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Division of Microbiology, Calgary Laboratory Services
Departments of Pathology and Laboratory Medicine
Medicine, University of Calgary, Calgary, Alberta, Canada
Service de Bacteriologie-Virologie, Hpital de Bicêtre, Assistance Publique/Hpitaux de Paris, Faculte de Medecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France
ABSTRACT
Metallo--lactamases (MBLs) have been increasingly recognized from clinical isolates worldwide, but the laboratory detection of these strains is not well defined. We report a study that developed an EDTA disk screen test and a molecular diagnostic assay for the detection of MBL-producing Pseudomonas aeruginosa. Using NCCLS disk methodology, inhibition zone diameters were determined in tests with imipenem (IPM) and meropenem (MEM) disks alone and in combination with 930 μg of EDTA. This test was compared with the MBL Etest. The duplex PCR assay showed 100% sensitivity and specificity for detecting MBL-producing control strains. Of the 241 clinical strains of IPM-nonsusceptible P. aeruginosa from the Calgary Health Region isolated from 2002 to 2004, 110/241 (46%) were MBL positive using phenotypic methods while 107/241 (45%) were PCR positive for MBL genes: 103/241 (43%) for blaVIM and 4/241 (2%) for blaIMP. The EDTA disk screen test using MEM showed 100% sensitivity and 97% specificity for detecting MBLs in control and clinical strains. The EDTA disk screen test is simple to perform and to interpret and can easily be introduced into the workflow of a clinical laboratory. We recommend that all IPM-nonsusceptible P. aeruginosa isolates be routinely screened for MBL production using the EDTA disk screen test and that PCR confirmation be performed at a regional laboratory.
INTRODUCTION
Pseudomonas aeruginosa producing metallo--lactamases (MBLs) was first reported from Japan in 1991 (43) and since then has been described from various parts of the world, including Asia (19, 44, 48), Europe (16, 21, 29, 32), Australia (30), South America (10), and North America (39). Metallo--lactamases belong to Ambler class B and have the ability to hydrolyze a wide variety of -lactam agents, such as penicillins, cephalosporins, and carbapenems (23). These enzymes require zinc for their catalytic activity and are inhibited by metal chelators, such as EDTA and thiol-based compounds (23). The genes responsible for the production of MBLs are typically part of an integron structure and are carried on transferable plasmids but can also be part of the chromosome (33). Therefore, because of the integron-associated gene cassettes, P. aeruginosa isolates producing MBLs are often resistant to different groups of antimicrobial agents, which can be transferred to various types of bacteria (27). Acquired MBLs can be divided into four categories according to their molecular structures, namely, the IMP, VIM, GIM, and SPM types (2, 17, 28, 40). On the basis of their sequences, three subclasses of class B -lactamses (B1 to B3) were identified, and a standard numbering scheme was proposed (11, 12).
Metallo--lactamase-producing P. aeruginosa isolates have been responsible for several nosocomial outbreaks in tertiary centers in different parts of the world, illustrating the need for proper infection control practices (6, 7, 34, 41). These isolates have also been responsible for serious infections, such as septicemia and pneumonia (6), and have been associated with failure of therapy with carbapenems (27).
Resistance to the carbapenems in P. aeruginosa is often due to impermeability, which arises via the loss of the OprD porin (22), the up regulation of an active efflux pump system present in the cytoplasmic membrane of these organisms (15), or the production of MBLs (23). Currently, there are no recommendations available from the NCCLS or elsewhere for the detection of organisms producing MBLs. The NCCLS has established guidelines for the detection of organisms producing extended-spectrum -lactamases. These include a confirmation test using both cefotaxime and ceftazidime tested alone and in combination with clavulanate (25).
Several phenotypic methods are available for the detection of MBL-producing bacteria. All these methods are based on the ability of metal chelators, such as EDTA and thiol-based compounds, to inhibit the activity of MBLs. These tests include the double-disk synergy tests using EDTA with imipenem (IPM) or ceftazidime (CAZ) (18, 20, 47), 2-mercaptopropionic acid with CAZ or IPM (1), the Hodge test (18, 20), a combined disk test using EDTA with CAZ or IPM (47, 50), the MBL Etest (AB BioDisk company, Solna, Sweden) (42), and a microdilution method using EDTA and 1,10-phenanthroline with IPM (24). A PCR detection assay was published in 1996 for the detection of gram-negative bacteria producing IMP-1 (36), and a PCR typing scheme for the identification of integron-associated MBLs was described in 2003 (38). Most of the current phenotypic methods described for the detection of MBLs, especially the double-disk synergy test and the Hodge test, are often difficult and subjective to interpret. These tests can also be technically demanding and time-consuming, since optimal disk spacing and reincubation of plates are sometimes required to obtain ideal results (18, 47). Furthermore, 2-mercaptopropionic acid and 1,10-phenanthroline are toxic for routine handling, and special precautions have to be taken when working with these compounds. These methods are thus unsuitable for clinical laboratories to perform on a routine basis. The IPM-EDTA disk method described by Yong et al. used 750 μg EDTA in combination with IPM disks with a zone difference of 7 mm between IPM alone and with EDTA (50). They reported excellent sensitivity and specificity to detect VIM-2- and IMI-1-producing P. aeruginosa and Acinetobacter spp. (50). Another method for the detection of MBL-producing bacteria is the MBL Etest, which is widely available but rather expensive and gives variable results (42, 47).
Pseudomonas aeruginosa producing IMP-7 was previously responsible for an outbreak at a tertiary-care center in our region, the Calgary Health Region (CHR) (13). The CHR is a fully integrated, publicly funded regional health system that provides health care to all the residents of the cities of Calgary and Airdrie and approximately 20 nearby small towns, villages, and hamlets. In the CHR, Calgary Laboratory Services (CLS) receives all clinical specimens submitted for bacteriological testing, including those from all hospitals, nursing homes, physicians' offices, and community collection sites (4). We needed to establish a cost-effective diagnostic approach at CLS for the accurate detection of P. aeruginosa producing MBLs. We report a study that evaluated the diagnostic utility of the metal chelator EDTA in search of a simple test for the screening of P. aeruginosa producing MBLs using methodology similar to the NCCLS guidelines for the extended-spectrum -lactamase confirmation disk test. We also developed and evaluated a molecular diagnostic assay for the confirmation of VIM and IMP types of MBLs and investigated the prevalence of these enzymes among clinical isolates of imipenem-intermediate or -resistant (nonsusceptible) P. aeruginosa from the CHR during 2002 to 2004.
MATERIALS AND METHODS
Bacterial strains. Strains with well-described -lactamases were used as positive and negative controls (Table 1). Consecutive nonduplicate isolates of P. aeruginosa nonsusceptible to imipenem (MIC > 8 μg/ml) collected at Calgary Laboratory Services from April 2002 to May 2004 were included in the study. Strains were identified to the species level with Vitek (Vitek AMS; bioMerieux Vitek Systems Inc., Hazelwood, MO).
Antimicrobial susceptibility testing. MICs of the following drugs were determined by Vitek: piperacillin (PIP), CAZ, IPM, ciprofloxacin (CIP), gentamicin (GEN), and tobramycin (TOB). The quality control strains used for this part of the study were Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Throughout the study, results were interpreted using NCCLS criteria for broth dilution (25).
Screening for and confirmation of MBLs. A disk screen test for the detection of MBLs was developed using different quantities of EDTA with IPM and meropenem (MEM) disks. Inhibition zones of the strains with well-described resistance mechanisms (Table 2) were determined by NCCLS disk methodology (25) on Mueller-Hinton agar (Oxoid Ltd., Basingstoke, Hampshire, England). The inocula used for the disk test corresponded to the 0.5 McFarland standard, which is approximately 108 CFU/ml. To determine the most suitable quantity of EDTA that would distinguish MBL-producing strains from IPM-nonsusceptible strains not producing MBLs, we tested antibiotic disks containing 10 μg IPM and 10 μg MEM alone and in combination with 744 μg, 930 μg, and 1,302 μg of EDTA (Sigma Chemicals, St. Louis, MO) (Table 2). This was accomplished by adding 4 μl (744 μg), 5 μl (930 μg), and 7 μl (1,302 μg) of 0.5 M EDTA to the IPM and MEM disks. Since EDTA has some bactericidal activity, a blank disk without an antibiotic was also inoculated with the different quantities of EDTA. The procedure was repeated twice to ensure the reproducibility of the results. The presence of MBLs was then evaluated in the clinical isolates of IPM-nonsusceptible P. aeruginosa using both the EDTA screen test and the MBL Etest according to the manufacturer's instructions (Table 3). Disks for MBL screen tests were obtained from Oxoid Inc. (Nepean, Ontario, Canada). P. aeruginosa PA105663 producing IMP-7; P. aeruginosa PS679/00 producing VIM-2; P. aeruginosa PA100609A, an efflux mutant; and P. aeruginosa ATCC 27853 were used as positive and negative controls (Table 2).
-Lactamase gene identification. DNA template preparation was performed as follows. The organisms were inoculated into 5 ml of Trypticase soy broth (Difco, Detroit, Mich.) and incubated for 20 h at 37°C with shaking. Cells from 1.5 ml of an overnight culture were harvested by centrifugation at 17,310 x g in a Hermle centrifuge for 5 min. After the supernatant was decanted, the pellet was resuspended in 500 μl of distilled water. The cells were lysed by heating them at 95°C for 10 min, and cellular debris was removed by centrifugation at 17,310 x g for 5 min. The supernatant was used as a source of template for amplification. Duplex PCR amplification for the simultaneous detection of blaIMP and blaVIM -lactamase genes were carried out on a Thermal Cycler 9600 instrument (Applied Biosystems, Norwalk, Conn.) with the Platinium Taq DNA polymerase kits and 10 mM deoxynucleoside triphosphate mix (Invitrogen Corporation, Carlsbad, CA). The composition of the reaction mixture was as follows: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, each of the three deoxynucleoside triphosphates at a concentration of 0.25 mM, dUTP (Roche Diagnostics, Laval, Quebec, Canada) at 0.75 mM, 0.125 U of UNG, and 2 U of Platinum Taq in a total volume of 23 μl. A total of 2 μl of sample lysate was added to the reaction mixture. The PCR program consisted of an initial incubation of 10 min at 37°C and an initial denaturation step at 94°C for 5 min, followed by 30 cycles of DNA denaturation at 94°C for 1 min, primer annealing at 54°C for 1 min, and primer extension at 72°C for 1.5 min. After the last cycle, the products were stored at 4°C. The PCR products (1/10 volume) were analyzed by electrophoresis with 1.4% agarose gels in TAE buffer (0.04 M Tris-acetate, 0.002 M EDTA [pH 8.5]). The gels were stained with ethidium bromide, and the PCR products were visualized with UV light. The primers, at a concentration of 0.16 μM each, used for PCR amplification of IMP types of MBLs were IMP-A (5'-GAA GGY GTT TAT GTT CAT AC-3') and IMP-B (5'-GTA MGT TTC AAG AGT GAT GC-3'), which amplified a 587-bp amplicon (33) (Y = C or T and M = A or C). The primers, at a concentration of 0.16 μM each, used for PCR amplification of VIM types of MBLs were VIM2004A (5'-GTT TGG TCG CAT ATC GCA AC-3') and VIM2004B (5'-AAT GCG CAG CAC CAG GAT AG-3'), which amplified a 382-bp amplicon (L. Poirel, personal communication). The sensitivities and specificities of the primers were evaluated against strains with well-described resistance mechanisms (Table 1). The presence of blaVIM and blaIMP was detected in the clinical isolates of IPM-nonsusceptible P. aeruginosa. Those isolates that were positive by the EDTA disk screen test and/or the MBL Etest and that were PCR negative for either blaVIM or blaIMP genes (three in total) were further investigated for the presence of SPM MBLs using primers SPM-1A (5'-CTG CTT GGA TTC ATG GGC GC-3') and SPM-1B (5'-CCT TTT CCG CGA CCT TGA TC-3') and conditions as previously described (31). We verified and validated our procedure extensively with positive and negative controls using different thermocyclers on different days. Confirmation of the VIM and IMP genes was performed using the class 1 integron primers 5CS (5'-GGC ATC CAA GCA GCA AG-3') and 3CS (5'-AAG CAG ACT TGA CCT GA-3') in combination with IMP-A-IMP-B and VIM2004A-VIM2004B, respectively, using the conditions described before.
RESULTS
Clinical bacterial strains. Of the 241 IPM-nonsusceptible P. aeruginosa strains, 103 (43%) were isolated from urine, 17 (7%) from blood, 50 (21%) from wounds (purulent), 48 (20%) from respiratory tract specimens, and the remaining 23 (9%) from various other specimens. The isolates producing MBLs were more prevalent in blood cultures (13% versus 3%) and less prevalent in respiratory specimens (12% versus 23%) than IPM-nonsusceptible isolates that do not produce MBLs.
Antimicrobial susceptibilities of clinical strains. Of the 241 P. aeruginosa clinical isolates included in this study, 47 (20%) were resistant to PIP, 141 (59%) to CAZ, 159 (66%) to TOB, 174 (72%) to GEN, and 149 (62%) to CIP. The isolates producing MBLs were more resistant to CAZ, TOB, CIP, and GEN and less resistant to PIP than IPM-nonsusceptible isolates not producing MBLs (Fig. 1). A particularly important feature was that all the MBL producers were resistant to CIP compared to 32% of the IPM-nonsusceptible isolates not producing MBLs (Fig. 1).
Screening for MBLs using EDTA disk screen tests and MBL Etest. The differences in inhibition zones for IPM and MEM disks alone and in combination with different quantities of EDTA for the strains with well-described resistance mechanisms are shown in Table 2. The zone diameters were similar and reproducible when the procedure was repeated. No clear breakpoint was evident that distinguished MBL producers from IPM-resistant isolates not producing MBLs when 744 μg of EDTA was used with IPM or MEM disks (Table 2). EDTA at 1,302 μg gave inhibition zones on the blank disk that were difficult to interpret. These inhibition zones with the blank disk were not evident when 744 μg or 930 μg of EDTA was used. An increase of 7 mm in zone diameter in the presence of 930 μg EDTA compared to those with both IPM and MEM tested alone was considered to be a positive test for the presence of an MBL (Table 2). The EDTA disk screen test using 930 μg EDTA with IPM and MEM disks with the above-mentioned criteria was then performed on all the recent clinical strains of P. aeruginosa isolates intermediate or resistant to IPM (Table 3). These results were compared to those of the MBL Etest (Table 3). Of the 241 P. aeruginosa clinical isolates included in this study, 110 (46%) were MBL positive using phenotypic methods, and 107 of these tested positive for MBL genes (see "Molecular detection of blaVIM and blaIMP genes" below). These strains were considered to be false positive using phenotypic tests. The EDTA disk screen test using IPM with and without EDTA (using the PCR results as a gold standard) showed a sensitivity of 96% (95% confidence interval [CI], 90 to 99%) and a specificity of 91% (95% CI, 86 to 95%), and MEM with and without EDTA showed a sensitivity of 100% (95% CI, 96 to 100%) and a specificity of 97% (95% CI, 92 to 99%). The MBL Etest showed a sensitivity of 96% (95% CI, 90 to 99%) and a specificity of 91% (95% CI, 86 to 95%) (Table 3).
Molecular detection of blaVIM and blaIMP genes. The designed primers were tested for specificity and sensitivity using DNA templates prepared from control strains known to produce specific VIM and IMP -lactamases or strains producing -lactamases other than MBLs (Table 1 and Fig. 2). The primers showed 100% sensitivity and 100% specificity for detecting VIM-2 and -3 and IMP-1, -4, -7, and -8 MBLs and did not cross-react with strains producing NMCA-1, KPC-1, CTX-M-14, SHV-2, and TEM-3 (Table 1 and Fig. 2). All the clinical P. aeruginosa isolates that were nonsusceptible to IPM were then examined by PCR for the presence of blaVIM and blaIMP genes (Table 3). Of the 241 P. aeruginosa isolates isolated during the study period, 103 (43%) were positive for blaVIM genes and 4 (2%) were positive for blaIMP genes. The remaining 134 (55%) were negative for VIM and IMP MBLs. PCR with primers 5CS, 3CS, and the different combinations of IMPA, IMPB, VIM2004A, and VIM2004B amplified various amplicons ranging from 900 bp to 2 kb. The three isolates that were positive by the EDTA disk screen test and/or MBL Etest and that were PCR negative for either the blaVIM or blaIMP gene were also negative for the presence of SPM MBLs.
DISCUSSION
MBLs have been identified from clinical isolates worldwide with increasing frequency over the past few years, and strains producing these enzymes have been responsible for prolonged nosocomial outbreaks that were accompanied by serious infections (6, 27, 37). A case-controlled study from Japan showed that patients infected with MBL-producing P. aeruginosa were more likely to receive multiple antibiotics and, more importantly, that infection-related deaths due to IMP-producing P. aeruginosa were more frequent than deaths caused by blaIMP-negative P. aeruginosa (14). The occurrence of an MBL-positive isolate in a hospital setting poses a therapeutic problem, as well as a serious concern for infection control management. The accurate identification and reporting of MBL-producing P. aeruginosa will aid infection control practitioners in preventing the spread of these multidrug-resistant isolates (27, 37).
Since P. aeruginosa producing IMP-7 was responsible for an outbreak in the 1990s in the CHR (13), we needed to establish a reliable and accurate diagnostic approach at CLS to detect these isolates. We report a study using NCCLS disk methodology that developed an EDTA disk screen test with IPM and MEM disks alone and in combination with 930 μg of EDTA. In our study, 744 μg of EDTA, as reported by Yong et al. (50), did not detect PA105663 producing IMP-7 (Table 2) or the clinical strains with blaIMP MBLs. None of the published phenotypic diagnostic methods for the detection of MBL-producing strains used MEM as a substrate. Our results showed that MEM alone and in combination with EDTA showed 100% sensitivity and 97% specificity in detecting well-characterized MBL-producing clinical strains of P. aeruginosa and performed better than IPM and the MBL Etest (Table 3). It needs to be emphasized that IMP-harboring strains accounted for only 1.6% of clinical test isolates compared to 43% of test isolates harboring VIMs. We do recommend, however, that both IPM and MEM be used as substrates for the EDTA disk screen test. The reason for this is that one of the four MBL-negative strains that tested false positive with MEM was indeed negative with IPM (Table 3). The IPM-EDTA disks can be stored at 4°C or –20°C for 12 to 16 weeks without significant loss of activity (50). The EDTA disk screen test is simple to perform and interpret, and since it uses NCCLS methodology, it can be easily introduced into the workflow of a clinical laboratory. The MBL Etest was less sensitive and more expensive than the EDTA disk screen test. With the advent of new and increasing numbers of MBLs, our EDTA disk screen test must be continually evaluated for sensitivity and specificity using different MBLs, including SPM and GIM types. We did not evaluate our EDTA disk screen test for the detection of Enterobacteriaceae producing MBLs.
We also developed a duplex PCR assay with excellent sensitivity and specificity for the simultaneous confirmation of VIM- and IMP-producing Pseudomonas aeruginosa that was verified and validated extensively in our setting. We also confirmed the presence of the different MBL alleles by using primers specific for class I integron in combination with the MBL primers. We did not include primers for SPM-1 and GIM-1 MBLs in our PCR, since these enzymes are confined to Brazil and Germany, respectively (2, 10). Our test is more suitable for a reference clinical microbiology laboratory, since the PCR-typing scheme proposed by Shibata et al. involves sequencing of the amplicons (38). The PCR results of the clinical strains of IPM-nonsusceptible or -resistant P. aeruginosa were a surprise, since it seems that isolates with blaVIM have replaced those producing IMP-7 that were identified in the 1990s (13). A study is under way to identify the types of VIM and IMP MBLs and to characterize the genetic support and environment of the genes encoding these enzymes. The simultaneous occurrence of two different groups of MBLs in a single hospital or region, as described in this study, is rare and has been previously described in Brazil (35). This suggests that the evolution, maintenance, and dissemination of MBL resistance genes among P. aeruginosa populations in larger geographic regions is a complex and dynamic field that needs to be studied in detail.
This study illustrates that MBL-producing isolates of P. aeruginosa are important causes of IPM resistance among this species isolated in the CHR (Table 3). The MBL-producing P. aeruginosa isolates were more resistant to various antimicrobial agents and were more prevalent in blood cultures than IPM-resistant isolates not producing MBLs (Fig. 1). This suggests that MBL-producing isolates in the CHR are responsible for serious infections, which was illustrated when these strains were responsible for a nosocomial outbreak during 2003 that resulted in several deaths at a tertiary center in the CHR (D. B. Gregson, T. Louie, J. D. D. Pitout, K. Laupland, S. Elsayed, P. Le, K. Sisson, C. Ross, and D. L. Church, Abstr. 43rd Intersci Conf. Antimicrob. Agents Chemother., abstr. K-1098, 2003). Our results support the notion that clinical microbiology laboratories must be able to distinguish MBL-producing P. aeruginosa from strains with other mechanisms responsible for carbapenem resistance. In the absence of novel agents for the treatment of infections caused by multidrug-resistant gram-negative bacteria in the near future, the uncontrolled spread of MBL producers may lead to treatment failures with increased morbidity and mortality. The early detection of MBL-producing P. aeruginosa may avoid the future spread of these multidrug-resistant isolates. We recommend that all IPM-nonsusceptible or -resistant P. aeruginosa isolates be routinely screened for MBL production using the EDTA disk screen test as described in this study. PCR confirmation for MBLs can be performed at a regional laboratory, such as CLS, using the methods reported in this study. PCR confirmation is an important step, since EDTA can give false-positive results in IPM-resistant P. aeruginosa isolates due to altered OprD levels (5). These steps will ensure the early recognition of an outbreak of organisms producing MBLs and identify the type of MBL involved, as well as the possible mechanism(s) for the spread of resistance. Routine detection of MBLs will ensure optimal patient care and the timely introduction of appropriate infection control procedures.
ACKNOWLEDGMENTS
We thank Barbara Chow, Lorraine Campbell, Harjinder Gill, and Terry Ross, Calgary Laboratory Services, Calgary, Alberta, Canada, for their technical support of this study and Richard Bonnet for providing the control strain CF1; R. A. Moore for PA105663, PA100609A, and PA100609B; J. J. Yan for NTU92/99, EB 464, KP1159/99, PS679/00, and NTU39/00; Y. W. Chu for 127091 and 74510; and E. S. Moland for Ent162 and Klebs265.
This work was supported by a grant from the Calgary Laboratory Services (73-1357).
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