Molecular characterization of Enterobacteriaceae resistant to carbapenem antimicrobials

Caracterização molecular de enterobactérias resistentes aos carbapenêmicos

ABSTRACT

The present study aimed to genotypically and phenotypically characterize clinical isolates of carbapenem-resistant Enterobacteriaceae collected from inpatients at the University Hospital of Santa Maria, during seven months. Among the clinical isolates subjected to the modified Hodge test (MHT), 62.5% were positive, indicating possible production of carbapenemase. Polymerase chain reaction (PCR) demonstrated that bla_KPC was the most frequently found gene (31%), followed by bla_IMP (12.5%). Combined use of the methods is needed to identify carbapenem resistance in enterobacteria to prevent their spread and control the infections caused by these organisms.

Key words:
Enterobacteriaceae ; carbapenemase; phenotypic methods; molecular methods

RESUMO

Objetivou-se caracterizar fenotípica e genotipicamente isolados clínicos de enterobactérias resistentes aos carbapenêmicos (CRE) provenientes do Hospital Universitário de Santa Maria (RS). Entre os isolados clínicos submetidos ao teste modificado de Hodge (MHT), 62,5% apresentaram positividade, indicando possível produção de carbapenemase. A reação em cadeia da polimerase (PCR) demonstrou que o bla_KPC foi o gene mais encontrado (31%), seguido de bla_IMP (12,5%). O uso conjunto de distintas metodologias faz-se necessário para identificar a resistência aos carbapenêmicos produzida pelas enterobactérias, de modo a auxiliar o controle de infecção prevenindo a disseminação desses microrganismos.

Unitermos:
enterobactérias; carbapenemases; métodos fenotípicos; métodos moleculares

INTRODUCTION

Antimicrobial resistance in Enterobacteriaceae is a serious threat to public health due to the association of different resistance mechanisms and the insufficient development of new drugs. Carbapenemase production, added to other resistance mechanisms already described in enterobacteria - such as the extended-spectrum betalactamases (ESBL) -, makes these microorganisms resistant to almost all available antimicrobials⁽¹⁾. Thus, they are frequently associated with pandemic outbreaks, being responsible for thousands of deaths from infections that persist in spite of treatment^{(2, 3)}.

Three major classes of carbapenemases are known: 1) metallo-beta-lactamases (imipenemase [IMP], Verona imipenemase [VIM], New Delhi metallo-beta-lactamase [NDM]); 2) oxa-carbapenemases (OXA-48); and 3) Klebsiella pneumoniae carbapenemase (KPC)-type serine carbapenemases⁽²⁾. From an epidemiologic point of view, these enzymes are extremely relevant because of their rapid and wide dissemination, once they are encoded by genes located in mobile genetic elements, such as plasmids and transposons⁽⁴⁾.

In Brazil, since the first detection of KPC in 2006, dissemination throughout the country is described, in outbreaks caused by K. pneumoniae and other enterobacteria. Other enzymes have already been found, such as IMP, VIM and, more recently, NDM^{(5, 6)}.

Although phenotypic tests are widely used in many clinical laboratories, it is necessary to confirm the production of enzymes that confer resistance to carbapenems by means of molecular tests, such as the polymerase chain reaction (PCR), which presents high sensitivity and specificity. The detection of carbapenemase-producing enterobacteria is extremely important to control infection and prevent dissemination of these resistance mechanisms^{(2, 4)}.

MATERIALS AND METHODS

Clinical isolates

Clinical isolates of carbapenem-resistant Enterobacteriaceae (CRE) were collected at the clinical laboratory of Hospital Universitário de Santa Maria (HUSM), from June to December 2013. Thirty-two positive culture samples obtained from any infection site were analyzed, identified by the automated method Vitek^® 2 (BioMérieux) and/or by disk diffusion assay. For the conduction of experiments, from patients who had more than one sample testing positive for CRE, only the first positive culture was included.

This study was approved by the research ethics committee of Universidade Federal de Santa Maria (UFSM) under report nº 27094514.7000-5346.

Modified Hodge test

The modified Hodge test (MHT) was carried out as described by the Clinical and Laboratory Standards Institute (CLSI) (2013)⁽⁷⁾. The test was conducted using ertapenem (10 µg) or meropenem (10 µg) disks, the standard strain Escherichia coli (ATCC 25922) and the KPC-type carbapenemase-producing K. pneumoniae strain. The latter was kindly provided by Prof. Dr. Anna Sara Levin, of the medical investigation laboratory (LIM54), bacteriology laboratory of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP).

PCR for detection of carbapenemase-encoding genes

The extraction of deoxyribonucleic acid (DNA) was done by the boiling technique described by Woodford and Johnson (1998)⁽⁸⁾ . All enterobacteria isolates with carbapenem resistance profile underwent PCR for the investigation of the following carbapenemase-encoding genes: bla_KPC, bla_OXA-48, bla_NDM,bla_SPM, bla_IMP, bla_VIM and bla_GIM. The identification of bla_KPC, bla_OXA-48 and bla_NDM genes was made by PCR; the detection of bla_SPM, bla_IMP, bla_VIM and bla_GIM genes, by multiplex PCR. The primers used in the detection of serine carbapenemases were those described by Lomaestro et al. (2006)⁽⁹⁾ and Poirel et al. (2011)⁽¹⁰⁾; for the detection of metallo-beta-lactamases, those indicated by Nordmann et al. (2010)⁽¹¹⁾ and Mendes et al. (2007)⁽¹²⁾. In both reactions, the 2x PCR kit LGC Biotecnologia was used. The strains used as positive control for reactions were provided by Prof. Dr. Anna Sara Levin, and had been obtained from the LIM54/FMUSP culture collection.

The PCR conditions for the detection of bla_KPC, bla_OXA-48, bla_NDM genes and other metallo-beta-lactamases were: denaturation for five minutes at 94ºC, 35 cycles of five minutes at 94ºC, annealing for 30 seconds at 58ºC, and extension for one minute at 72ºC, followed by a final extension step of 10 minutes at 72ºC.

The PCR products were added to GelRed^TM (Biotium) and gel-loading buffer (6x) and underwent electrophoresis on 1.5% agarose gel run at 110 V/cm. The gels were visualized under an ultraviolet (UV) transilluminator (Hoefer MacroVue UV20) and photographed using the Loccus Biotecnologia LPix^® and software L-Pix Image ST^® (data not exhibited).

RESULTS

Based on the analysis of patients' records, it is possible to associate Enterobacteriaceae species with their respective isolation sites. The most frequent species, K. pneumoniae, was mainly isolated from stool and urine; K. oxytoca, from blood, tracheal secretion, and sputum. Data on resistance to ertapenem, imipenem and meropenem by each clinical isolate were associated with the MHT results and the presence of bla_KPC gene. These pieces of information are displayed in the Table, in which 12 different resistance profiles are observed.

Among the isolates subjected to MHT, 62.5% (n = 20) were positive, indicating a possible carbapenemase production. About the number of studied microorganisms, the genotypic tests evidenced that bla_KPC was the most encountered gene, in 31% (n = 10) of the samples, followed by bla_IMP, in 12.5% (n = 4). MHT was associated with the detection of resistance genes, and four main groups were observed, what can be seen in the Table.

TABLE
Profiles of isolates according to the modified Hodge test and bla_RPC gene detection

Species	MHT + blaKPC+	MHT+blaKPC-	MHT - blaKPC+	MHT - blaKPC-	Total
Enterobacter aerogenes		1		1	2
Enterobacter cloacae		3		1	4
Escherichia coli	1				1
Klebsiella oxytoca	4	1	1	1	7
Klebsiella pneumoniae	4	6		8	18
Total	9	11	1	11	32

• KPC: K. pneumoniae carbapenemase; MHT: modified Hodge test.

DISCUSSION

Enterobacteria present multiple mechanisms of antimicrobial resistance, of which the production of carbapenemases stands out, as its dissemination is made easy by resistance gene transfer through plasmids and transposons⁽³⁾. Rapid identification of these enzymes is, thus, essential, and molecular tests able to identify specific carbapenemase-encoding genes must be included in laboratory routines⁽¹³⁾.

Analysis of the different groups after association of MTH results and detection of resistance genes allows the discussion of some aspects. The first group encompasses the samples in which there was agreement between phenotypic (MHT) and genotypic methods. The second comprises samples that were positive in MHT, but negative in PCR. The third group includes a small number of samples, for which MHT resulted in low sensitivity, not detecting genes found when molecular tests were performed. The fourth group includes samples with negative results, both in MHT and PCR, evidencing that these isolates may show another mechanism of resistance that does not involve carbapenemases, as they show resistance to at least one carbapenem in the sensitivity test. In the cases encompassed by the second and fourth groups, it is possible that the isolates present a combination of resistance mechanisms, such as AmpC beta-lactamase hyperproduction, presence of efflux pumps or porin loss. However, in the first and third groups, it is not possible to exclude the existence of mechanisms other than carbapenemase production^{(14, 15)}.

Even if new CLSI guidelines have minimized the importance of phenotypic tests for clinical purposes, their use for identification of carbapenem resistance mechanisms may help in infection control, as they are sensitive and able to detect the activity of carbapenemases in K. pneumoniae, K. oxytoca and E. coli species⁽¹⁶⁾. Confirming other studies, the disk diffusion method, using ertapenem disks, proved more sensitive than that using meropenem in screening for resistant samples, demonstrating that screening may be optimized by the use of two carbapenems in combination^{(15, 17- 19)}.

In Brazil, most reported CRE isolates contain the bla_KPC^{(5, 6, 12)} gene. However, this study revealed low prevalence of this gene in the tested isolates. In addition, the concomitant presence of bla_IMP and bla_KPC genes was found in four isolates of K. oxytoca, K. pneumoniae and E. cloacae. The coexistence of these genes was reported by Wei et al. (2011) ⁽²⁰⁾. Pournaras et al. (2010) ⁽²¹⁾ verified a clinical isolate with simultaneous presence of bla_VIM and bla_KPC genes, which evidenced a higher minimal inhibitory concentration (MIC) for meropenem and imipenem. The authors point that the spread of these associated genes may have even more serious consequences for the treatment of such infections.

The variability demonstrated by phenotypic tests, both with ertapenem and MHT, means that it is not advisable to employ a single screening criterion for carbapenemase, but to associate methods to the confirmatory PCR test⁽¹⁵⁾. Similarly, it is necessary to research other genes and mechanisms of resistance, as the concomitant presence of several types of beta-lactamases in enterobacteria makes it difficult to identify individual resistance mechanisms⁽¹⁸⁾.

In spite of its limitations, this study issues an alert to health professionals and clinical laboratories, so that they establish new criteria for the detection of carbapenemases. With the application of molecular techniques associated with traditional phenotypic tests, the detection of carbapenemases will become faster and reliable, aiding diagnosis and treatment, as well as providing information that directly influences the control measures for infections caused by these microorganisms.

• ¹
  Li H, Zhang J, Liu Y, et al. Molecular characteristics of carbapenemaseproducing Enterobacteriaceae in China from 2008 to 2011: predominance of KPC-2 enzyme. Diagn Microbiol Infect Dis. 2014; 78: 63-5.
• ²
  Agência Nacional de Vigilância Sanitária. Nota Técnica nº 01/2013. Medidas de prevenção e controle de infecção por enterobactérias multirresistentes. Brasília (DF); 2013.
• ³
  Nordmann P. Carbapenemase-producing Enterobacteriaceae: overview of a major public health challenge. Med Mal Infect. 2014; 44(2): 51-6.
• ⁴
  Lupo A, Papp-Wallace KM, Sendi P, et al. Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae. Diagn Microbiol Infect Dis. 2013; 77(3): 179-94.
• ⁵
  Peirano G, Seki LM, Val Passos VL, et al. Carbapenem-hydrolysing betalactamase KPC-2 in Klebsiella pneumoniae isolated in Rio de Janeiro, Brazil. J Antimicrob Chemother. 2009; 63(2):265-8.
• ⁶
  Pavez M, Mamizuka EM, Lincopan N, et al. Early dissemination of KPC-2-producing Klebsiella pneumoniae strains in Brazil. Antimicrob Agents Chemother. 2009; 53(6): 2702.
• ⁷
  Clinical and Laboratory Standards Institute. CLSI document M-100, S 23. Approved standard. Wayne [PA]: Clinical and Laboratory Standards Institute; 2013.
• ⁸
  Woodford N, Johnson AP. Molecular bacteriology and clinical application. Humana Press. 1998; 1: 17-33.
• ⁹
  Lomaestro BM, Tobin EH, Shang W, Gootz T, et al. The spread of Klebsiella pneumoniae carbapenemase - producing K. pneumoniae to upstate New York. Clin Infect Dis. 2006; 43(3): 26-8.
• ¹⁰
  Poirel L, Walsh TR, Cuvillier V, et al. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis. 2010; 70(1): 119-23.
• ¹¹
  Nordmann P, Cuzon G, Naas T, et al. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis. 2010; 9(4): 228-36.
• ¹²
  Mendes RE, Kiyota KA, Monteiro J, et al. Rapid detection and identification of metallo-ß-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol. 2007; 45(2): 544-7.
• ¹³
  Doyle D, Peirano G, Lascols C, et al. Laboratory detection of Enterobacteriaceae that produce carbapenemases. J Clin Microbiol. 2012; 50(12): 3877-80.
• ¹⁴
  Gazin M, Paasch F, Goossens H, et al. Current trends in culture-based and molecular detection of extended-spectrum-ß-lactamaseharboring and carbapenem-resistant Enterobacteriaceae. J Clin Microbiol. 2012; 50(4): 1140-6.
• ¹⁵
  Tzouvelekis LS, Markogiannakis A, Psichogiou M, et al. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev. 2012; 25(4): 682-707.
• ¹⁶
  Seah C, Low DE, Patel SN, et al. Comparative evaluation of a chromogenic agar medium, the modified Hodge test, and a battery of meropenem-inhibitor discs for detection of carbapenemase activity in Enterobacteriaceae. J Clin Microbiol. 2011; 49(5): 1965-9.
• ¹⁷
  Anderson KF, Lonsway DR, Rasheed JK, et al. Evaluation of methods to identify the Klebsiella pneumonia carbapenemase in Enterobacteriaceae. J Microbiol. 2007; 45(8): 2723-5.
• ¹⁸
  Birgy A, Bidet P, Genel N, et al. Phenotypic screening of carbapenemases and associated ß-lactamases in carbapenem-resistant Enterobacteriaceae. J Clin Microbiol. 2012; 50(4): 1295-1302.
• ¹⁹
  Pasteran F, Mendez T, Guerriero L, et al. Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae. J Clin Microbiol. 2009; 47(6): 1631-9.
• ²⁰
  Wei Z, Yu T, Qi Y, et al. Coexistence of plasmid-mediated KPC-2 and IMP-4 carbapenemases in isolates of Klebsiella pneumoniae from China. J Antimicrob Chemother. 2011.
• ²¹
  Pournaras S, Poulou A, Voulgari E, et al. Detection of the new metallo-beta-lactamase VIM-19 along with KPC-2, CMY-2 and CTX-M-15 in Klebsiella pneumoniae. J Antimicrob Chemother. 2010; 65(8): 1604-7.

Publication Dates

• Publication in this collection
  May-Jun 2015

History

• Received
  01 Mar 2015
• Accepted
  07 Apr 2015