CTX-M β-Lactamase–producing Klebsiella pneumoniae in Suburban New York City, New York, USA

CTX-M extended-spectrum β-lactamase (ESBL)–producing Klebsiella pneumoniae isolates are infrequently reported in the United States. In this study, we analyzed nonduplicate ESBL-producing K. pneumoniae and Escherichia coli clinical isolates collected during 2005–2012 at a tertiary care medical center in suburban New York City, USA, for the presence of blaCTX-M, blaSHV, blaTEM, and blaKPC genes. Despite a high prevalence of blaCTX-M genes in ESBL-producing E. coli since 2005, blaCTX-M genes were not detected in K. pneumoniae until 2009. The prevalence of CTX-M–producing K. pneumoniae increased significantly over time from 1.7% during 2005–2009 to 26.4% during 2010–2012 (p<0.0001). CTX-M-15 was the dominant CTX-M genotype. Pulsed-field gel electrophoresis and multilocus sequence typing revealed high genetic heterogeneities in CTX-M–producing K. pneumoniae isolates. This study demonstrates the recent emergence and polyclonal spread of multidrug resistant CTX-M–producing K. pneumoniae isolates among patients in a hospital setting in the United States.


8-year period
. Microbiological characteristics of CTX-M ESBL-producing K. pneumoniae isolates were examined, and certain clinical/epidemiologic features of patients with these isolates were analyzed.

Bacterial Isolates and Phenotypic Detection of ESBLs
Nonduplicate K. pneumoniae clinical isolates were recovered from patient specimens during January 2005-July 2012 at the clinical microbiology laboratory of Westchester Medical Center. These included 208 bla KPC -negative non-K. pneumoniae carbapenemase (non-KPC) ESBL-producing or third-generation cephalosporin-resistant K. pneumoniae isolates and 228 KPC (bla KPC -positive)-producing K. pneumoniae isolates. In addition, 163 nonduplicate ES-BL-producing E. coli clinical isolates from the same period were also analyzed for comparison. Isolates were randomly selected to span the entire study year with an approximately equal number of isolates per quarter; only 1 isolate from each patient was chosen and tested. The center is a 643bed academic tertiary-care medical center in Westchester County, New York. The Institutional Review Board of New York Medical College approved this study.
The bacterial isolates were identified and evaluated for antimicrobial drug susceptibility with the MicroScan Walk-Away 96 system (Siemens, Sacramento, CA, USA). ESBL production was phenotypically confirmed by a double-disk or broth microdilution method for suspected ESBL isolates according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (19). The antimicrobial drug susceptibility of CTX-M-producing K. pneumoniae isolates against selected antimicrobial drugs was also assessed with standardized CLSI disk diffusion and Etest methods. Bacterial isolates were refrigerated on nutrient agar slants or were frozen (-80°C) in MicroBank cryovials containing 20% glycerol (Pro-Lab Diagnostics, Round Rock, TX, USA). For antimicrobial drug susceptibility testing of frozen isolates, fresh subcultures were used per CLSI guidelines.

PCR Detection of bla CTX-M , bla SHV , bla TEM , and bla KPC Genes
For PCR, bacterial genomic DNA was extracted directly from colonies on nutrient slants or from fresh subcultures grown on Trypticase soy agar with 5% sheep blood (TSA II, BBL, Sparks, MD, USA) by boiling a dense suspension of an approximately no. 1 McFarland standard in sterile distilled water. As the DNA template in the PCR assays, 2-3 μL of the boiled cell suspension was used. PCR amplification of bla CTX-M , bla SHV , bla TEM , and bla KPC genes in K. pneumoniae and E. coli clinical isolates was performed by using a consensus primer pair specific to each type of β-lactamase as described (20)(21)(22). A multiplex PCR was developed and used for simultaneous detection of bla CTX-M (551 bp) and bla TEM (972 bp) genes. Two PCRs were performed for bla SHV -ESBL and bla KPC, respectively. PCRs were carried out by using the HotStart DNA polymerase master mix (QIA-GEN, Germantown, MD, USA) with 30-35 cycles at an annealing temperature of 50°C for bla CTX-M and bla TEM, and 52°C for bla SHV and bla KPC. PCR products were analyzed by agarose gel electrophoresis or by using the QIAxcel system (QIAGEN). The specificity of PCR amplicons on representative isolates was confirmed by DNA sequencing.

DNA Sequencing
For DNA sequencing, PCR products were purified by using the PCR Purification kit (QIAGEN) or the ExoSAP-IT PCR Clean-up kit (Affymetrix, Cleveland, OH, USA), according to the manufacturer's instructions. The purified DNA amplicons were sequenced by using an ABI Prism BigDye Terminator (version 1.1) cycle sequencing ready reaction kit on the ABI Prism 3730xl or ABI 3500xl DNA Analyzers (Applied Biosystems, Foster City, CA, USA) in-house, or by a commercial facility (GeneWiz, South Plainfield, NJ, USA). The CTX-M, TEM, and SHV gene sequences were compared with sequences in GenBank by using the NCBI basic local alignment search tool (www. ncbi.nlm.nih.gov/BLAST).

Multilocus Sequence Typing
Multilocus sequence typing (MLST) was performed by using primers and conditions as described by Diancourt et al. (23). PCR products from MLST were sequenced as described above. Allelic profiling and sequence types (STs) were determined by querying the K. pneumoniae MLST database maintained by the Pasteur Institute (www.pasteur.fr/ recherche/genopole/PF8/mlst/Kpneumoniae.html).

Pulsed-field Gel Electrophoresis (PFGE)
Pulsed-field gel electrophoresis (PFGE) on CTX-M ESBL-producing K. pneumoniae isolates representing each CTX-M genotype was performed as described (24). The GelCompare II software, (version 2.0; Applied Maths, Austin, TX, USA) was used to calculate the Dice similarity coefficients and generate dendrograms by cluster analysis with the unweighted-pair group method using average linkages. Pulsotype designations were assigned at the ≥80% profile similarity level.

CTX-M in KPC-producing K. pneumoniae Clinical Isolates
Two hundred twenty-eight KPC-producing K. pneumoniae isolates from 2005 to 2012 were examined by PCR for detection of bla CTX-M genes. All K. pneumoniae isolates were positive for the bla KPC gene by PCR as described (22). None was positive for the bla CTX-M gene.

Clinical and Microbiological Characteristics of CTX-M-producing K. pneumoniae
Selected clinical/epidemiologic features of the 25 patients with CTX-M ESBL-producing K. pneumonia and certain microbiological characteristics of the isolates are shown in Table 3, Appendix (wwwnc.cdc.gov/EID/ article/19/11/12-1470-T3.htm). Mean patient age was 56 years, and 13 (52%) of the patients were male. Sixteen patients (64%) had bacteriuria. CTX-M-producing K. pneumoniae isolates were recovered from 13 (52%) patients within 72 hours of hospital admission; however, 18 (72%) of these patients had been hospitalized in the 8 months before the current admission.
All 25 CTX-M-producing K. pneumoniae isolates examined were resistant to cefotaxime, and all but 1 isolate  showed higher MICs of cefotaxime than of ceftazidime. The 50% minimum inhibitory concentration (MIC 50 ) for cefotaxime among these isolates was >256 µg/mL. By contrast, the MIC 50 and 90% inhibitory concentration for ceftazidime were 16 µg/mL and 128 µg/mL, respectively. Two CTX-M-producing K. pneumoniae isolates (8.0%) were susceptible (MIC ≤4 µg/mL) and 5 isolates (20%) were intermediate in susceptibility (8 µg/mL) to ceftazidime according to the 2010 revised CLSI breakpoints ( Figure 1). In addition, we determined the susceptibilities of 22 CTX-M-producing K. pneumoniae isolates against cefotaxime and ceftazidime by using the standard disk diffusion method. All CTX-M-producing K. pneumoniae isolates examined were resistant to cefotaxime by disk diffusion (mean inhibitory zone size 8.3 mm; range 6-13 mm). Two of these isolates were susceptible (≥21 mm) and 5 had intermediate (18-20 mm) susceptibility to ceftazidime by disk diffusion (Table 3, Appendix). The disk diffusion results showed a category agreement with the Etest MIC of 100% for cefotaxime and 90.9% for ceftazidime with 2 minor errors.

PFGE and MLST Analysis of CTX-M-producing K. pneumoniae
Of 17 representative CTX-M-producing K. pneumoniae isolates analyzed by PFGE, 8 different pulsotypes (PF1-8) were identified with Dice coefficients of ≥80% similarity ( Figure 2). Ten of 17 K. pneumoniae isolates examined belonged to 3 major groups (PF3, PF4, PF5) with 3-4 isolates in each group. The remaining pulsotypes contained only 1 or 2 K. pneumoniae isolates. No clear temporal relationship was shown among the highly related isolates.
MLST was performed on 18 CTX-M-producing K. pneumoniae isolates. These isolates were selected to represent different CTX-M genotypes, pulsotypes, antimicrobial susceptibility profiles, and years of isolation. Twelve STs were recognized for the K. pneumoniae isolates examined (Table 3,
Our data provide strong evidence for the recent, rapid emergence, and polyclonal spread of the CTX-M-1 group, especially CTX-M-15 ESBL-producing K. pneumoniae in a US hospital setting. In this study, 24 (32). Why CTX-M-14 is absent in the ESBL-producing E. coli and K. pneumoniae isolates from the New York, NY, metropolitan area is unknown. Because CTX-M-15producing K. pneumoniae isolates may exhibit significantly higher resistance rates to ciprofloxacin and pipercillin-tazobactam than CTX-M-14-producing isolates (27,28), CTX-M genotypes and their antimicrobial drug profiles should be monitored among CTX-M-producing E. coli and K. pneumoniae isolates in regions where they are emerging.
We investigated the genetic relatedness of CTX-Mproducing K. pneumoniae isolates by PFGE and MLST. Of the 17 representative isolates examined by PFGE, 8 different pulsotypes were determined. Similarly, 12 MLST STs were identified for the 18 CTX-M-producing isolates analyzed. Our data, in combination with findings from other groups (1), suggest that CTX-M-producing K. pneumoniae isolates are genetically heterogeneous. The emergence and polyclonal spread of CTX-M-producing K. pneumoniae likely occurred among isolates with diverse genetic backgrounds. This hypothesis contrasts with findings regarding KPC-producing K. pneumoniae: a clonal spread of KPCproducing K. pneumoniae isolates belonging to the ST258 lineage was observed by us (33) and Pournaras et al. (34). In clinical strains, CTX-M-encoding genes have commonly been located on plasmids that vary in size from 7 kb to 160 kb (2). Plasmid-mediated transmission of CTX-M genes in Enterobacteriaceae that involves several motile  genetic elements has been described (2,35,36). Given the dominance of CTX-M-15 genotypes among genetically heterogeneous K. pneumoniae isolates, our study also implies the probable horizontal transfer of a genetic element carrying bla CTX-M among K. pneumoniae isolates. Of the 12 STs determined for the CTX-M ESBL-producing K. pneumoniae isolates, ST11, ST15, ST17, ST48, ST147, and ST258 have been reported in CTX-M-positive K. pneumoniae in Spain, Hungary, or Korea (28,37,38). Among these, only ST17 was reported among CTX-Mproducing K. pneumoniae isolates in Canada (39). In this study, we determined the STs among CTX-M-producing K. pneumoniae in the United States and document the existence of 6 STs (ST16, ST252, ST280, ST392, ST437, ST792) in CTX-M-producing K. pneumoniae not previously described.
The CTX-M-producing K. pneumoniae isolates evaluated in this study showed several notable epidemiologic, clinical, and microbiological features. First, most CTX-M-producing isolates were recovered from patients with bacteriuria, which is similar to that observed for infections caused by CTX-M-producing E. coli in New York, NY, (9,10). Although CTX-M-producing K. pneumoniae was isolated in clinical specimens collected within 72 hours of hospitalization in about half of the patients, 18 (72%) of 25 patients had been hospitalized in the prior 8 months. This factor highlights the potential for acquiring CTX-M-producing K. pneumoniae in health care settings and differs from the experience with CTX-M-producing E. coli that are associated with infections arising in the community setting unrelated to exposure to health care facilities (26). Second, the CTX-M-producing K. pneumoniae study isolates exhibited high rates of resistance to gentamicin (68%), trimethoprim-sulfamethoxazole (96%), and tetracycline (80%), in addition to resistance to ciprofloxacin (88%) and pipercillin-tazobactam (64%) as described previously in Europe and Asia (27,28,37). Whether such high rates of resistance are associated with the dominant spread of CTX-M-15-producing, rather than CTX-M-14-producing, K. pneumoniae, in these patients is not known. The coexistence of CTX-M ESBL and TEM-1 and SHV-type β-lactamases in these isolates may have also contributed to the observed high rate of antimicrobial drug resistance. All except 1 of our CTX-M-positive K.   pneumoniae isolates produced SHV-and CTX-M-type ESBLs. These findings have clinical implications for selecting empiric antimicrobial drug therapy when infection caused by ESBL-producing K. pneumoniae is suspected. The rapid emergence of such CTX-M-producing K. pneumoniae isolates, mainly in US hospitals, is also raising new concerns for public health and infection control practice. Third, none of the 228 KPC-producing K. pneumoniae isolates examined carried bla CTX-M . Coexistence of bla KPC and bla CTX-M has only been reported in KPC-producing K. pneumoniae in China (40). Whether certain genetic mechanisms prevent KPC-producing K. pneumoniae from acquiring bla CTX-M is unclear.
This study reveals the rapid emergence and polyclonal spread of CTX-M-producing K. pneumoniae in patients in Westchester County, New York. A limitation of our study is that the clinical isolates were collected from patients at a single tertiary-care medical center. Investigations of CTX-M-producing K. pneumoniae isolates from a variety of geographic regions should be undertaken to clarify the epidemiology and clinical and public health effects of the emergence of CTX-M-producing K. pneumoniae in the United States.