Bacteriology midterm I

Conventional classification of bacteria

Based on morphology (colony appearance, shape, size, G+-), physiology (substrate usage test, enzyme rxns), or immunology (serotyping of surface antigens).

Numerical / phenetic classification of bacteria

Using biochemical, morphological, & cultural characteristics to create a phenogram (diagram of taxonomic relationships).

Phylogenetic classification of bacteria

The study of evolutionary relatedness using molecular sequencing data.

Characteristics of prokaryotic cells

No membrane-bound organelles, circular DNA, 70S ribosomes (30S / 50S). No sterols in membranes. Presence of D-amino acid, diaminopimelic acid, muramic acid.

Characteristics of eukaryotic cells

Membrane-bound organelles, linear DNA, 80S ribosomes (40S / 60S). Sterols in membranes. No presence of D-amino acid, diaminopimelic acid, muramic acid.

What characteristics differentiate Bacteria from Archaea & Eukarya?

Peptidoglycan in the cell wall. Ester linkages of glycerol & fatty acids (shared with Eukarya). First amino acid synthesised is formylmethionine, as opposed to methionine.

Polyphasic taxonomy

Uses all phenotypic & genotypic data to form a consensus. Genotypic methods include 16s rRNA, DNA hybridisation, multi locus sequence typing (MLST), etc…

16s ribosomal RNA gene (16s rRNA)

Has conserved regions between species & hypervariable regions that are species specific. If >97% similar, they are same species.

T/F: Fungi also use the 16s rRNA gene for identification?

FALSE, they use 18s rRNA.

LR1: Genus and species names are written in bold?

False, they are written in italics.

LR1: Phylogenetics studies the relationships of organisms based on evolutionary similarities & differences?

True.

LR1: The 16s ribosomal RNA gene is the standard for classification and identification of bacteria and fungi?

False, fungi use 18s rRNA.

LR1: Bacteria and fungi are prokaryotes?

False, fungi are eukaryotes.

LR1: Prokaryote cells do not have a membrane-bound nucleus?

True.

LR1: Gram-stain reaction and colony appearance are examples of bacterial genotypic characteristics?

False, these are phenotypic characteristics.

Binary fission

Asexual, vertical form of reproduction. Only requires one parent cell, is rapid, and creates an abundant number of clones. Measured by generation time (avg time for one bacterium to divide in two).

Three broad categories of bacterial structure?

Cell envelope (membrane, wall).

Cytoplasmic structures (nuclear body, ribosomes, inclusion bodies, spores).

Surface structures (capsule, flagella, fimbriae/pili).

Shapes of bacterial cells

Coccus (spherical), bacilli (rod), spiral.

Gram-positive vs. Gram-negative envelopes

G+: thick layer of peptidoglycan threaded with teichoic acids.

G-: lipopolysaccharides (LPS) in capsule, thin layer of peptidoglycan with lipoproteins, inner & outer membranes with porin. Outer layer of "O antigen” sugars are species-specific, used to I.D.

Why do bacteria have enzymes & proteins in their cell walls?

Enzymes break down complex nutrients for easier transport. Proteins can act as selective barriers (porins in G-s for small mols., teichoic acids in G+s for cations), or for adhesion / immune system evasion or stimulation.

Reasons for differences in Gram-staining colour?

Crystal violet & iodine form complexes that are trapped in the thick peptidoglycan layer of Gram+ bacteria, turning them purple.

The thinner peptidoglycan layer of Gram- bacteria means they are decolorised & recolorised with safranin, turning them pink.

Why might Gram-staining not work?

User error during staining process. Some organisms are Gram-variable (+ & -), some such as anaerobes or older colonies stain poorly or not at all.

Flagellum

Filamentous appendages of Gram-negative bacteria used for chemotaxis in liquids.

Pilus, what are the types?

Thread-like appendages of Gram-negative bacteria. Usually, there are numerous short attachment pili (fimbrae), and few longer conjugation pili.

Type I (adherence), Type III (secretory apparatus for virulence proteins), Type IV (gliding & twitching motility for solid surface chemotaxis), Conjugative Type IV (plasmid transfer).

Extracellular polymeric matrices secreted by bacteria

Capsule: well-organised, thick gelatinous covering the cell wall.

Slime layer: loose, unorganised & easily removable glycocalyx around the cell wall.

Biofilm: structured community of bacteria in slime-matrix attached to a surface.

Bacterial capsule

Protective, surrounding mass made of polysaccharides. Usually hydrophilic & negatively charged (acidic), which helps to block immune recognition & phagocytosis. Causes mucoid colonies!

Microbiota / microbiome

Community of microorganisms in an environment (microbiota), plus their genetic material / metagenome, metabolites, & environment (microbiome). Internal microbiomes significantly influence host health.

Endospores

Resistant, dormant (metabolically inactive, long-lived) structures that allow bacteria to survive extreme conditions. Germinate into vegetative cells in favourable conditions.

Structures of endospores

Core (DNA, ribosomes, calcium, acid): stability, resistance.

Cortex (thick peptidoglycan): dehydration.

Spore coat (proteins): resistance.

Exosporium: barrier against cell damage.

LR2: The structure of the bacterial cell envelope determines whether the organism is Gram-positive or Gram-negative?

True

LR2: Gram-positive bacteria have a thin layer of peptidoglycan in their cell wall and an extra outer membrane?

False, this is Gram-negatives.

LR2: Gram-negative bacterial cell walls resist decolorisation with acid alcohol?

False, this is Gram-positives.

LR2: The outermost layer of the Gram-positive cell envelope is called LPS?

False, this is Gram-negatives.

LR2: Bacteria use flagella and pili for movement in different environments?

True.

LR2: Microbiome is a monomicrobial community?

False, polymicrobial.

LR2: Endospores can germinate into vegetative cells when conditions become favourable?

True.

LR2: Bacterial capsules facilitate host immune recognition and phagocytic killing?

False, they inhibit this.

Extracellular pathogens

Proliferate in extracellular environment, cause disease by secreting toxins.

Intracellular pathogens

Facultative: use multiple strategies, virulence factors.

Obligate: need host energy, can’t be grown on artificial media, require cell culture or embryonated egg cultures.

What is septicemia, sepsis, septic shock?

Septicemia is the prescence & multiplication of bacteria in the blood. Sepsis is a life-threatening organ dysfunction caused by dysregulated host immune response that can lead to septic shock (low blood pressure, organ failure).

What are Koch’s postulates?

A microorganism must be found in diseased, but not healthy organisms. It should be isolated & grown in culture, then cause disease when introduced in healthy organisms. It should be re isolated and be identified as the causative agent of the disease.

What are the molecular Koch’s postulates?

Gene must only be found in disease-causing strains, be isolated by cloning & sequencing. Disrupting it in virulent strains should reduce virulence (or introduction to avirulent strains causes virulence). Pathogenicity is restored when gene is re-introduced, & expressed by the bacteria during infection.

Steps of infection & disease process

Entry, attachment and colonisation, spread into host tissue, damage to & survival in host, transmission to new hosts.

How do host cells recognise bacteria?

They have pathogen recognition receptors (PRRs), such as Toll-like receptors, which recognise pathogen-associated molecular patterns (PAMPs) of bacteria. Bacterial DNA also has unmethylated cytosine on cytosine-guanine bonds (motifs).

How does a macrophage destroy a bacterium?

Catch via pseudopodia. Ingested, forming phagosome that fuses with lysosome, resulting in digestion of bacteria by lysosomal enzymes. The digestion products are released from the cell.

What ways do bacteria evade the immune system?

Disrupting Toll-like receptor signalling, coating (complement, fibrinogen, hydrophilic capsule…), secreting toxins or attachment proteins.

How do facultative intracellular pathogens evade the innate immune system?

Inhibition of interleukin production (autophagy). Prevention of lysosome-phagosomefusion. Resistance to lysosomal enzymes. Escape into cytoplasm.

Quorum sensing

Cell-cell communication, allowing bacteria to share cell density information & adjust their virulence gene expression, ensuring it occurs at the appropriate stage of infection.

How do bacteria invade the skin & blood?

Skin: produce hyaluronidase & collagenase, breaking down collagen between cells to allow for deeper tissue invasion.

Blood: produce coagulase, forming a perivascular bacterial clot. Later produce kinase, dissolving the clot & releasing the bacteria into the bloodstream.

Exotoxins (protein toxins)

Released from the cell. Effector proteins are injected into their target by specialised secretion systems, such as type III. Specific, potent toxins that are highly antigenic & become toxoids (induce neutralising antibodies).

Endotoxins

Released from the cell wall of G-s after death. Non-specific, moderate toxins that are weakly antigenic (but highly pyrogenic), do not become toxoids / do not produce neutralising antibodies.

Gram-negative associated endotoxemia

Endotoxins can lead to complement & tissue factor activation, leading to cytokine storm that leads to DIC, organ failure, & death.

Superantigens

Cause excessive, nonspecific activation of T-cells & APCs, leading to massive cytokine release & systemic inflammation.

Mobile genetic elements

DNA sequences that can be transposed between genomic sites or between bacteria. Plasmids, jumping genes (transposons, integrons, genomic islands). Carry transposition genes or additional genes (antibiotic resistance, toxins).

Horizontal gene transfer via conjugation

Donor draws recipient into contact via pilus, transfers one strand of plasmid DNA. Each cell synthesises a complementary strand to restore the complete plasmid, making them both F+ cells.

Horizontal gene transfer via transduction

A phage injects its DNA as its enzymes degrade the host DNA. The cell synthesises new phages with phage & host DNA. A transducing phage injects this donor DNA into a new cell, which incorporates it by recombination.

Horizontal gene transfer via transformation

Recipient cell takes up loose fragments of donor DNA, then recombines it into its own genome.

LR3: Dual intracellular / extracellular pathogens use multiple virulence mechanisms to survive and grow inside or outside host cells?

True

LR3: Attachment to & colonisation of host surfaces are key steps of bacterial infection?

True

LR3: Host immune cell PRRs recognise bacterial PAMPs?

True

LR3: Gram-positive cell LPS can cause endotoxemia with massive cytokine release, resulting in sepsis & septic shock?

False, this is Gram-negatives.

LR3: Bacteria are not able to share genes with different bacterial species?

False, this is called horizontal gene transfer.

LR3: Plasmids, integrons, and transposons are mobile genetic elements?

True

LR3: Horizontal gene transfer allows bacteria to evolve and adapt rapidly?

True

Why would one submit a bacterial sample to a lab?

To confirm bacterial infection, identify which organism, gain antimicrobial susceptibility data & response to therapy. “Certification, surveillance, monitoring”.

T/F: Sampling blood once from a single vein is enough to submit.

FALSE, at least two samples from different sites within the same 24 hour period.

Five stages of specimen submission

1. Stage: collect early, before antibiotics.

2. Site: collect from actual infection site.

3. Collect: tissue or fluid, or swabs with transport medium.

4. Label: label, fill out Sample Submission Form.

5. Store and Ship: ship chilled, except anaerobic & blood cultures.

What kind of organisms use Stuart medium?

Aerobic / aerotolerant, fastidious organisms.

What kind of organisms use Cary-Blair medium?

Fecal samples, such as Salmonella.

What kind of organisms use Amies medium with charcoal?

Toxic organisms, such as Campylobacter, Taylorella equigenitalis (CEM), Moraxella (pink eye).

What kind of organisms use Amies medium without charcoal?

Mycoplasma & Ureaplasma… contains antimicrobial agents to minimise contamination.

What kind of organisms use Thioglycollate medium?

Anaerobes.

How should urine samples be collected?

Cystocentesis, but if catching is needed try to do mid-stream to limit skin contaminants.

How should Dermatophyte specimens be collected?

If there are obvious lesions, remove hairs with forceps & skin scales at the edge of the active inflammation & store in dry container.

If there are no obvious lesions (as in cats), rub them with a toothbrush & send it in a plastic bag.

What kind of pathogens can’t be tested via cultures?

Unculturable / RG3 pathogens (PCR or serology), old partially decomposed tissues (PCR), fecal/serum toxins (ELISA).

LR4: Clinical specimens should be collected at early stage of infection?

True

LR4: Amies medium with charcoal is preferred for sampling pink eye?

True

LR4: Urine samples can be stored safely at room temperature prior to shipping to diagnostic lab?

False, only anaerobic & blood cultures can be.

LR4: Double-guarded swab is used for uterine sampling?

True

LR4: Ear swab is typically submitted for anaerobic culture?

False, submitted for aerobic culture.

LR4: A single blood sample is good enough to detect bacteria?

False, at least two from different sites within 24 hours needed.

LR4: A wound sample can yield both aerobic and anaerobic pathogens?

True

LR4: The preferred method of urine collection is ___?

Cytocentesis.

LR4: Besides diagnosis and antibiotic sensitivity confirmation, reason for submitting specimens to diagnostic lab is for ___?

Identification, surveillance, monitoring.

LR4: While sampling, donning of gloves is optional?

False, always required.

Characteristics of Staphylococcus

Facultative anaerobes, Gram-positive, cocci, clusters. Commensal flora of skin / mucosa.

Coagulase tests

Coagulase enzyme clots plasma into fibrin, which hides bacteria from PMNs. Coagulase-positive Staph includes aureus, pseudintermedius, coagulans, hyicus. Coagulase-negative Staph includes epidermis, felis, chromogenes.

Result of coagulase test?

POSITIVE: double-zone hemolysis.

NEGATIVE: nonhemolytic.

Virulence factors used by Staphylococcus aureus

Many, making it a successful pathogen. For example: capsule, protein A, extracellular toxins, free coagulase.

Methicillin-resistant Staphylococcus (MRSA)

Acquisition of mobile genetic element SCCmec, which confers resistance to all beta-lactam antimicrobials.

Clones

Group of strains with same genotype & a common ancestor. Can have different virulence factors, antimicrobial resistance, or affect different hosts.

MRSA clones

Zoonotic, easily colonise & spread between species. Pigs serve as major reservoir, as can horses.

Characteristics of S. aureus

Primary, opportunistic, zoonotic pathogen. Often nosocomial & a multidrugresistant (MDR) superbug.

S. aureus associated septicemia, tick pyaemia, Morel’s disease

Septicemia: causes embolic microabscesses, myocarditis, pneumonia in lambs.

Tick pyaemia (British): opportunistic infection as a result of Anaplasma associated immunosuppression, causes abscesses in joints & organs.

Morel’s disease: cervical lymph node & lung abscesses, can combine with caseous lymphadenitis (Corynebacterium) to cause abscess disease.

S. aureus associated pododermatitis

Pododermatitis: chronic ulcers, deep granulomas in avian feet (or guinea pigs), predisposed by trauma or poor husbandry.

S. aureus associated bovine mastitis

Contagious mastitis pathogen, usually subclinical. Hides in microabscesses within the udder (poor Abx response, negative culture). Can cause botryomycosis (chronic, purulent granulomatous lesions), or acute gangrenous masitis (cytotoxins, superantigens).

Caused by S. aureus clone ET3, subtype ST151 (cytolytic toxins).

Coagulase-negative Staphylococcus & bovine mastitis

Normal udder flora, mainly cause subclinical persistent infections with increased somatic cell counts (SCCs). Has increased antimicrobial resistance vs. S. aureus. Includes S. chromogenes, haemolyticus, simulans, xylosus, epidermis…

Staphylococcus pseudintermedius

Commensal flora of dogs. Has coagulase, catalase, & produces exfoliative toxins (damage epidermis, important in pyoderma).

Co-selection

Treatment with one antimicrobial can select for resistance across classes, due to SCCmec carrying multiple resistance genes. An example is Cephalexin selecting for MRSP in dogs.

Cause of canine chronic otitis externa

Coagulase-positive Staph, including both S. pseudintermedius & S. coagulans.