broth microdilution
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The broth microdilution (BMD) method is widely used for the determination of minimum inhibitory concentrations of antimicrobial agents, including volatile oils and their components. In this series of various experiments, we have demonstrated the influence of thymoquinone (TQ) vapor on the results of the BMD test performed with Staphylococcus aureus as a model organism. The spread of vapor from the TQ containing wells (32-512 μg/mL) caused the complete inhibition of staphylococcal growth in adjoining wells initially containing bacterium-inoculated pure Mueller-Hinton broth only and thus produced false positive results of the test. The ability of TQ to pass into the adjoined wells was subsequently confirmed by gas chromatography-mass spectrometry, whereas TQ at concentrations up to 84 μg/mL was detected in these wells after five hours. Based on these results, we suppose that vapors of TQ as well as of other naturally occurring volatile compounds and their mixtures (for example essential oils and plant extracts) can significantly influence results of the standard BMD assay. These observations, therefore, call for development of new appropriate BMD method suitable for assessment of antimicrobial activity of volatile substances.
- MeSH
- antiinfekční látky chemie farmakologie MeSH
- benzochinony chemie farmakologie MeSH
- mikrobiální testy citlivosti metody MeSH
- oleje prchavé chemie farmakologie MeSH
- oleje rostlin chemie farmakologie MeSH
- oxacilin chemie farmakologie MeSH
- Staphylococcus aureus účinky léků růst a vývoj MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
With aim to develop effective proof-of-concept approach which can be used in a development of new preparations for the inhalation therapy, we designed a new screening method for simple and rapid simultaneous determination of antibacterial potential of plant volatiles in the liquid and the vapour phase at different concentrations. In addition, EVA (ethylene vinyl acetate) capmat™ as vapour barrier cover was used as reliable modification of thiazolyl blue tetrazolium bromide (MTT) assay for cytotoxicity testing of volatiles on microtiter plates. Antibacterial activity of carvacrol, cinnamaldehyde, eugenol, 8-hydroxyquinoline, thymol and thymoquinone was determined against Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae using new broth microdilution volatilization method. The cytotoxicity of these compounds was evaluated using MTT test in lung fibroblast cells MRC-5. The most effective antibacterial agents were 8-hydroxyquinoline and thymoquinone with the lowest minimum inhibitory concentrations (MICs) ranging from 2 to 128μg/mL, but they also possessed the highest toxicity in lung cell lines with half maximal inhibitory concentration (IC50) values 0.86-2.95μg/mL. The lowest cytotoxicity effect was identified for eugenol with IC50 295.71μg/mL, however this compound produced only weak antibacterial potency with MICs 512-1024μg/mL. The results demonstrate validity of our novel broth microdilution volatilization method, which allows cost and labour effective high-throughput antimicrobial screening of volatile agents without need of special apparatus. In our opinion, this assay can also potentially be used for development of various medicinal, agricultural, and food applications that are based on volatile antimicrobials.
- MeSH
- akrolein analogy a deriváty chemie MeSH
- antibakteriální látky chemie MeSH
- benzochinony chemie MeSH
- buněčné linie MeSH
- eugenol chemie MeSH
- fytonutrienty chemie MeSH
- Haemophilus influenzae účinky léků MeSH
- lidé MeSH
- mikrobiální testy citlivosti metody MeSH
- monoterpeny chemie MeSH
- oxychinolin chemie MeSH
- Staphylococcus aureus účinky léků MeSH
- Streptococcus pneumoniae účinky léků MeSH
- těkavé organické sloučeniny chemie MeSH
- tetrazoliové soli MeSH
- thiazoly MeSH
- thymol chemie MeSH
- volatilizace * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Carvacrol and thymol, both plant-derived volatile compounds, have extensively been studied individually as well as in combination with other agents for their antimicrobial activity in liquid phase. However, in contrast to well-established assays for testing of antimicrobial combinatory effects in liquid media, there are no standardized methods for evaluation of interactions between volatile compounds in vapour phase. The objective of this study was to verify new broth volatilization chequerboard method by testing the combination of carvacrol and thymol and to determine in vitro inhibitory effect of these compounds in liquid and vapour phase against twelve Staphylococcus aureus strains. The new method, based on combination of standard microdilution chequerboard and new broth volatilization tests allowing calculation of fractional inhibitory concentrations (FICs), was used. Combination of carvacrol and thymol produced the additive antimicrobial effect against all strains tested. In several cases, they reached ΣFIC values lower than 0.6, which can be considered as a strong additive interaction. The best result was found in vapour phase against one standard strain at combination of 128 μg/mL of carvacrol and 16-256 μg/mL of thymol (ΣFIC = 0.51) and in liquid phase against one clinical isolate at combination of 256 μg/mL of carvacrol and 256 μg/mL of thymol (ΣFIC = 0.53). The study verified that the new technique is suitable for simple and rapid high-throughput combinatory antimicrobial screening of volatile compounds simultaneously in vapour and liquid phase and that it allows determination and comparison of MIC and FIC values in both, liquid and solid media.
The susceptibility of 25 Stenotrophomonas maltophilia (S. maltophilia) clinical isolates to four different antimicrobials (trimethoprim/sulfomethoxazole, piperacillin/tazobactam, ceftazidime, ciprofloxacin) were investigated by disk diffusion, E-test and commercial Sensititre and PASCO broth microdilution techniques. Discrepancies between the results of broth microdilution and the other methods studied were characterized as very major, major and minor errors. Using the broth microdilution as the reference method, 24% of the isolates were found susceptible to trimethoprim/sulfamethoxazole, 20% to ceftazidime, 0% to piperacillin/tazobactam and 12% to ciprofloxacin. Good correlation was observed between the two broth microdilution Sensititre and PASCO for all antibiotics tested. Disc diffusion and E-test generated inconsistent results for all agents except trimethoprim/sulfamethoxazole. A great genomic diversity was demonstrated within the S. maltophilia strains tested. Although our results confirm that trimethoprim-sulfamethoxazole had some in vitro activity against S. maltophilia, further clinical studies are necessary to evaluate the clinical efficacy of these compounds for the treatment of S. maltophilia infections, since no randomized controlled trials have been carried out and no correlation between the clinical response and susceptibility testing results has been reported. Furthermore, the high genomic diversity observed in the S. maltophilia strains indicates the need for careful epidemiological evaluation especially in nosocomial outbreaks.
- Klíčová slova
- E-test, disk diffusion, broth microdilution, epidemiological analysis,
- MeSH
- gramnegativní bakteriální infekce farmakoterapie MeSH
- kombinace léků trimethoprim a sulfamethoxazol farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti metody MeSH
- pulzní gelová elektroforéza MeSH
- Stenotrophomonas maltophilia genetika klasifikace účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
The in vitro biological activity of N-benzylsalicylthioamides was evaluated against eight fungal strains by the broth microdilution method and the results were compared with those obtained with fluconazole. The compounds exhibited an in vitro antifungal activity against the fluconazole-susceptible as well as the fluconazole-resistant fungal strains. The biological activity was analyzed by quantitative structure-activity relationship (QSAR).
BACKGROUND: Colistin belongs to the last-resort antibiotics. The discovery of plasmid-bound colistin resistance mediated by the mcr-gene(s) is of great concern because, given its biological potential, there is a risk of its rapid spread. OBJECTIVES: To discuss the current literature on the methods for the screening for mcr-mediated resistance to colistin. SOURCES: Literature was drawn from a search of PubMed from 1 January 2016 to 26 April 2021. CONTENT: The selective culture-based or culture-independent approach can be used for the screening of mcr-mediated resistance to colistin in clinical samples. Rapid Polymyxin NP, Colistin Drop or Colistin Agar Spot tests are applicable for the selection of isolates with a suspected resistance to colistin that has to be confirmed by broth microdilution. The mcr-mediated resistance to colistin can be confirmed by the detection of the causal gene(s) or by phenotype using EDTA-colistin broth disc elution; production of the MCR-1 enzyme can be confirmed with lateral flow immunoassay, using matrix-assisted laser desorption/ionization time-of flight or liquid chromatography-based mass spectrometry. Whole-genome sequencing (WGS) is the ultimate typing method. When a WGS platform is not available at a healthcare facility, a WGS-outsourced service, in combination with freely available bioinformatics tools, allows for the characterization of the mcr-gene(s) carrying isolates. IMPLICATIONS: mcr-mediated colistin resistance should be monitored through active targeted screening. The broth microdilution method is required for colistin susceptibility testing but as only a selected number of clinical isolates are tested, colistin resistance, including mcr-mediated, may remain undetected. In mcr-1-positive Escherichia coli isolates, the MIC to colistin can range from 2 to 8 mg/L, so it is proposed that Enterobacterales with a colistin MIC of 2 mg/L should also be included in the mcr-mediated colistin resistance screening and those with a confirmed mcr-genotype and/or MCR-phenotype should be considered to be colistin-resistant.
The in-vitro biological activity of N-benzylsalicylthioamides against 8 bacterial strains was determined by broth microdilution method; results were compared with those obtained with neomycin, penicillin G, ciprofloxacin and penicillin V. The compounds showed moderate to high activity against G(+) bacteria; especially compounds 4, 6, 13, 16-21 and 24 exhibited comparable or higher activity than reference drugs. The antibacterial activity was analyzed by quantitative structure-activity relationship (QSAR). The antibacterial activity increased with lipophilicity, with the presence of halogens and with increasing value of Hammet substituent constant ?.
Staphylococcus xylosus, Staphylococcus equorum, and Staphylococcus epidermidis strains were isolated from Bryndza cheese and identified using PCR method. The antimicrobial susceptibility of these strains was assessed using disc diffusion method and broth microdilution method. The highest percentage of resistance was detected for ampicillin and oxacillin, and in contrary, isolates were susceptible or intermediate resistant to ciprofloxacin and chloramphenicol. Fourteen of the S. xylosus isolates (45%) and eleven of the S. equorum isolates (41%) exhibited multidrug resistance. None of the S. epidermidis isolate was multiresistant. The phenotypic resistance to oxacillin was verified by PCR amplification of the gene mecA.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence * MeSH
- mikrobiální testy citlivosti MeSH
- potravinářská mikrobiologie * MeSH
- rezistence na methicilin MeSH
- Staphylococcus klasifikace účinky léků izolace a purifikace MeSH
- sýr mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
During our investigation on the endophytic fungi of Azadirachta indica, the strain YM 311593 was obtained from the fruit of the plant. The culture extract of the strain showed antifungal activities against four phytopathogenic fungi. Based on the morphological features and phylogenetic definition, the strain YM 311593 was identified as Paraconiothyrium sp. Four xanthones and one anthraquinone were obtained from the extract of the fermentation broth of the strain. They were characterized to be globosuxanthone A (1), vertixanthone (2), hydroxyvertixanthone (3), 3,8-dihydroxy-1-methy1-9H- xanthen-9-one (4), and danthron (5), respectively, by spectroscopic elucidation. Furthermore, the absolute configuration of 1 was deduced by X-ray diffraction analysis. Besides, compound 4 was firstly found from natural sources. The antifungal activities of compounds 1-5 towards four phytopathogens were assayed using broth microdilution method. Among them, globosuxanthone A (1) showed obvious antifungal activity towards Fusarium graminearum, Fusarium solani, and Botrytis cinerea with MIC values of 4, 8, and 16 μg/mL, respectively.
- MeSH
- Ascomycota chemie izolace a purifikace MeSH
- Azadirachta mikrobiologie MeSH
- endofyty chemie izolace a purifikace MeSH
- fylogeneze MeSH
- houby účinky léků MeSH
- kultivační média chemie MeSH
- ovoce mikrobiologie MeSH
- průmyslové fungicidy izolace a purifikace farmakologie MeSH
- xantony izolace a purifikace farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
