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1
Q

Typical Gram positive

A

Typical Gram-positive bacteria
1.

staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is a common cause of boils

2.

streptococci such as the many species of oral streptococci, Streptococcus pyogenes which causes many a sore throat and scarlet fever and Streptococcus pneumoniae which causes lobar pneumonia

3.

clostridia such as Clostridium tetani which cause tetanus (lockjaw)

4.

actinomyces such as Actinomyces odontolyticus which is found in mouths

5.

species of the genus Bacillus such as Bacillus subtilis which are common microbes living in soil

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2
Q

Typical gram negative bacteria

A

Typical Gram-negative bacteria
the bacilli that cause

1.

whooping cough, Bordetella pertussis

2.

typhoid, Salmonella typhi

3.

cholera, Vibrio cholerae

4.

the normally benign, ubiquitous, gut-dwelling Escherichia coli

Generally cocci are Gram-positive but there are exceptions. The most significant from a clinical point of view is the gonococcus, Neisseria gonorrhoea which typically appears as a Gram-negative diplococcus looking very much like a pair of kidney bean.

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3
Q

What are the major groups of microorganisms?

A 
Viruses
Mycoplasma
Rickettsia
Bacteria
Fungi
Protozoa
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4
Q

What are the differences between prokaryotic and eukaryotic microorganisms?

A

Prokaryotic: lack defined nucleus or membrane bound organelle
Eukaryotic:nuclear envelop + microtubules in cell devision

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5
Q

What is the difference between streptococcus and staphylococcus?

A

Staph: coccus in clusters
Strep: coccus in chain

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6
Q

What are the characterisitcs of a gram positive cell wall?

A

Thickness: 20-80nm thick
No outer membrane/capsule (which is resistant to Ax) - source of endotoxin
Provides structural integrity + transportation of nutrients via protein channels
Attach mechanisms to surfaces

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7
Q

What are the main structural components of a bacterial cell?

A

Cell wall
Outer membrane
Nucelus + ribosome + mitochondria + pilli + flagelle + cell membrane + cytoplasm

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8
Q

What is a bacterial spore?

A

From gram positive bacilli
complicated morphological and biochemical process (7 stages)
Result in thick walled, highly resistant endospore with low water content and metabolic activity
Improve Env factors - germinate and return to vegetative form

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9
Q

What are the main phases of microbial growth curve?

A 
Multiply by binary fission
lag phase
Log phase
Stationary phase
Death phase
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10
Q

Describe the process of carrying out Gram stain.

A 
Glass slide + culture - dried
Crystal violet (1min) - stain gram +ive purple
wash with DIW
Iodine (mordant) (1min)
Wash with DIW
Alcohol treatment (5-10sec)
wash with DIW
neutral red (1min) - stain gram -ive pink
wash with DIW
Dry
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11
Q

What are the key characteristics of common bacteria found in the pharmaceutical env?

A

Staphylococcus (staph. Epidermidis)
Human skin
Staph aureus less commonly found - nasal passage + wound infection + boils
Gram positive cocci
Bacillus
Widespread in the env: soil + water + cardboard, paper and wood
Spore forming
Gram positive bacili
Pseudomonas (Ps aeruginosa)
Water: drains and surface water + U bends of sinks + water sys deadlegs + bore hole water supply + Mop heads and disinfectant buckets + equip stored wet
Large number - musty smell
Sensitive to lack of moisture + temp over 45-50 dec
Other water source - acinetobacter, Achromobacter and enterobacter
Gram negative bacilli

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12
Q

What are the key characteristics of common fungi found in the pharmaceutical env?

A

Eukaryotic microorganisms
Cell wall composed of chitin - not peptidoglycan
Degradation of organic matter
Moulds vs yeasts
Moulds
Spore forming - thoudsands at a time - less resistant than bac spore
requires longer period of incubation + specialised method of staining
Cardboard shippers, exposed plaster and damaged pipework lagging
Yeasts
Large gram positive spheres/ovoids
moderately resistant spores - less resistant buds
require higher level of nutrition and moisture
Spoilage of product

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13
Q

What is a typical formula of a broth? What are limitations?

A 
Tryptone soya broth:
Tryptone
Soy peptone
NaCl
Dipotassium phosphate
Dextrose
Cannot give any indication of the range of organisms present
Does not give any indication of the number of organisms at the start of the growth
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14
Q

What are the different types of solid growth media?

A

General purpose: Tryptone Soy Agar
Enhanced media: Chocolate Blood Agar - Factor X+Y - Neisseria sp
Selective media: Cetrimide agar (contains Ax)
Differential media:Baird Parker (staph aureus and other) - colour change + MacConkey Agar (Bile salts)
Enrichment media: enhance nutrition - sub-lethally damaged

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15
Q

What are the different method of microbiological enumeration?

A

Miles misra Technique
Serial dilution until 20 -80 bacterial per 0.5mL
4 dilutions are plated and 4 drops per dilution
Useful when accurate count required
starting innoculum level unkown
Use a lot of materials
Pour plate technique
1mL sample pipetted into empty plate then pour media
1 to 500 bacteria per mL
dilution needed if more concentrated
high temp may kill bacteria
colonies may obscure
The Spread Plate Technique
0.1mL sample spread onto the surface of an agar plate
homogenate would obscure the count
10-100 cfu
higher count need furhter dilution
Most Probrable Number Technique
Serial dilution 1 in 10
sample innoculated into tubes containing nutrient broth
Turbitity is then used to calculate and compared with statistical tables
MPN cacluated
The Membrane Filtration Technique
Most widely used in Pharm industry
disolve sample in a neutral carrier liquid
pass the solution through a bacteria retentive membrane filter
Place membrane onto a nutrient agar
Bac retained forms visible cfu
Inhibitory aspects removed by filtration

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16
Q

What are the considerations when validating microbiological methods?

A

Product toxicity:
antimicrobial / bacteristatic qualties must be neutralised
neutralising agents dependant on nature of the inhibition
membrane filtration adv - common to include a neutralising rinse as part of the test
Contamination recovery:
Innoculate with known numbers of a range of bacteria
Compare results with innoculant level
Assess neutralisation efficacy as well as contamination recovery
Operator validation
replicate sample testings to evaluate operator consistency
Paired operator sample testing
External QA systems

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17
Q

What are the automated methods for identification?

A 
Substrate ultilisation:
Reduce medium volume 
Enhancing sensitivity of result detection
Need pure culture to give good results
i.e. Biolog
Gas chromatography of fatty acids
discrimination between organisms on the basis of their fatty acid content as measured by GC
i.e. MIDI MIS
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18
Q

What are the types of rapid methods for detection?

A

detect specific organisms in mixed cultures
PCR based methods
Applies to DNA and RNA
Amplified nucleic acid by PCR - detected using labelled cpmlementary DNA probes
Target: chromosomal DNA and Ribosomal RNA
Adv: Specific + sensitive + quick
Weak: not robust + expensive

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19
Q

What are the main types of rapid enumeration methods?

A 
ATP Bioluminescence
Use enzyme luciferase - uses energy of ATP to produce light
Sesitivity: 10^3 /mL
seconds to get results
DEFT
Direct epifluorescence filtration testing
filtrate through a black filter
stain organisms with fluorescent stain
Can't tell if it was dead or alive
Chemiluminescence
Absorption of fluorochrome by organsims
detect by laser scanning analysis
Impedence
Measure impedence in medium due to increase in microorganism numbers
20
Q

What are the common microorganisms in the atmospheric habitat?

A

Commonly organisms which can withstand extended periods of desiccation
Spore forming bacteria: Bacillus spp, Clostridium spp
Non spore forming G+ive: Staphylococcus spp + corynebacterium spp
Sporing mould: Penicillum spp + Aspergillus spp, Mucor spp
Yeasts: Rodotorula spp
Enter via vehicle i.e. particles

21
Q

What are the common microorganisms resides on human bodies?

A

Skin: Staph + Strep + Corynebacteria + Coliforms (E coli, Enterobacter species + mycobacteria + fungi including yeasts
Eye: Staph + strep + Neisseria + Corynebacteria + branhamella catarrhalis
URT: Acinetobacter + Staph + strep + Branhamella catarrhalis + neisseria + Corynebacteria + candida alb + Spirochetes + Actinomyces
Mouth: Staph + Strep + Lactobacillus + Fusobacterium + Bacteriodes + spirochetes + Veillonella
Intestine: G-ive anaerobic rods (Bacteroides, fusobacterium) Pseudomonas aeruginosa + Lactobacilli +

22
Q

What are the main reasons for identifying isolates?

A

Assess risk
ID source
Prepare strategy for control
Recognition of repeated isolation

23
Q

What are the controls in place to prevent contamination of water sampling?

A

IMS sample point -contact time
Flush sample point for 2 min - splash back
Sampling procedure must reflect in use procedure - if hose is used, use hose
Container: sterile? Pyrogen free? Label for traceability
transfer to lab 4-6hrs
Staff training

24
Q

How does LAL test work?

A

LAL is an aq extract obtained after lysis of blood cells of the horseshoe crab limulus polyphemus
Endotoxin: G-ive bacteria outer memberane lipopolysaccharide + cell death
Unit of measurement: EU/mL
No direct link between endotoxin level and number of bacteria present

25
What is the structure of LPS?
Polysacchride side chains Core polysaccharide Lipid A: di-glucosamine backbone - ling chain fatty acid linked
26
What are the methods available for the deactivation of endotoxin?
Acid base hydrolysis Strong acid break linkages between lipd A and KDO Strong alkalis saponisation of the fatty acids Oxidation: H2O2: 0.1% 2 hrs at 100 deg Dry Heat: By incineration purified endotoxin - twice more resistant hence worst case 250 deg
27
What are the methods of removal of endotoxin?
Rinsing Rinse with pyrogen free water i.e. components for filling and eqiupment leaving the cleanroom Distillation dobule phase change: liquid - vapour - liquid Ultrafiltration Molecular wt cut off: 100,000 delton Reverse osmosis micorbiological vulnerability of the sys - not 100% fool proof Activated carbon Efficient means produce fines - need filtration Electrostatic attraction to charged media Endotoxin: negatively charged - attract to positively charged matrices Hydrophobic attraction Aliphatic polymers: PVDF, PTFE
28
Describe a LAL test
Ph Eur:2.6.14 Endotoxin + Ca2+ / MG 2+: activate clotting factor stadardised, freeze dried lysate redissolve in pyrogen free water Mix with serial diluted test solution Incluabe at 37 deg for 60 min last dilution at which coagulation is observed - corresponds to an endotoxin conc equivalent to the stated sensitity of the lysate
29
How do you test for inhibition of test solution to LAL coagulation activity?
Extreme pH Protein denaturants Enzyme inhibitors Substrate interfere with human blood clotting Spike test solution with amount twice stated sensitivity of the lysate As the solution dilute out - if inhibit, neat solution will have no clot
30
How do you test for enhancement in LAL test?
Test cleanest sample and compare with pyrogen free water
31
What are the factors to be considered when looking at microbial contamination of the product?
Type of organisms: pathogenic? Number of organisms present: Patient's susceptibility Route of administration
32
What are the ideal characteristics of a preservative?
Broad spectrum Rapid action Chemically stable and effective under all pH conditions Compatible with excipients and packaging materials Physically undetectable Safe to use Cost effective
33
What are the strategies to preservative system?
Available water Most organism req over 70% AW = vapour press of product / water pH Majority of microorganisms grow best aroudn neutrality Limited by physiological acceptability and formulation stability Temperature Chemical and microbiological stability Excipient choice choose excipient resistant to microbial degradation Chemical synthetic preservatives Acids + salts: Benzoic acid + sodium metabisulphite Alcohols: Benzyl alcohol + ethanol Hydroxybenzoates: para hydroxybenzoates and ester Mercurial: phenyl mercuric acetate (not common) Phenols: Phenol + cresol + chlorocresol Quarternary ammonium compounds: Cetrimide + benzalkonium chloride Biguanides: chorhexidine Natural Essential oils and perfumes: tea tree oil + thyme oil Enzymes and proteains: lactoferrin - bind iron + oral use only
34
Describe PET
``` per Ph Eur: 5.1.3 Prepare inocula Candida albicans Aspergillus niger E coli Pseudomonas aeruginosa Staph aureus Optional contaminants Soybean Casen Digest - bac + Sabouraud dextrose agar for yeasts/mould - final conc 10^8 organisms per mL Innoculate product Confirm culture counts single culture inocula used Remove residual antimicrobial activity (dilution / filtration / neutraliser) inoculate each of 5 individual containers 0.1mL to 20mL product mix well Incubation of inoculated product incubate at 20-25 deg Examine containers at 7 + 14 + 21 + 28 days for TVC Calculate percentage changes Interpretation of results Concentratin of viable organisms should be reduced by 1-3 logs by 14th daywith no subsequent recoery/increase ```
35
Describe common types of disinfectant used in cleanroom.
Phenolic compounds: Chloroxylenol Adv: Fungicidal + broad spectrum + soluble Dis: reduced efficacy with hard water + organic maters + natural soap + not sporicidal + not good cleansing agent QUATs: Cetrimide Adv: G+ive + stable + solume + compatible with detergents + fungicidal + odourless Dis: not sporidical + hard water + organic material + inactivate natural and man made materials Iodophors: Adv: Quick microbial kill + G+ive + less irritant to skin Dis: not good cleaners + stain/discolour + mucous membrane irritant + orgnaic mater + >40deg release iodine Alcohols: Adv: Broad spectrum + rapid action + easy to use + min residue + not affected by organic matter + cleaning effect Dis: not sporicidal + costly in large quantities Chlorine compound: sodium hypochlorite Adv: Broad spectrum + sporicidal + not affected by hard water + little residue Dis: inactivated by orgnaics + loss activity on prolonged storage and UV + strong odour + corrosive Gluteraldehyde: Tegodor Adv: non staingin + relatively non corrosive + stable + broad spectrum Dis: not stable in solution + irritating to skin + inactivate by orgnics
36
What are the main BI use in sterilisation methods?
Steam: Geobacillus stearothermophillus D Value (121): >1.5min Z value: 10 min Dry Heat: Bacillus atrophaeus D Value (160) 2.5min Z value: 20 min EtO: Bacillus atrophaeus H2O2: G.StearothermophillusD Value 2.5min 600mg/L 54 deg 60 RH Radiation: Bacilus pumilis D Value (>1.9kGy)
37
What is LRV in the context of filter sterilisation?
LRV = log reduction value LVR = Log challenge org / Log filtrate org Normally >10
38
What are the main filter testing methods?
Bubble point Wet filter surface with water water retained in pores by surface tension Air pressure to displace water from pore = inversely related to the diameter of the largest pore There pressure indicates the largest pore size For smaller filters Flow test based on the diffusion of gas through the wetted membrane Require all pores full of liquid Air flow rate controlled by Fick's law Measure of gas diffusion - characterise membrane in relation to pore size and geometry Gas pressure s ability to retain an applied presure for a detined perio dof time, per unit area of filter at a given pressure Hydrophilic membrane Water intrusion test Hydrophobic membrances Measure pressure req to blow water into the membrane
39
What are the ideal properties of a BI?
Inherent resistant to sterilisation method Stable and reproducible Efficient recovery Nonpathogenicity characteristic growth features - easy to ID Uncommon in the natural bioburden
40
Describe sterility testing procedure
Per Ph Eur: 2.6.1 Media Soya bean casein digest (20-25 deg) + Fluid thoglycollate (30-35 deg) Direct innoculation removal of sample transfer contents to suitable media incluabe for 14 days Dis: product may inhibit growth + turbid product - can't see + Dilution of test media ( system Adv: large vol no dilution + wash x 3 remove inhibitory bits + any number of test containers can be filtered
41
Management of microbial failure
To consider: What is the ID of the organism? Likely source? Number of bacteria present? Type of sample taken? Other out of limit for the day of sampling or since? Localised issue? Links to real time data? i.e. non viable + LAF air flows + Press diff + temp + RH? Activity at time of sampling? Batch in progress? Cleaning? Interventions? Engineering? Media fill? Go and look at the area in question Trend data review: sampler + room + specific location + plant Action taken: Notify plant immediately Quarentine product: batches in progress during sampling + any more batches Additional sampling/cleaning? Investigate sampling operation and related lab procedures Investigate operator related issue
42
How do you use HACCP to enhance microbiological control of medicines?
``` HACCP - hazard analysis and critical control point Define product and process Identify potential hazards Identify potential control measures Determination of CCP Establish critical limits for each CCP Establish monitoring system for each CCP Establish CAPA plan Establish verification procedure to demonstrate compliance ```
43
Describe what you would find in a disinfectant SOP
``` Type Contact time Application technique Temperature Concentration and how to mix Training Documentation Compatibility Frequencies Rotation ```
44
Describe aldehydes used as a disinfectant
``` Sporocidal action Eg.2%gluteraldehyde Non corrosive, non staining Broad spec Harmful to skin and mucosa ```
45
Describe chlorine and sodium hypochlorite as a disinfectant
``` Sporocidal 0.5% sodium hypochlorite Non foaming Little residue Broad spec Inactivated after long storage and UV Corrosive Skin irritations ```
46
Describe two general purpose disinfectants
``` 1-Alcohol eg. 70% IPA Broad spec Easy to use Rapid action Expensive Not sporocidal Flammable ``` 2- quarternary ammonium compounds eg, BAK or cetrimide Broad spec Active against gram + (staph,strep) Cheap Fungicidal Not sporocidal Affected by hard water
47
Tell me about resistance of some clinically important microorganism to disinfectants
Most resistant- bacterial spores eg. Bacillus subtilis or clostridium sporogenes 2- mycobacteria: M. Tuberculosis 3- non lipid coated viruses: polio 4- fungal spores: candida spp 5-vegetative bacteria: ps. Aeruginosa, staph aureus, Salmonella 6- lipid coated bacteria : HIV, hep b

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