A Silent Threat of Emerging Resistance Based on Antifungal Susceptibility Pattern of Filamentous Fungi by Microdilution, E Test and, Disc Diffusion Method: A Critical Constructive Analysis

For more than two decades, antifungal susceptibility testing and interpretation haunted the medical professionals in diagnostics and management. This article mainly focuses on the three most widely used methods: broth microdilution, E test, and disc diffusion. It also focuses on the fact that clinicians should switch from empirical treatment to susceptible drugs as early as possible to combat antifungal resistance and newer mutations that horrify us every single day with poor patient outcomes. Many factors need to be taken into account during the interpretation of results but the positive side of the story is that they have been well documented in the literature. Though many methods have come up in testing antifungal susceptibility, still there is a scope for a rapid yet accurate testing modality to flourish and take the lead

GC-MS Analysis and Antimicrobial Activity of the Extract and Fractions of Bacillus subtilis subsp. subtilis 168 Isolated from a River Bank in Nigeria

The menace of drug resistant pathogens is increasing and their level of evading conventional antimicrobials is rising. It is therefore important to discover new antimicrobials to counter the current challenges. Our preliminary investigation identified Bacillus subtilis subsp. subtilis 168 isolated from soil sample sourced from a river bank in Abuja, Nigeria, as the most potent antibiotic-producing bacteria among the other identified producers. The current study screened for the antimicrobial activity of the extract and fractions of the isolate by broth microdilution method. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the ratio of the MBC/MIC were determined. All the tested pathogens were susceptible to the ethyl-acetate extract (MIC between 28.70 mg/ml and 57.40 mg/ml). The extract displayed bactericidal activity against all tested pathogens (MBC/MIC between 1.00 and 2.00) while Proteus mirabilis was least susceptible. The extract was purified by vacuum liquid chromatography and the fractions challenged with pathogenic strains. The fraction E was the most potent (MIC between 0.09 mg/ml and 0.75 mg/ml) and also bactericidal against all the test microbes (MBC/MIC between 2.00 and 2.11). GC-MS analysis of the purified sub fraction obtained from fraction E identified 13 compounds with different Retention time and peak areas. Among these were three major compounds which include: (i) bis(2-ethylhexyl) phthalate (ii) 1,4-epoxynaphthalene-1(2H)-methanol, 4,5,7-tris(1,1-dimethylethyl)-3,4-dihydro- (iii) D:B-Friedo-B':A'-neogammacer-5-en-3-ol, (3.beta.)-. Our findings suggest that Bacillus subtilis subsp. subtilis 168 isolated locally could serve as a valuable source of lead compounds for pharmaceutical and biotechnological purposes.

Evaluación de la elución de sensidiscos para la determinación de susceptibilidad a colistín en bacilos gramnegativos multi-resistentes / Evaluation of sensi-disk elution for colistin susceptibility determination in multidrug resistant gramnegative bacilli

Resumen Utilizando cepas clínicas de bacilos gramnegativos multi-resistentes (MDR), comparamos las CIM obtenidas de la microdilución en caldo, el método de referencia y el método de elución de sensidiscos. Encontramos que, con la excepción de A. baumannii, los resultados fueron muy similares. El método de elución de sensidiscos podría ser una buena alternativa y confiable para la determinación de la resistencia a colistín.

Abstract Using clinical strains of multidrug resistant (MDR) Gram negative bacilli, we compared MICs obtained from both broth microdilution, the reference method, and sensi-disk elution method. We found that, with A. baumannii exception, results were very similar. Sensi-disk elution method could be a good and reliable alternative for colistin resistance determination.

Antibacterial Property of Synsepalum dulcificum Leaves Aqueous Extract against Oral Pathogens and its Chemical Compounds

@#Synsepalum dulcificum (S. dulcificum) commonly known as “miracle fruit” because its berries have the capability to modify sour taste to the sweet taste when eaten. Beside the berries, S. dulcificum leaves were also known to possess biological properties such as antioxidant, antimutagenic and antidiabetic activities. However, the study of its antimicrobial activity against oral pathogen is still lacking. Thus, this study aimed to evaluate the antibacterial activity of its leaves against cariogenic bacteria and to analyse its phytochemical compounds. The samples of S. dulcificum leaves were collected in Kelantan, the east coast region of Peninsular Malaysia and extracted with distilled water using a Soxhlet technique. The antibacterial activity of the S. dulcificum leaves aqueous extract against Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus) and Lactobacillus salivarius (L. salivarius) was evaluated using the broth microdilution assay. The identification of the phytochemical compounds was performed using gas chromatography-mass spectrometry (GC-MS). The antibacterial study showed the minimum inhibitory concentration of S. dulcificum leaves aqueous extract against S. mutans and S. sobrinus were 16 mg/mL and 8 mg/mL, respectively. Interestingly, there was no inhibitory effect of S. dulcificum leaves aqueous extract against L. salivarius. A total of 42 chemical compounds were identified and major identified bioactive compounds groups were heterocyclic and phenolic compounds. Our results suggested S. dulcificum leaves aqueous extract has antimicrobial properties against S. mutans and S. sobrinus, but no inhibitory activity against oral normal flora, with the presence of bioactive compounds has potential in oral care products application.

Isolation, identification and antifungal susceptibility of Candida in patients with fungal sepsis

Background: In this study, our aim was to identify and isolate Candida species from patients admitted in ICU,s of our hospital and to determine their susceptibilities to various antifungal agents so as to find the local resistance pattern and guide for empirical treatment.Methods: In our study 37 strains of candida were isolated (4 Candida albicans, 33 Non-albicans Candida strains). Candida species were identified by conventional, biochemical and molecular methods. Antifungal susceptibility tests for amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole were performed with broth microdilution method and E- tests as described by National Committee for Clinical Laboratory Standards (NCCLS).Results: Out of 37 Candida strains, the most prevalent species were C. tropicalis (43.2%), C. parapsilosis (24.3%), C. krusei (16.2%), C. albicans (10.8%), and C. glabrata (2.7%). Among all strains four strains (10.8 %) were resistant, two Candida albicans where found resistant to fluconazole one Candida krusei and one Candida parapsilosis were found to be resistant to all azoles.Conclusions: Candidemia continues to be associated with substantial morbidity and mortality and non albicans Candida species are the commonly isolated pathogen from those patients admitted in tertiary care hospitals in Indian scenario. Thus, it is imperative to perform antifungal susceptibility to select appropriate and effective antifungal therapy.

Antimicrobial efficacy of fermented mango leaves beverage towards selected foodborne pathogens

Aims@#Fermented mango leaves of Chokanan variety was produced using selected symbiotic culture of bacteria and yeast (SCOBY) from MARDI’s Collection of Functional Food Cultures (CFFC). The aim of this work was to investigate its functional benefits as food remedy to reduce the risk of food poisoning illness incidence. @*Methodology and results@#Five species of foodborne pathogens: Escherichia coli O157:H7 UPMEC32 (local isolate), Salmonella typhimurium ATCC®53648™, Salmonella enteritidis MDC15 (local isolate), Listeria monocytogenes ATCC®51772™ and Streptococcus gallolyticus (ATCC®9809™) were selected to examine the antimicrobial effect of fermented mango leaves beverage by means of agar well diffusion assay and broth microdilution method to determine its minimum bactericidal concentration (MBC>99). In comparison with chemical inhibitor (acetic acid, 1%) and antibiotic (Penicillin streptomycin, 1%), the agar diffusion assay results confirmed the inhibition efficacy of fermented mango leaves beverage against all five foodborne pathogens tested. Particularly, fermented mango leaves beverage was showing a significant inhibitory effect (P<0.05) against S. gallolyticus, whereas both acetic acid and penicillin streptomycin have no inhibitory activities at all towards this pathogen. Another antimicrobial activity assay using broth microdilution method also confirmed the 100% inhibition effect of fermented mango leaves beverage against these selected pathogenic microorganisms. Furthermore, the efficacy retained 100% inhibitory activities even though the fermented mango leaves beverage has been diluted to 50%. Synergetic effect of significant amount of multiple organic acids present in fermented mango leaves beverage were the main factors contributing to its potent antimicrobial properties and improvement taste after fermentation. On the contrary, little or no antimicrobial inhibitory activity was observed in all non-fermented mango leaves beverages treated samples. @*Conclusion, significance and impact of study@#This finding indicates that the potential of fermented mango leaves beverages as prophylaxis measures to reduce the risk of food poisoning incidence as it has shown a good antimicrobial effect against selected foodborne pathogens. Moreover, this fermented mango leaves beverage are more tasteful after gone through the microbial fermentation process. It is recommended to consume daily to reduce the incidence of food poisoning illness.

Deciphering polymyxin B minimum inhibitory concentration from colistin minimum inhibitory concentration and vice versa: An analysis on 156 carbapenem-resistant Enterobacteriaceae isolates

The susceptibility determination to polymyxins (colistin and polymyxin B) remains a challenge for clinical microbiology laboratories. We evaluated the minimum inhibitory concentration (MIC) of both antimicrobials by the broth microdilution method in a selected subset of 156 carbapenem-resistant Enterobacteriaceae (CRE) isolates. Good concordance between polymyxin B and colistin MIC values was obtained, and there was 98% categorical agreement in CRE isolates. Future large-scale multicentre study is needed to draw conclusion if the MIC of colistin can be used to extrapolate the MIC of polymyxin B and vice versa.

Phytochemistry and antimicrobial activity of Campomanesia adamantium

ABSTRACT Campomanesia adamantium (Cambess.) O. Berg., Myrtaceae, is a plant popularly used for its anti-inflammatory, anti-diarrhoeal and urinary antiseptic activities. The aims of this study were to obtain the crude ethanolic extract and the hexane, dichloromethane, ethyl acetate, aqueous and concentrated aqueous tannin fractions from C. adamantium leaves, perform biomonitored fractionation to isolate and identify chemical compounds, study the chemical composition of the volatile oils of the leaves and flowers and test the antimicrobial activity of the ethanolic extract, fractions, isolated substances and volatile oils. Phytochemical screening and chromatographic and spectrometric techniques were used. Volatile oils were isolated by hydrodistillation in a Clevenger apparatus and analyzed by gas chromatography/mass spectrometry. The antimicrobial activity was tested by a broth microdilution test. The component stictane-3,22-diol was isolated and identified from the hexane fraction, while valoneic and gallic acid were isolated and identified from the concentrated aqueous tannin fraction. The major constituents of the volatile oils of the leaves were verbenene (13.91%), β-funebrene (12.05%) and limonene (10.32%), while those of the volatile oils of the flowers were sabinene (20.45%), limonene (19.33%), α-thujene (8.86%) and methyl salicylate (8.66%). Antibacterial activity was verified for the hexane fraction, while antifungal activity was observed for the aqueous fraction and concentrated aqueous tannin fraction and for vanoleic acid. These results may justify the popular use of C. adamantium.

Comparación y evaluación de métodos cuantitativos para determinar la susceptibilidad antimicrobiana de cepas del complejo Mycobacterium abscessus / Comparison and Evaluation of Quantitative Methods for Determining Antimicrobial Susceptibility of Complex Mycobacterium Abscessus Strains / Comparação e avaliação de métodos quantitativos para determinar a susceptibilidade antimicrobiana de cepas do complexo Mycobacterium abscessos

Resumen Introducción: el complejo Mycobacterium abscessus incluye especies patógenas emergentes multirresistentes, lo cual limita las opciones terapéuticas para tratar las infecciones causadas por dichos microorganismos. En este estudio se compararon las concentraciones inhibitorias mínimas (CIM) obtenidas mediante dos métodos cuantitativos, se establecieron los puntos de corte empleados en el micrométodo colorimétrico (MMC) y se evaluó la susceptibilidad antimicrobiana. Materiales y métodos: la CIM de nueve antibióticos fue determinada mediante el MMC y la microdilución en caldo (MDC) para 19 cepas del complejo M. abscessus. El test F de Snedecor se utilizó para establecer la diferencia significativa de las CIM entre los dos métodos y se determinaron los puntos de corte mediante la técnica de distribución de la probabilidad para el MMC. Resultados: se encontró una correlación de los resultados de la CIM del 50% entre MMC y MDC para los antibióticos ensayados. Probablemente esta discrepancia en los resultados se deba a diferencias en algunos parámetros técnicos de cada procedimiento. Todas las cepas fueron sensibles a la amikacina y resistentes a meropenem y ampicilina-sulbactam. Independientemente de la especie del complejo M. abscessus, las fluoroquinolonas mostraron una baja actividad inhibitoria (0-25%) sobre los aislados clínicos, resultados que son similares a los reportados por otros autores. Conclusión: Los patrones de multirresistencia observados en las cepas analizadas sugieren la necesidad de utilizar las pruebas de susceptibilidad como herramientas que permitan orientar y optimizar las conductas terapéuticas en infecciones producidas por M. abscessus.

Abstract Introduction: The Mycobacterium abscessus complex includes multidrug resistant emerging pathogens, which limit therapeutic options for treating infections caused by these microorganisms. In this study, the minimum inhibitory concentrations (MICS) obtained by 2 quantitative methods were compared, the cut-off points used in the colorimetric micromethod (CMM) were established and the antimicrobial susceptibility was evaluated. Materials and Methods: The MIC for nine antibiotics was determined by CMM and broth microdilution (BMD) for 19 strains of M. abscessus complex. The Snedecor F test was used to establish the significant difference in the CIM between the methods, cutoff points were determined by the probability distribution method for the CMM. Discussion: A correlation of 50% between CMM and BMD for antibiotics tested was found. Probably, this discrepancy in the results is due to differences in some technical parameters of each procedure. All strains were susceptible to amikacin and were resistant to meropenem and ampicillin-sulbactam. Independently of the species of M. abscessus complex, fluoroquinolones showed a low inhibitory activity (0-25%) on clinical isolates, results that are similar to those reported by other authors. Conclussion: The Multidrug resistance patterns observed in the strains tested suggest the need for susceptibility testing as tools to guide and optimize the therapeutic behavior in infections caused by M. abscessus.

Resumo Introdução: o complexo Mycobacterium abscessus inclui espécies patógenas emergentes multirresistentes, o qual limita as opções terapêuticas para tratar as infeções causadas por estes microrganismos. Neste estudo compararam-se as concentrações inibitórias mínimas (CIMS) obtidas mediante 2 métodos quantitativos, se estabeleceram os pontos de corte empregados no micrométodo colorimétrico (MMC) e se avaliou a susceptibilidade antimicrobiana. Materiais e métodos: a CIM de 9 antibióticos foi determinada mediante o MMC e microdiluição em caldo (MDC) para 19 cepas do complexo M. abscessus. O teste F de Snedecor utilizou-se para estabelecer a diferença significativa das CIMS entre os dois métodos e determinaram-se os pontos de corte mediante a técnica de distribuição da probabilidade para o MMC. Resultados: se encontrou uma correlação dos resultados da CIM do 50% entre MMC e MDC para os antibióticos testados. Provavelmente, esta discrepância nos resultados se deve a diferenças em alguns parâmetros técnicos de cada procedimento. Todas as cepas foram sensíveis à amikacina e resistentes a meropenem e ampicilina-sulbactam. Independentemente da espécie do complexo M. abscessos, as fluoroquinolonas mostraram uma baixa atividade inibitória (0-25%) sobre os isolados clínicos, resultados que são similares aos reportados por outros autores. Conclussão: Os patrões de multirresistência observados nas cepas analisadas, sugerem a necessidade de utilizar as provas de susceptibilidade como ferramentas que permitam orientar e otimizar as condutas terapêuticas em infeções produzidas por M. abscessus.

Evaluation of colorimetric nitrate reductase assay for rapid detection of methicillin resistance in clinical isolates of Staphylococcus aureus.

Background & objectives: Methicillin resistant Staphylococcus aureus (MRSA) remains a major cause of health care-associated infections. Rapid detection of MRSA facilitates the early initiation of appropriate treatment and infection control. Hence, the present study was undertaken to standardize and evaluate the performance of rapid colorimetric nitrate reductase assay (NRA) for determining methicillin resistance in S.aureus. Methods: A total of 160 clinical isolates of S. aureus, (80 each of methicillin susceptible and methicillin resistant) were included in the study. Minimum inhibitory concentration (MIC) was determined by NRA and reference broth micro dilution (BMD) methods. Results of NRA were compared with BMD and analyzed. Results: For MRSA, the MIC values ranged from 4 to ≥ 16 μg/ml and for MSSA, ≤ 0.5 to 2 μg/ml. Category and essential agreement for NRA as compared with BMD were found to be 99.4 and 89.7 per cent, respectively. No minor or major discrepancy was observed. A single resistant isolate showed very major discrepancy. Interpretation & conclusions: Colorimetric NRA being an inexpensive test requiring no special equipment can be employed as an alternative method for rapid detection of MRSA in resource limited settings.

Comparison of two methods testing synergistic action of Reduning and cefoperazone sodium/sulbactam sodium on extensive drug resistant Acinetobacter bauman / 国际检验医学杂志

Objective To observe the results of broth dilution method and disc diffusion method to test the synergistic effect of Reduning and cefoperazone sodium / sulbactam sodium(SCF) on extensive drug resistant Acinetobacter bauman (XDR-AB) in vitro environment ,and compare their compliance to guide the clinical medication .Methods A total of 12 strains of XDR-AB from infec-tion patients in our hospital in 2015 were collected ,the strain was sub cultured .Firstly ,observe the minimum inhibitory concentra-tion (MIC) of SCF and Reduning on XDR-AB alone and in combination by broth dilution method .And then judge the synergy effects through calculation .Secondly ,the inhibition ring diameter and the synergy effects was detected using the disc diffusion meth-od .Results The MIC of Reduning and SCF in combination on XDR-AB was declined compared with them alone .The Fractional in-hibitory concentration of Reduning and SCF in combination on XDR-AB were equal or less than 0 .5 ,they had synergistic effect on XDR-AB .The inhibition ring diameter of Reduning was 10 mm tested by disk diffusion method .Different strains of XDR-AB on SCF bacteriostatic annulus diameter difference ,5 strains were 15 mm ,3 strains were 16 mm ,and 4 strains were 17 mm .Reduning and SCF appeared synergistic effect according to the inhibition ring diameter expanded when they effected on XDR -AB in combina-tion .Conclusion In vitro ,Reduning combined with SCF on XDR-AB has good synergistic effect .Compared with broth microdilution checkerboard dilution method ,disk diffusion method is more simple and convenient ,but it has a certain subjective on judging re-sults ,which is better to operate by experienced person .

Differences in the antimicrobial susceptibility profiles of Moraxella bovis, M. bovoculi and M. ovis

The aim of this study was to determine the differences in the antimicrobial susceptibility profiles of Moraxella bovis, M. bovoculi and M. ovis. Thirty-two strains of Moraxella spp. isolated from cattle and sheep with infectious keratoconjunctivitis were tested via broth microdilution method to determine their susceptibility to ampicillin, cefoperazone, ceftiofur, cloxacillin, enrofloxacin, florfenicol, gentamicin, neomycin, oxytetracycline and penicillin. The results demonstrated that Moraxella spp. strains could be considered sensitive for most of the antimicrobials tested in this study, but differences between the antimicrobial susceptibility profiles of these three Moraxella species were found. M. bovis might differ from other species due to the higher MIC and MBC values it presented.

Evaluation of the capabilities of disc diffusion and Vitek2-compact GN13 methods for testing antimi-crobial susceptibility and screening of ESBLs Enterobacteriaceae clinical isolates / 中华微生物学和免疫学杂志

Objective To evaluate the capabilities of disc diffusion and Vitek2-compact GN13 methods for testing antimicrobial susceptibility of screening ESBLs ( extended-spectrumβ-lactamase) in En-terobacteriaceae clinical isolates.Methods A total of 93 Enterobacteriaceae strains were isolated from pa-tients with intra-abdominal infections in 21 hospitals during 2011 to 2012.The in vitro minimum inhibition concentration ( MIC ) values of ampicillin-sulbactam, piperacillin-tazobactam, ertapenem, ceftazidime, ceftriaxone, cefepime, imipenem, amikacin, ciprofloxacin and levofloxacin were determined by disc diffu-sion, Vitek2-compact GN13 and broth microdilution methods, respectively.Categorical agreement ( CA ) rates of disc diffusion and Vitek2-compact GN13 methods were determined by using broth microdilution meth-od as the reference method.The genes encoding ESBLs were screened in Escherichia coli (E.coli), Kleb-siella pneumoniae (K.pneumonia), Klebsiella oxytoca (K.oxytoca) and Proteus mirabilis (P.mirabilis) strains by using PCR analysis and gene sequencing.Disc diffusion and Vitek2-compact GN13 methods were used for the phenotypic confirmatory test of ESBLs and the sensitivity, specificity, positive predictive value and negative predictive value of the two tests were evaluated.Results The CA values of disc diffusion and Vitek2-compact GN13 methods for the 10 antibiotics were all >90% as compared with broth microdilution method.The major error (ME) rate for ertapenem was 3.2%and the very major error (VME) rates for am- picillin-sulbactam, ceftazidime and cefepime tests were all 2.2% by using Vitek2-compact GN13 method. The sensitivity, specificity, positive predictive value and negative predictive value of disc diffusion and Vitek2-compact GN13 methods in the phenotypic confirmatory test of ESBLs were 96.7%(29/30), 100%(20/20), 100%(30/30) and 95%(19/20), respectively.Conclusion Both disc diffusion and Vitek2-compact GN13 methods could be used for testing the antimicrobial susceptibility and the detection of ESBLs in Enterobacteriaceae clinical isolates with the advantage of accuarcy.Attention should be paid to the posibil-lity of oaurance of ME and VME when testing ertapenem, ampicillin-sulbactam, ceftazidime and cefepime by using Vitek2-compact GN13 method.

Study on disk diffusion method for antimicrobial susceptibility test of Candida isolated from patients with lung cancer / 肿瘤研究与临床

Objective To compare the ROSCO disk diffusion method with broth microdilution method (CLSI, M27-A) for antimicrobial susceptibility test of Candida species isolated from patients with lung cancer. Methods Danish ROSCO company disk diffusion testing method and bio Merieux ATB FUNGUR2 were applied to test 5-flucytosine, fluconazole, itraconazole and amphotericin B antimicrobial susceptibility for 78 Candida species strains isolated from patients with lung cancer. Results Through evaluating the susceptibility to 5-flucytosine, amphotericin B, fluconazole and itraconazole by disk diffusion method, the Kappa value was 0.89. The sensitive strains detected by one method did not show resistance in another method. The sensitive rates of 78 strains of Candida species to 5-flucytosine, amphotericin B, fluconazole and itraconazole were 88.20 %, 89.17 %, 56.34 % and 52.12 %. The susceptibility of C.albicans, C.tropicalis, C.glabrata and C.krusei to four kinds of antifungal agents was 90.95 %, 85.71 %, 67.50 % and 41.67 %respectively. Conclusions Results of disk diffusion method coincide well with broth microdilution method. It can be chosen as a clinical routine method for antimicrobial susceptibility test.

Actividad antifúngica de extractos de biomasa celular de neem sobre aislamientos de dermatofitos / Antifungal activity of neem cellular biomass extracts on dermatophytes isolates

Las líneas celulares de neem (Azadirachta indica A. Juss.) cultivadas en suspensión líquida han demostrado producir metabolitos secundarios bioactivos, particularmente triterpenoides. En consecuencia, se han realizado estudios para el control de microorganismos de importancia médica, como los hongos dermatofitos. El objetivo principal de este trabajo fue evaluar a través de un método de referencia in vitro la actividad antifúngica de diferentes extractos de cultivos celulares de neem sobre varios aislamientos de Trichophyton mentagrophytes, Trichophyton rubrum y Epidermophyton floccosum. Se realizó un escalado de cultivos de suspensiones celulares de neem, a partir de los cuales se obtuvo un extracto crudo metanólico. Éste extracto fue fraccionado posteriormente por cromatografía en columna de silica gel. Con los extractos obtenidos se determinó la Concentración Mínima Inhibitoria (CMI), siguiendo el método de microdilución en caldo M38-2, con cinco aislamientos de T. mentagrophytes, cinco de T. rubrum y tres de E. floccosum. Se usó como control positivo el antimicótico Terbinafina. Los resultados mostraron que el extracto crudo de biomasa celular de neem inhibe el crecimiento hasta en 100 % de T. mentagrophytes, T. rubrum y E. floccosum. Al evaluar las fracciones por separado, se observó que las de menor polaridad exhibieron en general mayor actividad antifúngica (CMI=109 μg/mL) que el extracto crudo per se (CMI=2500 μg/ mL) y las fracciones más polares (CMI=7000 μg/mL). Lo anterior indica que las células de neem cultivadas en suspensión producen compuestos con actividad antifúngica, siendo más bioactivos los presentes en las fracciones de menor polaridad.

Cell lines of neem (Azadirachta indica A. Juss.) grown in liquid suspension have shown to produce bioactive secondary metabolites, particularly triterpenoids. In consequence, its use as a control of medical microorganisms (like dermatophytes) is proposed. The main goal of this study was to assess the antifungal activity of methanolic extracts from neem cultured cell suspensions on several isolates of Trichophyton mentagrophytes (five isolates), Trichophyton rubrum (five isolates) and Epidermophyton floccosum (three isolates). Neem cell suspension cultures were scaled up, from which a raw methanolic extract was obtained. This extract was fractionated by silica gel column chromatography. The raw methanolic extract and its fractions were used in order to determine the Minimal Inhibitory Concentration (MIC) on the dermatophytes isolates by following M38-A2 broth microdilution method. Antimycotic Terbinafine was used as positive control. The results shown that neem raw cellular biomass extract inhibits the growth of T. mentagrophytes, T. rubrum and E. floccosum in at least 100%. In the evaluations of the separated fractions, it was observed that the low polarity fractions had higher antifungal activity (MIC=109 μg/mL) than the raw extract per se (MIC=2500 μg/mL) and the most polar ones (MIC=7000 μg/mL). The latter suggest that neem cells cultured in liquid suspension produces compounds with antifungal activity, being more active those present in the low polarity fractions.

Study of the anti-MRSA activity of Rhizoma coptidis by chemical fingerprinting and broth microdilution methods / 中国天然药物

AIM@#Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes both hospital- and community-acquired infections, and for which single-drug treatments are becoming less efficient. Rhizoma coptidis has been used for more than two thousand years in China to treat diarrhea, fever, and jaundice. In this study, the anti-MRSA activity of Rhizoma coptidis is examined and its effective components sought.@*METHODS@#The mecA and norA genes were determined by PCR amplification and sequencing. Drug susceptibility of Staphylococcus aureus ATCC43300 was performed using the VITEK2 compact system. The chemical fingerprint of Rhizoma coptidis was investigated using HPLC and preparative liquid chromatography, and the anti-MRSA activity was determined using an improved broth microdilution method.@*RESULTS@#The drug susceptibility test revealed that the penicillin-binding protein phenotype of the strain changed in comparison to penicillin-sensitive Staphylococcus aureus. Ten batches of Rhizoma coptidis showed anti-MRSA activity on the norA-negative Staphylococcus aureus strain, as well as the strain that contained a norA gene. The spectrum-effect relationship revealed that the berberine alkaloids were the effective components, within which berberine, coptisine, palmatine, epiberberine, and jatrorrhizine were the major components.@*CONCLUSION@#This study lays a foundation for in vivo studies of Rhizoma coptidis and for the development of multi-component drugs.

Estudio de la susceptibilidad a los antibióticos de aislados clínicos de Mycobacterium abscessus: Reporte preliminar / Study of antibiotic susceptibility of Mycobacterium abscessus clinical isolates: Preliminary report

Mycobacterium abscessus posee una elevada resistencia a los antibióticos, lo cual limita las opciones terapéuticas para tratar las infecciones causadas por este microorganismo. En este estudio se determinó la susceptibilidad antimicrobiana de 26 cepas de M. abscessus de origen clínico frente a 14 antibióticos, mediante el método de microdilución en caldo (MDC) de acuerdo al procedimiento descrito por el CLSI (2011). Todas las cepas fueron sensibles a la amikacina, seguidas de claritromicina (62%) e imipenem (46%), mientras que el porcentaje de sensibilidad a ciprofloxacina, ampicilina/sulbactam, meropenem, ceftriaxona, amoxacilina y doxiciclina osciló entre 0 y 8%. Nueve diferentes patrones de resistencia fueron observados, representados por la asociación de 4 a 12 antibióticos. La combinación de 8 y 10 marcadores de resistencia constituyeron los patrones más frecuentes (28% cada uno). La amikacina fue el antibiótico con mayor actividad inhibitoria frente a las cepas estudiadas (0,5 - 16 µg/mL). Los patrones de resistencia observados sugieren la necesidad de utilizar las pruebas de susceptibilidad como herramientas que permitan orientar y optimizar las conductas terapéuticas en infecciones producidas por M. abscessus.

Mycobacterium abscessus has a high antibiotic resistance, which limits therapeutic options for treating infections produced by this microorganism. In this study we determined the antimicrobial susceptibility of 26 M. abscessus strain of clinical origin towards 14 antibiotics through the broth microdilution method (BMD) according to the procedure described by the CLSI (2011). All the strains were sensitive to amikacine, followed by clarithromycin (62%), and imipenem (46%), while the percentage sensitivity to ciprofloxacin, ampicillin/sulbactam, meropenem, ceftriaxone, amoxicycillin, and doxicycline varied between 0 and 8%. Nine different resistant patterns were observed, represented by the association of 4 to 12 antibiotics. The combination of 8 and 10 resistance markers constituted the most frequent patterns (28% each). Amikacin was the antibiotic with the highest inhibitory activity towards all the strains studied (0.5 - 16 µg/mL). The resistance patterns observed indicate the need of using susceptibility tests as tools that allow to guide and optimize the therapeutic approach to M. abscessus produced infections.

Combined antimicrobial susceptibility test against pan-drug-resistant Acinetobacter baumannii with E-test and microdilution checkerboard assay / 中华微生物学和免疫学杂志

Objective To evaluate the activity of antibiotics against pan-drug-resistant (PDR) Acinetobacter baumannii by combination antimicrobial susceptibility test in viro with epsilometric methods (Etest method) and microdilution checkerboard (CB method),and to detect a good correlation between timekill curve with the above mentioned two assays.Methods Thirty-one clinical isolates of PDR Acinetobacter baumannii were selected for mono and combination antimicrobial susceptibility test in vitro by E-test and CB method,then a comparison was conducted between the test results and the time-kill curve.Mono drugs involved tigecycline,colistin,imipenem and amikacin,and combinations involved two of drugs above,and three drugs involved imipenem/tigecycline,plus amikacin combination.Results Synergistic effect was detected in imipenem plus colistin and tigecycline plus imipenem combination.A high comparability was revealed between the E-test method with antimicrobial drugs added into the culture medium and the time-kill curves.Synergy in the combination of imipenem/tigecycline,plus amikacin was detected by the CB method and time-kill curves.Conclusion The results showed that the effect of specific combination of antibiotics against PDR Acinetobacter baumannii could be predicted by testing their synergistic effect with combination antimicrobial susceptibility test.

Comparison of disc and MIC reduction methods with polymerase chain reaction for the detection of metallo-β-lactamase in Pseudomonas aeruginosa.

Purpose: The present study was undertaken to evaluate the screening antibiotic, confirmatory phenotypic test and agent against PCR as gold standard and to detect the prevalent MBL gene. Materials and Methods: Three hundred and twenty-six Pseudomonas aeruginosa isolates were screened for resistance to Imipenem (IPM), Meropemem (MEM) and Ceftazidime (CAZ) by disc diffusion. Isolates resistant to any of these were considered screen test-positive for MBL and were subjected to Double disc synergy test (DDST) and Disc potentiation test (DPT: Using IPM, MEM and CAZ alone and with EDTA), Minimum inhibitory concentration (MIC) reduction [four-fold or more reduction in MIC of IPM and MEM in presence of chelators: EDTA and 1,10-phenanthroline (EPI/EPM: EDTA-phenanthroline- Imipenem/Meropenem Broth Microdilution method)] and polymerase chain reaction (PCR) for blaIMP and blaVIM . Results: Screen test-positives by MEM and CAZ were 19.3% as against 17.8% by IPM. MEMDDST, DPT and EPM confirmed 100% screen-test positives as against 93.7% by CAZ DDST and DPT-2, 76.2% by CAZ DPT-1, 88.9% by IPM DDST, 85.7% by IPM DPT-1 and 92.1% by EPI. IPMand CAZ DDST together confirmed 100% while IPM and CAZ DPT-2 confirmed 96.8%. All 63 screen-test positives showed the presence of blaVIM . Conclusions: MEM was found to be the best screening and confirmatory agent for MBL detection and blaVIM was found to be the prevalent MBL gene in this part of the country.

Comparison of the activities of amphotericin B, itraconazole, and voriconazole against clinical and environmental isolates of Aspergillus species.

Background: Invasive fungal infections are a significant cause of morbidity and mortality in immunocompromised populations. Aims: To evaluate the susceptibility pattern of our isolates against amphotericin B, itraconazole, and voriconazole and to compare the antifungal activities of these agents with each other against the Aspergillus species tested. Settings and Design: A prospective study was designed to include clinical and environmental isolates of Aspergillus species. Materials and Methods: 420 sputum samples, 70 bronchoalveolar lavage fluids, 160 oral washings, and 47 environmental samples were collected. Direct microscopy by potassium hydroxide and lactophenol cotton blue mounts followed by culture on Sabourad`s dextrose agar (SDA) was done. Susceptibility testing was performed by the broth microdilution technique as per Clinical Laboratory Standards Institute standards (M-38A). Additionally, all the isolates were also tested by the colorimetric microdilution technique using Alamar Blue dye. Statistical Analysis: It was done by the Chi-square test and Z-test using SPSS statistical software version 12.0. Results and Conclusion: Twenty-seven isolates (47.3%) were recovered from patients with chronic bronchial asthma followed by fibrocavitary pulmonary tuberculosis in 9 (15.7%), allergic bronchopulmonary aspergillosis (ABPA) in 6 cases (10.5%), bronchiectasis in 3 (5.2%), bronchogenic carcinoma in 5 (8.7%) and those receiving radiotherapy for head and neck cancer 7 (12.2%). Thirteen environmental isolates were also included in the study. The most common isolate was A. fumigatus 28 (40%), followed by A. niger 22 (31%), A. flavus 13 (19%), and A. terreus 7(10%). All isolates were susceptible to amphotericin B, itraconazole, and voriconazole. Among the three agents tested, voriconazole exhibited lowest MICs (≤1 μg/ml) against all Aspergillus species.