterial taxa have been isolated from. 52. the oral cavity.4 Dental plaque is a microbial biofilm formed by multiple organisms bound. 53. tightly to the tooth surface. An increase in anaerobic Gram-negative rods such as. 54. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis is closely. 55. associated with the etiology of periodontitis.5. In biofilm, the susceptibility of. These factors are major contributors to the etiology of infectious disease.. 56. The susceptibility of periodontopathic bacteria to antimicrobial agents changes. 57. with formation of biofilm. Wright et al.6 examined the in vitro effects of metronidazole on. 58. P. gingivalis, and found that biofilm cells had a 160-times greater MIC than planktonic. 59. cells. Larsen7 also investigated the susceptibility of P. gingivalis biofilm cells to. 60. amoxicillin, doxycycline, and metronidazole, and found that the MBC for these agents. 61. was 2-8 times greater, or in the case of doxycycline, 64 times greater than that for. 62. planktonic cultures.. 63. A. actinomycetemcomitans is frequently isolated from aggressive periodontitis.8. 64. Fresh isolates of this microorganism form rough-type colonies on agar plates, and also. 65. form biofilms on the glass surfaces of test tubes by using fimbriae.9,. 66. subculture results in conversion from rough morphological type to smooth type, which. 67. results in turbid growth in broth.10 This microorganism has also been reported to invade. 10. Repeated.

(5) epithelial. cells.11. 68. host. Moreover,. another. study. found. that. A.. 69. actinomycetemcomitans-positive sites were at greater risk of further attachment loss,12. 70. while absence of this microorganism had a negative predictive value for further. 71. attachment loss.13 Periodontal therapies which involve mechanical cleaning such as. 72. scaling and root planing and/or open-flap surgery are essential in eliminating biofilm. 73. containing periodontopathogens from periodontal lesions. However, mechanical treatment. 74. alone cannot reliably eliminate A. actinomycetemcomitans.14 These reports suggest that. 75. the eradication of this microorganism requires a combination of mechanical therapy and. 76. chemotherapy. The primary objective of this study was to clarify the efficacy of various. 77. antibiotics against rough type A. actinomycetemcomitans biofilm formation at different. 78. phases of growth and determine which was the most effective in eradicating this. 79. microorganism.. 80. MATERIALS AND METHODS. 81. Bacterial strains and culture conditions. A. actinomycetemcomitans clinical isolates. 82. 310a (provided by Dr. H. Ohta, Ibaraki University, Japan). Rough- and smooth-type A.. 83. actinomycetemcomitans 310a have been described previously.9 Strains were grown on. 84. blood agar plates containing Tryptic soy agar (Becton Dickinson Microbiology System,. 85. Cockeysville, MD) supplemented with 10% defibrinated horse blood, haemin (5 μg/mL;. 86. Sigma Chemical Co., St. Louis, MO) and menadione (0.5 μg/mL; Wako Pure Chemical. 87. Industries, Osaka, Japan), or Tryptic soy broth (Becton Dickinson Microbiology System). 88. supplemented with 0.1% Yeast-Extract (Difco Laboratories, Detroit, MI) （TSBYE）in an. 89. anaerobic chamber (N2: 80%, H2: 10%, CO2: 10%) at 37 C. Rough-type colony. 90. morphology was confirmed by observing the wrinkled colony morphology on the agar. 91. plates. Smooth-type 310a was obtained by repeated subculture of the rough types of these. 92. strains.. o.

(6) 93. Staining of extracellular polysaccharide (EPS) of A. actinomycetemcomitans. The. 94. extracellular polysaccharide (EPS) of the bacterial cells was stained with Alcian blue. 95. solution (pH 7.0, Nacalai Tesque, INC., Kyoto, Japan), which binds to acidic. 96. polysaccharides, according to the method of Sauer et al.15 A. actinomycetemcomitans 310a. 97. was precultured for about 40 h. Aliquots of 200 μL of each preculture were inoculated. 98. into glass bottom dishes (Matsunami Glass IND., LTD., Kishiwada, Japan) containing 4. 99. mL TSBYE. After 48 h incubation, the culture medium was removed, and the. 100. microcolonies on the glass surface were washed with phosphate-buffered saline (PBS, pH. 101. 7.2). Then they were stained with the Alcian blue solution for 30 min at room. 102. temperature. After rinsing with distilled water, the specimens were observed under a. 103. microscope. The bacteria were also stained with 0.1% crystal violet (Difco) for 15 min. 104. and processed for microscopic observation.. 105. Antimicrobial agents and MIC determinations. The antibiotics used in this study were. 106. ofloxacin (LKT Laboratories, Inc., MI), cefalexin monohydrate (ICN Biomedical, Inc.,. 107. Aurora,. 108. hydrochloride (Wako), and minocycline hydrochloride (Wako).. OH),. ampicillin. sodium. (Wako),. erythromycin. (Wako),. tetracycline. 109. The MICs for planktonic cells were determined by duplicate tests to compare the. 110. susceptibility of the biofilms. Aliquots of 50 μL A. actinomycetemcomitans 310a smooth. 111. type were inoculated into 1 mL TSBYE containing each antibiotic and incubated at 37 C. 112. in an anaerobic chamber containing 80% N2, 10% H2, and 10% CO2 for 48 h. MIC was. 113. determined as the lowest concentration of the antibiotic inhibiting visible growth of the. 114. bacteria.. 115. Quantification of biofilms. Quantification of biofilms was achieved by staining with. 116. crystal violet.. 117. wells of 96-well (flat-bottom) cell culture plates (Sumitomo Bakelite Co, Ltd, Tokyo,. o. Five μL A. actinomycetemcomitans 310a rough type were inoculated into.

(7) 118. Japan) containing 95 μL TSBYE in each well. After the designated incubation time (12,. 119. 18, 24 and 48 h), the culture medium containing planktonic cells was removed, and the. 120. wells were washed with 200 μL distilled water. The adherent bacteria were stained with. 121. 50 μL of 0.1% crystal violet for 15 min at room temperature. After rinsing twice with 200. 122. μL distilled water, the dye bound to the biofilms was extracted with 200 μL of 99%. 123. ethanol for 20 min. The extracted dye was then quantified by measuring the absorbance at. 124. 595 nm with a microplate reader (Model 3550, Bio-Rad Laboratories, Hercules, CA).. 125. Effects of antimicrobial agents on biofilm formation by A. actinomycetemcomitans.. 126. As described above, 5 μL aliquots of precultured cells were inoculated into the wells of. 127. cell culture plates containing 95 μL TSBYE. The plates were incubated under anaerobic. 128. conditions at 37 C for 12, 18, 24, and 48 h, and biofilm formation was assayed. The. 129. results of the preliminary experiment were used to confirm that the biofilm grew. 130. continuously from 0 to 48 h. To determine the relationship between antibiotic resistance. 131. and biofilm formation, we evaluated the resistance of these cells to the various antibiotics. 132. at two phases of biofilm formation: early phase (the period of microcolony formation) and. 133. mature phase (the period after thin biofilm formation).. 134. after inoculation; mature phase indicates 24 h to 48 h after inoculation.. o. Early phase indicates 0 h to 24 h. 135. To determine the susceptibility of the early-phase biofilm to antibiotics,. 136. precultured cells were inoculated into the wells of the plates with TSBYE supplemented. 137. with each antibiotic. The plates were incubated under anaerobic conditions at 37 C for 24. 138. h and then assayed for quantification of biofilm formation as described above, and for. 139. quantification of viability of biofilm as described below.. o. 140. To determine the antibiotic susceptibility of mature-phase biofilm, precultured. 141. cells were inoculated into the wells of the plates containing 95 μL TSBYE without. 142. antibiotics. After 24 h incubation, 100 μL TSBYE containing a specified concentration of.

(8) 143. each antibiotic was added to each well. The plates were incubated for a further 24 h and. 144. then assayed for biofilm formation and quantification of viability of biofilm as described. 145. below.. 146. Evaluation of viability of biofilm. To measure the number of viable cells in the biofilms,. 147. the bioactivity of A. actinomycetemcomitans was evaluated by an ATP-bioluminescence. 148. assay with the Kinshiro KKT-100 (TOYO B-Net CO. LTD., Tokyo, Japan) based on the. 149. method of Lundin et al.16. Briefly, grown A. actinomycetemcomitans cells were washed. 150. with ATP buffer (50 mM HEPES and 5 mM MgSO4, pH 7.75). Then an ATP extractant. 151. solution (TOYO B-Net CO. LTD.) was added, and the mixture was incubated for 10 sec.. 152. After addition of a bioluminescent reagent, bioluminescence was measured with a. 153. luminometer (Model Lumat LB9507, Berthold Technologies, Bad Wildbad, Germany) for. 154. 60 sec. The relationship between the adenosine triphosphate (ATP) content and the viable. 155. cell count [cfu/mL] in the liquid aliquots was examined before evaluation of cell viability.. 156. After. 157. actinomycetemcomitans 310a rough type on the 96-well plate was then evaluated using. 158. bioactivity. The bioactivity of the biofilms after exposure to each antibiotic was. 159. calculated and expressed as the relative ATP content. Relative ATP content = ATP levels. 160. of antibiotic-treated samples/ATP levels of controls (without antibiotics).. 161. Scanning electron microscopic observation of biofilm. Aliquots of 50 μl of precultured. 162. A. actinomycetemcomitans 310a rough type were inoculated into 12-well plates. 163. (Sumitomo Bakelite Co, Ltd.). Each well contained 1 mL TSBYE and a 12 mm diameter. 164. circular glass coverslip (Matsunami Glass IIND, Tokyo, Japan). After 24 h incubation. 165. under anaerobic conditions at 37 C, 1 mL TSBYE supplemented with various. 166. concentrations of each antibiotic was added to each well, and the plates were incubated. 167. again for 24 h. The biofilms formed on the coverslips were fixed in 2% glutaraldehyde in. determining. the. relationship,. the. number. of. viable. cells. of. A.. o.

(9) 168. PBS at room temperature for 1 h. After washing with PBS, the cells were dehydrated. 169. through a graded series of ethanol, dried at the critical point of t-butyl alcohol, and then. 170. coated with osmium. The samples were observed with a scanning electron microscope. 171. (SEM, Field Emission Scanning Microscopy, JSM-6340F, JEOL. Ltd., Tokyo, Japan) at. 172. an acceleration voltage of 15 kV.. 173. Statistics. Each experiment using the 96-well plates was performed more than three. 174. times, with each conducted in triplicate. The Mann-Whitney U test was used for. 175. quantification of the biofilms, and the ATP-bioluminescence assays to identify. 176. statistically significant differences.. 177. RESULTS. 178. Morphology and growth of A. actinomycetemcomitans biofilm. Rough-type A.. 179. actinomycetemcomitans 310a formed microcolonies at the bottoms of the wells in the. 180. culture plates. In contrast, smooth-type A. actinomycetemcomitans 310a attached weakly. 181. and uniformly (Fig. 1, A and C). A. actinomycetemcomitans 310a rough-type strains. 182. stained with Alcian blue, but strain 310a smooth type did not (Fig. 1, B and D).. 183. Biofilm growth of rough-type A. actinomycetemcomitans 310a in a 96-well plate. 184. is shown in Fig. 2. Growth continued until 48 h of culture. We chose 24 h as the. 185. incubation time as that time point occurs well before the plateau of biofilm maturation.. 186. MICs for A. actinomycetemcomitans 310a smooth type. The. 187. actinomycetemcomitans 310a smooth types are presented in Table 1. Ofloxacin was the. 188. most effective, and tetracycline and minocycline were moderately effective among the. 189. antibiotics tested. Ampicillin, erythromycin, and cefalexin exhibited a weaker. 190. antimicrobial effect against the strains tested. The bacterial strain was not completely. 191. resistant to any of the antibiotics used in this study.. 192. Effects of antibiotics on biofilm formation by A. actinomycetemcomitans 310a rough. MICs. for. A..

(10) 193. type. We investigated the effects of these antibiotics during the early phase of A.. 194. actinomycetemcomitans 310a rough-type biofilm formation in 96-well cell culture plates. 195. for 24-h (Fig. 3A). When the cells were cultured together with 10 mg/L of any of the. 196. antibiotics, they all showed significantly reduced biofilm formation compared to that of. 197. the controls (p<0.05). Ofloxacin significantly inhibited biofilm formation at all. 198. concentrations used in this study. Tetracycline and minocycline reduced biofilm. 199. formation at concentrations of 1-10 mg/L. Erythromycin, ampicillin, and cefalexin. 200. reduced biofilm formation only at the highest concentration of 10 mg/L. In contrast, at a. 201. low concentration of 0.1 mg/L, erythromycin and ampicillin both increased biofilm. 202. formation significantly compared to the controls (p<0.05).. 203. The effects of the antibiotics on mature-phase biofilm formation are summarized. 204. in Fig. 3B. Ofloxacin completely inhibited biofilm growth to the level where ofloxacin. 205. was added.. 206. effect on biofilm growth, even in the mature phase, while cefalexin and ampicillin did. 207. not. The mature phase was not affected by addition of 10 mg/L ampicillin, 10 mg/L. 208. cefalexin, 0.1 or 1 mg/L tetracycline, or 0.1 mg/L minocycline, all of which showed. 209. significant inhibitory effects in the early phase of biofilm formation. Relative mass of. 210. mature-phase biofilm at 48 h from 24 h after treatment with 10 mg/L of each antibiotic,. 211. apart from ofloxacin and minocycline, showed an increase from 24 h onwards, although a. 212. reduction was observed in the 48-h control.. 213. Viability of A. actinomycetemcomitans 310a rough-type biofilms. At both. 214. quantification points, ofloxacin showed significant inhibitory effects on the early- and. 215. mature-phase biofilms, while tetracycline, and minocycline showed significant inhibitory. 216. effects on the early-phase biofilms alone (Fig. 3A). However, the assays did not reveal the. 217. actual bioactivity of the microorganisms in these biofilms as quantification with crystal. Erythromycin, tetracycline, and minocycline exhibited a moderate inhibitory.

(11) 218. violet stained both live and dead cells. It is inherently difficult to evaluate viable cells in. 219. biofilms with reproducibility, as releasing and dispersing biofilm cells from hard surfaces. 220. is difficult. Therefore, to determine the effects of these antibiotics on the viability of the. 221. bacterial cells in the biofilm, we used an ATP-bioluminescence assay to measure of. 222. viability (bioactivity). The relationship between viable cell count (cfu) and. 223. ATP-bioluminescence is shown in Fig. 4. This result confirms that viable cell number can. 224. be quantified by ATP-bioluminescence. The. 225. quantity. of. viable. cells. in. the. biofilm. was. evaluated. by. 226. ATP-bioluminescence. When 10 mg/L of each antibiotic was added to the early-phase. 227. biofilm, the relative ATP content of all the 310a biofilms decreased significantly. 228. compared to that of the controls (p<0.05) (Fig. 5A). In contrast, when 10 mg/L of each. 229. antibiotic was added at the mature phase of 310a biofilm formation, only ofloxacin. 230. showed a significant decrease in relative ATP content (p<0.05). ATP levels in the biofilm. 231. increased after addition of ampicillin or cefalexin indicating susceptibility. Unexpectedly,. 232. erythromycin, tetracycline, and minocycline showed significantly increased ATP (p<0.05). 233. (Fig. 5B), suggesting that the viable cells existing inside the biofilm were not negatively. 234. affected, even if biofilm formation somewhat decreased, as revealed by crystal violet. 235. staining.. 236. SEM observations. Fig. 6 shows scanning electron micrographs of the A.. 237. actinomycetemcomitans 310a rough-type biofilms that formed on the glass coverslips.. 238. The most striking feature of the 310a rough type was that it consisted of individual cells. 239. bound tightly to each other, forming a biofilm structure resembling a ball (Fig. 6A). This. 240. colonial morphology and size were significantly affected by exposure to ofloxacin (Fig.. 241. 6B). The structure changed into a honeycomb pattern in some parts of the biofilm. In. 242. other sections, the cells expanded extraordinarily, until the sizes were about three times.

(12) 243. larger than normal. Swelling of the cells was observed in cefalexin- and ampcillin-treated. 244. biofilm (Fig. 6, D and E). Minocycline and minocycline treatment yielded only a small. 245. effect on colony and cell morphology (Fig. 6, E-G).. 246 247. DISCUSSION. 248. Antibiotic administration in conjunction with mechanical cleansing, such as. 249. scaling and root planing, or with periodontal surgery is essential in eradicating A.. 250. actinomycetemcomitans from periodontal lesions.12, 13 Biofilm bacteria exhibit a distinct. 251. mode of growth which differs from that of planktonic cells.2 However, most evaluations. 252. of the susceptibility of A. actinomycetemcomitans to antibiotics have been performed with. 253. planktonic organisms, and there are few reports on the susceptibility of rough-type strain. 254. to antimicrobial agents.17 The purpose of this study was to characterize the susceptibility. 255. of rough type A. actinomycetemcomitans biofilms to various antibiotics. Biofilms consist. 256. of cells and EPS, and their accumulation is a net result of planktonic cell attachment,. 257. biofilm cell growth, detachment and EPS production.1,. 258. staining indicated that A. actinomycetemcomitans 310a rough type produced EPS. This. 259. result agrees with the report of Kaplan et al.19 demonstrating that a linear polymer of. 260. N-acetyl-D-glucosamine residues in the β (1,6) linkage was a major matrix component of. 261. biofilms produced by A. actinomycetemcomitans.. 18. The results of Alcian blue. 262. In this study, we also demonstrated differences in the susceptibilities of A.. 263. actinomycetemcomitans biofilm cells to ofloxacin, cefalexin, ampicillin, erythromycin,. 264. tetracycline, and minocycline. These antibiotics were chosen for their different kinetics in. 265. drug activity. Some of them are clinically used in the treatment of periodontitis.20 As. 266. rough-type strains of A. actinomycetemcomitans form biofilms on glass surfaces of test. 267. tubes with no turbidity when cultured in broth,9 we could not use turbidity to define the.

(13) 268. MICs of the rough type in test tubes. Throughout the study, we demonstrated that the. 269. most effective antibiotic against A. actinomycetemcomitans was ofloxacin. This. 270. susceptibility to ofloxiacin was similar to that in the reports of earlier investigations using. 271. planktonic cells.21 We compared the effects of various antibiotics at the early and mature phases of. 272 273. biofilm formation by using crystal violet staining (Fig. 3).. Our results suggest that the. 274. susceptibility of A. actinomycetemcomitans rough type decreases with maturation of the. 275. biofilms. Antibiotic resistance within biofilms has been demonstrated to change in several. 276. microorganisms.18, 22 The following reasons for such resistance have been suggested: the. 277. antibiotics penetrate slowly or incompletely into a biofilm; microbial nutrients and waste. 278. products modify the local environment inside a biofilm; the growth rates of the bacteria. 279. decrease inside a biofilm; biofilm/attachment-specific phenotypes develop inside a. 280. biofilm.. 281. observe: the first occurring after tooth brushing or professional mechanical tooth. 282. cleaning, and the second occurring after biofilm has grown over a period of several hours,. 283. after which, it reaches maturation.. 284. phase” and “mature phase”. In preliminary tests, we also observed a decrease in the. 285. susceptibility of mature phase biofilm using rough-type A. actinomycetemcomitans AB55. 286. (data not shown). These results suggest that mechanical removal of biofilm prior to. 287. chemotherapy is required to eradicate A. actinomycetemcomitans from the oral cavity.. 288. We used SEM to visually investigate the effects of antibiotics on mature-phase A.. 289. actinomycetemcomitans 310a biofilms, and found that colonial morphology was. 290. distinctively affected depending on the antibiotic. As shown in Fig. 6B, ofloxacin induced. 291. the most dramatic morphological changes, indicating that ofloxacin damages A.. 292. actinomycetemcomitans in biofilm. Among all the antibiotics used in this study, ofloxacin. There were two phases in the lifecycle of oral biofilms that we wished to. In our simulation, these were designated as “early.

(14) 293. had the greatest inhibitory effects in both the early and mature phases of biofilm formation. 294. through all experiments. Kleinfelder et al.. 295. an adjunct to open flap surgery was able to suppress A. actinomycetemcomitans to below. 296. detectable levels in patients. Our present results agree with those of their report.. 23. demonstrated that systemic ofloxacin used as. 297. The ATP-bioluminescence assay has been reported to be a useful tool in. 298. evaluating the quantity of bacterial biofilms.24 We used this assay in conjunction with. 299. crystal violet staining to evaluate the biofilm formation of A. actinomycetemcomitans. All. 300. of the antibiotics used in this study showed an inhibitory effect on the viability of the. 301. biofilm cells at the early phase (Fig. 5A). After exposure of mature-phase biofilm to. 302. ampicillin or cefalexin, ATP increased significantly. These antibiotics did not affect the. 303. growth of A. actinomycetemcomitans. It is possible that this discrepancy between. 304. production and consumption of ATP was due to weak inhibition of cell-wall organization. 305. by antibiotics under MIC level. Another possibility, however, is that this temporary. 306. increase was due to an influx of ATP released from dying microorganisms. Erythromycin,. 307. tetracycline, and minocycline enhanced the bioactivity of the 310a cells at the mature. 308. phase, in spite of a reduction in biofilm formation revealed by crystal violet staining.. 309. They exerted a bacteriostatic effect, but no bactericidal effect. It is possible that the. 310. accumulation of ATP in biofilm cells is involved in a survival response to antibiotics . To. 311. clarify these issues, further analysis employing other methods of evaluation is required to. 312. determine live cell number.. 313. mature phases of biofilm formation. Several reports have indicated that the macrolide. 314. family affects bacterial quorum sensing at sub-MICs, and that this results in an. 315. attenuation of biofilm formation.25 It has also been reported that auto-inducer 2 (AI-2). 316. signals in A. actinomycetemcomitans may modulate aspects of virulence, including the. 317. uptake of iron, and that they may control cellular adaptation to growth under iron-limiting. Erythromycin showed no effect, either at the early or.

(15) 318. conditions26. Our results showed that sub-MICs of erythromycin did not affect biofilm. 319. formation, which suggests that AI-2 in A. actinomycetemcomitans is not affected by. 320. erythromycin, or that the gene induced by AI-2 exerts no effect on susceptibility to. 321. antibiotics. Further analyses such as evaluation of AI-2 production are required to clarify. 322. this point.. 323. The results of our study demonstrated that the susceptibility of rough-type A.. 324. actinomycetemcomitans to antibiotics decreases during maturation of the biofilm. These. 325. data highlight the difficulty of designing antibiotic therapies for periodontitis. Further. 326. study is required to determine the effect of dose in the gingival crevice in chemotherapy.. 327. Periodontal pockets harbor a variety of different microbial species with different. 328. susceptibilities to different antimicrobials in vivo. Therefore, it is essential to determine. 329. the antibiotic susceptibility of biofilms containing different species of subgingival. 330. bacteria.. 331. ACKNOWLEDGMENTS. 332. We wish to thank K. Tadokoro for his assistance with the SEM observations. Part. 333. of this work was supported by Grant 16591837 from the Ministry of Education, Science,. 334. Sport, and Culture (MEXT) of Japan and Grant HRC7 from the Oral Health Science Center. 335. of Tokyo Dental College and a “High-Tech Research Center” Project for Private. 336. Universities: matcching fund subsidy from MEXT, 2006-2010... 337. TRANSPARENCY DECLARATIONS. 338. None to declare.. 339 340 341 342.

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(18) 393. 21. Miyake, Y., Tsuruda, K., Okuda, K. et al. In vitro activity of tetracyclines, macrolides,. 394. quinolones, clindamycin and metronidazole against periodontopathic bacteria. J. 395. Periodontal Res 1995; 30: 290-3.. 396 397. 22. Gilbert, P., Das, J. & Foley, I. Biofilm susceptibility to antimicrobials. Adv Dent Res 1997; 11: 160-7.. 398. 23. Kleinfelder, J. W., Mueller, R. F. & Lange, D. E. Fluoroquinolones in the treatment of. 399. Actinobacillus actinomycetemcomitans-associated periodontitis. J Periodontol 2000;. 400. 71: 202-8.. 401. 24. Kumon, H., Ono, N., Iida, M. et al. Combination effect of fosfomycin and ofloxacin. 402. against Pseudomonas aeruginosa growing in a biofilm. Antimicrob Agents Chemother. 403. 1995; 39: 1038-44.. 404 405. 25. Wozniak, D. J. & Keyser, R. Effects of subinhibitory concentrations of macrolide antibiotics on Pseudomonas aeruginosa. Chest 2004; 125: 62S-9S; quiz 9S.. 406. 26. Fong, K. P., Gao, L. & Demuth, D. R. luxS and arcB control aerobic growth of. 407. Actinobacillus actinomycetemcomitans under iron limitation. Infect Immun 2003; 71:. 408. 298-308.. 409 410.

(19) 411. Table 1. MICs for A. actinomycetemcomitans 310a and AB55 smooth type. MIC (mg/l) Strain. OFX. LEX. AMP. ERY. TET. 310a. 0.03. 6.0. 1.0. 2.0. 0.13. MIN 0.25. 412 413. MICs for smooth-type (planktonic-type) A. actinomycetemcomitans performed under. 414. same time schedule as early-phase. 50 μL or 200 μL A. actinomycetemcomitans 310a or. 415. AB55 smooth type were inoculated into TSBYE medium containing antibiotics and then. 416. incubated at 37 C in an anaerobic chamber for 48 h. MICs were determined as lowest. 417. concentration of antibiotic inhibiting visible growth of bacteria. Experiments were. 418. performed in duplicate.. o.

(20) 419. FIGURE LEGENDS. 420. Fig. 1. Microscopical observations of A. actinomycetemcomitans 310a biofilm.. 421. Microscopy of 48-h cultured A. actinomycetemcomitans 310a (magnification×1000).. 422. Pictures: A and B show A. actinomycetemcomitans 310a rough type. Pictures: C and D. 423. show A. actinomycetemcomitans 310a smooth type. Pictures: A and C show staining with. 424. crystal violet. Pictures: B and D show staining with Alcian blue.. 425 426. Fig. 2. Biofilm formation of A. actinomycetemcomitans 310a rough type on 96-well. 427. plates. Aliquots of 5 μL cell suspension were inoculated into wells of 96-well plates. 428. containing 95 μL medium and incubated for designated times. After incubation, biofilm. 429. formation assays were performed. Experiments were performed in triplicate. 430 431. Fig. 3. A: Effects of antibiotics on A. actinomycetemcomitans 310a rough-type early. 432. phase biofilm formation. Cells were inoculated into wells of 96-well plate and incubated. 433. for 24-h with each antibiotic. B: Antibiotic susceptibility in mature phase biofilm. Cells. 434. were inoculated into wells of 96-well plate. After 24-h incubation, antibiotics were added,. 435. and culture was continued for further 24-h. Polka-dot pattern indicates control without. 436. antibiotics incubated for 48 h. Striped-pattern indicates biofilm formation at time. 437. antibiotics added (24 h). Experiments were performed more than three times, and each. 438. was conducted in triplicate. Effects of antibiotics were statistically analyzed with. 439. Mann-Whitney U test, * p<0.05 vs. biofilm level after 48 h incubation [polka-dot pattern].. 440. † p<0.05 vs. biofilm level at time antibiotics added.. 441 442. Fig. 4. Relationship between ATP-content and cfu/mL. Aliquots of 200 μL cell. 443. suspension of A. actinomycetemcomitans 310a smooth type were inoculated into test.

(21) 444. tubes containing 2 ml medium and incubated for 24 h. After incubation, ATP content and. 445. bacterial viability (cfu/mL) were measured.. 446 447. Fig. 5. Effects of antibiotics on A. actinomycetemcomitans 310a rough type bioactivity.. 448. A: Effects in early phase of biofilms. Cells were inoculated into wells of 96-well plate. 449. and incubated for 24-h with each antibiotic (10 mg/L). B: Effects in mature phase of. 450. biofilms. Cells were inoculated into wells of 96-well plate. After 24-h incubation,. 451. antibiotics (10 mg/L) were added, and culture was continued for further 24-h. Polka-dot. 452. pattern indicates control without antibiotics incubated for 48 h. Striped-pattern indicates. 453. biofilm formation at time antibiotics added (24 h). Experiments were performed more. 454. than three times, and each was conducted in triplicate. Effects of antibiotics were. 455. statistically analyzed with Mann-Whitney U test, * p<0.05 vs. biofilm level after 48 h. 456. incubation without antibiotics [polka-dot pattern].. 457. antibiotics added.. † p<0.05 vs. biofilm level at time. 458 459. Fig. 6. Scanning electron micrographs of biofilms formed by A. actinomycetemcomitans. 460. 310a rough type on glass coverslips. After 24-h incubation, cells were added to medium. 461. supplemented with or without the following antibiotics (10 mg/L each) and incubated. 462. further for 24-h; Panels: A, control (without antibiotics); B, ofloxacin; C, cefalexin; D,. 463. ampicillin; E, erythromycin; F, tetracycline; and G, minocycline.. 464 465.

(22) 466 467.

(23) 468 469.

(24) 470 471. 472. 473. Fig. 3.

(25) 474.

(26) 475. 476. 477. Fig. 5.

(27) 478.

(28) Fig. 1.

(29) Absorbance (595 nm). Fig. 2. 1.2 1 0.8 0.6 0.4 0.2 0 12. 18. 24. Incubation time (hours). 48.

(30) Fig. 3. A. Absorbance (595 nm). 1.4 **. 1.2. OFX LEX AMP ERY TET MIN. 1.0 0.8. * *. *. 0.6. *. 0.4 *. 0.2. *. *. *. 0.0 0 (control). 0.1. *. *. 1. Concentration (mg/L). 10. **.

(31) Fig. 3. B 2.5 Absorbance (595 nm). † * † †. † * † † †† *. 2.0 1.5. † *. 1.0. † † * *. † † * * *. † † * **. *. 0.5 0.0 0 48-h 24-h Control. 0.1. 1. Concentration (mg/L). 10. OFX LEX AMP ERY TET MIN.

(32) Fig. 4. -8. ATP contents (×10 ) M. 3 2.5 2. R = 0.86. 2 1.5 1 0.5 0 0. 1. 2. 3. Viable bacteria (×108 cfu/ml). 4. 5.

(33) Fig. 5. Relative ATP Content ( antibiotics/control ). A 1.5 1 *. 0.5 *. 0 0 (control). *. *. 10. Concentration (mg/L). *. *. OFX LEX AMP ERY TET MIN.

(34) Fig. 5. B Relative ATP Content ( antibiotics/control ). 7. † † * *. 6 5. † *. 4 3. * *. 2 1 0. * 0 48-h 24-h Control. 10. Concentration (mg/L). OFX LEX AMP ERY TET MIN.

(35) A. B. C. D. E. F. G. Fig. 6.

(36)