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LETTERS TO THE EDITOR

Novel Chromosome-Encoded CTX-M-78 β-Lactamase from a Kluyvera georgiana Clinical Isolate as a Putative Origin of CTX-M-25 Subgroup

María Margarita Rodríguez, Pablo Power, Helio Sader, Moreno Galleni, Gabriel Gutkind
María Margarita Rodríguez
Cátedra de Microbiología Facultad de Farmacia y Bioquímica Universidad de Buenos Aires Buenos Aires, Argentina
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Pablo Power
Cátedra de Microbiología Facultad de Farmacia y Bioquímica Universidad de Buenos Aires Buenos Aires, Argentina
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Helio Sader
JMI Laboratories North Liberty, Iowa
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Moreno Galleni
Macromolécules Biologiques Centre d'Ingénierie des Protéines Institut de Chimie Université de Liège Liège, Belgium
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Gabriel Gutkind
Cátedra de Microbiología Facultad de Farmacia y Bioquímica Universidad de Buenos Aires Junin 954 Buenos Aires 1113, Argentina
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  • For correspondence: ggutkind@ffyb.uba.ar
DOI: 10.1128/AAC.01615-09
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While TEM- and SHV-derived extended-spectrum β-lactamases (ESBLs) arose by point mutations in genes encoding broad-spectrum variants that were extensively disseminated into mobile elements, CTX-M β-lactamases are derived from natural (chromosomal) counterparts that already have oxyimino-cephalosporinase activity (11). To date, the CTX-M/KLU β-lactamases account for nearly 110 representatives clustered in five main groups (http://www.lahey.org/studies/webt.asp ): CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. A hypothetical chromosomal counterpart for the CTX-M-25 subfamily is still missing.

Kluyvera georgiana 14751 was isolated from a bloodstream infection in a 62-year-old male patient through the SENTRY Antimicrobial Surveillance Program in Louisville, KY, on 2 December 2002. Biochemical classification, ambiguous between Kluyverageorgiana and Kluyveraascorbata, was resolved by 16S rRNA gene sequencing (GenBank accession no. AM933755), displaying 99.7% identity with the sole available sequence from K. georgiana ATCC 51603 (GenBank accession no. AF047186). Identity to all deposited K. ascorbata sequences ranged from 97.4 to 98.5% (6).

The strain was resistant to ampicillin, gentamicin, nalidixic acid (Table 1) and trimethoprim-sulfamethoxazole (TMP-SMX) (by disc diffusion). Conjugation to Escherichiacoli CAG12177 resulted in E. coli CK10 harboring an IncFII group conjugative plasmid of ca. 30 kb (pTC10; partial sequence deposited in EMBL under accession no. FN568351), responsible for TMP-SMX and aminoglycoside resistance (Table 1) by a class 1 integron harboring dfrA17 and aadA5 gene cassettes (previously reported individually in different Kluyveraintermedia isolates; EMBL accession no. EU523051 and EU523052). Upstream of the integron we found a blaTEM-1b gene followed by a region, including the Tn3-tnpR, the first 91 bp of Tn3-tnpA, and an IS26 which is also found in plasmids carrying some blaCTX-M genes. However, no CTX-M could be recovered from the transferred plasmid.

Partially EcoRI-digested chromosomal DNA from K. georgiana 14751 was cloned in a pK19 vector (Kanr) and transformed into E. coli Top10F′ (E. coli TKE14751-1KA2), yielding pTKE-1KA2 with an 8-kb insert (Fig. 1) harboring a bla gene (876 bp) which encodes a novel CTX-M-78 (EMBL accession no. AM982522) closely related to CTX-M-39 (96.2% amino acid identity) (1) and clustered in the CTX-M-25 subgroup. As seen in Table 1, the MIC increases for cefotaxime, while that of ceftazidime is almost unaffected, correlating with the expected (and experimental [data not shown]) kinetics for most CTX-M enzymes.

As in other chromosome-encoded blaCTX-M/KLU, a 1,227-bp att-like gene encoding a putative aspartate aminotransferase is located upstream, with at least 96% identity with other entries from Kluyvera. Downstream, an orf3-like gene similar to a putative ttrR response regulator from Kluyverageorgiana (5) and similar to the orf3 version from the complex class 1 integrons associated with blaCTX-M-2 (found as a fusion gene orf3::qacEΔ1) (7), and an orf4-like gene encoding a putative autotransporter in Kluyvera ascorbata (2), were also found. In contrast to other kluyveras, in which sequences similar to IRr-ISEcp1 were found downstream of blaCTX-M/KLU (8), we did not observe any sequence that could serve as putative right inverted repeats (IRrs) there (Fig. 1). A 35-bp sequence seems to represent the boundary between the chromosome-derived information and the plasmid-acquired information. In blaCTX-M-25 and blaCTX-M-26 (GenBank accession no. AF518567 and AY455830, respectively) (4), the immediate upstream region is occupied by ISEcp1, also associated with other blaCTX-M genes (3). A putative 5-bp target site for ISEcp1B (AATAC) was found immediately upstream of the 35-bp sequence in the chromosomal DNA from K. georgiana 14751.

CTX-M-78 possesses high similarity with members of the CTX-M-25 subgroup, making this enzyme the closest representative to be considered one of the probable progenitors of the subfamily.

FIG. 1.
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FIG. 1.

Schematic representation of the insert of pTKE-1KA2 from Kluyvera georgiana 14751 (top) and comparison with available sequences harboring blaCTX-M-25 (middle) and blaCTX-M-26 (bottom).

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TABLE 1.

MICs of antibiotics for Kluyvera georgiana 14751, the recipient strain, and the derived recombinant clone

ACKNOWLEDGMENTS

This work was supported by grants from UBACYT and ANPCyT to G.G. and the αLFA program of the European Union to M.G. Doctoral fellowships from UBA and αLFA to M. M. Rodríguez are also acknowledged. P.P. and G.G. are members of “Carrera del Investigador Científico” CONICET.

We thank I. Thamm (Centre d'Ingénierie des Protéines, Université de Liège, Belgium) for technical assistance, C. Vay and M. Almuzara (University of Buenos Aires) for their expert advice in biochemical taxonomy, and J. W. Snyder (University of Louisville, Louisville, KY) and R. N. Jones (JMI Laboratories, North Liberty, IA) for kindly providing the bacterial strain for study.

  • Copyright © 2010 American Society for Microbiology

REFERENCES

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    Chmelnitsky, I., Y. Carmeli, A. Leavitt, M. J. Schwaber, and S. Navon-Venezia. 2005. CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum β-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel. Antimicrob. Agents Chemother.49:4745-4750.
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    Humeniuk, C., G. Arlet, V. Gautier, P. Grimont, R. Labia, and A. Philippon. 2002. β-Lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types. Antimicrob. Agents Chemother.46:3045-3049.
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    Lartigue, M. F., L. Poirel, D. Aubert, and P. Nordmann. 2006. In vitro analysis of ISEcp1B-mediated mobilization of naturally occurring β-lactamase gene blaCTX-M of Kluyvera ascorbata. Antimicrob. Agents Chemother.50:1282-1286.
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    Olson, A. B., M. Silverman, D. A. Boyd, A. McGeer, B. M. Willey, V. Pong-Porter, N. Daneman, and M. R. Mulvey. 2005. Identification of a progenitor of the CTX-M-9 group of extended-spectrum β-lactamases from Kluyvera georgiana isolated in Guyana. Antimicrob. Agents Chemother.49:2112-2115.
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    Pavan, M. E., R. J. Franco, J. M. Rodriguez, P. Gadaleta, S. L. Abbott, J. M. Janda, and J. Zorzopulos. 2005. Phylogenetic relationships of the genus Kluyvera: transfer of Enterobacter intermedius Izard et al. 1980 to the genus Kluyvera as Kluyvera intermedia comb. nov. and reclassification of Kluyvera cochleae as a later synonym of K. intermedia. Int. J. Syst. Evol. Microbiol.55:437-442.
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    Power, P., M. Galleni, J. Di Conza, J. A. Ayala, and G. Gutkind. 2005. Description of In116, the first blaCTX-M-2-containing complex class 1 integron found in Morganella morganii isolates from Buenos Aires, Argentina. J. Antimicrob. Chemother.55:461-465.
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    Rodriguez, M. M., P. Power, M. Radice, C. Vay, A. Famiglietti, M. Galleni, J. A. Ayala, and G. Gutkind. 2004. Chromosome-encoded CTX-M-3 from Kluyvera ascorbata: a possible origin of plasmid-borne CTX-M-1-derived cefotaximases. Antimicrob. Agents Chemother.48:4895-4897.
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Novel Chromosome-Encoded CTX-M-78 β-Lactamase from a Kluyvera georgiana Clinical Isolate as a Putative Origin of CTX-M-25 Subgroup
María Margarita Rodríguez, Pablo Power, Helio Sader, Moreno Galleni, Gabriel Gutkind
Antimicrobial Agents and Chemotherapy Jun 2010, 54 (7) 3070-3071; DOI: 10.1128/AAC.01615-09

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Novel Chromosome-Encoded CTX-M-78 β-Lactamase from a Kluyvera georgiana Clinical Isolate as a Putative Origin of CTX-M-25 Subgroup
María Margarita Rodríguez, Pablo Power, Helio Sader, Moreno Galleni, Gabriel Gutkind
Antimicrobial Agents and Chemotherapy Jun 2010, 54 (7) 3070-3071; DOI: 10.1128/AAC.01615-09
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KEYWORDS

Chromosomes, Bacterial
Kluyvera
beta-lactamases

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