PAGES: 163-167 DOI: 10.1590/S0074-02762010000200009 Full paper
blaCTX-M-2 and blaCTX-M-28 extended-spectrum ?-lactamase genes and class 1 integrons in clinical isolates of Klebsiella pneumoniae from Brazil

Ana Catarina S LopesI, II, +; Dyana Leal VerasII; Alexsandra Mariá S LimaII; Rita de Cássia Andrade MeloI; Juan AyalaIII

IDepartamento de Medicina Tropical
IILaboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Av. Prof. Morais Rego s/n, 50.732-970 Recife, PE, Brasil
IIICentro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Madrid, Spain


Twenty-eight Klebsiella pneumoniae clinical isolates that exhibited an extended-spectrum cephalosporin-resistance profile from a city in the Northeast of Brazil were analysed by PCR and DNA sequencing in order to determine the occurrence of blaCTX-M genes and class 1 integrons. We determined the occurrence of the blaCTX-M-2 gene in six K. pneumoniae isolates and describe the first detection of the blaCTX-M-28 gene in South America. Seven isolates carried class 1 integrons. Partial sequencing analysis of the 5'-3'CS variable region in the class 1 integrons of three isolates revealed the presence of aadA1, blaOXA-2 and dfr22 gene cassettes.

Klebsiella pneumoniae is a Gram-negative bacterium that accounts for a significant portion of hospital and community acquired infections worldwide (Souza Lopes et al. 2005, Minarini et al. 2007, Yu et al. 2007). The introduction of oxyimino-cephalosporins into clinical practice for the treatment of resistant gram-negative bacterial infections was soon followed by the emergence of extended-spectrum ?-lactamases (ESBLs). The CTX-M-type enzymes, which are non-TEM and non-SHV derivatives, represent a rapidly growing family of ESBLs. The first CTX-M ?-lactamase (CTX-M-1/ MEN-1) was characterised in Escherichia colistrains isolated from German and Italian patients (Bauernfeind et al. 1990, Barthélémy et al. 1992). In the past 15 years, more than 60 different CTX-M-type ?-lactamases have been identified in most parts of the world ( (Livermore et al. 2007). On the basis of amino acid sequence similarities, CTX-M enzymes have been classified into five distinct phylogenetic groups: CTX-M-1 (> 97% identity), CTX-M-2 (> 94% identity), CTX-M-8 (> 98% identity), CTX-M-9 (> 98% identity) and CTX-M-25 (> 98% identity) (Bonnet 2004).

Members of CTX-M groups evolved by the capture of chromosomal genes from various Kluyvera species (Rodriguez et al. 2004). Once mobilised, blaCTX-M genes can be hosted by many mobile elements, most often by large multiresistance plasmids that are responsible for the rapid dissemination of these genes. Insertion sequences (IS), such as ISEcp1, IS10, IS26 and IS903, might be involved in the mobilisation of blaCTX-M genes (Bonnet et al. 2000, Saladin et al. 2002). blaCTX-M genes have also been associated with ISCR1 (IS common region 1, previously also called orf513), which is often found downstream of complex class 1 integrons (Partridge & Hall 2003).

CTX-M enzymes are replacing TEM and SHV mutants in many European countries, with E. coli joining K. pneumoniae as a major host (Livermore et al. 2007). In South America, studies from Argentina have shown that blaCTX-M-2 seems to be the most frequent ESBL among Enterobacteriaceae (Quinteros et al. 2003). In the Brazilian Southeast Region, blaCTX-M genes were identified in E. coli, Citrobacter amalonaticus, Enterobacter aerogenes and Enterobacter cloacae (Bonnet et al. 2000, 2001, Minarini et al. 2007), but blaCTX-M was only recently detected in K. pneumoniae (Do Carmo et al. 2008, Garcia et al. 2008). In this paper we describe the first detection of blaCTX-M genes and class 1 integrons in clinical isolates of K. pneumoniae from the Northeast of Brazil.



Bacterial isolates - Bacterial strains analysed in this study included 28 K. pneumoniae clinical isolates that have an ESBL phenotype (resistant isolates to cefotaxime and ceftazidime or aztreonam) (Table I), selected from a collection of 50 resistant isolates from different patients from the city of Recife, state of Pernambuco (PE), from 1998-2008. The isolates were collected from two public hospitals, one private hospital and one private laboratory. All isolates were identified using the API ID 20E (bioMérieux, Marcy l'Etoile, France).

Antibiotic susceptibility - The susceptibility to antimicrobial agents was tested on Mueller-Hinton agar by the disk diffusion method (CLSI 2006). Commercially available disks (Oxoid) loaded with the following antibiotics were used: ampicillin, amoxicillin, amoxicillin-clavulanate, chloramphenicol, tetracycline, nalidixic acid, streptomycin, amikacin, trimethoprim/sulfamethoxazole, ciprofloxacin, cefoxitin, cefotaxime, ceftazidime, aztreonam, meropenem and imipenem. Minimum inhibitory concentrations of ceftazidime, cefotaxime and aztreonam (Sigma Aldrich) were determined by a microdilution test in accordance with the criteria of the CLSI (2006).

DNA preparation and identification by PCR of the blaCTX-M genes - Genomic DNA was extracted from direct colony suspensions in 200 ?L of distilled water. The suspensions were heated to 100ºC for 10 min, centrifuged (5 min/10,000 g) and 150 ?L of the recovered supernatant was frozen at -20ºC until use.

blaCTX-M genes were first investigated by PCR using group-specific primers (Table II). The blaCTX-M gene was investigated in 28 isolates of K. pneumoniae that were resistant to either third generation cephalosporin or aztreonam. The amplification reactions were prepared in a total volume of 25 ?L containing 1 ng of genomic DNA, 2.0 U of Taq DNA polymerase (Promega), 200 ?M of deoxynucleoside triphosphates (Invitrogen), 1.5 mM of MgCl2, 1 ?M of each primer and 1X reaction buffer (final concentration). The PCR amplifications of the blaCTX-M gene were performed in a thermocycler (MJ Research) as follows: 95ºC for 5 min and 30 cycles of 1 min at 95ºC, 1 min at 60ºC and 1 min at 72ºC. A final extension step of 10 min at 72ºC was performed. Afterwards, more specific primers were used for each group of blaCTX-M genes, as described in Table II, using the same PCR conditions described above.

Detection of class 1 integron - The presence of class 1 integron was investigated using PCR amplification and sequencing of the 5' and 3' CS variable regions in the blaCTX-M-positive isolates. These primers (Table II) amplify the region between the 5'-CS-3'-CS conserved segments, generating products of variable sizes depending on the numbers and lengths of the inserted gene cassettes.

DNA sequencing and analysis of blaCTX-M and class 1 integron genes - All positive amplicons were purified with Wizard SV Gel and PCR Clean-up System (Promega, USA) and sequenced. DNA sequences were determined in both strands with an automated sequencer ABI Prism 3700 (Applied Biosystems), using the same primers as for the PCR (Table II). The sequencing was partial for the integron variable regions. The nucleotide sequences and deduced protein sequences were analysed by the programs BLAST ( and Clustal W of the European Bioinformatics Institute ( /). The sequences of the blaCTX-M-2 and blaCTX-M-28 genes and class 1 integron genes (blaOXA-2, aadA1 and dfr22) were deposited in the GenBank Database under the following accession: FJ973565, FJ973566, FJ973567, FJ973568, FJ973570, FJ973571, FJ973572, FJ985262, FJ976897, FJ976898.



Twenty-eight clinical isolates of K. pneumoniae from the Brazilian Northeast Region (Recife, PE) that exhibited resistance to extended-spectrum cephalosporins or aztreonam were also resistant to ampicillin and amoxicillin and showed a high rate of resistance to amoxicillin-clavulanate, chloramphenicol, tetracycline and cefotaxime. No isolate exhibited resistance to amikacin, imipenem or meropenem (Table I). Three blaCTX-M-positive isolates were more resistant to cefotaxime than to ceftazidime and aztreonam (Table III).

The results of the PCR with consensus primers for the 28 isolates of K. pneumoniae showed that seven were positive for the presence of blaCTX-M genes. The PCR results with more specific primers for each group of the blaCTX-M genes demonstrated that six isolates were positive for the group blaCTX-M-2 and one isolate was positive for blaCTX-M-1. None of the isolates were positive for the groups blaCTX-M-8, blaCTX-M-9 or blaCTX-M-25. The entire coding sequences of the seven blaCTX-M genes were subsequently sequenced with the specific primers for groups blaCTX-M-2 and blaCTX-M-1. The analysis of the nucleotide sequences and deduced protein sequences with BLAST and Clustal W showed that six isolates of K. pneumoniae harboured blaCTX-M-2 genes and one isolate had blaCTX-M-28 (Table III).

The investigation of the class 1 integron in the seven blaCTX-M-positive isolates of K. pneumoniae by PCR revealed that they carried the following structure. Two isolates (K11P and K18P) had two intregons each, with sizes ranging from 750-2000 bp, while the other five positive isolates amplified only one class 1 integron with sizes ranging from 1800-2000 bp (Table III). Analysis of the partial sequence of integrons 5'-3' CS variable regions in three isolates revealed that one isolate contained the aadA1 gene cassette, one isolate had blaOXA-2, which confers resistance to aminoglycosides and ?-lactams, respectively and the isolate K11P harboured a gene cassette (dfr22) coding for trimethoprim resistance (Table III). It is very likely that the integrons variable regions with a size of 1800 bp and 2000 bp harboured more than one gene cassette.



The blaCTX-M genes have been increasingly detected worldwide, but this is the first study on the occurrence of blaCTX-M in the Brazilian Northeast. The data presented in this work demonstrate the presence of blaCTX-M-2 and blaCTX-M -28 genes in K. pneumoniae from Recife, Brazil and show that blaCTX-M-2 genes were present in K. pneumoniae from 1998 until at least 2008.

Recent studies in the Brazilian Southeast Region have demonstrated the presence of blaCTX-M-2 and blaCTX-M-59 genes in K. pneumoniae (Do Carmo et al. 2008, Garcia et al. 2008). Clinical isolates of Proteus mirabilis, C. amalonaticus, E. cloacae and E. aerogenes collected from hospitals in Rio de Janeiro (Brazil) also demonstrated blaCTX-M-2 (Bonnet et al. 2000, Bonnet et al. 2001). In South America, the blaCTX-M-2 appears to be dominant in Argentina (Quinteros et al. 2003). Studies carried out in Europe have shown that the occurrence of the blaCTX-M-2 group is rare. However, there is a high prevalence of group 1, predominantly blaCTX-M-15, in Europe (Livermore et al. 2007).

In the present study, we also report the first blaCTX-M-28 gene found in Brazil and, to our knowledge, the first isolation of this gene in South America. blaCTX-M-28 was first described in 2003, in clinical isolates of E. coli in France (GenBank accession AJ549244.1) and recently it was also detected in China (Yu et al. 2007) and India (Kingsley & Verghese 2008) in K. pneumoniae and E. coli nosocomial isolates, respectively. Thus, blaCTX-M-28, a gene that was detected mainly in Asia is also present in Brazil.

Most blaCTX-M positive isolates exhibited resistance to non-?-lactams antibiotics, mainly streptomycin, sulphonamides and trimethoprim. Among Enterobacteriaceae, these resistances were commonly associated with a few types of integrons, mainly class 1 and 2 (Bissonnette & Roy 1992, Partridge & Hall 2003). In this study, two isolates that carried the gene blaCTX-M-2 (K16R and K18P) also contained class 1 integrons with the gene cassettes aadA1 or blaOXA-2, which encode the enzymes aminoglycoside-3´-adenyltransferase and oxacillinase type 2, respectively. In Brazil, this is the first report of the occurrence of blaOXA-2 in K. pneumoniae. This gene has also been described in Shigella spp isolates from Brazil (Peirano et al. 2005). The isolate K11P, which contained the gene blaCTX-M-28, also harboured the gene cassette dfr22 that encodes resistance to trimethoprim. This gene showed 100% identity with the gene dfr22 described in a class 1 integron of K. pneumoniae originating from São Paulo, Brazil, in 2008 (GenBank accession EU622041). The different sizes and gene cassettes inserted between the conserved segment regions of the strains studied demonstrate the variable nature of these structures.

In conclusion, the data presented herein illustrate the complexity and extent of the spread of blaCTX-M genes in K. pneumoniae and exemplify the diverse mode of spreading of this emerging resistance mechanism all over the world.



To Dr. Vera Magalhães and Dr. Maria Amélia V Maciel, for supplying some isolates.



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Received 6 August 2009
Accepted 3 February 2010
Financial support: Spanish Ministry of Science and Innovation (BFU2006-04574) (to JA), Banco Santander Central Hispano, Fundación Carolina (to ACSL), CBMSO, Fundación Ramon Areces, FACEPE

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