PATHOLOGY

1 57
2 58
3 59
4
5
In Vitro Investigation of the 60
61
6
7 Antimicrobial Effect of Three 62
63
8
9 Bisphosphonates Against Different 64
65

Bacterial Strains
10 66
11 67
12 68
13 Q5 Michael A. Ermer, MD, DDS,* Simon C. Kottmann, DDS,y J€ org-Elard Otten, MD, DDS, 69
14 Q1 PhD,z Annette Wittmer,x Philipp Poxleitner, MD, DDS,k and Klaus Pelz, MD{ 70
15 71
Purpose: Since the first descriptions of medication-related osteonecrosis of the jaw (MRONJ) in 2003,
16 72
17 the pathogenesis has remained unanswered. Recent histomorphometric studies have found several micro- 73
18 organisms, including Actinomyces, Bacillus, Fusobacterium, Staphylococcus, Streptococcus, Selenomo- 74
19 nas, Treponema, and Candida albicans in necrotic bone. Polymerase chain reaction studies have 75
20 recently confirmed the occurrence of 48 genera. Only a few studies have examined the antimicrobial effect 76
21 of bisphosphonates (BPs). The influence of bacterial growth on the etiology remains unclear. The aim of 77
22 the present study was the in vitro investigation of the antimicrobial effect of 3 BPs against different bac- 78
23 terial strains. 79
24 Materials and Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal con- 80
25 centration (MBC) of 48 strains from 40 species were determined in microdilution assays against pami- 81
26 dronic, ibandronic, and zoledronic acid. 82
27 Results: Growth of gram-positive oral microbiota, which account for most microorganisms in MRONJ, 83
28 was present for 2 of 22 species; 6 of 26 gram-negative species and 9 of 13 anaerobes were inhibited. 84
29 The MIC values were compared with the BP bone concentrations from previous reports. Of the 48 strains, 85
30 9 had an MIC or MBC less than the bone concentrations. 86
31 87
Conclusions: The results of the present study have demonstrated that BPs have an inhibitory effect on
32 88
33 selected bacterial species and might inhibit the growth of some relevant pathogens in osteonecrosis. How- 89
34 ever, most of the species tested were unaffected at the concentration levels assumed present in the human 90
35 jawbone. The clinical relevance of these in vitro data will better be clarified with reliable data on the BP 91
36 concentrations in the human jawbone. The present study has provided a first approach toward the assess- 92
37 ment of the interaction of oral bacteria and BPs. 93
38 Ó 2017 Published by Elsevier Inc on behalf of the American Association of Oral and Maxillofacial 94
39 Surgeons 95
40 J Oral Maxillofac Surg -:1-8, 2017 96
41 97
42 98
43 99
44 100
45 *Consultant, Department of Oral and Maxillofacial Surgery, Conflict of Interest Disclosures: None of the authors have any 101
46 University Medical Center Freiburg, Freiburg, Germany. relevant financial relationship(s) with a commercial interest. 102
47 yPrivate Practitioner, Department of Oral and Maxillofacial Address correspondence and reprint requests to Dr Ermer: 103
48 Surgery, University Medical Center Freiburg, Freiburg, Germany. Department of Oral and Maxillofacial Surgery, University Medical 104
49 zProfessor, Department of Oral and Maxillofacial Surgery, Center Freiburg, Hugstetter Straße 55, Freiburg 79106, Germany; 105
50 University Medical Center Freiburg, Freiburg, Germany. e-mail: michael.ermer@uniklinik-freiburg.de 106
51 xBiologist, Department of Hygiene and Microbiology, Albert- Received May 1 2017 107
52 Ludwigs-Universit€at, Freiburg, Freiburg, Germany. Accepted August 9 2017 108
53 jjResident, Department of Oral and Maxillofacial Surgery, Ó 2017 Published by Elsevier Inc on behalf of the American Association of Oral 109
54 University Medical Center Freiburg, Freiburg, Germany. and Maxillofacial Surgeons 110
55 {Consultant, Department of Hygiene and Microbiology, Albert- 0278-2391/17/31142-4 111
56 Ludwigs-Universit€at, Freiburg, Freiburg, Germany. http://dx.doi.org/10.1016/j.joms.2017.08.019 112

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113 Bisphosphonates (BPs) and monoclonal RANK-L (re- with 100 mL M€ uller-Hinton broth (MHB) for the inocu- 169
114 ceptor activator of nuclear factor kappa-B ligand) anti- lation of all aerobes, facultative aerobes, and C. albi- 170
115 bodies such as denosumab are widely administered to cans. Wilkins-Chalgren broth (WCB) was used for all 171
116 patients with benign and malignant bone disorders, anaerobes and for some facultative anaerobes, which 172
117 including osteoporosis, Paget disease, multiple did not grow in MHB. Wells A1 and B1 were filled 173
118 myeloma, bone metastases of various tumors, and with WCB or MHB with a pH of 9.05 owing to the 174
119 tumor-induced hypercalcemia.1,2 As antiresorptive acidic pH (pH 4) of ibandronic acid to achieve a 175
120 agents, BPs serve to reduce pain, fracture risk, and physiologic pH of 7. Wells A11 to H11 served as 176
121 disease progression. As a pyrophosphate structure growth controls, and wells A12 to H12 as medium 177
122 analogue, BPs influence the bone turnover by controls. The 3 BPs were used in their original concen- 178
123 inhibiting osteoclasts and angiogenesis.3 Medication- trations: ibandronic acid, 1 mg/mL; pamidronic acid, 179
124 related osteonecrosis of the jaw (MRONJ) is the most 2,527 mg/mL; and zoledronic acid, 0.8 mg/mL. Thus, 180
125 serious side effect. Since the first descriptions in 100 mL were pipetted into wells A1 to F1, resulting 181
126 2003, the cases have increased steadily.4 The etiopatho- in a dilution of 1:2. Using a multichannel pipette, dilu- 182
127 genesis remains unclear. In addition to the known toxic tions from 1:2 up to 1:1,024 were made. From each 183
128 effects against osteoclasts, the antiangiogenic effect, bacterial and fungal strain, suspensions were prepared 184
129 and the negative effect on soft tissue healing, the according to the McFarland standard, 0.5 to 1 in sterile 185
130 involvement of the oral biofilm has been discussed.3-7 0.9% NaCl to achieve 5  105 cells/mL and 186
131 Several microorganisms, including Actinomyces, 5  106 cells/mL for anaerobes (EN ISO 20776- 187
132 Fusobacteria, Streptococcus, Staphylococcus, 1:2006). All wells, except for A12 to H12, were inocu- 188
133 Treponema, and Candida albicans, were found in lated with 5 of the respective microbial suspensions. 189
134 MRONJ by histomorphometric and histologic All assays were performed in duplicate. Before incuba- 190
135 studies.8-10 These results were supplemented and tion, the inoculum size was controlled by removing 191
136 confirmed by the findings from polymerase chain 10 mL from wells A11 to H12. Of these, 1:10 and 192
137 reaction (PCR) studies, in which a specific microbiota 1:100 dilutions were inoculated on Columbia-blood 193
138 composition was identified and compared in patients agar (CBA) or yeast-cysteine-blood agar (HCBA) to 194
139 taking BPs with and without MRONJ.11 obtain countable colonies. 195
140 To date, few studies have examined the antimicro- Agar plates and the microtiter plates with aerobes 196
141 bial effect of BPs. Furthermore, the influence of bacte- and facultative anaerobes were incubated for 197
142 ria on the pathomechanism remains unclear. It is not 24 hours at 36 C and 5 to 10% carbon dioxide. 198
143 known whether the oral microbiota or variations in The anaerobes were as quickly as possible bagged 199
144 the spectrum are responsible, singly or together, for in Anaerocult IS (Merck Millipore, Darmstadt, Ger- 200
145 the development or aggravation of MRONJ. To gain many) and incubated for 48 hours at 36 C or until 201
146 more knowledge regarding whether clinically used growth was visible. 202
147 BPs provide antimicrobial activity on known oral mi- The MIC was evaluated by comparing the turbidity 203
148 crobiota, the minimum inhibitory concentration between the growth controls and the turbidity of the 204
149 (MIC) and minimum bactericidal concentration wells with the increasing BP or CHX concentrations. 205
150 (MBC) were determined in microdilution assays for pa- The MIC is the lowest concentration able to 206
151 midronic, ibandronic, and zoledronic acid. Chlorhexi- completely inhibit visible growth. After assessing the 207
152 dine (CHX) was used as the control. MICs, a digital photograph of each microtiter plate 208
153 The aim of the present study was the in vitro was taken. This enabled secondary assessment by 209
154 investigation of the antimicrobial activity of 3 magnification and a second researcher. 210
155 frequently prescribed BPs against different bacte- 211
156 rial strains. SUBCULTIVATION 212
157 For determination of the MBC, 10 mL was removed 213
158 from the wells without visible growth and inoculated 214
159
Materials and Methods 215
on CBA or HCB. The aerobes were incubated for at
160 BROTH MICRODILUTION ASSAY least 24 hours at 36 C and 5 to 10% carbon dioxide. 216
161 The MIC and MBC of 48 strains from 40 species Anaerobes were incubated in an anaerobic chamber 217
162 were determined in 67 microdilution assays against (GENbox; BioMerieux SA, Marcy/Etoile, France) for 218
163 pamidronic, ibandronic, and zoledronic acid. CHX at least 48 hours at 36 C. After incubation, the agar 219
164 was tested in parallel as a control of the method. plates were searched for growth of colonies. The col- 220
165 Bacterial and fungal strains were cultivated as over- ony forming units (CFUs) were counted and recorded. 221
166 night cultures. Next, 96-well microtiter plates were The MBC is defined as the concentration at which 222
167 prepared. Each well, except for A1 and B1, was filled either no colony growth occurs or fewer than 223
168 224

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225 Table 1. MICRODILUTION ASSAY RESULTS OF GRAM-POSITIVE AEROBES

281
226 282
227 Ibandronate Zoledronate 283
228 (mg/mL) Pamidronate (mg/mL) (mg/mL) CHX (mg/mL) 284
229 285
Sample MIC MBC MIC MBC MIC MBC MIC MBC
230 286
231 287
Actinomyces naelundii* DSM >500 >500 >1,263 >1,263 >400 >400 <1.95 3.91
232 288
Actinomyces oris* A22-3 >500 >500 >1,263 >1,263 >400 >400 <1.95 3.91
233 Actinomyces odontolyticus* >500 >500 >1,263 >1,263 >400 >400 1.95 1.95 289
234 P12-7 290
235 Bacillus subtilisy ATCC 6633 500 500 >1,263 >1,263 >400 >400 <1.95 <1.95 291
236 Candida albicansy DSM 1386 >500 >500 >1,263 >1,263 >400 >400 15.63 15.63 292
237 Enterococcus faecalisy ATCC >500 >500 >1,263 >1,263 >400 >400 7.81 7.81 293
238 29212 294
239 Lactobacillus salivarius* DSM >500 >500 >1,263 >1,263 >400 >400 <0.48 0.98 295
240 2055 296
241 Lactobacillus caseiy DSM >500 >500 78.97 158 400 >400 <0.48 <0.48 297
20011
242 298
Micrococcus luteusy DSM348 500 500 >1,263 >1,263 400 >400 <1.95 1.95
243 299
Staphylococcus epidermidisy >500 >500 158 >1,263 >400 >400 1.95 3.91
244 DSM1798 300
245 Staphylococcus aureusy ATCC 500 >500 >1,263 >1,263 >400 >400 1.95 3.91 301
246 25423 302
247 Streptococcus sanguinis* DSM >500 >500 >1,263 >1,263 >400 >400 7.81 7.81 303
248 20068 304
249 Streptococcus salivariusy DSM 125 250 >1,263 >1,263 100 200 <0.48 <0.48 305
250 20067 306
251 Streptococcus gordoniiy DSM 250 500 >1,263 >1,263 400 400 <0.48 <0.48 307
252 6777 308
Streptococcus anginosus* 1218 >500 >500 >1,263 >1,263 >400 >400 1.95 1.95
253 309
Streptococcus anginosus* 1218 >500 >500 >1,263 >1,263 >400 >400 1.95 1.95
254 310
Streptococcus anginosus* R10- >500 >500 >1,263 >1,263 >400 >400 1.95 1.95
255 1-A-2 311
256 Streptococcus anginosus* R10- >500 >500 >1,263 >1,263 >400 >400 1.95 1.95 312
257 1-A-2 313
258 Streptococcus sobrinusy DSM >500 >500 158 316 >400 >400 <1.95 >3.91 314
259 20381 315
260 Streptococcus oralisy ATCC 250 >500 316 1,263 200 400 3.91 7.81 316
261 35037 317
262 Streptococcus mutansy DSM 250 250 316 1,263 100 400 <1.95 >3.91 318
263 20523 319
Streptococcus mutans* DSM 250 500 316 1,263 200 400 <1.95 3.91
264 320
20523
265 321
Vagococcus fluvialisy DSM >500 >500 39.48 39.48 >400 >400 <0.48 0.98
266 5731 322
267 323
268 The data include results from repeated analyses of the same species on different days. 324
269 Abbreviations: CHX, chlorhexidine; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration. 325
270 * Wilkins-Chalgren broth as growth medium. 326
y M€
uller-Hinton broth as growth medium.
271 327
272 Ermer et al. IMF Screw Morbidity in Mandibular Fractures. J Oral Maxillofac Surg 2017. 328
273 329
274 330
10 CFUs are countable, indicating that 99.9% of the BACTERIAL AND FUNGAL STRAINS
275 331
276 inoculum of the tested strain was killed. Performing A total of 48 different bacterial strains and C. albi- 332
277 the determination of MBC on solid media also allowed cans of 40 different species were evaluated in 67 mi- 333
278 for control of contamination. crodilution assays. The microorganisms were 334
279 335
280 336

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337 Table 2. MICRODILUTION ASSAY RESULTS OF GRAM-NEGATIVE AEROBES

393
338 394
339 Ibandronate (mg/ Pamidronate (mg/ Zoledronate (mg/ 395
340 mL) mL) mL) CHX (mg/mL) 396
341 397
Sample MIC MBC MIC MBC MIC MBC MIC MBC
342 398
343 399
A. actinomycetemcomitans* 7.81 7.81 158 158 3.13 3.13 <1.95 <1.95
344 400
Fr260102
345 A. actinomycetemcomitans* 7.81 7.81 316 316 6.25 6.25 1.95 1.95 401
346 Fr260102 402
347 A. actinomycetemcomitans* 62.5 250 158 >1,263 6.25 25 0.98 1.95 403
348 Fr129922 404
349 Capnocytophaga ochracea* 7.81 >500 39.48 >1,263 3.13 >400 <0.48 >3.91 405
350 P12-2 406
351 Capnocytophaga ochracea* 7.81 >500 78.97 >1,263 6.25 >400 <0.48 >3.91 407
352 P12-2 408
353 Eikenella corrodens* FB72-11-3 31.25 62.5 78.97 158 12.5 12.5 3.91 3.91 409
Eikenella corrodens* FB72-11-3 31.25 62.5 39.48 158 6.25 12.5 <0.48 <0.48
354 410
Eikenella corrodens* Fr39712/ 62.5 >500 158 >1,263 12.5 50 0.98 0.98
355 411
99
356 Escherichia coliy ATCC 25922 >500 >500 >1,263 >1,263 >400 >400 3.91 - 412
357 Haemophilus aphrophilus* 62.5 250 316 632 12.5 >400 <0.48 <0.48 413
358 B68/32-5 414
359 Haemophilus aphrophilus* 62.5 >500 632 >1,263 12.5 >400 3.91 3.91 415
360 B68/32-5 416
361 Haemophilus aphrophilus* 250 >500 1,263 1,263 400 >400 1.95 1.95 417
362 E1299-4/96 418
363 Moraxella osloensis* CT36-SO- 125 125 316 316 50 50 <0.48 <0.48 419
364 8 420
Neisseria subflava* A18-3 31.25 >500 632 >1,263 50 100 7.81 7.81
365 421
Pseudomonas aeruginosay 125 250 >1,263 >1,263 200 >400 15.63 15.63
366 422
ATCC27853
367 Pseudomonas aeruginosay 500 >500 >1,263 >1,263 200 >400 7.81 15.63 423
368 ATCC27853 424
369 P. aeruginosay DSM1128 500 >500 >1,263 >1,263 200 200 7.81 7.81 425
370 426
371 The data include results from repeated analyses of the same species on different days. 427
372 Abbreviations: ATCC, American Type Culture Collection; CHX, chlorhexidine; MBC, minimum bactericidal concentration; 428
MIC, minimum inhibitory concentration.
373 * Wilkins-Chalgren broth as growth medium.
429
374 y M€
uller-Hinton broth as growth medium. 430
375 Ermer et al. IMF Screw Morbidity in Mandibular Fractures. J Oral Maxillofac Surg 2017.
431
376 432
377 433
378 provided by the Institute of Medical Microbiology and Streptococcus mutans was 250 mg/mL and was 434
379 Hygiene (Albert-Ludwigs University, Freiburg, Ger- 500 mg/mL for B. subtilis, Micrococcus luteus, Strepto- 435
380 many). The species are listed in detail in Tables 1 to 3. coccus gorodonii, and S. mutans. 436
381 Pamidronate had an MIC of 39.48 mg/mL for Vago- 437
382
Results coccus fluvialis and 316 mg/mL for S. mutans and 438
383 Streptococcus oralis. The MBC was 39.48 mg/mL for 439
384 GRAM-POSITIVE AEROBES AND FACULTATIVE V. fluvialis and 1,263 mg/mL for S. oralis and 440
385 ANAEROBES S. mutans. 441
386 Q2 In 23 experiments, 19 different gram-positive aero- The MIC of zoledronate was 100 mg/mL for S. sali- 442
387 bic species were tested (Table 1). Ibandronate had varius and S. mutans and 400 mg/mL for Lactobacillus 443
388 an MIC of 125 mg/mL against Streptococcus salivarius casei, M. luteus, and Streptococcus gordonii. The MBC 444
389 and 500 mg/mL against Bacillus subtilis and Staphylo- was 200 mg/mL for S. salivarius and 400 mg/mL for S. 445
390 coccus aureus. The MBC for S. salivarius and gordonii, S. oralis, and S. mutans. 446
391 447
392 448

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449 Table 3. MICRODILUTION ASSAY RESULTS OF THE ANAEROBES

505
450 506
451 Ibandronate Pamidronate (mg/ Zoledronate (mg/ 507
452 (mg/mL) mL) mL) CHX (mg/mL) 508
453 509
Sample MIC MBC MIC MBC MIC MBC MIC MBC
454 510
455 511
Campylobacter rectus* B65/15- 15.63 250 78.97 632 12.5 200 1.95 1.95
456 512
4b
457 Campylobacter rectus* B65/15- 3.91 15.63 158 158 25 25 1.95 1.95 513
458 4b 514
459 Campylobacter concisus* 500 500 >1,263 >1,263 200 400 3.91 3.91 515
460 DSM5360 516
461 Campylobacter gracilis* 62.5 62.5 1,263 >1,263 50 100 3.91 3.91 517
462 MCCM 01196 518
463 Campylobacter gracilis* 62.5 >500 >1,263 >1,263 200 >400 <0.48 1.95 519
464 MCCM 01196 520
465 Fusobacterium nucleatum* 500 500 >1,263 >1,263 400 400 3.91 3.91 521
ATCC 25586
466 522
Fusobacterium nucleatum* >500 >500 >1,263 >1,263 >400 >400 3.91 3.91
467 523
ATCC 25586
468 Fusobacterium nucleatum* >500 >500 >1,263 >1,263 >400 >400 3.91 3.91 524
469 ATCC 25586 525
470 Fusobacterium nucleatum* 250 250 1,263 1,263 200 200 0.98 0.98 526
471 KN20-2b 527
472 Porphyromonas gingivalis* 250 500 632 1,263 200 200 1.95 1.95 528
473 W381 529
474 Porphyromonas gingivalis* 250 500 1,263 1,263 200 200 1.95 1.95 530
475 W381 531
476 Porphyromonas gingivalis* 250 500 1,263 1,263 200 200 1.95 1.95 532
W83
477 533
Porphyromonas gingivalis* 125 125 316 632 25 50 0.98 1.95
478 534
1493
479 Prevotella intermedia* ATCC 31.25 125 78.97 1,263 50 100 1.95 1.95 535
480 25611 536
481 Prevotella intermedia* ATCC 125 250 316 632 100 100 <0.48 1.95 537
482 25611 538
483 Prevotella intermedia* 1493 125 250 158 1,263 100 100 <0.48 1.95 539
484 Prevotella nigrescens* NCTC 500 500 >1,263 >1,263 400 400 3.91 3.91 540
485 Prevotella corporis* 10,283 250 250 1,263 1,263 200 200 0.98 1.95 541
486 Prevotella oralis* 43,707 500 500 >1,263 >1,263 400 400 7.81 7.81 542
487 Veillonella parvula* DSM 2008 >500 >500 >1,263 >1,263 400 400 1.95 1.95 543
Veillonella parvula* DSM 2008 >500 >500 >1,263 >1,263 400 400 0.98 0.98
488 544
Parvimonas micra* ATCC 250 500 316 632 200 400 1.95 7.81
489 545
23195
490 Parvimonas micra* ATCC 250 500 632 632 200 200 1.95 1.95 546
491 23195 547
492 Parvimonas micra* ATCC 250 250 316 632 200 200 1.95 3.91 548
493 23195 549
494 Slackia exigua* 260469 >500 >500 >1,263 >1,263 >400 >400 0.48 0.98 550
495 Slackia exigua* 260469 >500 >500 >1,263 >1,263 400 400 <0.48 <0.48 551
496 Propionibacterium acnes* >500 >500 >1,263 >1,263 >400 >400 1.95 1.95 552
497 DSM 1897 553
498 554
The data include results from repeated analyses of the same species on different days.
499 Abbreviations: CHX, chlorhexidine; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration. 555
500 * Wilkins-Chalgren broth as growth medium. 556
501 Ermer et al. IMF Screw Morbidity in Mandibular Fractures. J Oral Maxillofac Surg 2017. 557
502 558
503 559
504 560

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561 Most gram-positive aerobes showed no inhibition of The results of CHX tested parallel with each micro- 617
562 growth in the tested concentration range. Only in case dilution assay showed the required accordance 618
563 of L. casei and V. Fluvialis did the 3 BPs show growth compared with the published data.12-14 619
564 inhibitory or bactericidal activity. Comparing the MIC or MBC values of the different 620
565 BP concentrations, only a few values were less than 621
566 GRAM-NEGATIVE AEROBES AND FACULTATIVE the known achievable BP bone concentrations with 622
567 ANAEROBES long-term application. These were selected from re- 623
568 ported studies (ibandronate 15 mg/mL, pamidronate 624
The antimicrobial activity of 3 BPs was tested in 17
569 81.6 mg/mL, and zoledronate 24.45 mg/mL). 625
tests; 12 different bacterial strains of 8 gram-negative
570 In the case of the gram-negative aerobes, A. actino- 626
aerobic species were investigated (Table 2). The high-
571 mycetemcomitans, C. ochracea, E. corrodens, and H. 627
est tested concentrations (ibandronate, 500 mg/mL;
572 aphrophilus had MIC and MBC values less than the 628
pamidronate, 1,263 mg/mL; and zoledronate, 400 mg/
573 calculated bone concentrations. Ibandronate inhibited 629
mL) proved to be growth inhibiting against all gram-
574 A. actinomycetemcomitans at a MIC of 7.81 mg/mL. In 630
negative aerobes, except for Escherichia coli.
575 the case of pamidronate, C. ochracea (39.48 mg/mL) 631
For ibandronate, the MIC for Aggregatibacter acti-
576 and E. corrodens (78.97 mg/mL) were inhibited but 632
nomycetemcomitans was 7.81 mg/mL, and for Pseu-
577 not killed. In the case of zoledronate, A. actinomyce- 633
domonas aeruginosa, it was 125 to 500 mg/mL. The
578 temcomitans, C. ochracea (3.13 and 6.25 mg/mL), H. 634
MBC ranged from 7.81 mg/mL (A. actinomycetemco-
579 aphrophilus, and E. corrodens (6.25 and 12.5 mg/ 635
mitans) to 250 mg/mL (Capnocytophaga ochracea,
580 mL) were inhibited, and A. actinomycetemcomitans 636
Haemophilus aphrophilus, and once to P.
581 and E. corrodens were killed. 637
aeruginosa).
582 Among the gram-positive aerobes, V. fluvialis 638
In the case of pamidronate, the MIC ranged from
583 (39.48 mg/mL) and L. casei (78.97 mg/mL) had MIC 639
39.48 mg/mL for Eikenella corrodens and C. ochracea
584 values for pamidronate that were less than the calcu- 640
to 1,263 mg/mL for H. aphrophilus. The MBC was
585 lated bone concentration. 641
158 mg/mL for A. actinomycetemcomitans and E. cor-
586 The anaerobe species C. rectus was inhibited with 642
rodens and 1,263 mg/mL for H. aphrophilus.
587 the calculated bone concentrations of ibandronate, pa- 643
The MIC of zoledronate was 3.13 to 6.25 mg/mL for
588 midronate, and zoledronate and killed by ibandronate 644
A. actinomycetemcomitans and C. ochracea. For A.
589 and zoledronate (MBC 15.63 mg/mL and 25 mg/mL, 645
actinomycetemcomitans, the MBC was between
590 respectively). Pamidronate inhibited P. intermedia 646
3.13 and 6.25 mg/mL. In the case of P. aeruginosa
591 (78.97 mg/mL), and zoledronate inhibited P. gingivalis 647
DSM 1,128, the MIC and MBC were both 200 mg/mL.
592 (25 mg/mL). 648
593 Of the 17 oral species, 5 (C. rectus, A. actinomyce- 649
594 ANAEROBES temcomitans, C. ochracea, E. corrodens, and H. aph- 650
595 We investigated 13 different anaerobic bacterial spe- rophilus) had MIC and MBC values that were less than 651
596 cies in 27 experiments (Table 3). Ibandronate had a the calculated bone concentration. The MIC and MBC 652
597 growth inhibitory effect on Campylobacter rectus values of the tested oral microorganisms (C. albicans, 653
598 with an MIC of 3.91 mg/mL. Campylobacter concisus, S. gordonii, Streptococcus anginosus, S. mutans, C. 654
599 Fusobacterium nucleatum, Prevotella nigrescens gracilis, F. nucleatum, P. gingivalis, P. intermedia, P. 655
600 and Prevotella oralis were inhibited at a concentra- corporis, P. oralis, P. micra, S. exigua) were greater 656
601 tion of 500 mg/mL. The MBC was 15.63 mg/mL for C. than the calculated bone concentrations. 657
602 rectus and 500 mg/mL for C. concisus, F. nucleatum, 658
603 Porphyromonas gingivalis, P. nigrescens, P. oralis, 659
Discussion
604 and Parvimonas micra. 660
605 Pamidronate had a growth inhibitory effect of The most frequently administered BPs, ibandronate, 661
606 78.97 mg/mL for C. rectus and Prevotella intermedia pamidronate, and zoledronate, were tested in the 662
607 and 1,263 mg/mL for Centruroides gracilis, F. nuclea- broth microdilution assay, with a preference for oral 663
608 tum, P. gingivalis, and P. corporis. The MBC was bacterial species. MRONJ is a disease of the jaw usually 664
609 158 mg/mL for C. rectus and 1,263 mg/mL for F. nucle- contaminated or infected with oral bacteria. Knowl- 665
610 atum, P. gingivalis, P. intermedia, and P. corporis. edge of the interactions between the 3 most frequently 666
611 Zoledronate showed a MIC of 12.5 mg/mL for C. prescribed BPs with physiologic and pathogenic oral 667
612 rectus and 400 mg/mL for F. nucleatum, P. nigrescens, bacteria is very limited in the scientific data. The deter- 668
613 Q3 P. oralis, Veillonella parvula, Slackia exigua. The mination of MIC and MBC can provide information 669
614 MBC ranged from 25 mg/mL for C. rectus to 400 mg/ about the possible interactions, such as selection pres- 670
615 mL for C. concisus, F. nucleatum, P. nigrescens, P. ora- sure or a shift to a more virulent bacterial spectrum. 671
616 lis, V. parvula, P. micra, and S. exigua. For the interpretation of the values determined 672

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673 in vitro, the expected bone concentrations for the 3 humans at the development of MRONJ on average. The 729
674 substances tested were taken from the published BP concentration levels we chose were 15 mg/mL for 730
675 data and partly calculated for the human bone. With ibandronate, 81.6 mg/mL for pamidronate, and 731
676 this assumption, the effects of ibandronate, pamidro- 24.45 mg/mL for zoledronate. 732
677 nate, and zoledronate on the physiologic, orally patho- When regarding these values as the so-called critical 733
678 genic, and some other bacterial species could bone concentration, ibandronate would inhibit 3 bacte- 734
679 be assessed. rial strains, pamidronate 6 bacterial strains, and zoledro- 735
680 The stimulus for the present study were reports on nate 8 bacterial strains in MRONJ. The species inhibited 736
681 the effect of BPs on protozoa-like toxoplasma, Trypa- by all 3 BPs were C. ochracea and C. rectus. E. corrodens 737
682 nosoma, and Leishmania.15,16 Laboratory-generated was inhibited by ibandronate and zoledronate and A. ac- 738
683 BPs without clinical approval have also been reported tinomycetemcomitans by ibandronate and zoledronate. 739
684 to be effective against bacteria.17 The selection of Pamidronate also inhibited L. casei and V. fluvialis, zo- 740
685 strains was determined from the microorganisms ledronate inhibited H. aphrophilus and P. gingivalis, 741
686 found in samples of necrosis reported in published and pamidronate inhibited P. intermedia. With the 742
687 studies.8-11 In particular, we considered the bacterial exception of Prevotella and Lactobacillus, the 7 remain- 743
688 spectrum of the oral microbiota. Nonoral bacteria ing strains had not been previously detected in samples 744
689 were chosen to complete the range of species. In of MRONJ.11 The unique spectrum of bacteria found in 745
690 particular, the study by Pushalkar et al,11 which identi- MRONJ might be characteristic for this disease and loca- 746
691 fied microorganisms using PCR and not exclusively us- tion11; however, the inhibiting effect and the noneffect of 747
692 ing histomorphology, confirmed our selection of BPs on bacterial growth should be considered when per- 748
693 oral bacteria. forming further studies. 749
694 The greatest concentration of the BPs tested was the In conclusion, the results of the present study have 750
695 original concentration of the respective drug for med- demonstrated that BPs have an inhibitory effect on 751
696 ical use. Therefore, we did not consider it useful to test selected bacterial species. In addition, most species 752
697 higher concentrations. The gram-positive bacteria tested were unaffected at the concentration levels 753
698 showed high MIC values, and the gram-negative bacte- assumed present in the human jawbone. Therefore, 754
699 ria were more sensitive. Of the 49 strains, 25 with it was difficult to assess the clinical relevance of these 755
700 ibandronate, 27 with pamidronate, and 17 with zoledr- in vitro data without knowing the exact BP concentra- 756
701 onate had MIC values at the greatest concentration or tions in human bone. Our results did not suggest a 757
702 even higher. The most sensitive strains were the rod- unique selective advantage or disadvantage for the 758
703 shaped gram-negative bacteria: A. actinomycetemco- selected single bacterial species tested. In future 759
704 mitans, C. ochracea, and C. rectus. The MIC values studies, these results could vary when evaluating bio- 760
705 were the 64th part of the greatest concentration film models. For the assessment of the interaction of 761
706 Q4 tested, that means 7 dilution steps lower. oral bacteria and BPs, the present study has at least 762
707 A. actinomycetemcomitans is a well-known path- provided a first approach. 763
708 ogen in periodontitis. With the concentrations we 764
709 measured in the present study, no selection advantage References 765
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