KPC-2-producing Klebsiella pneumoniae in a hospital in the Midwest region of Brazil

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 bla KPC-2 gene. Thus, KPC-2producing Klebsiella pneumoniae has been present in a Brazilian hospital located in the Midwest region since at least 2009.


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 b-lactamase enzymes, including Klebsiella pneumoniae carbapenemase (KPC).
KPC-type enzymes inactivate b-lactam antibiotics, including cephalosporins, monobactams, and carbapenems, complicating the treatment of infections caused by these bacteria (Hirsch and Tam, 2010). 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., 2012).
The aim of this study was to investigate the presence of the bla KPC gene in Klebsiella spp. carbapenemnonsusceptible isolates collected from a tertiary hospital in Mato Grosso do Sul, a Brazilian state in the Midwest region.

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-offlight (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, 2011).

Carbapenemase production
The modified Hodge test (MHT) with ertapenem and imipenem disks (10 mg each) was employed for the phenotypic detection of carbapenemase production (CLSI, 2011). A molecular investigation of the bla KPC 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) with minor modifications. DNA extraction was made by boiling method. One or two colonies were transferred to a microcentrifuge tube containing 300 mL 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.

PCR reaction
A master mix solution containing 1.0 mL of each primer (10 mmol), 12.5 mL of Go Taq ® Green Master Mix 2X (Promega, Madison, USA) and 8.5 mL of sterile MilliQ water was prepared. Then, 2 mL of DNA was added to achieve a final reaction volume of 25 mL. 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.

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, 2011). 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 bla KPC-2 gene.
The PCR amplification profile of the bla KPC gene (800 bp) from the K. pneumoniae isolates is shown in Figure 1.
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., 2012;Nordmann et al., 2011). 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-2producing K. pneumoniae. This subtype is one of the most frequently occurring worldwide (Nadkarni et al., 2009;Nordmann et al., 2011), and the prevalence of this subtype in other Brazilian regions has been described previously (Castanheira et al., 2012;Monteiro et al., 2009;Nicoletti et al., 2012). KPC-producing K. pneumoniae in Brazil was first described in 2006 by Monteiro et al. (2009) 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., 2012;Nicoletti et al., 2012). Table 1, resistance to ertapenem (MIC ³ 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, 2012). The MHT demonstrated accurate results, with 100% sensitivity and 100% specificity, compared with PCR, corroborating a study by Fehlberg et al. (2012).

As shown in
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 MbL) alone or in combination with the loss of porins (Doumith et al., 2009.), ESBL (TEM, SHV, CTX-M) and/or AmpC enzymes associated with porin loss, and the presence of efflux pumps (Carvalhaes et al., 2009;Fehlberg et al., 2012, Queenam et al., 2007. 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., 2011;Nordmann et al., 2011).
A rapid and effective method for detecting KPCproducing 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, 2010).
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., 2011).  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 bla KPC gene. Finally, we conclude that this study provides evidence of the presence of K. pneumoniae isolates carrying the bla KPC-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.