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VOLUME 9 - NUMBER 2 - 2018
Photodynamic therapy on a biofilm monospecie of candida albicans: an in vitro study



 Palaia G., Tenore G., Del Vecchio A., Pergolini D., Tramutola L., Berlutti F., Romeo U. 
Original Article, 65-71
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Antimicrobial photodynamic therapy (aPDT) is a medical treatment based on the use of a light source at a specific wavelength that activates a photosensitive molecule causing the formation of oxidizing agents that provokes the death of the target cells. The aim of this study was to evaluate the activity of aPDT against biofilms of Candida albicans. Biofilms of C.albicans were subjected to aPDT using a diode GaAlAs (λ:635nm) and a toluidin blue solution (0.1mg/ml) as photosensitizer. Three different protocols (1 minute, fluence 24J/cm2; 2 minutes, fluence 48J/cm2 and 3 minutes, fluence 72J/cm2) were tested. To evaluate the number of Candida cells in biofilm before and after treatment, Bio Timer Assay was used. Candida biofilms treated with aPDT showed significant reductions of the microbial population as compared to the control groups. aPDT showed good antifungal properties in vitro. Clinical trials are needed to test its efficacy in vivo and to propose it as an adjuvant or alternative therapy to conventional medical treatment.




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Annali di stomatologia
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Photodynamic therapy on a biofilm monospecie of candida albicans: an in vitro study
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Photodynamic therapy on a biofilm monospecie of candida albicans: an in vitro study
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                                                                                                Original article



    Photodynamic therapy on a biofilm monospecie of
    candida albicans: an in vitro study




                                                                                             li
                                                                                           na
    Gaspare Palaia1 DDS, PhD                              Key words: photodynamic therapy, oral candidia-
    Gianluca Tenore1 DDS, PhD                             sis, Bio Timer Assay, antifungals.
    Alessandro Del Vecchio1 DDS, PhD
    Daniele Pergolini1 DDS
                                                          Introduction




                                                                                         io
    Lorenzo Tramutola1 DDS
    Francesca Berlutti2 BScD
    Umberto Romeo1 DDS                                    The incidence of fungal infections has been increas-




                                                                    az
                                                          ing since the 1980s, especially for systemic forms in
                                                          immunocompromised and/or hospitalized populations
                                                          with other systemic diseases. (1) This increase is
    1 Department of Oral and Maxillofacial Sciences,      partially due to medical interventions, resulting in im-




                                                                  rn
     “Sapienza” University of Rome, Rome, Italy           munosuppression that leaves patients at risk for de-
    2 Department of Public Health Sciences,               veloping fungal infections (e.g., patients undergoing
    “Sapienza” University of Rome, Rome, Italy            bone marrow transplants, blood transfusions, im-
                                                          munosuppressive therapy, invasive surgery, use of




                                                         te
                                                          broad-spectrum antibiotics, anti-cancer chemothera-
                                                          py, and AIDS patients). Moreover, fungal pathogens
    Corresponding author:                                 have many virulence features, such as the ability to
                                                       In
    Gaspare Palaia                                        switch themselves among different morphological
    Department of Oral and Maxillofacial Sciences,        states, biofilm formation and resistance to antifungal
    “Sapienza” University of Rome                         drugs (2, 3).
    Via Feronia 148                                       Candida albicans, a polymorphic microorganism, has
    00157 Rome, Italy                                     become one of the most common agents of nosoco-
                               ni

    Tel.: +39 3394522515                                  mial infection in immunocompromised patients and
    Fax: +39 067806974                                    those undergoing long-term treatment with antibiotics
    E-mail: gaspare.palaia@uniroma1.it                    or other immunosuppressive therapies. In these pa-
                             io


                                                          tients, C. albicans invades the deeper tissues and
                                                          can cause life-threatening systemic infections.
    Summary                                               Candidemia occurs with an infection rate of 8-10 out
                           iz



                                                          of 100,000 people a year and is associated with a
    Antimicrobial photodynamic therapy (aPDT) is a        mortality rate of 30-50% (4-6). However, the available
    medical treatment based on the use of a light         epidemiological data are rather heterogeneous be-
            Ed




    source at a specific wavelength that activates a      cause of the clinical features of infections and the
    photosensitive molecule causing the formation of      lack of official records or statistics.
    oxidizing agents that provokes the death of the       Candidiasis and oral candidiasis are opportunistic in-
    target cells. The aim of this study was to evaluate   fections of the skin and oral cavity. They are common
    the activity of aPDT against biofilms of Candida      in old people, especially denture wearers, children and
    albicans. Biofilms of C.albicans were subjected to    immunocompromised patients. C. albicans is the causal
 IC




    aPDT using a diode GaAlAs (λ:635nm) and a tolu-       agent in 50% of oral mycosis cases, although in recent
    idin blue solution (0.1mg/ml) as photosensitizer.     years, a high incidence of non-albicans species (C.
    Three different protocols (1 minute, fluence          tropicalis, C. krusei, C. glabrata, C. parapsilosis and C.
    24J/cm 2; 2 minutes, fluence 48J/cm 2 and 3 min-      dublinensis) has been documented (7, 8).
C




    utes, fluence 72J/cm 2) were tested. To evaluate      Candidiasis is the most common intra-oral infection in
    the number of Candida cells in biofilm before and     AIDS patients, and it is caused by an overgrowth of
    after treatment, Bio Timer Assay was used. Candi-     Candida species. In fact, 84% of patients with HIV in-
    da biofilms treated with aPDT showed significant      fection develops candidiasis (9). Patients with can-
©




    reductions of the microbial population as com-        didiasis often report other disorders such as burning
    pared to the control groups. aPDT showed good         and taste alteration, as well as pain, dysphagia, nau-
    antifungal properties in vitro. Clinical trials are   sea, vomit and diarrhea at times. Such symptoms can
    needed to test its efficacy in vivo and to propose    disrupt feeding, worsening quality of life. Treatment
    it as an adjuvant or alternative therapy to conven-   with topical or oral antifungal agents, such as nys-
    tional medical treatment.                             tatin, amphotericin B or ﬂuconazole can achieve only

    Annali di Stomatologia 2018;IX (2):65-71                                                                     65
    G. Palaia et al.


    transient responses during treatment, generally for 15      Before proceeding to aPDT, the wells were washed
    days (10). Recurrences are very common, consider-           three times with phosphate buffer saline (PBS) to re-
    ing the multifactorial etiology of candidiasis, and         move the non-filmy adherent fungus.
    widespread use of fungicides has resulted in the de-
    velopment of resistant species, especially C. albi-         Photodynamic treatment
    cans.                                                       The aPDT was performed using a prototype diode
    In addition, antifungal agents can reduce the effec-        laser device GaAlAsat with a wavelength (λ) of 635




                                                                                                   li
    tiveness of antiretroviral drugs, and this reduction is a   nm (Doctor Smile, Brendola, Italy), using a power of




                                                                                                 na
    serious problem considering the systemic diseases           0.1 mW, in continuous wave mode, with an optical
    that often affect these patients (11).                      fiber of 300 μm.
    Antimicrobial photodynamic therapy (aPDT) is a med-         Irradiations were performed maintaining the fiber at
    ical treatment based on the use of a light source at a      the distance of 11 mm from the bottom of the wells
    specific wavelength that actives a specific photosen-       using the fiber sheath that blocked the fiber itself in a




                                                                                               io
    sitive substance, which reacts in the presence of oxy-      perforated cover plate. The irradiated area corre-
    gen causing the formation of oxidizing agents, such         sponded to about 50 mm2.
    as singlet oxygen and free radicals. Therefore, PDT         When required, the wells containing C. albicans




                                                                          az
    causes the death of the target cells through mem-           biofilm were treated using the laser light for 1, 2 or 3
    brane lysis and protein inactivation.                       minutes. In these conditions, keeping constant the
    The use of aPDT for inactivating microorganisms was         other parameters, three different energies of 12, 24
    first demonstrated more than 100 years ago by Oscar         and 36 J, respectively, and a fluence of 24, 48 and 72




                                                                        rn
    Raab when he reported the lethal effect of acrinide         J/cm2, respectively, were obtained.
    hydrochloride and visible light on Paramecia cauda-         Toluidine blue (TB) (Sigma-Aldrich Co. St. Louis MO-
    tum (12). However, it is only in recent times that          USA) was used as the photo-activating substance. A
    aPDT has been studied as a medical treatment.               total of 50 μl of 0.1 mg/ml TB solution was added to




                                                         te
    Photodynamic therapy has been approved in many              the wells containing C. albicans biofilm. After 5 min-
    countries for oncological clinical treatment, especially    utes, the TB solution was removed, and then, the
    in head and neck tumors, Several studies have               laser irradiation was performed. The wells were divid-
                                                       In
    shown the antimicrobial properties of aPDT against          ed into 8 groups and treated as follows:
    Gram positive and Gram negative bacteria, fungi and         Group 1: biofilm subjected to laser irradiation with TB
    viruses (13, 14); therefore, it may be considered an        for 1 minute (L1+P+)
    effective alternative to the use of antimicrobial           Group 2: biofilm subjected to laser irradiation with TB
    agents.                                                     for 2 minutes (L2+P+)
                             ni

    Despite the promising advantages that aPDT has,             Group 3: biofilm subjected to laser irradiation with TB
    such as the lack of selection of resistant strains, the     for 3 minutes (L3+P+)
    absence of mutagenic effects, and the almost total          Group 4: biofilm subjected to laser irradiation for 1
                           io


    absence of side effects and drugs interactions in topi-     minute in absence of TB (L1+P-)
    cal applications (15), a standardized protocol for the      Group 5: biofilm subjected to laser irradiation for 2
    application of such therapy in patients being treated       minutes in absence of TB (L2+P-)
                         iz



    for superficial fungal infection is lacking.                Group 6: biofilm subjected to laser irradiation for 3
    The aim of this work was to test, in vitro, the efficacy    minutes in absence of TB (L3+P-)
    of aPDT against C. albicans biofilm using a diode           Group 7: biofilm exposed to TB in the absence of
           Ed




    laser as a light source and toluidine blue (TB) as a        laser irradiation (L-P+)
    photo-activating agent.                                     Group 8: biofilm not subjected to any treatment (L-P).
                                                                After the treatment, the wells were washed once with
                                                                PBS to remove the dead fungal cells.
    Materials and methods
                                                                Bio Timer Assay
 IC




    Biofilm production                                          BTA is an indirect method that measures the concen-
    In this study, Candida albicans strain ATCC 24433           tration of microbes in biofilm (16-21). BTA employs
    was used. To obtain C. albicans biofilm, the fungus         BioTimer medium with red phenol (BT-PR medium)
    was plated on Brain Heart Infusion (BHI) agar medi-         prepared as follows: BHI 37 g/L, glucose 5 g/L phenol
C




    um and incubated at 37°C for 24 h. After incubation,        red 25 mg/L and distilled water to 1000 ml. After ster-
    10 isolated colonies were picked from the plate with a      ilization at 121°C for 15 minutes, the pH was checked
    sterile loop and placed into 10 ml of BHI broth medi-       and adjusted to 7.2±0.1. The final medium appeared
    um. After 24 h of incubation at 37°C, the optical den-      clear and red. BTA measures the microbial metabo-
©




    sity (λ 600 nm) of the culture was adjusted to obtain a     lism: the time required for color switching of the phe-
    microbial suspension of approximately 1x105 CFU/ml.         nol red indicator in BT-PR medium (red-to-yellow)
    A volume of 0.1 ml was used to inoculate a sterile 96-      (Fig. 1), due to fungal metabolism, is correlated to the
    well plate. The 96-well plate was incubated in a hu-        initial fungal concentration. Therefore, the time re-
    mid atmosphere at 37°C for 72 h to obtain a biofilm         quired for a color switch determined the number of
    adherent to the walls of the wells.                         fungi present in a sample at Time 0 through a correla-

    66                                                                              Annali di Stomatologia 2018;IX (2):65-71
    Photodynamic therapy on a biofilm monospecie of candida albicans: an in vitro study


                                                                                      Figure 1. Reagent with phenol red
                                                                                      (BT-PR): initial color (red on the left)
                                                                                      and after the “switching” (yellow on the
                                                                                      right).




                                                                                                        li
                                                                                                      na
                                                                                                    io
                                                                             az
                                                                           rn
    tion line. To draw the correlation line specific for Can-     equation: t = y = -0.3436x + 7.3616 with r2= 0.9957
    dida spp., 0.02 ml of BHI-overnight broth cultures            (Fig. 2).




                                                         te
    were mixed with 0.18 ml of BT-PR medium. Serial               To evaluate the number of C. albicans cells in the
    two-fold dilutions in 0.1 ml of BT-PR medium were             biofilm, the colonized wells were supplemented with
    performed in 96-well plates (BD, Italy) and simultane-        BT-PR medium. The time required for a color switch
    ously counted using the colony forming unit (CFU)             in each well was recorded using the Tecan apparatus
                                                       In
    method. The time required for color switching of inoc-        and it was used to determine the number of viable
    ulated 96-well plates was recorded using Tecan Sun-           fungi by the correlation line. As the correlation line
    rise, a fully automatized spectrophotometer appara-           was constructed on planktonic cultures, the number
    tus. The apparatus allowed the incubation at 37°C of          of Candida in the biofilm is defined as the planktonic-
                               ni

    the 96-well plates and the recording of the ODs (λ            equivalent CFUs (PE-CFUs).
    570 and 450 nm) of each well every 15 minutes for
    24 hours. The time for a color switch was plotted ver-        Statistics
                             io


    sus the log10 of CFUs to calculate the correlation line       All experiments were repeated at least three times to
    that relates the time for a color switch with the num-        obtain the mean value and standard deviation. Statis-
    ber of fungi. The correlation line was described by the       tical analysis was performed using Student’s T test,
                           iz



                                                                                          Figure 2. Correlation curve.
            Ed
 IC
C
©




    Annali di Stomatologia 2018;IX (2):65-71                                                                               67
    G. Palaia et al.




                                                                                                         li
                                                                                                       na
                                                                                                     io
                                                                                 az
                                                                               rn
                                                            te
    Figure 3. Overall results for the group in terms of logCFU/ml with relative DS.
                                                          In
    comparing the groups by their average values, and P               Discussion
    (probability) values lower than 0.05 (P≤0.05) were
    considered significant. The correlation line was ob-              C. albicans is a saprophytic fungus that is present in
    tained using linear regression analysis, and the linear
                               ni

                                                                      the oral cavity of many people (>80%), and it is re-
    correlation coefficient was calculated with the equa-             sponsible for superficial infections of the mucous
    tion: r=(nΣxy−ΣxΣy)/{sqrt[nΣx2−(Σx)2][nΣy2−(Σy)2]}.               membranes (90%) and systemic infection (44-62% of
                                                                      cases) (22). C. albicans can be found in both plank-
                             io


                                                                      tonic and biofilm lifestyles.
    Results                                                           Increasing resistance by fungi versus the various
                                                                      conventional medical therapies, the appearance of
                           iz



    Results are expressed as the mean value ± standard                new emerging pathogens and the antagonism of
    deviations of the CFU log obtained from at least three            drugs in patients are the reasons that push the scien-
    independent experiments (Fig. 3).                                 tific community to look for alternative therapies.
           Ed




    Laser treatments without photoactivator did not effi-             Currently, most studies assessing the performance of
    ciently kill the C. albicans biofilm population, and the          anti-Candida laser light at low fluence (LLLT) em-
    reduction values (of approximately 24%) were not                  ployed cultures of fungus in the planktonic state.
    significant according to the length of the treatment (1,          The main objective of this study was to evaluate the
    2, or 3 minutes). Similarly, the treatment with the pho-          effects of three protocols of laser irradiation against
    toactivator alone did not decrease significantly the              C. albicans grown in biofilm, and precisely, the three
 IC




    number of CFUs.                                                   protocols were as follows: low level laser irradiation in
    By contrast, the aPDT treatments reduced the C.                   association with the Toluidine Blu (TB) as photo acti-
    albicans biofilm population to different extents, de-             vator (aPDT protocol), low level laser irradiation in
    pending on the time of treatment. Comparing values                the absence of TB (L), and only TB in the absence of
C




    of the population before and after treatment, aPTD                any laser irradiation. TB was chosen according to
    significantly reduced the number of C. candida cells              Souza et al. (2010) (24), which demonstrated that
    already after 1 minute of laser irradiation. Reduc-               employing a diode laser (Ga-Al-As) 660 nm, 0.035W
    tions ranging from 65 to 79% were recovered, and                  with fluence of 15.8, 26.3 and 39.5 J/cm2 with 171,
©




    the highest rate of reduction was observed after 2                285 and 428 seconds of irradiation, respectively, had
    and 3 minutes of treatment (Fig. 4). However, the                 the highest efficacy against planktonic C. albicans
    differences in values of Candida reduction between                when using TB as a photoactivator.
    the different times of treatments were not signifi-               In our study, a Bio Timer Assay (BTA) was used for
    cant.                                                             the first time to counts of C. albicans biofilms. The


    68                                                                                    Annali di Stomatologia 2018;IX (2):65-71
    Photodynamic therapy on a biofilm monospecie of candida albicans: an in vitro study


                                                                                          Figure 4. Percent reductions of Can-
                                                                                          dida albicans ATCC 24433 popula-
                                                                                          tion in biofilm after laser treatment in
                                                                                          the absence or in the presence tolui-
                                                                                          dine blue as photoactivator. C.albi-
                                                                                          cans biofilm was treated for 1, 2, or
                                                                                          3 minutes with laser in the absence




                                                                                                        li
                                                                                          (L1, L2, L3) or in the presence of
                                                                                          photoactivator toluidine blue (LP1,




                                                                                                      na
                                                                                          LP2, LP3).




                                                                                                    io
                                                                             az
    method, already used for counting Staphylococcus,             power of 100 mw for 98 seconds with a fluence of
    Streptococcus and Enterococcus in biofilms (19-21),           350 J/cm 2 alone or in combination with methylene
    is the only method that is able to count biofilm sam-         blue as a photoactivator on single, double and triple




                                                                           rn
    ples without any manipulation of the samples (7). For         biofilms formed by C. albicans, Sthaphylococcus au-
    this purpose, a correlation line showing the time of          reus and Streptococcus mutans. The study demon-
    color switches of the BTA medium and the number of            strated a significant reduction in all species of mi-
    planktonic C. albican cells was constructed, and the



                                                         te
                                                                  croorganisms after the application of laser in combi-
    correlation line was described by a linear equation.          nation with photosensitizer; in particular, mono-
                                                                  species biofilms were more susceptible compared to
    The results of the present study showed that laser            multispecies of biofilms.
                                                       In
    treatment without a photoactivator was inefficient in         However, it should be underscored that the fluence
    significantly reducing the Candida biofilm indepen-           employed in the abovementioned studies on both
    dently of the length of treatment. These results              planktonic and biofilm Candida were from 5.5- to 30-
    agreed with Wilson and Mia (1993) (23), which                 fold higher than the lower fluence used in the present
                               ni

    showed that the He-Neon laser at a wavelength of              study (24, 48 and 72 J/cm 2 ). In fact, our results
    632.8 nm and 66.36 J/cm2 of fluence in the absence            showed that a significant reduction can be achieved
    of the photoactivator was unable to kill planktonic C.        even when using 24 J/cm2 fluence and that the in-
                             io


    albicans. In contrast, the combined use of laser and          crease in the fluence was not able to raise the killing
    photoactivator (i.e., methylene blue, crystal violet and      rate of Candida biofilm. The explanation for this phe-
    thionine) reduced planktonic C. albicans by approxi-          nomenon could be the different photoactivator con-
                           iz



    mately 77%. Similar results were obtained against             centrations, the intrinsic photoactivator properties, or
    planktonic C. tropicalis, and C. stellatoidea whose           even the responsiveness of the specific strain of C.
    killing rate was 65 and 63%, respectively. Similar re-        albicans. Another possible explanation for this phe-
            Ed




    sults were reported by Queiroga et al. (2011) (25);           nomenon could be offered assuming the peak of
    testing the effects of irradiation with a diode laser 640     maximum effectiveness was reached in a time of ap-
    nm on planktonic Candida ssp using 3 different flu-           proximately 60 seconds.
    ences, i.e., 60, 120 and 180 J/cm2 in the presence of         According to previous works on a murine model of
    methylene blue, showed that the higher reduction of           oral candida infection (1, 28-30) and on the treatment
    Candida was obtained at a fluence of 180 J/cm2.               of HIV positive patients with aPDT (10), this study
 IC




    When laser treatment was carried out in the presence          confirmed the efficacy of photodynamic therapy
    of the TB photoactivator, significant reductions in the       against C. albicans.
    C. albicans biofilm were observed. The duration of            The observation that the killing rate did not change
    the treatment did not influence the killing rate, and no      with increases in fluence is important in the case of
C




    significant differences were recorded after 1, 2 or 3         laser application on patients for the clinical treatment
    minutes of treatment.                                         of C. albicans oral infections. In fact, the previous
    The efficacy of laser treatment in the presence of the        studies are applicable for clinical use, especially for
    photoactivator confirmed the efficacy of LLLT in com-         the excessively long times of irradiation that are not
©




    bination with photosensitizing agents against plank-          reproducible in patients. Moreover, even if the side ef-
    tonic Candida. However, Authors are focused, nowa-            fects of laser irradiation were not described, putative
    days, on the evaluation the killing rate of laser treat-      cell damage cannot be excluded due to the duration of
    ment against Candida biofilm (26). Our results are in         laser applications. Consequently, the possibility of us-
    agreement with Pereira et al. (2010) (27). The Au-            ing a true LLLT by decreasing the putative side effects
    thors tested the effects of diode laser 660 nm at a           may increase the compliance of the patients.

    Annali di Stomatologia 2018;IX (2):65-71                                                                                   69
    G. Palaia et al.


    In this study, we verified the framework that emerged                           of oral candidiasis with methylene blue-mediated photody-
    in the literature on the effectiveness of photodynamic                          namic therapy in an immunodeficient murine model.Oral Surg
    protocols implemented as antifungal treatments.                                 Oral Med Oral Pathol Oral Radiol Endod. 2002;93(2):155-
                                                                                    160.
    Here, we showed the efficacy of aPDT in significantly
                                                                              10.   Scwingel AR, Barcessat AR, Núñez SC, Ribeiro MS. An-
    reducing the fungal population of C. albicans ATCC
                                                                                    timicrobial photodynamic therapy in the treatment of oral can-
    24433 grown in biofilm using a diode laser l 635 nm                             didiasis in HIV-infected patients. Photomed Laser Surg.
    as a light source, already at the fluence of 24 J/cm2.




                                                                                                                          li
                                                                                    2012;30(8):429-432.
                                                                              11.   Gallè F, Sanguinetti M, Colella G, Di Onofrio V, Torelli R,




                                                                                                                        na
                                                                                    Rossano F, Liguori G. Oral candidosis: characterization of
    Conclusions                                                                     a sample of recurrent infections and study of resistance de-
                                                                                    terminants. New Microbiol. 2011;34:379-389.
    Even if more studies are needed to optimize the anti-                     12.   Nikolaus S, Soukos & J. Max Goodson Photodynamic ther-
                                                                                    apy in the control of oral biofilms Periodontology.
    Candida protocol, this study suggests a possible use




                                                                                                                      io
                                                                                    2000;55:2011, 143-166.
    of aPDT in “in vivo infection”. For this purpose, it will                 13.   Demidova TN, Hamblin MR. Photodynamic therapy target-
    be necessary to carry out clinical trials that could                            ed to pathogens. Int J Immunopathol Pharmacol. 2004;17
    show the real evidence for fungicidal effects of aPDT                           (3):245-254.




                                                                                          az
    to propose this procedure as an alternative or adju-                      14.   Sardi JC1, Scorzoni L, Bernardi T, Fusco-Almeida AM,
    vant to traditional drug therapies currently in use for                         Mendes Giannini MJ.Candida species: current epidemiolo-
    the treatment of oral infections.                                               gy, pathogenicity, biofilm formation, natural antifungal prod-
                                                                                    ucts and new therapeutic options. J Med Microbiol. 2013;62(Pt




                                                                                        rn
                                                                                    1):10-24.
    Conflict of interest
                                                                              15.   Baltazar LM, Ray A, Santos DA, Cisalpino PS, Friedman AJ,
                                                                                    Nosanchuk JD. Antimicrobial photodynamic therapy: an ef-
                                                                                    fective alternative approach to control fungal infections. Front
    The Authors declare that they have no conflict of in-




                                                                     te
                                                                                    Microbiol. 2015;6:202.
    terest.                                                                   16.   Konopka K, Goslinski T. Photodynamic therapy in dentistry.
                                                                                    J Dent Res. 2007;86(8):694-707.
                                                                              17.   Pantanella F, Valenti P, Frioni A, Natalizi T, Coltella L, Berlut-
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                                                                   In
                                                                                    ti F. BioTimer Assay, a new method for counting Staphylo-
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                                                                                    to evaluate antibiotic susceptibility of biofilm. J Microbiol Meth-
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                                                                                    ods. 2008;75(3):478-484.
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                                  ni

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    Annali di Stomatologia 2018;IX (2):65-71                                                                                        71
