Date Published: April 6, 2017
Publisher: Public Library of Science
Author(s): Adriana Calderaro, Mirko Buttrini, Maddalena Piergianni, Sara Montecchini, Monica Martinelli, Silvia Covan, Giovanna Piccolo, Maria Cristina Medici, Maria Cristina Arcangeletti, Carlo Chezzi, Flora De Conto, Alex Friedrich.
Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. The introduction of rapid and sensitive methods for the detection of carbapenemase-producing bacteria is of increasing importance. The carbapenemase production can be detected using non-molecular methods (such as the modified Hodge test, the synergy test, the Carba NP test and the antibiotic hydrolysis assays) and DNA-based methods. In this study, we propose a modified version of a previously described meropenem hydrolysis assay (MHA) by MALDI-TOF MS for the phenotypic detection in 2h of carbapenemase-producing Enterobacteriaceae. The MHA was successfully applied to detect carbapenemase activity in 981 well-characterized Enterobacteriaceae strains producing KPC or VIM carbapenemases, and in 146 carbapenem fully susceptible strains. This assay, applied also to NDM and OXA-48-producing strains and to CRE with resistance mechanisms other than carbapenemase production, has proved to be able to distinguish between carbapenemase-producing and -nonproducing Enterobacteriaceae.
Multidrug resistance is now emerging at an alarming rate among a variety of bacterial species, especially Gram-negative rods (GNR) as Enterobacteriaceae (EB), Pseudomonas spp. and Acinetobacter spp., causing both nosocomial and community-acquired infections [1, 2]. One of the most important emerging traits is the resistance to extended-spectrum β–lactams in GNR. Increasing resistance to carbapenems, which are most often the last line of therapy, has recently been reported worldwide . In 2013, the Centers for Disease Control and Prevention assigned the highest threat level to carbapenem-resistant Enterobacteriaceae (CRE) and declared that CRE require urgent public health attention [3, 4]. Carbapenemases are classified by Ambler system into class A (mostly Klebsiella pneumoniae carbapenemase, KPC), class B or metallo-β-lactamases (mostly Verona Integron-encoded metallo-β-lactamase, VIM, New Delhi metallo-β-lactamase, NDM, and to a lesser extent Imipenem metallo-β-lactamase, IMP) and class D (mostly Oxacillinase-48, OXA-48, and to a lesser extent OXA-162 and OXA-181), based on their aminoacid sequence homology [5, 6]. Acquired KPC, VIM, NDM, and OXA-48 are the prevalent carbapenemases in Enterobacteriaceae in Europe . In particular, in Italy, VIM-producing Enterobacteriaceae have been detected since the early 2000s, with recent epidemic diffusion of KPC producers and occasional detection of NDM and OXA-48 producers . Moreover, the dramatic increase of carbapenem-resistant K. pneumoniae has been documented by the European Antimicrobial Resistance Surveillance Network (EARS-Net), which showed that the percentage of invasive isolates of carbapenem-resistant K. pneumoniae increased to 15% in 2010 to reach 35% in 2013 vs 2% before 2009 . The carbapenemase production can be detected through culture-based methods, such as the modified Hodge test (MHT) and the disk diffusion inhibition test (synergy test, ST) used for years  or DNA-based methods (e.g., Polymerase Chain Reaction—PCR). The results of culture-based methods are available in 18–24 h, whereas those of PCR assays in few hours but at higher cost [10, 11]. Moreover, PCR, due to its specificity, can only detect known genetic targets encoding carbapenem-resistance genes . More recently other non-molecular tests able to differentiate between carbapenemase-producing and carbapenemase-nonproducing isolates have been proposed: the Carba NP test  and the antibiotic hydrolysis assays. Although, the Carba NP test is a rapid biochemical technique, developed in 2012  and recently suggested by Clinical and Laboratory Standards Institute (CLSI) instead of ST as one of the confirmatory tests for suspected carbapenemase production in Enterobacteriaceae and other Gram negative rods (such as Pseudomonas aeruginosa and Acinetobacter spp.), it cannot detect certain carbapenemase types (i.e. OXA-type, chromosomally encoded) . The antibiotic hydrolysis assays could be performed by UV spectrophotometry, a cheap technique allowing to accurately achieve the purpose but with a time-consuming process, which can be shortened by the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) . As a matter of fact MALDI-TOF MS, recently introduced into routine microbiology laboratories for the identification of bacteria and fungi, may be potentially applied in this complex diagnostic process .
In all the experiments performed by the five drug distributions arrived with ice, the spectra obtained for the pure meropenem did not show peaks referring to degraded molecule and those obtained for the carbapenemase-nonproducing (ATCC BAA-1706) and carbapenemase-producing K. pneumoniae (ATCC BAA-1705, ATCC BAA-2146) reference strains showed the presence of the peaks corresponding only to the intact or to the hydrolyzed meropenem, respectively (Fig 1A). When the antibiotic received with the distribution delivered at room temperature was used, the spectra of meropenem alone as well as those obtained for the K. pneumoniae carbapenemase-nonproducing (ATCC BAA-1706), AmpC (NEQAS 2832 and NEQAS 3419), and ESBL (NEQAS 3253 and NEQAS 3341) reference strains showed the co-existence of the peaks of intact and degraded drug with CAV ≤ 0.17. On the contrary, the CAVs for the carbapenemase-producing reference strains (ATCC BAA-1705 and NCTC 13442) were ≥ 0.56.
Infections caused by CPE are now emerging worldwide and are difficult to treat . Previous data have shown that strict epidemiological intervention based on the rapid detection of carbapenemase production can prevent the spread of those bacteria . Therefore, the introduction and the evaluation of rapid and sensitive methodologies for the detection of carbapenemase-producing bacteria is of increasing importance. Phenotypic and DNA-based techniques are able to identify these carbapenemase producers, although with variable efficiencies . In particular, MHT and ST are able to directly detect the carbapenemases but they need a time to results of at least 18h, are difficult to standardize and can give rise to false-positive results [11, 20]. On the other hand, the Carba NP test is recognized as a rapid assay but it suffers from a limited ability to detect OXA-type-producing isolates and some weak carbapenemases of class A, like GES-5 and SME-1 . Conversely, PCR assays are rapid and highly sensitive and specific but, detecting only known enzyme-encoding genetic targets, they could be affected by the increasing number of types of carbapenemases [11, 22].