Bacterial Infections Flashcards
Is it an infectious disease?
Which pathogen is?
What is the antimicrobial sensitivity profile? Does it have any antibiotic resistance? Which treatment plan should I recommend? Any prevention measurements?
How about control plan?
Is it a zoonosis?
Is it notifiable disease?
Regular ? of clinical specimens for ?, ? and ? testing will improve and ? this knowledge
Regular submission of clinical specimens for isolation, identification and susceptibility knowledge testing will improve and update this knowledge
Pre-analytical phase:
* Animal species
* Clinical characteristics
* Disease state
* Epidemiology
* Biospecimen type
* Anatomical site
Analytical phase (lab stuff)
* ?
* ?
* Processing
* Preparation
* Analysis
* Result & review
Post-analytical phase
* Reporting
* Interpretation
* Diagnosis
* Treatment
which phase is the MOST IMP.?
V V IMP step -> pre-analytical phase (if make mistake here then like domino falling, next steps fail)
Analytical phase (lab stuff)
* SAMPLE COLLECTION
* SELECTION OF METHODOLOGY
* Processing
* Preparation
* Analysis
* Result & review
SAMPLE COLLECTION
Choice for specimen collection depends on
■ ? symptoms
■ Type of ?
■ ? of infection
■ ? of infection
■ ? tests to be performed
Common specimens for bacterial infections include: ?
** Proper specimen collection is the ? and ? step for accurate diagnostic testing! **
SAMPLE COLLECTION
Choice for specimen collection depends on
■ clinical symptoms
■ Type of pathogen
■ location of infection
■ duration of infection
■ diagnostic tests to be performed
Common specimens for bacterial infections include: skin, ear swab, milk, urine, wound swabs, blood
** Proper specimen collection is the FIRST and KEY step for accurate diagnostic testing! **
SAMPLE COLLECTION
SPECIMEN TYPES:
Blood
Scraps/Swabs/Impression
?/Exudates
Urine/Misc. fluids
Vomitus/sputum
Feces
Tissues
SELECTION OF METHODOLOGY
Detection of the agent
1. Direct detection of the ?
2. ? cultivation
3. ? of pathogen
OR
Detection of host immune response
1. Serology: detection of ? immunity
2. detection of ? immunity
✓ Infection type? ✓ Test availability? ✓ Sensitivity?
✓ Specificity?
✓ Time and costs?
SAMPLE COLLECTION
SPECIMEN TYPES:
Blood
Scraps/Swabs/Impression
transudate/Exudates
Urine/Misc. fluids
Vomitus/sputum
Feces
Tissues
SELECTION OF METHODOLOGY
Detection of the agent
1. Direct detection of the bacteria
2. sample cultivation
3. taxonomic identification of pathogen
OR
Detection of host immune response
1. Serology: detection of humoral immunity
2. detection of cell-mediated immunity
humoral: relating to the body fluids, especially with regard to immune responses involving antibodies in body fluids as distinct from cells.
- Direct detection of the bacteria
■ ? and ? staining
■ ? staining
Examination of ? or ? preparations (‘smears’)
Single most important and cost-effective procedure
Gives an idea of:
* Bacterial ? (estimation of bacterial numbers)
* Bacterial ?
* ? response (cellular reaction)
Common staining:
* ?-staining
* ? staining (acid-fast staining) -> heat?
* ? antibody staining
* ? microscopy (generally used only for research)
Mycobacterium sp. revealed by fluorescent microscopy in a sputum sample. Using ?, the sensitivity to detect the bacteria is improved, and it is easy to differentiate them from other artifacts or cells.
- Direct detection of the bacteria
■ ** microscopy and differential staining
■ fluorescent antibody staining **
Examination of stained or unstained preparations (‘smears’)
Single most important and cost-effective procedure
Gives an idea of:
* Bacterial density (estimation of bacterial numbers)
* Bacterial morphology
* host immune response (cellular reaction)
Common staining:
* gram-staining
* Ziehl Neelsen staining (acid-fast staining) -> heat? = heat the smear to enhance the dye penetration (steam helps to loosen up the waxy layer and promotes entry of the primary stain inside the cell); (to highlight acid-alcoholic resistant bacilli (AARB)
* Fluorescent antibody staining
* Electron microscopy (generally used only for research)
Mycobacterium sp. revealed by fluorescent microscopy in a sputum sample. Using fluorescence, the sensitivity to detect the bacteria is improved, and it is easy to differentiate them from other artifacts or cells.
SAMPLE CULTIVATION
* Many pathogens can be isolated from clinical samples in the laboratory, using ?
* This process is known as ? or ? of bacteria.
* Media can be ? (broth medium) or ? (agar medium).
* To successfully isolate bacteria, we must provide the bacteria their ?, ? and ?.
* Bacteria grow faster on ? media. Nevertheless, in ? medium, bacteria can be distinguished by phenotypic characteristics.
SAMPLE CULTIVATION
* Many pathogens can be isolated from clinical samples in the laboratory, using simple media.
* This process is known as the cultivation or isolation of bacteria.
* Media can be liquid (broth medium) or solid (agar medium).
* To successfully isolate bacteria, we must provide the bacteria with their optimal nutrients, atmosphere and temperature.
* Bacteria grow faster on liquid media. Nevertheless, in solid medium, bacteria can be distinguished by phenotypic characteristics.
formation of discrete colonies on agar
single colony originated from a single bacteria -> forms a bacterial colony (colony forming unit CFU = 1 bacterium)
they appear differently depending on the type of bacteria;
E creamy small white
F bigger even though same type of incubation etc.
G: hemolysis (bubbles) so something inbacteria is being released and eating whats inside RBCs so membrane being destroyed of RBCs
H bacteria that made a capsule so capsule get together altho looks like a water
I 4 huge colonies, so 2 organisms there so contaminated so have to keep isolting or that bacterium has ..some of the population dont grow too well and in 24 hours some behave some way ..
General- purpose media
Allows growth of a large number of non-? bacteria
Selective media
Contains ? compounds for certain groups. ? medium can become selective (just add an ? substance)
Differential media
Allow the ? of different bacterial groups
WHY IS MacConkey Agar both selective and differential medium?
[Energy source: Lactose or Peptones
Inhibitory compounds: Bile salts and crystal violet which largely inhibit the growth of the Gram-? bacteria.
Differentiation: ? indicator (if lactose is consumed pH will be ?; if peptones (color yellow or red) are consumed, pH will be ?)]
(fastidious: very attentive to and concerned about accuracy and detail)
General- purpose media
Allows growth of a large number of non-fastidious bacteria
Selective media
Contains inhibitory compounds for certain groups. Any medium can become selective (just add an inhibitory substance)
[Selective media generally selects for the growth of a desired organism, stopping the growth of or altogether killing non-desired organisms.]
Differential media
Allow the distinguishment of different bacterial groups -> (so something like color or shape or precipitation or sum so SUM THAT MAKES IT DISTINGUISH FROM ANOTHER)
[Differential media takes advantage of “ biochemical” properties of target organisms, often leading to a visible change when growth of target organisms are present.]
WHY IS MacConkey Agar both selective and differential medium?
-> because it is selective due to its selectivity for gram -
[Energy source: Lactose or Peptones
Inhibitory compounds: Bile salts and crystal violet which largely inhibit the growth of the Gram + bacteria.
Differentiation: pH indicator (if LACTOSE is consumed pH will be ACIDIC indicating that lactose fermenting bacteria are present; if PEPTONES (color yellow or red) are consumed, pH will be BASIC)]
Blood agar is a ?, enriched medium often used to grow ? organisms and to differentiate bacteria based on their ? properties
Some ? medium may give extra information for differentiating the bacteria
Routine samples are cultured on three different solid media:
1. ? or enriched medium
2. ? and ? medium for ?
3. ? and/or ? medium for ?
Incubation:
Most pathogens grow at ?°C (mesophiles) Incubation time range from 18h-48h
Primary culture:
Mixed culture, with pathogen and microbiota (so ? -> pathogen)
2nd incubation and then:
2nd culture:
May have the ? pathogenic bacteria
fastidious: very attentive to and concerned about accuracy and detail
Blood agar is a general purpose, enriched medium often used to grow fastidious organisms and to differentiate bacteria based on their hemolytic properties
Some general proposed medium may give extra information for differentiating the bacteria
Routine samples are cultured on three different solid media:
1. general purpose or enriched medium
2. differential and selective medium for gram -
3. differential and selective medium for gram +
Incubation:
Most pathogens grow at 37°C (mesophiles) Incubation time range from 18h-48h
Primary culture:
Mixed culture, with pathogen and microbiota (so not enough of what we need -> pathogen)
2nd incubation and then:
2nd culture:
May have the pure pathogenic bacteria
CULTURE OF FASTIDIOUS ORGANISM:
chocolate agar (lyse black? hemolysis: describe them?
chopped meat broth
atmospheric jar. gas converted is added to change the composition of the atmosphere.
anaerobic chamber to work and incubate “strict anaerobic bacteria”
incubator with controlled CO2%
- chocolate agar or chocolate blood agar is a non-selective, enriched growth medium used for isolation of pathogenic bacteria. it is a variant of the blood agar plate, containing RBCs that have been lysed by slowly heating to 80 C
- chopped meat medium will support the growth of most non-sporeforming and spore-forming anaerobes associated with human and animal infections. may also be used for holding media for stock cultures.
- anaerobic jars are used to provide an oxygen-free atmosphere for work with oxygen-sensitive materials.
IDENTIFICATION
2nd culture:
May have the pure pathogenic bacteria
2nd culture:
? characterization
? characterization (MALDI-TOF)
? characterization (GC)
?
? PCR
? antibody staining
? (ELISA)
Monoclonal antibodies for ? for detection and/or identification
Goals: ? & Antigen detection
IDENTIFICATION
2nd culture:
May have the pure pathogenic bacteria
2nd culture:
biochemical characterization
proteomic characterization (MALDI-TOF)
fatty acid characterization (GC)
PCR (for DNA)
Real-time PCR
fluoroscent antibody staining
Enzyme-linked immunosorbent assay (ELISA)
agglutination tests (antibodies cause agglutination, clumping of pathogens!)
Enzyme-linked immunosorbent assay (ELISA): common use is to detect and measure antibodies (mostly diagnose virus infection esp of blood-borne viruses like HIV
real time PCR or quantitative PCR is a PCR-based technique that couples the amplification of a DNA sequence with quantification of the concentration of that DNA species in the reaction.
Monoclonal antibodies for antigen-recognition for detection and/or identification
Goals: SEROTYPING & Antigen detection
available in many places are proteomics! -> have laser to destroy bacteria and what is released are ribosomes so that ribosomes get to detector and time of it reaching the detector is MALDI-TOF
ribosomes are used for phylogeny as they are rlly concerned?;
some people use FATTY ACID CHARACTERIZATION)
Out of 100% only #%? are culturable.
The term ‘ ? ’ is used to describe bacteria that are not grown on artificial media till date
Reasons y we can’t culture them?
Out of 100% only 1% are culturable.
The term ‘ unculturable ’ is used to describe bacteria that are not grown on artificial media till date
Reasons y we can’t culture them:
- other stresses
- siderophores not working properly
- common nutrients not present
- other growth factors absent
Detection of host immune response
- Serology: detection of ? immunity
- Detection of ? immunity
animal that is primarily exposed to pathogen illicit diff. types of immune response (depend on ? type)
MATURE HELPER T CELL (Th1 or Th2):
- involve “immature CD#? T cell” involved w/ antigen-presenting cell
- If it’s usually a bacteria that was phagocytes by antigen-presenting cell e.g. ? cell or ? then pathogens gets destroyed in small pieces and those pieces will present it’s antigen to the immune system (antigens are present on surface of pathogens), and then many different T cells will come.
- So one that is completely complementary to that antigen, will bind to it. Then more T cells of that same type will arrive at the same site and elicit two different kinds of response.
- Th1: One is ? which means antibody response mainly
- Th2: more ?
MATURE CYTOTOXIC T CELL: (usually ?), if it is a virus, there is another kind of receptor that the antigen present you can use.
The “CD#? T cell”, which is a ? cell, get excited to start to kill anyone who looks like the ?.
notes: immature CD8 KILLS ALL PATHOGEN RELATED STUFFS (rutheless) -> mature cytoxic T cell
CD4T cell with antigen presenting cell -> Mature helper T cell (Th1 or Th2)
Detection of host immune response
- Serology: detection of humoral immunity
- Detection of cell-mediated immunity
animal that is primarily exposed to pathogen illicit diff. types of immune response (depend on ? type)
MATURE HELPER T CELL (Th1 or Th2):
- “immature CD4 T cell” involved w/ antigen-presenting cell
- If it’s usually a bacteria that was phagocyteD by antigen-presenting cell e.g. by dendritic cell or macrophage then gets destroyed in small pieces and those pieces will present it to the immune system (those antigens present on surface of pathogens), And then many different T cells will come and.
- So one that is completely complementary to that antigen, will bind to it. Then more T cells of that same type will arrive at the same site and elicit two different kinds of response.
- Th1: One is humoral which means antibody response mainly
- Th2: more cell mediated
MATURE CYTOTOXIC T CELL: (usually viruses), if it is a virus, there is another kind of receptor that the antigen present you can use.
The “CD8 T cell”, which is a cytotoxic cell, get excited to start to kill anyone who looks like the pathogen.
notes: immature CD8 KILLS EVERYONE (rutheless) -> mature cytoxic T cell
CD4T cell with antigen presenting cell -> Mature helper T cell (Th1 or Th2)
1st pic
So the bacteria can enter, let’s call it port of entry -> Could be the respiratory tract, the gastrointestinal tract, the skin, etc.
So it gets to the lungs and can undergo ? or taken by a dendritic cell which will move his way to the ? where all the T cells are.
INNATE IMMUNE RESPONSE: Then naive T cell (adaptive immune response) and the antigen presenting cell with MHCII will come apart. And then what I just mentioned before,
2nd route in pic: also the bacteria can be more invasive.
And through the port of entry can go to the lymph nodes and undergo phagocytosis by a ? cell, and then the B cell can present it to the T cell (CD#? T cell)
So the bacteria can enter, let’s call it port of entry -> Could be the respiratory tract, the gastrointestinal tract, the skin, etc.
So it gets to the lungs and can undergo phagocytosis or taken by a dendritic cell which will move his way to the lymph node where all the T cells are.
Then naive T cell (adaptive immune response) and the antigen presenting cell with MHCII will come apart. And then what I just mentioned before, (CD4 T cells are MHC-II restricted and pre-programmed for helper functions; CD4 T lymphocytes help coordinate the immune response by stimulating other immune cells such as macrophages, B lymphocyte (B cells) whereas CD8 T cells are MHC I-restricted and pre-programmed for cytotoxic functions; CD8 T lymphocytes also helps fight infection; HIV weakens the immune system by destroying CD4 cells)
2nd route in pic: also the bacteria can be more invasive.
And through the port of entry can go to the lymph nodes and undergo phagocytosis by a B cell, and then the B cell can present it to the T cell (CD4 T cell)
2nd pic
Again, the response. Once the T cell, he got excited because he’s activated
He does two things.
1. production of ? to call the B cells and ?,
- and the other one is to ? itself.
So Its type will be more effective.
- So he calls the ? cell which transfers to ? cells
which is just a B cell producing plenty of antibodies inside and then ultimately releases its antibodies to kill the enemy.
Okay, then memory happens while few of them will become ?
second time will be the response will be faster. and then the 2nd time the response will be faster.
we can check antibodies was produced: That will be evidence that the anti pathogen was there in the host or also the cellular reaction.
OR
also the cellular reaction for instance the macrophages; what is calling the macrophages
2nd pic
Again, the response. Once the T cell, he got excited because he’s activated
He does two things.
1. production of cytokines to call the B cells and macrophages,
- and the other one is to clone itself.
So Its type will be more effective.
- So he calls the B cell which transfers to plasma cells
which is just a B cell producing plenty of antibodies inside and then ultimately releases its antibodies to kill the enemy.
Okay, then memory happens while few of them will become t memory second time will be the response will be faster. and then the 2nd time the response will be faster..
SEROLOGY
- Collection of 2 serum samples with 2 weeks apart and a four- fold rise of antibody titer are indicative for recent exposure to an infectious agent (SEROCONVERSION)
[Does the first one. Then there is recombination, changes on that, on that plasma cell and then become immunoglobulin G or
primary infection: igM
- in acute infection more igM than igG
secondary infection: v little igM produced and more of igG
Thus, after long time (sample 2) more igG detected and after looong time (sample 3) then ONLY igG found!
from the graph we can say that pathogen was exposed to it and immune response was developed.
how recent it was we don’t know as we don’t have any evidence of the primary igM
** Seroconversion: when level of igG changes significantly. Thus evidence of seroconversion means that our first sample was in the acute phase **
If we instead took only a sample two and three, then no seroconversion so can say no acute infection so for example can say that the animal was exposed to it LONG time ago as no acute infection detected!]
Then there is this dendritic type of cell (langerhans cell) presenting the antigen to a T cell.
One of the things that this cell is producing is interferon gamma
This calls macrophages which will come there and try to capture any other bacteria that are in the tissue.
And the macrophage itself will produce some cytokines which will call all their macrophages.
Their macrophage, which is in the bloodstream, is called monocytes.
The monocytes will enter to the very, very old tissue or the tissue affected, and then they will become activated as a macrophage.
You will start to eat anything because he wants to capture the pathogen that is inside. He received the alarm and the alarm says there is a pathogen here so come as many as u can
then that calls the other Th cells and then this a cycle of immunity. And this is the cellular version!
DELAY TYPE OF SENSITIVITY: after second exposure all the cells are more sensitive -> so will produce more cytokines and many more macrophages come leading to an inflammation on that site: only some cells are able to produce cell-meditated response
Cellular mediated response. Some of them again use listeria or Mycobacterium avium Bobbie in cattle.
And then we have an inflammation on the site. Of course, only when the animal was exposed before to the pathogen. Not the primary infection. And this is what happened.
This is an illustration of inflame swelling tissue or skin.
DELAY TYPE OF SENSITIVITY: after second exposure all the cells are more sensitive -> so will produce more cytokines and many more macrophages come leading to an inflammation on that site: only some cells are able to produce cell-meditated response
Cellular mediated response. Some of them again use listeria or Mycobacterium avium Bobbie in cattle.
And then we have an inflammation on the site. Of course, only when the animal was exposed before to the pathogen. Not the primary infection. And this is what happened.
This is an illustration of inflamed swelling tissue or skin.
So we take an animal and we see if he was exposed to Mycobacterium how we do that, we cut a little bit and we put an antigen of the mycobacterium.
If there is a cell mediated response hypersensitivity response,
then we will have a swelling swell in an inflamed area 48 to 72 hours after very fast.
TUBERCULIN SKIN TEST
* Testing for bovine tuberculosis (Mycobacterium bovis)
* Measure of cell-mediated immunity (delayed hypersensitivity)
* Obligatory in the EU
Procedure
* Day 1: two sites are clipped, and tuberculin injected (M. bovis and M. avium antigens)
* Day 4: measure skin reaction ➔ if bovine site > 4 mm = animal is reactor
accumulation of macrophages in 3rd pic
what is happening there is we introduce a protein from Mycobacterium.
And then there is an inflammation dermatitis after three days.
And this is the th1 cells reacting in the late type 48 to 72 hours.
And it’s called type four hypersensitivity reaction.
Not all of the bacterial causes, okay, is just part of it.
Then we can also measure the production of that ?.
? is another reaction that will mediate a cell’s reaction.
what do they do?
IFN gamma:
IL-1 and TNF-alpha:
IL-12:
accumulated macrophages release ? and ? to remove ?
however in infections like TUBERCULOSIS, despite inflammation the offending agents still persist and this leads to accumulating macrophages caused by the activation of macrophages through Interferon (IFN)-gamma and these accumulated macrophages turn into epitheloid cells (Langerhans, foreign body giant cell) surrounded by fibrosis, lymphocytes and this is called GRANULOMA
So it’s a cytokine i.e., interferon GAMMA we can measure and there are some tests where we can put ? cells isolated in the lab and expose it to the serum of this animal and see how much ? is there - another way to identify it -> its slower or faster?
Then we can also measure the production of that interferon.
Gamma is another reaction that will mediate a cell’s reaction.
what do they do?
IFN gamma: will activate macrophages (thus better antigen presenting cells, better MHC II expression, better phagocytosis
IL-1 and TNF-alpha: they cause leukocytes (macrophages in nearby blood vessels: monocytes) to leak out of blood vessels
IL-12: activation of more Th 1 cells (cell-mediated immune response, intracellular bacteria and virus)
accumulated macrophages release ? and ? to remove ?
So it’s a cytokine we can measure and there are some tests where we can put lymphoid cells isolated in the lab and expose it to the serum of this animal and see how much interferon-gamma is there - another way to identify it -> its faster. You don’t have to wait two days or three days thats the advantage.
