scholarly journals The Role of fosA in Challenges with Fosfomycin Susceptibility Testing of Multispecies Klebsiella pneumoniae Carbapenemase-Producing Clinical Isolates

2019 ◽  
Vol 57 (10) ◽  
Author(s):  
Zachary S. Elliott ◽  
Katie E. Barry ◽  
Heather L. Cox ◽  
Nicole Stoesser ◽  
Joanne Carroll ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Clinical Microbiology Laboratory ◽  
Content Type ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Agar Dilution ◽  
Species Specific ◽  
The Impact

ABSTRACT With multidrug-resistant (MDR) Enterobacterales on the rise, a nontoxic antimicrobial agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-Escherichia coli isolates in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with 96 KPC-producing clinical isolates of multiple strains and species collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole-genome sequencing (WGS) data using NCBI’s AMRFinder and custom HMM search. Susceptibility testing was performed using a glucose-6-phosphate-supplemented fosfomycin Etest and Kirby-Bauer disk diffusion (DD) assays, and the results were compared to those obtained by agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by Etest, 70% (67/96) by DD, and 88% (84/96) by agar dilution. fosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA positive compared to those that were fosA negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes.

scholarly journals The Role offosAin Challenges with Fosfomycin Susceptibility Testing of MultispeciesKlebsiella pneumoniaeCarbapenemase-Producing Clinical Isolates

2019 ◽  
Author(s):  
Zachary S. Elliott ◽  
Katie E. Barry ◽  
Heather L. Cox ◽  
Nicole Stoesser ◽  
Joanne Carroll ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Clinical Microbiology Laboratory ◽  
Toxic Agent ◽  
E Coli ◽  
Agar Dilution ◽  
Species Specific ◽  
The Impact

AbstractWith multidrug resistant (MDR) Enterobacteriales on the rise, a non-toxic agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-E. coliin a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with multiple strains and species of KPC-producing clinical isolates collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole genome sequencing (WGS) data using NCBI’s AMRFinder and custom HMM search. Susceptibility testing was performed using glucose-6-phosphate supplemented fosfomycin E-Test and Kirby-Bauer disk diffusion (DD) assays, and compared to agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints forE. coliwere applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by E-Test, 70% (67/96) by DD, and 88% (84/96) by agar dilution.FosAwas detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, andfosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibitfosAand showed significant increases in the zone diameter of DD testing for isolates that werefosA-positive compared tofosA-negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate thatfosAinfluences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory when compared to agar dilution. Further research is needed to determine the impact offosAon clinical outcomes.

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 Variability in Azithromycin Susceptibility Results for Neisseria gonorrhoeae Obtained Using Gradient MIC Strip and Agar Dilution Techniques

2019 ◽  
Vol 57 (12) ◽  
Author(s):  
Gary N. McAuliffe ◽  
Marian Smith ◽  
Gavin Cooper ◽  
Rose F. Forster ◽  
Sally A. Roberts
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Susceptibility Testing ◽  
Geometric Mean ◽  
Test Strip ◽  
Clinical Isolates ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Content Type ◽  
Test Strips ◽  
Agar Dilution

ABSTRACT Azithromycin is a component of empirical treatment regimens for Neisseria gonorrhoeae infections, but antimicrobial susceptibility testing for this agent is technically challenging. We compared the intertest variability, MIC values, and CLSI/EUCAST categorization of clinical and reference isolates of N. gonorrhoeae treated with azithromycin by testing 107 clinical isolates and nine reference isolates by agar dilution and in duplicates using MIC test strips (Liofilchem, Italy) and Etests (bioMérieux, France). Replicate isolate agreement within 1 log2 between duplicate tests was 87% for MIC test strips and 100% for Etests (P < 0.001). Essential agreement with the agar dilution method was higher for Etests (91%) than for MIC test strips (44%, P < 0.001). The geometric mean MIC was highest for MIC test strips (0.8 mg/liter) and significantly higher than both Etest (0.47 mg/liter, P < 0.001) and agar dilution (0.26 mg/liter, P < 0.001) methods. Etest MICs were higher than those obtained with agar dilution (P < 0.001). Agar dilution, MIC test strip, and Etest methods categorized 96%, 85%, and 95% (P = 0.003) of clinical isolates, respectively, as susceptible/wild type according to CLSI/EUCAST criteria. Our results illustrate the difficulties underlying azithromycin susceptibility testing for N. gonorrhoeae and demonstrate that results can vary using different methods. This variability could influence antimicrobial resistance reporting between laboratories involved in N. gonorrhoeae surveillance programs.

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 Impact of Variations in Test Method Parameters onIn VitroActivity of Surotomycin against Clostridium difficile and Surotomycin Quality Control Limits for Broth Microdilution and Agar Dilution Susceptibility Testing

2015 ◽  
Vol 54 (3) ◽  
pp. 749-753 ◽  
Author(s):  
Maria M. Traczewski ◽  
Jennifer Deane ◽  
Daniel Sahm ◽  
Steven D. Brown ◽  
Laurent Chesnel
Keyword(s):  
Quality Control ◽  
Clostridium Difficile ◽  
Calcium Concentration ◽  
Susceptibility Testing ◽  
Test Method ◽  
Test Parameter ◽  
Content Type ◽  
Parameter Variations ◽  
Agar Dilution ◽  
Control Limits

Test parameter variations were evaluated for their effects on surotomycin MICs. Calcium concentration was the only variable that influenced MICs; therefore, 50 μg/ml (standard for lipopeptide testing) is recommended. Quality control ranges forClostridium difficile(0.12 to 1 μg/ml) andEggerthella lenta(broth, 1 to 4 μg/ml; agar, 1 to 8 μg/ml) were approved by the Clinical and Laboratory Standards Institute based on these data.

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 Preexposure to Isavuconazole Increases the Virulence of Mucorales but Not Aspergillus fumigatus in a Drosophila melanogaster Infection Model

2018 ◽  
Vol 63 (2) ◽  
pp. e01896-18 ◽  
Author(s):  
Sebastian Wurster ◽  
Russell E. Lewis ◽  
Nathaniel D. Albert ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Drosophila Melanogaster ◽  
Aspergillus Fumigatus ◽  
Clinical Isolates ◽  
Infection Model ◽  
Content Type ◽  
The Impact

ABSTRACT Breakthrough mucormycosis in patients receiving isavuconazole prophylaxis or therapy has been reported. We compared the impact of isavuconazole and voriconazole exposure on the virulence of clinical isolates of Aspergillus fumigatus and different Mucorales species in a Drosophila melanogaster infection model. In contrast to A. fumigatus, a hypervirulent phenotype was found in all tested Mucorales upon preexposure to either voriconazole or isavuconazole. These findings may contribute to the explanation of breakthrough mucormycosis in isavuconazole-treated patients.

scholarly journals Burkholderia pseudomallei clinical isolates are highly susceptible in vitro to cefiderocol, a novel siderophore cephalosporin

2020 ◽  
Author(s):  
Delaney Burnard ◽  
Gemma Robertson ◽  
Andrew Henderson ◽  
Caitlin Falconer ◽  
Michelle Bauer-Leo ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Transport Systems ◽  
Gram Negative ◽  
Highly Active ◽  
Amoxicillin Clavulanate ◽  
Clinical Breakpoints ◽  
Species Specific

AbstractCefiderocol is a novel cephalosporin designed to treat multidrug resistant Gram-negative infections. By forming a chelated complex with ferric iron, cefiderocol is transported into the periplasmic space via bacterial iron transport systems and primarily binds to penicillin-binding protein 3 (PBP3) to inhibit peptidoglycan synthesis. This mode of action results in cefiderocol having greater in vitro activity against many Gram-negative bacilli than currently used carbapenems, β-lactam/β-lactamase inhibitor combinations, and cephalosporins. Thus, we investigated the in vitro activity of cefiderocol (S-649266) against a total of 271 clinical isolates of Burkholderia pseudomallei from Australia. The collection was comprised of primary isolates (92.3%) and subsequent isolates (7.7%). Minimum inhibitory concentrations (MIC) of cefiderocol ranged from ≤0.03 to 32 mg/L, where the MIC90 was 1 mg/L and 16 mg/L for primary and subsequent isolates, respectively. Based upon non-species specific (Gram-negative bacilli) clinical breakpoints for cefiderocol (MIC ≤ 4 mg/L), twelve isolates (4.4%) would be classified as non-susceptible. Further testing for co-resistance to meropenem, ceftazidime, trimethoprim-sulfamethoxazole, amoxicillin-clavulanate and doxycycline was performed on a subset of isolates with elevated cefiderocol MICs (≥2 mg/L, 4.8%) and 84.6% of these isolates exhibited resistance to at least one of these antimicrobials. Cefiderocol was found to be highly active in vitro against B. pseudomallei primary clinical isolates. This novel compound shows great potential for the treatment of melioidosis in endemic countries and should be explored further.

scholarly journals In Vitro Activity of Sulbactam-Durlobactam against Acinetobacter baumannii-calcoaceticus Complex Isolates Collected Globally in 2016 and 2017

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Sarah M. McLeod ◽  
Samir H. Moussa ◽  
Meredith A. Hackel ◽  
Alita A. Miller
Keyword(s):  
Acinetobacter Baumannii ◽  
Antibacterial Activities ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Content Type ◽  
Level Of Activity ◽  
Severe Infections ◽  
Sources Of Infection

ABSTRACT Acinetobacter baumannii-calcoaceticus complex (ABC) organisms cause severe infections that are difficult to treat due to preexisting antibiotic resistance. Sulbactam-durlobactam (formerly sulbactam-ETX2514) (SUL-DUR) is a β-lactam–β-lactamase inhibitor combination antibiotic designed to treat serious infections caused by ABC organisms, including multidrug-resistant (MDR) strains. The in vitro antibacterial activities of SUL-DUR and comparator agents were determined by broth microdilution against 1,722 clinical isolates of ABC organisms collected in 2016 and 2017 from 31 countries across Asia/South Pacific, Europe, Latin America, the Middle East, and North America. Over 50% of these isolates were resistant to carbapenems. Against this collection of global isolates, SUL-DUR had a MIC50/MIC90 of 1/2 μg/ml compared to a MIC50/MIC90 of 8/64 μg/ml for sulbactam alone. This level of activity was found to be consistent across organisms, regions, sources of infection, and subsets of resistance phenotypes, including MDR and extensively drug-resistant isolates. The SUL-DUR activity was superior to those of the tested comparators, with only colistin having similar potency. Whole-genome sequencing of the 39 isolates (2.3%) with a SUL-DUR MIC of >4 μg/ml revealed that these strains encoded either the metallo-β-lactamase NDM-1, which durlobactam does not inhibit, or single amino acid substitutions near the active site of penicillin binding protein 3 (PBP3), the primary target of sulbactam. In summary, SUL-DUR demonstrated potent antibacterial activity against recent, geographically diverse clinical isolates of ABC organisms, including MDR isolates.

scholarly journals Simultaneous Infection with Enterobacteriaceae and Pseudomonas aeruginosa Harboring Multiple Carbapenemases in a Returning Traveler Colonized with Candida auris

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ayesha Khan ◽  
William C. Shropshire ◽  
Blake Hanson ◽  
An Q. Dinh ◽  
Audrey Wanger ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Susceptibility Testing ◽  
Multidrug Resistant ◽  
Critically Ill Patient ◽  
Whole Genome ◽  
Candida Auris ◽  
Content Type ◽  
Simultaneous Infection ◽  
Polymicrobial Infections ◽  
Selection Of

ABSTRACT We report our clinical experience treating a critically ill patient with polymicrobial infections due to multidrug-resistant Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in a 56-year-old woman who received health care in India and was also colonized by Candida auris. A precision medicine approach using whole-genome sequencing revealed a multiplicity of mobile elements associated with NDM-1, NDM-5, and OXA-181 and, supplemented with susceptibility testing, guided the selection of rational antimicrobial therapy.

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