Isolation and identification of exosomes from mouse primary peritoneal macrophages / 中国生物制品学杂志

@#Objective To isolate,purify and identify exosomes secreted by mouse primary peritoneal macrophages.Methods Five male C57BL/6 mice were intraperitoneally injected with 3% mercaptoacetate broth respectively,and the primary peritoneal macrophages were obtained by lavage,and then the purity was analyzed by flow cytometry.The exosomes of mouse primary peritoneal macrophages were extracted by ExoQuick TC exosome kit,which were measured for the protein content with BCA kit,observed for the morphology by transmission electron microscopy,detected for the particle size and distribution with nanoparticle tracking analyzer,and determined for the expression of exosome-specific markers(CD9,CD63 and TSG101) by Western blot.Results About 5 × 10~6 peritoneal macrophages with the purity of(99.17±0.65)%were obtained from each mouse.Approximately 869 μg of exosomal protein was extracted from 5 mL of mouse primary peritoneal macrophage culture supernatant.The exosomes of mouse primary peritoneal macrophages were typical tea saucerlike vesicles with strong refraction under electron microscopy,and highly expressed the exosome-specific markers TSG101,CD63 and CD9.The particle size distribution was concentrated between 100 and 200 nm,with an average particle size of175.2 nm.Conclusion Intraperitoneal injection of mercaptoacetate broth can improve the yield of mouse primary peritoneal macrophages.ExoQuick TC.exosome kit can extract sufficient amount of exosomes with high purity from mouse primary peritoneal macrophages.

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.

Assessment of colistin resistance in Gram negative bacteria from clinical samples in resource-limited settings

Objective: To find a proper method to assess colistin resistance in multidrug resistant Gram negative bacteria (MDR-GNB) on a routine basis in resource limited settings. Methods: Clinical samples were processed. MDR-GNB were identified and were examined for colistin resistance by colistin broth elution method, colistin agar method, and colistin disk elution screening method. Broth microdilution method was used the gold standard. Results: A total of 10 235 clinical samples were processed, in which 857 (8.4%) MDR-GNB were identified. The very significant errors, categorical agreement, major errors, positive predictive values, negative predictive values, specificity and sensitivity of all the phenotypic methods were 5.5%, 0%, 94.4%, 100%, 99.6%, 100% and 94.4%, respectively for the detection of colistin resistance. The colistin elution screening method was cheap and easy to perform with similar results to broth microdilution method. Conclusions: All the evaluation methods for colistin resistance showed similar results. So the laboratories can choose any method for detection of colistin resistance. However, we recommend colistin disk elution screening method because, it is easy and cheap and can be performed in limited resources.

Comparison of well diffusion, disc diffusion and broth dilution methods for antimicrobial activity of Andrographis paniculata herbal gel against acne-associated pathogen

Aims@#Acne is a common skin disease among teenagers and also affects other ages. It occurs when the oil and dead skin cells plug into the hair follicles and causing pimples or whitehead. Although antibiotics have been used for many years in treating acne, the widespread use of it has led to the development of bacterial resistant, which resulted in unsuccessful treatment. Thus, in this study, Andrographis paniculata (AP) herbal formulation gel is proposed in order to determine its effectiveness in treating acne. Three different methodologies were used to compare the antimicrobial effect of A. paniculata herbal gel against acne-associated pathogens. @*Methodology and results@#Well diffusion, disc diffusion and broth dilution methods were applied to evaluate the antimicrobial effect of AP herbal gel at concentrations of 1.5% (w/w), 2.5% (w/w) and 5.0% (w/w) onto selected pathogens associated with acne which consisted of Staphylococcus epidermidis, Staphylococcus aureus, Propionibacterium acnes and Candida albicans. Among the three methods, broth dilution showed the best antimicrobial effect towards all microorganisms used. AP herbal gel at concentration 2.5% (w/w) showed the optimum antimicrobial effect of S. aureus and C. albicans, while 5.0% (w/w) exhibited the best antimicrobial activities for P. acnes and S. epidermidis. @*Conclusion, significance and impact of study@#Broth dilution method appears to be a reliable method for the determination of antimicrobial effects for the pathogens tested. In addition, AP herbal formulation gel has great potential to treat acne effectively.

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.

Enhanced production of prodigiosin by Serratia marcescens FZSF02 in the form of pigment pellets

Background: Prodigiosin has been demonstrated to be an important candidate in investigating anticancer drugs and in many other applications in recent years. However, industrial production of prodigiosin has not been achieved. In this study, we found a prodigiosin-producing strain, Serratia marcescens FZSF02, and its fermentation strategies were studied to achieve the maximum yield of prodigiosin. Results: When the culture medium consisted of 16.97 g/L of peanut powder, 16.02 g/L of beef extract, and 11.29 mL/L of olive oil, prodigiosin reached a yield of 13.622 ± 236 mg/L after culturing at 26 °C for 72 h. Furthermore, when 10 mL/L olive oil was added to the fermentation broth at the 24th hour of fermentation, the maximum prodigiosin production of 15,420.9 mg/L was obtained, which was 9.3-fold higher than the initial level before medium optimization. More than 60% of the prodigiosin produced with this optimized fermentation strategy was in the form of pigment pellets. To the best of our knowledge, this is the first report on this phenomenon of pigment pellet formation, which made it much easier to extract prodigiosin at low cost. Prodigiosin was then purified and identified by absorption spectroscopy, HPLC, and LCMS. Purified prodigiosin obtained in this study showed anticancer activity in separate experiments on several human cell cultures: A549, K562, HL60, HepG2, and HCT116. Conclusions: This is a promising strain for producing prodigiosin. The prodigiosin has potential in anticancer medicine studies.

Characterization of biofilm formation by Escherichia coli: An in vitro study

Objective: The purpose of the present study was to characterize the effects of medium, optical density (OD), andincubation time on biofilm formation by Escherichia coli in brain heart infusion (BHI) and Luria-Bertani broth(LB). Methods: The main procedure involved fixing the bacterial film with 95% ethanol, staining with 0.1% crystalviolet, releasing the bound dye with 33% glacial acetic acid, and measuring the OD of the solution at 570 nm using amicroplate reader. Results: It was found that 3 and 5 days of incubation are critical for biofilm formation as indicatedby the OD values of 0.55–0.35 and 0.70–0.39 in BHI and LB, respectively, at OD 0.05. Similarly, pattern in resultswas noted for OD 0.1 in both media BHI and LB. Conclusion: It is confirmed that 3 days (72 h) are required forobtaining effective biofilm formation in both BHI and LB at 37°C at OD 0.05 and 0.1.

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.

Preliminary study on antibacterial activity of artemisinin and its derivatives / 中国中药杂志

In this study,in order to detect the antimicrobial activity of artemisinin and its derivatives artesunate and dihydroartemisinin,two methods including broth dilution and plate punching method were used to detect the antibacterial activity against gram-negative bacteria(Escherichia coli)and gram-positive bacteria(Staphylococcus aureus)of artemisinin,dihydroartemisinin and artesunate at various concentrations within 5 mmol·L~(-1)and at four time points(8,16,24,32 h).Two antibacterial positive drugs,streptomycin against E.coli and penicillin against S.aureus,were used as positive controls.Plate punching method showed that,unlike the results of 5 mmol·L~(-1)dihydroartemisinin or artesunate,no inhibition zone was detected at the same concentration of artemisinin after 24 h-treatment against E.coli.Broth dilution method showed that,the antibacterial activity of dihydroartemisinin against E.coli.was stronger than those of both artesunate and artemisinin;IC_(50)at24 h-treatment was 155.9μmol·L~(-1)for dihydroartemisinin,370.0μmol·L~(-1)for artesunate and none for artemisinin.Interestingly,dihydroartemisinin and artesunate showed the strongest antibacterial activity between 16-24 h,while artemisinin showed relatively stronger antibacterial activity between 8-16 h.Dihydroartermisinin showed no antibacterial activity against S.aureus.Above all,the antibacterial activity of artemisinins against E.coli is dihydroartemisinin>artesunate>artemisinin.Artemisinin and its derivatives have showed different antibacterial kinetics,and no antibacterial activity against S.aureus.has been detected with dihydroartemisinin.

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.

Research on New Experimental Scheme of Mycoplasma Solid Culture Combined with Liquid Culture / 现代检验医学杂志

Objective To investigate the best experimental scheme of Mycoplasma solid culture combined with liquid culture.Methods A total of 961 samples of urogenital tract excretion were collected from March 2016 to June 2016.Both solid culture and liquid culture were performed for detection of Mycoplasma,false positive broths were picked out after 48 h,20 μl of each one was transformed to solid media for subculture,final results were read after 48 h.The experimental data was analyzed to find an optimal combination culture scheme.Results The positive rate of solid culture was 38.7％ (372/961) for UU and 7.8％ (75/961) for MH.The positive rate of liquid culture was 45.27％ (435/961) for UU and 7.08％ (68/961) for MH.The different rate between both methods was 9.47％ (91/961) for UU (x2 =43.61,P＜0.005),and 3.02％ (29/ 961) for MH (x2 =1.24,P＞ 0.05).The different rate was 53.5 ％ (46/86) between primary solid culture and subculture.The positive rate of total (primary solid culture and subculture) solid culture was 41.9％ (403/961) for UU and 8.6％ (83/961) for M H,which produced higher sensitivity than single (primary solid culture or subculture) solid culture.The different rate between the total solid culture and the liquid method was 6.24 ％ (60/961) for UU (x2 =17.067,P＜0.05),and 3.43 ％ (33/961) for MH (x2=5.94,P＜0.05).Conclusion Perform both solid culture and liquid culture for Mycoplasma,pick out those false positive broths for subculture.Total solid culture could be more reliable as final result,for which could combine specificity of solid culture with sensitivity of liquid culture.

Synthesis and antifungal activities of N-1,3,4-thiadiazol-2-yl-4-oxo-thiochroman-2-yl-formamide derivatives / 药学学报

Thiochromanones and 1,3,4-thiadazoles as heterocyclic compounds have broad biological activities. In order to find novel compounds with antifungal bioactivity, substituted thiophenol and maleic anhydride were used to synthesize the intermediate 4-oxothiochromane-2-carboxylic acid. It was reacted with 2-amino-1,3,4-thiadiazol to get fourteen target compounds containing 1,3,4-thiadazole moiety. The structures of the obtained compounds were confirmed by 1H NMR, 13C NMR and HR-MS. All compounds were investigated for antifungal activity via microdilution broth method. The results showed that the target compounds 3a and 3c to Epidermophyton floccosum and Mucor racemosus exhibited better antifungal activity than the positive control fluconazole, in which the minimum inhibition concentration can reach 8 μg·mL-1 and 16 μg·mL-1. Compound 3e showed significant inhibitory activity to Helminthosporium maydis, Sclerotinia sclerotiorum and Botrytis cinerea compared with that of the positive control carbendazim. Compound 3b exhibited inhibitory activity to Helminthosporium maydis better than the positive control carbendazim.

Drug susceptibility of rapid and slow growing non‑tuberculous mycobacteria isolated from symptomatics for pulmonary tuberculosis, Central India.

Background: Non‑tuberculous mycobacteria (NTM) are emerging as important pathogens. Their treatment also differs from that of Mycobacterium tuberculosis. In India, any datum on them is scarce as species identification and drug susceptibility are not performed in most laboratories. Susceptibility also differs from one geographic area to another, and in our country, there are no data even to guide the clinicians to start treatment empirically. Methodology: The present study endeavours to generate drug susceptibility data on NTM isolated from sputum samples collected and stored from 6445 symptomatics for pulmonary tuberculosis during a prevalence survey and from specimens received from the hospital. Isolates were not necessarily associated with the disease. Species were identified and antibiotic susceptibility was performed using micro‑broth dilution technique as per the standard Clinical and Laboratory Standards Institute guidelines. Results: A total of 65 NTM with 11 species were identified, of which 27 belonged to Mycobacterium fortuitum complex, 14 Mycobacterium gordonae, 9 Mycobacterium avium, 7 Mycobacterium flavescens, 4 Mycobacterium scrofulaceum and one each of others. Sensitivity to amikacin for M. fortuitum was 95.22% (20 out of 21), followed by ciprofloxacin (76.19%) and clarithromycin (71.42%). All the 9 M. avium isolates, 11 of M. gordonae (78.57%), 5 of M. flavescens and 2 of M. scrofulaceum were sensitive to clarithromycin. All NTM were resistant to first‑line antitubercular drugs except 8, which were sensitive to streptomycin. Conclusions: Drug sensitivity of NTM varies from species to species. While amikacin was the best for rapidly growing mycobacteria, clarithromycin was the most active drug against M. avium and other slow growers.

To Evaluate The Antimicrobial Efficacy Of Green Coffee Bean Extract On Periopathogens- A Clinico-microbiological Study.

Aim - To evaluate the Antibacterial efficacy of three different dilution of green coffee bean extract on periopathogens. Objective- To achieve a dilution of green coffee bean extract which has the maximum inhibitory effect on P. gingivalis and A. actinomycetocomitans. Methods- The sterilised blood agar culture plate was prepared on which colonies of P. gingivalis and A. actinomycetocomitans were cultured by subgingival sample taken from Chronic Generalized Periodontitis cases. Three dilution of green coffee bean extract were prepared i.e 10 -8, 10 -9 and 10 -10 with the serial dilution method using Distilled water. Then streaking of colonies was done on three different areas of culture plate on which different dilution was added. Minimum inhibitory concentration (MIC) on culture plates was observed to see the inhibitory effect of Green Coffee Bean extract on periopathogens by Agar Diffusion Method.Result- 10-9 was found have maximum inhibitory effect on periopathogens especially P.Gingivalis Conclusion- P.Gingivalis is more susceptible to 10-9 concentration of Green Coffee Bean Extract [Sachin M NJIRM 2016; 7(5):56-59]

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.