Convenient Test Using a Combination of Chelating Agents for Detection of Metallo-{beta}-Lactamases in the Clinical Laboratory

Although transmissible metallo-ß-lactamases (MBLs)are a serious threat to ß-lactam antibiotic therapy,the CLSI currently does not recommend testing methods for thedetection of MBLs. The aim of this study was to evaluate thecapability of double-disk tests (DDTs) by using disks containinga combination of the chelators 2-mercaptopropionic acid (MPA)and Tris-EDTA (TE) to detect MBLs. Sixteen isolates (4 Acinetobacterbaumannii isolates, 6 Pseudomonas aeruginosa isolates, 1 Serratiamarcescens isolate, 1 Aeromonas hydrophila isolate, 1 Aeromonasveronii isolate, 2 Chryseobacterium meningosepticum isolates,and 1 Stenotrophomonas maltophilia isolate) producing IMP-1,IMP-1-like, IMP-18, GIM-1, SPM-1, VIM-2, VIM-2-like, and chromosomalMBLs and 20 isolates (7 Klebsiella pneumoniae isolates, 3 Escherichiacoli isolates, 5 Enterobacter cloacae isolates, 2 S. marcescensisolates, 1 Proteus mirabilis isolate, and 2 A. baumannii isolates)producing non-MBL carbapenemases, AmpC ß-lactamases,and extended-spectrum ß-lactamases were tested. TheDDT method was evaluated by using four types of chelator disks(TE, high-strength TE, MPA, and TE plus 20 µl of MPA [atvarious concentrations]) and the ß-lactams imipenem(IPM), meropenem (MEM), ertapenem (ERT), and ceftazidime (CAZ).DDTs with IPM and a TE disk supplemented with 1:320 MPA detectedall MBLs and yielded no false-positive results. Some, but notall, MBL producers were detected in IPM-based tests involvingthe single chelator TE or MPA alone or by ERT- or CAZ-basedtests. IPM-based tests with MPA concentrations other than 1:320and all MEM-based tests had suboptimal sensitivities or specificities.DDT with IPM and a TE disk supplemented with 20 µl of1:320 MPA appears to be convenient for the detection of MBLsin the clinical laboratory.

INTRODUCTION

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INTRODUCTION

Carbapenems are the drugs of choice for the treatment of infectionscaused by multiresistant gram-negative bacilli. Carbapenemasesinvolved in acquired resistance are of Ambler molecular classesA, B, and D (4). The class B enzymes, metallo-ß-lactamases(MBLs), are the most clinically threatening carbapenemases,because they are capable of hydrolyzing all ß-lactamsexcept aztreonam. Gram-negative bacilli producing acquired MBLshave been reported in many countries and are sometimes carbapenemsusceptible in vitro under standard conditions, making themdifficult to recognize (4, 7). Therefore, accurate detectionis important for optimal therapy and infection control precautions.Recently, an E-test MBL strip (AB Biodisk, Solna, Sweden) wasrecommended for use for screening for MBLs in clinical laboratories,but it has been reported to be unable to detect all MBL-positivemembers of the family Enterobacteriaceae and also to give false-positiveresults with some Pseudomonas aeruginosa and Acinetobacter baumanniistrains (5, 7). Although various MBL detection techniques havebeen investigated, there are currently no perfect phenotypicmethods for the detection of all transferable MBLs (7).

Here we report on a study performed to evaluate the utilityof double-disk tests (DDTs) involving disks containing imipenem(IPM), meropenem (MEM), ertapenem, (ERT), and ceftazidime (CAZ);disks containing low and high concentrations of Tris-EDTA (TE);disks containing 2-mercaptopropionic acid (MPA); and TE diskssupplemented with MPA for the detection of MBL producers.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains. Sixteen isolates (4 A. baumannii isolates, 6 P. aeruginosa isolates,1 Serratia marcescens isolate, 1 Aeromonas hydrophila isolate,1 Aeromonas veronii isolate, 2 Chryseobacterium meningosepticumisolates, and 1 Stenotrophomonas maltophilia isolate) producingIMP-1, IMP-1-like, IMP-18, GIM-1, SPM-1, VIM-2, VIM-2-like,and chromosomal MBLs and 20 isolates (7 Klebsiella pneumoniaeisolates, 3 Escherichia coli isolates, 5 Enterobacter cloacaeisolates, 2 S. marcescens isolates, 1 Proteus mirabilis isolate,and 2 A. baumannii isolates) producing class A and D carbapenemases(IMI-1, KPC-2, KPC-3, NMC-A, SME-like, and OXA-23), AmpC ß-lactamases,and extended-spectrum ß-lactamases (ESBLs) were studied(Table 1).

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TABLE 1. Comparison of methods for MBL detection

Detection of MBLs. Four types of disks containing chelating agents were investigated.These were (i) TE disks (BD Diagnostic Systems, Sparks, MD)premoistened with 20 µl saline; (ii) TE disks supplementedwith 20 µl of 1:80, 1:160, 1:320, 1:640, and 1:1,280 dilutionsof MPA (T31003-5G; Sigma-Aldrich Corp., St. Louis, MO); (iii)high-strength TE disks, i.e., TE disks supplemented with 20µl of either undiluted TE or a 1:16 dilution of 100x TE(T-9285; Sigma-Aldrich Corp.); and (iv) MPA disks, i.e., blankdisks supplemented with 20 µl of a 1:80 or 1:320 dilutionof MPA. The test strains were adjusted to a McFarland 0.5 standardand inoculated as lawns onto Mueller-Hinton agar plates (OxoidLtd., Basingstoke, England). Disks containing 10 µg IPM,MEM, or ERT or 30 µg CAZ (BD Diagnostic Systems) wereplaced on the surface, and the chelator disk was placed 10 mm(edge to edge) from the ß-lactam disk. After incubationovernight at 37°C, any clear extension of the inhibitionzone around the carbapenem disk toward the TE disk was interpretedas a positive result.

RESULTS

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RESULTS

As shown in Table 1, the IPM-based test with a TE disk supplementedwith 1:320 MPA provided the most sensitive (100%) and specific(100%) test for MBL detection. In tests with IPM, the unsupplementedor high-strength TE disks did not detect IMP-1 in two isolatesof A. baumannii (Fig. 1) and SPM-1 in one isolate of P. aeruginosa.MPA disks alone failed to detect a VIM-2-like MBL in one isolateof P. aeruginosa (Fig. 2A). Three of the five chromosomal MBL-producingisolates (two C. meningosepticum isolates and one S. maltophiliaisolate) yielded strongly positive results with IPM and a TEdisk supplemented with 1:320 MPA, but two isolates (one A. hydrophilaisolate and one A. veronii isolate) yielded only weakly positiveresults. Higher MPA concentrations (1:80 and 1:160) were associatedwith false-positive results in tests with IPM and some classA carbapenemase-, AmpC-, and ESBL-producing isolates; and lowerMPA concentrations (1:640 and 1:1,280) reduced the sensitivityof the test. MEM-based tests with lower MPA concentrations (1:320to 1:1,280) had suboptimal sensitivities (63.6 to 81.8%), whilethose with higher MPA concentrations (1:80 to 1:320) had suboptimalspecificities (50 to 75%) (Table 1 and Fig. 3). ERT-based testswere too insensitive (54.5%) with the highest MPA concentration(1:80). CAZ-based tests exhibited larger inhibition zones intests with five P. aeruginosa isolates, including one VIM-2-likeMBL-producing P. aeruginosa isolate, than the correspondingIPM-based tests (Fig. 2), but were less sensitive because theyfailed to detect one IMP-1-producing A. baumannii isolate (Fig.1).

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FIG. 1. IMP-1-producing A. baumannii. Positive test results (expanded inhibition zones) were obtained with MPA-supplemented TE disks in tests with IMP, MEM, and ERT. The results of the tests with the MPA-supplemented TE disk with CAZ and the high-strength TE disk with IMP were negative.

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FIG. 2. VIM-2-like MBL-producing P. aeruginosa. (A) IPM-based tests showed positive results with different chelating agents, excluding MPA alone. (B) CAZ-based tests showed positive results with all chelating agents used and inhibition zones larger than those obtained by the IPM-based tests.

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FIG. 3. False-positive results in tests with NMC-A-producing E. cloacae by using MEM and various concentrations of MPA added to the TE disks. Tests with high concentrations of MPA ranging from 1:80 to 1:320 showed false-positive results.

DISCUSSION

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DISCUSSION

It is necessary to perform MBL detection tests with suspiciousisolates because these enzymes cannot be identified by routinesusceptibility tests. Suspicious isolates are those with significantlyreduced susceptibility to carbapenems, e.g., E. coli and Klebsiellaisolates with imipenem MICs of 1 µg/ml or higher; Enterobacter,Citrobacter freundii, and S. marcescens isolates with imipenemMICs of 4 µg/ml or higher; and P. aeruginosa isolateswith imipenem MICs of 8 µg/ml or higher. Tests involvingchelating agents such as EDTA and MPA are useful because thechelators are specific inhibitors of MBLs but not of other ß-lactamases(1-3, 5-8). A single chelating agent may sometimes not adequatelyinhibit all MBLs in certain pathogens, making it necessary touse a mixture of chelating agents for the reliable detectionof MBLs.

The IPM-based test involving TE alone did not detect two IMP-1-producingA. baumannii isolates (Fig. 1) and one SPM-1-producing P. aeruginosaisolate, whereas The IPM-based test involving MPA alone failedto detect one VIM-2-like MBL-producing P. aeruginosa isolate.Positive tests with these isolates were obtained only when anMPA-supplemented TE disk was used. The finding that two chelatorswere needed was consistent with the findings presented in thereport of Lee et al. (3) that EDTA disks were more sensitivethan sodium mercaptoacetic acid (SMA) disks for the detectionof MBL-producing pseudomonads, while SMA disks provided moresensitive tests for the detection of MBL-producing acinetobacters.The MPA concentration used for supplementation of the TE diskswas critical, with a 1:320 dilution providing optimal specificityand sensitivity.

The tests with the MEM, ERT, and CAZ disks yielded some inaccurateresults and were less satisfactory than the tests with IPM disks.Arakawa et al. (1) reported that CAZ disks used in conjunctionwith MPA provided optimal MBL detection because MBL producerswere usually highly resistant to CAZ (MIC, >64 µg/ml).However, Lee et al. (3) reported that MBL production by someAcinetobacter isolates was difficult to detect by tests basedon CAZ disks and attributed this to the possibility that theisolates may have additional CAZ resistance mechanisms, suchas ESBL and AmpC production. In our study, the MBL activityof one Acinetobacter isolate was not detected with the CAZ disk,although this isolate was highly resistant to CAZ (MIC, >64µg/ml).

Our findings with 16 MBL-producing isolates that produced IMP-1,IMP-1-like, IMP-18, GIM-1, SPM-1, VIM-2, VIM-2-like, and chromosomalMBLs suggest that a convenient and accurate screening methodsuitable for use in clinical laboratories for the detectionof MBL producers may be provided by use of the IPM and TE disksupplemented with 20 µl of 1:320 MPA. Because only a modestnumber of isolates that produced MBLs were included in thisstudy, it is necessary for further evaluation of this methodto be undertaken.

ACKNOWLEDGMENTS

We thank the following microbiologists for isolates used inthis study: K. Lee, Yonsei University College of Medicine, Seoul,Korea; M. Inoue, Kitasato University School of Medicine, Sagamihara,Japan; and L. M. Deshpande, JMI Laboratories, North Liberty,IA.

FOOTNOTES

* Corresponding author. Mailing address: Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology & Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-1881. Fax: (402) 280-1225. E-mail: kstaac{at}creighton.edu

Published ahead of print on 27 June 2007.

REFERENCES

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