JAC Advance Access published online on May 2, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn185
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Original research |
A convenient microbiological assay employing cell-free extracts for the rapid characterization of Gram-negative carbapenemase producers
,*1 Departamento de Microbiología, Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina 2 Hospital Intendente Carrasco, Departamento Bioquímico Municipal, Secretaría de Salud Pública, Municipalidad de Rosario, 2000 Rosario, Argentina 3 Departamento de Química Biológica, Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina
* Corresponding author. Tel: +54-341-4350661; Fax: +54-341-4390465; E-mail: limansky{at}ibr.gov.ar
Received 16 November 2007; returned 4 January 2008; revised 31 March 2008; accepted 2 April 2008
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Abstract |
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Objectives: The dissemination of metallo and serine carbapenem-hydrolysing β-lactamases among Gram-negative nosocomial bacteria represents an acute problem worldwide. Here, we present a rapid and sensitive assay for the characterization of carbapenemase producers to aid in infection control and prevention.
Methods: The assay involves a rapid disruption of bacterial isolates with silicon dioxide microbeads, followed by the testing in cell-free extracts of hydrolytic activity towards various β-lactams including two carbapenems (imipenem and meropenem) and a cephalosporin (ceftazidime). A parallel testing of the effects of selective β-lactamase inhibitors such as EDTA and clavulanic acid allows differentiation of metallo carbapenemases from serine carbapenemases, and also clavulanic-acid-sensitive from -resistant enzymes among the latter.
Results: The efficiency of bacterial disruption using silicon dioxide microbeads was identical to that of ultrasonic treatment. The subsequent microbiological assay aimed to evaluate both substrate specificity and inhibitor profile of carbapenem-hydrolysing enzymes present in the extracts and allowed an accurate differentiation of A, B and D types, as judged by the analysis of 24 well-characterized clinical strains that included metallo-β-lactamase producers (i.e. VIM-, IMP- and SPM-type Pseudomonas producers; an L1 Stenotrophomonas maltophilia producer; and a GOB-18 Elizabethkingia meningoseptica producer) as well as serine carbapenemase producers (i.e. an SME-type Serratia marcescens producer, a GES-2 Pseudomonas aeruginosa producer, Klebsiella pneumoniae and Citrobacter freundii KPC-2 producers and OXA-type Acinetobacter baumannii producers).
Conclusions: We have developed a convenient microbiological assay aimed to more accurately and in a short time characterize carbapenem-hydrolysing enzymes produced by Gram-negative bacteria. The assay possesses broad applicability in the clinical setting.
Key Words: carbapenem resistance , β-lactamase detection , bacterial disruption
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Introduction |
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Carbapenem antimicrobials such as imipenem and meropenem, due to their broad activity spectrum and stability against most common β-lactamases, generally represent last resources for the treatment of nosocomial infections produced by multidrug- resistant Gram-negative bacteria.1 Worryingly, this scenario is now being threatened by the emergence of strains worldwide, displaying increased resistance to these antibiotics.1–4 One main cause is represented by the production of carbapenemases derived either from horizontal gene transfer (‘acquired’ carbapenemases) or by overexpression of endogenous β-lactamase genes (‘intrinsic’ carbapenemases).3 These enzymes belong to a rather heterogeneous group, and many of them are capable of hydrolysing almost all clinically relevant β-lactam antibiotics distributed among classes A, B and D in the molecular classification scheme (corresponding to groups 2f, 3 and 2d, respectively, in the functional classification scheme).3–5 Class A and D enzymes contain serine at the active sites (serine enzymes), whereas class B enzymes require zinc ions for activity and are inhibited by EDTA (metalloenzymes).1,3–7
The early recognition of carbapenemase producers among pathogenic isolates and a preliminary characterization of the type of enzyme produced are considered essential steps to control infections and to prevent generalized spread.3,7 Although substantial clinical evidence is lacking, data from different sources tend to Support the view that carbapenemase-positive strains must be held as resistant to all carbapenems and reported as such.4,7 Controlling the use of antibiotics that may favour the spread of carbapenemase producers also holds an important role in infection-control measures.7
The detection of carbapenemase activity in a clinical isolate can be challenging for a clinical microbiology laboratory.3,4,6 Accurate identification of carbapenemase types requires genotypic approaches and methodologies that are mainly designed to detect previously characterized genes and that are generally restricted to specialized laboratories.4 However, phenotypic approaches designed for a rapid characterization of bacterial isolates actively producing carbapenem-hydrolysing enzymes are largely demanded by the diagnostic microbiology laboratory.6,7 In this context, several authors have emphasized that an extensive validation and standardization of ancillary phenotypic tests still remain open and urgent issues,7 but neither the CLSI nor similar international committees have issued standardized recommendations for the phenotypic screening of Gram-negative carbapenemase producers.4 This situation admits several explanations: first, conventional susceptibility tests lack enough sensitivity and specificity.3,6–8 Second, different phenotypic procedures are proposed as ancillary tests for the detection of metallo or serine carbapenemases based on the diffusion or dilution schemes that rely on the synergy between carbapenemase inhibitors (EDTA for metalloenzymes and clavulanic acid for serine enzymes) and a carbapenem or an oxyimino-cephalosporin4,7 display several drawbacks.4,9 Third, increased carbapenem resistance due to specific reductions in outer membrane permeability, efflux pumps and/or alteration of penicillin-binding proteins (PBPs), rather than the presence of carbapenem-hydrolysing enzymes, may lead to an erroneous interpretation of the actual causes of resistance.8,10 The latter problem can be circumvented by analysing cell-free extracts instead of conducting tests on whole cultures of the suspected isolates, and in this context, different authors (including us) have proposed a number of combined microbiological approaches to more precisely characterize carbapenemase producers.7,11,12 The requirement of equipment such as ultrasonic disruptors, however, limits the general implementation of these assays in the routine microbiological laboratory.7
We propose here a simple microbiological assay designed to rapidly characterize both the substrate spectra and inhibitor profiles of carbapenemases produced by Gram-negative bacterial isolates. This assay involves bacterial disruption with silicon dioxide microparticles (microbeads), followed by the simultaneous testing of imipenemase, meropenemase and ceftazidimase activities in cell-free extracts, and also includes testing of EDTA and clavulanic acid sensitivity on the detected enzymes. The whole procedure, designated AIM (for Antimicrobials and Inhibitors Microbiological assay), allows differentiation of metallo from serine carbapenemase producers and discrimination among the latter.
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Materials and methods |
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Bacterial strains
The usefulness of the AIM assay proposed here was tested with 24 different well-characterized Gram-negative carbapenemase producers belonging to various species, including 22 strains resistant to imipenem and/or meropenem and 2 strains susceptible to both carbapenems, as judged by a disc susceptibility test13 (see Table 1 for details of strains and sources). Eleven strains characterized previously as non-carbapenemase producers were also incorporated for control purposes, which included: (i) four strains showing susceptibility to imipenem or meropenem, including the type strains Pseudomonas aeruginosa ATCC 27853 and the SHV-18 producer Klebsiella pneumoniae ATCC 700603, as well as two P. aeruginosa clinical strains resistant to ceftazidime and phenotypically characterized as AmpC overproducers;14 (ii) P. aeruginosa PASE1, a strain imipenem-resistant due to a deficiency in the outer membrane specific channel OprD;15 (iii) five P. aeruginosa clinical strains showing increased carbapenem resistance from our collection; and (iv) Acinetobacter baumannii 22540, a strain resistant to both imipenem and meropenem due to deficiencies in the outer membrane protein CarO16 and also an OXA-51-like oxacillinase producer (P. Marciaro, V. Ballerini, G. Cera, A. Viale and A. Limansky, unpublished data). Strain identification was conducted by both conventional procedures17 and the API 20NE system (bioMérieux, Marcy l'Étoile, France).
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Metallo-β-lactamases were identified spectrophotometrically employing crude bacterial extracts using imipenem as a substrate and the subsequent testing of whether the detected activity was sensitive to EDTA inhibition.11 Isoelectric focusing (including the overlay of gels run in parallel with specific inhibitors) was also used to more accurately discriminate between class C, B and A enzymes.3 The presence of blaVIM-type, blaIMP-type, blaSPM-1 and blaL1 or blaGOB-type genes was tested by PCR using the specific primers described previously.11 In addition, genes encoding members of the blaOXAsubgroup 1, blaOXAsubgroup 2, blaOXA-5818 and blaOXAsubgroup 3,18 as well as blaSME,19 blaGES20 and blaKPC,19 were detected using the primers described in Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)].
Cells derived from liquid or solid cultures were used to test carbapenemase production. For liquid cultures, bacteria obtained from an isolated colony were grown overnight at 37°C in 20 mL of Luria–Bertani (LB) broth under gentle agitation, harvested by centrifugation at 5000 g for 10 min at 4°C, rinsed once with 3 mL of 50 mM Tris–HCl (pH 8.0) and collected again by centrifugation, and the resulting pellet (equivalent to 
50 mg of wet cell weight) was finally resuspended in 0.3 mL of the same buffer for disruption purposes (see below). For a more practical approach, bacterial colonies were directly collected from the surface of overnight-grown solid cultures obtained on LB or Mueller–Hinton (MH) agar media. In this case, a thick bacterial paste (comparable to a quarter of confluent growth over the surface of the plate) taken from the agar surface was carefully resuspended in 1 mL of Tris–HCl (pH 8.0). The cells were collected by centrifugation, rinsed once and finally resuspended in 0.3 mL of the same buffer for disruption purposes as above.
Preparation of cell-free extracts (CE)
Bacterial cells derived from each of the different carbapenemase-producing strains shown in Table 1 were disrupted with the aid of silicon dioxide microbeads. Similar results were obtained with the use of different commercially available microbeads such as zirconia/silica (0.1 mm diameter, BioSpec Products Inc., Bartlesville, OK, USA, Cat. no. 11079101z) or glass beads (75–150 µm diameter, Sigma-Aldrich, Saint Louis, MO, USA, Cat. no. G8893). Zirconia/silica microbeads were used directly as provided by the manufacturer, whereas glass beads were treated with a 1 M HCl solution, thoroughly rinsed with distilled water to eliminate HCl traces and dried overnight in an oven at 70°C before use. For bacterial disruption purposes, 0.3 mL of the bacterial suspension was added to 1.5 mL microcentrifuge tubes containing 0.3 g of the corresponding microbeads (which roughly corresponds to an equal volume ratio between microbeads and bacterial suspension). The tubes (up to 12) were placed in a special holding device and subjected to gentle agitation for 3 min using a Vortex-Genie 2 Mixer® set at maximum speed. Both the vortexing apparatus and 12-tube holding adapters for 1.5 or 2.0 mL tubes are commercially available (Genie® Cell Disruptor, Scientific Industries, Inc., Bohemia, NY, USA). The treated samples were clarified by centrifugation (10 000 g for 10 min at 4°C) and kept on ice before use (normally within 1 h after cell disruption).
AIM assay using cell-free extracts
The assay, which is based on a procedure described previously for the detection of metallo-β-lactamase producers,11 now extends the range of substrates tested by including two carbapenems (i.e. imipenem and meropenem) and also a cephalosporin, i.e. ceftazidime. It also analyses the effects of different carbapenemase inhibitors such as EDTA (used to distinguish metallo from serine enzymes)4,7 and clavulanic acid (used to both identify serine carbapenemases and differentiate clavulanic-acid-sensitive from -resistant enzymes among them).3,6
In short, MH agar plates were inoculated with an overnight culture of an indicator strain, Escherichia coli ATCC 25922, previously adjusted to 0.5 McFarland standard turbidity using fresh culture medium or saline solution, following the CLSI recommendations.13 Antimicrobial commercial discs (BBL, Cockeysville, MD, USA) containing 10 µg of imipenem, 10 µg of meropenem or 30 µg of ceftazidime were placed on the agar surface of separate plates as described in the figures below. To evaluate metallo-β-lactamase producers (panel A in all of the figures), four filter discs (C, C/Zn, C/E and B, respectively) were placed at the periphery of each of the antibiotic discs within the expected zone of inhibition (3 mm from edge to edge between the antibiotic disc and each of the other filter discs). Disc C was loaded with 20 µL of cell-free bacterial extract prepared as described earlier. The same volume of extract previously supplemented with 0.1 mM ZnSO4 was added to the C/Zn disc (see below). In turn, disc C/E (cell extract plus EDTA) was also loaded with the same volume of extract previously supplemented with 20 mM EDTA. Finally, disc B (buffer) was loaded with 20 µL of 50 mM Tris–HCl (pH 8.0) and was used as a growth control of the indicator E. coli strain without alterations derived from the bacterial extract under evaluation.
The C/Zn disc was incorporated in the assay for the following reason: the Zn2+ content of metallo-β-lactamases in the periplasm is largely unknown at present.5 Based on different experimental evidence, some authors have proposed that in vivo these enzymes are mainly metal-free and that they bind Zn2+ only when confronted with their substrates.21 Zn2+ supplementation of culture media or cell extracts, in fact, has been found to significantly improve the performance of tests designed for their detection.5,22 Therefore, to reduce possible inconsistencies in the detection of these metalloenzymes resulting from differential Zn2+ losses during cell or extract preparation, we tested both the effects of Zn2+ supplementation and EDTA as a specific metallo-β-lactamase inhibitor.
Serine carbapenemase producers were characterized in parallel as shown in panel B in all of the figures. For this purpose, three filter discs were placed on separate plates at the margins of each of the antibiotic-containing discs as described earlier. In all cases, disc C was loaded with 20 µL of cell-free extract, disc C/clavulanic acid with 20 µL of extract previously supplemented with 30 µM clavulanic acid and disc B with 20 µL of 50 mM Tris–HCl (pH 8.0).
All plates were incubated overnight at 37°C, and the growth of the indicator E. coli cells around the different discs was analysed. The ability of the enzymes present in the extracts of the tested bacteria to hydrolyse imipenem, meropenem and ceftazidime was judged by observing the growth of the indicator E. coli cells around discs C (panels A and B) and C/Zn (panel A). Metallo-β-lactamase producers were identified by comparing growth of the indicator E. coli cells around discs C, C/Zn and C/E (panel A). In these cases, inhibition of growth around disc C/E in comparison with much appreciable growth around disc C or C/Zn indicates lack of antimicrobial inactivation and therefore inhibition of the corresponding enzyme by EDTA. The presence of clavulanic-acid-sensitive serine enzymes was judged by a reduced growth of the indicator E. coli cells around disc C/clavulanic acid when compared with disc C (panel B). The latter test also allows the differentiation of clavulanic-acid-sensitive from -resistant carbapenemases (see below). The whole AIM assay, starting from a pure culture of a given bacterial isolate, can easily be completed in <24 h.
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Results and discussion |
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Disruption of clinical Gram-negative strains with silicon dioxide microbeads
We previously reported11 a microbiological assay for the detection of Gram-negative metallo-β-lactamase producers based on testing EDTA-sensitive imipenemase activity in cell-free extracts of suspected isolates. This procedure, however, required the use of ultrasonic disruptors that are not commonly accessible to the routine clinical microbiological laboratory. We therefore evaluated a more practical approach to efficiently disrupt bacterial strains of clinical origin such as the treatment with silicon-dioxide-based microbeads. We analysed the efficiency of disruption of 24 Gram-negative carbapenemase producers (Table 1) with different commercially available microbeads by evaluating imipenemase activity in the cell-free extracts when compared with that obtained after ultrasonic disruption of the same bacterial strains.11 No significant differences in disruption efficiency were observed between zirconia/silica or glass microbeads when compared with ultrasonic treatment when processing bacteria derived from either liquid or solid cultures. Thus, we found it convenient to use either of these microbeads and a fixed treatment time of 3 min for the routine disruption of bacterial isolates obtained from solid cultures for the detection of carbapenemase producers.
The proposed procedure tests the presence of imipenem-, meropenem- and ceftazidime-hydrolyzing activities in cell-free extracts of the analysed bacteria and, in parallel, evaluates the behaviour of the identified enzymes towards specific carbapenemase inhibitors such as EDTA and clavulanic acid. The proposed assay allowed the correct discrimination into class A, B or D of carbapenemases produced by 24 selected Gram-negative clinical strains (Table 1), including variants among each class (see below). Representative results are shown below.
Metallo-β-lactamase producers. Figure 1 illustrates the representative pattern generated by a VIM producer (in this case P. aeruginosa strain 5109; Table 1). As seen in the figure, the hydrolysis of imipenem, meropenem and ceftazidime by the extracts was revealed by the growth of the indicator E. coli strain around discs C (panels A and B) and C/Zn (panel A) (white arrows at the top). The presence of a metallo-β-lactamase was revealed by EDTA inhibition, as inferred from the absence of growth of the indicator E. coli strain around disc C/E (black arrows) for all β-lactam substrates (panel A). In turn, comparable growth around discs C and C/clavulanic acid for all substrates (panel B, white arrows) indicated the absence of a clavulanic acid-sensitive serine carbapenemase.
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Class A serine enzyme producers. The inclusion of ceftazidime in the AIM assay provides a useful means to differentiate chromosomally encoded carbapenemases from plasmid-encoded carbapenemases among this particular class. For instance, while chromosomally encoded SME-like enzymes show null hydrolytic activity towards ceftazidime, plasmid-encoded KPC- or GES-like enzymes on the other hand can readily hydrolyse this cephalosporin.3 Representative AIM patterns for class A producers are seen in Figures 2–4. Figure 2 shows a typical situation found for a GES-2 producer (in this case P. aeruginosa strain 5200; Table 1). As seen in the figure, hydrolysis of imipenem and ceftazidime was revealed by the growth of the indicator E. coli strain around discs C (panels A and B) and C/Zn (panel A) for these substrates (white arrows at the top). In contrast, null hydrolytic activity towards meropenem could be detected. The absence of metallo-β-lactamase activity (i.e. no inhibition by EDTA) was judged by the similar growth for the reference strain around discs C and C/E for imipenem and ceftazidime (panel A, white arrows). In turn, the reduced growth of the indicator strain in the periphery of disc C/clavulanic acid (black arrows) when compared with disc C (panel B) indicated a clavulanic acid-sensitive serine enzyme in this strain. Both the obtained substrate and inhibitor profiles represent characteristic features of GES-like enzymes.20
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Figure 3 shows the typical pattern of an SME producer (in this case Serratia marcescens strain 5635; Table 1). As shown in the figure, both imipenem and meropenem were hydrolysed by the extracts, which was not the case for ceftazidime, indicating selectivity of the enzyme towards carbapenems (disc C in panels A and B, and disc C/Zn in panel A, white arrows at the top). Lack of EDTA inhibition (as judged by the comparison of growth of the indicator strain around discs C and C/E for both imipenem and meropenem in panel A) indicates the absence of a metalloenzyme. In turn, the clear inhibition of both imipenem and meropenem hydrolysis by clavulanic acid (black arrows, panel B), as judged by the comparison of growth of the indicator strain around discs C and C/clavulanic acid, revealed a clavulanic-acid-sensitive serine carbapenemase. All the above features are characteristic of all members of the SME family of carbapenemases.6
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Figure 4(a) illustrates the situation found in the case of K. pneumoniae strain 9171, a KPC carbapenemase (KPC-2) producer and also an extended-spectrum β-lactamase (PER-2) producer (Table 1). As seen in the figure, hydrolysis of imipenem and meropenem, and ceftazidime to a lesser extent, was judged by the different extents of growth of the indicator strain around discs C and C/Zn for the corresponding antimicrobials (white arrows at the top). The lack of inhibition by EDTA in all cases (panel A) indicated no detectable metallo-β-lactamase activity. In contrast, the inhibition of the hydrolysis of both imipenem and meropenem by clavulanic acid (panel B, black arrows) clearly revealed the presence of a class A serine carbapenemase in these extracts. All these features are clearly compatible with a KPC carbapenemase producer.3 In this case, ceftazidime hydrolysis most probably resulted from the contribution of both KPC-2 and PER-2 enzymes.
Figure 4(b) shows the AIM pattern obtained for Citrobacter freundii strain 9169, which produces both KPC-2 and AmpC enzymes (Table 1). Relevant growth of the indicator strain was observed for both carbapenems around discs C (panels A and B) and C/Zn (panel A), whereas a much reduced growth occurred in the vicinity of the equivalent discs in the case of ceftazidime (white arrows at the top). The hydrolytic pattern for imipenem and meropenem as well as the inhibitory action of clavulanic acid on these activities (panel B, black arrows) indicated the presence of a class A serine carbapenemase in these extracts. In turn, the similar growth pattern of the indicator strain around discs C and C/clavulanic acid for ceftazidime (Figure 4b, panel B, white arrow) indicated the additional occurrence of a clavulanic-acid-resistant serine enzyme, a situation compatible with the presence of AmpC. For comparison purposes, the AIM pattern generated by a P. aeruginosa clinical strain overproducing an AmpC enzyme was also included in this figure (panel B*). It is generally accepted that ceftazidime is labile, imipenem is marginally labile and meropenem is stable under conditions of AmpC overproduction.23 As shown in panel B*, a slight hydrolysis of ceftazidime, of imipenem to a much lesser extent and almost null meropenem hydrolysis, could be judged by the growth of the indicator strain in the vicinity of the corresponding C disc (white arrows at the top). A similar growth observed for the indicator strain around discs C and C/clavulanic acid for each of the corresponding antimicrobials (white arrows) indicated the presence of a clavulanic-acid-resistant β-lactamase in these extracts. These results are thus compatible with a strain overproducing an AmpC β-lactamase displaying almost null levels of carbapenemase activity.23
The combined data provided by the AIM assay could thus uncover the presence of a clavulanic-acid-sensitive enzyme such as KPC-2 in the two different KPC producers shown in Figure 4, with no significant interference from the different β-lactamases that co-exist with the carbapenemase in these strains.
Class D serine carbapenemase producers (oxacillinases). The pattern obtained in the case of an OXA-72 producer, A. baumannii strain 20706 (Table 1), is shown in Figure 5. As seen in the figure, significant growth of the indicator strain around discs C (panels A and B, white arrows at the top) and C/Zn (panel A) for both imipenem and meropenem indicated reduction of the antimicrobial activity of these carbapenems by the extracts. In contrast, much reduced growth of the indicator strain was observed around the corresponding discs in the case of ceftazidime (panels A and B, white arrows at the top). In turn, a similar growth of the indicator strain observed for all β-lactam substrates around discs C and C/E (panel A, white arrows) and discs C and C/clavulanic acid (panel B, white arrows) indicated no inhibition of the antimicrobial inactivating activity by either EDTA or clavulanic acid. The above represent the characteristic features of class D carbapenemases.3,8
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Patterns similar to those shown in Figure 5 were generated by the A. baumannii strains producing OXA-23 and OXA-58 oxacillinases (Table 1, data not shown). It is worth noting that A. baumannii strains producing oxacillinases belonging to the OXA-51 family showed no detectable hydrolytic ability towards imipenem, meropenem or ceftazidime by the AIM assay (data not shown), reinforcing observations made previously by other authors using other approaches.8,24
Non-carbapenemase producers. Finally, it is worth noting that control strains previously identified as non-carbapenemase producers (for details, see the Materials and methods section) generated noticeable carbapenemase-negative patterns when tested by the AIM assay (data not shown).
The AIM assay allows a correct discrimination of carbapenemases produced by different Gram-negative clinical isolates into class A, B or D, including variants among each class. The use of bacterial extracts instead of cell cultures helps circumvent other mechanisms of antimicrobial resistance such as reduced outer membrane permeability, efflux pumps or altered PBPs,11 as well as problems related to the induction of endogenous β-lactamase genes (e.g. ampC) during the assay mediated either by β-lactam substrates10 or inhibitors25 which may obscure detection of co-existing carbapenemases. The AIM assay tests the presence of both imipenem- and meropenem-hydrolysing activities in the extracts, allowing the identification of enzymes with radically different affinities4,16,26,27 for these two carbapenems (Figure 2). The parallel testing of ceftazidime hydrolysis has additional advantages, such as the differentiation within class A carbapenemase producers (Figures 2 and 3) or the more accurate identification of the typical hydrolytic profile of A. baumannii oxacillinases (Figure 5). In the latter case, the AIM assay correctly characterized as oxacillinases all variants produced by the different A. baumannii strains tested (detailed in Table 1), therefore providing a useful alternative to identify this emerging mechanism of carbapenem resistance.3,8,28 Other procedures such as Etest MβL strips, in contrast, have been found to produce false-positives for OXA-23-producing A. baumannii,3 and a similar observation was also made by us for OXA-58 and OXA-72 producers.
The few available therapeutic options for the treatment of infections caused by carbapenemase producers mandate that all efforts be made to limit their spread and associated clinical threat. Phenotypic approaches currently in use show different failures to consistently detect carbapenemase-producing strains, and improved methodologies are needed.3,4,6,7 In this context, by simultaneously testing the presence of imipenem- and meropenem-hydrolysing activities, the AIM assay provides a useful tool for the diagnostic microbiological laboratory to rapidly identify as carbapenemase-positive isolates presenting any of these activities.3 Although substantial clinical evidence is lacking, the use of carbapenems for the treatment of infections due to these strains would be contraindicated even though they test as sensitive by routine disc testing.4,7 Moreover, and although subsequent confirmation by molecular methods is certainly required, the AIM assay provides an early indication of the type of carbapenemase present in these strains, all in all essential steps to help prevent their generalized spread.
The high phenotypic diversity exposed by carbapenemase producers has promoted different discussions and suggestions on the candidates to be screened by the diagnostic microbiology laboratory.3,7,8 At present, we routinely screen by the AIM assay all enterobacterial, Pseudomonas and Acinetobacter nosocomial isolates showing non-susceptibility towards imipenem and/or meropenem, as well as those susceptible to both carbapenems but resistant to most non-carbapenem β-lactams, as detected by the routine disc testing.13
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Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)
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Funding |
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TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary dataFundingTransparency declarationsReferences |
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This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina); Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Howard Hughes Medical Institute (HHMI); Comisión Nacional de Programas de Investigación Sanitaria (Becas Carrillo-Oñativia 2004–05), Ministerio de Salud y Ambiente de la Nación Argentina; and Departamento de Salud Pública, Municipalidad de Rosario.
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Transparency declarations |
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None to declare.
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Footnotes |
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These authors contributed equally to this work. 
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Acknowledgements |
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We are indebted to M. Castanheira and M. Toleman for kindly providing P. aeruginosa 48-1997, to A. P. Gibb for P. aeruginosa 105663 and to P. Nordmann for P. aeruginosa COL-1. We are also indebted to M. Galas and F. Pasteran for the gift of Pseudomonas, Klebsiella, Citrobacter and Serratia strains of the Malbrán Institute collection. We are grateful to personnel from the Bacteriology Service, Hospital Provincial Centenario, Facultad de Ciencias Bioquímicas y Farmacéuticas, for kindly providing clinical strains. We are also grateful to R. Morbidoni for generous discussions and advice.
A. M. V. and A. J. V. are Staff Members of CONICET. A. J. V. is also an International Scholar of HHMI. P. M. and A. S. L. are Researchers of the National University of Rosario. V. B. is a Researcher of the Departamento de Salud Pública, Municipalidad de Rosario. T. S. and G. C. are fellows of the National University of Rosario. M. A. M. and J. M. B. are post-doctoral fellows of CONICET.
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References |
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