Microdilution Research Articles

Investigation of Cytotoxic Effects and Antimicrobial Activities of Light-cured and Self-cured Universal Adhesive Systems

Objective: This study aimed to compare the cytotoxicity and antimicrobial activity of a light-cured adhesive system and a self-cured adhesive system from the same company. Materials and Methods: A Tokuyama BOND force II (Light-cured) adhesive system (TF2B) and a Tokuyama Universal Bond (Self-cured) adhesive system (TUB) were selected for the study. The cytotoxicity evaluation of these two systems on cell cultures was performed using MTT assay and Agar Diffusion assay in L929 fibroblast cells. Disk diffusion method and liquid microdilution (MIC) method were used to evaluate their antimicrobial activity. The experiments were performed on 6 pathogenic bacteria and 1 yeast fungus. Results: According to MTT test results, both adhesive systems have no significant toxic effect on healthy cells (L929). However, when TUB and TF2B were compared with each other, it was found that TF2B had almost no toxic effect. In the agar diffusion test, when the two bonds were compared with each other, a weak color lightening was observed only around the first concentration of TUB. No visible melting was detected in other concentrations of TUB and TF2B. Both adhesive systems failed to reach MIC values effectively on the test microorganisms. Since the results were far above the MIC values of the reference antibiotics, it was determined that they did not have antimicrobial effects. Disk diffusion results similarly showed that both bonds did not form an inhibition zone on the test microorganisms. Conclusions: In dentistry, cytotoxic effects of universal adhesive systems on living cells can be observed. Self-cured and Light-cured adhesive systems did not show toxic effects on L929 cells. In addition, antimicrobial effects on test microorganisms were not detected. The cytotoxicity of the materials can be tested on different cells.

Synergistic action of 6-gingerol as an adjuvant to colistin for susceptibility enhancement in multidrug-resistant Klebsiella pneumoniae isolates.

The growing threat to human health posed by multidrug-resistant Klebsiella pneumoniae (MDR-KP) indicates an urgent need to develop alternative therapeutic options. The emergence of colistin resistance further adds to the complexity. The study aims to explore in silico-screened phytomolecule 6-gingerol, the most potent active constituent of ginger, as an adjuvant to restore sensitivity in MDR-KP isolates to colistin. The screening of phytocompounds of Zingiber officinale were obtained from the spiceRx database, and molecular docking with efflux pump protein AcrB was performed using Schrödinger's Glide program. The synergistic and bactericidal effects of 6-gingerol in combination with colistin against MDR-KP isolates were determined following broth micro-dilution (MIC), checkerboard assay, and time-kill study. 6-Gingerol showed a good binding affinity with AcrB protein (-9.32 kcal mol-1) and followed the Lipinski rule of (RO5), demonstrating favourable drug-like properties. Further, the synergistic interaction of 6-gingerol with colistin observed from checkerboard assays against efflux-mediated colistin resistance MDR-KP isolates reveals it to be a prospectus adjuvant. The time-killing assays showed the effect of 6-gingerol in combination with colistin to be bactericidal against MSK9 and bacteriostatic against MSK4 and MSK7. Overall, the study provides insights into the potential use of 6-gingerol as a safe and easily available natural product to treat multidrug-resistant K. pneumoniae infections combined with colistin but needs in vivo toxicity evaluation before further recommendations can be made.

Antibacterial and Antioxidant Activity of Ziziphora clinopodioid Lam. (Lamiaceae) Essential Oil.

The genus Ziziphora belongs to medicinal plants. It is often used as a stomach tonic, carminative, antimicrobial, and expectorant; the extracted essential oils can be used as a second line of defence against pathogens. This study aimed to determine the antioxidant activity of essential oils of Z. clinopodioides as well as antibacterial activity against foodborne pathogens (Bacillus sp., Escherichia coli, Staphylococcus aureus and Pseudomonas sp.). The antibacterial activity of Z. clinopodioides essential oil was determined using the microdilution (M.D.) method in the nutritional broth medium and the agar disk diffusion assay. The result demonstrated that essential oil exhibit solid antibacterial properties against both gram-positive and gram-negative bacteria. Sequentially regarding MIC and MBC values, Escherichia coli was a higher level of resistance to the essential oil compared to Bacillus sp. Our findings suggested that the essential oil of Z. clinopodioides could be used as an antibacterial agent. The total antioxidant capacity of Z. clinopodioides leaves was assessed as ascorbic acid equivalents per gram of the leaves essential oil extract. The total antioxidant capacity was determined using ascorbic acid (y=0.1185x + 49.508, R2=0.3877). While the result of Z. clinopodioides was (y=0.1372x + 40.032, R2=0.4503).

In Silico Study for Algerian Essential Oils as Antimicrobial Agents against Multidrug-Resistant Bacteria Isolated from Pus Samples.

In the context of the globally growing problem of resistance to most used antibacterial agents, essential oils offer promising solutions against multidrug-resistant (MDR) bacterial pathogens. The present study aimed to evaluate the prevalence, etiology, and antibiotic-resistance profiles of bacteria responsible for pyogenic infections in Regional Military University Hospital of Constantine. Disc diffusion and broth microdilution (MIC) methods were used to evaluate the antimicrobial activity of essential oils from five Algerian aromatic plants growing wild in the north of Algeria—Salvia officinalis (Sage), Thymus vulgaris (Thyme), Mentha pulegium L. (Mentha), Rosmarinus officinalis (Rosemary), and Pelargonium roseum (Geranium)—against reference and MDR strains. During three months of the prospective study, 112 isolates out of 431 pus samples were identified. Staphylococcus aureus was the most predominant species (25%), followed by Klebsiella pneumoniae (21.42%), Pseudomonas aeruginosa (21%), and Escherichia coli (17.95%). Among pus isolates, 65 were MDR (58.03%). The radial streak-line assay showed that R. officinalis and M. pulegium L. had weak activity against the tested strains, whereas P. roseum showed no activity at all. Meanwhile, T. vulgaris was the most potent, with an inhibition zone of 12–26 mm and an MIC value ranging between 0.25 and 1.25%, followed by S. officinalis with an inhibition zone of 8–12 mm and an MIC value ranging between 0.62 and 2.5%. Generally, A. baumannii and S. aureus ATCC6538P were the most sensitive strains, whereas P. aeruginosa ATCC27853 was the most resistant strain to the oils. Gas chromatography–mass spectrometry analysis of chemical composition revealed the presence of borneol (76.42%) and thymol (17.69%) as predominant in thyme, whereas camphor (36.92%) and α- thujone (34.91%) were the major volatiles in sage. The in-silico study revealed that sesquiterpenes and thymol had the highest binding free energies against the vital enzymes involved in biosynthesis and repair of cell walls, proteins, and nucleic acids compared to monoterpenes. The results demonstrated that T. vulgaris and S. officinalis are ideal candidates for developing future potentially active remedies against MDR strains.

Potential antifungal impact of citral and linalool administered individually or combined with fluconazole against clinical isolates of Candida krusei

Introduction: Candida krusei is recognized as a major fungal pathogen in patients with immunodeficiency disorders. The present study aimed at investigating the anticandidal activities of citral and linalool combined with fluconazole (FLZ) against FLZ-resistant C. krusei strains. Methods: Antifungal activities were evaluated by the broth microdilution (MD) method to determine the minimum inhibitory and fungicidal concentrations (namely, MICs and MFCs) according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document. The interactions were further evaluated using fractional inhibitory concentration indices (FICIs) for combinations of citral+FLZ and linalool+FLZ, calculated from checkerboard MD assays. Results: The mean ± standard deviation (SD) MIC values of citral, linalool, and FLZ against the C. krusei isolates were 70.23 ± 17, 150 ± 38.73, and 74.66 ± 36.95 μg/mL, respectively. Some fungicidal activities were also observed for citral (2.5) and linalool (1.53) against the C. krusei isolates. The FICI values of citral+FLZ and linalool+FLZ for the C. krusei isolates ranged from 0.4 to 1.00 and 0.19 to 0.63, respectively. The additive and synergistic interactions of linalool + FLZ were further observed in 12 (57.1%) and 9 (42.9%) C. krusei isolates. However, there was an additive interaction for citral + FLZ in 17 (80.9%) isolates. They also showed a synergistic interaction in only four (19.1%) isolates. Moreover, linalool and citral plus FLZ did not have any antagonistic effect on any isolates. Conclusion: The study findings support the possible capabilities of citral and linalool, as anticandidal agents, and FLZ might be supplemented with citral and/or linalool for treating FLZ-resistant C. krusei infections.

Antioxidant, antibacterial, and antifungal activity of Hymenochaete rubiginosa

The use of fungi in alternative medicine and their use as pharmacological agents is gradually increasing today. Hymenochaete rubiginosa (Dicks.) Lév, locally known as Crust, is a species that belongs to the Polyporales family. In this study, it was aimed to determine the antimicrobial and antioxidant activity of H. rubiginosa extracts obtained from ethanol solvent. Its antimicrobial activity has been determined using the microdilution (MIC) method. In this study, bacteria such as Staphylococcus aureus ATCC 29213, Staphylococcus aureus MRSA ATCC 43300, Enterococcus faecalis ATCC 29212. Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606 and Candida albicans ATCC 10231, Candida krusei ATCC 34135 and Candida glabrata ATCC 9003 have been used as fungi. Total antioxidant level (TAS), total oxidant level (TOS) and oxidative stress index (OSI) values were calculated for antioxidant activity. According to the results obtained, it has been determined that the ethanol extract of H. rubiginosa showed antimicrobial effect against test microorganisms at concentrations of 25-200 µg/mL. The TAS value of H. rubiginosa has been determined as 6.313±0.050 mmol/L, TOS value as 14.358±0.202 µmol and OSI value as 0.227±0.002. As a result, it has been determined that H. rubiginosa can be used as an antioxidant and antimicrobial agent in pharmacological designs.

Proteomics characterization of Staphylococcus spp. from goat mastitis and phenogeno-typical assessment of resistance to beta-lactamics

ABSTRACT: Mastitis occupies a prominent place among the diseases that affect dairy herds due to economic problems and public health. Staphylococcus spp. are infectious agents more involved in the etiology of caprine mastites, especially coagulase-negative Staphylococcus. Nineteen isolates of Staphylococcus spp. were obtained from subclinical caprine mastitis. All isolates were characterized by MALDI-TOF MS, being 47.36% (9/19) identified for S. epidermidis, 15.78% (3/19) for S. warneri, 10.52% (2/19) for S. aureus and S. caprae and 5.26% (1/19) for S. lugdunensis, S. simulans, and S. cohnii. All isolates characterized by MALDI-TOF were subjected a to polymerase chain reaction (PCR) for the 16S rRNA gene of Staphylococcus spp. to confirm the gender. After determining the species, tests for phenotypic detection of resistance to beta-lactams were carried out simple disk diffusion oxacillin, cefoxitin, penicillin G and amoxicillin + clavulanic acid, agar “screen” oxacillin and microdilution (MIC) cefoxitin. The disk diffusion test showed a strength of 58% (11/19) for penicillin G, 26.31% (5/19) for cefoxitin and 26.31% (5/19) for oxacillin. All strains were susceptible to amoxicillin + clavulanic acid and agar “screen” oxacillin. In the MIC, 63.15% (12/19) of the samples were cefoxitin resistant (MIC >4.0μg/ml). Then isolates were subjected to detection of the mecA resistance genes and regulators (mecl and mecRI), mecC and blaZ. Two samples of Staphylococcus epidermidis had the mecA gene. All isolates were negative for the mecA gene variant, mecl, mecRI, mecC and blaZ. These findings reinforce the importance of this group of microorganisms in the etiology of subclinical mastitis in goats and open perspectives for future research to investigate the epidemiology of the disease.

Comparison of Microdilution Method with Agar Dilution Method for Antibiotic Susceptibility Test of Neisseria gonorrhoeae.

IntroductionAntimicrobial resistance (AMR) of Neisseria gonorrhoeae (N. gonorrhoeae) becomes a grave public health problem in the world. A strengthened Antimicrobial Resistance Surveillance Program is needed to track the trend of AMR development. However, the lack of a proper antimicrobial susceptibility test (AST) method is a barrier to expand the AMR surveillance in China. Traditional agar dilution (AD) method is laborious and E-test strips have no approval license for clinical use. Herein, a Chinese group modified the microdilution (MD) method for clinical ASTs. The objective of this study is to compare the MD method with the AD method for N. gonorrhoeae AST.Materials and MethodsA total of 166 clinical isolates were tested for antimicrobial susceptibility of ceftriaxone, spectinomycin, azithromycin, ciprofloxacin, tetracycline, and penicillin using MD and AD method simultaneously. Results of MD method were read manually or automatically. Rates of essential agreement (EA), category agreement (CA), minor error, and very major error were compared.ResultsThe total EAs (compared with results read manually) of penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, and azithromycin were 90.4%, 97.0%, 85.5%, 100.0%, 94%, and 72.3%; and CAs were 82.5%, 94.0%, 100%, 100%, 95.2%, and 94%, respectively.ConclusionWe conclude that the MD method might be an alternative for clinical AST of N. gonorrhoeae in China. In particular, MD method has the potency of accurate differentiation of isolates resistant to ceftriaxone or azithromycin, which were empirically recommended for gonococcal treatment, but its quality remained suboptimal, and further improvement is needed for clinical use.

English

In this study, the chemical composition of the peel and pulp of Mauritia flexuosa fruits were analyzed and the antimicrobial activity of ethanolic extracts from these fruits was evaluated using in vitro tests. Chemical composition analysis with gas chromatography-mass spectrometry (GC-MS) indicated the presence of saturated and unsaturated fatty acids. The peel extracts (ECBU) presented 54.41% and the pulp (EPBU) presented 94.05% of the saturated fatty acids lauric, myristic, palmitic, stearic, oleic and linoleic acids. The antimicrobial activities were performed using the diffusion and micro-dilution (MIC) methods. ECBU was active against the bacteria Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus at a concentration of 200 mg mL-1, but it was not active against the yeasts Candida albicans and Candida parapsilosis using the diffusion method. The MIC results showed that ECBU was active against the tested bacteria at concentrations > 12.5 mg mL-1 and EPBU was active at concentrations > 25.0 mg mL-1. This was probably due to higher sensibility of the method. The results indicated that the peel and pulp extracts of M. flexuosa present antibacterial activity and that ECBU is an especially promising potential candidate for the prospection of new pharmaceutical compounds. Key words: Mauritia flexuosa, Buriti, anti-bacterial agents, fatty acids.

Green synthesis of silver nanoparticles using <i>Arbutus andrachne</i> leaf extract and its antimicrobial activity

Purpose: To synthesize silver nanoparticles (AgNPs) of Arbutus andrachne leaf water extract (LE) and to evaluate the antimicrobial activity of both LE and AgNPs.Methods: The synthesized AgNPs were characterized using the following techniques: ultraviolet-visible spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), X-ray diffraction (XRD) analysis, and analysis of particle size (PS) and zeta potential (ZP). The antimicrobial activities of LE and NPs were assessed by Kirby-Bauer disc diffusion (DD) and broth microdilution (MD) methods according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). LE and AgNPs were examined against fresh cultures of four Gram-positive and five Gram-negative bacteria, and three yeast strains.Results: AgNPs were successfully synthesized and characterized using Arbutus andrachne LE. The AgNPs showed moderate antibacterial activity against Staphylococcus aureus ATCC 6538p, S.epidermidis ATCC 12228, Escherichia coli ATCC 29998, Klebsiella pnemoniae ATCC 13883 and Pseudomonas aeruginosa ATCC 27853, and also antifungal activity against Candida albicans ATCC 10239 and C. krusei ATCC 6258.Conclusions: Due to the potent activity of AgNPs against Gram-positive and Gram-negative bacteria, and yeast strains, it is suggested that AgNPs are potential broad spectrum antimicrobial agents.Keywords: Arbutus andrachne, Silver nanoparticles, Antimicrobial activity

In Vitro Antimicrobial and Antiproliferative Activity of Amphipterygium adstringens.

Amphipterygium adstringens is a plant widely used in Mexican traditional medicine for its known anti-inflammatory and antiulcer properties. In this work, we evaluated the in vitro antimicrobial and antiproliferative activities of the methanolic extract of A. adstringens against oral pathogens such as Streptococcus mutans, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Candida albicans, and Candida dubliniensis, using microdilution (MIC) and agar diffusion methods (MBC), and the antiproliferative activity evaluating total growth inhibition (TGI) by staining the protein content with sulforhodamine B (SRB), using nine human cancer cell lines. Crude extract (CE) of A. adstringens showed some degree of activity against one or more of the strains with a MIC from 0.125 mg/mL to 63 mg/mL and MBC from 1.6 to 6.3 mg/mL and cytotoxic activity, particularly against NCI-ADR/RES, an ovarian cell line expressing multiple resistance drugs phenotype. The CE is a complex mixture of possible multitarget metabolites that could be responsible for both antimicrobial and antiproliferative activities, and further investigation is required to elucidate the identity of active compounds. Nevertheless the CE itself is useful in the development of new antimicrobial treatment based on natural products to prevent oral diseases and as alternative natural source for cancer treatment and prevention.

Characterisation of a collection of Streptococcus pneumoniae isolates from patients suffering from acute exacerbations of chronic bronchitis: In vitro susceptibility to antibiotics and biofilm formation in relation to antibiotic efflux and serotypes/serogroups

The correlation between Streptococcus pneumoniae serotypes, biofilm production, antibiotic susceptibility and drug efflux in isolates from patients suffering from acute exacerbations of chronic bronchitis (AECB) remains largely unexplored. Using 101 isolates collected from AECB patients for whom partial (n=51) or full (n=50) medical details were available, we determined serotypes (ST)/serogroups (SG) (Quellung reaction), antibiotic susceptibility patterns [MIC (microdilution) using EUCAST and CLSI criteria] and ability to produce biofilm in vitro (10-day model; crystal violet staining). The majority of patients were 55–75 years old and <5% were vaccinated against S. pneumoniae. Moreover, 54% showed high severity scores (GOLD 3–4), and comorbidities were frequent including hypertension (60%), cancer (24%) and diabetes (20%). Alcohol and/or tobacco dependence was >30%. Isolates of SG6-11-15-23, known for large biofilm production and causing chronic infections, were the most prevalent (>15% each), but other isolates also produced biofilm (SG9-18-22-27 and ST8-20 being most productive), except SG7, SG29 and ST5 (<2% of isolates each). Resistance (EUCAST breakpoints) was 8–13% for amoxicillin and cefuroxime, 35–39% for macrolides, 2–8% for fluoroquinolones and 2% for telithromycin. ST19A isolates showed resistance to all antibiotics, ST14 to all except moxifloxacin, and SG9 and SG19 to all except telithromycin, moxifloxacin and ceftriaxone (SG19 only). Solithromycin and telithromycin MICs were similar. No correlation was observed between biofilm production and MIC or efflux (macrolides, fluoroquinolones). S. pneumoniae serotyping may improve AECB treatment by avoiding antibiotics with predictable low activity, but it is not predictive of biofilm production.

ONE POT PREPARATIONS 1-AMIDOALKYL-2-NAPHTHOLS DERIVATIVE CATALYZED BY NANO-TICL4.SIO2 WITH ANTIMICROBIAL STUDIES OF SOME PRODUCTS

Nano - TiCl 4 ·SiO 2 has been introduced to be an extremely efficient cataly st for the preparation of 1 - amidoalkyl - 2 - naphthols from various aldehydes and amides under mild conditions. We synthesized this solid Lewis acid catalyst by the reaction of nano - SiO 2 and TiCl 4 . The processor was simple and environmentally benign with high to excellent yields. Our method has the advantages of high yields, simple methodology, and easy work - up. We studied the different properties of the catalyst by FT - IR (ATR), X - ray diffraction (XRD), scanning electron microscope (SEM), transmission electr on microscopy (TEM) and thermal gravimetric analysis (TGA) . It was stable at high temperature and also it was reusable for at least three times. The antimicrobial and antifungal activities of some of the synthetic compounds were determined by broth microd ilution methods as recommended by Clinical Laboratory Standard Institute. Further studies still needed to investigate the other biological activities of the compounds.

Caspofungin MICs Correlate with Treatment Outcomes among Patients with Candida glabrata Invasive Candidiasis and Prior Echinocandin Exposure

Mutations in Candida glabrata FKS genes, which encode the echinocandin target enzyme, are independent risk factors for treatment failures during invasive candidiasis. We retrospectively compared the ability of caspofungin susceptibility testing methods to identify C. glabrata FKS mutant isolates and predict outcomes among patients at our center. Eight percent (10/120) of sterile-site C. glabrata isolates harbored FKS1 (n = 3) or FKS2 (n = 7) mutations, including 32% (10/32) recovered from patients with prior echinocandin exposure. Median echinocandin exposures for mutant and nonmutant isolates were 55 (range, 7 to 188) and 13 (3 to 84) days, respectively (P = 0.004). Sensitivity and specificity of the CLSI caspofungin resistance breakpoint MIC (>0.12 μg/ml by broth microdilution using RPMI medium [BMD-RPMI]) were 90% (9/10) and 3% (3/110), respectively, for identifying FKS mutants. Sensitivity and specificity of receiver-operator characteristic (ROC) curve-derived breakpoints by BMD-RPMI, BMD-antibiotic medium 3, Etest, and YeastOne ranged from 70 to 100% and 89 to 95%, respectively; susceptibility rates varied from 83 to 90%. The 14-day echinocandin treatment success rate was 67% (44/66); failure was more likely with prior echinocandin exposure (P = 0.002) or infection with an FKS mutant (P = 0.0001) or echinocandin-resistant isolates by BMD-AM3, Etest, and YeastOne (P ≤ 0.03). The failure rate among patients with prior exposure and infection with a resistant isolate was 91% (10/11); it was 22% (12/55) among others (P < 0.0001). In conclusion, ROC-derived caspofungin MIC breakpoints by several methods were sensitive and specific for identifying C. glabrata FKS mutant isolates. Mutations were seen exclusively among patients with prior echinocandin exposure. A paradigm that considers prior echinocandin exposure and caspofungin MICs accurately classified treatment outcomes for C. glabrata invasive candidiasis.

Ultrasonication‐Induced Synthesis and Antimicrobial Evaluation of Some Multifluorinated Pyrazolone Derivatives

A series of novel fluorine containing pyrazole‐pyrazolone (4a–j) and chromone‐pyrazolone (5a–i) was synthesized from multifluorinated pyrazolone by the Knoevenagel condensation reaction. All compounds were synthesized by conventional heating as well as ultrasound irradiation technique. It was found that ultrasonication method was more efficient than conventional heating method. The newly synthesized compounds were subjected for in vitro antimicrobial screening against four bacterial pathogens, namely, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa and three fungal pathogens Candida albicans, Aspergillus niger, and Aspergillus clavatus, using broth microdilution (MIC) method (CLSI guidelines). Among them, some compounds exhibited promising antibacterial activity against the tested strains. All synthesized compounds were characterized by IR, 1H‐NMR, mass, and elemental analysis.

Antifungal susceptibility of the aspergillus species by Etest and CLSI reference methods.

Because resistance to antifungal drugs is seen in patients, susceptibility testing of these drugs aids in choosing the appropriate drug and respective epidemiology. This study has investigated and compared susceptibility patterns of the Aspergillus species isolated from patients by the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (MD) assay and Etest method. The minimum inhibitory concentrations (MICs) of various antifungal agents (amphotericin B, ketoconazole, itraconazole, and voriconazole) for 108 Aspergillus species isolated from patients were determined by CLSI M38-A broth MD and Etest. The isolates were obtained from clinical samples that included tissues, sputum, bronchoalveolar lavage, abdominal tap, and cerebrospinal fluid. As revealed by the MD method, 63.9% of the isolates were sensitive to amphotericin B and 36.1% were resistant.Etest revealed that 61.1% were sensitive to amphotericin B and 38.9% were resistant. As for ketoconazole, 108 isolates (100%) were shown to be sensitive through the MD method; while the Etest revealedan 88.9% sensitivity and 11.1% were resistant. All species were susceptible to voriconazole, according to both methods. The measure of agreement (Kappa Index) for these three drugs was satisfactory (≥0.6). According to the MD method, 69.4% of the species were susceptible to itraconazole, whereas 30.6% were not.For this drug, the Etest showed 86.1% susceptible and 13.9% resistant. Voriconazole was the most effective agent against isolates. Using RPMI agar, we found the Etest to be helpful, readily available, and easy to use for determining invitro susceptibilities of Aspergillus species to voriconazole, amphotericin B, ketoconazole, and itraconazole in the region of this study.

Susceptibility tests of oropharyngeal Candida albicans from egyptian patients to fluconazole determined by three methods.

Candida albicans frequently cause oropharyngeal candidiasis in immunocompromised patients. As some of these isolates show resistance against azoles, the clinician is wary of initiating therapy with fluconazole (FZ) until a final susceptibility report is generated. We aimed to evaluate the efficacy of rapid flow cytometry (FCM) and disc diffusion (DD) methods in comparison to reference microdilution (MD) of Clinical and Laboratory Standards Institute (CLSI) method for FZ. Thirty seven Candida albicans isolates were tested by the three methods. By both MD and FCM, 26/37 (70.3%) were sensitive with minimal inhibitory concentration (MIC) ≤ 8μg/ml, 5/37 (13.5%) were susceptible dose dependant (S-DD) with MIC 16–32 μg/ml and 6/37 (16.2%) were resistant with MIC ≥64μg/ml. More than 92% of isolates susceptible to FZ by the MD were susceptible by the DD methods with good agreement (81.08%, P = 0.000). However, 4/5 isolates diagnosed as S-DD by MD were resistant by DD. Interestingly, the MIC by FCM at 4 h showed excellent agreement (95.59%, P = 0.000) to that obtained by MD method at 24 h. Overall, FCM antifungal susceptibility testing provided rapid, reproducible results that are valuable alternative to MD. The DD test is recommended as a simple and reliable screening test for the detection of susceptible Candida albicans isolates to FZ.

A Challenge for Clinical Laboratories: Detection of Antifungal Resistance inCandidaSpecies Causing Vulvovaginal Candidiasis

Background: To determine the in vitro susceptibility of vaginal yeasts against 7 antifungals by using 2 different methods and to evaluate if there is a possibility to use diskdiffusion (DD) method in the daily routine. Methods: Eighty-eight vaginal yeasts were tested against 5 antifungal azoles and 2 polyenes, according to Clinical and Laboratory Standards Institute (CLSI) documents DD (M44-A) and broth microdilution (MD) (M27-A3). Results: Resistance was recorded for ketoconazole (KETO), itraconazole (ITR), micoconazole amphotericin B (AMB), and nystatin (NYS). Between DD and MD, higher rates of agreement were observed for AmB (98.9%), voriconazole (VOR) (84.1%), and NYS (77.3%). For the other antifungals, the agreement varied from 34.1% (KETO) to 71.0% fluconazole (FLU). Conclusion: While the DD method may be a useful tool to determine the antifungal susceptibility profile in clinical laboratories in the future, it still requires improvements in its standardization since it was not reliable in detecting resistance in vitro.

Correlation between Etest®, disk diffusion, and microdilution methods for antifungal susceptibility testing of Candida species from infection and colonization

The correlation between the microdilution (MD), Etest (ET), and disk diffusion (DD) methods was determined for amphotericin B, itraconazole and fluconazole. The minimal inhibitory concentration (MIC) of those antifungal agents was established for a total of 70 Candida spp. isolates from colonization and infection. The species distribution was: Candida albicans (n=27), C. tropicalis (n=17), C. glabrata (n=16), C. parapsilosis (n=8), and C. lusitaniae (n=2). Non-Candida albicans Candida species showed higher MICs for the three antifungal agents when compared with C. albicans isolates. The overall concordance (based on the MIC value obtained within two dilutions) between the ET and the MD method was 83% for amphotericin B, 63% for itraconazole, and 64% for fluconazole. Considering the breakpoint, the agreement between the DD and MD methods was 71% for itraconazole and 67% for fluconazole. The DD zone diameters are highly reproducible and correlate well with the MD method, making agar-based methods a viable alternative to MD for susceptibility testing. However, data on agar-based tests for itraconazole and amphotericin B are yet scarce. Thus, further research must still be carried out to ensure the standardization to other antifungal agents.

Comparación de tres métodos para determinar la sensibilidad a imipenem y meropenem en Acinetobacter baumannii con fenotipo heterorresistente a carbapenemes

We assessed agreement among 3 assays for determining susceptibility to imipenem (IMP) and meropenem (MPM) of Acinetobacter baumannii (Ab) heteroresistant to carbapenems (Hr-CP). Thirty Ab clinical isolates belonging to 6 clones (REP-PCR) were studied, in which IMP and MPM MICs were between < or = 1 and > or = 8 mg/l by Wider system. MICs determined by Wider were compared with those obtained by microdilution (MD) and E-test. Errors in the clinical category were determined considering MD as the reference method. Twenty-five Ab were Hr-CP (growth of resistant colonies within the inhibition area of disks and E-test strips). Agreement for the MICs (+/- 1log(2)) in Hr-CP colonies was as follows: Wider vs. MD (96% IMP, 100% MPM), Wider vs. E-test (50% IMP, 64% MPM), MD vs. E-test (64% IMP, 60% MPM). Major errors were not observed. Minor errors and moderate errors for IMP included Wider vs. MD (40% and 0%), E-test vs. MD (40% and 12%), and disks vs. MD (36% and 8%). Agreement for the MICs considering colonies growing within the inhibition areas (E-test and disks) was Wider vs. E-test (8% IMP, 12% MPM), and MD vs. E-test (8% IMP and MPM). Major errors were not observed in this case either; minor errors and moderate errors for IMP were seen in E-test vs. MD (40% and 48%), and disks vs. MD (40% and 48%). Susceptibility testing methods based on microdilution (Wider system and MD) are not useful to detect subpopulations of Ab Hr-CP.