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Journal of Antimicrobial Chemotherapy logoLink to Journal of Antimicrobial Chemotherapy
. 2009 Jan 20;63(3):427–437. doi: 10.1093/jac/dkn547

Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA

Andrea Endimiani 1,2, Andrea M Hujer 1, Federico Perez 3, Christopher R Bethel 1, Kristine M Hujer 1,2, Jennifer Kroeger 4, Margret Oethinger 5, David L Paterson 6, Mark D Adams 7, Michael R Jacobs 8, Daniel J Diekema 4, Gerri S Hall 5, Stephen G Jenkins 9, Louis B Rice 1, Fred C Tenover 10, Robert A Bonomo 1,11,12,*
PMCID: PMC2640158  PMID: 19155227

Abstract

Background

The emergence of blaKPC-containing Klebsiella pneumoniae (KPC-Kp) isolates is attracting significant attention. Outbreaks in the Eastern USA have created serious treatment and infection control problems. A comparative multi-institutional analysis of these strains has not yet been performed.

Methods

We analysed 42 KPC-Kp recovered during 2006–07 from five institutions located in the Eastern USA. Antimicrobial susceptibility tests, analytical isoelectric focusing (aIEF), PCR and sequencing of bla genes, PFGE and rep-PCR were performed.

Results

By in vitro testing, KPC-Kp isolates were highly resistant to all non-carbapenem β-lactams (MIC90s ≥ 128 mg/L). Among carbapenems, MIC50/90s were 4/64 mg/L for imipenem and meropenem, 4/32 mg/L for doripenem and 8/128 for ertapenem. Combinations of clavulanate or tazobactam with a carbapenem or cefepime did not significantly lower the MIC values. Genetic analysis revealed that the isolates possessed the following bla genes: blaKPC-2 (59.5%), blaKPC-3 (40.5%), blaTEM-1 (90.5%), blaSHV-11 (95.2%) and blaSHV-12 (50.0%). aIEF of crude β-lactamase extracts from these strains supported our findings, showing β-lactamases at pIs of 5.4, 7.6 and 8.2. The mean number of β-lactamases was 3.5 (range 3–5). PFGE demonstrated that 32 (76.2%) isolates were clonally related (type A). Type A KPC-Kp isolates (20 blaKPC-2 and 12 blaKPC-3) were detected in each of the five institutions. rep-PCR showed patterns consistent with PFGE.

Conclusions

We demonstrated the complex β-lactamase background of KPC-Kp isolates that are emerging in multiple centres in the Eastern USA. The prevalence of a single dominant clone suggests that interstate transmission has occurred.

Keywords: carbapenemases, ESBLs, Enterobacteriaceae, PFGE, rep-PCR

Introduction

The emergence of KPC carbapenemases in strains of Enterobacteriaceae and Pseudomonas aeruginosa is attracting significant attention. In current surveys, Klebsiella pneumoniae is the most common pathogen harbouring blaKPC genes.1–5 Additionally, blaKPC-containing K. pneumoniae (KPC-Kp) isolates are becoming endemic in certain hospitals and are responsible for increasing numbers of outbreaks in several healthcare facilities located in the Eastern USA,6–8 Israel9,10 and Greece.11,12 Recently, sporadic KPC-Kp isolates were detected in Central and South America,5,13,14 the Far East15–17 and Europe.18–20

In the clinical laboratory, KPC-Kp isolates demonstrate resistance or reduced susceptibility to all β-lactams as well as many other classes of antimicrobial agents (i.e. fluoroquinolones, aminoglycosides and sometimes polymyxins).4,7,21 Therefore, the emergence and spread of this multidrug-resistant (MDR) phenotype has created serious treatment and infection control problems.22,23 Detection of KPC-mediated carbapenem resistance in these isolates also poses a significant challenge for many clinical laboratories.21,24,25

Studies that examine the clonal relationship of large collections of KPC-Kp isolates from geographically separate institutions are still lacking. Earlier efforts investigating the clonal relatedness of KPC-Kp used isolates that were obtained from single hospitals or from the same geographic region (e.g. Tel Aviv and New York City).6–10 Moreover, previous analyses were performed using PFGE, ribotyping or randomly amplified DNA.6–9 The DiversiLab™ Strain Typing System, (Bacterial BarCodes, bioMérieux, Athens, GA, USA) is a semi-automated rep-PCR method able to analyse large numbers of isolates rapidly.26 Despite the literature regarding the use of the rep-PCR for epidemiological studies, the DiversiLab™ System has not yet been tested for K. pneumoniae as well as for KPC-Kp isolates.

Little is known regarding the presence of other β-lactamases or the genetic environment of isolates possessing blaKPC genes. Moland et al.27 reported a unique KPC-Kp strain producing at least eight different β-lactamases. Other studies have discovered a limited number of bla genes in addition to blaKPC.7,10,15,24,28,29 In a study performed by Bradford et al.,29 the β-lactamases possessed by 18 KPC-Kp isolates were characterized by PCR and cloning. Although three strains possessed two TEM-type enzymes, only one SHV-type β-lactamase was found in each of the 18 strains.

Taken together, the above considerations compelled us to characterize the β-lactamase background of KPC-Kp isolates from several institutions in order to: (i) understand the evolution of the molecular epidemiology of KPC-Kp in the Eastern USA; (ii) assess the in vitro efficacy of antibiotic combinations (i.e. β-lactam/β-lactamase inhibitor combinations); and (iii) compare the ability of DiversiLab™ Strain Typing System versus PFGE to characterize KPC-Kp isolates. To these ends, we determined the β-lactam resistance phenotypes, β-lactamase genotypes and clonality of a large set of KPC-Kp isolates recently collected from five major healthcare institutions located in the Eastern USA. We discovered that KPC-Kp isolates frequently belong to a single genotype and possess a complex β-lactamase background, in the context of significant resistance to penicillins, cephalosporins, carbapenems and β-lactam/β-lactamase inhibitor combinations, limiting the choices available for antibiotic therapy.

Materials and methods

Clinical isolates

Forty-two KPC-Kp strains collected during a previous study were analysed.30 Isolates were detected from January 2006 to October 2007 at Mount Sinai Medical Center in New York City (MSMCNY, n = 24), University of Pittsburgh Medical Center (UPMC, n = 4) and three Cleveland institutions including University Hospitals Case Medical Center (UHCMC, n = 2), Cleveland Clinic (CC, n = 9) and Louis Stokes Veterans Affairs Medical Center (LSVAMC, n = 3).

PCR and sequencing of β-lactamase genes

Specific primers were used to amplify and sequence the blaKPC genes [Table S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. KPC-Kp isolates were further investigated by PCR amplification to detect other resistance determinants, including those for other carbapenemases, blaESBL genes (where ESBL stands for extended-spectrum β-lactamase) and blaAmpC genes. The complete list of genes investigated and the corresponding set of primers used for PCR and DNA sequencing analysis are reported in Table S1.

DNA was obtained by suspending two to three colonies of each test isolate grown on MacConkey agar plates in 500 µL of nuclease-free water (UBS Corporation, Cleveland, OH, USA) and heating at 90°C for 10 min using a dry bath incubator. Samples were spun at 10 000 rpm for 10 min and the resulting supernatant was diluted 1:10 with nuclease-free water. These samples were used as the bacterial DNA template for PCR assays. Amplification reactions were performed using the high-fidelity rTth DNA Polymerase XL (GeneAmp XL PCR Kit; Applied Biosystems, Foster City, CA, USA). Temperature cycling conditions included an initial denaturation at 94°C for 1 min followed by 30 cycles of 94°C for 30 s, annealing for 30 s (see Table S1 for the temperature of each primer set), with an extension at 72°C for 30 s. Cycling was followed by a final extension at 72°C for 10 min. Samples were incubated in the PTC-225 Peltier Thermal Cycler (MJ Research, Waltham, MA, USA). Reaction mixture (5 µL) containing the PCR product was mixed with 2 µL of UltraClean Gel Dye (Mo Bio Laboratories Inc., Carlsbad, CA, USA) and analysed by electrophoresis in a 1% Ultrapure MB Grade agarose gel (USB Corporation).

The remaining amplified product (20 µL) was purified using Exonuclease I and Shrimp Alkaline Phosphatase before DNA sequencing, according to the manufacturer's instructions (ExoSAP-IT protocol; USB Corporation). Samples were incubated at 37°C for 30 min, at 45°C for 15 min and at 80°C for 15 min to degrade the unincorporated primers and nucleotides. Sequencing reactions were performed using BigDye v1.1 Sequencing Kits (Applied Biosystems, Foster City, CA, USA) and sequence data were acquired on a 3730xl DNA Analyzer (Applied Biosystems). DNA traces were interpreted by Lasergene 7.2 (DNASTAR, Madison, WI, USA). The final β-lactamase amino acid sequences were determined using the ExPASy Proteomics Server (http://ca.expasy.org) and compared with those previously described (http://www.lahey.org/studies/webt.htm).

Cloning and sequencing of multiple β-lactamase alleles

In cases where multiple alleles were suspected (i.e. double ‘spikes’ in the DNA sequencing traces at a single location), a new PCR product for each isolate was obtained as described above; however, the reactions were limited to 18 cycles of amplification. The product was purified using the QIAquick PCR Purification Kit (Qiagen Sciences, Valencia, CA, USA) and subsequently cloned into a pCR-XL-TOPO vector, according to the manufacturer's instructions (TOPO XL PCR Cloning Kit; Invitrogen Corporation, Carlsbad, CA, USA). Plasmids were electroporated into competent ElectroMAX Escherichia coli DH10B cells (Invitrogen Corporation) and selected on Luria–Bertani (LB) agar containing kanamycin sulphate (50 mg/L; Invitrogen Corporation). Ten random colonies for each cloned PCR product were selected and cultures were grown overnight at 37°C in 3 mL of LB broth containing kanamycin sulphate (50 mg/L). Plasmids were isolated using the Wizard Plus Miniprep Kit (Promega, Madison, WI, USA). Direct sequencing of the plasmid preparations was done using the primers previously listed (Table S1). Furthermore, specific primers for the TOPO vector were also used (M13-forward, 5′-GTAAAACGACGGCCAG-3′; M13-reverse, 5′-CAGGAAACAGCTATGAC-3′).

Three E. coli DH10B isolates containing blaSHV-11 (VA-401-t5), blaSHV-12 (VA-389-t4) or blaTEM-1 (VA-402-t3) cloned into the pCR-XL-TOPO vector and one blaKPC-2-containing K. pneumoniae strain (VA-361) were used as controls to test the reliability of the above cloning method.

Antimicrobial susceptibility testing

MICs of β-lactams were determined using the agar dilution method on cation-adjusted Mueller–Hinton agar (BBL, Becton Dickinson, Sparks, MD, USA) using a Steer's ReplicatorTM that delivers 104 cfu/10 µL spot. We tested the following antibiotics: cefoxitin (Abraxis Pharmaceutical Products, Schaumburg, IL, USA), piperacillin (Sigma Chemical Co., St Louis, MO, USA), piperacillin/tazobactam (Wyeth Pharmaceuticals Inc., Philadelphia, PA, USA), cefotaxime (Sigma Chemical Co.), ceftazidime (Sandoz GmbH, Kundl, Austria), cefepime (Bristol-Myers Squibb, Princeton, NJ, USA), aztreonam (Bristol-Myers Squibb), imipenem/cilastatin (Merck & Co. Inc., Whitehouse Station, NJ, USA), meropenem (AstraZeneca Pharmaceuticals, Italy), ertapenem (Merck & Co. Inc.) and doripenem (Ortho-McNeil Pharmaceutical Inc., Raritan, NJ, USA). Lithium clavulanate (GlaxoSmithKline, Research Triangle Park, NC, USA) and sodium tazobactam (Wyeth Pharmaceuticals Inc., Pearl River, NY, USA) were tested at a constant concentration of 4 mg/L in combination with cefepime, imipenem, meropenem, ertapenem and doripenem. For non-β-lactam antibiotics (e.g. aminoglycosides and quinolones), antimicrobial susceptibility testing was performed using the Microscan Autoscan System (Siemens, West Sacramento, CA, USA). Results were interpreted according to the CLSI criteria31 except for doripenem, for which the FDA breakpoint for Enterobacteriaceae (i.e. ≤0.5 mg/L susceptible) was used. The following ATCC control strains were used: E. coli 25922, P. aeruginosa 27853 and K. pneumoniae 700603.

Analytical isoelectric focusing (aIEF)

aIEF was performed to identify and determine the pIs of β-lactamases expressed by the KPC-Kp isolates. Crude cell lysates were prepared using a previously described method.32 Enzyme extracts were loaded onto 5% polyacrylamide gels containing ampholines (pH range, 3.5–9.5; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and electrophoresed using a Multiphor II apparatus (Amersham Biosciences, Piscataway, NJ, USA). Gels were focused at 4°C with 8 W for 150 min. The detection of β-lactamases was performed by the addition of 1 mM nitrocefin (Becton Dickinson Biosciences, Cockeysville, MD, USA) onto the gel. The following purified β-lactamase enzymes prepared by our laboratory were used as controls: TEM-1 (pI, 5.4), SHV-1 (pI, 7.6), KPC-2 (pI, 6.7) and CMY-2 (pI, 9.0).

Clonal analysis by PFGE

PFGE was performed as described previously.33,34 Whole chromosomal DNA in agarose was digested with SpeI (Sigma-Aldrich, St Louis, MO, USA) and the restriction fragments were separated in a CHEF DRII apparatus (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis, the gels were stained with ethidium bromide, illuminated under UV light and photographed. Computer-assisted (BioNumerics; Applied Maths, Kortrijk, Belgium) analysis of PFGE patterns was performed. The Dice correlation coefficient was used to analyse the similarity of the banding patterns. Strains with similarity coefficients of >80% were considered to belong to the same PFGE type (i.e. clonally related), while those with indistinguishable PFGE banding patterns (>97% similarity coefficient) were considered to be of the same subtype.35 Clustering was based on the unweighted pair-group method with arithmetic averages. The tolerance position was 1%.

rep-PCR

Genomic similarity of KPC-Kp isolates was also investigated with the DiversiLab™ Strain Typing System (Bacterial BarCodes, bioMérieux). Genomic DNA was extracted using the UltraClean™ Microbial DNA Isolation Kit (Mo Bio Laboratories). Rep-PCR was performed using the DiversiLab Klebsiella Kit (Bacterial BarCodes). Results were interpreted with DiversiLab Web-Based Software using the Pearson correlation and the modified Kullback–Leibler methods. In this study, clonally related isolates were defined as a ≥95% homology as recorded by software provided by the manufacturer.

Results

Molecular characterization of β-lactamase genes

As summarized in Table 1, sequencing analysis revealed that 25 (59.5%) strains harboured blaKPC-2 and the remaining 17 (40.5%) carried blaKPC-3. The majority of KPC-Kp strains also contained blaTEM-1 (38/42, 90.5%) and blaSHV-11 (40/42, 95.2%); blaSHV-12 was detected in 50.0% (21/42) of KPC-Kp isolates. In general, seven isolates contained other SHV variants (VA-368, blaSHV-5 and blaSHV-68; VA-373, VA-375 and VA-384, blaSHV-14; VA-391, blaSHV-77; VA-398, blaSHV-27; VA-417, blaSHV-26). The aIEF results were consistent with these findings, showing β-lactamase bands of pI 5.4 (TEM-1 enzyme), 6.7 (KPC-2,-3), 7.6 (SHV-11 enzyme) and 8.2 (SHV-12 enzyme) (Table 1). β-Lactamases migrating with pIs >8.2 were not detected. The average number of β-lactamases in KPC-Kp isolates was 3.5 (range 3–5) (Table 1). We did not detect blaCTX-M, blaIMP, blaVIM, blaCMY-2-like, blaACT-1, blaP99, blaPER-1, blaPSE, blaFOX, blaMIR or blaDHA genes among the 42 KPC-Kp isolates (Table 1).

Table 1.

Sequencing of β-lactamase genes and aIEF results for the 42 blaKPC-containing (KPC-Kp) isolates detected in five institutions in the Eastern USA

β-Lactamase genotype
Isolate KPC type TEM type SHV typea aIEF bands (pI)
VA-184 KPC-2 — SHV-11, SHV-12 5.2, 6.7, 7.6, 8.2
VA-237 KPC-2 — SHV-11, SHV-12 5.2, 6.7, 7.6, 8.2
VA-267 KPC-3 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6, 8.2
VA-357 KPC-2 — SHV-11 5.4, 6.7, 7.0, 7.6
VA-360 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6, 8.2
VA-361 KPC-2 TEM-1 SHV-11 5.4, 6.7, 7.6, 8.2
VA-362 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.0, 7.6, 8.2
VA-364 KPC-2 TEM-1 SHV-11 5.4, 6.7, 7.0, 7.6
VA-367 KPC-3 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6, 8.2
VA-368 KPC-2 TEM-1 SHV-5, SHV-68b 5.4, 6.7
VA-373 KPC-2 — SHV-11, SHV-14c 6.7, 7.0, 7.6
VA-375 KPC-3 TEM-1d SHV-11, SHV-14 5.4, 5.8, 6.7, 7.0, 7.6
VA-376 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6, 8.2
VA-378 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6, 8.2
VA-380 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6, 8.2
VA-383 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6, 8.2
VA-384 KPC-2 TEM-1 SHV-11, SHV-12, SHV-14c 5.4, 6.7, 7.6, 8.2
VA-387 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6, 8.2
VA-388 KPC-3 TEM-1 SHV-11 5.4, 6.7
VA-389 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6
VA-390 KPC-2 TEM-1 SHV-11 5.4, 6.7, 7.6
VA-391 KPC-3 TEM-1 SHV-11, SHV-12, SHV-77e 5.4, 6.7, 7.6, 8.2
VA-392 KPC-3 TEM-1 SHV-11 5.4, 6.7, 7.6, 8.2
VA-394 KPC-3 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6
VA-395 KPC-2 TEM-1d SHV-11, SHV-12 5.4, 5.8, 6.7, 7.6, 8.2
VA-396 KPC-2 TEM-1d SHV-11 5.4, 5.6, 6.7, 7.6
VA-397 KPC-3 TEM-1 SHV-11 5.4, 5.6, 6.7, 7.6
VA-398 KPC-3 TEM-1 SHV-11, SHV-12, SHV-27f 5.4, 6.7, 7.6
VA-400 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6
VA-401 KPC-3 TEM-1 SHV-11 5.4, 6.7, 7.6
VA-402 KPC-3 TEM-1 SHV-11 5.4, 5.8, 6.7, 7.6
VA-403 KPC-3 TEM-1 SHV-11 5.4, 6.7, 7.6
VA-404 KPC-2 TEM-1 SHV-11, SHV-12 5.4, 6.7, 7.6
VA-406 KPC-2 TEM-1d SHV-11, SHV-12 5.4, 5.8, 6.7, 7.6
VA-408 KPC-2 TEM-1d SHV-11, SHV-12 5.4, 5.8, 6.7, 7.6
VA-409 KPC-3 TEM-1 SHV-11 5.4, 5.8, 6.7, 7.6
VA-410 KPC-3 TEM-1d SHV-11 5.4, 5.8, 6.7, 7.6
VA-412 KPC-2 TEM-1 SHV-11 5.2, 5.4, 5.8, 6.7, 7.6, 8.2
VA-413 KPC-2 TEM-1d SHV-11 5.4, 5.8, 6.7, 7.6
VA-414 KPC-3 TEM-1d SHV-11 5.4, 5.8, 6.7, 7.6
VA-416 KPC-3 TEM-1 SHV-11 5.4, 6.7, 7.6
VA-417 KPC-3 TEM-1 SHV-26 5.4, 6.7, 7.6
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aFor all KPC-Kp isolates, the SHV PCR product was cloned and sequenced.

bSHV-68 is not characterized.

cSHV-14 is not an ESBL.52

dKPC-Kp isolates for which the TEM PCR product was cloned and sequenced.

eSHV-77 is not characterized.

fSHV-27 possesses a pI of 8.2.

Antimicrobial susceptibility tests

As shown in Table 2, KPC-Kp isolates expressed high-level resistance to all non-carbapenem β-lactams (MIC90s ≥128 mg/L), except for three isolates (VA-237, VA-387 and VA-398) that were susceptible to cefepime (MICs of 8 mg/L).

Table 2.

Antimicrobial susceptibility test results for the 42 KPC-Kp isolates detected in five institutions in the Eastern USA

MIC (mg/L)a
Isolate FOX PIP TZP CAZ CTX ATM FEP FEP/CLA FEP/TZB IPM IPM/CLA IPM/TZB MEM MEM/CLA MEM/TZB ERT ERT/CLA ERT/TZB DORb DOR/CLA DOR/TZB
VA-184 128 2048 2048 512 128 1024 32 8 16 4 2 4 4 4 4 16 8 8 4 2 4
VA-237 128 512 512 256 32 512 8 4 8 4 1 2 2 2 2 4 8 8 4 2 4
VA-267 256 2048 1024 512 64 1024 32 16 16 4 1 2 2 2 2 8 8 4 4 2 4
VA-357 256 >2048 2048 128 128 512 32 32 32 4 2 2 4 4 4 16 16 16 4 4 4
VA-360 512 >2048 2048 512 256 2048 128 64 32 32 8 16 32 16 32 128 128 128 32 32 32
VA-361 256 >2048 1024 256 64 512 32 8 16 4 2 4 4 2 4 16 16 8 4 4 4
VA-362 256 >2048 512 128 64 256 16 8 16 4 2 4 2 2 1 2 2 2 4 4 4
VA-364 256 >2048 2048 128 64 128 32 8 32 8 2 8 8 4 8 4 1 1 4 2 4
VA-367 256 1024 512 512 64 1024 16 8 16 2 0.5 0.5 1 1 0.5 4 2 2 1 1 1
VA-368 512 1024 512 32 32 512 32 32 32 4 4 4 4 4 4 16 8 8 4 4 4
VA-373 256 >2048 2048 64 32 512 16 16 16 2 2 2 4 4 4 4 4 4 4 4 4
VA-375 512 1024 1024 64 32 512 16 16 16 2 2 2 2 2 2 4 4 4 4 2 4
VA-376 1024 2048 2048 512 256 1024 128 64 64 32 8 16 32 16 32 128 128 128 32 32 32
VA-378 256 2048 1024 256 64 1024 32 8 16 4 2 4 4 2 4 16 8 8 4 2 4
VA-380 256 2048 1024 256 128 512 64 32 64 8 2 8 16 8 16 16 8 8 8 8 8
VA-383 256 2048 512 256 64 1024 16 8 8 4 1 2 4 1 2 8 4 4 2 2 2
VA-384 1024 2048 1024 256 256 1024 128 64 64 32 16 16 64 64 64 128 128 128 32 32 32
VA-387 2048 >2048 1024 128 512 2048 256 128 128 64 64 64 128 128 128 256 256 256 256 128 128
VA-388 64 2048 512 512 64 256 8 4 8 4 2 4 2 2 2 4 4 4 2 2 2
VA-389 256 2048 512 1024 64 1024 16 8 16 2 1 2 2 1 2 8 4 4 2 2 2
VA-390 1024 1024 512 256 128 512 64 64 64 32 16 16 64 64 64 128 128 128 32 32 32
VA-391 256 1024 1024 512 128 2048 16 16 16 4 2 2 4 2 4 8 4 8 4 4 4
VA-392 256 1024 512 512 64 1024 16 16 16 4 2 2 4 2 2 8 4 8 4 2 4
VA-394 128 1024 512 512 64 1024 16 16 16 4 2 2 4 2 4 8 4 4 4 4 4
VA-395 256 >2048 1024 1024 256 2048 16 16 16 4 2 2 4 2 4 8 4 8 4 2 4
VA-396 256 1024 512 512 64 256 16 16 16 2 1 2 2 2 2 8 8 8 4 2 4
VA-397 512 2048 1024 512 128 512 32 32 32 4 2 2 4 4 4 8 8 8 4 4 4
VA-398 64 1024 64 128 32 512 8 4 4 4 2 2 2 1 2 2 2 2 2 2 2
VA-400 256 1024 256 512 128 2048 16 16 16 4 2 2 8 1 4 8 8 8 4 4 2
VA-401 1024 >2048 1024 1024 512 1024 128 128 128 64 32 64 64 64 64 128 128 128 64 64 64
VA-402 256 2048 1024 128 16 512 32 32 32 8 8 2 4 4 4 8 8 8 4 4 4
VA-403 1024 2048 512 1024 256 512 128 64 64 16 16 16 16 16 16 32 32 32 16 16 16
VA-404 128 2048 1024 512 32 512 16 8 8 8 2 2 4 4 4 16 8 8 4 4 4
VA-406 1024 2048 1024 512 512 512 256 128 128 256 128 256 256 128 128 512 256 256 256 128 128
VA-408 256 2048 1024 512 64 512 16 8 16 8 1 1 4 2 2 8 4 8 4 4 4
VA-409 256 2048 1024 1024 256 512 32 16 16 2 2 2 4 4 4 8 8 4 4 4 4
VA-410 128 1024 512 256 64 256 32 16 16 4 1 2 4 1 4 8 2 4 4 2 4
VA-412 256 2048 1024 1024 64 1024 16 8 16 8 1 2 8 4 4 16 4 8 4 4 2
VA-413 1024 2048 1024 64 64 512 128 64 64 64 32 32 64 64 64 128 128 128 64 64 64
VA-414 1024 2048 1024 >1024 512 2048 128 256 128 64 16 16 64 32 32 64 64 64 32 32 32
VA-416 512 2048 1024 1024 512 512 128 64 64 64 32 32 16 16 8 32 32 32 32 32 32
VA-417 128 2048 512 128 64 512 32 16 16 8 4 8 8 4 8 8 4 4 4 2 4
MIC50 256 2048 1024 512 64 512 32 16 16 4 2 2 4 4 4 8 8 8 4 4 4
MIC90 1024 >2048 2048 1024 512 2048 128 64 64 64 32 32 64 64 64 128 128 128 32 32 32
S (%) 0.0 0.0 0.0 0.0 0.0 0.0 7.1 33.3 11.9 57.1 71.4 64.3 61.9 71.4 66.7 4.8 11.9 9.5 0.0 0.0 0.0
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FOX, cefoxitin; PIP, piperacillin; TZP, piperacillin/tazobactam; CAZ, ceftazidime; CTX, cefotaxime; ATM, aztreonam; FEP, cefepime; IPM, imipenem; MEM, meropenem; ERT, ertapenem; DOR, doripenem; CLA, clavulanate (constant concentration of 4 mg/L); TZB, tazobactam (constant concentration of 4 mg/L); S, susceptible. MICs were interpreted according to the CLSI criteria.31

aResults of β-lactam/β-lactamase inhibitor combinations were interpreted as the CLSI criteria established for the drug alone.

bInterpretative criteria have not yet been established by CLSI; therefore, we interpreted the results according to the US FDA criteria (S ≤ 0.5 mg/L).

MIC50/90 values of carbapenems were 4/64 mg/L for imipenem and meropenem, 4/32 mg/L for doripenem and 8/128 mg/L for ertapenem. Approximately two-thirds of the isolates tested were susceptible to imipenem and meropenem. All isolates were doripenem-resistant according to the US FDA breakpoints, while 57.1% were susceptible to imipenem, 61.9% to meropenem and 4.8% to ertapenem according to the CLSI criteria. The carbapenem MIC cut-off values able to accurately predict KPC production in these isolates were: imipenem, ≥2 mg/L; meropenem, ≥1 mg/L; ertapenem, ≥2 mg/L; and doripenem, ≥1 mg/L (Table 2). Combining either of the β-lactamase inhibitors, clavulanate or tazobactam, with cefepime or the carbapenems did not decrease the MICs by more than two doubling dilutions in most cases (Table 2).

Percentages of non-susceptible KPC-Kp isolates for the remaining antibiotics were as follows: gentamicin, 57.1%; tobramycin, 92.9%; amikacin, 59.5%; ciprofloxacin, 88.1%; levofloxacin, 88.1%; moxifloxacin, 85.7%; tetracycline, 11.9%; and trimethoprim/sulfamethoxazole, 97.6%. Colistin was tested at 4 mg/L, and all isolates were susceptible at this concentration.

PFGE

Using the Dice similarity coefficient to assess clonality by PFGE, we found that 32 KPC-Kp isolates belonged to the same genotype (type A), whereas the remaining 10 isolates showed <80% similarity (Figure 1). Isolates of PFGE type A were detected in collections from all five institutions. Thirteen subtypes of type A were observed, of which subtypes A1, A4, A9 and A13 were the most common. Isolates with subtypes A1, A4 and A9 were detected at the MSMCNY, whereas isolates of subtype A13 were collected at CC, UHCMC and MSMCNY (Figure 1). Interestingly, type A contains K. pneumoniae isolates possessing either blaKPC-2 or blaKPC-3 genes. However, each subtype includes strains that uniformly possess only a single blaKPC gene type. For example, subtypes A13 and A9 possess blaKPC-2, whereas subtypes A1 and A4 carry blaKPC-3.

Figure 1.

Figure 1

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PFGE patterns of the 42 KPC-Kp isolates detected in five institutions located in the Eastern USA from January 2006 to October 2007 (MSMCNY, UPMC, UHCMC, CC, LSVAMC). Thirty-two isolates showed a genomic similarity >80% according to the Dice coefficient (type A). The remaining 10 isolates showed a similarity <80% when compared with type A isolates.

rep-PCR

As shown in Figure 2(a), using the Pearson correlation, two KPC-Kp genotypes were present. The major genotype included 25 KPC-Kp isolates collected in all five institutions, whereas the second included five strains collected at the MSMCNY only. The two genotypes shared a genomic homology of 80% to 95%. In contrast, using the modified Kullback–Leibler method, only one major KPC-Kp clone (n = 34) was observed (Figure 2b).

Figure 2.

Figure 2

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Genotype analysis of the 42 KPC-Kp isolates performed with the DiversiLab™ Strain Typing System (Bacterial BarCodes, bioMérieux). (a) rep-PCR results interpreted using the Pearson correlation. Twenty-five isolates (from #3 to #27) were included in a single genotype, whereas five (from #31 to #35) were included in another clone type. (b) rep-PCR results interpreted using the modified Kullback–Leibler method. A single major KPC-Kp clone was identified among the 42 isolates analysed. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Discussion

The CDC estimates that K. pneumoniae is responsible for 8% of hospital-acquired infections and 3% of nosocomial outbreaks.36 In endemic areas, such as New York City, it is reported that more than one-third of K. pneumoniae isolates possess blaKPC genes.3 Therefore, the spread of KPC-Kp is a significant clinical and public health concern.1,2,37,38 In the present study, we analysed 42 KPC-Kp isolates recently collected in five institutions from a large geographic region to determine the number of β-lactamases that were expressed in the isolates and the genomic relatedness of them using both PFGE and rep-PCR.

Overall, four important findings emerged from our study: (i) contemporary KPC-Kp isolates possess a complex β-lactamase background (an average of 3.5 β-lactamase genes per isolate); (ii) clavulanate and tazobactam are unable to restore the activity of β-lactams against these isolates; (iii) there is evidence that KPC-Kp isolates from the five institutions in the Eastern USA are clonally related; and (iv) rep-PCR is a quick and reliable method to study KPC-Kp outbreaks.

Complex β-lactamase background

Survey studies are showing that the β-lactamase background of K. pneumoniae is becoming more complex.39–41 In our analysis, the blaKPC-2/3 carbapenemase genes detected among KPC-Kp were often accompanied by the blaTEM-1 and blaSHV-11 genes; the blaSHV-12 ESBL gene was also frequently found (50% of strains). This was previously observed by Essack et al. among blaKPC-negative K. pneumoniae isolates.39 However, it should be noted that using a standard DNA sequencing analysis method, many of the blaSHV-12-producing organisms would have been missed (during our study, 15 out of 21 blaSHV-12-positive KPC-Kp were identified as blaSHV-11-containing only; data not shown). While SHV-11 is a broad-spectrum β-lactamase (group 2b), SHV-12 is an ESBL able to confer resistance to all cephalosporins (group 2be).42 Thus, finding an additional SHV ESBL might have important clinical and epidemiological relevance. It should be noted that SHV-12 is one of the most prominent ESBLs found in the Eastern USA.43 Standard β-lactamase inhibitors were unable to lower the MICs of β-lactams for our clinical KPC-Kp isolates. However, in previous studies, clones expressing only the blaKPC gene showed MICs of β-lactam/β-lactamase inhibitor combinations three or four dilutions lower than the antibiotic alone.44,45 Therefore, we could speculate that the additional β-lactamases (e.g. SHV and TEM types) expressed by KPC-Kp isolates could counteract the activity of the commercially used β-lactamase inhibitors. For similar reasons, the escalating number of β-lactamases could also contribute to the impaired ability of clinical laboratories to identify KPC-producing isolates.21,24,25

It is interesting to observe that blaCTX-M genes were not detected in our KPC-Kp isolates. This is in agreement with the known relatively low prevalence of CTX-M ESBLs among K. pneumoniae isolates in the USA compared with Asia, Europe, Canada and South America.46 To the best of our knowledge, KPC-Kp isolates possessing blaCTX-M genes have been detected only in China, Brazil and Puerto Rico.13,14,17 It is also interesting to note that production of AmpC enzymes could be reasonably excluded on the basis of our molecular and aIEF results.

In vitro activity

The antimicrobial susceptibility testing results show that KPC-Kp isolates are frequently resistant to aminoglycosides and quinolones and extremely resistant to all non-carbapenem β-lactams (MIC90s ≥ 128 mg/L). In addition, combinations of cefepime or carbapenems with clavulanate or tazobactam are unable to lower significantly the MICs. Our data indicate that the complexity of the class A β-lactamase background limits the clinical efficacy of novel combinations of β-lactam/β-lactamase inhibitors using currently available clavulanate or tazobactam for treating infections caused by KPC-producers.

Our analysis also shows the limitations of our current CLSI breakpoints for imipenem and meropenem. Two-thirds of imipenem and meropenem MIC results for KPC-Kp strains were in the susceptible range, and 95% of ertapenem MIC results were resistant. Having some KPC-containing isolates that were ‘missed’ by an ertapenem screen is also disturbing as an important clinical predictor may be lost. Clinicians should be aware of these important limitations and, when necessary, request further testing.

Using the US FDA Enterobacteriaceae breakpoints, all isolates were doripenem-resistant. Thus, in our opinion, isolates of K. pneumoniae that exhibit imipenem, meropenem or doripenem MICs ≥1 mg/L or ertapenem MICs ≥2 mg/L should be screened by PCR for blaKPC genes or with a phenotypic method (i.e. modified Hodge test) for carbapenemase production.25

It should be noted that 10 KPC-Kp isolates (24%) included in PFGE type A expressed high-level resistance to carbapenems (i.e. MICs of imipenem ≥32 mg/L). Since production of other common carbapenemases has been excluded by molecular analysis and aIEF, this result may be due to alterations of outer membrane proteins such as OmpK35, OmpK36 and OmpK37.47,48 Strains lacking OmpK35 and OmpK36 have been reported to occur frequently among KPC-Kp strains.6,17,24,44

Clonal analysis

PFGE results show that 76% of KPC-Kp isolates were clonally related and were included in one common type (type A). Surprisingly, type A KPC-Kp was present in all five institutions studied. We also performed genotyping using rep-PCR. In our case, the modified Kullback–Leibler method of analysis of genotype data seemed to be more consistent than the Pearson correlation when compared with PFGE.

As in the case of Staphylococcus aureus,49 clonal analysis of KPC-Kp isolates is important for understanding how these organisms may be responsible for outbreaks and how these isolates are spreading in our healthcare system. We demonstrated that the majority of KPC-Kp isolates that are spreading from institution to institution in the Eastern USA originated from a single K. pneumoniae clone. We also demonstrated that the DiversiLab™ Strain Typing System can be used to quickly analyse large numbers of K. pneumoniae isolates.

Conclusions

In summary, the above results show why K. pneumoniae containing blaKPC represent a formidable therapeutic, diagnostic and clinical challenge. The complex β-lactamase background contributes to the incapacity of current β-lactams and β-lactam/β-lactamase inhibitor combinations to inhibit KPC-Kp strains. It is most concerning that a single clone may be contributing to the current outbreaks in the Eastern USA. Further studies are warranted to assess if these related isolates possess transferable resistance determinants in common (e.g. Tn4401, plasmids and the KQ element), which are important in the dissemination of the MDR phenotype in KPC-Kp.50,51

Funding

This work was supported in part by AstraZeneca (to A. E. and R. A. B.), the National Institutes of Health (grant RO1-AI063517 to R. A. B. and grant RO1-AI045626 to L. B. R.), the Veterans Affairs Merit Review Program (L. B. R. and R. A. B.) and the Geriatric Research Education and Clinical Care VISN 10 (R. A. B.).

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Transparency declarations

R. A. B. received research and speaking invites from various pharmaceutical companies. None of these poses a conflict of interest with the present work. Other authors: none to declare.

Supplementary data

Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).

Supplementary Material

[Supplementary Data]
dkn547_index.html (833B, html)

Acknowledgements

We thank Dr Philip N. Rather for his critical evaluation of our cloning and sequencing methods.

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[Supplementary Data]
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