MECHANISM OF RESISTANCEThe bacterial cells produce resistance against antibiotics by three mechanisms:Preventing the antibiotic attack to the target site by mechanism of altered permeability(via efflux pumps) By transforming the target site By the inactivation of antibiotics (by enzymes)11. ENZYMATIC ACTION: The ?-lactamses classes of enzymes are responsible for this activity. They are also called as pencillinase or cephalosporinase. Penicillinase is mostly active against penicillin whereas cephalosporinase are active against cephalosporins. Sometimes both types are present in the same organism. It is one of the defense mechanism against antibiotics in gram negative bacteria. ?- lactamases cleaves the ?-lactam ring of pencillin and other antibiotics1. They are classified into four classes (A to D). They all have mechanism that involves the nucleopilic attack of ?- lactam antibiotics on the active site serine which results in acyl-enzyme intermediate 3. This is a type nucleophilic substitution reaction in which ?-lactam ring act as a nucleophile. This intermediate is hydrolyzed by base activated water molecule. Generally ?-lactamases inhibitors are given in combination with ?-lactam antibiotics to counteract this resistance.Extended spectrum beta lactamases (ESBLs) intercede protection from all pencillins, third generation cephalosporins. They are for the most part distinguished in E.coli yet additionally have been found in enterobacteriaceae. 2. TARGET ALTERATION:The second mechanism includes target modification so that the antibiotic can’t bind legitimately. Due to the imperative cell elements of the objective locales, creatures can’t sidestep antimicrobial activity by abstaining from them completely. Generally the target for ?-lactam antibiotics is to inhibit cell wall synthesis. This is achieved by their capacity to bind covalently with radiolabelled penicillin. These proteins to which antibiotics (?-lactam antibiotics) bind are called Pencillin Binding Protein (PBP). A given life form contains four to eight PBPs with sub-atomic sizes of 35 to 120 kDa. There is no fundamental connection between identically numbered PBPs of two random organisms, although taxonomically related organisms have comparable PBPs. There are two types of PBPs in an organism based on their molecular weight- Low molecular weight and high molecular weight PBP. Most of the microorganisms are resistant to penicillins because of the alteration of PBPs. This alteration mostly occurs due to procured mutation or due to any gene transfer. Resistance to macrolides is due to the attainment of one of 21 erm genes. These genes codes for rRNA methylases which are the enzymes for the methylation of adenine residues in 23s rRNA. These inhibits the binding of macrolides to 50s ribosomal subunit 4. PBP2a belong to the class of high molecular weight PBPs. Most of the PBPs have high transpeptidase activity which is required for the crosslinking of membranes proteins. This transpeptidase activity of PBPS has been inhibited by methicillin but they were not able to inhibit PBP2a because they largely depend on trans glycosylase activity. PBP2a is encoded by a mecA gene which is present at the 30kb DNA segment. These are present in the chromosome of MRSA. This is an additional gene present in MRSA which are found to be absent in MSSA (methicillin susceptible staphylococcus aureus). Vancomycin was one of the important drugs which were used for the treatment against MRSA infections, but unfortunately in the last few years MRSA has acquired resistance against vancomycin. This is due to presence of van A which caused alteration in the vancomycin binding site. It is accompanied with three fold increase in the production PBP2a and PBP2′. Similarly most of the drugs including oxacillin became ineffective due to the presence of PBP2a 5. Their major fuction is to provide the transpeptidase and transglycosylase activity which are important for the cross-linking of cell wall. These are inhibited by ?-lactam antibiotics in susceptible species.The other target alterations include alteration in protein synthesis, alteration DNA synthesis. The mechanism of action of macrolides and lincosamide is different from that of ?-lactam antibiotics. They inhibit the protein synthesis by binding with 50s ribosomal subunit. But MRSA shows resistance toward macrolides and lincosamide by the alteration in 23s ribosomal component of 50s ribosomal subunit. These occurs during the post transcriptional period where the 23s rRNA component get altered and therefore the macrolides cannot bind to the 50s subunit. The drugs which act by inhibiting the dna replication and transcription are the quinolines especially fluoroquiolines. They mainly inhibit the dna gyrase and topoisomerase enzymes. The resisitance to quinolines in mrsa occurs by mutation in the structural gene which alters both the enzymes. This reduces the affinity of enzymes to quinolines 1.PREVENTING THE ANTIBIOTIC ATTACK TO THE TARGET SITE (Efflux Pumps)A drug has to reach its specific target to produce its therapeutic action. Drug resistance can be acquired by preventing the antibiotic from reaching its target site. This can be achieved by means of efflux pumps. Efflux pumps are membrane proteins that export antibiotics out of the cell. A drug concentration inside the cell depends on its permeability through the cell membrane. Resistance to penicillin in gram negative bacteria occurs mainly by this mechanism. The outer membrane of bacteria contains aqueous channels formed by proteins called porins which allows the rapid entry of most of the antibiotics. Mutations in the gene coding for porins can prevent the entry antibiotics into the cell. Efflux pumps are one of the important systems which are involved in the expulsion of tetracycline from the cell 6. It is one of the most identified and studied mechanism by which tetracycline resistance is caused. The tetracycline accumulation in bacteria is related with energy supplied in the cell. The studies have shown that tetracycline expelled out of the cell in presence of energy and its accumulation increases when energy is not supplied. This was carried out by specific proteins present in the outer membrane called tet protein. The efflux system by the tet protein is indeed a antiport system which export one molecule of tetracycline out of the cell for one proton which is entering into the cell. Efflux pumps may be of single or multi component pumps. The presence of multi drug efflux pumps is the reason for multi-drug resistance in most of the bacteria. Efflux pumps are regulated by specific genes which can themselves be a target for the antibacterial agents. They are mostly regulated by global receptors which include marA, soxS, rob. They are also regulated by two component system. Any mutations in these genes can reduce the resistance and therefore it increases the susceptibility of organisms to antibiotics. Efflux inhibitors can be used for this purpose.