scholarly journals Klebsiella pneumoniae and Colistin Susceptibility Testing: Performance Evaluation for Broth Microdilution, Agar Dilution and Minimum Inhibitory Concentration Test Strips and Impact of the “Skipped Well” Phenomenon

Diagnostics ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 2352
Author(s):  
Rita Elias ◽  
José Melo-Cristino ◽  
Luís Lito ◽  
Margarida Pinto ◽  
Luísa Gonçalves ◽  
...  
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Klebsiella Pneumoniae ◽  
Susceptibility Testing ◽  
Inhibitory Concentration ◽  
Quantitative Description ◽  
Reference Method ◽  
Broth Microdilution ◽  
Test Strips ◽  
Agar Dilution ◽  
The Impact

The emergence of multidrug resistant Gram-negative pathogens, particularly carbapenemase producers, has forced clinicians to use last line antibiotics, such as colistin. Since colistin susceptibility testing presents several challenges, this study aimed at evaluating the performance of two alternative susceptibility methods for Klebsiella pneumoniae, namely, agar dilution (AD) and MIC test strips (MTS). These approaches were compared with the reference method, broth microdilution (BMD), and provide a quantitative description for the “skipped well” (SW) phenomenon. Colistin susceptibility was evaluated by BMD and AD in parallel and triplicate, using 141 K. pneumoniae clinical isolates while MTS performance was evaluated only for a subset (n = 121). Minimum inhibitory concentration analysis revealed that a substantial part (n = 26/141; 18.4%) of the initial isolates was deemed undetermined by BMD due to the following: discordance between replicates (1.4%); presence of multiple SWs (7.8%); and the combination of both events (9.2%). Both AD and MTS revealed a high number of false-susceptible strains (“very major errors”), 37.5% and 68.8%, respectively. However, AD agreement indices were reasonably high (EA = 71.3% and CA = 94.8%). For MTS these indices were lower, in particular EA (EA = 41.7% and CA = 89.6), but the approach enabled the detection of distinct sub-populations for four isolates. In conclusion, this study provides the most comprehensive study on the performance of AD and MTS for colistin susceptibility testing in K. pneumoniae, highlighting its limitations, and stressing the importance of sample size and composition. Further, this study highlights the impact of the SW phenomenon associated with the BMD method for K. pneumoniae.

scholarly journals Broth Microdilution and Gradient Diffusion Strips vs. Reference Agar Dilution Method: First Evaluation for Clostridiales Species Antimicrobial Susceptibility Testing

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 975
Author(s):  
Florian Baquer ◽  
Asma Ali Sawan ◽  
Michel Auzou ◽  
Antoine Grillon ◽  
Benoît Jaulhac ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Antimicrobial Susceptibility Testing ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Broth Microdilution ◽  
Multicenter Studies ◽  
Gradient Diffusion ◽  
Agar Dilution

Antimicrobial susceptibility testing of anaerobes is challenging. Because MIC determination is recommended by both CLSI and EUCAST, commercial broth microdilution and diffusion strip tests have been developed. The reliability of broth microdilution methods has not been assessed yet using the agar dilution reference method. In this work, we evaluated two broth microdilution kits (MICRONAUT-S Anaerobes® MIC and Sensititre Anaerobe MIC®) and one gradient diffusion strip method (Liofilchem®) for antimicrobial susceptibility testing of 47 Clostridiales isolates (Clostridium, Clostridioides and Hungatella species) using the agar dilution method as a reference. The evaluation focused on comparing six antimicrobial molecules available in both microdilution kits. Analytical performances were evaluated according to the Food and Drug Administration (FDA) recommendations. Essential agreements (EA) and categorical agreements (CA) varied greatly according to the molecule and the evaluated method. Vancomycin had values of essential and categorical agreements above 90% for the three methods. The CA fulfilled the FDA criteria for three major molecules in the treatment of Gram-positive anaerobic infections (metronidazole, piperacillin/tazobactam and vancomycin). The highest rate of error was observed for clindamycin. Multicenter studies are needed to further validate these results.

scholarly journals Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates

2015 ◽  
Vol 59 (8) ◽  
pp. 4625-4630 ◽  
Author(s):  
Konstantina Dafopoulou ◽  
Olympia Zarkotou ◽  
Evangelia Dimitroulia ◽  
Christos Hadjichristodoulou ◽  
Vasiliki Gennimata ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Acinetobacter Baumannii ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Test Strip ◽  
Clinical Isolates ◽  
Content Type ◽  
Gradient Diffusion ◽  
Agar Dilution ◽  
Resistant Strains

ABSTRACTWe compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptibleKlebsiella pneumoniae(n= 41) andAcinetobacter baumannii(n= 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for bothK. pneumoniaeandA. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.

scholarly journals Does sub-culturing of positive MRSA blood cultures affect vancomycin MICs?

2020 ◽  
Vol 69 (8) ◽  
pp. 1095-1099
Author(s):  
Izumo Kanesaka ◽  
Takamitsu Ito ◽  
Ritsuko Shishido ◽  
Makoto Nagashima ◽  
Akiko Kanayama Katsuse ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Minimum Inhibitory Concentration ◽  
Type Species ◽  
Susceptibility Testing ◽  
Inhibitory Concentration ◽  
Blood Cultures ◽  
Content Type ◽  
Vancomycin Mics ◽  
Agar Dilution ◽  
Mrsa Strains

Introduction. Empirical vancomycin (VAN) treatment failure for methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia, with significantly higher mortality, has been reported for MRSA strains with reduced VAN susceptibility. Aim. Our goal was to study the effect of sub-culture on VAN minimum inhibitory concentration (MIC) values compared to direct susceptibility of MRSA-positive blood cultures. Methodology. Using 19 MRSA-positive blood cultures and 19 seeded MRSA-positive blood cultures, we compared the VAN MICs from direct susceptibility testing of MRSA-positive blood cultures and MRSA sub-cultured from positive blood cultures. Results. In comparing direct VAN MICs from MRSA-positive blood cultures and standard agar dilution, nearly half of the MICs from agar dilution were lower, with one sample decreasing from 1.5 to 0.75 µg ml−1. Furthermore, in seeded blood cultures, 80 % or more showed lower values from standard agar dilution compared to direct VAN MICs. Conclusion. Our results reveal a trend towards lower MICs after positive blood culture isolates are sub-cultured. Some clinical failures among MRSA infections treated with VAN may result from this phenomenon.

scholarly journals Comparison of in vitro activity of five antifungal agents against dermatophytes, using the agar dilution and broth microdilution methods

2009 ◽  
Vol 42 (3) ◽  
pp. 250-254 ◽  
Author(s):  
Crystiane Rodrigues Araújo Mota ◽  
Karla Carvalho Miranda ◽  
Janine de Aquino Lemos ◽  
Carolina Rodrigues Costa ◽  
Lúcia Kioko Hasimoto e Souza ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Reference Method ◽  
Trichophyton Mentagrophytes ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Microsporum Canis ◽  
Broth Microdilution ◽  
In Vitro Susceptibility ◽  
Agar Dilution

The purpose of this study was to compare the agar dilution and broth microdilution methods for determining the minimum inhibitory concentration (MIC) of fluconazole, itraconazole, ketoconazole, griseofulvin and terbinafine for 60 dermatophyte samples belonging to the species Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. The percentage agreement between the two methods, for all the isolates with < 2 dilutions that were tested was 91.6% for ketoconazole and griseofulvin, 88.3% for itraconazole, 81.6% for terbinafine and 73.3% for fluconazole. One hundred percent agreement was obtained for Trichophyton mentagrophytes isolates evaluated with ketoconazole and griseofulvin. Thus, until a reference method for testing the in vitro susceptibility of dermatophytes is standardized, the similarity of the results between the two methods means that the agar dilution method may be useful for susceptibility testing on these filamentous fungi.

Comparison of minimum inhibitory concentration results for gepotidacin obtained using agar dilution and broth microdilution methods

2020 ◽  
Vol 98 (2) ◽  
pp. 115107
Author(s):  
S.J. Ryan Arends ◽  
Michele A. Canino ◽  
Rodrigo Mendes ◽  
Nicole E. Scangarella-Oman ◽  
Robert K. Flamm
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Broth Microdilution ◽  
Agar Dilution

scholarly journals 2166. Performance Study on the New ETEST® Piperacillin/Tazobactam (P/T) MIC Strip

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S735-S735
Author(s):  
Roland Martelin ◽  
Edwige Pillon ◽  
Emilie Cutivet ◽  
Marion Pompilio ◽  
Diane Halimi ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Inhibitory Concentration ◽  
Reference Method ◽  
Error Rates ◽  
Performance Study ◽  
First Line ◽  
Severe Infections ◽  
Agar Dilution ◽  
Resistant Strains ◽  
High Level

Abstract Background Piperacillin/Tazobactam combination is a first-line antibiotic and carbapenem sparing option for severe infections due to Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. ETEST® strips allow to determine antimicrobial Minimum Inhibitory Concentration (MIC). ETEST® Piperacillin/Tazobactam (Ptc) was developped in 1995 against agar dilution reference method. Since then, resistance to Piperacillin/Tazobactam has been increasing and broth microdilution (BMD) substituted for agar dilution as the reference method. The new ETEST® P/T strip for determining MIC of Enterobacteriaceae, P. aeruginosa and A. baumannii was developed against BMD using a panel of recent strains well genotypically characterized. The aim of this study was to compare the performance of both strips on a panel of challenging strains harboring different-resistant mechanisms. Methods A total of 64 strains were tested using ETEST® P/T, ETEST® PTc and BMD: 48 Enterobacteriaceae including 25 resistant strains and 16 P. aeruginosa including 11 resistant strains. The results were analyzed for essential (EA) and category (CA) agreements, minor (mE), major (ME) and very major (VME) error rates using FDA/CLSI 2019 breakpoints (Enterobacteriaceae, P. aeruginosa: ≤ 16/4(S); ≥ 128/4(R) µg/mL). Results Although the panel of strains was challenging including different resistant mechanisms (acquired penicillinase, high-level cephalosporinase, acquired cephalosporinase, ESBL, carbapenemase), the new ETEST® P/T performance was significantly improved for Enterobacteriaceae with an EA at 92,2% without ME or VME. This improvement was also linked to the easiest reading (significant decrease of microcolonies in the ellipse zone). For P. aeruginosa, the performance was similar between the two strips but the new ETEST® P/T was better correlated with the BMD and showed an EA of 100%. The results are summarized in the table. Conclusion The new ETEST® P/T improved the MIC determination and resistance detection, as well as the reading of MIC end points for the routine use. This study emphasizes the need to check the performance of the antimicrobial susceptibility testing products by testing strains reflecting the current epidemiology. Disclosures All authors: No reported disclosures.

scholarly journals Detection of Antibiotic Susceptibility by Colorimetric Minimum Inhibitory Concentration in Staphylococcal Isolates

QJM ◽  
2020 ◽  
Vol 113 (Supplement_1) ◽  
Author(s):  
B S Mahmoud ◽  
S A ElMasry ◽  
N A Fahim ◽  
O A Shaker ◽  
M A Abdelsattar
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Antimicrobial Susceptibility ◽  
Antibiotic Susceptibility ◽  
Susceptibility Testing ◽  
Inhibitory Concentration ◽  
Antimicrobial Susceptibility Testing ◽  
Bacterial Concentration ◽  
Tetrazolium Chloride ◽  
Wide Range ◽  
Agar Dilution

Abstract Background Staphylococcus species are implicated in a wide range of community and hospital-acquired infections, which necessitates rapid provision of antibiotic susceptibility results for patients at risk. The current study evaluated the usefulness of applying broth microdilution (BMD) and agar dilution methods for antimicrobial susceptibility testing of Staphylococci using tetrazolium salts as redox indicators. Methods Minimum inhibitory concentration (MICs) of eight antimicrobials representing different antibiotic classes as recommended by CLSI; vancomycin (VA), linezolid (LZD), oxacillin (OX), gentamicin (CN), tetracycline (TE), ciprofloxacin (CIP), erythromycin (E) and clindamycin (DA) was investigated for 80 isolates of Staphylococci by a modified BMD with the addition of dimethyl thiazole diphenyl tetrazolium bromide (MTT). As well as agar dilution with the addition of MTT & triphenyl tetrazolium chloride (TTC) at the standard bacterial concentration together with addition of MTT at an experimental bacterial concentration. Results BMD (MTT) showed the highest agreement regarding most antibiotics among different isolates in comparison with the standard BMD. All performed methods showed significant agreement except agar dilution (TTC) using TE, DA, E, and agar dilution (MTT 108) using DA as well as agar dilution (MTT 104) using E among S. aureus. Conclusions Colorimetric BMD was rapid, easy to interpret and showed the highest agreement with the standard BMD. Colorimetric agar dilution (MTT) was less tedious than BMD. The colorimetric MIC method using MTT may be a useful surrogate of antimicrobial susceptibility testing among Staphylococcus isolates.

scholarly journals 701. Comparison of MIC Results for Gepotidacin by Agar Dilution and Broth Microdilution Methods

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S317-S317
Author(s):  
S J Ryan Arends ◽  
Michele A Canino ◽  
Brieanna M Roth ◽  
Paul R Rhomberg ◽  
Robert K Flamm ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Reference Method ◽  
Broth Microdilution ◽  
Topoisomerase Iv ◽  
Acute Cystitis ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Agar Dilution ◽  
Gram Negative Organisms

Abstract Background Gepotidacin (GSK2140944) is a novel triazaacenaphthylene bacterial type II topoisomerase inhibitor in clinical development for the treatment of gonorrhea and uncomplicated UTI (acute cystitis). Gepotidacin selectively inhibits bacterial DNA gyrase and topoisomerase IV by a unique mechanism not utilized by any currently approved therapeutic agent and demonstrates in vitro activity against most target pathogens resistant to established antibacterials, including fluoroquinolones. This study tested the equivalency of minimal inhibitory concentrations (MICs) obtained by 2 reference susceptibility testing methods, agar dilution (AD) and broth microdilution (BMD), for gepotidacin against Gram-positive and Gram-negative organisms. Methods Susceptibility testing for both methods was performed on a total of 733 clinical isolates recovered largely in 2016 from over 120 medical centers worldwide. For N. gonorrhoeae, only the AD method is recommended by CLSI, therefore BMD was performed using Fastidious Broth for comparison purposes. Essential agreement (EA) based on evaluable results was calculated as the number of isolates with MICs within one 2-fold dilution of the reference method divided by the total number of results. Equivalency was defined using the 95% criteria from the FDA’s class II controls document. Results The EA observed for gepotidacin with these 2 methods was 85.8% overall and 98.3% when H. influenzae and N. gonorrhoeae isolates were excluded. Slightly higher gepotidacin MICs were observed when tested by BMD for each of these species/groups; this trend was especially prominent for E. coli and S. pyogenes. Gepotidacin tested against H. influenzae (73.1%) or N. gonorrhoeae (28.6%) species had much lower EAs. Conclusion Equivalency (EA >95%) was established between AD and BMD methods for determining gepotidacin susceptibility results against Staphylococcus spp., Streptococcus spp. and E. coli. However, for N. gonorrhoeae and H. influenzae, equivalency between the 2 methods was not established; therefore, future antimicrobial susceptibility testing for gepotidacin against these organisms should adhere to the methods for which quality control ranges and breakpoints are approved. Disclosures All authors: No reported disclosures.

scholarly journals Evaluation of Reference Dilution Test Methods for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa Strains Isolated from Patients with Cystic Fibrosis

1999 ◽  
Vol 37 (9) ◽  
pp. 2987-2991 ◽  
Author(s):  
Lisa Saiman ◽  
Jane L. Burns ◽  
Susan Whittier ◽  
Jay Krzewinski ◽  
Steve A. Marshall ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Susceptibility ◽  
Susceptibility Testing ◽  
Prolonged Exposure ◽  
Reference Method ◽  
Antibiotic Susceptibility Testing ◽  
Broth Microdilution ◽  
Optimal Method ◽  
Agar Dilution

The development of multidrug-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is most likely a consequence of increasing life expectancy and more prolonged exposure to antibiotics. The optimal method for antibiotic susceptibility testing of CF strains, particularly mucoid P. aeruginosastrains, is unknown. Antimicrobial susceptibilities of 48 CF strains (25 mucoid) and 50 non-CF strains to 12 anti-Pseudomonasagents were tested by both agar dilution and commercially custom-prepared broth microdilution plates (PML Microbiologicals, Portland, Oreg.) in three laboratories simultaneously to determine if broth microdilution could substitute for agar dilution as the reference method in subsequent studies. Comparison of MICs generated by agar dilution and broth microdilution demonstrated correlation coefficients (r) exceeding 0.85 for all agents tested; correlation was excellent for aminoglycosides (r ≥ 0.92) and very good for β-lactam agents including agents paired with a β-lactamase inhibitor (r ≥ 0.87) and for ciprofloxacin (r = 0.86). Correlation was not improved by 48-h readings, but correlation between 24- and 48-h readings ranged between 0.91 and 0.98 for both methods. Interlaboratory variations were minimal, as the percentage of acceptable variations was 94% for both methods, and serious discords were infrequent (<2% of comparisons). However, CF strains were more likely to have serious discords than were non-CF strains (P < 0.0001), although mucoid strains were not more likely to have serious discords than were nonmucoid strains. In this study, MICs determined by custom-prepared broth microdilution compared favorably with MICs determined by agar dilution. Thus, this broth microdilution assay can serve as a reference method and facilitate future studies to determine the optimal method for antibiotic susceptibility testing of CF strains.

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