Friday, September 2, 2011

Resistent to Carbeneme: Carbapenemases

Carbapenemases represent the most versatile family of β-lactamases, with a breadth of spectrum unrivaled by other β-lactam-hydrolyzing enzymes. The term “carbapenemases” suggesting that carbapenems are but one segment of their substrate spectrum (Rasmussen and Bush, ‎1997).
Classification of β-lactamases can be defined according to two properties, functional and molecular. A new β-lactamase was analyzed biochemically by isolating the protein and determining its isoelectric point, followed by enzymatic studies to determine substrate hydrolysis and inhibition characteristics (Sykes and Matthew, ‎1976 ). The relative rates of hydrolysis for a broad spectrum of β-lactam substrates, and inhibitor profiles, allowed for the classification of the new β-lactamase. This functional classification process evolved over many years into a widely accepted scheme currently dividing the known β-lactamases into four major functional groups (groups 1 to 4), with multiple subgroups under group 2 that are differentiated according to group-specific substrate or inhibitor profiles (Bush et al., ‎1995). In this functional classification scheme, carbapenemases are found primarily in groups 2f and 3 (Queenan and Bush, 2007).
Classification based on amino acid homology has resulted in four major classes (Ambler, 1980), which correlate well with the functional scheme but lack the detail concerning the enzymatic activity of the enzyme. Molecular classes A, C, and D include the β-lactamases with serine at their active site, whereas molecular class B β-lactamases are all metalloenzymes with an active-site zinc. Carbapenemases, β-lactamases with catalytic efficiencies for carbapenem hydrolysis, resulting in elevated carbapenem MICs, include enzymes from classes A, B, and D (Queenan and Bush, 2007).
I- Molecular class A carbapenemases
Class A serine carbapenemases of functional group 2f have appeared sporadically in clinical isolates since their first discovery over 20 years ago (Medeiros and Hare, 1986). These β-lactamases have been detected in Enterobacter cloacae, Serratia marcescens, and Klebsiella spp. as single isolates or in small outbreaks (Nordmann et al., ‎1993). Bacteria expressing these enzymes are characterized by reduced susceptibility to imipenem, but MICs can range from mildly elevated (e.g., imipenem MICs of ≤ 4 μg/ml) to fully resistant (Queenan and Bush, 2007).
Three major families of class A serine carbapenemases include the NMC/IMI, SME, and KPC enzymes. Their hydrolytic mechanism requires an active-site serine at position 70 in the Ambler numbering system for class A β-lactamases (Ambler et al., ‎1991). All have the ability to hydrolyze a broad variety of β-lactams, including carbapenems, cephalosporins, penicillins, and aztreonam, and all are inhibited by clavulanate and tazobactam, placing them in the group 2f functional subgroup of β-lactamases. A fourth member of this class, the GES β-lactamases, was originally identified as an ESBL family, but over time variants were discovered that had low, but measurable, imipenem hydrolysis. This subgroup of GES enzymes is also classified as functional group 2f carbapenemases (Queenan and Bush, 2007).
a. Chromosomally encoded enzymes: SME, NMC, and IMI
The antibiotic resistance profile of strains expressing the chromosomal group 2f β-lactamases is distinctive: carbapenem resistance coupled with susceptibility to extended spectrum cephalosporins. SME-1 (for “Serratia marcescens enzyme”) was first detected in England from two S. marcescens isolates that were collected in 1982 (Yang et al., 1990). The IMI (for “imipenem-hydrolyzing β-lactamase”) and NMC-A (for “not metalloenzyme carbapenemase”) enzymes have been detected in rare clinical isolates of E. cloacae in the United States, France, and Argentina (Pottumarthy et al., ‎2003).
The genes for these three β-lactamases are all chromosomally located, with no evidence of mobile element association, a fact that may have contributed to their rarity. More recently, however, genes encoding IMI-2 β-lactamases were found on plasmids in Enterobacter asburiae from United States rivers and on a plasmid from an E. cloacae isolate from China (Yu et al., 2006).
b. Plasmid-encoded enzymes: KPC and GES
Two characteristics separate the KPC (for “Klebsiella pneumoniae carbapenemase”) carbapenemases from the other functional group 2f enzymes. First, the KPC enzymes are found on transferable plasmids; second, their substrate hydrolysis spectrum includes the aminothiazoleoxime cephalosporins, such as cefotaxime. Although the KPC β-lactamases are predominantly found in K. pneumoniae, there have been reports of these enzymes in Enterobacter spp. and in Salmonella spp. (Bratu et al., 2005). KPC carbapenemases hydrolyze β-lactams of all classes, with the most efficient hydrolysis observed for nitrocefin, cephalothin, cephaloridine, benzylpenicillin, ampicillin, and piperacillin. Imipenem and meropenem, as well as cefotaxime and aztreonam, were hydrolyzed 10-fold-less efficiently than the penicillins and early cephalosporins. Weak but measurable hydrolysis was observed for cefoxitin and ceftazidime, giving the KPC family a broad hydrolysis spectrum that includes most β-lactam antibiotics (Queenan and Bush, 2007).
The GES/IBC family of β-lactamases is an infrequently encountered family that was first described in 2000 with reports of IBC-1 (for “integron-borne cephalosporinase”) from an E. cloacae isolate in Greece (Giakkoupi et al., 2000) and GES-1 (for “Guiana extended spectrum”) in a K. pneumoniae isolate from French Guiana (Poirel et al., 2000). The genes encoding the GES family of enzymes were located in integrons on plasmids. Because the enzymes had a broad hydrolysis spectrum that included penicillins and extended-spectrum cephalosporins, they were initially classified as extended-spectrum β-lactamases (Giakkoupi et al., 2000). Their hydrolysis spectrum was expanded in 2001 to include imipenem, with the report of GES-2 in a clinical isolate of Ps. aeruginosa (Poirel et al., 2001).
II- Class B metallo-β-lactamases
This class of β-lactamases is characterized by the ability to hydrolyze carbapenems and by its resistance to the commercially available β-lactamase inhibitors but susceptibility to inhibition by metal ion chelators. The substrate spectrum is quite broad; in addition to the carbapenems, most of these enzymes hydrolyze cephalosporins and penicillins but lack the ability to hydrolyze aztreonam (Queenan and Bush, 2007). The common feature of the MBLs is the principal zinc-binding motif histidine-X-histidine-X-aspartic acid (H-X-H-X-N). The binding of zinc to the active site coordinates the arrangement of two H2O molecules which are important for the hydrolysis. Hence, chelation of zinc, which can be performed with substances such as EDTA or mercaptopropionic acid (MPA), impairs β-lactam hydrolysis and therefore restores susceptibility to the carbapenem (Lee et al., 2003).
a. Chromosomally encoded metallo-β-lactamases
Chromosomally encoding MBL bacteria include Bacillus cereus, Bacillus anthracis, Stenotrophomonas maltophilia, Aeromonas hydrophilia, Chryseobacterium meningosepticum, Chryseobacterium indologenes, Legionella gormannii, Caulobacter crescentus, Myroides spp., and Janthinobacterium lividium, Flavobacterium johnsoniae and S. fonticola (Walsh et al., 2005).
b. Transferable metallo-β-lactamases
The most common metallo-β-lactamase families include the VIM, IMP, GIM, and SIM enzymes, which are located within a variety of integron structures, where they have been incorporated as gene cassettes. The genes encoding MBLs are in nearly all cases located on class 1 integrons, although class 3 integrons harboring IMP-type enzymes have been reported (Walsh et al., 2005). Integrons are genetic elements consisting of two conserved regions (5’CS and 3’CS), as well as a variable region where gene cassettes encoding resistance determinants can be inserted. The 5’CS part consists of an integrase (intI), an adjacent recombination site (attI) and a common promoter for all gene cassettes in the variable region, whereas the 3’CS region usually consists of a partially deleted gene encoding a quaternary ammonium compound efflux pump (qacEΔ1) fused with a sulfonamide resistance gene (sul1). The promoter region consist of two promotors that can exist in strong or weak variants. Apart from the combination of promotors, the distance between the promoter and the respective gene cassettes will also influence the impact of the promoter on gene transcription. In the variable region MBL and aminoglycoside resistance genes are uasually found, and between the genes recombination sites (attC) which is known also by (59 be) (be: base element) can be found (fig 8). Integration of new gene cassettes, which is mediated by the integrase, can take place either between a gene cassette and att1 or between two gene cassettes containing attC, although the later event probably ocuurs rarely. Integrons are not themselves mobile genetic structures, but they are usually located on transposons or plasmids (Hall and Collis, 1995). When these integrons become associated with plasmids or transposons, transfer between bacteria is readily facilitated. Transferable imipenem resistance was first detected in Japan, initially in a Ps. aeruginosa isolate, in 1990 (Watanabe et al., ‎1991), followed by a second report of a transferable carbapenemase in B. fragilis (Bandoh et al., 1992).
Read more at Manual of Antibiotics: Method of Actions, Mechanisms of Resistance and Relations to Health Care associated Infections [Kindle Edition]

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