碳青霉烯酶表型检测方法

A simple phenotypic method for the differentiation of metallo-b -lactamases and class A KPC carbapenemases inEnterobacteriaceae clinical isolatesAthanassios Tsakris 1*,Aggeliki Poulou 1,2,Spyros Pournaras 3,Evangelia Voulgari 1,Georgia Vrioni 1,Katerina Themeli-Digalaki 4,Dimitra Petropoulou 5and Danai Sofianou 61Department of Microbiology,Medical School,University of Athens,Athens,Greece;2Department of Microbiology,Serres General Hospital,Serres,Greece;3Department of Microbiology,Medical School,University of Thessaly,Larissa,Greece;4Department of Microbiology,Tzaneion General Hospital,Piraeus,Greece;5Department of Microbiology,Saint Panteleimon Hospital,Nicea,Greece;6Department of Microbiology,Hippokration University Hospital,Thessaloniki,Greece*Corresponding author.Tel:+30-210-7462011;Fax:+30-210-7462210;E-mail:atsakris@med.uoa.grReceived 12March 2010;returned 15April 2010;revised 27April 2010;accepted 11May 2010Background:The increasing frequency of class A KPC enzymes and class B metallo-b -lactamases (MBLs)among Enterobacteriaceae as well as their possible co-production makes their early differentiation urgent.Methods:A simple phenotypic algorithm employing three combined-disc tests consisting of meropenem alone and with phenylboronic acid (PBA),EDTA,or both PBA and EDTA was designed for the differentiation of KPC and MBL enzymes.Augmentation of the zone of inhibition by ≥5mm was considered a positive combined-disc test result.A total of 141genotypically confirmed carbapenemase-positive Enterobacteriaceae clinical isolates (63KPC producers,47MBL producers,and 31KPC and MBL producers)with various carbapenem MICs were exam-ined.For comparison,84genotypically confirmed carbapenemase-negative Enterobacteriaceae clinical isolates [39extended-spectrum b -lactamase (ESBL)producers,22AmpC producers,and 23ESBL and AmpC producers]were also tested.Results:The phenotypic algorithm was able to differentiate MBL from KPC producers as well as to detect the possible co-production of both carbapenemases (positive result only with the combined-disc test using mero-penem alone and with both PBA and EDTA).The method detected all KPC or MBL producers (sensitivity 100%)as well as 30of the KPC and MBL producers (sensitivity 96.8%).All three combined-disc tests were negative for non-carbapenemase-producing isolates,except two ESBL and AmpC producers that gave positive combined-disc tests using meropenem alone and with PBA and both PBA and EDTA (specificity for KPC detection 98.8%).Conclusions:This phenotypic method is very helpful to detect carbapenemase production and provides a simple algorithm for the differentiation of KPC and MBL enzymes,especially in regions where KPC-and MBL-pos-sessing Enterobacteriaceae are highly prevalent.Keywords:class A carbapenemases,MBLs,extended-spectrum b -lactamases,plasmid-mediated AmpC,combined-disc test,EDTA,boronic acidIntroductionCarbapenems are often considered last resort antibiotics in the treatment of infections due to multidrug-resistant Enterobacter-iaceae clinical isolates,since they are stable even in response to extended-spectrum b -lactamases (ESBLs)and AmpC enzymes.However,during the last decade carbapenem resistance has been increasingly reported among Enterobacteriaceae and is largely attributed to the production of Ambler class B acquired metallo-b -lactamases (MBLs).1These enzymes efficientlyhydrolyse all b -lactams except monobactams.They have shown a worldwide dissemination although they are more fre-quently reported in southern Europe and the Asiatic-Pacific region.1,2More recently,a new type of class A b -lactamase,the Klebsiella pneumoniae carbapenemase (KPC),has also spread among K.pneumoniae isolates and other Enterobacteria-ceae.3These enzymes confer various levels of resistance to all b -lactams including carbapenems,even though cephamycins and ceftazidime are only weakly hydrolysed.3,4They have become widespread in several regions of North and South#The Author 2010.Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.All rights reserved.For Permissions,please e-mail:journals.permissions@J Antimicrob Chemother 2010;65:1664–1671doi:10.1093/jac/dkq210Advance Access publication 11June 20101664by guest on March 18, 2013/Downloaded fromAmerica as well as in Israel,China and Greece.5–10K.pneumo-niae isolates that co-produce both MBL and KPC carbapene-mases have also been documented 11and recently they have become widespread in several Greek hospitals (A.Tsakris,A.Poulou,E.Voulgari,S.Pournaras and D.Petropoulou,unpub-lished data).Such co-existence of carbapenem-hydrolysing enzymes in enterobacterial pathogens may further compromise the therapeutic alternatives not only due to the carbapenemase-mediated resistance to every b -lactam,but also due to the linkage with non-b -lactam resistance determinants.1,7It also has several epidemiological implications,since MBLs and KPCs are mostly transposon-and/or integron-encoded determinants that can easily disseminate to other enterobacterial strains.1,12,13Therefore,practical and accurate phenotypic approaches are urgently needed to differentiate the horizontally acquired mech-anisms of reduced susceptibility to carbapenems among Entero-bacteriaceae in the clinical laboratory.These assays may provide substantial information before application of the more expensive molecular techniques,which usually are used only by reference laboratories.Several non-molecular methods have been studied for the identification of carbapenemases,and in many cases their detection is based on the use of specific inhibitors.Thus,the detection of MBLs is based on the enzyme’s zinc dependence by using as inhibitors chelating agents such as EDTA.1,14Combined-disc tests based on the use of a KPC inhibitor,boronic acid,have also recently been described as promising methods for the detection of organisms with KPC enzymes.15–17However,the laboratory identification of carbapenemases needs to be adjusted as an ever-increasing variety of b -lactamases is reported in members of the family Enterobacteriaceae.In our hospitals K.pneumoniae isolates that harbour both MBL and KPC carbapenemases are increasingly recovered from clinical specimens,and this has led to difficulty differentiating and iden-tifying these enzymes by phenotypic testing.MBL and KPC enzymes hydrolyse almost all b -lactam antibiotics,and hence the phenotypic detection of each one of them can be masked by the expression of the other.The present study reports on the implementation of a phenotypic method that uses both the inhibitors EDTA and boronic acid.It describes the screening criteria,and a simple algorithm is recommended to detect and differentiate class A and B carbapenemases among enterobac-terial isolates that exhibit reduced susceptibility to broad-spectrum b -lactams.Materials and methodsClinical isolatesA total of 141genotypically confirmed carbapenemase-positive Entero-bacteriaceae clinical isolates (63KPC producers,47MBL producers,and 31KPC and MBL producers)with various carbapenem MICs were included in this study.The isolates were collected during 2007–10from separate patients who were hospitalized in five tertiary care hospitals located in four distinct Greek regions (two hospitals in the broad region of Athens and one hospital each in Thessaloniki,Larissa and Serres).The presence of bla KPC and bla VIM was determined using previously described oligonu-cleotide primers and cycling conditions.2,18For comparison,84genotypi-cally confirmed carbapenemase-negative Enterobacteriaceae clinical isolates (39ESBL producers,22AmpC producers,and 23ESBL and AmpC producers)were randomly selected among those exhibiting reduced susceptibility to either cefoxitin (MIC .8mg/L),third-generationcephalosporins (cefotaxime or ceftazidime,MIC .8mg/L)or carbapenems (imipenem or meropenem,MIC .4mg/L;ertapenem,MIC .2mg/L).All 84isolates were negative for known carbapenemase genes by PCR.These 84isolates came from collections held at the clinical laboratories providing KPC-positive isolates for the present study.The identification of all isolates was confirmed by using the API20E system(bioMe´rieux,Marcy l’E ´toile,France).Antimicrobial susceptibility testing and phenotypicscreeningDetailed susceptibility analysis was carried out by the agar dilution method following the CLSI guidelines and interpretative criteria.19ESBL production was tested with the CLSI ESBL confirmatory test 19and,in Enterobacteriaceae that produced AmpC or KPC enzymes,was tested with modified CLSI ESBL confirmatory tests using clavulanate in combi-nation with boronic acid.20,21Molecular testing for b -lactamase genesb -Lactamase genes were amplified using a panel of primers for detection of all known types of MBLs,2KPCs,18OXA-type carbapenemases,22plasmid-mediated AmpCs in single PCRs for each gene 23and ESBLs,including SHV,TEM,CTX-M and GES/IBC enzymes.24For Enterobacter aerogenes isolates,total RNA from logarithmic-phase-growth cultures was extracted with TRI Reagent (Ambion,Austin,TX,USA)and reverse transcription of 1m g of total RNA was performed with the ThermoScript RT-PCR System (Invitrogen,Carlsbad,CA,USA)according to the manufac-turer’s instructions.Derepressed AmpC-hyperproducing E.aerogenes iso-lates were identified by quantitative real-time PCR using the Quanti Test SYBR Green (Qiagen,Hilden,Germany)and primers 5′-TGCGTGTCATA ACATTATCCG-3′and 5′-AACCCGTAGCCCAGGTAAAC-3′.25Positive controls used were previously characterized isolates from our collection carrying all the types of tested b -lactamases.The PCR products were subjected to direct sequencing.PCR products were purified using ExoSAP-IT reagent (USB Corporation,Cleveland,OH,USA)and used as templates for sequencing on both strands with an ABI Prism 377DNA sequencer (Applied Biosystems,Foster City,CA,USA).Phenotypic testing for the differentiation of MBLs and class A KPC carbapenemasesA phenotypic detection method employing combined-disc tests of mero-penem alone and with 400m g of phenylboronic acid (PBA)or 292m g of EDTA or both 400m g of PBA and 292m g of EDTA was evaluated for the detection of carbapenemase production and the differentiation of KPC and MBL enzymes.The stock solution of PBA was prepared as previously recommended 17,26by dissolving PBA (benzeneboronic acid;Sigma-Aldrich,Steinheim,Germany)in DMSO at a concentration of 20mg/mL.From this solution 20m L (containing 400m g of PBA)was dispensed onto commercially available meropenem discs.The stock solution of EDTA was prepared by dissolving anhydrous EDTA (Sigma-Aldrich)in dis-tilled water at a concentration of 0.1M.14From this solution 10m L (con-taining 292m g of EDTA)was dispensed onto meropenem discs.The discs were then dried and used within 60min.The test was performed by inoculating Mueller–Hinton agar as given for the standard diffusion method 19and placing onto agar one disc of meropenem without any inhibitor and three discs of meropenem containing 400m g of PBA,292m g of EDTA or both 400m g of PBA and 292m g of EDTA.The agar plates were incubated at 378C overnight.The diameter of the growth-inhibitory zone around the meropenem disc with PBA,EDTA,or PBA plus EDTA was compared with that around the plain meropenem disc.Production of KPC was considered when the growth-inhibitory zone diam-eter around the meropenem disc with PBA and the meropenem disc withPhenotypic method for differentiating MBLs and KPCs in Enterobacteriaceae1665JACby guest on March 18, 2013/Downloaded fromboth PBA and EDTA was increased≥5mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone.Production of MBL was considered when the growth-inhibitory zone diameter around the meropenem disc with EDTA and the merope-nem disc with both PBA and EDTA was increased≥5mm compared with the growth-inhibitory zone diameter around the disc containing merope-nem alone.Production of both KPC and MBL enzymes was considered when the growth-inhibitory zone diameter around the meropenem disc with both PBA and EDTA was increased≥5mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone while the growth-inhibitory zone diameters around the merope-nem disc with PBA and the meropenem disc with EDTA were increased ,5mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone.Finally,when none of the three combined-disc tests was positive,the isolate was considered negative for MBL and KPC carbapenemase production.It should be noted that the concentration of PBA and EDTA employed in the present study did not show any detectable effect on bacterial growth.Sensitivity and specificityThe performance of the various tests of the algorithm for the detection of KPC and/or MBL producers was evaluated using PCR as the gold standard. For each interpretation of the algorithm the sensitivity was calculated from the number of MBL-and/or KPC-possessing organisms that were correctly determined,while the specificity was calculated from the number of non-KPC-and/or non-MBL-possessing organisms that were correctly determined.ResultsSpecies identification and susceptibilities to carbapenems of the bacterial isolates used in this study The speciation of the studied isolates is presented in Table1.The 63KPC producers belonged to six different enterobacterial species,the47MBL producers to four enterobacterial species, while all31isolates that co-produced KPC and MBL carbapene-mases were identified as K.pneumoniae.The84KPC-and MBL-negative isolates were identified as K.pneumoniae, Escherichia coli or E.aerogenes.Carbapenem MICs varied substantially among the141 carbapenemase-possessing isolates(Table2).The63KPC-positive isolates had carbapenem MICs that ranged from2to 64,1to64and4to.128mg/L for imipenem,meropenem and ertapenem,respectively.Seventeen of these isolates were susceptible to imipenem and meropenem,andfive additional isolates were susceptible only to meropenem.Slightly higher carbapenem MIC50s,MIC90s and mean MICs were detected among the47VIM-positive isolates as well as the31isolates that produced both carbapenemases.Among the84 carbapenemase-negative isolates,carbapenem MICs ranged from,1to8,,1to4and,1to32mg/L for imipenem,mer-openem and ertapenem,respectively(Table2).Sixteen of the latter isolates were non-susceptible(imipenem and merope-nem,MIC.4mg/L;ertapenem,MIC.2mg/L)to at least one of the carbapenems.Phenotypic and molecular screeningAmong the genotypically KPC-positive isolates,sequencing analysis identified the KPC-2variant in15randomly selected iso-lates.Phenotypic testing in combination with molecular testing showed that43of the63KPC-positive isolates were additionally ESBL producers.An SHV-type ESBL was detected in38isolates and a CTX-M-type ESBL in the remaining5isolates(Table1). Among the genotypically VIM-positive isolates sequencing analy-sis identified the VIM-1variant in15randomly selected isolates. Phenotypic testing in combination with molecular testing showed that21of the47VIM-positive isolates were additionallyTable1.Strain groups and genotypes of bacterial isolates used in this studyNo.of isolatesKlebsiella pneumoniae Klebsiella oxytoca Escherichia coli Enterobacter spp.f Serratia marcescens total KPC producers654520KPC/ESBL a producers392243VIM producers174526VIM/ESBL b producers18321 VIM/KPC producers2020 VIM/KPC/ESBL c producers1111 AmpC derepressed producers77 Plasmidic AmpC producers10515 Plasmidic AmpC/ESBL d producers131023 ESBL e producers122739 Total146647215225a blaCTX-M-15(n¼5),bla SHV-5(n¼2),bla SHV-12(n¼36).b blaCTX-M-15(n¼6),bla SHV-5(n¼15).c blaCTX-M-15(n¼3),bla SHV-5(n¼9).d blaCTX-M-3(n¼6),bla CTX-M-15(n¼4),bla SHV-5(n¼13).e blaCTX-M-3(n¼13),bla CTX-M-15(n¼16),bla SHV-5(n¼9),bla GES/IBC-1(n¼1).f All Enterobacter spp.isolates were identified as E.aerogenes except two KPC producers and four VIM producers that were identified as E.cloacae. Tsakris et al.1666 by guest on March 18, 2013 / Downloaded fromESBL producers.An SHV-type ESBL was detected in 15isolates and a CTX-M-type ESBL in the remaining 6isolates (Table 1).Among the genotypically KPC-and VIM-positive isolates,sequen-cing analysis identified KPC-2and VIM-1variants in 10randomly selected isolates.Twelve of the 31KPC-and VIM-positive isolates were additionally ESBL producers;9produced an SHV-type ESBL and 3a CTX-M type ESBL (Table 1).PCR testing for other groups of ESBL genes (TEM,GES/IBC)as well as for all known clusters of plasmid-mediated AmpC genes was consistently negative in all KPC-and/or VIM-positive isolates.However,the quantitative real-time PCR showed that all 8KPC-or VIM-possessing E.aerogenes isolates contained stably derepressed AmpCs in comparison with control E.aerogenes isolates which contained inducible AmpC enzymes.Molecular testing in combination with phenotypic testing of the 84carbapenemase-negative isolates showed that 62harboured ESBLs (22SHV-types,39CTX-M-types and 1GES/IBC-type)and 38harboured plasmid-mediated AmpC b -lactamases,which belonged to three of the six plasmid-mediated clusters of AmpCs (22belonged to the cluster MOX-1,MOX-2,CMY-1,CMY-8to CMY-11;14belonged to the cluster LAT-1to LAT-4,CMY-2to CMY-7,BIL-1;and 2to the cluster DHA-1,DHA-2).It should be noted that 23of the above isolates contained both ESBL and plasmidic AmpC enzymes.The seven E.aerogenes isolates were genotypically ESBL and plasmidic AmpC negative.However,all of them contained stably derepressed AmpCs in comparison with control E.aerogenes isolates that had inducible chromoso-mal AmpC enzymes.Combined-disc tests for the differentiation of MBL and KPC carbapenemasesDetailed results of the combined-disc tests are shown in Table 3.All 63KPC-possessing isolates showed a ≥5mm increase in the zone diameters of the combined discs with PBA or both PBA and EDTA compared with meropenem alone,whereas the combined-disc test that uses meropenem with and without EDTA was clearly negative in all of them (sensitivity 100%;Table 4).The activity of meropenem was enhanced remarkably by PBA (mean increase 9mm)or PBA plus EDTA (mean increase 10mm)in all KPC producers and irrespective of the carbapenem MICs or the co-production of an ESBL.All 47VIM-possessing isolates showed a ≥5mm increase in the zone diameters of the combined discs with EDTA or both PBA and EDTA compared with meropenem alone,whereas the combined-disc test that uses meropenem with and without PBA was clearly negative in all VIM producers (sensitivity 100%;Table 4).The activity of meropenem was enhanced remarkably by EDTA (mean increase 10mm)or PBA plus EDTA (mean increase 11mm)in all 47VIM producers and irre-spective of the carbapenem MICs or the co-production of an ESBL.All but one of the 31KPC-and VIM-possessing isolates showed a ≥5mm increase in the zone diameter of the combined-disc test using meropenem with and without both PBA and EDTA,whereas the combined-disc tests using meropenem with and without PBA or EDTA were negative in all KPC and VIM producers (sensitivity 96.8%;Table 4).The activity of meropenem was enhanced remarkably by the combination of PBA and EDTATable 2.Distribution of carbapenem MICs for the carbapenemase-positive and carbapenemase-negative clinical isolates of this studyPCR-confirmed strain group/antimicrobial Number of isolates with an MIC (mg/L)of:,11248163264128.128KPC and KPC/ESBL positive (n ¼63)imipenem 5121914103meropenem 2713181193ertapenem7121715921VIM and VIM/ESBL positive (n ¼47)imipenem 49141541meropenem 25917122ertapenem3611131031KPC/VIM and KPC/VIM/ESBL positive (n ¼31)imipenem 1581241meropenem 257134ertapenem3612541AmpC and AmpC/ESBL positive (n ¼45)imipenem 2110563meropenem 26784ertapenem 18745443ESBL positive (n ¼39)imipenem 2910meropenem 318ertapenem2883Phenotypic method for differentiating MBLs and KPCs in Enterobacteriaceae1667JACby guest on March 18, 2013/Downloaded from(mean increase 10mm)in all 30positive isolates and irrespective of the carbapenem MICs or the co-production of an ESBL,clearly indicating the combined inhibitory activity of the two inhibitors against the production of both carbapenemases.Characteristi-cally,the increase in the zone diameters of the combined-disc test using meropenem with and without both PBA and EDTA was similar among KPC-possessing,VIM-possessing,as well as KPC-and VIM-possessing clinical isolates (Figure 1).Regarding the 84carbapenemase-negative isolates,all but two gave negative results in the combined-disc tests using mer-openem alone and with PBA,EDTA,or PBA and EDTA.Two AmpC-and ESBL-possessing isolates gave positive results in the combined-disc tests using meropenem alone and with PBA or PBA and EDTA,showing an augmentation in the zone diameters at the cut-off of 5mm (Table 3;specificity for KPC detection 98.8%;Table 4).In the remaining 82carbapenemase-negative isolates,the activity of meropenem was not enhanced by either PBA or EDTA irrespective of the carbapenem MICs or the co-production of AmpC and ESBL enzymes,indicating the specific activity of PBA and EDTA against KPC and VIM enzymes,respect-ively.It should be noted that all the above results were consist-ent between two different batches of Mueller–Hinton agar.Figure 2shows the growth patterns of representative isolates possessing KPC and VIM,KPC,VIM or none of the carbapen-emases using the three combined-disc tests of this study.DiscussionCurrently MBLs and KPCs are considered a major threat in Enter-obacteriaceae,representing a potential source of clinical failure in patients treated with almost all b -lactam regimens.1,3Although MBL and KPC carbapenemases are independently dis-seminated in different regions worldwide,1,3their co-production has been reported in K.pneumoniae 11and currently is widely detected among Enterobacteriaceae in tertiary care Greek hospi-tals (A.Tsakris, A.Poulou, E.Voulgari,S.Pournaras and D.Petropoulou,unpublished data).The production of both enzymes might contribute to their hydrolytic activity and levels of resistance to broad-spectrum b -lactams,as well as to the possible co-migration of both enzymes.Moreover,KPC and MBL genes are often co-transferred with plasmid-mediated ESBL,fluoroquinolone and aminoglycoside resistance genes,1,7,27,28T a b l e 3.D e t a i l e d r e s u l t s o f t h e t h r e e c o m b i n e d -d i s c t e s t s e m p l o y i n g m e r o p e n e m w i t h a n d w i t h o u t E D T A ,P B A ,o r P B A p l u s E D T A a g a i n s t t h e 141c a r b a p e n e m a s e -p o s i t i v e a n d 84c a r b a p e n e m a s e -n e g a t i v e c l i n i c a l i s o l a t e s i n t h i s s t u d yA u g m e n t a t i o n i n t h e z o n e d i a m e t e r s a n d n u m b e r o f p o s i t i v e /n e g a t i v e i s o l a t e s i n e a c h o f t h e t h r e e c o m b i n e d -d i s c t e s t sE D T AP B AP B A p l u s E D T Ar a n g e (m m )m e a n i n c r e a s e (m m )n o .o f i s o l a t e sr a n g e (m m )m e a n i n c r e a s e (m m )n o .o f i s o l a t e sr a n g e (m m )m e a n i n c r e a s e (m m )n o .o f i s o l a t e sp o s i t i v en e g a t i v e p o s i t i v en e g a t i v ep o s i t i v en e g a t i v eK P C (n ¼63)0–100636–1196306–1210630V I M (n ¼47)7–14104700–310478–1511470V I M +K P C (n ¼31)1–320311–420314–1310301A m p C a n d A m p C /E S B L (n ¼45)0–10450–522430–52243E S B L (n ¼39)0–200390–210390–21039Table 4.Sensitivities and specificities using the algorithm for detection of KPC,MBL or KPC/MBL b -lactamases Combined-disc testb -Lactamase detectedSensitivity Specificity PBA positive KPC100%98.8%EDTA negativePBA plus EDTA positive PBA negative MBL 100%100%EDTA positivePBA plus EDTA positive PBA negative MBL/KPC 96.8%100%EDTA negativePBA plus EDTA positiveTsakris et al.1668by guest on March 18, 2013/Downloaded fromN o . o f i s o l a t e s012345678Increase in inhibition zone diameter of meropenem (in mm) in the presence ofPBA and EDTA910111213141516Figure 1.Increase in the inhibition zone diameters of meropenem (in mm)in the presence of PBA and EDTA for 63isolates producing KPC,47isolates producing VIM and 31isolates producing both KPC and VIM carbapenemases.MEMMEM+EDTA +EDTA + boronic+boronic +EDTA +boronic MEMMEMMEMMEM+EDTA +EDTA + boronic+boronic MEMMEMMEMMEM+EDTA+EDTA + boronic+boronicMEMMEM MEM+EDTA + boronicMEMMEMMEMFigure 2.Representative results of the three combined-disc tests using discs of meropenem (MEM)alone and with EDTA,phenylboronic acid,or EDTA plus phenylboronic acid for a KPC/VIM/ESBL-possessing isolate (a),a KPC/ESBL-possessing isolate (b),a VIM-possessing isolate (c)and an AmpC/ESBL-possessing isolate (d).Phenotypic method for differentiating MBLs and KPCs in Enterobacteriaceae1669JACby guest on March 18, 2013/Downloaded fromand this may possibly contribute to the dissemination of additional resistance mechanisms among MBL and/or KPC pro-ducers.Therefore,the accurate phenotypic detection of MBL and KPC carbapenemases is important for clinical and epidemio-logical purposes,adding to studies on the preliminary character-ization of the antimicrobial resistance mechanisms.It is suggested that the modified Hodge test should be used as a confirmatory test for carbapenemase production when the initial screening tests are indicative(carbapenem MICs .1mg/L).29However,this test is often difficult to interpret,is only indicative of enzymatic activity of carbapenemase and cannot differentiate class A carbapenemases from class B MBLs.In the present study three combined-disc tests employing meropenem alone and with PBA,EDTA,or both PBA and EDTA were tested for the differentiation of KPCs and MBLs using a large collection of Enterobacteriaceae strains producing well-characterized broad-spectrum b-lactamases.Disc tests based on the inhibitory activity of EDTA were originally described for the identification of MBL-producing Enterobacteriaceae using imipenem as antibiotic substrate.14Disc tests based on the inhibitory activity of boronic acid were initially proposed for the identification of AmpC-type b-lactamase-producing enterobac-terial pathogens26,30and subsequently using carbapenems as substrates for the identification of KPC-possessing K.pneumoniae isolates.15–17,28The latter tests were found to considerably enhance growth-inhibitory zones around discs of carbapenems, allowing the differentiation of KPC-possessing K.pneumoniae iso-lates.Remarkably,meropenem demonstrated the largest differ-ences in inhibition zone diameters between KPC-possessing and non-KPC-possessing K.pneumoniae strains17and this prompted us to use meropenem as the antibiotic substrate in the current phenotypic algorithm.The present study gave additional indications that the combined-disc test using a boronic acid compound as inhibitor and meropenem as the antibiotic substrate can be successfully used for the identification of KPC enzymes not only in K.pneumoniae but also among other members of the family Enter-obacteriaceae such as E.coli,Klebsiella oxytoca,Enterobacter cloacae,E.aerogenes and Serratia marcescens.The current assay has also proposed an additional combined-disc test based on the simultaneous use of both inhibitors,PBA and EDTA,in order to detect the co-production of both MBLs and KPCs.The concurrent use of both inhibitors seems to restrain the activity of both carba-penemases against meropenem,allowing the detection of iso-lates that co-produce these enzymes in almost all cases. Combinations of inhibitors and substrates have been previously used for the differentiation of multiple broad-spectrum b-lactamases in enterobacterial isolates.Characteristically,clavu-lanate and boronic acid in combination have been employed as b-lactamase inhibitors for the detection of ESBLs among AmpC producers20,30as well as among KPC producers.21The present study has clearly shown that in the clinical laboratory the com-bined use of two inhibitors can clearly detect carbapenemase activity.The method was able to identify all MBL or KPC PCR-positive isolates and gave false-positive results only for two AmpC-and ESBL-possessing isolates that had elevated carba-penem MICs.These two isolates gave false-positive results in the combined-disc tests using meropenem alone and with PBA or PBA plus EDTA,and according to our algorithm were categorized as KPC producers.It is noteworthy that the possible co-production of an ESBL did not diminish the ability of the proposed algorithm to detect MBL and/or KPC production among Enterobacteriaceae.Finally, the phenotypic method successfully detected MBL or KPC activity among Enterobacter spp.isolates harbouring derepressed AmpC mutants.This might indicate that the proposed algorithm could detect carbapenemase activity even among Enterobacteriaceae that harbour MBLs and/or KPCs in the presence of hyperproduced AmpC enzymes.In conclusion,our results demonstrate that in regions where prevalence data suggest dissemination of MBL and KPC carbapenemases,boronic acid compounds are useful tools in the clinical laboratory not only for the detection of KPCs but also in combination with EDTA for the identification of possibly co-produced MBLs,by means of a combined-disc method using meropenem alone and with PBA,EDTA,or both PBA and EDTA.FundingThis study was supported by internal funding.Transparency declarationsNone to declare.References1Queenan AM,Bush K.Carbapenemases:the versatile b-lactamases. 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