Imipenem Resistance in a Salmonella Clinical Strain Due to Plasmid-Mediated Class A Carbapenemase KPC-2

A Salmonella enterica serotype Cubana isolate exhibiting resistanceto most ß-lactam antibiotics, including oxyimino-cephalosporinsand imipenem, was isolated from a 4-year-old boy with gastroenteritisin Maryland. ß-Lactam resistance was mediated by aconjugative plasmid that encoded KPC-2, a class A carbapenemasepreviously found in a Klebsiella pneumoniae isolate from theMaryland area as well. Sequence analysis of the flanking regionsindicated a potential association of bla_KPC-2 with mobile structures.

INTRODUCTION

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Introduction

Resistance to expanded-spectrum oxyimino-cephalosporins amongSalmonella strains is mostly due to acquisition of plasmidsencoding various class A extended-spectrum ß-lactamases(1, 8, 13, 17, 19-21). Production of plasmid-mediated classC ß-lactamases by Salmonella isolates has also beendescribed previously (4, 22, 23). The emergence of such strainsmay have serious implications because of the limitation of therapeuticchoices for patients with invasive Salmonella infections andby facilitation of the spread of bla genes in the community.We describe here an imipenem-resistant Salmonella enterica serotypeCubana isolate that produces the recently described KPC-2 ß-lactamase(E. S. Moland, J. Johnson, J. A. Black, T. J. Lockhart, A. Hossain,V. L. Herrera, N. D. Hanson, and K. S. Thomson, Abstr. 41stIntersci. Conf. Antimicrob. Agents Chemother., abstr. 2226,2001).

MATERIALS AND METHODS

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Materials and Methods

Salmonella serotype Cubana was isolated in December 1998 froma stool specimen of a 4-year-old boy with diarrhea in a hospitalin Maryland. The patient was chronically ill with Wiskott-Aldrichsyndrome. In the 6 months before isolation of serotype Cubana,he had been hospitalized three times and had received intravenousantibiotics. During each of those hospitalizations he had receivedintravenous ß-lactams, including ceftriaxone and ceftazidime,but not carbapenems. There was no history of recent travel.The isolate (AM04707) was submitted to the Centers for DiseaseControl and Prevention as part of the National AntimicrobialResistance Monitoring System for enteric bacteria (http://www.cdc.gov/NARMS/)and was subsequently forwarded to the Hellenic Pasteur Institute.

Escherichia coli K-12 strain 14R525 (Nal^r) was used as the recipientin conjugation experiments. E. coli DH5 ${alpha}$ (GIBCO-BRL, Carlsbad,Calif.) was used for transformation. Chloramphenicol-resistantplasmid pBCSK(+) (Stratagene, La Jolla, Calif.) was used forcloning and expression of bla genes.

The MICs of ß-lactams were determined by an agar dilutiontechnique (10). Susceptibilities to other antimicrobial agentswere assessed by a disk diffusion method (11) and by a partial-rangebroth microdilution method (Sensititre; Trek Diagnostics; Westlake,Ohio), according to the instructions of the manufacturer.

Conjugation experiments were carried out in mixed broth culturesas described previously (5). Transconjugant clones were selectedon Mueller-Hinton agar containing ampicillin (50 µg/ml)plus nalidixic acid (200 µg/ml). Plasmid DNA preparationswere obtained by an alkaline lysis technique and resolved in0.8% (wt/vol) agarose gels. Individual plasmids were excisedas discrete bands from low-melting-point agarose (0.8%) andwere subjected to partial digestion with various restrictionenzymes including HindIII. Digests were ligated into the multicloningsite of pBCSK(+). The resulting recombinant plasmids were usedto transform E. coli DH5 ${alpha}$ competent cells. ß-Lactam-resistanttransformants were selected on Luria-Bertani agar containingchloramphenicol (20 µg/ml) and ampicillin (50 µg/ml).The nucleotide sequences of the cloned fragments were determinedwith an ABI Prism 377 DNA sequencer (Perkin-Elmer, Applied BiosystemsDivision, Foster City, Calif.). Sequence similarity searcheswere performed with the BLAST program (available at the websiteof the National Center for Biotechnology Information).

ß-Lactamases were extracted in one of two ways. Onemethod involved ultrasonic treatment of bacterial cells, followedby suspension in phosphate buffer (100 mM; pH 7.0) and clarificationby centrifugation (Fig. 1A). A small-scale freeze-thaw methodwith modifications provided by J. W. Biddle and J. K. Rasheedwas used for comparison of KPC-1 and KPC-2 (3) (Fig. 1B). Detectionof carbapenemase activity was performed by bioassay as describedpreviously (24). Analytical isoelectric focusing was performedin polyacrylamide gels containing ampholytes (pH range, 3.5to 9.5; APBiotech, Piscataway, N.J.). ß-Lactamaseactivity was visualized with nitrocefin (Oxoid Ltd., Basingstoke,United Kingdom, or Becton-Dickinson, Lexington, Ky.). In situinhibition of ß-lactamase activity was performed bysoaking the gels in solutions containing clavulanate (0.5 mM),tazobactam (0.5 mM), or EDTA (5 mM). Maximum hydrolysis ratesfor various ß-lactam substrates were estimated byUV spectrophotometry, and inhibition by clavulanate and tazobactamwas assessed by using nitrocefin as the reporter substrate.The respective procedures were as described previously (5).

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FIG. 1. (A) Isoelectric focusing of ß-lactamase preparations from Salmonella serotype Cubana 4707, E. coli(pST4707), and E. coli(pST-H1) (lanes 2, 3, and 4, respectively). ß-Lactamases with known pIs are in lane 1 (TEM-1, pI 5.4; PSE-3, pI 6.9; SHV-1, pI 7.6; SHV-5, pI 8.2). (B) Comparison of preparations from K. pneumoniae 1534 (lane 2) and Salmonella serotype Cubana 4707 (lane 3). ß-Lactamases with known pIs are in lane 1 (TEM-12, pI 5.25; TEM-2, pI 5.6; SHV-3, pI 7.0; MIR-1, pI 8.4).

Nucleotide sequence accession number. The sequence of the 5.2-kb fragment of pST-H1 containing bla_KPC-2has been submitted to the GenBank database and assigned accessionnumber AF481906.

RESULTS

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Results

Salmonella serotype Cubana 4707 exhibited either resistanceor decreased susceptibility to all ß-lactams testedincluding ß-lactam-ß-lactamase inhibitorcombinations, oxyimino-cephalosporins, aztreonam, and carbapenems.In the presence of clavulanic acid, the MICs of ceftazidimeand imipenem were decreased by 4 and 2 doubling dilutions, respectively(Table 1). The isolate was also resistant to streptomycin, trimethoprim,and sulfamethoxazole. The isolate was susceptible to nalidixicacid (MIC, $<=$ 4 µg/ml) and ciprofloxacin (MIC, $<=$ 0.015 µg/ml).

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TABLE 1. Susceptibilities to antibiotics of Salmonella serotype Cubana 4707, an E. coli transconjugant containing wild-type plasmid pST4707, and an E. coli DH5 ${alpha}$ transconjugant containing plasmid pSTH-1

Transfer of ß-lactam resistance to E. coli by conjugationwas successful. Transconjugants exhibited a phenotype of resistanceto ß-lactams similar to that of Salmonella serotypeCubana 4707 (Table 1). They were also resistant to streptomycin,trimethoprim, and sulfonamides. Analysis of plasmid DNA indicatedtransfer of a plasmid (pST4707) that, according to conventionalgel electrophoresis in a 0.8% gel, was between 24.5 and 42 MDa(data not shown).

ß-Lactamase extracts from Salmonella serotype Cubana4707 and E. coli(pST4707) were positive in a carbapenemase bioassayperformed as described by Yigit et al. (24; data not shown).Isoelectric focusing demonstrated that the extracts containedtwo ß-lactamases with apparent pIs of 5.4 and 6.7,respectively (Fig. 1). Both enzymes were inhibited in situ byclavulanic acid and tazobactam but not by EDTA. The ß-lactamasewith a pI of 5.4 is consistent with a TEM-1 enzyme, the presenceof which was supported by sequencing of 95% of the bla_TEM-1-codingregion (data not shown).

Cloning of a 5.2-kb HindIII fragment of pST4707 yielded a pBCSK(+)derivative (pST-H1) which mediated resistance only to ß-lactams.E. coli(pST-H1) exhibited resistance or decreased susceptibilityto all ß-lactam antibiotics tested, including carbapenems(Table 1). Also, it was positive in the carbapenemase bioassayand produced a single ß-lactamase with a pI of 6.7(Fig. 1). Hydrolysis experiments performed with extracts fromE. coli(pST-H1) showed that the rate of imipenem hydrolysisrelative to that of penicillin G, which was set at 100, was45 ± 5. The value for cefotaxime was 58 ± 3. Hydrolysisrates for ceftazidime and cefoxitin were too low to obtain reliablevalues, although the slightly elevated cefoxitin MIC indicatessome degree of hydrolysis (Table 1). The hydrolysis rates fornitrocefin and cephalothin were significantly higher than thatfor penicillin G (2.4- and 3.1-fold, respectively). The 50%inhibitory concentrations of clavulanate and tazobactam were1.4 and 0.08 µM, respectively.

The 5.2-kb HindIII fragment included an 882-bp open readingframe (ORF) that differed by only 1 bp (nucleotide 520) frombla_KPC-1 found in Klebsiella pneumoniae (24). An identical sequence(bla_KPC-2), also from K. pneumoniae, has recently appeared inGenBank (accession number AY034847; Moland et al., 41st ICAAC).The deduced protein, KPC-2, differs from KPC-1 by one aminoacid residue (KPC-2 contains Gly instead of Ser at position175). Gly-175 is also found in SME-1, NMC-A, and IMI-1 (9, 12,16). KPC-2 contained the motifs typical for class A ß-lactamases(7) as well as residues characteristic for the carbapenem-hydrolyzingenzymes of this molecular class, including Cys at positions69 and 238, His at position 105, and a hydroxylated residue(Thr) at position 237 (14, 15, 18). The putative secretory signalsequence comprises 24 residues. The mature KPC-2 has a predictedmolecular weight of 28,480. The calculated pI (6.4) differsslightly from the apparent pI of the native ß-lactamase.The pI reported for KPC-1 was 6.7 (24). When isoelectric focusingwas done with both ß-lactamases, the pI of K. pneumoniaeKPC-1 was very similar to that of KPC-2 from Salmonella serotypeCubana (Fig. 1).

The flanking sequences of bla_KPC-2 (120 bp upstream, includingthe ribosome binding site and the -10 and -35 regions, and 300bp downstream) were identical to those reported for bla_KPC-1(24). Analysis of the rest of the sequence of the 5.2-kb fragmentshowed at least four additional ORFs (orf1 to orf4) (Fig. 2).The respective putative peptides (P1 through P4) were significantlysimilar to proteins associated with transposable elements (Table2). P1 was highly similar to the C terminus of a transposaseA (TnpA) found in the Tn3 family of transposons from Pseudomonasputida (6). P2 and P3 displayed similarity to a TnpA and a transpositionhelper protein, respectively, found in Ralstonia solanacearum.P4 shared a moderate degree of similarity with putative transposasesdescribed in Burkholderia cepacia and Ralstonia spp.

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FIG. 2. Schematic representation of the cloned HindIII fragment (GenBank accession number AF481906). Note that an additional HindIII site is located within the bla_KPC-2 gene.

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TABLE 2. ORFs, including bla_KPC-2, of the 5.2-kb HindIII fragment of plasmid pST4707

DISCUSSION

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Discussion

KPC-2, along with KPC-1 (24), SME-1 (9), NMC-A (12), and IMI-1(16), comprise a small group of class A ß-lactamases(functional group 2f [2, 15]) with potent carbapenemase activities.The KPC enzymes seem to differ from the rest of the group inthat they hydrolyze oxyimino-cephalosporins more efficiently.KPC-2 conferred levels of resistance to ß-lactamsthat were comparable to those conferred by KPC-1 (24). The twoenzymes, however, were expressed in different systems, and theyalso differed by an amino acid residue of the putative omegaloop. The latter structure is significant in determining thecatalytic properties of class A ß-lactamases (7).Hence, differences in the substrate specificity cannot be ruledout.

The high degree of homology between bla_KPC-1 and bla_KPC-2 andthe identity of their flanking sequences indicate that thesevariant bla genes could be parts of a single structure. However,KPC-2-encoding plasmid pST4707 was self-transferable, whilethe bla_KPC-1-encoding plasmid could not be conjugated into E.coli (24). Also, the bla_KPC-1-encoding plasmid, unlike pST4707,neither encoded TEM nor mediated resistance to antibiotics otherthan ß-lactams. Therefore, it can be speculated thatthe bla_KPC variants resulted from insertion of a similar orcommon mobile element into different plasmid backgrounds. Sequencingdata are compatible with this notion, although the existenceof a transposable element was not proven.

KPC-1 was found in a K. pneumoniae isolate from a hospital inNorth Carolina. KPC-producing K. pneumoniae strains have alsobeen found in a Maryland hospital (Moland et al., 41st ICAAC).The isolation of Salmonella serotype Cubana 4707, also in Maryland,indicates a certain degree of spread of bla_KPC genes in theUnited States. The plasmid locations of these genes and theirassociation with possibly mobile structures may facilitate theirspread. Continued surveillance is essential to monitor for thepotential spread of bla_KPC genes among Salmonella strains.

ACKNOWLEDGMENTS

We thank Jennifer Nelson, David Blythe, Dipti Shah, and DavidI. Wheeler for contributions to this work.

FOOTNOTES

* Corresponding author. Mailing address: National Center for Infectious Diseases, MS G29, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-2000. Fax: (404) 639-4290. E-mail: zyr3{at}cdc.gov.

REFERENCES

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