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Journal of Clinical Microbiology, December 2008, p. 4083-4086, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.01408-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Simple Disk-Based Method for Detection of Klebsiella pneumoniae Carbapenemase-Type β-Lactamase by Use of a Boronic Acid Compound{triangledown}

Yohei Doi,1* Brian A. Potoski,1,2 Jennifer M. Adams-Haduch,1 Hanna E. Sidjabat,1 Anthony W. Pasculle,1,3 and David L. Paterson1,4

Division of Infectious Diseases,1 Antibiotic Management Program,2 Clinical Microbiology Laboratory, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania,3 University of Queensland, UQ Centre for Clinical Research, and Royal Brisbane and Women's Hospital, Brisbane, Australia4

Received 22 July 2008/ Returned for modification 19 September 2008/ Accepted 8 October 2008


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ABSTRACT
 
A disk potentiation method using carbapenems as substrates and 3-aminophenyl boronic acid as an inhibitor was evaluated for the detection of Klebsiella pneumoniae carbapenemase (KPC)-type β-lactamases. When combined with nonsusceptibility to ertapenem, the method was easy to perform and reliably differentiated isolates producing KPC-type β-lactamases from those producing other types of β-lactamases.


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TEXT
 
Since the initial report in 2001 (24), carbapenem resistance mediated by the production of Klebsiella pneumoniae carbapenemase (KPC)-type β-lactamases is increasingly identified in the United States and more recently worldwide (3, 22). Initially confined mostly to K. pneumoniae, these enzymes are now reported in various species of Enterobacteriaceae as well as Pseudomonas aeruginosa (7, 16, 21). Clinically, infections due to KPC-producing strains of Enterobacter spp. have been associated with increased mortality in affected patients (14). From the infection control standpoint, KPC-producing K. pneumoniae and Escherichia coli have caused outbreaks in acute-care hospitals and long-term-care facilities, respectively (4, 19, 22). However, laboratory detection of these organisms is difficult (5, 20). Several methods have been developed specifically for the detection of KPC-producing Enterobacteriaceae. A method that utilizes nonsusceptibility to ertapenem for screening followed by confirmation using the modified Hodge test has been proposed (1). In the modified Hodge test, a carbapenem disk is placed on a Mueller-Hinton agar plate inoculated with E. coli ATCC 25922. The test strain is then streaked radially from the edge of the disk to the periphery of the plate. After an overnight incubation, the presence of a distorted inhibition zone indicates the carbapenem-hydrolyzing activity of the test strain. This method is relatively easy to perform and feasible in clinical laboratories but requires some experience in interpreting the results. A method measuring the hydrolysis of carbapenems by cell extracts has been reported as well, though this method is technically demanding and requires specialized laboratory equipment (15).

Boronic acid compounds are known to be excellent inhibitors of class C β-lactamases (2, 13). One such compound, 3-aminophenyl boronic acid (APB), has recently been used successfully in detecting the production of plasmid-mediated class C β-lactamases in Enterobacteriaceae (6, 10, 12, 23). We have subsequently undertaken a study investigating the effects of APB on zone diameters of carbapenem-containing disks in a set of isolates producing KPC-type β-lactamases as well as other non-KPC broad-spectrum β-lactamases.

A total of 23 epidemiologically unrelated K. pneumoniae and E. coli clinical isolates (10 producing KPC-type β-lactamase, 3 ertapenem-resistant isolates without KPC-type β-lactamase, 5 producing extended-spectrum β-lactamase [ESBLs], and 5 producing plasmid-mediated class C β-lactamase) were included in the study. Specifically, the KPC-producing isolates originated from five hospitals in three states. We also included E. coli DH10B strains carrying recombinant plasmids that bear the genes for the metallo-β-lactamases (MBLs) IMP-1 and VIM-2. The β-lactamase types were determined by PCR analysis and nucleotide sequencing as appropriate. For the detection and sequencing of the KPC gene, primers KPC-1-F (5'-GGC TTG CCG CTC GGT GAT ATT-3') and KPC-1-R (5'-TAT CTG TGA GGG CGA AGG TTA-3') were used at an annealing temperature of 62°C. ESBL genes and plasmid-mediated class C β-lactamase genes were amplified as described previously (11, 18). The isolates were suspended in and diluted with normal saline to 108 CFU/ml by comparison with a McFarland 0.5 turbidity standard and spread on a Mueller-Hinton agar plate (BD Microbiology Systems, Sparks, MD) as recommended by the Clinical and Laboratory Standards Institute (CLSI) (8). The following disks (BD Microbiology Systems) were tested: ertapenem (10 µg), ertapenem (10 µg) with APB (300 µg), imipenem (10 µg), imipenem (10 µg) with APB (300 µg), meropenem (10 µg), meropenem (10 µg) with APB (300 µg), ceftazidime (30 µg), and ceftazidime (30 µg) with APB (300 µg). APB (3-aminophenyl boronic acid hydrochloride; Sigma-Aldrich, St. Louis, MO) was dissolved in water at 50 mg/ml, and 6 µl was applied per disk. The amount of APB to be applied to the disks was determined based on the following observation: when the inhibitory effects of APB on representative KPC-positive and -negative isolates were examined at 100, 200, 300, 450, and 600 µg per disk, 300 µg was found to provide optimal sensitivity and specificity in detecting the presence of KPC-type β-lactamase when combined with ertapenem or meropenem, and a cutoff of a 5-mm difference in zone diameter was used. The zone diameters were read by at least two microbiologists. The modified Hodge test was performed to confirm the production of carbapenem-hydrolyzing β-lactamase as described previously (1).

The results are summarized in Table 1. The modified Hodge test was positive for all 10 KPC-producing clinical isolates and 2 MBL-producing laboratory strains, confirming the presence of significant carbapenem-hydrolyzing activity. None of the other isolates had positive results with the modified Hodge test. All 10 KPC-producing isolates were resistant to ertapenem, intermediate or resistant to meropenem, and variably resistant to imipenem. Ertapenem and meropenem were both sensitive substrates for potentiation by APB. When APB was added to ertapenem or meropenem disks, an increase in zone diameter of ≥5 mm was observed for all KPC-producing isolates (Table 1; Fig. 1). Potentiation of ≥5 mm was observed in 6 of the 10 isolates for imipenem. On the other hand, none of the three ertapenem-resistant isolates without KPC-type β-lactamase or the two MBL-producing strains produced a ≥5-mm increase in zone diameter with any of the carbapenems when combined with APB. Of note, all five isolates producing plasmid-mediated class C β-lactamase gave a ≥5-mm increase with ertapenem when combined with APB but not with imipenem or meropenem. However, they were all susceptible to ertapenem and were thus unlikely to be screened for the presence of KPC-type β-lactamase.


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  		TABLE 1. Zone diameters of the carbapenem disks tested against the study isolates


Figure 1
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  		FIG. 1. Potentiation of carbapenems by APB in K. pneumoniae producing KPC-2. (A) Ertapenem (10 µg); (B) ertapenem plus APB (300 µg); (C) meropenem (10 µg); (D) meropenem plus APB (300 µg).

The mechanism for inhibition of KPC-type β-lactamase by APB is not known. A recent report from Argentina described the inhibition of the carbapenem-hydrolyzing activity of crude extracts from KPC-producing K. pneumoniae by APB (17). The authors cautioned that synergism between APB and KPC could lead to a false assumption of class C β-lactamase production. However, our results suggest that APB may have a role in the phenotypic confirmation of KPC production. Nonsusceptibility to ertapenem has been reported to be an excellent indicator of the presence of KPC-type β-lactamase with sensitivity exceeding 90% (1). Therefore, a two-tiered detection method, i.e., screening for ertapenem nonsusceptibility followed by confirmation with a disk potentiation test between ertapenem or meropenem and APB, appears to be a viable approach in identifying KPC-producing K. pneumoniae and E. coli. Our study is limited by the relatively small number of isolates assessed, especially ertapenem-resistant isolates without KPC-type β-lactamase. However, if replicated on large, diverse samples of KPC producers and nonproducers, this method holds promise for the busy clinical microbiology laboratory since the algorithm is analogous to the ESBL detection method using disk diffusion as defined by the CLSI (9), thus making it easy for the laboratory staff to incorporate it into daily practice.


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ACKNOWLEDGMENTS
 
Y.D. was supported by NIH training grant T32AI007333. D.L.P. is supported in part by NIH research grant R01AI070896.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases, University of Pittsburgh Medical Center, S829 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261. Phone: (412) 648-9445. Fax: (412) 648-8521. E-mail: doiy{at}dom.pitt.edu Back

{triangledown} Published ahead of print on 15 October 2008. Back


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REFERENCES
 
    1
  1. Anderson, K. F., D. R. Lonsway, J. K. Rasheed, J. Biddle, B. Jensen, L. K. McDougal, R. B. Carey, A. Thompson, S. Stocker, B. Limbago, and J. B. Patel. 2007. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J. Clin. Microbiol. 45:2723-2725.[Abstract/Free Full Text]
    2. 2
  2. Beesley, T., N. Gascoyne, V. Knott-Hunziker, S. Petursson, S. G. Waley, B. Jaurin, and T. Grundstrom. 1983. The inhibition of class C β-lactamases by boronic acids. Biochem. J. 209:229-233.[Medline]
    3. 3
  3. Bradford, P. A., S. Bratu, C. Urban, M. Visalli, N. Mariano, D. Landman, J. J. Rahal, S. Brooks, S. Cebular, and J. Quale. 2004. Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 β-lactamases in New York City. Clin. Infect. Dis. 39:55-60.[CrossRef][Medline]
    4. 4
  4. Bratu, S., S. Brooks, S. Burney, S. Kochar, J. Gupta, D. Landman, and J. Quale. 2007. Detection and spread of Escherichia coli possessing the plasmid-borne carbapenemase KPC-2 in Brooklyn, New York. Clin. Infect. Dis. 44:972-975.[CrossRef][Medline]
    5. 5
  5. Bratu, S., M. Mooty, S. Nichani, D. Landman, C. Gullans, B. Pettinato, U. Karumudi, P. Tolaney, and J. Quale. 2005. Emergence of KPC-possessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection. Antimicrob. Agents Chemother. 49:3018-3020.[Abstract/Free Full Text]
    6. 6
  6. Brenwald, N. P., G. Jevons, J. Andrews, L. Ang, and A. P. Fraise. 2005. Disc methods for detecting AmpC β-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae. J. Antimicrob. Chemother. 56:600-601.[Free Full Text]
    7. 7
  7. Cai, J. C., H. W. Zhou, R. Zhang, and G. X. Chen. 2008. Emergence of Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli isolates possessing the plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in intensive care units of a Chinese hospital. Antimicrob. Agents Chemother. 52:2014-2018.[Abstract/Free Full Text]
    8. 8
  8. Clinical and Laboratory Standards Institute. 2006. Performance standards for antimicrobial disk susceptibility tests, 9th ed. Approved standard M2-A9. Clinical and Laboratory Standards Institute, Wayne, PA.
    9. 9
  9. Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; 17th informational supplement M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA.
    10. 10
  10. Coudron, P. E. 2005. Inhibitor-based methods for detection of plasmid-mediated AmpC β-lactamases in Klebsiella spp., Escherichia coli, and Proteus mirabilis. J. Clin. Microbiol. 43:4163-4167.[Abstract/Free Full Text]
    11. 11
  11. de Oliveira Garcia, D., Y. Doi, D. Szabo, J. M. Adams-Haduch, T. M. I. Vaz, D. Leite, M. C. Padoveze, M. P. Freire, F. P. Silveira, and D. L. Paterson. 2008. Multiclonal outbreak of Klebsiella pneumoniae producing extended-spectrum β-lactamase CTX-M-2 and novel variant CTX-M-59 in a neonatal intensive care unit in Brazil. Antimicrob. Agents Chemother. 52:1790-1793.[Abstract/Free Full Text]
    12. 12
  12. Jacoby, G. A., K. E. Walsh, and V. J. Walker. 2006. Identification of extended-spectrum, AmpC, and carbapenem-hydrolyzing β-lactamases in Escherichia coli and Klebsiella pneumoniae by disk tests. J. Clin. Microbiol. 44:1971-1976.[Abstract/Free Full Text]
    13. 13
  13. Kiener, P. A., and S. G. Waley. 1978. Reversible inhibitors of penicillinases. Biochem. J. 169:197-204.[Medline]
    14. 14
  14. Marchaim, D., S. Navon-Venezia, M. J. Schwaber, and Y. Carmeli. 2008. Isolation of imipenem-resistant Enterobacter species: emergence of KPC-2 carbapenemase, molecular characterization, epidemiology, and outcomes. Antimicrob. Agents Chemother. 52:1413-1418.[Abstract/Free Full Text]
    15. 15
  15. Marchiaro, P., V. Ballerini, T. Spalding, G. Cera, M. A. Mussi, J. Moran-Barrio, A. J. Vila, A. M. Viale, and A. S. Limansky. 2008. A convenient microbiological assay employing cell-free extracts for the rapid characterization of Gram-negative carbapenemase producers. J. Antimicrob. Chemother. 62:336-344.[Abstract/Free Full Text]
    16. 16
  16. Navon-Venezia, S., I. Chmelnitsky, A. Leavitt, M. J. Schwaber, D. Schwartz, and Y. Carmeli. 2006. Plasmid-mediated imipenem-hydrolyzing enzyme KPC-2 among multiple carbapenem-resistant Escherichia coli clones in Israel. Antimicrob. Agents Chemother. 50:3098-3101.[Abstract/Free Full Text]
    17. 17
  17. Pasteran, F. G., L. Otaegui, L. Guerriero, G. Radice, R. Maggiora, M. Rapoport, D. Faccone, A. Di Martino, and M. Galas. 2008. Klebsiella pneumoniae carbapenemase-2, Buenos Aires, Argentina. Emerg. Infect. Dis. 14:1178-1180.[CrossRef][Medline]
    18. 18
  18. Pérez-Pérez, F. J., and N. D. Hanson. 2002. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol. 40:2153-2162.[Abstract/Free Full Text]
    19. 19
  19. Samra, Z., O. Ofir, Y. Lishtzinsky, L. Madar-Shapiro, and J. Bishara. 2007. Outbreak of carbapenem-resistant Klebsiella pneumoniae producing KPC-3 in a tertiary medical centre in Israel. Int. J. Antimicrob. Agents 30:525-529.[CrossRef][Medline]
    20. 20
  20. Tenover, F. C., R. K. Kalsi, P. P. Williams, R. B. Carey, S. Stocker, D. Lonsway, J. K. Rasheed, J. W. Biddle, J. E. McGowan, Jr., and B. Hanna. 2006. Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing. Emerg. Infect. Dis. 12:1209-1213.[Medline]
    21. 21
  21. Villegas, M. V., K. Lolans, A. Correa, J. N. Kattan, J. A. Lopez, and J. P. Quinn. 2007. First identification of Pseudomonas aeruginosa isolates producing a KPC-type carbapenem-hydrolyzing β-lactamase. Antimicrob. Agents Chemother. 51:1553-1555.[Abstract/Free Full Text]
    22. 22
  22. Woodford, N., P. M. Tierno, Jr., K. Young, L. Tysall, M. F. Palepou, E. Ward, R. E. Painter, D. F. Suber, D. Shungu, L. L. Silver, K. Inglima, J. Kornblum, and D. M. Livermore. 2004. Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A β-lactamase, KPC-3, in a New York Medical Center. Antimicrob. Agents Chemother. 48:4793-4799.[Abstract/Free Full Text]
    23. 23
  23. Yagi, T., J. Wachino, H. Kurokawa, S. Suzuki, K. Yamane, Y. Doi, N. Shibata, H. Kato, K. Shibayama, and Y. Arakawa. 2005. Practical methods using boronic acid compounds for identification of class C β-lactamase-producing Klebsiella pneumoniae and Escherichia coli. J. Clin. Microbiol. 43:2551-2558.[Abstract/Free Full Text]
    24. 24
  24. Yigit, H., A. M. Queenan, G. J. Anderson, A. Domenech-Sanchez, J. W. Biddle, C. D. Steward, S. Alberti, K. Bush, and F. C. Tenover. 2001. Novel carbapenem-hydrolyzing β-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45:1151-1161.[Abstract/Free Full Text]

Journal of Clinical Microbiology, December 2008, p. 4083-4086, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.01408-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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  • Pasteran, F., Mendez, T., Guerriero, L., Rapoport, M., Corso, A. (2009). Sensitive Screening Tests for Suspected Class A Carbapenemase Production in Species of Enterobacteriaceae. J. Clin. Microbiol. 47: 1631-1639 [Abstract] [Full Text]  

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