Effects of Nd:YAG laser irradiation on the growth of Candida albicans and Streptococcus mutans: in vitro study

The human oral cavity is a conducive environment to unrestricted formation of natural microbial biofilm. However, in a distorted equilibrium balance of the oral health, infectious pathogens may gain access into the dental tissues and gingival area. Infectious pathogens from the oral cavity cause oral diseases such as caries, gingivitis, periodontitis, endodontic infections, and alveolar osteitis, and sometimes are concomitant to systemic diseases, including cardiovascular disorders, stroke, preterm birth, diabetes, and pneumonia, among others [1, 2]. Streptococcus mutans is one of the main bacterial strains colonizing the oral cavity and a major contributor of tooth decay, which in turn may affect the overall health of the host [3]. The fungal species of Candida albicans is by far the most commonly detected fungal organism in humans, part of the healthy human microbiota, but in immunocompromised hosts, it may cause a number of infections, ranging from superficial infections of the mucosa and skin to life-threatening systemic infections. C. albicans cells are frequently found along with S. mutans-derived plaque biofilms. Recent studies indicate high prevalence of S. mutans in dental biofilm where the fungal pathogen C. albicans resides, suggesting that this association is involved in the enhancement of biofilm virulence [4, 5]. C. albicans coadheres with S. mutans in the presence of sucrose [5‐7]. Such bacterium-fungus association may enhance S. mutans infection [8] and augment fungal carriage and infectivity of mucosal disease [9].

Management of infections caused by bacteria and fungi is a viable challenge in various medical fields, including dentistry. It cannot be emphasized enough that we need to focus on the search for alternative methods to manage rapidly developing drug resistance and recurrent candidiasis [10]. The development of laser medicine has provided a number of new therapy modalities capable of damaging pathogenic organisms. Photoantimicrobial therapy is safe, effective, and easy to implement and its activity spectrum covers bacteria, fungi, viruses, and protozoa, which make it superior to conventional therapies [11]. There are studies reporting antimicrobial and bactericidal effects of laser application [10]. Among them, one promising treatment modality is low-level laser therapy (LLLT), in which non-thermal laser irradiation is applied to the target site. LLLT uses mainly semiconductor lasers at 685 and 830 nm wavelengths, which are considered safe and effective against biofilm-associated infections [10]. In this study, Nd:YAG laser at 1064 nm was used based on the assumption that its characteristic qualities, high scattering effect, and deep soft tissue penetration could be more effective in reduction of C. albicans and S. mutans.

Antibacterial effects of Nd:YAG laser in patients treated for oral diseases have been confirmed in previous research, where pulsed Nd:YAG laser was applied with power settings over 2 W to induce a photothermal effect [12‐14]. The efficacy of laser irradiation has been demonstrated against Escherichia coli, Staphylococcus aureus, Actinomyces naeslundii, Pseudomonas aeruginosa, Enterococcus faecalis, and Streptococcus anginosus [12, 15, 16].

The present study aimed at evaluating the effects of laser therapy on pathogenic organisms commonly inhabiting the oral cavity using Nd:YAG with a flat-top handpiece. Flat-top handpiece spreads the energy of the laser beam evenly over the treated area in a no contact mode. It can be used at a variable distance of up to 100 cm without modifying the irradiation energy over a 1-cm spot diameter. The handpiece is most commonly used for biostimulation and anti-inflammatory treatment. The present study explored the potential of Nd:YAG laser with flat-top handpiece to affect the in vitro growth of S. mutans and C. albicans—the two oral microbials which may become pathogenic in the presence of predisposing factors, producing infections that range from local to systemic.

To evaluate the effect of laser on selected cultures, quantitative culture technique (indicating the number of colony-forming units [cfu]/ml) and microscopic analysis with Janus green stain were used. All tested and control samples were subjected to triplicate procedure.

Numerous studies on low-level laser therapy in combination with infrared lasers over the last 20 years provided positive results and thus a basis for the introduction of this technology into clinical practice to stimulate wound healing, treat inflammation, and reduce pain [17‐19]. However, development of new optical systems and handpieces to photons transport into a beam generated by Nd:YAG laser to the point of application demands verification of the knowledge of using LLLI on cells and tissues. Laser light has also been used in antimicrobial therapy. Studies indicate that laser application can effectively reduce or eliminate pathogenic organisms [20‐23]. The study aimed at evaluating the effects of Nd:YAG irradiation on the in vitro growth of Candida albicans and Streptococcus mutans. The major finding of the study is that Nd:YAG laser with a flat-top handpiece resulted in the reduction of C. albicans and S. mutans at fluence below the soft tissue ablation threshold (0.03 and 0.13 J/cm²). Compared to the control group, irradiation using flat-top handpiece of Nd:YAG laser resulted in the decrease of the cfu/ml value for both C. albicans and S. mutans. These findings indicate that 1064 nm infrared wavelength is efficient and safe in reduction of the total number of irradiated microorganisms.

Our review of the available literature provides no record of using Nd:YAG laser with the flat-top handpiece in both C. albicans and S. mutans irradiation. The analysis of the effects of laser irradiation revealed a statistically significant reduction of both C. albicans and S. mutans cultures for both sets of parameters of laser application. In both cases with a lower dose (3 J at 0.25 W, 10 Hz) or higher dose (59 J at 1 W, 10 Hz), the number of pathogens was reduced.

A recent in vivo study by Piccolo et al. evaluated the effect of Nd:YAG irradiation to fight fungal overgrowth in the nail plate and indicated a great potential for safe and effective clinical application. The use of long-pulsed 1064 nm Nd:YAG laser (neodymium-doped yttrium aluminum garnet) for the treatment of Onychomycosis (caused by Candida species, among other pathogenic fungi) has demonstrated promising results [21]. However, the authors used the laser with 5 msec pulse duration and 1 Hz repetition rate at the fluence of 30 J/cm², thus the fungal destruction was evoked strictly by photothermal effects what authors underline in the conclusion.

The use of LLLT with wavelength below 900 nm for C. albicans eradication was described in literature [20, 22‐24]. Risovic et al. [20] observed the wavelength-dependent eradication efficiency of C. albicans, where most effective wavelength for elimination of C. albicans was UV-C 254 nm (Δ = 6.1 mJ/cm⁻², ET99.99 = 56 mJ/cm⁻²) and least efficient 406 nm (Δ = 11.4 J/cm⁻², ET99.99 = 104 J/cm⁻²). Gupta et al. [24] and Rosa et al. [22] confirmed that violet-blue visible light (400–490 nm) does not require the use of photosensitizer to kill C. albicans cells in biofilms. Other studies [10, 23] proved that laser light at 685 nm and 830 nm might be considered a promising treatment for C. albicans infections. Also, our present study showed the ability of 1064-nm Nd:YAG laser in eradication C.albicans at dose 3.0 J/cm² (0.25 W) and 59 J/cm² (1 W).

Seyedmousavi et al. [10] observed that LLLT with energies > 10 J at both 685 and 830 nm wavelengths produced statistically significant effects in vitro on the pathogenicity of C. albicans, and in vivo on the survival rate of mice (p value ≤ 0.05). In turn, Maver-Biscanin et al. [23] in their in vivo study reduction of C. albicans species in the treated areas were irradiated with different exposure times—5 min (830 nm, 3.0 J/cm², 60 mW) and 10 min (685 nm, 3.0 J/cm², 30 mW). In contrast to above-mentioned studies [10, 23] efficient eradication of C. albicans and S. mutans was found with an exposure time 15 s (3.0 J/cm², 250 mW) and 60 s (59 J/cm², 1000 mW).

In the literature was described very well that the laser light generated by the infrared lasers with wavelengths in the range of 600–1100 nm affects a wider cell-light response [25]. The dose-dependent effects of LLLT are described by the Arndt-Schultz’s curve [25, 26]. It suggests that different types of stimuli evoke different reactions of cells, e.g., increased stimulus inhibits activity [26]. On the other hand, the bacterial eradication can be obtained in a different process by photodynamic therapy where the irradiated photosensitizer generates induction of reactive oxygen species (ROS) which have a high killing potential for bacteria, fungus, and viruses. Despite the above-mentioned photodynamic effects of LLLT, literature review provides numerous studies on the influence of light on stimulation and inhibition of bacterial growth in the dose range of 1–10 J/cm² without administration of photosensitizers [10]. This effect was defined by the generation of ROS elicited by activation of endogenous chromophores in cells by the infrared laser light [10]. Thus, the results of our study where the irradiation with the two different doses (3 J at 0.25 W/10 Hz or 59 J at 1 W/10 Hz) resulted in a reduction in S. mutans and C. albicans amount in the same session can be explained by this above-mentioned phenomenon. The effect of laser on bacterial/fungal destruction was also described by Sommer where the expansion and contraction of the intracellular water volume and fluidity generate bidirectional flow [27].

In our opinion, Nd:YAG laser light at 1064 nm which is closed to the third peak wavelength of 1000 nm causes photoexcitation of endogenous microbial porphyrin molecules contained in S. mutans and C. albicans evoking oxidative damage through reactive oxygen species (ROS).

Translating our data into the clinical setting, we suggest that laser-based antimicrobial treatment can significantly reduce the quantity of Streptococcus mutans and Candida albicans especially in the treatment of caries and candidiasis.

More clinical studies are needed to evaluate the efficacy of a direct laser-based approach in vivo. Further study of Nd:YAG application with the Genova handpiece to treat bacterial and fungal infection is still needed to assess the efficacy of Nd:YAG 1064 nm laser therapy as a valid treatment for this frequent pathology.

The present data suggest that laser light at specific wavelengths might have some positive effects on the reduction of S. mutans and C. albicans infections; however, the clinical studies should be performed. Laser irradiation merits further attention for the many advantages it offers: shorter treatment duration, lower cost of treatment, minimal or no side effects, and an alternative to the systemic administration of antibiotics. Both low- and high-intensity Nd:YAG laser can significantly reduce the quantity of Streptococcus mutans and Candida albicans, which can be obtained after further clinical studies.