Antimicrobial Chemotherapeutic Agents

Drug Resistance

In the clinical context an organism is said to be resistant: If it is not killed or inhibited by drug concentrations readily attainable in the patient; this usually means blood and tissue concentrations. However, an organism resistant to these may of course be sensitive to the higher concentrations attainable in urine or by topical application. Even the broadest of broad-spectrum antibacterial drugs is ineffective against some bacterial genera, against some species of other genera, and usually against some strains of species that are in general sensitive to it.

Resistance Inherent (non specific) Acquired

Inherent (non specific) Resistance Certain bacteria are, and as far as we know always have been, more or less resistant to some antibiotics. For example, gram-negative bacteria, especially Ps. aeruginosa, are inherently resistant to a number of antibiotics that are very effective against gram-positive bacteria such as penicillin G, erythromycin, lincomycin…….

Acquired Resistance When a bacterial population adapts to the presence of an antibiotic, sensitive cells are gradually replaced by resistant cells as in the presence of antibiotic. Resistant cells continue to grow at the expense of sensitive cells. When a new antibiotic is introduced into clinical practice for the treatment of infections caused by bacteria that are not inherently resistant to the drug, the majority of infections respond to the new drug.

But following months or years of continuous use, resistant strains are reported. The degree of resistance and the speed with which it develops varies with:  The organism &  The drug. Generally, the development of acquired bacterial resistance is common and must be usually expected with some exceptions.

Streptococcus pyogenes has remained sensitive to Penicillin G after 40 year's exposure to the drug. Staph aureus develops slow or multisteps resistance to penicillin, chloramphenicol and tetracycline. While Mycobacterium tuberculosis and various organisms develops sudden or one step resistance to Streptomycin.

Biochemical Mechanisms of Resistance Production of drug-inactivating enzymes Switch to alternative metabolic pathways unaffected by the drug:   Change in the antibiotic target site  Increased production of essential metabolite  . Reduction in cellular permeability to the antibiotic: 

Production of drug-inactivating enzymes  Inactivation of Aminoglycosides Inactivation of ß-lactams Inactivation of Chloramphenicol

Inactivation of Aminoglycosides Inactivation of aminoglycosides by plasmid controlled adenylating , phosphorylating or acetylating intracellular enzymes of drug resistant gram negative bacteria. Although the inactivating enzymes vary considerably in their substrate and specificities, known modifications are restricted to acetylation of amino groups and adenylylation or phosphorylation of hydroxyl groups.

Inactivation of ß-lactams Inactivation of β-lactams by β -lactamases ( penicillinase, cephalosporinase ) into penicilloic or cephalosporoic acid. β -lactamases are: Gram-positive Gram-negative Chromosomally Plasmid Constitutive Inducible

Penicilloic acid

The synthesis of gram positive β-lactamase is induced by the antibiotic themselves and is released extracellularly and destroy antibiotic in the external environment. Most strains of gram-negative cells, by contrast, synthesize β-lactamases constitutively. i.e. continuously and are not released into the external environment (cell-bound or intracellular).

 Chromosomally-mediaied β-lactamases of gram negative hydrolyze cephalosporins more rapidly than penicillins and are inhibited by cloxacillin but not by clavulanic acid. However, those of Aeromonas spp. and Klebsiella spp. are more active against penicillins and not inhibited by cloxacillin.

R1= cl , R2 = H (Cloxacillin)

Inducible types are found in microorganisms such as Pseudomonas spp Inducible types are found in microorganisms such as Pseudomonas spp., Proteus and other gram negative bacteria but infrequently in E. coli in which, as many enterobacter species, constitutive types could be isolated.

 Different types of plasmid-mediated β-lactamases were isolated. TEM type enzymes are present in almost all gram negative bacteria. These enzymes were first isolated from E. coli strains isolated, in Athens, from a young girl called Temoniera and was referred to as TEM enzyme. Electrophoretically different type was then isolated from Pseudomonas aeruginosa (TEM-2).

E. coli Klebsiella spp. H. influenza Aeromonas spp. Ps. aeruginosa OXA Klebsiella spp. SHV H. influenza ROB Aeromonas spp. AER Ps. aeruginosa LCR

Inactivation of chloramphenicol Inactivation of chloramphenicol by chloramphenicol acetyl transferase (CAT). Usually they are a plasmid-mediated enzymes which are inducible type in gram-positive bacteria but constitutive in gram-negative bacteria. These enzymes acetylates the OH groups in the side chain of the drug.

Replacement of the terminal - OH group of this side chain, which is normally the first to be acetylated by an inert fluorine atom, yields a chloramphenicol derivative that is not susceptible to attack by CAT.

Change in the antibiotic target site  Chromosomal Resistance to Aminoglycosides Resistance to Erythromycin Resistance to Sulfonamides & Trimethoprim Resistance- to some Penicillins

Chromosomal Resistance to Aminoglycosides It is associated with the loss or alteration of a specific protein in the 30S subunit of the bacterial ribosome that serve as a binding site in the susceptible organisms. Resistance to Erythromycin It is associated with alteration, of its receptor on the 5OS subunit of the ribosome.

Resistance- to some Penicillins Resistance- to some penicillins due to loss or alteration of Penicillin Binding Proteins (PBPs). Resistance to Sulfonamides & Trimethoprim Occurs by alteration of the tetrahydropteroat synthetase and tetrahydrofolate reductase, respectively, that have a much higher affinity for PABA than these drugs.

Reduction in cellular permeability to the antibiotic  Bacterial cells altering the permeability of their cell membrane making it difficult for antimicrobials to enter. This type of resistance is found in bacteria resistant to: Polymyxins. Tetracyclines. Amikacin & some aminoglycosides. Streptococci have a natural permeability barrier to aminoglycosides. This can be partly overcome by combination with cell wall active drug, e.g. (penicillin).

Switch to alternative metabolic pathways unaffected by the drug:  The organism develop an altered metabolic pathway that bypasses the reaction inhibited by the drug e.g. some sulfonamide-resistant bacteria do not require extra-cellular PABA but, like mammalian cells, can utilize preformed folic acid.

Increased production of essential metabolite :  That is competitively antagonized by the drug in sensitive cells e.g. resistance to sulfonamides may be associated with high level of bacterial synthesis of PABA.

The origin of drug resistance Non Genetic Origin Genetic Origin

Non Genetic Origin This involves metabolically inactive cells or loss of target sites. Most antimicrobial agents act effectively only on replicating cells. Mycobacteria survive for many years in tissue yet are restrained by the host's defenses and do not multiply. Such persisting organisms are resistant to treatment and cannot be eradicated by drugs. When they start to multiply they are fully susceptible to the drugs.

Loss of a particular target structure, often induced by the drug, may result in antimicrobial resistance. Exposure of some gram-positive bacteria to penicillin results in the formation of cell lacking cell wall (i.e. L-forms). These cells then are penicillin resistant, having lost the structural target site of the drug. When these organisms revert to their bacterial parent forms resuming cell wall production, they are again fully susceptible to penicillin.

Genetic Origin Chromosomal Resistance Extra Chromosomal Resistance Most drug-resistant microbes emerge as a result of genetic change and subsequent selection processes by antimicrobial drugs. The mechanisms by which genetic change occur are: Chromosomal Resistance Extra Chromosomal Resistance

Chromosomal Resistance This develops as a result of mutation in a gene locus that controls susceptibility to a given antimicrobial drug. The presence of the drug serves as a selecting mechanism to suppress susceptible organisms and favor the growth of drug resistant mutant. Spontaneous mutation occurs at a frequency of 10 7 to 10 12.

Chromosomal mutants are most commonly resistant by virtue of a change in a structural receptor for a drug as in bacterial resistance to erythromycin, lincomycin, aminoglycosides and others by alteration of their target site in susceptible cells. Prevention of the emergence of resistant mutants is one of the main indications for the clinical use of combinations of drugs.

But provided that the mechanisms of action of the two drugs are unrelated. Therefore if both drugs are given in adequate dosage, the risk of the emergence of a resistant strain is very much less than if either is used alone.

Extra Chromosomal Resistance Plasmids Transposons

Plasmids Bacteria often contain extra chromosomal DNA units known as plasmids. Some of which alternate between being free and being integrated into the chromosome. R factors are a class of plasmids that carry genes for resistance to one and often several antimicrobial drugs and heavy metals.

Plasmid genes for antimicrobial resistance often control the formation of enzymes that inactivate the antimicrobial drugs such as β-lactamases, CAT and enzymes that inactivates aminoglycosides; or enzymes that determine the active transport of tetracyclines across the cell membrane, and for others.

Transposons The drug resistance (R) genes are often part of highly mobile short DNA sequences known as transposons (Transposable elements or jumping genes) that is able to move, from one position to another, between one plasmid and another or between a plasmid and a portion of the bacterial chromosome within a bacterial cell.

Thus, transposons are able to insert themselves into many different genomic sites with no homology with them. Simple transposons (IS) only carry information concerned with the insertion function. Simple transposons (IS) (i.e. insertion sequences) have no known effects beyond transposition and inactivation of the gene (or operon) into which they may insert.

Complex or composite transposons (Tn) contain additional genetic material unrelated to transposition, such as drug-resistance genes. Such as penicillin, kanamycin, streptomycin, sulfonamides, tetracyclines, chloramphenicol ,and trimethoprim.

Mechanisms of Transmission of Genetic Material and Plasmids Transduction Transformation Conjugation Transposition

Transduction This is the main mechanism for transmission of antibiotic resistance between gram-positive cocci, and occurs in other bacterial groups. Plasmid DNA is enclosed in a bacteriophage and transferred by the virus to another bacterium of the same species e.g. the plasmid carrying the gene for β-lactamase production can be transferred from a penicillin-resistant to a susceptible staphylococcus if carried by a suitable bacteriophage.

Transformation Naked DNA passes from one cell of a species to another cell, thus altering its genotype. This can occur through laboratory manipulation and perhaps spontaneously.

Conjugation This is the commonest method by which, multi-drug resistance spreads among different genera of gram-negative bacteria. But also occurs among some gram-positive cocci.

The usual plasmid found in resistant gram-negative bacteria consists of two distinct but frequently linked elements: One or more linked genes each conferring resistance to a specific antibacterial drug (resistance determinants). A resistance transfer factor (RTF) that enable the cell to conjugate with a sensitive bacterium and to transfer to It a copy of the entire plasmid.

R-factor

The entire linked complex of RTF and resistance determinants is known as R-factor and takes the form of a double stranded circular molecule of DNA. A proportion of R-factor-bearing cells (R* cells) possess hair-like structures that extend out from the bacterial surface known as pili. The pili, whose synthesis is under the control of the RTF component of the R-factor are essential to the conjugation phenomenon with R' bacteria and the transfer of an R- factor.

Transposition A transfer of short DNA sequences (transposon) occur between a plasmid and another or between a plasmid and a portion of the bacterial chromosome within a bacterial cell.

Transferable or infective drug resistance is important for the following reasons The transferable plasmids commonly determine resistance to several unrelated drugs. Such plasmids are transferable not merely to related strains of the same species but to strains of other species and genera; for example, antibiotic-resistant but harmless organism in human or animal intestine (E. coli) can confer antibiotic resistance, by plasmid transfer, on pathogenic but previously antibiotic-sensitive bacteria of other genera which the host happens to ingest (such as typhoid or dysentery bacilli).

It is possible for multiple-resistant enterobacteria to develop in farm animals and be transmitted to man. Development of this resistance is due to the widespread use of antibiotics especially cheap types, as food supplements for young animal to accelerate their growth by partial suppression of their intestinal flora.

Specific and Cross Resistance Specific resistance: When the organism acquire resistance to a certain drug but Its susceptibility to other drugs is unaffected. Cross resistance: Microorganisms resistant to a certain drug may also be resistant to other drugs that share a mechanism of action.

Such relationship exist mainly between agents that are closely related Polymyxin B and Polymyxin E. Erythromycin and Oleandomycin. Neomycin and kanamycin. However, it may also exist between unrelated chemicals  Erythromycin-Lincomycin.

When the active nucleus of the chemicals is so similar, extensive cross-resistance is to be expected e.g. resistance to one of the tetracyclines imparts resistance to the other members of the group e.g. resistance to one sulfonamide cause resistance to hundreds of other sulfonamides.

Antibiotic Policies Abuse of antibiotics, is avoided for many reasons including the following: To prevent the emergence of antibiotic resistance. To reduce the cost of antibiotic use. To prevent antibiotic toxicity.

1 General Principles of Optimal Antibacterial Therapy Unless there is a valid reason for giving an antibiotic, the patient would probably be better off without it. Treatment of Known or Suspected Infection Prevention of Bacterial Infection Peri-operative Prophylaxis Patients at Special Risk

2 3 4 5 In cases when immediate drug treatment is necessary. It is bad treatment to use broad-spectrum antibiotics when an infective condition can be treated with a more specific agent. 4 It is essential to use bactericidal and not bacteriostatic therapy. 5 It is essential to use combination of antimicrobial drugs in certain situations.

6 For treatment of superficial infections it is important to use either antiseptic or antibiotics which are rarely or never used systemically. 7 Give enough, for long enough, and then stop treatment with the antibiotic. 8 To reduce the spread of microbial resistance, avoid the use of antibiotics as food supplement for animals or for preservation of human food stuffs, avoid liberation of antibiotic powders and solutions into the environment.

9 To reduce the emergence of antibiotic-resistant strains, an antibiotic policy has to be introduced for a hospital or area e.g., using antibiotics in rotation, keeping a particular antibiotics and permitting their use only on rare and special occasions, or insisting on combined therapy.

Drug combination To provide broad coverage. For initial (blind) therapy when the patient is seriously ill and results of cultures are pending. To provide synergism when organisms are not effectively eradicated with a single agent alone e.g., in enterococcal endocarditis both penicillin and an aminoglycoside are given because their combined effect is greater than the sum of their independent activities.

To prevent emergence of resistance, as in the treatment of tuberculosis. Inappropriate use of combinations could result in: Antagonism it occurs when a bactericidal agent is used with a bacteriostatic one as in penicillins plus tetracycline or Chloramphenicol. Sulphonamides do not antagonize penicillins, possibly because their bacteriostatic action is too low. An increase in the number or severity of adverse reactions. Increased coast.