James W. Biddle

Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae

ABSTRACT A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of blaSHV andblaTEM genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies withEscherichia coli showed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene,blaKPC-1, was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, fromSerratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role.

Evaluation of Methods To Identify the Klebsiella pneumoniae Carbapenemase in Enterobacteriaceae

ABSTRACT The Klebsiella pneumoniae carbapenem (KPC) β-lactamase occurs in Enterobacteriaceae and can confer resistance to all β-lactam agents including carbapenems. The enzyme may confer low-level carbapenem resistance, and the failure of susceptibility methods to identify this resistance has been reported. Automated and nonautomated methods for carbapenem susceptibility were evaluated for identification of KPC-mediated resistance. Ertapenem was a more sensitive indicator of KPC resistance than meropenem and imipenem independently of the method used. Carbapenemase production could be confirmed with the modified Hodge test.

CHARACTERIZATION OF CLINICAL ISOLATES OF KLEBSIELLA PNEUMONIAE FROM 19 LABORATORIES USING THE NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS EXTENDED-SPECTRUM BETA-LACTAMASE DETECTION METHODS

ABSTRACT Extended-spectrum β-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were ≥2 μg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of ≥5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by ≥3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were ≥32 μg/ml, suggesting either the presence of an AmpC-type β-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a β-lactamase with a pI of ≥8.3 suggestive of an AmpC-type β-lactamase; 6 of the 7 isolates were shown by PCR to contain bothampC-type and blaOXA genes. The IEF profiles of the remaining 16 isolates showed a variety of β-lactamase bands, all of which had pIs of ≤7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type,blaOXA, and blaCTX-Mgenes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained β-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained β-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.

Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing.

Detecting β-lactamase–mediated carbapenem resistance among Klebsiella pneumoniae isolates and other Enterobacteriaceae is an emerging problem. In this study, 15 blaKPC-positive Klebsiella pneumoniae that showed discrepant results for imipenem and meropenem from 4 New York City hospitals were characterized by isoelectric focusing; broth microdilution (BMD); disk diffusion (DD); and MicroScan, Phoenix, Sensititre, VITEK, and VITEK 2 automated systems. All 15 isolates were either intermediate or resistant to imipenem and meropenem by BMD; 1 was susceptible to imipenem by DD. MicroScan and Phoenix reported 1 (6.7%) and 2 (13.3%) isolates, respectively, as imipenem susceptible. VITEK and VITEK 2 reported 10 (67%) and 5 (33%) isolates, respectively, as imipenem susceptible. By Sensititre, 13 (87%) isolates were susceptible to imipenem, and 12 (80%) were susceptible to meropenem. The VITEK 2 Advanced Expert System changed 2 imipenem MIC results from >16 μg/mL to <2 μg/mL but kept the interpretation as resistant. The recognition of carbapenem-resistant K. pneumoniae continues to challenge automated susceptibility systems.

High-level tetracycline resistance in Neisseria gonorrhoeae is result of acquisition of streptococcal tetM determinant.

Recently, strains of Neisseria gonorrhoeae have been isolated which are highly resistant to tetracycline (MICs of 16 to 64 micrograms/ml). This resistance was due to the acquisition of the resistance determinant tetM, a transposon-borne determinant initially found in the genus Streptococcus and more recently in Mycoplasma hominis, Ureaplasma urealyticum, and Gardnerella vaginalis. In N. gonorrhoeae, the tetM determinant was located on a 25.2-megadalton plasmid. This plasmid arose from the insertion of tetM into the 24.5-megadalton gonococcal conjugative plasmid. The tetM determinant could be transferred to suitable recipient strains of N. gonorrhoeae by both genetic transformation and conjugation. Images

Carbapenem-Resistant Strain of Klebsiella oxytoca Harboring Carbapenem-Hydrolyzing beta-Lactamase KPC-2

ABSTRACT We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32μ g/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five β-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the blaSHV and blaTEM genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the β-lactamase with a pI of 6.5. The blaKPC-2 determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing β-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase KPC-2. This β-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination.

Detection of the Klebsiella pneumoniae Carbapenemase Type 2 Carbapenem-Hydrolyzing Enzyme in Clinical Isolates of Citrobacter freundii and K. oxytoca Carrying a Common Plasmid

ABSTRACT The Klebsiella pneumoniae carbapenemase (KPC) was detected in carbapenem-resistant isolates of Citrobacter freundii and Klebsiella oxytoca recovered from different patients in a Michigan hospital. Restriction analysis and hybridization with a KPC-specific probe showed the blaKPC-2 genes of these two genera of the family Enterobacteriaceae are carried on a common plasmid.

Identification of multiple clones of extended-spectrum cephalosporin-resistant Streptococcus pneumoniae isolates in the United States.

We characterized 12 isolates of Streptococcus pneumoniae with various levels of susceptibility of penicillin and extended-spectrum cephalosporins by antimicrobial susceptibility patterns, serotypes, ribotypes, chromosomal DNA restriction patterns by pulsed-field gel electrophoresis, multilocus enzyme electrophoresis patterns, penicillin-binding protein (PBP) profiles, and DNA restriction endonuclease cleavage profiles of pbp1a, pbp2x, and pbp2b. Seven cefotaxime-resistant (MIC, > or = 2 micrograms/ml) serotype 23F isolates were related on the basis of ribotyping, pulsed-field gel electrophoresis, and multilocus enzyme electrophoresis, but they had two slightly different PBP patterns: one unique to strains for which the MIC of penicillin is high (4.0 micrograms/ml) and one unique to strains for which the MIC of penicillin is low (0.12 to 1.0 micrograms/ml). The pbp1a and pbp2x fingerprints were identical for the seven isolates; however, the pbp2b fingerprints were different. An eighth serotype 23F isolate with high-level resistance to cephalosporins was not related to the other seven isolates by typing data but was a variant of the widespread, multiresistant serotype 23F Spanish clone. The PBP profiles and fingerprints of pbp1a, pbp2x, and pbp2b were identical to those of the Spanish clone isolate. An additional serotype 6B isolate with high-level resistance to cephalosporins had unique typing profiles and was unrelated to the serotype 23F cephalosporin-resistant isolates but was related on the basis of genetic typing methods to a second serotype 6B isolate that was cephalosporin susceptible. The serotype 6B isolates had different PBP profiles and fingerprints for pbp1a, but the fingerprints for pbp2x and pbp2b were the same.

Characterization of the Extended-Spectrum β-Lactamase Reference Strain, Klebsiella pneumoniae K6 (ATCC 700603), Which Produces the Novel Enzyme SHV-18

ABSTRACT Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-β-lactams. A consistent reduction in the MICs of oxyimino-β-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum β-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single β-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of ablaSHV gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a blaSHV-specific probe indicated that a blaSHV gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this blaSHV-related gene revealed that it differs from blaSHV-1 at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.

Evidence of interhospital transmission of extended-spectrum β-lactam-resistant Klebsiella pneumoniae in the United States, 1986 to 1993

Background: In addition to single-hospital outbreaks, interhospital transmission of extended-spectrum β-lactam-resistant (ESBLR) Klebsiella pneumoniae has been suspected in some reports. However, these studies lacked sufficient epidemiological information to confirm such an occurrence. Methods: We reviewed the surveillance data reported to the National Nosocomial Infections Surveillance (NNIS) System during 1986 to 1993 for K pneumoniae isolates and their susceptibility to either ceftazidime, cefotaxime, ceftriaxone, or aztreonam. Pulsed-field gel electrophoresis (PFGE) was used to study available ESBLR K pneumoniae isolates. Results: Among 8,319 K pneumoniae isolates associated with nosocomial infections, 727 (8.7%) were resistant or had intermediate-level resistance to at least one of these antibiotics. One hospital (hospital A) accounted for 321 isolates (44.2%) of ESBLR K pneumoniae . During 1986 to 1993, the percentage of K pneumoniae isolates that were ESBLR increased from 0 to 57.7% in hospital A, from 0 to 35.6% in NNIS hospitals 0 to 20 miles from hospital A (area B), and from 1.6 to 7.3% in NNIS hospitals more than 20 miles from hospital A, including hospitals located throughout the United States. Analysis of PFGE restriction profiles showed a genetic relationship between a cluster of isolates from hospital A and some isolates from one hospital in area B, and consecutive admission in these two hospitals was confirmed for two patients from whom isolates were available. Conclusions: These data provide evidence of interhospital transmission of ESBLR K pneumoniae in one region of the United States and stress the interrelationship between hospitals when trying to control antimicrobial resistance.