Sensitive Screening Tests for Suspected Class A Carbapenemase Production in Species of Enterobacteriaceae

Abstract

The detection of class A serine-carbapenemases among species of Enterobacteriaceae remains a challenging issue. Methods of identification for routine use in clinical microbiology laboratories have not been standardized to date. We developed a novel screening methodology suitable for countries with high basal levels of carbapenem resistance due to non-carbapenemase-mediated mechanisms and standardized several simple confirmatory methods that allow the recognition of bacteria producing class A carbapenemases, including KPC, Sme, IMI, NMC-A, and GES, by using boronic acid (BA) derivatives. A total of 28 genetically unrelated Enterobacteriaceae strains producing several class A carbapenemases were tested. Thirty-eight genetically unrelated negative controls were included. The isolates were tested against imipenem (IPM), meropenem (MEM), and ertapenem (ETP) by MIC and disk diffusion assays in order to select appropriate tools to screen for suspected carbapenemase production. It was possible to differentiate class A carbapenemase-producing bacteria from non-carbapenemase-producing bacteria by using solely the routine IPM susceptibility tests. The modified Hodge test was evaluated and found to be highly sensitive, although false-positive results were documented. Novel BA-based methods (a double-disk synergy test and combined-disk and MIC tests) using IPM, MEM, and ETP, in combination with 3-aminophenylboronic acid as an inhibitor, were designed as confirmatory tools. On the basis of the performance of these methods, a sensitive flow chart for suspicion and confirmation of class A carbapenemase production in species of Enterobacteriaceae was designed. By using this methodology, isolates producing KPC, GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of ␤-lactamases (extended-spectrum ␤-lactamases, AmpCs, and metallo-␤-lactamases, etc). These methods will rapidly provide useful information needed for targeting antimicrobial therapy and appropriate infection control.

The combined-disk boronic acid test as an accurate strategy for the detection of KPC carbapenemase in Central America

“…Previously, a flowchart for the detection of KPC, using the VITEK 2 system, determined that a minimum inhibitory concentration for imipenem ≥ 2 and meropenem ≥ 1 provided a sensitivity of 98%, a specificity of 94%, and a PPV of 95% to detect KPC [16]. Several studies have shown that the boronic acid test has a sensitivity of 89%-100%, a specificity of 100%, a PPV of 100%, and a NPV of 100% for the detection of KPC [14,21]. However, very few reports have described the pragmatic application of the boronic acid test during outbreaks of KPC.…”

“…The primers used were the following: 5′-AACAAGGAATATCGTTGATG-3′ and reverse primer 5′-AGATGATTTTCAGAGCCTTA-3′ (Integrated DNA Technologies, Coralville, USA). The combined-disk boronic acid test, containing 300 µg of 3-aminophenyl-boronic acid (APBA), was used as a confirmatory test in phase two for the detection of KPC (catalogue code B1241627, Britania Laboratory, Buenos Aires, Argentina) [14], only after the diagnostic accuracy was determined from January 2011-May 2011 (phase one) by comparing it to the PCR test results in 131 samples. An imipenem disk (10 µg) with a distance of 20 mm was used for APBA inhibition.…”

“…In phase one, PCR for blaKPC was performed in all samples (131) since the combined-disk boronic acid test required evaluation for diagnostic accuracy and a national algorithm was not available in this phase. The combined-disk boronic acid test was used according to the recommendations by the Carlos G. Malbran Laboratory [14].…”

Relación clonal y detección del gen blaKPC en cepas de Klebsiella pneumoniae resistentes a carbapenémicos, en un hospital de Venezuela

“…Se realizó mediante los métodos de Hodge modificado 19 , sinergia con ácido fenil borónico (AFB) 21 23 . Los productos de amplificación se sometieron a electroforesis en gel de agarosa al 2%, luego se colorearon con bromuro de etidio (0,5 μg.ml -1 ) en tampón TBE 1X, durante 30 min a 10 V.cm -1 .…”

Caracterização molecular da resistência aos carbapenêmicos em enterobactérias isoladas em hospitais brasileiros

“…Pasteran e colaboradores no ano 2009 pesquisaram a efetividade desse método para a deteção de todas as carbapenemases de classe A, obtendo novas recomendações para a triagem destas enzimas (Pasteran, et al, 2009 (Yigit et al, 2003, Wolter, et al, 2008, Girlich et al, 2008, Woodford et al, 2007. A cepa Kp-13 teve este comportamento característico associado à presença da enzima KPC, porém a cepa Kp-35 apresentou uma resistência maior para o imipenem (CIM = 128 µg/mL), ertapenem e meropenem (CIM ≥512 µg/ml).…”