Journal of Antimicrobial Chemotherapy (2009) 63, 265 268 doi:10.

1093/jac/dkn484 Advance Access publication 20 November 2008

Carbapenem-hydrolysing b-lactamase KPC-2 in Klebsiella pneumoniae isolated in Rio de Janeiro, Brazil

Gisele Peirano1*, Liliane M. Seki1, Vera Lucia Val Passos2, Maria Cristina F. G. Pinto3, Llia R. Guerra3 and Marise D. Asensi1
1

Oswaldo Cruz Institute, Avenida Brasil 4365, 21040-360 Rio de Janeiro, Brazil; 2Hospital Geral de Bonsucesso (HGB), Avenida Londres 616, 21041-030 Rio de Janeiro, Brazil; 3Hospital Universitario Antonio Pedro (HUAP)Universidade Federal Fluminense, Rua Marques de Parana 303, 24033-900 Niteroi, Brazil
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Received 17 July 2008; returned 27 August 2008; revised 30 October 2008; accepted 31 October 2008 Objectives: The aim of this study was to characterize the KPC-type carbapenem-hydrolysing b-lactamase, extended-spectrum b-lactamases (ESBLs) and class 1 integrons among nosocomial Klebsiella pneumoniae isolated in Rio de Janeiro, Brazil. Methods: MICs were determined and isolates were screened for ESBLs, metallo-b-lactamases (MBLs) and class A carbapenemase-producing phenotypes. The main b-lactamases resistance genes (blaTEM, blaSHV, blaCTX-M, blaKPC, blaIMP and blaVIM) and class 1 integrons were detected by PCR followed by DNA sequencing. The genetic relatedness of isolates was determined by PFGE. Results: All K. pneumoniae isolates were positive for ESBL and class A carbapenemase production and negative for MBL production. All isolates were resistant to all b-lactam antibiotics, ciprooxacin and gentamicin, being susceptible only to tigecycline and polymyxin B. The blaKPC-2, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8 and blaSHV-11 genes were detected. PFGE analysis revealed two clonal types among KPC-producing isolates, both identied in the same hospital. Conclusions: Our ndings should alert medical authorities to implement stringent methods for the detection and spread control of emerging KPC-2 carbapenemases in the hospital setting in Brazil. Keywords: multidrug resistance, class 1 integrons, PCR, PFGE

Introduction
Carbapenems currently represent the drugs of choice for the treatment of serious infections caused by the multidrug-resistant isolates that are prevalent in many Gram-negative bacterial species, especially those producing extended-spectrum b-lactamases (ESBLs) and/or derepressed AmpC b-lactamase. However, resistance to carbapenems is being increasingly detected and is mainly related to the action of carbapenemasetype enzymes. Three major classes of acquired carbapenemhydrolysing b-lactamases have been reported, namely: the molecular Ambler class A serine-b-lactamases of the IMI-, SME-, GES-, NMCA- and KPC-types; the class B metallo-b-lactamases (MBLs) of the IMP and VIM-types; and the class D oxacillinases of the OXA-types.1 Within class A, the KPCs are the most frequently encountered, primarily among Enterobacteriaceae; carbapenemase-producing

Klebsiella pneumoniae is a recent addition to the pool of multidrug-resistant nosocomial pathogens. Seven blaKPC variants have been detected: KPC-1 to KPC-7 in Enterobacteriaceae, except for KPC-5, which was described in a Pseudomonas aeruginosa isolate. KPC-type b-lactamases efciently hydrolyse penicillins, cephalosporins and aztreonam in addition to carbapenems and are inhibited by clavulanic acid and tazobactam.1,2 Until now, KPC-carbapenemase-producing K. pneumoniae have been recovered from hospitalized patients with prolonged hospital stays (usually in intensive care units), those given multiple antimicrobial drug courses and those mechanically ventilated. The strains can colonize the urinary, intestinal and respiratory tracts, as well as wounds; bloodstream infections are associated with higher mortality rates than infections at other sites.3 KPC-carbapenemase-producing K. pneumoniae have been reported in the USA since 1996,4 but now have an expanding

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*Corresponding author. Tel: 55-21-2598-4277, ext. 319; Fax: 55-21-2270-6565; E-mail: peirano@ioc.ocruz.br
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Peirano et al.
TIGa POLa PFGE

geographic range, including Israel, China, Europe, Central and South America and, recently, Brazil.5 In this report, we describe the detection of KPC-producing K. pneumoniae isolates recovered from two hospitals in Rio de Janeiro, Brazil.
Table 1. Clinical data, resistance genes, class 1 integron cassette array, antimicrobial susceptibility patterns and PFGE types of K. pneumoniae KPC-producing isolates

Bacterial isolates and phenotypic tests

The Laboratory of Enterobacteria (LGB), located at Oswaldo Cruz Institute, Rio de Janeiro, Brazil, routinely receives clinical bacterial isolates from hospitals that are part of a Bacterial Nosocomial Infection Resistance Surveillance network. Recently, K. pneumoniae clinical isolates exhibiting reduced susceptibility or resistance to carbapenems, broad-spectrum cephalosporins and multiple other antimicrobials were sent from two hospitals in Rio de Janeiro for the determination of the underlying resistance mechanism. Non-duplicate isolates were recovered from blood (n 4), urine (n 1) and tracheal aspirate (n 1) from six patients hospitalized between September and November 2007, and later in April and May 2008. Initial species identication and antimicrobial susceptibility testing were performed with the automated Vitek 2 system (bioMerieux, Marcy lEtoile, France) and later conrmed at LGB using standard biochemical tests and Etests (AB Biodisk, Solna, Sweden) for carbapenems (imipenem, meropenem and ertapenem), tigecycline and polymyxin B. Isolates were screened for the ESBL and carbapenemase-producing phenotypes by the standard doubledisc synergy test, Etest and a modied Hodge test. Tigecycline was evaluated using breakpoints for Enterobacteriaceae recommended by the US Food and Drug Administration (2 and 8 mg/L for susceptible and resistant, respectively).

MEMa ETPa AMK ATM FEP CTX CAZ CIP GEN

MIC (mg/L)

32 16 32 32 32 32

64 64 64 64 64 64

32 32 32 32 32 32

4 4 4 4 4 4

16 16 16 16 16 16

128/4 128/4 128/4 128/4 128/4 128/4

Materials and methods

IPM, imipenem; MEM, meropenem; ETP, ertapenem; AMK, amikacin; ATM, aztreonam; FEP, cefepime; CTX, cefotaxime; CAZ, ceftazidime; CIP, ciprooxacin; GEN, gentamicin; TZP, piperacillin/ tazobactam; TIG, tigecycline; POL, polymyxin B. a Etest MIC.

TZP

2 2 1 2 2 2

2 2 2 2 2 2

A A A B B A

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Molecular investigations
Screening for resistance genes was performed by PCR using previously reported conditions and primers for blaKPC, blaTEM, blaSHV, blaCTX-M, blaIMP, blaVIM and class 1 integrase. Amplication products were puried using the GFX PCR DNA and the Gel Band Purication Kit (GE Healthcare, UK) according to the manufacturers instructions. Sequencing was performed with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit on a 3730 DNA Analyser (Applied Biosystems, CA, USA) at the PDTIS-IOC DNA Sequencing Platform. Sequences were compared with those in GenBank. Clonal relatedness of isolates was examined by the PFGE of SpeI-digested genomic DNA (Invitrogen, CA, USA). Banding patterns were analysed with BioNumerics software (Applied Maths, Kortrijk, Belgium).

Results and discussion

In this study, we described the detection of KPC-2 among six ESBL-producing K. pneumoniae clinical isolates recovered from two hospitals in Rio de Janeiro, Brazil, from September 2007 to May 2008. According to the records of the hospitals microbiology laboratories, automated antimicrobial susceptibility testing resulted in broad resistance to b-lactams and its combinations, ciprooxacin and gentamicin. K. pneumoniae isolates exhibited reduced susceptibility or resistance to imipenem and meropenem when tested by the Etest method. The MICs of 266

513LGB-blood/Sep 07-HUAP 616LGB-blood/Oct 07-HUAP 779LGB-urine/Nov 07-HGB 1337LGB-blood/Apr 08-HGB 1382LGB-tracheal aspirate/Apr 08-HGB 1383LGB-blood/May 08-HGB

Strain-site/period of isolation-hospital

KPC-2, CTX-M-1, SHV-11/dfrA5, aadA2 4 KPC-2, CTX-M-1/dfrA5, aadA2 4 KPC-2, CTX-M-1, SHV-11, TEM-1/dhfrV 0.5 KPC-2, CTX-M-8, SHV-1/arr-5 64 KPC-2, CTX-M-2, SHV-1, TEM-1/arr-5 64 KPC-2, CTX-M-1, SHV-11, TEM-1/dhfrV 64

bla genes/class 1 integron cassette array

IPMa

4 1 2 32 32 32

32 32 32 32 32 32

64 2 8 64 32 8

32 32 32 32 32 32

KPC-2 in K. pneumoniae from Brazil

% similarity
100 70 75 80 85 90 95

PFGE 616 KPC 513 KPC 1383 KPC 779 KPC 1382 KPC 1337 KPC HUAP HUAP HGB HGB HGB HGB

Figure 1. PFGE of KPC-producing K. pneumoniae isolates. Dendrogram generated by Dice coefcient with clustering by UPGMA.

these drugs were between 0.5 and 4 mg/L, except for the 1337LGB, 1382LGB and 1383LGB isolates, whose MICs were 64 mg/L (imipenem) and 32 mg/L (meropenem). Isolates 616LGB, 779LGB and 1383LGB remained susceptible to amikacin. Most isolates were susceptible only to tigecycline and polymyxin B (Table 1). It was recently observed that some KPC-producing strains had imipenem and/or meropenem MIC results within the CLSI susceptible range despite carbapenemase production. Current evidence suggests that the signicance and prevalence of carbapenemases may be clinically unrecognized due to the fact that the KPC carbapenemases may not confer resistance to carbapenems but only reduced susceptibility, and also to the fact that most KPC producers are ESBL producers as well. Such intermediate resistance may not be consistently detected by automated antimicrobial susceptibility testing systems, the panels of which usually comprise imipenem and meropenem. Ertapenem, which is not available in most manufactured antimicrobial panels, has been considered as a sensitive indicator for KPCmediated carbapenem resistance.6 So, it is suggested that clinical laboratories may consider using the ertapenem disc diffusion test routinely. Isolates were positive for KPC and ESBL phenotype production, but negative for class B carbapenemases (MBL). ESBL-encoding genes blaKPC-2 (six isolates) carbapenemase, blaCTX-M-1 group (four isolates), blaCTX-M-2 (one isolate), blaCTX-M-8 (one isolate) and blaSHV-11 (three isolates) were detected among isolates. These ndings are in agreement with the recent ones observed among K. pneumoniae KPC-producing Brazilian isolates.5 CTX-M-2, CTX-M-8 and CTX-M-9 groups are the most frequently detected CTX-M-type enzymes among ESBL-producing Enterobacteriaceae isolates from Brazil7 and most South American countries.8,9 To our best knowledge, this is the rst description of the blaCTX-M-1 group in Brazil. The resulting PFGE gel of KPC-producing K. pneumoniae isolates showed two clonal types, A and B, both identied at Hospital Geral de Bonsucesso (HGB). Also, two isolates from HGB shared close similarity (96.6% to 100%) to those from Hospital Universitario Antonio Pedro (HUAP), suggesting that they form a genetically related cluster. The similarity between the two clonal groups was 67.03% (Figure 1). KPC-producing strains from North-eastern Brazil exhibited two PFGE patterns as well.5 Although both studies comprised a low number of isolates, the clonal dissemination of carbapenemase-producing isolates was observed in and between medical centres. All isolates were positive for a class 1 integron element. dfrA5 and aadA2 gene cassettes were identied in two isolates, and dhfrV and arr-5 in another two isolates, separately (Table 1). Both dfrA5 and dhfrV code for trimethoprim resistance. aadA2 codes for streptomycin/spectinomycin resistance

and arr-5 codes for rifampicin resistance. The gene cassettes inserted in class 1 integrons carried by these isolates had already been considered as integron-borne co-resistance genes reported to occur with class A b-lactamases. However, the new arr-5 allele was recently described as a gene cassette in class 1 integrons present in four clinical strains of K. pneumoniae recovered from a hospital located in Rio de Janeiro, Brazil.10 Our ndings should alert medical authorities to institute stringent methods for the detection and spread control of emerging carbapenemases in hospital settings. The identication of resistance mechanisms will help determine the epidemiology, risk factors and appropriate therapeutic strategies for carbapenemresistant isolates.

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Acknowledgements
We are grateful to PDTIS-IOC platform for DNA sequencing and to Mr Evaldo Soares for technical assistance in culture media preparation.

Funding
This work was supported by grants from FAPERJ (Fundacao de ` Amparo a Pesquisa do Estado do Rio de JaneiroE-26/ 170.232/2007) and Oswaldo Cruz Institute.

Transparency declarations
None to declare.

References
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