Emergence of Two Klebsiella pneumoniae Isolates Harboring Plasmid-Mediated CTX-M-15 β-Lactamase in Taiwan

In recent years, cefotaximase (CTX-M)-type extended-spectrum β-lactamase-producing Enterobacteriaceae has been increasingly discovered worldwide (7). These β-lactamases confer a typical phenotype for which MICs of ceftriaxone and cefotaxime are higher than those of ceftazidime (7). In Taiwan, CTX-M-14 β-lactamase has been found in Klebsiella pneumoniae (9), and CTX-M-3 enzyme has been discovered in Escherichia coli (8), K. pneumoniae (9), and Serratia marcescens (11). In this report, we described the first isolation of Taiwanese CTX-M-15-producing K. pneumoniae isolates for which MICs of ceftazidime were high (≥128 μg/ml).

Among 211 extended-spectrum β-lactamase-producing K. pneumoniae isolates collected from the whole country between January 1998 and June 2000, two isolates (KPN 154, a urine isolate from hospital N03, and KPN 176, a blood isolate from hospital N04) containing an uncharacterized enzyme with a pI of 8.8 were studied. Both clinical isolates of K. pneumoniae were susceptible to imipenem (MIC, 0.12 μg/ml) and meropenem (MICs, 0.064 to 0.094 μg/ml) but were resistant to cefotaxime (MIC, >256 μg/ml), ceftazidime (MIC, 128 to >256 μg/ml), and cefepime (MIC, 32 to 64 μg/ml), as determined by using the E-test (Table 1). Clavulanic acid totally restored the activities of cefotaxime, ceftazidime, and cefepime to levels at which the isolates were susceptible (MICs, 0.047 to 2 μg/ml).

TABLE 1.

Activity of various antimicrobial agents^a against two isolates of K. pneumoniae (KPN 154 and 176), two transconjugants (E154 and E176), and the recipient strain (E. coli J53-2) and their ribotypes, pIs, and bla gene characterization

Isolate	Site	Ribotype	pI	MICb (μg/ml)					β-lactamase
				FOX	CT/CTL	TZ/TZL	PM/PML	IPM/MEM
154	NO3	255.3	5.4	>32	>256/0.5	>256/2.0	64/0.125	0.12/0.094	TEM-1
			7.6						SHV-1
			8.2						SHV-5
			8.8						CTX-M-15
E154	Nil	NTc	5.4	1.5	>256/0.25	128/2.0	128/0.125	0.12/0.047	TEM-1
			8.8						CTX-M-15
176	NO4	691.5	5.4	16	>256/0.5	128/1.0	32/0.047	0.12/0.064	TEM-1
			7.6						SHV-1
			8.2						SHV-5
			8.8						CTX-M-15
E176	Nil	NTc	5.4	2.0	>256/0.25	128/1.0	64/0.094	0.12/0.064	TEM-1
			8.2						SHV-5
			8.8						CTX-M-15
E. coli J53-2	Nil	NTc	Nil	8.0	0.12/0.25	0.5/1.0	0.094/0.064	0.25/0.047	Nil

^aCT, cefotaxime; CTL, cefotaxime-clavulanate; TZ, ceftazidime; TZL, ceftazidime-clavulanate; PM, cefepime; PML, cefepime-clavulanate; FOX, cefoxitine; IPM, imipenem; MEM, meropenem.

^bMICs were determined by E-test according to the manufacturer's (AB BIODISK) package insert.

^cNT, not tested.

Plasmid DNA templates were prepared and purified for PCR. Amplification was performed using PCR primers for the bla genes encoding TEMs, SHVs, or CTX-M-3 β-lactamases (5, 9). Isoelectric focusing analysis revealed four enzymes with pIs of 5.4, 7.6, 8.2, and 8.8 in both isolates, which were matched to TEM-1, SHV-1, SHV-5, and CTX-M-15, respectively, by PCR and DNA sequence analysis (Table 1).

Automated ribotyping using the RiboPrinter Microbial Characterization system (Qualicon, Inc., Wilmington, Del.) revealed nonclonal relatedness of both K. pneumoniae strains, but each belonged to two different major clones in Taiwan (KPN 154, ribotype 255.3; KPN 176, ribotype 691.5).

Conjugation experiments provided evidence of transferable plasmids containing bla genes encoding SHV-5 (pI, 8.2) and/or CTX-M-15 (pI, 8.8). Both transconjugants revealed MIC profiles that were similar to those of the parent strains. The transconjugant E154 contained the gene bla_CTX-M-15 without bla_SHV-5, possibly indicating the failure of bla_SHV-5 gene transference. Therefore, the presence of CTX-M-15 conferred to transconjugant E154 the ceftazidime resistance characterized in previous papers (1, 2, 6). This spectrum of hydrolysis has rarely been observed in other members of the CTX-M enzyme family (7). The mechanism of cefepime resistance may be the cumulative effects of CTX-M-15 and SHV-5 (as in KPN 154, KPN 176, and E176) or the presence of CTX-M-15 alone (as in E154).

CTX-M-15, an Asp-240-Gly variant of CTX-M-3, increased the catalytic efficiency against ceftazidime (6). CTX-M-15 β-lactamase has already been identified in several countries, including India (2), Poland (1), Turkey (3), and the United Kingdom (4). Since CTX-M-15 differs from CTX-M-3 by only a single amino acid change and CTX-M-3 has also been widely disseminated among isolates of Enterobacteriaceae in Taiwan (8, 9, 11), it is not surprising that CTX-M-15 emerged in the Taiwan environment.

Although not clonally related, these two emerging CTX-M-15-producing strains were documented to evolve from different major K. pneumoniae clones in Taiwan. Ribotype 691.5 has been recognized as an epidemic clone in northern Taiwan, and ribotype 255.3 has been recognized as another nationwide-epidemic clone (10). Although still rare, strains producing CTX-M-15 in Taiwan could potentially further disseminate, either clonally or by plasmid-related transmission.