Farmakologi dan Terapi II

Aktivitas Antibakterial dari Moxifloxacin pada Bakteri

terkait Periodontitis dalam Biofilm

Oleh:

Senitza Anisa Salsabilla

NIM 021511133116

Departemen Biologi Oral

Fakultas Kedokteran Gigi Universitas Airlangga
Surabaya
2017

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 KATA PENGANTAR

Dengan menyebut nama Allah SWT yang Maha Pengasih lagi Maha Penyayang. Kami
panjatkan puja dan puji syukur atas kehadirat-Nya, yang telah melimpahkan rahmat dan
hidayah kepada kami, sehingga saya dapat menyelasaikan tugas mandiri farmakologi dan
terapi II dan semoga dapat memberikan manfaatnya bagi masyarakat.

Makalah ini disusun dengan mendapatkan bantuan dari berbagai pihak sehingga dapat
memperlancar pembuatan makalah ini. Untuk itu saya menyampaikan banyak terima
kasih kepada semua pihak yang telah berkontribusi dalam pembuatan makalah ini.

Terlepas dari semua itu, penulis menyadari sepenuhnya bahwa masih ada kekurangan
dalam makalah ini. Oleh karena itu penulis menerima segala saran dan kritik dari
pembaca agar kami dapat memperbaiki makalah ilmiah ini.

Akhir kata kami berharap semoga tugas mandiri tentang Aktivitas Antibakterial dari
Moxifloxacin pada Bakteri terkait Periodontitis dalam Biofilm dapat memberikan
manfaatnya kepada masyarakat maupun inpirasi terhadap pembaca.

Surabaya, Juni 2017

Penulis

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 DAFTAR ISI

Cover ................................................................................................................... i

Kata Pengantar .................................................................................................... ii

Daftar Isi ............................................................................................................ iii

Abstrak................................................................................................................ 1

BAB I : PENDAHULUAN ................................................................................ 3

BAB II : TINJAUAN PUSTAKA ...................................................................... 5

BAB III : METODE PENELITIAN ................................................................... 9

BAB IV : PEMBAHASAN .............................................................................. 12

BAB V : PENUTUP ......................................................................................... 21

DAFTAR PUSTAKA ....................................................................................... 22

iii
Abstract

The activity of moxifloxacin was compared with ofloxacin and doxycycline against
bacteria associated with periodontitis within a biofilm (single strain and mixed
population) in vitro. MICs and minimal bactericidal concentrations (MBCs) of
moxifloxacin, ofloxacin and doxycyline were determined against single strains and mixed
populations in a planktonic state. Single-species biofilms of two Porphyromonas
gingivalis and two Aggregatibacter actinomycetemcomitans strains and a multispecies
biofilm consisting of 12 species were formed for 3 days. The minimal biofilm eradication
concentrations (MBECs) were determined after exposing the biofilms to the antibacterials
(0.002512 mg ml1) for 18 h, addition of nutrient broth for 3 days and subsequent
subcultivation. Photographs were taken using confocal laser-scanning microscopy and
scanning electron microscopy. The MICs and MBCs did not differ between ofloxacin and
moxifloxacin against A. actinomycetemcomitans, whilst moxifloxacin was more active
than the other tested antibacterials against anaerobes and the mixed population. The
single-species biofilms were eradicated by moderate concentrations of the antibacterials,
and the lowest MBECs were always found for moxifloxacin (28 mg ml-1). MBECs
against the multispecies biofilms were 128, .512 and .512 mg ml-1 for moxifloxacin,
ofloxacin and doxycycline, respectively. In summary, moxifloxacin in a topical
formulation may have potential as an adjunct to mechanical removal of the biofilms.

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Abstrak

Aktivitas dari moxifloxacin dibandingkan dengan ofloxacin dan doxyxyxline melawan

bakteri yang berhubungan dengan periodontitis pada lingkungan biofilm in vitro (single
strain dan populasi campur). MIC dan konsentrasi minimal bakterisidal (MBCs) dari
moxifloxacin, ofloxacin, dan doxycycline telah ditentukan terhadap single strain dan
populasi campur dalam keadasan planktonic. Species tunggal biofilm dari dua rangkaian
Porphyromonas gingivalis dan dua rangkaian Aggregatibacter actinomycetemcomitans
dan biofilm multi spesies yang terdiri dari 12 spesies yang terbentuk dalam 3 hari.
Konsentrasi minimal pemberantasan biofilm (MBECs) telah terbentuk setelah
memaparkan biofilm ke antibakteri (0.002-512 mg mll) selama 18 jam, dengan
tambahan nutrisi kaldu selama 3 hari dan subkultivasi berikutnya. Fotografi diambil
menggunakan convocal laser-scanning mikroskop. Dan scanning electron mikroskop.
MICs dan MBCs tidak berbeda antara ofloxacin dan moxifloxacin terhadap A.
actinomycetemcomitans, sementara moxifloxacin lebih aktif dari pada antibacterial
lainnya yang telah dites dalam melawan anaerob dan populasi campur. Spesies tunggal
biofilm telah diberantas dengan konsentrasi sedang antibakteri, dan MBECs paling
rendah selalu ditemukan pada moxifloxacin (2-8 mg mll). MBECs terhadap biofilm
multispesies adalah 128, .512 dan .512 mg mll untuk moxifloxacin, ofloxacin dan
doxycycline, berturutan. Dapat disimpulkan, moxifloxacin pada formulasi topikal dapat
mempunyai potensi sebagai tambahan untuk pengangkatan biofilm secara mekanis.

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 BAB I
PENDAHULUAN

1.1 Latar Belakang

Menurut Pllnen et al., Periodontitis merupakan penyakit multifaktoral dan
berefek pada perubahan struktur gigi yang disebabkan oleh bakteri pada biofilm
gingiva. Pada umumnya, bakteri penyebab periodontitis adalah bakteri gram
negatif yang berkolonisasi pada plak sub gingival (Carranza, 2012). Bakteri
patogen utama yang menyebabkan periodontitis adalah sebagai berikut :
Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia,
Aggregatibacter actinomycetemcomitans dan juga Filifactor alocis,
Staphylococcus aureus dan Genus desulfobulbus (Pllnen et al., 2013). Pada
studi ini digunakan bakteri Porphyromonas gingivalis, Aggregatibacter
actinomycetemcomitans, dan 12 spesies campuran lainnya.
Terapi yang dilakukan untuk penyakit periodontal adalah terapi mekanis
dan penggunaan antimikroba atau antibakteri. Obat-obatan juga dapat membantu
perawatan periodontitis adalah antiinflamasi, antibiotik, analgetik, dan antipiretik
(Manson et al, 2013). Antibiotik mampu menembus jaringan periodontal dan
mencapai konsentrasi tertinggi pada cairan krevikular (Pejcic et al., 2013)
meskipun begitu tetap akan ada bakteri yang dapat bertahan hidup sehingga perlu
peningkatan alternatif lain untuk meningkatkan daya kerja antibiotik dalam
melawan bakteri periodontitis. Antibiotik yang digunakan sebagai perbandingan
dalam studi ini adalah; Moxifloxacin, Ofloxacin, dan Doxycycline.

1.2. Rumusan Masalah

1. Mengetahui aktivitas moxifloxacin, ofloxacin dan doxycycline terhadap

melawan bakteri periodontitis dalam biofilm

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1.3 Tujuan

Mengetahui aktivitas moxifloxacin dengan ofloxacin dan doxycycline

dalam melawan bakteri yang berhubungan dengan periodontitis dalam biofilm.

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 BAB II

TINJAUAN PUSTAKA

2.1. Periodontitis

2.1.1. Definisi

Penyakit periodontal yang menyerang struktur gigi yang lebih dalam

dibandingkan gingivitis yang hanya sebatas gingiva. Periodontitis juga inflamasi
jarringan periodontal yang ditandai adanya perpindahan epitel junctional ke arah
apikal, hilangnya perlekatan tulang, dan resorpsi tulang alveolar. (Charles, 2008)

2.1.2. Faktor Penyebab Periodontitis

2.1.2.1. Faktor Primer

Iritasi bakteri merupakan penyebab primer dari penyakit

periodontal. Menurut teori non-spesifik, bakteri mulut terkolonisasi pada
gingiva dan membentuk plak karena kebersihan mulut yang baik. Seluruh
bakteri memungkinkan mempunyai faktor virulensi penyebab inflamasi
gingival dan kerusakan periodontal. (Manson, 1993)

2.1.2.2. Faktor Sekunder

Faktor sekunder dapat dibagi menjadi dua faktor yaitu lokal dan
sistemik. Faktor lokal meliputi plak dan hal-hal yang mendukung
pertumbuhan plak, sedangkan faktor sistemik merupakan kondisi
kesehatan yang mempengaruhi tumbuhnya kelainan ataupun penyakit
daerah periodontal. (Manson, 1993)

2.2 Biofilm

Biofilm merupakan mikroorganisme yang melekat erat pada suatu permukaan.

Pada rongga mulut, biofilm sering disebut dengan plak, suatu lapisan lunak membentuk
biofilm yang melekat pada seluruh permukaan gigi dan keras termasuk restorasi tetap
ataupun sementara dalam rongga mulut. Akumulasi plak pada margin gingiva adalah
penyebab utama terjadinya penyakit periodontal. (Haake, et al 2002)

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2.2.1 Tahap Pembentukan Biofilm

1. Pembentukan pelikel pada permukaan gigi

Tahap pertama pembentukkan biofilm supragingiva adalah deposit

komponen saliva (acquired pellicle) pada permukaan gigi. Pelikel ini
mempermudah bakteri spesifik berkoloni dan mudah melekat. Kelenjar
saliva pun turut berkontribusi dalam pembuatan biofilm dengan menghasil
protein dan peptida. (Haake, et al 2002)

Pelikel mempunyai fungsi sebagai pertahanan yang memberikan

lubrikasi pada permukaan serta mencegah pengeringan jaringan.Namun,
pelikel juga menyediakan permukaan substrat yang menyebabkan
perlekatan bakteri pada permukaan terluarnya. Oleh karena itu, akumulasi
populasi bakteri semakin bertambah dan seterusnya terbentuk plak pada
permukaan itu. Mekanisme perlekatan bakteri pada permukaan pelikel
adalah dengan tekanan elektrostatis, tekanan van der Waals dan tekanan
hidrofobik. (Marsh, et al. 2009)

2. Kolonisasi awal bakteri

Tahap kedua dari pembentukan plak adalah kolonisasi awal bakteri

pada permukaan gigi. Bakteri bertumbuh dan jumlahnya meningkat
dengan cepat dan akan dijumpai pada pelikel gigi.17Mula-mula, bakteri
aerob gram positif dari kelompok Streptococci, Lactobacilli dan
Actinomyces misalnya, Streptococcus sanguinis, Streptococcus mitis dan
Streptococcus mutans yang merupakan bakteri awal yang berkolonisasi
dan melekat pada permukaan gigi yang berlapisan pelikel.

Pelikel mempunyai dua permukaan adhesif yaitu permukaan yang

melekat pada permukaan gigi dan permukaan untuk menfasilitasi
perlekatan bakteri. Bakteri tersebut melekat ke pelikel dengan fimbriae.
Fimbriae adalah satu struktur protein fibrous yang ada pada permukaan sel
bakteri. Adhesi protein pada fimbriae dapat berikatan dengan proline-rich
protein yang ada dalam pelikel untuk menimbulkan perlekatan bakteri ke

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permukaan gigi pada lapisan pelikel. Selain itu, bakteri yang berkolonisasi
awal ini akan melekat pada bacterial extracellular slime dan polisakarida
serta dengan permukaan absorpsi tambahan dari protein saliva dan
glikoprotein. Oleh karena itu, bakteri gram positif dapat berinteraksi
dengan menggunakan glikoprotein saliva sebagai substrat dalam
perlekatan melalui aktivitas glikosidase. Massa plak akan menjadi matang
apabila kolonisasi bakteri semakin bertambah dan terus-menerus berikatan
dengan spesies bakteri yang lain (Marsh, et al. 2009)

3. Kolonisasi sekunder dan pematangan plak

Tahap terakhir dari pembentukan plak adalah kolonisasi sekunder

dan pematangan plak. Setelah 2-4 hari, terjadi kolonisasi sekunder oleh
bakteri gram negatif seperti Prevotella intermedia, Prevotella loescheii,
Capnocytophaga spp., Fusobacterium nucleatum, dan Porphyromonas
gingivalis yang berperan dalam perkembangan plak.

Bakteri-bakteri ini akan melekat pada plak bakteri yang telah

melekat lebih dahulu pada massa plak. Proses perlekatan yang
menimbulkan perlekatan bakteri pengkoloni sekunder ke bakteri
pengkoloni awal dinamakan sebagai proses koagregasi. Proses perlekatan
tersebut berupa interaksi stereokimia spesifik yang tinggi antara molekul
protein dengan karbohidrat pada permukaan sel bakteri, sedangkan untuk
interaksi spesifik yang rendah adalah dari interaksi hidrofobik,
elektrostatik dan gaya van der Waals. (Marsh, et al. 2009)

Pembentukan plak terjadi apabila interaksi koagregasi antara

bakteri koloni sekunder yaitu spesies gram-negatif dengan bakteri koloni
awal seperti spesies gram positf seperti koagregasi bakteri Fusobacterium
nucleatum dengan Streptococcus sanguis, Provotella loescheii dengan
Actinomyces visvosus dan Capnocytophaga ochracea dengan
Actinomyces viscosus. Pematangan plak terjadi setelah hari ke-4. Bakteri
yang dominan adalah Spiral filamentus dan Spirochete sp.17Biofilm yang

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 matang menyebabkan bakteri tersebut tertumpuk dalam sulkus periodontal
pada waktu jangka panjang. Jadi biofilm yang sudah terbentuk dan tidak
disingkirkan akan menyebabkan inflamasi pada daerah margin gingiva.
Keadaan inflamasi dapat bertambah berat sehingga mengakibatkan
kedalaman sulkus gingiva bertambah dan menyebabkan biofilm meluas ke
daerah subgingiva. Plak subgingiva yang didominasi bakteri anaerob gram
negatif akan terbentuk yang merupakan kondisi optimum untuk bakteri,
misalnya Tanerella forsythis, Porphyromonas gingivalis dan Treponema
denticola yang sangat berperan dalam periodontitis. (Bansal, et al. 2012)

2.3 Obat Antibiotik

2.3.1. Pengertian Antibiotik

Antibiotika adalah zat kimia yang dihasilkan oleh fungi dan bakteri yang
berfungsi untuk menghambat ataupun mematikan pertumbuhan kuman, dan
mempunyai kadar toksisitas rendah pada manusia. (Tjay et al, 2007)

Antibiotik adalah zat biokimia yang diproduksi oleh mikroorganisme,

dalam jumlah tertentu dapat menghambat dan membunuh pertumbuhan
mikroorganisme lainnya. (Harmita, 2008)

Penggolongan antibiotika diklasifikasikan berdasarkan struktur kimia

antibiotik; Golongan Beta-Laktam, golongan aminoglikosida, golongan
tetrasiklin, golongan makrolida, golongan linkomisin, golongan kuinolon,
golongan kloramfenikol. Antibiotik juga dapat dibedakan berdasarkan toksistas
selektif, terdapat antibiotik bersifat bakteriostatik dan bakterisid.

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 BAB III

METODE PENELITIAN

Kemoterapi Antibakteria

Moxifloxacin (Bayer Innovation GmbH), Ofloxacin (Sigma-Aldrich) dan

Doxycycline (Bayer Innovation) telah diujicobakan. Larutan konsentrat (stock) 1024 g
ml-1 dalam Dh2O telah disiapkan dan diencerkan dua kali dengan A stock solution of
1024 mg ml 21 in Dh2O turun hingga konsentrasi 0,004 g ml-1. Pengenceran ini selalu
ditambahkan ke kaldu nutrisi dengan rasio 1:1. Uji final dari konsentrasi berkisar dari
512 g ml-1 hingga 0.002 g ml-1 . Dh2O dijadikan sebagai control 9uspense.

Mikroorganisme

Pada eksperimen biofilm, terpilih dua rantai Aggregatibacter

actinomycetemcomitans (Y4 dan isolasi klinis J7), termasuk dua rantai Porphyromonas
gingivalis (ATCC 33277 dan isolasi klinis M5-1-2). Bahan tambahan, Populasi campur
yang terdiri dari 12 spesies; Streptococcus gordonii ATCC 10558, Actinomyces
naeslundii ATCC 12104, Fusobacterium nucleatum ATCC 25586, Campylobacter rectus
ATCC 33238, Eubacterium nodatum ATCC 33099, Eikenella corrodens ATCC 23834,
Prevotella intermedia ATCC 25611, Parvimonas micra ATCC 33270, Porphyromonas
gingivalis ATCC 33277, Tannerella forsythia ATCC 43037, Treponema denticola ATCC
35405, Aggregatibacter actinomycetemcomitans Y4 turut digunakan dalam eksperimen.

Seluruh rantai telah dipre-kultivasi selama 24-72 jam sebelum eksperimen.

Menggunakan media kaldu mycoplasma (BD) dengan tambahan 1 mg glukosa ml -1, 400
g niacinaide ml-1, 150 g spermine tetrahydrochloride ml-1, and 20 g sodium
isobutyrate ml-1 diperkaya dengan 1 mg cysteine ml-1 dan 5 g cocarboxylase (Sigma-
Aldrich) ml-1. Kultivasi selalu dilakukan pada 37C. Kandungan 5% CO2 pada atmosfer
untuk rantai Aggregatibacter actinomycetemcomitans dan S. gordonii ATCC 10558.
Rantai lainnya diinkubasi secara anaerob.

Dari seluruh kultur, telah disiapkan 9uspense 9uspense9 menggunakan standar

McFarland 0,5 (~ 1.56108 mikroorganisme). Suspensi bakteri (1 ml) telah ditambahkan

9
dengan 15 ml kaldu nutrisi. Untuk populasi campur, 25 g 10uspense S. Gordonii ATCC
10558, 50 g 10uspense Actinomyces naeslundii ATCC 12104, dan 100 g suspense dari
rantai lainnya telah dicampur dan dipipet ke dalam 15 ml kaldu nutrisi.

Penentuan konsentrasi inhibisi minimal (MICs) dan konsentrasi bakterisida

minimal (MBCs) bakteri plankton

Menggunakan plat mikrotitre dan 100 l dilusi antibakteri pun juga ditambahkan.
Setelah itu 100 l kaldu yang mengandung bakteria ditambahkan. Kaldu tersebut
mengandung konsentrasi ganda kaldu Wilkins Chalgren (Oxoid) yang disuplementasi
dengan 5% darah domba (dan 5 g cocarboxylase ml-1 untuk kultur campur).

Mikrotitre diinkubasi dengan atmosfer yang sesuai selama semalam (18 jam). Plat
kemudian kekeruhan diperiksa dan setiap 1 l media dikultivasi dalam agar tryptic soy
yang telah dimodifikasi selama 3 hari. MIC ditentukan sebagai konsentrasi terendah tanpa
kekeruhan yang terlihat dari kaldu yang telah dikultivasi pada plat agar. Penilaian MICs
dan MBCs dari antibakterial kemoterapi terhadap Treponema denticola, antibakteria
dimasukkan pada media kultivasi. MBCs terdapat paling konsentrasi paling rendah tanpa
adanya pertumbuhan dari subkultivasi pada plat agar. (setara dengan 99.9% reduksi dari
inoculum inisial).

Penentuan konsentrasi minimal untuk pemberantasan biofilm (MBECs)

MBECs dievaluasi pada 2 rantai Aggregatibacter actinomycetemcomitans,

Porphyromonas gingivalis dan populasi 12 spesies campur. Pertama kali ditemukannya
sumur kecil pada permukaan dasar yang datar dari mikrotitre adalah dengan
menggunakan 10 l dari 25% (v/v) serum manusia inaktif (Sigma-Aldrich) per sumur
dalam 1 jam. Kemudian, 200 l suspense bakteri ditambahkan. Media yang digunakan
adalah kaldu brain-heart infusion (Oxoid) dengan 5% (v/v) darah (dan 5 g
cocarboxylase ml-1 untuk populasi campur). Plat mikrotiter diinkubasi dengan atmosfer
yang diperlukan (5% CO2 untuk Aggregatibacter actinomycetemcomitans spesies tunggal
biofilm, dan anaerob untuk lainnya).

Setelah 48 jam, media ditukar, ditambahkan Porphyromonas gingivalis ATCC

33277, Tannerella forsythia ATCC 43037 dan Treponema denticola ATCC 35405 pada

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media nutrisi sebelum diaplikasikan ke dalam sumur. Kemudian, diinkubasi selama 24
jam (hari 3), media dipindahkan, biofilm dibersihkan perlahan dan 100 l cairan
antibakterial yang dicampurkan dengan 100 l kaldu Wilkins Chalgren konsentrasi ganda
yang ditambahkan suplemen 5% darah domba (dan 5 g cocarboxylase ml-1 untuk
populasi campur). Plat mikrotiter telah diinkubasi selama 18 jam dengan atmosfer yang
ditentukan. Hari berikutnya (hari 4), media dipindahkan, biofilm dibersihkan perlahan
dan menambahkan kaldu nutrisi yang tidak menggunakan antibakterial baru (Kaldu
brain-heart infusion dengan 5% darah dan 5 g cocarboxylase ml-1) selama 72 jam. Pada
hari terakhir (hari 7), setelah memindahkan media dan membersihkannya, biofilm dikikis
dan dicampur menggunakan pipet, dan 10 l telah disubkultivasi dalam agar tryptic soy
yang telah dimodifikasi selama 3 hari. Nilai MBEC yang terlihat adalah konsentrasi
paling rendah tanpa ada ada pertumbuhan setelah subkultivasi.

Pada awal eksperimen, bakteri dapat dihitung dalam biofilm control (tanpa
tambahan antibakteri) kemudian dievaluasi oleh daftar c.f.u. untuk spesies yang termasuk
pada hari ketiga (sebelum ditambahkan antibakterial dalam uji biofilm), hari keempat
(pada saat pembersihan dari antibakteri dalam uji biofilm) dan pada hari ketujuh (hari
terakhir eksperimen). Untuk mengonfirmasi daftar c.f.u. dalam menghitung jumlah
sebagian besar spesies Treponema denticola.

Confocal laser-scanning microscopy (CLSM) dan scanning electron microscopy

(SEM)

Hasil visualisasi biofilm multispesies diambil menggunakan fotografi CLSM dan

SEM. Menurut deskripsi pabrik, fotografi menggunakan CLSM disiapkan menggunakan
pewarnaan hidup/mati (Live/dead BacLight Bacterial Viability kit; Invitrogen). Sampel
diujikan dengan Zeiss LSM150 Exiter confocal microscope (Carl Zeiss NTS). Untuk
fotografi SEM, sampel difiksasi dengan 2% glutaraldehyde dalam buffer cacodylate
selama 30 menit, di cuci dua kali dengan buffer cacodylate, dan dihidrasi menggunakan
graded ethanol series (10 menit untuk setiap konsentrasi). Setelah mongering, sampel
dilapisi dengan emas dan diperiksa dengan ZEISS LEO-1530 Gemini scanning electron
microscope (Carl Zeiss NTS) dilengkapi dengan field emission electron gun pada 10KeV.

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 BAB IV

HASIL

Rantai Aggregatibacter actinomycetecomitans termasuk rantai yang rentan

terhadap florokuinolon, sedangkan pada spesies lainnya dihambat oleh konsentrasi
rendah moxifloxacin daripada ofloxacin. MICs dari moxifloxacin berkisara dari 0.032
hingga 2 g ml-1, sementara ofloxacin dan doxycycline masing-masing berkisar antara
0.032 hingga 16 dan 0.125 hingga 128 g ml-1. Pada umumnya, MBCs moxifloxacin
adalah satu hingga enam titrasi lebih tinggi. Pada ofloxacin, satu hingga delapan titrasi
lebih tinggi, dan pada doxycycline satu hingga lima titrasi lebih tinggi.

Pada umumnya MBCs satu tingkat lebih tinggi daripada MICs dengan
pengecualian terhadap doxycycline, dimana MBCs sama dengan MICs. MICs dan MBCs
dari doxycycline lebih tinggi daripada rantai ATCC dengan perbandingan pada isolasi
klinis. Moxifloxacin lebih aktif terhadap rantai Porphyromonas gingivalis jika
dibandingkan dengan ofloxacin. Seluruh MBCs lebih tinggi daripada MICs.

Dalam melawan campuran multispesies, MIC dari moxifloxacin sama tingginya

dengan yang tertinggi dalam melawan rantai tunggal yang terdapat dalam campuran, i.e
2 g ml-1. MIC dari ofloxacin (4 g ml-1) dan doxycycline (4 g ml-1) lebih rendah
daripada MIC tertinggi terhadap rantai tunggal. Pada kasus ofloxacin, ditentukan MIC
lebih tinggi terhadap dua rantai untuk perbandingan dengan campuran. Seluruh MBCs
beberapa tingkat lebih tinggi daripada MICs. Doxycycline tidak mampu mengeliminasi
semua bakteri hingga konsentrasi 512 4 g ml-1.

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Tabel 4.1 MBCs dan MICs dari ketiga antibiotik dalam melawan rantai bakterial
monokultur terpilih yang berhubungan dengan periodontitis dan populasi
campuran yang berisi 12 spesies tingkat plantonik.

Pada awal eksperimen, Evaluasi mode pertumbuhan biofilm dilakukan secara

khusus hingga bakteri yang tumbuh pada biofilm bersentuhan dengan bahan antibakteri
selama 19 jam pada hari ketiga dan jumlahnya dapat dihitung pada hari terakhir.
Perhitung c.f.u. pada biofilm spesies tunggal dalam kisaran 106-107 per sumur setiap poin
waktu. Jumlah bakteri dalam populasi campur adalah 7.13 0.23 log10 c.f.u. per sumur
pada hari ketiga, 7.13 0.15 log10 pada hari keempat, dan 7.07 0.03 log10 c.f.u. pada
hari ketujuh (hari terakhir). Bakteri terhitung pada 4log10 per sumur pada Eubacterium
nodatum, Prevotella intermedia dan Eikenella corrodens, dan spesies lain berada diantar
5 hingga 6log10 c.f.u per sumur. Jumlah layak tidak berbeda jauh diantara waktu
berselang.

13
Gambar 4.1 Hitungan bakteri per sumur pada biofilm spesies tunggal (a) dan pada
biofilm multispesies (b) tanpa paparan dari antibakteri pada hari ketiga (sebelum
ditambahkan antibiotic), hari keempat (pada saat pembersihan antibakteri) dan
hari ketujuh (hari terakhir percobaan). A actinom., Aggregatibacter
actinomycetemcomitans.

Konsentrasi minimal pemberantasan biofilm (MBEC) ditentukan pada dua

eksperimen independen dengan dua variasi langkah titrasi. Seperti yang dieharapkan,
MBECs dari bakteri yang tumbuh dalam biofilm mencapai tujuh kali pengenceran lebih
tinggi daripada masing-masing MBCs untuk bakteri plantonik.

Golongan quinolone memberantas Aggregatibacter actinomycetemcomitans

dalam biofilm pada konsentrasi medium. Moxifloxacin lebih aktif daripada ofloxacin
terhadap Aggregatibacter actinomycetemcomitans pada biofilm. MBEC doxycycline

14
lebih tinggi daripada golongan quinolone, sesuai dengan MBCs lebih tinggi terhadap
bakteri plantonik.

MBECs terhadap biofilm Porphyromonas gingivalis lebih tinggi dibandingkan

masing-masing MBCs terhadap bakteri plantonik. Perbedaan antara MBEC 50 dan MBCs
pada doxycycline adalah tiga hingga empat kali tahap pengenceran dan dua kali
pengenceran untuk golongan quinolone. Terkait dengan rendahnya MBCs, moxifloxacin
adalah zat paling aktif dari antibakteri yang telah diujikan terhadap biofilm
Porphyromonas gingivalis.

MBECs dalam melawan biofilm multispesies memiliki efek sangat tinggi. Hanya
moxifloxacin dalam konsentrasi tinggi ( 128 g ml-1) yang mampu memberantas bakteri
dalam biofilm.

Analisa CLSM dan SEM

Pewarnaan hidup/mati menunjukan prosentase tinggi pada bakteri layak. Tidak

terdapat bakteri mati yang terlihat pada control dan paparan berikutnya pada antibakteri
4 g ml-1. Tidak terdapat bakteri mati yang dapat dilihat setelah terpapar ofloxacin pada
konsentrasi diatas 256 g ml-1. Doxycycline dan moxifloxacin menunjukan aktivitas
consentration-dependent.

Fotografi SEM dengan jelas menggarisbawahi fakta bahwa antibakteri tidak dapat
menghancurkan biofilm multispesies, meskipun lebih banyak matriks ekstraseluler yang
terlihat pada biofilm yang tidak diberi perlakuan.

15
 BAB V

PEMBAHASAN

Hasil dari golongan quinolone dibandingkan lebih jauh dengan doxycycline

sebagai well-established agen pada antibakterial topical untuk pengobatan periodontitis.
Aggregatibacter actinomycetemcomitans memiliki kepekaan yang sama terhadap
moxifloxacin dan ofloxacin. MICs dan MBCs moxifloxacin terhadap bakteri anaerob
terlihat lebih rendah daripada ofloxacin. Dengan demikian, aktivitas in vitro moxifloxacin
yang baik melawan oral anaerob dan Aggregatibacter actinomycetemcomitans dapat
terkonfirmasi. Populasi campuran menjadi perhatian pada kasus ini, MICs terdapat dalam
kisaran MICs individual. Belakangan ini, telah terlihat kombinasi dari dua rantai dapat
memungkinkan menjadi kurang atau lebih sensitif daripada MICs rantai tunggal. MBCs
dua rantai pun lebih tinggi daripada rantai tunggal. Golongan quinolone termasuk
bactericidal pada konsentrasi 256 g ml-1, dan doxycycline tidak dapat memberantas
bakteria pada konsentrasi hingga 512 g ml -1.

Bakteri pada rongga mulut sebagian besar membentuk biofilm. Plak subgingival
terdiri dari ratusan taxa yang berbeda, banyak agregrasi yang berbeda terjadi, komunikasi
bakteri dan transfer DNA. Salah satu fenomena umum dari biofilm adalah antibakteri
tidak seberapa aktif dalam melawan bakteri plantonik. Resistensi yang lebih tinggi
dikaitkan dengan pertumbuhan terbatas, sintesis protein, aktivitas metabolik, dan
peningkatan frekuensi mutasi serta matriks polimer di sekitar mikrokoloni.

Pada percobaan ini, mencoba untuk mengetahui situasi in vitro ketika

antibakterial diberikan secara topical pada pocket periodontal. Konsentrasi gingival
crevicular fluid menjadi lebih tinggi setelah aplikasi topical disbanding yang sistemik,
dan konsentrasi doxycycline menjadi lebih dari 1000 g ml-1 ketika diukur sehari setelah
pengaplikasian, dimana setelah aplikasi sistemik tingkat doxycycline tidak lebih dari 3
g ml-1. Gingival crevicular fluid dikenal dengan kemampuannya untuk high turnover.
Dengan rate flow 44 l h-1 dan resting volume 1.5 l pada periodontitis parah. Perantara
pengantar digunakan untuk menjaga tingkat kadar tinggi antibakteri pada situs
penempatan. Pada percobaan ini, antibakteri diaplikasikan pada konsentrasi untuk

16
diujikan selama 18 jam yang kemudian ditambahkan kaldu nutrisi untuk meniru high
turnover dari gingival crevicular fluid. Tindakan ini pun juga memungkinkan replikasi
dari bakteri yang tidak terbunuh.

Tabel 4.2 MBEC50 dan jarak MBECs dari tiga antibakteri dalam melawan
biofilm spesies tunggal dari Aggregatibacter actinomycetemcomitans dan
Porphyromonas gingivalis dan biofilm multispesies yang terdiri dari 12 spesies

Pada biofilm spesies tunggal, MBECs beberapa langkah lebih tinggi daripada
MBCs yang sesuai pada bakteri plantonik. Pada model biofilm Aggregatibacter
actinomycetemcomitans dibandingkan dengan antibiotic beta-laktam, tetrasiklin,
erithromisin, levofloxacin dan ofloxacin, hanya ofloxacin yang memberikan aktivitas
penghambat pada biofilm dewasa. Moxifloxacin terlihat dapat mereduksi biomass dari
biofilm Staphylococcus aureus. Ia menginduksi gangguan lendir pada biofilm yang
diproduksi oleh salah satu spesimen pernafasan. Pada biofilm spesies tunggal dari
periodontopatogen, moxifloxacin tidak terlihat dapat menghancurkan struktur dari
biofilm multispesies meskipun pada fotograf SEM terlihat kerusakan dari sel bakteri dan
matriks ekstraselullar yang berkurang.

Hanya moxifloxacin yang dapat membunuh seluruh bakteri pada konsentrasi 128
g ml-1 dalam biofilm multispesies. Sementara, ofloxacin dan doxycycline terlihat tidak
mungkin untuk mengeliminasi seluruh bakteri meski pada konsentrasi lebih dari 512 g
ml-1.

17
Gambar 4.2. fotografi CLSM pada efek tiga antibakteri pada konsentrasi berbeda
terhadap biofilm 12 spesies; (a) 4 g ml-1 ;(b) 32 g ml-1; (c) 256 g ml-1. Pada
(a-c) dilihatkan bakteri dengan pewarnaan hidup/live (green) dan mati/dead (red);
pada (d), replica dari (c), hanya bakteri mati yang terwarna.

Fotograf CLSM menunjukan aktivitas ketergantungan konsentrasi pada

prosentase bakteri terbunuh yang dianalisa dengan pewarnaan hidup/mati. Namun, foto
tidak membuktikan hasil yang ditentukan dari kultivasi; prosentase dari jumlah bakteri
selalu tinggi. Metode pewarnaan hidup/mati (live/dead staining) (merah/hijau) digunakan
berdasarkan integritas membrane. SYTO 9 (green fluorescence) melabel bakteri dengan
membran utuh dan propidium iodide (red fluorescence) akan masuk melabel bakteri
dengan membran yang rusak. Bakteri yang telah ternoda dapat dibilang tidak dapat
dikultivasi lagi. Dimana, 100% kelayakan dari banyak biofilm berperan pada bagian hasil

18
false-positive pada bakteri vital. Dengan membandingkan perbedaan metode pewarnaan
untuk penentuan vitalitas bakteri streptococci plantonik, tingkat vitalitas tertinggi telah
ditemukan pada pewarnaan jenis ini (live/dead staining). Dengan tambahan komponen
biofilm dapat mencegah uptake dari propidium iodide pada bakteri mati.

Gambar 4.3. fotografi SEM pada efek antibakteri terhadap biofilm 12 spesies
tanpa antibiotic (a) dan setelah paparan 256 g ml-1 doxycycline (b), 256 g ml-1
ofloxacin (c), dan 256 g ml-1 moxifloxacin (d).

Biofilm spesies tunggal tidak terlalu resisten terhadi aktivitas antibakteri.

Aktivitas antibakteri diujikan terhadap biofilm 12 spesies. Pada studi ini, hanya mewakili
sebagian kecil dari kompleksitas biofilm. Demikian, ini dapat diajukan sebagai efisiensi
dari antibakteri in vivo lebih kurang daripada yang diperlihatkan pada in vitro model.

Moxifloxacin mempunyai potensi untuk menjadi obat topical untuk pengobatan

residual pocket pada periodontitis. Sebelumnya, telah dibuktikan bahwa moxifloxacin

19
aktif secara in vitro dalam melawan infeksi intraseluler. Terlebih lagi pada percobaan
klinis secara acak, moxifloxacin menjadi obat paling efektif sebagai tambahan dalam
tindakan scalling dan root planing, dengan beranggapan hasil klinis dan mikrobiologis,
dan parameter immunologi dibandingkan dengan penggunaan adjunctive doxycycline
sendiri. Terdapat Pengurangan kedalaman pocket tambahan, dengan aplikasi
moxifloxacin lokal sebagai tambahan untuk scalling dan root planning pada pasien
periodontitis kronis dengan pocket residu. Permasalahan umum yang berhubungan
dengan penggunaan antibakteri dikembangkan sebagai perlawanan. Secara in vitro, telah
dibuktikan bahwa Porphyromonas gingivalis dapat mengembangkan pertahanan;
padahal, berdasarkan studi klinis, pertahanan rantai tidak ditemukan. Namun,
pengaplikasian antibakteri seharusnya dibatasi untuk kasus tertentu. Pertahanan
antibakteri tergantung pada penggunaan umum dari obat kemoterapi antibakteri, dengan
contohnya, pertahanan periodontopatogen lebih prevalen di Spanyol, sama dengan
Negara-negara dengan tingkat konsumsi antibakteri tinggi dibandingkan Belanda, dimana
aplikasi antibakteri dibatasi.

Penggunaan antibakteri dapat berpengaruh terhadap resistensi ataupun pertahanan

bakteri tersebut. Karena semakin tinggi penggunaan, semakin besar kesempatan bakteri
agar dapat bertransformasi menjadi lebih resisten.

20
 BAB VI

PENUTUP

Kesimpulan

Moxifloxacin menjadi obat yang lebih aktif melawan bakteri anaerob

dibandingkan ofloxacin dan juga aktivitas yang dilakukan melawan bakteri dalam
biofilm. Diantara antibakteri yang diujikan, moxifloxacin menjadi satu yang dapat
memberantas bakteri dalam biofilm multispesies, meskipun konsentrasi yang diperlukan
tinggi. Dalam kombinasi dengan penghilangan mekanis biofilm, moxifloxacin dapat
menjadi obat kemoterapi antibakteri topikal yang baik untuk kasus periodontitis.

Saran

Diperlukan penelitian lebih lanjut terhadap penggunaan antibiotic golongan

kuinolon terhadap bakteri penyebab periodontitis pada biofilm.

21
 DAFTAR PUSTAKA

Bansal S, Rastogi S, Bajpai M, 2012, Mechanical, Chemical and herbal aspects of

periodontitis: A review. International Journal of Pharmaceutical Sciences and Research;
3(5):1261

Carranza, 2012, Clinical Periodontology 11th Edition, Singapore: ELSEIVIER

Charles M.Cobb, Charles M. (2008) Microbes, Inflammation, Scaling and Root
Planning, and the Periodontal Condition. Journal of dental hygiene: JDH/American
Dental Hygienists' Association 3: 4-9
Haake SK, Newman MG, etal. 2002. Periodontal Microbiology. In: Newman MG,
Takei HH, Carranza FA. Clinical Periodontology. Edisi 9. Massachusetts: WB Saunders
Company: 97-100,651.
Harmita dan Radji, M., 2008. Kepekaan Terhadap Antibiotik. Dalam: Buku Ajar
Analisis Hayati, Ed.3. EGC, Jakartar: 1-5

Manson, J. D., Eley, B. M., 2013, Buku Ajar Periodonti, Jakarta : EGC.
Marsh PD, Martin MV, Lewis MAO, Williams DW. Oral Microbiology. Edisi 5. China:
Elsevier, 2009: 78-84.

Pejcic, A., Kesic, L., Obradovic, R., Mirkovic, D., 2010, Antibiotics in The
Management of Periodontal Disease, Scientific Journal of Medicine in Nis, 27 (2): 85-
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Pllnen,M.T,. Paino,A., dan Ihalin,R., 2013. Review Environmental Stimuli Shape
Biofilm Formation and the Virulence of Periodontal Pathogens.Int.J.Mol.Sci., 14,
17221-17237

Tjay, T. H. dan Rahardja, K. (2008). Obat-Obat Penting, Khasiat, Penggunaan dan

Efek-Efek Sampingnya. Edisi Keenam. Jakarta : Penerbit PT. Elex Media Komputindo.
578 - 580.

22
Journal of Medical Microbiology (2014), 63, 284292 DOI 10.1099/jmm.0.065441-0

Antibacterial activity of moxifloxacin on bacteria

associated with periodontitis within a biofilm
Phoebus Tsaousoglou,1 Sandor Nietzsche,2 Georg Cachovan,3
Anton Sculean1 and Sigrun Eick1
Correspondence 1
School of Dental Medicine, Department of Periodontology, University of Bern, Bern, Switzerland
Sigrun Eick 2
Department of Electron Microscopy, University Hospital of Jena, Jena, Germany
sigrun.eick@zmk.unibe.ch
3
Department of Restorative and Preventive Dentistry, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany

The activity of moxifloxacin was compared with ofloxacin and doxycycline against bacteria
associated with periodontitis within a biofilm (single strain and mixed population) in vitro. MICs
and minimal bactericidal concentrations (MBCs) of moxifloxacin, ofloxacin and doxycyline were
determined against single strains and mixed populations in a planktonic state. Single-species
biofilms of two Porphyromonas gingivalis and two Aggregatibacter actinomycetemcomitans
strains and a multispecies biofilm consisting of 12 species were formed for 3 days. The minimal
biofilm eradication concentrations (MBECs) were determined after exposing the biofilms to the
antibacterials (0.002512 mg ml1) for 18 h, addition of nutrient broth for 3 days and subsequent
subcultivation. Photographs were taken using confocal laser-scanning microscopy and scanning
electron microscopy. The MICs and MBCs did not differ between ofloxacin and moxifloxacin
against A. actinomycetemcomitans, whilst moxifloxacin was more active than the other tested
antibacterials against anaerobes and the mixed population. The single-species biofilms were
eradicated by moderate concentrations of the antibacterials, and the lowest MBECs were always
found for moxifloxacin (28 mg ml1). MBECs against the multispecies biofilms were 128, .512
and .512 mg ml1 for moxifloxacin, ofloxacin and doxycycline, respectively. In summary,
moxifloxacin in a topical formulation may have potential as an adjunct to mechanical removal of the
Received 10 July 2013
biofilms.
Accepted 10 November 2013

INTRODUCTION proteolytic activity of Porphyromonas gingivalis, an asac-

Tooth loss is mostly a consequence of plaque-caused
diseases such as periodontitis and caries, followed by
endodontic infections. Oral microbial-plaque communities
are biofilms composed of numerous bacteria on host
surfaces. Streptococci and actinomycetes are the major
initial colonizers. Fusobacteria play a central role as bridges
that promote co-aggregation to anaerobic bacteria
(Kolenbrander, 2000). Periodontal disease status impacts
markedly on biofilm composition (Socransky & Haffajee,
2005). It is generally accepted that a small group of
predominantly Gram-negative anaerobic or microaerophi-
lic bacteria is associated with initiation and progression of
periodontitis. Organisms strongly implicated as aetiological
agents of periodontitis include Aggregatibacter actinomyce-
temcomitans, Porphyromonas gingivalis, Tannerella forsythia
and Treponema denticola (Anonymous, 1996). The high
Abbreviations: CLSM, confocal laser-scanning microscopy; MBC,
minimal bactericidal concentration; MBEC, minimal biofilm eradication
concentration; SEM, scanning electron microscopy.
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charolytic anaerobe, is mainly the result of gingipains
(arginine- and lysine-specific cysteine proteases), which are
considered to be one of the most important virulence factors
(Guo et al., 2010). Virulence of Aggregatibacter actinomyce-
temcomitans is associated with synthesis of many toxins such
as leukotoxin and cytotoxin (Henderson et al., 2003).
Controlling the total oral bacterial load and especially the
periodontopathogenic bacteria is an essential component
of any periodontitis treatment and is associated with an
improvement and a stability of a post-therapeutic out-
come. Plaque is removed mechanically by scaling and root
planing (Van der Weijden & Timmerman, 2002; Apatzidou
& Kinane, 2010). In particular cases, antibacterial che-
motherapeutics might be useful when applied systemically
(Herrera et al., 2008). Systematic reviews underline a
probable positive effect for metronidazole combined with
amoxicillin on clinical parameters in the treatment of
aggressive and chronic periodontitis (Sgolastra et al., 2011,
2012). Recently, azithromycin has been tested to be
effective in aggressive periodontitis patients (Haas et al.,
065441 G 2014 SGM Printed in Great Britain

2008), whereas the results were contradictory in chronic
periodontitis patients (Yashima et al., 2009; Han et al.,
2012). Tetracyclines are mostly used as topical antibacterial
chemotherapeutics (Bosco et al., 2009; Bland et al., 2010).
Quinolones were only rarely included in clinical trials of
periodontitis. Ciprofloxacin and ofloxacin were system-
ically applied to eradicate Aggregatibacter actinomycetem-
comitans (Muller et al., 1998; Kleinfelder et al., 2000).
Moreover, ofloxacin was tested in a topical formulation
(Yamagami et al., 1992). In vitro, moxifloxacin has a better
activity towards anaerobes (including Porphyromonas
gingivalis) than ofloxacin and ciprofloxacin (Behra-
Miellet et al., 2002). In vivo, it shows superiority over
doxycycline in systemic application (Guentsch et al., 2008)
and seems to be able to reduce pocket depths when applied
topically (Flemmig et al., 2011).
Our hypothesis was that moxifloxacin has a superior
activity against bacteria associated with periodontitis in
multispecies biofilm compared with doxycycline and
ofloxacin. Thus, the aim of this in vitro study was to
obtain more information about the antimicrobial activity
of moxifloxacin in comparison with other antibacterial
chemotherapeutics against selected bacteria in a planktonic
form and within biofilms. These experiments were
undertaken using monocultures and a mixed population
consisting of 12 different species normally present in a
periodontopathogenic biofilm to find differences among
planktonic bacteria and within biofilms as well as between
monocultures and mixed cultures.
METHODS
Antibacterial chemotherapeutics. Moxifloxacin (Bayer Innovation
GmbH), ofloxacin (Sigma-Aldrich) and doxycycline (Bayer Innovation)
were tested. A stock solution of 1024 mg ml21 in dH2O was prepared
and diluted twice with dH2O down to a concentration of 0.004 mg ml21.
These dilutions were always added in a ratio of 1 : 1 to the nutrient
broth. Thus, the final tested concentrations ranged from 512 mg ml21 to
0.002 mg ml21. dH2O served as the negative control.
Micro-organisms. For the biofilm experiments, two Aggregatibacter
actinomycetemcomitans strains (Y4 and clinical isolate J7) were
chosen. Two Porphyromonas gingivalis strains (ATCC 33277 and
clinical isolate M5-1-2) were also included. Additionally, a mixed
population consisting of 12 species (Streptococcus gordonii ATCC
10558, Actinomyces naeslundii ATCC 12104, Fusobacterium nucleatum
ATCC 25586, Campylobacter rectus ATCC 33238, Eubacterium
nodatum ATCC 33099, Eikenella corrodens ATCC 23834, Prevotella
intermedia ATCC 25611, Parvimonas micra ATCC 33270, Porphyromonas
gingivalis ATCC 33277, Tannerella forsythia ATCC 43037, Treponema
denticola ATCC 35405, Aggregatibacter actinomycetemcomitans Y4) was
used.
All the strains were pre-cultivated for 2472 h prior to the
experiments. Modified tryptic soy agar (Feres et al., 2002) was used
except for Treponema denticola ATCC 35405. Here, the medium was
mycoplasma broth (BD) with the addition of 1 mg glucose ml21,
400 mg niacinamide ml21, 150 mg spermine tetrahydrochloride ml21,
and 20 mg sodium isobutyrate ml21 and enriched by 1 mg cysteine
ml21 and 5 mg cocarboxylase (Sigma-Aldrich) ml21. Cultivation was
always carried out at 37 uC. The atmosphere was 5 % CO2 for the
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Moxifloxacin and biofilms
Aggregatibacter actinomycetemcomitans strains and for S. gordonii
ATCC 10558. The other strains were incubated anaerobically.
From all cultures, a microbial suspension of approximately
McFarland standard 0.5 (~1.56108 micro-organisms) was prepared.
Bacterial suspension (1 ml) was added to 15 ml nutrient broth. For
the mixed population, 25 ml of S. gordonii ATCC 10558 suspension,
50 ml of Actinomyces naeslundii ATCC 12104 suspension and 100 ml of
the suspensions of the other strains were mixed and pipetted into
15 ml nutrient broth.
Determination of MICs and minimal bactericidal concentra-
tions (MBCs) of planktonic bacteria. MICs and MBCs (against
planktonic bacteria) of the agents were determined for the
monocultures of all single strains and for the mixed population.
Microtitre plates were used and 100 ml of the antibacterial dilutions
was added per well. Thereafter, 100 ml of bacteria-containing broth
was added. The broth used was double-concentrated Wilkins
Chalgren broth (Oxoid) supplemented with 5 % sheep blood (and
5 mg cocarboxylase ml21 for the mixed culture). After careful mixing,
the microtitre plates were incubated in the appropriate atmosphere
overnight (18 h). The plates were then checked for turbidity and each
1 ml medium was subcultivated on modified tryptic soy agar for
3 days. The MIC was defined as the lowest concentration without
visible turbidity of the broth confirmed by subcultivation on agar
plates. For the assessment of MICs and MBCs of the antibacterial
chemotherapeutics against Treponema denticola, the antibacterials
were added to the cultivation medium. The MBC was the lowest
concentration without any growth of the subcultivations on the agar
plates (equivalent to a reduction of 99.9 % of the initial inoculum).
All experiments were carried out in independent duplicates.
Determination of minimal biofilm eradication concentrations
(MBECs). MBECs were evaluated for the two Aggregatibacter
actinomycetemcomitans and Porphyromonas gingivalis strains and the
12-species mixed population. The wells of flat-bottomed microtitre
plates were first covered with 10 ml of 25 % (v/v) inactivated human
serum (Sigma-Aldrich) per well for 1 h. Next, 200 ml of bacterial
suspension was added. The medium used was brainheart infusion
broth (Oxoid) with 5 % (v/v) blood (and 5 mg cocarboxylase ml21 for
the mixed population). The microtitre plates were incubated in the
appropriate atmosphere (5 % CO2 for the Aggregatibacter actinomyce-
temcomitans single-species biofilms, and anaerobic for the others).
After 48 h, the medium was carefully exchanged. In the case of the
mixed biofilm, Porphyromonas gingivalis ATCC 33277, Tannerella
forsythia ATCC 43037 and Treponema denticola ATCC 35405 were
again added to the nutrient medium before application to the wells.
Further addition of selected bacterial strains guaranteed a sufficient
number of these species within the biofilms. After an additional
incubation for 24 h (day 3), the medium was removed, the biofilms
were carefully washed and 100 ml of antibacterial dilution mixed with
100 ml of doubled-concentrated Wilkins Chalgren broth and supple-
mented with 5 % sheep blood (and 5 mg cocarboxylase ml21 for the
mixed population) was added. The microtitre plates were incubated in
the appropriate atmosphere overnight (18 h). The following day (day
4), the medium was removed, the biofilms were carefully washed and
fresh antibacterial-free nutrient broth (brainheart infusion broth with
5 % blood (and 5 mg cocarboxylase ml21) was added for 72 h. Finally
(day 7), after removing the medium and washing, the biofilm was
scraped and mixed by pipetting, and 10 ml was subcultivated on
modified tryptic soy agar for 3 days. The MBEC was the lowest
concentration without any growth after subcultivation. These experi-
ments were carried out in two independent experiments, each in
duplicate.
In preliminary experiments, the bacterial counts within the control
biofilms (without the addition of antibacterials) were evaluated by
285
P. Tsaousoglou and others
enumeration of c.f.u. for the included species at day 3 (time before
addition of antibacterials in test biofilms), day 4 (time after removal
of antibacterials in test biofilms) and day 7 (end of the experiments).
To confirm the c.f.u. counts of most of the species and to enumerate
Treponema denticola, real-time PCR was carried out using primers
and a protocol described previously (Ashimoto et al., 1996; Eick et al.,
2011).
Confocal laser-scanning microscopy (CLSM) and scanning
electron microscopy (SEM). CLSM and SEM photographs were
taken to visualize the multispecies biofilm results. Biofilms were
formed on glass slides in 24-well plates as described above. The
biofilms were exposed to 4, 32 and 256 mg antibacterial chemother-
apeutics ml21. dH2O was used as negative control. The medium was
changed 18 h later for an antibacterial-free medium and the plates
were incubated for a further 3 days. The photographs for CLSM were
prepared by using live/dead staining (Live/dead BacLight Bacterial
Viability kit; Invitrogen) according to the manufacturers description.
The samples were examined with a Zeiss LSM510 Exciter confocal
microscope (Carl Zeiss NTS). For SEM, samples were fixed in 2 %
glutaraldehyde in cacodylate buffer for 30 min, washed twice with
cacodylate buffer and dehydrated using a graded ethanol series
(10 min for each concentration). Following critical-point drying,
samples were sputter coated with gold and examined with a ZEISS
LEO-1530 Gemini scanning electron microscope (Carl Zeiss NTS)
equipped with a field emission electron gun at 10 keV.
RESULTS
MICs and MBCs against planktonic bacteria
In monocultures, the Aggregatibacter actinomycetemcomi-
tans strains were equally susceptible to both fluoroquino-
lones studied, whereas the other species were inhibited by
lower concentrations of moxifloxacin than ofloxacin. The
MICs of moxifloxacin ranged from 0.032 to 2 mg ml21,
whilst those of ofloxacin and doxycycline ranged from
0.032 to 16 and 0.125 to 128 mg ml21, respectively. In
general, the MBCs were one to six titration steps higher
for moxifloxacin, one to eight titrations steps higher for
ofloxacin, and zero to five titration steps higher for
doxycycline.
The MBCs were generally one step higher than the MICs
with the exception of doxycycline where the MBCs were
the same as the MICs. The MIC and MBC of doxycycline
were higher against the ATCC strain in comparison with
the clinical isolates. Moxifloxacin was more active against
the Porphyromonas gingivalis strains in comparison with
ofloxacin. All MBCs were higher than the MICs.
Against the multispecies mixture, the MIC of moxifloxacin
was as high as the highest one against a single strain
involved in the mixture, i.e. 2 mg ml21. The MICs of
ofloxacin (4 mg ml21) and doxycycline (4 mg ml21) were
lower than the highest MIC against a single strain. In the
case of ofloxacin, for two strains a higher MIC (up to two
steps) was determined in comparison with the mixture.
All MBCs were several steps higher than the MICs.
Doxycycline was not able to eliminate all bacteria up to a
concentration of 512 mg ml21.
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All MICs and MBCs were reproducible. The MICs and
MBCs are given in Table 1.
Biofilms
In preliminary experiments, evaluation of the biofilm
mode of growth was carried out specifically in so far as
biofilm-grown bacteria were in contact with the antibac-
terials for 18 h only on day 3 of the experiment and that
viable counts were quantified on day 7.
The c.f.u. counts of the single-species biofilms were in the
range of 106107 per well at each time point. The numbers
of bacteria within the mixed population (c.f.u. except for
real-time PCR of Treponema denticola) were 7.130.23
log10 c.f.u. per well at day 3, 7.310.15 log10 c.f.u. per well
at day 4 and 7.070.03 log10 c.f.u. at day 7. The bacteria
counts for Eubacterium nodatum, Prevotella intermedia and
Eikenella corrodens were about 4 log10 c.f.u. per well, and
for the other species were between 5 and 6 log10 c.f.u. per
well. Viable counts did not differ among the time points
(Fig. 1).
MBECs
The MBECs determined in two independent experiments
varied by two titration steps. As expected, MBECs of
biofilm-grown bacteria were up to seven doubling dilutions
higher than the respective MBCs for planktonic bacteria.
The quinolones eradicated Aggregatibacter actinomycetemco-
mitans within a biofilm at moderate concentrations.
Moxifloxacin was more active than ofloxacin against
Aggregatibacter actinomycetemcomitans within a biofilm. The
MBEC of doxycycline was higher than those of the quinolones,
in accordance with higher MBCs against planktonic bacteria.
The MBECs against Porphyromonas gingivalis biofilms were
higher than the respective MBCs against planktonic
bacteria; the differences between the MBEC50 and the
MBCs were three to four doubling dilution steps for
doxycycline and twofold dilution steps for the quinolones.
Associated with the lower MBCs, moxifloxacin was the
most active agent of the tested antibacterials against
Porphyromonas gingivalis within a biofilm.
The MBECs against the multispecies biofilms were
extremely high. Only moxifloxacin in a high concentration
(128 mg ml21) was able to eradicate the bacteria within
the biofilm (Table 2).
CLSM and SEM analysis
The live/dead staining showed a high percentage of viable
bacteria. No dead bacteria were visible in the controls and
following exposure to any of the antibacterials at 4 mg
ml21. In addition, no dead bacteria could be seen after
exposure to ofloxacin at a concentration of up to 256 mg
ml21. Doxycycline and moxifloxacin showed a concentra-
tion-dependent activity (Fig. 2).
Journal of Medical Microbiology 63
 Moxifloxacin and biofilms

Table 1. MICs and MBCs of the three antibiotics against monocultures of selected bacterial strains associated with periodontitis and
a mixed population consisting of 12 species in a planktonic state

Bacterial strain MIC (mg ml1) MBC (mg ml1)

Ofloxacin Moxifloxacin Doxycycline Ofloxacin Moxifloxacin Doxycycline

Mono-culture
Aggregatibacter 0.032 0.032 1 0.064 0.064 1
actinomycetemcomitans Y4
Aggregatibacter 0.032 0.032 1 0.064 0.064 1
actinomycetemcomitans J7
Porphyromonas gingivalis ATCC 1 0.125 1 4 0.5 8
33277
Porphyromonas gingivalis M5-1-2 2 0.25 0.125 4 0.5 2
S. gordonii ATCC 10558 1 0.25 0.5 4 1 1
Actinomyces naeslundii ATCC 12104 4 1 0.25 4 1 1
F. nucleatum ATCC 25586 16 1 0.125 128 32 4
C. rectus ATCC 33238 4 2 16 128 16 32
Eubacterium nodatum ATCC 33099 2 2 128 64 16 256
Eikenella corrodens ATCC 23834 0.25 0.064 32 16 0.5 128
Prevotella intermedia ATCC 25611 2 0.25 0.125 2 0.5 0.125
Parvimonas micra ATCC 33270 1 0.125 1 256 64 32
Tannerella forsythia ATCC 43037 8 2 16 256 128 32
Treponema denticola ATCC35405 4 2 0.5 16 16 4
Mixed population* 4 2 4 256 256 .512

*Among the 14 strains tested in this study, only strains Aggregatibacter actinomycetemcomitans J7 and Porphyromonas gingivalis M5-1-2 were not
used in the mixed population.

SEM photographs clearly underlined the fact that the
antibacterials were not able to destroy the multispecies
biofilm, although more extracellular matrix was visible in
the untreated biofilms. Ruffling of cell walls as a possible
sign of dead bacteria was especially visible after exposure of
the biofilms to the quinolones (Fig. 3).
DISCUSSION
The aim of the present study was to evaluate and to
compare the activity of moxifloxacin with ofloxacin and
doxycycline against bacterial species associated with
periodontitis within a biofilm.
Moxifloxacin is a fourth-generation synthetic fluoro-
quinolone (8-methoxyquinolone) with improved activity
against Gram-positive bacteria as well as against micro-
aerophiles and anaerobes in comparison with earlier
fluoroquinolones, such as ofloxacin (Barman Balfour &
Wiseman, 1999; Ackermann et al., 2000; Hardy et al., 2000;
Speciale et al., 2002). Moxifloxacin shows favourable
pharmacokinetic properties, a high bioavailability, safety,
a long half-life time allowing a once-daily dosage, high
secretion in gingival crevicular fluid and saliva, and good
penetration into tissues and cells, e.g. polymorphonuclear
granulocytes and epithelial cells (Stass et al., 1998; Pascual
et al., 1999; Dalhoff & Schmitz, 2003). In dentistry,
moxifloxacin has been shown to be more effective than
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clindamycin as the antibacterial in the treatment of
inflammatory infiltrates and as effective as clindamycin in
the treatment of odontogenic abscesses (Cachovan et al.,
2011). Ofloxacin is a second-generation fluoroquinolone; it
is available in a topical formulation recommended in
ophthalmology (Ta et al., 2006) and in the treatment of
otitis externa (Torum et al., 2004). Considering the
recently published EUCAST (2013) clinical breakpoints,
Gram-positive (non-spore-forming) and Gram-negative
anaerobes are listed as poor targets for ofloxacin, whereas
for moxifloxacin the evidence is insufficient against these
species.
The results of the quinolones were compared further
with doxycycline as a well-established agent in topical
antibacterial treatment of periodontitis (Tomasi &
Wennstrom, 2011; Tonetti et al., 2012).
Aggregatibacter actinomycetemcomitans was equally sens-
itive to moxifloxacin and ofloxacin, and the MICs and
MBCs of moxifloxacin against the anaerobes were lower
compared with ofloxacin. Thus, the good in vitro activity of
moxifloxacin against both oral anaerobes and Aggregatibacter
actinomycetemcomitans (Ardila et al., 2010) was confirmed.
As far as a mixed population of the bacteria was concerned,
the MICs were in the range of the individual MICs. Recently,
it has been shown that a combination of two strains might be
more or less sensitive than the MICs of the single strains
(Mouratidou et al., 2011). MBCs were equal or higher than
287
P. Tsaousoglou and others

(a) 8 antibacterials are well known for good tissue penetration

7 (Kuck et al., 1998; Beringer et al., 2012), but these
concentrations are much higher than the levels within tissues
6 A. actinom. Y4 after systemic administration.
log10 c.f.u. per well

5
A. actinom. J7 Bacteria in oral cavities mostly form biofilms. Subgingival
4 plaque consists of hundreds of different taxa (Shchipkova
P. gingivalis ATCC et al., 2010), many different co-aggregations occur, and
3
33277 bacteria communicate and transfer DNA (Kolenbrander
2 P. gingivalis M5-1-2 et al., 2010). One general phenomenon of biofilms is that
1 antibacterials are not as active as against planktonic
bacteria. The higher resistance is associated with limited
0
Day 3 Day 4 Day 7
growth, protein synthesis and metabolic activity and an
increased mutation frequency as well as with a polymer
(b) 7 matrix around microcolonies (Hiby et al., 2010).
6 Single- and multispecies biofilms were studied. Recently, a
log10 c.f.u. per well

5 study determined the activity of antibacterials in concen-

4 trations found after systemic application in gingival
3 crevicular fluid on a multispecies biofilm in vitro; a
2 significant reduction in bacterial counts was not found
1 (Belibasakis & Thurnheer, 2013). Our study design tried to
0 mimic the in vivo situation, when an antibacterial is
0h Day 3 Day 4 Day 7 topically placed into a periodontal pocket. Concentrations
A. actinom A. naeslundii C. rectus within gingival crevicular fluid are much higher after
E. corrodens E. nodatum F. nucleatum topical application compared with a systemic one, and
P. gingivalis P. intermedia P. micra doxycycline concentrations of more than 1000 mg ml21
S. gordonii T. denticola T. forsythia
were measured for 1 day after topical application, whereas
after systemic application the levels did not exceed 3 mg ml21
(Stoller et al., 1998). Gingival crevicular fluid is known
Fig. 1. Counts of bacteria per well in single-species (a) and for its high turnover. The flow rate is about 44 ml h21 with a
multispecies (b) biofilms without exposure to the antibacterials at resting volume of 1.5 ml in severe periodontitis (Goodson,
day 3 (time before addition of antibiotics), day 4 (time after removal 2003). Delivery devices are used to maintain a high level
of the antibacterials) and day 7 (end of the experiments). A of the antibacterial at the site of placement (Zilberman &
actinom., Aggregatibacter actinomycetemcomitans. Elsner, 2008). Here, the antibacterial was applied at the
concentration to be tested for 18 h; later, nutrient broth
was added to mimic the high turnover of gingival crevicular
fluid. This also allowed replication of non-killed bacteria.
the single MBCs. The quinolones were bactericidal at a
concentration of 256 mg ml21, and doxycycline was not able In single-species biofilms, the MBECs were several steps
to eliminate all bacteria up to 512 mg ml21. All the studied higher than the corresponding MBCs to planktonic

Table 2. MBEC50 and range of MBECs of the three antibacterials against the Aggregatibacter actinomycetemcomitans and
Porphyromonas gingivalis single-species biofilms and a multispecies biofilm consisting of 12 species
MBEC50 was the value at which 50 % of bacteria the biofilms were not cultivable in comparison with the controls without exposure to antibacterials.

Biofilm MBEC50 (mg ml1) Range (mg ml1)

Ofloxacin Moxifloxacin Doxycycline Ofloxacin Moxifloxacin Doxycycline

Aggregatibacter 4 2 64 416 28 32128

actinomycetemcomitans Y4
Aggregatibacter 8 2 32 416 28 32128
actinomycetemcomitans J7
Porphyromonas gingivalis ATCC 16 2 8 1664 24 416
33277
Porphyromonas gingivalis M5-1-2 16 2 8 1632 24 8
Multispecies .512 128 .512 .512 128256 .512

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 Moxifloxacin and biofilms

(a) (b) (c) (d)

Ofloxacin
Moxifloxacin
Doxycycline

Fig. 2. CLSM photographs of the effect of the three antibacterials at different concentrations against 12-species biofilms: (a)
4 mg ml1; (b) 32 mg ml1; (c, d) 256 mg ml1. In (ac), staining is shown for both live (green) and dead (red) bacteria; in (d), a
replica of (c), only dead bacteria are stained.

bacteria. Moxifloxacin was most efficient, confirming an
earlier study where clindamycin, doxycycline, metronida-
zole and moxifloxacin were tested (Eick et al., 2004b). In an
Aggregatibacter actinomycetemcomitans biofilm model com-
paring b-lactam antibiotics, tetracyclines, erythromycin,
levofloxacin and ofloxacin, ofloxacin was the only one that
exerted an inhibitory activity in mature biofilms (Takahashi
et al., 2007). Moxifloxacin was shown to reduce the biomass
of Staphylococcus aureus biofilms (Di Bonaventura et al.,
2004). It induced slime disruption of biofilms produced by
selected respiratory specimens (Roveta et al., 2007). In
contrast to single-species biofilms of periodontopathogens
(Eick et al., 2004b), moxifloxacin seemed not to be able to
destroy the structure of a multispecies biofilm, although
SEM photographs showed damaged bacterial cells and less
extracellular matrix.
In multispecies biofilms, only moxifloxacin was able to kill
all bacteria at a concentration of 128 mg ml21, whilst it was
not possible to eliminate all bacteria with ofloxacin and
doxycycline at concentrations of up to 512 mg ml21.
CLSM photographs showed a concentration-dependent
activity on the percentage of dead bacteria analysed by live/
dead staining. Nevertheless, they did not confirm the
results determined by cultivation; the percentage of viable
bacteria was always extremely high (Fig. 1). The live/dead
combined staining (red/green) method used is based on
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membrane integrity. The SYTO 9 (green fluorescence)
labels bacteria with intact membranes and propidium
iodide (red fluorescence) enters bacteria with damaged
membranes. It can be said that viable stained bacteria are
not cultivable anymore. On the other hand, the 100 %
viability in many biofilms suggests in part false-positive
results for vital bacteria. By comparing different staining
methods for the vitality determination of planktonic
streptococci, the highest vitality rates have been found
for this kind of staining (Decker, 2001). In addition,
biofilm components might prevent an uptake of propi-
dium iodide in dead bacteria.
Single-species biofilms were less resistant against the
action of the antibacterial. The activity of antibacterials
was tested against a 12-species biofilm. Antimicrobials
against multispecies biofilms have rarely been tested
(Badet & Quero, 2011; Hofer et al., 2011; Belibasakis &
Thurnheer, 2013). Nevertheless, the model used in this
study represents only a small part of the complexity of
biofilms. Thus, it might be suggested that the efficacy of
antibacterials in vivo is much less than that shown in this
in vitro model.
Moxifloxacin may have potential as a topically applied
drug in the treatment of residual pockets in periodontitis.
Previously, it has been shown that it is active in vitro
against an intracellular infection (Eick & Pfister, 2004).
289
P. Tsaousoglou and others

(a) (b)

(c) (d)

Fig. 3. SEM photographs of the effect of the antibacterials against 12-species biofilms without antibiotics (a) and after exposure
to 256 mg doxycycline ml1 (b), 256 mg ofloxacin ml1 (c) and 256 mg moxifloxacin ml1 (d). Bars, 1 mm.

Moreover, in a randomized clinical trial, it was most develop resistance (Eick et al., 2004a); however, in a clinical
effective as an adjunct to scaling and root planing, with study (Guentsch et al., 2008), these resistant strains were
regard to the clinical outcomes and microbiological and not found (unpublished data). Nevertheless, application of
immunological parameters in comparison with adjunctive antibacterials should be limited to selected cases.
doxycycline use and scaling and root planing alone Antibacterial resistance depends on the general usage of
(Guentsch et al., 2008). Flemmig et al. (2011) showed antibacterial chemotherapeutics, e.g. resistance of period-
recently additional pocket depth reduction following local ontopathogens is much more prevalent in Spain as a
application of moxifloxacin as an adjunct to scaling and country with a high consumption of antibacterials in
root planing in chronic periodontitis patients with residual comparison with the Netherlands where the application of
pockets. Data about the level of moxifloxacin within antibacterials is restricted (van Winkelhoff et al., 2005).
gingival crevicular fluid are not available, but similar levels In conclusion, moxifloxacin was more active against an-
as measured for ofloxacin, as an example of another aerobes than ofloxacin and also exerted activity against
fluoroquinolone, might be assumed. Evaluation of a bacteria within a biofilm. Among the tested antibacterials, it
controlled-release insert consisting of 11 % ofloxacin was the only one that was able to eradicate bacteria in a
showed concentrations of 1700 mg ml21 at 3 h after multispecies biofilm, although the concentrations required
insertion, which decreased to 10 mg ml21 after 1 day were high. In combination with mechanical removal of the
(Higashi et al., 1989). A general problem associated with biofilms, moxifloxacin might be a favourable topical
antibacterials use is the development of resistance. In vitro, antibacterial chemotherapeutic for selected cases of period-
it has been shown that Porphyromonas gingivalis is able to ontitis.
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ACKNOWLEDGEMENTS
The authors would like to thank Regula Hirschi, Sabrina Ruggiero,
and Marianne Weibel (Department of Periodontology, Laboratory of
Oral Microbiology, School of Dental Medicine, University of Bern,
Switzerland) for technical assistance. The present study was supported
in part by Bayer Technology Services GmbH, Leverkusen, Germany.
The authors declare that they have no conflicts of interests.
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