• Gram-positive cocci can first be categorized as catalase positive or catalase negative.
  — Catalase-positive cocci include species of Staphylococcus.
  — Catalase-negative cocci include species of Streptococcusand Enterococcus.

Catalase-positive cocci: Staphylococcus

• Species of Staphylococcus can be categorized based on the presence of coagulase, which is a bacterial enzyme that induces blood or plasma coagulation:
  — The coagulase-positive group comprises Staphylococcus aureus.
  — Coagulase-negative species include Staphylococcus epidermidis and Staphylococcus saphrophyticus.

Staphylococcus aureus

• Named for its golden color.
• Some strains are resistant to the antibiotic Methicillin (these strains are called MRSA); infections caused by these strains are difficult to treat.
• Inflammatory Conditions caused by S. aureus
  — Skin infections include various purulent conditions such as impetigo, furuncles, and others.
  — Serious organ infections include endocarditis, pneumonia, and infections of the bones and joints that lead to osteomyelitis and septic arthritis.

• Toxin-mediated conditions caused by S. aureus
• Toxic shock syndrome
  — Formerly associated with tampon use, septic shock now occurs at least as often, if not more often, in non-menstruating individuals.
  — Toxic shock syndrome is characterized by acute onset of fever, gastrointestinal upset, sore throat, and diffuse erythroderma; desquamation occurs when the skin begins to peel and flake away.
• Scalded skin syndrome* is also a desquamating condition, is most common in infants and young children.
  — In our illustration, we’ve shown the characteristic red and flaky areas of skin.
• Food Poisoning

Staphylococcus epidermis

• An important source of medical device infections, particularly in individuals with prosthetic joints and valves or catheters and shunts; it is a significant cause of bacterial sepsis.

Staphylococcus saphrophyticus

• Common cause of urinary tract infections.

Catalase-negative cocci: Streptococci & Enterococci

• Hemolysis can be used to distinguish between species that are alpha-hemolytic, beta-hemolytic, and gamma-hemolytic.

Alpha-hemolytic strains
Can be further distinguished by their sensitivity to optochin:

• Streptococcus pneumoniae is sensitive to optochin, whereas Viridans group Streptococci are not.
  — Streptococcus pneumoniae is associated with pneumonia, otitis media and sinusitis, as well as meningitis.
  — Viridans group Streptococci are associated with subacute endocarditis and dental caries, as well as some other infections not listed here.

Beta-hemolytic strains
Can be distinguished by their sensitivity to Bacitracin:

• Group A Streptococcus is sensitive, whereas Group B streptococcus is not.
  — Group A Streptococcus, also known as Streptococcus pyogenes, causes pharyngitis with white exudate in the tonsils (strep throat); some people will also develop a rash over their bodies, called Scarlet fever.
  — Group A Streptococcus is also responsible for a variety of soft tissue infections, which can range from impetigo and erysipelas to the more serious cellulitis or even necrotizing fasciitis.
  — Group A streptococcus can cause toxic shock syndrome.
  — Delayed, anti-body mediated reaction to Group A Streptococcus infection can occur in some patients, and may produce post-streptococcal glomerulonephritis or rheumatic fever.
  — Group B Streptococcus, also called Streptococcus agalactiae, is associated with neonatal infections including meningitis, pneumonia, and bacteremia; because the neonates acquire the bacteria from their mothers, prenatal care should include screening for Group B Streptococcus. Post-pregnancy infections can also have serious consequences for the mother.
  — Adult infections can manifest similarly, including bacteremia, pneumonia, and bone, joint, and soft tissue infections.

Gamma-hemolytic strains
Strains that can grow in relatively high concentrations of salt and bile are categorized as Enterococcus
— These bacteria were formerly categorized as Group D Streptococcus, and are common commensals of the GI tract.

• Of particular concern are strains resistant to Vancomycin.
• Enterococci are a significant cause of nosocomial infections.
• Enterococci are commonly associated with urinary tract infections, as well as endocarditis, peritonitis, and bacteremia.
• Streptococcus bovis, which is also gamma-hemolytic but cannot thrive in high salt concentrations, causes similar illnesses as Enterococcus, and is also associated with colorectal cancer.

OVERVIEW

• Bacteria produce toxins to break down host tissues and promote their own growth.
• Toxins facilitate invasion, release nutrients from host cells, and resist destruction by the immune system.
• Furthermore, as bacteria colonize the host, they trigger immune and inflammatory responses; in fact, the symptoms of many infections are the result of host immune response.

ENDOTOXINS

• Endotoxins are part of the cell wall of Gram-negative bacteria
  – The lipopolysaccharide endotoxin extends from the outer membrane
  – Three important regions of the endotoxin, starting at the cell membrane: Lipid A, Core polysaccharides, and the O-antigen
• They have relatively low toxicity
• Upon infection and endotoxin release, the Lipid A portion of the lipopolysaccharide interacts with Toll-Like Receptor 4 on macrophage surfaces and triggers cytokine release, potentially inciting cytokine storms.
• Indicate four important consequences:
  – Complement activation, which results in neutrophil chemotaxis and inflammation.
  – Cytokines IL-1 and IL-6 induce fever.
  – Tissue factor activation leads to coagulation.
  – Tumor necrosis factor, nitric oxide, and bradykinin induce hypotension (low blood pressure) via vasodilation.

• Recognize that, in acute, local conditions, these reactions can protect the host from infection; however, in large quantities, endotoxin can be fatal.
• Endotoxin is a major cause of septic shock.

EXOTOXINS

• Polypeptides secreted by both gram-positive and gram-negative bacteria.
• Many are dimeric in structure, with A and B subunits.
  – The A subunit is the toxic active Portion; the B subunit is the Binding portion that attaches to the host cell.
• Highly toxic, even in small quantities.
• Directly kill or alter host cell functions.

Mechanisms:

• ADP-ribosylation adds ADP-ribose to proteins in the host cell.
  – Diptheria toxin inhibits protein synthesis, leading to cell death; other toxins that act via ADP-ribosylation can hyperactivate protein synthesis.

• Increase Cyclic AMP
  – In the case of heat-labile enterotoxin, this results in fluid and electrolyte loss into the lumen of the gastrointestinal tract, which causes watery diarrhea.

• Proteases
  – Botulinum toxin is a neurotoxin that blocks acetylcholine release, producing paralysis.

• Super antigens
  – For example, Toxic Shock Syndrome toxin overstimulates T cells, triggering cytokine storms.

INFLAMMATION

Two main types of inflammation associated with bacterial pathogens:

• Purulent inflammation is characterized by neutrophilinfiltration and pus formation from liquefied tissues.
• Granulomatous inflammation, which occurs in chronic inflammation, is characterized by aggregates of macrophages and epithelioid cells, called granulomas, aka, tubercles (as in tuberculosis).

Recall that inflammation is characteristic of early immune responses:

• When controlled and acute, it has protective effects for the host: eradication of microbes and tissue healing.
• However, when uncontrolled or chronic, inflammatory and immune responses cause significant damage to the host.
  – For example, let’s consider two disease states that can occur after Group A streptococcus (Streptococcus pyogenes) infection.

1. Post-streptococcal glomerulonephritis:
   Neutrophil infiltration and deposition of antigen-antibody complexes in the basement membrane, which damage the renal filtration system. Be aware that the initial infection occurred elsewhere in the body, such as the skin or throat, leading to circulating immune complexes that became fixed to the glomeruli.
2. Rheumatic fever and heart disease, which develop in the weeks following untreated pharyngeal infection by Group A streptococcus. The host’s innate and adaptive responses lead to valve thickening, which can cause severe valvular stenosis. In the histological sample, we can see areas of calcification and fibrosis have damaged the valve tissue.

OVERVIEW

• The host provides shelter, warmth, moisture, and food for bacteria; as we learn elsewhere, there are several microorganisms that take advantage of these benefits without harming the host – these commensals comprise the microbiome.
• Virulence factors increase a bacterial strain’s ability to colonize and cause disease.
  – The genes for virulence factors are often clustered together in pathogenicity islands; thus, they are easily transferred via plasmids, bacteriophages, and other gene-sharing mechanisms.
  – Furthermore, the genes for many virulence factors are regulated via quorum sensing; as we learn elsewhere, quorum sensing allows for bacterial behaviors to change with group density.

ADHESION TO HOST CELLS & ECM

This early step in colonization unleashes specific pathogen behaviors and host responses.

• Adhesins are molecules that facilitate adhesion to other pathogens or host structures; indicate that they can be located on the tips of pili or on the bacterial cell surface.
• A bacterium can have one or several types of pili and surface adhesions.
  – Different strains of the same bacteria can have different pili types, which can influence their virulence in different host environments.

Gram-Negative bacteria:

• P pili, Type I pili, Curli pili, and Type IV pili.
  – Uropathogenic strains of Escherichia coli use both P pili and Type I pili to adhere to the urothelium of the urinary tract; without these pili, the bacteria would be physically removed by the flow of urine.
  – Some strains of E. coli have curli pili, which, in addition to adhesion, provoke the host inflammatory response.
  – Type IV pili confer twitching motility to some species, independent of flagella; Neisseria gonorrhoeae and Pseudomonas aeruginosa are examples of bacteria that “walk” via retraction of Type IV pili.

Gram-Positive bacteria

• Also have pili-like structures; though assembled differently the pili of Gram-negative bacteria, they perform similar functions.
• Spa, GAS M1, PI-1, PI-2
  – Spa pili, which are long and flexible, facilitate adherence of Corynebacterium diphtheriae, the causative agent of diphtheria, to epithelial cells of the pharynx.
  – Similarly, GAS M1 facilitates adherence of Group A Streptococcus (aka, Streptococcus pyogenes) to pharyngeal epithelial cells.
  – PI-1 and PI-2 facilitate adherence of Group B Streptococcus (Streptococcus agalactiae) to the cells of the lungs.
  PI = Pilus Island, which refers to the gene loci. Group B streptococcus causes neonatal sepsis, pneumonia, and meningitis.

MSCRAMMs

Non-pilus adhesins on the bacterial cell surface that attach pathogens to host structures.

• MSCRAMMs – Microbial Surface Components Recognizing Adhesive Matrix Molecules – are proteins that facilitate colonization by Gram positive bacteria.
• Staphylococcus aureus adheres to fibrinogen via Clumping factor A, and to fibronectin via Fibronectin Binding Protein (FnBP).
• In turn, these ECM components make their own connections to platelets and host cells, thereby establishing secure associations between S. aureus and the host.
• Furthermore, S. aureus can take advantage these associations and enter host cells to either lie latent or act as a super-antigen (for more, see our tutorial on bacterial endocarditis).

Biofilm

• To further secure adherence to the host, and to protect themselves from the immune system and antibiotics, pili and surface adhesins contribute to the formation of biofilms.
• Comprises bacterial cells, in some cases of multiple strains or species, surrounded by matrix. Biofilm formation is an example of a virulence factor regulated by quorum sensing.
  – Dental plaque is an example of a biofilm, which we can see in the image as purple-stained areas.
  – Biofilm production by S. aureus in endocarditis facilitates the growth of large bacterial vegetations, which can damage the heart or, if they break free, cause stroke.

ENTRY INTO HOST CELLS

2 examples of how some bacteria enter into host cells.

Complement Opsonization

• Mycobacterium tuberculosis, the causative of tuberculosis, makes use of complement opsonization.
• Recall that opsonization by C3b typically results in phagocytosis and microbe destruction; however, M. tuberculosis, once taken up by macrophages, avoids destruction and instead replicates inside the host cell.
• Ultimately, pathogen-host interactions result in the formation of granulomas, aka, tubercles, which harbor M. tuberculosis.

Type III secretion system

• The Type III secretion system uses a needle-like structure to inject effectors into host cells.
  – The effectors and their actions vary by bacterial strain.
• In the case of salmonella, the effectors trigger cytoskeleton reorganization of host cells such that the pathogen can enter it; once inside, the bacteria can make use of host cell machinery and replicate.

EVASION OF HOST IMMUNE SYSTEM

Mechanisms to evade phagocytosis

• The polysaccharide capsule on Gram positive bacteria inhibits phagocyte adhesion.
  – Thus, anticapsular antibodies are important preventative measures against infection by Streptococcus pneumoniae and Neisseria meningitides.
• The M protein of Group A Streptococcus (aka, Streptococcus pyogenes) resists opsonization and phagocytosis.
• Protein A, found in the cell wall of Staphylococcus aureus, binds immunoglobulins M and G, preventing complement activation and, therefore, phagocytosis.
• Leukocidins, which are pore-forming cytotoxins released by staphylococcus bacteria, kill leukocytes, including phagocytic neutrophils and macrophages.

Immunoglobin A protease degrades IgA

• This allows the causative agents of bacterial meningitis, Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, to adhere to mucous membranes.

Operon

Cluster of genes that are transcribed into one long mRNA allowing the genes of a single pathway to be controlled with a single on/off switch

Operator

Segment of DNA that acts as a switch to control access of RNA polymerase to the gene

Activator

Protein that binds to DNA and stimulates gene transcription

Negative regulation

Bound repressor protein blocks transcription

Positive regulation

Bound activator protein promotes transcription

LAC OPERON

• Set of genes that code for proteins necessary for the bacterium to use the sugar lactose as an energy source

Structure

• 3 genes: lacZ (beta-galactosidase), lacY (lactose permease) and lacA (galactoside acetyltransferase)
• Promoter region:

1. CAP-binding site
2. Operator

• lacI gene – prior to CAP-binding site; codes for repressor protein; under control of a different promoter

High glucose, no lactose

• CAP-binding site empty (inactive catabolite activator protein due to low cAMP levels)
• Repressor is bound to operator (no allolactose present to inactivate repressor)
• No transcription

No glucose, no lactose

• CAP is bound to CAP-binding site (low glucose means high levels of cAMP)
• Repressor is bound to operator
• No transcription

High glucose, lactose available

• Cap-binding site empty
• Operator is empty (allolactose present inactivates repressor protein)
• Low-level transcription

No glucose, lactose available

• CAP is bound to CAP-binding site
• Operator is empty
• High levels of transcription

OVERVIEW

• Growth refers to the increase in number of bacterial cells, which occurs via binary fission.
• Ultimately, growth can produce a colony of millions of bacterial cells.
• The time it takes for takes for the cell population to double is the “generation time”.
  — This time varies by species, and is moderated by environmental factors such as pH, nutrient availability, temperature, etc.
  — For example, the generation time for Staphylococcus aureus grown in heart infusion broth is about 30 minutes.
• Bacteria are haploid
  — E. coli, which we use in our diagram, have chromosomal DNA organized into circular, double-stranded structures.
• Pathogenicity islands refer to the distinct regions of some bacterial chromosomes that code for virulence factors; these islands are absent in non-virulent strains.
• Extrachromosomal genetic elements* may also be present.
  For example, plasmids and bacteriophages may engage in horizontal DNA transfer.
• Quorum sensing is a type of bacterial communication that arises when cell population density is high.
  — Quorum sensing is mediated by autoinducers, which are molecules released by bacterial cells.
  — As cell density increases, so does autoinducer concentration.
  — In different species, quorum sensing moderates virulence factor secretion, biofilm production, sporulation, and other behaviors.
  — For example, consider the bacteria Staphylococcus aureus, which produces autoinducer peptides (AIPs).
  — When the density of S. aureus increases, so does the concentration of autoinducer peptides. In turn, this induces bacterial release of virulence factors, including several toxins.
  Furthermore, the cells are stimulated to release additional AIPs, thus creating a positive feedback loop.
  Because quorum sensing facilitates some of the harmful effects of acute S. aureus infection, researchers are investigating treatments that prohibit or moderate this type of cellular communication.

BACTERIAL GROWTH CURVE

Tracks the stages of cell population growth.
— We’ll plot time along the x-axis, and the log number of cells along the y-axis.

• Lag stage
  — Bacterial cells engage in metabolic activity but not cell division; during this stage, the bacteria acclimate to the growth conditions.
• Logarithmic
  — Aka, exponential stage.
  — Rapid cell division occurs.
  — Beta-lactam antibiotics, such as penicillin are effective during this period, because they interfere with cell wall production.
• Stationary stage
  — The curve plateaus during the stationary phase because proliferation and cell death are in balance; this steady state is reached when nutrients are running low and/or toxin levels are elevated.
• Death stage
  — Finally, the number of bacteria declines.
  — However, some bacteria may remain viable during this stage.

Important points regarding the growth cycle:

• Growth requirements include carbon, nitrogen, energy sources, water, and ions; though specific requirements vary by species.
  — Iron is so important for growth that some bacteria can secrete siderophores that “steal” iron from the host.
• Obligate anaerobes cannot grow in the presence of oxygen.
  — Clostridium is an example.
• Obligate aerobes can only grow in the presence of oxygen.
  — Mycobacterium tuberculosis
• Facultative anaerobes can grow with or without oxygen.
  — Most common; Staphylococcus, which contributes to the normal flora of the nares, is an example of this.
• Obligate intracellular pathogens they can only grow within living cells because they rely on ATP derived from the host
  — Chlamydia

CHROMOSOME REPLICATION IN E. COLI

• Occurs via binary fission – chromosome replication triggers initiation of cell division.
• We illustrate this in Escherichia coli; be aware that not all bacteria replicate in this exact manner.

First drawing

• Single circular chromosome with inner and outer strands.
  — In reality, bacterial DNA is arranged in loops. Recall that there is no distinct nucleus, as in eukaryotic cells; instead, DNA lies in the nucleoid region.
• Origin of replication is marked by oriC; this is where the replication initiator proteins bind.
• Terminus is located opposite oriC; this is the region where DNA replication terminates.

Second drawing
After DNA replication begins:

• Outer and inner parental strands begin to separate in bidirectional replication.
• Replication “bubble,” is the space between the strands.
• Two y-shaped replication forks, one on each side of the bubble.
• DNA helicases separate the parental DNA strands in short segments, thus creating the replication forks; if the two strands separated all at once, excessive DNA damage could occur.
• Developing daughter strands: each one begins at the origin of its parental strand and grows towards its terminal end.
  — These complementary daughter strands are synthesized by the replisome, which comprises DNA polymerase III and other components.

Third drawing

• The parental and growing daughter strands are further along in the replication process.
  — The parental strands have further separated from each other as the daughter strands grow towards the terminal region.

Fourth drawing

• Finally, the chromosomes separate after the daughter strands are complete.
• DNA replication is semiconservative:
  — Each new chromosome comprises one strand of DNA from the parental chromosome and one complementary daughter strand.

BASIC COMPONENTS OF A BACTERIAL CELL

• Be aware that different strains of bacteria have special anatomical and physiological traits, which we address elsewhere as these features relate to infectious disease.
• Cell wall
• Plasmic/cytoplasmic membrane is deep to the cell wall.
  – It has an area of infolding. The membrane comprises a lipid bilayer that serves multiple functions, including transport of molecules into the cell, secretion of toxins and enzymes, and energy generation.
• Cytoplasm
  – Ribosomes are sites of protein synthesis.
• Nucleoid is the region with bacterial DNA; there is no nuclear membrane.
• Outer capsule, which most often comprises gelatinous polysaccharides.
  – Variable; When present, the capsule contributes to the serologic type and enhances virulence.
• Pili, aka, fimbriae, which are short filaments found primarily on gram-negative strains.
  – Variable; When present, they participate in bacterial attachment to host cells.
  – The sex pilus attaches donor and recipient bacteria during conjugation.
• Flagella
  – Variable; when present, propels the cell; the number of flagella varies.

STAINING

• Most bacteria associated with infectious disease can be categorized as gram-positive or gram-negative, which refers to whether they retain crystal violet stain, and depends on the composition of their cell walls.

Gram-positive

• In a microscopic sample, we can see the bright purple stain – think Purple Positive.
• Cell wall has thick layer of peptidoglyclan (aka, murein and mucopeptide)
  – It comprises a network of sugars and amino acids, and is the target of some antibiotic drugs.
  – We indicate two acids that are present in the cell wall: teichoic acids, which attach to the peptidoglycan, and, lipoteichoic acid, which are attached to the cytoplasmic membrane.
  – These surface acids contribute to the cell wall structure and charge.

Gram-negative

• In a microscopic sample, gram negative bacteria appear reddish-pink.
• They have a thin peptidoglycan layer, which is covered by the outer membrane.
• The outer membrane is unique to gram negative bacteria and comprises lipopolysaccharide, aka, endotoxin.
  – Endotoxin contributes to disease symptoms such as fever and shock.

Other stain types

• Gram-staining is not appropriate for all bacterial strains.
• We show an image of mycobacteria, which does not have a cell wall and is visualized with acid-fast staining methods.
• We show Treponema pallidum, which is a bacterial strain that has very thin cell wall but can be seen with dark-field microscopy, as in our image, or fluorescent antibodies.
• Some strains, such as the intracellular Chlamydiae, can be seen with hematoxylin and eosin (H&E) staining.

MORPHOLOGY

• The bacterial cell wall contributes to its morphology, which is also used for classification purposes.

Cocci – Spherical

• Clusters
  – Ex: Staphylococcus species
• Chains
  – Ex: Streptococcus pyogenes
• Pairs
  – Ex: Pointed, like Streptococcus pneumoniae
  – Ex: Coffee-bean shaped, like Neisseria gonorrhoeae.

Bacilli – Rods

• Rectangular ends
  – Ex: Bacillus anthracis
• Rounded ends
  – Ex: Salmonella
• Club-shaped
  – Ex: Corynebacterium diptheriae (“Coryne” means “club”)
• Fusiform-shaped
  – Ex: Fusobacterium nucleatum (associated with periodontal disease)
• Bent/comma-shaped
  – Ex: Desulfovibrio desulfuricans (associated with the gut)

Coccobacilli – Intermediate

• Short, rounded rods
  – Ex: Coxiella burnetii (Q fever)

Spirochetes – Spirals

• Ex: Treponema pallidum (Syphilis)

Be aware that there is intertextual variation in regards to bacterial classification schema, and some authors describe more or fewer morphological types.