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Introduction: In fixed orthodontics, formation of white spot lesions, enamel demineralization, and tooth decay around appliances are common complications which mar the outcome. Aim: The aims of this study were the determination of the shear bond strength (SBS) and adhesive remnant index (ARI) of orthodontic adhesive doped with N-GO, as well as the assessment of antimicrobial activities of the modified orthodontic adhesive against Streptococcus mutans. Materials and methods: N-GO was characterized by a scanning electron microscope (SEM), Fourier transformation infrared (FTIR), X-ray diffraction (XRD), and Zeta potential. The SBS and ARI of modified orthodontics adhesive containing different concentrations of N-GO (0, 1, 2, 5, and 10 wt%) were then measured. The influences of adding N-GO on antimicrobial properties of orthodontic adhesive were determined against S. mutans by disc agar diffusion (DAD) testing and biofilm formation inhibition assay. Results: The SEM, FTIR, XRD, and Zeta potential analysis indicated the successful synthesis of N-GO. Orthodontics adhesive doped with 5 wt% N-GO showed the highest concentration of N-GO and SBS value (21.71±7.45 MPa, p Conclusions: It could be concluded that 5 wt% of N-GO can be considered as an orthodontic adhesive additive to reduce the microbial count and biofilm with no adverse effect on SBS and ARI.

Background. The present study evaluated the antimicrobial efficacy of composite resins containing nano-silver (NAg) particles used in fixed orthodontic retainers. Methods. Nano-composite resin samples with 1%, 2%, and 5% concentrations of NAg were prepared. The antimicrobial effectiveness of NAg was assessed against Streptococcus mutans, Streptococcus sanguis, and Lactobacillus acidophilus by the biofilm inhibition test (three-day-old biofilms), eluted components test (on days 3, 15, and 30), and disk-diffusion agar test after 48 hours. Measures of central tendency and index of dispersion were used to determine colony-forming units. Kruskal-Wallis test and Mann-Whitney U test were also used. Results. The biofilm inhibition test showed a significant decrease in the colonies of S. mutans (87.64%, 96.47%, and 99.76% decrease), S. sanguis (98.13%, 99.47%, and 99.93% decrease), and L. acidophilus (81.59%, 90.90%, and 99.61% decrease) at 1%, 2%, and 5% concentrations of Nag, respectively, compared to the control groups. The colony-forming unit (CFU)/mL of tested microorganisms continuously decreased with increased NAg concentration. In the eluted component test, no significant differences were noted in the 3rd, 15th, and 30th days between the different concentrations of Nag-containing composite resin disks and control samples. According to the disk-diffusion agar test, there was no growth inhibition zone for the composite resin disks containing 1% and 2% concentrations of Nag. However, the growth inhibition zone was seen with a 5% concentration, with a diameter of 9.5±0.71 mm for S. mutans, 8.5±0.71 mm for S. sanguis, and 8±1.41 for L. acidophilus. Conclusion. The incorporation of NAg into composite resins has antibacterial effects, possibly preventing dental caries around fixed orthodontic retainers.

This study aimed to focus on the simultaneous use of antimicrobial photodynamic therapy (aPDT) and sonodynamic antimicrobial chemotherapy (SACT), which is called photo-sonodynamic antimicrobial chemotherapy (PSACT) to attenuate the virulence of Aggregatibacter actinomycetemcomitans. Following the synthesis of Curcumin-decorated nanophytosomes (Cur-NPhs) as a novel photo-sonosensitizer, its particle size, polydispersity, ζ-potential surface morphology, physical stability, drug release, and entrapment efficiency were determined. In the Cur-NPhs-PSACT, the antimicrobial activities of Cur-NPhs against A. actinomycetemcomitans were investigated using cell viability, biofilm killing/degradation, metabolic activity, expression of quorum-sensing-associated qseB and qseC genes, and biofilm-associated rcpA gene under blue laser irradiation plus ultrasonic waves. Characterization tests showed the presence of a sphere-shaped vesicle and the self-closed structure of Cur-NPhs, resulting in a high drug-loading content and encapsulation efficiency. However, the antimicrobial effect of Cur-NPhs-PSACT was dose-dependent, PSACT using the high concentrations of Cur-NPhs (50 × 10–4 g/L) showed significant reductions (P 10 CFU/mL), biofilm killing/degradation (65%), metabolic activity (89.6%,), and mRNA levels of virulence determinant genes (qseB; 9.8-fold, qseC; 10.2-fold, and recA; 10.2-fold). This study concludes that the Cur-NPhs-PSACT had antimicrobial activities against A. actinomycetemcomitans by downregulating the expression of virulence genes, and may attenuate this bacterium that decreases periodontal disease severity in patients.

Background Biofilm formation is an important causative factor in the expansion of the carious lesions in the enamel. Hence, new approaches to efficient antibacterial agents are highly demanded. This study was conducted to evaluate the antimicrobial-biofilm activity of chitosan hydrogel (CS gel), zinc oxide/ zeolite nanocomposite (ZnONC) either separately or combined together [ZnONC / CS gel (ZnONC-CS)] against Streptococcus mutans biofilm. Results MTT assay demonstrated that the ZnONC-CS exhibits a non-cytotoxic effect (> 90% cell viability) toward human gingival fibroblast cells at different dosages (78.1–625 μg/mL) within 72 h. In comparison with CS gel and ZnONC, ZnONC-CS was superior at biofilm formation and metabolic activity reduction by 33 and 45%, respectively; (P gtfB, gtfC, and ftf significantly decreased. Conclusions Taken together, excellent compatibility coupled with an enhanced antimicrobial effect against S. mutans biofilm has equipped ZnONC-CS as a promising candidate for dental biofilm control.

Introduction: Microbial biofilm accumulation around orthodontic brackets and composite is a common complication of fixed orth-odontic treatment. This study assessed the antibacterial effects of orthodontic primer containing chitosan nanoparticles (CNPs) against the multispecies biofilm of cariogenic bacteria in а rat model. Materials and methods: Transbond XT orthodontic primer containing 0%, 1%, 5%, and 10% CNPs was experimentally prepared. The Wistar rats were randomly divided into four groups (n=7) of control (0% CNPs), 1%, 5% and 10% CNPs. The oral cavities of the rats were infected with cariogenic bacteria. After anesthetizing the rats, 1 drop (10 µL) of primer with different concentrations of CNPs was applied to their central incisor and light-cured for 20 seconds. Transbond XT orthodontic adhesive (2 × 2 mm) was applied on the primer. Another drop (10 µL) of primer was applied and light-cured for 40 seconds. The number of Streptococcus mutans, Streptococcus sanguinis, and Lactobacillus acidophilus colonies in the saliva of rats was quantified at 24 hours, 4 days and 7 days. Results: Adding 1% (p=0.005), 5% (p Conclusions: The addition of 5% CNPs to orthodontic primer significantly decreased the colony count of cariogenic bacteria in rats.

Introduction: Acinetobacter baumannii strains are important agents causing serious nosocomial infections including soft-tissue and skin infections in patients with burn wounds which have become resistant to several classes of antibiotics. Antimicrobial photodynamic therapy (aPDT) as an alternative antimicrobial procedure is suggested for the treatment of these kinds of infections. The aim of the current study is to evaluate the antibacterial and anti-biofilm efficiency of aPDT by the utilization of an improved form of indocyanine green (ICG) which is encapsulated in chitosan nanoparticles (NCs@ICG). Methods: NCs@ICG were synthesized and confirmed by the scanning electron microscope (SEM). aPDT was performed using NCs@ICG with an 810 nm wavelength of the diode laser at the fluency of 31.2 J/cm2 on 50 A. baumannii strains isolated from burn wounds. The antibacterial and antibiofilm potential of NCs@ICG-aPDT was determined via the colony forming unit (CFU)/mL and crystal violet assays, respectively. In addition, microbial biofilm degradation was evaluated by the SEM. Results: According to the results, NCs@ICG-aPDT showed a significant reduction of 93.2% on the CFU/ mL of planktonic A. baumannii strains compared to the control group (untreated group; P < 0.05). In addition, the biofilm formation of A. baumannii strains was significantly reduced by 55.3% when the bacteria were exposed to NCs@ICG-aPDT (P < 0.05). In contrast, NCs@ICG, ICG, and the diode laser alone were not able to inhibit the CFU/mL and biofilm of A. baumannii strains (P > 0.05). Based on the results of SEM images, NCs@ICG-aPDT disrupted the biofilm structure of A. baumannii strains more than other groups. Conclusion: NCs@ICG-aPDT demonstrates a promising treatment candidate for exploitation in wound infections against both planktonic and biofilm forms of A. baumannii strains

Aim: To evaluate the antimicrobial effects of photoexcited silver sulfadiazine nanoliposomes (AgSD-NLs) doped by curcumin (AgSD-NLs@Cur) on Acinetobacter baumannii. Materials & methods: Following characterization, the cytotoxic and hemolytic activities of AgSD-NLs@Cur were evaluated. The antimicrobial activities of AgSD-NLs@Cur-mediated antimicrobial photodynamic therapy (aPDT) were determined. Histopathological examination of the burn wound sites of infected mice treated with photoexcited AgSD-NLs@Cur was assessed. Results: No significant cytotoxic and hemolytic activities were observed. There was a decrease in the Acinetobacter baumannii count in planktonic and biofilm forms and the gene expression level using AgSD-NLs@Cur-aPDT (p < 0.05). Histopathological analysis indicated the epidermis developed markedly and the bacterial load decreased significantly after aPDT. Conclusion: Photoexcited AgSD-NLs@Cur has an antimicrobial potential against A. baumannii.

Aim This study aimed to assess the effect of addition of nano-titanium oxide (nano-TiO2), nano-silicon dioxide (nano-SiO2), and a combination of both, on antimicrobial activity of an orthodontic composite. Materials and methods Molds measuring 0.64 × 0.5 mm were used for the fabrication of composite disks. For this purpose, 0.5% and 1% nano-TiO2, nano-SiO2, and a combination of both (0.5% nano-TiO2 and 0.5% nano-SiO2), were mixed with Transbond XT composite (3M Unitek). A total of 180 composite disks were fabricated for eluted component, disk agar diffusion (DAD), and biofilm inhibition tests. The colony counts of Streptococcus mutans (S. mutans), Streptococcus sanguinis (S. sanguinis), and Lactobacillus acidophilus (L. acidophilus) and the diameters of growth inhibition zones were measured at 3, 15, and 30 days after exposure to the materials. Data were analyzed using one-way ANOVA and a post hoc test. Results None of the nano-TiO2 and nano-SiO2 concentrations had any significant effect on the growth inhibition zone. All tested concentrations of nano-TiO2 and nano-SiO2 decreased the colony count of all bacteria. The composite sample containing both nano-TiO2 and nano-SiO2 had the greatest efficacy for reduction of S. mutans and S. sanguinis colony counts at all three time points. Also, 1% nano-TiO2 and 1% nano-SiO2 had similar effects on L. acidophilus in eluted component test. Conclusion Addition of TiO2 and SiO2 nanoparticles conferred antimicrobial property to the tested orthodontic composite. Clinical significance Using orthodontic composite containing nonoparticles with antibacterial activity may prevent dental caries.

The limitations of conventional chemo-mechanic therapy in endodontic have given rise to introduce and develop photoactivated disinfection (PAD) as an adjunctive treatment. The objective of this ex vivo study was to evaluate the effects of PAD using C-Phycocyanin, as a natural photosensitizer, on biological activities and expression of quorum-sensing system gene fsrB of Enterococcus faecalis.A total of 52 extracted single-rooted human premolar teeth were divided into five groups (n = 10) and were incubated with E. faecalis for biofilm formation, and they were treated according to following groups; A. C-PC only; B. Diode laser only (at wavelengths of 635 nm); C. PAD (C-PC plus diode laser); and D. Sodium hypochlorite (NaOCl). E. Control group (not treatment). Two remaining teeth were used to confirm E. faecalis biofilm formation in the root canal system by scanning electron microscopic. The effects of each treatment were measured on viability and expression level of fsrB gene of E. faecalis using microbial cell viability and quantitative real-time PCR assays, respectively. Additionally, intracellular reactive oxygen species (ROS) production was measured using 2',7'-dichlorfluorescein-diacetate (DCFH-DA) fluorescent probe.The C-PC alone and C-PC-PAD reduced the culture viability of E. faecalis by 38.1% and 89.45%, respectively (both; P 0.05). According to the Bonferroni post hoc test, a significant difference in the reduction of E. faecalis count was observed between C-PC-PAD and other groups (P 0.05), except the NaOCl group (P 0.05). As well as, photoexcited C-PC in PAD could increase (3.8- fold) the intracellular ROS production in E. faecalis compared to the control group (P 0.05). C-PC-PAD at sub-significant inhibitory concentration and NaOCl at sub-lethal dose could significantly decrease the expression levels of fsrB by 10.8- and 11.4-fold, respectively.Our data support that C-PC alone and C-PC-PAD can serve as a potent irrigation solution and a disinfection method, respectively for microbial reduction and promising adjunct therapy in endodontic infection treatment against E. faecalis with clinical applications for infection control in endodontics.

Background: Photodynamic therapy (PDT) is alternative treatment of cutaneous leishmaniasis (CL), and phenolthiazine dyes such as Toluidine Blue O (TBO) have the potential role in PDT and notably affect parasites inactivation. This study aimed to evaluate the effectiveness of PDT by using TBO and a light-emitting diode (LED) in the treatment of zoonotic CL (ZCL). Methods: The study was conducted in Iran University of Medical Sciences, Tehran, Iran in 2018-2020. Different concentration (7.8 µg/ mL up to 500 µg/ mL) of TBO as a photosensitizer and a 630 nm LED light as a source of light were used for antileishmanial activity against both forms of Leishmania major promastigotes and intracellular amastigotes. Effective concentration (EC50) and cell cytotoxicity (CC50) were calculated in both infected and non-infected J774.A1 macrophages, respectively. As well as inhibitory concentration (IC50) was quantified in L. major promastigotes for 2 h, 24 h, and 48 h after incubation using a MTT colorimetric assay. Results: TBO dye in combination with the PDT significantly decreases the L. major promastigotes and intracellular amastigotes viability when compared with TBO alone. Both TBO dye in combination with the PDT and TBO alone had no toxic effects on the mice macrophages; however, it significantly killed the entered parasites inside the cells. Our results in the current study established satisfactory findings in clearing intracellular L. major parasites in in-vitro conditions. Conclusion: TBO dye in combination with the PDT can be considered as a harmless, effective and importantly perfect treatment against L. major, causative agent of ZCL, in an in-vitro situation without any negative toxicity to the mice macrophages.

Background Antimicrobial photodynamic therapy (aPDT) is an effective adjunctive therapeutic modality for the treatment of local infections, including periodontitis and peri-implantitis. After receiving aPDT, microbial cells in the biofilm structure may produce and/ or release soluble biofilm-derived effectors (BDEs), which may affect the biology of the host cells in the community context of their surrounding microenvironment. Given the fact that no study has investigated the role of BDEs following aPDT in the pathogenesis of infectious diseases, the aim of the current study was to determine the effect of BDEs of Aggregatibacter actinomycetemcomitans following exposure to sub-lethal doses of indocyanine green (ICG)-aPDT on human gingival fibroblasts (HGFs) in terms of cytokines produced. Materials and methods In this study, we evaluated the effect of biofilm-conditioned medium (BCM) resulting from the treatment of A. actinomycetemcomitans biofilm with a sub-lethal dose of aPDT on cytokines production, including IL-6, IL-8, CXCL10, TGF-β, and bFGF of HGFs using enzyme-linked immunoassays (ELISA). The sensitivity of cytokines to BDEs was determined by micro-titer plates. Results The maximal sub-lethal dose of ICG-PDT was 20.15 μM/mL ICG at a fluence of 31.2 J/cm2. The BCM of ICG-PDT-treated viable A. actinomycetemcomitans significantly reduced IL-6, IL-8, and CXCL10 levels compared to the BCM of untreated viable A. actinomycetemcomitans (78-, 93-, and 61.6-fold reduction, respectively; all P Conclusion The results of the current study revealed that a sub-lethal dose of ICG-aPDT through the effect of BCM on HGFs could not only significantly reduce the production of pro-inflammatory cytokines but also promoted their role in periodontal regeneration due to increasing the bFGF level. Altogether, ICG-aPDT, with it's antimicrobial effects reduces inflammation and induces of tissue regeneration resulting from BCM, can be considered an efficient adjunctive therapeutic method for the treatment of local infections.

Background This study sought to evaluates the efficiency of anti-microbial activity of acrylic resins doped with different concentrations of Undaria pinnatifida after activation with light-emitting diode (LED) at producing photodynamic damage to multispecies biofilm-producing microbiome. Material and methods In this study, bioinformatics tools and computer simulation molecular modeling were used to evaluate the capacity of ferredoxin (FDX), an electron acceptor in metabolic pathways of U. pinnatifida, which can discharge electrons produced from photo-excited chlorophyll-a (Chl-a) by LED irradiation. Acrylic resin discs containing different concentration of U. pinnatifida (0, 0.5, 1, and 2%) were fabricated and were subjected to LED irradiation immediately before each experiment. After continuously rinsed (up to 30 days), the antimicrobial activity of acrylic resins doped with U. pinnatifida following photo-activation was determined by disc agar diffusion, biofilm formation inhibition, and eluted component assays versus bacterial species linked to caries that constitute a mixed biofilm including Streptococcus mutans, S. sanguinis, and Lactobacillus acidophilus, as well as Candida albicans as main etiology of candidal stomatitis. Results Modeling and a virtual screening analysis of FDX indicated that it is a stable protein with an iron-sulfur center that can discharge electrons produced from photo-excited Chl-a and transfers them to FDX–NADP+ reductase for NADP+ reduction in photosystem I, which is essential in the Calvin cycle for carbon assimilation. FDX acts as an electron transfer agent in the redox reactions. The results showed that growth inhibition zones were not seen around acrylic resin discs in any group. In biofilm test, the colony counts of all test microorganisms significantly decreased (36%–87%) by an increase in the percentage of U. pinnatifida in acrylic resins after photo-activation (P Conclusion Based on the results presented here, an acrylic resin containing U. pinnatifida, even at the lowest concentration, following photo-activation using LED have antimicrobial properties against planktonic and biofilm forms of the cariogenic microorganisms as well as C. albicans.

BACKGROUND Acinetobacter baumannii has emerged as a major cause of nosocomial infections. Various resistance mechanisms of A. baumannii against antibiotics have transformed it into a successful nosocomial pathogen. Because of the limited number of available antibiotics, we used a medicinal plant with an antibacterial effect. Zataria multiflora Boiss (ZMB) extract and its components were used for the treatment of pneumonic mice infected with A. baumannii. The biological effects of this extract and the regulation of the outer membrane protein A (ompA) gene were used in a mouse model. METHODS A pneumonic mouse model was prepared using clinical and standard strains (1.5 × 108 colony-forming units/mL) of A. baumannii. BALB/c mice groups were treated with a ZMB extract, carvacrol, thymol, and sensitive antibiotics. The lung tissues of the treated mice were cultured for 5 days and each day, bacterial clearance and the ompA gene expression were assessed by quantitative real-time polymerase chain reaction. RESULTS In the lung tissue culture of pneumonic mice infected with standard or clinical isolate, no colony was detected when treated with the ZMB extract after 2 and 3 days (P

Objectives: Eradication of Aggregatibacter actinomycetemcomitans (A. action-mycetemcomitans), as an opportunistic periodontopathogen, and inhibition of its virulence factor expression require a new adjunctive therapeutic method. In this study, we accessed the expression level of rcpA gene, as a virulence factor associated with A. actinomycetemcomitans biofilm formation, following treatment by antimicrobial photodynamic therapy (aPDT) using indocyanine green (ICG) doped with chitosan nanoparticles (CS-NPs@ICG). Materials and Methods: CS-NPs@ICG was synthesized and examined using scanning electron microscopy (SEM). A. actinomycetemcomitans ATCC 33384 strain was treated with CS-NPs@ICG, as a photosensitizer, which was excited with a diode laser at the wavelength of 810 nm with the energy density of 31.2 J/cm2. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the changes in rcpA gene expression level. Results: Synthetized CS-NPs@ICG was confirmed via SEM. The results revealed that CS-NPs@ICG-mediated aPDT could significantly decrease rcpA gene expression to 13.2-fold (P0.05). Conclusion: aPDT with CS-NPs@ICG leads to a decrease of the virulence factor of A. actinomycetemcomitans and can be used as an adjunct to routine treatments for successful periodontal therapy in vivo.

Background and aims The emergence of drug-resistant infections is a global problem. Acinetobacter baumannii has attracted much attention over the last few years because of resistance to a wide range of antibiotics. Applying new non-antibiotic methods can save lives of many people around the world. Antimicrobial photodynamic therapy (aPDT) technique can be used as a new method for controlling the infections. In this study we investigated the effect of aPDT on the expression of pathogenic genes in colistin-resistance A. baumannii isolated from a burn patient. Materials and methods The suspension of colistin-resistance A. baumannii was incubated with 0.01 mg/ml of toluidine blue O (TBO) in the dark; then the light emitting diode device with a wavelength of 630 ± 10 nm and output intensity of 2000-4000 mW /cm2 was irradiated to the suspension at room temperature. Subsequently, after the aPDT, genes expression of ompA and pilZ was investigated by using real-time polymerase chain reaction technique. Result Among the genes studied, the transcript of the ompA gene after aPDT was increased significantly in comparison with control groups (P 0.05). Conclusions It can be concluded from the results that the ompA as an outer membrane of A. baumannii is degraded after exposing aPDT and it will probably be done the penetration of antibiotics into cells of this bacterium easily.

Background During antimicrobial photodynamic therapy (aPDT) in the treatment of endodontic intracanal infection, it is extremely likely that microorganisms would be exposed to sub-lethal doses of PDT (sPDT). Although sPDT would not result in microorganism death, it can considerably influence microbial virulence. This study evaluated the effect of sPDT on gene expression of Enterococci surface protein (esp) as a major virulence factor for biofilm formation in Enterococcus faecalis. Materials and Methods The lethal and sub-lethal potential of aPDT using indocyanine green (ICG), toluidine blue O (TBO), and methylene blue (MB), as the photosensitizers (PSs) and 660, 635, and 810 nm diode laser against E. faecalis was analyzed using colony-forming unit assays. Considering sub-lethal doses of PSs and photo-irradiation time of the diode laser, the expression of esp was evaluated through quantitative Real-time PCR (qRT-PCR). Results ICG at a concentration of 31.2–1000 μg/mL and both TBO and MB at 6.2–100 μg/mL significantly reducedE. faecalis growth. 660, 635, and 810 nm diode laser with energy density of 93.75–140.62, 137.5–206.25, and 31.25–62.5 J/cm2, reduced the bacterial count. ICG with a concentration of 15.6 μg/mL and irradiation time of 0.5 min, TBO with a concentration of 3.1 μg/mL and irradiation time of 3 min, and MB with a concentration of 3.1 μg/mL and irradiation time of 3 min were found as a sPDT dose against E. faecalis. The esp expression was significantly reduced at sub-lethal doses of ICG compared to TBO (2 fold) and MB (2.4 fold). Conclusion Although all tested PSs showed bacterial reduction, ICG may be considered as the best PS in treating endodontic infection due to its higher efficacy in reduction of esp expression which has a main role in biofilm formation of E. faecalis.

Background Dental caries is a disease caused by a group of oral streptococcal microorganisms, primarily Strptococcus mutans. Photoactivated disinfection (PAD) has been proposed as a conservative method for treatment of carious lesions. Therefore, the aim of this study was to assess the effectiveness of indocyanine green (ICG)- and toluidine blue O (TBO)-PAD as supplementary tools in reducing the bacterial load and expression profiling of the gene associated with the biofilm formation in S. mutans. Materials and methods S. mutans strains were isolated from dental plaque samples collected from 212 patients through conventional biochemical tests and molecular methods. After identification, S. mutans strains were photosensitized in vitro with TBO and ICG, which were excited at a specific light wavelength based on the photosensitizer. After evaluating doses of TBO- and ICG-PAD contributing to sub-significant reduction of CFU/mL, ROS levels were measured with DCFH-DA fluorescent probe. Eventually, TBO- and ICG-PAD effects on the gtfB gene expression were assessed using real-time quantitative reverse transcription polymerase chain reaction. Results S. mutans strains were isolated from 50 (23.5%) patients. In this study, maximum doses of TBO- and ICG-PAD contributing to sub-significant reduction of CFU/mL against S. mutans strains were 23.12 μM/mL TBO at fluence of 68.75 J/cm2 and 20.15 μM/mL ICG at fluence of 31.2 J/cm2. According to the results, 3.8- and 6.1-fold increases in the fluorescence were observed in ICG and TBO treated cells compared to the control, respectively. The expression of gtfB was down regulated to approximately 3.9- and 8.25-fold following treatment with TBO- and ICG-PAD, respectively. Conclusions The gtfB gene expression profiling decreased in the bacterial cells, with ICG-PAD causing a greater reduction. PAD may be a promising therapy for dentinal carious lesions.

Background: Despite the high success rate of endodontic treatment, failure may occur in some cases. In this case, Enterococcus faecalis is the most common species in endodontic treatment failure and post-treatment apical periodontitis. Therefore, a new adjunctive strategy is needed for the prevention of endodontic infections due to E. faecalis. The aim of the present study was to compare the antimicrobial and anti-biofilm activities of different common photosensitizers (PSs) for use in antimicrobial photodynamic therapy (aPDT) against E. faecalis. Materials and methods: E. faecalis strain ATCC 29212 was used as the tested strain and methylene blue (MB), toluidine blue O (TBO), indocyanine green (ICG), and curcumin (CUR) were used as PSs. Irradiation was carried out using diode laser and light emitting diode (LED) at wavelengths related to the above PSs. Then, antimicrobial and anti-biofilm activities were measured using the microbial viability assay and crystal violet test, respectively. Results: aPDT with using the above PSs significantly decreased the CFU/mL count of E. faecalis compared to the control group (P < 0.05). The killing percentage of E. faecalis via PS mediated aPDT was 99.6%, 98.2%, 85.1%, and 65.0% for CUR, ICG, TBO, and MB, respectively. aPDT using the above PSs significantly decreased the biofilm formation ability of E. faecalis compared to the control group (P < 0.05). The biofilm reduction percentage of the PSs was 68.4%, 62.9%, 59.0%, and 47.6% for CUR, ICG, TBO, and MB, respectively. Conclusion: CUR and ICG mediated aPDT exhibited considerably more antimicrobial activity than other PSs, while TBO and MB demonstrated weaker anti-biofilm effects against E. faecalis compared to other PSs.

Recent advancements in DNA-based approaches have led to the identification of uncommon and rare bacterial pathogens. In this study, by utilizing a DNA-based approach, a total of 1043 clinical specimens were processed for the identification of actinobacteria targeting the 16S rRNA and gyrB genes. Drug susceptibility testing was also conducted using micro-broth dilution and PCR. Two isolates of Nocardia flavorosea and Rhodococcus erythropolis were reported for the first time in Iran. Also, Nocardiopsis dassonvillei, Streptomyces olivaceus, and Streptomyces griseus were reported for the first time in Asia. Infections caused by Nocardia caishijiensis and Prauserella muralis have also been reported in this study. The first Asian case of pulmonary infection caused by Nocardia ignorata and the first global case of brain abscess caused by Nocardia ninae and Nocardia neocaledoniensis have been reported in this study. Overall 30 isolates belonging to 6 genera (Nocardia, Streptomyces, Rodoccoccus, Nocardiopsis, Rothia, and Prauserella) were detected in 30 patients. All 30 isolates were susceptible to amikacin and linezolid. Three isolates including Nocardia otitidiscaviarum (n = 2) and Nocardia flavorosea (n = 1) were resistant to trimethoprim-sulfamethoxazole which were the first trimethoprim-sulfamethoxazole resistant clinical actinomycetes in Iran. Isolation of rare species of actinomycetes particularly Nocardia spp. requires urgent action before they spread clinically particularly among immunocompromised patients.

Background Antimicrobial sonodynamic therapy (aSDT) is an adjunctive modality, which uses ultrasound irradiation to kill microbial cells by the activation of a sonosensitizer. The aim of this study was to evaluated the synergistic biocidal effects of zinc oxide nanoparticles (ZnO NPs) and titanium dioxide nanoparticles (TiO2 NPs) as the metal oxide nanoparticles (MONPs)-assisted ultrasound irradiation against Streptococcus mutans biofilms. Materials and methods Following preparation and characterization of MONPs, cellular uptake and generation of intracellular reactive oxygen species (ROS) were assessed. After determination of the sub-significant reduction (SSR) doses of ZnO NPs, TiO2 NPs, ZnO/TiO2 NPs, and ultrasound intensity against S. mutans, anti-biofilm effects of aSDT were assessed using colorimetric assay, plate counting, and field emission scanning electron microscope (FESEM) analysis. Also, the metabolic activity of S. mutans and the expression levels of glucosyltransferase B (gtfB) as a main virulence factor of S. mutans were evaluated by XTT assay and quantitative real-time polymerase chain reaction following ZnO/TiO2 NPsSSR- mediated aSDT. Results The finding of this study showed that an incubation time of 5 min was sufficient to achieve maximal uptake of MONPs. The ROS production following aSDT using ZnO NPs, TiO2 NPs, and ZnO/TiO2 NPs were ~ 4.1-, 5.6-, and 11.7-fold increase, respectively. The dose-dependent reduction in cell viability of S. mutans was revealed by increasing the concentrations of ZnO NPs, TiO2 NPs, ZnO/TiO2, as well as ultrasound intensities. According to the data, 1.5 µg/mL, 3.1 µg/mL, 25 µg/mL, and 0.75 W/cm2 were considered as the SSR doses of ZnO/TiO2 NPs, ZnO NPs, TiO2 NPs, and ultrasound intensity, respectively (P>0.05). ZnO/TiO2 NPsSSR-mediated aSDT showed a significantly higher biofilm inhibitory activity than the other treatment groups (P 0.05). Conclusion The results revealed anti-metabolic and anti-biofilm potential activities of ZnO/TiO2 NPs-mediated aSDT against S. mutans with the highest cellular uptake and ROS generation.

Uncontrolled accumulation of microbial plaque and formation of biofilm on the surface orthodontic acrylic removable appliances increases the risk of enamel decalcification and periodontal diseases. The purpose of the present study was to evaluate antimicrobial activities, anti-virulence potencies, and mechanical properties of orthodontic acrylic resin containing different concentrations of Ulva lactuca (a green marine macroalga) following photo-activation against Streptococcus mutans.Minimum inhibitory concentration (MIC) of U. lactuca was determined against S. mutans. Acrylic resin specimens with different concentrations of U. lactuca (0.2 %, 0.5 %, 1%, 2.5 %, 5%, and 10 % weight/weight) were fabricated. Flexural strength values, antimicrobial effects, and anti-biofilm activities of samples were assessed in comparison with original acrylic resin as the control group. Also, the expression of the virulence-associated genes was assessed by quantitative real-time polymerase chain reaction.U. lactuca at concentrations of 1-10% significantly reduced the S. mutans growth rate by 20.3%-63.3% in comparison to the control group (P0.05). Therefore, the concentration of 1% of U. lactuca was considered as a MIC. The highest and lowest flexural strength values were observed in the control group (43.5 ± 2.4 MPa) and the group with a 10 % concentration of U. lactuca (19.2 ± 1.8 MPa), respectively. Flexural strength values decreased in samples containing 2.5 %, 5%, and 10 % concentrations of U. lactuca in comparison to the control group significantly (P0.05). In the disc agar diffusion test, the growth inhabitation zones around samples containing different concentrations of photo-activated U. lactuca ranged from 13 mm to 25 mm in diameter. Interestingly, the anti-biofilm activity of U. lactuca-mediated aPDT against S. mutans was dose-dependent. Additionally, the sub-MIC dose of U. lactuca (0.5 %) following photo-activation could significantly decrease the expression levels of gtfB, gtfC, and gtfD to 4.1-, 5.3-, and 7.4-fold, respectively.Adding photo-activated U. lactuca to the orthodontic acrylic resin at a concentration of 1% increases its antibacterial and anti-biofilm activities besides not detrimentally affects its flexural strength.

Objectives. The present study evaluated the antimicrobial effects of fissure sealants containing chitosan nanoparticles. Materials and Methods. Antibacterial effect of Master Dent fissure sealant alone and after incorporating chitosan nanoparticles was evaluated on Streptococcus mutans, sanguis, and Lactobacillus acidophilus. Biofilm growth was evaluated by determining colony counts. Antimicrobial effect was determined on days 3, 15, and 30 by counting microbial colonies using eluted components test. One-way ANOVA, Tukey HSD tests, t test, and two-way ANOVA were used for statistical analyses (α = 0.05). Results. Biofilm inhibition test showed that fissure sealant containing 1 wt.% chitosan decreased colony counts significantly ( P < 0.05 ). Eluted components test with S. mutans and sanguis showed significant decrease in colony counts during the first 15 days in chitosan containing group; however, from day 30, antimicrobial activity decreased noticeably, with no significant difference from control group ( P > 0.05 ). Antimicrobial activity against L. acidophilus was maintained in chitosan group up to 30 days, and decrease in colony counts was significant ( P < 0.05 ). Conclusion. According to the results of this study, incorporation of 1 wt.% chitosan into fissure sealant induced an antimicrobial activity. Antibacterial effect on L. acidophilus persisted for longer time (30 days) compared to the two other bacterial species (15 days).

Considering the antibacterial properties of nano-curcumin (nCur) reinforced with antimicrobial photodynamic therapy (aPDT), this study aimed to assess the antibacterial activity and durability of Activa BioActive Base/Liner (ABBL) containing nCur (nCur-ABBL) as a pulp capping agent against Streptococcus mutans, the most common cause of secondary caries.In this in vitro experimental study, ABBL discs containing 0.5 %, 1%, 2%, and 5% (w/w) concentrations of nCur were fabricated. After aPDT using light emitting diode (LED) at 435 ± 20 nm wavelength for 5 min, the discs were undergone aging in artificial saliva for 90 days. The antibacterial activity of the discs against S. mutans was evaluated by the disc agar diffusion test, and the number of bacterial colonies present in the biofilm formed on the disc surfaces was counted after 0, 15, 30, and 60 days of aging.The maximum growth inhibition zone was noted around the 5% nCur-ABBL discs. Increasing the concentration of nCur from 0.5 % to 5% combined with aPDT significantly decreased the number of S. mutans colonies in the biofilm over time (P0.05). nCur-ABBL discs containing 2% and 5% nCur had no difference in antibacterial activity at any time point up to 60 days (P0.05).According to our data, 5% nCur-ABBL revealed the largest growth inhibition zone in S. mutans culture. Moreover, 5% nCur can serve as an excellent ABBL additive in aPDT producer against S. mutans biofilms up to 60 days of aging period.

Objectives: This study assessed the antimicrobial efficacy of silver nanoparticles (AgNPs) incorporated in Transbond XT orthodontic adhesive used in rats. Materials and Methods: Transbond XT orthodontic adhesive containing 0%, 1%, 5% and 10% AgNPs was experimentally produced. Twenty-eight male Wistar rats were randomly divided into four groups (n=7) of control (0% AgNPs), 1% AgNPs, 5% AgNPs and 10% AgNPs. After anesthetizing the rats, one drop (10 μm) of the adhesive was applied on the central incisor, and light-cured for 20 s. Transbond XT composite (1×1×1 mm) was also applied. Another 10-μm drop was applied over it, and light-cured for 40 s. Biofilm test was carried out, and the number of colony forming units (CFUs) of Streptococcus sanguinis (S. sanguinis), Streptococcus mutans (S. mutans) and Lactobacillus acidophilus (L. acidophilus) in the saliva of rats was counted at baseline and 24 h after the application of adhesive. The data were analyzed using one-way ANOVA and Tukey’s test. Results: In presence of 5% and 10% AgNPs, S. sanguinis and L. acidophilus counts were significantly lower than those in the control and 1% AgNP groups (P

Less invasive removal approaches have been recommended for deep caries lesions. Antimicrobial photodynamic therapy (aPDT) and propolis nanoparticle (PNP) are highlighted for the caries management plan. Evidence is lacking for an additive effect of combination PNP with photosensitizer (PS) in aPDT. This study aimed to investigate the individual and synergistic effects of chlorophyllin-phycocyanin mixture (PhotoActive+) and toluidine blue O (TBO) as PSs in combination with PNP in the aPDT process (aPDTplus) against major important virulence factors of Streptococcus mutans. Following characterization, biocompatibility of the PSs alone, or in combination with PNP were investigated on human gingival fibroblast cell. The in vitro synergy of PhotoActive+ or TBO and PNP was evaluated by the checkerboard method. The bacteria's virulence properties were surveyed in the presence of the PSs, individually as well as in combination. When the PSs were examined in combination (synergistic effect, FIC Index + or TBO with PNP with the least cytotoxicity effects and the highest antimicrobial activites would improve aPDT outcomes, leading to synergistic effects and impairing the virulence of S. mutans.

This study aimed to compare the antibacterial effect of antimicrobial photodynamic therapy (aPDT) by use of light emitting diode (LED) and diode laser light sources with toluidine blue (TBO) photosensitizer on Enterococcus faecalis (E. faecalis) biofilm in root canals of extracted single-canal human teeth.Eighty-five sound human single-canal teeth were chosen for this study and standardized to have 15 mm of root length. The root canals were prepared with ProTaper rotary files and inoculated and incubated with E. faecalis for one week. Samples were divided into five experimental (n = 15) groups of PDT with TBO/LED, TBO/diode laser, LED, TBO and diode laser and one negative (NaOCl) and one positive (no treatment) control group. Dentin chip samples were collected from inside the canals using size 40 hedstrom file. The number of colony forming units (CFUs) in each group was calculated.Irrespective of the light source used for activation of photosensitizer (diode or LED), PDT was significantly more effective than other experimental groups (P0.001). No significant difference was noted between aPDT with diode laser or LED (P0.05). No significant difference was noted in colony count among other groups (LED, TBO, diode; P0.05).The results showed that aPDT significantly decreased residual bacteria in the canal. Thus, it may be used as an adjunct for root canal disinfection. Both diode and LED are suitable light sources for this purpose and can be used alternatively.

Porphyromonas gingivalis (P. gingivalis), a Gram-negative periodontal pathogen, is frequently detected in subgingival plaques in patients with chronic periodontitis. Considering the insufficient results achieved by the current antimicrobial approaches, this study aimed to compare the susceptibility of P. gingivalis to the different modes of photo-activated disinfection in planktonic culture.Cultures of P. gingivalis were assigned into three main groups of photothermal disinfection with 810 nm laser in continuous mode + EmunDo® (indocyanine green based photosensitizer), 808 nm laser in pulse mode + Perio green® (indocyanine green based photosensitizer); and antibacterial photodynamic therapy with 662 nm laser + Radachlorin® photosensitizer. Then, the bacterial viability was determined by serial dilution method by counting the number of colonies forming units. The results were analyzed by one-way analysis of variance and Tukey test (p 0.05).All three groups showed significant reduction in the viability of P. gingivalis (p 0.001). However; this reduction was significantly higher in photothermal therapy with EmunDo®.Both methods of photodynamic and photothermal therapy were successful in Significant eradication of P. gingivalis. Although, photothermal approach was more efficient with EmunDo® in combination with 810 nm laser irradiation.

Background: Photoactivated disinfection (PAD) has been indicated as one of the potential methods for the initial periodontal therapy in periodontitis and peri-implantitis, which may have bystander effects on the viability of off-target cells. Objective: In this article, we evaluated the effects of PAD-induced bystander on biofilm formation ability, metabolic activity, and gene expression level of Aggregatibacter actinomycetemcomitans as one of the bacteria involved in causing periodontitis and peri-implantitis. Methods: Bystander effects induced by bacterial cell suspension (BCST) obtained from treated A. actinomycetemcomitans culture with indocyanine green (ICG)-PAD on biofilm formation of A. actinomycetemcomitans were determined using microbial viability assay. Additionally, metabolic activity and changes in gene expression of fimA were assessed using the XTT reduction method and quantitative real-time PCR (qRT-PCR) assay, respectively. Results: The results revealed that 1000 and 125 μg/mL of ICG combined with diode laser irradiation for 1 min were significant and sub-significant reduction doses of ICG-PAD, respectively, versus A. actinomycetemcomitans. The biofilm formation ability, metabolic activity, as well as, expression level of fimA gene in A. actinomycetemcomitans were significantly reduced after treatment by a significant reduction dose of ICG-PAD-induced bystander effects (P Conclusions: ICG-PAD could significantly reduce the microbial population, cell metabolic activity, and gene expression through the bystander effects and could enhance the efficiency of PAD as an adjunct approach for the nonsurgical treatment of periodontitis and peri-implantitis.

Introduction: Antimicrobial orthodontic adhesives aim to reduce enamel demineralization, white spot lesions, and incipient tooth decay around bonded orthodontic brackets, but they should not imperil its mechanical properties. Aim: To evaluate the antimicrobial and physico-mechanical properties of acrylic containing different concentrations of C-phycocyanin on Streptococcus mutans, Lactobacillus acidophilus, and Candida albicans. Materials and methods: The mechanical properties of acrylic resins were measured by flexural strength test after preparation of acrylic resin samples with concentrations of 1%, 2%, 5%, 7.5%, and 10% of C-phycocyanin. Then we evaluated the antimicrobial effects of acrylic resin containing the maximum concentration of C-phycocyanin with clinically acceptable flexural strength and the changes in expression of virulence factors. Results: The highest and lowest means of flexural strength were obtained in acrylic resins containing 0% and 10% concentrations of C-phycocyanin at 50.2±4.5 and 30.1±3.3 MPa, respectively. Adding 1%, 2%, and 5% of C-phycocyanin showed no significant decrease in flexural strength (p>0.05). The maximum mean diameter of the growth inhibition zone was observed around discs containing 5% of C-phycocyanin. Until day 30 of the study, no microbial biofilms were formed on any acrylic disc. Only microbial biofilms of C. albicans were able to form on discs containing 5% of C-phycocyanin at 90 days. 5% C-phycocyanin could significantly decrease the expression levels of gtfB, hsp16, and ALS9 6.1-, 7.3-, and 3.9-fold, respectively. Conclusions: It can be concluded that the most acceptable concentration of C-phycocyanin in acrylic resin is 5% based on the results of flexural strength tests and antimicrobial activities of acrylic resin containing various concentrations of C-phycocyanin.

Background The appearance of bacterial resistance to antibiotics a are major health problems. Antimicrobial photodynamic therapy (aPDT) has emerged as an adjunctive treatment for drug-resistant infections. Titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles have recently been proposed as effective carriers for photosensitizers. The present study was designed to evaluate the antibacterial activity of ZnO and TiO2-nanoparticles accompanying aPDT and antimicrobial photocatalysis approaches against extensively drug-resistant colistin resistant Acinetobacter baumannii (XDR−CO-Ab). Materials and methods A XDR−CO-Ab isolated from burn-wound infection was used in this study. The bacterial suspension was exposed to TBO (Toluidine blue O), TiO2, and ZnO-nanoparticles in different groups. Ultraviolet A (UVA) (320 nm) and blue laser light (405 nm) were used to activate the photosensitizer and nanoparticles. The bacterial viability was then determined by counting the number of colonies forming units (CFUs)/mL. Results Using both aPDT and antimicrobial photocatalysis methods simultaneously, it had an amazing result; so that 99.32 % of XDR−CO-Ab was killed. According to the results, the bactericidal effects of activated TBO using UVA and laser lights was 5.1 % more than TBO was excited with laser light alone. Conclusions It was found that the combination of photocatalyst (TiO2+ZnO-nanoparticles) and TBO as photosensitizer at two-wavelength irradiation with blue laser and UVA lights have been shown to have significant antimicrobial effects.

Summary Introduction Biofilm accumulation around orthodontic brackets and composite is a common complication of orthodontic treatment. Objective A systematic review and meta-analysis were done to find out whether the association of nanoparticles with the orthodontic adhesives compromises its properties and whether there are exceptional nanoparticles exhibiting excellent antimicrobial potential against cariogenic bacteria along with remarkable mechanical properties. Materials and methods Electronic databases were searched using the following keywords; orthodontic or orthodontics and antimicrobial or antibacterial and adhesive and nanoparticles and shear bond strength. Thirteen studies were included and meta-analysis was performed. Results The results indicated no drastic changes in mechanical properties (0.812, 95% CI [0.750, 0.861], P = 0.000). The Ag-HA, Cur, Cur-ZnO, and TiO2 in concentration ≥ 1% showed a statistically significant difference, where the control groups had higher shear bond strength. Nine studies assessed the antimicrobial properties of nanoparticles. 1 wt% Cu and 5 wt% TiO2 not only did not affect shear bond strength but also showed more antimicrobial activity against Streptococcus mutans. The analysis demonstrated the absence of heterogeneity (Q value = 44.014; df (Q) = 12; and I2 = 72.736) in shear bond strength of orthodontic adhesives with nanoparticles, with low risk of bias. Conclusions Adding ≤ 5 wt% antimicrobial nanoparticles to an orthodontic adhesive is less conducive to microbial growth than unmodified adhesive and does not influence bracket-enamel bond strength.

Background As the increase in the prevalence of antimicrobial resistant bacterial strains, development of adjuvant antimicrobial approach for the treatment of burn wound infection is important. Therefore, the aim of this study was to assess the effectiveness of antimicrobial photodynamic therapy (aPDT) using indocyanine green (ICG) in reducing the bacterial load and expression profiling of the quorum sensing (QS) system associated with the biofilm formation in multi-species bacterial biofilms. Materials and methods Multi-species bacterial suspension including Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus were photosensitized with ICG, which was excited at a wavelength of 810 nm. After evaluating the dose of ICG-aPDT contributing to the sub-significant reduction of colony forming unit (CFU)/mL, the gene expression levels of abaI, agrA, and lasI were assessed using real-time quantitative reverse transcription polymerase chain reaction. As well as, lipid peroxidation, superoxide radicals and reactive oxygen species (ROS) were measured and the morphological changes of multi-species bacteria were evaluated by scanning electron microscopy (SEM). Results According to the results, maximal sub-significant reduction dose of aPDT against CFUs/mL of multi-species bacterial cells was found with a combination of 31.2 μg/mL of ICG and irradiation by the diode laser for 1 min, with an estimated average output light energy 31.2 J/cm2. The expression levels of abaI, agrA, and lasI in A. baumannii, P. aeruginosa, and S. aureus were down-regulated to approximately 1.9-, 3.7-, and 4.9-fold, respectively. The amount of lipid peroxidation, superoxide radical production, and ROS generation significantly increased (P Conclusions ICG-aPDT with antimicrobial, anti-biofilm, and gene expression inhibitor characteristics, as well as, ROS generation can be used for the treatment of burn wound infections in vivo.

Potential complications during fixed orthodontic procedures are white spot lesions (WSLs) and tooth decay. This study evaluated the anti-biofilm activity of an orthodontic adhesive (OA) incorporating curcumin (Cur) doped Poly lactic-co-glycolic acid nanoparticles (Cur-PLGA-NPs), which can have the highest concentration of Cur-PLGA-NPs and shear bond strength (SBS) value simultaneously, against cariogenic bacteria i.e., Streptococcus mutans.Following synthesis and confirmation of Cur-PLGA-NPs, SBS and adhesive remnant index (ARI) of the modified orthodontic adhesives (MOA) containing Cur-PLGA-NPs (3, 5, 7, and 10 % wt.) were measured using universal testing machine and stereomicroscope, respectively. After artificial aging (continuously rinsed up to 180 days), the residual anti-biofilm ability of MOA which can have the highest concentration of Cur-PLGA-NPs and SBS value simultaneously were determined by anti-biofilm assay following photoexcited enamel slab bonded brackets by MOA containing Cur-PLGA-NPs against S. mutans biofilms using crystal violet assay.Adhesive with 7 % wt. Cur-PLGA-NPs revealed the highest concentration of Cur-PLGA-NPs and SBS value (16.19 ± 2.69 MPa, P0.05) simultaneously. No statistically significant difference in ARI scores was observed between the MOA and control (Transbond XT without the Cur-PLGA-NPs). On days 15, 30, 60, 90 and 120 there was a considerable decrease in optical density (OD) of preformed S. mutans biofilms on photoexcited enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs, to 94.1 %, 79.6 %, 69.6 %, 69.4 %, and, 55.1 % respectively in comparison to the control group (all, P0.05). From days 150 onwards, microbial biofilm formation was progressively increased on enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs compared to the control group (OA). Although chlorhexidine (2 %; as positive control) showed significant activity against pre-formed S. mutans biofilms on enamel slab bonded brackets using OA (99.1 % biofilm reduction; P = 0.001), its activity was slightly higher but not significant than photoexcited enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs on the days 15 and 30 (both, P0.05).The 7 % wt. Cur-PLGA-NPs can serve as an orthodontic adhesive antimicrobial additive as exposure to blue laser provides an acceptable antimicrobial effect against cariogenic bacteria for a considerable time.

Resistant infections essentially cause mortality in a burn unit. Several bacteria contribute to burn infections; among these, Pseudomonas aeruginosa majorly contributes to these infections revealing significant drug resistance. Similar to other bacteria, P. aeruginosa reveals various mechanisms to attain highest pathogenicity and resistance; among these, efflux pumps and quorum sensing are crucial. Quorum sensing enables effective communication between bacteria and synchronizes their gene expression resulting in optimum effect of the secreted proteins; alternatively, efflux pumps increase the bacterial resistance by pumping out the antimicrobial factors as well as the QS signals and precursors. Of recent, increasing episodes of drug resistance led to new findings and approaches for killing pathogenic bacteria without inducing the drug-resistant species. Photodynamic therapy (PDT), considered as an adjuvant and innovative method for conventional antibiotic therapy, is a photochemical reaction that includes visible light, oxygen, and a photosensitizer (PS). In this therapy, after exposure to visible light, the PS generates reactive oxygen species (ROS) that are bacteriostatic or bactericidal. Furthermore, this oxidative stress can disrupt the coordination of gene expression and alter the bacterial behavior. Considering the fact that the adaption and several gene expression patterns of microorganisms within the biofilm make them notably resistant to the recent antimicrobial treatments, this study aimed to emphasize the relationship between the efflux pump and QS under oxidative stress and their role in P. aeruginosa's reaction to PDT.

Background It has clearly been demonstrated that Enterococcus faecalis, as a persistent microorganism, is the major agent in the etiopatogeny of endodontic infections. Recently, the limitations of conventional endodontic therapy have given rise to many attempts to introduce antimicrobial photodynamic therapy (aPDT) as an alternative treatment. The aim of this study was to analyze the ex vivo effect of aPDT in combination with 2.0% chlorhexidine (CHX) as a conventional therapy on colony count and expression patterns of genes associated with biofilm formation of E. faecalis. Materials and methods A total of 125 extracted human single-rooted teeth were divide into six groups (A-F; n = 20) and were incubated with E. faecalis. Group A- photosensitizer (indocyanine green [ICG]); B- diode laser; C- aPDT; D- 2.0% CHX; E- aPDT with photosensitizer modified by 2.0% CHX; and F- control group (no procedure was performed). Five remaining teeth were used to confirm the presence of E. faecalis biofilm via scanning electron microscope. Counts of colony forming units (CFUs) in each group were evaluated separately and quantitative real-time PCR (qRT-PCR) was then applied to monitor genes expression of fsrC, efa, and gelE involved in E. faecalis biofilm. Results The results showed that none of the tested groups achieved eradication or inhibition of biofilm. On the other hand, aPDT + 2.0% CHX, 2.0% CHX, and ICG- mediated aPDT groups showed significantly less CFU/mL than ICG and diode laser groups. The group with the lowest CFU/mL count was the aPDT + 2.0% CHX, being statistically different from all other groups that could decrease the expression levels of efa, gelE, and fsrC genes 6.8-, 8.3-, and 12.1-fold, respectively. Conclusion Based on the results, the synergism effect of ICG-aPDT with 2.0% CHX leads to modulation of the virulence of E. faecalis strains biofilm model by suppressing the expression of the genes associated with biofilm formation.

Introduction Porphyromonas gingivalis, an important endodontic pathogen, may be exposed to sublethal doses of photo-activated disinfection (sPAD) during root canal therapy. Such an exposure can affect bacterial survival and virulence features. In this study, we evaluated the effect of sPAD-related oxidative stresses on the expression of oxidative stress response gene (oxyR) in P. gingivalis clinical isolates surviving in vitro photodynamic treatment. Materials and methods To determine the sPAD, 16 clinical P. gingivalis isolates photosensitized with toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) were irradiated with specific wavelength and energy density of diode laser corresponding to the photosensitizers following bacterial viability measurements. The effects of sPAD on the expression ratio of oxyR of 16 clinical P. gingivalis isolates were then assessed using quantitative real-time PCR (qRT-PCR) assay. Results Maximum values of sPAD against P. gingivalis were 6.25, 15.6, and 25 μg/mL at fluencies of 171.87, 15.6, and 93.75 J/cm2, respectively, for TBO-, ICG-, and MB-sPAD (P>0.05). ICG-, MB-, and TBO-sPAD could increase the oxyR gene expression of the clinical P. gingivalis isolates 12.3-, 5.6-, and 8.5-fold, respectively. ICG-sPAD increased the expression of oxyR gene in clinical isolates of P. gingivalis ~1.5- and 2-fold higher than TBO- and MB-sPAD, respectively. Conclusion Our results showed that upregulation of oxyR during sPAD may lead to better survival and increased pathogenicity of P. gingivalis isolates. Therefore, selection of appropriate photo-activated disinfection dosage should be considered for the successful treatment of endodontic infection.

Background The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. Materials and methods Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. Results Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). Conclusion As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively.

Background The endodontic disinfection therapy majorly aims to eradicate microbial pathogens. Photo-activated disinfection (PAD), also known as antimicrobial photodynamic therapy, is an alternative antimicrobial modality used to control the microorganisms causing endodontic infections. Notably, microorganisms may be exposed to sub-lethal doses of PAD (sPAD), influencing microbial virulence. The present study assessed the effects of sPAD on expression profiling of the gene associated with the biofilm formation being the most essential virulence factor in Porphyromonas gingivalis strain cells that survive the photodynamic reatment in vivo. Materialas and methods Sixteen clinical strains of PAD resistant P. gingivalis that were isolated in vivo, were further photosensitized in vitro with toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) as the photosensitizing agents, which were excited with specific wavelength of light based on the photosensitizer. After evaluating sPAD, its effects on the fimA gene expression were assessed using real-time quantitative reverse transcription PCR (qRT-PCR). Results In this study, maximum values of sPAD against P. gingivalis were 6.25 μg/mL TBO at a fluency of 171.87 J/cm2, 15.6 μg/mL ICG at fluency of 15.6 J/cm2, and 25 μg/mL MB at fluency of 93.75 J/cm2. MB-, TBO-, and ICG-sPAD could cause about 4.6-, 14.4-, and 17.3-fold suppression of fimA expression, respectively. P. gingivalis strains expressed less virulence in cells surviving PAD. Conclusions In conclusion, the gene expression profiling was reduced in the bacterial cells, wherein greater reduction was observed for ICG-sPAD than TBO- and MB-sPAD.

Background Management of burn wound infections (BWIs) is difficult due to the emergence of multidrug-resistant microorganisms. This study aimed to explore the anti-biofilm efficacy of sonodynamic therapy (SDT) using nano-emodin (N-EMO) against multi-species bacterial biofilms containing Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii. Methods Following synthesis and confirmation of N-EMO as a sonosensitizer, the anti-biofilm efficacy of SDT against multi-species bacterial biofilms was determined using minimum inhibitory concentrations (MIC), minimum biofilm inhibitory concentration (MBIC), and minimal biofilm eradication concentration (MBEC) of N-EMO. The reduction of multi-species bacterial biofilms was then evaluated following the treatments using Log reduction and crystal violet (CV) assays. In addition, the expression profiling of abaI, agrA, and lasI genes using SDT with sub-MIC, sub-MBIC, and sub-MBEC of N-EMO was assessed. Results Successful synthesis of N-EMO was confirmed through several characterization tests. As the results demonstrated, the MIC value of N-EMO for the multi-species bacterial suspension was 0.15 × 10–4 g/L, as well as, the MBEC value of N-EMO was 2.5 × 10–4 g/L, approximately 4-fold higher than that of MBIC (0.62 × 10–4 g/L). According to the CV assay, there were 57.8 %, 71.0 %, and 81.5 % reduction in the biofilm of multi-species bacterial growth following SDT using 1/128 MBEC, 1/16 MBIC, and 1/2 MIC of N-EMO, respectively. Log reductions analysis demonstrated that 1/2 MIC of N-EMO was more potent in inhibiting the biofilm growth of multi-species test bacteria by 5.725 ± 0.12 (99.9993 %). In this study, N-EMO-mediated SDT could obviously downregulate the gene expression of virulence factors (P Conclusion These results highlight the potential of N-EMO-mediated SDT in inhibition of biofilm formation, degradation of formed biofilms, and reduction of virulence factor associated with biofilms of multi-species bacterial biofilms in BWIs.

To elucidate the association between asymptomatic infections caused by Mycoplasma genitalium and male infertility, and evaluate the role of antibiotic therapy in treatment of this failure. A total of 165 infertile males having abnormal semen parameters (study group) and 165 healthy fertile men (control group) were included. Semen samples were taken from all participants and after analyzing for semen parameters, undergone real-time PCR, microbial culture, and reactive oxygen species (ROS), as well as total antioxidant capacity (TAC) assays. Infected individuals of study group were treated with antibiotic. One month after the treatment completion, second semen samples were taken and subjected to all the tests mentioned. The data were analyzed using SPSS statistical software, version 22.0. The frequency of M. genitalium was significantly higher in the infertile men compared with the fertile ones (9.7% vs. 1.2%; p = 0.001). Mean cycle threshold (C t) value was lower in infected infertile than infected fertile men (p

Background Acinetobacter baumannii is an important human pathogen which has recently gained increased attention due to the occurrence of drug-resistant nosocomial infections in patients suffering from immune system disorders, and those in hospital intensive care units. The aim of this research was to identify and isolate A. baumannii strains resistant to colistin, determine antibiotic resistance pattern of this bacteria, investigate the presence of colistin-resistant genes, and finally assess the effect of expression changes in pmrA and pmrB genes resistant to A. baumannii against colistin via real-time polymerase chain reaction. Methods The samples were initially purified and isolated using biochemical tests and Micro-gen kit. Later, the resistance pattern evaluation of validated samples to different antibiotics and colistin was carried out using two methods viz., disc diffusion and E-test. This was followed by the assessment of genes resistant to colistin via polymerase chain reaction besides gene expression changes via real-time polymerase chain reaction. Results The results of this study indicated that eleven strains of A. baumannii isolated from Shahid Rajaee Trauma Hospital were resistant to colistin. However, in the resistance pattern evaluation of A. baumannii isolated from Ali Asghar Hospital, all the strains were sensitive to colistin. In the evaluation of genes resistant to pmrA and pmrB, most of the strains resistant to colistin were carriers of these genes. Besides, in the expression assessment of these genes, it was demonstrated that expression of pmrA in the strains resistant to colistin significantly increased in relation to sensitive strains, but the expression of pmrB increased at a lower rate in the strains resistant to colistin as compared to the sensitive strains. Conclusion Thus, it can be safely mentioned that increased expression of pmrA was due to the resistance of A. baumannii to colistin.

Aggregatibacter actinomycetemcomitans is an important pathogen that is frequently found in various infections, particularly aggressive periodontitis. In this study, we described the outcome of the expression level of A. actinomycetemcomitans virulence factor following treatment by antimicrobial photodynamic therapy (aPDT) with indocyanine green (ICG) as a photosensitizing agent.To determine the aPDT effect on the cell-surviving assay and expression ratio of the rcpA gene in A. actinomycetemcomitans by a colony-forming unit and relative quantitative (q) real-time PCR (qRT-PCR) assays, respectively, the proper dosing of sub-lethal aPDT was specified.The results of the current study showed that ICG-mediated aPDT, using 250-1000μg/mL, showed a significant reduction in A. actinomycetemcomitans growth when compared to the control group (P0.05). Also, a sub-lethal dose of aPDT against A. actinomycetemcomitans was 125μg/mL ICG, with a 30s diode laser irradiation time at fluency of 15.6J/cmaPDT with ICG could reduce the cell survival and the virulence agent of A. actinomycetemcomitans. Thus, use of the appropriate aPDT dosage can be used for the successful treatment of periodontitis in vivo.

The role of asymptomatic infections caused by Ureaplasma species in male infertility and the efficacy of antibiotics in treatment of this failure is not yet definitely determined. A total of 165 infertile males having abnormal semen parameters (study group) as well as 165 healthy fertile men (control group) were included in this study. Semen samples were taken from all participants and after analyzing, undergone real-time PCR, microbial culture, and reactive oxygen species (ROS) as well as total antioxidant capacity (TAC) assays. Infected individuals of study group were treated with antibiotic. One month after the treatment completion, second semen samples were taken and undergone all the tests mentioned. The frequency of Ureaplasma spp. was significantly higher in the infertile men compared with the fertile ones (36.4% versus 11.5%; p < 0.001). Most of semen parameters were improved (p < 0.05) and reached their normal range, the level of TAC elevated (p < 0.001), and ROS level (p = 0.003) as well as ROS/TAC ratio (p = 0.003) reduced after antibiotic treatment. Moreover, wives of 37 infertile men (61.7%) became pregnant six months after the treatment completion. These findings indicate that Ureaplasma species are correlated with male infertility and that antibiotic therapy can improve the semen parameters and treat the male infertility.

Here we studied the prevalence and mechanisms of simultaneous resistance to carbapenem and tigecycline and accumulation of resistance determinants reservoirs in genome of Acinetobacter baumannii (A. baumannii) clinical isolates. Susceptibility of the isolates were measured to 18 antimicrobial agents. Genetic diversity of the microbial population was determined using the International Clonal lineage typing (IC typing), multiple locus VNTR analysis (MLVA) and plasmid profiling methods. To detect the AbaRs, Carbapenem-Hydrolyzing Class D β-Lactamases (CHDLs) genes, AdeABC efflux pump genes and resistance determinants, PCR was used. Filter mating experiments were used to prove that if carbapenem resistance genes are located on conjugative plasmids or not. Among the A. baumannii clinical isolates, 40.8% were carbapenem-tigecycline resistant and in this population, 46.9% were belonging to IC I, IC II or IC III and 53.1% were IC variants. These isolates had fallen in 40 MLVA types and were harboring plasmids in multiple numbers and sizes. In this study, blaOXA-23-like was the most prevalent CHDL and conjugation analysis proved that the carbapenem resistance genes are located on conjugative plasmids. All efflux pump genes, except for adeC, were detected in all carbapenem-tigecycline resistant A. baumannii (CTRAb) isolates. Resistance determinants were distributed in both TnAbaRs and R plasmids with a shift toward the R plasmids. Emerging of carbapenem resistant A. baumannii (CRAB) with simultaneous resistance to the last line therapy including tigecycline represent emerging of extensively drug resistance (XDR) and pandrug resistance (PDR) phenotypes that would be a great threat to our public health system.

Background The alternative antibacterial treatments of photodynamic therapy (PDT) and photothermal therapy (PTT) significantly affect microbiota inactivation. The aim of the present research was the assessment of the antimicrobial and anti-biofilm effects of PDT with toluidine blue O (TBO) and PTT with indocyanine green (ICG) on Streptococcus mutans as a cariogenic bacterium. Materials and methods The S. mutans ATCC 35668 strain was treated with final concentrations of 0.1 mg/mL TBO and 1 mg/mL ICG with energy densities of 17.18 and 15.62 J/cm2, respectively. Cell viability was evaluated after culturing and anti-biofilm potential was analyzed using crystal violet assay and scanning electron microscopy. Results The number of S. mutans colony forming unit (CFU)/mL was significantly lower in the groups submitted to PDT (12.5–100 μg/mL TBO) and PTT (62.5–1000 μg/mL) compared to the control (untreated group). 0.1 mg/mL TBO-PDT and 1 mg/mL ICG-PTT showed stronger inhibitory effects on biofilm formation in S. mutans than other concentration levels, with a reduction of 63.87% and 67.3%, respectively. Conclusion Photo-elimination by high concentrations of TBO-PDT and ICG-PTT exhibited significantly stronger inhibitory effects on biofilm formation and cell viability in S. mutans.

Background Porphyromonas gingivalis is one of the etiological agents in the initiation of combined periodontal-endodontic (perio-endo) lesions. Successful treatment of perio-endo lesions with photo-activated disinfection (PAD) as a novel therapeutic approach depends on the selection of an appropriate target site, specific photosensitizer (PS), and wavelength of light. Since the fimbrillin ( FimA ) is the most important pathogenic agent in P. gingivalis it was evaluated as a target site against PAD with toluidine blue O (TBO) as a PS. Materials and methods We evaluated the potential of FimA using a number of bioinformatic tools and computer simulation molecular modeling. In silico analysis was done to predict the hierarchical nature of structure, topology, backbone, physicochemical properties, and functional characterization of FimA . Results The predicted structure of FimA exhibited that it is an anionic protein with negative charge (−7.00) in pH7 and random coil which dominates other secondary structures located outside the cell. It has three motifs with seven ligand binding site residues and one active site residue which can be a site of interaction for cationic TBO. Conclusion Based on the results of this study cationic TBO tends to interact with FimA during PAD as a major target to increase the efficiency of treatment of perio-endo lesions.

Introduction: Plaque accumulation and bond failure are drawbacks of orthodontic treatment, which requires composite for bonding of brackets. As the antimicrobial properties of TiO2 nanoparticles (NPs) have been proven, the aim of this study was to evaluate the antimicrobial and mechanical properties of composite resins modified by the addition of TiO2 NPs. Methods: Orthodontics composite containing 0%, 1%, 5% and 10% NPs were prepared. 180 composite disks were prepared for elution test, disk agar diffusion test and biofilm inhibition test to collect the counts of microorganisms on three days, measure the inhibition diameter and quantify the viable counts of colonies consequently. For shear bond strength (SBS) test, 48 intact bovine incisors were divided into four groups. Composites containing 0%, 1%, 5% and 10% NPs were used for bonding of bracket. The bracket/tooth SBS was measured by using an universal testing machine. Results: All concentration of TiO2 NPs had a significant effect on creation and extension of inhibition zone. For S. mutans and S. sanguinis, all concentration of TiO2 NPs caused reduction of the colony counts. Composite containing 10% TiO2 NPs had significant effect on reduction of colony counts for S. mutans and S. sanguinis in all three days. The highest mean shear bond strength belonged to the control group, while the lowest value was seen in 10% NPs composite. Conclusions: Incorporating TiO2 nanoparticles into composite resins confer antibacterial properties to adhesives, while the mean shear bond of composite containing 1% and 5% NPs still in an acceptable range. RESUMO Introdução: o acúmulo de placa e as descolagens de braquetes são algumas desvantagens presentes no tratamento ortodôntico, no qual se requer o uso de materiais compósitos para a colagem dos braquetes. Objetivo: tendo em vista que as propriedades antimicrobianas das nanopartículas (NPs) de TiO2 já foram confirmadas, o objetivo do presente estudo foi avaliar as propriedades antimicrobianas e mecânicas de resinas compostas modificadas pela adição de NPs de TiO2. Métodos: compósitos ortodônticos contendo 0%, 1%, 5% e 10% de NPs foram preparados. Cento e oitenta discos de compósito foram preparados para o teste de eluição, o ensaio de difusão em ágar por disco, e o ensaio de inibição da formação de biofilme, para se calcular as contagens de microrganismos ao longo de três dias, medir o diâmetro da inibição e, consequentemente, quantificar as contagens de colônias viáveis. Para o teste de resistência da colagem ao cisalhamento (SBS), 48 incisivos bovinos intactos foram divididos em quatro grupos, nos quais os compósitos contendo 0%, 1%, 5% e 10% de NPs foram utilizados para colagem dos braquetes. A SBS da interface braquete/dente foi medida em uma máquina universal de ensaios. Resultados: todas as concentrações de NPs de TiO2 apresentaram efeito significativo na formação e na extensão da zona de inibição. Para o S. mutans e o S. sanguinis, todas as concentrações de NPs de TiO2 causaram redução na contagem das colônias. O compósito contendo 10% de NPs de TiO2 apresentou uma diminuição significativa na contagem de colônias de S. mutans e S. sanguinis durante os três dias. A média mais alta da SBS foi observada no grupo controle, enquanto o valor mais baixo foi observado para o compósito com 10% de NPs. Conclusões: a incorporação de nanopartículas de TiO2 nas resinas compostas lhes conferiu propriedades antibacterianas, e o valor médio da SBS das resinas contendo 1% e 5% de NPs apresentou-se dentro de uma faixa aceitável.

Nocardia ignorata, which was first described in 2001, is a rare human pathogen. We report a case of pulmonary nocardiosis caused by this bacterium in a 55-year-old man from Iran. The patient, a gardener, had frequent exposure to soil and may have acquired the infection from that source.

Background Periodontitis is a polymicrobial, chronic, and degenerative disease that can lead to destruction of the teeth-supporting tissues and eventually to loss of teeth. Aggregatibacter actinomycetemcomitans is predominantly associated with periodontal diseases. Outer membrane protein (Omp) 100 is a more important virulence factor of A. actinomycetemcomitans due to the effect of adhesion and invasion into human gingival epithelial cells. Attachment of A. actinomycetemcomitans inhibition is significant in the treatment process. Methods We evaluated the capacity of Omp100 in A. actinomycetemcomitans as a novel target for photodynamic therapy (PDT) using a range of bioinformatic tools. In silico analysis was used to predict molecular modeling, the hierarchical nature of protein structure and backbone, and sub-cellular localization. Results The results showed that Omp100 is most similar to thiamine-phosphate pyrophosphorylase [Haemophilus influenzae PittGG], with a 74% similarity. The predicted structure of Omp100 displayed that it is a protein with positive charge (10.4) in pH 7 and alpha helix dominates other secondary structures located outside the cell. Protein-protein interaction network showed that Omp100 interacted with extracellular matrix protein adhesion, glycoside hydrolase, Omp 64, phospholipase D/Transphosphatidylase, Flp pilus assembly protein, and heme acquisition system receptor. Conclusion According to the results, anionic indocyanine green tends to interact with Omp100 during PDT as a major target.

Hossein Kazemian,1 Jalil Kardan-Yamchi,2 Abbas Bahador,1 Shadi Khonsari,3 Mahshid Nasehi,4 Gholamreza Hamzehloo,5 Farzam Vaziri,6 Mohammad Reza Salehi,7 Mohammad Mehdi Feizabadi1,8 1Department of Medical Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; 2Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran; 3Natural Science Department, School of Science and Technology, Middlesex University, London, UK; 4Department of Epidemiology and Biostatistics, Iran University of Medical Sciences, Tehran, Iran; 5Tehran Regional Reference Laboratory for Tuberculosis, Tehran, Iran; 6Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran; 7Department of Infectious Disease, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran; 8Thoracic Research Center, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, IranCorrespondence: Mohammad Mehdi FeizabadiDepartment of Medical Microbiology, School of Medicine, Tehran University of Medical Sciences, Poursina Street, Engelab-e-Eslami Avenue, Tehran, IranTel +989121767987Fax +982188955810Email mfeizabadi@tums.ac.irBackground: Successful treatment of tuberculosis depends on early diagnosis and use of appropriate drug susceptibility testing in a timely manner. In the present study, LPA efficacy was assayed in detection and drug susceptibility testing of pulmonary tuberculosis in comparison to available methods in Iran and phylogenetic analyses of isolated cases carried outby MIRU-VNTR.Methods: This study was conducted at the Tehran Regional Reference Laboratory for Tuberculosis. All sputum specimens were subjected to smear, culture, and drug susceptibility testing (DST), GeneXpert, and LPA. Finally, 15-locus-based MIRU-VNTR was used for molecular genotyping.Results: From a total of 920 sputum specimens, 6.08% (n=56) were identified as MTBC by culture, 6.8% (n=63) by GeneXpert, and 6.5% (n=60) by LPA. Phenotype DST and LPA methods confirmed the resistance of 4 and 14 specimens to rifampin (RIF) and isoniazid (INH); two cases were considered as multidrug-resistant (MDR). Using GeneXpert, four cases were identified as RIF-resistant. Based on LPA results, inhA and katG mutations were detected in 100% and 21.4% of INH-resistant cases, respectively. All 56 culture positive Mycobacterium tuberculosis isolates were placed in 29 different clusters using MIRU-VNTR genotyping. Two MDR-TB, 2 RIF mono-resistant, and 12 INH mono-resistant cases were placed in different clusters.Conclusion: LPA is an appropriate method for early detection and accurate diagnosis of TB and drug-resistant cases that makes it possible to distinguish INH mono-resistant cases from MDR cases in Iran.Keywords: tuberculosis, line probe assay, early diagnosis, drug resistance, mutation