MAJOR ARTICLE
Emergence of Carbapenem-Resistant Klebsiella Species Possessing the Class A CarbapenemHydrolyzing KPC-2 and Inhibitor-Resistant TEM-30 b-Lactamases in New York City Patricia A. Bradford,1 Simona Bratu,2 Carl Urban,4 Melissa Visalli,1 Noriel Mariano,4 David Landman,2 James J. Rahal,4 Steven Brooks,3 Sanda Cebular,2 and John Quale2 1
Wyeth Research, Pearl River, 2State University of New York–Downstate and 3Kingsbrook Jewish Medical Center, Brooklyn, and 4New York Hospital Queens, Flushing, New York
Nineteen isolates of carbapenem-resistant Klebsiella species were recovered from 7 hospitals in New York City. Most K. pneumoniae belonged to a single ribotype. Nucleotide sequencing identified KPC-2, a carbapenemhydrolyzing b-lactamase. In 3 strains, TEM-30, an inhibitor-resistant b-lactamase, was detected. Carbapenemresistant Klebsiella species possessing KPC-2 are endemic in New York City. This study documents the identification of an inhibitor-resistant TEM b-lactamase in the United States. During the past 2 decades, increased use of cephalosporins has been accompanied by the emergence of Enterobacteriaceae possessing extended-spectrum blactamases (ESBLs). The emergence of ESBL-producing Klebsiella species has been especially problematic in New York City [1–3]. Although several genera have been found to carry ESBLs, Klebsiella pneumoniae account for the majority of isolates. Klebsiella species carrying these enzymes are frequently resistant to other classes of antibiotics, including aminoglycosides, fluoroquinolones, and sulfonamides [3, 4]. Carbapenems are considered to be the agents of choice for the treatment of serious infections due to isolates possessing ESBLs. Carbapenem resistance has been reported in only a small number of Enterobacteriaceae and is caused by a number of mechanisms. First, the combination of the high-level production of an AmpC b-lactamase and loss
Received 11 December 2003; accepted 7 February 2004; electronically published 14 June 2004. Presented in part: 43rd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, 14–17 September 2003, Chicago, Illinois (abstract C2-35). Reprints or correspondence: Dr. John Quale, SUNY Downstate, Box 77, Division of Infectious Diseases, 450 Clarkson Ave, Brooklyn, NY 11203 (jquale@ downstate.edu). Clinical Infectious Diseases 2004; 39:55–60 2004 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2004/3901-0010$15.00
of outer membrane proteins has been associated with carbapenem resistance in K. pneumoniae and Enterobacter species [5–9]. Second, the finding of efficient carbapenem-hydrolyzing b-lactamases in Enterobacteriaceae remains unusual but appears to be increasing. Two distinct classes of b-lactamases have the ability to hydrolyze carbapenems. Although class B metallob-lactamases are usually associated with Pseudomonas aeruginosa and Acinetobacter baumannii, they may also be found in K. pneumoniae [10]. A small number of class A b-lactamases have been found to possess hydrolytic activity against carbapenems. SME-1 and SME2 have been recovered from carbapenem-resistant isolates of Serratia marcescens [11], and GES-2 has been recovered from P. aeruginosa [12]. IMI-1 and NMC-A have been recovered in isolates of Enterobacter cloacae [13, 14]. One of the more frequently encountered class A carbapenem-hydrolyzing enzymes are the KPC-type blactamases. These enzymes are capable of efficiently hydrolyzing penicillins, cephalosporins, aztreonam, and carbapenems and are inhibited by clavulanic acid and tazobactam. The initial report of one of these b-lactamases, KPC-1, involved a carbapenem-resistant strain of K. pneumoniae recovered in North Carolina [15]. This enzyme was found to reside on a transferable plasmid [15]. Soon afterward, reports from the northeastCarbapenem-Resistant Klebsiella Species • CID 2004:39 (1 July) • 55
ern United States of K. pneumoniae [16, 17], Klebsiella oxytoca [18], Salmonella enterica [19], and E. cloacae [20] expressing KPC-2 surfaced. The blaKPC-2 gene differs from blaKPC-1 by a single nucleotide and also confers reduced susceptibility to carbapenems. Recently, carbapenem-resistant isolates of Escherichia coli in New Jersey [21] and K. pneumoniae in New York [22] possessing blaKPC-3 (which differs from blaKPC-2 by a single nucleotide) have been recovered. In some of these reports, diminished production of outer membrane porins has been noted [15, 22] and may also contribute to carbapenem resistance. We now report the emergence of Klebsiella species possessing the carbapenem-hydrolyzing class A b-lactamases KPC-2 in New York City, as well as the first report of an inhibitor-resistant TEM-type b-lactamase in the United States. MATERIALS AND METHODS Isolates of Klebsiella species with reduced susceptibility to carbapenems were collected during 1997–2001 from 1 hospital in Queens (9 isolates) and 6 hospitals in Brooklyn (10 isolates), New York. The 9 isolates from Queens were selected because reduced susceptibility to imipenem was identified during initial testing. Seven of the 10 isolates in Brooklyn were collected during citywide surveillances assessing rates of bacterial resistance [2, 3]; the remaining 3 isolates were sent to the investigators because of their unusual resistance pattern. Isolates were identified by means of standard criteria, and susceptibility
testing was performed according to NCCLS methodology [23]. All isolates of K. pneumoniae underwent ribotyping with the Riboprinter Microbial Characterization System (Qualicon) (figure 1). b-Lactamases were characterized by isoelectric focusing according to established methods [24]. In brief, cells grown to a logarithmic phase were disrupted by 3 cycles of freeze-thaw in 0.2 mol/L sodium acetate and centrifuged. Supernatants were used for isoelectric focusing with an LKB Multiphor apparatus with prepared PAGplates (pH, 3.5–9.5; Pharmacia LKB) and b-lactamases determined after overlay with nitrocephin. The isoelectric point (pI) was determined after comparison to known standard b-lactamases. Supernatants from selected isolates were also analyzed for spectrophotometric evidence of carbapenemase activity in 50 mmol/L HEPES (pH, 7.0) with 1 mmol/L ZnCl2 [25]. Inhibition of hydrolytic activity was also assessed spectrophotometrically after the addition of 80 mmol/ L clavulanic acid or 10 mmol/L EDTA. b-Lactamases were identified by amplification by means of established protocols [26] with the following primers: TEM forward, 5-GTGCGCGGAACCCCTATT-3; TEM reverse, 5-TTACCAATGCTTAATCAGTGAGGC-3; SHV forward, 5-CTTTACTCGCCTTTATCGGC3; SHV reverse, 5-TTACCGACCGGCATCTTTCC3; KPC forward, 5-ATGTCACTGTATCGCCGTCT-3; KPC reverse, 5TTTTCAGAGCCTTACTGCCC-3. Amplified DNA fragments were cloned into pCR2.1 with the TOPO TA kit (Invitrogen).
Figure 1. Ribotyping of Klebsiella pneumoniae clinical isolates expressing KPC-2 performed with the RiboPrinter System (Qualicon) with EcoRI as the restriction enzyme. Twelve of the isolates were grouped programmatically into a common ribogroup. This included strains that were isolated from both Brooklyn and Queens, New York. Similar results were obtained with a second restriction enzyme (PvuII, data not shown). 56 • CID 2004:39 (1 July) • Bradford et al.
Nucleotide sequencing was performed on both the strands of the DNA from clones resulting from at least 3 independent PCR reactions with the Big Dye sequencing kit and an automated DNA sequencing system 3700 (Applied Biosystems), as described elsewhere [27]. The DNA sequences were identified by the BLAST program from the National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov/ BLAST). Medical records were reviewed to obtain essential demographic and clinical information. Particular attention was paid to the location of the patient, duration of hospitalization, and antibiotic therapy before recovery of the carbapenem-resistant isolate. RESULTS A total of 19 isolates of Klebsiella species producing carbapenem-hydrolyzing b-lactamase were recovered from November 1997 to July 2002 from 7 different hospitals in New York City. Eighteen were identified as K. pneumoniae, and the remaining one was K. oxytoca (table 1). The MIC of imipenem was 4 mg/mL for one isolate and 8 to 164 mg/mL for all others. Four isolates from 2 hospitals were identified originally by the clinical microbiology laboratories as susceptible to imipenem. These isolates were collected during the citywide surveillance study in Brooklyn. One of these hospitals used the Dade Microscan Walkaway System; the second used the Vitek System (bioMe´rieux). These isolates were later confirmed to Table 1.
be resistant by agar dilution and E-test techniques in the research laboratory of one of the principal investigators. The actual MIC of imipenem was 16–64 mg/mL for these isolates. Most isolates were also resistant to aminoglycosides and fluoroquinolones (table 1). Of the 18 isolates of K. pneumoniae, 4 ribotypes were identified (table 1). However, 12 isolates belonged to a single ribotype and were recovered from 6 different hospitals. The 9 isolates recovered from one hospital in Queens belonged to 1 of 2 ribogroups, including the dominant ribotype. The 9 isolates of K. pneumoniae collected from Brooklyn were distributed among 6 hospitals; the predominant ribotype accounted for 7 of these isolates. There was no obvious epidemiological association linking these isolates. Isoelectric focusing characterization of the b-lactamases revealed multiple enzymes in all isolates (table 1). Not all blactamases present in these strains were identified by PCR and nucleotide sequencing. One enzyme with a pI of 6.7 was uniformly found in all 19 isolates and was subsequently identified as KPC-2 by nucleotide sequencing. Supernatants containing b-lactamases from the 10 Brooklyn isolates demonstrated efficient spectrophotometric hydrolysis of imipenem that was inhibited by clavulanic acid but not EDTA (data not shown). In addition to the KPC-2 enzyme, several different TEM and SHV-type b-lactamases were identified in the strains. Fifteen of the strains possessed an ESBL, 14 of which were identified to be SHV-12. Although the published pI of SHV-12 is 8.2, in
b-Lactamase and susceptibility data for Klebsiella isolates possessing KPC-2 carbapenemase.
Isolate
Ribotype
MIC of drug, mg/mL
Isoelectric points of b -lactamases (identification confirmed by nucleotide sequencing)
Klebsiella pneumoniae 7628
B
5.4 (TEM-1), 6.7 (KPC-2), 8.2, 8.5
K. pneumoniae 7635
C
6.7 (KPC-2), 7.6 (SHV-1)
Imipenem
Piperacillintazobactam
Ceftazidime
Amikacin
Ciprofloxacin
8
⭓128
16
4
1
16
⭓128
32
32
⭓4
K. pneumoniae 7636
C
5.4 (TEM-1), 6.7 (KPC-2), 7.6 (SHV-7), 8.2, 8.8
⭓64
⭓128
⭓64
⭓64
⭓4
K. pneumoniae 7629
A
5.2, 5.4 (TEM-1), 6.7 (KPC-2), 7.4, 8.8 (SHV-12)
32
⭓128
⭓64
32
⭓4
K. pneumoniae 7630
A
5.2 (TEM-30), 5.4 (TEM-1), 6.7 (KPC-2), 7.6, 8.8 (SHV-12)
16
⭓128
⭓64
32
⭓4
K. pneumoniae 7631
A
5.2 (TEM-30), 5.4 (TEM-1), 6.7 (KPC-2), 7.4, 8.2, 8.8 (SHV-12)
16
⭓128
⭓64
32
⭓4
K. pneumoniae 7632
A
5.4 (TEM-1), 6.7 (KPC-2), 8.8 (SHV-12)
64
⭓128
⭓64
32
⭓4
K. pneumoniae 7633
A
5.4, 6.7 (KPC-2), 7.6, 8.8 (SHV-12)
32
⭓128
⭓64
32
⭓4
4
⭓128
⭓256
32
⭓4
32
⭓128
⭓64
⭓64
⭓4
K. pneumoniae 7650
A
5.4, 6.7
K. pneumoniae 7640
A
5.4 (TEM-1), 6.7 (KPC-2), 7.6, 8.8 (SHV-12)
K. pneumoniae 7641
A
5.2, 5.4 (TEM-1), 6.7 (KPC-2), 7.6, 8.8 (SHV-12)
32
⭓128
⭓64
16
⭓4
K. pneumoniae 7642
A
5.2, 5.4 (TEM-1), 6.7 (KPC-2), 7.6, 8.8 (SHV-12)
⭓64
⭓128
⭓64
32
⭓4
K. pneumoniae 7643
A
5.2, 5.4 (TEM-1), 6.7 (KPC-2), 8.2, 8.8 (SHV-12)
⭓64
⭓128
⭓64
⭓64
⭓4
K. pneumoniae 7645
A
5.2 (TEM-30), 5.4 (TEM-1), 6.7 (KPC-2), 7.6, 8.8 (SHV-12)
⭓64
⭓128
⭓64
32
⭓4
K. pneumoniae 7646
A
6.7 (KPC-2), 8.2, 8.8 (SHV-12)
64
⭓128
⭓64
16
⭓4
K. pneumoniae 7637
D
6.7 (KPC-2), 7.6 (SHV-11), 8.2, 8.8 (SHV-12)
64
⭓128
⭓64
32
⭓4
K. pneumoniae 7638
D
6.7 (KPC-2), 8.2, 8.8 (SHV-12)
K. pneumoniae 7639
D
6.7 (KPC-2), 7.6, 8.2, 8.8 (SHV-12)
K. oxytoca 7627
5.4 (TEM-1), 6.7 (KPC-2), 8.2
⭓64
⭓128
⭓64
32
⭓4
32
⭓128
⭓64
32
⭓4
8
⭓128
16
4
0.5
Carbapenem-Resistant Klebsiella Species • CID 2004:39 (1 July) • 57
this particular study, the experimental pI was 8.8. In addition, a b-lactamase with a pI of 5.2 was identified as TEM-30 in 3 of the K. pneumoniae isolates. The median age of the 17 patients from whom KPC-2 possessing Klebsiella species were isolated was 73 years. Nine isolates were recovered from urine cultures, with 2 representing active infection. The remaining 8 isolates were recovered from sputum samples (4 isolates, 3 representing colonization), blood samples (1 isolate), and miscellaneous sources (3 isolates, all representing colonization). Clinical information was available for 13 of the patients. All isolates were acquired nosocomially, with a median duration of hospitalization of 28 days before culture yielded the organism. Ten isolates were acquired during the hospitalization; 3 isolates were acquired either in the hospital or in a nursing home. Although previous carbapenem use was documented in only 3 of the 13 cases that were reviewed, previous broad-spectrum b-lactam administration (b-lactam/ b-lactamase–inhibitor combination antibiotics or cephalosporins) was evident in 8 cases. Three of the 4 patients with true infection survived hospitalization. Of the 2 patients with urinary tract infection, one was successfully treated with a regimen that included piperacillin-tazobactam and imipenem, and the other was successfully treated with imipenem and polymyxin. The patient with a bloodstream infection was successfully treated with imipenem and amikacin, and the patient with a lower respiratory tract infection did not survive, despite receiving therapy with ciprofloxacin and polymyxin. DISCUSSION With increasing rates of resistance of gram-negative bacilli to cephalosporins and fluoroquinolones [28], greater emphasis has been placed on carbapenems for the treatment of serious nosocomial infections. The emergence of carbapenem-resistant gram-negative nosocomial pathogens has been generally confined to P. aeruginosa and A. baumannii. To date, only rare isolates of carbapenem-resistant P. aeruginosa from the United States have been found to possess a carbapenem-hydrolyzing b-lactamase [29]. The finding of an enzyme with efficient activity against carbapenems in isolates of Klebsiella species from several hospitals in New York City further threatens the usefulness of this class of antibiotic. Identifying isolates possessing KPC-2 may be difficult when current standard methods of testing antibiotic susceptibility are used. The presence of KPC-2 in K. pneumoniae may increase the MIC of imipenem, but not to the level of frank resistance [17]. Therefore, strains carrying this enzyme may only be recognized as ESBL-producing isolates. In fact, 4 imipenemresistant isolates from our report were not identified as resistant by 2 clinical microbiology laboratories. In most isolates of Klebsiella possessing KPC-2, MICs of imipenem have been 2–8 mg/
58 • CID 2004:39 (1 July) • Bradford et al.
mL [16, 17], which is around the NCCLS breakpoint of 4 mg/ mL. However, use of an imipenem breakpoint of 2 mg/mL to screen for these isolates has been suggested [16]. The fact that many of our isolates were nosocomially acquired in hospitals that were not experiencing overt outbreaks of infection due to carbapenem-resistant Klebsiella species suggests that decreased carbapenem susceptibility due to KPC-2 is underrecognized. KPC-2 has also been recovered in isolates from Maryland and Boston [16, 17, 19, 20], and the closely related enzyme KPC-3 has been recovered in Manhattan and New Jersey [21, 22]. These findings suggest that the evolution of carbapenemases is a regional problem. The genetic strain typing data in this study demonstrated spread of a predominant strain of K. pneumoniae carrying KPC-2 to multiple hospitals in our area; this strain accounts for 15% of ESBL-possessing K. pneumoniae in Brooklyn [3]. However, several unique strains were also recovered, and other studies have confirmed that KPC-2 resides on a transmissible plasmid [17–20]. The development of carbapenem resistance in Klebsiella species typically involves several mechanisms. The presence of an efficient carbapenem-hydrolyzing b-lactamase is certainly an important contributing factor. All of our isolates also contained other b-lactamases, although none is known to possess significant hydrolytic activity against carbapenems. Loss of outer membrane proteins, together with class A or class C b-lactamases, also contributes to carbapenem resistance in Klebsiella species [5–7, 15, 22]. One particular porin, OmpK37, may be important for entry of carbapenems into Klebsiella species [30]. Carbapenem resistance may also be mediated by efflux systems (in P. aeruginosa) [31] and altered penicillin-binding proteins (in Proteus mirabilis) [32]; whether these are contributory factors in Klebsiella species will require further study. In addition to the KPC-2 enzyme, several ESBLs were detected in our isolates of K. pneumoniae. Furthermore, TEM30, an inhibitor-resistant TEM variant, was detected in 3 strains. To our knowledge, this is the first report of an inhibitor-resistant enzyme found in isolates originating in the United States. Inhibitor-resistant TEM-type b-lactamases have been reported widely in Europe but less so elsewhere [33]. In this study, the K. pneumoniae strains that possessed TEM-30 had multiple blactamases, including the ESBL SHV-12 and the class A carbapenem-hydrolyzing b-lactamase KPC-2. The combination of multiple b-lactamases can often render a strain resistant to the b-lactam/b-lactamase combinations [5]. Therefore, it is likely that inhibitor-resistant TEM variants are present and are underreported in the United States among multidrug-resistant isolates, such as our K. pneumoniae strains. Therapy for infections caused by imipenem-resistant Klebsiella species will be difficult. The isolates in this report were resistant to all b-lactam antibiotics, and most were resistant to
aminoglycosides and fluoroquinolones. Whether polymyxin will provide an effective therapy remains to be determined. The emergence and spread of this enzyme in our region has created serious dilemmas regarding detection, therapy, and control.
15.
16.
Acknowledgments Financial support. Research funding from Merck, Pfizer, and Wyeth Pharmaceuticals (D.L. and J.Q.); financial support from BMA Medical Foundation, the Beatrice Snyder Foundation, and Agnes Varis (C.U. and N.M.).
17.
18.
19.
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