Bacterial Genetics Lectures PDF

7/7/2024 Bacterial Genetics  One of the most important differences between bacteria and other living organisms is how genetic material is located in the cell.  While all living things contain two or more chromosome...

7/7/2024 Bacterial Genetics  One of the most important differences between bacteria and other living organisms is how genetic material is located in the cell.  While all living things contain two or more chromosomes inside a nucleus surrounded by a membrane, we find that the genetic material in bacteria is a single, circular chromosome composed of double-stranded DNA that exists irregularly in a specific shape and is called the nucleoid, the nuclear body, the chromatin body, or Nuclear zone.  The nuclear region can be seen after staining with Feulgen's stain, which reacts specifically with DNA.  A bacterial cell when dividing can contain more than one nuclear region. 1  7/7/2024  Chemical analysis of the nuclear region showed that it contains 60% DNA, RNA and a few proteins (different from proteins called histones that characterize eukaryotic chromosomes).  In E.coli bacteria (about 2 - 6 μm in length), we find that the DNA ring is about 1400 μm; thus, this DNA must be packed very efficiently to fit this nuclear region inside this bacteria.  The bacterial chromosome contains all the genes necessary for various life functions. Plasmids:  In addition to the basic chromosome, many bacterial species contain a circular piece of double-stranded DNA that replicates independently of the chromosome or may integrate with it.  It can be inherited or transmitted from parental to daughter cells in both cases.  This extra piece of DNA is called a plasmid. 2  7/7/2024 5  The genes on the plasmid are non-essential genes, meaning they are not necessary for basic life functions (reproduction and growth) and can even be lost without affecting the growth of the bacterial cell.  But plasmids certainly give bacteria a selective advantage – for example, making the bacteria resistant to certain toxins or giving them new metabolic capabilities. 3  7/7/2024  Plasmids contain a small number of genes - usually fewer than 30 genes.  There may be one copy of the plasmid in each cell (single copy plasmid) or several copies in one cell (multi-copy plasmid).  Plasmids that can be found attached or unattached to the bacterial chromosome are called episomes.  Plasmids can be removed by a process known as Curing, which can occur randomly or be induced by processes that inhibit plasmid copying but do not affect the copying of the bacterial chromosome. 4  7/7/2024 Pathogenicity Islands (PAI)  Unique genetic elements on the chromosomes of a some bacterial pathogens encoding various virulence factors.  Characteristics of PAI: 1. A distinct class of genomic clusters. 2. Acquired by horizontal transfer. 3. Contains virulence factors; responsible for pathogenicity. 4. Present in pathogenic strains. 5. Important in the evolution of pathogenesis. Example: Helicobacter pylori (cag PAI). Staphylococcus aureus SCCmec 5  7/7/2024 Exchange of genetic materials (Horizontal gene transfer) 1. Transformation. 2. Conjugation. 3. Transduction. Transformation  Uptake (acquisition) of free (naked) DNA by a competent bacterial cell.  The ability to take up DNA from the environment is called competence.  Natural transformable organisms:  Streptococcus pneumoniae.  Haemophilus influenzae.  Neisseria gonorrhoea.  Artificial transformation:  E.coli for gene cloning 6  7/7/2024 13 Conjugation  the transfer of genetic information (usually carried on plasmids) from donor to recipient bacterial cell in a process that requires intimate cell contact. 7  7/7/2024 Transduction  Virus-mediated (Bacteriophage) transfer of genetic information from donor to recipient cell.  Bacteriophages can infect, coexist, and lyse bacterial cells.  Virulent or lytic phages: cause lysis of the host bacterium as a result of the synthesis of many new viruses within the infected cell.  Temperate phage: may initiate a lytic growth process of this sort or can enter a latent form (called a prophage). 8  7/7/2024  Lysogen: The bacterial cell that harbors a latent prophage. This condition is referred to as lysogeny.  Lysogenic conversion: It is when a temperate bacteriophage induces a change in the phenotype of the infected bacteria that is not part of a usual phage cycle. Transposition  It is the transfer of genetic elements from one location to another on the same chromosome, or between chromosome and plasmid.  It relies on a site-specific recombination enzyme, called Transposase. 9  7/7/2024 Microbial Interactions with the host  Mutualism: Relationship between two organisms biologically interact where each individual derives a fitness benefit.  Commensalism: Relationship between two organisms where one organism benefits but the other is neutral (there is no harm or benefit).  Parasitism:  relationship between organisms of different species where one organism, the parasite, benefits at the expense of the other, the host.  Pathogen:  Microorganism that is capable of causing disease. 10  7/7/2024 Bacterial Interactions with the host  Virulence: quantitative measure of pathogenicity and is measured by the number of organisms required to cause disease.  The 50% lethal dose (LD50): the number of organisms needed to kill half the exposed hosts.  the 50% infectious dose (ID50): the number needed to cause infection in half the exposed hosts. 11  7/7/2024 Factors determining virulence  Number of infecting bacteria.  Route of entry into the body.  Specific and nonspecific host defense mechanisms.  Virulence factors of the bacterium. Virulence Factors  Are factors that help bacteria to: 1. Invade the host. 2. Cause disease (pathology). 3. Survive host defenses.  The virulence of an organism is determined by its ability to produce various virulence factors. 12  7/7/2024 Types of virulence factors  Adherence Factors: Interaction between surface of the microbe and specific host cell receptors. E.g. Glycocalyx (slime layer) e.g. (Streptococcus mutans). Pili e.g. (Neisseria gonorrhoea). Adherence proteins e.g. (M protein Streptococcus pyogenes). Lipoteichoic acid e.g. (Streptococcus pyogenes).  Capsules: protect bacteria from opsonization & phagocytosis. e.g. (Streptococcus pneumoniae)  Secretion systems: Mostly Type III and Type IV. Needle like structures used by some bacteria to secrete proteins into host cells. e.g. Pseudomonas aeruginosa 13  7/7/2024  Invasion Factors: bacterial components that allow the bacterium to invade host cells. Invade  colonize  spread.  Examples: hyaluronidase (spreading factor). Collagenase. proteases, nucleases, lipases.  Exotoxins: Proteins released extra-cellularly as the organism grows. Arise from focus of infection to other parts of the body.  Types: Cytolytic toxins: Act on cell membranes e.g. Haemolysins. Leucocidins. 14  7/7/2024 A-B toxins: Composed of: B subunit: binding portion A subunit: active" portion  Examples: o Cytotoxin e.g. (Diphtheria toxin) o Neurotoxin e.g. (Tetanus toxin). o Enterotoxin e.g. (Cholera toxin ) Super-antigen toxins: Stimulate large numbers of lymphocytes; Induce massive release of cytokines. Causes fever, systemic toxicity and immune suppression.  Example: o TSST (Staphylococcus aureus); and Erythrogenic toxin (Streptococcus pyogenes).  Endotoxin (only in G-ve bacteria)  Lipopolysaccharide: Released when organisms die or bacterial cells are lysed Cause : Fever, Circulatory collapse, septic shock. 15  7/7/2024 Exotoxins and Endotoxins Property Exotoxin Endotoxin Source G+ve and G-ve Cell wall of G-ve Secretion from the Yes No cell Chemistry Polypeptide Lipopolysaccharide Gene location Plasmid & chromosome bacteriophage Toxicity Usually high (very Usually low (not specific) specific) Clinical effect Various fever, shock Mode of action Various Induces TNF and IL-1 Antigenicity Highly antigenic Poorly antigenic Vaccine design Available for some Not available (Toxoids) Heat stability Labile (60°C) Stable (at 100°C) Infectious diseases  Infection: microbes gain access to or colonize a part of the human body; avoid host defenses; invade tissues, multiply and set up an infection. Major cause of human illness. Major cause of death.  Infectious disease: when the changes are caused by a microorganism or its toxins. 16  7/7/2024 Bacterial Infectivity Is the results of the disturbance of the balance between bacterial virulence and host resistance.  Primary pathogen: Cause disease upon infection, not normally associated with host and is capable of causing disease in healthy individuals (immunocompetent).  Opportunistic pathogen: Infectious agent is capable of causing disease only when host resistance is impaired (immunocompromised hosts), usually is member of normal flora.  Toxicity: capacity of organism to cause disease by means of toxins production.  Invasiveness: ability of organism to multiply to high levels in tissues and cause disease. 17  7/7/2024 Pathogenesis of Bacterial infection  Bacteria cause disease by two major mechanisms: 1. Toxin production. 2. Invasion and inflammation.  Two types of inflammatory reactions produced by the bacteria: Acute; Pyogenic (pus forming). Chronic; Granulomatous.  Stages of bacterial pathogenesis: 1. Transmission. 2. Evasion of primary host defenses. 3. Adherence to mucous membranes. 4. Colonization: by growth of the bacteria at the site of adherence. 5. Disease symptoms caused by toxin production or invasion + inflammation 6. Host responses, both nonspecific and specific immunity. 7. Progression or resolution of the disease. 18  7/7/2024 Blood invasion  Bacteremia: is the presence of viable bacteria in the circulating blood; usually transient and asymptomatic.  Toxemia: Presence of bacterial Toxin in the blood  Septicaemia: Presence of active multiplicating bacteria in the blood that result in evoking severe inflammatory response. Usually results in fever, circulatory collapse and multisystem involvement. Transmission  Human to human: 1. Direct contact: sexual. 2. Fecal oral route. 3. Trans-placental. 4. Blood borne.  Non-human sources: 1. Soil. 2. Water source. 3. Zoonotic. 4. Through vector (insect borne). 19  7/7/2024 Types of bacterial infection Type of bacterial Description Examples infection Asymptomatic In apparent No detectable clinical gonorrhoea in (subclinical) symptoms and signs women and men Dormant Carrier state Typhoid carrier (latent) Zoonosis & Environmental Anthrax , Accidental Exposure Lab exposure Infection caused by Streptococcus normal flora or transient pneumoniae Opportunistic bacteria when the immune infection in AIDS system is compromised. patients Types of bacterial infection Type of bacterial Description Examples infection Clinically apparent Primary (organism multiply in body Dysentery tissues cause local injury) Bacterial Microbial invasion pneumonia Secondary subsequent to primary following viral infection lung infection Anaerobic Two or more microbe abscess Mixed infecting the same tissue (E.coli & Bacteroides) 20  7/7/2024 Types of bacterial infection Type of bacterial Description Examples infection Acute Rapid onset (hours or days) Diphtheria Sub acute Brief duration (days or weeks) Brucellosis Prolonged duration (months Chronic Tuberculosis or years) Types of bacterial infection Type of bacterial Description Examples infection Confined to small area or an Staphylococcal Localized organ abscess Disseminated in many body Generalized Septicemia tissues Infection that occurs Pneumonic Fulminant suddenly and intensely plague 21  7/7/2024 Antibiotics and Antimicrobial Agents 43 Antibiotics and Antimicrobial Agents  Antibiotics: Small molecular weight substances; are produced to kill bacteria or inhibit their growth. Antibiotics can be natural, semi-synthetic, or synthetic.  Antimicrobial: any substance with sufficient antimicrobial activity that can be used in the treatment of infectious diseases.  Chemotherapeutic: broad definition that includes antibiotics, antimicrobials, and drugs used in the treatment of cancer. 22  7/7/2024 History of Antibiotics  1st antibiotic discovered by scientist Alexander Fleming is PENICILLIN in 1929.  He was working in his lab, trying to kill a deadly bacteria, when he noticed a blue mold growing on the dish. 45 Antimicrobial effect  Bactericidal antibiotic: antimicrobial that is lethal to bacteria.  Bacteriostatic antibiotic: an antimicrobial that inhibits bacterial growth but does not kill the organisms. The microorganism is eliminated by the immune system. 23  7/7/2024  Minimum bactericidal Concentration (MBC): laboratory term that defines the lowest concentration (μg/mL) able to kill 99% of the organism.  Minimum inhibitory concentration (MIC): laboratory term that defines the lowest concentration (μg/mL) able to inhibit growth of the microorganism. Source of antibiotics  Fungi o Penicillin, Griseofulvin, Cephalosporin  Bacteria o Polymyxin B, Colistin, Bacitracin, Aztreonam.  Actinomycetes. o Aminoglycosides, Macrolides, Tetracyclines, Polyenes, Chloramphenicol 48 24  7/7/2024 Antibiotics Classification  Based on the spectrum of antimicrobial activity:  Narrow-spectrum:  the antibiotic has activity against only a few organisms.  Extended-spectrum:  the antibiotic has activity against the main category (Gram-positive) but some effect against another category (Gram-negative).  Broad-spectrum:  the antibiotic has activity against organisms of diverse types (e.g. Gram-positive, Gram- negative, anaerobes). Based on the chemical structure: A) Beta-lactam antibiotics e.g. Benzylpenicillin, Ampicillin, Amoxicillin.  Uses-Meningitis, Aspiration pneumonia, Diphtheria B) Non beta-lactam antibiotics e.g. Tetracyclin, Streptomycin.  Uses-Plague, Ricketsial infections C) Miscellaneous antibiotics e.g. Chloramphenicol.  Uses-Typhoid, Meningitis, Fever, UTI 25  7/7/2024 Mechanism of action of the antibiotics  Structures and processes targeted by the antibiotics: Cell wall. Cell membrane. Ribosome (Translation). Nucleic acid: Replication. Transcription. Synthesis. Mechanism of Action  Agents that inhibit synthesis of bacterial cell walls (e.g. Penicillins & cephalosporins).  Agents that act directly on the cell membranes of the microorganisms (e.g. Polymixin, Polyene antifungal agents: Nystatin, Amphotericin B).  Agents that affect the function of ribosomal subunits to cause a reversible inhibition of protein synthesis (e.g. Bacteriostatic drugs: Chloramphenicol, Tetracyclines). 52 26  7/7/2024  Agents that bind to 30S ribosomal subunit & alter protein synthesis, which eventually leads to cell death (e.g. Aminoglycosides).  Agents that affect bacterial nucleic acid metabolism (e.g. Rifamycins which inhibit RNA polymerase).  Anti-metabolites (including trimethoprim & sulphonamides  Antiviral agents (Nucleic acid analogues, Non- nucleoside reverse transcriptase inhibitors, Inhibitors of viral enzymes). 53  Basic Classes of Antibiotics According to their Target  Although a large number of antibiotics exist, they fall into only a few classes with an even more limited number of targets. –β-lactams (penicillins) – cell wall biosynthesis –Glycopeptide (vancomycin) – cell wall biosynthesis –Aminoglycosides (gentamycin) – protein synthesis –Macrolides (erythromycin) – protein synthesis –Quinolones (ciprofloxacin) – nucleic acid synthesis –Sulfonamides (sulfamethoxazole) – folic acid metabolism 54 27  7/7/2024 Antibiotics Synergism Antibiotic B  Beneficial interaction in which antibiotics work together to produce an effect more potent than if each antibiotic were applied singly. Causes of this interaction: 1. Antibiotic A enhances the activity of Antibiotic B at a biochemical level. 2. Antibiotic A assists Antibiotic B to penetrate the bacterial cell. 3. Antibiotic A Protects Antibiotic B from inactivation. 4. Antibiotic A overcomes spontaneous mutation that leads Antibiotic A to resistance to Antibiotic B. Bacterial growth Bacterial growth inhibition Therapeutic index  is the ratio of the dose that produces toxicity to the dose that produces a clinically desired or effective response.  Therapeutic index = toxic dose/effective dose  The therapeutic index is a measure of the drug's safety. 28  7/7/2024 Therapeutic index  Large value indicates that there is a wide margin between doses that are effective and doses that are toxic (safe).  Small value indicates that there is a narrow margin between doses that are effective and doses that are toxic (toxic). Successful Antimicrobial Therapy  Concentration: site of infection Concentration should inhibit microorganisms. Simultaneously it should be below the level toxic to human beings.  Host Defenses: Immunity intact - Bacteriostatic Agents. Impaired immunity - Bactericidal Agents. 29  7/7/2024 Antimicrobial resistance  Resistant organisms are those that cannot be inhibited by clinically achievable concentrations of an antimicrobial agent.  Intrinsic resistance: Natural features of the bacterial species usually expressed by chromosomal genes.  Acquired resistance: Resistant strains emerge from previously sensitive bacterial populations either by mutation or by acquisition of plasmids or transposons. Bacterial Resistance to Antimicrobial Agents  3 general categories Drug does not reach its target. Drug is not active. Target is altered. 30  7/7/2024 Drug does not reach its target  Porins  Absence/mutation.  Reduce drug entry.  Reduced effective drug concentration at the target site.  Efflux pumps  Transport drugs out of the cell.  Resistance to tetracyclines & β-lactam antibiotics. Inactivation of Drug  Second general mechanism of drug resistance  β-lactam antibiotics - β-lactamase.  Aminoglycosides - Aminoglycoside modifying enzymes.  Variant: failure of bacterial cell to convert an inactive drug to its active metabolite. Resistance to isoniazid in mycobacterium TB. 31  7/7/2024 Alteration of the Target  Mutation of natural target.  Target modification  The new target does not bind the drug for native target resulting in resistance to antibiotic.  Components mediating resistance to β-lactam antibiotics in Pseudomonas aeruginosa. β –lactam antibiotics hydrophilic must cross outer membrane barrier of the cell via outer membrane protein (Omp) channel or porins.  Mutation/missing/deleted Drug entry slow or prevented. 64 32  7/7/2024  β - lactamase concentrated between the inner & outer membrane in the periplasmic space constitutes an enzymatic barrier.  Drug destroyed.  Effective concentration not achieved. 65 66 33  7/7/2024 Common Highly Resistant Bacteria Staphylococcus aureus - MRSA, VRSA Enterococcus faecalis - VRE Pseudomonas aeruginosa. Klebsiella sp. Acinetobacter baumanni. Mycobacterium tuberculosis 68 34