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. {type:entrez-nucleotide,attrs:{text:AM933755,term_id:193227311,term_text:AM933755}}AM933755), displaying 99.7% identity with the sole available sequence from K. georgiana ATCC 51603 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF047186,term_id:3273703,term_text:AF047186}}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 {type:entrez-protein,attrs:{text:CAG12177,term_id:47220029,term_text:CAG12177}}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. {type:entrez-nucleotide,attrs:{text:FN568351,term_id:290964838,term_text:FN568351}}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. {type:entrez-nucleotide,attrs:{text:EU523051,term_id:171188351,term_text:EU523051}}EU523051 and {type:entrez-nucleotide,attrs:{text:EU523052,term_id:171188353,term_text:EU523052}}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. TABLE 1. MICs of antibiotics for Kluyvera georgiana 14751, the recipient strain, and the derived recombinant clone 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. {type:entrez-nucleotide,attrs:{text:AM982522,term_id:209413817,term_text:AM982522}}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. 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). 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. {type:entrez-nucleotide,attrs:{text:AF518567,term_id:37789822,term_text:AF518567}}AF518567 and {type:entrez-nucleotide,attrs:{text:AY455830,term_id:38373216,term_text:AY455830}}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.
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Sep 6, 2017
J Jneid + 34
Open Access
