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Trimethoprim/sulfamethoxazole (TMP/SMZ) is considered the treatment of choice for infections caused by Stenotrophomonas maltophilia, but limited pharmacodynamic data are available to support current susceptibility breakpoints or guide optimal dosing. Time-kill studies using a TMP/SMZ concentration of 4/40 μg/mL were conducted to compare 4 S. maltophilia with 4 Escherichia coli isolates having the same MICs (0.25/4.75 to 4/76 μg/mL) in cation-adjusted Mueller-Hinton broth (CAMHB) and ISO-Sensitest broth (ISO broth). With the exception of the resistant isolates (4/76 μg/mL), which resulted in regrowth approaching the growth of the control, TMP/SMZ displayed significantly greater killing for E. coli than for S. maltophilia at each MIC. Against E. coli, the mean changes at 24 h were -4.49, -1.73, -1.59, and +1.83 log10 CFU for isolates with MICs of 0.25/4.75, 1/19, 2/39, and 4/74 μg/mL, respectively. The area under the concentration-time curve for the free, unbound fraction of the drug (fAUC)/MIC ratio required for stasis and 1-log10 and 2-log10 CFU reductions were 40.7, 59.5, and 86.3, respectively. In contrast, TMP/SMZ displayed no stasis or CFU reductions against any S. maltophilia isolate regardless of the MIC, and no pharmacodynamic thresholds were quantifiable. Observations were consistent in both CAMHB and ISO broth. These data add increasing evidence that current TMP/SMZ susceptibility breakpoints against S. maltophilia should be reassessed.
Pan-drug-resistant (PDR) Acinetobacter baumannii is an important nosocomial pathogen that poses therapeutic challenges. Tigecycline alone or in combination with agents such as colestimethate, imipenem, and/or amikacin is being used clinically to treat PDR A. baumannii infections. The purpose of this study was to compare in vitro susceptibility testing by epsilometric (Etest) methods and the checkerboard (CB) method with testing by time-kill analysis. PDR A. baumannii clinical strains representing eight unique pulsed-field gel electrophoresis clones selected from a total of 32 isolates were tested in vitro with tigecycline, colestimethate, imipenem, and amikacin in single- and two-drug combinations by using two different methods of Etest (with a fixed ratio method [method 1] and with the incorporation of the active drug in medium [method 2]) and by using CB. The three-drug combination of imipenem, tigecycline, and amikacin was also tested by CB. These results were compared to time-kill results. Synergy was consistently detected with the imipenem plus colestimethate and tigecycline plus imipenem combinations. The Etest method with active drug incorporated into the agar allowed us to detect synergy even in the presence of the active drug and was more comparable to CB and time-kill tests. Synergy was detected with the three-drug combination of imipenem, tigecycline, and amikacin by both CB and time-kill methods among several tested clones. These findings indicate the utility of synergy testing to predict activity of specific antibiotic combinations against PDR A. baumannii.
The rapid rise of antimicrobial resistance is a worldwide problem. This has necessitated the need to search for new antimicrobial agents. Mushrooms are rich sources of potential antimicrobial agents. This study investigated the antimicrobial properties of methanol extracts of Trametes gibbosa, Trametes elegans, Schizophyllum commune, and Volvariella volvacea. Agar well diffusion, broth microdilution, and time-kill kinetic assays were used to determine the antimicrobial activity of the extracts against selected test organisms. Preliminary mycochemical screening revealed the presence of tannins, flavonoids, triterpenoids, anthraquinones, and alkaloids in the extracts. Methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea showed mean zone of growth inhibition of 10.00 0.0 to 21.50 0.84, 10.00 0.0 to 22.00 1.10, 9.00 0.63 to 21.83 1.17, and 12.00 0.0 to 21.17 1.00 mm, respectively. The minimum inhibitory concentration of methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea ranged from 4.0 to 20, 6.0 to 30.0, 8.0 to 10.0, and 6.0 to 20.0 mg/mL, respectively. Time-kill kinetics studies showed that the extracts possess bacteriostatic action. Methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea exhibited antimicrobial activity and may contain bioactive compounds which may serve as potential antibacterial and antifungal agents.
The Time-kill kinetics assay is used to study the activity of an antimicrobial agent against a bacterial strain and can determine the bactericidal or bacteriostatic activity of an agent over time.
Bactericidal activity is defined as greater than 3 log10 -fold decrease in colony forming units (surviving bacteria), which is equivalent to 99.9% killing of the inoculum. The time kill analysis can monitor the effect of various concentrations of an antimicrobial agent over time in relation to the stages of the growth of the bacteria (lag, exponential, stationary phase). Time-kill kinetics assays for agents such as antiseptics require a shorter time-kill kinetics study and follow different methodology. In contrast to the multiple time points in a time-kill kinetics assay, the minimal bactericidal concentration (MBC) test is defined as a 99.9% or greater killing efficacy at a specified time.
Figure Legend. A sample time-kill kinetics assay is depicted. Three different test compounds, Compound 1, Compound 2, and Compound 3 are added to media containing a starting culture of bacteria. A vehicle only control and a growth control are included as negative controls. The log CFU/mL for all groups is determined at time 0 and at subsequent time points up to 24 hours, depending on the bacteria strain and the media used. In this example, Compound 1 exhibited bactericidal effect, reducing the starting log CFU/mL by greater than 3 logs. Compound 2 exhibited bacteriostatic effect, as the log CFU/mL over time remained roughly the same as the starting log CFU/mL concentration. Compound 3 exhibited little antimicrobial effect, as the bacteria in the presence of this compound grew over time to a level similar to the vehicle only control.
Emery Pharma has successfully performed several Time-Kill studies for multiple clients in pharmaceutical industry. All testing follows the guidelines set by the Clinical & Laboratory Standards Institute (CLSI).
Tea tree oil has recently emerged as an effective topical antimicrobial agent active against a wide range of organisms. Tea tree oil may have a clinical application in both the hospital and community, especially for clearance of methicillin-resistant Staphylococcus aureus (MRSA) carriage or as a hand disinfectant to prevent cross-infection with Gram-positive and Gramnegative epidemic organisms. Our study, based on the time-kill approach, determined the kill rate of tea tree oil against several multidrug-resistant organisms, including MRSA, glycopeptide-resistant enterococci, aminoglycoside-resistant klebsiellae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, and also against sensitive microorganisms. The study was performed with two chemically different tea tree oils. One was a standard oil and the other was Clone 88 extracted from a specially bred tree, which has been selected and bred for increased activity and decreased skin irritation. Our results confirm that the cloned oil had increased antimicrobial activity when compared with the standard oil. Most results indicated that the susceptibility pattern and Gram reaction of the organism did not influence the kill rate. A rapid killing time (less than 60 min) was achieved with both tea tree oils with most isolates, but MRSA was killed more slowly than other organisms.
Increasing global antibiotic resistance has resulted in more use of antibiotic combinations. There is a lack of a gold standard for in vitro testing of these combinations for synergy or antagonism. Time-kill assay (TKA) may be used but is labor intensive and not practical for clinical use. Etest synergy methods are more rapid and easier to perform, but there is no agreement regarding which method is best. We tested 31 clinical genetically unique Klebsiella pneumoniae carbapenemase-producing Klebsiella isolates with the combination of meropenem and polymyxin B by TKA and 3 Etest methods, each in triplicate: Method 1, MIC:MIC; Method 2, direct overlay; and Method 3, cross. Overall, testing with Etest synergy methods showed the following agreement with TKA: Method 1: 25/31 (80.6%), Method 2: 7/31 (22.6%), and Method 3: 8/31 (25.8%). The MIC:MIC method had the highest agreement (80.6%, κ = 0.59, P < 0.001) and should be evaluated more extensively.
One of those traits is that extended leaf time, creating a thick canopy of leaves that shades out light from native plants and can last from early spring into early winter. But that very trait also makes an opening for homeowners and landowners to attack it.
The best time is late October and early November, when all the other plants are bare and the honeysuckle is still hanging on to its leaves. By spraying it with an herbicide such as glycosophate (the active ingredient in Round-Up), McEwan said the leaves take up the herbicide and transport it into the plant, while minimizing the impact on native plants. So, when the plant finally drops its leaves, they don't come back in the spring.
That method works, along with staying ahead of small invasions by pulling young plants, he said. And it's worth the effort.
"The level of energy and investment it takes to eradicate it and then to restore the native species is enormous," he said.
To learn more about Amur honeysuckle, McEwan recommends visiting the Ohio Invasive Plants Council and the USDA Amur honeysuckle sites.