time-kill assay
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In consideration of high production costs of new antimicrobial drugs, a more convenient and economical method for time-kill study is urgently required. In the present experiment, we attempted to demonstrate the feasibility of microplate method as an alternative measure of macrodilution method for time-kill study. Three conventional antibiotics (ciprofloxacin, ceftazidime, and levofloxacin) and two antimicrobial peptides [A-thanatin and K(4)-S4(1-16)a] were used to determine time-kill curves against Escherichia coli ATCC 25922 and Staphylococcus epidermidis ATCC 14990. Meanwhile, both methods were also performed with three antisense peptide nucleic acids (PNA3, PNA4, and PNA5) targeting ropD gene of Staphylococcus aureus ATCC 29213 and MRSA WHO-2. In order to study the correlation between the two methods, the growth inhibition rate of PNAs, antimicrobial peptides, and antibiotics for the tested strains were evaluated. A strong agreement between the results obtained from the two methods has been demonstrated. Although microplate method required longer incubation time for a significant result than macrodilution method, the former provides a more convenient, economical, and stable way to perform time-kill test for these agents. Thus, we concluded that microplate method was an available measure for time-kill study of new antimicrobial drugs.
The effect of combinations of the crude acetone and aqueous extracts of Helichrysum pedunculatum leaves and eight antibiotics was determined by means of checkerboard and time-kill methods. In the checkerboard method, synergy of 45.8% was observed, being independent of Gram reaction, with combinations in the aqueous extract yielding largely (18.8%) antagonistic interactions. The time-kill assay detected synergy (45.8%) that was also independent of Gram reaction with a potentiation of more than 3 orders of the bactericidal activity of the test antibiotics. The crude leaf extracts of H. pedunculatum could thus be considered to be potential source of a broad-spectrum antibiotic-resistance-modifying compounds.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacteria účinky léků MeSH
- financování organizované MeSH
- Helichrysum chemie MeSH
- infekce v ráně farmakoterapie mikrobiologie MeSH
- kombinovaná farmakoterapie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- rostlinné extrakty farmakologie MeSH
- synergismus léků MeSH
- Check Tag
- lidé MeSH
Caenorhabditis elegans has been increasingly used to study the innate immunity and for the screening of microbe/host-specific pathogenic factors. Staphylococcus aureus-mediated infections with live C. elegans were performed on solid (full-lawn) and liquid assays. S. aureus required 90 ± 10 h for the complete killing of C. elegans, but the infection was started only after 32 h of exposure with 20% inoculum of S. aureus. The short time exposure studies revealed that, in 20% of inoculum, continuous exposure to the pathogen was required for the killing of nematode. In 100% of inoculum, only 8 h of exposure was sufficient to kill the C. elegans. To evaluate kinetically at the innate immune level, the regulation of representative candidate antimicrobial genes was investigated. Both semi-quantitative reverse transcriptase polymerase chain reaction (PCR) and real-time PCR analyses indicated the regulation of candidate immune regulatory genes of lysozyme (lys-7), cysteine protease (cpr-2), and C-type lectin (clec-60 and clec-87) family members during the course of S. aureus infections, indicating the possible contribution of the above players during the host immune response against S. aureus exposures.
- MeSH
- bakteriální léková rezistence MeSH
- Caenorhabditis elegans genetika imunologie metabolismus MeSH
- cysteinové proteasy genetika imunologie metabolismus MeSH
- exprese genu MeSH
- interakce hostitele a patogenu MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lektiny typu C genetika imunologie metabolismus MeSH
- muramidasa genetika imunologie metabolismus MeSH
- počet mikrobiálních kolonií MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- přirozená imunita MeSH
- proteiny Caenorhabditis elegans genetika imunologie metabolismus MeSH
- stafylokokové infekce genetika imunologie mikrobiologie MeSH
- Staphylococcus aureus fyziologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A series of twenty-one salicylanilide N-alkylcarbamates was assessed for novel antibacterial characteristics against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum inhibitory concentration was determined by the broth dilution micro-method with subsequent subcultivation of aliquots to assess minimum bactericidal concentration. The bactericidal kinetics was established by time-kill assay. Ampicillin, ciprofloxacin and vancomycin were used as reference antibacterial drugs. All the tested compounds exhibited highly potent anti-MRSA activity (⩽ 0.008-4 μg/mL) comparable or up to 250× higher than that of vancomycin, the standard in the treatment of serious MRSA infections. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl butylcarbamate and 4-chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl ethylcarbamate were the most active compounds. In most cases, compounds provided reliable bacteriostatic activity, except for 4-chloro-2-(4-chlorophenylcarbamoyl)phenyl decylcarbamate exhibiting bactericidal effect at 8h (for clinical isolate of MRSA 63718) and at 24h (for clinical isolates of MRSA SA 630 and MRSA SA 3202) at 4× MIC. Structure-activity relationships are discussed.
The efficacy of the agents was evaluated on the basis of minimum inhibitory concentration (MIC) and time-kill curve analysis in Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Mycobacterium smegmatis, and Citrobacter braakii; MIC was found to be 0.5, 4.0, 0.015625, 0.0078125, and 0.0625 mg/L in FDC of ceftazidime + sulbactam, respectively, which is lower than ceftazidime and sulbactam individually. Time-kill curve analysis demonstrated maximum killing of bacteria after 4 h. A fixed dose combination of ceftazidime + sulbactam was found to have stronger antibacterial properties than ceftazidime and sulbactam alone at in vitro analysis.
Recently, the interest is increasing to find alternatives to replace the usage of antibiotics since their massive and improper usage enhance the antibiotic resistance in human pathogens. In this study, for the first time we showed that the soil proteins have very high antibacterial activity (98% of growth inhibition) against methicillin resistant Staphylococcus aureus (MRSA), one of the most threatening human pathogens. We found that the protein extract (C3) from the forest with past intensive management showed higher antibacterial activity than that of unmanaged forest. The MIC and IC50 were found to be 30 and 15.0 μg protein g-1 dry soil respectively. C3 was found to kill the bacteria by cell wall disruption and genotoxicity which was confirmed by optical and fluorescent microscopy and comet assay. According to qPCR study, the mecA (the antibiotic resistant gene) expression in MRSA was found to be down-regulated after C3 treatment. In contrast, C3 showed no hemolytic toxicity on human red blood cells which was confirmed by hemolytic assay. According to ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), 144 proteins were identified in C3 among which the majority belonged to Gram negative bacteria (45.8%). Altogether, our results will help to develop novel, cost-effective, non-toxic and highly efficient antibacterial medicines from natural sources against antibiotic resistant infections.
Carbapenem resistance observed in Klebsiella pneumoniae strains limits treatment options. Therefore, use of antibiotics combined with bioactive compounds may be an important strategy to control K. pneumoniae. The purpose of this study was to evaluate the activity of combination of carvacrol and meropenem on carbapenem-resistant K. pneumoniae (CRKP) strains. The presence of blaOXA-48 carbapenemase in all 25 CRKP strains was identified using the PCR technique. The combination of carvacrol and meropenem was tested for antimicrobial activity on CRKP strains. The minimum inhibitory concentrations of carvacrol and meropenem were detected within a range of 32-128 μg/mL using the broth microdilution method. Synergy between carvacrol and meropenem was observed on 8 of the 25 CRKP strains by checkerboard assay (FICI = 0.5) and confirmed by time-kill assay. According to the live-dead test results, the viability percentage of the cells exposed to synergistic combination was 35.47% at the end of 24 h. The membrane damage caused by the synergistic combination was spectrophotometrically measured (A = 0.21) and further confirmed by SEM analysis. According to the MTT assay, both carvacrol and meropenem did not show any statistically significant cytotoxic effect on Vero cells (p > 0.05). In conclusion, the results suggest that carvacrol and meropenem can act synergistically to inhibit the growth of CRKP.
- MeSH
- antibakteriální látky farmakologie MeSH
- beta-laktamasy * genetika MeSH
- Cercopithecus aethiops MeSH
- cymeny MeSH
- karbapenemy farmakologie MeSH
- Klebsiella pneumoniae * MeSH
- meropenem farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- synergismus léků MeSH
- Vero buňky MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
A series of 13 salicylamide derivatives was assessed for antibacterial activity against three isolates of vancomycin-resistant Enterococcus faecalis (VRE) and Enterococcus faecalis ATCC 29212 as a quality standard. The minimum inhibitory concentration was determined by the broth microdilution method with subsequent subcultivation of aliquots to assess minimum bactericidal concentration. The growth kinetics was established by the time-kill assay. Ampicillin, ciprofloxacin, tetracycline and vancomycin were used as the reference antibacterial drugs. Three of the investigated compounds showed strong bacteriostatic activity against VRE (0.199-25 µM) comparable to or more potent than ampicillin and ciprofloxacin. In addition, these compounds were tested for synergistic effect with vancomycin, ciprofloxacin and tetracycline, while 5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide showed the highest potency as well as synergistic activity with vancomycin against VRE 368. Screening of the cytotoxicity of the most effective compounds was performed using human monocytic leukemia THP-1 cells, and based on LD50 values, it can be stated that the compounds have insignificant toxicity against human cells.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- Enterococcus faecalis účinky léků MeSH
- enterokoky rezistentní vůči vankomycinu účinky léků MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- vankomycin chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Oncolytic viruses are among the most powerful and selective cancer therapeutics under development and are showing robust activity in clinical trials, particularly when administered directly into tumor nodules. However, their intravenous administration to treat metastatic disease has been stymied by unfavorable pharmacokinetics and inefficient accumulation in and penetration through tumors. METHODS: Adenovirus (Ad) was "stealthed" with a new N-(2-hydroxypropyl)methacrylamide polymer, and circulation kinetics were characterized in Balb/C SCID mice (n = 8 per group) bearing human ZR-75-1 xenograft tumors. Then, to noninvasively increase extravasation of the circulating polymer-coated Ad into the tumor, it was coinjected with gas microbubbles and the tumor was exposed to 0.5 MHz focused ultrasound at peak rarefactional pressure of 1.2 MPa. These ultrasound exposure conditions were designed to trigger inertial cavitation, an acoustic phenomenon that produces shock waves and can be remotely monitored in real-time. Groups were compared with Student t test or one-way analysis of variance with Tukey correction where groups were greater than two. All statistical tests were two-sided. RESULTS: Polymer-coating of Ad reduced hepatic sequestration, infection (>8000-fold; P < .001), and toxicity and improved circulation half-life (>50-fold; P = .001). Combination of polymer-coated Ad, gas bubbles, and focused ultrasound enhanced tumor infection >30-fold; (4 × 10(6) photons/sec/cm(2); standard deviation = 3 × 10(6) with ultrasound vs 1.3 × 10(5); standard deviation = 1 × 10(5) without ultrasound; P = .03) and penetration, enabling kill of cells more than 100 microns from the nearest blood vessel. This led to substantial and statistically significant retardation of tumor growth and increased survival. CONCLUSIONS: Combining drug stealthing and ultrasound-induced cavitation may ultimately enhance the efficacy of a range of powerful therapeutics, thereby improving the treatment of metastatic cancer.
- MeSH
- Adenoviridae * MeSH
- adjuvancia farmaceutická aplikace a dávkování MeSH
- akrylamidy aplikace a dávkování MeSH
- analýza rozptylu MeSH
- farmaceutická vehikula aplikace a dávkování MeSH
- kombinovaná terapie metody MeSH
- lidé MeSH
- mikrobubliny MeSH
- myši inbrední BALB C MeSH
- myši SCID MeSH
- myši MeSH
- nádory prsu metabolismus patologie terapie MeSH
- onkolytická viroterapie metody MeSH
- onkolytické viry * MeSH
- ultrazvuková terapie * MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
α-Terpineol, terpinen-4-ol, and δ-terpineol, isomers of terpineol, are among the compounds that give Cinnamomum longepaniculatum leaf oil its distinguished pleasant smell. The objective of this study was to evaluate the antimicrobial activity of these three isomeric terpineols. The determination of antibacterial activity was based on the minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC). Changes in time-kill curve, alkaline phosphatase (AKP), UV-absorbing material, membrane potential, and scanning electron microscopy (SEM) were measured to elucidate the possible antimicrobial mechanism. α-Terpineol, terpinen-4-ol, and δ-terpineol demonstrated good inhibitory effects against several gram-negative bacteria, particularly Shigella flexneri. MIC and MBC of α-terpineol and terpinen-4-ol were similar (0.766 mg/mL and 1.531 mg/mL, respectively) for S. flexneri, while the MIC and MBC values of δ-terpineol were 0.780 mg/mL and 3.125 mg/mL, respectively. Time-kill curves showed that the antibacterial activities of the tested compounds were in a concentration-dependent manner. Release of nucleic acids and proteins along with a decrease in membrane potential proved that α-terpineol, terpinen-4-ol, and δ-terpineol could increase the membrane permeability of Shigella flexneri. Additionally, the release of AKP suggested that the cell wall was destroyed. SEM analysis further confirmed that S. flexneri cell membranes were damaged by α-terpineol, terpinen-4-ol, and δ-terpineol. Our research suggests that these three isomeric terpineols have the potential of being used as natural antibacterial agents by destroying the cell membrane and wall, resulting in cell death. However, the specific antibacterial activity differences need further investigation.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- buněčná stěna účinky léků MeSH
- gramnegativní bakterie účinky léků MeSH
- isomerie MeSH
- listy rostlin chemie MeSH
- membránové potenciály účinky léků MeSH
- mikrobiální testy citlivosti MeSH
- oleje prchavé chemie farmakologie MeSH
- skořicovník chemie MeSH
- terpeny chemie farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
