scholarly journals Highly Reproducible Bactericidal Activity Test Results by Using a Modified National Committee for Clinical Laboratory Standards Broth Macrodilution Technique

1999 ◽  
Vol 37 (6) ◽  
pp. 1881-1884 ◽  
Author(s):  
Donna M. Hacek ◽  
Dana C. Dressel ◽  
Lance R. Peterson
Keyword(s):  
Bactericidal Activity ◽  
Minimum Bactericidal Concentration ◽  
Clinical Laboratory ◽  
National Committee ◽  
Test Results ◽  
Activity Test ◽  
Mueller Hinton ◽  
Broth Macrodilution ◽  
High Level ◽  
Mueller Hinton Broth

Bactericidal testing historically has exhibited variable reproducibility, even when prior standardized methods were employed. Several modifications to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method are proposed to improve reproducibility. Recommended changes from the approved NCCLS guidelines (M21-A and M26-A) include omitting serum supplementation of Mueller-Hinton broth, incubating tubes at 35°C for 24 h with no agitation until they are sampled, running all tests in duplicate with six dilutions instead of nine, reincubating the test for an additional 24 h to resolve discrepant bactericidal activity test results, using a single 0.1-ml sample from each clear tube for subculture, and adopting an alternate method for calculating endpoint determination. In order to test these recommendations in a clinical laboratory setting, we used the modified methodology on 224 separate tests for bactericidal activity. There were 102 serum bactericidal titer (SBT) and 122 minimum bactericidal concentration (MBC) assays performed. By defining reproducibility as agreement between duplicate tests ± 1 dilution, we found 207 of 224 tests (92%) were reproducible at the 24-h subculture point (94% for the SBT assay and 91% for the MBC assay). When the 17 assays with discrepant results were incubated an additional 24 h for a second subculture, only 1 of 224 tests (0.4%) remained discrepant. The method used is practical for a clinical laboratory that chooses to perform bactericidal activity testing and assures a high level of reproducibility between duplicate assays. The total cost of a test was approximately $25.00.

scholarly journals In Vitro and In Vivo Activities of Tigecycline (GAR-936), Daptomycin, and Comparative Antimicrobial Agents against Glycopeptide-Intermediate Staphylococcus aureus and Other Resistant Gram-Positive Pathogens

2002 ◽  
Vol 46 (8) ◽  
pp. 2595-2601 ◽  
Author(s):  
Peter J. Petersen ◽  
Patricia A. Bradford ◽  
William J. Weiss ◽  
Timothy M. Murphy ◽  
P. E. Sum ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Clinical Laboratory ◽  
Infection Model ◽  
National Committee ◽  
Gram Positive ◽  
Mueller Hinton ◽  
Mueller Hinton Broth

ABSTRACT Tigecycline (GAR-936) and daptomycin are potent antibacterial compounds in advanced stages of clinical trials. These novel agents target multiply resistant pathogenic bacteria. Daptomycin is principally active against gram-positive bacteria, while tigecycline has broad-spectrum activity. When tested by the standard protocols of the National Committee for Clinical Laboratory Standards in Mueller-Hinton broth II, tigecycline was more active than daptomycin (MICs at which 90% of isolates tested are inhibited, 0.12 to 1 and 0.5 to 16 μg/ml, respectively) against staphylococcal, enterococcal, and streptococcal pathogens. Daptomycin demonstrated a stepwise increase in activity corresponding to an increase in the supplemental concentration of calcium. When tested in base Mueller-Hinton broth supplemented with 50 mg of calcium per liter, daptomycin demonstrated improved activity (MIC90s, 0.015 to 4 μg/ml). The activity of daptomycin, however, equaled that of tigecycline against the glycopeptide-intermediate Staphylococcus aureus (GISA) strains only when the test medium was supplemented with excess calcium (75 mg/liter). Tigecycline and daptomycin demonstrated in vivo efficacies against GISA, methicillin-resistant S. aureus, and methicillin-susceptible S. aureus strains in an intraperitoneal systemic murine infection model. These data suggest that tigecycline and daptomycin may offer therapeutic options against clinically relevant resistant pathogens for which current alternatives for treatment are limited.

scholarly journals The frequency of resistance to antibiotics of most frequently isolated bacteria from blood cultures during the period 1997-2002

2004 ◽  
Vol 61 (4) ◽  
pp. 391-397
Author(s):  
Veljko Mirovic ◽  
Branka Tomanovic ◽  
Sonja Konstantinovic
Keyword(s):  
Clinical Laboratory ◽  
Blood Cultures ◽  
Diffusion Method ◽  
National Committee ◽  
Disk Diffusion Method ◽  
Beta Lactamases ◽  
Resistance To Antibiotics ◽  
Extended Spectrum Beta Lactamases ◽  
High Level

The aim of this study was to determine the frequency of resistance to antibiotics of the most frequently isolated bacteria from blood cultures of hospitalized patients during the period 1997-2002. The resistance to antibiotics was determined by disk diffusion method according to National Committee for Clinical Laboratory Standards procedures. The majority of staphylococci isolates were resistant to methicillin, and the proportion of methicillin-resistant Staphylococcus aureus was stable (76.8-81.6%), during the follow-up period. None of the staphylococci isolates were resistant to vancomycin, but there was a very high incidence of high-level resistance of enterococci to aminoglycosides (47.2-72.2%). In 1998, only one strain among enterococci was resistant to vancomycin (Enterococcus faecium, VanA fenotype). Enterococcus spp isolates expressed variable frequency of resistance to ampicillin (15-40.1%) during the follow-up period. Among Enterobacteriaceae there were no isolates resistant to imipenem, but dramatic increase of the resistance to ceftriaxone was found from 35.9% in 1997 to 95.9% in 2002 (p<0.001). Extended spectrum beta-lactamases production was found in all the species of enterobacteria isolates. Resistance to imipenem was observed in Acinetobacter spp isolates in 2002 for the first time. Pseudomonas spp isolates expressed high and very variable resistance to all antibiotics tested during the follow-up period.

scholarly journals Synergy of Daptomycin with Oxacillin and Other β-Lactams against Methicillin-Resistant Staphylococcus aureus

2004 ◽  
Vol 48 (8) ◽  
pp. 2871-2875 ◽  
Author(s):  
Kenneth H. Rand ◽  
Herbert J. Houck
Keyword(s):  
Staphylococcus Aureus ◽  
Bactericidal Activity ◽  
Screening Method ◽  
Methicillin Resistant ◽  
Drug Concentrations ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
High Level ◽  
Time Kill

ABSTRACT We previously observed marked synergy between daptomycin and both rifampin and ampicillin against vancomycin-resistant enterococci (VRE). Because the synergy between daptomycin and ampicillin was observed for 100% of VRE strains with high-level ampicillin resistance (ampicillin MIC of ≥128 μg/ml), we looked for synergy between daptomycin and other β-lactams against 18 strains of methicillin-resistant Staphylococcus aureus (MRSA) by employing a time-kill method using Mueller-Hinton broth supplemented to 50 mg of Ca2+/liter. All strains were resistant to oxacillin (16 of 18 strains were resistant at drug concentrations of ≥256 μg/ml), and all strains were susceptible to daptomycin (the MIC at which 90% of the tested isolates were inhibited was 1 μg/ml). Daptomycin was tested at concentrations of 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg/ml alone or in combination with oxacillin at a fixed concentration of 32 μg/ml. Synergy was found for all 18 strains with daptomycin at one-half the MIC in combination with 32 μg of oxacillin/ml, and synergy was found for 11 of 18 strains (61%) with daptomycin at one-fourth the MIC or less in combination with oxacillin. At 24 h, the daptomycin-oxacillin combination with daptomycin at one-half the MIC showed bactericidal activity against all 18 strains, and the combination with one-fourth the daptomycin MIC showed bactericidal activity against 9 of 18 strains. We also used a novel screening method to look for synergy between daptomycin and other β-lactams. In this approach, daptomycin was incorporated into Ca2+-supplemented Mueller-Hinton agar at subinhibitory concentrations, and synergy was screened by comparing test antibiotic Kirby-Bauer disks on agar with and without daptomycin. By this method, daptomycin with ampicillin-sulbactam, ticarcillin-clavulanate, or piperacillin-tazobactam showed synergy comparable to or greater than daptomycin with oxacillin. For seven of the eight strains tested, time-kill studies confirmed synergy between daptomycin and ampicillin-sulbactam with ampicillin in the range of 2 to 8 μg/ml. The combination of daptomycin and β-lactams may be useful for the treatment of MRSA infection, but further studies are needed to elucidate the mechanisms and to determine the in vivo efficacy of the combination.

scholarly journals Emergence of Penicillin-ResistantStreptococcus Pneumoniaein Southern Ontario, 1993–94

1995 ◽  
Vol 6 (3) ◽  
pp. 157-160 ◽  
Author(s):  
Andrew E Simor ◽  
Anita Rachlis ◽  
Lisa Louie ◽  
Janet Goodfellow ◽  
Marie Louie
Keyword(s):  
Antimicrobial Agents ◽  
Clinical Laboratory ◽  
Tertiary Care ◽  
National Committee ◽  
In Vitro Susceptibility ◽  
Southern Ontario ◽  
Recent Emergence ◽  
Resistant Strains ◽  
High Level

Objective: To determine the prevalence of resistance ofStreptococcus pneumoniaeto penicillin and other antimicrobial agents in metropolitan Toronto.Design: Consecutive pneumococcal isolates from different patients were obtained from two private community-based laboratories and from patients assessed in the emergency department of a tertiary-care teaching hospital in Toronto, Ontario between June and December 1993, and between March and October 1994. In vitro susceptibility testing was done by broth microdilution in accordance with National Committee for Clinical Laboratory Standards guidelines.Results: Twenty (7.3±3.1%) of 274 pneumococcal isolates were resistant to penicillin; six (30%) isolates had high-level resistance (minimal inhibitory concentration [mic] 2.0 μg/mL or greater); and 14 isolates had intermediate resistance (mic0.1 to 1.0 μg/mL). Penicillin-resistant strains were also frequently resistant to tetracycline (55%), cotrimoxazole (50%), erythromycin (40%) and cefuroxime (35%). Resistant strains comprised several serotypes: 19F (six isolates), 9V (three), 23F (three), and one each of 6A, 6B, 14, and 19A; four isolates were nontypeable.Conclusions: There has been a recent emergence of penicillin-resistantS pneumoniaein southern Ontario. National and regional surveillance is warranted to determine the extent of the problem elsewhere in Canada.

scholarly journals In Vitro Activities of Free and Lipid Formulations of Amphotericin B and Nystatin against Clinical Isolates of Coccidioides immitis at Various Saprobic Stages

2002 ◽  
Vol 46 (5) ◽  
pp. 1583-1585 ◽  
Author(s):  
Gloria M. González ◽  
Rolando Tijerina ◽  
Deanna A. Sutton ◽  
John R. Graybill ◽  
Michael G. Rinaldi
Keyword(s):  
Amphotericin B ◽  
Clinical Laboratory ◽  
National Committee ◽  
Growth Stages ◽  
Coccidioides Immitis ◽  
In Vitro Susceptibility ◽  
Broth Macrodilution ◽  
Lipid Formulations ◽  
Different Growth Stages

ABSTRACT We investigated the susceptibilities of hyphal, mixed hyphal, ungerminated arthroconidial, and germinated arthroconidial populations of Coccidioides immitis to lipid formulations of amphotericin B and nystatin and their conventional preparations, utilizing the National Committee for Clinical Laboratory Standards M38-P broth macrodilution method. The differences in effects of the three different growth stages of the saprobic phase of C. immitis on the MIC/minimum lethal concentration (MLC) ratio were not statistically significant for any of the antifungal agents tested. These results suggest that either inocula could be used for in vitro susceptibility studies with C. immitis.

scholarly journals Ability of Bicarbonate Supplementation to Sensitize Selected Methicillin-Resistant Staphylococcus aureus (MRSA) Strains to β-Lactam Antibiotics in an Ex Vivo Simulated Endocardial Vegetation Model

2019 ◽  
Author(s):  
Warren E. Rose ◽  
Ana M. Bienvenida ◽  
Yan Q. Xiong ◽  
Henry F. Chambers ◽  
Arnold S. Bayer ◽  
...  
Keyword(s):  
Bactericidal Activity ◽  
Ex Vivo ◽  
Rabbit Model ◽  
Growth Media ◽  
Highly Active ◽  
Mueller Hinton ◽  
Mrsa Strains ◽  
High Level ◽  
Susceptibility Breakpoint

ABSTRACTSupplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO3) significantly increases β-lactam susceptibility of selected MRSA strains (“NaHCO3-responsive”). This “sensitization” phenomenon translated to enhanced β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO3-mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro “NaHCO3-responsive” or “NaHCO3-nonresponsive” phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB±NaHCO3. Intra-SEV MRSA killing was determined over 72 hr exposure. In both NaHCO3-responsive strains, supplementation with 25 mM or 44 mM NaHCO3 significantly reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤ 4 mg/L) resulting in bactericidal activity (≥ 3 log kill) in the model for both OXA and CFZ. In contrast, neither in vitro-defined NaHCO3-nonresponsive MRSA strains showed significant sensitization in the SEV model to either β-lactam. At both NaHCO3 concentrations, the fractional time-above-MIC was >50% for both CFZ and OXA in the NaHCO3-responsive MRSA. Also, in RPMI+10% LB media (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO3), both CFZ and OXA exhibited enhanced bactericidal activity against each NaHCO3-responsive strain in the SEV model. Neither CFZ nor OXA exposures selected for high-level β-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO3 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO3-supplemented media in vitro.

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