Quizlet Import MICROM442 Exam 1

How would you treat a patient with an Enterococci infection prior to having susceptibility results back?

- E. faecalis- ampicillin
- E. faecium - vancomycin and hope it's susceptible or if really severe daptomycin

What two drugs have the same target and are concerning for cross resistance?

-Erythromycin and clindamycin

Why do you not want to treat Enterobacter with ceftriaxone?

Potential for rapid development of resistance by mutations and up regulation in AmpC

Why are infections of mechanical heart valves and prosthetic joints "surgical solutions"?

- Biofilms provide physical barriers to antimicrobials
- organisms are often not rapidly growing, reducing the efficacy of many antimicrobials

What are the four resistance mechanisms

- restricted access to target, efflux, drug inactivation, target alteration.

Prokaryotes: do they have the following- 1) nucleus 2) extrachromosomal DNA 3) cytoplasmic organelles 4) ribosomes 5) cytoplasmic membrane 6) cell wall 7) sterols

1) no (nucleoid)
2) yes, plasmids
3) no
4) 70S in cytoplasm
5) yes, respiration, secretion, DNA syn, phospholipid syn
6) rigid peptidoglycan (PG)
7) no

Eukaryotes: do they have the following- 1) nucleus 2) extrachromosomal DNA 3) cytoplasmic organelles 4) ribosomes 5) cytoplasmic membrane 6) cell wall 7) sterols

1) yes
2) yes, organelles
3) yes
4) 80S in cytoplasm
5) yes, semi-permeable layer, no specialized function
6) no PG, some cases cellulose
7) yes

size of spherical bacteria

0.2 to 2 µm diameter

size of rods

0.2 - 2 µm wide by 1-10 µm long

shapes of bacteria

cocci (coccus), bacilli (bacillus), coccobacilli, fusiform, vibrio, spirilla, spirochete

multicellular arrangements of bacteria

diplococci, chains, clusters (biofilms)

Differences between Gram negative and gram positive bacteria

-GN has LPS
-GN has periplasm
- GN doesn't have an endospore whereas GP may have one

Who said "Humanity has but three great enemies: fever, famine and war; of these by far the greatest, by far the most terrible, is fever."

Sir William Osler, 1896

Who developed single lensed microscopes?

Robert Hooke and antoni Van Leeuwenhoek (1600s)

\_______ reports cowpox vaccination against smallpox

Edward Jenner (1700s)

\_________: hygiene is a great way to avoid infection!

Florence Nightingale (1800s)

\______: microbes as causative agents of disease

Pasteur and Koch - 1800's

Who invented the Gram's stain?

Hans Christian Gram 1800's

Microbiology breakthroughs in the 1900's

Paul Erlich: "606" salvarsan or arsphenamine
Alexander Fleming: penicillin; Ernst Chain and Howard Florey: purification/production
Gerhard Domagk: sulfonamides

Avery, MacLeod, McCarty: DNA as a transforming principle
Sanger: protein sequencing
Watson, Crick, Rosalind Franklin: DNA structure
Sanger: DNA sequencing
Rich Roberts: restriction enzymes
Herb Boyer, Stanley Cohen: constructing recombinant DNA
Kary Mullis: PCR

Microbiology in the 2000's

Massively parallel, high throughput, next generation sequencing

microbial pathogen relationship with host

Microbial pathogen: colonize (\----\> multiply \-----\>) transmission
Human host: infection disease

T/F fetus is microbiologically sterile

T

Factors determining the nature of microbiota

local physiology and ecology
microbial attributes
microbial interactions (competition)

Microbiota at skin

Staphylococcus epidermidis; other Staph; Propionibacterium; diphtheroids (contam of Cx's!?)

microbiota in conjunctiva

1. s. epidermidis
2. non pathogenic corynebacteria

Microbiota of the intestinal tract
- Mouth
- stomach and small intestine
- colon

Mouth: 10e8 orgs/ ml in saliva; Streptococcus mutans specifically adhere to teeth; Neisseria and Moraxella; strict anaerobes and microaerophilic organisms associated with gingival crevice
stomach and small intestine normally sparsely inhabited
colon: 10e10 to 10e11 bacteria/ml; feces are about 25% bacteria by weight
90% are anaerobes: Bacteroides and Fusobacterium, Clostridium perfringens can be found
10% facultative: E. coli, other Enterobacteriaceae, enterococci, yeasts such as Candida
populations vary with diet and age
Example: Bifidobacterium in human infants

microbiota of the respiratory tract

Nares: similar to skin, importantly includes Staphylococcus aureus
Nasopharynx: similar to mouth, but also Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae
Larynx and below, middle ear and sinuses protected by mucociliary escalator

microbiota of genitourinary tract

Urinary tract: scanty microbiota from perineum in first 1 cm of urethra, sterile above this in health
Vagina: Before puberty/after menopause: mixed, non-specific from skin, colon, perineum.
Child bearing years: Lactobacillus, anaerobic GNRs, GPC, Gardnerella, Mycoplasma, Ureaplasma

role of microbiota in disease

opportunists: quantity, genetic attributes

beneficial effects of microbiota

- priming immune system
- exclusionary effect, demonstrated by antibiotic treatment
- nutritional: digestion, malabsorption, vitamin K

Next generation sequencing or massively parallel sequencing

- extract template
- limited PCR amplification of 16S rRNA target sequence
- prepare library (addition of adaptors for capture, sequencing, barcoding (multiplexed))
- immobilize single molecule templates on a lawn of capture oligonucleotides (100's of millions)
- isothermal bridge amplification and cluster formation (Illumina technology)
- templates hybridize to adjacent oligonucleotides to form bridges
- amplified dsDNA are denatured to form two ssDNA strands
- repeated denaturation/amplification yields dense clusters of ~2,000 molecules each
- sequence clusters created by amplifying captured templates
- each dsDNA cluster is denatured to ssDNA
- reverse strand can be removed by specific cleavage
- 3'-ends of forward strand are chemically blocked from interfering with sequencing
- flow cell contains \>200 million clusters with ~1,000 molecules/cluster
(consider the similarity to single colonies on a plate)
- sequencing primer hybridizes to adapter sequence
- sequence by synthesis in presence of 4 labeled reversible terminators
- process sequencing data (substantial effort and the most labor intensive part of process!)
- multiplexed approach provides economy of scale: barcodes in library primers
- compare with sequences obtained from phenotypically validated isolates
- establish identification, relatedness to known pathogens
- deconvolute polymicrobial samples to determine constituent members of microbiome

diacrisis synonym

diagnosis

diagnosis

the determination of the nature of a disease. Involves:
-history
-physical exam
- imaging
- lab testing
- treatment
- formulate hypothesis and test it

identification of etiological agents

- determine nature of disease
- predict course and potential outcome(s)
- tailor therapy: apply specific intervention(s) to a clearly defined problem
- exclude non-infectious causes of symptoms

specimen collection

• sterile
• non-sterile
i) indirect: specimen collected thru a site containing normal microbiota
ii) at a site with normal microbiota
• description

Examination: microscopy
i) bright field
ii) dark field
iii) fluorescence microscopy

i) bright field: Gram stain, acid-fast stain
ii) dark field: thin organisms, e.g., spirochete
iii) fluorescence microscopy: very sensitive but artifacts can cause problems

Types of culture

• nutrient
• selective
• indicator

incubation

•temperature
•oxygen rich (aerobic), low oxygen, or no oxygen (anaerobic)
•presence of carbon dioxide

Conventional identification - two tools of microbiology?

• 9 log amplification: one bacterium can form a colony on a plate:109 colony forming units
• ability to isolate single cells on a plate: single colony isolation

What does conventional identification determine? (6)

1) gross phenotype
2) biochemical characteristics
catalase: H2O2 --\> O2 + H2O
urease: (NH2)2C\=O --\>NH3 + CO2
coagulase: fibrinogen --\> fibrin clot
3) Antigenic structures, e.g., streptococcal polysaccharide
4) Toxin production
5) Nucleic acid sequences, including genomic approaches:
note the significance of "functional genomics"
6) Flow of information: DNA --\> RNA --\> enzymatic functions \---\> structures

Serological detection of infection

in most cases, identification of host immunoglobulins specifically recognizing antigens from pathogenic organisms
recall the kinetics of a humoral immune response in a immunocompetent host (Fig 2-16).
i) measured by titer
ii) acute disease: antibody titer increases

iii) significance:
•acute disease vs convalescent disease
•4-fold increase in titer
•IgM/IgG: primary vs secondary infection
iv) ELISA, EIA and agglutination fig 4-13; fig 4-10
v) Western Blot (Chapter 4. Immunological Systems. Western Blot)

What is the detection of pathogen-specific nucleic acid sequences useful for?
- technological advantage?
- drawbacks?

organisms that are difficult or impossible to cultivate
- Technological advantage: problems not previously identifiable
- Drawbacks include false-positive results due to contamination, TAT, limited ability to assess properties of pathogen

Methods for molecular detection of pathogen specific nucleic acid sequences

1) PCR (polymerase chain reaction) 106-109 enzymatic amplification of target sequences (see Fig 4-18)
2) Traditional PCR amplification + Sanger sequencing [dye terminator sequencing]
Traditional PCR amplification + melting curve analysis
3) Next Generation Sequencing

sequencing platform requirements

- accuracy for identification (taxonomy)
- informative about phylogeny
- accurate molecule chronometer or evolutionary clock

What does disease result from?

Lack of resistance (elimination of pathogen) and tolerance (reduction in tissue damage

innate immunity

-First line of defense
-Rapid responses to a broad range of microbes
-Germline encoded receptors and responses
- Early warning and defense system

external defenses of innate immunity

- physical and chemical barriers
-mucous membranes

internal defenses of innate immunity

- complement
- phagocytic cells
-Pattern recognition receptors
- inflammatory response

Adaptive immunity

- primary response takes time
-targeted to specific microbes
- clonally rearranged receptors
- provides immunologic memory
e.g.
Humoral response (antibodies)
Cell mediated response (T cells)

physical and chemical barriers

Skin
Tears - contain lysozyme, an enzyme that digests peptidoglycan
Saliva - antibacterial enzymes
Respiratory - mucus and cilia trap and remove organisms Microbiota compete for nutrients, produce antimicrobials and influence immune system

GI tract mucosa
how the mucus barrier is created

-Goblet cells secrete gel-forming mucins (prevent pathogen binding)
-Paneth cells secrete antimicrobial defensins and other proteins
-M-cells transport antigens from the gut lumen to cells of the immune system
- causes inner mucus layer to be essentially sterile

Pathways of complement activation

1. classical pathway: antigen-antibody complexes
2. alternative pathway: binds pathogen surfaces
3. lectin pathway: mannose binding lectin and proteases bind to microbe

outcomes of complement activation

1. inflammation and chemotaxis
2. opsonization
3. pathogen lysis (membrane attack complex)

terminal complement component deficiencies predispose to \______ infections

Neisseria

Chemotaxis

Cell movement that occurs in response to chemical stimulus

opsonization and phagocytosis

-complement coats bacterium to target particles for uptake or phagocytosis
- opsonization independent mechanisms can also trigger uptake

neutrophils

A type of white blood cell that engulfs invading microbes and contributes to the nonspecific defenses of the body against disease.

macrophages and dendritic cells

Phagocytosis of foreign substances
Antigen presentation to lymphocytes to initiate immune response

Pattern Recognition Receptors (PRRs)

Recognize pathogen associated molecular patterns - conserved and essential microbial components
-Pattern recognition receptors are present: on the cell surface in the cytoplasm in intracellular compartments

Toll-like receptors (TLRs)

-Family of pattern recognition receptors present on the cell surface and intracellular compartments
-Recognize specific pathogen associated molecular patterns (PAMPs)
-Activate signaling cascades for cellular activation and cytokine production

How bacteria evade Toll-like receptors

- modulating structure to prevent recognition
- interfering with signaling pathways

how infection triggers an inflammatory response

1) bacteria trigger complement, cytokine, and chemokine release
2) vasodilation and increased vascular permeability
3) inflammatory cells migrate into tissue

T/F TLR4 recognizes bacterial lipopolysaccharide (LPS).

T

T/F Antigen-antibody complexes activate the classical complement pathway.

T

Humoral immunity

-specific immunity produced by B cells that produce antibodies that circulate in body fluids
- each B cell receptor/antibody has a unique antigenic specificity

IgM vs IgG when it comes to viral testing

IgM - means you have an acute virus

IgG - means you were exposed and had the infection, your body produced antibodies against it but you don't have it now

antibody functions

-neutralization, opsonization, complement activation
-Haemophilus influenzae produces a protease that cleaves IgA

What do T cells bind?

- peptides presented on MHC
- MHC class I binds CD8 T cells
-MHC class II binds CD4 T cells

CD4 Helper T cells

-Bind antigen presented on MHC Class II
-Activate B Cells and Macrophages
-Produce cytokines

CD8 Cytotoxic T cells

-Bind antigen presented on MHC Class I
-Kill infected cells
-Produce cytokines

T/F IgM is the first antibody isotype produced during an infection

T

Pathogen

organism endowed with the capacity to cause disease in a particular host

primary pathogen

infects competent host

opportunistic pathogen

infects compromised host:
i) loss of specific defense mechanisms
ii) loss of non specific defense mechanism

infection

occurs after colonization, when multiplication is sufficient to induce damage to the host or alter host physiology. This level of multiplication can ultimately result in "disease", and is usually accompanied by a clinically detectable host response.

Inflammation

rubor, tumor, calor, dolor, and loss of function

Carrier state/latency

successful colonization WITHOUT sufficient multiplication to evoke the changes characteristic of the disease state. Latency is defined by Stedman's Medical Dictionary as "not manifest, but potentially discernible".

disease

often a manifestation of the transmission mechanism utilized by the pathogen

colonize -\> multiply -\> transmission: mechanisms dictated by \_______ determinants

virulence

virulence determinants

unique attributes that permit a microbe to successfully establish infection and cause subsequent disease.

virulence

quantitative measure of pathogenicity or likelihood of causing disease.

infectivity

quantitative measure of a pathogen's ability to infect another susceptible host (also known as attack rate, expressed as %).
\#infected/\#susceptible and exposed x100% \= attack rate

How can we change the infectivity?

vaccination, limiting exposure (i.e. washing hands, social distancing)
- decreases number susceptible and exposed

Colonization

1. adherence: pili: PAP pili of uropathogenic E. coli
2. motility: flagella: many enteric organisms
chemotaxis: same
3. survival or fitness in an environment outside host

How microbes multiply

1. nutrition
2. avoiding host immune surveillance
3. survival outside host

nutrition: iron acquisition

siderophores: enterics
lactoferrin/transferrin receptors: N. gonorrhoeae \---\> these receptors increase affinity and avidity for iron

How microbes avoid host immune surveillance intracellularly

a. breaking out of the phagolysosome or life in the cytoplasm: Listeria, Shigella
b. life in the vacuole:
i) modify phagolysosome: Salmonella, MTB
ii) block phagolysosome fusion: Toxoplasma

How microbes avoid host immune surveillance extracellularly

a. inhibit phagocytosis
capsules: S. pneumoniae
IgA proteases: H. influenzae
Bind host proteins: fibrinogen by M-protein of group A strep
b. block complement mediated lysis: S. typhimurium LPS
c. antigenic variation: Borrelia

How microbes avoid host immune surveillance by altering host immune response

a. exotoxin: pertussis toxin alters lymphocyte function
b. endotoxin: can overstimulate host resulting in DIC
(disseminated intravascular coagulation)
c. antigenic variation
d. superantigens: polyclonal T-cell proliferation

Polyclonal T-cell proliferation by staphylococcal enterotoxin H

binds to the side of TCR and MHC molecules so T cells respond and proliferate, not directed at staphylococcus and it's to the pathogen's benefit because the immune system is misled (no specificity)

Exotoxins

A. classical A B subunit toxins: PT, DT, CT, etc
A catalytic subunit; ADP ribosylates G-proteins
B membrane-binding subunit; translocating A subunit into host cytoplasm
B. RTX (repeats in toxin); hemolysins

Endotoxins

Gram negative (GN): "endotoxin" or LPS (lipopolysaccharide)
Gram positive (GP): lipoteichoic acids
Both GN and GP: peptidoglycan

expression of virulence determinants

pathogens adapt to, and alter gene expression in response to changes in their environment

mobile virulence determinants

i) carried by bacteriophages: Diphtheria toxin
ii) plasmids: Yersinia adhesins, invasins and effectors
iii) transposons: drug resistance

Regulation of virulence determinant gene expression

1. Altered expression of determinants in response to:
temperature
ionic conditions (iron, calcium)
oxygen concentration
pH
2. Two component regulatory systems: R S
S: sensor of environmental stimuli
R: response regulator, regulates protein fxn/gene expression

What is Epidemiology?

The study of the distribution and determinants of health related states and events in specified populations
- data driven
-public health
- Provides insight regarding the nature, causes, and extent of health and disease in a population
• Provides information needed to plan and target resources appropriately

Who founded Epidemiology

John Snow

epidemiological triad

agent, host, and environmental factors that affect health

epidemiological triad: agent

Disease producing microorganism (bacteria, virus, fungus, parasite) Characteristics: • Virulence, dose, toxicity • Ability to survive in different environments • Antibiotic susceptibility Interventions: • Control or eliminate the infection at its source

epidemiological triad: host

Human or animal who can get the disease Characteristics: • Exposure: • Behavior - age, sex, sexual practices, hygiene • Susceptibility and response: • Host genetics • Immunological status • Anatomic structure • Disease or medications Interventions: • Treat infection • Immunize • Behavior modification