Foodborne illnesses can be caused by microbes two different ways: poisoning and infection. Food poisoning results from ingesting a bacterial exotoxin. Onset of food poisoning symptoms is rapid. Food infection results from ingesting a food contaminated with bacteria. Onset of food infection symptoms is relatively slow (sometimes several days).

Virulence factors are cellular properties which promote pathogenesis. Without these virulence factors, pathogenesis is attenuated or eliminated. These virulence factors can be:

Introduction & History

The outcome of a microbe-host encounter depends on host defense and the strain of microbe. Disease is not the desire outcome: pathogens evolved to cause disease, but are evolving away from virulence. Humans are accidental hosts.

Louis Pasteur (1822-1895), the father of microbiology, developed:

• The germ theory of disease
• Rabies, anthrax, chicken cholera
• Heat based sterilization techniques
• Pasteurization
• Vaccines and immunization

Robert Koch (1843-1910), the father of microbial pathogenesis, developed:

• Methylene blue staining of bacterial cells
• Solid bacterial cultures
• Chemical disinfectants
• Discovered the tuberculosis bacillus (1882)
• Discovered the cholera bacillus (1883)
• 1905 Nobel Laureate in Medicine for the discoveries on tuberculosis.
• Koch’s Postulates

Alexander Fleming discovered penicillin in 1929, thereby beginning the era of antibiotics. He won the 1945 Nobel Prize for this discovery.

• “Fleming kept his cultures 2-3 weeks before discarding them. When he looked at
  one set he noticed that the staphylococcus bacteria seemed to be dissolving. The
  mold that contaminated the culture was a rare organism called penicillium. He left
  the culture on the lab bench and went on vacation. While he was away the culture
  was subjected to a cold spell followed by a warm one – the only conditions under
  which the discovery could be made.”

Joshua Lederberg discered bacterial conjugation in 1946. Bacterial conjugation and mating has become the basis for genetic mapping, recombination, and gene discovery. He won the 1958 Nobel Prize for this discovery.

Since 1953, a molecular revolution has ensued due to discoveries and developments:

• Watson and Crick: DNA double helix
• Genetic code
• Central dogma
• Enzymes to manipulate DNA (DNAP, restriction enzymes, ligases)
• Gene cloning and genetic engineering

See Koch’s Postulates

There are 3 major categories of pathogenic bacteria:

• Gram-Positive and -Negative
• Cocci/Rods
• Spirochetes (helically shaped & neither gram-positive or -negative)

Importance of Bacteriopathology Research

A human body has 10¹³ eukaryotic cells and 10¹⁴ prokaryotic cells. Microbial infections include:

• Oral microbial infections (cavity and gum disease)
• Diarrhea and enteric bacteria
• Tuberculosis and mycobacterium
• Ulcer and helicobacter infection
• Urinary tract infection
• STD

There an 800% chance for a person to contract a microbial infection during their lifetime. $200 billion are spent annually treating microbial infections, with $55 billion spent annually treating oral microbial infections (cavities and gum disease), $19 billion spent annually treating ulceres, and $10 billion annually spent treating non-HIV/AIDS STDs. Microbial infections accounted for 57 million deaths in 2002 according to the World Heath Organization (microbial shown in red and non-microbial in blue):

• Tuberculosis = 11 million
• Pertussis/tetanus = 0.6 million
• Diarrheal diseases = 1.8 million
• Respiratory infections = 3.8 million
• Malaria = 1.2 million
• Injuries (car accidents to war) = 5.2 million
• Cancer = 7.1 million
• Cardiovascular disease = 16.6 million

Overview

It is important to approach and study bacterial pathogenesis at both a cellular (diagnosis using Koch’s postulates and treatment by killing pathogen and blocking transmission) and molecular level (using molecular Koch’s postulates and treatment by targeting virulence factors).

Evading and suppressing host immune response
•Protective capsule
•Phase variation
•Type III secretion system
•Immunotoxins
•Repelled by or resistance to nitric oxide
•Surviving phagocytosis
•Degrade complement
•Ig degradation or Ig binding ability
•Bacteria induced apoptosis
•etc

To study bacterial infection at the cellular level, there are 3 forms of cultures:

Detecting and monitoring bacterial infections. Classical methods innclude bacterial culture of infected tissues and em analysis of infected tissues. Detecting and monitoring bacterial infection, like modern in vivo imaging, whole body fluorecsent imaigng of E. coli GFP in various organs.

Virulence Factors

Virulence Gene Regulation

There are several ways a strain can become pathogenic: by gaining genes specific to the pathogen, absence of a suppressor locus in the pathogen, allelic differences between genes shared by pathogen and nonpathogen, and by differential regulation of the same genes.

Virulence genes can be regulated by:

• Environmental cues (CO₂, temperature, iron, pH, etc)
• 2-component regulatory systems
• Quorum sensing
• Genetic rearrangement: fimA & RecA- dependent pilin antigenic variation
• Contact-dependent regulation

Secretory Pathways

Gram (+) and Gram (-) cells have different secretory pathways. This is due to morphological difference, with Gram (+) cells having 3 compartments (cytoplasm, membrane, and extracellular) and Gram (-) cells having 5 compartments (cytoplasm, inner membrane, periplasm, outer membrane, and extracellular). Gram (+) cells use the sec-dependent general secretory pathway (types II and V), and Gram (-) cells use the sec-independent pathway (types I, III, and V) as well as the autotransporter (type V).

Competition assay tests virulence factor. Mix mutant and WT strains of bacteria. If mutant has defect in virulence, this will be detected by examining ratio of mutant:WT at assay endpoint.

1. Underproduction of proteins required for drug activation.
2. Underproduction of a membrane protein that is necessary for uptake of the drug (methotrexate resistance in Leishmania).
3. Overproduction of the target enzyme resulting from DNA amplification: Dihydrofolate reductase-thymidylate synthetase (DHFR-TS) overproduction leading to methotrexate resistance.
4. Mutation in a cellular protein that results in decreased affinity of the drug and its target.
5. Overproduction of a membrane glycoprotein that pumps drugs out of the cell.

Introduction & History

Alexander Fleming discovered penicillin (the 1^st antibiotic) in 1929. As more antibiotics have been developed, selective pressure for antibiotic resistance has increased. As genes encoding resistance are transferred between bacteria, antibiotics gradually become effective against fewer bacteria. As a result, new antibiotics must perpetually be discovered.

Approximately 2 million nosocomial infections/yr occur, resulting in ~80,000 patient deaths. Strains of Staph. aureus and Enterococcus resistant to conventional antibiotics are widespread, and the total cost of additional health care due to resistant strains is ~$5 billion.

Agriculture and aquaculture account for ~40-50% of all antibiotic use in the US. Tetracycline derivatives are used at subtherapeutic doses to augment the size of pigs and chickens, and to reduce farm animal infection rates. As a result, though, groundwater and manure from farms oftentimes carry Tet^r (tetracycline resistant) bacteria which contaminate the ecosystem.

Currently, there are three precautions implemented to abate the development of antibiotic resistance:

• Educating the medical community about infection prevention
• Conservative use of antibiotics like vancomycin to slow evolution of antibiotic^r strains.

Spread of Resistance Genes

An example of a drug resistance plasmid is pB10. It was isolated in a waste water treatment plant and confers resistance to antimicrobials (amoxicillin, streptomycin, sulfonamides, and tetracycline) and mercury. It is a mosaic plasmid, meaning it has regions from several plasmids combined via recombination. It has at least 5 distinct mobile genetic elements (most conferring resistance).

Resistance genes can get around by gene transfer (plasmids, transposons, and conjugative transposons) or bacterial intermediaries (non-pathogenic strains are resistance gene reservoirs).

Example: Tetracyclines

Tetracycline antibiotics include doxycycline (clinical use) and tetracycline, chlortetracycline, and oxytetracycline (agricultural use). Tetracycline readily crosses the peptidoglycan layer of Gram-positive and outer membrane of Gram-negative bacteria and inhibits translation. Tetracycline is classified as a “broad spectrum”antibiotic because it is effective against a wide range of organisms. In Gram negative bacteria, tetracycline is transported though the outer membrane as a metal-chelatedcomplex (probably Mg²⁺) by the OmpFand OmpC porin channels. Accumulation of the cationic tetracycline-Mg complex in the periplasmis facilitated by the Donnanpotential of the membrane. Dissociation of the cationic complex allows the low molecular weight, lipophilictetracyclinesto penetrate the cytoplasmic membrane. In Gram-positive cells the peptidoglycan layer is porous to low molecular weight compounds allowing entry into the cell and bind the ribosome.

Mechanisms of resistance against tetracylines:

• Effluxers: transporters move tetracyclines across cell membrane (often antiporters, with tetracycline out and H⁺ in). Effluxer proteins may have broad specificity, meaning they are mutlidrug transporters.
• Ribosomal Protection: Proteins bind the ribosome to prevent tetracyclines from binding. tetM resembles EF-G and displaces tetracycline, but does not replace EF-G. Mutations to rRNA alter binding pockets where tetracyclines bind (16s rRNA).
• Enzymatic: enzymes modify and inactivate tetracyclines

There are many Tet^r genes. Those marked with asterisks are restricted to Gram-positive cells. Gene families are separated by semicolons.

• Efflux: tetA, tetB, tetD, tetE, tetG, tetH, tetI, tetJ, tetZ*, tet30, tet31, tet33*, tet38, tet39; tetK*, tetL*; otrB*, tcr3*, otrC*; tetP(A); tetV; tetY; tet35
• Ribosomal protection: tetM*, tetO*, tetS*, tetW*; tetQ*, tetT*, tet36*; otrA*, tetP(B), tet*; tet32*
• Enzymatic: tetX, tet34, tet37