LETTER
The increase in carbapenemase producing Enterobacteriaceae (CPE) is a serious concern worldwide (1–7). However, not all CPE isolates show reduced susceptibility to carbapenems (6, 8–11). Some CPE isolates also produce other beta-lactamases, such as extended-spectrum and/or AmpC-type beta-lactamases (12–14). For these reasons, screening for CPE by antibiotic susceptibility testing is challenging. The specific phenotypic detection methods for CPE currently in use include the carbapenem inactivation method (CIM) (15), the Carba NP test (15, 16), and the Cica-beta test (17). The CIM is based on the disk diffusion method. The Carba NP and Cica-beta tests are able to identify some beta-lactamase classes by using specific inhibitors. However, specific inhibitors that work against OXA-48 group class D carbapenem-hydrolyzing beta-lactamases are not available (18, 19). A screening technique for CPE before a second confirmatory assay by CIM, Carba NP test, Cica-beta test, or genetic detection test by PCR would be useful. Here, we demonstrate the efficiency of a simple screening technique for CPE using moxalactam.
Nonduplicate isolates including CPE and non-CPE were identified and characterized at Toho University (Table 1). The types of beta-lactamase genes were confirmed by PCR amplification and DNA sequencing. All isolates were stored in a freezer at −80°C until use. Antibiotic susceptibility testing was performed by the Clinical and Laboratory Standards Institute-recommended microdilution method (M07-A10) (20). Customized frozen plates for microdilution testing were purchased from Eiken Chemical Co., Ltd. (Tokyo, Japan). The Clinical and Laboratory Standards Institute interpretative criteria in document M100-S25 (21) were applied. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as the quality control strains for antibiotic susceptibility testing.
TABLE 1.
Antibiotic activities of imipenem, meropenem, ceftazidime, and moxalactam against members of the family Enterobacteriaceae
| Enzyme(s) produced (no. of isolates) | No. of isolates of: |
Antibiotic | MIC (mg/liter) |
%S/%Ra | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Escherichia coli | Klebsiella pneumoniae | Klebsiella oxytoca | Salmonella sp. | Enterobacter sp. | Citrobacter sp. | Proteus mirabilis | Morganella morganii | Range | MIC50 | MIC90 | |||
| Carbapenemases | |||||||||||||
| IMP type (44) | 0 | 0 | 1 | 0 | 43 | 0 | 0 | 0 | Imipenem | 0.25 to 8 | 0.5 | 2 | 81.8/6.8 |
| Meropenem | ≤0.12 to 8 | 0.5 | 2 | 77.3/4.5 | |||||||||
| Ceftazidime | 32 to >256 | 128 | >256 | 0.0/100 | |||||||||
| Moxalactam | 32 to >256 | 256 | >256 | 0.0/93.2 | |||||||||
| IMP and CTX-M types (19) | 7 | 5 | 4 | 0 | 3 | 0 | 0 | 0 | Imipenem | ≤0.12 to 2 | 0.25 | 1 | 94.7/0.0 |
| Meropenem | ≤0.12 to 8 | 1 | 4 | 63.2/26.3 | |||||||||
| Ceftazidime | 4 to 128 | 32 | 64 | 5.3/89.5 | |||||||||
| Moxalactam | 16 to >256 | 256 | >256 | 0.0/84.2 | |||||||||
| NDM-1 (11) | 4 | 6 | 0 | 0 | 1 | 0 | 0 | 0 | Imipenem | 2 to 64 | 8 | 64 | 0.0/90.9 |
| Meropenem | 2 to 64 | 16 | 64 | 0.0/90.9 | |||||||||
| Ceftazidime | >256 | >256 | >256 | 0.0/100 | |||||||||
| Moxalactam | >256 | >256 | >256 | 0.0/100 | |||||||||
| KPC type (12) | 3 | 4 | 0 | 0 | 5 | 0 | 0 | 0 | Imipenem | 0.25 to 32 | 4 | 8 | 16.7/58.3 |
| Meropenem | ≤0.12 to 32 | 1 | 8 | 58.3/25 | |||||||||
| Ceftazidime | 4 to 256 | 64 | 256 | 16.7/66.7 | |||||||||
| Moxalactam | 1 to 32 | 2 | 32 | 75/0.0 | |||||||||
| GES-4 (3) | 0 | 1 | 0 | 0 | 2 | 0 | 0 | 0 | Imipenem | 16 to 64 | |||
| Meropenem | 16 to 64 | ||||||||||||
| Ceftazidime | 4 to >256 | ||||||||||||
| Moxalactam | 16 to >256 | ||||||||||||
| OXA-48 (11) | 3 | 6 | 0 | 0 | 1 | 1 | 0 | 0 | Imipenem | 0.5 to 128 | 4 | 16 | 18.2/54.5 |
| Meropenem | 0.25 to 128 | 0.5 | 32 | 63.6/27.3 | |||||||||
| Ceftazidime | 0.25 to 256 | 64 | 256 | 27.3/72.7 | |||||||||
| Moxalactam | 2 to >256 | 8 | >256 | 54.5/36.4 | |||||||||
| Total (100) | 17 | 22 | 5 | 0 | 55 | 1 | 0 | 0 | Imipenem | ≤0.12 to 128 | 1 | 16 | 58.0/29.0 |
| Meropenem | ≤0.12 to 128 | 1 | 16 | 60.0/26.0 | |||||||||
| Ceftazidime | 0.25 to >256 | 128 | >256 | 8.0/89.0 | |||||||||
| Moxalactam | 1 to >256 | 128 | >256 | 15.0/74.0 | |||||||||
| Noncarbapenemases | |||||||||||||
| CTX-M type (57) | 57 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | Imipenem | ≤0.12 to 0.5 | ≤0.12 | 0.25 | 100/0.0 |
| Meropenem | ≤0.12 | ≤0.12 | ≤0.12 | 100/0.0 | |||||||||
| Ceftazidime | 0.5 to 256 | 4 | 64 | 57.9/24.6 | |||||||||
| Moxalactam | ≤0.12 to 16 | 0.25 | 1 | 98.2/0.0 | |||||||||
| Chromosomal AmpC (7) | 0 | 0 | 0 | 0 | 5 | 1 | 0 | 1 | Imipenem | ≤0.12 to 2 | |||
| Meropenem | ≤0.12 to 0.25 | ||||||||||||
| Ceftazidime | 0.25 to 256 | ||||||||||||
| Moxalactam | ≤0.12 to 64 | ||||||||||||
| External AmpC (11) | 5 | 4 | 0 | 1 | 0 | 0 | 1 | 0 | Imipenem | ≤0.12 to 64 | 0.25 | 4 | 81.8/18.2 |
| Meropenem | ≤0.12 to 16 | ≤0.12 | ≤0.12 | 90.9/9.1 | |||||||||
| Ceftazidime | 1 to >256 | 64 | 256 | 9.1/90.9 | |||||||||
| Moxalactam | ≤0.12 to >256 | 4 | >256 | 63.6/18.2 | |||||||||
| Total (75) | 62 | 4 | 0 | 1 | 5 | 1 | 1 | 1 | Imipenem | ≤0.12 to 64 | ≤0.12 | 0.25 | 96.0/2.7 |
| Meropenem | ≤0.12 to 16 | ≤0.12 | ≤0.12 | 98.7/1.3 | |||||||||
| Ceftazidime | 0.25 to >256 | 8 | 128 | 46.5/40 | |||||||||
| Moxalactam | ≤0.12 to >256 | 0.25 | 16 | 88.0/6.7 | |||||||||
S, susceptible; R, resistant.
The positive predictive values (PPVs) of CPE detection by using CLSI resistance criteria for imipenem, meropenem, ceftazidime, and moxalactam were 93.5, 96.3, 74.8, and 93.7%, respectively. The negative predictive values (NPVs) of CPE detection by using the nonsusceptibility criteria for imipenem, meropenem, ceftazidime, and moxalactam were 50.7, 50.0, 80.4, and 72.9%, respectively. The NPV increased from 72.9% to 81.5% when the criterion for moxalactam (≥16 mg/liter) was used, but the PPV decreased from 93.7% to 90.4% (Table 2). Five false-positive results were observed in AmpC producers, and 26 false-negative results were observed in 12 KPC-type, 7 OXA-type, 6 IMP-type, and 1 GES-4-like enzyme-producing members of the family Enterobacteriaceae.
TABLE 2.
Results of screening of carbapenemase-producing members of the family Enterobacteriaceae by interpretation criteriaa
| Antibiotic | % PPVb | % NPVc |
|---|---|---|
| Imipenem | 93.5 (29/31) | 50.7 (73/144) |
| Meropenem | 96.3 (26/27) | 50.0 (74/148) |
| Ceftazidime | 74.8 (89/119) | 80.4 (45/56) |
| Moxalactam | 93.7 (74/79) | 72.9 (70/96) |
| Moxalactam (≥16 mg/liter) | 90.4 (85/94) | 81.5 (66/81) |
| Ceftazidimed | 67.0 (61/91) | 95.7 (45/47) |
The interpretation criteria used were those in reference 21, except for moxalactam (≥16 mg/liter).
The values in parentheses are the number of carbapenem producers/number of resistant isolates.
The values in parentheses are the number of non-carbapenem producers/number of susceptible and nonsusceptible isolates.
IMP-type enzyme producers, n = 63.
A limitation of this study is that we were unable to test a comprehensive range of CPE isolates because of a limited number of KPC-type, OXA-48, OXA-181, NDM-type, VIM-type, and VEB-type enzyme-producing CPE isolates. In Japan, the major carbapenemase is of the IMP type. Further testing to assess performance with chromosomal or acquired AmpC-producing Enterobacteriaceae isolates is in progress.
In conclusion, moxalactam at ≥16 mg/liter may be a useful, cheap, and simple primary screening method for detecting CPE in the clinical laboratory but requires follow-up confirmatory testing.
ACKNOWLEDGMENTS
We thank Shionogi & Co., Ltd., and Sumitomo Dainippon Pharma Co., Ltd., for providing moxalactam powder and meropenem powder, respectively.
This work was supported by a grant from the Ministry of Health, Labor and Welfare of Japan (H26-Tokubetsu-Shitei-005 to Y.I.) and by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22591113 to Y.I.). The research reported in this publication was supported in part by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under awards R01AI100560, R01AI063517, R21AI114508, and R01AI072219 to R.A.B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This study was supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, (award 1I01BX001974 to R.A.B.) and by the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development and the Geriatric Research Education and Clinical Center (VISN 10 to R.A.B.).
We have no conflicts of interest to declare.
REFERENCES
- 1.Abraham S, Wong HS, Turnidge J, Johnson JR, Trott DJ. 2014. Carbapenemase-producing bacteria in companion animals: a public health concern on the horizon. J Antimicrob Chemother 69:1155–1157. doi: 10.1093/jac/dkt518. [DOI] [PubMed] [Google Scholar]
- 2.Falagas ME, Tansarli GS, Karageorgopoulos DE, Vardakas KZ. 2014. Deaths attributable to carbapenem-resistant Enterobacteriaceae infections. Emerg Infect Dis 20:1170–1175. doi: 10.3201/eid2007.121004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Nordmann P. 2014. Carbapenemase-producing Enterobacteriaceae: overview of a major public health challenge. Med Mal Infect 44:51–56. doi: 10.1016/j.medmal.2013.11.007. [DOI] [PubMed] [Google Scholar]
- 4.Savard P, Perl RM. 2014. Combating the spread of carbapenemases in Enterobacteriaceae: a battle that infection prevention should not lose. Clin Microbiol Infect 20:854–861. doi: 10.1111/1469-0691.12748. [DOI] [PubMed] [Google Scholar]
- 5.Tängdén T, Giske CG. 2015. Global dissemination of extensively drug-resistant carbapenemase-producing Enterobacteriaceae: clinical perspectives on detection, treatment and infection control. J Intern Med 277:501–512. doi: 10.1111/joim.12342. [DOI] [PubMed] [Google Scholar]
- 6.Voulgari E, Poulou A, Koumaki V, Tsakris A. 2013. Carbapenemase-producing Enterobacteriaceae: now that the storm is finally here, how will timely detection help us fight back? Future Microbiol 8:27–39. doi: 10.2217/fmb.12.130. [DOI] [PubMed] [Google Scholar]
- 7.Woodford N, Wareham DW, Guerra B, Teale C. 2014. Carbapenemase-producing Enterobacteriaceae and non-Enterobacteriaceae from animals and the environment: an emerging public health risk of our own making? J Antimicrob Chemother 69:287–291. doi: 10.1093/jac/dkt392. [DOI] [PubMed] [Google Scholar]
- 8.Birgy A, Bidet P, Genel N, Doit C, Decre D, Arlet G, Bingen E. 2012. Phenotypic screening of carbapenemases and associated beta-lactamases in carbapenem-resistant Enterobacteriaceae. J Clin Microbiol 50:1295–1302. doi: 10.1128/JCM.06131-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Cohen Stuart J, Leverstein-Van Hall MA, Dutch Working Party on the Detection of Highly Resistant Microorganisms. 2010. Guideline for phenotypic screening and confirmation of carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents 36:205–210. doi: 10.1016/j.ijantimicag.2010.05.014. [DOI] [PubMed] [Google Scholar]
- 10.Nordmann P, Poirel L. 2002. Emerging carbapenemases in Gram-negative aerobes. Clin Microbiol Infect 8:321–331. doi: 10.1046/j.1469-0691.2002.00401.x. [DOI] [PubMed] [Google Scholar]
- 11.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. doi: 10.1128/JCM.00130-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Carvalho-Assef AP, Pereira PS, Albano RM, Beriao GC, Tavares CP, Chagas TP, Marques EA, Timm LN, Da Silva RC, Falci DR, Asensi MD. 2014. Detection of NDM-1-, CTX-M-15-, and qnrB4-producing Enterobacter hormaechei isolates in Brazil. Antimicrob Agents Chemother 58:2475–2476. doi: 10.1128/AAC.02804-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Seiffert SN, Marschall J, Perreten V, Carattoli A, Furrer H, Endimiani A. 2014. Emergence of Klebsiella pneumoniae co-producing NDM-1, OXA-48, CTX-M-15, CMY-16, QnrA and ArmA in Switzerland. Int J Antimicrob Agents 44:260–262. doi: 10.1016/j.ijantimicag.2014.05.008. [DOI] [PubMed] [Google Scholar]
- 14.Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR. 2009. Characterization of a new metallo-beta-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 53:5046–5054. doi: 10.1128/AAC.00774-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.van der Zwaluw K, de Haan A, Pluister GN, Bootsma HJ, de Neeling AJ, Schouls LM. 2015. The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in Gram-negative rods. PLoS One 10:e0123690. doi: 10.1371/journal.pone.0123690. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Poirel L, Nordmann P. 2015. Rapidec Carba NP test for rapid detection of carbapenemase producers. J Clin Microbiol 53:3003–3008. doi: 10.1128/JCM.00977-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Lavigne JP, Pfeiffer C, Vidal L, Sotto A. 2011. Rapid detection of multidrug resistant Gram-negative bacilli by Cica-Beta-test strips. Pathol Biol (Paris) 59:e7–e11. doi: 10.1016/j.patbio.2010.08.004. [DOI] [PubMed] [Google Scholar]
- 18.Harris PN, Tambyah PA, Paterson DL. 2015. Beta-lactam and beta-lactamase inhibitor combinations in the treatment of extended-spectrum beta-lactamase producing Enterobacteriaceae: time for a reappraisal in the era of few antibiotic options? Lancet Infect Dis 15:475–485. doi: 10.1016/S1473-3099(14)70950-8. [DOI] [PubMed] [Google Scholar]
- 19.Shlaes DM. 2013. New beta-lactam–beta-lactamase inhibitor combinations in clinical development. Ann N Y Acad Sci 1277:105–114. doi: 10.1111/nyas.12010. [DOI] [PubMed] [Google Scholar]
- 20.Clinical and Laboratory Standards Institute. 2015. Performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement. M100-S25. CLSI, Wayne, PA. [Google Scholar]
- 21.Clinical and Laboratory Standards Institute. 2015. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard tenth edition. M07-A10. CLSI, Wayne, PA. [Google Scholar]