Rapid Flow Cytometry Test for Identification of Different Carbapenemases in Enterobacteriaceae

Silva, Ana T.; Faria‐Ramos, Isabel; Ricardo, Elisabete; Miranda, Isabel M.; Espinar, Maria José; Costa-de-Oliveira, Sofia; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

doi:10.1128/aac.02947-15

Cited by 12 publications

(12 citation statements)

References 18 publications

(11 reference statements)

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“…We previously described microbiological applications of FC, including susceptibility evaluation of antifungals, as well as the elucidation of antifungal resistance mechanisms (Pina-Vaz and Rodrigues, 2010 ). Moreover, we developed AST cytometric protocols regarding the main mechanisms of resistance to betalactams with the most important bacteria involved in clinical infections (Faria-Ramos et al, 2013 ; Silva et al, 2016 ). In this study we evaluate the performance of FASTinov ® kit for AST of Gram negative bacteria from positive blood cultures.…”

Section: Introductionmentioning

confidence: 99%

Potential Impact of Flow Cytometry Antimicrobial Susceptibility Testing on the Clinical Management of Gram-Negative Bacteremia Using the FASTinov® Kit

et al. 2017

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Laboratory assessment of antimicrobial susceptibility is a prerequisite for adequate management of infections. The aim of this research was to evaluate the performance of the novel FASTinov® kit for antimicrobial susceptibility testing (AST) of Gram negative bacilli directly on positive blood cultures. One hundred and two positive blood cultures from patients of a Portuguese University Hospital were included. AST were performed with routine method, Vitek2, with FASTinov® kit, and with the gold standard microdilution. Bacteria directly extracted from blood cultures were used to inoculate the FASTinov® kit. Time-to-result as well as the number of patients receiving initially inappropriate therapy (and those in whom de-escalation would have been done) and length of stay (LOS) was recorded. Seventy percent of patients were over 70 years old and 18.6% were admitted in intensive care units. Regarding the isolates, 88.2% were Enterobacteriaceae, 9.8% Pseudomonas spp. and 1% Acinetobacter spp. Extended spectrum β-lactamases producing-Enterobacteriaceae were found in 7.8% of cases and 10.8% were multi-drug resistant. Fifty-one hours was the mean of time-to-result for routine test (Vitek2) vs. 2 h response regarding Fastinov® test. The overall agreement between FASTinov® and the reference microdilution method was 98%. According to the susceptibility phenotype, 16.7% of patients received initially inappropriate therapy and the mean hospital LOS of these patients was significantly higher. FASTinov® kit revealed an excellent correlation with the AST standard method and provided much earlier results than Vitek2.

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Section: Introductionmentioning

confidence: 99%

Potential Impact of Flow Cytometry Antimicrobial Susceptibility Testing on the Clinical Management of Gram-Negative Bacteremia Using the FASTinov® Kit

et al. 2017

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“…In the future, this assay can be eventually compared with ceftazidime/avibactam combination (only with KPC or OXA-48-like enzymes) ( Chahine et al, 2015 ). Flow cytometry is an excellent tool, still unexplored in Microbiology, allowing to study antimicrobial activity ( Pina-Vaz et al, 2005 ; Pina-Vaz and Rodrigues, 2010 ) drug associations ( Teixeira-Santos et al, 2012 ), and mechanisms of resistance ( Faria-Ramos et al, 2013 ; Silva et al, 2016 ). Flow cytometric results showed that even at a low concentration and a short incubation time, a synergistic effect could be easily observed and quantified.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, we studied the association between ertapenem and the other three carbapenems: imipenem, meropenem, and doripenem, against different types of carbapenemases producing by Enterobacteriaceae , belonging to Amber classe A, B, and D. Those drugs associations were studied by the time-killing kinetic curves as well as a new flow cytometry cell analysis. We have recently developed a protocol for identification of carbapenemases in 1 hour based on flow cytometry analysis ( Silva et al, 2016 ) with great accuracy. Additionally, a computational protein–ligand docking analysis allowed us to understand the molecular affinity of the different drugs regarding different enzymes.…”

Section: Introductionmentioning

confidence: 99%

A Flow Cytometric and Computational Approaches to Carbapenems Affinity to the Different Types of Carbapenemases

et al. 2016

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The synergy of carbapenem combinations regarding Enterobacteriaceae producing different types of carbapenemases was study through different approaches: flow cytometry and computational analysis. Ten well characterized Enterobacteriaceae (KPC, verona integron-encoded metallo-β-lactamases –VIM and OXA-48-like enzymes) were selected for the study. The cells were incubated with a combination of ertapenem with imipenem, meropenem, or doripenem and killing kinetic curves performed with and without reinforcements of the drugs. A cephalosporin was also used in combination with ertapenem. A flow cytometric assay with DiBAC4-(3), a membrane potential dye, was developed in order to evaluate the cellular lesion after 2 h incubation. A chemical computational study was performed to understand the affinity of the different drugs to the different types of enzymes. Flow cytometric analysis and time-kill assays showed a synergic effect against KPC and OXA-48 producing-bacteria with all combinations; only ertapenem with imipenem was synergic against VIM producing-bacteria. A bactericidal effect was observed in OXA-48-like enzymes. Ceftazidime plus ertapenem was synergic against ESBL-negative KPC producing-bacteria. Ertapenem had the highest affinity for those enzymes according to chemical computational study. The synergic effect between ertapenem and others carbapenems against different carbapenemase-producing bacteria, representing a therapeutic choice, was described for the first time. Easier and faster laboratorial methods for carbapenemase characterization are urgently needed. The design of an ertapenem derivative with similar affinity to carbapenemases but exhibiting more stable bonds was demonstrated as highly desirable.

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“…For each selected antibiotic concentration, a staining index (SI) was calculated as the ratio of fluorescence intensity of bacterial cells treated with antibiotic to that of non-treated cells. An increased median intensity of the green fluorescence (530/30 nm), of at least twice corresponded to dead bacterial cells (depolarized cells) [ 3 ].…”

Section: Methodsmentioning

confidence: 99%

“…The standard AST methods based on the assessment of microbial growth inhibition include manual (e.g., disk diffusion, broth microdilution) and automated AST, such as the Microscan Walkaway (Beckman Coulter), Phoenix Automated Microbiology System (BD), and Vitek 2 (bioMérieux) [ 1 ]. Several analysis protocols based on flow cytometry (FC) were developed for microbial detection and species identification, as well as for AST and elucidation of antimicrobial resistance mechanisms [ 2 , 3 ]. The major advantage of using FC directly on clinical specimens is that it could provide early data on antibiotic susceptibility profiles, thereby allowing the implementation of an adequate empirical treatment [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection and Antibiotic Susceptibility of Uropathogenic Escherichia coli by Flow Cytometry

et al. 2020

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Background: Early preliminary data on antibiotic resistance patterns available before starting the empiric therapy of urinary tract infections (UTIs) in patients with risk factors for acquiring antibiotic resistance could improve both clinical and epidemiological outcomes. The aim of the present study was two-fold: (i) to assess the antibiotic susceptibility of uropathogenic Escherichia coli isolates, exhibiting different antibiotic resistance phenotypes, directly in artificially contaminated urine samples using a flow cytometry (FC) based protocol; (ii) to optimize the protocol on urine samples deliberately contaminated with bacterial suspensions prepared from uropathogenic E. coli strains. Results: The results of the FC based antimicrobial susceptibility testing (AST) protocol were compared with the reference AST methods results (disk diffusion and broth microdilution) for establishing the sensitivity and specificity. The proposed FC protocol allowed the detection and quantification of uropathogenic E. coli strains susceptibility to nitrofurantoin, trimethoprim–sulfamethoxazole, ciprofloxacin, and ceftriaxone within 4 h after the inoculation of urine specimens. The early availability of preliminary antibiotic susceptibility results provided by direct analysis of clinical specimens could essentially contribute to a more targeted emergency therapy of UTIs in the anticipation of AST results obtained by reference methodology. Conclusions: This method will increase the therapeutic success rate and help to prevent the emergence and dissemination of drug resistant pathogens.

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Rapid Flow Cytometry Test for Identification of Different Carbapenemases in Enterobacteriaceae

Cited by 12 publications

References 18 publications

Potential Impact of Flow Cytometry Antimicrobial Susceptibility Testing on the Clinical Management of Gram-Negative Bacteremia Using the FASTinov® Kit

Potential Impact of Flow Cytometry Antimicrobial Susceptibility Testing on the Clinical Management of Gram-Negative Bacteremia Using the FASTinov® Kit

A Flow Cytometric and Computational Approaches to Carbapenems Affinity to the Different Types of Carbapenemases

Rapid Detection and Antibiotic Susceptibility of Uropathogenic Escherichia coli by Flow Cytometry

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