Colonizers
non-pathogenic organisms that live in gut/skin
prevent pathogenic organisms from overtaking systems and harming host
provide essential functions, eg. fermentation of carbs, Vit K
OpportunistsOpportunists
Organisms that normalyl do not cause harm.
Can cause disease when “given the opportunity”; eg in catherizations for Staphlococci
Pathogen
Organism that is always harmful to host; eg Ebola
Methods of Identification of MIcrobes
- naked eye
- light microscope (w/ and w/o stains)
- electron microscope
- antigen recognition: flourescent dyes, serology
- biochemical properties eg. testing of enzymatic activity
- PCR or gene sequencing
- protein detection
- culture
Differential Diagnosis
List of possible diagnoses for the signs/symptoms of the patient
–>involves figuring out infectious state, infectious agent (bacterial v viral)
–>takes into account pattern recognition, exposure risks, incubation times and epidemiology
–>confirmed by microbiological diagnosis
Koch’s Postulates
Critique
Purpose: to identify the organism causing an infectious disease
- The microorganism must be found in abundance in all organisms suffering from the disease, but not in healthy organisms.
Some people are carriers
- The microorganism must be isolated from a diseased organism and grown in
pure culture.
Viruses/prions don’t can’t be grown in pure culture
- The cultured microorganism should cause disease when introduced into a
healthy organism.
Very difficult to test without animal models
- The microorganism must be re-isolated from the inoculated, diseased
experimental host and identified as being the same as the original causative agent.
Some diseases can be caused by multiple organisms
Koch’s Postulates–when they’re not met, what do you need to implicate a particular organism as the cause of disease?
- biostatistical tests of association
- serological surveys in human populations
- epidemiologic studies (case controlled)
- molecular pathogenesis postulates
Molecular Koch’s Postulates
Purpose: to identify whether a factor produced by a pathogen is important for causing disease
–>Identify virulence factors
a) the phenotype or property encoeded by the virsulence gene should be associated with pathogenic strains
b) specific inactivation of the virulence gene encoding the suspected virulence strain should lead to measurable loss of virulence
c) addition of a cloned copy of the wild type gene to the mutant should restore virulence
Microbiologic Testing Methods
a) Qualitative
Negative or positive
Negative result takes 5-42 days to process (growth rate dependent); high flase positive rate
Used for sterile body fluid analsysis
**b) Semi quantitative **
Specimen is directly stained w/ result expressed in a gradient (eg. low, med, high)
**c) Quantitative **
Specimen is direclty stained and known volume is pltated
CFU/mL calculated
Blood culture collection
2 sets of 2 bottles are collected (one for aerobe, one for anaerobe)
different sites are used
Purpose: to differentiate between contamination and true bacteremia
**Blood should be sterile**
Urine culture collection
Skin around urethral meatus may be contaminated. To check for true infection look for:
WBC, absence of epithelial cells, >100k bacteria/mL, one kind of bacteria in culture
CSF Culture collection
CSF should be sterile; can be tested via lumbar puncture
–>look for WBC, high protein, low glucose as infection markers
Virulence Factors
Factor produced by a pathogen that causes disease
eg. Toxins, adhesins, capsules
General Infectious Life Cycle
Entry
Adherence/Colonization
Invasion
Evasion of host defenses
Damage
Dissemination
Virulence
the degree of pathogenicity as indicased by case fatality rates and/or the ability of the organism to invade the tissues of the host
Transposons
contain genes required for movement as well as additional genes such as antibiotic resistance, toxin production, etc.
“Jumping gene” –> can disrupt virulence gene thereby eliminating virulence factor production-
have interverted repeats
Bacteria with sterols in plasma membrane
Bacteria w/o cell walls of peptidoglycan
Bacteria that do not make their own energy
Gram pos bacteria w/ endotoxin
Mycoplasma, H. pylori, Borrelia burgdorferi
Mycoplasma, Chlamydiae
Rickettsia, Chlamydiae
Listeria monocytogenes
Transpeptidases
AKA pencillin binding proteins
Carry out peptidoglycan crosslinking (cell wall synthesis)
Gram neg/gram pos
O Antigen
Provides bile resistance
–>LOS lacks O antigen and is found in non-enteric Gram-neg bacteria
Provides resistance to lysis by complement
used for epidemiologic serotyping
Porins
Allows movement of molecules across outer membrane in Gram neg bacteria
Alterations/loss of porins is a mechanism of antibiotic resistance
Important Factors for Bacterial Growth
Temperature
pH
Oxygen (Redox) conditions
Nutrients (eg iron)
Osmolarity
Why are some bacteria anaerobic
- often lack catalase
- often lack superoxide dismutase
- often have sensitive enzymes that must be in a reduced environment to function properly
**IOW: anaerobes must be in a low redox environment
Endospores
Resistant to destruction via heat, radiation, chemicals and drying
–> due to low water content and DNA stabilizing proteins
Sporulation is not a reproductive strategy: it’s survival
Pharmacokinetics v Pharmacodynamics
Pharmacokinetics
–the ways the body manipulates a drugs:
absorption: bioavailabitlity
distribution: measured by Vd
metabolism
elimination: renal v hepatic
Pharmacodynamics
–the biochemical and physiologic effects of the drug and its mechanism of action: MOA, toxicities, dosing strategies for optimal kills, static v cidal activity
Minimum inhibitory concentration
the lowest concentration of antibiotic that results in no visible growth
Bacteristatic (5)
an antibiotic which only inhibits growth at usual achievable concentrations
eg tetracyclines, macrolides, clindamycin, linezolid, tigecycline
Bactericidal (7)
An antibiotic which kills organism at usual achievable serum concentrations
eg: penicillins, cephalosporins, aminoglycosides, vancomycin, fluroquinolones, monobactams, daptomycin
*Critical for life threatening infections
Antibiotic v Antimicrobial
TL;DR: Antimicrobials include antibiotics
Antibiotics
Produced by a microorganism
Kill or inhibit the growth of other microorganisms (usually bacteria)
Beta-lactams, aminoglycosides, etc.
Antimicrobials
Any product that kill or inhibit the growth of microorganisms (bacteria, fungi, viruses, parasites)
Includes all antibiotics
Some are fully synthetic, e.g. fluoroquinolones
Types of Antimicrobial Therapy (5)
Prophylaxis: use of antimicrobial agents to prevent infection eg. before surgery
Pre-emptive antimicrobial therapy: starting antimicrobial therapy when lab markers indicate infection or ractivity but NO clinical signs of disease are present eg. bone marrow transplant patient with increasing CMV titer
Empric therapy: use of antibiotics in a patient w/ a suspected infection before the etiology of the infection is known; not usually done in chronic infections
Pathogen directed therapy: the organism is known but the antibiotic susceptibility is not
Susceptibility guided therapy: organism is known, antibiotic susceptibility is know: opportunity to streamline therapy o be most effective, least toxic, w/ narrowest spectrum, cheapest
Disk diffusion
Clear zone size correlates with susceptibility
MIC’s E test
combines disc diffusion methods w/ MIC methods through use of rectangle paper impregnanted w/ various doses of drug
Concentration dependent killing
Maximal killing at maximal concentrations
Upper concentration limit is the concentration that will produce toxicity
aminoglycosides, fluoroquinolones
Time-dependent killing
Do not achieve greater killing at high concentrations
Time above MIC is the important consideration
beta lactams (except those w/ long half life) and vancomycin
Horizontal Gene Transfer in Bacteria
Genetic transformation: transfer of naked DNA between cells; Natural transformation occurs in bacteria that produce a protein called the “competence” factor. Homologus recombination of the DNA occurs via the RecA protein
Transduction: DNA transfer mediated by a bacterial virus (phage)
Conjugation: DNA transfer involving direct cell to cell contact mediated by conjugative plasmids
Bacterial Plasmids
Extrachromosomal elements that exist in bacterial cells and have the ability to confer new genetic properties on the bacterial cell. They are usually circular, double stranded DNA molecules.
–l>inear plasmids are also found in nature
Often contain drug resistance genes
Very stable and rarely lost from bacterial cells
Carry genes that allow them to replicate independently of the bacterial chromosome
Require host proteins and enzymes for replication
R plasmid
AKA Resistance factors
Conjugative
have a broad host range and can transer b/n different bacterial species
Structure: tra gene (sex pilus gene), drug resistance genes, replication region, inserstion sequences and transposons
is conjugative
Virulence Plasmids
Carry genes that encode toxins and other virulence factors
eg. toxin proteins carried on plasmid (anthrax and tetanus)
Structure of a Conjugative R plasmid
Initiate their own transfer from cell to cell
Contain tra genes that encode the sex pilus through which the DNA is transferred
Contain oriT which is nicked by a relaxase to initiate the transfer of a ss of plasmid DNA
Usually contain insertion sequences and transposons
Can also promote the conjugative transfer of mobilizable plasmids containing oriT
Are highly promiscuous
eg R plasmids
Transposable Elements
Transposable (Tn) elements are specific DNA segments that can repeatedly insert at many sites in a genome. Carry one or more genes encoding drug resistant, toxin production, etc to a host cell. Normal contituients of bacterial plasmids and chromosomes. Are always part of a genome.
- *Insertion sequences (IS):** Small elements (~ 750 bp-1.5 kb) which only carry genes that are required for movement
- *Transposons (Tn):** contain genes required for movement as well as additional genes such as antibiotic resistance, toxin production, etc.
Replicative transposition involves replication of the Tn and results in duplication of the element
Conservative transposition involves simple cut-and-paste insertion of the Tn at the target site
Transposition can be Intermolecular (between two genomes) or intramolecular (within the same genome)
Viruses
- Obligate intracellular pathogens (they require the host cell for replication)
- Have DNA or RNA, not both.
- No independent energy production or protein synthesis
- Usually show selectivity for infecting particular types and/or species of host cells 5. Not sensitive to antibacterial/antifungal antibiotics (e.g. penicillins)
H antigen
found on flagella
K antigen
capsular antigen
Sterilization
all microorganisms are killed
Disinfection
pathogens are killed but some other organisms may remain
Pasteurization
brief exposure to heat, kills all pathogens
Antiseptic
disinfectant that kills/inhibits microorganisms when applied topically
Antimicrobial Chemicals
a. Agents that affect membranes:
i) 70% ethanol (bactericidal)
ii) Quaternary ammonium detergents (bactericidal)
iii) Phenols: used in disinfectant solutions (e.g., Lysol).
b. Agents that modify proteins:
i) Chlorine: water purification, active component of hypochlorite solutions used as disinfectants at hospitals. Cross-links cysteines to inactivate proteins. Bactericidal.
ii) Iodine: used as skin antiseptics prior to surgery. Often bactericidal.
iii) Hydrogen peroxide: weak antiseptic often used topically.
iv) Ethylene oxide: gas used extensively in hospitals to sterilize heat-sensitive items. Inactivates proteins.
Antimicrobial Physical Agents
a. Heat
i) Autoclaving: sterilizes using heat under pressure to achieve high temperatures.
ii) Pasteurization: removes most pathogens by heating to 60-65°C.
iii) Dry heat: can be used to sterilize objects sensitive to moist heat.
b. Radiation
i) Ultraviolet: can be bactericidal but doesn’t penetrate well; sometimes used to disinfect hospital rooms.
ii) Ionizing: bactericidal; used to sterilize disposable surgical/medical supplies.
c. Filtration
i) Used to sterile very heat-labile substances
ii) Bacteria are too large to pass through filter pores and are thus remove from the filtered solution.
iii) Filters do not remove viruses.
Conjugative Plasmids
Found in both Gram negative and gram positive bacteria
Plasmid Replicon
A plasmid replicon consists of:
a. Origin - the site of initiation of replication.
b. rep gene - encodes an initiator protein that is required for
plasmid replication.
c. Copy control gene (cop) - involved in controlling the
replication and copy number of a plasmid.
Copy Number
the number of plasmid molecules per chromosome in a cell; the copy number for a given pladmi is fixed
–>large plamids usually have a low copy # and small plasmids a high copy #
Tn3–the typical transposon
trpA gene: encodes the transposase protein required for transposition
tnpR gene: encodes repressor of transposition
Amp gene: beta lactamase which makes bacteria resistant to ampicillin
interverted repeasts
NDM-1: New Delhi metallo-beta-lactamase 1
refers to a transmissible genetic element encoding multiple resistance genes
intrinscic ability to destroy most beta lactams including carbapenems
Sources of Antibiotic Resistance
Introduction of resistant strains from outside the “community”
Acquisition of resistance from another strain
Emergence of resistance as a result of mutation
Selection of resistant strain by antibiotic pressure
Mechanisms of Antibacterial Resistance: Enzymes That Destroy or Inactivate Antibiotics
beta-lactamases:
- can have a narrow or extended spectrum
- gene acquired from plasmid or bacterial chromosome
- does not result in a “fitness cost” to organism
- enzymatically hydrolysis the beta-lactam ring of antibiotic–>inability of hydrolyzed product to bind to PCP–>resistance
EXAMPLES:
ESBL: extended spectrum beta lactamase–>notable for ability to affect advanced gens of Beta lactams including 3/4th gen cephalosporins
AmpC: tend to be inducible and can coade for broad spectrum beta lactamase activity (does not act on cefepime) and are not fully inhibited by beta lactamase inhibitors
carbapenemase
Mechanisms of Antibacterial Resistance:Development of Altered Targets
- Diminished ability of antibiotics to attach to bacteral target
eg. PCP and beta lactam: PBPs are responsible for cross-linking of the cell wall; they bind beta-lactams which leads to inability of cell wall to form–>death. If a PBP has low afinity for beta-lactams, then the bacteria will be able to make the cell wall in the prescense of beta lactam antibiotics
eg2. mutant peptidoglycan precursors and vancomycin-resistance
eg3. alteration of ribosome and macrolide resistance
eg4. quinolone resistance and alterations in DNA Gyrase: quinolones extert antibacterial effect by binding to DNA gyrase and preventing normal function–>mutation of DNA gyrase gene selected by exposure to quinolones–>alterations of DNA gyrase prevents binding of quinlones and conveys resistance
Mechanisms of Antibacterial Resistance:Alterations in permeability of bacterial cells
Modification of porins may prevent or slowantibiotic entrance into cell
eg. Porins and P. aeruginosa
Quinolones/other antibiotics enter P. aeruginosa via the porin “OprD”–>decreased OprD expression–>altered permeability
Mechanisms of Antibacterial Resistance: Prescence of Pumps which remove antibiotics
“Efflux” pumps: remove antibiotics from cell cytoplasm before it is able to bind the target
increased importance of pumps if there is already decreased susceptibility
Mechanisms of Antibacterial Resistance: Highlights
- there are 4 mechanisms
- more than one mechanism can be working at the same time
- multiple mechanisms are synergistic
Mechanisms of Antibacterial Resistance: biofilms
in vivo mechanism of resistance
–>channels, quorum sensing, etc
Vancomycin-intermediate S. aureus (VISA)
Mechanism is synthesis of unusually thickened cell wall containing dipeptides (D-Ala-D-Ala) capable of binding vancomycin which reduces availability of drug to reach intracellular target molecules
Heterogenous vancomycin-intermediate S. aureus (hVISA)
subpopulations display variable rather than uniform susceptibility to vancomycin
hVISA populations withstand vancomycin by means of an unusually thickened cell wall
High-level vancomycin-resistant S. aureus (VRSA)
Mechanism due to transfer of plasmid-mediated transfer of VanA gene cluster on transposon
Fungi 101
Eukaryotic organisms w. more than one chromosome, mitochondria, ER, and 80S ribosomes.
Chemotrophic: secrete enzymes that degrade a wide variety of organic substrates into soluble nutrients which are then passively absorbed or taken into the cell by active transport
Reproduce sexually
non-motile
May produce spores
Rigid cell wall: chitin and glucan composition; also contains sterols and mannan
Stain blue w/ calcoflour, green with silver stains (cell wall), pink with PAS (only on living fungal cell walls)
Dimorphic in some species
Fungal growth forms
Molds aka filamentous fungi
- multicellular forms composed of hyphae (mycelium=many hyphae)
- hyphae grow by branching and longitudinal extension; they are branched/tubular
- fuzzy colonies
- hyphae may be septate or non septate
- speciated based on colonial morphology, color and microscopic appearance
- saprophytic stage of dimorphic fungi
Yeast
- independent single cells–>round to oval
- form smooth flat colonies
- Propagate by budding
- speciated by biochemical test
- parasitic stage of dimorphic fungi
Pseudohyphae
- several elongated yeast cells chained together and resembling true hyphae
Dimoprhic Fungi
- can grow either the yeast (in tissue) or mycelium (in environment) forms depending on environmental conditions
Fungal disease
Overview
AKA mycoses–>superficial, cutaneous, subcutaneous, systemic and opportunistic variants
important resistance factor: phagocytosis by neutrophils and macrophages
non specific resistance: mechanical, humoral and cellular factor
specific resistance: cellular immunity
Allergies
due to spores
allergic rhinitis, bronchial asthma or allergic alveolitis
Toxicoses
due to mycotoxins
effect is indepenent of infection or viability
i.e. aflatoxins–>Aflatoxin B1: most potent carcinogen known to man (hepatocellular carcinoma)
Types of Parasites
Endoparasites
live inside host; protozoa or helminths
Protozoa
unicellular, free living organisms
motile w/ flagella, cilia, pseudopods or apical microtubule complex
Helminths (worms): cause disease based on mechanical effects (intestinal obstruction), competition for nutrients or invasion of host tissues
1) Roundworms
intestinal nematode
tissue nematode (filaria)
2) Flatworms
flukes (trematodes)
tapeworms (cestodes)
Ectoparasites
arthropods or arachnids that fuilfill a life cycle requirement by being on the skin or hair of the host
sometimes disease is simply due to ectoparasite prescense, other times, the ectoparasite serves as a vector for bacteria/virus/protozoa
Parasite Life Cycles
Parasites can take different forms (eggs, larvae, mature organisms, etc)
Parasites may require more than one host species within which they complete their life cycle
- *Definitive host:** where the parasite reproduces sexually
- *Intermediate host:** where the parasite reproduces asexually/larval stage
Parasitism
type of symbiotic relationship where one organism depends on the other for survival at some expense to the host
Common modes of parasitic transmission
- fecal oral route
- skin penetration
- insect vectors
- vertical transmission
- inhalation of eggs
- sexual intercourse
Viral Diagnosis
incoculation of cell culture: plaque assay, transformation assay
immunohistochemical staining for antibodies
ID of virus particle or antigens–>eg through ELISA
detection of viral nucleic acids->PCR
Detection of viral (CPE) cytopathic effect (e.g. rounding of cells, cell lysis, syncytia formation = fused cells).
EM for detection of viral paricles
hemagglutination assay for erythrocyte lattice formation
Viral Structure
- Genome (DNA or RNA) enclosed within a protein shell called a capsid
- Capsid symmetry: helical/rod shaped, spherical w/ icosahedral symmetry (w/ 2, 3 or 5 points of symmetry)
Note: helical viruses are always enveloped except TMV
- Capsid composition: may or may not have capsomeres
- Virus can be:
1) enveloped-composed of a nucleocapsid surrounded by a lipid containing envelope
2) non-enveloped- composed of a viral genome enclosed within a capsid (nucleocapsid)
Virion
the intact infectious particle
Viral genome
the viral nucleic acid (either DNA or RNA)
Capsid
the protein shell surrounding the viral nucleic acid
Capsomere
a clustering of capsid proteins discernible by electron microscopy
**capsomeres are basically building blocks that come together to make a capsid; not all capsids are composed of capsomeres
Nucleocapsid
the viral genome + the capsid
Non-enveloped virus
AKA naked nucleocapsids
have only the viral genome and capside
eg. Adenovirus
Stable against: temperature, drying, acid/detergents
Flourish in GI tract
Enveloped viruses
have a lipoprotein membrane external to the nucleocapsid
eg Herpesvirus
**the envelope is an essential part of the infectious virion for these sorts of viruses–>w/o it, the viron is not infectious. This means that enveloped viruses are easier to destroy than non-enveloped viruses
Environmentally unstable–>need moisture to transmit (wet hands, snot, sexual transmission)
do not survive in GI tract
Viral tropism
- what cell types a virus can infect
- determined by proteins on the viral surface–>iow, often the cell surface proteins that a specific virus binds is restricted to a specific cell type:
• Factors affecting viral tropism.
- The proteins on the cell surface (presence of a viral receptor). - The proteins on the virus surface that interact with the cell surface receptor. - capsid protein (non-enveloped virus) or glycoprotein (enveloped virus).
Nuetralizing antibodies for viruses
can recognize and bind to proteins on the viral surface and interefere with the ability of a virus to enter a cell, thus nuetralizing the infectivity of a virus
- Enveloped: neutralizing antibodies recognize the outer envelope proteins, typically glycoproteins that stick out from the lipoprotein membrane
- Non-eveloped: nuetralizing antibodies recognize the capsid proteins
DNA viruses: General Characteristics and Exceptions, and diseases caused
HHAPPPI: hepadna (HBV), herpes, adeno, pox, papova(HPV), parvo(B19, AAV), irido
- double stranded DNA
- linear genome
- icosahedral capsid
- replicate in the nucleus
-Enveloped: hepadna, pox, herpes, irido
-Naked: parvo, adeno, papova
Exceptions
Parvovirus–>single strand DNA
Papovavirus and Hepadnavirus–>circular genome
Poxvirus–>not icosahedral and replicates in the cytoplasm
Iridoviridae–>no known human diseases
Reovirus
Double stranded RNA virus
naked (non enveloped)
segmented genome
cause of rotavirus
RNA viruses that replicate in the nucleus
Influenza
Retroviruses
Naked RNA viruses
Calicivirus
Picornavirus
Reovirus
ssRNA + viruses
–>enveloped/naked, diseases caused, segmented genomes?
None segmented!
Enveloped
Togaviridae–Rubella
Coronaviridae–SARS
Flaviviridae–HCV, West Nile, Dengue
Retroviridae–HIV, HTLV
Note: retroviruses bring in RT, integrease and protease in virion
Naked
Picornaviridae–Polio, Hep A, Rhinovirus
Caliciviridae–Norovirus, Hep E
ssRNA - viruses
–>enveloped/naked, diseases caused, segmented genomes?
–All enveloped!
-bring in a RNA polymerase in the virus particle
Segmented
Arenaviridae–Lasa Fever virus
Bunyaviridae–Hantavirus
Orthomyxoviridae–Influenza
Non-segmented
Filoviridae–ebola, marburg
Paramyxoviridae–RSV, PIV, measles, mumps
Rhabdoviridae–rabies
Viral Life Cycle
1) Attachment
- involves finding the appropiate host cells, adsorbing/sticking to charged molecules/charged lipids on host cell surface and binding to other cell surface receptors that faciliate entry across the membrane
2) Penetration
- fusion of viral evelope and plasma membrane
- receptor mediated endocytosis (can occur w/ both naked and enveloped viruses)
3) Uncoating
can occur at the plasma membrane or within endosomes
4) Gene expression–>transcription and translation
5) Replication
Involved transcription (viral mRNA synthesis) and translation (viral protein synthesis)
Some DNA viruses have early and late phase of gene expression
Viruses encode their own polymerases (except parvovirus) to do this
6) Assembly / Maturation
Concerted Assembly: the nucleic acid is incorporated into the capsid as it’s being assembled; mechanism used by majority of viruses
Sequential assembly: the nucleic acid is interested into a preformed capsid shell.
7) Release
- infected cell may or may not die
- enveloped viruses may exist through budding or fusion of secretory vesicles
Viruses that use host cell polymerase for replication
Parvovirus
single stranded DNA virus
RNA to RNA enzyme
RNA to DNA enzyme
RNA dependent RNA polymerase; used by RNA viruses
RNA dependent DNA polymerase; used by retroviruses
—Plus v Minus Strand—
What is a Plus strand?
What is a Minus strand?
mRNA is defined as the plus (+) strand because it contains immediately translatable infromation
A strand of DNA of equaivalent polarity is also a plus strand–>iow, if this DNA was RNA, it would be translated into protein
5-3 polarity
the RNA and DNA complement of the + strand is the - strand
the - strand CANNOT be translated; it must first be copied into a + strand
3-5 polarity
“Early” v “Late” viral proteins
**Early Viral Proteins: **
important for altering the host cell and for viral genome replication (eg polymerase)
Made BEFORE genome replication
**Late Viral Proteins: **
important for virus structure and assembly (eg nucleocapsid and structural proteins)
made AFTER genome replication
**some large DNA viruses also have an “immediate early” phase before the early phase
Hepatitis B virus
Hepadnavirus
incompletely double stranded DNA virus
unique DNA virus in that it uses RT for replication:
Gapped DNA repaired in the nucleus – covalently closed circle
Viral mRNA and Pregenomic RNA are made using host cell machinery.
Pregenomic RNA is exported to the cytoplasm where it serves as template for RT to form dsDNA within newly formed viral particles.
If enough envelope proteins available the particle is released from the cell if not the particle is directed back to the nucleus.
Stages of Viral Pathogenesis
1) Entry into human host
2) Primary replication
3) Primary viremia (spread throughout the bloodstream)
4) Secondary replication
5) Spread to target organisms
6) Further replicaiton, cell injuary and clinical disease
Routes of viral acquisition
- Fecal/Oral: localized/systemic
- Respiratory: upper/lower localized or systemic
- Urogenital/sexual: localized or systemic
Eyes: systemic
Parenteral
Insect vector/animal bite
**Entry may be through one method but it may cause disease elsewhere/systemic disease
Routes of Viral Dissemination
- -Hematogenous spread: viremia–>BLOOD
- -Localized spread: remain in close proximity to site of entry
- -Neural spread: infect and spread throughout the nervous system
Factors that Affect Dissemination
- Portal of entry
- Host immunity (innate and adaptive)
- Cell specific virus receptors
Outcomes of viral infection
- Lysis of the infected cell (cell death).
- No morphological or deleterious change.
- Cell dysfunction/morphologic changes without cell death.
Hyperplasia of cells Excessive mucous secretion by cells Syncytia formation Inclusion body formation transformation of cell into immoratalized phenotype
Latent/Chronic infections
-In latency: no production of infectious virus –>no replication of viral genome, no production of capisd strutural proteins
Latent viruses have the potential to undergo reactivation
Reactivation of latent viruses is in response to a stimuli (stress, injury, better cellular environment)
Mechanisms of Viral Adaptation
- point mutations: viral genomic replication is more error prone than cellular genomic replication
- recombination of omologus nucleic acid sequences b/n related viral strains
- reassortment of viral genes–>only with segmented RNA viruses
antigenic shift: sudden dramatic shift in viral antigen due to reassortment
antigenic drift: more subtle change in viral antigens due to point mutations
Strategies of viral immune evasion
Latency. Minimal to no expression of viral antigens.
Infection of sites not readily accessible to the immune system
Antigenic variation: virus rapidly evolves and mutates antigenic sites that are critical for immune recognition
Antigenic variation: reassortment of viral genes (antigenic shift)
Viral “defense” molecules that interfere with immune function, such as antigen processing and presentation
Infection of immune cells with destruction or alteration of immune cell function
Viral Diseases
Immediately Lethal
Self Limited
Contained-Not Eradicted
Rare viral diseases; cause intense vascular inflammation
eg Ebola, YF, Hanta virus
Most viral infections
Virus is eradicated by host immunity (T/B cell response)
- Life-long immunity: Hep A
- Short duration immunity: RSV
Chronic persistent: HIV, Hep C
Laten: Herpes
Active Immunization Vaccine
- Elicit a protective immune response: nuetralizing antibodies and cytotoxic T cells
1) Live attenuated - weakened virus that can produce immunity w/o causing disease in the normal host
eg) MMR, Flumist, OPV, Chickenpox, YF, smallpox, rotavirus
2) Inactivated - viral antigens or recombinant proteins; safe in immunocompromised patients
eg) Hep A, Hep B, Flu shot, HPV, IPV, rabies
Passive immunization
- Hyperimmune IG from human serusm: nuetralize virus and block infectivity by prohibiting viral entry into self,short half life, not widely used due to shortages and expense
- Available: Hep A, Hep B, Chickenpox, rabies, CMV, RSV
Virucides
Directly inactivate intact viruses
eg. detergents, UV lights
- limited use: muscocutaneous HSV< warts
Immunomodulation (Viruses)
**stimulate the host immune response to contain or eradicate an infection **
1) inducing host factors w/ antiviral properties:
interferon-degrade viral RNA, inhibit viral protein synthesis and enchance CTL, NK activity and cytokine relsease
2) Decreasing immuno suppression:
treat HIV in AIDS patient w/ Kaposi sarcome
decrease dose of immunosuppressive agents in a kidney strandplant patient w/ EBC related lymphoma
Antivirals
- Target virus specific proteins: low affinity for host encoded proteins
- act only on replicating viruses: viruses in latent/quiescent phase are not susceptible
- effective in early phase of infection: often used to late to make an impact
- development of drug resistance
Mechanism of Action of Antivirals
**Polymerase inhibitors **
- nucleoside/nucleotide analogues
- pyrophosphate analogues
used for herpes viruses
Ion channel blockers
used for influenza
Neuraminidase inhibitors
used for influenza
+antivirals for Hep B and C
Retrovirus Replication
Retrovirus Replication
- +RNA is converted to dsDNA using reverse transcriptase (RT) brought into the cell as part of the virus particle
- Integration of the viral dsDNA genome into host cell DNA to generate the provirus - integrase brought into the cell as part of the virus particle carries out this step.
- Viral mRNAs are made from the provirus genome using the host cell machinery.
- Viral mRNAs are translated by the host cell machinery
- make capsid, envelope, RT, protease and integrase plus other viral proteins. - Some of the viral RNA made in step 3 is packaged into new virus particles along with RT, integrase and protease to generate new infectious virus.
Replication of - strand RNA viruses
- Negative Strand Viruses Require a packaged RNA polymerase.
(-)RNA is converted to (+)RNA using RNA polymerase brought into the cell as part of the virus particle. - Viral (+)RNA are translated by the host cell machinery
- make capsid, envelope, RNA polymerase plus other viral proteins. - RNA polymerase makes (-) RNA which is packaged into virus particles along RNA polymerase to generate new infectious virus.
Replication of + Strand RNA viruses
- Viral RNA genome brought in with the virus can function as mRNA to encode viral proteins using host cell translation machinery.
- Viral RNA polymerase made in step 1 used to make complementary copies of the viral genome
Incubation period
time from exposure to development of disease
Infectious period
length of time a person can transmit disease
Latent period
period of infection without being infectious
–this may occur right after exposure or late in the disease
Epidemic
Endemic
Pandemic
occurence of cases of illness in excess of expectancy
epidemic whose incidence remains stable for a long time
global outbreak
Incidence
Incidence rate
number of NEW CASES in a given time period within a given population
number of NEW CASES PER POPULATION at risk for the disease over time
Comparing incidence and incidence rates can help determine if an epidemic is occuring
Prevalence
total number of cases of a disease/# of individuals in population
**Important if an infection is of long duration
**can be broken down by groups of interest (race, gender, etc)
Attack Rate (infectivity)
proportion of exposed individuals who become ill
Primary / secondary cases
primary: the person who infects a poplation
secondary: those who subsequently contract the infection
Case fatality rate
proportion of infected individuals who die of the infection. this is a function of the severity of the infection and is heavily influenced by how many mild cases are not diagnosed
Reservoir
ecological niche of pathogen: where it normally lives and replicates
Vector
any organism, usually an arthropod, which transmits pathogen to susceptible individuals
i.e. tick, skeetos
Zoonosis
infection that can spread from vertebrate animals to man
basic reproductive rate (Ro) the number of secondary cases following a single introduction into a fully susceptible population
the number of secondary cases following a single introduction into a fully susceptible population
If Ro < 1, then every new generation of infection will affect fewer individuals and the disease will die out. Population that is vaccinated or became immune affects disease spread
If Ro = 1 then approximately the same number of individuals are infected with every new generation causing endemicity
If Ro > 1 then there is an ever increasing number of infected individuals causing epidemic or pandemic
Herd Immunity Threshold
the fraction of a population that must be immune to a given microorganism to prevent an outbreak beyond one index case
–>the higher the Ro of infection, the higher the herd immunity threshold
Modes of Disease Transmission
Airborne: long distance respiratory transmission
Droplet: medium to short distance respiratory transmission
Contact: transmission via environment
Sexual: body fluids are exchanged
Fecal/Oral: food supply or direct contact
Steps to evaluate/control an outbreak
Verify index cases & diagnosis.
Draw an epidemic curve (chronological histogram)
Develop a case definition.
Perform additional case finding.
Create a line listing of cases, exposures, other features than can point to an etiology
Develop a hypothesis.
Test the hypothesis.
Refine and repeat #6-7
Implement control measures.
Communicate findings.
Intoxication
ingestion of preformed toxinis sufficient to obtain disease
Food born intoxications often involve heat and pH stable toxins
Symptoms develop quickly since toxin is already present in body
Symptoms often include GI tract: vomitting, diarrhea, cramps
Exception: food borne botulism
Ingestions acquired infection
Typically cause inflammation–>increase in fecal leukocytes
GI symptoms–>cramps, diarrhea, fever
May remain in GI tract or disseminate
Symptoms tend to develop slowly
Important Factors in emerging infections
- Interspecies crossing
Close human proximity to birds, pigs (Influenza) and bats Encroachment to new reservoirs (humans handling jungle meat-Ebola) - Adaptation of a virus to the human host
Change (human) as incidental host to reservoir (SARS) Improved human-human transmission (HIV)
Tissue tropism can determine pathogenicity (diarrhea in SARS) - Susceptible hosts
International travel increases the size of population at risk (SARS) Antigenic shift exposes non immune humans to a “new” disease (H5N1) Decreased childhood vaccination rates due to concerned parents (measles) - Global warming
Expansion of mosquito habitats (Dengue, chikungunya) - Improved diagnostic tests
Reveal new pathogens of existing syndromes (Metapneumovirus)
Category A Biological Weapons
can be easily disseminated or transmitted from person to person
result in high mortality rates and have the potential for major public health impact
might cause public panic and social disruption
require special action for public health preparedness.
anthrax
plague
botulism
smallpox
tularemia
viral hmorrhagic fevers
Category B Biological Weapons
are moderately easy to disseminate
result in moderate morbidity rates and low mortality rates
require specific enhancements of CDC’s diagnostic capacity and enhanced disease surveillance.
Category C Biological Weapons
Third highest priority agents include emerging pathogens that could be engineered for mass dissemination in the future because of
availability
Ease of production and dissemination
Potential for high morbidity and mortality rates and major health impact
Antigenic variation
systematic changes or variations in surface molecules to avoid elimination by the immune response
avoidance of antibodies usually
usually seen in extracellular pathogens or those that are extracellular part of the time
T cell epitopes change as well, but this is not usually in a pattern. –>“immune escape” such as HIV
Types of Antigenic variation
1) Phase variation: switching on/off genes that produce a phenotpe
–>must be able to go in both directions
eg. E.coli fimbraie
2) Antigenic drift: accumulation of mutations that alters antigen composition and changes recogniton by the immune system; often the result of error prone replication
eg influenza, HIV
3) Antigenic shift: abrupt and major change in surface antigen due to gene conversion, rearrangement, reassortment
eg influenza, gonorrhea, t. brucei
Why does antigenic variation occur?
Evolution of the population of microbes in response to the immune response of the host
Selective pressure by (usually) antibodies to change surface molecules to avoid being killed
Need successive changes to avoid being eliminated by pre-existing antibodies
Invasins:
Invasins:
bacterial surface proteins
- provoke phagocytic ingestion of bacterium by host cell
- some act as adhesins (binding), or work with adhesins to bind
- cause cytoskeletal rearrangements that lead to engulfment
- actin rearrangements and formation of pseudopod-like structures
- can involve Type III secretion systems on bacteriabacterial surface proteins
- provoke phagocytic ingestion of bacterium by host cell
- some act as adhesins (binding), or work with adhesins to bind
- cause cytoskeletal rearrangements that lead to engulfment
- actin rearrangements and formation of pseudopod-like structures
- can involve Type III secretion systems on bacteria
Enterotoxin Actions
Alter intestinal cyclic nucleotide levels–>cholera toxin, LT of ETEC (cAMP), ST of ETEC (cGMP)
Kill cells (shiga toxin)
Act as superantigen to induce an inflammatory response (S.aureus enterotoxins)
Affect tight junction permeability by inducing signal tranduction changes (C diff)
Characteristics of extotoxins
Most are proteins
b. Active in smaller amounts than endotoxin
c. One bacterial cell can make >1 toxin
d. Gram-positive bacteria are generally better toxin producers, but there are
some good toxin-producing Gram-negatives
Tetanus toxin
works on CNS in spinral cord (travels up motor nueron into the CNS)
blocks release of inhibitory neurotransmitters like glycine
results in constant stimulation of the motor neuron
motor neuron constantly exceites muscle
Botulinum toxin
acts at neuromuscular junction
blocks release of acetylcholine (excitatory neurotransmitters)
w/o acetylcholine, muscles don’t contract
results in flaccid paralysis
coordinate regulation
all virulence genes necessary for one step in the pathogenic cycle are expressed at the same time
global regulator
coordinate regulation is achieved with just the single regulatory element
Mechanisms to sense environemental changes used by bacteria
1) two component regulatory systems
- one bacteria can have multiple two component regulatory systems
- one two component regulatory system can regulate another
2) quorum sensing systems
two component regulatory systems
- These systems regulate many bacterial functions, including virulence factor expression.
- These systems are found in both Gram+ and Gram- bacteria
.•Components include:
–Sensor protein: transmembrane protein that, when stimulated, undergoes a conformational change that activates a histidine kinase domain.
–Transcriptional regulator: cytoplasmic protein that is phosphorylated by the activated sensor protein. When phosphorylated, it causes increased expression of some genes but decreased expression of others.
quorum sensing
•Bacterial pathogens often must reach a critical density in the human body before causing disease.•Some pathogens sense their population density, and then adjust their virulence factor expression, using quorum sensing.•Quorum sensing involves secretion of a small molecule (a peptide or homoserine lactone) called an autoinducer.
Persistence
ability of a pathogen to remain long term within the host
–result of failure of host defense to clear the pathogen
Latency
form of persistence infection in which the pathogen is quiesant
–causes no overt disease
Reactivation
persistent/latent pathogen reenters productive replication
usually causes overt disease
carrier state
infected individual that displays no overt disease
-serves as a reservoir for spread of disease
Toxoid
bacterial toxin rendered non toxic; used to induce anti-toxin antibodies