Background
Multidrug resistance among
Enterobacteriaceae has become a worldwide major public health issue. The most worrisome emerging resistance feature corresponds to the production of carbapenem-hydrolysing beta-lactamases [
1]. Carbapenems are considered the last line of effective therapy available for the treatment of severe infections [
2,
3]; resistance to these agents reduces clinical therapeutic choices and frequently leads to treatment failure. Carbapenemase enzymes include class A carbapenemases (KPC types), class B or metallo-beta-lactamases (MBLs) (VIM, IPM, and NDM types), and class D oxacillinases (e.g., OXA-48-like enzymes). In addition, decreased susceptibility to carbapenems in
Enterobacteriaceae may be caused by either extended spectrum beta-lactamases (ESBLs) or AmpC enzymes combined with drug decreased permeability, due to loss of porins [
4]. The association of multiple resistance determinants, comprehending carbapenemase, cephalosporinase enzymes and ESBLs, poses a challenge in the routine microbiologic diagnostic of
Enterobacteriaceae[
4,
5]. Different phenotypic and molecular screening and confirmatory tests have been described [
6‐
8]. Currently, the resistance detection workflow involves an initial screening step followed by phenotypic and/or genotypic confirmation [
8‐
10]. The screening step is based on evaluation of strain susceptibility to carbapenems, measured as MIC and compared to MIC values of ATCC control strains. Current EUCAST clinical breakpoints for the detection of carbapenemase-producing
Enterobacteriaceae are set to 0.5 mg/L for ertapenem and 2 g/ml for meropenem and imipenem, while the screening cutoff are set to 0.12 mg/L for meropenem and ertapenem and 1 g/L for imipenem [
9,
10]. This choice reflects the presence of carbapenemase producers that poorly express resistance determinants and would thus be categorized as susceptible by clinical breakpoints, while they need further screening for correct identification.
The phenotypic confirmation of carbapenemase production is based on the detection of diffusible carbapenemases (evaluated in the Modified Hodge Test (MHT)) or on inhibition of carbapenemase activity, detected in phenotypic assays based on the synergy between MBL or KPC inhibitors and carbapenems [
11,
12]. The most widely used inhibitors are the metal-chelating agent EDTA and dipicolinic acid against MBL, boronic acid for Ambler class A carbapenemases and cloxacillin against AmpC. These are used in different formats which include the double-disk synergy test, the combined disk test, and carbapenem/carbapenem-EDTA or cefotetan/cefotetan-cloxacillin Etest strips [
13‐
15]. In contrast, the genotypic confirmation is based on PCR-based techniques.
In this work we aimed at establishing the best workflow based on phenotypic analysis in the routine detection and characterization of carbapenem resistant Enterobacteriaceae isolates. We compared the efficiency of six different phenotypic assays: 1) MHT, 2) MBL Etest, 3) Double disk test with EDTA, 4) Rosco Diagnostica KPC and MBL confirm kit (RDCK™), 5) AmpC Etest and 6) Cloxacillin inhibition test in detecting resistance determinants on a panel of Enterobacteriaceae isolates with reduced susceptibility to carbapenems. Results were validated by comparison with genotypic data. The RDCK™ was the most reliable assay in our hands; however, all phenotypic tests failed to detect multiple carbapenem resistance mechanisms, which needed to be assessed by genotypic analysis. Therefore, the optimized workflow to diagnose and control the spread of pathogens with complicated resistance patterns must involve both genotypic and phenotypic analysis.
Discussion and conclusions
The massive worldwide spreading of carbapenemase-producers, mainly
Enterobacteriaceae, has forced routine analysis to elaborate reliable detection methods. High sensitivity and specificity together with a rapid workflow has become mandatory to delineate the treatability of dangerous pathogens and to control and hinder their spread. The epidemiology of carbapenemases had been widely discussed as it has become a major health issue, especially in countries, such as Greece, Israel, USA and many others, where carbapenemase producers are becoming endemic [
1]. A large variety of carbapenem-hydrolyzing enzymes has been identified in Gram negative bacilli [
22]. The Ambler class A (KPC-type) and class B (VIM- and NDM-type) are the most relevant carbapenemases in the clinic; class D (OXA-48-like) are gaining increasing importance, due to their recent spread and to their peculiar hydrolysis profile [
23]. Moreover a particular class of plasmidic cephalosporinases (AmpC, Ambler class C) displays a slightly extended inducible hydrolytic activity towards carbapenems and therefore has to be taken into consideration during putative carbapenemase detection and subsequent antimicrobial treatment [
24]. In the present work we analyzed six different phenotypic tests for their ability to correctly identify carbapenem resistance mechanisms, to provide the most accurate, reliable and easy to set up workflow for the detection of carbapenemase and carbapenem hydrolyzing AmpC producers in clinical specimens.
The threshold of susceptibility to the three major carbapenems (imipenem, meropenem or ertapenem) was set to 0.5 mg/L: considering the latest EUCAST document on “Detection of resistance mechanisms” [
9,
10], the chosen screening cutoff offers a broad and safe probability of detection of carbapenemase producers. A similar threshold has been set by other groups (MIC of meropenem ≥ 0.5 mg/L as point of suspicion of carbapenemase production [
20]). Although a low threshold leads to inclusion in routine diagnostic of isolates that may result negative in subsequent characterization of carbapenem-resistance, this choice maximizes detection sensitivity and should be recommended for epidemiology and spreading control purposes. In addition, our results suggest to take into consideration the MIC of the three main carbapenems used in the clinical practice, i.e. imipenem, meropenem and ertapenem, as each of them displays both positive and negative features: reduced screening breakpoints to imipenem and meropenem increase sensitivity but decrease specificity of carbapenemase detection, as the MIC distribution of the wild-type population is very variable and may be several times higher than the breakpoint. Meropenem offers the best compromise between sensitivity and specificity in terms of detection of carbapenemase-producers. Ertapenem exhibits low efficiency in indicating the presence of non-KPC class A carbapenemases and in general low specificity for carbapenemase detection, since AmpC/ESBL positive Enterobacter spp isolates, have higher MICs of ertapenem than of imipenem and meropenem [
13,
25]. To avoid resistance spreading, we would not recommend setting threshold or screening cutoff higher than those indicated by EUCAST.
Among the tested phenotypic assays, we found important differences in terms of sensitivity and specificity. To summarize, the MHT (1) worked well for the detection of KPC, while it was not able to consistently recognize MBLs. In addition, it has been reported that high levels of expression of AmpC coupled with decreased permeability may be interpreted as carbapenem hydrolyzing enzyme and therefore may yield false positive results [
19,
21]. The MHT has been the gold standard technique in the past years [
26]; however, the massive spreading of both MBL and OXA-48-like enzymes, coupled with the diffusion of Ambler class A carbapenemases in Gram negative bacilli, such as
Proteus spp. and
Pseudomonas spp., makes it less reliable nowadays [
27]. The successful detection of MBLs was mainly achieved by the EDTA inhibition test, using indifferently Etest strips (2) or the double disk test (3). The main limit of these assays is that they may fail to detect positive isolates with low level resistance [
26]. Moreover, in both assays only KPC was detected in samples positive for both KPC and MBL. To our experience, the strips are less sensitive than the double disk test; on the other hand, EDTA containing discs are manually prepared which increases the statistical error. The RDCK™ (4) was the best choice for the phenotypic detection of carbapenemase producers which expressed enzymes belonging to Ambler class A, B, C and D. The latest EUCAST guidelines recommend the use of this kit in routine diagnostic [
9,
10]. Results were achieved within 24 hours from the first identification of diminished susceptibility to carbapenems at the Vitek II. However, we found interpretation of results disputable in the case of OXA-48-like enzymes and AmpCs, which thus needed to be confirmed by other techniques. Results obtained by this method may be improved introducing temocillin or combining two different inhibitors, such as boronic acid and dipicolinic acid [
7,
20].
Other groups have also recommended the improvement of RDCK™ in terms of sensitivity of the meropenem/DPA combination, in particular for the detection of IMP enzymes (IMP-8), and the evaluation of local epidemiology prior to utilization of this phenotypic test in the routine diagnostic [
28,
29]. The identification of overexpressed AmpC enzymes, which display a broadened hydrolysis spectrum conferring carbapenem resistance [
30], resulted sensitive and specific using the cloxacillin-containing medium (6), as previously reported [
13]. In contrast, the Etest strips (5) lacked sensitivity as they failed to detect 50% of positive isolates. To note that none of the tested phenotypic assays was able to detect isolates positive for two resistance determinants, i.e. KPC and VIM. This problem has to be taken into serious consideration, since bacilli with multiple carbapenem resistant mechanisms are increasingly encountered. Only two studies have so far addressed this issue reporting that clinical isolates expressing more than one carbapenemase in association with other beta-lactamases, such as ESBLs, display composite phenotypic resistance profiles that available phenotypic assays are unable to correctly dissect [
7,
26]; the EUCAST guidelines do not thoroughly cover this aspect yet.
On the whole, phenotypic analysis was less reliable than genotypic characterization in the identification of carbapenem resistant strains. However, PCR-based molecular assays have their own limitations: they need expensive equipment and reagents, and expert personnel which are not always available to the diagnostic laboratory. In addition, primers have to be designed within low mutation rate regions and the use of specific primers hinders the identification of novel resistance genes, possibly reporting false negative results.
Therefore, among phenotypic methods for the detection of carbapenemase producers, in our hands RDCK™ reported the most reliable results. This method, as indicated in the EUCAST guidelines, should be applied in the routine screening of all samples with reduced susceptibility to carbapenems. In case of negative results in carbapenem non-susceptible strains, further analysis by means of cloxacillin inhibition test is suggested for the unambiguous detection of AmpC enzymes. In addition, if RDCK™ gives doubtful results, the simultaneous presence of two or more carbapenem resistance mechanisms, or an OXA-48-like enzyme, should be suspected and the molecular tests are the methods of choice in this instance. Therefore, where possible, the concomitant use of both phenotypic and genotypic analysis is highly recommended. Molecular tests can be applied straightforward to screen putative infected/colonized patients/carriers in intensive care or transplant units and immunocompromised patients. Quickness to achieve results, obtained by PCR-based methods, is crucial in terms of clinical management, implementation of infection control measures and for antibiotic stewardship [
31]. In conclusion, to our experience, the best workflow applicable in routine phenotypic diagnostics of carbapenem non-susceptible
Enterobacteriaceae involves the application of the updated EUCAST clinical breakpoints and the use of RDCK™ as first line analysis, followed by, in case of doubtful results, AmpC (i.e. cloxacillin containing medium) and OXA-48 (i.e. evaluation of temocillin resistance) confirmation tests. Molecular assays are also recommended due to their rapidity and sensitivity in the characterization of isolates displaying a complex phenotype, whose identification of resistance mechanisms would be time-consuming and challenging at the phenotypic level.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AB carried out the phenotypic assays; IF carried out the genotypic analysis and drafted the manuscript; AB and IF analyzed the collected data; AC conceived of the study and participated in its design and coordination; SNR participated in study design and coordination and drafted/revised the manuscript; GP participated in the study coordination. All authors read and approved the final manuscript.