Colorimetric Microdilution Research Articles

Implication of genotypes for prognosis of Candida glabrata bloodstream infections.

Genotyping isolates of a specific pathogen may demonstrate unique patterns of antimicrobial resistance, virulence or outcomes. However, evidence for genotype-outcome association in Candida glabrata is scarce. We aimed to characterize the mycological and clinical relevance of genotypes on C. glabrata bloodstream infections (BSIs). Non-duplicated C. glabrata blood isolates from hospitalized adults were genotyped by MLST, and further clustered by the unweighted pair group method with arithmetic averages (UPGMA). A clonal complex (CC) was defined by UPGMA similarities of >90%. Antifungal susceptibility testing was performed by a colorimetric microdilution method and interpreted following CLSI criteria. Of 48 blood isolates evaluated, 13 STs were identified. CC7 was the leading CC (n = 14; 29.2%), including 13 ST7. The overall fluconazole and echinocandin resistance rates were 6.6% and 0%, respectively. No specific resistance patterns were associated with CC7 or other CCs. Charlson comorbidity index (adjusted OR, 1.49; 95% CI, 1.05-3.11) was the only predictor for CC7. By multivariable Cox regression analyses, CC7 was independently associated with 28 day mortality [adjusted HR (aHR), 3.28; 95% CI, 1.31-8.23], even after considering potential interaction with neutropenia (aHR, 3.41; 95% CI, 1.23-9.42; P for interaction, 0.24) or limited to 34 patients with monomicrobial BSIs (aHR, 2.85; 95% CI, 1.15-7.08). Also, the Kaplan-Meier estimate showed greater mortality with CC7 (P = 0.003). Fluconazole resistance or echinocandin therapy had no significant impact on mortality. Our data suggested comorbid patients were at risk of developing CC7 BSIs. Further, CC7 was independently associated with worse outcomes.

Antifungal susceptibility of clinically isolated invasive Candida tropicalis in East China from 2017 to 2021

Objective: To explore the epidemiological characteristics of sample distribution and antifungal susceptibilities of clinically invasive C. tropicalis isolates from 2017 to 2021 in East China. Methods: Using a retrospective analysis, the East China Invasive Fungal Infection Group (ECIFIG) collected C. tropicalis clinically isolated from 32 hospitals in East China between January 2017 and December 2021. The identification results of the strains were reviewed using mass spectrometry by the central laboratory of the Shanghai East Hospital. The minimum inhibitory concentrations (MICs) of the strains against fluconazole (FLU), voriconazole (VOR), itraconazole (ITR), Posaconazole (POS), isavuconazole (ISA), anidulafungin (ANI), caspofungin (CAS), micafungin (MICA) and 5-fluorocytosine (FCT) were tested by the ThermoFisher CMC1JHY colorimetric microdilution method. The MIC of amphotericin B (AMB) was tested by the broth microdilution method. The MIC results were analyzed based on the clinical breakpoints and epidemiological cutoff values (ECV) published by the Clinical and Laboratory Standards Institute (CLSI) M27 Ed3 and M57 Ed4 documents. Data analysis was conducted using the Kruskal-Wallis test and paired t-test. Results: In total, 305 C. tropicalis isolates were collected. There were 38.0% (116/305) strains isolated from blood, 11.5% (35/305) ascites, 8.9% (27/305) catheter and 8.9% (27/305) drainage fluid. The resistance rate of C. tropicalis to FLU was 32.5%, to VOR was 28.5%, and the cross-resistance rate to FLU and VOR was 28.5%. The wild-type proportions for ITR and POS were 79.3% and 29.2% respectively. There was no significant difference in resistance rates, MIC50, and MIC90 of FLU and VOR, or in the wild-type rates of ITR and POS over five years. More than 95.0% of the isolates were susceptible to echinocandins. However, one strain was identified as being multi-drug resistant. In azole antifungals, voriconazole, itraconazole, posaconazole, and isavuconazole have similar GM MIC values. The GM MIC of fluconazole is significantly higher than that of itraconazole (t=9.95, P<0.05), posaconazole (t=9.99, P<0.05), and voriconazole (t=10.01, P<0.05), Meanwhile, among echinocandins, the GM MIC of ANI was comparable to that of CAS (t=1.17, P>0.05), both of which were significantly higher than MICA (t=11.56, P<0.05; t=4.15, P<0.05). Conclusion: The clinical isolates of C. tropicalis in East China from 2017 to 2021 were relatively susceptible to echinocandins. However, there was consistently high resistance to fluconazole and voriconazole. More intensive efforts should be made on the monitoring of drug resistance in C. tropicalis.

Inhibitory Activity of Essential Oils of Mentha spicata and Eucalyptus globulus on Biofilms of Streptococcus mutans in an In Vitro Model.

The aim of this study was to evaluate the inhibitory activity of the commercially available essential oils of Mentha spicata (spearmint) and Eucalyptus globulus (eucalyptus) on Streptococcus mutans ATCC 25175 biofilms in vitro, emulating dental plaque conditions. The composition of the essential oils (EOs) was determined using gas chromatography coupled with mass spectrometry (GC-MS), with the main metabolites being Carvone (57.93%) and Limonene (12.91%) for Mentha spicata and 1,8-Cineole (Eucalyptol) (65.83%) for Eucalyptus globulus. The inhibitory activity was evaluated using the methods of agar-well diffusion and colorimetric microdilution. The inhibition halos were 18.3 ± 0.47 mm and 27.0 ± 0.82 mm, and the MICs were 1.8484 mg/mL and 1.9168 mg/mL for the EOs of Mentha spicata and Eucalyptus globulus, respectively. The activity against the biofilms was evaluated on a substrate of bovine enamel pieces using a basal mucin medium (BMM) in anaerobic conditions with daily sucrose exposition cycles in order to emulate oral cavity conditions. The EOs were applied in a concentration of 0.5% in a sterile saline vehicle with 1% polysorbate 20. After 72 h of cultivation, a significant reduction was observed (p < 0.001%) on the biofilm biomass, which was evaluated by its turbidity in suspension and using a count of the recoverable organisms with regards to the control. The effects of the Eos were not significantly distinct from each other. The EOs showed antimicrobial activity against both the Streptococcus mutans planktonic and biofilm cultures. Thus, EOs may have great potential for the development of pharmaceutical and sanitary products for oral health.

The investigation of antifungal susceptibilities of Kluyveromyces marxianus and Clavispora lusitaniae strains isolated from various clinical specimens

Background/Aim: C. albicans remains the most common pathogen responsible for invasive candidiasis. On the other hand, increased rates of candidiasis have been reported worldwide, caused by other Candida species (such as K. marxianus and C. lusitaniae). Considering these uncommon yeasts may be crucial pathogens in the future, it is preferable to describe the in-vitro activities of antifungal agents as potential options for their treatments. This study aimed to evaluate the in-vitro activity of nine different antifungal agents that are routinely used to contribute to the treatment of the infections caused by K. marxianus and C. lusitaniae. Methods: The study included 21 K. marxianus and eight C.lusitaniae strains isolated from various clinical specimens of patients with suspected invasive fungal infection. Conventional identification was confirmed using the molecular methodology of DNA sequencing analysis. Antifungal susceptibilities of the isolates were tested using the Sensititer Yeast One Test Panel Y06 kit, a colorimetric microdilution test. Results: For K. marxianus, amphotericin B had the highest geometric mean MIC (1 μg/mL) and voriconazole had the lowest geometric mean MIC (0.010 μg/mL). For C. lusitaniae, flucytosine had the highest geometric mean MIC (8 μg/mL) and voriconazole had the lowest geometric mean MIC (0.011 μg/mL). Conclusion: Considering that these two species, rare causes of invasive candidiasis nowadays, may become important pathogens in the future, it is reasonable to investigate the in-vitro activities of antifungal agents that can be used in their treatment.

Antagonistic Potential of Soil Streptomyces Isolates from Southern Thailand to Inhibit Foodborne Bacterial Pathogens.

Streptomyces are well known for their competence to produce thousands of bioactive secondary metabolites and enzymes. This study aimed to assess the inhibitory activities of crude extracts from diverse Streptomyces collected from rice soils in Narathiwat, Thailand, against foodborne bacterial pathogens. In total, 136 Actinomycete isolates were screened using a cross-streak method for the ability to produce effective metabolites against 5 pathogenic bacteria. Out of these, 19 (13.97%) isolates had antibacterial activity against at least one tested bacterium. Most of the isolates could strongly suppress the growth of S. aureus ATCC25923 and B. cereus MTCC430 except P. aeruginosa ATCC27853. On the basis of morphological, cultural, and biochemical characteristics, all potent isolates exhibited typical features that fitted the genus Streptomyces. Two of the 7 selected ethyl acetate crude extracts had good antagonistic activity against S. aureus ATCC25923 and B. cereus MTCC430 when tested using the agar well diffusion assay. Furthermore, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the 2 extracts evaluated using the colorimetric broth microdilution method ranged from 256 to >1,024 μg/ml against the tested bacteria. The partial nucleotide sequences of the 16S rRNA gene led to identifying both active isolates as Streptomyces species. These active Streptomyces isolates could provide an interesting source for generating innumerable natural compounds with antibacterial activity that can presumably be developed to fight bacterial pathogens in the near future.

Molecular identification, virulence factors and antifungal susceptibility patterns of Candida parapsilosis complex species isolated from clinical samples.

The aim of this study was to identify C. parapsilosis complex strains isolated from various clinical samples by sequence analysis and to investigate whether there are any differences between the species in terms of virulence factors and antifungal susceptibility. The study included a total of 42 isolates identified as C. parapsilosis complex based on the color they formed in chromogenic medium, colony morphology, and microscopic appearance in Corn Meal-Tween 80 Agar and they were confirmed with API 20 C AUX. For the DNA sequence analysis of clinical isolates, V9G forward and LS reverse primers were used as well as internal transcribed spacers (ITS1 and ITS4). Biofilm formation, esterase, phospholipase, and protease activities were evaluated as virulence factors. Antifungal susceptibility was investigated via colorimetric microdilution method. A total of 75 non-C. albicans isolates were obtained from various clinical samples between 2016 and 2017 in a Turkish Tertiary Care Hospital. Of them, 42 were identified as members of the C. parapsilosis complex. Of the 42 strains, 41 were identified as C. parapsilosis sensu stricto (CpSS), while only one was identified as C. orthopsilosis. Of the CpSS strains, 31 (75.6%) were biofilm-positive, six (14.6%) were esterase-positive, nine (21.9%) were positive for phospholipase activity, and 31 (75.6%) were positive for protease formation, whereas all virulence factors of C. orthopsilosis strain were found to be negative. All CpSS strains were found susceptible to amphotericin B, echinocandins, and flucytosine. It has been concluded that the use of molecular methods to identify CpSS would not be effective in routine laboratory practices as it is the most commonly isolated species from the C. parapsilosis complex and there are no significant differences between species in terms of antifungal susceptibility.

Antifungal susceptibility of clinical mould isolates in New Zealand, 2001–2019

The objective of this study was to review the antifungal susceptibility of clinical mould isolates performed by the New Zealand Mycology Reference Laboratory. Isolates were either local or referred for testing from other New Zealand laboratories. All isolates were tested by the broth colorimetric microdilution method, Sensititre YeastOne (SYO). Epidemiological cut-off values (ECVs) derived from either the Clinical and Laboratory Standards Institute (CLSI) method or SYO were used to determine the proportion of non-wild type (non-WT) isolates, i.e., those with an increased likelihood to harbour acquired mechanisms of resistance. A total of 614 isolates were tested. Most isolates (55%) were from the respiratory tract followed by musculoskeletal tissue (17%), eye (10%) and abdomen (5%). The azoles had similar activity except for voriconazole which was less active against the Mucorales. The echinocandins had good activity against Aspergillus spp., other hyaline moulds and dematiaceous isolates but were inactive against Fusarium spp., Lomentospora prolificans and the Mucorales. Amphotericin B had best activity against the Mucorales. The two least susceptible groups were Fusarium spp. and L.prolificans isolates. Three Aspergillus isolates were non-WT for amphotericin B, and four non-WT for azoles. Non-WT were not encountered for caspofungin. Non-Aspergillus isolates in New Zealand have susceptibility patterns similar to those reported elsewhere. In contrast to a growing number of other countries, azole resistance was rare in A.fumigatus sensu stricto. Non-WT isolates were uncommon. The results provide a baseline for monitoring emerging antifungal resistance in New Zealand and support current Australasian treatment guidelines for invasive fungal infections.

Antibacterial activity of methanol extracts of the leaves of\xa0three medicinal plants against selected bacteria isolated from wounds of lymphoedema patients

BackgroundPatients with lymphoedema are at high risk of getting bacterial and fungal wound infections leading to acute inflammatory episodes associated with cellulitis and erysipelas. In Ethiopia, wound infections are traditionally treated with medicinal plants.MethodsAgar well diffusion and colorimetric microdilution methods were used to determine the antibacterial activity of methanol extracts of the three medicinal plants against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Shewanella alage, methicillin-resistant S. aureus ATCC®43300TM, Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, and Pseudomonas aeruginosa ATCC37853.ResultsThe methanol extract of L. inermis leaves showed high activity against all tested bacterial species, which was comparable to the standard drugs. Similarly, the extracts of A. indica showed activity against all tested species though at higher concentrations, and higher activity was recorded against Streptococcus pyogenes isolates at all concentrations. However, the extract of A. aspera showed the lowest activity against all tested species except Streptococcus pyogenes isolates. The lowest minimum inhibitory concentration (MIC) was recorded with the extract of L. inermis against E. coli isolate and S. aureus ATCC 25923.ConclusionMethanol extracts of L. inermis, A. indica, and A. aspera leaves exhibited antimicrobial activity against selected bacterial isolates involved in wound infections, of which the methanol extracts of L. inermis exhibited the highest activity. The results of the present study support the traditional use of plants against microbial infections, which could potentially be exploited for the treatment of wound infections associated with lymphoedema.

Prevalence and Antifungal Susceptibility of Pathogenic Yeasts in China: A 10-Year Retrospective Study in a Teaching Hospital.

To determine the dynamic changes of pathogenic yeast prevalence and antifungal susceptibility patterns in tertiary hospitals in China, we analyzed 527 yeast isolates preserved in the Research Center for Medical Mycology at Peking University, Beijing, China, between Jan 2010 and Dec 2019 and correctly identified 19 yeast species by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ribosomal DNA sequencing. Antifungal susceptibility testing was performed following a Sensititre YeastOne colorimetric microdilution panel with nine clinically available antifungals. The Clinical and Laboratory Standards Institute (CLSI)-approved standard M27-A3 (S4) and newly revised clinical breakpoints or species-specific and method-specific epidemiological cutoff values were used for the interpretation of susceptibility test data. In this study, although Candida albicans was the predominant single species, non-C. albicans species constituted >50% of isolates in 6 out of 10 years, and more rare species were present in the recent 5 years. The non-C. albicans species identified most frequently were Candida parapsilosis sensu stricto, Candida tropicalis, and Candida glabrata. The prevalence of fluconazole and voriconazole resistance in the C. parapsilosis sensu stricto population was <3%, but C. tropicalis exhibited decreased susceptibility to fluconazole (42, 57.5%) and voriconazole (31, 42.5%), and 22 (30.1%) C. tropicalis isolates exhibited wild-type minimum inhibitory concentrations (MICs) to posaconazole. Furthermore, fluconazole and voriconazole cross-resistance prevalence in C. tropicalis was 19 (26.1%). The overall prevalence of fluconazole resistance in the C. glabrata population was 14 (26.9%), and prevalence of isolates exhibiting voriconazole non-wild-type MICs was 33 (63.5%). High-level echinocandin resistance was mainly observed in C. glabrata, and the prevalence rates of isolate resistance to anidulafungin, micafungin, and caspofungin were 5 (9.6%), 5 (9.6%), and 4 (7.7%), respectively. Moreover, one C. glabrata isolate showed multidrug resistant to azoles, echinocandins, and flucytosine. Overall, the 10-year surveillance study showed the increasing prevalence of non-C. albicans species over time; the emergence of azole resistance in C. tropicalis and multidrug resistance in C. glabrata over the years reinforced the need for epidemiological surveillance and monitoring.

Colorimetric microdilution assay: Validation of a standard method for determination of MIC, IC50%, and IC90% of antimicrobial compounds

The emergence of multiresistant bacteria directly impacts on the search for new compounds with antimicrobial activity, and it is important the improvement of new techniques are able to determine the minimum inhibitory concentration (MIC) of antimicrobial compounds. The microdilution technique is widely used for saving culture media, reagents and compounds to be tested. However, the literature does not describe a colorimetric method capable of correlating absorbance with concentration of viable microorganisms (CFU mL−1). Therefore, the novelty of this work was the standardization and validation of a colorimetric and quantitative method capable of determining the MIC of several compounds with antimicrobial activity and the conversion of absorbance values to CFU mL−1. The conditions carried out for the method were: the use of 0.125% (w/v) 2,3,5-triphenyltetrazolium chloride (TTC) solution added after 22 h of incubation at 35 °C, followed by 2 more hours of incubation and subsequent reading in a spectrophotometer. The tested microorganisms were: Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and Candida albicans (ATCC 10231). The method was validated and showed linearity (R2 > 0.95), precision (RSD <26%), accuracy (75% to 122%) and robustness (p > 0.05). The validated parameters ensured the harmonization of methodology to determine not only MIC as well as inhibitory concentrations of 50% (IC50%) and 90% (IC90%) of the antimicrobial compounds.

Antileishmanial Potentialities of Croton linearis Leaf Essential Oil

The essential oil was obtained from Croton linearis Jacq. leaves. Its chemical composition was analyzed by Gas Chromatography-Mass Spectrometry. A total of 82 compounds were identified, being the principal components: guaiol (7.93%); eudesma-4(15),7-dien-1β-ol (4.94%) and guaia-3,10(14)-dien-11-ol (4.52%). Antimicrobial activity was determined against six Candida spp., three bacteria ( Staphylococcus aureus, Escherichia coli and Pseudomona aeruginosa) and two parasites ( Leishmania amazonensis and Trypanosoma cruzi). The selectivity index (SI) was computed through the determination of it cytotoxicity on peritoneal macrophages from BALB/c mice and murine fibroblasts L929 cells. Colorimetric microdilution methods as well as direct counting in optical microscopes were performed. The essential oil showed remarkable activity against L. amazonensis (IC50Promastigote: 20.0 ± 4.9 μg/mL; IC50Amastigote: 13.8 ± 4.3 μg/mL), moderate activity against T. cruzi (IC50Trypomastigote: 197.26 ± 8.7 μg/mL) and weak activity against P. aureginosa. Cytotoxicity values were CC50= 89.1 ± 3.4 μg/mL for macrophages BALB/c and CC50= 306 ± 110 μg/mL for L929 cells, rendering a SI of 6 for amastigote form of L. amazonensis. Croton linearis Jacq. leaf essential oil, therefore, could be a potential candidate for future investigations regarding Leishmaniasis treatment.

ANTIMICROBIAL, ANTIVIRAL AND CYTOTOXIC ACTIVITIES OF SELECTED MARINE ORGANISMS COLLECTED FROM THE COASTAL AREAS OF MALAYSIA

Many marine organisms have developed the capability of producing unique metabolites and thus are highly likely to contain anti-infective agents. This study was conducted to investigate extracts of three seaweeds (Caulerpa racemosa, Caulerpa sertularioides, Kappaphycus alvarezii), two soft corals (Lobophytum microlobulatum, Sarcophyton auritum) and a marine sponge (Spheciospongia vagabunda) collected from Malaysian coast for antibacterial, antifungal, antiviral and cytotoxic activities. The samples were subjected to sequential solvent extraction in order to obtain hexane, chloroform, ethyl acetate, ethanol, methanol and water extracts. The antibacterial and antifungal activities were studied using a colorimetric broth microdilution method. The hexane extract of L. microlobulatum had the strongest antibacterial activity and exhibited the lowest minimum inhibitory concentration (0.04 mg/mL) and minimum bactericidal concentration (0.08 mg/mL) against Staphylococcus aureus and Bacillus cereus, respectively. For antifungal activity, the lowest MIC and minimum fungicidal concentration values were produced by the hexane extract of S. auritum against the dimorphic yeast Cryptococcus neoformans, both with 0.04 mg/mL. None of the extracts were active against the filamentous fungus Aspergillus fumigatus. Only the hexane and ethanol extracts of L. microlobulatum and the ethyl acetate extract of S. auritum exhibited strong inhibition on the cytopathic effect induced by the Chikungunya virus (a re-emerging mosquito-borne virus) with 50% effective concentrations of 14.3 ± 0.2, 124.3 ± 1.9 and 176.6 ± 9.7 μg/mL, respectively. Extracts from the two soft corals, L. microlobulatum and S. auritum possessed stronger antibacterial, antifungal and antiviral activities compared to the seaweeds and the sponge.

Differences in antibacterial effectiveness between the whole extract and high-performance liquid chromatography-separated constituents from the cultivated mushroom Agaricus bisporus

There is inadequate data regarding how components interact in the whole extract of mushrooms to confer antibacterial activities. This study evaluated differences in the antibacterial capability (both potency and spectrum of action) between the crude extract, an EtOAc/MeOH fraction, and reverse-phase high performance liquid chromatography (RP-HPLC)-separated constituents from the cultivated button mushroom using a colorimetric micro-dilution method. The total extract showed excellent activities at doses < 1 mg/mL towards Bacillus subtilis only. In contrast, the fraction inhibited a more range of the tested bacteria at concentrations higher than did the extract (p < 0.05). The only active HPLC-separated compound exhibited degrees of inhibition against all the Gram-negative and Gram-positive bacteria at concentrations higher than 5 mg/mL. These findings may point to antibacterial synergism in the crude extract, implying high nutritional values for the whole mushroom as a functional food. Conversely, the broader spectrum of action of individual components warrants further investigations to determine its efficacy against antibiotic-resistant or Gram-negative bacteria in combination with commercial antibiotics.

Antituberculosis activity, phytochemical identification of Costus speciosus (J. Koenig) Sm., Cymbopogon citratus (DC. Ex Nees) Stapf., and Tabernaemontana coronaria (L.) Willd. and their effects on the growth kinetics and cellular integrity of Mycobacterium tuberculosis H37Rv

BackgroundCostus speciosus, Cymbopogon citratus, and Tabernaemontana coronaria are herbal plants traditionally used as remedies for symptoms of tuberculosis (TB) including cough. The aims of the present study were to evaluate the in vitro anti-TB activity of different solvent partitions of these plants, to identify the phytochemical compounds, and to assess the effects of the most active partitions on the growth kinetics and cellular integrity of the tubercle organism.MethodsThe in vitro anti-TB activity of different solvent partitions of the plant materials was determined against M. tuberculosis H37Rv using a tetrazolium colorimetric microdilution assay. The phytochemical compounds in the most active partition of each plant were identified using gas chromatography-mass spectrometry (GC-MS) analysis. The effects of these partitions on the growth kinetics of the mycobacteria were evaluated over 7-day treatment period in a batch culture system. Their effects on the mycobacterial cellular integrity were observed under a scanning electron microscope (SEM).ResultsThe respective n-hexane partition of C. speciosus, C. citratus, and T. coronaria exhibited the highest anti-TB activity with minimum inhibitory concentrations (MICs) of 100–200 μg/mL and minimum bactericidal concentration (MBC) of 200 μg/mL. GC-MS phytochemical analysis of these active partitions revealed that majority of the identified compounds belonged to lipophilic fatty acid groups. The active partitions of C. speciosus and T. coronaria exhibited high cidal activity in relation to time, killing more than 99% of the cell population. SEM observations showed that these active plant partitions caused multiple structural changes indicating massive cellular damages.ConclusionsThe n-hexane partition of the plant materials exhibited promising in vitro anti-TB activity against M. tuberculosis H37Rv. Their anti-TB activity was supported by their destructive effects on the integrity of the mycobacterial cellular structure.

Design, characterization, in vitro antibacterial, antitubercular evaluation and structure–activity relationships of new hydrazinyl thiazolyl coumarin derivatives

Herein, we describe the synthesis of 11new thiazolyl coumarin derivatives and evaluation of their potential role as antibacterial and antituberculosis agents. The structures of the synthesized compounds were established by extensive spectroscopic studies (Fourier transform infrared spectroscopy, 1H-nuclear magnetic resonance, 13C-nuclear magnetic resonance, 2D-nuclear magnetic resonance and liquid chromatography–mass spectrometry) and elemental analysis. All synthesized compounds were assayed for their in vitro antibacterial activity against a few gram positive and gram negative bacteria and antituberculosis activity against Mycobacterium tuberculosis H37Rv ATCC 25618 by using colorimetric microdilution assay method. Nine derivatives showed moderate anti-bacterial and anti-tuberculosis activities against all the tested strains. The highest activity against all the pathogens including Mycobacterium tuberculosis was observed by compound 7c with MIC values ranging between 31.25–62.5 μg/mL, indicating that coumarin skeleton could indeed provide useful scaffold for the development of new anti-microbial drugs.

Identification of Candida species and susceptibility testing with Sensititre YeastOne microdilution panel to 9 antifungal agents.

Objectives:To determine the species incidence and susceptibility pattern to 9 antifungal agents of yeasts isolated from various clinical specimens of colonized or infected patients treated in the coronary and surgical intensive care units (ICU).Methods:A total of 421 ICU patients were treated at the Cardiology Institute, Istanbul University, Istanbul, Turkey between June 2013 and May 2014, and 44 Candida species were isolated from blood, urine, endotracheal aspiration fluid, sputum, and wounds of 16 ICU patients. Identification of Candida was performed using CHROMagar. Antifungal susceptibility was determined by a Sensititre YeastOne colorimetric microdilution panel.Results:Candida albicans (C. albicans) was the most commonly observed microorganism 23 (54%); the other microorganisms isolated were Candida tropicalis 12 (27%), Candida glabrata 5 (11%), Candida parapsilosis 1 (2%), Candida lusitaniae 1 (2%), Candida sake 1 (2%), and Geotrichum capitatum 1 (2%). All isolates were susceptible to amphotericin B and 5-flucytosine. Geotrichum capitatum excepted, the other isolates were also susceptible to anidulafungin, micafungin, and caspofungin. Candida parapsilosis was found to be susceptible to all the studied antifungals. High MIC rates for azole group of antifungal drugs were found for C. albicans, C. tropicalis, and C. glabrata. The rate of colonisation was 3.8% (16/421). Only 0.7% (3/421) patients out of a total of 421 developed candidemia.Conclusion:We found that the yeast colonization and infection rates of patients in our ICUs are very low. Candida albicans is still the most common species. We detected a decreasing susceptibility to azole compounds.

Anti-Bacterial from &lt;i&gt;Moringa oleifera&lt;/i&gt; Lamk Crude Extracts

Moringa oleiferaLamk (Moringaceae) is a worldwide use for food and traditional medicine. In this study, plants were separated into seven parts: 1) Leaf, 2) Drum, 3) Branching, 4) Seed, 5) Outer stem, 6) Stem bark, and 7) Root, respectively. Then, they were extracted by two different methods: methanol soxhlet extraction and ethanol maceration. All crude extracts were tested for anti-bacterial activity by agar diffusion method and colorimetric micro-dilution plate technique. The results showed that 7 parts of Moringa crude extracts had low anti-bacterial activity againstStaphylococcus aureus,Escherichia coli, andPseudomonas aeruginosawhen compared with standard antibiotic (ampicillin or gentamycin). In conclusion, anti-bacterial effects ofM. oleiferacrude extracts had low anti-bacterial activityin vitrotest.

Bioactivity-guided isolation and structural characterization of the antifungal compound, plumbagin, from Nepenthes gracilis

Context: Despite several phytochemical studies of Nepenthes gracilis Korth (Nepenthaceae), the biological activities of this pitcher plant remain to be explored.Objective: This study evaluates the antifungal activity of N. gracilis extracts, isolates, and characterizes its bioactive compound and evaluates the cytotoxicity of the isolated compound.Materials and methods: Fresh leaves of N. gracilis were sequentially extracted. The fungistatic and fungicidal activities of the extracts were evaluated against six species of fungi of medical importance using a colorimetric broth microdilution method. The most active extract was fractionated by liquid–liquid partitioning and further purified by a preparative thin layer chromatography. Structural elucidation was carried out using FT-IR, GC-MS, and NMR. Cytotoxicity testing against rhesus monkey kidney epithelial cells (LLC-MK2) was assessed by a neutral red uptake (NRU) assay.Results: The hexane extract, which showed the lowest minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), both at 20 μg/mL against Candida albicans, Issatchenkia orientalis, and Trichophyton mentagrophytes, was subjected to bioactivity-guided fractionation. The isolated compound exhibited potent activity with the MIC values ranging from 2 to 31 μg/mL against all the fungi. The active compound was identified as plumbagin (5-hydroxy-2-methyl-naphthalene-1,4-dione). The 50% cytotoxicity concentration (CC50) of plumbagin was 0.60 μg/mL.Discussion and conclusion: The selectivity indices of plumbagin against all the fungi were less than 1.0, indicating that plumbagin is more toxic to mammalian than fungal cells. This study provides information on the antifungal properties of N. gracilis leaf extracts, as well as the antifungal and cytotoxicity properties of plumbagin.

ANTIFUNGAL AND ANTIBACTERIAL PROPERTIES OF THREE MEDICINAL PLANTS FROM MALAYSIA

Introduction: The increase of opportunistic fungal infections and the escalation of bacterial resistance have seriously reduced the efficacy of chemotherapeutic agents available. Thus the search for new antimicrobial agents from natural sources such as medicinal plants becomes necessary. Methods: The aerial parts of Diplazium esculentum and Sechium edule, and the fruits of Solanum muricatum were used, and extracted sequentially using hexane, chloroform, ethyl acetate, ethanol, methanol, and water. The extracts were then evaluated, in triplicate, against a panel of 12 medically-important microorganisms for microbiostatic and microbiocidal activities using colorimetric broth microdilution methods. Results: The total percentage yield obtained were 1.20%, 1.84% and 3.53% (w/w, based on fresh weight) for D. esculentum, S. edule and S. muricatum, respectively. All plant extracts showed antifungal activity with 66% and 49% of the bioassays demonstrating fungistatic and fungicidal activity, respectively. Two yeasts, Cryptococcus neoformans and Issatchenkia orientalis were found to be susceptible to all extracts. The lowest minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was exhibited by the hexane extracts of S. edule and S. muricatum against C. neoformans, both with values of 0.08 mg/mL. In the antibacterial screening assays, 49% of the bioassays exhibited bacteriostatic activity while only 21% of them showed bactericidal activity. The lowest MIC and minimum bactericidal concentration (MBC) was recorded for the hexane extract of S. muricatum against Bacillus cereus and Klebsiella pneumoniae, both with values of 0.31 mg/mL. The susceptibility of bacteria towards the plant extracts evaluated was species-dependent, with the susceptibility indices ranging from 0% for Escherichia coli to 72% for Pseudomonas aeruginosa. Conclusions: The results from this study show that extracts from these plants have significant antimicrobial activity, which corroborates their use in traditional medicine.

Design, synthesis and activity of thio-linked arabinofuranosyl disaccharides against mycobacterial tuberculosis (MTB) and Mycobacterium avium complex (MAC)

We report the chemical synthesis of a series of disaccharides of arabinofuranose with a glycosidic sulfur linker as mimics of the acceptor for arabinofuranosyltransferases with and without using any activator to avoid any complex reactions. These analogs were tested for in vitro activity against MTB strain H37Ra and 3 MAC clinical isolates. MICs were determined using a colorimetric microdilution broth assay. Bactericidal activity was studied with kill curves over a period of seven days. Intracellular activity against MTB H37Ra was determined in the Mono Mac 6 (MM6) human monocytic cell line.