MICROM 442 Final (Quizlet Comp)

Fuck my life, MICROM final that could have around 80 questions

1600s microbiological developments

Robert Hooke and Antoni Van Leeuwenhoek develop single-lensed microscopes

1700s microbiological developments

Edward Jenner reports cowpox vaccination against smallpox

1800s microbiological developments

Florence Nightingale: hygiene prevents infection
Pasteur and Koch: microbes are causative agents of disease
Hans Christian Gram: Gram's stain developed

1900s microbiological developments

Paul Ehrlich: 606 salvarsan or arsphenamine
Alexander Fleming: penicillin; Ernst Chain and Howard Florey purify and production of penicillin
Gerhard Domagk: sulfonamides
Avery, MacLeod, McCarty: DNA transformation
Sanger: protein sequencing
Watson, Crick, Franklin: DNA structure
Sanger: DNA sequencing
Rich Roberts: restriction enzymes
Herb Boyer, Stanley Cohen: recombinant DNA
Kary Mullis: PCR

2000s microbiological developments

Massively parallel, high throughput, next generation sequencing

Shapes of bacteria

cocci, bacilli, coccobacilli, fusiform, vibrio, spirilla, spirochete

Multicellular arrangements

diplococci, chains, clusters, biofilms

Gram negative organisms

outer membrane of proteins, LPS, and phospholipids, Peptidoglycan, no teichoic acids, and a periplasm

Gram positive organisms

No outer membrane, has peptidoglycan (thick layer), has teichoic acids, and no periplasm

Teichoic acid

polymer associated with the cell wall of Gram-positive bacteria
unique to Gram positive bacteria --\> potential antibiotic target

Lipopolysaccharide (LPS)

Molecule that makes up the outer layer of the outer membrane of Gram-negative bacteria
contain O antigen that can be recognized by immune molecules
lipid A - base of lipid in membrane; polysarrcharide+O antigen - reaches into outer membrane space, etc

Taxonomy

theory and practice of classification of individuals into groups

phylogeny

study of evolution and ancestry of an organism

phenotype

morphology, biochemical capabilities, fatty acid composition (membrane and by-product), mycolic acid analysis

Genotype

%G+C, DNA:DNA hybridization, sequence and whole genome sequence analyses

microbiome

cataloguing constituent members of microbial populations

indigenous microbiota

microbes that live on and in our bodies
commensal/beneficial relationship with host when associated with proper niche within the body

Symbiosis

biological association of two or more species that may, but does not necessarily, benefit each

Commensal

one spcies derives benefit and the other is unharmed

Mutualism

both species derive benefit
most microbes fit here (microbiota)

parasitism

one species (parasite) benefits at expense of the other (host)

transients and residents

microbes that stay short term and others that colonize and are permanent in a normal environment

carrier state

person harbors organism but does not show any signs of the disease, may pass it on
transmission of infectious agents (vehicle)

Origin of normal microbiota

fetus is microbiologically sterile but receives skin and respiratory microbiota from birth canal, environment, personnel
neonate narrows group by ability to maintain colonization

factors determining the nature of microbiota

local physiology and ecology; microbiological attributes; microbiological interactions (competition)
competitive exclusion, etc

Microbiota of skin

Staphylococcus epidermidis, other staphylococci; Propionibacterium (Cutibacterium); diphteroids (contaminants of cultures); micrococcus

Microbiota of Conjunctiva

S. epidermidis; non-pathogenic corynebacteria

Microbiota of Intestinal tract

10e8 organisms/mL in saliva
Streptococcus mutans (adhere to teeth)
Neisseria and Moraxella
anaerobes and microaerophilic organisms associated with gingival crevice

microbiota of stomach and small intestine

normally sparsely inhabited
stomach has very low pH 2 (highly acidic(
Small intestine under intense immune surveillance

microbiota of colon

10e10-10e11 bacteria/mL
90% anaerobes --\> Bacterioides and Fusobacterium; Clostridium perfringens
10% facultative anaerobes --\> E. coli, Enterobacteriaceae, enterococci, yeasts (Candida)
infants (Bifidobacterium)

Bifidobacterium

some strains (probiotic) are protective from E. coli O157 disease
secrete molecule protective for epithelial integrity of GI tract (lnbX)

lacto-N-biosidase (lnbX)

degrade lacto-N-tetraose (abundant in human milk oligosaccharide (HMO))

Microbes in Nares

Staphylococcus aureus
similar to skin

Microbiota of Nasopharynx

similar to mouth
Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae

Microbiota in Larynx and below, middle ear, and sinuses

protected by mucociliary escalator rather than microbes

Microbiota of Urinary Tract

urine usually sterile
scanty microbiota from perineum in first 1cm of urethra

Microbiota of Vagina

before puberty and after menopause: mixed, non-specific from skin, colon, perineum
child bearing years: lactobacillus, anaerobic GNRs, GPC, Garnerella, Mycoplasma, Ureaplasma

Opportunists

Microorganisms that produce disease only under favorable conditions
attributes that cause disease in defects in immune system or in abnormal but favorable niches

Benefits of microbiota

prime immune system, exclusionary effect, nutrition (digestion, malabsorption, vitamin K)

exclusionary effect

exclude pathogenic organism from colonizing certain locations by outcompeting
demonstrated by antibiotic treatment --\> less normal microbiota, more susceptibility to disease

Next Generation Sequencing

entire genomes sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences.

Interrelated processes that contribute to infection

Pathogen: colonize --\> multiply --\> transmission
Human host: infection --\> disease

Diacrisis

Diagnosis
determination of the nature of a disease
History --\> formulate hypothesis --\> test hypothesis

Identification of etiological agents

determine nature of disease
predict course and potential outcomes
tailor therapy: specific interventions to clearly defined problem
exclude non-infectious causes of symptoms

Specimens

sterile: urine, blood, cerebral spinal fluid
non-sterile: indirect --\> specimen collected through a site containing normal microbiota (collected through skin, oral cavity)
direct: at a site with normal microbiota (colon, throat, etc)

Gram stain

Used to classify prokaryotes based on cell wall composition. Important for antibiotics; some antibiotics work on one but not the other.
presence of peptidoglycan (thickness)

Acid-fast stain

A staining procedure for identifying bacteria that have a waxy cell wall.

bright field microscopy

generates a dark image of an object over a light background
Gram stains and acid-fast stain

dark field microscopy

thin organisms, e.g., spriochete
bright bacteria on a dark background

fluorescence microscopy

Uses UV light
Fluorescent substances absorb UV light and emit visible light
Cells may be stained with fluorescent dyes (fluorochromes)
very sensitive but can cause problems with artifacts

fluorophore

A fluorescent molecule used to stain specimens for fluorescence microscopy.
direct: antigen is labeled with fluorescent molecule
indirect: need secondary antibody with fluorescent molecule to recognize primary antibody

Nutrient media

culture media that are complex and made of extracts of meat or soybeans
grows all kinds of bacteria

selective media

allows for growth of certain pathogens and prevent others from growing
sort pathogen from normal microbiota

indicator media

allows organisms to grow and reveal aspects of its physiology
ex: utilize carbohydrate through fermentation

Nutrient broth

commonly used liquid complex medium
used for culture of blood and all direct tissue samples from sites that are normally sterile to obtain maximum culture sensitivity

Blood agar

Used as an enrichment medium for fastidious microbes as well as differential media
enhances growth of some bacteria such as streptococci
indicator of hemolytic zone (alpha and beta)

Chocolate agar

Complex medium used to culture fastidious bacteria, particularly those found in clinical specimens. Not selective or differential.
H. influenzae
red blood cells gently lysed in media

Martin-Lewis Media

variant of chocolate agar
selective of pathogenic Neisseria
contains antimicrobial agents to prevent other bacteria from genitourinary tract

MacConkey Agar

selective and indicator media for Gram negative rods
Enterobacteria and Pseudomonas
bile salts, crystal violet, lactose, and neutral red as pH indictor

Hektoen Enteric Agar

highly selective for Salmonella and Shigella from stool specimens
selective and indicator media - mixture of bile, thiosulfate, citrate salts to inhibit Gram + and other enterobacteriae

Anaerobic media

Obligate anaerobes must be cultured in the absence of free oxygen
Reducing media contain compounds that combine with free oxygen and remove it from the medium
Petri plates are incubated in anaerobic culture vessels (Sealable containers that contain reducing chemicals)

Highly selective media

media specific to the isolation of every important pathogen and is specific to said organism
prevents normal microbiota to grow

Conventional identification

relies on two tools of microbiology
9 log amplification
ability to isolate single cells on plate --\> single colony isolation

9 log amplification

one bacterium can form a colony on a plate
1 colony forms 10e9 colony forming units

Identification tools

gross phenotype
biochemical characteristics (catalase, urease, coagulase)
Antigenic structures
toxin production
nucleic acid sequences
flow of information (DNA --\> RNA --\> enzymatic functions --\> structures)

serological detection of infection

identification of host immunoglobulins specifically recognizing antigens from pathogenic organisms
specific to pathogen
humoral immune response (IgM, IgG, etc)
measured by titer, acute disease (antibody titer increases, significance (acute versus convalescent disease, 4-fold increase in titer, IgM vs IgG)

Antibody based diagnostic strategies

ELIA, EIA, agglutination
Western Blot
antibodies are incredibly specific --\> can be made to any structure

direct immunosorbant assay

antibody absorbed onto well, antigen added, enzyme-linked antibody added and binds to antigen to form double-antibody sandwich
enzyme substrate added and measured spectrophotometrically for enzyme activity (color change)

indirect immunosorbent assay

antigen absorbed onto well and antiserum added (with primary antibody) and binds to antigen
enzyme-linked anti-gamma globulin (anti-antibody) added and binds to bound antibody
enzyme substrate added and produces visible color change

Agglutination

clumping of red blood cells
test sera placed in wells along with RBCs
sufficient antibodies --\> sink as mat
insufficient antibody --\> sink as pellet

detection of pathogen-specific nucleic acid sequences

particularly useful for organisms that are difficult or impossible to culture

Molecular Detection

used with organisms that are difficult or impossible to cultivate; critical samples
sometime false positives from contaminations (during diagnosis), limited ability to assess properties of pathogen
PCR, PCR + Sanger sequencing, melting curve analysis, next gen seq

16S rRNA

acts as molecular clock for sequencing and informs about phylogeny of organism
some variable and conserved regions

Resistance

host is able to prevent organism from causing infection and disease
able to eliminate the pathogen

Tolerance

Organisms can exist in the host but not make it sick

Susceptible host

failure of resistance and tolerance
failure to clear the pathogen or tolerate immune-mediated damage

innate immunity

first line of defense
rapid response to a broad range of microbes
germ-line encoded receptors and responses
early warning and defense system

external defenses

physical and chemical barriers
mucous membranes

internal defenses

complement
phagocytic cells
pattern recognition receptors
inflammatory response

adaptive immunity

primary response takes time
targeted to specific microbes
clonally rearranged receptors
provides immunologic memory

humoral response

antibodies

cell-mediated response

T cells

physical and chemical barriers

skin - thick layer of dead cells
tears - lysozyme
saliva - antibacterial enzymes
respiratory - mucus and cilia trap and remove organisms
microbiota - compete for nutrients, produce antimicrobials, and influence immune system

lysozyme

enzyme that digests peptidoglycan

Goblet cells

secrete gel-forming mucins --\> form mucosa

Paneth cells

secrete antimicrobial defensins and other proteins

M-cells

transport antigens from the gut lumen to cells of the immune system
sample contents to determine if there are any potential pathogens

Complement

family of proteins that all work together in a cascade
present in blood and tissues
includes enzymes, receptors, and regulatory proteins

Classical pathway of complement activation

antibody binds to antigen and is recognized by complement

lectin pathway of complement activation

activated by microbe binding to lectin

alternative pathway of complement activation

Microbial products directly activate complement

Outcomes of Complement Pathway

inflammation and chemotaxis
opsonization
pathogen lysis (membrane attack complex)

chemotaxis

immune cells detect soluble signal and move up concentration gradient
allows immune cells to know where the infection is
movement towards complement

Opsonization and phagocytosis

targets particles for uptake or phagocytosis
complement activated on surface of pathogen and binds to complement receptor on phagocytic cell

Neutrophils

phagolysosome

Intracellular vesicle formed by fusion of a phagosome with a lysosome, in which the phagocytosed material is broken down by degradative lysosomal enzymes.
NADPH shoots in reactive oxygen species

Chronic granulomatous disease

NADPH oxidase deficiency
predisposition to certain types of infection

macrophages and dendritic cells

antigen presenting cells
phagocytosis and cytotoxicity - present antigens to T cells to stimulate activation

Pattern Recognition Receptors

recognize pathogen associated molecular patterns
recognize elements of conserved microbial structures
PAMPs --\> immune system evolved to recognize commonalities of pathogens

PAMPs

pathogen associated molecular patterns
recognized by receptors (external, within the cytoplasm, within organelles)