IAP Journal Jan 2011 Issue

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Journal of the International

Academy of PeriodontologyThe official journal of the International Academy of Periodontology

Volume 13 Number 1 January 2011

Published by

Stephen Hancocks Ltd


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Acknowledgements and Conflict of InterestInclude acknowledgement of those individuals who contributed to the publication,source of financial support, and any financial relationships of any of the authorswhich may pose a perceived conflict of interest.

ReferencesIn the text the authors (authors) name(s) and date of publication should be used aseither: in a similar study (Anderson and Morgan, 1992), or Conversely, Blinkhorn(1994) found that. If there are more than two authors, the first author and year arecited in the text; for example, (Spencer et al.,1995). Citation of authors of morethan one paper in a single year are shown as 1995a; 1995b; etc. Multiple referencesin the text should appear in chronological order separated by semicolons. Authorsof unreferenced work should appear in the text only.

The list of references at the end of the text should be double-spaced and

arranged alphabetically by name of first author. All authors should be listed unlessthere are more than six, when only the first three should be given followed by et al.This should be followed by the title of the article, the full name of the journal (initalics); the year of publication; the volume number (in bold); and the first and lastpage numbers in full. Please follow the punctuation used in the examples below.Papers submitted with an incoreectly formatted reference list will be held withoutreview until a corrected reference list is provided.

Examples:Reference to an article:Shiloah, J. and Patters, M.R. Repopulation of Periodontal

Pockets by Microbial Pathogens in the Absence of Supportive Therapy.Journalof Periodontology1996; 67:130139.

Reference to a book:Schuster, G.S. Oral Microbiology and Infectious Diseases, 3rd ed.Philadelphia: B. C. Decker, 1990; 516522.

Reference to a chapter in a book: Chesney, J., Patters, M.R. and Budreau-Patters, A.Oral Infections. In Long, S., Prober, C. and Pickering, L. (Eds): Principles and

Practice of Pediatric Infectious Disease. New York. John Wiley and Sons, 1996.Reference to a dissertation or thesis:James, A. On the Immune Response to GTRMembranes in Periodontics. PhD, Liverpool, UK. 1994; 1525.

Reference to a report: Committee on Mercury Hazards in Dentistry. Code ofPractice for Dental Mercury Hygiene. London: Department of Health andSocial Security, 1979; Publication No. DHSS 79-F372.

Reference to an abstract: Patters, M.R., Shiloah, J., Dean, J.W., Bland, P. andToledo, G. The Consequence of Infection of Treated Periodontal Pocketsby Microbial Pathogens.Journal of Dental Research1997; 76 (special issue),111 (Abst).

TablesEach table must be submitted on a separate sheet of paper, double spaced throughout,including column heads, footnotes, and data. They should be numbered with arabicnumerals according to their order of mention in the text. Tables should be self-ex-planatory and supplement, not duplicate, the text. All footnotes should immediately

follow the table, and all abbreviations should be defined in the footnote.

IllustrationsIllustrations should be numbered with arabic numerals in order of their mention inthe text. They may be submitted in colour or black and white, but colour will be usedat the Editors discretion where it enhances the text. In general, the Editor will requirethat clinical photographs and stained histologic specimens be submitted and publishedin colour. In such cases, the authors must agree in advance to pay the increasedprinting costs for publishing the colour figures. If submitting electronically, figuresshould be submitted in jpeg format. Glossy photographic prints, transparencies, orgood quality laser prints are also acceptable if submitting via post. Lettering must beclear and large enough to remain legible in the event of reduction of the figure in re-production. One set of original illustrations must accompany the original manuscript.Copies of the illustrations, if legible, can be submitted with the additional copies ofthe manuscript. They should be labeled with the authors name, figure number andan arrow to indicate the top edge using adhesive labels in the margin or on the back

of the illustration. Typed labels are preferred as ink and pen or pencil pressure maydamage the emulsion of carefully prepared photographic prints.

LegendsAll illustrations should be listed by legend on a separate sheet of paper, type-written,double-spaced, and numbered to correspond with the numbering in the text.

Permissions

Direct quotations, tables, and illustrations that have appeared in copyright materialmust be accompanied by written permission for their use from the copyright ownerand the original author, along with complete information as to the source. Photo-graphs of identifiable persons must be accompanied by signed releases showinginformed consent. If an illustration is taken from previously published material, thelegend must give full credit to the original source.

Statements and opinions expressed in the articles and communications hereinare those of the author(s) and not necessarily those of the Editor(s) or publisher, andthe Editor(s) and publisher disclaim any responsibility or liability for such material.Authors must disclose to the Editor any financial interest they may have in productsmentioned in their article.

The Journal will endeavour to provide a decision to the authors within 16 weeksof arrival of the paper at the editorial office, provided that the corresponding authorhas a functioning e-mail address. Reliance on overseas mail may delay the decisionfrom reaching the author within the above time frame. Manuscripts submittedfor review that do not follow these instructions may be delayed until corrected orreturned unreviewed.

Manuscripts

Manuscripts for publication and all correspondence should be sent to Dr. Mark R.Patters, Editor, Journal of the International Academy of Periodontology, Universityof Tennessee College of Dentistry, Office of Academic Affairs, 875 Union Avenue,Memphis, TN USA, 38163, e-mail: [email protected]. Printed submissions sentto the above address must be prepared as described below and accompanied by astandard floppy disk containing an electronic copy of the manuscript. The floppydisk should be labelled with the manuscript title, author(s) and specific versionof the word processing program used. In lieu of printed manuscripts, electronicsubmissions done entirely in Microsoft Word (PC or Mac) will be accepted at theabove e-mail address. Effective 1 January 2006, the corresponding author of amanuscript submitted for publication in the Journal of the International Academyof Periodontology must be a member of the International Academy of Periodontol-ogy or, in lieu of membership, pay a submission fee of US$100.An application for

membership can be found at http://www.perioiap.org/join_iap.htm. Those authorschoosing to pay the submission fee should contact the Editor at [email protected] submitting the manuscript.. All submissions must be written in English andwill be subject to peer and editorial review.

Articles for publication will be considered under the following headings:original research, clinical case reports and review articles relevant to all aspects ofperiodontology and implantology. Articles must be original and may not have beensubmitted or accepted for publication elsewhere, with the exception of presentationat a scientific meeting and publication as an abstract. A signed statement to this effectshould be included with the submission of the manuscript. Research that involvesstudies on humans must conform to the Declaration of Helsinki and the authorsmust indicate that appropriate informed consent was obtained.

Research reportsState the problem and objectives clearly, describe the methods and materials indetail, report the results clearly using the minimum number of figures and tables;

and, bearing in mind previously published work, discuss the results, the conclusions,and the clinical implications.

Clinical case reportsDiscuss a clinical challenge; describe the treatment method and discuss the results inlight of previously published methods of treatment of individual patients.

Literature reviewsRecord the sequence of development of a particular aspect of periodontology indetail, as briefly and succinctly as possible. The review should cover the topic com-pletely and be thoroughly referenced. At least one contributing author of a reviewmust have personal experience with relevant research.

Letters to the EditorLetters may address relevant matters of concern to the membership of the Interna-tional Academy of Periodontology or offer constructive criticism of articles publishedby JIAP. Letters must be concise and signed. If the letter comments on a publishedarticle, it should contain appropriate references. The letter will be referred to the

author(s) of the original work so that they will have an opportunity to respond.

EditorialsEditorials may be solicited from authorities to provide a unique perspective on pub-lished articles, or to comment on other items of interest to the membership.

Copyright statement

A copyright transfer statement will accompany the galley proofs of accepted, typesetmanuscripts. The form must be signed by at least one of the authors and returnedwith the corrected proofs.

Manuscript preparation

Manuscripts must be submitted in quadruplicate (one original and three copies) andshould be typewritten, double-spaced on one side only of A4 or 8.5 x 11 inch paper,with at least 25 millimetre (1 inch) margins on all four sides. Articles generally should

not exceed 1012 pages (excluding references, tables, figure legends and figures)and should be limited to no more than six authors. Additional contributing authorswill be listed as an addendum to the manuscript. Abbreviations should be placed inparenthesis after the first complete use of the term(s) to be abbreviated. Use genericnames for drugs and for dental materials. Give trade names and manufacturers namesand addresses in parentheses.

Title pageShould include for each author the full name and title, academic degrees, and insti-tutional affiliations. The corresponding author should also include a street address,telephone and fax numbers, and e-mail address. Only individuals who have made asubstantial contribution to the work and who agree to take public responsibility forthe content of the work should be included. If the work was supported by a grant,the name of the supporting organization and the grant number should appear onthe title page. The address for reprint requests will be assumed to that of the cor-responding author unless otherwise specified.

Abstract and Key WordsAbstracts are required for all articles, and should be limited to 250 words typeddouble-spaced on a separate page. The abstract should serve as a concise sum-mary of the manuscript, including objective, methods, results and conclusions.Abbreviations should not be used in the abstract. Please provide three to six keywords (Dental Descriptors, Index to Dental Literature and/or Index Medicus) tobe used for indexing purposes.

TextThe body of the manuscript should contain an Introduction, a detailed review of theMaterials and Methods, a description in logical sequence of Results, and a Discussionsection with Conclusions


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Journal

of the

International Academy ofPeriodontology

Volume 13

Number 1January 2011

ISSN 14662094

EDITORIAL BOARD

Mark R PattersEditor

Memphis, TN, USA

Andrea B PattersAssociate Editor

Sultan Al MubarakRiyadh, Saudi Arabia

P Mark BartoldAdelaide, SA, Australia

Michael BralNew York, NY, USA

Nadine BrodalaChapel Hill, NC, USA

Cai-Fang Caoeijing, Peoples Republic of China

Chong-Pyoung ChungSeoul, Korea

Daniel EtienneParis, France

Erhan FiratliIstanbul, Turkey

Kohji HasegawaTokyo, Japan

Vincent J Iacono

Stony Brook, NY, USA

Isao IshikawaTokyo, Japan

Georges KrygierParis, France

Yoji MurayamaOkayama, Japan

Hamdy NassarCairo, Egypt

Angela R C PackDunedin, New Zealand

David PaquetteChapel Hill, NC, USA

Stephen PolinsBoston, MA, USA

Rok ScharaLjubljana, Slovenia

Lior ShapiraJerusalem, Israel

Uros SkalericLjubljana, Slovenia

Aubrey SoskolneJerusalem, Israel

Thomas E Van DykeBoston, MA, USA

The Effect of Different Interdental Cleaning Devices on Gingival Bleeding

Nanning A. M. Rosema, Nienke L. Hennequin-Hoenderdos, Claire E. Berchier,

Dagmar E. Slot, Deborah M. Lyle and Godefridus A. van der Weijden 2

Additive or Synergistic Antimicrobial Effects of Amoxicillin and

Metronidazole on Whole Plaque Samples: A Preliminary Report

Clemens Walter, Eva M. Kulik, Roland Weiger, Nicola U. Zitzmann and

Tuomas Waltimo 11

Clinical and Microbiological Comparison of Three Non-surgical

Protocols for the Initial Treatment of Chronic Periodontitis

Carlos Serrano, Nidia Torres, Angela Bejarano, Marcela Caviedes and

Mara Eugenia Castellanos 17

13th International Biennial Congress - Registration Form 27

The Journal of the Inte rna tional Academy of Perio dontolog y is the official journal of th e International Academy ofPeriodontology and is published quarterly (January, April, July and October) by Stephen Hancocks Ltd in association with DennisBarber Ltd.

Manuscripts,prepared in accordance with the Information for Authors should be submitted to the Editor, Dr. Mark R. Patters, Universityof Tennessee, Department of Periodontology, 875 Union Avenue, Memphis, TN 38163, USA. Electronic submissions will be accepted andshould be sent to [email protected]. Instructions to Authors are available at: http://www.perioiap.org/publications.htm#JOURNAL

All enquiries concerning advertising, subscriptions, inspection copies and back issuesshould be addressed to Mrs. Kelly OgilvieMcLean, Goldman School of Graduate Dentistry, Boston University, 100 E. Newton Street, Boston, MA, USA 02118. Tel: +1 617638-4758; Fax: +1 617 638-4799. Email: [email protected]. Whilst every effort is made by the publishers and Editorial Board to see

that no inaccurate or misleading opinion or statement appears in this Journal they wish to make clear that the opinions expressed inthe articles, correspondence, advertisements etc., herein are the responsibility of the contributor or advertiser concerned. Accord-ingly, the publishers and the Editorial Board and their respective employees, ofces and agents accept no liability whatsoever for theconsequences of any such inaccurate or misleading opinion or statement.

2011 International Academy of Periodontology.All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by anymeans, electronic, mechanical, photocopying, or otherwise, without permission of the Academy.

Produced in Great Britain by Dennis Barber Limited, Lowestoft, Suffolk


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International Academy of Periodontology

Journal of the International Academy of Periodontology 2011 13/1:210

Correspondence to: N.A.M. Rosema. Department of Period-ontology, Academic Centre for Dentistry Amsterdam (ACTA),University of Amsterdam and VU University Amsterdam, Gus-tav Mahlerlaan 3004 1081 LA Amsterdam, The Netherlands.E-mail: [email protected]

The Effect of Different Interdental CleaningDevices on Gingival BleedingNanning A. M. Rosema1, Nienke L. Hennequin-Hoenderdos1,Claire E. Berchier1, Dagmar E. Slot1, Deborah M. Lyle2and

Godefridus A. van der Weijden11Department of Periodontology, Academic Centre for DentistryAmsterdam ACTA, University of Amsterdam and VU UniversityAmsterdam,The Netherlands, and 2Water Pik Inc, Fort Collins, CO, USA

Abstract

Objective: To compare the effectiveness of an oral irrigator (OI) with a prototype jettip or a standard jet tip to oss as adjunct to daily toothbrushing on gingival bleeding.Methods: In this single masked, 3-group parallel, 4-week home use experiment, 108subjects were randomly assigned to one of three groups: 1) OI with a prototype jet tip;

2) OI with a standard jet tip; 3) waxed dental oss. All groups used their assigned prod-uct once a day as adjunct to twice daily toothbrushing for two minutes with a standardADA reference toothbrush. Professional instructions were given by a dental hygienist inOI use or oss use according to written instructions. All subjects also received a tooth-brush instruction leaet (Bass technique). Subjects were assessed for both bleeding andplaque at baseline and after two weeks and four weeks and were instructed to brushtheir teeth approximately 2 to 3 hours prior to their assessment. Results: With respect tomean bleeding scores the ANCOVA analysis with baseline as covariate and week 4 asdependent variable showed a signicant difference between groups in favor of both theoral irrigator groups. For plaque, however, no signicant difference among groups wasobserved. Conclusion: When combined with manual toothbrushing the daily use of anoral irrigator, either with prototype or standard jet tip, is signicantly more effective inreducing gingival bleeding scores than is the use of dental oss, as determined withinthe limits of this 4-week study design.

Key words: Floss, dental water jet, oral irrigator, water fosser, gingivitis, bleeding,

plaque, toothbrush

Introduction

Biolms are 3-dimensional arrangements of bacteriathat are loosely or more rmly adherent to teeth andtissue (Costerton et al.,1994). Biolms consist of micro-colonies of bacteria embedded in slimy matrices and areself-sufcient, dynamic communities that can survive in

hostile environments (Marsh and Bradshaw,1995) Theregular removal of dental plaque biolm, which containsthe bacteria responsible for caries formation and forthe etiology of gingivitis and periodontitis, is the well-accepted conditio sine qua nonof dental health (Gorur et al.,2009). Mechanical removal is considered the most effec-tive method to control the growth of the oral biolm. Themost common device used for mechanical plaque control

is either a manual or power toothbrush. As toothbrushefcacy is limited to the surfaces of the teeth it can access(facial, lingual, and occlusal), another device is needed toclean the interdental area and the proximal surfaces ofthe teeth and surrounding gingivae. Other factors thataffect the efcacy of mechanical plaque biolm removalinclude brushing frequency, brushing time, toothbrush

design, and brushing technique (Jepsen et al., 1998; Vander Weijden et al., 1993).

For most people, however, total plaque biolmremoval is not a realistic goal. It is difcult for patientsto effectively remove or disrupt the biolm from allsurfaces of the teeth on a daily basis (Douglass et al.,1993; Brown et al., 1993). On average, people reducetheir plaque scores by approximately 50% by brushing(Jepsen et al., 1998). Therefore, compliance with instruc-tions is a major consideration when recommending anyself-care device. To be truly patient-centered, practitio-

ners must shift to recommending available tools that,besides having demonstrated efcacy in reducing inam-mation based on scientic evidence, are also preferredby patients (Slot et al., 2008).


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The Effect of Different Interdental Cleaning Devices on Gingival Bleeding 3

A dental water jet or water osser or oral irrigator(OI) is an electric device that delivers a pulsating uidvia controlled pressure which is aimed at the removalof interdental and subgingival plaque biolm on toothsurfaces to reduce inammation as a supplement totoothbrushing (Lobene, 1969; Drisko et al., 1987; Cobb etal., 1988; Flemmig et al., 1990; Chaves et al., 1994; Flem-

mig et al.,1995; Barnes et al., 2005; Gorur et al., 2009).The OI was introduced to the dental profession in 1962and has been studied extensively for the past decades.Clinical studies demonstrate that an OI is safe and cansignicantly reduce bleeding and gingivitis in a varietyof cohorts (Lobene et al., 1969; Flemmig et al., 1990;Brownstein et al., 1990; Burch et al., 1994; Newman etal., 1994; Flemmig et al., 1995; Felo et al., 1997; Barneset al., 2005; Sharma et al., 2008). However, erythrosine-based plaque indices have yielded equivocal data. Somestudies have shown a reduction in plaque indices with

the use of the OI compared to toothbrushing alone(Burch et al., 1994; Felo et al., 1997; Cutler et al., 2000;Al Mubarak et al., 2002; Sharma et al., 2008), while otherstudies showed no signicant differences (Ciancio et al,1989; Brownstein et al., 1990; Walsh et al., 1992; Chaveset al., 1994; Fine et al., 1994).

The OI is likely to provide a particular benet interms of gingival health to a large part of the generalpublic that does not clean the interproximal spaceson a regular basis (Research, Science and TherapyCommittee, 2005). In 2001 the American Academy of

Periodontology stated, Among individuals who do notperform excellent oral hygiene, supragingival irrigationwith or without medicaments is capable of reducinggingival inammation beyond that normally achievedby toothbrushing alone. This effect is likely due to theushing out of subgingival bacteria (Research, Scienceand Therapy Committee, 2001). In a 2005 positionpaper, the American Academy of Periodontology statedthat supragingival lavage can assist individuals withgingivitis or poor oral hygiene. The greatest benet isseen in patients who perform inadequate interproximal

cleansing. Patients report that the OI facilitates theremoval of food debris in posterior areas, especiallyin cases of xed bridges or orthodontic appliances,when the proper use of interdental cleaning devices isdifcult (Research, Science and Therapy Committee,2005). However, anecdotal discussions and commentarycontinue concerning the appropriate use and efcacyof this instrument. OI devices can be used with waterbut also with antimicrobial agents (Flemmig et al., 1990,Brownstein et al., 1990, Jolkovsky et al., 1990; Newmanet al., 1994, Fine et al., 1994; Chaves et al., 1994; Flemmiget al., 1995; Felo et al., 1997).

The objective of the present study was to test theadjunctive effect to toothbrushing of an OI with eithera prototype jet tip or a standard tip in the potential to

improve gingival health over a 4-week period. This pro-totype tip, which is congured with laments, may helpthe user guide the tip along the gingival margin and theinterproximal area.Both OI tips were compared to theuse of dental oss. These treatments were combinedwith the use of a regular at trimmed manual toothbrushtogether with a standard dentifrice.

Materials and methods

Study population

One hundred seventy-two subjects (non-dental students)from different universities and colleges in and aroundAmsterdam responded to an e-mail advertisement andreported for a screening appointment. The volunteerswere informed about the study, rst in a recruitmentletter and secondly at the screening. Participation wasnot limited by race or gender. Subjects received a written

explanation of the background of the study, its objec-tives and their involvement. Before screening for theirsuitability they were all requested to give their writteninformed consent. Subjects were required to fulll thefollowing criteria: 18 years of age, a minimum ofve evaluable teeth in each quadrant (with no partialdentures, orthodontic banding or wires); moderategingivitis (50% bleeding on marginal probing, Galgut etal., 1998), an absence of oral lesions and/or periodontalpockets > 5 mm and/or generalized recession, and theabsence of pregnancy and systemic diseases such asAIDS, cirrhosis, diabetes, any adverse medical historyor long-term medication, or any physical condition thatlimits manual dexterity. All subjects received oral andwritten information about the products and purposeof the study. One hundred eight subjects met the in-clusion criteria and were enrolled into the study, whichwas conducted in accordance to the ethical principlesthat have their origin in the Declaration of Helsinki andwas consistent with Good Clinical Practice guidelines.Medical Ethics Committee approval was obtained priorto the start of the study (MEC 09/198 #09.17.1322).All assessments took place at the Department of Peri-

odontology at ACTA, Amsterdam, The Netherlands inSeptember and October, 2009.

Study products

Three different interdental products were tested in thisstudy, one product per group, with 36 subjects enrolledin each group. All subjects received a standard tooth-brush (Oral-B Indicator 35, Procter & Gamble, Cincin-nati, OH, USA, Figure 1) and standard uoride dentifrice(Everclean, HEMA, Amsterdam, The Netherlands).In addition, subjects were randomized (see below for

details) into one of three groups for assignment of aninterdental cleaning device:


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4 Journal of the International Academy of Periodontology (2011) 13/1

Group 1 (OIP): OI (DWJ-Waterpik Ultra WaterFlosser, Fort Collins, CO, USA) with a prototype jet tip(Figure 2, test group).

Group 2 (OIS): OI (DWJ-Waterpik Ultra WaterFlosser, Fort Collins, CO, USA) with a standard jet tip(Figure 3, benchmark control group, Husseini et al., 2008).

Group 3 (DF): standard waxed oss (Johnson &

Johnson, New Brunswick, NJ, USA) (Figure 4, controlgroup).

Clinical assessment

Clinical parameters were assessed at baseline (S1), week2 (S2), and week 4 (S3). First gingivitis and then plaquewas scored. All gingivitis assessments were carried outby the same trained examiner (NLH). All plaque assess-ments were carried out by a second trained examiner(CEB). All examinations were carried out under thesame conditions. All teeth were examined for both

indices at six sites per tooth (disto-buccal, mid-buccal,mesio-buccal, disto-lingual, mid-lingual, mesio-lingual)except for 3rdmolars.

Criteria

Gingivitis was assessed as the primary outcome usingthe bleeding on marginal probing index (BOMP) asdescribed by Van der Weijden et al. (1994a, 1994b) andLie et al. (1998). In short, the gingival margin is probedat an angle of approximately 60to the longitudinal axisof the tooth and the absence or presence of bleedingis scored within 30 seconds of probing on a scale 0 - 2(0 = no bleeding, 1 = pinprick bleeding, 2 = excessivebleeding).

Plaque was assessed as a secondary outcome usingthe Turesky (1970) modication of the Quigley & Hein(1962) plaque index (TQHPI) as described in detail by

Figure 1. Toothbrush - Oral-B indicator 35

Figure 2. OIP - oral irrigator with prototype tip

Figure 3. OIS - oral irrigator with standard tip

Figure 4. DF - standard waxed dental foss


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Figure 5. Flowchart

Flowchart

Screening

Baseline (S1)

2 Weeks (S2)

4 Weeks (S3)

StatisticalAnalyses(ITT)

Screening172 subjects

Excluded64 subjects

Total: 108 subjects

Group 1: 36 subjectsGroup 2: 36 subjectsGroup 3: 36 subjects

BOMP & TQHPIProfessional instruction

Total: 106 subjects


BOMP & TQHPI

Drop-out2 subjects

Drop-out2 subjects

Total: 104 subjects

Group 1: 34 subjects

Group 2: 34 subjectsGroup 3: 36 subjects

BOMP & TQHPI

Total: 104 subjects


Paraskevas et al. (2007). Briey, the teeth were dyed us-ing a new cotton swab with fresh disclosing solution(Mira-2-Ton; Hager & Werken GmbH & Co. KG.Duisburg, Germany) for each quadrant in order to dis-close the plaque. Subsequently, the absence or presenceof plaque was recorded on a 6-point scale (0-5, 0 = noplaque, 5 = plaque covering more than two-thirds ofthe tooth surface).

Study design

This study was designed as single masked, 3-group paral-lel, 4-week home use experiment. After meeting the inclu-

sion criteria, completion of a medical questionnaire andinformed consent, subjects returned to the clinic for theirrst (baseline) assessment (S1) for both clinical param-eters (bleeding on marginal probing and plaque). At thestart of the experiment all subjects received a unique trialnumber. Subjects were randomly assigned to one of threegroups according to a randomization list (www.random.org). The allocation of products was carried out by the

study coordinator, who was responsible for allocationconcealment. All products were distributed in such a waythat blindness of the examiners was assured. At the lastvisit (S3) the study coordinator assured blindness of the


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examiners by collecting the study products in a separateroom from where the clinical examinations took place.Subjects were also instructed not to mention anythingto the examiners that could lead to allocation disclosure.

During the 4-week experimental phase OIS andOIP subjects used the OI once a day in the eveningwith lukewarm tap water and were instructed to nish

one container of 500 ml at each occasion. Subjects inthe control group (DF) used standard waxed dentaloss once a day in the evening. At the baseline visit(S1), immediately following the baseline assessment,subjects used their allocated product for the rst time.The study coordinator (NAMR) was present to providedetailed verbal instruction, a demonstration to ensurecorrect use, and aid with further personal instructionwhen necessary. Subjects in both OI groups wereinstructed to use the OI according to the instructionleaet provided by the manufacturer. Subjects in the DF

group were instructed to use their product accordingto the description of Van der Weijden et al. (2008). Allsubjects in each group were instructed to brush twice aday in their normal manner, once in the morning afterbreakfast and once in the evening. In the evening theysubsequently used their assigned product (OI or DF).All participants were instructed to refrain from usingany other oral hygiene product or device such as tooth-picks, interdental brushes, mouthrinses, etc., during thestudy period. To check for compliance, subjects wereasked to register the time of use of the products onto

a calendar record chart.After two weeks (S2), subjects returned to the clinic

for the second clinical assessment for both gingivitisand plaque. After four weeks (S3), subjects visited theclinic for their nal assessment for both parameters.Subjects were asked to return all products provided forthis study as well as the calendar record chart. On eachoccasion subjects were instructed to brush between 2and 3 hours prior to their appointments to avoid therisk of increased bleeding on probing as a result oftoothbrushing (Abbas et al., 1990). The day prior to each

appointment all subjects received an SMS-message as areminder with the following text: Remember that you havean appointment at ACTA! Note that you need to brush your

teeth 2-3 hours prior to your visit. See you tomorrow! ACTA.After the nal assessment habitual oral hygiene proce-dures were resumed.

Data analyses

The unit of analysis was the subject and collected datawere analyzed as intention to treat. The bleeding scoreswere used as the main response variable (Galgut et al.,1998) and plaque scores as secondary response variable.A prioricalculations with an alpha of 0.05, a differenceof 0.0883 (between groups) of the bleeding index with80% power, based on a pooled SD of 0.13 as derived

from previous studies supported a sample size of 105.An analysis of covariance (ANCOVA) with S1 as cov-ariate and S3 as dependent variable was performed tocompare groups over time (Heynderickx et al., 2005).Analyses comparing differences between the test andcontrol groups at each time point were performedusing non-parametric tests. Explorative analyses were

performed to investigate the origin of the overall differ-ences. Pvalues of < 0.05 were accepted as statisticallysignicant.

Results

Of 108 subjects who started the trial, four subjects didnot complete the protocol. One chose not to continuethe trial for personal reasons. Another left the countryand moved abroad. Two did not attend the second visitbecause of scheduling conicts. This resulted in a study

population of 104 subjects providing evaluable data(Figure 5). The study population data on demographicsand pre-study oss habits are presented in Table 1. Noadverse events were reported by any of the subjects whoparticipated in this study.

Results for bleeding on probing are presented in Table2. The overall ANCOVA analysis showed a statisticallysignicant difference between the three groups (p =0.007). Mean overall reductions after four weeks of use(S1 to S3) were 0.15 for the OIP group, 0.17 for the OISgroup, and 0.02 for the DF group. The mean bleedingscores of the three groups did not differ signicantly atbaseline. At session 2 the scores decreased for all threegroups. Post testing showed that both the OI groupsprovided signicantly lower bleeding scores as comparedto the DF group. At session 3 a statistically signicantdifference could be detected among the three groups.Post testing showed that again both the OI groups hadsignicantly lower bleeding scores as compared to theDF group. The 95% condence interval of the differ-ence compared to the DF group at S3 was -0.27 -0.04for the OIP group and -0.28 -0.05 for the OIS group.

Results for plaque index are presented in Table 3.

With regard to the plaque scores the overall ANCOVAanalysis showed no statistically signicant differencesamong the three groups (p= 0.126). Mean overall re-ductions after four weeks of use (S1 to S3) were -0.09for the OIP group, 0.06 for the OIS group, and 0.01for the DF group.

Discussion

Effective brushing remains the most obvious way ofmaintaining low levels of plaque and good gingivalhealth. Gingivitis is known to be associated with theonset of periodontitis, and although the relationship be-tween these two conditions may not be fully understood,the importance of maintaining good gingival health and


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this with traditional dental oss (Asadoorian, 2006).Thus, compliance with oss is low (Warren and Chater,1996), and various adjuncts for interdental cleaning havebeen studied. Dental oss, toothpicks, woodsticks andinterdental brushes have all been recommended forthis purpose.

The present study focussed on the ability to reducegingival inammation in a population of young individu-als with moderate gingivitis using an OI. The OI worksthrough the direct application of a pulsed stream ofwater or other solution. A study duration of four weekswas chosen to monitor the changes in the bleeding index,which meets the ADA guidelines on OIs for studies as-sessing the effects of adjunctive therapies on reductionof gingivitis (ADA, 2008). Studies of longer durationwill more clearly demonstrate the clinical benet thatsubjects will obtain from this product.

The efcacy of use of oss on the bleeding indexwas considered inconclusive in a systematic review byBerchier et al. (2008). The results of the present study are

Table 1.Demographic data and pre-study ossing habits of the study population.

OIP, oral irrigation device with prototype jet tip; OIS, oral irrigation device with standard jettip; DF, dental oss

Total OIP OIS DF

N 104 34 34 36Female 74 24 27 23Male 30 10 7 13Age [range] (SD) 21.8 [18-36] 21.9 (3.2) 21.1 (2.3) 22.4 (3.1)Daily oss users 6 2 1 3Weekly oss users 16 7 4 5Monthly oss users 20 9 7 4Seldom/never oss users 62 16 22 24

Table 2.Mean bleeding index (BOMP) and mean % bleeding scores for all groups at all sessions.

Standard deviation in parentheses. Univariate analyses of covariance with session 1 as covariate and session 3 as depen-

dent variable. (p= 0.007). *Statistically signicant difference compared to DF group, p< 0.05 (Mann-Whitney). Statisti-cally signicant difference compared to DF group, p= 0.020 (Mann-Whitney). OIP, oral irrigation device with prototypejet tip; OIS, oral irrigation device with standard jet tip; DF, dental oss

N Session 1 Session 2 Session 3 Relative Reduction Relative Reduction

S1 S2 S1 S3OIP - index 34 0.82 (0.25) 0.65 (0.24) 0.67 (0.26)

% 46 % 37 % 39 % 20 % 15 %

OIS index 34 0.83 (0.23) 0.61 (0.27)* 0.66 (0.26)*% 46 % 34 % 38 % 26 % 17 %

DF - index 36 0.86 (0.26) 0.74 (0.26) 0.84 (0.30)

% 47 % 41 % 47 % 13 % 0 %

p- value(Kruskal Wallis)

0.579 0.084 0.016

Univariate analyses of covariance with session 1 as covariateand session 3 as dependent variable. (p= 0.126). OIP, oralirrigation device with prototype jet tip; OIS, oral irrigationdevice with standard jet tip; DF, dental oss

Table 3.Mean Quigley & Hein plaque scores standarddeviation for all groups at all sessions.

N Session 1 Session 2 Session 3

OIP 34 1.64 0.43 1.61 0.34 1.73 0.37OIS 34 1.79 0.34 1.74 0.29 1.73 0.28

DF 36 1.60 0.26 1.51 0.27 1.59 0.27

preventing periodontitis is well recognised (Van Dyke etal., 1999).As the interproximal area is known as wherethe onset of gingival inammation is likely to occur,the reason for interproximal plaque control seems clear.Although it is universally recognized that interproximalcleansing is essential for controlling periodontal disease(Le, 1979), many people have difculty accomplishing


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in support of this statement. In contrast, in the presentstudy both OI groups did show statistically signicantimprovements after four weeks. At the end of the studyboth OI groups show a signicant 15 - 17% reductionof the bleeding index as compared to baseline. For theDF group this difference was not observed. Compari-sons among groups showed a signicant difference at

four weeks between the DF group and both OI groups.The absolute difference of 8% and 9% at four weeksfor both OI groups as compared to the oss group re-veals a relative effect of 17% (OIP) and 19% (OIS). Inconsideration of the ADA guidelines for oral irrigators,the results of the present study do not reach the lowerlimit of superiority of 20% as estimated proportionatereduction related to clinical relevance as compared tostandard oral hygiene procedures (ADA, 2008). How-ever, the ADA also has guidelines on adjunctive dentaltherapies (ADA, 1997). In those guidelines a lower limit

of 15% is applied. The study outcomes of the presentstudy do comply with this guideline, indicating a poten-tial benecial effect for the OI.

With respect to plaque, the DF group started with amarkedly lower score as compared to both OI groups.All subjects were instructed to brush 2-3 hours priorto examination, to reduce the risk of greater bleedingtendency (Abbas et al., 1990). As the difference in PIscores was consistent throughout the study and was notreected in bleeding index scores, it seems that subjectswho were randomly allocated to the oss group coin-

cidently performed better instant plaque removal bybrushing at visit days. In a study carried out by Galgutet al. (2000) the effect of unevenly distributed baselinedata is discussed and it was concluded that this mightnot inuence the results and the conclusions drawn.Historically, plaque reductions are considered a pre-requisite for an oral hygiene device to be consideredeffective (Le et al., 1965). A recent systematic review(Husseini et al., 2008) reported no statistically signicantreduction in plaque when the OI was used as an adjunctto toothbrushing when compared to toothbrushing only.

Despite a lack of effect on plaque index, the studies thatwere included in this review did nd a signicant effecton bleeding and gingival indices. The mechanisms ofactions underlying these clinical changes for the bleedingindex in the absence of a clear effect on plaque are notunderstood, although different hypotheses have beenput forward (Husseini et al., 2008). One of the hypoth-eses is that supragingival irrigation alters the popula-tion of key pathogens, reducing gingival inammation(Flemming et al.,1995). Another hypothesis is that thewater-pulsation may alter the specic host-microbialinteraction in the subgingival environment (Chaves etal., 1994). There is also the possibility that the benecialaction of an OI is at least partly because of the removalof loosely adherent soft deposits interfering with plaque

maturation and stimulation of the immune response(Frascella et al., 2000). Other explanations could be amechanical stimulation of the gingiva or a combina-tion of the above-mentioned factors (Frascella et al.,2000; Flemmig et al., 1990). Furthermore, irrigation mayreduce the thickness of the plaque, which may not beeasily detectable using 2-dimensional scoring systems

(Jolkovsky et al., 1990).The absence of an effect for DF at four weeks may

also seem surprising. A transient effect of 6% BI re-duction was observed at two weeks. However, a recentsystematic review supports this nding that dental osshas no signicant effect on plaque or bleeding indices(Berchier et al., 2008). The small effect observed at twoweeks is most likely the result of a novelty or Hawthorneeffect.The Hawthorne effect is a reaction of subjects tothe realization they are in a study and are being observed(Adair et al., 1984). The novelty effect and Hawthorne

effect can be considered as certain placebo effects. Theimpact of a placebo effect should not be underestimated(Finniss et al., 2010). In a study by Feil et al. (2002), theHawthorne effect was intentionally used and shown toimprove oral health. The novelty effect is somethingthat could have inuenced all groups within this model.Subjects were pre-selected on having no experiencewith an OI, whereas only six out of the 104 were regularossers (Table 1). The rebound that is observed fromthe 2-week to the 4-week follow-up is, however, mostevident in the oss users. With respect to the Hawthorne

effect, this is probably not only present in the DF groupbut also in both OI groups, as subjects were selected onhaving a bleeding index of > 50%. However at session1 the bleeding index was already reduced to 46-47%for all three groups. This indicates that subjects alreadyacted as if they were entered into the protocol beforethe rst assessment of the primary response variable.

The results of the present study add to the existingdata and clearly show a reduction in inammation fromusing an OI. Interestingly, the reduction in bleedingcould not be linked to plaque removal. This is similar

to data presented by Flemmig et al. (1990) showing nochange in plaque scores for either the brushing groupor the brushing and irrigation group from baseline to 6months, but a signicant difference in bleeding on prob-ing and gingival index scores in favor of the irrigationgroup. Likewise, Flemmig et al. (1995) reported that thewater irrigation group was signicantly better at reducingbleeding on probing and gingival index scores comparedto the regular oral hygiene group at six months. Alsoin this study there were no statistically signicant dif-ferences detected in plaque scores among the groups.Chaves et al. (1994) found similar reductions in plaquescores for water irrigation compared to toothbrush-ing alone, and a signicant difference for bleeding onprobing in favor of the irrigation group at six months.


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These studies support the present data in nding nocorrelation between reduction of plaque biolm andinammation in 3-6 months.

Conclusion

There is a long-standing, well-documented body of

evidence supporting the use of an oral irrigator. An oralirrigator is at least as effective as dental oss for reducinggingival bleeding and gingivitis. When combined withmanual toothbrushing the use of an oral irrigator, eitherwith a prototype or standard jet tip, is signicantly moreeffective in reducing gingival bleeding scores as com-pared to the use of dental oss, as determined withinthe limits of this 4-week study design.

Acknowledgment

The study was performed in commission of ACTA

Research BV.Waterpik Inc, Fort Collins, CO, USA initiated thestudy project and provided study products. ACTAResearch BV received nancial support for their com-mitment to appoint this project to the Department ofPeriodontology of ACTA.

D.M. Lyle is the director of professional and clini-cal affairs for Water Pik, Inc. The authors employed byACTA declare that they have no conict of interest.

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Correspondence to: Prof. Dr. Nicola U. Zitzmann, Depart-ment of Periodontology, Endodontology and Cariology,School of Dentistry, University Basel, Hebelstrasse 3, CH-4056 Basel, Switzerland. Email: [email protected]

Additive or Synergistic Antimicrobial Effectsof Amoxicillin and Metronidazole on WholePlaque Samples: A Preliminary ReportClemens Walter1,2, Eva M. Kulik3, Roland Weiger1, Nicola U.Zitzmann1and Tuomas Waltimo3

1Department of Periodontology, Endodontology and Cariology,School of Dentistry, University Basel, Switzerland; 2Department ofOral Surgery, School of Dentistry, University of Birmingham, UnitedKingdom; 3Institute for Preventive Dentistry and Oral Microbiology,School of Dentistry, University Basel, Switzerland

Abstract

Objective:In vitro

data on the susceptibility of oral bacteria to the combination of met-ronidazole and amoxicillin is limited. The aim of this preliminary study was to determinethe susceptibility of whole subgingival plaque samples to amoxicillin and metronidazoleand to their combination. Methods: Prior to any treatment procedures subgingivalplaque samples from patients with severe generalized periodontitis were taken. Appro-priate dilutions were plated on Columbia blood agar supplemented with the followingagents: 3 g/mL amoxicillin, 8 g/mL amoxicillin, 8 g/mL metronidazole, 16 g/mLmetronidazole, 3 g/mL amoxicillin plus 8 g/mL metronidazole or 8 g/mL amoxicil-lin plus 16 g/mL metronidazole. All plates were incubated anaerobically at 36 C for14 days and the colony forming units (CFU) were determined. Results:Both appliedmetronidazole concentrations were able to decrease the CFU counts by approximatelyone order of magnitude in a log10 scale. Amoxicillin 3 g/mL revealed a reduction of2.4 log10 CFU, whereas 50% of the samples did not grow on the plates supplementedwith 8 g/mL of amoxicillin. There was no anaerobic bacterial growth on agar plates

supplemented with the combination of amoxicillin and metronidazole even at the lowerantibiotic concentrations. Conclusion:Susceptibility screening of subgingival samples tometronidazole and amoxicillin and to their combination seems to offer a rational basisfor the selection of adjunctive antibiotic therapy

Key words: Antibiotics, synergistic effect, aggressive periodontitis, metronidazole,

amoxicillin

Introduction

Periodontal diseases are multifactorial biolm-associatedinfections. A distinct differentiation between aggressive

and chronic forms is difcult

(Meyer et al., 2004), evenon the basis of microbiological ndings (Mombelli et al.,2002; Ximenez-Fyvie et al., 2006; Schacher et al., 2007).Hence, the diagnosis of aggressive periodontitis isprimarily based on clinical and radiological character-istics, on patients age, and on ndings derived duringclinical follow-up. Due to the infection-induced natureof periodontal diseases, antimicrobial therapies basedon microbiological examinations may improve the treat-

ment outcome of advanced and/or aggressive formsof periodontitis.

First attempts to control periodontal diseases withthe adjunctive use of antibiotics included systemic ad-

ministration of tetracyclines, amoxicillin with or withoutclavulanic acid, clindamycin and metronidazole (Listgar-ten et al., 1978; Lekovic et al., 1983; Gordon et al., 1985;Magnusson et al.,1989). Another adjunctive treatmentapproach was topical administration of various antibiot-ics or antiseptics (Lindhe et al., 1979; Needleman andWatts, 1989; Stabholz et al., 2000). Two decades ago,the combination of metronidazole and amoxicillin - socalled van Winkelhoff-Cocktail - was introduced asan adjunctive systemic therapy for periodontitis treat-ment (Van Winkelhoff et al., 1989). This regimen was

specically designed for treatment of diseases associatedwithAggregatibacter (Actinobaccillus) actinomycetemcomitans,for which a synergistic in vitro effect between the twosubstances or their metabolites has been reported


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(Pavicic et al., 1994a; Pavicic et al., 1994b).Clinical stud-ies on aggressive forms of periodontitis have revealedimproved outcomes within observation periods up tove years, provided that the adjunctive treatment withthe combination of amoxicillin and metronidazole wasstrictly combined with mechanical biolm removal(Buchmann et al., 2002; Guerrero et al., 2005; Kaner et al.,

2007a; Kaner et al., 2007b). Moreover, improved clinicaladvantages of this regimen were found in a placebo-controlled study comparing the antibiotic combinationto the agents alone, again as adjunctive to mechanical,non-surgical periodontal treatment (Rooney et al., 2002).

In this report, the treatment outcomes of subjects withadvanced chronic periodontal disease were independentfrom the initial microbiological ndings. Recently, thisstrategy of combining amoxicillin and metronidazolewas used for the treatment of generalized aggressiveperiodontitis without targeting against specic micro-

organisms (Guerrero et al., 2005).Whenever antibiotics are administrated as an adjunc-

tive periodontal treatment, existing or possibly develop-ing resistance of the associated microora should becarefully considered. In vitrondings have suggested thatthere are remarkable differences in resistance proles ofcertain oral bacterial species (Van Winkelhoff et al., 2005;Lakhssassi et al., 2005). Recent ndings in microbiologi-cal susceptibility testing have indicated the rationale ofthe examination of mixed microbial cultures instead of,or in addition to, the individual disease-associated strains

(Karbach et al., 2007). Such in vitrodata about bacterialsusceptibility to the combination of amoxicillin andmetronidazole is hitherto scarce.

The aim of the present preliminary study was todetermine the susceptibility of whole subgingival plaquesamples to amoxicillin and metronidazole and to theircombination.

Materials and methods

Patients and sampling

Four generally healthy patients with severe generalized

chronic or aggressive periodontitis were recruited fromthe pool of patients from the Department of Period-ontology, Endodontology and Cariology at the Schoolof Dental Medicine, University of Basel, Switzerland.Diagnosis was based on clinical and radiographic nd-ings, related to age and the severity of destruction (Table1, Figure 1a-b,Armitage, 1999). Clinical measurementsof probing pocket depth and attachment level wereperformed with the probe PCPUNC-15 (Hu-Friedy,Chicago, IL, USA). All recruited patients (one femaleand three males with a mean age of 40.8 years) were cur-

rent or former heavy smokers and had neither receivedany earlier periodontal treatment nor systemic or topicalantibiotics one year prior to the sampling. The femalepatient was not pregnant.

Subgingival plaque samples were taken for antibioticresistance analysis. At least the two deepest periodontalpockets with bleeding on probing were selected formicrobiological sampling. Supragingival plaque wasremoved, the sampling site was isolated using cottonrolls and gently dried with air. A sterile paper point wasinserted to the bottom of the pocket, left in place for

20 s and placed in 0.5 ml of thioglycolate broth (bi-oMrieux, Genf, Switzerland; Casas et al., 2007).

Microbiological procedures

Immediately after sampling, pooled paper points werevortexed for one minute and serially diluted in thioglyco-late broth. For the determination of the total anaerobicbacterial count, 100 mL of the dilutions were plated onColumbia blood agar plates (Columbia Agar Base [BBLBecton Dickinson, Allschwil, Switzerland] enrichedwith 4 mg/L hemin, 1 mg/L menadione, and 50 ml/L

human blood).For quantication of the proportion of microorgan-

isms resistant to either amoxicillin and/or metronida-zole, Columbia blood agar plates supplemented with thefollowing concentrations of the respective antimicrobialagent were used: 3 mg/mL amoxicillin (Fluka, Buchs,Switzerland), 8 mg/mL amoxicillin, 8 mg/mL metro-nidazole (Fluka), 16 mg/mL metronidazole, 3 mg/mLamoxicillin plus 8 mg/mL metronidazole or 8 mg/mLamoxicillin plus 16 mg/mL metronidazole. The con-centrations of the antibiotics were adopted from van

Winkelhoff et al.(2000) and/or the Clinical Laboratoryand Standards Institute (2007). All plates were incubatedanaerobically (10% CO

2, 10% H

2, 80% N

2) at 36C

for 14 days and the colony forming units (CFUs) weredetermined.

Results

Microbial ndings

Microbiological data are presented in Table 2. Thetotal anaerobic plaque count (CFU) ranged from 3.1x 106to 7.2 x 107among the plaque samples, and the

percentage of black-pigmented bacteria ranged from40 to 80%. All samples showed a decrease of bacte-rial growth on agar by approximately 1 log with bothconcentrations of the antibiotic agent (8 mg/mL and16 mg/mL). All agar plates supplemented with 3 mg/mL amoxicillin showed a reduced bacterial growth bylog 2.4, whereas two out of four samples revealed nogrowth on the plates supplemented with 8 mg/mL ofamoxicillin (Table 2). On agar plates supplemented withthe combination of amoxicillin and metronidazole, noanaerobic bacterial growth was detected even at lower

antibiotic concentrations.


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Additive and/or synergistic in vitroeffect of antibiotics 13

Figure 1. Patient N 2 was diagnosed with generalized aggressive periodontitis due to extensive bone loss at theage of 32 years. a) Clinical intraoral photographs; b) Full-mouth periapical radiographs

Figure 1a.

Figure 1b.

Table 1. Prole of study patients and clinical characteristics.

BOP, bleeding on probing; GAgP, generalized aggressive periodontitis; GChP, generalized chronic periodontitis

Patient Age Periodontaldiagnosis

Smoking status Number ofteeth

Number of sites with PPD 6 mm

Number ofsites with BOP+

1 45 GAgP Former smoker30 pack years

29 165 174

2 32 GAgP Current smoker15 pack years

29 92 133

3 38 GAgP Current smoker17 pack years

25 69 96

4 48 GChP Current smoker30 pack years

27 25 46

BPB, black-pigmented bacteria; CFU, colony forming units

Table 2.Microbiological characteristics and results of the antibiotic susceptibility analyses.

Bacterial growth onagar plates(control)

Bacterial growth on agar plates supplemented with different concentrations of metronidazoleor amoxicillin

Patient CFU % BPB CFUamoxicillin

3 g/mL

CFUamoxicillin

8 g/mL

CFUmetronida-

zole8 g/mL

CFUmetronida-

zole16 g/mL

CFUamoxicillin 3 g/mL

+metronidazole8 g/mL

CFUamoxicillin 8 g/mL

+ metronidazole16 g/mL

1 4.0 x 107 40 9.0 x 104 - 6.0 x 106 1.5 x 107 - -

2 1.0 x 107

50 8.0 x 103

1.0 x 104

6.0 x 105

6.0 x 105

- -3 3.1 x 106 40 8.4 x 104 - 6.0 x 105 1.3 x 106 - -4 7.2 x 107 80 4.0 x 105 3.0 x 105 1.3 x 106 1.5 x 106 - -


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Discussion

The present preliminary study using subgingival plaquesamples demonstrated reduced bacterial growth in thepresence of low concentrations of metronidazole oramoxicillin, while higher amoxicillin concentrationsinhibited bacterial growth in two out of four samples.

Interestingly, the combination of metronidazole andamoxicillin was effective against microorganisms in allsubgingival plaque samples at lower antibiotic concen-trations. This in vitroobservation suggests an additiveor synergistic mode of action for these agents, whichis likely to be benecial for infection control, as dem-onstrated by recent clinical studies (van Winkelhoff etal., 1989; Buchmann et al., 2002; Rooney et al., 2002;Guerrero et al., 2005; Kaner et al., 2007a; Kaner et al.,2007b).It may be hypothesized that the targeted use ofthis additive/synergistic effect, which is either based on

growth inhibition or on bacteriocidal effects, may offera strategy against the development and/or the controlof resistant strains.

The introduced method testing microbial suscep-tibility to a frequently administrated combination ofantibiotics is a novel approach, which enlightens thecapacity of additive and/or synergistic effects betweenthe two substances. A synergistic effect of two anti-biotics needs to be evaluated on a species level, andwas documented forAggregatibacter actinomycetemcomitans(Pavicic et al., 1994a; Pavicic et al., 1994b).The authorssuggested a higher rate of metronidazole uptake bybacterial cells simultaneously incubated with amoxicil-lin. Resistance of anaerobic bacteria to metronidazolehardly ever occurred (Seifert and Dalhoff, 2010). Inthe current material, bacterial growth was detected inall four subgingival plaque samples, which is indicativeof metronidazole-resistant strains and emphasizes theneed for susceptibility testing in selected patients withinfections involving anaerobic bacteria. The results ofthe current study should be, however, interpreted withcaution due to the limited number of subjects included,and the lack of specic bacterial strain characterisation.

However, the mixed subgingival plaque samples usedhere represented the expected general characteristicsin terms of relative proportions of back-pigmentedanaerobes in the total culturable ora.

This preliminary study was restricted to currentor former heavy smokers, who have an increased riskfor the onset and progression of periodontal diseases(Warnakulasuriya et al., 2010). Cigarette smoking is likelyto affect the composition of the oral microora due toa decrease in oxygen tension in periodontal pockets,and may promotes a selection of anaerobic bacteria

(Hanioka et al., 2000). However, the literature has beenindecisive as to whether a specic smoking-associatedmicrobial prole exists (van Winkelhoff et al., 2001; vander Velden et al.,2003). Interestingly, recent evidence

from a randomized controlled trial suggests a benetof adjunctive antimicrobial therapy with metronidazoleand amoxicillin in the non-surgical periodontal treat-ment of smokers with chronic periodontitis (Matarazzoet al., 2008).

The culture technique used in the current inves-tigation may have some shortcomings: (i) restricted

to growth of viable bacteria, (ii) strict sampling andtransport conditions essential, (iii) specic laboratoryequipment and experienced personnel required forbacterial culturing, (iv) time needed for bacterial growthon appropriate media, (v) specic pathogens in thesubgingival plaque may not be detected. However, themain advantage of the technique used is the probabilityof an analysis of bacterial resistance against the com-bination of antibiotics, in particular against amoxicillinand metronidazole. The diversity of the oral microora,reaching up to 700 different bacterial species (Kazor et

al., 2003), makes it impossible to analyze every singlebacterial strain regarding a genetic prole encoding forantibiotic resistance. In addition, the molecular mecha-nisms of bacterial resistance to antibiotics are quite farfrom being completely understood. Therefore, the an-tibiotic susceptibility of a subgingival plaque sample orof putative periodontal pathogens needs to be analyzedby conventional culture techniques (Armitage, 2003).

A major concern of the presented approach is thenatural biolm association of the subgingival bacterialsamples analysed. A biolm is a difcult therapeutic

target because of its three-dimensional structure, whichprotects the bacteria from the host response as well asfrom antimicrobial agents (Socransky and Haffajee,2002; Eick and Pster, 2004). The methodology ofthe present report allowed the interactions betweenculturable microorganisms, but no attempt was madeto mimic other characteristics of the subgingival plaque.Different results may be expected when a biolm ofmixed microbial samples is formed on an appropriatesubstrate prior to their susceptibility testing. However,such an approach is currently not available. The chosen

methodology aims to provide an approach for clinicallyrelevant susceptibility testing.According to the contemporary understanding of

the pathogenesis, periodontal diseases are caused byan opportunistic infection with a conglomerate ofpotentially periopathogenic microorganisms organizedin the subgingival biolm. A number of different testmethods and procedures are available for qualitativeand quantitative microbiological diagnostics of putativeperiopathogens. However, the pathogenic potential ofa certain putative periodontal pathogen against the hostcan hitherto not be determined. Moreover, major indi-vidual differences in the immune response are causedby a number of acquired or genetic factors. Althoughspecic bacteria have a periopathogenic potential or


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Additive and/or synergistic in vitroeffect of antibiotics 15

may initiate periodontal inammation, it is still difcultto determine the microbiota responsible for the onsetand progression of disease in the individual subject.Thus, in the diagnosis and therapy of periodontal dis-eases, microbiological identication and susceptibilitytesting of single disease-associated strains may be oflimited value(Mombelli et al., 2002; Sanz et al., 2004).

Instead or in addition to the conventional approach ofmicrobiological diagnostics, susceptibility testing of theentire subgingival plaque sample may offer additionalvaluable information for the choice of the antibiotic tobe administered adjunctively.

Improved therapy outcomes indicate that patientswith periodontal diseases - particularly those with highlydestructive forms (aggressive and/or advanced) - mayprot from an adjunctive antibiotic therapy using amoxi-cillin and metronidazole (Guerrero et al., 2005; Kaner etal., 2007a). However, due to the increased use of antibi-

otics and the alarming development of resistant strains,antibiotics should be administered with care, and test-ing the susceptibility of a given individuals microoramay have an increasing importance (Walker, 1996; VanWinkelhoff et al.2005; Lakhssassi et al., 2005; Walterand Weiger, 2006). Therefore, microbial testing can notbe recommended for routine dental practise. However,some patients, in particular those in need of adjunctiveantimicrobial therapy, may prot from the informationabout potential therapeutic targets (Armitage, 2003).Susceptibility testing of whole subgingival samples to

metronidazole and amoxicillin and to their combinationseems to offer a rational diagnostic tool to the selectionof adjunctive antibiotic therapy. In the event of an un-favorable response, i.e. bacterial growth on agar platessupplemented with amoxicillin and metronidazole,another antibiotic has to be tested and subsequentlyapplied for adjunctive antimicrobial therapy.

The current report about susceptibility analyses ofsubgingival plaque samples was initiated as a proof-of-principle study. The microbial results derived from theaudit of four cases may indicate a potential benet for

further analysis in a larger clinical microbiological trial.

Acknowledgements

We gratefully acknowledge the technical assistance ofMrs. Krystyna Lenkeit (Dental School, University Basel,Switzerland) and the constructive criticism of Prof. em.Jrg Meyer (Dental School, University Basel, Switzer-land). There is no conict of interest.

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Correspondence to: Carlos Serrano, MSc., Specialist in Peri-odontology, Department of Periodontal System, School ofDentistry, Ponticia Universidad Javeriana, Carrera 7 # 40-62,Edicio de Odontologa, Bogot, Colombia. E-mail: [email protected]

Clinical and Microbiological Comparison ofThree Non-surgical Protocols for the InitialTreatment of Chronic PeriodontitisCarlos Serrano, Nidia Torres, Angela Bejarano, Marcela Cavie-des and Mara Eugenia CastellanosSchool of Dentistry, Ponticia Universidad Javeriana, Bogot, Colombia

Abstract

Objective: To compare the clinical and microbiological effects of three protocols for non-surgical periodontal therapy, including full-mouth scaling and root planing plus systemicantibiotics, on the treatment of chronic periodontitis patients. Methods: Twenty-ninepatients diagnosed with moderate to severe chronic periodontitis, selected accordingto specic criteria, were randomly assigned to one of three treatment groups: quadrant

scaling, full-mouth scaling, and full-mouth scaling supplemented by systemic antibiot-ics. Antibiotic selection was based on the results of individual susceptibility testing.Oral hygiene instructions and reinforcement were given during the study. All patientsreceived a clinical periodontal and microbiological examination at baseline and at re-examination, 4-6 weeks after therapy. Means and standard deviations were calculatedand differences between groups were analyzed via the Kruskal-Wallis test, p< 0.05.Results: The mean age of the study sample was 49.1 11.6 years old, and there were17 men and 12 women. Patients treated with antibiotics showed antimicrobial suscep-tibility for amoxicillin and doxycycline. All study groups showed a similar signicantimprovement in periodontal parameters. Plaque scores were reduced in a range of29.0% to 42.6%. Bleeding on probing was reduced by 34.8% to 55.0%; the reductionfor the full-mouth scaling group was larger. Mean reduction in pocket depth was 1.2to 1.3 mm in all groups. Mean bacterial counts were reduced in the groups receivingfull-mouth treatment, but not in the quadrant treatment group. Conclusion: The three

protocols for non-surgical periodontal treatment demonstrated a similar positive effecton clinical parameters; however, only full-mouth treatment groups showed a reductionin anaerobic microbial counts at re-examination.

Key words: Chronic periodontitis, scaling and root planing, antibiotics, full-mouth scaling

Introduction

The main goal of periodontal therapy is to control theinfection associated with chronically inamed tissuesthrough a series of activities aimed at reducing thebacterial destructive effect, such as oral hygiene in-

struction, subgingival debridement and surgical pocketreduction. These protective measures, when reinforcedby meticulous self-performed oral hygiene and regularprofessional maintenance, lead to the re-establishmentof periodontal health (Tunkel et al.,2002; van der Wejdenand Timmerman, 2002). Initial therapy for disease in-cludes root surface instrumentation procedures, scalingand root planing, usually performed on jaw quadrants

during a series of appointments. Systematic reviews onnon-surgical periodontal therapy have considered scal-ing and root planing an effective treatment, measuredby clinical parameters such as reduction of bleedingupon probing, reduction of probing pocket depth and

gain in probing attachment level (van der Wejden andTimmerman, 2002; Hung and Douglass, 2002; Hallmonand Rees, 2003). Studies on the microbiological effectof scaling and root planing have shown that it producesa marked disruption of the subgingival biolm, leadingto decreased levels and proportion of sites colonized byperi

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