TUGAS REFRAT shtttt

TUGAS REFRAT

SALIVA DAN KELENJAR SALIVA

Disusun Oleh :

1. M. Iqbal J5201100222. Yusifa Edo R J5201100233. Kurnia Indah P J520110027

FAKULTAS KEDOKTERANPROGRAM STUDI KEDOKTERAN GIGI

UNIVERSITAS MUHAMMADIYAH SURAKARTA 2012

GMS Krankenhhyg Interdiszip. 2012;7(1):Doc06. Epub 2012 Apr 4.

XTT assay of ex vivo saliva biofilms to test antimicrobial influences.

Koban I, Matthes R, Hübner NO, Welk A, Sietmann R, Lademann J, Kramer A, Kocher T.

Source

Unit of Periodontology, Dental School, University of Greifswald, Greifswald, Germany.

Abstract

Objective: Many dental diseases are attributable to biofilms. The screening of antimicrobial

substances, in particular, requires a high sample throughput and a realistic model, the evaluation

must be as quick and as simple as possible. For this purpose, a colorimetric assay of the

tetrazolium salt XTT (sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-

nitro)benzene-sulfonic acid hydrate) converted by saliva biofilms is recommended. Cleavage of

XTT by dehydrogenase enzymes of metabolically active cells in biofilms yields a highly colored

formazan product which is measured photometrically.Materials and method: The suitability of

the XTT assay for detecting the vitality of ex vivo saliva biofilms was tested to determine the

efficacy of chlorhexidine and ozone versus saliva biofilms grown on titanium discs.Results: The

XTT method lends itself to testing the vitality of microorganisms in saliva biofilms. The

sensitivity of the arrays requires a specific minimum number of pathogens, this number being

different for planktonic bacteria and those occurring in biofilms. The antibacterial effect after

treatment with chlorhexidine or ozone was measured by XTT conversion that was significantly

reduced. The antimicrobial efficacy of 60 s 0.5% and 0.1% chlorhexidine treatment was equal

and comparable with 60 s ozone treatment. Conclusion: The XTT assay is a suitable method to

determine the vitality in saliva biofilms, permitting assessment of the efficacy of antimicrobial

substances. Its quick and easy applicability renders it especially suitable for screening.

GMS Krankenhhyg Interdiszip. 2012; 7(1): Doc06.

Published online 2012 April 4. doi:  10.3205/dgkh000190

PMCID: PMC3334957

XTT assay of ex vivo saliva biofilms to test antimicrobial influences

Ina Koban,*,1 Rutger Matthes,2 Nils-Olaf Hübner,2 Alexander Welk,1 Rabea Sietmann,3 Jürgen

Lademann,4 Axel Kramer,2 and Thomas Kocher1

Author information   ►  Copyright and License information   ►

Abstract

Objective: Many dental diseases are attributable to biofilms. The screening of antimicrobial

substances, in particular, requires a high sample throughput and a realistic model, the evaluation

must be as quick and as simple as possible. For this purpose, a colorimetric assay of the

tetrazolium salt XTT (sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-

nitro)benzene-sulfonic acid hydrate) converted by saliva biofilms is recommended. Cleavage of

XTT by dehydrogenase enzymes of metabolically active cells in biofilms yields a highly colored

formazan product which is measured photometrically.

Materials and method: The suitability of the XTT assay for detecting the vitality of ex

vivo saliva biofilms was tested to determine the efficacy of chlorhexidine and ozone versus saliva

biofilms grown on titanium discs.

Results: The XTT method lends itself to testing the vitality of microorganisms in saliva

biofilms. The sensitivity of the arrays requires a specific minimum number of pathogens, this

number being different for planktonic bacteria and those occurring in biofilms. The antibacterial

effect after treatment with chlorhexidine or ozone was measured by XTT conversion that was

significantly reduced. The antimicrobial efficacy of 60 s 0.5% and 0.1% chlorhexidine treatment

was equal and comparable with 60 s ozone treatment.

Conclusion: The XTT assay is a suitable method to determine the vitality in saliva biofilms,

permitting assessment of the efficacy of antimicrobial substances. Its quick and easy

applicability renders it especially suitable for screening.

Keywords: biofilm model, saliva, S. mutans, ozone, chlorhexidine, XTT assay

Introduction

Bacterial infections play a specific role in dentistry. After the supragingival tooth surfaces and

mucous membranes have been wetted with saliva, microbes settle there and form a biofilm [1].

This biofilm accommodates dental pathogens and protects them against environmental stress

factors, such as chemotherapeutics, the immune system, acids, hunger periods, and reactive

oxygen products [2], [3].

Antimicrobially effective substances and techniques should be tested on a suitable biofilm

model, as the efficacy against planktonic pathogens has little predictive value for the efficacy

against biofilms.

For a number of years, several mono-species biofilm models have been available which

accommodate typical oral microbes [4], [5]. Streptococci are frequently used as a caries model,

although other scientists found out that they do not represent the etiological pathogens of the

disease [6]. For the treatment of periodontal diseases, anaerobic periodontal marker pathogens

are important [7]. But in vivo plaque microbiota is highly diverse and complex [8]. The oral

cavity harbors more than 1,000 different microorganisms, which join to form multispecies

biofilms [9]. Guggenheim et al. used biofilms consisting of a maximum of six different

pathogens [10]. The oral fluid, too, is an essential component in the formation of dental biofilms.

The proteins in the saliva are a significant source of food for microbes. Pellicle proteins settle on

the dental surfaces forming the so-called conditioning film. This conditioning film forms the

basis for the development of biofilms, as the adhesins of the bacteria directly bind to these

oligosaccharide-containing glycoproteins [11], [12]. In addition salivary antimicrobial factors are

important stressors that can enhance biofilm formation [13], [14]. Many artificial saliva

formulations have been designed that attempt to imitate this process in order to ensure realistic

biofilm formation. In most cases, however, artificial saliva fails to provide all the organic and

inorganic components that exist in natural saliva. Moreover, no evidence has been supplied that

artificial saliva promotes biofilm formation [15].

For their biofilm models, other researchers used volunteers’ saliva which was filtered under

sterile conditions, diluted and centrifuged [16].

A simple, more realistic multispecies biofilm model can be obtained by culturing the saliva of

volunteers, without prior filtration under sterile conditions [17]. Of course, this is only a model

because the circadian rhythm of salivation and the correspondingly variable composition of

saliva cannot be imitated, nor can regular food intake [18], [19]. Furthermore, the oral conditions

are different in each patient [20]. A subsequent detection of biofilm formation is difficult. Even

when using a non-specific agar, such as Columbia blood agar, it is not possible to definitely

detect every species in the culture. Alternatively, colorimetric methods, e.g., the XTT assay, are

appropriate to measure metabolic activity and vitality. XTT is a yellow salt that is reduced by

dehydrogenases of metabolically active cells to a colored formazan product. Colorimetric

methods are attractive because they have the potential to generate clear-cut endpoints based on a

visible color change.

The objective of this study, therefore, was the development of a method suitable for testing

saliva biofilms using XTT.

Materials and method

Cultivation of biofilms

Biofilms were cultured on titanium discs 5 mm in diameter and 1 mm thick (Straumann, Basel,

Switzerland).

The sterile titanium discs were positioned in 96-well microtitre plates (Techno Plastic Products

AG, Trasadingen, Switzerland), covered with 100 µl fresh, unstimulated saliva of healthy

volunteers (aged between 20 and 30 years, non-smokers), and incubated aerobically at 37°C. The

donors did not take any medication three months prior the study and did not have active carious

lesions or periodontal disease. After 24 h, the saliva was drawn off and replaced by sterile brain-

heart infusion broth (BD, BBL™, Heidelberg, Germany), as a highly nutritious general-purpose

growth medium. After 48 h, the medium was drawn off, and the discs were washed with 0.9%

NaCl solution and transferred onto a new, sterile microtitre plate.

Antiseptic treatment with chlorhexidine

Chlorhexidine digluconate was used as a 0.1% and 0.05% aqueous solution. The discs were

covered with 100 µl of the antiseptic and incubated for 1 min.

After this exposure, the chlorhexidine was drawn off, and the antiseptic effect was stopped by

adding 1 ml of inactivator (Lipofundin MCT 20%, B.Braun, Melsungen, Germany). The

inactivation of chlorhexidine by the inactivator was validated by the quantitative suspension test

according to EN 1040. Physiological saline was used for control.

Application of ozone

The test objects were direct treated for 20 s, 30 s, 40 s, or 60 s with gaseous ozone provided by a

HealOzone device (KAVO, Biberach, Germany). The ozone is delivered via a hose into a

disposable sterile cup at a concentration of 2,100 ppm ± 10%. The ozone gas is refreshed in this

disposable cup at a rate of 615 cc/minute changing the volume of gas inside the cup over 300

times every second [21].

Inactivation was unnecessary as the device suctions off any residual ozone after application.

Vitality measurement by XTT assay

Bioreduction of XTT could be potentiated by addition of electron coupling agents such as

phenazine methosulfate (PMS) or menadione (Men) [22].

To optimize the staining solution, 200 µl of the XTT solution was added to each disc bearing the

grown biofilm after 24 h and 48 h, respectively. The added XTT solution was composed of the

following:

1. XTT (180 mg/l) (AppliChem, Darmstadt, Germany) and menadione (0.688 mg/l) (Sigma-

Aldrich, Munich, Germany) (hereafter called „XTT+Men“)

2. XTT (180 mg/l) and phenazine methosulfate (20 mg/l) (PMS, AppliChem, Darmstadt,

Germany) (hereafter called „XTT+PMS“)

3. XTT (180 mg/l), menadione (0,688 mg/l) and phenazine methosulfate (20 mg/l)

(hereafter called „XTT+Men+PMS”)

To determine the measuring range, saliva was diluted with physiological saline and incubated

with the XTT staining solution specified in test 1 (XTT+PMS+menadione).

To test the antimicrobial efficacy of chlorhexidine and gaseous ozone, the treated discs were also

incubated with 200 µl of the staining solution (XTT+PMS+menadione).

After 3 h of incubation while shaking (Titramax, Heidolph Instruments, Schwabach, Germany)

at 37°C, 100 µl of all solutions were transferred onto a new sterile microtitre plate and analyzed

at 450 nm (reference wavelength 620 nm) using a photometer (anthos Mikrosysteme, Krefeld,

Germany) [23].

Determination of CFU

After treatment, the titanium discs were placed into wells with 200 µl 0.9% NaCl solution and

the biofilm was removed by ultrasonic scaling (Branson 2510, 130 W, 42 kHz, Dietzenbach,

Germany). Serial dilutions were made by transferring 0.1 ml of the resultant suspension to 0.9 ml

of fresh 0.9% NaCl solution. After that, an aliquot portion of 0.1 ml from each dilution was

plated on Columbia sheep blood agar (BBL™, BD, Heidelberg, Germany) and incubated aerobe

at 37°C for 48 h. The colonies were counted and expressed as colony forming units (CFU). The

CFU values were log transformed.

Confocal laser scanning microscopy

Live/Dead-Staining (BacLight, Invitrogen, Darmstadt, Germany) was used for microscopical

analysis of the bacterial vitality. The biofilms were incubated immediately with the dye

according to manufacturer’s instructions. After incubation the discs were rinsed with 0.9% NaCl

solution to remove dye residues from the biofilm. The samples were evaluated in the confocal

laser scanning microscope (CLSM510 Exciter, Zeiss, Jena, Germany).

Scanning electron microscopy

For electron microscopy, the biofilms were prepared as follows: after a fixation step (1 h in 1%

glutaraldehyde, 2% paraformaldehyde, 0.2% picric acid, 5 mM HEPES (pH 7.4), and 50 mM

NaN3), the samples were treated with 2% tannic acid for 1 h, 1% osmium tetroxide for 1 h, 1%

thiocarbohydrazide for 30 min, 1% osmium tetroxide at 4°C overnight, and with 2% uranyl

acetate for 2 h with washing steps in between. The samples were dehydrated in a graded series of

acetone solutions (10 to 100%) and then critical-point dried. Finally, samples were mounted on

aluminum stubs, sputtered with gold-palladium and examined in an EVO LS10 (Zeiss,

Oberkochen, Germany) (Figure 1 (Fig. 1)).

Figure 1

Schematic view of the experiment

Analysis

For all experiments, at least eight test objects each were used. The chlorhexidine treatment

required 23 discs. In addition, eight test objects each were available for control tests. Continuous

data are presented as mean ± standard deviation. Statistical analyses were performed with

STATVIEW® 5.0 (SAS, Cary, NC) software. Graphs were also created using STATVIEW.

Medians are given with their standard errors. Nonparametric correlations (Mann-Whitney U-test)

were estimated for comparison of absorptions. P-values below 0.05 were considered statistically

significant.

Results

Cultivation of biofilms

The cultivation procedure was constantly checked for cultures by determining the CFU and for

reproducibility by microscopy. Cell densities of 108 CFU/ml were regularly be removed from the

test objects by ultrasound scaling.

Optimization of the staining solution

The absorption of the colored formazan derivate of XTT converted by the microbes is a measure

of the cellular vitality.

A high absorption value indicates high metabolic activity. Figure 2 (Fig. 2) shows the absorption

values (at 450 nm) of the different staining solutions (XTT+Men, XTT+PMS and

XTT+Men+PMS) on 24-h- and 48-h-old biofilms. For all staining solutions, significant (p<0.01)

differences were visible between the 24-h and 48-h biofilms.

Figure 2

Diagram of the absorption at 450 nm (reference wavelength 620 nm) of XTT metabolized

by saliva biofilm (XTT+ menadione, XTT+ phenazine methosulfate, XTT+ menadione+ N-

methyl-phenazinium methylsulfate).

In the case of XTT+Men, the absorption at 450 nm increased to a value an average of 30 times

higher (from 0.01 to 0.30) after 48 h than after 24 h. Within these 24 h, the absorption value of

XTT+PMS increased to five times (from 0.10 to 0.50) the original value, while the absorption of

XTT+Men+PMS was increased three times from 0.20 to 0.65.

The absorption values of XTT+Men+PMS were always significantly (p<0.01) higher than those

of the other straining solutions.

Determination of the measuring range

Figure 3 (Fig. 3) shows the absorption values at 450 nm determined by dilution of the saliva and

subsequent staining with XTT+Men+PMS. There are significant differences (p<0.01) between

the absorption values of the biofilms up to increments of 4.5 log10. Absorption was no longer

measurable in concentrations from 3.5 log10 (CFU/ml), except for negative controls.

Figure 3

Diagram of the absorption at 450 nm (reference wavelength 620 nm) of XTT + menadione

+ phenazine methosulfate metabolized by various saliva dilutions.

Antimicrobial treatment

A reduced XTT conversion was observed in saliva biofilms which had been subjected to

antimicrobial treatment with gaseous ozone and chlorhexidine. This reduction was significant

after a 60-s or 120-s ozone treatment (p<0.02) and a one-minute chlorhexidine treatment

(p<0.01) (see Figure 4 (Fig. 4)). There was no difference in the conversion of XTT using a

0.05% or a 0.1% chlorhexidine solution. No difference could be seen in the scanning electron

micrographs between the untreated biofilm and the biofilm treated with ozone. In both samples,

the cells appear plump and the biofilm has a loosely bound structure. The bacteria in the biofilm

treated with chlorhexidine are damaged and the overall structure appears to be tighter (Figure

5 (Fig. 5)). The CLSM images confirmed these observations (Figure 6 (Fig. 6)). After treatment

with ozone, parts of the biofilm were dyed red (cells with damaged membranes). After CHX

treatment, no green colored areas (cells with intact membranes) were identified.

Figure 4

Diagram of the absorption at 450 nm (reference wavelength 620 nm) of XTT+ menadione+

phenazine methosulfate, metabolized by saliva biofilms for 48 h after treatment with 0.9%

NaCl solution (control), gaseous ozone and chlorhexidine.

Figure 5

SEM micrograph: 48-h mature saliva biofilm: (A) untreated, (B) after ozone treatment, (C)

after chlorhexidine treatment. Magnification 10,000 x

Figure 6

CLSM micrographs: 48-h mature saliva biofilm: (A) untreated, (B) after ozone treatment,

(C) after chlorhexidine treatment. Magnification 100 x

Discussion

Causing typical dental diseases, such as caries and periodontitis, biofilms complicate the

elimination of microbes responsible for forming the biofilms by antimicrobial substances. The

objective of this study was to develop a biofilm model suitable for testing the efficacy of

antimicrobial substances with non-culture-based detection of the vitality of saliva biofilms using

XTT, and to prepare a suitable XTT assay.

Our study had several limitations. We used saliva of volunteers to create a practically relevant

biofilm model. For better understanding of the interactions between bacteria and XTT, we

performed our experiments on machined titanium discs to exclude material hydrophobicity,

retention niches such as cavities and porosities into which the biofilm could adhere. Titanium

implants have been successfully used in dentistry and biofilms on titanium are the central

problem in peri-implantitis. Peri-implantitis of osseointegrated oral implants is not a

monoinfection by single pathogens; rather, they show the characteristics of mixed infections.

Whereas the single species of a mixture of bacteria could not induce experimental abscesses, the

combination of these species could do it [24]. Plaque biofilms containing multiple species of

appropriate bacteria should be more relevant for studying dental diseases and antimicrobial

efficacies [25]. The first step was the use of only aerobe cultivation methods. Most XTT assays

are carried out aerobically. In the next step we will use a subgingival plaque biofilm under

anaerobic conditions for XTT assays. But studies also showed that the same microbiota that can

be found around implants (under anaerobic conditions) also can be found around teeth (under

aerobic conditions) [26], [27].

XTT is a colorless tetrazolium salt, which is converted into a colored, water-soluble formazan

derivate by dehydrogenases, with succinate dehydrogenase being particularly important [23] as it

plays a major role in the energy supply of each individual living cell [28]. Unlike other

tetrazolium salts (e.g., CTC and TTC), XTT does not require any insoluble formazans to be

extracted. Since the color change in the solution can be directly determined by photometry, the

XTT test permits a large number of test objects to be tested for their vitality very quickly. The

incubation of bacteria with XTT for 3 h at 37°C has already been the subject of several

publications [23]. What is new is the composition of the XTT staining solution applied. The

saliva biofilm contains a large variety of different microbes (Gram-positive, Gram-negative

bacteria and fungi). To convert XTT, these microbes require various additives which function as

electron carriers. The standard additive for Candida spp. is menadione [29], [30], [31], [32], but

PMS can be applied as well [33]. For Gram-positive cocci bacteria, PMS is used in most

instances [34], but sometimes menadione is also used [35]. In the case of Gram-negative rod-

shaped bacteria, mainly menadione is applied [35], [36]. Our own preliminary (unpublished)

investigations confirm these results on the suitability of menadione for Candida albicans and

PMS for Streptococcus mutans andStreptococcus sanguinis as representatives in dentistry. As

for Pseudomonas aeruginosa, the combined application of menadione and PMS turned out to be

suitable [37]. By means of a culture-based analysis of the saliva, Gram-positive cocci bacteria, i.

a., could be isolated. Therefore, the addition of PMS seemed to be indispensable for the

colorimetric detection using XTT. McCluskey et al. also exclusively used PMS for the

colorimetric detection of microbes occurring in activated sludge, and were able to prove that

there is a direct correlation between formazan production and oxygen consumption [23].

In a direct comparison, the combined PMS-menadione electron mediators showed the highest

XTT conversion in saliva biofilms (see Figure 2 (Fig. 2)). It turned out, however, that a high cell

count of at least 4.5 log10 (CFU/ml) is required to detect the XTT reduction (see Figure 4 (Fig.

4)). At 5.5 log10(CFU/ml), a very high absorption (1.91) was detected at 450 nm. The higher the

number of metabolically active cells, the higher the colorimetric signal. In addition, the higher

the metabolism of cells, the higher the signal. Obviously, there is no linear relation between the

number of cells and the colorimetric signal [38]. When other dyes (FDA und Syto9) were used,

the adsorption even remained constant [39]. After 48 h of biofilm formation, cell densities of ca.

8 log10 (CFU/ml) were reached, but following the XTT test, the absorption in the biofilm was

0.65, i.e., less than in planktonic bacteria although the cell density was higher. The amount of

retained product may vary between planktonic bacteria and biofilms [38]. Moreover, biofilms are

subjected to other conditions than are fresh bacteria suspensions. Many pathogens are persistent

and, therefore, exhibit lower metabolic activity [40]. For this reason, a minimum number of

pathogens cannot be determined from suspensions and biofilms in exactly the same manner. In

addition, planktonic cells can invest more energy in routine metabolism [30]. In our experiments,

the absorption was 30 times higher after 48 h than after 24 h, i.e., the conversion of XTT

increased due to the growth of the biomass. However, the different metabolic levels do not lead

to a logarithmic increase of the XTT reduction. This XTT-related observation was also made by

other research groups [39].

Reduced formazan formation was observed due to the antimicrobial treatment of the saliva

biofilm. Consequently, the XTT test is suitable for determining the efficacy of antimicrobial

substances, especially for screening. The low chlorhexidine concentration used has already been

investigated by other researchers, who noted insufficient antimicrobial efficacy in the biofilms

[41]. On the other hand, chlorhexidine proved to be slightly superior to ozone. This has also been

published by other research groups who applied alternative methods [42]. However, in

vivo Hauser-Gerspach et al. could not find significant antimicrobial effects of CHX and ozone

[43]. The scanning electron micrographs confirmed this result. Following the ozone treatment,

the morphology of the cells showed no differences. Although the biofilm treated with

chlorhexidine appeared to be damaged compared to the control, only a few cells were

morphologically deformed.

In spite of some disadvantages, XTT with the addition of menadione and PMS is a suitable

method for determining the vitality in bacterial saliva biofilms and permits assessment of the

efficacy of antimicrobial substances.

The assay is easy to perform, and allows a large number of test objects to be tested. It is

particularly suited to screening various factors influencing the biofilm, such as antiseptics or

other physical or chemical treatments, for instance, ozone, photodynamic therapy or atmospheric

pressure plasma.

Notes

Acknowledgements

This work was realized within the framework of the multi-disciplinary research cooperation

“Campus PlasmaMed”, particularly within the project “PlasmaDent”. The authors acknowledge

that this work was supported by a grant funded by the German Ministry of Education and

Research (BMBF, grant no, 13N9779).

We thank Tina Dornquast and Hartmut Fischer for their excellent technical assistance. We also

thank PD Dr. Lutz Netuschil for the critical discussions.

Competing interests

The authors declare that they have no conflict of interest.

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PEMBAHASAN JURNAL

Banyak penyakit gigi yang disebabkan biofilm. Pemutaran zat antimikroba, khususnya,

memerlukan throughput sampel tinggi dan model yang realistis, evaluasi harus secepat dan

sesederhana mungkin. Untuk tujuan ini, alat tes kolorimetri dari tetrazolium garam XTT (natrium

3'-[1 - [(phenylamino)-carbony] -3,4-tetrazolium]-bis (4-metoksi-6-nitro) benzena-sulfonat hidrat

asam ) dikonversi oleh biofilm air liur dianjurkan. Pembelahan oleh enzim dehidrogenase XTT

sel yang aktif secara metabolik dalam biofilm menghasilkan produk formazan sangat berwarna

yang diukur photometrically.

Kesesuaian uji XTT untuk mendeteksi vitalitas biofilm air liur ex vivo diuji untuk

menentukan kemanjuran dari klorheksidin dan ozon terhadap air liur biofilm tumbuh pada

cakram titanium. Metode XTT cocok untuk menguji vitalitas mikroorganisme dalam biofilm air

liur. Sensitivitas dari array membutuhkan jumlah minimum tertentu patogen, jumlah ini menjadi

berbeda bagi bakteri plankton dan yang terjadi dalam biofilm. Efek antibakteri setelah

pengobatan dengan klorheksidin atau ozon diukur dengan konversi XTT yang berkurang secara

signifikan. Efektivitas antimikroba dari 60 0,5% s dan pengobatan klorheksidin 0,1% adalah

sama dan sebanding dengan pengobatan ozon 60 detik. Uji XTT adalah metode yang cocok

untuk mencari vitalitas dalam biofilm air liur, yang memungkinkan penilaian efektivitas zat

antimikroba. Penerapannya cepat dan mudah menjadikan itu sangat cocok untuk penyaringan.

Infeksi bakteri memainkan peran khusus dalam kedokteran gigi.Setelah permukaan gigi

supragingiva dan selaput lendir telah dibasahi dengan air liur, mikroba menetap di sana dan

membentuk biofilm. Biofilm ini mengakomodasi patogen gigi dan melindungi mereka terhadap

faktor stres lingkungan, seperti kemoterapi, sistem kekebalan tubuh, asam, periode kelaparan,

dan produk oksigen reaktif .

Zat Antimicrobially efektif dan teknik harus diuji pada model biofilm yang cocok, sebagai

efektivitas terhadap patogen planktonik memiliki sedikit nilai prediktif untuk efektivitas terhadap

biofilm.

Selama beberapa tahun, beberapa mono-spesies model biofilm telah tersedia yang

mengakomodasi mikroba mulut khas. Streptococcus sering digunakan sebagai model karies,

meskipun para ilmuwan lainnya menemukan bahwa mereka tidak mewakili patogen etiologi

penyakit .Untuk pengobatan penyakit periodontal, anaerob patogen periodontal penanda penting.

Namun dalam mikrobiota plak vivo sangat beragam dan kompleks. Rongga mulut pelabuhan

lebih dari 1.000 mikroorganisme yang berbeda, yang bergabung untuk membentuk biofilm

spesies lain. Guggenheim dkk. biofilm digunakan terdiri dari maksimal enam patogen yang

berbeda. Cairan oral, juga merupakan komponen penting dalam pembentukan biofilm

gigi. Protein dalam air liur adalah sumber penting makanan untuk mikroba. Protein pelikel

menetap pada permukaan gigi membentuk film penyejuk disebut. Film pengkondisian

membentuk dasar untuk pengembangan biofilm, sebagai adhesins bakteri langsung mengikat ini

oligosakarida yang mengandung glikoprotein. Selain faktor antimikroba ludah adalah stressor

penting yang bisa meningkatkan pembentukan biofilm. Banyak formulasi saliva buatan telah

dirancang yang mencoba untuk meniru proses ini untuk memastikan pembentukan biofilm

realistis. Dalam kebanyakan kasus, bagaimanapun, saliva buatan gagal untuk menyediakan

semua komponen organik dan anorganik yang ada dalam air liur alam. Selain itu, tidak ada bukti

telah diberikan bahwa air liur buatan mempromosikan pembentukan biofilm.

Untuk model biofilm mereka, peneliti lainnya menggunakan air liur relawan yang

disaring dalam kondisi steril, dan disentrifugasi diencerkan.

Secara sederhana lebih realistis spesies lain Model biofilm dapat diperoleh dengan kultur

air liur relawan, tanpa filtrasi sebelumnya dalam kondisi steril. Tentu saja, ini hanya model

karena irama sirkadian dari air liur dan komposisi Sejalan variabel air liur tidak dapat ditiru, juga

tidak dapat asupan makanan biasa. Selanjutnya, kondisi lisan berbeda dalam setiap pasien.

Sebuah deteksi selanjutnya pembentukan biofilm sulit. Bahkan ketika menggunakan agar-agar

yang tidak spesifik, seperti agar darah Columbia, tidak mungkin untuk pasti mendeteksi setiap

spesies dalam budaya. Atau, metode kolorimetri, misalnya, uji XTT, sesuai untuk mengukur

aktivitas metabolisme dan vitalitas. XTT adalah garam kuning yang dikurangi dengan

dehydrogenases sel yang aktif secara metabolik untuk produk formazan berwarna.Metode

kolorimetri yang menarik karena mereka memiliki potensi untuk menghasilkan yang jelas titik

akhir berdasarkan perubahan warna terlihat.

Tujuan dari penelitian ini, oleh karena itu, adalah pengembangan metode yang cocok

untuk menguji air liur menggunakan biofilm XTT.

Budidaya biofilm

Biofilm dikultur pada cakram titanium diameter 5 mm dan 1 mm tebal (Straumann, Basel,

Swiss). Cakram titanium steril diposisikan dalam 96-well piring microtitre (Techno Produk

Plastik AG, Trasadingen, Swiss), ditutupi dengan 100 ml air liur segar, tidak distimulasi

sukarelawan sehat (berusia antara 20 dan 30 tahun, bukan perokok), dan diinkubasi aerobikpada

37 ° C. Para donor tidak mengambil obat tiga bulan sebelum studi dan tidak memiliki lesi karies

aktif atau penyakit periodontal.Setelah 24 jam, air liur tersebut diambil off dan digantikan oleh

steril otak-jantung kaldu infus (BD, BBL ™, Heidelberg, Jerman), sebagai media untuk tujuan

umum pertumbuhan yang sangat bergizi. Setelah 48 jam, medium ditarik off, dan cakram dicuci

dengan larutan NaCl 0,9% dan dipindahkan ke piring, microtitre baru steril.

Antiseptik pengobatan dengan klorheksidin

Chlorhexidine digluconate digunakan sebagai% 0,1 dan larutan 0,05%. Cakram ditutupi

dengan 100 ml antiseptik dan diinkubasi selama 1 menit. Setelah paparan ini, klorheksidin

tersebut diambil off, dan efek antiseptik dihentikan dengan menambahkan 1 ml inactivator

(Lipofundin MCT 20%, B.Braun, Melsungen, Jerman). Inaktivasi klorheksidin oleh inactivator

divalidasi dengan uji suspensi kuantitatif sesuai dengan EN 1040. Garam fisiologis digunakan

untuk kontrol.

Penerapan ozon

Benda uji langsung dirawat selama 20 s, 30 detik, 40 detik, atau 60 dengan ozon gas yang

disediakan oleh perangkat HealOzone (KAVO, Biberach, Jerman). Ozon ini disampaikan

melalui selang ke dalam cangkir steril sekali pakai pada konsentrasi 2.100 ppm ± 10%. Gas ozon

di-refresh dalam cangkir sekali pakai dengan kecepatan 615 cc / menit perubahan volume gas di

dalam cangkir lebih dari 300 kali setiap detik.

Inaktivasi itu tidak perlu sebagai perangkat suctions dari setiap ozon sisa setelah aplikasi.

Vitalitas pengukuran dengan alat tes XTT

Bioreduction dari XTT bisa diperkuat dengan penambahan agen kopling elektron seperti

methosulfate phenazine (PMS) atau menadione (Pria). Untuk mengoptimalkan solusi pewarnaan,

200 ml larutan XTT ditambahkan ke setiap disk menyandang biofilm tumbuh setelah 24 jam dan

48 jam, masing-masing. Solusi XTT menambahkan terdiri dari:

o XTT (180 mg / l) (AppliChem, Darmstadt, Jerman) dan menadione (0,688 mg / l)

(Sigma-Aldrich, Munich, Jerman) (selanjutnya disebut "XTT + Pria")

o XTT (180 mg / l) dan methosulfate phenazine (20 mg / l) (PMS, AppliChem, Darmstadt,

Jerman) (selanjutnya disebut "XTT + PMS")

o XTT (180 mg / l), menadione (0,688 mg / l) dan methosulfate phenazine (20 mg / l)

(selanjutnya disebut "XTT + Pria + PMS")

Untuk menentukan rentang pengukuran, air liur diencerkan dengan garam fisiologis dan

diinkubasi dengan larutan pewarnaan XTT ditentukan dalam tes 1 (XTT + PMS + menadione).

Untuk menguji efektivitas antimikroba dari klorheksidin dan ozon gas, cakram diobati juga

diinkubasi dengan 200 ml larutan pewarnaan (XTT + PMS + menadione).

Setelah 3 jam inkubasi sambil geleng-geleng (Titramax, Heidolph Instrumen, Schwabach,

Jerman) pada 37 ° C, 100 ml dari semua solusi dipindahkan ke piring microtitre baru steril dan

dianalisis pada 450 nm (referensi panjang gelombang 620 nm) dengan menggunakan fotometer

sebuah ( anthos Mikrosysteme, Krefeld, Jerman)

Penentuan CFU

Setelah pengobatan, cakram titanium ditempatkan ke dalam sumur dengan 200 ml larutan

NaCl 0,9% dan biofilm telah dihapus dengan skala ultrasonik (Branson 2510, 130 W, 42 kHz,

Dietzenbach, Jerman). Pengenceran Serial dibuat dengan mentransfer 0,1 ml suspensi yang

dihasilkan menjadi 0,9 ml larutan NaCl 0,9% segar.Setelah itu, porsi alikuot dari 0,1 ml dari

pengenceran masing-masing disebar pada agar darah domba Columbia (BBL ™, BD,

Heidelberg, Jerman) dan aerob diinkubasi pada 37 ° C selama 48 jam. Koloni dihitung dan

dinyatakan sebagai unit pembentuk koloni (CFU). Nilai CFU transformasi log.

Laser confocal pemindaian mikroskop

Live / Mati-Pewarnaan (BacLight, Invitrogen, Darmstadt, Jerman) digunakan untuk

analisis mikroskopis dari vitalitas bakteri. Biofilm diinkubasi segera dengan pewarna sesuai

dengan instruksi produsen. Setelah inkubasi cakram dibilas dengan larutan NaCl 0,9% untuk

menghilangkan residu zat warna dari biofilm. Sampel dievaluasi dalam mikroskop laser scanning

confocal (CLSM510 Exciter, Zeiss, Jena, Jerman).

Scanning elektron mikroskop

Untuk mikroskop elektron, biofilm disusun sebagai berikut: setelah langkah fiksasi (1

jam dalam 1% glutaraldehid, paraformaldehyde 2%, asam picric 0,2%, HEPES mM 5 (pH 7,4),

dan 50 mM NaN3), sampel diperlakukan dengan asam tanat 2% selama 1 jam, 1% osmium ferri

selama 1 jam, thiocarbohydrazide 1% selama 30 menit, 1% osmium ferri pada 4 ° C semalam,

dan dengan uranil asetat 2% selama 2 jam dengan langkah-langkah mencuci di

antaranya. Sampel penelitian adalah dehidrasi dalam serangkaian bergradasi solusi aseton (10

sampai 100%) dan kemudian kritis-point kering. Akhirnya, sampel yang dipasang di bertopik

aluminium, tergagap dengan emas-paladium dan diperiksa dalam LS10 EVO (Zeiss,

Oberkochen, Jerman)

gambar 1.

Skema pandangan percobaan

Analisa

Untuk semua percobaan, sedikitnya delapan objek setiap tes yang digunakan. Perlakuan

klorheksidin diperlukan 23 disc. Selain itu, benda uji masing-masing delapan yang tersedia untuk

tes kontrol.Data kontinu disajikan sebagai rata-rata ± standar deviasi. Analisis statistik dilakukan

dengan STATVIEW ® 5.0 (SAS, Cary, NC) perangkat lunak. Grafik juga dibuat menggunakan

STATVIEW.Median diberikan dengan kesalahan standar mereka. Korelasi nonparametrik

(Mann-Whitney U-test) diperkirakan untuk perbandingan serapan. P-nilai di bawah 0,05

dianggap signifikan secara statistik.

Hasil

Budidaya biofilm

Prosedur budidaya terus-menerus diperiksa budaya dengan menentukan CFU dan untuk

reproduktifitas dengan mikroskop. Sel kepadatan dari 108 CFU / ml secara teratur dihapus dari

benda uji dengan skala USG.

Optimasi solusi pewarnaan

Penyerapan turunan formazan berwarna XTT dikonversi oleh mikroba adalah ukuran vitalitas

sel. Nilai penyerapan yang tinggi menunjukkan aktivitas metabolisme tinggi. Gambar 2 (Gambar

2) menunjukkan nilai penyerapan (pada 450 nm) dari solusi pewarnaan yang berbeda (XTT +

Pria, XTT + PMS dan XTT + Pria + PMS) pada 24-h-dan 48-jam berusia biofilm. Untuk semua

solusi pewarnaan, bermakna (p <0,01) terlihat perbedaan antara 24-jam dan 48-jam biofilm.

Gambar 2

Diagram absorpsi pada 450 nm (referensi panjang gelombang 620 nm) dari XTT

dimetabolisme oleh biofilm air liur (XTT + menadione, XTT + phenazine methosulfate,

XTT + menadione + N-metil-phenazinium methylsulfate).

Dalam kasus XTT + Pria, penyerapan pada 450 nm meningkat menjadi nilai rata-rata 30 kali

lebih tinggi (0,01-0,30) setelah 48 jam dari setelah 24 jam. Di dalam 24 jam, nilai penyerapan

XTT + PMS meningkat menjadi lima kali (0,10-0,50) nilai asli, sementara penyerapan XTT Pria

+ + PMS meningkat tiga kali 0,20-0,65.

Nilai penyerapan dari XTT + Pria + PMS selalu nyata (p <0,01) lebih tinggi dibandingkan

dengan solusi yang tegang lainnya.

Penentuan rentang pengukuran

Gambar 3 (Gambar 3) menunjukkan nilai serapan pada 450 nm ditentukan oleh cairan air liur

dan pewarnaan selanjutnya dengan XTT + Pria + PMS. Ada perbedaan yang signifikan (p <0,01)

antara nilai penyerapan biofilm hingga penambahan sebesar 4,5 log10.Penyerapan tidak lagi

diukur dalam konsentrasi dari 3,5 log10 (CFU / ml), kecuali untuk kontrol negatif.

Gambar 3

Diagram penyerapan pada 450 nm (referensi panjang gelombang 620 nm) dari XTT + +

methosulfate menadione phenazine dimetabolisme oleh pengenceran air liur berbagai.

Antimikroba pengobatan

Sebuah konversi XTT berkurang diamati pada biofilm air liur yang telah mengalami

pengobatan antimikroba dengan ozon gas dan chlorhexidine. Penurunan ini signifikan setelah

pengobatan ozon 60-s atau 120-s (p <0,02) dan satu menit klorheksidin pengobatan (p <0,01)

(lihat Gambar 4 (Gbr. 4)). Tidak ada perbedaan dalam konversi XTT menggunakan 0,05% atau

larutan klorheksidin 0,1%.Tidak ada perbedaan terlihat di mikrograf elektron scanning antara

biofilm yang tidak dirawat dan biofilm diobati dengan ozon. Dalam kedua contoh, sel-sel muncul

montok dan biofilm memiliki struktur longgar terikat. Bakteri dalam biofilm diobati dengan

klorheksidin yang rusak dan struktur keseluruhan tampaknya lebih ketat (Gambar 5 (Gbr.

5)). Gambar CLSM dikonfirmasi pengamatan ini (Gambar 6 (Gbr. 6)). Setelah pengobatan

dengan ozon, bagian dari biofilm dicelup merah (sel dengan membran yang rusak). Setelah

pengobatan CHX, tidak ada bagian berwarna hijau (sel dengan membran utuh) diidentifikasi.

Gambar 4

Diagram penyerapan pada 450 nm (referensi panjang gelombang 620 nm) dari XTT + +

methosulfate menadione phenazine, dimetabolisme oleh biofilm air liur selama 48 jam

setelah pengobatan dengan larutan NaCl 0,9% (kontrol), ozon gas dan chlorhexidine.

Gambar 5

SEM mikrograf: 48-h biofilm air liur matang: (A) yang tidak diobati, (B) setelah

pengobatan ozon, (C) setelah pengobatan klorheksidin.Perbesaran 10.000 x

Gambar 6

CLSM mikrograf: 48-h air liur biofilm matang: (A) yang tidak diobati, (B) setelah

pengobatan ozon, (C) setelah pengobatan klorheksidin.Pembesaran 100 x

Diskusi

Menyebabkan penyakit gigi yang khas, seperti karies dan periodontitis, biofilm

menyulitkan penghapusan mikroba bertanggung jawab untuk pembentukan biofilm oleh zat

antimikroba. Tujuan dari penelitian ini adalah untuk mengembangkan model biofilm cocok

untuk menguji kemanjuran zat antimikroba dengan non-berbasis budaya deteksi vitalitas biofilm

air liur menggunakan XTT, dan untuk mempersiapkan alat tes XTT cocok.

Penelitian kami memiliki beberapa keterbatasan. Kami menggunakan air liur dari

sukarelawan untuk membuat model biofilm praktis yang relevan. Untuk lebih memahami

interaksi antara bakteri dan XTT, kami melakukan percobaan kami pada cakram titanium mesin

untuk mengecualikan bahan relung hidrofobik retensi, seperti gigi berlubang dan porositas di

mana biofilm bisa menempel. Implan titanium telah berhasil digunakan dalam kedokteran gigi

dan biofilm pada titanium adalah masalah sentral dalam peri-implantitis. Peri-implantitis implan

osseointegrasi lisan bukan monoinfeksi oleh patogen tunggal, melainkan, mereka menunjukkan

karakteristik infeksi campuran. Sedangkan spesies tunggal dari campuran bakteri tidak dapat

menyebabkan abses eksperimental, kombinasi dari spesies ini bisa melakukannya. Plak biofilm

yang mengandung beberapa spesies bakteri yang sesuai harus lebih relevan untuk mempelajari

penyakit gigi dan khasiat antimikroba. Langkah pertama adalah penggunaan metode budidaya

aerob saja. Tes paling XTT dilakukan aerobik.Pada langkah berikutnya kita akan menggunakan

biofilm plak subgingiva dalam kondisi anaerob untuk tes XTT. Tetapi penelitian juga

menunjukkan bahwa mikrobiota yang sama yang dapat ditemukan di sekitar implan (di bawah

kondisi anaerobik) juga dapat ditemukan di sekitar gigi (dalam kondisi aerobik).

XTT adalah garam tetrazolium tidak berwarna, yang diubah menjadi turunan, berwarna

larut air formazan oleh dehydrogenases, dengan dehidrogenase suksinat menjadi sangat penting

karena memainkan peran utama dalam penyediaan energi setiap sel hidup individu. Tidak seperti

garam tetrazolium lain (misalnya, CTC dan TTC), XTT tidak memerlukan formazans larut harus

diekstrak.Karena perubahan warna dalam larutan dapat langsung ditentukan oleh fotometri, tes

XTT memungkinkan sejumlah besar objek pengujian yang akan diuji untuk vitalitas mereka

sangat cepat.Inkubasi bakteri dengan XTT selama 3 jam pada 37 ° C telah menjadi subyek dari

beberapa publikasi. Apa yang baru adalah komposisi dari solusi pewarnaan XTT

diterapkan. Biofilm air liur mengandung berbagai macam mikroba yang berbeda (Gram positif,

bakteri Gram-negatif dan jamur). Untuk mengkonversi XTT, mikroba ini memerlukan berbagai

aditif yang berfungsi sebagai pembawa elektron. Aditif standar untuk Candida spp.adalah

menadione, tetapi PMS dapat diterapkan juga. Untuk bakteri Gram-positif cocci, PMS digunakan

dalam kebanyakan, tapi kadang-kadang menadione juga digunakan. Dalam kasus Gram-negatif

berbentuk batang bakteri, terutama menadione diterapkan. Awal kami sendiri (tidak diterbitkan)

investigasi mengkonfirmasi hasil ini pada kesesuaian menadione untuk Candida albicans dan

PMS untuk Streptococcus mutans dan Streptococcus sanguinis sebagai wakil dalam kedokteran

gigi. Adapun Pseudomonas aeruginosa, aplikasi gabungan dari menadione dan PMS ternyata

cocok. Dengan menggunakan analisis berbasis budaya dari air liur, Gram-positif cocci bakteri,

i. a., dapat diisolasi. Oleh karena itu, penambahan PMS tampaknya sangat diperlukan untuk

mendeteksi kolorimetri menggunakan XTT. McCluskey dkk. juga digunakan secara eksklusif

PMS untuk deteksi kolorimetri mikroba terjadi di lumpur aktif, dan mampu membuktikan bahwa

ada korelasi langsung antara produksi dan konsumsi oksigen formazan.

Dalam perbandingan langsung, PMS-menadione gabungan elektron mediator

menunjukkan konversi XTT tertinggi dalam biofilm air liur (lihat Gambar 2 (Gambar

2)). Ternyata, bagaimanapun, bahwa jumlah sel tinggi minimal 4,5 log10 (CFU / ml) diperlukan

untuk mendeteksi penurunan XTT (lihat Gambar 4 (Gbr. 4)). Sebesar 5,5 log10 (CFU / ml),

penyerapan sangat tinggi (1,91) dideteksi pada 450 nm. Semakin tinggi jumlah sel yang aktif

secara metabolik, sinyal semakin tinggi kolorimetri. Selain itu, semakin tinggi metabolisme sel,

semakin tinggi sinyal. Jelas, tidak ada hubungan linier antara jumlah sel-sel dan sinyal

kolorimetri.Ketika pewarna lainnya (FDA und Syto9) digunakan, adsorpsi bahkan tetap

konstan. Setelah 48 jam biofilm, kepadatan sel pembentukan ca. 8 log10 (CFU / ml) tercapai,

tetapi mengikuti tes XTT, penyerapan dalam biofilm adalah 0,65, yaitu, kurang dari pada bakteri

planktonik meskipun kepadatan sel lebih tinggi.Jumlah produk yang ditahan dapat bervariasi

antara bakteri planktonik dan biofilm. Selain itu, biofilm dikenakan kondisi selain segar bakteri

suspensi. Banyak patogen gigih dan, karenanya, menunjukkan aktivitas metabolik yang lebih

rendah.Untuk alasan ini, jumlah minimum patogen tidak dapat ditentukan dari suspensi dan

biofilm secara persis sama. Selain itu, sel planktonik dapat berinvestasi lebih banyak energi

dalam metabolisme rutin. Dalam percobaan kami, penyerapan adalah 30 kali lebih tinggi setelah

48 jam dari setelah 24 jam, yaitu konversi XTT meningkat karena pertumbuhan

biomassa. Namun, tingkat metabolisme yang berbeda tidak menyebabkan peningkatan

logaritmik dari pengurangan XTT. Pengamatan XTT terkait juga dibuat oleh kelompok

penelitian lain.

Mengurangi formazan pembentukan diamati karena pengobatan antimikroba dari biofilm

air liur. Akibatnya, tes XTT cocok untuk menentukan kemanjuran zat antimikroba, terutama

untuk penyaringan. Konsentrasi klorheksidin rendah digunakan telah diselidiki oleh peneliti lain,

yang mencatat khasiat antimikroba tidak cukup dalam biofilm. Di sisi lain, klorheksidin terbukti

menjadi sedikit lebih unggul ozon. Ini juga telah diterbitkan oleh kelompok penelitian lain yang

menerapkan metode alternatif. Namun, in vivo Hauser-Gerspach dkk. tidak menemukan efek

antimikroba yang signifikan dari CHX dan ozon. Para mikrograf elektron scanning

mengkonfirmasi hasil ini. Setelah pengobatan ozon, morfologi sel-sel menunjukkan tidak ada

perbedaan. Walaupun biofilm diobati dengan klorheksidin tampaknya rusak dibandingkan

dengan kontrol, hanya beberapa sel yang secara morfologis cacat.

Terlepas dari beberapa kelemahan, XTT dengan penambahan menadione dan PMS

adalah metode yang cocok untuk menentukan vitalitas dalam biofilm bakteri air liur dan

penilaian izin dari efektivitas zat antimikroba.

Pengujian adalah mudah dilakukan, dan memungkinkan sejumlah besar benda uji yang

akan diuji. Hal ini sangat cocok untuk skrining berbagai faktor yang mempengaruhi biofilm,

seperti antiseptik atau perawatan fisik atau kimia lainnya, misalnya, terapi ozon, photodynamic

atau plasma tekanan atmosfer.