Dissemination of blaIMP-1-carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23

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Diagnostic Microbiology and Infectious Disease 63 (2009) 87 – 91 www.elsevier.com/locate/diagmicrobio

Dissemination of blaIMP-1-carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23 Andréia P. Penteadoa , Mariana Castanheiraa,⁎, Antonio C.C. Pignataria , Thaís Guimarãesb , Elsa M. Mamizukac , Ana C. Galesa a

Laboratório ALERTA, Division of Infectious Diseases, Universidade Federal de São Paulo, São Paulo 04039-032, Brazil b Division of Infectious Diseases, Hospital Servidor Público Estadual, São Paulo 04039-901, Brazil c Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo 05508-900, Brazil Received 27 June 2008; accepted 17 September 2008

Abstract The genetic context of the blaIMP-1 gene was evaluated in 9 Klebsiella pneumoniae isolates recovered from 2 hospitals in São Paulo, Brazil. All isolates harbored a copy of In86 carrying blaIMP-1, aac(6′)-31, and aadA1. Eight strains from the same hospital also carried another class 1 integron harboring a new trimethoprim resistance gene (dfr23) that was chromosomally embedded. In86 was likely to be in a 30-kb nontransferable plasmid and was flanked upstream by a sequence identical to one identified in an IMP-1–producing Pseudomonas putida isolate. The blaIMP-1-carrying integron In86 was recently reported from nonfermentative bacilli isolated in São Paulo. These isolates appear to be the source of this integron now acquired by K. pneumoniae strains from different hospitals in the same city. Metallo-β-lactamase production is still rare among Enterobacteriaceae isolates in Brazil, but the acquisition of genetic structures carrying these mobile resistance determinants is worrisome and could lead to an increase in the prevalence of these phenotypes of resistance. © 2008 Elsevier Inc. All rights reserved. Keywords: Klebsiella pneumoniae; Metallo-β-lactamases; Trimethoprim resistance; Class 1 integrons

1. Introduction Carbapenems have become the most important therapeutic option for treatment of serious infections caused by extendedspectrum β-lactamases–producing Klebsiella pneumoniae. These antimicrobial agents rapidly cross the bacterial membrane and are stable against most β-lactamases. Carbapenem resistance among K. pneumoniae isolates is rare and basically restricted to determine geographic areas (Bratu et al., 2005; Koh et al., 1999; Livermore and Woodford, 2000, 2006; Woodford et al., 2004; Yan et al., 2001); however, an increase in this resistance phenotype has been observed, and it has been associated with the increased use of carbapenem compounds (Livermore and Woodford, 2000, 2006).

⁎ Corresponding author. JMI Laboratories, North Liberty, Iowa 52317, USA. Tel.: +1-319-665-3370; fax: +1-319-6653371. E-mail address: [email protected] (M. Castanheira). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.09.013

Resistance to carbapenems in Enterobacteriaceae, including K. pneumoniae, can be caused by overproduction of Amp-C β-lactamases associated with loss of outer membrane porins and/or overexpression of efflux pumps (Poirel et al., 2004) or by production of carbapenem-hydrolyzing enzymes. These carbapenemases can be divided in metalloβ-lactamases (MβLs; Ambler class B) and serine carbapenemases (class A or Bush class 2f) according to the functional requirements and structure of their active site (Queenan and Bush, 2007; Walsh, 2005). The genes encoding most MβLs are part of the gene cassettes residing on class 1 integrons that usually carry additional genes encoding resistance to other antimicrobial classes (Poirel and Nordmann, 2002). Plasmids and transposons harboring integrons can readily be acquired by Gramnegative pathogens facilitating the dissemination of these potent resistance mechanisms and, additionally, reducing significantly the treatment options for infections caused by MβL-producing isolates. Integrons also play an important role in the dissemination of trimethoprim resistance encoding

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genes (White and Rawlinson, 2001) because many of the dihydrofolate reductase (dfr)-encoding genes occur as gene cassettes in class 1 integrons. In addition, most class 1 integrons carry a sulfonamide resistance gene (sul1) that can be selected by the use of trimethoprim/sulfamethoxazole (TMP–SMZ), which exerts strong selective pressure for coresistance (Adrian et al., 2000). This study was initiated to evaluate the genetic content of class 1 integrons carried by IMP-1–producing K. pneumoniae isolated in the city of São Paulo, Brazil. In addition, the genetic relatedness of such isolates was assessed. 2. Materials and methods 2.1. Bacterial strains Nine IMP-1–producing K. pneumoniae isolates collected in 2 hospital of São Paulo, Brazil, from December 2003 and January 2004 were evaluated. The source of these isolates was summarized in Table 1. One K. pneumoniae strain included in this study was previously reported as the 1st IMP-1–producing Enterobacteriaceae isolate in Brazil (Lincopan et al., 2005). All isolates were resistant to ceftazidime (MIC, ≥256 μg/mL), imipenem (MIC, ≥32 μg/mL), and meropenem (MIC, ≥32 μg/mL) by the reference agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI, 2006). 2.2. Pulsed field gel electrophoresis (PFGE) Molecular typing was performed by PFGE. Genomic DNA was prepared in agarose blocks and digested with SpeI (New England, Beverly, MA). Electrophoresis was per-

Table 1 Characteristics and results obtained in this study for the 9 IMP-1–producing K. pneumoniae clinical isolates collected in the city of São Paulo, Brazil Isolate

Date of isolation

A13304 December 2003 A13305 December 2003 A13306 December 2003 A13307 January 2004 A13308 January 2004 A13309 January 2004 A13310 January 2004 A13311 January 2004 A11775 April 2003

Medical center

Source PFGE Integron type amplicons

HSPE

Blood A culture Urine A

800 bp, 4 kb Positive

Blood culture Blood culture Blood culture Urine

A

800 bp, 4 kb Positive

A

800 bp, 4 kb Positive

A

800 bp, 4 kb Positive

A

800 bp, 4 kb Positive

Blood A culture HSPE Blood A culture HU-USP Blood A culture

800 bp, 4 kb Positive

HSPE HSPE HSPE HSPE HSPE HSPE

Detection of dfr23

800 bp, 4 kb Positive

800 bp, 4 kb Positive 4 kb

Negative

HU-USP = Hospital Universitário da Faculdade de Medicina da Universidade de São Paulo, Brazil.

formed on the CHEF-DR III (BioRad, Richmond, CA) with the following conditions: 0.5× TBE, 1% agarose, 13 °C , 200 V, for 24 h with the switch time ramped from 5 to 90 s. Interpretation of the results was performed as previously described (Tenover et al., 1995). 2.3. Polymerase chain reaction (PCR) experiments and DNA sequencing Primers targeting the 5′ conserved sequence (CS) and the 3′ CS of class 1 integrons were used together or in combination with blaIMP-1 primers to amplify these genetic structures from IMP-1–producing K. pneumoniae isolates. The region upstream of the blaIMP-1-carrying integron was amplified from strain A11775 (carrying a single integron) by PCR using a degenerate primer approach using primers anchored in the integrase gene in combination with random primers (Mendes et al., 2007). Amplicons were sequenced on both strands using an ABI Prism 377 system (Applied Biosystems, Foster City, CA). The nucleotide sequences and deduced amino acid sequences were analyzed using the Lasergene software package (DNASTAR, Madison, WI). Sequences were compared with others available over the internet (http://www.ncbi.nlm.nih.gov/blast/). 2.4. Cloning of the dfr gene Amplicons containing the dfr gene were cloned into TOPO plasmid vector (Invitrogen, Carlsbad, CA) and transformed in Escherichia coli TOP10 cells according to the manufacturer's instructions. Colonies were selected in plates containing kanamycin 50 μg/mL, and the presence and orientation of the insert were confirmed by sequencing. The MIC for trimethoprim (Sigma-Aldrich, Steinheim, Germany) was determined by agar dilution for the E. coli TOP10 carrying pTOPOdfr23 plasmid and for the host strain carrying the same plasmid without an insert, according to the CLSI (2006) guidelines. 2.5. Plasmid analysis, hybridization, and transference of resistance genes Plasmid DNA was extracted using the Plasmid DNA Midi Kit (Qiagen, Hilden, Germany), and molecular weight was determined by comparison with strains carrying multiple plasmids with known molecular weights. Bacterial DNA was obtained with the cetyl trimethylammonium bromide method (Sambrook et al., 2001). Total DNA was treated with HindIII and resolved in agarose gels along with plasmid and undigested chromosomal DNA. DNA transference to a nylon membrane was performed by Southern hybridizations as described by Sambrook et al. (2001) Probes for blaIMP-1 and dfr23, generated by PCR, were labeled, and membranes were hybridized and revealed with nonradioactive ECL Direct Nucleic Acid Labeling and Detection System (GE Healthcare, Piscataway, NJ). E. coli J53 streptomycin-resistant derivative was used as recipient for conjugation experiments. Logarithmic phase

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of donor and the recipient strains were grown in trypticase soy broth and were mixed and incubated at 37 °C overnight. Transconjugants were selected on MacConkey agar supplemented with streptomycin (300 μg/mL) and ceftazidime (30 μg/mL). Transfer of the β-lactam resistance markers from strain A13304 into E. coli DH5α was also attempted with electroporation using Bio-Rad Gene Apparatus (Bio-Rad, Richmond, CA) set at 2.5 kV, 25 μF, and 400 Ω. Selection for transformants was carried out on nutrient agar plates containing 5 μg/mL ceftazidime. 2.6. Nucleotide sequence accession number The nucleotide sequence of the dfr23 gene reported in this study has been submitted to GenBank nucleotide database and assigned under accession number AJ968952. 3. Results 3.1. Clonality and class 1 integron analysis All IMP-1–producing K. pneumoniae isolates showed a single PFGE pattern, indicating clonal dissemination. Primers targeting specific class 1 structures revealed that all isolates harbored a 4-kb class 1 integron, but the 8 strains recovered in the Hospital do Servidor Público Estadual (HSPE, São Paulo, Brazil) also presented a 2nd amplicon of 800 bp, indicating the presence of an additional integron. DNA sequencing of amplicons obtained by anchoring blaIMP-1 primers with CS primers revealed that the MβL gene was carried in the 1st position of a class 1 integron that also harbored 2 aminoglycoside-modifying genes aac(6′)-31 and aadA1 (Fig. 1A). This integron, named In86, was previously described in Pseudomonas spp. and Acinetobacter spp. from another hospital of São Paulo (Mendes et al., 2007).

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The integron showing an 800-bp amplicon harbored a single-gene cassette and was named In87. This integron revealed all key genetic components found in class 1 integrons (Fig. 1B) and carried a new trimethoprim resistance gene, designated dfr23. 3.2. Characterization of the trimethoprim-resistant gene dfr23 encoded a protein of 165 amino acids and showed the length and the 12 N-termini conserved residues compatible with the DFR type A proteins (157–187 amino acids) (White and Rawlinson, 2001). DFR23 was closely related to DFR22 (99% homology) showing only 1 amino acid substitution in the position 121 (Ser-121→Tyr). dfr22 was characterized in urinary tract isolates of Gram-negative bacteria from Sweden (Grape et al., 2005). The trimethoprim MIC for the E. coli carrying pTOPOdfr23 was at least 16-fold higher than the E. coli host strain carrying only the TOPO plasmid (MICs, 4 and ≤0.12 μg/mL, respectively), confirming that this gene confers resistance to trimethoprim. 3.3. Genetic support of blaIMP-1 and dfr23 All IMP-producing K. pneumoniae harbored a unique plasmid estimated to be 30-kb in size. Hybridization showed that all isolates carried the MβL gene in this plasmid. Furthermore, hybridization with dfr23 probe showed that the trimethoprim resistance gene was carried on the chromosome of the 8 isolates from the HSPE hospital. Repeated attempts failed to transfer the blaIMP-1 gene from the index strain A13304 to E. coli recipient cell by conjugation or electroporation. The 1-kb DNA sequence located upstream of the blaIMP-1 in K. pneumoniae strain A11775 contained the integrase gene bound to a Tn402-like 25 inverted repeat (IRi);

Fig. 1. (A) Schematic representation of the class 1 integron In86 (containing blaIMP-1, aac(6′)-31 and aadA1) found in K. pneumoniae isolates from this study and in Acinetobacter baumannii and P. putida from another hospital in the city of São Paulo, Brazil. The region upstream of the integron containing Tn402-like 25 IRi and the DNA sequence (shaded element) showing the active domains of UvrB-like proteins is also represented. (B) Schematic representation of the new class 1 integron In87 harboring a single-gene cassette, dfr23. The horizontal arrows indicate the gene cassette and their respective translation orientation.

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however, 400 bp were 100% identical to the region flanking In86 unique to an IMP-1–producing Pseudomonas putida previously described (Mendes et al., 2007). The putative protein derived from this DNA sequence showed active domains of UvrB, a DNA excision repair enzyme found in many Gram-negative rods. The protein fragment showed 97% homology with the ATP-dependent UvrAC, an excinuclease UvrB-like protein from Herminiimonas arsenicoxydans (GenBank accession number CAL62189). The presence of the same structure was confirmed in the remaining isolates by PCR with primers annealing in the DNA fragment anchored in blaIMP-1. 4. Discussion IMP-1–producing isolates have been found in Brazil in clinically important pathogens, such as Pseudomonas spp. and Acinetobacter spp. since the early 2000s. In 2003, the 1st MβL was reported in an Enterobacteriaceae strain in Latin America. This strain, harboring blaIMP-1, was a multidrug-resistant K. pneumoniae isolated in the city of São Paulo (Lincopan et al., 2005). In 2003 and in the next year, other 8 IMP-1–producing K. pneumoniae isolates were identified in another hospital in São Paulo. The genetic environment of blaIMP-1 and the content of class 1 integrons harbored by these isolates were analyzed in the present study. All IMP-1–producing K. pneumoniae strains carried In86, recently reported in IMP-1–producing Acinetobacter spp. and P. putida also isolated in the city of São Paulo (Mendes et al., 2007). These Acinetobacter spp. and P. putida strains were collected in a 600-bed teaching hospital located only a few blocks away from HSPE, where 8 of the 9 K. pneumoniae strains were recovered. The DNA structure found upstream of the In86 in the K. pneumoniae strains was identical to the unique sequence flanking this integron in the P. putida strain. However, in the IMP-1– producing K. pneumoniae isolates, In86 was carried in a plasmid, whereas in the P. putida isolate, this integron seemed to be chromosomally located (Mendes et al., 2007). These findings suggest that In86 was recently mobilized into a plasmid that disseminated into different species in at least 3 hospitals in the city of São Paulo. The DNA sequence observed upstream of the In86 from the K. pneumoniae strains and P. putida previously described showed low homology with other DNA sequences in the GenBank database. However, comparisons of the protein encoded by this sequence showed similar features with a gene from an environmental Gram-negative rod from the order Burkholderiales that is metalloresistant and found in arsenic-rich environments (Muller et al., 2006). This relationship needs further investigation and corroborates for the hypothesis that MβL genes are derived from environmental species (Castanheira et al., 2007). Interestingly, the 9 blaIMP-1-carrying K. pneumoniae isolates belonged to a single molecular type, indicating

interhospital clonal spread. However, all strains from 1 hospital carried a chromosomally located dfr23, whereas the strain isolated from the other hospital did not, suggesting that although these strains are epidemiologically related, genetic events occurred locally for the acquisition and chromosomal mobilization of In87 carrying dfr23. High rates of resistance to TMP–SMZ have been observed in Brazil, and the selective pressure caused by 1 antimicrobial agent can simultaneously select genetic elements such as class 1 integrons that carry other genes mediating resistance to other compounds. The high consumption of TMP–SMZ can explain the high incidence of trimethoprim gene cassettes inserted in class 1 integrons, which normally harbor the sulfonamide resistance gene (sul1) in the 3′ CS region. Coselection of antibiotic resistance is possible among strains carrying integrons harboring several antibiotic resistance gene cassettes (Adrian et al., 2000); therefore, a simple reduction in the use of trimethoprim would probably not lead to a decrease in resistance to the antimicrobial agent in the near future. Metallo-β-lactamase production is still rare in Enterobacteriaceae strains, but Pseudomonas and Acinetobacter spp. seems to constitute a reservoir of MβL genes, and it is likely that the horizontal transfer of these mobile genes incorporated into class 1 integrons has occurred and disseminated to Enterobacteriaceae as observed in other countries (Walsh et al., 2005). In addition, the acquisition of MβL among the Enterobacteriaceae family is of an alarming concern because it severely limits treatment options, resulting in the use of older and more toxic drugs, such as polymyxins. Acknowledgments This work was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo—research grant 04/ 15339-4. Ana C. Gales has been partially supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq process number: 307714/2006-3). References Adrian PV, Thomson CJ, Klugman KP, Amyes SG (2000) New gene cassettes for trimethoprim resistance, dfr13streptomycinaadA4, inserted on a class 1 integron. Antimicrob Agents Chemother 44:355–361. Bratu S, Mooty M, Nichani S, Landman D, Gullans C, Pettinato B, Karumudi U, Tolaney P, Quale J (2005) Emergence of KPC-possessing Klebsiella pneumoniaeepidemiology and recommendations for detection. Antimicrob Agents Chemother 49:3018–3020. Castanheira M, Sader HS, Jones RN, Debbia E, Picao RC, Gales AC (2007) In71, an Enterobacter cloacaeblaVIM-1-Pseudomonas aeruginosa isolates: a SENTRY Antimicrobial Surveillance Program report. Microb Drug Resist 13:130–134. Clinical and Laboratory Standards Institute (CLSI) (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard, M7-A7, 7th ed. Wayne, PA: CLSI. Grape M, Farra A, Kronvall G, Sundstrom L (2005) Integrons and gene cassettes in clinical isolates of co-trimoxazole–resistant Gram-negative bacteria. Clin Microbiol Infect 11:185–192.

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