Resistance Plasmid

Bacteria produce chromosomady and R-plasmid (resistance factor) mediated P-lactamases. The plasmid-mediated enzymes can cross interspecific and intergeneric boundaries. This transfer of resistance via plasmid transfer between strains and even species has enhanced the problems of P-lactam antibiotic resistance. Many species previously controded by P-lactam antibiotics are now resistant. The chromosomal P-lactamases are species specific, but can be broadly classified by substrate profile, sensitivity to inhibitors, analytical isoelectric focusing, immunological studies, and molecular weight deterrnination. Individual enzymes may inactivate primarily penicillins, cephalosporins, or both, and the substrate specificity predeterrnines the antibiotic resistance of the producing strain. Some P-lactamases are produced only in the presence of the P-lactam antibiotic (inducible) and others are produced continuously (constitutive). 

More recently additional systems have been developed that rely on other DBDs. Golemis and co-workers developed a system that uses the DBD from the bacteriophage X repressor cl protein [25]. Interestingly, the plasmid encoding the cl fusion protein carries the zeoR gene, which makes both yeast and E. coli containing the plasmids resistant to zeocin. Also, a system has been developed that uses the DBD from the human estrogen receptor [26]. 

Table 2.1 Expression conditions for recombinant proteins Construct Plasmid Resistance...

Most plasmids are topologically closed circles of DNA. They can be separated from the bulk of the chromosomal DNA by virtue of their resistance to alkaline solution. The double-stranded stmcture of DNA is denatured at high pH, but because the two strands of the plasmid are topologically joined they are more readily renatured. This property is exploited in rapid procedures for the isolation of plasmid DNA from recombinant microorganisms (5,6). 

This resistance, inducible by low concentrations of dalbaheptides, is plasmid mediated and is transferable. Concomitant with the induction of resistance is the appearance or increased expression of a protein having a molecular weight of either 39,500 or 39,000. The enzymatic activity of this material has been postulated (112). Although the mechanism of resistance induction by dalbaheptides is unknown, different dalhabaheptides have different induction capacity. Vancomycin (39) is the most powerful inducer teicoplanin is a very weak inducer. 

Resistance to Tetracyclines. The tetracyclines stiU provide inexpensive and effective treatment for several microbial infections, but the emergence of acquired resistance to this class of antibiotic has limited their clinical usehilness. Studies to define the molecular basis of resistance are underway so that derivatives having improved antibacterial spectra and less susceptibiUty to bacterial resistance may be developed. Tetracyclines are antibiotics of choice for relatively few human infections encountered in daily clinical practice (104), largely as a result of the emergence of acquired tetracycline-resistance among clinically important bacteria (88,105,106). Acquired resistance occurs when resistant strains emerge from previously sensitive bacterial populations by acquisition of resistance genes which usually reside in plasmids and/or transposons (88,106,107). Furthermore, resistance deterrninants contained in transposons spread to, and become estabUshed in, diverse bacterial species (106). 

Over 4 decades, between 1960 and 2000, the development of new antibiotics used well characterized basic structures for partial synthetic modifications, primarily to overcome resistance by increasing the pharmacodynamic properties and, secondarily, to improve the pharmacokinetic profile of older compounds. However, bacteria rapidly responded by acquiring additional genetic alterations either as mutations or by accumulating resistance genes as part of mobile genetic elements ( integrons) on transferable resistance plasmids. 

Antibiotic Resistance. Figure 1 According to Bush, Jacoby and Medeiros [2] four molecular classes of (3-lactamases can be discriminated based upon biochemical and molecular features. Classes 1, 2, and 4 included serine-proteases, while metallo enzymes are included in class 3. The substrate spectrum varies between different subclasses and the corresponding genes can be part of an R-plasmid leading to a wider distribution or are encoded chromosomally in cells of specific species. 

Resistance to the A compound of streptogramins is mediated by products of the vat genes (virginiamycin acetyliransferase), of which at least five molecular classes are known (vatA-E). These genes are located on plasmids of Enterococcus faecium and frequently in combination with a gene encoding a streptogramin hydrolase (vga/vgb) in staphylococci. 

Bacteria can develop resistance to antimicrobial agents as a result of mutational changes in the chromosome or via the acquisition of genetic material (resistance genes carried on plasmids or transposons or the recombination of foreign DNA into the chromosome) (Fig. 2). 

Another group of antibiotics that can be inactivated by hydrolysis are 14- and 15- membered macrolides [2]. Esterases cleave the lactone ring. The plasmid encoded ere genes are found in members of the Enter-obacteriaceae and increase the intrinsic resistance. Furthermore, these esterases can also be found in some isolates of erythromycin resistant staphylococci. 

Resistance to streptogramin type B antibiotics can be mediated in staphylococci and enterococci by plasmids carrying a vgb gene [2]. The Vgb enzyme is a lyase that linearizes the cyclic hexadepsipeptide by cleavage of the ester bond via an elimination reaction. 

It has been shown that an AAC enzyme variant (AAC (6 )-Ib-cr, cr for ciprofloxacin resistance) found in various Enterobacteriaceae is capable of A-acetylating fluoroquinolones with an unmodified piperazinyl substituent at the amino nitrogen. Although the increase in MIC is low, this plasmid encoded quinolone resistance determinant can augment further development of clinically relevant resistance. 

Nucleotidylation - the addition of adenylate-residues by Lnu enzymes - can also be the cause of resistance to lincosamide antibiotics in staphylococci and enterococci. A plasmid encoded ADP-ribosylating transferase (Arr-2) that leads to rifampicin resistance has been detected in various Enterobacteriaceae as well as in Pseudomonas aeruginosa. 

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