Intra- and Interlaboratory Performances of Two Commercial Antimicrobial Susceptibility Testing Methods for Bifidobacteria and Nonenterococcal Lactic Acid Bacteria

ABSTRACT In a small-scale harmonization study involving nine laboratories in eight European countries, the intra- and interlaboratory performances of two commercially available systems, i.e., the VetMIC microplate system and Etest, for antimicrobial susceptibility testing of nonenterococcal lactic acid bacteria (NELAB) and bifidobacteria were analyzed. In addition, one laboratory also performed standard broth microdilution as a reference method. MICs of tetracycline, erythromycin, ampicillin, gentamicin, clindamycin, and streptomycin for the type strains of 25 species of NELAB and bifidobacteria and MICs of vancomycin for a selection of relevant taxa were determined. The previously described lactic acid bacterium susceptibility test medium (LSM) and related mixed-medium formulations, all including Iso-Sensitest broth as a basic component, were used as test media. The overall agreement of median MIC ranges ± 1 log2 dilution determined by the VetMIC and Etest methods with the median MICs determined by the reference method was very good for tetracycline, ampicillin, and streptomycin (92.3 to 100%) but low for erythromycin (19.5 to 30.7%) and clindamycin (50.0 to 80.8%). There was a consensus among the participating laboratories that VetMIC was preferred over Etest because of its lower cost, better growth support, and more uniform criteria for MIC end point reading. With the range for acceptable intralaboratory reproducibility being defined as the median MIC ± 1 log2 dilution, VetMIC results (with 69.2% of all data sets in the acceptable range) were shown to display greater reproducibility than Etest results (with 58.8% of all data sets in the acceptable range). Also at the interlaboratory level, the proportion of MIC values obtained with VetMIC that belonged to the complete agreement category (60.0%) was higher than the proportion of such values obtained with Etest (47.0%), which indicates a higher degree of interlaboratory reproducibility for the former method. Apart from some agent-specific effects, the majority of VetMIC and Etest replicate data sets were situated within a 1- to 2-log2 dilution range, suggesting that the two methods can be considered to be equivalent for recognizing resistance phenotypes. This multicenter study has further validated the standard use of LSM and related mixed-medium formulations with commercially available systems and formed the basis for the ongoing development of the ISO 10932/IDF 223 standard for susceptibility testing of NELAB and bifidobacteria.

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Antimicrobial susceptibility testing of lactic acid bacteria and bifidobacteria by broth microdilution method and Etest

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2009.03.012 ◽

2009 ◽

Vol 132 (1) ◽

pp. 54-58 ◽

Cited By ~ 17

Author(s):

Akira Kushiro ◽

Christian Chervaux ◽

Stephanie Cools-Portier ◽

Audrey Perony ◽

Sophie Legrain-Raspaud ◽

...

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Antimicrobial Susceptibility Testing ◽

Broth Microdilution ◽

Microdilution Method ◽

Broth Microdilution Method

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

Antibiotics ◽

10.3390/antibiotics10080975 ◽

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.

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Antimicrobial Susceptibility Testing of 59 Strains of Campylobacter fetus subsp. fetus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1847 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1847-1849 ◽

Cited By ~ 22

Author(s):

Carole Tremblay ◽

Christiane Gaudreau

Keyword(s):

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Antimicrobial Susceptibility Testing ◽

Disk Diffusion ◽

Complete Agreement ◽

Campylobacter Fetus ◽

Agar Dilution ◽

Fetus Subsp

The susceptibilities of 59 Campylobacter fetus subsp.fetus isolates to eight antibiotics were studied by the agar dilution, E-test, and disk diffusion methods. None of the isolates were β-lactamase producers. All were susceptible to ampicillin, gentamicin, imipenem, and meropenem as determined by the three methods, with MICs at which 90% of the isolates are inhibited (MIC90s) (determined by agar dilution) of 2, 1, ≤0.06, and 0.12 μg/ml, respectively. Twenty-seven percent of the isolates were resistant to tetracycline, with complete agreement between the agar dilution and disk diffusion results. The MIC90s determined by agar dilution were 2 μg/ml for erythromycin, 1 μg/ml for ciprofloxacin, and 8 μg/ml for cefotaxime.

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The Poisoned Well: Enhancing the Predictive Value of Antimicrobial Susceptibility Testing in the Era of Multidrug Resistance

Journal of Clinical Microbiology ◽

10.1128/jcm.00511-17 ◽

2017 ◽

Vol 55 (8) ◽

pp. 2304-2308 ◽

Cited By ~ 13

Author(s):

Thea Brennan-Krohn ◽

Kenneth P. Smith ◽

James E. Kirby

Keyword(s):

Antimicrobial Susceptibility ◽

Clinical Microbiology ◽

Predictive Power ◽

Susceptibility Testing ◽

Reference Method ◽

Antimicrobial Susceptibility Testing ◽

Clinical Microbiology Laboratory ◽

Microbiology Laboratory ◽

Laboratory Reference ◽

Doubling Dilution

ABSTRACTAntimicrobial susceptibility testing (AST) is a fundamental mission of the clinical microbiology laboratory. Reference AST methods are based on bacterial growth in antibiotic doubling dilution series, which means that any error in the reference method inherently represents at least a 2-fold difference. We describe the origins of current AST reference methodology, highlight the sources of AST variability, and propose ideas for improving AST predictive power.

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The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz548 ◽

2020 ◽

Vol 75 (4) ◽

pp. 968-978 ◽

Cited By ~ 7

Author(s):

Emma Jonasson ◽

Erika Matuschek ◽

Gunnar Kahlmeter

Keyword(s):

Blood Culture ◽

Positive Blood Culture ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Reference Method ◽

Bloodstream Infections ◽

Error Rates ◽

Antimicrobial Susceptibility Testing ◽

Diffusion Method ◽

Disc Diffusion

Abstract Objectives With increasing antimicrobial resistance, rapid antimicrobial susceptibility testing (RAST) becomes important, especially in patients with bloodstream infections. EUCAST decided to develop a standardized rapid method, based on EUCAST disc diffusion, to offer susceptibility reports within 4–8 h of a positive blood culture (BC). Methods BC bottles were spiked with clinical isolates (n = 332) of the seven most relevant sepsis pathogens with a variety of resistance mechanisms. RAST was performed directly from the bottle and zones read after 4, 6 and 8 h. Several variables were investigated, including the effect of using different BC bottles and of a 0–18 h delay between a positive signal and the performance of RAST. Results For five species, most inhibition zones could be read after 4 h. The proportion of results that could be interpreted increased from 75% at 4 h to 84% after 8 h. Categorical agreement against the reference method was good, with error rates of false susceptibility of 0.2%, 0.2% and 0.2% at 4, 6 and 8 h and false resistance of 1.2%, 0.2% and 0.1% at 4, 6 and 8 h, respectively. Conclusions With the EUCAST RAST method, reliable AST results can be delivered within 4–8 h of positivity of BC bottles for seven important bloodstream infection pathogens. To reduce the occurrence of errors and to absorb the variability caused by using a non-standardized inoculum, material from different manufacturers and workflow-related delays, we have introduced an area in which interpretation is not permitted, the Area of Technical Uncertainty.

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Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – the EUCAST broth microdilution reference method for MIC determination

Clinical Microbiology and Infection ◽

10.1016/j.cmi.2020.07.036 ◽

2020 ◽

Vol 26 (11) ◽

pp. 1488-1492 ◽

Cited By ~ 1

Author(s):

Thomas Schön ◽

Jim Werngren ◽

Diana Machado ◽

Emanuele Borroni ◽

Maria Wijkander ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Mycobacterium Tuberculosis Complex ◽

Reference Method ◽

Antimicrobial Susceptibility Testing ◽

Broth Microdilution ◽

Tuberculosis Complex

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A Simplified Method for Testing Bordetella pertussisfor Resistance to Erythromycin and Other Antimicrobial Agents

Journal of Clinical Microbiology ◽

10.1128/jcm.38.3.1151-1155.2000 ◽

2000 ◽

Vol 38 (3) ◽

pp. 1151-1155 ◽

Cited By ~ 20

Author(s):

Bertha C. Hill ◽

Carolyn N. Baker ◽

Fred C. Tenover

Keyword(s):

Antimicrobial Susceptibility ◽

Antimicrobial Agents ◽

Susceptibility Testing ◽

Reference Method ◽

Antimicrobial Susceptibility Testing ◽

Disk Diffusion ◽

Mueller Hinton ◽

Disk Diffusion Testing ◽

Direct Inoculation ◽

Agar Dilution

Present methods of antimicrobial susceptibility testing ofBordetella pertussis are time consuming and require specialized media that are not commercially available. We tested 52 isolates of B. pertussis for resistance to erythromycin, trimethoprim-sulfamethoxazole, chloramphenicol, and rifampin by agar dilution with Bordet-Gengou agar (BGA) containing 20% horse blood (reference method), Etest using BGA and Regan-Lowe agar without cephalexin (RL−C), and disk diffusion using BGA and RL−C. The organisms tested included four erythromycin-resistant isolates ofB. pertussis from a single patient, a second erythromycin-resistant strain of B. pertussis from an unrelated patient in another state, and 47 nasopharyngeal surveillance isolates of B. pertussis from children in the western United States. The results of agar dilution testing using direct inoculation of the organisms suspended in Mueller-Hinton broth were within ±1 dilution of those obtained after overnight passage of the inoculum in Stainer-Scholte medium, which is the traditional method of testing B. pertussis. The Etest method produced MICs similar to those of the agar dilution reference method for three of the four antimicrobial agents tested; the trimethoprim-sulfamethoxazole results were lower with Etest, particularly when the direct suspension method was used. Most of the Etest MICs, except for that of erythromycin, were on scale. Disk diffusion testing using RL−C medium was helpful in identifying the erythromycin-resistant strains, which produced no zone of inhibition around the disk; susceptible isolates produced zones of at least 42 mm. Thus, the antimicrobial susceptibility testing of B. pertussis can be simplified by using the Etest or disk diffusion on RL−C to screen for erythromycin-resistant isolates of B. pertussis.

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Comparison of novel rapid diagnostic of blood culture identification and antimicrobial susceptibility testing by Accelerate Pheno system and BioFire FilmArray Blood Culture Identification and BioFire FilmArray Blood Culture Identification 2 panels

BMC Microbiology ◽

10.1186/s12866-021-02403-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Dorothy T. T. Sze ◽

Candy C. Y. Lau ◽

Tsz-Ming Chan ◽

Edmond S. K. Ma ◽

Bone S. F. Tang

Keyword(s):

Blood Culture ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Reference Method ◽

Turnaround Time ◽

Antimicrobial Susceptibility Testing ◽

Performance Factors ◽

Hands On ◽

Culture Identification ◽

Sensitivity Specificity

Abstract Background Conventional turnaround time (TAT) for positive blood culture (PBC) identification (ID) and antimicrobial susceptibility testing (AST) is 2–3 days. We evaluated the TAT and ID/AST performance using clinical and seeded samples directly from PBC bottles with different commercial approaches: (1) Accelerate Pheno® system (Pheno) for ID/AST; (2) BioFire® FilmArray® Blood Culture Identification (BCID) Panel and/ or BCID2 for ID; (3) direct AST by VITEK® 2 (direct AST); and (4) overnight culture using VITEK® 2 colony AST. Results A total of 141 PBC samples were included in this evaluation. Using MALDI-TOF (Bruker MALDI Biotyper) as the reference method for ID, the overall monomicrobial ID sensitivity/specificity are as follows: Pheno 97.9/99.9%; BCID 100/100%; and BCID2 100/100%, respectively. For AST performance, broth microdilution (BMD) was used as the reference method. For gram-negatives, overall categorical and essential agreements (CA/EA) for each method were: Pheno 90.3/93.2%; direct AST 92.6/88.5%; colony AST 94.4/89.5%, respectively. For gram-positives, the overall CA/EAs were as follows: Pheno 97.2/98.89%; direct AST 97.2/100%; colony AST 97.2/100%, respectively. The BCID/BCID2 and direct AST TATs were around 9–20 h (1/9-19 h for ID with resistance markers/AST), with 15 min/sample hands-on time. In comparison, Pheno TATs were around 8–10 h (1.5/7 h for ID/AST) with 2 min/sample hands-on time, maintains a clinically relevant fast report of antibiotic minimal inhibitory concentration (MIC) and allows for less TAT and hands-on time. Conclusion In conclusion, to the best of our knowledge, this is the first study conducted as such in Asia; all studied approaches achieved satisfactory performance, factors such as TAT, panel of antibiotics choices and hands-on time should be considered for the selection of appropriate rapid ID and AST of PBC methods in different laboratory settings.

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Validation of a Gradient Diffusion Method (Etest®) for Antimicrobial Susceptibility Testing of Aerococcus urinae to Fluoroquinolones

Journal of Clinical Microbiology ◽

10.1128/jcm.00259-21 ◽

2021 ◽

Author(s):

France Emilie Roy ◽

Tammy Berteau ◽

Julie Bestman-Smith ◽

Simon Grandjean Lapierre ◽

Simon Frédéric Dufresne ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Reference Method ◽

Error Rates ◽

Antimicrobial Susceptibility Testing ◽

Diffusion Method ◽

Sheep Blood ◽

Gradient Diffusion ◽

Reference Methods ◽

Aerococcus Urinae

Aerococcus urinae is a urinary pathogen with well-described resistance to fluoroquinolones. This study aimed to validate the gradient diffusion (GD) method (Etest®) on cation-adjusted Mueller-Hinton agar with 5% sheep blood for Aerococcus urinae antimicrobial susceptibility testing (AST) to ciprofloxacin and levofloxacin and compare it to the broth microdilution (BMD) method from CLSI M45-A3. Agar dilution (AD), as recommended by EUCAST, was used as an alternate reference method to arbitrate discrepancies or address technical issues. Aerococcus urinae isolates from urinary specimens were prospectively collected between June 2016 and December 2017 from six Quebec hospitals (Canada) and identifications were confirmed using Vitek MS® with IVD 3.0 database. Of the 207 isolates tested using BMD, 37 (17.9%) showed trailing and 19 (9.2%) showed insufficient growth and were tested using AD. Also, 38 isolates (18.4%) for ciprofloxacin and 13 isolates (6.3%) for levofloxacin showed a lack of essential or categorical agreement between Etest® and BMD and were also tested by AD. Using a combined reference method (BMD or AD), susceptibility rate of Aerococcus urinae was 82.6% and 81.6% for ciprofloxacin and levofloxacin, respectively. Categorial agreement between GD and the combined reference methods was 95.2% for ciprofloxacin and 97.1% for levofloxacin, with no very major error identified. Major and minor error rates were 0.6% and 4.3% for ciprofloxacin, and 1.2% and 1.9% for levofloxacin, respectively. Overall, AST using Etest® on sheep blood agar showed a good agreement with reference methods and can be considered by clinical laboratories wishing to perform AST on Aerococcus urinae isolates.

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