First Detection of the Plasmid-Mediated Class A Carbapenemase KPC-2 in Clinical Isolates of Klebsiella pneumoniae from South America

Abstract

The plasmid-mediated class A carbapenemase KPC-2 was isolated from unrelated Klebsiella pneumoniae isolates in Medellin, Colombia. These KPC enzymes are the first from South America and the second isolation outside of the United States. The expanding geographic spread of KPC carbapenemases underscores the importance of clinical recognition of these enzymes.

A number of resistance mechanisms can circumvent the efficacy of carbapenems. Predominant among these is the production of carbapenem-hydrolyzing β-lactamases (carbapenemases). The small but potent group of molecular class A carbapenemases that includes KPC1-3, NmcA, Sme1-3, IMI-1, and GES-2 are sporadically characterized from enterobacterial species (14).

Within this class, the KPCs are most frequently encountered. While Klebsiella pneumoniae remains their most frequent host, its range has broadened to include Klebsiella oxytoca (3, 23), Salmonella enterica (12), and Enterobacter spp. (4, 9), and KPC-producing organisms are being increasingly detected. They are predominant in facilities in New York (3, 5, 21), where one report demonstrated an impressive 24% of K. pneumoniae producing KPCs (6). Their geographical distribution, once limited to the eastern United States, is gradually widening. A single KPC-2-producing K. pneumoniae isolate was recently reported in France (13), although a possible transfer from the United States was acknowledged in that case. We have identified two KPC-possessing K. pneumoniae isolates, collected from different facilities in Medellin, Colombia. These KPC enzymes are the second identified outside of the United States and are the first from Latin America.

During 2005, a surveillance network within Colombia identified two carbapenem-resistant K. pneumoniae isolates from different hospitals. Both patients had nosocomial infections and subsequently received meropenem. The first was diagnosed with an ultimately fatal bacteremic pneumonia. Isolate KPN2303 was recovered from blood, ascitic fluid, and urine cultures. The second patient had a urinary tract infection that resolved upon appropriate therapy, with isolate KPN633 recovered from urine cultures. No discernible linkage to the United States could be established from either patient. Isolate identification and antibiotic susceptibilities were determined by Vitek (bioMerieux, Lyon, France). Isolates were resistant to all tested antibiotics, except that KPN2303 remained susceptible to aminoglycosides while KPN633 remained susceptible to fluoroquinolones. Both were extended-spectrum beta-lactamase (ESBL) negative by Vitek. Susceptibilities by broth microdilution (7) confirmed that both isolates had unusually high-level resistance to all three carbapenems (MICs > 256 μg/ml) (Table 1), with no change or only a slight reduction in the MICs noted after the addition of clavulanic acid.

TABLE 1.

Antimicrobial susceptibility patterns of K. pneumoniae KPN2303, its E. coli XL-10 transformant, and K. pneumoniae KPN633

Antimicrobial agent(s)	MIC (μg/ml)
	KPN2303 (clinical)	E. coli XL-10 (pBC-Wilma)	E. coli XL-10	KPN633 (clinical)
Imipenem	>256	8	0.125	>256
Imipenem-CLAa	>256	2	0.125	>256
Meropenem	>256	2	0.015	>256
Meropenem-CLA	>256	2	0.015	>256
Ertapenem	>256	4	0.008	>256
Ampicillin	>256	>256	2	>256
Ampicillin-SULb	>256	>256	4	>256
Piperacillin	>256	>256	1	>256
Piperacillin-TZBc	>256	>256	0.5	>256
Ceftazidime	>256	64	0.25	>256
Cefotaxime	>256	128	0.06	>256
Ceftriaxone	>256	>256	0.06	>256
Cefepime	>256	16	0.03	>256
Aztreonam	>256	>256	0.125	>256
Aztreonam-CLA	>256	256	0.06	>256

^aCLA, clavulanic acid, inhibitor tested at a fixed concentration of 2 μg/ml.

^bSUL, sulbactam. Penicillin/inhibitor ratio, 2:1.

^cTZB, tazobactam, inhibitor tested at a fixed concentration of 4 μg/ml.

The first isolate identified was KPN2303. β-Lactamases in the cell extract were examined by isoelectric focusing (IEF) (10) with concurrent evaluation in a three-dimensional extract bioassay using imipenem (8). IEF demonstrated only a single experimental pI 7.0 β-lactamase. SHV-specific PCR on a genomic template (20) amplified bla_SHV in KPN2303. However, a positive bioassay result suggested β-lactamase involvement in imipenem hydrolysis (data not shown).

A shotgun cloning approach was used to identify the carbapenemase, and Escherichia coli XL-10 transformants were selected with ampicillin (50 μg/ml). Extracts of a transformant having a positive bioassay result revealed transfer of the pI 7.0 β-lactamase on IEF. The entire 4.67-kb insert of the plasmid (pBC-Wilma) was sequenced. Sequence homologies were determined using the BLAST program from the National Center for Biotechnology Information (1), revealing the presence of a KPC-type β-lactamase, bla_KPC-2 (Fig. 1). Nucleotides 1 through 2808 had a 99% identity to a cloned bla_KPC-2 containing a HindIII fragment as described by Miriagou et al. (12) (GenBank accession no. AF481906) from the Salmonella enterica subsp. enterica serovar Cubana plasmid, pST4707. The open reading frames (ORFs) flanking bla_KPC-2 on the BamHI fragment (orf1 and orf2) (Fig. 1) encode proteins associated with mobile genetic elements. orf1 had 100% identity to the terminal partial gene for a putative transposition helper protein (GenBank accession no. AAM10642.1). orf2 had 100% identity with the proximal partial gene for a putative transposase (GenBank accession no. AAM10644.1). No additional ORFs were identified downstream. Interestingly, there did exist downstream a 155-bp region identical at the nucleotide level to Tn5708 (GenBank accession no. AJ010745) that may represent the now-defunct vestiges of a transposon.

FIG. 1.

Schematic representation (not to scale) of the cloned plasmid 4.67-kb BamHI insert from KPN2303. Open reading frames and transcriptional orientation are represented by arrows. GenBank sequences listed below ORFs represent those with the most significant similarity. The dashed line represents the upstream portion of AF481906 not included in our cloned fragment.

The transformant showed only moderately reduced carbapenem susceptibilities compared to those of the clinical strain (Table 1), suggesting that additional resistance mechanisms may contribute to that high-level resistance. Although not examined, alterations in porin expression are known to affect the MICs of these antibiotics in Klebsiella (2, 22).

This evaluation of KPN2303 was completed before identification of KPN633. Thus, PCR analysis with KPC primers and conditions (22) as well as sequencing of the pCR-XL-TOPO (Invitrogen, Carlsbad, CA) cloned amplicon confirmed bla_KPC-2 in KPN633. Chromosomal DNA was fingerprinted by pulsed-field gel electrophoresis with XbaI digestion (Invitrogen, Carlsbad, CA) (11) and indicated that these isolates were unrelated (data not shown).

Our experimental pI of 7.0 differed from the published KPC-2 pI of 6.7. This discrepancy led to the cloning approach utilized and recognition of similarities to the Salmonella plasmid that might not have been otherwise ascertained. Other notable similarities to pST4707 identified include a New York K. oxytoca isolate (23) and the recent KPC-2-producing French K. pneumoniae isolate (13). While the precise relationship of our isolates to these strains has not been investigated, the resemblance of genetic structures from multiple strains and hosts suggests dissemination of the KPC-2 β-lactamase through conjugal plasmid transfer and transposition.

Southern transfer of plasmids to a positively charged nylon membrane (Roche, Indianapolis, IN) (15) was performed. A bla_KPC-2 probe hybridized under high-stringency conditions to single plasmids of 15.5 and ∼30 Kb in KPN633 and KPN2303, respectively (data not shown). Attempts to transfer β-lactam resistance from both K. pneumoniae strains to Escherichia coli J53 Az^R by a mixed broth mating procedure (17) were unsuccessful.

In areas with a high prevalence of ESBL producers, as in Colombia (32.6%) (19), carbapenems remain one of the few therapies for Klebsiella infections (16). As KPC-producing organisms are frequently resistant to multiple antibiotic classes (3, 6, 23), the detection of this plasmid-mediated resistance mechanism is worrisome. Current evidence suggests that the significance and prevalence of these carbapenemases may be clinically unrecognized. Multiple factors can complicate the detection process. First, carriage of the bla_KPC allele may confer only reduced carbapenem susceptibility but not frank resistance (18). Second, KPCs have been associated with positive ESBL confirmation tests (18), leading to their misidentification as ESBL-producing strains. Third, an inoculum effect exists (6) for some isolates in the low-inoculum type tests employed by some automated systems. Accurate detection of KPC enzymes will be crucial in controlling their spread, especially in isolates remaining susceptible in vitro.

Nucleotide sequence accession number.

The nucleotide sequence of the BamHI fragment obtained from K. pneumoniae KPN2303 was assigned GenBank accession no. DQ523564.