scholarly journals Comprehensive Statistical Evaluation of Etest®, UMIC®, MicroScan and Disc Diffusion versus Standard Broth Microdilution: Workflow for an Accurate Detection of Colistin-Resistant and Mcr-Positive E. coli

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 861
Author(s):  
Isidro García-Meniño ◽  
Pilar Lumbreras ◽  
Pablo Valledor ◽  
Dafne Díaz-Jiménez ◽  
Luz Lestón ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Statistical Evaluation ◽  
Disc Diffusion ◽  
Broth Microdilution ◽  
Colistin Resistance ◽  
Testing Methods ◽  
Disc Diffusion Assay ◽  
E Coli ◽  
Diffusion Assay ◽  
Microscan System

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.

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

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.

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

2018 ◽  
Vol 12 (06) ◽  
pp. 504-507 ◽  
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.

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

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.

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

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.

scholarly journals Enaminone-Derived Pyrazoles with Antimicrobial Activity

2019 ◽  
Vol 2019 ◽  
pp. 1-10
Author(s):  
Mashooq Ahmad Bhat ◽  
Mohamed A. Al-Omar ◽  
Ahmed M. Naglah ◽  
Abdul Arif Khan
Keyword(s):  
Antimicrobial Activity ◽  
Gram Negative Bacteria ◽  
Significant Activity ◽  
Disc Diffusion ◽  
Initial Screening ◽  
Gram Positive ◽  
Gram Negative ◽  
Disc Diffusion Assay ◽  
Gram Positive Bacteria ◽  
Diffusion Assay

A series of pyrazoles derived from the substituted enaminones were synthesized and were evaluated for antimicrobial activity. All the compounds were characterized by the spectral data and elemental analysis. The synthesized compounds were initially screened for their antimicrobial activity against ATCC 6538, NCTC 10400, NCTC 10418, and ATCC 27853. During initial screening, compounds (P1, P6, and P11) presented significant antimicrobial activity through disc diffusion assay. These compounds were further evaluated for antimicrobial activity at different time points against Gram-positive and Gram-negative bacteria and presented significant activity for 6 hours. The activity was found to be greater against Gram-positive bacteria. In contrast at 24 hours, the activity was found only against Gram-positive bacteria except compound (P11), showing activity against both types of bacteria. Compound (P11) was found to have highest activity against both Gram-positive and Gram-negative bacteria.

scholarly journals An Improved Medium for Colistin Susceptibility Testing

2018 ◽  
Vol 56 (5) ◽  
Author(s):  
Konrad Gwozdzinski ◽  
Saina Azarderakhsh ◽  
Can Imirzalioglu ◽  
Linda Falgenhauer ◽  
Trinad Chakraborty
Keyword(s):  
Susceptibility Testing ◽  
Acquired Resistance ◽  
Multidrug Resistant ◽  
Colistin Resistance ◽  
Reliable Determination ◽  
Content Type ◽  
E Coli ◽  
Resistant Species ◽  
Mueller Hinton ◽  
Serovar Typhimurium

ABSTRACTThe plasmid-located colistin resistance genemcr-1confers low-level resistance to colistin, a last-line antibiotic against multidrug-resistant Gram-negative bacteria. Current CLSI-EUCAST recommendations require the use of a broth microdilution (BMD) method with cation-adjusted Mueller-Hinton (CA-MH) medium for colistin susceptibility testing, but approximately 15% of all MCR-1 producers are classified as sensitive in that broth. Here we report on an improved calcium-enhanced Mueller-Hinton (CE-MH) medium that permits simple and reliable determination ofmcr-1-containingEnterobacteriaceae. Colistin susceptibility testing was performed for 50mcr-1-containingEscherichia coliandKlebsiella pneumoniaeisolates, 7 intrinsically polymyxin-resistant species,K. pneumoniaeandE. coliisolates with acquired resistance to polymyxins due tomgrBandpmrBmutations, respectively, and 32mcr-1-negative, colistin-susceptible isolates ofAcinetobacter baumannii,Citrobacter freundii,Enterobacter cloacae,E. coli,K. pneumoniae, andSalmonella entericaserovar Typhimurium. A comparison of the colistin MICs determined in CA-MH medium and those obtained in CE-MH medium was performed using both the BMD and strip-based susceptibility test formats. We validated the data using an isogenic IncX4 plasmid lackingmcr-1. Use of the CE-MH broth provides clear separation between resistant and susceptible isolates in both BMD and gradient diffusion assays; this is true for bothmcr-1-containingEnterobacteriaceaeisolates and those exhibiting either intrinsic or acquired colistin resistance. CE-MH medium is simple to prepare and overcomes current problems associated with BMD and strip-based colistin susceptibility testing, and use of the medium is easy to implement in routine diagnostic laboratories, even in resource-poor settings.

scholarly journals Agreement Assessment of Tigecycline Susceptibilities Determined by the Disk Diffusion and Broth Microdilution Methods among Commonly Encountered Resistant Bacterial Isolates: Results from the TigecyclineIn VitroSurveillance in Taiwan (TIST) Study, 2008 to 2010

2011 ◽  
Vol 56 (3) ◽  
pp. 1414-1417 ◽  
Author(s):  
Jien-Wei Liu ◽  
Wen-Chien Ko ◽  
Cheng-Hua Huang ◽  
Chun-Hsing Liao ◽  
Chin-Te Lu ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Total Error ◽  
Error Rates ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Broth Microdilution ◽  
Content Type ◽  
E Coli ◽  
Drug Resistant Bacteria

ABSTRACTThe TigecyclineIn VitroSurveillance in Taiwan (TIST) study, initiated in 2006, is a nationwide surveillance program designed to longitudinally monitor thein vitroactivity of tigecycline against commonly encountered drug-resistant bacteria. This study compared thein vitroactivity of tigecycline against 3,014 isolates of clinically important drug-resistant bacteria using the standard broth microdilution and disk diffusion methods. Species studied included methicillin-resistantStaphylococcus aureus(MRSA;n= 759), vancomycin-resistantEnterococcus faecium(VRE;n= 191), extended-spectrum β-lactamase (ESBL)-producingEscherichia coli(n= 602), ESBL-producingKlebsiella pneumoniae(n= 736), andAcinetobacter baumannii(n= 726) that had been collected from patients treated between 2008 and 2010 at 20 hospitals in Taiwan. MICs and inhibition zone diameters were interpreted according to the currently recommended U.S. Food and Drug Administration (FDA) criteria and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. The MIC90values of tigecycline against MRSA, VRE, ESBL-producingE. coli, ESBL-producingK. pneumoniae, andA. baumanniiwere 0.5, 0.125, 0.5, 2, and 8 μg/ml, respectively. The total error rates between the two methods using the FDA criteria were high: 38.4% for ESBL-producingK. pneumoniaeand 33.8% forA. baumannii. Using the EUCAST criteria, the total error rate was also high (54.6%) forA. baumanniiisolates. The total error rates between these two methods were <5% for MRSA, VRE, and ESBL-producingE. coli. For routine susceptibility testing of ESBL-producingK. pneumoniaeandA. baumanniiagainst tigecycline, the broth microdilution method should be used because of the poor correlation of results between these two methods.

Two Novel Mutations Associated with Polymyxin-B Resistance in a Pandemic Lineage of Uropathogenic Escherichia coli of the Sequence Type 69

Chemotherapy ◽  
2021 ◽  
pp. 1-7
Author(s):  
Carla Adriana dos Santos ◽  
Rodrigo Tavanelli Hernandes ◽  
Marcos Paulo Vieira Cunha ◽  
Filipe Onishi Nagamori ◽  
Claudia Regina Gonçalves ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Susceptibility Testing ◽  
Polymyxin B ◽  
Antimicrobial Treatment ◽  
Sequence Type ◽  
Broth Microdilution ◽  
Economic Costs ◽  
Novel Mutations ◽  
E Coli ◽  
Total Inhibition

<b><i>Background:</i></b> Uropathogenic <i>Escherichia coli</i> (UPEC) are frequent pathogens worldwide, impacting on the morbidity and economic costs associated with antimicrobial treatment. <b><i>Objectives:</i></b> We report two novel mutations associated with polymyxin-B resistance in an UPEC isolate collected in 2019. <b><i>Methods:</i></b> Isolate was submitted to antimicrobial susceptibility testing including broth microdilution for polymyxin B. Whole genome was sequenced and analyzed. <b><i>Results:</i></b> Polymyxin-B total inhibition occurred at 16 mg/L (resistant). UPEC isolate was assigned to the phylogroup D, serotype O117:H4, and Sequence Type 69. <i>mcr</i> genes were not detected, but two novel mutations in the <i>pmrA/basS</i> (A80S) and <i>pmrB/</i>basR (D149N) genes were identified. <b><i>Conclusions:</i></b> The occurrence of non-<i>mcr</i> polymyxin resistance in <i>E. coli</i> from extraintestinal infections underscores the need of a continuous surveillance of this evolving pathogen.

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