Chromosome-Encoded Ambler Class A -Lactamase of Kluyvera georgiana, a Probable Progenitor of a Subgroup of CTX-M Extended-Spectrum -Lactamases

Plasmid-mediated CTX-M-type extended-spectrum -lactamases (ESBLs) are being increasingly reported worldwide in enterobacterial isolates (20). They may be grouped according to their amino acid identity in four clusters: CTX-M-1 (CTXM-1, -3, -10, -11, -12, -15, and -22), CTX-M-2 (CTX-M-2, -4, -5, -6, -7, and -20 and Toho-1), CTX-M-8, and CTX-M-9 (CTXM-9, -13, -14/-18, -16, -19, and -21 and Toho-2) (2–4, 6, 10, 11, 13, 15–18, 20) (GenBank accession no. AY080894). These -lactamases do not hydrolyze ceftazidime at a high level except for CTX-M-15, CTX-M-16, and CTX-M-19 (2, 10, 15). The natural producers of CTX-M enzymes have been identified in rare cases. The chromosome-encoded -lactamases KLUC-1 from Kluyvera cryocrescens (5) and KLUA-1 from K. ascorbata (8) share 86 and 99% amino acid identity with the plasmid-borne CTX-M-1 and CTX-M-2 subgroups, respectively. To elucidate further natural producers of CTX-M-type -lactamase genes, we studied the -lactamase content of K. georgiana, which belongs to the genus Kluyvera, which includes such species as K. cochleae, K. ascorbata, and K. cryocrescens (12). Kluyvera spp. have been rarely reported from clinical specimens (19). Detailed susceptibility of K. georgiana to -lactams is not known and a preliminary disk diffusion antibiogram indicated that reference strain K. georgiana CUETM 4246-74 displayed a weakly expressed clavulanic acid-inhibited penicillinase phenotype (data not shown). Cloning and sequencing identified a chromosomally encoded Ambler class A enzyme that shared 99% identity with CTX-M-8, the single representative of one of the four CTX-M clusters. Bacterial strains and plasmid analysis. K. georgiana reference strain CUETM 4246-74 (DSM 9408) has been identified previously (12). The Escherichia coli DH10B reference strain was used for cloning experiments. Plasmid DNA extractions, performed according to several methods as described previously (10, 14), failed to identify a plasmid. Cloning and sequence analysis of the -lactamase gene from K. georgiana. Whole-cell DNA of K. georgiana CUETM 4246-74 was extracted as described previously (14), digested with Sau3AI restriction enzyme, and ligated into the BamHI site of pBK-CMV phagemid (14). Ten E. coli DH10B recombinant clones were obtained after selection onto Mueller-Hinton plates containing 30 g of kanamycin and 50 g of amoxicillin per ml. The recombinant plasmid (pKG-2) that had the shortest insert (1,781 bp) was sequenced as described previously (14). An open reading frame of 876 bp was identified. Within the deduced protein of this open reading frame (291 amino acids), named KLUG-1 (for “Kluyvera georgiana”), characteristic elements of Ambler class A -lactamases were identified (Fig. 1) (9). Computer analysis indicated a putative cleavage site for the mature protein located at the same position compared to that of -lactamase KLUC-1 of K. cryocrescens (Fig. 1) (9). Isoelectric focusing analysis, performed as previously reported (14), showed that homogenates of K. georgiana 4246-74 and of E. coli DH10B (pKG-2) gave a single and identical -lactamase with a pI value of 7.6. The -lactamase KLUG-1 shared 99% amino acid identity with the plasmid-mediated CTX-M-8 enzyme identified in Brazilian isolates of Citrobacter amalonaticus, Enterobacter cloacae, and Enterobacter aerogenes (3) (Fig. 1). Only one amino acid change (290) was noted between CTX-M-8 and KLUG-1 sequences that was located in the C termini. In addition, two amino acid changes were identified in the leader peptide sequence (Fig. 1). KLUG-1 shared 83 and 77% amino acid identity with the chromosomally encoded -lactamases KLUA-1 from K. ascorbata (8) and KLUC-1 from K. cryocrescens (5). Just upstream of blaKLUG-1, a 533-bp DNA fragment was identified from recombinant plasmid pKG-2. Within this fragment, the 389-bp 5 -end located sequence shared 87% identity with DNA sequence upstream of blaKLUA-1 (8), whereas the 336-bp 5 -end located sequence shared 88% identity with DNA sequence upstream of blaKLUC-1 (data not shown) (5). This result indicated similarities in the DNA * Corresponding author. Mailing address: Service de BactériologieVirologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-145-21-63-40. E-mail: [email protected].