Bacteriology exam prep topics 10-12

History of Antimicrobial Chemotherapy

• The term "antibiotic" originated from the word "antibiose" coined by Paul Vuillemin in 1890, describing the antagonist action between microorganisms.
• Mycophenolic acid, the first antibiotic discovered from nature, was first reported by Bartolomeo Gosio in 1893 and isolated from Penicillium brevicompactum.
• Mycophenolic acid showed bacteriostatic, anti-viral, anti-fungal, and anti-tumor properties, but remained undiscovered until its rediscovery in 1913 in the USA.
• Paul Ehrlich, a German physician and scientist, founded antibacterial chemotherapy and proposed the concept of the "magic bullet" (Zauberkugel).
• Ehrlich and his group synthesized and tested compounds from arselenic acid against bacterial strains, leading to the development of Arsphenamine (Salvarsan) in 1909.
• Arsphenamine was the first man-made antibacterial agent, effective against syphilis, and remained the most effective treatment until penicillin became available in the 1940s.

Discovery of Penicillin

• Alexander Fleming discovered penicillin in 1928 when a Staphylococcus aureus colony contaminated with Penicillium chrysogenum showed lysis.
• Fleming attempted to isolate and purify penicillin but was unsuccessful until 1940.
• Penicillin was first used for treatment in 1941, but the patient died, and the drug supply ran out.
• Penicillin became available to the public in 1945, and its structure was discovered, classifying it as the first member of the beta-lactam family of antibiotics.

Development of Antimicrobial Chemotherapy

• The current era of antimicrobial chemotherapy began in 1935 with the discovery of sulfonamides.
• Notable discoveries in the following years:
  • 1939: First polypeptide antibiotic discovered
  • 1943: First aminoglycoside antibiotic discovered (streptomycin)
  • 1945: Tetracyclines discovered
  • 1947: Chloramphenicol discovered
  • 1950s: Azomycin, Metronidazole, macrolides, glycopeptides, and streptogramins discovered

Classification of Antimicrobial Drugs

• Classification based on spectrum of activity against class of microorganism:
  • Antibacterial (e.g., penicillin)
  • Antifungal
  • Antiprotozoal (e.g., sulfanomides, trimadoprime)
• Antibacterial activity:
  • Narrow spectrum (e.g., bacitracin, vacomycin; polymyxin)
  • Broad spectrum (e.g., tetracyclines)
• Pharmacodynamic activity: antibacterial action depends on concentration and time.
• Mechanism of action depends on drug class:
  • Inhibition of protein synthesis
  • Inhibition of nucleic acid synthesis
  • Inhibition of cell wall synthesis
  • Disruption of membrane function
  • Block pathways and inhibit metabolism

Inhibition of Cell Wall Synthesis

• Antibiotics that interfere with cell wall synthesis include β-lactam antibiotics (penicillins, cephalosporins), bacitracin, and vancomycin.

Bacterial Cell Wall

• The bacterial cell wall is a thick envelope that gives shape to the cell and distinguishes it from mammalian cells.
• In Gram-positive (G+) bacteria, the cell wall is thick, consisting of a layer of peptidoglycans that provides rigidity and maintains high internal osmotic pressure.
• In Gram-negative (G-) bacteria, the layer is thinner, resulting in lower osmotic pressure.

Peptidoglycan Layer

• The peptidoglycan layer consists of a polysaccharide chain made up of a repeating disaccharide backbone of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).
• The sugars are connected through a glucosidic linkage, and a tetrapeptide is attached to N-acetylmuramic acid, with peptide interbridges connecting one peptide to another.
• The disaccharide backbone is cross-linked both within and between layers, giving the cell wall remarkable strength.

β-Lactam Antibiotics

• β-Lactam antibiotics bind to penicillin-binding proteins (PBPs), many of which are transpeptidase enzymes responsible for cell wall formation and remodeling during growth and division.
• Different penicillin-binding proteins have different affinities for drugs, explaining the variation in the spectrum of action of different β-lactam antibiotics.
• By binding to PBPs, β-lactam antibiotics prevent the final cross-linking in the cell wall, inhibiting cell division and creating weak points.

Effects of β-Lactam Antibiotics

• β-Lactam antibiotics decrease normal inhibition of autolysins and block peptidoglycan synthesis, weakening the cell wall and promoting cell lysis.
• They are active against growing cells, bactericidal, and have greater activity against G+ bacteria due to the higher quantity of peptidoglycan and high osmotic pressure.
• The activity of β-lactam antibiotics can be expanded by combining them with β-lactamase-inhibiting drugs, such as clavulanic acid and sulbactam, to neutralize enzymes that might otherwise inactivate them.

Nitroimidazoles

• Possess antiprotozoal and antibacterial properties
• Affect only anaerobes and microaerophiles, which reduce the drug
• Reduced products cause DNA strand breakage by inhibiting DNA repair enzyme - DNase 1 or forming complexes with nucleotide bases
• Bactericidal against anaerobic G- and many G+ bacteria, and active against protozoa such as Trichomonas fetus, Giardia lamblia, and Histomonas meleagridis

Fluoroquinolones

• Active against G- bacteria
• Selectively inhibit bacterial DNA synthesis by inhibiting DNA gyrase (topoisomerase II) and DNA topoisomerase IV
• DNA gyrase is involved in packing DNA into bacterial cells, while topoisomerase IV is involved in relaxing supercoiled DNA
• Bactericidal, with newer fluoroquinolones being broader spectrum and active against some G+ bacteria, including mycobacteria, mycoplasmas, and rickettsia

Sulfonamides and Trimethoprim

• Synthetic drugs with antibacterial and antiprotozoan properties
• Interfere with biosynthesis of folic acid, necessary for nucleic acid production in bacteria
• Sulfonamides competitively inhibit the enzyme dihydropteroate synthase and block folic acid synthesis
• Active against G+ and G- bacteria and protozoa, with bacteriostatic effects
• Trimethoprim inhibits the enzyme dihydrofolate reductase, producing a sequential blockade of folic acid synthesis
• Bacteriostatic, with selective toxicity due to mammalian cells absorbing folic acid from the intestine, while bacteria must synthesize it

Inhibition of Protein Synthesis

• Examples include tetracyclines, aminoglycosides, chloramphenicol, lincosamides, and macrolides
• Can be divided into those affecting the 30S ribosome (tetracyclines, aminoglycosides) and those affecting the 50S ribosome (chloramphenicol, macrolides, lincosamides)

Tetracyclines

• Interfere with protein synthesis by inhibiting the binding of aminoacyl tRNA to the recognition site
• Bacteriostatic, with broad-spectrum activity against G+, G-, including rickettsia, chlamydia, some mycoplasmas, and protozoa (such as Theileria)

Aminoglycosides

• Complex effects in the bacterial cell, including:
  • Binding to a specific receptor protein in the 30S ribosomal subunit, distorting the anti-code interaction at recognition site
  • Causing misreading of the genetic code, leading to faulty proteins being produced
  • Binding to "initiating" ribosomes, preventing the formation of 70S ribosomes
  • Inhibiting the elongation reaction of protein synthesis
• Bactericidal, particularly active against G- bacteria, mycobacteria, and some mycoplasmas
• Anaerobes are resistant, often combined with β-lactams

Chloramphenicol and Macrolides

• Chloramphenicol is broad-spectrum, bacteriostatic, and binds the 50S ribosome, distorting the region and inhibiting the peptidyl transferase reaction
• Active against G+ and G- bacteria, including chlamydia, rickettsia, and some mycoplasmas
• Macrolides are bacteriostatic, particularly active against G+ bacteria and Mycoplasma
• Mechanism of action is the same as for chloramphenicol and lincosamides

Antimicrobial Resistance

• Prior to the commercialization of antibiotics, main causes of death were pneumonia, flu, tuberculosis, and GI infections.
• During WWI, infectious diseases caused more deaths than battle wounds.
• The discovery of penicillin in 1938 marked a new era of medicine, helping to develop new antibiotics and changing the entire drug discovery process.

Emergence of Antimicrobial Resistance

• Overreliance on antibiotics led to the emergence of antimicrobial resistance, which was not unexpected.
• A. Fleming predicted the emergence of resistance and expressed concern about the improper use of penicillin in his Nobel Prize acceptance speech in 1945.

Resistance Mechanisms

• Antimicrobial resistance is defined as the ability of a microorganism to withstand the effect of a normally active concentration of an antimicrobial agent.
• Five major categories of antimicrobial resistance mechanisms:
  • Antimicrobial agent (AMA) is prevented from reaching its target by reducing its penetration into the bacterial cell.
  • AMAs are expelled out of the cell by general or specific efflux pumps.
  • AMAs can be inactivated by modification or degradation.
  • Antimicrobial targets can be modified or protected by another molecule preventing access of antibiotic to its target.
  • Microorganisms can develop alternate pathways that bypass the reaction inhibited by the drug.

Types of Resistance

• Intrinsic resistance: chromosomally encoded, relates to the general physiology of an organism, natural to all members of a specific bacterial taxonomic group.
• Examples of intrinsic resistance: G- bacteria are resistant to macrolides, aminoglycosides have reduced activity in anaerobes, G+ bacteria are resistant to polymyxins.
• Acquired (extrinsic) resistance: mutation in a resident gene, transfer of genetic material (via plasmids, bacteriophages, transposons).
• Acquired resistance can result from the mutation of genes involved in normal physiological processes and cellular structures, or from the acquisition of foreign resistance genes.

Mutation and Resistance

• Gene mutations occur continuously at a relatively low frequency (10-8-10-9).
• Mutation-based resistance is often a gradual stepwise process where a transferable resistance is often high-level, all-or-none resistance.
• Mutations can lead to other changes that leave the cell at a disadvantage, and in the absence of antibiotic selection, these mutants may gradually be lost.

Horizontal Gene Transfer

• Foreign DNA can be acquired by bacteria in three different ways: transformation, transduction, and conjugation.
• Mobilome: all mobile genetic elements that can move around within or between genomes in a cell (plasmids, transposons, bacteriophages, self-splicing molecular parasites).
• DNA can be transferred from one organism to another through horizontal gene transfer, permanently changing the recipient's genetic composition.

Horizontal Gene Transfer

• Transformation: a process by which DNA is taken up from the environment by bacteria, resulting in the emergence of new forms of existing genes
• Example: emergence of penicillin resistance in Streptococcus pneumoniae through the development of novel penicillin-binding proteins

Transduction

• Process by which bacteriophages (bacterial viruses) transfer resistant genes between bacteria
• Example: transfer of β-lactamase genes from penicillin-resistant to previously penicillin-susceptible Staphylococci

Conjugation

• Plasmids and conjugation are major players in the global spread of antimicrobial resistance genes in bacteria
• During conjugation, a donor bacterium synthesizes a sex pilus, which attaches to a recipient bacterium, resulting in the transfer of plasmid genes
• Conjugation can occur between different species and families, allowing for the spread of antimicrobial resistance genes

Origin of Resistance Genes

• Resistance genes originated long before the introduction of therapeutic antimicrobials into medicine
• Antibiotic-producing microorganisms developed resistance mechanisms to protect themselves from their own antibiotics
• Evidence suggests that resistance genes in human pathogens originated from environmental, non-pathogenic bacteria via horizontal gene exchange

Factors Contributing to Antibiotic Resistance

• Lack of regulation in antibiotic use, particularly in agriculture and the environment
• Use of antibiotics as growth promoters in agriculture
• Over-prescription of antibiotics, including for viral infections
• Economic hurdles that have stalled the development of new antibiotics by pharmaceutical industries

Controlling Antimicrobial Resistance

• Establishing effective surveillance systems for collecting data on resistant organisms
• Monitoring the supply and use of antimicrobial drugs
• Enforcing strict adherence to recommended therapeutic doses and drug withdrawal periods
• Not using antimicrobial agents for growth promotion
• Greater reliance on better hygiene, disinfection, and vaccination for prevention and control of infectious diseases

Bacteria and Ecological Associations with Animals

• Bacteria can be classified into three categories based on their ecological associations with animals: parasites, commensals, and saprophytes.
• Parasites live in permanent association with an animal host and at the expense of the host.
• Commensals are parasites that cause no discernible harm to the host.
• Saprophytes normally inhabit inanimate environments shared with animals.

Identifying Bacteria that Cause Disease

• Robert Koch proposed a series of postulates in 1884 to determine whether a specific bacterial species causes a particular disease.
• Koch's postulates:
  • The suspected agent is present in all cases of the disease.
  • The agent is isolated from the disease and propagated serially in pure culture, apart from its natural host.
  • Upon introduction into an experimental host, the isolate produces the original disease.
  • The agent can be reisolated from this experimental infection.
• Many microorganisms do not meet the criteria of Koch's postulates but have been shown to cause disease.

Infection and Infectious Disease

• Infection refers to the multiplication of an infectious agent within the body.
• Infectious agents can be pathogenic microorganisms such as bacteria, viruses, parasites, or fungi.
• Infection does not necessarily imply disease, as it may not lead to clinical signs or impairment of normal function.
• Infectious disease refers to the clinical manifestation of the infectious process.
• Infections can be exogenous (occurring after direct or indirect transmission from an infected animal or the environment) or endogenous (caused by commensal bacteria when an animal is subjected to stressful environmental factors).

Transmission of Infection

• Modes of transmission:
  • Contact: direct or indirect transmission from one animal to another.
  • Vectors: mechanical (insects) or passive (fomites).
  • Iatrogenic: after medical activity.
  • Vertical: from mother to baby.
  • By ingestion, inhalation, or inoculation.

Portals of Entry

• Skin provides a protection barrier for pathogens, which must overcome these barriers.
• Most frequent portals of entry:
  • Damaged skin (cuts, burns, and other injuries).
  • Damaged mucous membranes (upper, lower respiratory tract, gastrointestinal tract, genital, urinary tracts, conjunctiva).
  • Injuries (including surgical sites).
  • Teats, umbilicus.

Definitions

• Bacteremia: presence of bacteria in the blood (transient).
• Septicemia: presence and multiplication of bacteria in the blood (usually leads to sepsis).
• Sepsis: the body's extreme response to an infection that damages its own tissues.
• Pathogen: a microorganism capable of causing disease.
• Virulence: the quantitative ability of an agent to cause disease.
• Virulence factors: toxins, enzymes, and other bacteria-produced molecules and substances that alter normal growth.

Pathogenesis of Bacterial Infection

• Characteristics of pathogenic bacteria:
  • Transmissibility.
  • Adherence to host cells.
  • Persistence.
  • Invasion of host cells and tissues.
  • Toxigenicity.
  • Ability to evade or survive the host's immune system.

Adherence Factors

• Adhesins: specific surface molecules that interact with host cells (receptors).
• Examples: pili, fimbriae, lipoteichoic acid.

Invasion Factors

• Invasion: the entry of bacteria into host cells and further spread into host tissues.
• Invasion may be facilitated through breakdown of host tissues by different enzymes.
• Host cells play an active role in the invasion process.

Immune Evasion Strategies

• Bacteria employ tactics to avoid or inactivate host defenses, ensuring their survival within a host
• Tactics include modulating their cell surfaces, releasing proteins to inhibit or degrade host immune factors, and mimicking host molecules
• Some bacteria can escape phagocytosis by having a capsule, and some can even multiply in phagocytes

Toxigenicity Factors

• Toxins are specific virulence factors produced by some bacterial pathogens, in the form of substances poisonous to the host
• Toxigenicity refers to an organism's ability to make toxins
• Bacteria can produce:
  • Enzymes (e.g., urease and decarboxylase) that break down certain substances, producing toxic metabolites
  • Exotoxins
  • Endotoxins

Endotoxins

• Integral part of gram-negative bacteria
• Toxicity resides in the lipid-A portion of the lipopolysaccharide molecule
• Released when bacterial cells are lysed
• Effects on the host: fever, diarrhea, weakness, blood coagulation

Exotoxins

• Heat-sensitive soluble proteins released by living organisms
• Can be produced by both gram-positive and gram-negative bacteria
• Incredibly potent and can spread throughout the host body, causing damage distant from the original site of infection
• Associated with specific diseases, such as:
  • Botulism (produced by Clostridium botulinum)
  • Tetanus (produced by Clostridium tetani)
  • Diphtheria

Bacterial Biofilms

• An aggregate of interactive bacteria attached to a solid surface or to each other, encased in an exopolysaccharide matrix
• Form a slimy coat on solid surfaces, occurring throughout nature
• Play a critical role in bacterial pathogenesis and environmental quality
• Can cause equipment damage, product contamination, and aid attachment and survival
• Protect from phagocytosis, antibodies, and antibiotics
• Examples: colonization of heart valves, catheter medical devices, and prostheses

Animal Microflora

• The assemblage of microorganisms that constantly inhabit the animal body
• Includes bacteria, fungi, and archaea
• Majority have no known beneficial or harmful effect on the animal
• Some have important tasks that are useful for the host
• Estimated 500-1000 species of bacteria live in the animal gut and a similar number on the skin
• Bacteria are vital for the maintenance of animal health, synthesizing vitamins, fermenting complex carbohydrates, and converting lactose to lactic acid
• Presence of bacterial colonies also inhibits the growth of potentially pathogenic bacteria and trains the animal's immune system