Controlling the false positive results of the Hodge and Masuda assays for class A. carbapenemase detection in species of Enterobacteriaceae

Transcription

1 JCM Accepts, published online ahead of print on February 0 J. Clin. Microbiol. doi:./jcm.0-0 Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Controlling the false positive results of the Hodge and Masuda assays for class A carbapenemase detection in species of Enterobacteriaceae Running Title: Hodge and Masuda tests for class A carbapenemases Authors: Fernando Pasteran, Tania Mendez, Melina Rapoport, Leonor Guerriero and Alejandra Corso Author s affiliation: Servicio Antimicrobianos, Departamento Bacteriología, Instituto Nacional de Enfermedades Infecciosas (INEI)- ANLIS Dr. Carlos G. Malbrán, Ministerio de Salud y Ambiente, Ciudad Autónoma de Buenos Aires, Argentina. Address of the Institution at which the work was performed: Servicio Antimicrobianos, INEI-ANLIS Dr. Carlos G. Malbrán, Av. Velez Sarsfield (C1AFF), Buenos Aires, Argentina. Corresponding author: Alejandra Corso Address: Servicio Antimicrobianos, INEI - ANLIS Dr. Carlos G. Malbrán, Velez Sarsfield Ave. (C1AFF), Buenos Aires, Argentina. acorso@anlis.gov.ar Phone/FAX:

2 The modified Hodge method (MHT) has been recommended by the CLSI for the confirmation of suspected class A carbapenemase production in species of Enterobacteriaceae. This test and the Masuda method (MAS), have advantages over traditional phenotypic methods by directly analyzing the carbapenemase activity. In order to identify the potential interferences of these tests, we designed a panel composed by diverse bacterial genera with distinct carbapenem susceptibility patterns ( carbapenemase producers and nonproducers). About % of false positives results among carbapenemase nonproducers, mainly strains harboring CTX-M and AmpC hyperproducers, could be observed. Subsequently, we developed an optimized approach for a more accurate detection of suspicious isolates of carbapenemase by the addition of boronic acid (BA) derivatives (reversible inhibitor of Class A carbapenemases and AmpC cephalosporinases) and oxacillin (inhibitor of AmpCs enzymes). The modified BA- and oxacilin-based MHT and MAS resulted highly sensitive (>0%) and specific (0%) for class A carbapenemase detection. By using these methodologies, isolates producing, KPC, GES, Sme, IMI, NMC-A carbapenemases, were successfully distinguished from those producing other classes of ß-lactamases (ESBLs, AmpCs, MBLs, etc). These methods will provide fast and useful information needed for targeting antimicrobial therapy and appropriate infection control.

3 Class A carbapenemases (KPCs Sme, NMC-A, IMI and some allelic variants of GES/IBC) have become more prevalent within the Enterobacteriaceae family (, ). Early recognition of carbapenemase producers has become mandatory as clinical failure associated with these enzymes had been described (). Therefore, different strategies are needed for their detection in any attempt aimed for their control and eradication. An accurate identification of Class A carbapenemases will therefore rely on the availability of specific, sensitive and simple assays able to differentiate carbapenemase producers from nonproducers. The modified Hodge test (MHT) (), and similar ones, as the Masuda assay (MAS) (), directly analyzes the carbapenemase activity in unbroken cells and enzyme crude extracts, respectively. These tests preformed better than routine phenotypic methods for the detection of carbapenemase producing bacteria, especially when combined mechanisms are present. Additionally, a practical and fast approach that efficiently disrupts bacteria cells has become available to the routine clinical microbiological laboratory, making MAS even easier to perform (1). Thus, given these features, these methods started to gain more adherents among microbiologists. Moreover, CLSI has issued recommendations for the phenotypic screening of carbapenemase producers among species of Enterobacteriaceae and included the MHT as a confirmatory assay (). Thus, MICs of ertapenem (ETP), meropenem (MEM), and imipenem (IPM) of, to, and to µg/ml, respectively (or a zone of inhibition by ETP or MEM of 1 mm in diameter in the disk diffusion assay), may indicate isolates with carbapenemase production, and this phenotype should be confirmed by the MHT (). However, we have reported up to a quarter of false positive MHT results (carbapenemase-like pattern) among carbapenemase nonproducers (as defined by spectrophometric assays and molecular methods) (1). These unexpected outcomes were observed among both, CTX-M producing bacteria with a uniformly decreased

4 susceptibility to ETP and AmpC hyperproducers. Recent reports has also questioned the specificity of the MHT, being AmpC hyperproducers and several members of the CTX- M ESBL family (mostly CTX-M-, but also CTX-M-1 and CTX-M-) the main cause of the unexpected performance (1,, 1). Cloxacillin, oxacillin (OXA) and boronic acids (BA) have long been known as reversible inhibitors of Class C enzymes (,, ). More recently, it has been shown an additional property of the BA derivatives, but not of OXA, which includes their ability to inhibit KPC enzymes (, 1, ) and the remaining members of the class A carbapenemase family (GES, Sme, IMI, NMC-A) (1). Using a panel composed by diverse bacterial genera with distinct carbapenem susceptibility patterns we design strategies to improve the efficiency of the MHT and MAS for the phenotypic confirmation of carbapenemase producers among species of Enterobacteriaceae by the addition of BA and OXA as inhibitors. The proposed procedures designated boronicbased modified Hodge test (BA-MHT) plus the oxacillin-based modified Hodge test (OXA-MHT), and the boronic-based Masuda assay (BA-MAS) plus the oxacillin-based Masuda assay (OXA-MAS) could be suitable as confirmatory tests as they displayed high sensitivity (SN) and specificity (SP) for detection of class A carbapenemase producers in addition to be relatively simple and inexpensive. Materials and Methods. Bacterial strains: Tests were performed with a panel consisting of diverse bacterial genera with distinct carbapenem susceptibility patterns. A total of carbapenemase-producing isolates (including producing class A carbapenemases) and non-carbapenemase-producing isolates of Enterobacteriaceae were included in the panel (Table 1). The resistance mechanisms of the strains employed in this work, carbapenem MICs for the strains, and

5 the sources of the strains are described in detail in Table 1. All isolates were nonduplicates (molecular typing to ensure that they were genetically unrelated was performed previously), and except for ATCC isolates and Salmonella species transconjugants, all were isolated from clinical specimens. All isolates were previously identified by conventional techniques (1) and by use of an API 0E system (biomérieux, Marcy l Etoile, France). The genotypes of the isolates were characterized previously by molecular techniques (1). In addition, to exclude the possible presence of other, not-yet-described carbapenemases in the negative control panel, the imipenemase activities of cell extracts from overnight broth cultures were previously determined by spectrophotometric assays (1). Microbiological assay: MHT, BA-MHT and OXA-MHT: The MHT was performed as described previously (). Briefly, a 1/ dilution of an inoculum of the indicator organism E. coli ATCC, adjusted to a 0. McFarland turbidity standard, was used to inoculate the surface of plates (diameter, 1 mm; Greiner Laboratory, Germany) of Mueller-Hinton agar (Difco, BBL) by swabbing. After the plates were allowed to stand for min at room temperature, disks (Difco, BBL), one with IPM ( µg), MEM ( µg) and ETP ( µg) (MHT) and one of each carbapenem disks supplemented with either µl of a - aminophenyl-boronic acid (APB) solution (BA-MHT) or µl of OXA solution (OXA- MHT) were placed onto the agar plates. We evaluated two APB formulations: a monohydrate, soluble in DMSO (Sigma Chemicals, St. Louis, MO) and the hemisulfate salt, water soluble (Boron Molecular Inc., Research Triangle Park, NC). Several APB concentrations of both formulations were tested: 0.,, 0, 0, 00 and 00 mg/ml, rendering, 0, 00, 00, 000 and 000 µg/disk of APB, respectively. As well, several OXA concentrations (Oxacillin sodium salt, Sigma Chemicals, St. Louis, MO)

6 were tested:, 0, 0 and 0 mg/ml, rendering 0, 00, 00 and 00 µg/disk of OXA, respectively. Subsequently, by using a -µl loop, three to five colonies of the test organisms, grown overnight on an agar plate, were inoculated onto the plate in a straight line from the edge of one disk to another, containing the same carbapenem (Figure 1). The presence of growth of the indicator strain towards the carbapenem disks was interpreted as a positive result for carbapenem hydrolysis screening (carbapenemase-like pattern). A BA-inhibited or an OXA-inhibited carbapenemasepattern was distinguished due to the absence of growth of the indicator strain towards the carbapenem plus APB or OXA disks, respectively, when compared to the corresponding disk without inhibitor. More information for the interpretation for a BA and/or OXA-inhibited carbapenemase pattern is described in the result section. Blank disks containing less than 00 µg of APB and 00 µg of OXA per disks did not inhibit or distort E. coli ATCC growth in preliminary assays. Higher concentrations of APB and OXA produced an inhibition of the growth of the indicator (inhibition zone less than mm). This effect did not interfere with the MHT because the inhibition zones produced by the inhibitors were by far within the zone of inhibition expected for the carbapenems. As APB monohydrate was dissolved in DMSO (Sigma Chemicals, St. Louis, MO), we tested also the effect of DMSO by itself and had no effect on the growth of the indicator. MAS, BA-MAS and OXA-MAS: the procedure described here was based on an adaptation of the assay originally developed by Masuda et al. and ulterior modifications (, 1). Crude extracts were prepared by subjecting the cell suspension to mechanic rupture as described previously (1). Briefly, bacteria were grown overnight at C in a blood agar plate. Two complete µl loops of the test strain were harvested and then suspended in 00 µl of 0 mm Tris-HCl (ph ) supplemented with 0.01 mm ZnSO

7 (ICN Biochemicals Ltd, Thame, Berks, UK). Next, 00 mg of 0.1 mm zirconia/silica beads (Biospect Products, Bartlesville, OK) were added. Crude extracts were prepared by subjecting the cell suspension to mechanic rupture by minutes vortex at maximal speed in a Maxi-Mix II mixer processor (Barnstead/Thermolyne, Inc., Waltham, MA). The cell extracts were clarified by centrifugation at 1,000 rpm for 1 min at C and supernatants stored at -0 C until processing. Before used, crude extracts were thawed at room temperature. A set of two filter disks were loaded with µl and l µl of crude extract of a test strain. The same volumes of extract were loaded on each of two blank disks previously supplemented with either µl of an APB or OXA solutions. Several APB concentrations of both, the monohydrate and the hemisulfate salt, were tested:, 0, 0 and 00 mg/ml, rendering 0, 00, 00 and 000 µg/disk of APB, respectively. As well, several OXA concentrations were tested:, 0 and 0 mg/ml, rendering 0, 00 and 00 µg/disk of OXA, respectively. APB and OXA disks were prepared previously and stored at -0 C. Disks were allowed to dry at room temperature for 1 minutes. Then, an inoculum of the indicator organism E. coli ATCC, adjusted to a 0. McFarland turbidity standard, was used to inoculate the surface of a plate of Mueller-Hinton agar by swabbing. After the plate was allowed to stand for min at room temperature, disks (Difco, BBL) with IPM ( µg), MEM ( µg) and ETP ( µg) were placed onto the agar plate. Subsequently, each carbapenem disk was surrounded by extracted-loaded disks that were applied within the expected susceptibility zone of the carbapenem tested (Figure ). The plates were incubated overnight at C, and the growth patterns of the indicator E. coli ATCC strain were evaluated. The presence in the extract of any ß-lactamase that could hydrolyze carbapenems (carbapenemase-like pattern) was revealed by the growth of the indicator E. coli cells around the blank disks loaded with the crude extract (with one or both of

8 the volumes used). The interpretation for a BA-inhibited and/or OXA-inhibited carbapenemase-like pattern is described in detail in the result section. Characterization of the mechanisms of resistance. i) PCR amplification and DNA sequencing. PCR analyses of all isolates were performed to confirm the presence of known carbapenemase genes. The isolation of total DNA was performed as described previously (1). Strains were analyzed for bla VIM, bla IMP, bla SPM, bla KPC, bla Sme, bla IMI, bla NMC-A, bla CTX-M, bla TEM, bla PER, bla SHV, bla AmpC and bla OXA genes (subgroups I, II, and III) as described previously (1). ii) Outer membrane protein (OMP): OMPs analysis was performed by SDS-PAGE as previously reported (1, 1). iii) AmpC production: First, suspected isolates were screening by isolectric focusing (IEF) using a substrate-base development method as published previously (1). Those crude extracts with IEF bands that displayed in situ inhibition with OXA (1 mm) were subjected to spectrophotometrically analysis. The ß-lactamase activities (nanomoles of cephalotin Laboratorios Richet, Argentina- hydrolyzed per minute per milligram of protein) were measured spectrophotometrically (SmartSpec 000, Bio-Rad, US) on selected crude extracts previously incubated for 1 min in µg/ml of clavulanate (Laboratorios Roemmers, Argentina) as described previously (). A >% reduction of cephalotin hydrolysis after OXA treatment (by incubation of crude extracts for 1 min in 0 µm OXA) was considered a positive result for class C ß-lactamases (). Acinetobacter baumannii (bla OXA- ) and A. baumannii FAV-1 (bla OXA- plus bla PER- ) were used as control strains (). Increments of AmpC ß -lactamase activity > fold when compared with the baseline activity derived from ceftazidme suscepitible of the panel, was considered as an AmpC hyperproduction. SN and SP. Sensitivity (SN) and specificity (SP) calculations were done essentially as described in reference. The SN value is based on the ratio a/(a + c), where a represents

9 the number of strains that were correctly identified as producers of group class A carbapenemases by the tested assay and c represents the number of true class A carbapenemase producers incorrectly identified as nonproducing strains. The SP value is based on the ratio d/(b + d), where d is the number of isolates correctly identified by the tested assay as nonproducers of group class A carbapenemases and b is the number of strains that were incorrectly identified as class A carbapenemase producers. The positive (PPV) and negative predictive (NPV) values are represented by a/(a + b) and d/(c + d), respectively. Results and discussion. MHT, BA-MHT and OXA-MHT The MHT detects the activity of carbapenem-hydrolyzing enzyme in the unbroken cells of the analyzed bacteria. Besides the substrates recommended by CLSI (ETP and MEM) we also tested IPM on basis of the promising performance obtained previously (1). For standardization purposes, all strains (except two with an indeterminate MHT results due to the inhibition of growth of the indicator strain) were subjected to APB and OXA inhibition regardless they exhibited a positive or a negative MHT (Table 1). For clinical detection will be adequate to test these inhibitors only in strains with a positive MHT. The optimal inhibitor concentrations for this assay (highest SN and SP) were 00 mg/ml of APB (or 000 µg APB/disk) and 0 mg/ml of OXA (or 00 µg OXA/disk). As mentioned previously, these APB and OXA concentrations affected the growth of the indicator strain only by few millimeters being overtaken by the inhibition zone of the carbapenems, thus, not affecting the performance of the method. Both APB formulations performed equivalent. The MHT displayed high efficiency for the detection of Class A carbapenemaseproducing strains (Table 1) with a SN of 0., regardless of the substrate used (Table ),

10 corroborating our previous results (1). However, the major limitation of the MHT was the presence of discernible carbapenemase-like patterns among carbapenemase nonproducers, as was previously noticed (1,, 1, 1) (Table 1 and Figure 1). These false-positive results corresponded mainly to strains producing CTX-M- (n=) and those with hyperproduction of AmpC (,, and strains with MEM, ETP and IPM, respectively) (Table 1). Thus, SP showed slight differences according the substrate used, being as low as 0., 0. and 0. for IPM, ETP and MEM, respectively (Table ). Thus, we designed the BA-MHT and the OXA-MHT to improve the performance of the MHT. Typical results observed of the proposed inhibitor-based MHT assay are shown in Figure 1: a growth of the indicator E. coli strain towards the carbapenem disk, indicative of carbapenem hydrolysis (carbapenemase-like pattern), was observed in four representative strains. Class A carbapenemases were identified by comparing the growth of the indicator E. coli cells towards the disk containing the carbapenem alone, with that observed towards the disks supplemented with the inhibitors. The figure shows the results with the ETP disk, as representative of the carbapenems, since the results of BA- MHT and OXA-MHT were identical regardless of carbapenem tested. The typical situation found for a class A carbapenemase is shown for a representative KPC producing strain (M), which was revealed by APB inhibition, as inferred from the absence of growth of the indicator E. coli strain toward the ETP plus APB disk only (Figure 1). OXA failed to inhibit the carbapenemase-pattern as inferred from the growth of the indicator E. coli strain toward the ETP plus OXA disk. This pattern (inhibition by APB and refractory action with OXA) corresponded unequivocally to a Class A carbapenemases.

11 The typical pattern for class A carbapenemease nonproducers is shown for a VIM (M) and CTX-M (M1) representative strains where the indicator strain grew towards the ETP, ETP plus APB and ETP plus OXA disks as well (Figure 1). This revealed the presence of a non-class A carbapenemase enzyme due to the lack of APB inhibition. As no APB effect was observed, OXA results became irrelevant. Strains showing these carbapenemase-patterns (no inhibition by OXA and APB) should be screening for Class B carbapenemases by using appropriate methodologies, as those previously published (1, 1). Finally, several AmpC hyperproducer strains included in the panel displayed a carbapenemase-like pattern (Table 1), as shown for M1, a representative strain, where the growth of the indicator strain observed towards the ETP but not the ETP plus APB disk could indicate the presence of class A carbapenemase. However, the strain also exhibited OXA inhibition as revealed by the lack of growth of indicator strain toward the ETP plus OXA disk (Figure 1). This unique pattern (inhibition by both, APB and OXA) corresponded unequivocally to AmpC strains. When analyzing the whole the panel, all KPCs, Sme, IMI and NMC-A, but only half of the GES-producing strains displayed a MHT inhibited only by APB (Table 1). Thus, the SN of the BA-MHT was of 0. (Table ). The low performance for GES was observed only among strains of clinical origin but not among the respective tranconjugant strains, indicating that these enzymes were not refractory to APB inhibition. Probably the mucoid phenotype observed for the parental strains (no other carbapenemase producing strain of the panel had this phenotype) that might interfere with the enzyme or/and APB diffusion from the unbroken cells, could be responsible for these results for GESproducing strains. Among the 1 strains of the control group with a positive MHT result, of them (CTX-M and MBL producers) remained with a positive

12 carbapenemase pattern after the addition of APB, being properly classified as class A carbapenemases nonproducers (Table 1). The remaining strains (AmpC hyperproducer) were finally resolved as class A carbapenemases nonproducers with the incorporation of OXA as inhibitor (Table 1). Thus, with the simultaneous use of both inhibitors, a major increase in the SP of the MHT was observed (from 0.0s to 1.0) (Table ). MAS, BA-MAS and OXA-MAS. This procedure tested the presence of carbapenem hydrolyzing activities using cells-free crude extracts of the analyzed bacteria. We evaluated the behavior of the identified enzymes towards the inhibitors by addition of the crude extract onto APB or OXA disks. We found some limitations for the development of this technique: i) it was not possible to use disks with 1,000 µg or 00 µg of APB and OXA, respectively, because the zone of inhibition produced around these disks became relevant for the interpretation of the MAS. Therefore, we had to use disks with 00 µg of either, APB and OXA for the standardization of the BA-MAS and OXA-MAS, respectively, a significantly lower concentration of that required by the MHT to achieve its best performance. ii) A high variability in the crude extract protein content was observed (data not shown). Thus, due to the competitive, mass-dependent nature of APB/OXA inhibition, and with the restriction to increase the disk load, we were forced to analyze simultaneously two volumes of the crude extract ( and 1 µl) to try to achieve adequate inhibition. The different APB formulations performed identical. Characteristic results observed of the proposed assay are shown in Figure. : the observation of the typical carbapenemase pattern is indicated by the growth of the indicator E. coli strain around a blank disk charged with either or 1 µl of extract, as showed for representative isolates (highly-loaded crude extract disks tends to produce carbapenemase-like patterns more frequently than disks with µl). Class A 1

13 carbapenemase were identified by comparing growth of the indicator E. coli cells around blank and OXA disks but not around those with APB (comparison should be done between disks loaded with the same volume of crude extract and if inhibition of the carbapenemase-like pattern was observed with at least one of the volumes of crude extract used, the test was considered positive for that inhibitor). The Figure shows the results with the ETP disk, as representative of the carbapenem, since the results of BA- MAS and OXA-MAS were identical regardless of carbapenem tested. The most typical situation found for a class A carbapenemase is shown for M, a representative KPC producing strain: hydrolysis of ETP was revealed by the growth of the indicator E. coli strain around disks loaded with both, or 1 µl of extract. The presence of a class A carbapenemase was exposed by the presence of the characteristic profile that included both, APB inhibition, as inferred from the absence of growth of the indicator E. coli strain around APB disks loaded with either or 1 µl of extract, respectively, and, the absence of OXA inhibition as observed by the growth of the indicator E. coli strain around the OXA disks. This major pattern was observed for all the Sme, most of the KPCs (1/0 strains) and GES (/ strains), and, one IMI-1. Other situation for class A carbapenemase was observed among of the 0 KPCs, as shown for M1, other representative KPC strain: hydrolysis of ETP was revealed again around blank disks with either or 1 µl of extract, but in this case, the presence of the class A carbapenemase characteristic pattern was deduced only around disks loaded with µl of extract, as indicated by the absence of growth of the indicator strain around the corresponding APB disk but not around that with OXA. Appreciable growth around the 1 µl supplemented APB disk, similar to that of the corresponding control (1 µl of bacterial extract alone) was observed, probably indicating an excess of ß- lactamase over the APB load. Finally, another situation was found for one GES and one 1

14 NMC-A, as shown for M representative strain (Figure 1B): null hydrolytic activity towards ETP was obtained using µl of extract, as an absence of growth of the indicator E. coli strain was observed around the disk. But ETP hydrolysis was visible when disks loaded with 1 µl of extract were used, suggesting a weak or poorly extracted ß- lactamase. Class A carbapenemase was therefore revealed only among disks loaded with 1 µl of crude extract by the typical profile of absence zone of E. coli around APB disk (APB inhibition) together with the growth of the indicator strains toward de OXA disk. Because of these two situations, a potential excess of beta lactamase over the APB load or the presence of a weak or poorly extracted ß-lactamase, we strongly recommended the simultaneously use of two volumes ( and 1 µl) of extract in the tests. Among the control group, the typical pattern for MBLs producers is shown for M, a representative strain: the growth of the indicator E. coli strain was observed around both blank disks ( and 1 µl) and the similar growth for the reference strain was maintained around both, APB and OXA disks. These reveal the lack of APB and OXA inhibition due to the presence of a non-class A carbapenemase enzyme. Other representative pattern for class A carbapenemase nonproducers was observed for several CTX-M- producers (Table 1), as shown in Figure for M1, a representative strain: it showed null hydrolytic activity towards imipenem using µl of extract but a significant imipenem hydrolysis was observed with 1 µl. However, growth of the indicator strain was also observed around the corresponding APB and OXA disks (1 µl), revealing the lack of APB and OXA inhibition due to the presence of a non-class A carbapenemase enzyme. As this lack of inhibition with APB and OXA of CTX-M producers is share with MBLs, strains with this typical profile should be screened for class B carbapenemases (1, 1). Several AmpC hyperproducer strains (Table 1) 1

15 showed a carbapenemase-like pattern but only with 1 µl extract disks, as showed for M1 representative strain (Figure ). In parallel, a significantly reduced growth of the indicator strain occurred around the corresponding 1 µl APB disk, initially suggesting the presence of a class A carbapenemase. However, the lack of growth of indicator strain toward the OXA disk revealed the presence of a dual inhibited (OXA and APB) enzyme, a feature that rules out class A carbapenemases. After assessing the panel, we obtained a SN of 1.0 for the MAS together with a noticeably improvement of the SP of the classic assay from 0. and 0. for IPM and MEM/ETP, respectively, up to 1.0 for the three substrates when the combined BA/OXA-MAS was used (Table ). Although in the present study, the MAS combo performed with slightly greater SN values than the MHT one, for a voluminous work burden, we find the MHT more practical because both, fewer disks are required and there is no need to prepare crude extracts. However, MAS becomes mandatory for strains with indeterminate MHT results and for strains with marked mucoid phenotype that might have difficult to achieve APB inhibition on the MHT. Finally, it should be noted that the MHT an MAS did not included a strict standardization of the size of the inoculum or the enzymes concentration used, respectively. Although, this seemingly appears as a major design flaw of these tests as it may be one of the factors causing false positive results with the original techniques, it not resulted critical when using the BA/OXA-MHT/MAS under the methodological standardization described in this work. Concluding remark. Given the clinical and public health impact involved in the discovery of a strain carrying carbapenemase, screening and confirmation in clinical microbiology laboratories of 1

16 carbapenemases producers should include efficient and inexpensive methods that cover the different needs and complexities of the local and regional epidemiology. The MHT and MAS, as are able to expose easily the carbapenemase activity of suspected isolates, have gained adherents throughout the world. Moreover, the MHT has been included in the last CLSI edition for phenotypic confirmation of suspected carbapenemase producing strains (). However, these methods, as known to date, should be used with caution because of the presence of false positives results, especially in those scenarios with high proportions of strains expressing non-carbapenemase- mediated carbapenem non-suceptibility (such as that generated by endemic CTX-M or AmpC hyperproduction) (1,, 1, 0). Thus, for improvement of their performances for carbapenemase confirmation, we standardized several strategies, with the addition of BA and OXA as inhibitors, to circumvent these main interferences. On basis of the results in this work, the revised inhibitor-based methods (BA/OXA-MHT and BA/OXA-MAS) resulted in a significant reduction of the false positive results in addition to distinguished class A from those strains producing other classes of carbapenemases. Thus, with these modifications, these methods can be used by routine clinical laboratories as confirmatory tests with performances comparable to the reference (molecular) methods. The proposed methodology will provide fast and useful information needed for targeting antimicrobial therapy and appropriate infection control, especially for regions where carbapenem resistance mediated by carbapenemase nonproducers is high or is increasing. Acknowledgements. We are indebted to the valuable collaboration of Monica Piedrabuena and the cooperation of WHONET-Argentina Network for their proficiency to detect bacteria with unusual resistance mechanism, Laboratorios Britania, Argentina for gently 1

17 providing APB hemisulfate and Dr. R. Melano, Dr. A. Medeiros, Dr. D. Marcano, Dr. L. Martinez-Martinez and Dr. L. Fernandez Canigia, for providing reference strains for this study. 1

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20 1. Martínez-Martínez, L., S. Hernández-Allés, S. Abertí, J. M. Tomás, V. J. Benedi and G. A. Jacoby. 1. In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expandedspectrum cephalosporins. Antimicrob. Agents Chemother. 0: Miriagou, V., G. Cornaglia, M. Edelstein, I. Galani, C. G. Giske, M. Gniadkowski, E. Malamou-Lada E, L. Martinez-MartinezL, F. Navarro, P. Nordmann, L. Peixe, S. Pournaras, G. Rossolini, A. Tsakris, A. Vatopoulos, and R. Cantón. 0. Clin. Microbiol. Infect. 1:- 1. Melano, R., A. Corso, A. Petroni, D. Centrón, B. Orman, A. Pereyra, N. Moreno, and M. Galas. 00. Multiple antibiotic-resistance mechanisms including a novel combination of extended-spectrum ß-lactamases in a Klebsiella pneumoniae clinical strain isolated in Argentina. J. Antimicrob. Chemother. :-. 1. Moland E.S., N. Hanson, S. Overman, S. Shepherd, J. Ribes, and K. Thomson. 00. Abstr. th Intersci. Conf. Antimicrob. Agents Chemother., abstr. D-. 1. Murray, P. R., E. J. Baron, M. A. Pfaller, J. H. Jorgensen, and R. H. Yolken (ed.), Manual of clinical microbiology, th ed. ASM Press, Washington, DC. 0. Papanicolaou, G. A., A. Medeiros, and G. Jacoby.. Novel plasmidmediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alphamethoxy beta-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. :00-. 0

21 1. Pasteran, F., T. Mendez, L. Guerriero, M. Rapoport, and, A. Corso. 00. Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae. J. Clin. Microbiol. : Poirel, L., S. Corvec, M. Rapoport, P. Mugnier, A. Petroni, F. Pasteran, D. Faccone, M. Galas, H. Drugeon, V. Cattoir, and P. Nordmann. 00. Identification of the novel narrow-spectrum beta-lactamase SCO-1 in Acinetobacter spp. from Argentina. Antimicrob. Agents Chemother. 1:1-.. Queenan, A. M., and K. Bush. 00. Carbapenemases: the versatile ß- lactamases. Clin. Microbiol. Rev. 0:0-.. Tsakris, A., I. Kristo, A. Poulou, K. Themeli-Digalaki, A. Ikonomidis, D. Petropoulou, S. Pournaras, and D. Sofianou. 00. Evaluation of boronic acid disk tests for differentiating KPC-Possessing Klebsiella pneumoniae isolates in the clinical laboratory. J. Clin. Microbiol. :-.. Walther-Rasmussen, J., and N. Høiby. 00. Class A carbapenemases. J. Antimicrob. Chemother. 0:0-.. Yagi, T., J. Wachino, H. Kurokawa, S. Suzuki, K. Yamane, Y. Doi, N. Shibata, H. Kato, K. Shibayama, and Y. Arakawa. 00. Practical methods using boronic acid compounds for identification of class C ß-lactamaseproducing Klebsiella pneumoniae and Escherichia coli. J. Clin. Microbiol. :1-. 1

22 Table 1. Resistance mechanisms, MICs and microbiological methods results of the panel of strains used in this study as positive and negative controls. β-lactamase groups / Strain No. a Bacterial Specie b β-lactamases/decreased porins c MIC (µg/ml) d Microbiological assays (carbapenemase activity) e Reference or source IPM MEM ETP MHT MHT MHT MAS MAS BA- OXA- BA- OXA- MAS KPC producers (n = 0) 1 Cfr KPC-; TEM Kpn KPC-; PER; TEM-1; SHV-1 D0/0 Kpn KPC CAP QC Assurance Kpn KPC Sal f KPC (TC) 0 Sal f KPC (TC) Ecl KPC- ; Hyper. AmpC Eco KPC Cfr KPC- ; Ind. AmpC Eco KPC- ; TEM Pmi KPC- ; TEM Pmi KPC- ; TEM Ecl KPC- ; CTX-M Sma KPC Kpn KPC- ; CTX-M

23 1 Kpn KPC-, PER Kpn KPC Kpn KPC Kpn KPC R. Melano R. Melano R. Melano R. 1 Kpn KPC Melano Sme producers (n = ) 1 Sma Sme-1b 1 0. ND g ND ND Sma Sme-1b; CTX-M- 0. ND ND ND ;OXA- 1 Sma Sme-1b;OXA Sma Sme-1b; CTX-M-; 1 0, OXA-; SHV-1 01 Sma Sme-1b Sma Sme-1b Sme Sme Sma Sme Sma Sme Sma Sme IMI or NMC- A producers (n = ) 0 Ecl IMI Medieros A.

24 Ecl NMC-A GES producers (n = ) 01 Ent GES Kpn GES-; CTX-M-; Kpn GES Martinez- (derepressed or plasmid mediated) (n = 1) C Kpn FOX L. 0 Sal f GES (TC ) G1 Sal f GES This work (TC 01) G Sal f GES This work (TC ) MBL producers (n = ) VIM-;CTX-M- Kpn ;TEM-1;SHV-1, Kpn VIM; TEM-1, SHV Ecl IMP-, PER Pre VIM-, PER-, hyperproduced AmpC Producers of AmpC

25 Martinez L. Eco AAC Martinez L. Eco CMY (I) - - Martinez Martinez- Martinez- Kpn DHA L. Martinez- Martinez Sfl CMY Pmi CMY Kpn CMY POS Mmo Hyperproduced AmpC POS1 Cfr Hyperproduced AmpC Cfr Hyperproduced AmpC Ecl Hyperproduced AmpC Ecl Hyperproduced AmpC/ decreased porins P Ecl Hyperproduced AmpC Eco MIR (I) Ecl Hyperproduced AmpC (I, E) This work Ecl Hyperproduced AmpC (I) - - This work Ecl Eae Hyperproduced AmpC/ decreased porins Hyperproduced AmpC/ decreased porins This work This work Eco CMY/ decreased porins This work 1 Sal CMY This work

26 (TC 1) 1 Ecl CMY; inducible AmpC This work ESBL producers (n = ) PER-; CTX-M-; Kpn SHV-1; TEM-1; CTX-M-; SHV-1 Kpn h TEM-1; 1 Kpn CTX-M-; SHV-1 TEM-1/ decreased porins 1 Kpn CTXM-; SHV TEM-1;porins Kpn CTX-M-; SHV-1; TEM-1/ decreased porins /C Eco CTX-M R. Melano 1/C Kpn CTX-M R. Melano H Kpn CTX-M R. Melano 000 Kpn SHV-1/ decreased porin ATCC Cos1 Eco PER; TEM OSBL producer Eco TEM ATCC strain 1

27 Producers of low levels of K1 or penicillinase or cefuroximase (n = ) 0.01 Cko Penicilinase HMC Ppe Cefuroximase FLE1 Kox Low K Producers of low basal levels of AmpC (inducible) (n = ) GUT Cfr Inducible AmpC CEN1 Sma Inducible AmpC HMI Ecl Inducible AmpC POS B Mmo Inducible AmpC Pst Inducible AmpC Producers of ESBL(s) plus inducible AmpC (n = ) 1 Sma CTX-M-;TEM- 1;inducible AmpC/ decreased porins Sma CTX-M-; inducible

28 0 Ecl Pst Strains with AmpC/ decreased porins PER-; inducible AmpC CTX-M-; inducible AmpC wild-type and/or marginal or low-level ß- lactamase activities (n = ) 1 Sal h None Ecl None (AmpC - mutant) ATCC HMI1 Pmi None Eco Basal AmpC ATCC 1 a ESBL, extended-spectrum ß-lactamase; OSBL, older-spectrum ß -lactamase. b Cfr, Citrobacter freundii; Kpn, K. pneumoniae; Sal, Salmonella sp.; Eco, E. coli; Pmi, Proteus mirabilis; Sma, S. marcescens; Ecl, E. cloacae; Ent, Enterobacter sp.; Sfl, Shigella flexneri; Mmo, Morganella morganii; Cko, Citrobacter koseri; Ppe, Proteus penneri; Kox, Klebsiella oxytoca; Pst, Providencia stuartii. c indicates decreased amount of the evaluated OMP as depicted in the references d MICs determined by agar dilution following CLSI recommendations; IPM: imipenem; MEM: meropenem; ETP: ertapenem

29 e Modified Hodge tests (MHT): + : if the tested strains produced carbapenemase activity; - : if the tested strains did not produced carbapenemase activity. Boronicbased MHT (BA-MHT) or OXA-based MHT (OXA-MHT): + : when comparing with the corresponding control, if APB or OXA did not produced inhibition of the carbapenemase activity (i.e., carbapenemase activity persisted under the presence of the inhibitor); - : when comparing with the corresponding control, if APB or OXA produced inhibition of the carbapenemase activity (i.e., carbapenemase activity disappeared under the presence of the inhibitor). Masuda test (MAS): + : if one or both of the volumes of crude extract tested produced carbapenemase activity; - : if both volumes of crude extract tested did not produce carbapenemase activity. Boronic-based MAS (BA-MAS) or OXA-based MAS (OXA-MAS): + : when the strain displayed carbapenemase activity only with 1 µl disks and this carbapenemase activity was not reverted with APB/OXA, or, when both volumes of extract tested ( µl and 1 µl) displayed carbapenemase activity and both carbapenemase activities were not inhibited by APB or OXA; - : if with one or both of the volumes of crude extract tested, carbapenemase activity was inhibited by APB/OXA when comparing with the corresponding blank disk. f Transconjugant strain; brackets referred to the parenteral strain g ND, not determined due to an indeterminate Hodge tests results due to the inhibition of growth of the indicator strain. 1 h The hemisulfate salt but not the monohydrate APB could produced a partial hydrolysis of the imipenem load of the disk, reducing the expected inhibition zone in about mm (not shown). The reduction of the IPM load potentiated a false positive pattern for this

30 carbapenemase nonproducer strain without interfering with the overall performance of the assay. i Recipient strain for the conjugation assays 0

31 TABLE. Performances analysis of the confirmatory test a Assay (n) SN SP NPV PPV Modified Hodge test (MHT) (0) 0. b 0. (IPM) (IPM) 0. (MEM) 0. (MEM) 0. (ETP) 0. (ETP) BA-MHT () c (IPM) (IPM) 0. (MEM) 0. (MEM) 0. (ETP) 0.0 (ETP) BA-MHT + OXA-MHT () c Masuda test (MAS) (0) (IPM) (IPM) 0. (MEM, ETP) 0. (MEM, ETP) BA-MAS (0) (IPM) (IPM) 0. (MEM, ETP) 0. (MEM, ETP) BA-MAS b + OXA-MAS (0) a The levels of performance of and the most favorable conditions for the phenotypic confirmatory tests described in this study for the detection of class A carbapenemases among species of Enterobacteriaceae are summarized. 1

32 b When the substrate is not expressly stated, means that the carbapenems (IPM, MEM and ETP) showed the same performance c A total of of carbapememase producer strains were analyzed (two strains with indeterminate Hodge tests results due to the inhibition of growth of the indicator strain were excluded)

33 FIGURES LEGENDS Figure 1. Results of the modified Hodge test (MHT), the boronic-based MHT (BA- MHT) and the OXA-based MHT (OXA-MHT) with ETP plus OXA (00 µg per disk; left disks), ETP (central disks) and ETP plus APB (000 µg per disk; right disks) for representative isolates (K. pneumoniae M [producing KPC-], K. pneumoniae M [producing VIM], Enterobacter cloacae M1 [producing high levels of AmpC]), K. pneumoniae M1 [producing CTX-M-] and E. coli ATCC 1 [producing TEM-1]. Letter p indicates that ETP was hydrolyzed by the streaked cells; letter n indicates that ETP was not hydrolyzed by the streaked cells. Final interpretation of the MHT, BA- MHT and OXA- MHT are shown below each strain Downloaded from on April, 01 by guest

34 Figure. Results of the Masuda assay (MAS), the boronic-based MAS (BA-MAS) and OXA-based MAS (OXA-MAS) with ETP (central disks). The left part of each photo corresponded to the following disks loaded with µl of the crude extract: a blank disk ( µl, left disks), OXA disk (00 µg per disk, right disk) and APB disk (00 µg per disk, lower disk); and in the right part of each photo, to the following disks loaded with 1 µl of the crude extract: a blank disk (1 µl, left disks), OXA disk (00 µg per disk, right disk) and APB disk (00 µg per disk, lower disk). Representative isolates (K. pneumoniae M [producing KPC-], K. pneumoniae M1 [producing KPC-], Salmonella spp M [producing GES-], K. pneumoniae M [producing VIM], K. pneumoniae M1 [producing CTX-M-], Enterobacter cloacae M1 [producing high levels of AmpC]) and E. coli ATCC 1 [producing TEM-1] are shown. Letter p indicates that ETP was hydrolyzed by the β-lactam content of the crude extract; letter n indicates that ETP was not hydrolyzed by the β-lactam content of the crude extract. NC: not correspond (the matching blank disk did not show carbapenemase activity). Final interpretation of the MAS, BA-MAS and OXA-MAS are shown below each strain.

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