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

Abstract

A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 g/ml. The ␤-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 ␤-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of bla SHV and bla TEM genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the ␤-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, bla KPC-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 ␤-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing ␤-lactamase, Sme-1, from Serratia 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 ␤-lactamase, KPC-1, although alterations in porin expression may also play a role.The carbapenems, such as imipenem and meropenem, are used with increasing frequency in the United States and elsewhere for the treatment of multiresistant gram-negative nosocomial pathogens (21,29,30). Resistance to carbapenems is uncommon in enteric organisms; however, resistance can arise by three known mechanisms. First, high-level production of a chromosomal AmpC cephalosporinase combined with decreased outer membrane permeability due to loss or alteration of porins can result in carbapenem resistance. This has been shown for Enterobacter cloacae (28, 54), Enterobacter aerogenes (9, 10, 13, 23), Proteus rettgeri (54), Citrobacter freundii (32), Escherichia coli (11, 64), and Klebsiella pneumoniae (5, 7, 16). The second mechanism is production of a ␤-lactamase that is capable of hydrolyzing carbapenems (8,30,58) The third mechanism of resistance involves changes in the affinity of the target enzymes, the penicillin binding proteins, for carbapenems (15,70).In this study, a K. pneumoniae strain manifesting carbapenem resistance was collected through project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) (4,20) and analyzed for its mechanism(s) of carbapenem resistance. The results presented suggest that the carbapenem resistance phen...

Prevalencia de bacterias Gram negativas portadoras del gen blaKPC en hospitales de Colombia

“…La presencia del gen bla KPC se determinó por medio de PCR según el protocolo de Yigit, et al (16). Se preparó una suspensión de una colonia en agua ultrapura y se usó 1 µl como plantilla para la reacción de PCR.…”

“…Desde su primera detección en Klebsiella pneumoniae en 1996 (16), las KPC se han reportado como causantes de epidemias dentro y fuera de Estados Unidos (17)(18)(19)(20)(21)(22)(23). En Colombia, el primer reporte de una KPC se hizo en K. pneumoniae en 2006 (14) y en 2007 se reportó por primera vez en el mundo en Pseudomonas aeruginosa (17).…”

Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

“…pneumoniae possessing bla KPC-2 and E. coli with the pBR322-catI-bla KPC-2 plasmid were a kind gift from Dr. Fred Tenover from the Centers for Disease Control and Prevention, Atlanta, GA. 9 The cloning of bla KPC-2 into pBC SK (þ) phagemid (Stratagene, La Jolla, CA) and into pET24a(þ) plasmid (Novagen, Darmstadt, Germany) is previously described. 14 WT and Trp105 variants of pBR322-catI-bla KPC-2 and pBC SK (þ) bla KPC-2 were expressed in E. coli DH10B cells (Invitrogen, Carlsbad, CA).…”

“…5,7,8 KPC-2 manifests a very broad substrate profile including penicillins, extended-spectrum cephalosporins, cephamycins, and carbapenems. 9,10 Moreover, Papp-Wallace et al showed that this enzyme is resistant to b-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam), which are typically given in combination with a b-lactam for the treatment of infections caused by b-lactamase producing pathogens. 11 Understanding the structural requirements that allow for this broad spectrum is critical for future b-lactam and b-lactamase inhibitor design.…”

Detection of bla KPC-2 in Proteus mirabilis in Brazil

“…Klebsiella pneumoniae carbapenemase (KPC)-type enzymes were fi rst reported in carbapenem-resistant K. pneumoniae strains in North Carolina, United States (1) . Until 2005, the geographical distribution of these enzymes in members of the family Enterobacteriaceae -K. pneumoniae, in particular -was limited to the eastern part of the United States.…”