Dataset Information

Multicenter Evaluation of the Etest Gradient Diffusion Method for Ceftolozane-Tazobactam Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa.

ABSTRACT: Ceftolozane-tazobactam (C/T) is a novel beta-lactam-beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration in 2014 for the treatment of complicated intra-abdominal infections (in combination with metronidazole) and complicated urinary tract infections. In this study, we evaluated the performance of the C/T Etest, a gradient diffusion method. C/T Etest was compared to broth microdilution (BMD) for 51 Enterobacteriaceae challenge isolates and 39 Pseudomonas aeruginosa challenge isolates at three clinical sites. Essential agreement (EA) between the methods ranged from 47 to 49/51 (92.2 to 96.1%) for the Enterobacteriaceae, and categorical agreement (CA) ranged from 49 to 51/51 (96.1 to 100.0%). EA and CA for P. aeruginosa were 100% at all sites. The C/T Etest was also compared to BMD for susceptibility testing on 966 clinical isolates (793 Enterobacteriaceae, including 167 Klebsiella pneumoniae and 159 Escherichia coli isolates, in addition to 173 P. aeruginosa isolates) collected at four clinical sites. EA between Etest and BMD was 96.9% for Enterobacteriaceae isolates and 98.8% for P. aeruginosa isolates. Within the Enterobacteriaceae, isolates from each species examined had >96% CA. For the clinical isolates, no very major errors were identified but two major errors were found (one for K. pneumoniae and one for Providencia rettgeri). By BMD, 47.0% of Enterobacteriaceae and 46.2% of P. aeruginosa challenge strains were nonsusceptible to C/T by CLSI breakpoint criteria; 8.2% of clinical Enterobacteriaceae isolates and 12.1% of clinical P. aeruginosa isolates were nonsusceptible to C/T by CLSI breakpoint criteria. In conclusion, Etest is accurate and reproducible for C/T susceptibility testing of Enterobacteriaceae and P. aeruginosa.

SUBMITTER: Bailey AL

PROVIDER: S-EPMC6113460 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Multicenter Evaluation of the Etest Gradient Diffusion Method for Ceftolozane-Tazobactam Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa.

Bailey Adam L AL Armstrong Tom T Dwivedi Hari-Prakash HP Denys Gerald A GA Hindler Janet J Campeau Shelley S Traczewski Maria M Humphries Romney R Burnham C A CA

Journal of clinical microbiology 20180827 9

Ceftolozane-tazobactam (C/T) is a novel beta-lactam-beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration in 2014 for the treatment of complicated intra-abdominal infections (in combination with metronidazole) and complicated urinary tract infections. In this study, we evaluated the performance of the C/T Etest, a gradient diffusion method. C/T Etest was compared to broth microdilution (BMD) for 51 <i>Enterobacteriaceae</i> challenge isolates and 39 <i ...[more]

PMID: 29976590

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Project description:Resistance mechanisms of Pseudomonas aeruginosa to ceftolozane/tazobactam (C/T) were assessed on a collection of 420 nonredundant strains nonsusceptible to ceftazidime (MIC > 8 μg/ml) and/or imipenem (>4 μg/ml), collected by 36 French hospital laboratories over a one-month period (the GERPA study). Rates of C/T resistance (MIC > 4/4 μg/ml) were equal to 10% in this population (42/420 strains), and 23.2% (26/112) among the isolates resistant to both ceftazidime and imipenem. A first group of 21 strains (50%) was found to harbor various extended-spectrum β-lactamases (1 OXA-14; 2 OXA-19; 1 OXA-35; 1 GES-9; and 3 PER-1), carbapenemases (2 GES-5; 1 IMP-8; and 8 VIM-2), or both (1 VIM-2/OXA-35 and 1 VIM-4/SHV-2a). All the strains of this group belonged to widely distributed epidemic clones (ST111, ST175, CC235, ST244, ST348, and ST654), and were highly resistant to almost all the antibiotics tested except colistin. A second group was composed of 16 (38%) isolates moderately resistant to C/T (MICs from 8/4 to 16/4 μg/ml), of which 7 were related to international clones (ST111, ST253, CC274, ST352, and ST386). As demonstrated by targeted mass spectrometry, cloxacillin-based inhibition tests, and gene blaPDC deletion experiments, this resistance phenotype was correlated with an extremely high production of cephalosporinase PDC. In part accounting for this strong PDC upregulation, genomic analyses revealed the presence of mutations in the regulator AmpR (D135N/G in 6 strains) and enzymes of the peptidoglycan recycling pathway, such as AmpD, PBP4, and Mpl (9 strains). Finally, all of the 5 (12%) remaining C/T-resistant strains (group 3) appeared to encode PDC variants with mutations known to improve the hydrolytic activity of the β-lactamase toward ceftazidime and C/T (F147L, ΔL223-Y226, E247K, and N373I). Collectively, our results highlight the importance of both intrinsic and transferable mechanisms in C/T-resistant P. aeruginosa Which mutational events lead some clinical strains to massively produce the natural cephalosporinase PDC remains incompletely understood.

Project description:This study established the in vitro activity of ceftolozane/tazobactam (C/T) and its genotypic resistance mechanisms by whole-genome sequencing (WGS) in 195 carbapenem-nonsusceptible Pseudomonas aeruginosa (CNSPA) clinical isolates recovered from Singapore between 2009 and 2020. C/T susceptibility rates were low, at 37.9%. Cross-resistance to ceftazidime/avibactam was observed, although susceptibility to the agent was slightly higher, at 41.0%. Whole-genome sequencing revealed that C/T resistance was largely mediated by the presence of horizontally acquired ?-lactamases, especially metallo-?-lactamases. These were primarily disseminated in well-recognized high-risk clones belonging to sequence types (ST) 235, 308, and 179. C/T resistance was also observed in several non-carbapenemase-producing isolates, in which resistance was likely mediated by ?-lactamases and, to a smaller extent, mutations in AmpC-related genes. There was no obvious mechanism of resistance observed in five isolates. The high C/T resistance highlights the limited utility of the agent as an empirical agent in our setting. Knowledge of local molecular epidemiology is crucial in determining the potential of therapy with novel agents.IMPORTANCE Pseudomonas aeruginosa infection is one of the most difficult health care-associated infections to treat due to the ability of the organism to acquire a multitude of resistance mechanisms and express the multidrug resistance phenotype. Ceftolozane/tazobactam (C/T), a novel ?-lactam/?-lactamase inhibitor combination, addresses an unmet medical need in patients with these multidrug-resistant P. aeruginosa infections. Our findings demonstrate geographical variation in C/T susceptibility owing to the distinct local molecular epidemiology. This study adds on to the growing knowledge of C/T resistance, particularly mutational resistance, and will aid in the design of future ?-lactams and ?-lactamase inhibitors. WGS proved to be a useful tool to understand the P. aeruginosa resistome and its contribution to emerging resistance in novel antimicrobial agents.

Project description:Pseudomonas aeruginosa produces a class C ?-lactamase (e.g., PDC-3) that robustly hydrolyzes early generation cephalosporins often at the diffusion limit; therefore, bacteria possessing these ?-lactamases are resistant to many ?-lactam antibiotics. In response to this significant clinical threat, ceftolozane, a 3' aminopyrazolium cephalosporin, was developed. Combined with tazobactam, ceftolozane promised to be effective against multidrug-resistant P. aeruginosa Alarmingly, ?-loop variants of the PDC ?-lactamase (V213A, G216R, E221K, E221G, and Y223H) were identified in ceftolozane/tazobactam-resistant P. aeruginosa clinical isolates. Herein, we demonstrate that the Escherichia coli strain expressing the E221K variant of PDC-3 had the highest minimum inhibitory concentrations (MICs) against a panel of ?-lactam antibiotics, including ceftolozane and ceftazidime, a cephalosporin that differs in structure largely in the R2 side chain. The k cat values of the E221K variant for both substrates were equivalent, whereas the Km for ceftolozane (341?±?64?µM) was higher than that for ceftazidime (174?±?20?µM). Timed mass spectrometry, thermal stability, and equilibrium unfolding studies revealed key mechanistic insights. Enhanced sampling molecular dynamics simulations identified conformational changes in the E221K variant ?-loop, where a hidden pocket adjacent to the catalytic site opens and stabilizes ceftolozane for efficient hydrolysis. Encouragingly, the diazabicyclooctane ?-lactamase inhibitor avibactam restored susceptibility to ceftolozane and ceftazidime in cells producing the E221K variant. In addition, a boronic acid transition state inhibitor, LP-06, lowered the ceftolozane and ceftazidime MICs by 8-fold for the E221K-expressing strain. Understanding these structural changes in evolutionarily selected variants is critical toward designing effective ?-lactam/?-lactamase inhibitor therapies for P. aeruginosa infections.IMPORTANCE The presence of ?-lactamases (e.g., PDC-3) that have naturally evolved and acquired the ability to break down ?-lactam antibiotics (e.g., ceftazidime and ceftolozane) leads to highly resistant and potentially lethal Pseudomonas aeruginosa infections. We show that wild-type PDC-3 ?-lactamase forms an acyl enzyme complex with ceftazidime, but it cannot accommodate the structurally similar ceftolozane that has a longer R2 side chain with increased basicity. A single amino acid substitution from a glutamate to a lysine at position 221 in PDC-3 (E221K) causes the tyrosine residue at 223 to adopt a new position poised for efficient hydrolysis of both cephalosporins. The importance of the mechanism of action of the E221K variant, in particular, is underscored by its evolutionary recurrences in multiple bacterial species. Understanding the biochemical and molecular basis for resistance is key to designing effective therapies and developing new ?-lactam/?-lactamase inhibitor combinations.

Project description:Guidelines for the treatment of sepsis, febrile neutropenia, and hospital-acquired pneumonia caused by Pseudomonas aeruginosa include empirical regimens incorporating two antibiotics from different classes with activity against P. aeruginosa for select at-risk patients to increase the likelihood that the organism will be susceptible to at least one agent. The activity against P. aeruginosa and the rates of cross-resistance of ceftolozane-tazobactam were compared to those of the ?-lactam comparators cefepime, ceftazidime, piperacillin-tazobactam, and meropenem alone and cumulatively with ciprofloxacin or tobramycin. Nonurine P. aeruginosa isolates were collected from adult inpatients at 44 geographically diverse U.S. hospitals. MICs were determined using reference broth microdilution methods. Of the 1,257 isolates collected, 29% were from patients in intensive care units and 39% were from respiratory sites. The overall rate of susceptibility to ceftolozane-tazobactam was high at 97%, whereas it was 72 to 76% for cefepime, ceftazidime, piperacillin-tazobactam, and meropenem. The rate of nonsusceptibility to all four comparator ?-lactams was 11%; of the isolates nonsusceptible to the four comparator ?-lactams, 80% remained susceptible to ceftolozane-tazobactam. Among the isolates nonsusceptible to the tested ?-lactam comparators, less than half were susceptible to ciprofloxacin. By comparison, approximately 80% of the ?-lactam-nonsusceptible isolates were susceptible to tobramycin, for overall cumulative susceptibility rates of 94 to 95%, nearly 10% higher than that of the ciprofloxacin-?-lactam combinations and approaching that of ceftolozane-tazobactam as a single agent. The rates of susceptibility to ceftolozane-tazobactam were consistently high, with little observable cross-resistance. Ceftolozane-tazobactam monotherapy performed at or above the level of commonly utilized combination therapies on the basis of in vitro susceptibilities. Ceftolozane-tazobactam should be considered for use in patients at high risk for resistant P. aeruginosa infection and as an alternative to empirical combination therapy, especially for patients unable to tolerate aminoglycosides.

Project description:We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ∆mutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 ug/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 ug/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.

Dataset Information

Multicenter Evaluation of the Etest Gradient Diffusion Method for Ceftolozane-Tazobactam Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa.

Publications

Multicenter Evaluation of the Etest Gradient Diffusion Method for Ceftolozane-Tazobactam Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa.

Similar Datasets

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