Bacteria identification Flashcards
Why is identification of microorganisms important?
If we can identify the pathogen, we can provide patient with an effective treatment for the infection caused by that microorganism. If pathogen is not identified, it will be more difficult to provide an effective treatment. In a product, it is important to identify the contaminant as there are some microorganisms which can’t be contained in a product.
What do we need to know to be able to combat an infection in a patient?
To be able to combat an infection , we need to know which microorganism causes the infection. THEREFORE IDENTIFICATION IS IMPORTANT.
What information does the BP give us regarding microorganisms?
BP gives us an idea of what methods we use to identify microorganisms and also about which microorganisms we don’t want in the pharmaceutical product.
Which 4 categories are used to measure microbiological quality of pharmaceutical products?
- preparations required to be sterile on the dosage form = Sterile products (the products injected into patients so injections, vaccines, total parental nutrition)
- Preparations for topical use and for use in the respiratory tract (you can’t have that many microorganisms in the respiratory tract)
- Preparations for oral and rectal administration
- herbal remedies (you can find contaminants in them but you can add boiling water)
Which pharmaceutical products have to be sterile and what does sterile mean?
Sterile= absence of microorganisms
You can’t have any microorganisms in TPN, vaccines and injections
Classification of bacteria
Classification of bacteria is more difficult than for eukaryotic organisms
Definition of eukaryotic organisms
For eukaryotic organisms(humans, protozoa) a species is defined as a group of closely related organisms, which reproduce sexually to produce fertile offspring
Difference bewteen bacteria and eukaryotes
Bacteria do not reproduce sexually whereas eukaryotes reproduce sexually
Where is Staphylococcus aureus found?
skin resident, found on the skin flora; can cause cellulitis, folliculitis, impetigo,boil
Where is Clostridium spp found?
foot and it can cause foot infection which can lead you to lose your foot
How does bacteria classification work?
- Based on the nucleotide sequence of rRNA (ribosomal RNA). So if we look at the nucleotide sequence of ribosomal RNA, we can identify a specific organism
- rRNA (RNA component of ribosome) is present in every cell; ribosomes are present in all living cells as they are required to build proteins in every cell (cells of plant, human, bacteria BUT NOT VIRUS)
- rRNA sequence forms the basis for phylogeny (=study of evolution of organisms)
How many domains are there and name them?
There are 3 domains of life: - Bacteria (Prokaryotes) -Archaea (Prokaryotes) -Eukarya (eukaryotes) They all have a common ancestor
Write the classification for S. aureus
Domain: Bacteria Kingdom: Bacteria Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Staphylococcaceae Genus: Staphylococcus Species:aureus
Describe Bacterial nomenclature
-Bacteria can be named after a person, most of the time the scientist who discovered it For example: -Salmonella:salmon -Escherichia:Escherich -Neisseria:Neisser - Bacteria can have a name which describes the bacterium itself: - Bacillus: rod in latin -Staphylo:grape -coccus: berry -aureus: gold colored
Methods for bacterial identification
- Cultivation and growth requirement on plate (simple and traditional)
- Cultivation on selective agar (traditional)
- BIochemical profiling (traditional)
- Serological testing (traditional) (rapid identification)
- Nucleic acid techniques (newer) (rapid identification)
- MALDI_TOFF (complex) (newer) (rapid identification)
Describe cultivation as a method for bacterial identification
- Some bacteria are grown in the laboratory and then you can look at them under the microscope
- Cultivation allows morphological characterization
- Electron microscopy is used to identify bacteria but it’s limited in terms of distinction (especially between similar looking bacteria)
Cultivation as a method for identification -evaluate
Morphology alone cannot distinguish between similar looking bacteria. It helps distinguish between different types of bacteria but it doesn’t give a name for it
Features to look for in bacteria when using cultivation as a method of identification
- Spore production, flagella ( this enables bacteria to move around -bacteria which can move around are called motile bacteria)
- Shape (cocci=spherical shape, bacilli=rod shapes)
- Staining characteristics (GRAM STAIN)
- Colony morphology (the colony formed by bacteria when they grow in rich media, they divide until there’s so any of them that you can see them with your eye)
- Oxygen requirements: aerobic/anaerobic/facultative anaerobe
- Temperature requirements-different types of microorganisms grow at different temperatures -(temperature for pathogens is 37 degrees Celsius -body temperature; Bacteria on fish will grow at low temperatures -in the fridge- fish will fluoresce
- salt tolerance
- Requirement for specific nutrients (aminoacids/sugars/sources of carbohydrates)
Describe Biochemical Profiling as a method of bacterial identification
- Bacteria can be identified based on their enzymatic activity (there is a specific enzyme for a specific type of bacteria)
- These tests can separate closely related microorganisms (this is due to the unique biochemical characteristics bacteria have)
-These tests can be performed in the traditional laboratory tests or in the form of kits
-This method requires a pure culture (= a colony made up of 1 type of microorganism). When you have the pure culture, you can resuspend bacteria - This method can help distinguish at a species level
Ability of bacteria is tested for they and localize it, we incubate it and when they grow in the media they form different colors
-Fermenting various sugars: Some bacteria can ferment a wide range of sugars, others are more restricted (for example if a bacteria is able to ferment lactose, it grows pink on the MacConkey agar = E coli bacteria) - Does the bacteria produce the enzyme oxidase? If it does, this shows the activity of cytochrome oxidase. This is done in a dehydrated media
- Hydrogen sulphide production (bacteria produce H2S which will react with iron salts to form a black precipitate
-These are unique characteristics of bacteria and if you put them together (API test strips) you can identify what bacteria you have (i.e build a biochemical profile of bacterium) - Color corresponds to growth or no growth
Describe bacterial identification using selective media
Vogel Johnson Agar
-used to detect coagulase positive and manitol fermenting strains of S.aureus in clinical specimes
- Tellurite and lithium chloride (selective)
-Tellurite - Coagulase reduces it to metallic tellurium -black colonies formed on the surface of the agar
- Manitol degradation alters pH-yellow halo
- Indicates S.aureus
MacConkey agar
-selective and differential medium designed to isolate and differentiate bacteria based on their ability to ferment lactose
- Growth of gram -ve bacteria (bile salts and crystal violet inhibit most gram positive bacteria)
-helps distinguish lactose fermentors: E.coli, Klebsiella form red/pink colonie, lac +ve; P.aeruginosa and S.typhimurium form white colonies, lac-ve
What are the positives and negatives for cultivation/biochemical profiling/ Selective media
Positives:
-easy to use
- can be automated
-doesn’t require specialized equipment
Negatives:
- miss-reading of the results (because you have to interpret color formation, sometimes it’s difficult to interpret the results which can lead to misidentification)
-kits can be expensive
-time consuming (it takes approximately 18-72 hours of incubation so 18=72 hours to get an answer)
-Not all bacteria can be cultivated (i.e grow on the agar) under normal laboratory conditions (as little as 0.1%)
Where do most pathogens grow?
Most pathogens grow on agar
Describe serological testing as a method for bacterial identification
- it uses highly specific antibody (Ab) antigen (Ag) interaction
- can be used to detect certain pathogens
- enzyme linked immunosorbent assay (ELISA) test
- if there is a marker on your antibody, you get a perfect identification of the pathogen
- you need a pure culture
positives and negatives of serological testing
POSITIVES
-ELISAs are available to test for E.coli, S.aureus, Salmonella and other organisms
-highly specific
-high sensitivity - signal is amplified by the conjugated enzyme
-very rapid
NEGATIVES
- expensive
-cannot identify unknown bacteria (can be used when you suspect a bacteria and you want to know exactly if you’re right i.e proof principle for confirming bacteria’s name)
-complex process (needs training)
Describe nucleic acid techniques as a method for bacteria identification
-PCR (Polymerase Chain Reaction) based techniques allow the amplification of a known gene of interest for nucleic acid sequencing
-sequencing of rRNA provides very accurate identification to genus or species level; DNA is unique for targetting bacteria
- can be used to identify unknown/uncultivable organisms
- not used to determine contaminant in pharmaceutical product because these contain odd organisms which are not in the database so you can’t design a DNA probe for them
-you don’t need a pure culture or to cultivate the microorganism
-you can take a direct sample from patient’s sputum rectal where there is a lot of flora
-Gene sequence is then compared with an online database NCBI- National center for Biotechnology Information)
-BLAST tool finds regions of similarity between your sequence and those identified previously
-it can also tell you the factors which enable bacteria to cause infection, metabolism and growth requirements, pathogenicity (=antibiotic resistance)
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Positives and Negatives of the nucleic acid techniques
POSITIVES
-Do not require the growth of bacteria for identification so you don’t need to cultivate the microorganism
-Very sensitive
- high accuracy
-if you have a sample with microorganisms, you can identify all microorganisms
NEGATIVES
- time consuming to prepare sample and also there is vast amount of data to analyse
- costly reagents although now nucleid acid techniques are becoming more cost effective
-specialised equipment
-only as good as your reference library: you can amplify DNA but if you don’t have that DNA in the library, you won’t know what microorganism you have.
Describe Microarrays as a method for bacterial identification
- thousands of ssDNA probes (rRNA or antibiotic resistant genes) are fixed to a solid support (chip)
- Labelled (fluorescent) target sample is hybridised with probes
- positive match shows as a bright dot
Describe MALDI-TOF MS as a method for bacterial identification
- unknown bacteria is mixed with matrix material
- mixture is then bombarded with laser pulses
- sample is then ionized and passed through electrostatic field (acceleration)
- ions pass thorough flight tube before hitting detector (smaller ions travel faster than large ones)
- generates a unique mass spectrum for different baceria-check against databse
Describe positives and negatives of MALDI-TOF MS
POSITIVE:
-COST EFFECTIVE (1.43 EUROS VS 4-8 EUROS PER SAMPLE)
-SHORT TURNAROUND TIMES (6-8 MIN VS 5-48 HOURS)
-PRECISE IDENTIFICATION TO SPECIES LEVEL
NEGATIVE:
-CAN’T IDENTIFY SPECIES IN MIXED BACTERIAL POPULATIONS
-RELIANT ON QUANTITY AND COVERAGE OF DATABASE USED
ADVANTAGES AND DISADVANTAGES OF TRADITIONAL METHODS VS NEWER METHODS
TRADITIONAL METHODS:
- NO SPECIALISED EQUIPMENT
- MINIMAL TRAINING
- 24-72 HOURS FOR RESULT
- CAN BE DONE IN MOST LABORATORIES
NEWER METHODS:
- SPECIALISED EQUIPMENT REQUIRED WHICH IS EXPENSIVE
- LOWER RUNNING COSTS (MALDI-TOF- MS)
- FASTER (APPROX 6 HOURS)
- HIGH THROUGHPUT (140000 SAMPLES PER YEAR)
