Preliminary Data on the Efficacy of Multi-Wave (Multi-Wavelength) Diode Laser on Bacteria in Superficial Canine Pyoderma. Energy for Health [18]

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Key words: Laser Therapy, superficial canine pyoderma, Staphylococcus pseudintermedius, antimicrobial activity, dermatology. Energy for Health [18]

folliculitis include a combination of both Preliminary data on topical and systemic antibiotic therapy (Hillier, 2014). In the initial step of the therapeutic the efficacy of treatment, topical therapy is considered a good approach, when lesions are localized, or in the early stages of generalized multi-wave superficial bacterial folliculitis (SBF) when lesions are mild. Moreover, local therapy can help prevent recurrence (Bajwa, 2016). (multi-wavelength) Laser therapy is an alternative treatment, since it has reported to be effective in the management of bacterial dermatitis. Laser diode laser on bacteria therapy may help the homeostasis of host tissue, but beside that, a direct activity on pathogen survival, host inflammatory response and repair mechanisms have in superficial canine been demonstrated. This study had a dual purpose: the first was to evaluate the ability of Near Infrared pyoderma. (NIR) laser emission (MLS® - Multiwave Locked System laser, Mphi, ASA Srl, 1* 2 1 1 3 Arcugnano, Italy) to decrease the bacterial C. S. Cabassi , G. Ghibaudo , S. Flisi , C. Spadini , M. Monici load present in the skin lesions of the 1Department of Veterinary Science, University of Parma, via del Taglio 10, 43126, Parma, examined dogs, before and after the in vivo Italy. laser treatment; the latter was to evaluate 2Clinica Veterinaria Malpensa, Samarate, Varese, Italy. 3ASAcampus Joint Laboratory, ASA Research Division, Department of Experimental and the in vitro bactericidal activity of the laser Clinical Biomedical Sciences, University of Florence, Florence, Italy. treatment on the isolated bacterial strains, after direct irradiation of the bacterial suspension in log phase of growth.

INTRODUCTION and transferred in the bacterial population. MATERIAL AND METHODS Superficial canine pyoderma is a Superficial bacterial folliculitis often Experimental design common bacterial skin infection which tends to become a recurrent condition This preliminary study was carried out on affects the superficial portion of the hair for inappropriate therapy (drugs used, a small group of patients (4 dogs). The follicle (Bajwa, 2016; Baumer, 2017). In duration of treatment), lack of diagnostics, study was conceived as a two-step work. dogs, this condition is associated with development of methicillin resistance in In the first step, the bacterial load present Staphylococcus pseudintermedius as the staphylococci population. in the superficial pyoderma lesions of the predominant pathogen. Other bacteria It is therefore of critical importance the dogs was evaluated before and after laser can be also isolated, such as E. coli application of a correct diagnostic protocol treatment. Moreover, the bacterial agents and others (Rantala, 2004). Antibiotic which include cytology, bacterial culture collected from the superficial pyoderma resistance is observed for Staphylococcus and antibiotic sensitivity evaluation. lesions of the patients were identified. Skin pseudintermedius for the presence of Since the acquisition of antimicrobial swabs were collected before (T0) and after transposon-borne resistance gene which resistance has become a common 30 minutes from multi-wave diode laser are incorporated into the chromosomal condition in the bacteria causing superficial (MLS®) treatment (T30). Bacteriological DNA; other mechanism of resistance in canine pyoderma, therapeutic options culture, strain identification and CFU Staphylococci and Gram negative bacteria usually require novel approaches. counts were performed. On each bacterial include plasmid-borne resistance genes. At date, guidelines for antimicrobial strain an evaluation of antibiotic sensitivity Resistance genes can be easily acquired therapy for canine superficial bacterial was performed by Kirby-Bauer agar disk

12 Preliminary data on the efficacy of multi-wave (multi-wavelength) diode laser on bacteria in superficial canine pyoderma. Energy for Health [18]

diffusion test. In vivo laser treatment and skin swabs To determine the antimicrobial activity of The second step of the study was aimed to collection the MLS® laser treatment in vivo, samples evaluate, on four of the previous bacterial After collection of skin swabs (T0), each collected before and after treatment isolates, the direct in vitro antimicrobial patient was treated by MLS® laser, using were compared. For each sample and activity of NIR laser treatment (MLS®). the specific program “contaminated each isolated bacterial strain, CFU were This step was performed by evaluating wounds”, with a radiant exposure of 2.05J/ counted. the CFU count of a standardized bacterial cm2. The laser exposure in each of the 6 suspension before and after laser treated points was of 6 seconds. After 30 Evaluation of antimicrobial activity of irradiation. minutes from the laser exposure, other direct in vitro MLS® laser treatment swabs were collected from the skin lesions To evaluate the direct bactericidal activity Enrollment of patients for the microbiological cultures. All swabs of the MLS® laser treatment, in vitro Dogs presenting clinical signs of superficial were maintained at 4°C and conferred tests were performed. Briefly, four of the generalized pyoderma were examined by to the Infectious Diseases laboratory of isolated bacterial strains were inoculated the clinician (Clinica Veterinaria Malpensa, the Department of Veterinary Science, in sterile Mueller Hinton Broth and Varese, Italy), who collected swabs from skin University of Parma, within 24 hours. incubated overnight at 37°C. The bacterial lesions to investigate the microbiological suspension was centrifuged 20 minutes flora. Cytological examination of each Evaluation of antimicrobial activity of at 2000 rpm and 4°C, then the pellet lesion was also performed and only 4 in vivo MLS® laser treatment resuspended in phosphate buffer. The patients with evidence of Gram positive Each swab was vortexed in 1ml of sterile turbidity of the bacterial suspension was cocci were included in the study. saline and this suspension further diluted immediately measured and adjusted by 1:10 and 1:100. One hundred microliters spectrophotometry. At 600 nm absorbance, Laser source of each dilution were plated on Columbia the OD range 0.08-0.13 was considered to Details of the laser source have been blood agar with 5% of bovine erythrocytes correspond to a bacterial concentration of previously described [Monici et al., 2013]. and MacConkey agar and incubated 108CFU/ml. The obtained suspension was Briefly, the treatments were performed overnight at 37°C. After incubation, further diluted 1:200 in phosphate buffer with a Multiwave Locked System (MLS®) bacterial growth was evaluated and to obtain a final bacterial concentration Laser (Mphi, ASA Srl, Arcugnano, colonies were isolated and amplified of 5*105CFU/ml. Then, 500µl of the Italy), a commercial laser source built in when necessary. Identification of bacterial bacterial suspension were transferred in a compliance with EC/EU rules, cleared by strains was based on colony morphology, 24 wells plate, according to Tab. 1. A black the US Food and Drug Administration Gram staining, catalase and oxidase paper was placed between contiguous (FDA). reactions. Species identification was wells to avoid the transmission of laser MLS laser is a class IV NIR laser with two carried out using the API biochemical test radiation through the walls . synchronized sources (laser diodes). The system (bioMérieux, France), as well as The bacterial suspensions were irradiated two sources have different wavelength, conventional biochemical tests (Markey BK, with two different exposure times (6 and peak power and emission mode. The 2013). Antimicrobial susceptibility test was 41 sec) with a radiant exposure of 2.05 first one is a pulsed 905nm laser diode performed by agar disk diffusion methods J/cm2. The laser source was placed in the with 25 W peak optical power. The pulse (Bauwe AW, 1966) on each isolated biosafety cabinet and fixed by a mechanical frequency may be varied in the range bacterial strain, according with the CLSI arm, which maintained the laser source 1-2000 Hz, thus varying the average guidelines (Clinical Laboratory Standards from a vertical distance of about 0.5 cm power delivered to the tissue. The second Institute, 2015). Tested antibiotics were from the top of the plate well containing laser diode (808nm) may operate in selected on the basis of the clinical use the bacterial suspension. continuous (power 1.1 W) or frequenced in the treatment of canine pyoderma. After the treatment, plates were incubated (repetition rate 1-2000 Hz, 550mW This list included Amikacin; Amoxicillin + at room temperature for two different mean optical power) mode, with a 50% Clavulanic acid; Ampicillin; Cephalexin; period (5 and 30 min). These incubation duty ratio independently of the repetition Cefovecin; Clindamycin; Chloramphenicol; times were selected to understand if rate. The two sources emit radiation Doxycycline; Enrofloxacin; Imipenem; the direct application of laser treatment synchronously and the propagation axes Marbofloxacin; Oxytetracycline; Oxacillin; required some time to cause bacteria killing, of the two laser beams are coincident. Sulfamethoxazole + Trimethoprim; depending on the bacterial structure and/ Rifaximin; Rifampin. or the parameters used in laser irradiation.

13 Preliminary data on the efficacy of multi-wave (multi-wavelength) diode laser on bacteria in superficial canine pyoderma. Energy for Health [18]

Then, for each incubation time, 10 µl of the Table I - Scheme of the plate used for one experiment for the determination of the direct antimicrobial activity irradiated bacterial suspension and its 1:10 of MLS® laser on different bacterial strains. Each test was performed in three replicates and three independent dilution were plated on Mueller Hinton experiments. agar plates. Sterility and growth controls were also diluted and plated in the same Sterility way. Agar plates were incubated at 37°C control overnight, then bacterial colonies (CFU) st st rd 1 growth were counted. Each test was performed 1 replicate 3 replicate control in triplicates and three independent 2nd experiments. 2nd growth replicate control 3rd growth control

Table II - Bacterial isolates and CFU count before and after MLS® laser treatment.

Pre-treatment Post-treatment Overall inhibition PRE- TREATMENT POST- TREATMENT Total CFU count Total CFU count percentage Case Staphylococcus 59.8*104 CFU/ml 3*104 CFU/ml 59,8*104 CFU/ml 3*104 CFU/ml 95% 1 pseudintermedius

Staphylococcus 0,28*104 CFU/ml 0.22*104 CFU/ml pseudintermedius

Proteus mirabilis 4.5*104 CFU/ml 0.035*104 CFU/ml swarming Case 6*104 0,56*104 CFU/ml 2 CFU/ml 90,6% Proteus mirabilis 1.1*104 CFU/ml 0.28*104 CFU/ml not swarming

Escherichia coli 0.125*104 CFU/ml 0.02*104 CFU/ml

Staphylococcus 4 4 pseudintermedius 273*10 CFU/ml 269*10 CFU/ml Case 2,75*106 CFU/ml 6 2,2% 3 2,69*10 CFU/ml Pseudomonas 2.29*104 CFU/ml fluorescens 0 CFU/ml

Case Staphylococcus 28200*104 CFU/ml 22700*104 CFU/ml 2,82*108 CFU/ml 2,27*108 CFU/ml 19,5% 4 pseudintermedius

RESULTS Staphylococcus pseudintermedius was Case n. 3 Evaluation of antimicrobial activity of isolated. The third case was a West Highland White in vivo MLS® laser treatment Terrier male dog, four years old, affected Four clinical cases were collected. Case n. 2 by generalized superficial pyoderma with The second clinical case was a Great crusts and papules localized on legs, Case n. 1 Dane male dog, seven years old, affected trunk and head. From the pre-treatment The first was a Bull Terrier male dog, by a generalized superficial pyoderma. swab Staphylococcus pseudintermedius eight years old, affected by a generalized From each swab, Staphylococcus and Pseudomonas fluorescens were superficial pyoderma with crusts and pseudintermedius, Proteus mirabilis isolated, while from post-treatment swab papules localized on legs, trunk and (swarming and not) and Escherichia coli only pure culture of Staphylococcus head. From each swab, a pure culture of were isolated. pseudintermedius was isolated.

14 Preliminary data on the efficacy of multi-wave (multi-wavelength) diode laser on bacteria in superficial canine pyoderma. Energy for Health [18]

Table III - Antimicrobial susceptibility test.

Sensitive Intermediate Resistant

Amikacin; Amoxicillin + Clavulanic acid; Cephalexin; Cefovecin; Clindamycin; Case Staphylococcus Chloramphenicol; Doxycycline; Sulfamethoxazole + Trimethoprim 1 pseudintermedius Enrofloxacin; Imipenem; Marbofloxacin; Ampicillin Oxytetracycline; Oxacillin; Rifaximin; Rifampin

Amoxicillin + Clavulanic acid; Cephalexin; Staphylococcus Cefovecin; Chloramphenicol; Doxycycline; Oxytetracycline; pseudintermedius Enrofloxacin; Imipenem; Marbofloxacin; Amikacin; Ampicillin; Clindamycin Sulfamethoxazole + Trimethoprim Oxacillin; Rifaximin; Rifampin

Amoxicillin + Clavulanic acid; Ampicillin; Proteus mirabilis Cephalexin; Cefovecin; Chloramphenicol; Amikacin; Clindamycin; Doxycycline; swarming Enrofloxacin; Imipenem; Marbofloxacin; Oxytetracycline; Oxacillin; Rifampin Rifaximin; Sulfamethoxazole + Trimethoprim Case 2 Amoxicillin + Clavulanic acid; Ampicillin; Proteus mirabilis Cephalexin; Cefovecin; Chloramphenicol; Amikacin; Clindamycin; Oxytetracycline; not swarming Enrofloxacin; Imipenem; Marbofloxacin; Doxycycline Oxacillin Rifaximin; Rifampin; Sulfamethoxazole + Trimethoprim

Amoxicillin + Clavulanic acid; Ampicillin; Cephalexin; Cefovecin; Chloramphenicol; Amikacin; Clindamycin; Doxycycline; Escherichia coli Enrofloxacin; Imipenem; Marbofloxacin; Rifaximin Oxacillin; Rifampin Oxytetracycline; Sulfamethoxazole + Trimethoprim

Amoxicillin + Clavulanic acid; Ampicillin; Cephalexin; Chloramphenicol; Staphylococcus Amikacin; Cefovecin; Clindamycin; Oxytetracycline; Doxycycline pseudintermedius Enrofloxacin; ; Marbofloxacin; Oxacillin; Rifaximin; Rifampin; Imipenem Sulfamethoxazole + Trimethoprim Case 3

Amikacin; Amoxicillin + Clavulanic Ampicillin; Cephalexin; Cefovecin; Pseudomonas Clindamycin; Oxacillin; Rifampin; fluorescens Enrofloxacin; Imipenem; Oxytetracycline acid; Chloramphenicol; Marbofloxacin; Doxycycline Sulfamethoxazole + Trimethoprim

Amoxicillin + Clavulanic acid; Cephalexin; Ampicillin; Clindamycin; Case Staphylococcus Cefovecin; Doxycycline; Enrofloxacin; Amikacin Chloramphenicol; Oxytetracycline; 4 pseudintermedius Imipenem; Marbofloxacin; Oxacillin; Sulfamethoxazole + Trimethoprim Rifaximin; Rifampin

15 Preliminary data on the efficacy of multi-wave (multi-wavelength) diode laser on bacteria in superficial canine pyoderma. Energy for Health [18]

Table IV - Antimicrobial activity of direct irradiation of bacterial isolates.

Seconds Minutes Growth control % Inhibition of exposition post- exposition Laser (CFU/ml) (CFU/ml)

5 1.17±0.20*105 1.31±0.18*105 11% 6 5 1.05±0.30*105 Staphylococcus 30 1.05±0.30*10 0% pseudintermedius Case 1 5 1.04±0.05*105 1.13±0.13*105 8% 41 30 1.51±0.99*105 1.51±0.99*105 0%

5 0.67±1.70*105 0.67±1.70*105 0% 6 5 0.63±1.49*105 Staphylococcus 30 0.63±1.49*10 0% pseudintermedius Case 2 5 0.26±0.04*105 0.26±0.04*105 0% 41 30 0.27±0.38*105 0.27±0.38*105 0%

5 3.31±0.37*105 3.63±0.42*105 9%

Bacterial strains Bacterial 6 30 3.36±0.09*105 3.39±0.25*105 1% Escherichia coli Case 2 5 2.36±0.97*105 2.56±1.22*105 8% 41 30 2.25±0.94*105 2.25±0.94*105 0%

5 1.64±0.17*105 1.67±0.14*105 2% 6 5 1.59±0.45*105 Pseudomonas 30 1.59±0.45*10 0% fluorescens Case 3 5 2.28±1.63*105 2.35±1.71*105 3% 41 30 2.05±1.61*105 2.21±1.56*105 7%

Case n. 4 Antimicrobial susceptibility test was MLS® laser, on the basis of their clinical The fourth case was a Poodle male dog, performed on each isolated bacterial importance (two Gram positive and two seven years old, affected by generalized strain. Results are shown in Table 3. Gram negative). superficial pyoderma with crusts and papules. From each swab Staphylococcus Evaluation of antimicrobial activity of Strain 1: Staphylococcus pseudintermedius pseudintermedius was isolated. direct in vitro MLS® laser treatment isolated from Case 1. Five minutes after CFU count was performed for each In the second step of the experimental laser treatment (irradiation time of six sample and each bacterial isolate. Results design, four bacterial strains from clinical seconds) growth inhibition percentage are shown in Table 2. and each bacterial cases were selected for the evaluation was 11%, while after thirty minutes isolate. Results are shown in Table 2. of direct in vitro antimicrobial activity of inhibition was 0%. The same bacterial

16 Preliminary data on the efficacy of multi-wave (multi-wavelength) diode laser on bacteria in superficial canine pyoderma. Energy for Health [18]

strain subjected to a laser exposure of 41 a significant reduction of CFU count platelet aggregation (Kreisler et al, 2002). seconds showed 8% of growth inhibition was observed for all isolates, except Moreover, an antibacterial effect on third- after incubation time of five minutes, the three strains of Staphylococcus degree burn wounds was proved (Bayat while after thirty minutes inhibition was 0%. pseudintermedius isolated from cases 2, et al, 2006). It has been also suggested 3 and 4. A higher effectiveness on Gram that the anti-inflammatory action of Strain 2: Staphylococcus pseudintermedius negative isolates was observed, according laser therapy in wounds, skin lesions and isolated from Case 2. This strain did to the literature, where it is reported that infectious diseases could be partly due to not show any growth inhibition, both laser antimicrobial activity depends on the reduction of the bacterial load at the after five minutes and thirty minutes of bacterial species. This may be attributable lesion site, thus reducing a major cause incubation from the irradiation step. to the peculiar structure of Gram positive of inflammation (Meyerholz et al, 2009; and Gram negative bacterial membranes Silva, 2013). Strain 3: Escherichia coli isolated from and wall (Schoop, 2004). Gram positive However, the results obtained in the Case 2. This strain showed growth wall is thicker, being structured in an second phase of the present study, inhibition after laser treatment of six outer cytoplasmic lipid membrane, thick where the antimicrobial effect of direct seconds, both after five minutes and peptidoglycan layer, lipoteichoic acids and in vitro NIR laser (MLS®) treatment thirty minutes of incubation (9% and 1%, a smaller periplasm than in Gram negative was evaluated, demonstrated a limited respectively), while laser irradiation of bacteria. Other studies have confirmed microbicidal activity. The growth inhibition 41 seconds resulted in 8% inhibition of that structural configuration of the cell was observed mostly with 6 sec exposure growth, only five minutes post-treatment. wall affects bacterial susceptibility to laser and 5 min incubation time. The findings irradiation. In different conditions, several suggest that there is no direct correlation Strain 4: Pseudomonas fluorescens cycles of laser irradiation are required to between effectiveness and exposure isolated from Case 3. This strain showed affect Gram-positive bacteria; whereas, time: in one case only a slight increase an inhibition of growth equal to 2% in Gram-negative bacteria are eliminated in growth inhibition was observed with the 6 seconds laser-treatment mode, faster and more easily (Moritz et al, 2000). increasing exposure time. after five minutes incubation, while Moreover, it has been demonstrated that This result suggests that the damage none inhibition was observed after thirty differences in the wavelengths, power, produced by laser treatment on the minutes. Furthermore, inhibition was of irradiation time, spot size and number bacterial population is slight, therefore 3% and 7%, respectively, after five and of cycles are responsible for the variable it can be detected immediately after thirty minutes of incubation from laser- efficacy of lasers reported in literature (De treatment but is easily diluted over a short treatment of 41 seconds. Paula, 2001). time. All the results of the direct irradiation of The anti-bacterial effect of laser-therapy It was beyond the scope of this study to bacterial strains are shown in Table 4. has been widely described in literature. evaluate the effect of laser treatment on IGAlAr laser emission was used against canine pyoderma from the clinical point DISCUSSION Staphylococcus aureus strains, and the of view. In fact, the clinical protocol for As expected, from all the clinical cases results revealed reduction in the number this application of MLS® therapy foresees of superficial pyoderma the most of colonies (Wilson et al, 1995). In several weekly sessions for a few weeks. isolated strain was Staphylococcus humans it was studied the effect of Therefore, in this study, which was based pseudintermedius. Other bacterial isolates laser-therapy with the wavelengths 630, on a single in vivo irradiation, the host (Pseudomonas fluorescens, E. coli, Proteus 660, 810 nm and 1-50 J/cm2 fluence on biological response to laser treatment was spp.) can be considered opportunistic, co- Staphylococcus aureus, Pseudomonas, not studied. infectious agents. Multi-drug resistance and E. coli which were collected from However, the higher antimicrobial activity was observed mostly in Gram negative infected wounds (Nussbaum et al, 2002). observed in vivo in comparison with the in isolates (Pseudomonas, Proteus, E. coli). A significantly reduction in the growth vitro irradiation suggests that in vivo laser Following in vivo laser treatment on the of bacteria was obtained in a study on irradiation induces a complex response skin lesions, significant results in bacterial diabetic wound healing in rats suffering which involves both microorganisms and growth inhibition, demonstrated by CFU from bacterial infection induced by host tissues. The final effects depend on count, were observed on the bacterial Staphylococcus aureus (Ranjbar, 2016). the absorption of laser radiation by the flora load isolated after 30 minutes A Diode laser source was used on dental host tissues and consequent biological from laser application. In particular, implants in order to avoid microbial response, which involves the immune cells

17 Preliminary data on the efficacy of multi-wave (multi-wavelength) diode laser on bacteria in superficial canine pyoderma. Energy for Health [18]

and their interaction with microrganisms different effectiveness shown in in REFERENCES (Clemente et al., 2015), the modulation vivo and in vitro conditions could be 1. Bajwa J. Canine superficial pyoderma of inflammation (Monici et al., 2013, explained with: and therapeutic considerations. Can Vet Squarzoni et al, 2017) the enhancement J. 2016 Feb;57(2):204-6. PubMed PMID: of anabolic processes in the tissues and 1) a higher absorption of laser 26834275; PubMed Central PMCID: activation of repair mechanisms (Monici radiation by the host tissues in PMC4713004 et al., 2013). comparison with bacterial 2. Bayat M, Vasheghani MM, Razavi N. Beyond the complexity of the in vivo suspensions (this implies that LLLT has antibacterial and angiogenic biological response, another explanation higher doses are required in vitro); properties in burns. J Photochem for the lower effectiveness of laser Photobiol B. 2006 Jan;30 treatment in inhibiting bacterial growth 2) a more complex biological 3. Bäumer W, Bizikova P, Jacob M, Linder in vitro could be the fluence (2.05J/cm2), response induced by the in vivo KE. Establishing a canine superficial which had deliberately been kept the irradiation, involving the pyoderma model. J Appl Microbiol. same as that used in vivo. interaction with the host tissues. 2017 Feb;122(2):331-337. doi: 10.1111/ However, the absorption of laser radiation jam.13362. Epub 2017 Jan 4. PubMed in the tissues is greater than that in In conclusion, in-depth studies on the PMID: 27891724. bacterial suspension in vitro. So, higher application of laser therapy in the 4. Bauer AW, Kirby WM, Sherris JC, Turck fluences are probably required in vitro. management of canine skin infections M. Antibiotic susceptibility testing by a This hypothesis is in agreement with what are required and further investigations standardized single disk method. Am J has been observed applying laser therapy are necessary, considering a wider Clin Pathol. 1966 Apr;45(4):493–6. to inhibit fungal growth. After trying bacterial sample and varying lasers 5. Clemente AM, Rizzetto L, Castronovo different energy densities, the maximum parameters to maximize their efficacy G, Perissi E, Tanturli M, Cozzolino F, effect in terms of mortality rate in Candida in vitro. Cavalieri D, Fusi F, Cialdai F, Vignali L, albicans suspensions treated in vitro was Torcia MG, Monici M. Effects of near- obtained with 7J/cm2 fluence (Clemente infrared laser radiation on the survival et al., 2015). When considering the and inflammatory potential of Candida treatment of oral mucositis in vivo, very spp. Involved in the pathogenesis of significant results are reported also with chemotherapy-induced oral mucositis. very low fluence (0.16J/cm2) (Squarzoni Eur J Clin Microbiol Infect Dis 2015 et al., 2017). Oct;34(10):1999-2007. doi: 10.1007/ s10096- 015-2443-5. CONCLUSION 6. Clinical Laboratory Standards Institute. In conclusion, this study shows that Performance Standards for Antimicrobial a single application of laser therapy Disk Susceptibility Tests, Approved carried out by MLS device effectively Standard, CLSI document M02-A11. reduced the microbial load of skin Clinical and Laboratory Standards lesions in superficial canine pyoderma. Institute; 2015. The antimicrobial action was higher on 7. De Paula Eduardo C, Gouw-Soares S the Gram negative bacteria than on the (2001): The Use of Lasers for Endodontic Gram positive ones. Applications in Dentistry. Medical Laser This effect was not reproduced by in Application, 16:231-243. vitro irradiation of bacterial suspensions 8. Hillier A, Lloyd DH, Weese JS, et prepared from skin swabs collected al. Guidelines for the diagnosis and before in vivo laser treatment of antimicrobial therapy of canine superficial patients affected by superficial canine bacterial folliculitis (Antimicrobial pyoderma. Guidelines Working Group of the In vitro, only a slight inhibition International Society for Companion of bacterial growth was observed Animal Infectious Diseases) Vet Dermatol. immediately after the treatment. The 2014;25:163–175.

18 Preliminary data on the efficacy of multi-wave (multi-wavelength) diode laser on bacteria in superficial canine pyoderma. Energy for Health [18]

9. Kreisler M, Kohnen W, Marinello C, Götz 17. Schoop U, Kluger W, Moritz A, Nedjelik H, Duschner H, Jansen B, d'Hoedt B. N, Georgopoulos A, Sperr W. Bactericidal Bactericidal effect of the Er:YAG laser on effect of different laser systems in the dental implant surfaces: an in vitro study. deep layers of dentin. Lasers Surg Med. J Periodontol. 2002 Nov;73(11):1292-8 2004;35(2):111-6.. 10. Markey BK, editor. Clinical veterinary 18. Schägger H. Respiratory chain microbiology. 2nd ed. Edinburgh: Elsevier; supercomplexes of mitochondria and 2013. 901 p. bacteria. Biochim Biophys Acta. 2002 Sep 11. Mojica FJ, Díez-Villaseñor C, Soria E, Juez 10;1555(1-3):154-9. Review. PubMed G. Biological significance of a family of PMID: 12206908. regularly spaced repeats in the genomes 19. Silva DC, Plapler H, Costa MM, Silva SR, of Archaea, Bacteria and mitochondria. Sá Mda C, Silva BS. Low level laser therapy Mol Microbiol. 2000 Apr;36(1):244-6. (AlGaInP) applied at 5 J/cm2 reduces the PubMed PMID: 10760181. proliferation of Staphylococcus aureus 12. Monici M, Cialdai F, Ranaldi F, Paoli P, MRSA in infected wounds and intact Boscaro F, Moneti G, Caselli A. Effect of skin in rats. An Bras Dermatol. 2013 Jan- IR laser on myoblasts: a proteomic study. Feb;88(1):50-5 Mol Biosyst. 2013; Jun;9(6):1147-61. doi: 20. Squarzoni P, Bani D, Cialdai F and Monici 10.1039/c2mb25398d. M. NIR Laser Therapy in the Management 13. Moritz A, Jakolitsch S, Goharkhay K, of Feline Stomatitis. SM Dermatolog J. Schoop U, Kluger W, Mallinger R, et al. 2017; 3(3): 1021. (2000): Morphologic changes correlating 21. Vacca RA, Marra E, Quagliariello E, Greco to different sensitivities of Escherichia M. Activation of mitochondrial DNA coli and enterococcus faecalis to Nd:YAG replication by He-Ne laser irradiation. laser irradiation through dentin. Lasers in Biochem Biophys Res Commun. 1993 surgery and medicine, 26:250-261 Sep 15;195(2):704-9. 14. Nussbaum EL, Lilge L, Mazzulli T. Effects 22. Von Bathen LC, Paula JB, Cielinski J, of 630-, 660-, 810-, and 905-nm laser Pilonetto M, von Bahten LC. Effects of irradiation delivering radiant exposure of low-level laser in in vitro bacterial culture 1-50 J/cm2 on three species of bacteria and in vivo infected wounds. Rev Col in vitro. J Clin Laser Med Surg. 2002 Bras Cir. 2014 Jan-Feb;41(1):49-55. Dec;20(6):325-33 23. Wilson M, Yianni C. Killing of methicillin- 15. Ranjbar R, Takhtfooladi MA. The resistant Staphylococcus aureus by low- effects of photobiomodulation therapy power laser light. J Med Microbiol. 1995 on Staphylococcus aureus infected Jan;42(1):62-6 surgical wounds in diabetic rats. A microbiological, histopathological, and biomechanical study. Acta Cir Bras. 2016 Aug;31(8):498-504. 16. Rantala M, Lahti E, Kuhalampil J, Pesonen S, Järvinen AK, Saijonmaa-Koulumies, Honkanen- Buzalski T. Antimicrobial resistance in Staphylococcus spp., Escherichia coli and Enterococcus spp. in dogs given antibiotics for chronic dermatological disorders, compared with non-treated control dogs. Acta Vet Scand. 2004;45(1-2):37-45.