MIMET-04634; No of Pages 2 Journal of Microbiological Methods xxx (2015) xxx–xxx

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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

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Marines Dalla Vale Martino a,c,⁎, Paula Célia Mariko Koga a, Jacyr Pasternak a, André Mario Doi a, Claudete Silvia Ciola b, Cely Barreto da Silva d, Irineu Francisco Delfino Silva Massaia c, Itacy Gonçalves Siqueira da Silva a, Maria Rita Elmor de Araújo b

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Article history: Received 24 February 2015 Received in revised form 1 May 2015 Accepted 2 May 2015 Available online xxxx

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Keywords: Carbapenemases Phenotypic method Enterobacteriaceae Resistance

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Hospital Israelita Albert Einstein, São Paulo, Brazil Hospital Beneficência Portuguesa de São Paulo, Brazil c Faculdade de Ciências Médicas da Santa Casa de São Paulo, Brazil d Irmandade da Santa Casa de Misericórdia de São Paulo, Brazil b

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We evaluated a new phenotypic test for carbapenemase detection. A total of 100 Enterobacteriaceae isolates were selected. The test was compared with conventional PCR for blaKPC and blaNDM detection. We found 100% sensitivity and specificity, suggesting that this test may be a feasible alternative for rapid carbapenemase detection. © 2015 Published by Elsevier B.V.

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Carbapenemases are the most important group of β-lactamases, because they can hydrolyze carbapenems, one of the last resources remaining for the treatment of Gram-negative infections (Bush and Jacoby, 2010; Nordmann and Poirel, 2014). According to Bush's classification, the most prevalent and important carbapenemases in Enterobacteriaceae are the Klebsiella pneumoniae carbapenemase — KPC (Ambler class A and Group 2f), metallo-β-lactamases (Ambler class B and Group 3) and OXA type carbapenemases (Ambler class D and Group 2df) (Bush and Jacoby, 2010). KPC is the most prevalent carbapenemase worldwide (Monteiro et al., 2009; Pereira et al., 2013). Other Enterobacteriaceae carbapenemases such as New Delhi metallo-β-lactamase (NDM), which was recently described in Brazil, and Imipenemase metallo-β-lactamase (IMP) have sporadically been reported (Carvalho-Assef et al., 2013; Rozales et al., 2014). Until now, there has been no report of OXA-48 carbapenemase in Enterobacteriaceae in Brazil. The gold standard for carbapenemase detection is PCR because phenotypic methods have low specificity. Further, molecular techniques are usually expensive, labor-intensive, time-consuming and not readily available in clinical laboratories.

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Evaluation of a new rapid test for carbapenemase detection in carbapenem resistant Enterobacteriaceae

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⁎ Corresponding author at: Hospital Israelita Albert Einstein, Albert Einstein Av. 627, São Paulo, Brazil. E-mail address: [email protected] (M.D.V. Martino).

The aim of this study was to propose a phenotypic and practical method for carbapenemase detection that was feasible for microbiological routines with high accuracy and specificity. This study was performed at Hospital Israelita Albert Einstein and Hospital Beneficencia Portuguesa de São Paulo, Brazil. A total of 100 isolates of Enterobacteriaceae family with different susceptibility profiles were selected. The evaluated test, called Cabapenembac, was compared with conventional PCR for blaKPC and blaNDM detection. Additionally, we compared this test with another phenotypic method for carbapenemase detection: inhibition with EDTA to detect metallo-β-lactamases. This new phenotypic method, based on the detection of carbapenem hydrolysis, consists of strips that are impregnated with a concentration of 100 μg carbapenem (Imipenem) and starch. The suspected strain is suspended in a Mueller–Hinton broth supplemented® (PROBAC do Brasil) with pH = 7.3 ± 0.1, adjusted to 7 to 10 MacFarland scale and inoculated over the strips (the volume of suspension deposited on the strips was 150 μL adjusted according with n.10 of McFarland scale). Then, these strips are incubated at 35– 37 °C for 10 min. In the next step, 200 μL of an iodine solution supplied by the manufacturer is added to the strip. The starch is 5% concentration, compounded by amyloses and amylopectin and the filter paper was 250 g/m2. When the iodine reacts with amyloses, a complex is formed that confers a dark color to the component.

http://dx.doi.org/10.1016/j.mimet.2015.05.001 0167-7012/© 2015 Published by Elsevier B.V.

Please cite this article as: Martino, M.D.V., et al., Evaluation of a new rapid test for carbapenemase detection in carbapenem resistant Enterobacteriaceae, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.05.001

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M.D.V. Martino et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx Table 1 Susceptibility profile of isolates for carbapenems (n = 100).

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References

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Bush, K., Jacoby, G.A., 2010. Updated functional classification of β-lactamases. Antimicrob. Agents Chemother. 54, 969–976. Carvalhaes, C.G., Picão, R.C., Nicoletti, A.G., Xavier, D.E., Gales, A.C., 2010. Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results. Antimicrob. Chemother. 65, 249–251. Carvalho-Assef, A.P., Pereira, P.S., Albano, R.M., Berião, G.C., Chagas, T.P., Timm, L.N., Da Silva, R.C., Falci, D.R., Asensi, M.D., 2013. Isolation of NDM-producing Providencia rettgeri in Brazil. J. Antimicrob. Chemother. 68, 2956–2957. Hindiyeh, M., Smollen, G., Grossman, Z., Ram, D., Davidson, Y., Mileguir, F., Vax, M., Ben, David D., Tal, I., Rahav, G., Shamiss, A., Mendelson, E., Keller, N., 2008. Rapid detection of blaKPC carbapenemase genes by real time PCR. J. Clin. Microbiol. 46, 2879–2883. Monteiro, J., Santo, A.F., Asensi, M.D., Peirano, G., Gales, A.C., 2009. First report of KPC-2producing Klebsiella pneumoniae strains in Brazil. Antimicrob. Agents Chemother. 53, 333–334. Nordmann, P., Poirel, L., 2014. The difficult-to-control spread of carbapenemase producers in Enterobacteriaceae worldwide. Biomed. Res. Int. 20, 821–830. Pereira, P.S., de Araujo, C.F., Seki, L.M., Zahner, V., Carvalho-Assef, A.P., Asensi, M.D., 2013. Update of the molecular epidemiology of KPC-2-producing Klebsiella pneumoniae in Brazil: spread of clonal complex 11 (ST11, ST437 and ST340). J. Antimicrob. Chemother. 68, 312–316. Rozales, F.P., Ribeiro, V.B., Magagnin, C.M., Pagano, M., Lutz, L., Falci, D.R., Machado, A., Barth, A.L., Zavascki, A.P., 2014. Emergence of NMD1-producing Enterobacteriaceae in Porto Alegre, Brazil. Int. J. Infect. Dis. 25, 79–81.

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interpret, leading to unreliable results (Carvalhaes et al., 2010). Additionally, the turnaround time for this test is high; generally, 24 h is required to obtain a final result. The PCR, however, is labor-intensive and requires skilled labor, specific infrastructure and high costs. In this study, we present a validation of a new method called CarbapenemBac, which is based on the detection of carbapenemase activity using iodine consumption (to incur a visual change of color). This method is easy to perform and does not require skilled labor. The results demonstrate that this method is highly sensitive and specific compared with PCR for the blaKPC gene, suggesting that it could be a feasible alternative for rapid carbapenemase detection. Despite the emergence of new techniques for detecting carbapenemase activity, such as mass spectrometry, CarbapenemBac does not require special equipment and can be performed in less than an hour. Due to the low prevalence of other carbapenemases in our setting, we could not evaluate the performance of this test for metallo βlactamases and OXA-48 carbapenemase, which are common Enterobacteriaceae in some parts of the world. These results demonstrate that this method is highly sensitive and specific compared with PCR for the blaKPC gene, suggesting that it may be a feasible alternative for rapid carbapenemase detection.

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When carbapenem hydrolysis occurs because of the presence of carbapenemases, the β-lactam ring (Imipenem) is broken, releasing hydrogen ions that acidify the medium. In the presence of an acid, the compound amyloses–iodine is broken, the dark color disappears, and the strip becomes clear. After 30 min of incubation at room temperature, the persistence of a dark coloration suggests a negative test (no carbapenemase). The dark color becoming clear at the edge of the stripes indicates a positive test and the presence of carbapenemase activity (Fig. 1). Escherichia coli ATCC 25922 was used as a negative control, and a known K. pneumoniae KPC and E. coli NDM positives were used as positive controls. The PCR multiplex for blaKPC and blaNDM followed the protocols from the literature (Hindiyeh et al., 2008; Carvalhaes et al., 2010). We tested a total of 100 isolates of Enterobacteriaceae with different susceptibility profiles for carbapenems, as demonstrated in Table 1, through the ETEST on cation-adjusted Mueller–Hinton agar. Of these, 90 isolates were identified as K. pneumoniae, 7 Enterobacter aerogenes and 3 E. coli. Concerning the molecular characterization of the resistance genes, 72 isolates were blaKPC-positives, and 28 were blaKPCnegatives. No amplification for blaNDM was detected. The phenotypic test for metalo-β-lactamase using EDTA inhibition was also performed, and no isolate was positive for this test. All blaKPC-positive isolates were also positive according to the phenotypic test, and all blaKPC-negative isolates were negative. The sensitivity and specificity of the new test, according to the reference test (PCR), were both 100%. Four isolates presented a minimal inhibitory concentration (MIC) that was lower or equal to 1 μg/mL, but the isolates were blaKPC positive. Despite the number of samples tested, we noted that the phenotypic test (CarbapenemBac) had 100% sensitivity and 100% specificity compared with PCR for the blaKPC gene. We conclude that this test has a good performance for KPC detection with high sensitivity and specificity, even between strains with low MIC for carbapenems. Although the Modified Hodge Test has good sensitivity for KPC, it is unspecific and provides false positive results due to other mechanisms, such as alteration of membrane permeability associated with the presence of ESBL. This test is also subjective and sometimes difficult to

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Fig. 1. CarbapenemBac test representation: positive control (blaKPC) and negative control.

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Please cite this article as: Martino, M.D.V., et al., Evaluation of a new rapid test for carbapenemase detection in carbapenem resistant Enterobacteriaceae, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.05.001

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