Evaluation of susceptibility testing methods for Burkholderia cepacia complex: a comparison of broth microdilution, agar dilution, gradient strip and EUCAST disc diffusion

Four colistin susceptibility testing methods were compared with the standard broth microdilution (BMD) in a collection of 75 colistin-susceptible and 75 mcr-positive E. coli, including ST131 isolates. Taking BMD as reference, all methods showed similar categorical agreement rates (CA) of circa 90%, and a low number of very major errors (VME) (0% for the MicroScan system and Etest®, 0.7% for UMIC®), except for the disc diffusion assay (breakpoint ≤ 11 mm), which yielded false-susceptible results for 8% of isolates. Of note is the number of mcr-positive isolates (17.3%) categorized as susceptible (≤2 mg/L) by the BMD method, but as resistant by the MicroScan system. ST131 mcr-positive E. coli were identified as colistin-resistant by all MIC-based methods. Our results show that applying the current clinical cut-off (>2 mg/L), many mcr-positive E. coli remain undetected, while applying a threshold of >1 mg/L the sensitivity of detection increases significantly without loss of specificity. We propose two possible workflows, both starting with the MicroScan system, since it is automated and, importantly, it categorized all mcr-positive isolates as colistin-resistant. MicroScan should be followed by either BMD or MIC-based commercial methods for colistin resistance detection; or, alternatively, MicroScan, followed by PCR for the mcr screening.

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Performance of Four Fosfomycin Susceptibility Testing Methods against an International Collection of Clinical Pseudomonas aeruginosa Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.01121-20 ◽

2020 ◽

Vol 58 (10) ◽

Author(s):

Elizabeth C. Smith ◽

Hunter V. Brigman ◽

Jadyn C. Anderson ◽

Christopher L. Emery ◽

Tiffany E. Bias ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Susceptibility Testing ◽

Wide Spectrum ◽

Multidrug Resistant ◽

Supporting Evidence ◽

Broth Microdilution ◽

Testing Methods ◽

Content Type ◽

E Coli ◽

Agar Dilution

ABSTRACT Fosfomycin has been shown to have a wide spectrum of activity against multidrug-resistant Gram-negative bacteria; however, breakpoints have been established only for Escherichia coli or Enterobacterales per the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), respectively. A lack of additional organism breakpoints limits clinical use of this agent and has prompted extrapolation of these interpretive categories to other organisms like Pseudomonas aeruginosa without supporting evidence. Further complicating the utility of fosfomycin is the specified method for MIC determination, namely, agar dilution, which is not widely available and is both labor and time intensive. We therefore sought to determine the susceptibility of a large international collection of P. aeruginosa isolates (n = 198) to fosfomycin and to compare testing agreement rates across four methods: agar dilution, broth microdilution, disk diffusion, and Etest. Results were interpreted according to CLSI E. coli breakpoints, with 49.0 to 85.8% considered susceptible, dependent upon the testing method used. Epidemiological cutoff values were calculated and determined to be 256 μg/ml and 512 μg/ml for agar dilution and broth microdilution, respectively. Agreement rates were analyzed using both agar dilution and broth microdilution with a resulting high essential agreement rate of 91.3% between the two susceptibility testing methods. These results indicate that broth microdilution may be a reliable method for fosfomycin susceptibility testing against P. aeruginosa and stress the need for P. aeruginosa-specific breakpoints.

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Comparison of Agar Dilution, Disk Diffusion, MicroScan, and Vitek Antimicrobial Susceptibility Testing Methods to Broth Microdilution for Detection of Fluoroquinolone-Resistant Isolates of the FamilyEnterobacteriaceae

Journal of Clinical Microbiology ◽

10.1128/jcm.37.3.544-547.1999 ◽

1999 ◽

Vol 37 (3) ◽

pp. 544-547 ◽

Cited By ~ 14

Author(s):

Christine D. Steward ◽

Sheila A. Stocker ◽

Jana M. Swenson ◽

Caroline M. O’Hara ◽

Jonathan R. Edwards ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Error Rates ◽

Disk Diffusion ◽

Fluoroquinolone Resistance ◽

Broth Microdilution ◽

Major Error ◽

Testing Methods ◽

Minor Error ◽

Agar Dilution

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the familyEnterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within ±1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.

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In vitro susceptibility of Burkholderia cepacia complex isolates: Comparison of disk diffusion, Etest®, agar dilution, and broth microdilution methods

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2016.08.015 ◽

2016 ◽

Vol 86 (4) ◽

pp. 422-427 ◽

Cited By ~ 6

Author(s):

Lorena Cristina Corrêa Fehlberg ◽

Adriana Gianinni Nicoletti ◽

Ana Carolina Ramos ◽

Fernanda Rodrigues-Costa ◽

Adriana Pereira de Matos ◽

...

Keyword(s):

Burkholderia Cepacia ◽

Burkholderia Cepacia Complex ◽

Disk Diffusion ◽

Broth Microdilution ◽

In Vitro Susceptibility ◽

Agar Dilution

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Comparative evaluation of susceptibility testing methods for colistin and polymyxin B among clinical isolates of carbapenem- resistant Klebsiella pneumoniae and Acinetobacter baumannii

The Journal of Infection in Developing Countries ◽

10.3855/jidc.9660 ◽

2018 ◽

Vol 12 (06) ◽

pp. 504-507 ◽

Cited By ~ 2

Author(s):

Yamuna Devi Bakthavatchalam ◽

Abirami Shankar ◽

Bhuvaneswari Thukaram ◽

Dhanabhagyam Naveena Krishnan ◽

Balaji Veeraraghavan

Keyword(s):

Comparative Evaluation ◽

Susceptibility Testing ◽

Reference Method ◽

Polymyxin B ◽

Disc Diffusion ◽

Broth Microdilution ◽

Testing Methods ◽

Carbapenem Resistant ◽

Clinical Laboratories ◽

Vitek 2

Susceptibility testing (ST) of colistin and polymyxin B is challenging. Disc diffusion testing is not reliable for polymyxin ST, because of poor diffusion. Currently, for polymyxin ST, the EUCAST-CLSI joint commission recommending broth microdilution (BMD) as the reference method. In this study, reliability of E-test and Vitek 2 was compared with BMD, using the susceptible breakpoint of ≤ 2μg/ml for both colistin and polymyxin B. Overall, essential agreement (EA) for colistin between E-test, Vitek2 and BMD were 37% and 74% respectively. EA for polymyxin B between E-test and BMD were 65%. Very major error (VME) for colistin and polymyxin B with E-test were 42% and 55% respectively. An unacceptable VME of 11% was seen for colistin with Vitek2. Major errors (MEs) were rather limited with both E-test and Vitek2. E-test and Vitek2 may lead to inappropriate decision-making for colistin/polymyxin B therapy. Thus, clinical laboratories should consider BMD for polymyxin ST.

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Detection of Colistin Resistance in Carbapenem Resistant Enterobacteriaceae by Reference Broth Microdilution and Comparative Evaluation of Three Other Methods

Journal of Laboratory Physicians ◽

10.1055/s-0041-1731137 ◽

2021 ◽

Author(s):

Punyatoya Kar ◽

Bijayini Behera ◽

Srujana Mohanty ◽

Jayanti Jena ◽

Ashoka Mahapatra

Keyword(s):

Susceptibility Testing ◽

Good Alternative ◽

Broth Microdilution ◽

Major Error ◽

Colistin Resistance ◽

Carbapenem Resistant ◽

Agar Dilution ◽

Moderate Sensitivity ◽

Phenotypic Methods ◽

User Friendly

Abstract Objective Challenges in susceptibility testing of colistin along with increase in the prevalence of colistin-resistant carbapenemase-producing Enterobacteriaceae (CRE) pathogens needs addressal. Evaluation of user-friendly methods is necessary as an alternative to broth microdilution (BMD), the reference susceptibility testing method, for routine implementation in diagnostic clinical microbiology laboratories. Genotypic detection of the plasmid-mediated colistin resistance is also needed for infection control purposes. Materials and Methods Colistin susceptibility of 200 nonduplicate clinical CRE isolates from December 2017 to June 2019 was determined by BMD, agar dilution (AD), E test, and rapid polymyxin NP test and interpreted as per the European Committee on Antimicrobial Susceptibility Testing. The results of AD, E test, and NP test were compared with that of BMD, considering minimal inhibitory concentration (MIC) ≤ 2 µg/mL as susceptible and > 2 µg/mL as resistant. Presence of any plasmid-mediated colistin resistance (mcr-1 and 2) was evaluated in 27 colistin-resistant CRE isolates by polymerase chain reaction. Statistical Analysis Performance of different phenotypic methods was analyzed by comparing MIC results of AD and E test with that of reference BMD method. Agreement between BMD and the other two methods was expressed in terms of categorical agreement and essential agreement. Errors were expressed as very major error (VME: false-susceptible) and major error (ME: false-resistance) by AD/E test. VME and ME of 3% disagreement were considered unacceptable. Results Colistin resistance was found in 27 (13.5%) isolates by BMD method. The VME rates of both AD (11%) and E test (37%) could not meet the Clinical and Laboratory Standards Institute recommendation (< 3% VME rate is acceptable) as alternative tests to the reference BMD. Colistin NP test showed sensitivity and specificity of 85% and 98%, respectively. The percentage discordant result in NP test was highest in Enterobacter spp. (17%). None of the 27 colistin resistant isolates showed presence of mcr-1 and mcr-2 genes. Conclusion High VME rate in AD and E tests precludes their use as alternatives to BMD for colistin susceptibility testing. NP test with moderate sensitivity but excellent specificity can be a good alternative for testing colistin susceptibility in CRE isolates, except in Enterobacter spp. Absence of mcr-1 and mcr-2 gene necessitates the exploration of other mechanisms of colistin resistance.

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Evaluation of methods for detection of β-lactamase production in MSSA

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab032 ◽

2021 ◽

Author(s):

Robert Skov ◽

David R Lonsway ◽

Jesper Larsen ◽

Anders Rhod Larsen ◽

Jurgita Samulioniené ◽

...

Keyword(s):

Susceptibility Testing ◽

Confirmatory Test ◽

Disc Diffusion ◽

Broth Microdilution ◽

Confirmatory Tests ◽

Correct Determination ◽

Primary Test ◽

Zone Edge ◽

Evaluation Of Methods

Abstract Objectives Correct determination of penicillin susceptibility is pivotal for using penicillin in the treatment of Staphylococcus aureus infections. This study examines the performance of MIC determination, disc diffusion and a range of confirmatory tests for detection of penicillin susceptibility in S. aureus. Methods A total of 286 consecutive penicillin-susceptible S. aureus blood culture isolates as well as a challenge set of 62 MSSA isolates were investigated for the presence of the blaZ gene by PCR and subjected to penicillin-susceptibility testing using broth microdilution MIC determination, disc diffusion including reading of the zone edge, two nitrocefin tests and the cloverleaf test. Results Using PCR-based detection of blaZ as the gold standard, both broth microdilution MIC testing and disc diffusion testing resulted in a relatively low accuracy (82%–93%) with a sensitivity ranging from 49%–93%. Among the confirmatory tests, the cloverleaf test performed with 100% accuracy, while zone edge interpretation and nitrocefin-based tests increased the sensitivity of β-lactamase detection to 96%–98% and 82%–96% when using MIC determination or disc diffusion as primary test, respectively. Conclusions This investigation showed that reliable and accurate detection of β-lactamase production in S. aureus can be obtained by MIC determination or penicillin disc diffusion followed by interpretation of the zone edge as a confirmatory test for apparently penicillin-susceptible isolates. The more cumbersome cloverleaf test can also be used. Nitrocefin-based tests should not be used as the only test for confirmation of a presumptive β-lactamase-negative isolate.

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Antimicrobial Susceptibility Testing for Staphylococcus lugdunensis

Journal of Clinical Microbiology ◽

10.1128/jcm.03202-20 ◽

2021 ◽

Author(s):

Joanne S.K. Teh ◽

Ioanna Pantelis ◽

Xiao Chen ◽

Tania Sadlon ◽

Kelly Papanaoum ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Resistant Isolate ◽

Disc Diffusion ◽

Broth Microdilution ◽

Major Error ◽

Staphylococcus Lugdunensis ◽

Zone Diameter ◽

Oxacillin Resistance ◽

Zone Edge

Evaluation of penicillin and oxacillin susceptibility testing was conducted on two hundred Staphylococcus lugdunensis isolates. Disc diffusion with penicillin 1 IU (P1, EUCAST) and penicillin 10 IU (P10, CLSI) was compared with nitrocefin discs (Cefinase®) and automated broth microdilution (Vitek2®). Oxacillin susceptibility was extrapolated from cefoxitin 30μg disc diffusion (FOX) and compared with Vitek2®. Reference methods were blaZ and mecA PCR. Penicillin zone diameter and zone edge correlated with blaZ in all except two P10 susceptible isolates (VME; very major error) and one P1 resistant isolate (ME). One hundred and forty-eight isolates were blaZ -negative of which one hundred and forty-six and one hundred and forty-nine isolates were susceptible by P1 and P10 respectively. One hundred and twenty-seven isolates were penicillin susceptible by Vitek2®. Vitek2® overcalled resistance in twenty-one blaZ -negative, twenty P1 and twenty-two P10 susceptible isolates (Vitek2® ME rate, 14.2%). Two mecA -positive isolates were oxacillin resistant by FOX and Vitek2® (categorical agreement). However, eighteen FOX susceptible, mecA -negative isolates tested resistant by Vitek2®. In conclusion, Vitek2® over-estimated penicillin and oxacillin resistance compared with disc diffusion and PCR. Disc diffusion with zone edge interpretation was more accurate and specific than automated broth microdilution for S. lugdunensis in our study.

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Variation in erythromycin and clindamycin susceptibilities of Streptococcus pneumoniae by four test methods.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.1.129 ◽

1997 ◽

Vol 41 (1) ◽

pp. 129-134 ◽

Cited By ~ 39

Author(s):

E L Fasola ◽

S Bajaksouzian ◽

P C Appelbaum ◽

M R Jacobs

Keyword(s):

Streptococcus Pneumoniae ◽

Susceptibility Testing ◽

Ambient Air ◽

Test Methods ◽

Disk Diffusion ◽

Broth Microdilution ◽

Microdilution Method ◽

Broth Microdilution Method ◽

Agar Dilution ◽

Resistant Strains

Susceptibilities of 124 strains of Streptococcus pneumoniae to erythromycin and clindamycin were determined by the National Committee for the Clinical Laboratory Standards (NCCLS) broth microdilution method, with incubation for 20 to 24 h in ambient air and with modifications of this method by incubation for up to 48 h in air and CO2. Strains were also tested by agar dilution, E-test, and disk diffusion; good correlation was obtained with these methods, with clear separation into bimodal populations of susceptible and resistant stains. The broth microdilution method, however, using incubation in air for 24 h (NCCLS method), misclassified 4 of 92 erythromycin-resistant strains (1 as susceptible and 3 as intermediate) and 25 of 58 clindamycin-resistant strains (all as susceptible). With the exception of one strain with clindamycin, susceptible and resistant strains were correctly classified by the microdilution method with incubation in CO2 for 24 h or in ambient air for 48 h. Disk diffusion, agar dilution, and E-test methods with incubation in 5% CO2 are therefore reliable methods for susceptibility testing of pneumococci against these agents. However, the NCCLS microdilution method, which specifies incubation for 20 to 24 h in ambient air, produced significant very major errors (43%) clindamycin. Modification of the microdilution method by incubation in 5% CO2 or by extension of incubation time in ambient air to 48 h corrected these errors. Disk diffusion, however, was shown to be a simple, convenient, and reliable method for susceptibility testing of pneumococci to erythromycin and clindamycin and is suggested as the method of choice for these agents.

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