P-lactamase gene

The synthesis of the carbapenam-3-carboxylic acid 36 <03JA15746> as well as a study on carbapenem biosynthesis have been documented <03JA8486>. The cephalosporin derivative 37 has been prepared and its use as a novel fluorogenic substrate for imaging P-lactamase gene expression demonstrated <03JA11146>. The nucleus of the carbacephem antibiotic loracarbef has been synthesized in a highly efficient and enantioselective fashion from 25,3iS-2-amino-3-hydroxy-6-heptenoic acid, which was derived from enzyme-catalyzed... 

Fig. 1. Schematic diagram of the human RBP expression vector, pOmp-RBP RBP, cDNA for human retmol-bmding protein, Omp, bacterial outer membrane signal sequence, RBS, nbosome-binding site AMPICILLIN, the p-lactamase gene. The arrow indicates the direction of the coding sequence.

P-Lactamase Sensitivity. Although carbapenems have remained relatively insensitive to the hydrolytic action of many clinically relevant P-lactamases, a number of carbapenem-hydrolyzing enzymes have been reported in recent years. Carbapenem-hydrolyzing p-lactamases include the more recently described penicillin-interactive proteins.(lO) The number of these enzymes relative to the variety of other p-lactamases remains low, only 17 carbapenem-hydrolyzing enzymes were included in the recent compilation of 190 functionally or molecularly distinct p-lactamases.(ll) Most carbapenem-hydrolyzing p-lactamases confer resistance not only to carbapenems, but also to other p-lactams. Since the majority of the carbapenem-hydrolyzing p-lactamase genes are chromosomally encoded this has certainly mediated the spread of these enzymes, with a concomitant slow increase in P-lactamase-mediated resistance to carbapenems in clinical applications. 

P-Lactamases are enzymes that hydrolyze the P-lactam ring of P-lactamantibiotics (penicillins, cephalosporins, monobactams and carbapenems). They are the most common cause of P-lactam resistance. Most enzymes use a serine residue in the active site that attacks the P-lactam-amid carbonyl group. The covalently formed acylester is then hydrolyzed to reactivate the P-lacta-mase and liberates the inactivated antibiotic. Metallo P-lactamases use Zn(II) bound water for hydrolysis of the P-lactam bond. P-Lactamases constitute a heterogeneous group of enzymes with differences in molecular structures, in substrate preferences and in the genetic localizations of the encoding gene (Table 1). 

Class C Serine p-lactamases AmpC enzymes of coti, Shigella spp., Enterobacterspp., C. freundii, M. morganii, Providencia spp. and Serratia spp. cephalos-porinases with wide spectrum of activity CMY, LAT, BIL, MOX, ACC, FOX and DHA types. All genes are ampC genes that have been mobilized by transfer to plasmid DNA. 

Resistance to antibiotics is usnally due to the acquisition of genes that express enzymes that can inactivate the antibiotics (e.g. P-lactamase degrades the lactam ring in penicillin (see below) or that can modify the structure of proteins that are necessary for the antibiotic to enter the... 

Genes encoding extended spectrum p-lactamases confer resistance to p-lactams... 

Gene encoding for the New Delhi metaUo-p-lactamase-1 confers resistance to almost all p-lactams British Society for Antimicrobial Chemotherapy Ciprofloxacin... 

The most common mechanism of bacterial resistance to P-lactam antibiotics such as the penicillins and cephalosporins is the synthesis of P-lactamases that cleave an amide bond in the antibiotics to generate inactive products (Wiedemann et al., 1989). Genes encoding P-lactamases can be found on the bacterial chromosome or on plasmids. The active site serine P-lactamases belong to a larger family of penicillin-recognizing enzymes that includes the penicillin binding proteins (Joris et al., 1988). All of these enzymes contain the active site serine as well as a conserved triad of K(S/T)G between the active site serine and the C-terminus (Joris et al, 1988). 

The class A P-lactamases are a subset of the active-site serine P-lactamases. TEM-1 P-lactamase is a class A enzyme encoded by the ft/ajEM-l gene that is present on the transposons Tn2 and Tn3 (Datta et al, 1965). Epidemiological studies have shown that TEM-1 is the most common plasmid-mediated P-lactamase and is therefore a major determinant of bacterial resistance to P-lactam antibiotics (Wiedemann et al, 1989). Compounding the problem of resistance is the discovery that TEM-1 mutant variants with altered substrate specificity have been identified in natural isolates (Jacoby and Medieros, 1991). These variant enzymes contain from one to three amino acid substitutions that enable the enzyme to hydrolyze the newer extended-spectrum cephalosporin antibiotics such as cefotaxime and ceftazidime (Jacoby and Medieros, 1991). Thus, the selective pressure of antibiotic therapy le s to die creation of new enzymes with expanded hydrolytic capabilities. 

Fig. 16. Map of the plasmid used for expression of flavocytochrome 62 in Escherichia coli. The flavocytochrome 62 coding region (CYB2) is located between a strong E. coli promoter (p) and terminator (t). Selection is based on ampicillin resistance conferred by the /3-lactamase gene (bla). coRI, BglU, and HindW. cleavage sites are indicated ori, origin of replication cat, chloramphenicol acetyltransferase coding sequence rbs, ribosome-binding site.

Continuing use of the third-generation cephalosporins and the introduction of p-lactamase inhibitor combinations (clavulanate with amoxycillin or ticarcillin, sulbactam with ampicillin, and tazobactam with piperacillin see section 4.2) resulted in the appearance of plasmids encoding class C P-lactamases. After several unconfirmed reports, the first proof that a class C P-lactamase had been captured on a plasmid came in 1990 when transmissible resistance to a-methoxy and oxyimino-P-lactams was shown to be mediated by an enzyme whose gene was 90% identical to the ampC gene of E. cloacae. They have subsequently been found worldwide. Strains with plasmid-mediated AmpC enzymes are typically resistant to aminopenicillins (ampicillin or amoxycillin), carboxypenicillins (carbenicillin or ticarcillin) and ureidopenicillins (piperacillin). The enzymes also provide resistance to the oxyimino cephalosporins (ceftazidime, cefo-... 

S. aureus transposons are small mobile elements that often encode resistance genes (P-lactamase, resistance to erythromycin and tetracycline). All the transposons encode a transposase gene, and the product of this gene catalyses excision and/or replication of the element, as well as integration. Horizontal transfer of transposons to other S. aureus cells is presumably mediated by another MGE that is transferred, most likely a plasmid transferred by transduction or conjugation. Conjugative transposons have also been described in S. aureus. However, it is not clear if native conjugative transposons are found in S. aureus (Novick 1990). 

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