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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 Kluyvera georgiana and Kluyvera ascorbata, 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 (Table1)1) and trimethoprim-sulfamethoxazole (TMP-SMX) (by disc diffusion). Conjugation to Escherichia coli 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 (Table1)1) by a class 1 integron harboring dfrA17 and aadA5 gene cassettes (previously reported individually in different Kluyvera intermedia 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. (Fig.1)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 Table1,1, 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 Kluyvera georgiana (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. (Fig.1).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.
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.
Published ahead of print on 26 April 2010.