Identification of a Progenitor of the CTX-M-9 Group of Extended-Spectrum β-Lactamases from Kluyvera georgiana Isolated in Guyana

Abstract

Chromosomal β-lactamase genes (bla_KLUY) from six Kluyvera georgiana strains isolated in Guyana were cloned and expressed in Escherichia coli. KLUY-1 exhibited 100% amino acid identity with the extended-spectrum β-lactamase CTX-M-14. We also show that a 2.7-kb Kluyvera chromosomal region exhibits 99% nucleotide identity to a portion of In60 that includes bla_CTX-M-9.

Extended-spectrum β-lactamases (ESBLs) mainly belong to the Ambler class A family (6). CTX-M-type ESBLs, characterized by their preference for hydrolysis of cefotaxime (3, 4, 12), have been grouped into five clusters (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25) based on amino acid identities (5). Chromosomal β-lactamases from Kluyvera ascorbata (KLUA) (10), K. ascorbata strain 69 (bla_CTX-M-3) (19), Kluyvera georgiana DSM 9408 (KLUG-1) (18), and Kluyvera cryocrescens (KLUC-1) (8) have been identified. KLUA and KLUG-1 share greater than 99% amino acid identity with the CTX-M-2 and CTX-M-8 groups, respectively, and are thought to be the progenitors for these groups. K. ascorbata strain 69 is likely the progenitor for the CTX-M-1 group (19). KLUC-1 may be a progenitor for an as yet unidentified CTX-M group of ESBLs (5).

Bacterial strains.

Six strains of Kluyvera spp. labeled N03-0457 to N03-0462 were isolated from human rectal swabs taken from nonhospitalized patients in Guyana, South America, in 2002 or 2003. The strains were isolated as part of a study on the carriage of antibiotic-resistant organisms among healthy individuals in the absence of recognized antibiotic pressure (M. Silverman, personal communication). The strains were identified as Kluyvera spp. by Vitek. Biochemical analysis and 16S rRNA sequence analysis confirmed the strains as K. georgiana. All strains had ≥99.6% rRNA nucleotide identity and exhibited 99.5% identity with the rRNA gene of K. georgiana ATCC 51603 (accession no. AF04716) (data not shown).

Cloning and sequence analysis of β-lactamase genes.

Each K. georgiana strain was positive with the CTX-U1-CTX-U2 primer set designed to amplify a fragment from CTX-M-type genes (CTX-U1, 5′-ATGTGCAGYACCAGTAARGTKATGGC; CTX-U2, 5′-TGGGTRAARTARGTSACCAGAAYCAGCGG; K is G or T, R is A or G, S is G or C, and Y is C or T) (13). EcoRI-digested DNA was ligated into the EcoRI site of pACYC184 and transformed into Escherichia coli DH10B, and clones were selected on ampicillin. Ten clones screened for each Kluyvera strain all harbored plasmids with 4.1-kb inserts. The insert from one plasmid for each strain was sequenced (DNA Core Facility, National Microbiology Laboratory), homology searches were carried out using BLAST (1), and open reading frames (ORFs) were analyzed by ORFinder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).

The size of the EcoRI fragments were 4,102 bp in N03-0457 and N03-0461, 4,100 bp in N03-0458, 4,099 bp in N03-0459 and N03-0460, and 4,098 bp in N03-0462, with identities ranging from 98.4 to 100%. The N03-0457 and N03-0461 sequences were identical. The size differences were due to single nucleotide insertions and/or deletions found in intergenic regions (data not shown). Analysis revealed four ORFs (Fig. 1). The first, bla_KLUY, was 876 bp and coded for a deduced protein of 291 amino acids with the characteristic sequence elements of Ambler class A β-lactamases (Fig. 2) (11). The amino acid sequences shared 98.3 to 100% amino acid identity with known ESBLs from the CTX-M-9 group of enzymes and were termed KLUY-1 (N03-0458), KLUY-2 (N03-0457 and N03-0461), KLUY-3 (N03-0459 and N03-0460), and KLUY-4 (N03-0462). KLUY-1 showed 100% amino acid identity to CTX-M-14 and 84.5, 80.4, and 80.1% identity with KLUG-1, KLUC-1, and KLUA-1, respectively (15). The results suggest that the bla_KLUY gene from these K. georgiana strains is likely the progenitor of the CTX-M-9 group of ESBLs. Located 0.46 kb downstream of bla_KLUY is orf3 (209 amino acids), whose product shares 75% amino acid identity to ORF3 proteins coded for by genes found downstream of bla_KLUA and bla_KLUC (8, 10). The K. ascorbata CIP82.95T orf3 is located 0.43 kb downstream of bla_KLUA, whereas in K. cryocrescens orf3 is located 0.99 kb downstream of bla_KLUC. Downstream of orf3 was orf339, encoding a putative protein of 112 amino acids that had no significant homology to proteins in GenBank. In K. ascorbata CIP82.95T, orf4 is located 198 bp downstream of orf3. We identified a partial ORF 219 bp downstream of K. cryocrescens orf3 coding for an orf4-like protein showing 34% amino acid identity to K. ascorbata orf4, though this was not annotated in the original GenBank entry (Fig. 1). Located 54 to 55 bp downstream of orf339 on the opposite strand was orf477 (158 amino acids), whose product shares 86% identity to the K. ascorbata strain 69 Orf477, found downstream of the chromosomal bla_CTX-M-3, and to Orf1, found downstream of bla_CTX-M-3 in the Citrobacter freundii plasmid pCTX-M3 (accession no. AF550415). We also identified orf477 in the bla_KLUC-1-orf3 intergenic region, though this was not annotated in the GenBank entry. There is limited sequence available surrounding bla_KLUG-1, which shares 75 to 77% nucleotide identity with the bla_KLUY genes, with sequence divergence even more extensive in the flanking regions (data not shown). Thus, the K. georgiana “group” may encompasses multiple subspecies. Further analysis of the bla_KLUY region revealed that a 2.7-kb region shared 99% nucleotide identity to a region of the complex class 1 integron In60 harboring bla_CTX-M-9 (Fig. 1) (20). Examination of the Kluyvera sequence revealed a 10-nucleotide direct repeat sequence, CACGGCGAGG, that overlaps the end junctions of this region. It seems likely the direct repeat sequences are involved in site-specific mobilization of the bla_KLUY region, possibly in association with ORF513, a putative recombinase (2). The complex integrons InS21 and In35 have a 2.2-kb region downstream of orf513 that harbors bla_CTX-M-2 and orf3 (Fig. 1) (2, 9). Further, the 347 bp of sequence downstream of bla_CTX-M-9a and bla_CTX-M-13 from plasmids pOZ173 and pOZ175, respectively, share 95.7 to 98.6% nucleotide identity with the region downstream of bla_KLUY genes (Fig. 1) (7). In addition, the 42 bp upstream of bla_CTX-M-14 in pOZ174 is 100% identical to the same region from the bla_KLUY genes (7); however, the bla_CTX-M-14 gene is located downstream of ISEcp1. Other bla_CTX-M genes are found associated with ISEcp1, and it is likely that they were mobilized by the insertion element as has been shown for bla_CTX-M-19 (17). Thus, bla_CTX-M genes from the same cluster may be mobilized by different methods.

FIG. 1.

Schematic diagram of the genetic environment of the bla_KLUY gene compared to similar regions as found in the GenBank database. The nucleotide sequences of the junctions of the nearly identical regions found in K. georgiana N03-0458 and In60 are shown. Accession numbers are indicated. Orf1 of pCTX-M-3 is a homolog of Orf477 and is not related to Orf1 of K. ascorbata strain 69. The K. georgiana DSM 9408 bla_KLUG-1 region is not shown, as no ORFs have been detected in the limited flanking sequence available (accession no. AF501233) (18).

FIG. 2.

Alignment of the amino acid sequences of KLUY-1, KLUY-2, KLUY-3, KLUY-4, CTX-M-9, and CTX-M-14. Dots represent identical amino acids. The vertical arrow indicates a putative cleavage site of the leader peptide. Five elements characteristic of Ambler class A β-lactamases are boxed in grey, and the omega loop is underlined (11).

Antibiotic susceptibilities.

The MICs of the K. georgiana strains and the E. coli transformants to β-lactams were determined by broth microdilution using recommended National Committee for Clinical Laboratory Standards methodology (Table 1) (14). In the E. coli transformants, significant synergy with clavulanic acid was observed with cefotaxime, as is typical for class A ESBLs (Table 1). MICs for ceftazidime were lower, ranging from 0.5 to 16 μg/ml, as is characteristic for most CTX-M enzymes (5). The cefoxitin resistance of N03-0460 is likely due to another β-lactamase and/or another mechanism, as E. coli (pKLUY-3a) had a cefoxitin MIC of 4 μg/ml. The bla_KLUY genes are weakly expressed, as found for other chromosomal β-lactamases from Kluyvera (8, 10, 18). Once mobilized, however, the plasmidic location and overexpression from a “foreign” promoter lead to the host strains exhibiting the classical CTX-M ESBL phenotype. The ISEcp1 element has been shown to provide a strong promoter for bla_CTX-M genes located downstream from it (17), and sequences downstream of orf513 have been postulated to act as a promoter for adjacent genes as suggested for bla_CTX-M2 (2) and dfrA10 (16). The resistance profile observed in the E. coli transformants is similar to those of plasmid-encoded CTX-M-9 and CTX-M-14 enzymes (15, 20, 21). We have identified the chromosomal β-lactamase of K. georgiana as the progenitor of the CTX-M-9 group of ESBLs. Mobilization of the gene may involve Orf513 or ISEcp1.

TABLE 1.

MICs of β-lactams for K. georgiana E. coli DH10B harboring cloned bla_KLUY genes, and E. coli DH10B

β-Lactam(s)a	MIC (μg/ml) for:
	K. georgiana						E. coli DH10B with cloned blaKLUY genesb						E. coli DH10B
	N03-0458 (KLUY-1)	N03-0457 (KLUY-2a)	N03-0461 (KLUY-2b)	N03-0460 (KLUY-3a)	N03-0459 (KLUY-3b)	N03-0462 (KLUY-4)	pKLUY- 1	pKLUY- 2a	pKLUY- 2b	pKLUY- 3b	pKLUY- 3a	pKLUY- 4
Cefpodoxime	4	2	2	2	4	2	>16	>16	>16	>16	>16	>16	1
Ceftriaxone	<1	<1	<1	<1	<1	<1	64	>64	64	16	>64	>64	<1
Cefotaxime	0.5	0.5	0.5	<0.25	0.5	0.5	32	>128	32	16	>128	>128	<0.26
Cefotaxime + CLA	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	0.5	<0.25	<0.25	<0.25
Ceftazidime	<0.25	<0.25	<0.25	<0.25	0.5	<0.25	2	8	2	2	16	4	0.5
Ceftazidime + CLA	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	1	<0.25	0.5	<0.25	0.5	0.5
Cefoxitin	8	4	4	64	4	4	4	16	4	8	4	4	8
Aztreonam	<1	<1	<1	<1	<1	<1	4	>16	4	4	>16	16	<1
Meropenem	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25

^aCLA, clavulanic acid at a fixed concentration of 4 μg/ml.

^bCloned in pACYC184.

Nucleotide sequence accession numbers.

The complete nucleotide sequences of the EcoRI fragments cloned from the K. georgiana strains used in this study have been deposited in the GenBank database with the accession numbers AY623932 (N03-0458, bla_KLUY-1), AY623935 (N03-0461, bla_KLUY-2), AY623934 (N03-0460, bla_KLUY-3), AY824948 (N03-0459, bla_KLUY-3), and AY623933 (N03-0462, bla_KLUY-4).