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
- Anti-Infective Agents chemistry pharmacology MeSH
- Benzoquinones chemistry pharmacology MeSH
- Microbial Sensitivity Tests methods MeSH
- Oils, Volatile chemistry pharmacology MeSH
- Plant Oils chemistry pharmacology MeSH
- Oxacillin chemistry pharmacology MeSH
- Staphylococcus aureus drug effects growth & development MeSH
- Dose-Response Relationship, Drug MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Acrolein analogs & derivatives chemistry MeSH
- Anti-Bacterial Agents chemistry MeSH
- Benzoquinones chemistry MeSH
- Cell Line MeSH
- Eugenol chemistry MeSH
- Phytochemicals chemistry MeSH
- Haemophilus influenzae drug effects MeSH
- Humans MeSH
- Microbial Sensitivity Tests methods MeSH
- Monoterpenes chemistry MeSH
- Oxyquinoline chemistry MeSH
- Staphylococcus aureus drug effects MeSH
- Streptococcus pneumoniae drug effects MeSH
- Volatile Organic Compounds chemistry MeSH
- Tetrazolium Salts MeSH
- Thiazoles MeSH
- Thymol chemistry MeSH
- Volatilization * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Antifungal drug resistance in dermatophytes was first reported shortly after the turn of the millennium and has today been reported in Trichophyton and occasionally in Microsporum, but not in Epidermophyton species. Although drug resistance in dermatophytes is not routinely investigated, resistance in Trichophyton spp. is increasingly reported worldwide. The highest rates are observed in India (36% and 68% for terbinafine (MIC ≥4 mg/L) and fluconazole (MICs ≥16 mg/L), respectively), and apparently involve the spread of a unique clade related to the Trichophyton mentagrophytes/Trichophyton interdigitale complex. OBJECTIVES: The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has released a new method (E.Def 11.0) for antifungal susceptibility testing against microconidia-forming dermatophytes including tentative MIC ranges for quality control strains and tentative breakpoints against Trichophyton rubrum and T. interdigitale. Here, the details of the new procedure E.Def 11.0 are described. SOURCES: This technical note is based on the multicentre validation of the EUCAST dermatophyte antifungal susceptibility testing method, the mould testing method (E.Def 9.3.2) and the updated quality control tables for antifungal susceptibility testing document, v 5.0 (available on the EUCAST website). CONTENTS: The method is based on the EUCAST microdilution method for moulds but significant differences include: (a) an altered test medium selective for dermatophytes; (b) an altered incubation time and temperature; and (c) a different end-point criterion (spectrophotometric determination) of fungal growth. It can easily be implemented in laboratories already performing EUCAST microdilution methods and has been validated for terbinafine, voriconazole, itraconazole and amorolfine against T. rubrum and T. interdigitale. IMPLICATIONS: This standardized procedure with automated end-point reading will allow broader implementation of susceptibility testing of dermatophytes and so facilitate earlier appropriate therapy. This is important, as resistance is rapidly emerging and largely underdiagnosed.
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.
- Keywords
- E-test, disk diffusion, broth microdilution, epidemiological analysis,
- MeSH
- Gram-Negative Bacterial Infections drug therapy MeSH
- Trimethoprim, Sulfamethoxazole Drug Combination pharmacology MeSH
- Humans MeSH
- Microbial Sensitivity Tests methods MeSH
- Electrophoresis, Gel, Pulsed-Field MeSH
- Stenotrophomonas maltophilia genetics classification drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Comparative Study MeSH
Cíl práce: Zjistit frekvenci výskytu druhů Campylobacter spp. izolovaných od lidí na území České republiky a standardní metodou EUCAST vyšetřit citlivost k antibiotikům, nejčastěji používaným k léčbě kampylobakterióz. Materiál a metody: Konsekutivní izoláty kampylobakterů, izolované v průběhu jednoho měsíce roku 2013 z klinických vzorků pacientů ve 49 mikrobiologických laboratořích, byly identifikovány metodou MALDI-TOF MS. Citlivost k erytromycinu, ciprofloxacinu a tetracyklinu byla vyšetřena diluční mikrometodou a výsledky byly interpretovány podle klinických breakpointů EUCAST pro rozlišení citlivých a rezistentních kmenů kampylobakterů. Výsledky: Soubor 769 kmenů Campylobacter spp. zahrnoval 90,1 % C. jejuni, 9,8 % C. coli a jediný kmen C. fetus (0,1 %). S výjimkou jednoho izolátu C. jejuni z krve byly všechny ostatní ze stolice. K ciprofloxacinu (MIC > 0,5 mg/l) bylo rezistentních 61,9 % kmenů C. jejuni a 72,0 % kmenů C. coli, k tetracyklinu (MIC > 2 mg/l) bylo shodně rezistentních 32,0 % kmenů obou druhů, a k erytromycinu bylo rezistentních 0,3 % kmenů C. jejuni (MIC > 8 mg/l) a 2,7 % kmenů C. coli (MIC > 4 mg/l). Jeden kmen C. coli byl multirezistentní (rezistentní ke všem třem antibiotikům). Závěry: Přesto, že většina humánních infekcí způsobených kampylobaktery je samoúzdravná, je znalost stavu rezistence k antibiotikům volby a jejich alternativám nezbytná pro léčbu případů se závažným průběhem, které vyžadují podávání antibiotik. Frekvence výskytu rezistence k makrolidům, zjištěná v této studii, byla nízká u kmenů C. jejuni (0,1 %) i C. coli (2,7 %). Znepokojivá je však rezistence k ciprofloxacinu, potvrzená u 61,9 % kmenů C. jejuni a 72,0 % kmenů C. coli. Vzhledem k tomu, že u druhu C. coli je frekvence výskytu antibiotické rezistence vyšší než u druhu C. jejuni, a ciprofloxacin s dalšími fluorochinolony slouží jako častá volba pro léčbu závažných alimentárních i celkových infekcí, je pro volbu účinné léčby nutno i v rutinní laboratoři identifikovat kampylobaktery do druhu a vyšetřovat jejich citlivost k relevantním antibiotikům validní a reprodukovatelnou metodou.
Study aim: To determine the frequency of Campylobacter spp. isolated from humans in the Czech Republic and to test their susceptibility to antimicrobials commonly used to treat campylobacteriosis by the standard EUCAST method. Material and methods: Consecutive Campylobacter isolates recovered from clinical specimens in 49 microbiological laboratories within one month in 2013 were identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Susceptibility to erythromycin, ciprofloxacin, and tetracycline was tested by the microdilution method and the results were interpreted based on the EUCAST clinical breakpoints to differentiate between susceptible and resistant strains. Results: Of the study set of 769 Campylobacter spp. strains, 90.1 % were assigned to C. jejuni, 9.8 % to C. coli, and a single strain to C. fetus (0.1 %). Except one blood isolate of C. jejuni, all other isolates were recovered from the stool. Ciprofloxacin resistance (MIC > 0.5 mg/l) was detected in 61.9 % strains of C. jejuni and in 72.0 % strains of C. coli, tetracycline resistance (MIC > 2 mg/l) was detected in 32.0 % of strains of both species, and erythromycin resistance was found in 0.3 % of strains of C. jejuni (MIC > 8 mg/l) and in 2.7 % of strains of C. coli (MIC > 4 mg/l). A C. coli strain was multidrug resistant (i.e. resistant to all three antimicrobials tested). Conclusions: Despite the fact that most Campylobacter infections in humans cure on their own, the resistance of the causative strains to the antimicrobials of choice and alternative agents needs to be studied because of its relevance to the treatment of severe cases that require antibiotics. Resistance to macrolides was found rather infrequently in this study in both C. jejuni (0.1 %) and C. coli (2.7 %) strains. Nevertheless, alarming is ciprofloxacin resistance confirmed in 61.9 % of C. jejuni strains and 72.0 % C. coli strains. As the species C. coli is more often resistant to antimicrobials than C. jejuni and ciprofloxacin along with other fluoroquinolones is commonly used to treat severe food-borne and generalized infections, it is crucial to identify the Campylobacter strains to the species level and to test their susceptibility to relevant antibiotics by a valid and reproducible method to be able to provide an effective therapy.
- MeSH
- Bacterial Infections microbiology MeSH
- Drug Resistance, Bacterial * MeSH
- Campylobacter coli genetics isolation & purification pathogenicity drug effects MeSH
- Campylobacter jejuni genetics isolation & purification pathogenicity drug effects MeSH
- Campylobacter * genetics isolation & purification pathogenicity drug effects MeSH
- Ciprofloxacin MeSH
- Enteritis etiology microbiology MeSH
- Erythromycin MeSH
- Campylobacter Infections * epidemiology etiology drug therapy MeSH
- Laboratories MeSH
- Humans MeSH
- Microbial Sensitivity Tests * methods utilization MeSH
- Tetracycline Resistance MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Geographicals
- Czech Republic MeSH
The results of the antimicrobial susceptibility testing of clinical isolates Streptococcus suis to amoxicillin and marbofloxacin obtained by the agar dilution method and broth microdilution method with the results obtained by the commercially available E-test were compared. Comparisons between the methods based on the determination of the minimal inhibitory concentration (MIC) of the antimicrobials were assessed based on the degree and frequency of the categorical agreement (Agar dilution method as a reference system) and the percentage of the categorical agreement and error rate. A statistical evaluation was determined using the Bland-Atman method. The presented MIC values, determined for the isolates in the E-test, were slightly different from the MIC values determined by the dilution tests, mainly due to the different defined testing concentrations. For the E-test as the test system and agar-dilution method as the reference system, no error of any class was detected (very major, major and minor error) and a complete categorical agreement was obtained between the evaluated methods for amoxicillin. For amoxicillin, the regression and correlation analysis show linear relationships between the E-test and the two dilution methods with significant coefficients of determination (0.62 and 0.75). The slopes of the equality and regression lines were not significantly different. However, the E-test tends to slightly overestimate the MIC values when compared to the microdilution. The reverse is true when compared with the agar dilution. There was good agreement between the E-test and the dilution methods with a low bias (0.001 3 and −0.005 0), all the experimental data were within the computed limits of agreement. For marbofloxacin, the same trends were observed with lower coefficients of determination (0.42 and 0.73) and a less favourable agreement. The E-test constantly underestimated the MIC values when compared to the two dilution methods. No significant difference between the microdilution and agar dilution was obtained.
Resistance of staphylococci to methicillin is important especially in the case of Staphylococcus aureus isolates. Its impact in veterinary medicine is not exactly specified in coagulase-negative staphylococci; however, these staphylococci may represent an important reservoir of resistance genes. The study aimed at detecting resistance to methicillin in coagulase-negative staphylococci from raw materials and foodstuffs of animal origin and assessing the tests frequently used to determine this resistance. Coagulase-negative staphylococci (198 isolates of 12 species) were tested. Resistance to methicillin was determined by the disk diffusion method using oxacillin and cefoxitin disks, microdilution method, detection of PBP2a and the mecA gene. Of the tested isolates, 109 (55.1%) were classified as resistant by the diffusion test with oxacillin, 32 isolates (16.2%) by the test with cefoxitin and 50 isolates (25.3%) on the basis of oxacillin minimum inhibitory concentration (MIC). No resistant isolates were incorrectly identified as susceptible when using the disk diffusion method with oxacillin (sensitivity of 100%). However, apart from 22 correctly classified resistant isolates, another 87 isolates were incorrectly identified as resistant as well (specificity of 50.6%). The test with cefoxitin showed the lowest (45.5%) sensitivity in determination of resistant isolates. By contrast, this test was the most precise in classification of resistant isolates (specificity of 87.5%). When using the microdilution method, resistant strains were identified with the sensitivity and specificity of 68.2% and 80.1%, respectively. The results revealed substantial variability of methicillin-resistant isolates ranging from 16.2% to 55.1%, depending on the phenotyping methods and recommended interpretation criteria used. Therefore, it is advisable to reconsider the current interpretation criteria in the case of coagulasenegative staphylococci of animal origin (with the exception of S. epidermidis).
- Keywords
- gen mecA, koaguláza negativní stafylokoky,
- MeSH
- Drug Resistance, Bacterial * genetics MeSH
- Cefoxitin MeSH
- Microbial Sensitivity Tests classification methods MeSH
- Dairy Products microbiology MeSH
- Milk microbiology MeSH
- Oxacillin MeSH
- Penicillin-Binding Proteins isolation & purification MeSH
- Methicillin Resistance * MeSH
- Sensitivity and Specificity MeSH
- Staphylococcus * genetics classification drug effects MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Tigecyklin je nové antibiotikum, jehož aktivita zasahuje gramnegativní i grampozitivní bakterie včetně kmenů rezistentních k jiným skupinám antibiotik. Laboratorní výsledky vyšetření citlivosti původců infekcí k tigecyklinu lze použít jako relevantní podklad pro léčbu pacientů nebo ke sledování stavu a trendů rezistence pouze za předpokladu jejich správné interpretace. Cílem této práce bylo prověřit podmínky, které umožní získat reprodukovatelné a hodnověrné výsledky vyšetření citlivosti k tigecyklinu v rutinní praxi. V 15 laboratořích České republiky bylo diskovou difuzní metodou vyšetřeno 807 kmenů bakterií izolovaných z příslušných vzorků odebraných pacientům s infekčním onemocněním. Inhibiční zónu kolem disku s 15 mg tigecyklinu menší, než je limit 19 mm pro citlivé kmeny, vytvářelo 59 kmenů (7,3 %); u nich byla v Národní referenční laboratoři pro antibiotika vyšetřena minimální inhibiční koncentrace (MIC) tigecyklinu. Při vyšetření MIC bujonovou diluční mikrometodou, která je metodou volby pro vyšetření citlivosti bakterií k antibiotikům pro vysokou reprodukovatelnost výsledků, se počet kmenů necitlivých k tigecyklinu snížil na 2,3 %, byl-li pro interpretaci citlivosti gramnegativních tyček použit limit (neboli break-point) FDA 2 mg/l, respektive na 4,8 %, byl-li použit pro interpretaci limit EUCAST 1 mg/l. V této práci bylo potvrzeno, že výsledky MIC tigecyklinu získané Etestem jsou o jednu až dvě koncentrace vyšší než výsledky získané bujonovou diluční mikrometodou. Koncentrace 1 mg/l rozděluje distribuci MIC tigecyklinu v divoké populaci gramnegativních tyček zejména při vyšetření MIC Etestem, a proto použití limitu FDA 2 mg/l snižuje počet chyb. Nově navrhovaný limit pro diskovou difuzní metodu 16 mm sice výrazně snížil počet diskrepancí ve srovnání s původním limitem 19 mm, ale k jeho zavedení do rutinní praxe je zapotřebí více údajů.
Tigecycline is a new antimicrobial agent with expanded broad-spectrum activity against both Gram-negative and Gram-positive aerobes and anaerobes, including multidrug resistant strains. If properly interpreted, laboratory results from tigecycline susceptibility testing can serve as background data relevant to the treatment and/or antibiotic resistance monitoring. The study objective was to test reproducibility and reliability of tigecycline susceptibility testing results for routine practice. Altogether 807 bacterial strains isolated from clinical specimens of patients with infectious diseases were tested by the disk diffusion method in 15 microbiology laboratories of the Czech Republic. An inhibition zone diameter of less than 19 mm, i.e. the susceptibility breakpoint, around the disk with 15 μ g of tigecycline was observed for 59 (7.3 %) strains subsequently referred to the National Referen- ce Laboratory for Antibiotics to be tested for the tigecycline minimum inhibitory concentration (MIC). In the broth microdilution test, which is the tool of choice for microbial susceptibility testing because of the high reproducibility of results, the number of strains non-susceptible to tigecycline decreased to 2.3 % when the FDA breakpoint of 2 mg/l was used in the interpretation of the susceptibility of Gram-nega- tive rods or to 4.8 % when the EUCAST breakpoint of 1 mg/l was used. This study has confirmed that the E-test MIC results for tigecyc line are one to twofold higher than those obtained by the broth microdilution test. The breakpoint of 1 mg/l divides the tigecycline MIC distribution in wild-type Gram-negative rods, in particular when the E-test is used, and using the FDA breakpoint of 2 mg/l reduces errors. The newly proposed disk diffusion breakpoint of 16 mm considerably reduced discrepancies in comparison with the initial one of 19 mm, but more data is needed for its introduction into routine practice.
- MeSH
- Drug Resistance, Bacterial drug effects MeSH
- Bacterial Proteins therapeutic use MeSH
- Disk Diffusion Antimicrobial Tests methods MeSH
- Microbial Sensitivity Tests methods MeSH
- Specimen Handling MeSH
- Mixed Function Oxygenases therapeutic use MeSH
- Serum Bactericidal Test methods MeSH
- Publication type
- Comparative Study MeSH
