Broth Dilution Test Research Articles

The Evaluation of Anti-Osteoclastic Activity of the Novel Calcium Hydroxide Biodegradable Nanoparticles as an Intracanal Medicament

ABSTRACT Objective: The present investigation looked into PLGA micron-sized particles or NPs, loaded with calcium hydroxide for canal disinfecting processes which have antimicrobial properties, given the significant rejection probabilities related to these kinds of treatments. Methodology: The MIC and inhibitory growing zones (n = 5) were found using the broth dilution and agar diffusion tests, accordingly. Employing confocal microscopy and live/dead labelling, cell viability was evaluated (n = 5). ANOVA was used to evaluate, and post hoc analysis came next. Results: Ca(OH)2 NPs showed a minimum inhibitory concentration of 5 μg/mL for Fn and 10 μg/mL for Pg and Ef (P < 0.001), following a 24-hour incubation period. Conclusion: To sum up, Ca(OH)2-contained PLGA NPs show encouraging ability to kill microbes during canal cleaning treatments.

Optimum ultrasonication duration for effective antimicrobial activity of Ti3C2-MXene

The characteristics of two-dimensional (2D) nanomaterials have attracted remarkable attention, particularly in the fields of environmental engineering, sciences and medicine. This study was focused on Ti3C2Tx-MXene, a novel 2D nanomaterial known for its antibacterial efficacy against both Gram-negative and Gram-positive bacteria. Our investigation has represented the initial exploration of the primary antifungal capabilities of Ti3C2Tx-MXene nanosheets, with variations in lateral size induced by ultrasonication time. The Ti3C2-MXene, characterized by negative surface potential, was tested against Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Enterobacter cloacae and fungal strains of Candida albicans. Ultrasonication was employed without the use of any chemical intercalant for delamination, separation, and exfoliation. The optimal ultrasonication duration was determined, revealing the minimum inhibitory concentrations of 8 μl/mL and 4 μl/mL against E. coli and S. aureus, respectively, as observed through the broth dilution test. The antifungal activity against Candida albicans has been assessed using the well diffusion method, revealing a substantial zone of inhibition measuring 14 mm. The demonstrated antimicrobial efficacy of ultrasonicated MXene sheets has shown a huge potential for serving as an effective anti-biofouling material.

Evaluation of Antibacterial Efficacy of Wax Apple (Syzygium samarangense) Fruit Extracts Against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans – An In vitro Study

Objective: The objective of this study was to evaluate the antibacterial efficacy of wax apple (Syzygium samarangense) fruit extracts (aqueous and ethanol) against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. Methodology: The stock solution was prepared for microbial analysis (100 mg/ml). A disc diffusion test to determine the zone of inhibition and the serial broth dilution test to determine the minimum inhibitory concentration were done to assess the antimicrobial activity. Data obtained were appraised observationally and interpreted. Results: When assessed for antibacterial activity of extracts by disc diffusion method, both aqueous and ethanolic extracts did not show a zone of inhibition on P. gingivalis and A. actinomycetemcomitans. However, the aqueous extract showed an inhibition zone of 8 mm at the concentration of 50 and 75 µl/ml against F. nucleatum. Similarly, the ethanolic extract showed an inhibition zone of 8 mm at 50 µl/ml and 9 mm at 75 µl/ml volume against F. nucleatum. When assessed for antibacterial activity of extracts by serial broth dilution method, only P. gingivalis (both aqueous and ethanolic extracts at 50 µg/ml) and F. nucleatum (aqueous extracts at 1. 6 µg/ml and ethanolic extract at 0.4 µg/ml) exhibited antibacterial effect, whereas A. actinomycetemcomitans was completely resistant to both extracts at all the concentrations. Conclusion: In the present in vitro study, S. samarangense (wax apple) showed its antibacterial activity against F. nucleatum only by exhibiting MIC at 1.6 µg/ml for aqueous and 0.4 µg/ml for ethanolic extract, but no antibacterial activity was shown against P. gingivalis and A. actinomycetemcomitans.

The antimicrobial activity of Hypericum perforatum L. flower extract against food pathogens and its non-enzymatic antioxidant activity

Foodborne pathogens pose a significant hazard to food safety. Most cases of foodborne illnesses are caused by bacterial pathogens that have infiltrated the food chain at some point, from farm to kitchen. According to the World Health Organization (WHO), approximately one-third of individuals in developed countries are affected by foodborne pathogens each year. Although there are studies on Hypericum perforatum L. in the literature, research in Turkey remains limited. Therefore, the aim is to contribute to the literature by studying H. perforatum samples from the Yaraş region of Muğla province in Turkey. This study specifically aims to investigate the antimicrobial activities against foodborne pathogens and the antioxidant activity of H. perforatum in Muğla. The in vitro antimicrobial activities of flower components from plants grown in Mugla were evaluated using the disc diffusion method and broth dilution test. Additionally, the extracts underwent ABTS (2,2'-azinobis-(3-ethyl benzothiazoline-6-sulfonic acid)) free-radical testing to evaluate their antioxidant activity. The extract exhibited a maximum inhibition zone of 16 mm against Staphylococcus aureus and Listeria monocytogenes. Notably, S. aureus and L. monocytogenes demonstrated the lowest sensitivity to H. perforatum methanol extract (1625 µg/mL). The methanol extract displayed moderate antioxidant activity, with a 53% ABTS radical scavenging capacity. Consequently, the extracts of H. perforatum exhibited both antimicrobial and antioxidant potential.

Determination of Vancomycin Minimum Inhibitory Concentration in Methicillin-resistant Staphylococcus Aureus by Various Phenotypic Methods: A Cross-sectional Study

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) has become a global threat, causing nosocomial and community-acquired infections, thus significantly contributing to morbidity and mortality. These organisms often exhibit Multidrug Resistance (MDR) to various groups of antimicrobials. The emergence of reduced susceptibility to vancomycin and resistance further exacerbates the problem, leaving clinicians with few therapeutic options to treat serious, life-threatening infections. Aim: To determine the vancomycin susceptibility of MRSA using various phenotypic methods. Materials and Methods: The present cross-sectional study was conducted in the Department of Microbiology, SHKM Government Medical College, Nalhar, Haryana, India, from February 2019 to January 2020. All MRSA isolates were included, totaling 66 MRSA isolates. MRSA isolates were screened using the cefoxitin disc (30 μg) according to the Clinical and Laboratory Standards Institute (CLSI) recommended disc diffusion method. The Minimum Inhibitory Concentration (MIC) of vancomycin was determined using phenotypic methods such as broth dilution, agar dilution and Epsilometer test (E-test). Demographic details such as age, sex, site of the sample and diagnosis were recorded. Statistical analysis was performed using the Pearson’s product-moment correlation. The significance level was set at a probability of <0.05 with a confidence interval of 95%. Results: A total of 66 (30.3%) isolates of Staphylococcus aureus (S. aureus) were identified as MRSA using standard laboratory tests and were studied for vancomycin MIC using phenotypic methods: Broth dilution, agar dilution and E-test. Vancomycin- intermediate S. aureus (VISA) was observed in 54.5%, 42.4% and 51.5% of MRSA by E-test, agar dilution and broth dilution, respectively. The presence of Vancomycin-resistant S. aureus (VRSA) was not detected in the region. When compared with broth dilution, E-test results demonstrated higher accuracy (97%) than agar dilution (90.9%). Conclusion: The present study highlights the emergence of VISA in more than 40% of the MRSA in the region. The higher MIC of vancomycin emphasises the need for strict adherence to antibiotic prescription guidelines to prevent further emergence of vancomycin resistance.

Chemical Profiling, Antibacterial Efficacy, and Synergistic Actions of Ptychotis verticillata Duby Essential Oil in Combination with Conventional Antibiotics

Introduction: The primary aim of the current investigation is to assess the chemical composition and antibacterial efficacy of Ptychotis verticillata essential oil (PVEO) against three Gram-positive bacteria ( Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis) and three Gram-negative bacteria ( Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae). Furthermore, this research endeavors to investigate the potential synergistic effects of PVEO when combined with established antibiotics (Amoxicillin, Erythromycin, and Ampicillin), with a focus on discerning their interaction dynamics. Methods: The phytochemical profiling of PVEO was performed via the Gas Chromatography-Mass Spectrometry technique. The broth dilution test determined the interaction effect between PVEO and these three antibiotics. Results: Components γ-terpinene (38.96%), p-cymene (19.38%), thymol (18.07%), and carvacrol (6.99%) are the major bioactive moleculs composing this studied essential oil. PVEO demonstrated significant antibacterial activity with minimum inhibitory concentration (MIC) values ranging from 0.2 against E coli to 2 mg/mL against M luteus. The assessment of the synergistic activity between PVEO and antibiotics was accomplished through the utilization of the Fractional Inhibitory Concentration Index (FICI). The combination of PVEO and amoxicillin against M luteus demonstrated the best synergistic action with a FICI value of 0.43. Conclusion: Significant reductions, ranging from two to sevenfold, in the MIC values of PVEO, and antibiotics were observed. This noteworthy synergistic interaction between highly potent essential oils and synthetic antibiotics holds the potential to open avenues for novel combination therapies aimed at addressing infections caused by multiresistant microorganisms, even at notably reduced concentrations within the pharmaceutical sector.

Ethanolic extract of propolis from Indonesian stingless bee (Tetragonula biroi) is ineffective against ESBL-producing Klebsiella pneumoniae: An in vitro study and a brief review of propolis antibacterial and anti-inflammatory activity.

The number of antimicrobial resistance which is increasing every year is a big problem that attracts attention. The prevalence of infections due to bacterial resistance is quite high, especially from nosocomial infections. This encourages the search for new materials that have the potential to fight resistant bacteria. Propolis is a product of bees that is not only used for structural support for their hives but also as an antiseptic to protect their hives from microorganisms. This study aims to determine whether the ethanolic extract of propolis (EEP) from the stingless bee Tetragonula biroi has an antibacterial effect against extended spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae. This study used the broth dilution test and agar-well diffusion test to test for antibacterial activity. EEP is generally inactive against ESBL-producing K. pneumoniae. It could not produce an inhibition zone in the agar-well diffusion test. The results of the dilution test for the minimum inhibitory concentration (MIC) are difficult to determine. Phytochemical qualitative test found EEP of T. biroi contains alkaloids, flavonoids, and phenols.

Characterization of Bacillus velezensis 32a metabolites and their synergistic bioactivity against crown gall disease

Crown gall disease caused by Agrobacterium tumefaciens is considered to be the main bacterial threat of stone fruit plants in Mediterranean countries. In a previous study, Bacillus velezensis strain 32a was isolated from Tunisian rhizosphere soil and revealed high antagonistic potential against A. tumefaciens strains. In order to better characterize the antagonistic activity of this strain against this important plant pathogen, the production of secondary metabolites was analyzed using liquid chromatography coupled with mass spectrometry. The results revealed the production of different compounds identified as surfactins, fengycins, iturins and bacillibactin belonging to the lipopeptide group, three polyketides (macrolactins, oxydifficidin and bacillaenes), bacilysin and its chlorinated derivative; chlorotetaine. The involvement of lipopeptides in this antagonistic activity was ruled out by performing agar and broth dilution tests with pure molecules. Thus, the construction of B. velezensis 32a mutants defective in polyketides and bacilysin biosynthesis and their antagonistic activity was performed and compared to a set of derivative mutants of a comparable strain, B. velezensis GA1. The defective difficidin mutants (△dfnA and △dfnD) were unable to inhibit the growth of A. tumefaciens, indicating the high-level contribution of difficidin in the antagonism process. While the macrolactin deficient mutant (∆mlnA) slightly decreased the activity, suggesting a synergetic effect with difficidin. Remarkably, the mutant △dhbC only deficient in bacillibactin production showed significant reduction in its capacity to inhibit the growth of Agrobacterium.Taken collectively, our results showed the strong synergetic effect of difficidin and macrolactins and the significant implication of siderophore to manage crown gall disease.

An In Vitro Evaluation of the Antibacterial Efficacy of Solanum xanthocarpum Extracts on Bacteria From Dental Plaque Biofilm.

The focus of research has recently shifted toward developing herbal-based medicines due to the emerging bacterial resistance and side effects of antibiotics. Solanum xanthocarpum (Sx) is a medicinal plant with potent pharmacological properties. This study aimed to evaluate the antibacterial activity of its crude extracts on bacteria isolated from dental plaque in patients with gingivitis. Aqueous, ethyl acetate, hexane, chloroform, and ethanolic extracts were prepared from Sx. Dental plaque samples were collected from patients with plaque-induced gingivitis. Disk diffusion assay was performed to determine the antibacterial activity of the extracts at concentrations of 25 mg/ml, 50 mg/ml, and 75 mg/ml with ampicillin 200 mg/ml as a positive control. The minimum inhibitory concentration (MIC) of the aqueous extract was also evaluated by broth dilution test against bacteria isolated from dental plaque biofilm. The antibacterial activity was estimated by measuring the zones of inhibition through the disc diffusion method. The Kruskal Wallis with Dunn post hoc test performed for intergroup comparison between the various extracts showed a statistically significant difference in inhibition of bacterial growth between 25 mg/ml and 75 mg/ml concentrations. There was no significant difference between the 75 mg/ml Sx concentration and the positive control. In addition, the MIC was elucidated to be 0.625 g/ml, at which there was maximum inhibition of bacterial growth. The Sx extract exhibited antibacterial activity against periodontal pathogens. Thus, it can be concluded that optimum concentrations of Sx could be used in therapeutic strategies to prevent and manage periodontal diseases.

Antimicrobial efficacy of chlorine agents against selected oral pathogens.

Method-dependent comparison of antimicrobial agents' efficacy against oral pathogens. Several sodium hypochlorite solutions (NaOCl)-Perisolv®, Carisolv® and Dakin's solution-were equated with chlorhexidine (CHX) and hydrogen peroxide (H2O2) against ten oral micro-organisms related to caries and periodontitis using different minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) techniques. Agents were adjusted to the final 70 mmol/L concentration of active chlorine molecule. Apart from H2O2 and the amino acids of Perisolv®, all the agents revealed an antimicrobial effect. Agar diffusion test ranked CHX (p < 0.05) as the most effective against all ten specimens, followed by the NaOCl of Perisolv® and Dakin's solution. Correspondingly, in broth microdilution on agar, CHX was the most effective in eradicating micro-organisms at 0.03 mmol/L compared with 2.2 mmol/L of Dakin's solution. In contrast, the bactericidal concentration of Dakin's solution was the most effective at 0.2 mmol/L, (p < 0.001), followed by Perisolv® (2.14 mmol/L), CHX (2.38 mmol/L) and Carisolv® (3.33 mmol/L) after 5 and 10 min in broth dilution test. In live/dead analysis, 60-min exposure to a 2-fold concentration of agents resulted in two-log Aggregatibacter actinomycetemcomitans inhibition by CHX (35 mmol/L) whilst Streptococcus mutans was more susceptible, in 0.8 and 8.8 mmol/L, after 10 min to CHX and Dakin's respectively. Replacement of CHX with tested hypochlorite agents showed evident potential and promoted rapid antimicrobial effect. Effective antimicrobial agents are crucial in controlling pathogen-induced oral infections increasing clinical possibilities to combat oral biofilms. Additionally, CHX substitution with hypochlorite agents could eliminate CHX's adverse effects.

Evaluation of the anti-bacterial activity of aqueous leaf extract of Phyllanthus amarus on Streptococcus pyogenes for the treatment of tonsillitis

Phyllanthus amarus belongs to the Euphorbiaceae family, it is a tiny herb that is known for its medical benefits and used widely globally. It is also an essential plant in the Indian Ayurveda medical system, and is used to treat stomach, genitourinary, liver and kidney problems and disorders. It is an astringent with bitter, stomachic, diuretic, febrifuge, and antiseptic in gonorrhea, menorrhagia, and other genital disorders, the entire plant is employed in eastern Asia for the treatment of infections. It helps with gastritis, diarrhea, dysentery, intermittent fevers, scabies, ulcers, and wounds. The present study aimed at addressing its activity against Streptococcus pyogenes for the treatment of tonsillitis and also it’s potential as a possible source of new antimicrobial product for tonsillitis. Distilled water (maceration method) was used in the extraction of aqueous leaf extracts of Phyllanthus amarus. Different phytochemical screening techniques were used to identify the phytochemicals present in the aqueous leaf extract of Phyllanthus amarus. The Agar well diffusion and broth dilution test methods were used to test for the in-vitro anti-bacterial activity of the aqueous leaf extract of Phyllanthus amarus against Streptococcus pyogenes. The percentage yield of Phyllanthus amarus aqueous leaf extracted was 13.88% (83.3mg). From the results of this research Phyllanthus amarus could be a good source for future drug developments, from the aqueous leaf extract of Phyllanthus amarus in order to address the issue of need for new antibiotics due to the alarming burden of antimicrobial resistance. Keywords: Phyllanthus amarus aqueous leaf extract, Tonsillitis, Streptococcus pyogenes.

Bioassay-guided isolation in Salvia abrotanoides Karel. stem based on its anti-fungal and anti-trichomonas activity.

Salvia abrotanoides is considered a medicinal plant and has a broad distribution in Iran. In Iran's traditional medicine, it is also used to treat leishmaniasis, malaria, atherosclerosis, cardiovascular disease, and as a disinfectant. This research aimed to determine the anti-Candida component from S. abratonoides and anti-Trichomonas natural compounds from the stems of this plant. The plant shoots were collected, dried, and after removing the leaves, grounded. Dried plant material was extracted in a maceration tank, concentrated by a Rotavap, degreased, and fractionated by normal column chromatography. Based on anti-fungal screening against Candida species, Fr. 4, with more anti-fungal activity, was selected for phytochemical analysis, by different chromatographic methods on the silica gel column and Sephadex LH-20. Isolated compounds were elucidated by NMR analysis, mass spectrum, and ultraviolet spectroscopy. Anti-fungal effects were investigated using the fungal suspension, incubation, and parasite-counting methods on purified compounds. Antibacterial effects were assessed using the Broth dilution test and reported according to the MIC parameter. Two diterpenoid compounds named carnosol (compound 1), 11-hydroxy-12-methoxy-20-norabiata-8, 11, 13-trien (compound 2), and a flavonoid: 6,7-dimethoxy-5, 4'-dihydroxyflavone (compound 3) were isolated and identified. Compound 1 had selective anti-fungal effects against C. albicans, C. glabrata, and C. parapsilosis, but weak toxicity against Trichomonas vaginalis with IC50 of 675.8 μg/mL, less than metronidazole with an IC50 of 13.2 μg/mL. Carnosol as the main component was assayed against Candida, Aspergillus, Rhizopus, and Trichomanas species. The results confirmed its effect on Candida compared to standard drugs.

Detection of virulence, antimicrobial resistance, and heavy metal resistance properties in Vibrio anguillarum isolated from mullet (Mugil cephalus) cultured in Korea.

In the present study, we identified and characterized 22 strains of V. anguillarum from 145 samples of mullets (Mugill cephallus) cultured in several fish farms in South Korea. They were subjected to pathogenicity tests, antimicrobial susceptibility test, and broth dilution test to detect virulence markers, antimicrobial resistance, and heavy metal resistance properties. All the isolates showed amylase and caseinase activity, followed by gelatinase (90.9%), DNase (45.5%), and hemolysis activities (α = 81.1% and β = 18.2%). The PCR assay revealed that isolates were positive for VAC, ctxAB, AtoxR, tdh, tlh, trh, Vfh, hupO, VPI, and FtoxR virulence genes at different percentages. All the isolates showed multi-drug resistance properties (MAR index ≥ 0.2), while 100% of the isolates were resistant to oxacillin, ticarcillin, streptomycin, and ciprofloxacin. Antimicrobial resistance genes, qnrS (95.5%), qnrB (86.4%), and StrAB (27.3%), were reported. In addition, 40.9% of the isolates were cadmium-tolerant, with the presence of CzcA (86.4%) heavy metal resistance gene. The results revealed potential pathogenicity associated with V. anguillarum in aquaculture and potential health risk associated with consumer health.

6His-tatritin promotes antimicrobial defense via regulating immune ability and intestinal microbial community in grass carp (Ctenopharyngodon idella)

Antimicrobial peptides are small, cationic, and amphiphilic peptides found in most organisms, and many of these peptides have broad antimicrobial activity against Gram-negative, -positive bacteria and fungi. In the present study, a derivative of antimicrobial peptide Tatritin, 6His-Tatritin, was designed and expressed by Pichia pastoris using a constitutive vector pGAPZαA with the promoter of pGAP. The 6His-Tatritin had a broad-spectrum antibacterial activity based on the Oxford cup method and the micro broth dilution test. In addition, to explore the role of 6His-Tatritin in vivo, grass carps (Ctenopharyngodon idellus) were infected with Aeromonas hydrophila after they were fed with 6His-Tatritin as feed additives for 28 days. The results revealed that 6His-Tatritin could significantly up-regulate the expression levels of Hepcidin, Leap-2b, Nrf-2, CuZn-SOD and LZM (P < 0.05). In addition, 6His-Tatritin could significantly reduce the mortality (P < 0.05) and the intestinal injury of grass carps infected with bacteria. The 16S sequencing analysis showed that the structure of microbial community in intestine of fish was more diversified compared with control after treatment with 6His-Tatritin. In summary, the peptide of 6His-Tatritin could promote antimicrobial defense via regulating immune ability and intestinal microbial community in grass carp. This study provides an effective method and approach for the application of antimicrobial peptide Tatritin in aquaculture, and also provides insights into the function of antimicrobial peptides in immunity against pathogens in fish.

WITHDRAWN: Combination of isothiocyanates and antibiotics increases susceptibility against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Serratia marcescens

The development and spread of multidrug-resistant organisms (MDRO) is evolving rapidly worldwide. Although effective antibiotics are still available, some infections are difficult to treat. Among MDROs, S. marcescens and A. baumannii are among the difficult-to-treat pathogens causing for instance sepsis and urinary tract infections (UTI). Isothiocyanates (ITC) are natural plant products. The antimicrobial properties of ITC appear to be more effective in combination with some antibiotics. We investigated a mixture of ITC from nature-identical plant extracts of nasturtium and horseradish in different combined concentrations with broad-spectrum antibiotics against S. marcescens, A. baumannii, E. coli, K. pneumoniae and P. mirabilis. First, we investigated the antibacterial activity of the compounds by standard agar disc diffusion test. Next, we evaluated the minimum inhibitory concentrations (MIC) via broth dilution tests. Checkerboard technique was used to assess antimicrobial effects against sulfamethoxazole-trimethoprim, ampicillin-sulbactam, nitrofurantoin, Fosfomycin, ciprofloxacin and imipenem combined with various concentrations of ITC. S. marcescens strains 1-3 showed reversal of resistance at >0.075 μg/mL ITC in combination with nitrofurantoin, whereas 0.05 μg/mL ITC increased antimicrobial susceptibility. Combination of fosfomycin with 0.003 μg/mL ITC resulted in increased efficacy against resistant S. marcescens strains, E. coli, K. pneumoniae and P. mirabilis. Remarkable additive antimicrobial activity was observed at 0.03 μg/mL and 0.06 μg/mL ITC in combination with 0.125 μg/mL imipenem (FIC > 0.5). The combination of ITC and antibiotics is a promising approach for the treatment of UTI caused by MDRO. The ITC combination with selected antibiotics had synergistic and additive effects.

An experimental study to evaluate the effect of polymixin E (Colistin) alone or in combination with gentamicin in McCarey-Kaufman corneal preservation medium on various drug resistant bacterial and fungal isolates.

To assess the efficacy of the addition of polymyxin E (colistin) in the McCarey-Kaufman (MK) corneal storage solution against multi-drug resistant strains of Enterobacteriaceae, Staphylococcus aureus, and Candida spp. A standard micro broth dilution test and a checkerboard assay were performed for five multi-drug resistant (MDR) clinical strains of P. aeruginosa and five clinical strains of methicillin-resistant S. aureus (MRSA) and C. albicans against colistin and gentamicin alone and in combination. The minimum inhibitory concentration (MIC) and the fractional inhibitory concentration index (FICI) were calculated to assess the efficacy of each combination. The MIC of colistin was in the range of 1-2 μg/mL for P. aeruginosa, whereas it was 256-1024 μg/mL against S. aureus. In comparison, the MIC of gentamicin was found to be 0.5-512 μg/mL and 0.5-8 μg/mL against P. aeruginosa and S. aureus, respectively. All five isolates of C. albicans did not exhibit any susceptibility to either colistin or gentamicin even at a concentration of ≥ 512 μg/mL each. The checkerboard assay was performed to evaluate the nature of the interaction of the combination of colistin and gentamicin. Based on the FICI, it was observed that the colistin and gentamicin combination has a maximum synergistic effect (FIC <0.5) in 80% (4/5) for S. aureus isolates, whereas the maximum additive effect (FIC >0.5-4) was 100% (5/5) for P. aeruginosa and the minimum additive effect was 20% (1/5) for S. aureus isolates. Antagonism (FIC ≥ 4) was not observed in any combination between the strains used in the study. Both colistin and gentamicin alone or in combination were, however, ineffective against Candida spp. The addition of colistin has an inhibitory effect on bacterial contamination that could be possibly caused by MDR strains and could potentially be considered as an additional additive in corneal storage media.

ASK1 is a novel molecular target for preventing aminoglycoside-induced hair cell death

Aminoglycoside antibiotics are lifesaving medicines, crucial for the treatment of chronic or drug resistant infections. However, aminoglycosides are toxic to the sensory hair cells in the inner ear. As a result, aminoglycoside-treated individuals can develop permanent hearing loss and vestibular impairment. There is considerable evidence that reactive oxygen species (ROS) production and the subsequent phosphorylation of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) drives apoptosis in aminoglycoside-treated hair cells. However, treatment strategies that directly inhibit ROS, JNK, or P38 are limited by the importance of these molecules for normal cellular function. Alternatively, the upstream regulator apoptosis signal-regulating kinase 1 (ASK1/MAP3K5) is a key mediator of ROS-induced JNK and P38 activation under pathologic but not homeostatic conditions. We investigated ASK1 as a mediator of drug-induced hair cell death using cochlear explants from Ask1 knockout mice, demonstrating that Ask1 deficiency attenuates neomycin-induced hair cell death. We then evaluated pharmacological inhibition of ASK1 with GS-444217 as a potential otoprotective therapy. GS-444217 significantly attenuated hair cell death in neomycin-treated explants but did not impact aminoglycoside efficacy against P. aeruginosa in the broth dilution test. Overall, we provide significant pre-clinical evidence that ASK1 inhibition represents a novel strategy for preventing aminoglycoside ototoxicity.Key messagesASK1 is an upstream, redox-sensitive regulator of P38 and JNK, which are known mediators of hair cell death.Ask1 knockout does not affect hair cell development in vivo, but significantly reduces aminoglycoside-induced hair cell death in vitro.A small-molecule inhibitor of ASK1 attenuates neomycin-induced hair cell death, and does not impact antibiotic efficacy in vitro.ASK1 may be a novel molecular target for preventing aminoglycoside-induced hearing loss.

Effects of Candida albicans on the metabolic activity and drug resistance of Streptococcus mutans based on D2O-labeled single-cell Raman microspectroscopy.

To evaluate the susceptibility of Streptococcus mutans (S. mutans) to NaF and the effects of Candida albicans (C. albicans) metabolites on the metabolic activity and drug resistance of S. mutans. The minimum inhibitory concentration (MIC) and MIC based on metabolic activity (MIC-MA) of NaF against S. mutans were measured through the broth dilution test and D2O-labeled single-cell Raman microspectroscopy, respectively, to explore the inhibitory effect of NaF on the growth and metabolism of S. mutans. The effects of different concentrations of C. albicans supernatant on the metabolic activity of S. mutans were evaluated by using D2O-labeled single-cell Raman microspectroscopy. The correlation between C. albicans and S. mutans was explored throughassessing the drug resistance of S. mutans against NaF in a mixed system containing C. albicans supernatant by using D2O-labeled single-cell Raman microspectroscopy. The MIC and MIC-MA of NaF against S. mutans were 0.4 g·L-1 and 1.2 g·L-1, respectively. At the MIC level, S. mutans showed completely inhibited growth but retained high metabolic activity. Therefore, S. mutans may regain its virulence. At the MIC-MA level, the metabolic activity of S. mutans was inhibited only when the NaF concentration reached 3×MIC. At the concentration of OD600≥0.5, the C. albicans supernatant significantly promoted the metabolic activity of S. mutans at different time points (P<0.05). In the mixed system, the susceptibility of S. mutans to NaF decreased, and statistical differences at different time points were observed (P<0.05). The metabolic activity of S. mutans was inhibited completely only at the concentration of 4×MIC (1.6 g·L-1). D2O-labeled single-cell Raman microspectroscopy is suitable for evaluating the effects of drugs on bacterial metabolic activity. In the mixed system, the metabolites in the C. albicans supernatant at the concentration of OD600≥0.5 significantly promoted the metabolic activity of S. mutans and reducedthe susceptibility of S. mutans to NaF.

Single‐cell Raman microspectroscopy‐based assessment of three intracanal disinfectants' effect on Enterococcus faecalis

AbstractEnterococcus faecalis (E. faecalis) is frequently encountered in asymptomatic, persistent endodontic infections; thus, its control and eradication via disinfectants are important. To explore a disinfectant formulation that is effective yet with minimal side effects, here, we evaluated the susceptibility of E. faecalis to three intracanal disinfectants. We quantitatively assessed and compared the growth‐ or metabolism‐inhibiting effects of intracanal disinfectants via minimum inhibitory concentration (MIC) and minimum inhibitory concentration based on metabolic activity (MIC‐MA), based on the broth dilution test and D2O‐probed single‐cell Raman spectra (SCRS), respectively. For sodium hypochlorite (NaClO), hydrogen peroxide (H2O2), and chlorhexidine gluconate (CHX), the corresponding MIC was 0.45 g/L, 110 mg/L, and 6 mg/L, respectively. Under their respective MIC doses, metabolic activity of bacterial cells was reduced to 2%, 4%, and 2% remained at 8 h, yet recovered to a retention level of 90%, 97%, and 2% at 24 h. Despite the halting of growth, the remained metabolic activity suggests a “nongrowing but metabolically active” (NGMA) state that may lead to potential recurring infections. In contrast, at their respective MIC‐MA doses of 0.9 g/L, 220 mg/L, and 12 mg/L, metabolic activities of all cells were completely inhibited throughout 24‐h exposure. Furthermore, lower combined concentration of above three intracanal disinfectants can elicit equivalent metabolism‐inhibiting effect with that of solo use of each one at the MIC‐MA dose. Thus, binary combined use of disinfectants can outperform their solo use in controlling infection of E. faecalis and reducing side effects. In conclusion, the MIC‐MA derived from D2O‐probed single‐cell Raman microspectroscopy is a promising approach in quantitatively assessing disinfectants' antimicrobial efficacy via metabolic activity.

Antimicrobial Activity of a Vaginal Gel Formulation: Considerations Related to Vaginal Infection and Dysbiosis.

Many non-prescription preparations intended to treat or alleviate symptoms of vaginal infection are available in American and European markets, but many have scant preclinical or clinical research underpinning. Respecta®Balance Gel (RBG) is marketed as an adjunct to probiotic treatment and its relevant antimicrobial properties were studied. Key findings with the manufacturer-supplied gel showed reduced turbidity in broth-dilution tests by 50% against Candida albicans and Candida glabrata at RBG concentrations 0.2–0.4% of neat product, respectively. A 50% reduction in turbidity of Escherichia coli, Streptococcus agalactiae, Enterococcus faecalis ranged from 1.6–2.2% and Gardnerella vaginalis was shown by flow cytometry counts to undergo a 50% reduction at 0.3% RBG. Propidium iodide staining indicated a rapid reduction of cell integrity of G. vaginalis almost immediately while after 4 h 45% of E. coli cells were stained. The lactic acid in BHI inhibited bacteria and yeast at concentrations ranging from 0.2–1.8% but inhibition was not solely due to pH since a 1:4 dilution of RBG resulted in a pH near neutral (6.75). Other findings showed biofilm accumulation assessed after 10-days exposure of Candida spp. to RBG and was reduced by an average of one-third (community strains) to one-half (drug-resistant strains). One excipient of the RBG, disodium EDTA, inhibited the growth of bacteria and yeast at concentrations below those present in RBG and may accentuate the activity of the host defense factor, lactoferrin. We conclude that RBG is a potent inhibitor of vaginal microorganisms relevant to vaginitis or intrapartum infections and contains excipients that may contribute to its antimicrobial activity.