Miriam Cordovana (Presenter)
University Hospital Sant'Orsola-Malpighi, Bologna
Bio: Laboratory technician degree at Florence University (2002). Molecular Biology degree at Urbino University (2012), thesis on antibiotic resistance. 2004-2005 sieroimmunology lab at Florence university hospital (viruses molecular diagnostics) 2005-2007 Human genetic lab at Florence university (diagnostic of hereditary human diseases) 2007-on Bacteriology section of Bologna University hospital 2010-on research projects about carbapenem resistance 2014-on research projects about applications of MALDI-TOF MS on the study of antibiotic resistances
Authorship: Cordovana M.(1), Pranada A.B.(2), Ambretti S.(1), Kostrzewa M.(3)
(1)University Hospital S.Orsola-Malpighi, Bologna – Unit of Microbiology (2) MVZ Dr. Eberhard & Partner Dortmund, Department of Medical Microbiology, Dortmund, Germany (3) Bruker Daltonik GmbH, Bremen, Germany
Carbapenem-resistance in Bacteroides fragilis is related to the cfiA-encoded metallo-beta-lactamase, and represents an emerging problem worldwide. Rapid detection of carbapenemase-producing strains is crucial for a proper treatment of patients, especially in bloodstream infections. While classical phenotypic and molecular methods present several key issues and limits, the latest applications of MALDI-TOF MS allow an approach that could perfectly fit this purpose.Here, n=302 Bacteroides fragilis strains were subtyped by MALDI-TOF MS (MALDI Biotyper, Bruker Daltonik) to detect cfiA-carrying strains, and the carbapenemase activity was verified and characterized by MBT STAR-CARBA hydrolysis assay. Further, 16 spiked positive blood cultures underwent the same approach, to evaluate a direct application of these methods on the primary sample.
Bacteroides fragilis is the most antibiotic-resistant among anaerobes, and carbapenem-resistance represents an emerging and underestimated problem. Strains belonging to the DNA homology group division II possess a class B carbapenemase encoded by the cfiA gene. The gene can be silent or expressed at different levels, resulting in a wide range of subsceptibility to carbapenems, nevertheless activation under therapy might result in therapeutic failures in “susceptible” isolates.
A rapid detection of carbapenemase-producing strains is crucial for a proper treatment of patients, especially in bloodstream infections. Classical and molecular methods for antimicrobial susceptibility testing of anaerobes present several key issues, as they are either laborious and slow or expensive.
In this study, an innovative diagnostic approach, completely MALDI-TOF MS based, was applied to investigate carbapenem-resistance in B. fragilis strains (clinical isolates and spiked blood cultures). Detection of cfiA by subtyping strains as belonging to division I/II was followed by investigating carbapenemase activity by observing carbapenem hydrolysis in division II members.
N=302 non-duplicated B. fragilis clinical isolates collected between May 2014 and May 2017, underwent detection of cfiA gene by MALDI-TOF MS subtyping, as described by Nagy et al. , and the presence of the gene was further confirmed by PCR.
The carbapenemase activity of the cfiA-positive strains was evaluated by MBT STAR-CARBA hydrolysis assay (Bruker Daltonik, Germany - RUO version), analyzing the decrease of intact imipenem molecules after a short incubation (30 min) with the bacteria tested.
The correlation between rate of imipenem hydrolysis and level of cfiA expression was evaluated comparing the hydrolysis results after 30 min and after a prolonged incubation (60 min) with the resistance level in terms of MIC values.
The same approach was further applied to 16 positive blood cultures spiked with a cfiA-positive strain.
The cfiA gene was detected by MALDI-TOF MS subtyping in 30/302 strains (9.93%), and all of them were confirmed by PCR.
All the n=30 cfiA-positive strains showed hydrolysis of imipenem in the MBT STAR-CARBA assay.
Moreover, the rate of hydrolysis showed a strong correlation with the MIC values of imipenem and meropenem, as the strains with high MIC values achieved a full imipenem hydrolysis after 30 min, while the strains with low MIC values after 60 min.
The bacterial pellet extracted from the positive blood culture bottles provided results identical to the strain with which the bottles had been spiked (16/16 subtyped as cfiA-positives, 16/16 imipenem-hydrolyzing, same correlation rate of hydrolysis/MIC).
Conclusions & Discussion
In this study, cfiA positive B. fragilis showed a significant prevalence among clinical isolates (9.93%), suggesting carbapenem-resistance may represent a real threat also in infections involving anaerobic bacteria. Therefore, its examination in routine diagnostics is advisable.
Overcoming the limits of classical and molecular methods for anaerobes, a completely MALDI-TOF MS based approach proved to be reliable and comprehensive in detection and characterization of carbapenemase-producing B. fragilis. Detecting the cfiA gene simply from the spectra recorded for routine identification, and verifying the carbapenemase-production in a very short time, it allows to significantly reduce the time of reporting resistant strains, enabling an earlier set up of adopted antibiotic therapy.
References & Acknowledgements:
 Differentiation of division I (cfiA-negative) and division II (cfiA-positive) Bacteroides fragilis strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Nagy E, Becker S, Sóki J, Urbán E, Kostrzewa M.
J Med Microbiol. 2011 Nov;60(Pt 11):1584-90. doi: 10.1099/jmm.0.031336-0. Epub 2011 Jun 16
IP Royalty: no
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||