Screening of Streptococcus mutans Sortase A Via Myricetin-Like Inhibitors: In Vitro Evaluation and Molecular Docking-Based Virtual

Background: The oral and dental infections that are mainly caused by bacterial biofilms are among the most prevalent human infections worldwide. Objectives: The present study aimed to investigate the in vitro and in vivo inhibitory and anti-biofilm effects of oregano essential oil on the Streptococcus mutans isolates obtained from elementary school students. Methods: This experimental study was conducted on 150 samples collected from the buccal and lingual surfaces of the posterior teeth of elementary school students. S. mutans strains were identified using conventional microbiological and biochemical tests, and biofilm formation was assessed using the microtiter plate assay. The minimum inhibitory concentration (MIC) of the oregano essential oil against the isolates was determined using the broth microdilution method. In addition, the effective constituents of the essential oil were measured via gas chromatography-mass spectroscopy. The in vitro and in vivo anti-biofilm activities of the oregano essential oil were also evaluated using the modified microtiter plate assay and on the tooth surfaces of male NMRI mice, respectively. Results: The frequency of S. mutans was 15.3%, 87% of which were capable of biofilm formation. The MIC of the oregano essential oil was 50 µl/ml against the S. mutans isolates, and 82% of the isolates did not grow at the concentrations of ≥ 512 µl/ml. However, none of the isolates were capable of biofilm formation at the MIC and sub-MIC concentrations of the essential oil. Limonene and myrcene were the most effective constituents of the essential oil. Furthermore, a significant correlation was observed between treatment with the oregano essential oil and biofilm formation by the streptococci isolates (P = 0.05). Conclusions: According to the results, the presence of biofilm and incidence of dental caries were significantly correlated. Moreover, the essential oil of oregano and its main constituents had potent anti-biofilm and antibacterial properties and could be utilized for the production of new plant-based mouthwashes.

Background and Objectives:The oral cavity harbors numerous Streptococcus mutans strains which display remarkable genotypic and phenotypic diversity. This study evaluated the genotypic and phenotypic diversity of 209 S. mutans strains isolated from 336 patients with dental caries and compared with the universal reference strain, UA159.Materials and Methods:Selective cultivation on mitis-salivaries-bacitracin agar and species-specific polymerase chain reaction (PCR) was carried out to isolate and identify the 209 S. mutans isolates from 336 patients with dental caries. Arbitrarily primed polymerase chain reaction (AP-PCR), PCR amplification of specific gene, acid production and biofilm formation capacity were performed to evaluate the genotypic and phenotypic variation. Student’s t-test and Chi-square test were used for analysis of variables and a probability (P) of <0.05 was considered as significant.Results:Our study revealed a high degree of genotypic and phenotypic variability among the clinical strains. We observed significant differences in colony morphology, generation time, biofilm formation, and acid production while growing in culture medium. All the clinical isolates were able to lower pH while growing in Todd-Hewitt broth. Consistent with phenotypic variations, we also observed genotypic variation by AP-PCR and gene specific PCR. AP-PCR analysis suggested that most of the patients with dental caries have distinct type of S. mutans strains. Genes related to various two component systems were highly conserved among the isolated strains, however, bacteriocin encoding genes such as nlmAB, nlmC were absent in nearly half of the clinical isolates.Conclusion:Our results support that S. mutans clinical isolates have wide genotypic diversity and show variation in growth kinetics, acid production, acid tolerance and biofilm formation capacity and indicates the presence of diverse mechanism to initiate and establish the biofilm lifestyle which leads to tooth decay.

The study explored the biofilm (BF) formation capacity, BF-related gene profiles, and the trends in antimicrobial resistance (AMR) of Salmonella pullorum (SP) strains over several years. A total of 627 SP strains were isolated from 4,540 samples collected from chicken farms in Guangxi, China during 2018-2022. The BF-forming capacity of these isolates was assessed using crystal violet staining, and the presence of eight BF-related genes (csgA, csgB, csgD, ompR, bapA, pfs, luxS, and rpoS) in BF formation-positive strains was determined through Polymerase Chain Reaction (PCR) analysis. Antimicrobial susceptibility test was conducted to investigate the AMR of the isolates. Minimum Inhibitory Concentration (MIC) and Minimal Biofilm Eradication Concentration (MBEC) of nine SP-BF strains were determined using the broth microdilution method to assess the impact of BF formation on AMR. Additionally, the Optimal Biofilm Formation Conditions (OBFC) were investigated. The results indicated that 36.8% (231/627) of the strains exhibited a positive BF-formation capacity. Among these, 24.7% (57/231) were strong BF producers, 23.4% (54/231) were moderate BF producers, and 51.9% (120/231) were weak BF producers. Analysis of the eight BF-related genes in SP-BF strains revealed that over 90% of them were positive for all the genes. Antimicrobial susceptibility test conducted on the isolates showed that 100% (231/231) of them exhibited resistance to at least one antibiotic, with 98.3% (227/231) demonstrating multidrug resistance (MDR). Both MIC and MBEC measurements indicated varying degrees of increased AMR after BF formation of the bacteria. The optimal conditions for BF formation were observed at 37°C after 48 h of incubation, with an initial bacterial concentration of 1.2 × 106 CFU/mL. Notably, NaCl had a significant inhibitory effect on BF formation, while glucose and Trypticase Soy Broth (TSB) positively influenced BF formation. The results of the study emphasized the need for effective preventive and control strategies to address the challenges posed by the BF formation and MDR of SP in the field.

Staphylococcus aureus is a widespread opportunistic pathogen, causing community-acquired and nosocomial infections with both acute and chronic recurrent courses. The process of chronicity of the disease is provided by biofilms. Features of the structure and functioning of biofilms, in particular the presence of matrix, quorum sensing systems, persistent cells, and efflux pumps, provide microbial communities with resistance to antimicrobial drugs under their action in therapeutic concentrations. The insufficient eff ectiveness of modern antimicrobial chemotherapy against biofi lm microorganisms indicates the urgency of the problem to search for compounds with antibiofilm activity that can affect various stages of the biofilm formation and the formed biofilm. The aim of the study is to establish the antibiofilm activity of 4-(adamantyl-1)-1-(1-aminobutyl) benzol against methicillin-resistant S. aureus (MRSA) and to determine the mechanism of its action. Methods. The ability of adamantane-containing compound 4-(adamantyl-1)-1-(1-aminobutyl) benzol (AM-166) to prevent biofilm formation and destroy the formed biofilm of S. aureus was investigated on polystyrene plates by the sorption of gentian violet on its structures followed with desorption of the dye into the organic solvent. The viability of S. aureus cells at the first stage of biofilm formation and in the composition of mature biofilms was evaluated using specific dyes for living (acridine orange) and non-viable (propidium iodide) cells. Detection of genes responsible for antibiotic resistance and biofi lm formation was performed by the polymerase chain reaction (PCR) with detection of PCR products in agarose gel. Evaluation of the effect of AM-166 on the expression of genes regulating the biofilm formation (ica, agrA, sarA, and sigB) was investigated by the real-time PCR and semi-quantitative PCR. Results. It was found that the compound AM-166 shows activity against S. aureus biofilm formation. The most pronounced effect was registered at a concentration of 5.0 minimum inhibitory concentration (MIC) (92.3%.) Under the action of AM-166 on the formed 2-day biofilms, their destruction was marked: the biomass decreases by 30.9% at 5.0 MIC. According to the results of fluorescence microscopy, the adamantane derivative at 5.0 MIC helps to reduce the number of viable cells at different stages of formation of the S. aureus biofilm. The results of molecular genetic studies indicate that the ica gene expression is significantly inhibited by the action of subinhibitory concentrations of the compound AM-116. No significant changes in the expression of sarA, agrA, and sigB genes were registered. Conclusions. Experiments on the effect of adamantane derivative on S. aureus biofilms showed that the most pronounced activity of AM-116 was observed at the stage of biofilm formation, as evidenced by the inhibition of transcriptional activity of the ica gene responsible for early stages of the biofilm formation, in particular the adhesion of planktonic cells to the substrate.

We aimed to investigate the synergistic effects of colistin and green-synthesized silver nanoparticles on the biofilm formation and expression of Quorum Sensing regulated and related genes in clinical isolates of P. aeruginosa. Ten clinical P. aeruginosa isolates collected from patients with burn wound infections were investigated. The antibiotic sensitivity pattern of the isolates was determined using disk diffusion and microbroth dilution tests. The silver nanoparticles (AgNPs) were synthesized using propolis and characterized. The microtiter plate method and scanning electron microscopy (SEM) were used to evaluate the synergistic effects of colistin and silver nanoparticles combination (AgNPs@CL) on the inhibition of biofilm formation. The effect of AgNPs@CL on the expression of genes controlled by QS was evaluated using RT-PCR. All isolates were strong biofilm formers. Confronting AgNPs@CL, all isolates were either synergistic or additive and effectively decrease the minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) values of Carbapenem-Resistant P. aeruginosa (CRPA) isolates. The SEM analysis corroborated the enhanced biofilm inhibition observed with the combined treatment compared to individual AgNPs or colistin treatments. When exposed to AgNPs@CL, the expression levels of lasI, lasR, rhlI, rhlR, pelA, and pslA genes significantly decreased in P. aeruginosa ATCC 27,853 and clinical isolate No. #354, which displayed synergistic activity. In contrast, with additive activity, clinical isolate No. #30 showed no significant decrease. Targeting critical components of QS could effectively inhibit biofilm production. The results of our study suggest AgNPs@CL as an auxiliary to antibiotic therapy.

Introduction: As Antimicrobial Resistance (AMR) continues to pose a global health crisis, rapid and accurate antimicrobial susceptibility testing is crucial. In Intensive Care Unit (ICU) patients with sepsis, the 48-hour delay associated with conventional culture-based reports can be critical. The present study aimed to compare the rapid Colistin NP test with conventional culture techniques as a key strategy to combat antibiotic resistance caused by Multidrug Resistant (MDR) organisms. Aim: To perform a comparative evaluation of three different methods for detecting colistin resistance: Broth Microdilution (BMD), Disc Elution (DE), and rapid colistin NP tests. in Enterobacterales And to determine the Minimum Inhibitory Concentration (MIC) of colistin using the BMD test, to determine the MIC of colistin. Materials and Methods: The present cross-sectional study was conducted over twelve months (January 2023 to December 2023) at SRM Medical College Hospital and Research Centre, Chengalpattu, Tamil Nadu, India. The primary inclusion criteria that was set for the study was that blood specimens from ICU patients testing positive for Gram Negative Bacilli (GNB) were included in the study. The sample size was determined to be 178. Blood samples positive only for GNB belonging to Enterobacterales were taken into consideration. BMD and DE tests were performed to determine the MIC of colistin. Additionally, the rapid colistin NP Test was conducted to assess antibiotic susceptibility.The assessment was conducted directly from BacT/ALERT bottles as well as from bacterial isolates. The blood samples were collected from patients above 18 years of age. Statistical analysis was performed using IBM Statistical Package for Social Sciences (SPSS) software. The Chi-square test was used to assess the correlation between BMD (the gold standard method) and other methods such as DE and the rapid colistin NP Test (from both isolates and BacT/ALERT bottles). A p-value of &lt;0.05 was considered statistically significant. Results: Over the study period, 178 GNB isolates were identified. Of these, 151/178 (84.8%) were found to be colistinsensitive by BMD and DE tests. Using the rapid colistin NP Test, 153/178 (85.9%) isolates from bacterial cultures and 154/178 (86.5%) from BacT/ALERT bottles were identified as colistin-sensitive and resistant, respectively. The sensitivity and specificity of the rapid colistin NP Test were 92.5% and 100% for bacterial isolates, and 88.9% and 100% for BacT/ALERT bottles, respectively. Conclusion: The present study demonstrates that the rapid colistin NP test is an effective and reliable method for the early detection of colistin resistance in GNB within the enterobacterales group. The test showed high sensitivity and specificity, offering rapid results that can significantly aid clinical decision-making. Its implementation can facilitate timely initiation of appropriate antimicrobial therapy, helping to curb the spread of resistant strains and improve patient outcomes. These findings support the routine use of the rapid colistin NP Test in clinical microbiology laboratories for the prompt identification of colistin-resistant pathogens.

Objective:This study aims to elucidate the effect of IgY anti ComD on the biological properties of Streptococcus mutans. (S. mutans) ComD is an interspecies quorum-sensing signaling receptor that plays an important role in biofilm formation by S. mutans.Materials and Methodology: Egg yolk IgY was produced by the immunization of chickens with a DNA vaccine containing the ComD DNA coding region. We evaluated the effect of the antibody on biofilm formation by S. mutans isolated from subjects with or without dental caries. We also assessed the immunoreactivity of the antibody against all isolates, and analyzed the protein profile of S. mutans by SDS-PAGE.Results:The ComD antibody was successfully induced in the hens’ eggs. It inhibited biofilm formation by all S. mutans isolates. In addition, the expression of some protein bands was affected after exposure to the antibody.Conclusion:IgY anti-S. mutans ComD reduces biofilm formation by this bacterium and alters the protein profile of S. mutans.

Multitarget mechanisms of Origanum vulgare L. essential oil against clinically isolated carbapenem-resistant Acinetobacter baumannii.

BackgroundAcinetobacter baumannii is a hospital-acquired opportunistic pathogen, with biofilm formation playing a crucial role in its multidrug resistance. Given the rise in antibiotic resistance, herbal medicines, including peppermint (Mentha piperita), have gained attention for their potential antibacterial properties.ObjectivesThis study aimed to evaluate the inhibitory effects of peppermint extract on biofilm formation in A. baumannii strains isolated from clinical samples.MethodsA total of 25 A. baumannii strains were isolated from clinical samples at the Faculty of Allied Medical Sciences, Tehran. Their biofilm-forming ability and antibiotic susceptibility against nine antibiotics were assessed using the disk diffusion method. The antibacterial activity of peppermint extract was evaluated by well diffusion, with its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) compared to ciprofloxacin. Synergistic effects between the extract and ciprofloxacin were analyzed, followed by a time-kill assay. Polymerase chain reaction (PCR) was used to detect the presence of PgaA and AbaI genes linked to biofilm formation.ResultsThe study found that 88% of A. baumannii strains exhibited strong biofilm formation. Peppermint extract effectively inhibited biofilm formation, with MIC values ranging from 1.5 to 6 mg/mL (mean MIC: 3.75 ± 1.38 mg/mL) and MBC values equivalent to their respective MIC concentrations. For ciprofloxacin, the MIC for all samples was greater than 2048 mg/mL. No significant synergistic effect was observed between peppermint extract and ciprofloxacin. Both PgaA (involved in biofilm matrix synthesis) and AbaI (quorum sensing-related autoinducer synthase) genes were present in all tested strains.ConclusionsPeppermint extract demonstrates biofilm-inhibitory properties against A. baumannii, suggesting its potential as an alternative therapeutic approach for combating biofilm-associated infections.

Biofilm-forming bacteria are associated with difficult-to-cure bacterial infections in veterinary patients. According to previous studies, N-acetyl-L-cysteine (NAC) showed an inhibitory effect on biofilm formation when it was applied in combination with beta-lactam antibiotics and fluoroquinolones. The lack of information about the effect of NAC on doxycycline activity against biofilm-forming strains was the reason for conducting this study. Staphylococcus aureus (S. aureus) ATCC 25923, Staphylococcus aureus O74, Escherichia coli (E. coli) ATCC 25922 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853 were used to evaluate the activity of doxycycline with and without addition of NAC on planktonic bacteria and on biofilm formation. The minimum inhibitory concentrations (MICs) of doxycycline were not affected by NAC for Gram-negative strains and were found to be two times higher for the strains of S. aureus. The minimum biofilm inhibitory concentrations (MBICs) for Gram-negative bacteria (2 μg/mL for E. coli ATCC 25922 and 32 μg/mL for P. aeruginosa ATCC 27853), determined using a standard safranin colorimetric assay, were higher than the MICs (0.5 and 4 μg/mL, respectively). The data suggest that the combinations of doxycycline and NAC could stimulate the growth of planktonic cells of S. aureus and biofilm-forming E. coli ATCC 25922. NAC did not affect the strong inhibitory effect of doxycycline on the biofilm formation by the strains of S. aureus.

β-Glycyrrhetinic acid (BGA) is a natural antibacterial agent. Previous studies reported that BGA has antibacterial effects against several bacteria. This study evaluated the effects of BGA on the regulation of supragingival plaque bacteria. First, the minimum inhibitory concentrations (MICs) of BGA against oral bacteria were measured. Next, the minimum concentrations for inhibition of biofilm formation were evaluated against Streptococcus mutans and Streptococcus sobrinus, possessing insoluble glucan synthesis abilities. The MICs of biofilm formation by these bacteria ranged from 1/8 to 2× MIC. Furthermore, the inhibition effects of BGA against the coaggregation of Porphyromonas gingivalis and Streptococcus gordonii were evaluated. BGA at 32 or 64 μg/mL inhibited the coaggregation of these bacteria after a 30 min incubation. Lastly, the inhibition effects of BGA against human supragingival plaque bacteria were evaluated. Human supragingival plaque samples were obtained from 12 healthy donors. The inhibition effects of BGA against biofilm formation by these plaque bacteria were evaluated. Of 12 samples, the biofilm formation by 11 was significantly attenuated by 128-256 μg/mL of BGA. The number of colony forming units in these biofilms was also significantly attenuated. In conclusion, it was revealed that BGA inhibits the growth and biofilm formation of bacteria, furthermore, the same effect was confirmed with supragingival plaque bacteria. BGA is a good candidate for a natural agent that prevents the outbreak and progression of periodontal disease because it suppresses not only the growth and biofilm formation of bacteria, but also the coaggregation of P. gingivalis with plaque bacteria.

Background: Streptococcus mutans is known as primary microorganism in the formation of dental plaque and tooth decay. Aims and Objectives: To determine the antibacterial activity of ant-nest plant ethanol extract to inhibit S. mutans clinical isolates growth in vitro. Materials and Methods: The ethanol extracts obtained from the ant-nest plant were studied for antibacterial activity against cariogenic S. mutans obtained from patients with clinically identified carious lesions, using the agar diffusion method. Chlorhexidine gluconate was using as a comparative drug. The simplisia of ant-nest plant was extracted using a maceration method. The phytochemical screening was taken using standard methods. The minimum inhibitory concentration (MIC) test was performed by a macrodilution method and following by subculturing the overnight result onto the surface of agar media. Results: Phytochemical screening of ant-nest plant ethanol extract revealed the presence of polyphenols, flavonoids, tannins, and saponins. The extract showed intensive activity and totally inhibited the growth of S. mutans clinical isolates at 1.25% w/v. In comparison, the chlorhexidine gluconate, essential oil of plants and povidone-iodine in a ranged tested concentration also had given inhibition effect to all hyaluronic acid resistant S. mutans isolates. Conclusion: It can be reasoned that the ethanolic extract of ant-nest plant gave potent and direct antibacterial effect on S. mutans.

ObjectiveTo conduct epidemiological analysis of Klebsiella pneumoniae (K. pneumoniae) with hypervirulence, and to investigate its drug resistance phenotype, Extended-spectrum β-lactamase (ESBLs) gene, virulence factor, capsular serotype and biofilm formation, so as to provide theoretical basis for further understanding of the drug resistance mechanism of K. pneumoniae with hypervirulence.MethodsK. Pneumoniae were isolated from clinical samples collected from inpatients. All strains were identified by VITEK2 Compact using fully automatic microbial analyzer, the minimal inhibitory concentration (MIC) of antibiotics was determined by microbroth dilution test. The double disk diffusion method was used to detect the production of ESBLs, modified carbapenem inactivation method (mCIM) was used to detect the production of carbapenemase, and hypermucoviscosity phenotype was detected by wire drawing test. PCR was used to detect ESBLs gene, virulence factor and capsular serotype. Crystal violet staining was used to detect the ability of biofilm formation.ResultsThe ESBLs genes detected in this study included strains blaTEM 35 (36.5%), blaSHV 51 (53.1%), and blaCTX-M 49 (51.0%). Most strains carried multiple ESBLs genes, but not all of them produce ESBLs. K1 and K2 accounted for 14.6% and 11.5% respectively. Most (91.7%) strains carried the fimH gene, and the other virulence genes were ybtS (53.1%), entB (46.9%), rmpA (41.7%), aerobactin (32.3%), allS (15.6%), kfu (15.6%). Of all the Klebsiella pneumoniae strains, 33 (34.4%) exhibited ESBLs phenotype, 16 (16.7%) were carbapenemase-producing, and 20 (20.8%) with ESBLs phenotype tested were resistant to all four drugs. The correlation between ESBLs-producing strains and biofilm formation was significantly increased compared to strains without ESBLs phenotype (P=0.035).ConclusionCompared to hypervirulent Klebsiella pneumoniae (hvKP), classical Klebsiella pneumoniae (cKP) has a tendency to acquire antibiotic resistance. Our study showed that genes encoding rmpA, K1 or K2, and kfu were highly associated with hvKP.

Dental caries and periodontal disease are widespread diseases for which microorganism infections have been identified as the main etiology. Silver nanoparticles (Ag Nps) were considered as potential control oral bacteria infection agent due to its excellent antimicrobial activity and non acute toxic effects on human cells. In this work, stable Ag Nps with different sizes (~5, 15 and 55nm mean values) were synthesized by using a simple reduction method or hydrothermal method. The Nps were characterized by powder X-ray diffraction, transmission electron microscopy and UV-vis absorption spectroscopy. The antibacterial activities were evaluated by colony counting assay and growth inhibition curve method, and corresponding minimum inhibitory concentration (MIC) against five anaerobic oral pathogenic bacteria and aerobic bacteria E. coli were determined. The results showed that Ag Nps had apparent antibacterial effects against the anaerobic oral pathogenic bacteria and aerobic bacteria. The MIC values of 5-nm Ag against anaerobic oral pathogenic bacteria A. actinomycetemcomitans, F. nuceatum, S. mitis, S. mutans and S. sanguis were 25, 25, 25, 50 and 50μg/mL, respectively. The aerobic bacteria were more susceptible to Ag NPs than the anaerobic oral pathogenic bacteria. In the mean time, Ag NPs displayed an obvious size-dependent antibacterial activity against the anaerobic bacteria. The 5-nm Ag presents the highest antibacterial activity. The results of this work indicated a potential application of Ag Nps in the inhibition of oral microorganism infections.

Streptococcus mutans is a major contributor to dental caries, and its biofilm formation is associated with treatment challenges. Therefore, identifying medicinal plants and natural compounds with antibacterial and anti-biofilm activities is important for the development of new therapeutic approaches. This study aimed to investigate the antibacterial and anti-biofilm effects of honey, aqueous extracts of sumac and Miswak fruit, royal jelly, lactogum, and silver diamine fluoride (SDF) against S. mutans. The antibacterial effects of the compounds and extracts were evaluated using a well diffusion assay. The minimum inhibitory concentration (MIC) was determined by broth microdilution. Biofilm formation was quantified using the crystal violet assay, and metabolic activity was assessed by the MTT assay. Morphology of the biofilms was examined by scanning electron microscopy (SEM). Additionally, the expression of key genes involved in biofilm formation (gtfB, gtfC, and gtfD) was measured by RT-PCR. The findings indicated that the antibacterial effects of honey, Miswak aqueous extract, sumac fruit extract, and SDF were comparable to chlorhexidine. MICs were 512mg/mL for honey, 512mg/mL for sumac fruit extract, 512mg/mL for royal jelly, and lactogum, with Miswak aqueous extract and SDF showing MICs of 256mg/mL and 32mg/mL, respectively. SDF, Miswak aqueous extract, and sumac fruit extract exhibited the strongest anti-biofilm effects, down-regulating gtfB, gtfC, and gtfD expression in S. mutans biofilms. Collectively, these results suggest that honey, Miswak and sumac aqueous extracts, and SDF have notable antibacterial activity against S. mutans and can inhibit biofilm formation through downregulation of gtfB, gtfC, and gtfD.