Correlation Between Etest (R), Disk Diffusion, and Microdilution Methods for Antifungal Susceptibility Testing of Candida Species From Infection and Colonization

The correlation between the microdilution (MD), Etest (R) (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 carded out to ensure the standardization to other antifungal agents. J. Clin. Lab. Anal. 23:324-330, 2009. (C) 2009 Wiley-Liss, Inc.

Candidemia neonatal, em hospital público do Mato Grosso do Sul

O objetivo de nosso estudo foi realizar tipagem molecular de 25 amostras clínicas de Candida spp, isoladas de crianças com candidemia, internadas na unidade de terapia intensiva neonatal de um Hospital Universitário entre 1998 a 2006. Dados demográficos e clínicos foram obtidos de prontuários para conhecimento dos aspectos clínicos e epidemiológicos. Identificação das leveduras foi feita por método convencional e a susceptibilidade antifúngica por método de microdiluição. O perfil genético foi determinado pela técnica de RAPD-PCR. Candida albicans (11; 44%) e Candida parapsilosis (10; 40%) foram as mais isoladas. Dezessete (68%) dos recém-nascidos tinham peso inferior a 1.500g. Prematuridade (92%), uso de cateter venoso central (100%), foram as condições de risco mais associados. Dezenove (76%) pacientes foram a óbito. Apenas uma cepa de Candida parapsilosis, mostrou ser sensível dose dependente ao fluconazol. Na análise molecular, foram observados 11 padrões genéticos distintos. Somente em dois casos foi observada relação epidemiológica, sugerindo mesma fonte de infecção.

Antimicrobial Activity of Diterpenes from Viguiera arenaria against Endodontic Bacteria

Six pimarane-type diterpenes isolated from Viguiera arenaria Baker and two semi-synthetic derivatives were evaluated in vitro against a panel of representative microorganisms responsible for dental root canal infections. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia, Prevotella buccae, Fusobacterium nucleatum, Bacteroides fragilis, Actinomyces naeslundii, Actinomyces viscosus, Peptostreptococcus micros, Enterococcus faecalis and Aggregatibacter actinomycetemcomitans. The compounds ent-pimara-8(14), 15-dien-19-oic acid, its sodium salt and ent-8(14), 15-pimaradien-3 beta-ol were the most active, displaying MIC values ranging from 1 to 10 mu g mL(-1). The results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new chemicals for use as a complement to instrumental endodontic procedures.

Antimicrobial activity of Piper arboreum and Piper tuberculatum (Piperaceae) against opportunistic yeasts

In the scope of our ongoing research on bioactive agents from natural sources, 24 extracts and fractions obtained from Piper arboreum Aub. and Piper tuberculatum Jacq. ( Piperaceae) were screened for antifungal activity by using broth microdilution method. The current investigation reveals that P. arboreum extracts and fractions were more effective against Candida krusei and Candida parapsilosis than Cryptococcus neoformans. The growth of Candida albicans was weakly affected by all the tested extracts and fractions. The strongest effects were observed for hexane and ethyl acetate fractions from leaves of P. arboreum, with MIC values ( in mu g/ml) of 15.6 and 31.2 mu g/ml against C. krusei, respectively. Additionally, phytochemical investigation of the hexane fraction of P. arboreum leaves furnished 3 pyrrolidine amides; piperyline, 4,5-dihydropiperyline and tetrahydropiperyline, which could be responsible, at least in part for the observed antifungal activity. The most active compound, tetrahydropiperyline, displayed MIC values of 15.6 mu g/ml against C. krusei, C. parapsilosis and C. neoformans.

Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay

Objectives: The resazurin microtitre plate assay (REMA) was evaluated to determine the susceptibility of Mycobacterium tuberculosis to pyrazinamide, and was compared with the broth microdilution method (BMM), the absolute concentration method (ACM) and pyrazinamidase (PZase) determination. Methods: Thirty-four M. tuberculosis clinical isolates (26 susceptible and 8 resistant to pyrazinamide) and reference strains M. tuberculosis H37Rv ATCC 27294 and Mycobacterium bovis AN5 were tested. Results: REMA and BMM showed 100% specificity and sensitivity when compared with ACM; BMM, however, demanded more reading time. The PZase determination assay showed 87.50% and 100% sensitivity and specificity, respectively. Conclusions: All tested methods in this preliminary study showed excellent sensitivity and specificity for the determination of pyrazinamide susceptibility of M. tuberculosis, but REMA was faster, low-cost and easy to perform and interpret. Additional studies evaluating REMA for differentiating pyrazinamide-resistant and-susceptible M. tuberculosis should be conducted on an extended panel of clinical isolates.

Antimicrobial activity of crude extratcs of Petiveria alliacea L.

Antimicrobial activity of crude extratcs of Petiveria alliacea L.. Petiveria alliacea L. (Phytolaccacea) is an herbaceous plant of great importance in traditional medicine. This species have been widely used in several applications such as antirheumatic, anticarcinogenic, anti-flu, antitussive, analgesic, insecticidal, acaricidal, as well as bactericide and fungicide. Currently, the pathogenic microorganisms are acquiring resistence against the traditional antibiotics, and the search for new herbal antimicrobial agents has been intensified. The objective of this study was to evaluate the antifungal and antibacterial activity of several leaf crude extracts of P. alliacea against several strains of bacterias and yeasts namely Bacilus subtilis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Candida parapsilosis, Candida kefyr and Candida albicans, using microdilution method. Promising results were observed for the 70% v/v ethanolic extract which presented minimum inhibitory concentration (MIC) from 250 to 760 mu g/mL for yeast. For the bacteria strains tested the MIC ranged between 240 to 3960 mu g/mL, depending of the extractive solution tested.

Antimicrobial Activity of Rosmarinus officinalis against Oral Pathogens: Relevance of Carnosic Acid and Carnosol

The in vitro inhibitory activity of crude EtOH/H(2)O extracts from the leaves and stems of Rosmarinus officinalis L. was evaluated against the following microorganisms responsible for initiating dental caries: Streptococcus mutans, salivarius, S. sobrinus, S. mitts 5 sanguinis, and Enterococcus faecalis. Minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The bioassay-guided fractionation of the leaf extract, which displayed the higher antibacterial activity than the stem extract, led to the identification of carnosic acid (2) and carnosol (3) as the major compounds in the fraction displaying the highest activity, as identified by HPLC analysis. Rosmarinic acid (1), detected in another fraction, did not display any activity against the selected microorganisms. HPLC Analysis revealed the presence of low amounts of ursolic acid (4) and oleanolic acid (5) in the obtained fractions. The results suggest that the antimicrobial activity of the extract from the leaves of R. officinalis may be ascribed mainly to the action of 2 and 3.

In vitro antifungal drug susceptibilities of dermatophytes microconidia and arthroconidia

Objectives: Arthroconidia have been considered as the primary cause of infection by dermatophytes. However, the in vitro antifungal testing evaluates the responses mainly of microconidia or hyphae, and dermatophytes in vivo often produce arthroconidia, a cellular structure presumably more resistant to antifungals. The aim of this study was to compare the in vitro susceptibility of microconidia and arthroconidia of Trichophyton rubrum, Trichophyton tonsurans and Trichophyton equinum to griseofulvin, itraconazole, terbinafine, fluconazole, amphotericin B and hygromycin B. Methods: Microconidia and arthroconidia were produced in vitro, and their susceptibility to each drug was evaluated by assessing the CLSI M38-A broth microdilution method. Results: Arthroconidia of all strains analysed appeared to be more resistant to fluconazole, griseofulvin and itraconazole than microconidia. The MIC of terbinafine was the same for microconidia and arthroconidia for all strains, and the MIC of amphotericin B for microconidia and arthroconidia was the same for isolates of T. equinum and T. tonsurans, but differed for T. rubrum. Finally, the level of resistance of microconidia for all strains towards the antibiotic hygromycin B was from 25 to 400 mg/L. Conclusions: The difference in the susceptibility between microconidia and arthroconidia depends on the drug and on the strain, and may be one of the causes of therapeutic failure. Also, the level of resistance to the antibiotic hygromycin B presented by microconidia of these isolates will allow the use of hygromycin resistance as a dominant marker in fungal transformation procedures in future studies of gene function.

Antimicrobial Activities of Ethanol Extract and Coumestans from Eclipta alba (L.) Hassk (Asteraceae)

Ethanol extract and fractions from aerial parts of Eclipta alba (L.) Hassk (Asteraceae) were screened for the antibacterial and antifungal activities against different species of human pathogenic bacterial ATCC, antibiotic-resistant clinical isolates and strains of the dermatophyte Trichophyton rubrum (wild and mutant for TruMDR2 gene) using a microdilution method. Demethylwedelolactone/wedelolactone (DWL/WL) and only wedelolactone (WL), both in a high homogeneity degree, were efficient to inhibit the ATCC strains of Staphylococus aureus (Minimal Inhibitory Concentration MIC = 75 mu g/mL), Staphylococcus epidemidis (MIC = 125 mu g/mL) and Escherichia coli (MIC = 125 mu g/mL) as well as antibiotic-resistant clinical isolates of Enterococcus spp (MIC = 250 mu g/mL) and S. aureus (MIC = 125 mu g/mL). Ethanol extract was more effective than the purified fractions against Trichophyton rubrum strains (MIC = 125 mu g/mL), suggesting that anti-fungal activity is not only related to demethylwedelolactone and wedelolactone, but also to a synergistic action between these coumestans and other compounds found in that extract. Thus, this work suggests that E. alba possesses a significant antimicrobial activity, including that against multi-drug resistant microorganisms, which could be of relevance for the treatment of infectious diseases.

Salmonella antibiotic-mutant strains reduce fecal shedding and organ invasion in broiler chicks

We investigated the exposure to antibiotics in the production of antibiotic-mutant strains of Salmonella. Ten isolates of poultry origin were assayed for antibiotic susceptibilities. One strain of Salmonella Enteritidis, one of Salmonella Heidelberg, and one of Salmonella Typhimurium were selected to induce antimicrobial resistance. Each strain was exposed to high concentrations of streptomycin, rifampicin, and nalidixic acid, respectively. Parent and antibiotic-mutant strains were assayed for antibiotic susceptibilities using a commercial microdilution test and the disk susceptibility test. The strains were assessed for virulence genes and evaluated for fecal shedding, cecal colonization, organ invasion, and mean Salmonella counts after inoculation in 1-day-old chicks. The study revealed that exposure to high concentrations of streptomycin produced the antibiotic-mutant strain SE/LABOR/USP/08 and the exposure to rifampicin produced the antibiotic-mutant SH/LABOR/USP/08. These strains showed significantly reduced fecal shedding (P = 0.05) and organ invasion, persisting less than the parental strains and showing no clinical signs in inoculated chicks. High concentrations of nalidixic acid produced the antibiotic-mutant strain ST/LABOR/USP/08, which did not show any differences compared with the parent strain. Likewise, SE/LABOR/USP/08 did not show the expression of plasmid-encoded fimbriae (pefA) and plasmid virulence protein (spvC), suggesting that after exposure to streptomycin, the parent isolate lost the original gene expression, reducing fecal shedding and organ invasion in inoculated chicks.

In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens

Aim: In the Amazon region of Brazil, the fruits of Caesalpinia ferrea Martius (Brazilian ironwood) are widely used as an antimicrobial and healing medicine in many situations including oral infections. This study aimed to evaluate the antimicrobial activity of Caesalpinia ferrea Martius fruit extract against oral pathogens. Materials and methods: Polyphenols estimation and spectral analysis ((1)H NMR) of the methanol extract were carried out. The microorganisms Candida albicans, Streptococcus mutans, Streptococcus salivarius, Streptococcus oralis and Lactobacillus casei were tested using the microdilution method for planktonic cells (MIC) and a multispecies biofilm model. Chlorhexidine was used as positive control. Results: Polyphenols in the extract were estimated at 7.3% and (1)H NMR analysis revealed hydroxy phenols and methoxilated compounds. MIC values for Candida albicans, Streptococcus mutans, Streptococcus salivarius, Streptococcus oralis and Lactobacillus casei were 25.0, 40.0, 66.0, 100.0, 66.0 mu g/mL, respectively. For the biofilm assay, chlorhexidine and plant extract showed no growth at 10(-4) and 10(-5) microbial dilution, respectively. At 10-4 and 10-5 the growth values (mean +/- SD) of the negative controls (DMSO and saline solution) for Streptococcus mutans, Streptococcus sp. and Candida albicans were 8.1 +/- 0.7, 7.0 +/- 0.6 and 5.9 +/- 0.9 x 10(6) CFU, respectively. Conclusion: Caesalpinia ferrea fruit extract can inhibit in vitro growth of oral pathogens in planktonic and biofilm models supporting its use for oral infections. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Dihydroisocoumarin from Xyris pterygoblephara active against dermatophyte fungi

The ethanol extract from Xyris pteygoblephara aerial parts was evaluated against five microorganism strains, by the microdilution and agar diffusion methods. Extract fractionation led to the isolation of three compounds, whose structures were assigned by spectrometric data (113 and 2D NMR, IR, MS and UV) as (3R,4R)-(-)-6-methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one (1), moronic acid and quercetin. The absolute configuration of I was defined by circular dichroism spectroscopy and comparison with data reported for other dihydroisocoumarins. Assay of 1 (100 mu g/disc) by the agar diffusion method against clinical isolates of the dermatophytes Epidermophyton floccosum (inhibition zone, mm +/- s.d.: 4.5 +/- 0.8), Trichophyton mentagrophytes (4.8 +/- 0.4) and Trichophyton rubrum (10.2 +/- 0.8) revealed similar inhibition zones to the positive control amphotericin B (32 mu g/disc; 5.0 +/- 0.2; 5.0 +/- 0.6 and 8.8 +/- 1.2, respectively). The result corroborates the ethnomedical use of Xyris species to treat dermatitis. (C) 2007 Elsevier Ltd. All rights reserved.