Plasmids enzyme inactivation

The lincosamides, lincomycin and clindamycin are active against Grampositive bacteria. Plasmid-mediated inactivation from enzymatic nucleo-tidylation occurs in some staphylococci. Plasmid-encoded enzymes can modify streptogramin A (O-acetyltransferase enzyme) and streptogramin B (hydrolase enzyme involved) in S. aureus [198, 199], There is no evidence that bacteria can circumvent the action of other antibiotics for example, mupirocin is not degraded [200]. 

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). 

Plasmid- ortransposon-mediated resistance occurs by inactivation ofthe antibiotic. Fosfomycin is combined with glutathione intracellularly to produce a compound lacking in antibacterial activity. The gene encoding the enzyme catalysing this reaction has been designated/or-r. 

Open the plasmid pEx-HltetO-CAG-tetR by digestion with Ascl and BsaBl and ligate annealed shA/B into the vector. Remove self-ligated empty vector by redigestion with Ascl and BsaBl. Heat inactivate enzymes by incubation for 15 min at 75°C. 

Chloramphenicol is able to inhibit the peptidyl transferase reaction and so bacterial protein synthesis by binding reversibly to the 50s ribosomal subunit. Resistance can occur due to the plasmid-mediated enzyme chloramphenicol acetyltransferase which inactivates the drug by acetylation. Such resistance is often a part of plasmid-mediated multidrug resistance. Resistance can also occur by an altered bacterial permeability. However in most instances resistance to chloramphenicol only develops slowly and remains partial. 

Resistance occurs as the result of one or more alterations in the cellular metabolism of the bacteria both mutation and plasmid-mediated resistance occurs. These changes, which can be irreversible, include alterations in the physical or enzymatic characteristics of the enzyme or enzymes that metabolize PABA and participate in the cellular synthesis of tetrahydrofolic acid. The appearance of alternative pathways for PABA synthesis within the bacteria or the development of an increased capacity to inactivate or eliminate the sulfonamide also may contribute to bacterial cell resistance. Bacteria that can use preformed folate are not inhibited by sulfonamides. 

Two microliters of pFlK-ORF plasmid solutions prepared with the Wizard SV 96 Plasmid DNA Purification System are treated with 5 U of S ifi and 5 U of Pmel in a 10 pL reaction volume containing lx Flexi Digest Buffer at 37°C for 60 min, and then the enzymes are inactivated by incubating at 65°C for 20 min. 

These substances resemble 3-lactam molecules (Figure 43-7) but they have very weak antibacterial action. They are potent inhibitors of many but not all bacterial 3 lactamases and can protect hydrolyzable penicillins from inactivation by these enzymes. -Lactamase inhibitors are most active against Ambler class A lactamases (plasmid-encoded transposable element [ ] lactamases in particular), such as those produced by staphylococci, H influenzae, N gonorrhoeae, salmonella, shigella, E coli, and pneumoniae. They are not good inhibitors of class lactamases, which typically are chromosomally encoded and inducible, produced by enterobacter, citrobacter, serratia, and pseudomonas, but they do inhibit chromosomal 3 lactamases of bacteroides and moraxella. 

Low-level resistance to chloramphenicol may emerge from large populations of chloramphenicol-susceptible cells by selection of mutants that are less permeable to the drug. Clinically significant resistance is due to production of chloramphenicol acetyltransferase, a plasmid-encoded enzyme that inactivates the drug. 

Structures of several important aminoglycoside antibiotics. Ring II is 2-deoxystreptamine. The resemblance between kanamycin and amikacin and between gentamicin, netilmicin, and tobramycin can be seen. The circled numerals on the kanamycin molecule indicate points of attack of plasmid-mediated bacterial transferase enzymes that can inactivate this drug. , , and , acetyltransferase , phosphotransferase , adenylyltransferase. Amikacin is resistant to modification at , , , and . 

Bacterial Resistance Mechanisms. The most common resistance mechanism involves the inactivation of the aminoglycoside by reactions cat alyzed by plasmid borne enzymes. In general, amikacin and isepam icin tend to be least susceptible to inactivation by this mechanism, while netilmicin and dibekacin are intermediate and gentamicin and tobramycin are most susceptible. Less common resistance mechanisms include decreased affinity for the antibiotic by the bacterial ribosome, and decreased rate of transport into the bacterial cytoplasm. 

Chromosomal metallo-beta-lactamases that hydrolyze carbapenem antibiotics, such as imipenem, meropenem, or biapenem, are present in some Stenotro-phomonas, Bacteroides, andAeromonas strains [26], Some clinical/5, aeruginosa and Serratia marcescens isolates have a plasmid that carries metallo-beta-lactamase genes [26], These enzymes are not inactivated by inhibitors of serine-based beta-lactamases such as clavulanic acid or sulbactam analogs. Enzyme-... 

DNA suitable for the insertion of foreign DNA is known as a vector the most commonly used vectors in bacteria are plasmids. Plasmids are small (2-3 kb) loops of DNA found in bacteria and yeast. They were first discovered when it was observed that bacteria could pass antibiotic resistance from one colony to another. This process was demonstrated to be mediated by plasmids containing genes for enzymes that inactivated the antibiotics. In addition to the work on plasmids, other research laboratories have isolated enzymes that cut DNA at specific sequences (restriction endonucleases) and other enzymes that can rejoin these cuts again (DNA ligases). 

Resistance can be caused by 1) decreased uptake of drug when the oxygen-dependent transport system for aminoglycosides is absent an altered receptor where the 30S ribosomal subunit binding site has a lowered affinity for aminoglycosides plasmid-associated synthesis of enzymes (for example, acetyltransferases, nucleotidyltransferases, and phosphotransferases) that modify and inactivate aminoglycoside... 

Plasmid (or transposon)-encoded enzymes are thus responsible for the degradation of several different types of antibiotics. The inactivation of several /J-lactams, AGACs, 14-membered macrolides, other macrolides, lin-cosamides and streptogramis (MLS) and chloramphenicol is a major resistance mechanism it has yet to be shown that inactivation of other antibiotics falls into this category. 

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