Comparative Evaluation of Five Assays for Detection of Carbapenemases with a Proposed Scheme for Their Precise Application
2020; Elsevier BV; Volume: 22; Issue: 9 Linguagem: Inglês
10.1016/j.jmoldx.2020.05.012
ISSN1943-7811
AutoresHazim O. Khalifa, Takashi Okanda, Amer Ali Abd El‐Hafeez, Amera Abd El Latif, Ahmed G. K. Habib, Hisakazu Yano, Yasuyuki Kato, Tetsuya Matsumoto,
Tópico(s)Vibrio bacteria research studies
ResumoThe escalating problem of the dissemination of carbapenemase-producing bacteria (CPB) has gained worldwide attention. The prompt diagnosis of CPB and precise identification of carbapenemases are imperative to enable specific antibiotic therapy and control the spread of these bacteria. The present study was designed to assess the performance of five important assays for the detection of carbapenemases. The modified carbapenem inactivation method (mCIM), CARBA-5, GeneXpert Carba-R, BD MAX Check-Points CPO, and GeneFields CPE assays were evaluated with an international collection of 159 bacterial isolates, including 93 CPB and 66 non-CPB isolates. The overall accuracy/sensitivity/specificity for carbapenemase detection were 100% (95% CI, 97.7%–100%)/100% (95% CI, 96.1%–100%)/100% (95% CI, 94.6%–100%) for mCIM, 98.7% (95% CI, 95.5%–99.9%)/97.9% (95% CI, 92.5%–99.7%)/100% (95% CI, 94.6%–100%) for CARBA-5, 96.9% (95% CI, 92.8%–99%)/95.7% (95% CI, 89.4%–98.8%)/98.5% (95% CI, 91.8%–99.9%) for GeneXpert Carba-R, 94.3% (95% CI, 89.5%–97.4%)/90.3% (95% CI, 82.4%–95.5%)/100% (95% CI, 94.6%–100%) for BD MAX Check-Points CPO, and 86.2% (95% CI, 79.8%–91.1%)/77.4% (95% CI, 67.6%–85.5%)/98.5% (95% CI, 91.8%–100%) for GeneFields CPE. Interestingly, mCIM and CARBA-5 assays showed 100% accuracy/sensitivity/specificity for detection of the target genes. Furthermore, all the other assays showed comparable high accuracy (96.9% to 100%), sensitivity (100%), and specificity (96.4% to 100%) for the detection of the target genes. On the basis of these results, a new scheme was proposed for their efficient application. These results confirmed the high sensitivity of the evaluated assays, and the proposed scheme is reliable and improves the overall sensitivity and specificity of the assays. The escalating problem of the dissemination of carbapenemase-producing bacteria (CPB) has gained worldwide attention. The prompt diagnosis of CPB and precise identification of carbapenemases are imperative to enable specific antibiotic therapy and control the spread of these bacteria. The present study was designed to assess the performance of five important assays for the detection of carbapenemases. The modified carbapenem inactivation method (mCIM), CARBA-5, GeneXpert Carba-R, BD MAX Check-Points CPO, and GeneFields CPE assays were evaluated with an international collection of 159 bacterial isolates, including 93 CPB and 66 non-CPB isolates. The overall accuracy/sensitivity/specificity for carbapenemase detection were 100% (95% CI, 97.7%–100%)/100% (95% CI, 96.1%–100%)/100% (95% CI, 94.6%–100%) for mCIM, 98.7% (95% CI, 95.5%–99.9%)/97.9% (95% CI, 92.5%–99.7%)/100% (95% CI, 94.6%–100%) for CARBA-5, 96.9% (95% CI, 92.8%–99%)/95.7% (95% CI, 89.4%–98.8%)/98.5% (95% CI, 91.8%–99.9%) for GeneXpert Carba-R, 94.3% (95% CI, 89.5%–97.4%)/90.3% (95% CI, 82.4%–95.5%)/100% (95% CI, 94.6%–100%) for BD MAX Check-Points CPO, and 86.2% (95% CI, 79.8%–91.1%)/77.4% (95% CI, 67.6%–85.5%)/98.5% (95% CI, 91.8%–100%) for GeneFields CPE. Interestingly, mCIM and CARBA-5 assays showed 100% accuracy/sensitivity/specificity for detection of the target genes. Furthermore, all the other assays showed comparable high accuracy (96.9% to 100%), sensitivity (100%), and specificity (96.4% to 100%) for the detection of the target genes. On the basis of these results, a new scheme was proposed for their efficient application. These results confirmed the high sensitivity of the evaluated assays, and the proposed scheme is reliable and improves the overall sensitivity and specificity of the assays. Carbapenems are a group of life-saving antibiotics that represent the last resort for the treatment of infection caused by multidrug-resistant bacteria.1Stewardson A.J. Marimuthu K. Sengupta S. Allignol A. El-Bouseary M. Carvalho M.J. Hassan B. Delgado-Ramirez M.A. Arora A. Bagga R. Owusu-Ofori A.K. Effect of carbapenem resistance on outcomes of bloodstream infection caused by Enterobacteriaceae in low-income and middle-income countries (PANORAMA): a multinational prospective cohort study.Lancet Infect Dis. 2019; 19: 601-610Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,2Khalifa H.O. Soliman A.M. Ahmed A.M. Shimamoto T. Hara T. Ikeda M. Kuroo Y. Kayama S. Sugai M. Shimamoto T. High carbapenem resistance in clinical Gram-negative pathogens isolated in Egypt.Microb Drug Resist. 2017; 23: 838-844Crossref PubMed Scopus (45) Google Scholar Carbapenem resistance is closely associated with increased hospitalization period and mortality rates with bloodstream-infected patients in low- and middle-income countries.1Stewardson A.J. Marimuthu K. Sengupta S. Allignol A. El-Bouseary M. Carvalho M.J. Hassan B. Delgado-Ramirez M.A. Arora A. Bagga R. Owusu-Ofori A.K. Effect of carbapenem resistance on outcomes of bloodstream infection caused by Enterobacteriaceae in low-income and middle-income countries (PANORAMA): a multinational prospective cohort study.Lancet Infect Dis. 2019; 19: 601-610Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Therefore, the emergence and dispersal of carbapenem resistance have gained worldwide attention to mitigate such problems and prevent epidemic spread.3Dortet L. Poirel L. Nordmann P. 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NDM-4-and NDM-5-producing Klebsiella pneumoniae coinfection in a 6-month-old infant.Antimicrob Agents Chemother. 2016; 60: 4416-4417Crossref PubMed Scopus (28) Google Scholar Carbapenemases are a unique group of β-lactamases that have interesting hydrolyzing activity against most β-lactams and are malleable against inhibition by nearly all β-lactamase inhibitors.5Queenan A.M. Carbapenemases B.K. The versatile β-lactamases.Clin Microbiol Rev. 2007; 20: 440-458Crossref PubMed Scopus (1833) Google Scholar During the last decade, a wide variety of carbapenemases have been reported worldwide. However, the most common belong to class B metallo-β-lactamases [New Delhi metallo-β-lactamase (NDM), Verona integron-encoded metallo-β-lactamase (VIM), and Imipenemase-type metallo-β-lactamase (IMP)], Ambler class A carbapenemases [K. pneumoniae carbapenemase (KPC)], and class D oxacillinases [oxacillinase group of β-lactamase (OXA)–48 and OXA-48 like].4Khalifa H.O. Soliman A.M. Ahmed A.M. 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PCR-dipstick chromatography for differential detection of carbapenemase genes directly in stool specimens.Antimicrob Agents Chemother. 2017; 6: e00067-e000617Google Scholar The developed techniques show considerable variation in their specificity and sensitivity, depending on the evaluated enzymes and bacterial species.6Lucena Baeza L. Pfennigwerth N. Greissl C. Göttig S. Saleh A. Stelzer Y. Gatermann S.G. Hamprecht A comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm.Clin Microbiol Infect. 2019; 25: 1286.e9-1286.e15Abstract Full Text Full Text PDF Scopus (65) Google Scholar,8Mancini S. Kieffer N. Poirel L. Nordmann P. Evaluation of the RAPIDEC(R)® CARBA NP and beta-CARBA(R)® tests for rapid detection of carbapenemase-producing Enterobacteriaceae.Diagn Microbiol Infect Dis. 2017; 88: 293-297Crossref PubMed Scopus (39) Google Scholar In addition, phenotypic tests can detect only carbapenem hydrolysis activity but not the specific gene involved, and most of the developed techniques are restricted to certain target enzymes, with a high possibility of missing nontarget or new enzymes.8Mancini S. Kieffer N. Poirel L. Nordmann P. Evaluation of the RAPIDEC(R)® CARBA NP and beta-CARBA(R)® tests for rapid detection of carbapenemase-producing Enterobacteriaceae.Diagn Microbiol Infect Dis. 2017; 88: 293-297Crossref PubMed Scopus (39) Google Scholar,10Shanmugakani R.K. Akeda Y. Yamamoto N. Sakamoto N. Hagiya H. Yoshida H. Takeuchi D. Sugawara Y. Kodera T. Kawase M. Laolerd W. PCR-dipstick chromatography for differential detection of carbapenemase genes directly in stool specimens.Antimicrob Agents Chemother. 2017; 6: e00067-e000617Google Scholar Therefore, it is necessary to compare the different methods available for the detection of carbapenemases to identify the one that is most accurate and effective. Furthermore, the absence of a single ideal method for detection of carbapenemases6Lucena Baeza L. Pfennigwerth N. Greissl C. Göttig S. Saleh A. Stelzer Y. Gatermann S.G. Hamprecht A comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm.Clin Microbiol Infect. 2019; 25: 1286.e9-1286.e15Abstract Full Text Full Text PDF Scopus (65) Google Scholar,11Lutgring J.D. Limbago B.M. The problem of carbapenemase-producing-carbapenem-resistant-Enterobacteriaceae detection.J Clin Microbiol. 2016; 54: 529-534Crossref PubMed Scopus (165) Google Scholar regenerates the interest to propose an approach for their precise use that could improve their potential application for carbapenemase detection. The present study was conducted to evaluate and compare the effectiveness of five carbapenemase detection methods. In this study, the phenotypic modified carbapenem inactivation method (mCIM), the immunochromatographic CARBA-5 assay (CARBA-5; NG Biotech, Guipry, France), the automated real-time quantitative PCR (qPCR)–based GeneXpert Carba-R assay (Cepheid, Frankfurt, Germany), the nucleic acid chromatography GeneFields CPE assay (KURABO, Tokyo, Japan), and the real-time PCR BD MAX Check-Points CPO assay (Check-Points Health, Wageningen, the Netherlands) using the BD MAX System (Becton, Dickinson and Company, Franklin Lakes, NJ) were evaluated and compared for the identification of carbapenemases. On the basis of these results, a proposed approach was developed for their accurate use for carbapenemase identification. A wide range of bacterial strains from an international collection of 159 Gram-negative isolates from Egypt, Japan, and Bangladesh were evaluated in this study (Table 1).4Khalifa H.O. Soliman A.M. Ahmed A.M. Shimamoto T. Shimamoto T. NDM-4-and NDM-5-producing Klebsiella pneumoniae coinfection in a 6-month-old infant.Antimicrob Agents Chemother. 2016; 60: 4416-4417Crossref PubMed Scopus (28) Google Scholar,12Ando S. Nakano R. Kuchibiro T. Yamasaki K. Suzuki Y. Nakano A. Mizuno T. Kasahara K. Yano H. Emergence of VIM-2-producing Citrobacter freundii in Japan.Infect Dis. 2018; 50: 862-863Crossref PubMed Scopus (4) Google Scholar The isolates were identified by matrix-assisted laser desorption/ionization–time-of-flight analysis and molecularly characterized by PCR and the sequencing of different carbapenemase-encoding genes (Table 2).13Poirel L. Walsh T.R. Cuvillier V. Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes.Diagn Microbiol Infect Dis. 2011; 70: 119-123Crossref PubMed Scopus (1243) Google Scholar, 14Poirel L. Héritier C. Tolün V. Nordmann P. 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Multiplex PCR for rapid detection of genes encoding class A carbapenemases.Ann Lab Med. 2012; 32: 359-361Crossref PubMed Scopus (52) Google Scholar The isolate collection consisted of 66 carbapenemase-negative isolates and 93 CPB encoding a total of 97 carbapenemases, including isolates belonging to Ambler classes A (n = 22), B (n = 58), and D (n = 17) (Table 1). For the comparison and evaluation of the test results, a fresh overnight bacterial culture on Mueller-Hinton agar plates was used. All the evaluated assays were performed according to the manufacturer's instructions and recommendations, unless reported otherwise.Table 1Characterization of the Carbapenemase-Producing Bacterial Isolates Evaluated in this StudyStrain no.Bacterial sppSourceSampleCountryDetected carbapenemases (PCR)1Enterobacter cloacaeClinicalSputumJapanIMP-62E. cloacaeClinicalSputumJapanIMP-63E. cloacaeClinicalSputumJapanIMP-64Klebsiella pneumoniaeAnimalsNasal swabEgyptOXA-485Enterobacter hormaecheiAnimalsNasal swabEgyptVIM-46E. hormaecheiAnimalsNasal swabEgyptVIM-47E. hormaecheiAnimalsNasal swabEgyptVIM-48E. hormaecheiAnimalsNasal swabEgyptVIM-49Escherichia coliAnimalsNasal swabEgyptOXA-24410E. hormaecheiAnimalsNasal swabEgyptVIM-411Enterobacter kobeiClinicalPusEgyptNDM-512K. pneumoniaeClinicalUrineEgyptOXA-4813∗Isolates from Khalifa et al.4K. pneumoniaeClinicalBloodEgyptNDM-514E. coliClinicalUnknownJapanIMP-115E. coliClinicalUnknownJapanIMP-116E. coliClinicalUnknownJapanIMP-117E. coliClinicalUnknownJapanIMP-118E. coliClinicalUnknownJapanIMP-619E. coliClinicalUnknownJapanIMP-620E. coliClinicalUnknownJapanIMP-621E. coliClinicalUnknownJapanIMP-622K. pneumoniaeClinicalSputumBangladeshNDM-123E. coliClinicalUrineBangladeshNDM-524E. coliClinicalPusBangladeshNDM-525E. cloacaeClinicalUrineBangladeshNDM-126K. pneumoniaeClinicalUnknownBangladeshOXA-181 + NDM-527E. coliClinicalUnknownBangladeshOXA-181 + NDM-528Klebsiella aerogenesClinicalUnknownBangladeshOXA-18129Proteus mirabilisClinicalUnknownBangladeshOXA-18130Citrobacter freundiiClinicalUnknownJapanVIM-231E. coliClinicalUnknownJapanKPC-232E. coliClinicalUnknownJapanKPC-233E. coliClinicalUnknownJapanKPC-234E. coliClinicalUnknownJapanKPC-235Klebsiella oxytocaClinicalUnknownJapanGES-2036K. pneumoniaeClinicalUnknownJapanOXA-4837K. pneumoniaeClinicalUnknownJapanOXA-24438K. pneumoniaeClinicalUnknownJapanKPC-239K. pneumoniaeClinicalUnknownJapanKPC-240K. pneumoniaeClinicalUnknownJapanKPC-241E. cloacaeClinicalUnknownJapanKPC-242E. cloacaeClinicalUnknownJapanKPC-243K. pneumoniaeClinicalUnknownJapanNDM-744E. cloacaeClinicalUnknownJapanNDM-745E. cloacaeClinicalUnknownJapanNDM-746∗Isolates from Khalifa et al.4K. pneumoniaeClinicalEndotracheal tube swabEgyptNDM-447K. pneumoniaeClinicalUnknownJapanNDM-148E. cloacaeClinicalUnknownJapanNDM-149K. pneumoniaeClinicalUnknownJapanIMP-150K. pneumoniaeClinicalUnknownJapanIMP-151E. cloacaeClinicalUnknownJapanIMP-152E. cloacaeClinicalUnknownJapanIMP-153K. pneumoniaeClinicalUnknownJapanIMP-654K. pneumoniaeClinicalUnknownJapanIMP-655K. pneumoniaeClinicalUnknownJapanIMP-656E. coliClinicalUnknownJapanOXA-4857E. coliClinicalUnknownJapanOXA-18158E. coliClinicalUnknownJapanOXA-18159K. pneumoniaeClinicalUnknownJapanOXA-4860K. pneumoniaeClinicalUnknownJapanNDM-161K. pneumoniaeClinicalUnknownJapanNDM-162K. pneumoniaeClinicalUnknownJapanNDM-563E. cloacaeClinicalUrineJapanIMP-664E. cloacaeClinicalUnknownJapanIMP-665E. cloacaeClinicalChest drainJapanIMP-666E. cloacaeClinicalDrainJapanIMP-667E. cloacaeClinicalBileJapanIMP-668E. cloacaeClinicalBileJapanIMP-669K. pneumoniaeClinicalSputumBangaladishOXA-4870K. pneumoniaeClinicalUnknownBangaladishOXA-23271K. pneumoniaeClinicalUnknownBangaladishOXA-23272E. coliClinicalUrineBangaladishNDM-473E. coliClinicalUrineBangaladishNDM-474K. pneumoniaeClinicalUnknownBangaladishOXA-232 + NDM-575E. coliUnknownUnknownJapanKPC-276E. cloacaeUnknownUnknownJapanKPC-277K. pneumoniaeUnknownUnknownJapanKPC-278C. freundiiUnknownUnknownJapanKPC-279K. pneumoniaeUnknownUnknownJapanKPC-280K. pneumoniaeUnknownUnknownJapanKPC-1981E. coliUnknownUnknownJapanKPC-1982K. pneumoniaeUnknownUnknownJapanKPC-283E. coliUnknownUnknownJapanKPC-284K. pneumoniaeUnknownUnknownJapanKPC-285E. coliUnknownUnknownJapanKPC-286Pseudomonas aeruginosaUnknownUnknownJapanVIM-287†Isolates from Ando et al.12 GES, Guiana extended-spectrum β-lactamase; IMP, Imipenemase-type metallo-β-lactamase; KPC, K. pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; OXA, oxacillinase group of β-lactamase; VIM, Verona integron-encoded metallo-β-lactamase.C. freundiiClinicalRetroperitoneal abscessJapanVIM-288Pseudomonas putidaEnvironmentWater sampleJapanVIM-289Pseudomonas mendocinaEnvironmentWater sampleJapanVIM-290Pseudomonas alcaligenesEnvironmentWater sampleJapanVIM-2, GES-191P. mendocinaEnvironmentWater sampleJapanVIM-292P. aeruginosaEnvironmentWater sampleJapanVIM-293P. putidaEnvironmentWater sampleJapanVIM-2Carbapenemase nonproducing isolates included 52 isolates that were recovered from Egypt, 13 isolates that were recovered from Japan, and a single isolate that was recovered from Bangladesh. The isolates recovered from Egypt include 40 Escherichia coli isolates (36 clinical isolates from urine, 2 clinical isolates from pus, and 2 animal isolates from nasal swabs), 6 clinical Klebsiella pneumoniae isolates from urine, 5 clinical Enterobacter cloacae isolates from urine, and 1 clinical Enterobacter kobei isolate from urine. Furthermore, 13 isolates were recovered from Japan, including 6 E. cloacae isolates (4 clinical isolates from urine, 1 clinical isolate from bile, and 1 unknown isolate), 3 E. coli isolates (1 clinical isolate from bile and 2 unknown isolates), 3 K. pneumoniae isolates (unknown), and 1 clinical Enterobacter aerogenes isolate from an abdominal drain. Finally, a single community Serratia plymuthica isolate was recovered from Bangladesh. All remaining isolates are from the current study.∗ Isolates from Khalifa et al.4Khalifa H.O. Soliman A.M. Ahmed A.M. Shimamoto T. Shimamoto T. NDM-4-and NDM-5-producing Klebsiella pneumoniae coinfection in a 6-month-old infant.Antimicrob Agents Chemother. 2016; 60: 4416-4417Crossref PubMed Scopus (28) Google Scholar† Isolates from Ando et al.12Ando S. Nakano R. Kuchibiro T. Yamasaki K. Suzuki Y. Nakano A. Mizuno T. Kasahara K. Yano H. Emergence of VIM-2-producing Citrobacter freundii in Japan.Infect Dis. 2018; 50: 862-863Crossref PubMed Scopus (4) Google ScholarGES, Guiana extended-spectrum β-lactamase; IMP, Imipenemase-type metallo-β-lactamase; KPC, K. pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; OXA, oxacillinase group of β-lactamase; VIM, Verona integron-encoded metallo-β-lactamase. Open table in a new tab Table 2Oligonucleotides Used for Identification of the Acquired Carbapenemase GenesPrimer nameSequence (5′ to 3′)TargetReferenceIMP-FIMP-R5′-GGAATAGAGTGGCTTAAYTCTC-3′5′-GGTTTAAYAAAACAACCACC-3′blaIMP13SPM-FSPM-R5′-AAAATCTGGGTACGCAAACG-3′5′-ACATTATCCGCTGGAACAGG-3′blaSPM13VIM-FVIM-R5′-GATGGTGTTTGGTCGCATA-3′5′-CGAATGCGCAGCACCAG-3′blaVIM13OXA-FOXA-R5′-GCGTGGTTAAGGATGAACAC-3′5′-CATCAAGTTCAACCCAACCG-3′blaOXA-48 like13OXA-48A5′-TTGGTGGCATCGATTATCGG-3′blaOXA-48 like14OXA-48B5′-GAGCACTTCTTTTGTGATGGC-3′BIC-FBIC-R5′-TATGCAGCTCCTTTAAGGGC-3′5′-TCATTGGCGGTGCCGTACAC-3′blaBIC13NDM-FNDM-R5′-GGTTTGGCGATCTGGTTTTC-3′5′-CGGAATGGCTCATCACGATC-3′blaNDM13NDM-full F5′-ATGGAATTGCCCAATATTATGCAC-3′Whole blaNDM15NDM-full R5′-TCAGCGCAGCTTGTCGGC-3′KPC-FKPC-R5′-CGTCTAGTTCTGCTGTCTTG-3′5′-CTTGTCATCCTTGTTAGGCG-3′blaKPC13KPC forward5′-ATGTCACTGTATCGCCGTCT-3′blaKPC16KPC reverse5′-TTTTCAGAGCCTTACTGCCC-3′AIM-FAIM-R5′-CTGAAGGTGTACGGAAACAC-3′5′-GTTCGGCCACCTCGAATTG-3′blaAIM13GIM-FGIM-R5′-TCGACACACCTTGGTCTGAA-3′5′-AACTTCCAACTTTGCCATGC-3′blaGIM13SIM-FSIM-R5′-TACAAGGGATTCGGCATCG-3′5′-TAATGGCCTGTTCCCATGTG-3′blaSIM13DIM-FDIM-R5′-GCTTGTCTTCGCTTGCTAACG-3′5′-CGTTCGGCTGGATTGATTTG-3′blaDIM13GES-FGES-MR5′-GCTTCATTCACGCACTATT-3′5′-CGATGCTAGAAACCGCTC-3′blaGES17AIM, Adelaide imipenemase; BIC, Bicêtre carbapenemase; DIM, Dutch imipenemase; F, forward; GES, Guiana extended-spectrum β-lactamase; GIM, German imipenemase; IMP, Imipenemase-type metallo-β-lactamase; KPC, K. pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; MR, reverse; OXA, oxacillinase group of β-lactamase; R, reverse; SIM, Seoul imipenemase; SPM, Sao Paulo metallo-β-lactamase; VIM, Verona integron-encoded metallo-β-lactamase. Open table in a new tab Carbapenemase nonproducing isolates included 52 isolates that were recovered from Egypt, 13 isolates that were recovered from Japan, and a single isolate that was recovered from Bangladesh. The isolates recovered from Egypt include 40 Escherichia coli isolates (36 clinical isolates from urine, 2 clinical isolates from pus, and 2 animal isolates from nasal swabs), 6 clinical Klebsiella pneumoniae isolates from urine, 5 clinical Enterobacter cloacae isolates from urine, and 1 clinical Enterobacter kobei isolate from urine. Furthermore, 13 isolates were recovered from Japan, including 6 E. cloacae isolates (4 clinical isolates from urine, 1 clinical isolate from bile, and 1 unknown isolate), 3 E. coli isolates (1 clinical isolate from bile and 2 unknown isolates), 3 K. pneumoniae isolates (unknown), and 1 clinical Enterobacter aerogenes isolate from an abdominal drain. Finally, a single community Serratia plymuthica isolate was recovered from Bangladesh. All remaining isolates are from the current study. AIM, Adelaide imipenemase; BIC, Bicêtre carbapenemase; DIM, Dutch imipenemase; F, forward; GES, Guiana extended-spectrum β-lactamase; GIM, German imipenemase; IMP, Imipenemase-type metallo-β-lactamase; KPC, K. pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; MR, reverse; OXA, oxacillinase group of β-lactamase; R, reverse; SIM, Seoul imipenemase; SPM, Sao Paulo metallo-β-lactamase; VIM, Verona integron-encoded metallo-β-lactamase. The phenotypic mCIM test was performed as described previosely.18Pierce V.M. Simner P.J. Lonsway D.R. Roe-Carpenter D.E. Johnson J.K. Brasso W.B. Bobenchik A.M. Lockett Z.C. Charnot-Katsikas A. Ferraro M.J. Thomson R.B. Modified carbapenem inactivation method for phenotypic detection of carbapenemase production among Enterobacteriaceae.J Clin Microbiol. 2017; 55: 2321-2333Crossref PubMed Scopus (215) Google Scholar Briefly, a 1-μL inoculation loop of the overnight cultured bacteria was added to a tube containing 2 mL of tryptic soy broth. A 10-μg meropenem disk was added to the bacterial suspension after vortex for 10 seconds, followed by incubation at 37°C for 4 hours. After the incubation time, the meropenem disk was placed on Mueller-Hinton agar previously inoculated with the 0.5 McFarland standard Escherichia coli 25922 (ATCC, Manassas, VA) as an indicator organism. The result was evaluated by measuring the inhibition zone around the meropenem disk after incubation at 37°C for 18 to 24 hours. In this assay, a full 1-μL inoculation loop of overnight bacteria cultured on Mueller-Hinton agar was mixed with five drops of CARBA-5 extraction buffer. After gentle vertexing, 100 μL of the mixture was transferred into the CARBA-5 cassette, and the results were evaluated after incubation for 15 minutes at room temperature. The qPCR-based GeneXpert Carba-R assay was performed as follows: a full 10-μL suspension of 0.5 McFarland standard harvested from bacteria and cultured overnight on Mueller-Hinton agar was mixed with the sample reagent in the GeneXpert Carba-R assay sample reagent vial. After 10 seconds of vortexing, the recommended volume was added to the GeneXpert Carba-R cartridge with a disposable transfer pipette and run on the GeneXpert IV system (Cepheid). All procedures, including sample preparation, DNA amplification, and gene detection, were automated, and the results were automatically explicated in <1 hour. A full 50 μL of 1:400-diluted 0.5 McFarland bacterial cell suspension was added to BD MAX Check-Points CPO sample buffer tubes. After vortexing for 10 seconds, the sample buffer tubes and the unitized reagent strip were placed into the BD MAX system rack together with the extraction and master mix tubes, and the BD MAX system was run as recommended by the company. The sample preparation, lysis, DNA extraction, and multiplex RT-PCR were automatically performed with the BD MAX system, and the results were automatically interpreted after approximately 2 hours. The bacterial DNA was extracted with DNA extraction kits (Cica Geneus Extraction Reagent, Tokyo, Japan), according to the manufacturer's instructions. The extracted DNA (2 μL) was used as a template for the nucleic acid chromatography assay in a 30-μL reaction mixture containing 15 μL of 2× MightyAmp Buffer Ver.3 (TAKARA BIO Inc, Shiga, Japan), 3 μL of 10× additive, 0.6 μL of MightyAmp Polymerase Ver.3 (TAKARA BIO Inc), and 5 μL of PCR Oligo Mix (KURABO). The thermal cycler was run at 98°C for 3 minutes and then for 35 cycles of 98°C for 30 seconds, 62°C for 30 seconds, and 72°C for 30 seconds, followed by a final step at 72°C for 7 minutes. For detection of the suspected carbapenemases, 5 μL of the DNA product was mixed with 11 μL of coloring buffer and 5 μL of sterilized nuclease-free water in a 1.5-mL tube. The DNA strip was immersed in the mixture, and the result was evalua
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