1
Q
What makes the cell wall of bacteria so special?
A
- chemically fairly inert
- composed of subunits found nowhere else in nature (good targets for antibacterial therapy0
- Made up of peptidoglycan
2
Q
Provide examples of gram positive and gram negative bacteria
A
-
3
Q
What feature of gram negative cell wall absent from positive cell wall?
A
Producing symptoms of disease via endotoxins
4
Q
What is the composition of the gram negative cell wall?
A
lipopolysaccharides and peptidoglycan
5
Q
How does the gram stain differentiate gram positive and gram negative?
A
- In gram positive, the low lipid concentration is important for the retention of the iodine crystal violet complex, thus the cells remain blue
- In gram negative, the high lipid Concentration found in the outer layers of the cell wall is dissolved which facilitates the release of the iodine crystal Violet complex leaving the cell colourless
6
Q
Why do we need to differentiate whether the bacteria is gram+/-?
A
Because they will respond differently to chemotherapeutic antibiotics.
7
Q
What is the primary stain in gram staining?
A
Crystal violet, used to identify the type of cell wall of the bacteria which will be determined in the decolorizing step, whereby if it loses the crystal violet complex it means that it its negative and if it doesn’t it is gram positive
8
Q
What is the decolorizing agent in the gram stain?
A
Alcohol, particularly ethanol 95%
9
Q
What is the counter-stain used for in gram staining?
A
As it is difficult to see colorless cells under the microscope, therefore, a counter-stain, usually safranin or red dye is used to color the bacteria, thereby negative becomes red and positive purple.
10
Q
What is the cell wall peptidoglycan made up of?
A
- Two major amino groups (N-acetyl muramic acid/N-acetyl glucosamine) that alternate to form a high weight polymer and a chain of several amino acids attached to each of the NAM molecules
11
Q
What is the function of the penicillin binding proteins?
A
Polymerize and modify peptidoglycan, the stress bearing component of the bacterial cell wall, in other words creates the morphology of the peptidoglycan exoskeleton together with cytoskeleton proteins that regulate septum formation and cell shape
12
Q
What antibiotics target the peptidoglycan?
A
Beta lactams, including penicillins, cephalosporins, monobactams, penems, carbapenems, beta lactamase inhibitors
13
Q
What are the classes of antibiotics?
A
- Intracellular
- Glycopeptides
- Beta lactams (pbp inhibitors)
14
Q
What is the mechanism of action of intracellular antibiotics?
A
Prevent the formation of the peptidoglycan inside the cell
15
Q
What is the mechanism of action of glycopeptide antibiotics?
A
Inhibit synthesis of cell wall peptidoglycan and inhibit bacterial cell membrane permeability
16
Q
What is the chemical composition of the plasma membrane ?(cytoplasmic membrane)
A
- 40% lipids (phospholipids)
- 60% proteins and some carbs
17
Q
The cytoplasmic membrane is a semi-permeable membrane, elaborate:
A
Only low molecular weight materials can penetrate to the inside of the cell, it acts as an effective permeability barrier of the cell regulating the inflow and outflow of metabolites to and from the protoplast
18
Q
The cytoplasmic membrane contains many proteins and enzymes essential for bacterium survival, provide one example of that:
A
The proton motive force that generates adenosine triphosphate (ATP) (cellular respiration)
19
Q
How are bacterial cells similar to mammalian cells?
A
- In terms of having to generate ATP
- Chemical composition of cytoplasmic membrane 40% lipids and 60% protein/carb
20
Q
Embedded in the cytoplasmic membrane, are efflux proteins, what is their function and what types are there? (Vomit pumps)
A
- System that enable anions and cations, or chemicals in and out of the cells
- Get rid of antibiotics and unwanted material
21
Q
what types of efflux pumps are there?
A
1- MATE family (Gram positive)
2- MFS (Gram positive)
3- SMR family (gram positive)
4- RND family (gram negative)
5- ABC super family (gram negative)
22
Q
Why is it that the more efflux pumps a bacteria has the more beneficial it is for them?
A
Because the role of the efflux pump is to pump out toxic and unwanted material, thereby ridding itself from damage and thus protecting the cell
23
Q
How are efflux proteins able to work with various types of substances?
A
They have loads of substrates that allow them to work with lots of types of chemicals
24
Q
What is difference between the ribosomes of bacteria and of eukaryotic cells?
A
- Bacterial ribosomes 70S: made up of (30S= 16S rRNA) and (50S= 23S rRNA + 5S rRNA)
- Eukaryotic ribosomes 80S:
25
What is the S in ribosomes?
unit of centrifugation
26
What is the function of the ribosomes?
Acts as a protein factory
27
Describe the chromosome of a bacterial cell
- Closed circle in all bacteria not surrounded by a nuclear membrane
- Extrachromosomal DNA in the form of plasmid
28
What is the function of plasmids?
Play a role in the transfer of genetic material between bacteria (conjugation), contain antimicrobial resistance genes
29
What is the role of inclusion bodies in the bacterial cell?
Stores granules in the form of high molecular weight polymers. For example
- Glycogen: to store carbon and energy
- polymer of beta-hydroxybutyric acid to store carbon and energy
- Polymeric phosphate volutin: phosphate
- Protein crystals: insecticides in bacillus thuringiensis
30
Provide examples of antibiotics that target the ribosomes of bacteria
- Tetracyclines 30s
- Aminoglycosides 30s
- Chloramphenicol 50s
- Macrolides 50s
31
What is the function of flagella in bacteria?
Offers bacterial motility, as the flagella offers bacterial motility, this means that it can move in the body from part to part making it virulent.
32
What is the difference between pili and fimbrae?
Fimbrae are usually longer than pili
33
Describe the structure of pili/fimbrae
- Fine hair like surface filaments
- Smaller than flagella
- Consist of protein subunits wound around one another
- Their number varies from 1-400
34
Pili can be separated into a number of types based on their function, list the functions of pili
- Adherence: to foreign cells (epithelial/RBC)
- Antigenic: useful for identify in culture and for immune system to fight the infection
- Genetic exchange: conjugation of F or sex pili (exchanging gene info)
- Attachment sites for bacteriophages
- Chemotaxis: respond to different sensory stimulus (oxygen/nutrients)
- Virulence: toxins
35
How can growth of bacteria be beneficial to us?
- Production of proteins, nutrients, or products for human consumption (fermentation)
- Identification
- Criteria of sterile dosage forms
36
what does CFU stand for?
colony forming units
37
How do bacteria divide?
Binary fission, increase of cell mass, duplication of genome, and cell membrane and wall separation producing identical daughter cells
1) Cell elongates and DNA is replicated
2) Cell wall and plasma membrane divide
3) Cross-wall forms around divided DNA
4) Cells separate
38
describe the phases of bacterial growth
39
At which phase is bacterial growth population measured?
Exponential growth phase, which is expressed as generation time or doubling time of bacterial population
40
How many cells are produced in binary fission from one bacterial cell?
2 daughter cells, and the increase in population is by geometric progression 1 2 4 8 16
41
What are the benefits of bioreactors?
- Control growth rate
- Optimised production
- Continuous fermentation
- Batch fermentation
42
Where do bacteria obtain their nitrogen source from?
- Atmospheric nitrogen
- Inorganic nitrogen from nitrate, nitrite, ammonium salts
- Organic compounds from amino acids
43
What are the chemical requirements for growth of bacteria?
- Energy: light and chemicals
- Electrons: reduced inorganic and organic compounds
- Carbon: from carbon dioxide and organic compounds
- Nitrogen: nitrate, nitrite, ammonium salts, amino acids
- Oxygen/sulfur/phosphorus
44
What are the physical requirements for the growth of bacteria?
- Temperature that differs based on the type of the organism but most pathogenic organisms are mesophiles so growth temp requirement is 25-40C
- pH optimum is 6.5-7.5
45
Thiobacillus thiooxidans grow best at a pH of 2-3.5 whereas alkaline spring isolates grow best at 9-9.5
keep that in mind
46
In terms of temperature, how can you suppress the growth of certain microorganisms?
- Deep freeze -20C for parenteral nutrition and raw materials
- 8-12C for reconstitued syrup and multi dose eyedrops
- 80C for WFI (regrowth of gram negative and release of toxins)
47
In terms of pH how can you suppress the growth of certain microorganisms?
- Extreme pH to suppress microbial growth
- Neutral pH for bacterial spoilage (antacid mixture/flavoured mouthwash)
- PH > 8 spoilage is rare
- Low pH spoilage by moulds and yeast
48
In terms of water activity, how can you limit the growth of microbes?
the greater the solute concentration the lower the water activity and the lower the Aw bacteria will be prevented from growing as they grow best in dilute solutions
49
Why is it that for sore throat syrup BP & tablets, doesn’t need additional antimicrobial?
Because for this particular formulation, the water activity is at 0.86, this means that it would unlikely to be spoiled by bacteria or fungus (self-preserving)
50
Give examples of non-sterile pharmaceutical products
- Topical use or resp use
- Oral or rectal administration
- Raw material, natural origin
- Herbal remedies
51
What are the viable count methods for bacteria?
- Colony count: single bacterium In the original culture being plated is assumed to give rise to a single viable colony, The type of medium used and conditions of incubation will alter bacterial growth and the formation of colonies.
- Plate count methods
52
What are the methods of plate count?
- Pour plate
- Spread plate
53
What is the difference between pour plate and spread plate?
The main difference between pour plate and spread plate is that the molten agar is poured onto the inoculum during the preparation of the pour plate, whereas the inoculum is spread on the surface of the solidified agar during preparation of the spread plate
54
What is membrane filtration used for?
It is used to determine the viable count, using a 0.45 mcg membrane filter of either cellulose nitrate for aqueous, oily and weekly alcoholic solutions or cellulose acetate for strongly alcoholic solutions, disallowing bacteria from passing through the filter
55
What is the least reliable enumeration methods?
Most probable number methods (MPN)
56
Provide examples of total count methods
- Cell count: microscopy or electronic particle counter
- Cell mass: weighing, measurement of nitrogen, measurement of dry weight
- Spectrophotometric measurement: measurement of turbidity (cloudiness) optical density
- Cell activity: relating degree of biochemical activity to size of the population
57
Give examples of Rapid Microbiology Methods (RMM)
- Growth-based: detectable signal by period of subculture
- Direct measurement: individual cells are differentiated and visualised
- Cell component analysis: Expression of specific cell components offers an indirect measure of microbial presence
58
In summary, what enumeration types are there?
- Viable count
- Total count
- RMM
59
How does contamination appear in emulsions and parenteral nutrition?
as Phase separation
60
What are the classes of life proposed by Carl Woese
Prokaryotes: bacteria/archaea
Eukaryotes: eukarya
61
Classes of life are classed based on which nucleic acid?
Ribosomal RNA (16S rRNA), as it is present in all living cells and conserved meaning it hasn’t evolved at all, and because of its specificity to bacteria
62
How do you write the name of bacteria?
genus followed by species in italics, capital initial letter for the genus and no capital on the initial letter of the species
63
What are the traditional and new bacterial identification methods?
64
What does pure culture tell you?
- Isolate a single colony
- Colony morphology
- Growth requirement (temp/oxygen requirement/salt requirement)
65
How would you promote the growth of a particular microorganism?
- By using selective media, as it inhibits other bacteria and promotes the growth of target bacteria with the development of indicative color (agar,colony), there are also chemical indicators to identify the use of sugars
For instance, for the growth of gram negative, bile salts and crystal violet inhibit most gram positive bacteria, and distinguish lactose fermentors such as E.coli and Klebsiella: in pink and Styphimurium and P auriginosa in white colonies
66
What is MacConkey agar used for?
Selective media to promote the growth of gram negative bacteria such as E.coli and Klebsiella, in addition, it contains lactose which helps in distinguishing lactose fermentors that produce acids forming pink colonies whereas the ones that arent fermentors remain colorless/white
67
What is Vogel Johnson agar used for?
Selective media to promote the growth of staphylococcus aures (gram positive), it contains tellurite and lithium chloride. S.aures are able to coagulase tellurite and reduce it to metallic tellurium which shows as black colonies, manitol sugar is used by s.aures to ferment thus altering the pH forming a yellow halo around the colonies
68
What is API test and what is it used for?
Test strips, a type of biochemical profiling, used for the identification of microorganisms based on growth requirement and enzymatic activity
69
What are the pros and cons of biochemical profiling
70
How does serological testing work?
Uses highly specific antibody-antigen interactions to detect certain pathogens
71
What is one type of serological testing?
Enzyme linked immunosorbent assay (ELISA), a highly sensitive test that amplifies signals by conjugate enzymes and very quantitive (linked to the concentration of antibodies) whereby the intensity of the color depends on antibody interaction
72
What are the pros and cons of serological testing?
73
Provide an example of nucleic acid techniques test
Polymerase chain reaction (PCR) test, these allow the amplification of a known gene of interest for nucleic acid sequencing, in particular via rRNA sequencing that provides a very accurate identification of the genus or species
74
How does a genome sequencing test work?
Gene sequence is compared with online database from National center for biotechnology information (NCBI), with BLAST tool that finds regions of similarity between your sequence and those identified previously
75
What are the pros and cons of nucleic acid techniques?
76
How do microarrays work in bacterial identification?
Thousands of ssDNA probes are fixed to a solid support chip, probes could be rRNA or antibiotic resistance genes, Labelled fluorescent target sample is hybridised with probes.positive March shows as a bright dot
77
What is the significance of whole genome sequencing?
- Bacterial identification
- Information about metabolism and growth requirements and pathogenicity
- Vast amounts of data coming out (time consuming analysis)
- Requires expensive equipment and reagents
- Most expensive than other methods
78
What does MALDI-TOF MS stand for?
Matrix assisted laser desorption ionisation time of flight mass spectrometry
79
How does MALDI-TOF MS work?
- Sample of microorganism, in pure culture
- Bombarded with laser pulses
- Sample is ionised and passed through electrostatic field
- Ions pass through flight tube before hitting detector and small ions travel faster
- Generates a unique mass spectrum of different bacter (database checking)
80
what are the pros and cons of traditional and newer methods?
81
define infectious disease
also known as a transmissible disease or communicable disease, is an illness resulting from an infection
82
What are the determinant factors of infectious disease?
- Direct and indirect transmission
- Adherence or colonization of host
- Multiply or complete life cycle in/on the host cells
- Evade host defence mechanism
- Possess mechanical, chemical, or molecular ability to damage host (production of symptoms)
83
What are the routes of infection, spread and transmission of disease?
- Contaminated water: conjunctive/ear infection/gi infection/wound
- Contaminated aerosol: conjunctiva/upper and lower resp infection
84
Provide examples of opportunstic pathogens
- Staphylococcus aureus
- Escherichia coli
- Proteus vulgaris
- Candida albicans
85
Provide examples of bacteria that can make you asymptomatic carrier
- Methicillin resistant staphylococcus aureus
- Clostridium difficile
86
Explain the concept of an asymptomatic carrier
Bacteria that are not part of the normal flora but present in a dormant non infectious form until certain conditions allow it to flourish causing diseases such CDAD from the clostridium dificile bacteria. (Can be transmitted to susceptible patients and thus automatically cause disease)
87
Why is MRSA difficult to treat?
Because not only is it resistant to methicillin but it is also resistant to a wide range of antibiotics
88
What are the virulence factors of E.Coli?
- Adhesin, a colonization factor linked to pili that enable the pathogen to remain attached cell causing UTI, making them persist in the urethra despite urinary flushing
- Endotoxin, lipopolysaccharide, particularly pyrogen
- Exotoxin, heat labile LT enterotoxin, allows invasion and multiplication in host cells
- Invasin, uropathogenic E.coli, allows invasion of the cell
- Capsule: structure that enables them to evade phagocytosis
89
Compare and contrast exotoxins and endotoxins
90
What is the mechanism of action of shigella dysenteriae exotoxin?
Produces shiga toxin C that causes diarrhea, it disturbs protein synthesis machinery in the host. It is encoded in a gene which can be transferred to other bacteria. Thus you can have Ecoli for instance expressing that toxin
91
What is endotoxin?
Gram negative bacterial cell wall’s outer layer is made up of lipopolysaccharides which are released upon cell death/lysis into the blood stream. LPS is composed of lipid A, core polysaccharide, and o-specific side chain. The latter allows identification bacteria through antigen-antibody reaction because it is highly antigenic.
92
What portion of the lipopolysaccharides causes most of the damage?
Lipid A, as it is highly pyrogenic. It shows as a rise in body temperature
93
What are the different names of the capsule?
- Slime layer
- Glycocalyx
94
What is the function of the capsule?
- Virulence, in that it prevents phagocytosis by host immune cells (macrophages)
- Protection against desiccation as it contains water, it also excludes bacterial viruses and most hydrophobic toxic materials such as detergents
95
What is meant by pathogenecity islands of bacteria?
Regions of bacterial genome where virulence genes can be found such as (plasmid and chromosomes).
96
What is the function of pathogenicity islands (plasmids/chromosomes)?
- Export of virulence factors directly into cells
- Confer virulence traits such as cell adherence, cell entry and toxins
- Provides sudden radical changes in bacterial-host interactions
97
What classes of antimicrobials are there?
- Antibacterial or antibiotics
- Anti-fungal
- Anti-viral
- Anti-protozoal
- Anti-Helmintics
98
What antibiotics have no sub-classes?
- Chloramphenicol
- Polypeptides
- Glycopeptides
99
What factors determine the antibiotic of choice?
- Toxicity
- Side effects to ensure compliance
- Rapid action
- Resistance
- Cost and duration
- Route of administration oral preferably started first
- Efficacy, bacteriostatic or bactericidal
100
higher the therapeutic index the more effective and less toxic the antibiotic
Ti=td/ed
keep it in ur 1-left-braincell
101
list 5 antibiotics safe in penicillin allergy
1- amikacin
2- chloramphenicol
3- azithromycin
4- nitrofurantoin
5- clindamycin
102
give examples of antibiotics with low risk of CDAD
amikacin
azithromycin
metronidazole
trimethoprim
103
What is the difference between intrinsic resistance and acquired resistance?
PH1123 Micro
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