Antibiotics & Bacterial Genetics PDF

ANTIMICROBIAL AGENT ❑ Any chemical or drug used to treat an infectious Antibiotics & Bacterial disease, either by inhibiting or killing the pathogens in vivo. Genetics ❑ Either kills microorganisms or stops their growth Fle...

ANTIMICROBIAL AGENT ❑ Any chemical or drug used to treat an infectious Antibiotics & Bacterial disease, either by inhibiting or killing the pathogens in vivo. Genetics ❑ Either kills microorganisms or stops their growth Fleming and Penicillin ANTIMICROBIAL AGENT 1. kill or inhibit the growth of pathogens 2. cause no damage to the host 3. cause no allergic reaction to the host 4. stable when stored in solid or liquid form 5.remain in specific tissues in the body long enough to be effective 6.kill the pathogens before they mutate and become resistant to it  ANTIBIOTICS – Classification 1- ANTIBIOTICS I. According to antimicrobial activity Substances derived from a microorganism or produced synthetically, that destroy or limit the growth of a 1. Bacteriostatic: prevents bacterial growth living organism. (i.e., it keeps them in the stationary phase of ❑ Active against bacteria, or bacterial infections. growth) 2. Bactericidal: kills bacteria. ❑ Antibiotics are not effective against viruses such as the common cold or influenza; drugs which inhibit viruses are termed antiviral drugs rather than antibiotics. ANTIBIOTICS – Sources ANTIBIOTICS – Classification 1. Natural: produced by microorganisms II. According to bacterial spectrum of activity a.Fungi – penicillin 1. Narrow spectrum: active against a selective group b.Bacteria –Actinomycetes (tetracycline, of bacterial types. chloramphenicol, streptomycin) 2. Synthetic: synthesized in the lab, chemically related to natural antibiotics. 2. Broad spectrum: active against a wider number of bacterial types and, thus, may be used to treat a variety of infectious diseases.  Antibiotic Spectrum of Activity ANTIBIOTICS – Classification III.According to absorbability from the site of administration to attain significant concentration for the treatment of systemic infection 1. Locally acting: affects a single organ or a part of the body, restricted to a specific location within the body of the host. 2. Systemic: affects the entire body, spread to several regions of the host. No antibiotic is effective against all microbes ANTIBIOTICS – Classification Mechanisms of Antimicrobial Action Bacteria have their own enzymes IV.According to mechanism of action 1. Inhibit bacterial cell wall synthesis for 2. Alter the function and permeability –Cell wall formation –Protein synthesis of the cell membrane 3. Inhibit protein synthesis (translation and transcription) –DNA replication –RNA synthesis 4. Inhibit nucleic acid synthesis –Synthesis of essential metabolites  Selective Toxicity Inhibition of cell wall synthesis Selective toxicity: A drug that kills harmful Target: block peptidoglycan (murein) synthesis microbes without damaging the host Bacteria have: Peptidoglycan Polysaccharide (repeating disaccharides of N- acetylglucosamine and N- – Cell wall acetylmuramic acid) – Transpeptidase and autolysin + cross-linked pentapeptide – D-ala-D-ala Pentapeptide with terminal D-alanyl-D-alanine unit required for cross-linking Humans do not have: Peptide cross-link formed between the free amine of the amino acid in the 3rd position – Cell wall of the peptide & the D-alanine in the 4th – Transpeptidase and autolysin position of another chain – D-ala-D-ala; have L-ala instead Inhibition of cell wall synthesis Inhibition of protein  -lactam antibiotics: the beta-lactam ring in their chemical synthesis structure,include the penicillins, cephalosporins and related ❑ Binds the ribosomes compounds. Result in: inhibit transpeptidation reaction (3rd stage) to block peptidoglycan synthesis 1. Failure to initiate protein synthesis Steps: 2. No elongation of protein a. activation of autolytic enzymes (murein hydrolases) 3. Misreading of tRNA-deformed protein in the cell wall b. degradation of peptidoglycan c. lysis of bacterial cell bactericidal but kills only when bacteria are actively growing inactivated by -lactamases: enzymes produced by bacteria that provide multi- resistance to β-lactam antibiotics  Mechanisms of Inhibitors of Protein Synthesis Antimicrobial Action Broad spectrum, toxicity problems Examples The more similar the pathogen and – Chloramphenicol (bone marrow) host enzymes, the more side – Aminoglycosides: Streptomycin, neomycin, gentamycin effects the antimicrobials will have (hearing, kidneys) – Tetracyclines (Rickettsias & Chlamydia; GI tract) – Macrolides: Erythromycin (gram +, used in children) Modes of Antimicrobial Action Antibiotic Resistance  Mechanisms of Antibiotic Antimicrobial Resistance Resistance Relative or complete lack of Enzymatic destruction effect of antimicrobial against a of drug previously susceptible microbe Prevention of penetration of drug Increase in MIC Alteration of antibiotic or target site Rapid ejection of the drug MIC versus MBC Antibiotic Selection for Minimum Inhibitory concentration (MIC): Resistant Bacteria - The lowest concentration of antimicrobial agent that inhibit growth/multiplication. Minimum bactericidal concentration (MBC) or Minimum lethal concentration (MLC): - The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive. The closer the MIC is to the MBC, the more bactericidal the compound.  What Factors Promote Antimicrobial Resistance? BACTERIAL GENETICS Exposure to sub-optimal levels of antimicrobial (Mutation & Recombination) Exposure to microbes carrying resistance genes Mutation Inappropriate Antimicrobial A heritable change in the nucleotide sequence of a gene is Use called a mutation. Prescription not taken correctly Mutations are usually detrimental, but they can also lead to Antibiotics for viral infections beneficial changes. Antibiotics sold without medical supervision A mutant is called an auxotroph if the mutation leads to a Spread of resistant microbes in new nutrient requirement. hospitals due to lack of hygiene  Mutation Genetic Recombination in Bacteria Two types – spontaneous and induced – Spontaneous mutation occurs naturally, about one in every Genetic recombination is the transfer of DNA from one million to one in every billion divisions, and is probably due to low organism to another. level natural mutagens present in the environment. The donor’s DNA may then be integrated into the recipient's – Induced mutation is caused by mutagens that cause a much higher rate of mutation; induced by chemicals or radiations DNA by various mechanisms. Radiations as mutagens General Features of Non-ionizing radiations (e.g. UV rays) Recombination Induce formation of thymine-thymine dimers, which does not form complementary base pair with the nucleotides and this terminates the Unidirectional replication of DNA strand. – Donor to recipient Ionizing Radiation (e.g. X-rays & γ-rays) ▪ has much more energy and penetrating power than ultraviolet Donor does not give an entire chromosome radiation; – Merozygotes ▪ ionizes water and other molecules to form free radicals that can break DNA strands and alter purine and pyrimidine bases. Gene transfer can occur between same or different species  Other Mechanisms of Genetic Recombination n Bacteria Mechanism of Transformation A bacterial cell dies or is degraded releasing its dsDNA molecule Transformation in environment. Nuclease enzymes cut the released DNA into fragments of usually Transduction about 20 genes long. Conjugation The fragments bind to DNA binding proteins present on the surface of a competent recipient bacterium and subsequently translocated in the cytoplasm of recipient bacteria NOTE THAT THESE ARE NOT METHODS OF REPRODUCTION The DNA fragment from the donor is then exchanged for a piece of the recipient's DNA by means of Rec A proteins. TRANSFORMATION Transformation – step I Transformation is a method of genetic recombination in which a naked DNA from a donor bacteria is transferred to a competent recipient bacteria and incorporated into chromosome of the latter, e.g. in Bacillus, Haemophilus, Neisseria, Pneumococcus. Transformation occurs in nature. It is widely used in recombinant DNA technology. In Gram+ve bacteria the DNA is taken up as a single stranded A donor bacterium dies and is degraded. molecule and the complementary strand is synthesized in the recipient. In Gram-ve bacteria double stranded DNA is transformed.  Transformation – step II Transformation – step IV Exchange is complete. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on cell wall of a competent live recipient bacterium Factors affecting Transformation – step III transformation – DNA size and state Sensitive to nucleases – Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus) The ability to take up DNA from the environment is known as competence only DNA from closely related bacteria (competent cells) would be successfully transformed Competence factor (a specific protein produced at a particular time in the growth cycle of competent bacteria and enable it to take up DNA naturally) The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA. Induced competence, e.g. by CaCl2  TRANSDUCTION Definition: Gene transfer from a donor to a recipient Bacterial Plasmids bacteria through a bacteriophage – Exrachromosomal pieces of DNA Often confer protection- resistance to drugs Bacteriophage (phage): A virus that infects bacteria – Tiny, circular – Free or integrated – Duplicate and are passed on to offspring Types of transduction – Used in genetic engineering – Generalized – Specialized Conjugation Types of plasmid Definition: Gene transfer from a Fertility-F-plasmids. They are capable of conjugation (transfer of genetic material between donor to a recipient by direct physical bacteria which are touching). contact between cells Resistance-(R)plasmids, which contain Mating types in bacteria genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. – Donor (Male/F+) Donor F factor (Fertility factor) Col-plasmids, which contain genes that determine the production of bacteriocins, proteins that can kill – F (sex) pilus other bacteria. – Recipient (Female/F-) Degradative plasmids, which enable the digestion of unusual substances Lacks an F factor Virulence plasmids, which turn the bacterium into a pathogen Recipient  Conjugation is of different Bacterial Conjugation types: Bacteria conjugation in E.coli was discovered by Lederberg and Tatum in 1946, when they observed sex like exchange 1- F+ conjugation: During conjugation the F+ bacteria between two mutants strains of E.coli called K12. synthesize a modified pilus (sex pilus) through which Conjugation involves the transfer of plasmid from a donor genetic material is transferred. (bacteria contain F plasmid) to the recipient (bacteria lacking This attached pilus is a temporary cytoplasmic bridge F plasmid). through which a replicating F plasmid is transferred from the male to the female. Conjugation in gram positive does not depend on sex pili, the donor cells form a protein called adhesin on the surface, that causes donor and recipient cells to aggregate. Eg. Bacillus subtillis, Streptococcus lactis. F+ conjugation: Bacterial Conjugation Conjugation is more common in gram negative between strains of same species or closely related species. Some plasmid like F plasmid (fertility factor or sex factor) they carry tra gene that mediate their own transfer through sex pilus. Bacteria that have a F plasmid are referred to as F+ or male. Those that do not have an F plasmid are F- of female. The F plasmid consists of 25 genes that mostly code for production of sex pili.  High frequency recombinant Resistant plasmid conjugation : (Hfr) conjugation: 2- Resistant plasmid conjugation : Some gram negative 3- High frequency recombinant (Hfr) conjugation: when bacteria (eg. E.coli, shigella) contain plasmids that contain the F+ plasmid is integrated within the bacterial antibiotic resistance gene called R factor. The R factor chromosome, the cell is called an Hfr cell. Plasmids that has two components: are capable of integrating into the chromosome is called i. RTF (resistance transfer factor) that codes for sex episome. transfer like F factor. HFR cells are able to transfer chromosomal gene to recipient ii. r determinant that codes for antibiotic resistance. with high frequency. Sometimes RTF may be dissociated from the r determinants The recipient F- cell usually remain F- after conjugation and the two components may exist as separate entities. because only a part of the F plasmid from the donor Hfr Under such condition, although the host cell remains drug cell to the recipient have been transferred. resistant, the drug resistance is not transferable. Resistant plasmid conjugation : Hfr conjugation