Exam #04 - Learning Objectives

How are animal viruses cultivated and isoalted?

Through tissue culture (using primary or immortalized cell lines with growth factors, glucose, amino acids, vitamins, and antibiotics) or embryonated chicken eggs

Viral infection is monitored by cell death (morphology change, detachment, trypan blue staining)

How are bacteriophages cultivated and isolated?

By infecting early exponential phase bacteria with phages at low MOI, using divalent cations (Ca2+ or Mg2+), growing overnight, releasing phages with chloroform, and purifying through low-speed centrifugation and filtration

How are fungi cultivated and isolated?

Most fungi grow easily on complex media at room temperature (25°C) or 37°C, often using specialized media like bird seed agar or potato dextrose agar; fungal spores are isolated via agar plates

How are pathogenic protozoans cultivated and isoalted?

Some grow in complex media (glucose, peptone, yeast extract, sometimes supplemented with liver extract or fetal calf serum); others require tissue culture or animal hosts—intestinal parasites are isolated by fecal flotation and centrifugation

How does temperature affect bacterial cultivation?

Bacteria have cardinal temperatures (minimum, optimum, maximum); most pathogens are mesophiles (20°C-45°C, optimum at 37°C)—special adaptations include cold enrichment for Listeria and pasteurization tolerance

How does pH affect bacterial cultivation?

Bacteria prefer near-neutral internal pH; pathogens typically grow best at pH 6.5-7.5

Acidophiles (pH 1.0-5.5); neutrophiles (pH 5.5-8.0); and alkalophiles (8.5-11.5)—acid adaptation helps survival through stomach acidity

How does oxygen tension affect bacterial cultivation?

Bacteria differ in oxygen requirements - obligate aerobes, facultative anaerobes, aerotolerant anaerobes, obligate anaerobes, and microaerophiles

Enzymes like superoxide dismutase (SOD) and catalase detoxify harmful oxygen radicals

How is enrichment media used to identify specific bacterial pathogens?

Favor the growth of certain bacteria by providing selective advantages, like tetrathionate broth for Salmonella or 4°C incubation for Listeria

How is selective media used to identify specific bacterial pathogens?

Inhibit unwanted microbes and allow target microbes to grow

• MacConkey agar - Gram-negatives

• PEA - Gram-positives

• XLT-4 agar - Salmonella and detects H2S production

Is a microscopy sufficient for identifying animal viruses, protozoans, helminths, fungi, or bacteria?

It is important but usually not sufficient for bacteria

For fungi, protozoans, and helminths, morphology (shape, structure, arrangement) seen by microscopy is critical for identification

What do you look for in microscopy to identify protozoans and helminths?

Look for cysts, trophozoites, oocysts, egg morphology, larval stages, and locomotion characteristics

What do you look for in microscopy to identify fungi?

Morphology like hyphae, mycelium, spores (conidia), yeast cells, and special structures like capsules (Cryptococcus neoformans)

What do you look for in microscopy to identify animal viruses?

By observing cytopathic effects (rounding, detachment, syncytium formation) is tissue culture, and by molecular tests like PCR or immunological assays (ELISA, immunofluorescence)

What is genotype?

The set of genes an organism has

What is phenotype?

The observable traits or characteristics, such as shape, staining, and metabolic behavior

How does genotype relate to phenotype in microbial identification?

Genotype underlies phenotype; observable traits (phenotype) are based on the organism’s genetic makeup (genotype)

What is a phenetic approach to bacterial identification?

Identifying bacteria based on observable traits like Gram stain, cell arrangement, colony morphology, metabolic reactions, and antigenic properties

What is a phylogenetic approach to microbial classification?

Classifying organisms based on evolutionary relationships (using 16S rRNA sequencing)

How does PCR work?

PCR mimics DNA replication

1. DNA is denatured (separated) by heat

2. Primers bind (anneal) to specific complementary sequences

3. DNA polymerase extends the primers, making new DNA strands

What does the specificity of a PCR test depend on?

Specific sequence of the primers—they must match the target DNA exactly to amplify the right sequence

What conditions are required for a good PCR target sequence?

Target gene must be unique to the species/strain, uniformly distributed across the population, and conserved

How can 16S rRNA sequencing be used for microbial identification?

PCR primers target conserved regions of 16S rRNA, allowing sequencing and matching to a database (BLAST) to identify bacteria to the species level

Describe how key enzymes are regulated in microbial metabolism.

Metabolic channeling

Altering enzyme activity (feedback inhibition, allosteric regulation, covalent modification)

Regulating gene expression

What is feedback inhibition?

Process where an end-product binds to an enzyme early in a pathway, decreasing its activity and thus regulating the entire pathway

What is allosteric regulation?

Molecules bind to a site other than the active site on an enzyme, causing a change in enzyme chape and activity

What is covalent modification in enzyme regulation?

Chemical groups are added to or removed from an enzyme, locking it into an active or inactive form

What are the properties of bacterial membranes that affect nutrient entry?

Membranes are made of phosphatidylethanolamine bilayers that block most molecules—specialized transport proteins allow selective nutrient entry

How do nutrients enter the bacterial cell?

Through specific transporters that recognize particular molecules; energy may be required if environmental concentrations are low

What is catabolism? Give an example of a pathway.

Breaks down molecules, decreases complexity, releases energy (exergonic)

Glycolysis - glucose is broken down into pyruvate with ATP production

What is anabolism? Give an example of a pathway.

Builds complex molecules from simpler ones, requires energy (endergonic)

Biosynthesis of amino acids from TCA cycle intermediates

How is NAD+ regenerated during fermentation (anaerobic)?

Organic molecules (like pyruvate) serve as electron acceptors, converting NADH back to NAD+ without using oxygen

How is NAD+ regenerated during respiration (aerobic)?

Electrons from NADH are transferred through an electron transport chain to oxygen, regenerating NAD+ and producing ATP

Discuss the features that make a pathway anabolic.

• Anabolism increases molecular complexity (‘builds’, ‘synthesis’)

• Requires free energy input (endergonic reactions)

• Coupled with exergonic reactions like ATP hydrolysis

• Involves reduction of carbon compounds (uses NADPH as reducing agent)

• Enzymes couple endergonic and exergonic reactions

Explain what is required for the synthesis of most bacterial polysaccharides like peptidoglycan or LPS.

• Synthesis of nucleosugars (NDP-sugars) like UDP-NAM and UDP-NAG

• Hydrolysis of phosphodiester bonds in nucleosugars provides energy for glycosidic bond formation (‘equivalent to 1 ATP per bond’)

• Use of a lipid carrier (bactoprenol) to transport building blocks across the membrane

• Special proteins are needed to export the polysaccharides outside the cell

List the steps in protein synthesis that expends the equivalent of ATP to incorporate just one amino acid into the growing polypeptide chain.

1. Formation of aminoacyl-tRNA costs 1 ATP equivalent

2. Ribosome initiation (30S + 50S subunits) costs 1 GTP (ATP equivalent)

3. Bringing aminoacyl-tRNA to the ribosome ‘A site’ costs 1 GTP (ATP equivalent)

4. Translocation of ribosome down mRNA (move 3 nucleotides) costs 1 GTP (ATP equivalent)

   • Total - About 3 ATP equivalents per amino acid incorporated

Describe the synthesis of peptidoglycan in bacteria.

Precursors made in cytoplasm

Assembled on bactoprenol lipid carrier (C55 undecaprenol)

Translocated across membrane; cross-linked by transpeptidation (Pbp)

Occurs in both Gram-positive and Gram-negative bacteria

Describe the synthesis of lipopolysaccharide (LPS) in bacteria.

O-antigen and core-lipid A made separately in cytoplasm

O-antigen uses bactoprenol lipid carrier; lipid A serves as its own carrier

Assembled and polymerized in periplasm

Unique to Gram-negative bacteria

Describe the synthesis of lipoteichoic acid (LTA) in bacteria.

Made only in Gram-positive bacteria

Precursors synthesized in cytoplasm

Assembled on a di-glycosyl diacylglycerol lipid carrier

Glycerol phosphate comes from membrane phospholipids

What features are unique to cell wall (peptidoglycan) synthesis?

Unique sugars - N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG)

Use of unusual D-amino acids and diminopimelic acid (DAP)

Covalent cross-linking by PBPs (penicillin-binding proteins)

What features are unique to lipopolysaccharide (LPS) synthesis?

Only found in Gram-negative bacteria

Build separately - O-antigen and core lipid A pathways

Outer membrane component; major PAMP

What features are unique to lipoteichoic acid (LTA) synthesis?

Only found in Gram-positive bacteria

Glycerol phosphate backbone from phosphatidylglycerol

Similar role to LPS but anchored in cytoplasmic membrane

Explain how PAMPs like peptidoglycan, LPS, or LTA are released from the bacterial cell.

• Lysis due to activation of latent bacteriophages

• Membrane attack complex (complement-mediated lysis)

• Phagocytosis and killing by neutrophils

• Antibiotic-induced lysis or stress-induced cell death

• Normal ‘blebbing’ of outer membrane vesicles (OMVs) during bacterial growth—outer membrane vesicles shed containing PAMPs

Explain how microbes survive environmentally stressful conditions like starvation.

• Produce exospores (fungi, bacteria, protozoans) to survive harsh conditions

• Form endospores (Gram-positive Bacillus and Clostridium) - resistant to heat, UV, desiccation

• Develop biofilms, using polysaccharides and proteins, becoming resistant to stress (including antibiotics)

• Adapt by scavenging nutrients (high-affinity transporters, siderophores)

What is the Lag phase of microbial growth and what factors contribute to it?

Cells introduced to fresh medium; no increase in number—synthesizing essential components (ribosomes)

What is the Exponential (Log) phase of microbial growth and what factors contribute to it?

Rapid, constant growth; depends on genetics and growth conditions (temperature, carbon source)

What is the Stationary phase of microbial growth and what factors contribute to it?

Growth ceases due to nutrient depletion, waste accumulation, oxygen limitation; cells enter starvation response—resistant to stressors and antibiotics

How do microbes respond to temperature stress?

Cardinal temperatures - each microbes has a minimum, optimum, and maximum temperature for growth

• Psychrophiles (0-15°C), mesophiles (20-45°C), and thermophiles (>55°C)

Heat shock proteins (chaperones DnaK, GroEL) refold damaged proteins

Stenothermal organisms tolerate narrow ranges; eurythermal organisms tolerate wide ranges

How do microbes respond to acid stress?

Maintain internal pH near neutrality (pH ~ 7)

Acid tolerance response - preadaptation to pH 5.0-6.0 enhances survival at pH <4.0

Use proton pumps (proton-translocating ATPase) to maintain pH

Produce enzymes like carbonic anhydrase, decarboxylases (release ammonium to neutralize acid)

Regulated by alternate sigma factors like σs

How do microbes respond to oxidative stress?

Oxygen radicals (O2-, H2O2, OH*) are toxic and damage DNA, proteins, and membranes

Produce detoxifying enzymes:

• Superoxide dismutase (SOD) converts superoxide to hydrogen peroxide

• Catalase breaks down hydrogen peroxide to water and oxygen

What are the growth categories for oxidative stress?

Obligate aerobes (require O2)

Facultative anaerobes (grow with or without O2)

Aerotolerant anaerobes (ignore O2)

Obligate anaerobes (O2 is toxic)

Microaerophiles (require low O2 levels)

Explain why antibiotics only affect bacteria and NOT fungi, protozoa, or animal viruses.

Antibiotics target features unique to bacteria, such as the cell wall, ribosomes, gyrases, or folate biosynthesis enzymes

Fungi, protozoa, and animal viruses lack these bacterial specific features, so antibiotics are ineffective against them

What is intrinsic resistance?

Natural property of a bacterial group

Gram-negative Enterobacteriaceae are naturally resistant to vancomycin due to their outer membrane acting as a barrier to drug diffusion

What is spontaneous mutation to resistance?

Bacteria mutate spontaneously in target genes, leading to reducing binding to antibiotics

Fluoroquinolone resistance caused by amino acid substitution in DNA gyrase (GyrA)

What is acquired resistance?

Bacteria gain resistance through genetic exchange via plasmids or transposons

Mechanisms include transformation, transduction (via phages), and conjugation (cell-to-cell transfer)

What is conditional resistance?

Resistance occurs under specific conditions (in stationary phase or biofilm formation)

What is silent resistance?

Resistance genes may remain inactive but are expressed under certain conditions or mutations in gene promoters

Explain how the rumen functions like a bioreactor. What is the media; how is pH modulated; how is the rumen made anaerobic?

Media:

• Rumen microbes use grasses provided by the host as nutrient media

• Fermentation is the primary catabolic process, generating volatile fatty acids (VFAs)

pH modulation:

• Saliva acts as a bicarbonate buffer to prevent excessive acidity

• VFAs are transported from the rumen epithelium to maintain pH balance

Anaerobic environment:

• Obligate anaerobic bacteria predominate, relying on fermentation

• Aerobic respiration by certain bacteria and mitochondrial consumption of oxygen help maintain anaerobic conditions

Discuss how a ruminant, not able to degrade plant cell wall, is capable of subsisting on grasses?

Ruminants rely on symbiotic microbes (eubacteria, fungi, and protozoans) in the rumen; these microbes produce carbohydrate-active enzymes (CAZymes) that break down cellulose, hemicellulose, pectin, and lignin into digestible components

The microbes convert plant cell wall polysaccharides into VFAs during fermentation; VFAs are absorbed and converted into glucose via gluconeogenesis in the ruminant’s liver, providing energy

How does Streptococcus mutans contribute to dental caries?

Metabolizes sucrose into glucans and fructans, forming sticky biofilms that colonize tooth enamel; produces lactic acid via fermentation of glucose and fructose, leading to tooth surface demineralization and cavities

A diet rich in sucrose supports the cariogenic activity of S. mutans

How does Porphyromonas gingivalis contribute to gingivitis?

Associated with a shift in oral microbiome towards a Gram-negative dominant population; releases outer membrane vesicles containing lipopolysaccharides (LPS), which trigger inflammation through TLR4 activation

LPS promotes immune responses and contributes to tissue damage in the gums

How do H2S-producing bacteria contribute to colorectal cancer?

Certain intestinal microbes metabolize dietary cysteine and taurine into hydrogen sulfide (H2S); H2S is genotoxic, causing DNA damage and promoting mutations associated with cancer development

Animal-based diets rich in cysteine and taurine lead to increased H2S production, heightening colorectal cancer risk

Discuss how diet contributes to dental caries.

High-sucrose diets fuel the activity of Streptococcus mutans, increasing production of lactic acid and enamel demineralization

Discuss how diet contributes to colorectal cancer.

Animal-based diets high in cysteine and taurine enhance H2S production by intestinal bacteria; elevated H2S levels disrupt mucin production, ion absorption, and cellular detoxification, promoting cancer

Plant-based diets result in lower H2S production and reduced cancer risk

Explain why a culture-based approach to characterizing an animal microbiome underrepresent the true community composition.

The Great Plate Count anomaly is a culture-based methods fail to capture the diversity because they select for ‘weeds’—microbes that easily grow under artificial conditions

• Growth conditions (media, atmosphere, temperature) and nutrient requirements restrict the types of microbes that can be cultivated

• Auxotrophic microbes or those reliant on helper microbes are missed because of their interdependence is not replicated in culture (symbiotic relationships)

• Oxygen sensitivity and nutritional specificity make isolation difficult (obligate anaerobes)

What is 16S rDNA’s approach to characterizing microbial communities? What are the advantages and disadvantages?

(A) Identifies microbes to genus/species level; relies on conserved and divergent regions

(D) Broad phyla characterization may not predict functional roles or metabolic interactions

What is metagenome’s approach to characterizing microbial communities? What are the advantages and disadvantages?

(A) Identifies species/genus and highlights potential metabolic pathways based on genome sequences

(D) Provides static data that does not capture active metabolism or expression

What is metatranscriptome’s approach to characterizing microbial communities? What are the advantages and disadvantages?

(A) Highlights active metabolism and enzyme pathways; identifies functional roles of specific microbes

(D) RNA degradation risks and the need for advanced sequencing methods

How can Koch’s postulate #3 be used to determine the contribution of the animal microbiome to disease?

“The cultured microorganism should cause disease when introduced into a healthy organism”

• Replacing ‘cultured microorganism’ with a fecal transplant from diseased animals

• Germ-free animals receive the microbiome from diseased donors; if these animals display the disease phenotype, it establishes the microbiome’s role in causing the disease

How are germ-free animals used to determine the contribution of the microbiome to diseases?

1. Germ-free animals are raised in sterile environments and lack of a microbiome

2. Fecal microbiota from healthy or diseased individuals is transplanted into these germ-free animals

3. If the animals receiving the diseased microbiota exhibit symptoms or physiological changes, it confirms the microbiome’s involvement in the disease

What evidence supports the intestinal microbiome’s contribution to obesity?

• Germ-free mice receiving microbiota from obese humans or mice develop increased body fat

• Differences in gut microbial composition between lean (Bacteroidetes-dominant) and obese (Firmicutes-dominant) individuals

• High-fat diets alter microbiome metabolism, leading to increased energy harvest, altered bile acid signaling, and fat storage

  • Bile salt hydrolase activity of gut microbes impacts triglyceride and cholesterol metabolism, promoting obesity

What evidence supports the intestinal microbiome’s contribution to atherosclerosis?

• Gut microbes metabolize nutrients like carnitine (from red meat) into trimethylamine-N-oxide (TMAO), which promotes inflammation and arterial plaque formation

• Antibiotics suppress gut microbiota activity, reducing TMAO levels and preventing atherosclerosis in experimental models

• TMAO production correlates with dietary intake of red meat and the presence of specific microbial enzymes (CutC and CntA)

What evidence supports the intestinal microbiome’s contribution to cancer?

• Diets rich in red meat increase levels of hydrogen sulfide (H2S), a genotoxic metabolite produced by gut microbes

• H2S damages DNA, inhibits butyrate metabolism, and decreases mucin production, contributing to colorectal cancer development

• Germ-free mice receiving microbiota from colon cancer patients develop tumors, linking microbiome composition to cancer

• Infusion of butyrate counters negative effects of H2S, highlighting the interplay between microbial metabolism and cancer

Discuss the evidence supporting the claim that the intestinal microbiome serves as a barrier for exclusion of the pathogens Salmonella and Clostridiodes difficile.

Inverse correlation between pathogen abundance and intestinal community diversity; higher diversity linked to exclusion of pathogens like Salmonella and Clostridiodes difficile

Salmonella -

• Chicks colonized with diverse microbiomes showed significant reductions in Salmonella colonization

• Mature intestinal microbiota rapidly reduces Salmonella abundance after 21 days

Clostridioides difficile -

• Antibiotic treatments reduce microbiome diversity, creating opportunities for C. difficile colonization

• Fecal microbiota transplantation (FMT) restores community diversity, excluding C. difficile and reducing toxin production

How does antimicrobial therapy determine the contribution of the intestinal microbiome in pathogen exclusion?

Salmonella - antibiotics like Streptomycin or Vancomycin disrupt microbiome diversity, permitting Salmonella colonization and colitis in mice

Clostridiodes difficile - Clindamycin treatment reduces microbiome diversity, leading to severe C. difficile infections and ‘super-shedder’ states

How do fecal microbiota transplantation (FMT) determine the contribution of the intestinal microbiome in pathogen exclusion?

Restores microbiome diversity, reducing pathogen colonization (Salmonella and C. difficile)

Clostridiodes difficile - FMT eliminates C. difficile, restores normal microbiota, and prevents relapsing disease

• Demonstrates the critical role of a healthy microbiome in excluding pathogens effectively without reliance on antibiotics