Abstract
The emergence of β-lactamase-producing Enterobacteriaceae in the last few decades has become major challenge faced by hospitals. In this study, isolates of Klebsiella pneumoniae carbapenemase-2 (KPC-2)-producing K. pneumoniae from a tertiary hospital in Mato Grosso do Sul, Brazil, were characterized. Bacterial identification was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF; Bruker Daltonics, Germany) mass spectrometry. The minimum inhibitory concentrations of carbapenems were determined using the agar dilution method as recommended by the Clinical Laboratory Standards Institute guidelines. Carbapenemase production was detected using the modified Hodge test (MHT) and polymerase chain reaction (PCR), followed by DNA sequencing. Of 360 (12.2%) K. pneumoniae isolates obtained between May 2009 and May 2010, 44 (12.2%) were carbapenem nonsusceptible. Of these 44 isolates, thirty-six K. pneumoniae isolates that were positive by MHT and PCR carried the blaKPC-2 gene. Thus, KPC-2producing Klebsiella pneumoniae has been present in a Brazilian hospital located in the Midwest region since at least 2009.
Klebsiella pneumoniae ; carbapenems; drug-resistant bacteria
Introduction
The increasing prevalence of bacterial resistance among Enterobacteriaceae isolated in hospitals is a global concern. The major mechanism of carbapenem resistance among these bacteria is the production of β-lactamase enzymes, including Klebsiella pneumoniae carbapenemase (KPC).
KPC-type enzymes inactivate β-lactam antibiotics, including cephalosporins, monobactams, and carbapenems, complicating the treatment of infections caused by these bacteria (Hirsch and Tam, 2010Hirsch EB, Tam VH (2010) Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection. J Antimicrob Chemother 65:1119–1125.). KPC-2-producing Enterobacteriaceae have been isolated in many Brazilian medical centers, most frequently in teaching hospitals in the southern and southeastern regions. No data regarding the epidemiology of KPC strains in hospitals in the Brazilian Midwest are available (Nicoletti et al., 2012Nicoletti AG, Fehlberg LCC, Picão RC American J Infect Control (2012) Clonal complex 258, the most frequently found multilocus sequence type complex in KPC-2-producing Klebsiella pneumoniae isolated in Brazilian hospitals. Antimicrob Agents Chemother 56:4563–4564.).
The aim of this study was to investigate the presence of the blaKPC gene in Klebsiella spp. carbapenem-nonsusceptible isolates collected from a tertiary hospital in Mato Grosso do Sul, a Brazilian state in the Midwest region.
Materials and Methods
Bacterial isolates
Klebsiella spp. isolates that were nonsusceptible to imipenem, meropenem, and/or ertapenem were collected from hospitalized patients at the Regional Hospital of Mato Grosso do Sul (RHMS) between May 2009 and May 2010. The bacterial isolates were recovered from urine, blood, surgical wound exudates, catheter tips, tracheal aspirates and spinal cerebrospinal fluid samples. Surveillance cultures were not included. Microbiology lab-books and patient medical records were consulted to obtain demographic and clinical data.
Identification and antimicrobial susceptibility
The Klebsiella spp. isolates were initially identified using conventional biochemical reactions at the RHMS clinical laboratory. Bacterial identification was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using the Microflex LT System and analysis by Biotyper 2.0 software (Bruker Daltonics, Germany) at the Universidade Federal de São Paulo. The minimum inhibitory concentrations (MICs) of carbapenems were determined using the agar dilution method as recommended by the Clinical Laboratory Standards Institute (CLSI) guidelines (CLSI, 2011Clinical and Laboratory Standards Institute. 2011. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. M100-S21, 30(1). Clinical and Laboratory Standards Institute, Wayne, PA.).
Carbapenemase production
The modified Hodge test (MHT) with ertapenem and imipenem disks (10 μg each) was employed for the phenotypic detection of carbapenemase production (CLSI, 2011Clinical and Laboratory Standards Institute. 2011. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. M100-S21, 30(1). Clinical and Laboratory Standards Institute, Wayne, PA.). A molecular investigation of the blaKPC gene was performed with all K. pneumoniae carbapenem nonsusceptible (resistant or intermediate) isolates.
DNA extraction, PCR and sequencing of the PCR products were performed according to Monteiro (2009)Monteiro J (2009) Caracterização molecular dos mecanismos de resistência aos antibióticos β-lactâmicos em Klebsiella spp. isoladas de infecções de corrente sanguínea do Projeto SCOPE Brasil. Tese de Doutorado, Universidade Federal de São Paulo, São Paulo, 145 p. with minor modifications. DNA extraction was made by boiling method. One or two colonies were transferred to a microcentrifuge tube containing 300 μL of sterile MilliQ water. The suspension was boiled for 5 min and subsequently centrifuged for 1 min at 12,000 rpm. The supernatant was carefully aspirated and transferred to a new sterile microtube.
The following primers were used to amplify the blaKPC gene: forward, 5′ TCGCTAAACTCGAACAGG 3′ and reverse, 5′ TTACTGCCCGTTGACGCCCAATCC 3′.
PCR reaction
A master mix solution containing 1.0 μL of each primer (10 μmol), 12.5 μL of Go Taq ® Green Master Mix 2× (Promega, Madison, USA) and 8.5 μL of sterile MilliQ water was prepared. Then, 2 μL of DNA was added to achieve a final reaction volume of 25 μL. The reactions were amplified in an Eppendorf AG System, Eppendorf Mastercycler (Hamburg, Germany).
The cycling parameters were as follows: 10 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 1 min. The PCR amplification was completed with a final extension cycle at 72 °C for 10 min.
The PCR products were sequenced after purification using a QIA quick Gel Extraction kit (Qiagen, Hilden, Alemanha) as described by the manufacturer. The amplified genomic DNA was quantified by optical density in a spectrophotometer (NanoDrop® ND-1000 UV-Vis, version 3.2.1; Thermo Fisher Scientific, Wilmington, DE, USA). Approximately 70 ng of DNA was prepared for sequencing using the Big Dye Terminator Cycle Sequencing (Applied Biosystems, Foster City, USA) kit. Sequencing was performed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA).
The resulting DNA sequences and their corresponding protein sequences were analyzed using the Lasergene Software Package (DNASTAR, Madison, WI) and compared with genetic databases available on the Internet (http://www.ebi.ac.uk/fasta33/ and http://www.ncbi.nlm.nih.gov/BLAST/).
This study was approved by the Research Ethics Committee of the Federal University of Mato Grosso do Sul.
Results
During the study period, 360 isolates of Klebsiella spp. were identified by the RHMS clinical laboratory, of which 44 (12.2%) were nonsusceptible to carbapenems according to the CLSI breakpoints (CLSI, 2011Clinical and Laboratory Standards Institute. 2011. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. M100-S21, 30(1). Clinical and Laboratory Standards Institute, Wayne, PA.). Identification as Klebsiella pneumoniae was confirmed by MALDI-TOF for all isolates. The antimicrobial susceptibility testing results for the carbapenems are reported in Table 1. Thirty-six of the forty-four carbapenem-nonsusceptible K. pneumoniae isolates were phenotypic carbapenemase producers as determined by the MHT, and all of those 36 isolates carried the blaKPC-2 gene.
The PCR amplification profile of the blaKPC gene (800 bp) from the K. pneumoniae isolates is shown in Figure 1.
PCR amplification profile of the blaKPC gene (800 bp) from the K. pneumoniae isolates. M: 100 bp DNA ladder marker. Samples 2, 4, 9, 7, 10, 11, 12, 15, 17, 22, 23, 24, 25, 28, 31, 32, 34, 40, 47, and 49: K. pneumoniae clinical isolates. Sample 7 was negative for the presence of the blaKPC gene.
Patients infected with KPC-producing K. pneumoniae were primarily admitted to the intensive care unit (ICU) (43%), followed by the internal medicine department and coronary care unit (13.6% each, respectively). The age of the patients ranged from 0 to 91 years, with a median age of 68 years. Of the 44 patients included in this study, 43.2% died. KPC-producing K. pneumoniae (36) was most frequently isolated from urine (41.7%), blood cultures (25%), surgical wound exudates (22.3%), catheter tips (5.5%) and tracheal aspirates (5.5%).
Discussion
The prevalence of carbapenem-resistant Enterobacteriaceae has increased substantially during the last decade (Castanheira et al., 2012Castanheira M, Costello AJ, Deshpande LM et al. (2012) Expansion of Clonal Complex 258 KPC-2-Producing Klebsiella pneumoniae in Latin American Hospitals: Report of the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother 56:1668–1669.; Nordmann et al., 2011Nordmann T, Naas T, Poirel L (2011) Global spread of carbapenemase- producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1797.). The rapid increase and dissemination of carbapenemases, such as KPC, is a major challenge for clinical laboratories and physicians. The identification of the bacterial mechanisms of resistance is critical for infection control and epidemiological studies. However, molecular biology techniques for the detection of resistance genes are not yet available in most Brazilian routine laboratories.
In this study, we detected the presence of KPC-2-producing K. pneumoniae. This subtype is one of the most frequently occurring worldwide (Nadkarni et al., 2009Nadkarni AB, Schliep T, Khan L et al. (2009) Cluster of bloodstream infections caused by KPC-2 carbapenemase-producing Klebsiella pneumoniae in Manhattan. Am J Infect Control 37:121–126.; Nordmann et al., 2011Nordmann T, Naas T, Poirel L (2011) Global spread of carbapenemase- producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1797.), and the prevalence of this subtype in other Brazilian regions has been described previously (Castanheira et al., 2012Castanheira M, Costello AJ, Deshpande LM et al. (2012) Expansion of Clonal Complex 258 KPC-2-Producing Klebsiella pneumoniae in Latin American Hospitals: Report of the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother 56:1668–1669.; Monteiro et al., 2009Monteiro J, Santos AF, Asensi MD et al. (2009) First Report of KPC-2-Producing Klebsiella pneumonia strains in Brazil. Antimicrob Agents Chemother 53:333–334.; Nicoletti et al., 2012Nicoletti AG, Fehlberg LCC, Picão RC American J Infect Control (2012) Clonal complex 258, the most frequently found multilocus sequence type complex in KPC-2-producing Klebsiella pneumoniae isolated in Brazilian hospitals. Antimicrob Agents Chemother 56:4563–4564.). KPC-producing K. pneumoniae in Brazil was first described in 2006 by Monteiro et al. (2009)Monteiro J (2009) Caracterização molecular dos mecanismos de resistência aos antibióticos β-lactâmicos em Klebsiella spp. isoladas de infecções de corrente sanguínea do Projeto SCOPE Brasil. Tese de Doutorado, Universidade Federal de São Paulo, São Paulo, 145 p. in a patient from the state of Pernambuco. An increasing number of cases were subsequently reported in geographically distant Brazilian cities, indicating the wide dissemination of KPC-2-producing isolates in Brazil (Castanheira et al., 2012Castanheira M, Costello AJ, Deshpande LM et al. (2012) Expansion of Clonal Complex 258 KPC-2-Producing Klebsiella pneumoniae in Latin American Hospitals: Report of the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother 56:1668–1669.; Nicoletti et al., 2012Nicoletti AG, Fehlberg LCC, Picão RC American J Infect Control (2012) Clonal complex 258, the most frequently found multilocus sequence type complex in KPC-2-producing Klebsiella pneumoniae isolated in Brazilian hospitals. Antimicrob Agents Chemother 56:4563–4564.).
As shown in Table 1, resistance to ertapenem (MIC ≥ 1) was more frequent (93%) than resistance to imipenem (16%). These findings are in agreement with data reported by the Centers for Disease Control and Prevention (CDC) indicating that ertapenem resistance is the best marker for carbapenemase production (CDC, 2012Centers for Diseases Control and Prevention (CDC). 2012. Department of health and human services centers for disease control and prevention. Available at: http://www.cdc.gov/maso/FACM/pdfs/HICPAC/2008061213_HICPAC_Minutes.pdf. Accessed 22 May 2012.
http://www.cdc.gov/maso/FACM/pdfs/HICPAC...
). The MHT demonstrated accurate results, with 100% sensitivity and 100% specificity, compared with PCR, corroborating a study by Fehlberg et al. (2012)Fehlberg LCC, Carvalho AMC, Campana EH et al. (2012) Emergence of Klebsiella pneumoniae-producing KPC-2 carbapenemase in Paraíba, Northeastern Brazil. Braz J Infect Dis 16:577–580..
In our study, eight K. pneumoniae isolates were KPC negative, suggesting the involvement of other resistance mechanisms. Carbapenem resistance may involve multiple mechanisms, such as production of carbapenemases (KPC, NDM, OXA, and MβL) alone or in combination with the loss of porins (Doumith et al., 2009Doumith M, Ellington MJ, Livermore DM et al. (2009) Molecular mechanisms disrupting porin expression in ertapenem- resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J Antimicrob Chemother 63:659–667..), ESBL (TEM, SHV, CTX-M) and/or AmpC enzymes associated with porin loss, and the presence of efflux pumps (Carvalhaes et al., 2009Carvalhaes CG, Picão RC, Nicoletti AG et al. (2009) Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results. J Antimicrob Chemother 65:1–3.; Fehlberg et al., 2012Fehlberg LCC, Carvalho AMC, Campana EH et al. (2012) Emergence of Klebsiella pneumoniae-producing KPC-2 carbapenemase in Paraíba, Northeastern Brazil. Braz J Infect Dis 16:577–580., Queenam et al., 2007Queenan AM, Bush K (2007) Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev 20:440–458.).
The high number of patients over 60 years of age (65.9%) and the high frequency of ICU admissions (43%) suggests that colonization by these multi-resistant bacteria is favored by the high number of invasive procedures and the prolonged use of broad-spectrum antibiotics associated with these units (Beirão et al., 2011Beirão EM, Furtado JJD, Girardello R et al. (2011) Clinical and microbiological characterization of KPC-producing Klebsiella pneumoniae infections in Brazil. Braz J Infect Dis 15:69–73.; Nordmann et al., 2011Nordmann T, Naas T, Poirel L (2011) Global spread of carbapenemase- producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1797.).
A rapid and effective method for detecting KPC-producing K. pneumoniae is needed to avoid therapeutic failures and introduce measures to prevent and control the dissemination of these multi-resistant microorganisms (Hirsch and Tam, 2010Hirsch EB, Tam VH (2010) Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection. J Antimicrob Chemother 65:1119–1125.).
Notably, 38.6% of the KPC-producing K. pneumoniae isolates were detected in urine cultures, and 31.8% were detected in blood cultures. These data confirm literature findings that Klebsiella spp. is an important causative agent of urinary tract infections in hospitalized patients (Beirão et al., 2011Beirão EM, Furtado JJD, Girardello R et al. (2011) Clinical and microbiological characterization of KPC-producing Klebsiella pneumoniae infections in Brazil. Braz J Infect Dis 15:69–73.).
Finally, we conclude that this study provides evidence of the presence of K. pneumoniae isolates carrying the blaKPC-2 gene in a Brazilian hospital located in the Midwest region since at least 2009. The results presented in this study further support the dissemination of this pathogen throughout the national territory. Understanding the mechanisms underlying resistance may facilitate the implementation of preventive measures, control of the dissemination of these pathogens, and the implementation of surveillance programs and effective therapies.
References
- Beirão EM, Furtado JJD, Girardello R et al. (2011) Clinical and microbiological characterization of KPC-producing Klebsiella pneumoniae infections in Brazil. Braz J Infect Dis 15:69–73.
- Carvalhaes CG, Picão RC, Nicoletti AG et al. (2009) Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results. J Antimicrob Chemother 65:1–3.
- Castanheira M, Costello AJ, Deshpande LM et al. (2012) Expansion of Clonal Complex 258 KPC-2-Producing Klebsiella pneumoniae in Latin American Hospitals: Report of the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother 56:1668–1669.
- Centers for Diseases Control and Prevention (CDC). 2012. Department of health and human services centers for disease control and prevention. Available at: http://www.cdc.gov/maso/FACM/pdfs/HICPAC/2008061213_HICPAC_Minutes.pdf Accessed 22 May 2012.
» http://www.cdc.gov/maso/FACM/pdfs/HICPAC/2008061213_HICPAC_Minutes.pdf - Clinical and Laboratory Standards Institute. 2011. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. M100-S21, 30(1). Clinical and Laboratory Standards Institute, Wayne, PA.
- Doumith M, Ellington MJ, Livermore DM et al. (2009) Molecular mechanisms disrupting porin expression in ertapenem- resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J Antimicrob Chemother 63:659–667.
- Fehlberg LCC, Carvalho AMC, Campana EH et al. (2012) Emergence of Klebsiella pneumoniae-producing KPC-2 carbapenemase in Paraíba, Northeastern Brazil. Braz J Infect Dis 16:577–580.
- Hirsch EB, Tam VH (2010) Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection. J Antimicrob Chemother 65:1119–1125.
- Monteiro J (2009) Caracterização molecular dos mecanismos de resistência aos antibióticos β-lactâmicos em Klebsiella spp. isoladas de infecções de corrente sanguínea do Projeto SCOPE Brasil. Tese de Doutorado, Universidade Federal de São Paulo, São Paulo, 145 p.
- Monteiro J, Santos AF, Asensi MD et al. (2009) First Report of KPC-2-Producing Klebsiella pneumonia strains in Brazil. Antimicrob Agents Chemother 53:333–334.
- Nadkarni AB, Schliep T, Khan L et al. (2009) Cluster of bloodstream infections caused by KPC-2 carbapenemase-producing Klebsiella pneumoniae in Manhattan. Am J Infect Control 37:121–126.
- Nicoletti AG, Fehlberg LCC, Picão RC American J Infect Control (2012) Clonal complex 258, the most frequently found multilocus sequence type complex in KPC-2-producing Klebsiella pneumoniae isolated in Brazilian hospitals. Antimicrob Agents Chemother 56:4563–4564.
- Nordmann T, Naas T, Poirel L (2011) Global spread of carbapenemase- producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1797.
- Queenan AM, Bush K (2007) Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev 20:440–458.
Publication Dates
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Publication in this collection
Apr-Jun 2015
History
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Received
21 Feb 2014 -
Accepted
07 Oct 2014