scholarly journals In vitro selection of aztreonam/avibactam resistance in dual-carbapenemase-producing Klebsiella pneumoniae

2019 ◽  
Vol 75 (3) ◽  
pp. 559-565 ◽  
Author(s):  
Siqiang Niu ◽  
Jie Wei ◽  
Chunhong Zou ◽  
Kalyan D Chavda ◽  
Jingnan Lv ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
In Vitro Selection ◽  
Fold Increase ◽  
Single Step ◽  
Mutant Selection ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Resistant Mutants ◽  
Selection Of

Abstract Objectives To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. Methods The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. Results We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. Conclusions Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.

scholarly journals In Vitro Selection of Resistance to Clinafloxacin, Ciprofloxacin, and Trovafloxacin in Streptococcus pneumoniae

2000 ◽  
Vol 44 (10) ◽  
pp. 2740-2746 ◽  
Author(s):  
Kensuke Nagai ◽  
Todd A. Davies ◽  
Glenn A. Pankuch ◽  
Bonifacio E. Dewasse ◽  
Michael R. Jacobs ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Single Step ◽  
Mutation Rates ◽  
Mutant Strains ◽  
Efflux Mechanism ◽  
Resistant Mutants ◽  
Time Kill ◽  
Selection Of ◽  
Selection Of Resistance

ABSTRACT Ability of daily sequential subcultures in subinhibitory concentrations of clinafloxacin, ciprofloxacin, and trovafloxacin to select resistant mutants was studied in 10 pneumococci (ciprofloxacin MICs, 1 to 4 μg/ml, and clinafloxacin and trovafloxacin MICs, 0.06 to 0.125 μg/ml [n = 9]; ciprofloxacin, clinafloxacin, and trovafloxacin MICs, 32, 0.5, and 2 μg/ml, respectively [n = 1]). Subculturing was done 50 times, or until MICs increased fourfold or more. Mutants for which MICs were fourfold (or more) higher than those for parent strains were selected in five strains by clinafloxacin, in six strains by trovafloxacin, and nine strains by ciprofloxacin. Sequence analysis of type II topoisomerase showed that most mutants had mutations in ParC at Ser79 or Asp83 and in GyrA at Ser81, while a few mutants had mutations in ParE or GyrB. In the presence of reserpine, the MICs of ciprofloxacin and clinafloxacin for most mutants were lower (four to eight times lower), but for none of the mutants were trovafloxacin MICs lower, suggesting an efflux mechanism affecting the first two agents but not trovafloxacin. Single-step mutation rates were also determined for eight strains for which the MICs were as follows: 0.06 μg/ml (clinafloxacin), 0.06 to 0.125 μg/ml (trovafloxacin), and 1 μg/ml (ciprofloxacin). Single-step mutation rates with drugs at the MIC were 2.0×10−9 to <1.1×10−11, 5.0×10−4 to 3.6×10−9, and 4.8×10−4 to 6.7×10−9, respectively. For two strains with clinafloxacin MICs of 0.125 to 0.5 μg/ml trovafloxacin MICs of 0.125 to 2 μg/ml, ciprofloxacin MICs of 4 to 32 μg/ml mutation rates with drugs at the MIC were 1.1×10−8−9.6×10−8, 3.3×10−6−6.7×10−8, and 2.3×10−5−2.4×10−7, respectively. Clinafloxacin was bactericidal at four times the MIC after 24 h against three parent and nine mutant strains by time-kill study. This study showed that single and multistep clinafloxacin exposure selected for resistant mutants less frequently than similar exposures to other drugs studied.

scholarly journals In Vitro Selection of Resistance in Haemophilus influenzae by Amoxicillin-Clavulanate, Cefpodoxime, Cefprozil, Azithromycin, and Clarithromycin

2002 ◽  
Vol 46 (9) ◽  
pp. 2956-2962 ◽  
Author(s):  
Catherine Clark ◽  
Bülent Bozdogan ◽  
Mihaela Peric ◽  
Bonifacio Dewasse ◽  
Michael R. Jacobs ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
In Vitro Selection ◽  
Fold Increase ◽  
Single Step ◽  
23S Rrna ◽  
Gene Encoding ◽  
Amoxicillin Clavulanate

ABSTRACT Abilities of amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin to select resistant mutants of Haemophilus influenzae were tested by multistep and single-step methodologies. For multistep studies, 10 random strains were tested: 5 of these were β-lactamase positive. After 50 daily subcultures in amoxicillin-clavulanate, MICs did not increase more than fourfold. However, cefprozil MICs increased eightfold for one strain. Clarithromycin and azithromycin gave a >4-fold increase in 8 and 10 strains after 14 to 46 and 20 to 50 days, respectively. Mutants selected by clarithromycin and azithromycin were associated with mutations in 23S rRNA and ribosomal proteins L4 and L22. Three mutants selected by clarithromycin or azithromycin had alterations in ribosomal protein L4, while five had alterations in ribosomal protein L22. Two mutants selected by azithromycin had mutations in the gene encoding 23S rRNA: one at position 2058 and the other at position 2059 (Escherichia coli numbering), with replacement of A by G. One clone selected by clarithromycin became hypersusceptible to macrolides. In single-step studies azithromycin and clarithromycin had the highest mutation rates, while amoxicillin-clavulanate had the lowest. All resistant clones were identical to parents as observed by pulsed-field gel electrophoresis. The MICs of azithromycin for azithromycin-resistant clones were 16 to >128 μg/ml, and those of clarithromycin for clarithromycin-resistant clones were 32 to >128 μg/ml in multistep studies. For strains selected by azithromycin, the MICs of clarithromycin were high and vice versa. After 50 daily subcultures in the presence of drugs, MICs of amoxicillin-clavulanate and cefpodoxime against H. influenzae did not rise more than fourfold, in contrast to cefprozil, azithromycin, and clarithromycin, whose MICs rose to variable degrees.

scholarly journals Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

2002 ◽  
Vol 46 (11) ◽  
pp. 3370-3380 ◽  
Author(s):  
Dilek Ince ◽  
Xiamei Zhang ◽  
L. Christine Silver ◽  
David C. Hooper
Keyword(s):  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Fold Increase ◽  
Single Step ◽  
The Novel ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dual Targeting ◽  
Resistant Mutants ◽  
Selection Of

ABSTRACT We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.

scholarly journals Influence of Inoculum Size on the Selection of Resistant Mutants of Escherichia coli in Relation to Mutant Prevention Concentrations of Marbofloxacin

2007 ◽  
Vol 51 (11) ◽  
pp. 4163-4166 ◽  
Author(s):  
Aude Ferran ◽  
Véronique Dupouy ◽  
Pierre-Louis Toutain ◽  
Alain Bousquet-Mélou
Keyword(s):  
Escherichia Coli ◽  
Bacterial Population ◽  
Inoculum Size ◽  
Pharmacodynamic Model ◽  
Initial Size ◽  
Mutant Selection ◽  
Selection Window ◽  
Resistant Mutants ◽  
Selection Of

ABSTRACT We demonstrate using an in vitro pharmacodynamic model that the likelihood of selection of Escherichia coli mutants resistant to a fluoroquinolone was increased when the initial size of the bacterial population, exposed to fluoroquinolone concentrations within the mutant selection window, was increased.

scholarly journals KPC-mediated resistance to ceftazidime-avibactam and collateral effects in Klebsiella pneumoniae

2021 ◽  
Author(s):  
Jacqueline Findlay ◽  
Laurent Poirel ◽  
Mario Juhas ◽  
Patrice Nordmann
Keyword(s):  
Klebsiella Pneumoniae ◽  
Broad Spectrum ◽  
In Vitro Selection ◽  
Multidrug Resistant ◽  
Single Step ◽  
Carbapenem Resistant ◽  
Rapid Spread ◽  
Collateral Effects ◽  
Novel Drug

Carbapenem-resistant Enterobacterales, such as KPC-producing Klebsiella pneumoniae , represent a major threat to public health due to their rapid spread. Novel drug combinations such as ceftazidime-avibactam (CZA), combining a broad-spectrum cephalosporin along with a broad-spectrum ß-lactamase inhibitor, have recently been introduced and have been shown to exhibit excellent activity towards multidrug-resistant KPC-producing Enterobacterale strains. However, CZA-resistant K. pneumoniae isolates are now being increasingly reported, mostly corresponding to producers of KPC variants. In this study, we evaluated in vitro the nature of the mutations in the KPC-2 and KPC-3 ß-lactamase sequences (the most frequent KPC-type enzymes) that lead to CZA resistance, and the subsequent effects of these mutations on susceptibility to other ß-lactam antibiotics. Single-step in vitro selection assays were conducted resulting in the identification of a series of mutations in the KPC sequence which conferred the ability to those mutated enzymes to confer resistance to CZA. Hence, 16 KPC-2 variants and 10 KPC-3 variants were obtained. Production of the KPC variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to broad-spectrum cephalosporins and carbapenems, with the exceptions of ceftazidime and piperacillin-tazobactam, compared to wild-type KPC enzymes. Enzymatic assays showed that all of the KPC variants identified exhibited an increased affinity toward ceftazidime and a slightly decreased sensitivity to avibactam, sustaining their impact on CZA resistance. However their respective carbapenemase activities were concurrently negatively impacted.

2-Tier Bacterial and In Vitro Selection of Active and Methotrexate-Resistant Variants of Human Dihydrofolate Reductase

2008 ◽  
Vol 13 (6) ◽  
pp. 504-514 ◽  
Author(s):  
Elena Fossati ◽  
Jordan P. Volpato ◽  
Lucie Poulin ◽  
Vanessa Guerrero ◽  
David-Antoine Dugas ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Drug Target ◽  
In Vitro Selection ◽  
Fold Increase ◽  
Active Site Residues ◽  
20 Nm ◽  
Human Dihydrofolate Reductase ◽  
Selection Of

We report a rapid and reliable 2-tier selection and screen for detection of activity as well as drug-resistance in mutated variants of a clinically-relevant drug-target enzyme. Human dihydrofolate reductase point-mutant libraries were subjected to a 1st-tier bacterial complementation assay, such that bacterial propagation served as an indicator of enzyme activity. Alternatively, when selection was performed in the presence of the inhibitor methotrexate (MTX), propagation indicated MTX resistance. The selected variants were then subjected to a 2nd-tier in vitro screen in 96-well plate format using crude bacterial lysate. Conditions were defined to establish a threshold for activity or for MTX resistance. The 2nd-tier assay allowed rapid detection of the best variants among the leads and provided reliable estimates of relative reactivity, ( kcat) and IC50MTX. Screening saturation libraries of active-site positions 7, 15, 24, 70, and 115 revealed a variety of novel mutations compatible with reactivity as well as 2 novel MTX-resistant variants: V115A and V115C. Both variants displayed KiMTX = 20 nM, a 600-fold increase relative to the wild-type. We also present preliminary results from screening against further antifolates following simple modifications of the protocol. The flexibility and robustness of this method will provide new insights into interactions between ligands and active-site residues of this clinically relevant human enzyme. ( Journal of Biomolecular Screening 2008:504-514)

scholarly journals Searching for the Optimal Predictor of Ciprofloxacin Resistance in Klebsiella pneumoniae by UsingIn VitroDynamic Models

2015 ◽  
Vol 60 (3) ◽  
pp. 1208-1215 ◽  
Author(s):  
Elena N. Strukova ◽  
Yury A. Portnoy ◽  
Andrey V. Romanov ◽  
Mikhail V. Edelstein ◽  
Stephen H. Zinner ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Area Under The Curve ◽  
Mutant Selection ◽  
Content Type ◽  
Selection Window ◽  
Mutant Prevention Concentration ◽  
Ciprofloxacin Resistance ◽  
Resistant Mutants ◽  
Time Courses ◽  
Selection Of

There is growing evidence of applicability of the hypothesis of the mutant selection window (MSW), i.e., the range between the MIC and the mutant prevention concentration (MPC), within which the enrichment of resistant mutants is most probable. However, it is not clear if MPC-based pharmacokinetic variables are preferable to the respective MIC-based variables as interstrain predictors of resistance. To examine the predictive power of the ratios of the area under the curve (AUC24) to the MPC and to the MIC, the selection of ciprofloxacin-resistant mutants of threeKlebsiella pneumoniaestrains with different MPC/MIC ratios was studied. Each organism was exposed to twice-daily ciprofloxacin for 3 days at AUC24/MIC ratios that provide peak antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC. ResistantK. pneumoniaemutants were intensively enriched at an AUC24/MIC ratio of 60 to 360 h (AUC24/MPC ratio from 2.5 to 15 h) but not at the lower or higher AUC24/MIC and AUC24/MPC ratios, in accordance with the MSW hypothesis. AUC24/MPC and AUC24/MIC relationships with areas under the time courses of ciprofloxacin-resistantK. pneumoniae(AUBCM) were bell shaped. These relationships predict highly variable “antimutant” AUC24/MPC ratios (20 to 290 h) compared to AUC24/MIC ratios (1,310 to 2,610 h). These findings suggest that the potential of the AUC24/MPC ratio as an interstrain predictor ofK. pneumoniaeresistance is lower than that of the AUC24/MIC ratio.

scholarly journals CTX-M Expression and Selection of Ertapenem Resistance in Klebsiella pneumoniae and Escherichia coli

2008 ◽  
Vol 53 (2) ◽  
pp. 832-834 ◽  
Author(s):  
Delphine Girlich ◽  
Laurent Poirel ◽  
Patrice Nordmann
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
In Vitro Selection ◽  
Gene Frequencies ◽  
Clinical Strains ◽  
Selection Of

ABSTRACT In vitro selection of mutants with decreased susceptibility to ertapenem was performed using Escherichia coli and Klebsiella pneumoniae clinical strains producing either the bla CTX-M-2, bla CTX-M-3, bla CTX-M-9, or bla CTX-M-15 gene. Frequencies of mutants with decreased susceptibilities to ertapenem were similar for all β-lactamases expressed.

scholarly journals In Vitro Evaluation of the activity of Ceftazidime-avibactam and Aztreonam-Avibactam against 76 Stenotrophomonas maltophilia isolates from a teaching hospital in Chongqing

2020 ◽  
Author(s):  
Qiuxia Lin ◽  
Hua Zou ◽  
Xian Chen ◽  
Menglu Wu ◽  
Deyu Ma ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Combined Therapy ◽  
Treatment Options ◽  
Clinical Isolates ◽  
Broth Microdilution ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Medical University ◽  
Selection Of

Abstract Background: Treatment options for Stenotrophomonas maltophilia (S. maltophilia) infections were limited. We assessed the efficacy of ceftazidime-avibactam (CAZ-AVI) and aztreonam-avibactam (ATM-AVI) against a selection of 76 S. maltophilia out of the 1179 strains isolated from the First Affiliated Hospital of Chongqing Medical University during 2011-2018. Methods: We investigated the antimicrobial resistance profiles of the 1179 S. maltophilia clinical isolates from the first affiliated hospital of Chongqing Medical University during 2011-2018, a collection of 76 isolates of which were available for further study of microbiological characterization. Minimum inhibitory concentrations (MICs) of ceftazidime (CAZ), ceftazidime-avibactam (CAZ-AVI), aztreonam (ATM) and aztreonam-avibactam (ATM-AVI) were determined via the broth microdilution method. We deemed that CAZ-AVI or ATM-AVI was more effective in vitro than CAZ or ATM alone when CAZ-AVI or ATM-AVI led to a category change from “Resistant” with CAZ or ATM alone to “Susceptible” or “Intermediate” with CAZ-AVI or ATM-AVI, or if the MIC of CAZ-AVI or ATM-AVI was at least 2-fold lower than the MIC of CAZ or ATM alone. Results: For the 76 clinical isolates included in the study, MICs of CAZ, ATM, CAZ-AVI and ATM-AVI ranged from 0.03-64, 1-1024, 0.016-64, and 0.06-64 μg/mL, respectively. In combined therapy, AVI was effective at restoring the susceptibility of 48.48% (16/33) and 89.71% (61/68) of S. maltophilia to CAZ and ATM, respectively. Furthermore, CAZ-AVI showed better results in terms of the proportion of susceptible isolates (77.63% vs.56.58%, P<0.001), MIC50 (2μg/mL vs. 8μg/mL, P<0.05), and MIC distribution (P<0.001) when compared to CAZ. According to our definition, CAZ-AVI was more effective in vitro than CAZ alone for 84.21% of the isolates. Similarly, ATM-AVI also showed better results in terms of the proportion of susceptible isolates (90.79%vs. 10.53%, P<0.001), MIC50 (2μg/mL vs. 64μg/mL, P<0.001), and MIC distribution (P<0.001) when compared to ATM. According to our definition, ATM-AVI was also more effective in vitro than ATM alone for 97.37% of the isolates. Conclusions: AVI potentiated the activity of both CAZ and ATM against S. maltophilia clinical isolates in vitro. We demonstrated that CAZ-AVI and ATM-AVI are both useful therapeutic options to treat infections caused by S. maltophilia.

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