Microbial contamination of pharmaceutical products can lead to spoilage or health hazards. Contamination can occur during manufacturing from water, the environment or packaging. It can be prevented through good manufacturing practices and controlling moisture, temperature, pH and packaging design. Spoilage results from microbial growth and causes product deterioration or infection. Factors influencing spoilage include the contaminant type/level, nutrients, moisture, temperature, pH and packaging. Strict control of these factors is needed to prevent issues.

1. `

2. Microbial contamination:
Spoilage and hazards

3. Microbial Contamination
Types of contamination
Sources of contamination
Prevention and control of contamination
Spoilage
Types of spoilage
Factors affecting microbial spoilage
Hazards
Causes of hazards
Prevention of hazards
Microbial Contamination
Types of contamination
Sources of contamination
Prevention and control of contamination

4. Microbial
Contamination

5. Microbial Contamination
“Microbiological contamination refers to the non-
intended or accidental introduction of infectious
material like bacteria, yeast, mould, fungi, virus,
prions, protozoa or their toxins and by-products.”

6. Reasons of Contamination
Pharmaceutical products contain wide range of excipients in
addition to API in order to make the formula easy to
manufacture, stable, effective & convenient to the patient.
Products in pharmaceutical industry must meet high
microbiological specs i.e if they are not sterile, they should
have no more than a minimal microbial population at the
time of product release & afterwards
Occasionally some product batches with unacceptable
contamination (level or type of organism) escape QC net.

7. Consequences will be:
Product spoilage rendering it unsuitable for use
Product spoilage will result in financial problems for
the manufacturer through direct loss of faulty product
& loss of publicity through product recall
Potential health hazard to patients perhaps resulting in
outbreaks of medicament-related infections.

8. Example
 Most commonly, Pseudomonas spp has resulted
in the spread of nosocomial infections in
compromised patients.
 Less common contamination with Salmonella &
products contaminated with toxic microbial
metabolites.

9. Types of Contamination
1. Some of the contaminants may be
pathogenic.
2. Others may grow even in the presence of
preservatives and spoil the product.
3. Or which are destroyed by in-process heat
treatment may still leave cell residues which may
be toxic or pyrogenic.

10. Sources of Contamination
in Manufacturing
1. Water:
Water for pharmaceutical manufacture requires
some further treatment, usually by distillation, reverse
osmosis or deionization or a combination of these,
depending on the intended use of water.

11. Cont.…
2. Environment:
The microbial flora of the
pharmacy environment is a reflection of the general
environment and the activities undertaken there.
Contamination levels in the production environment
may, however, be minimized by observing good
manufacturing practices.

12. Cont.…
3. Packaging:
Sacking, cardboard, card liners, corks
and paper are unsuitable for packaging
pharmaceuticals, as they are heavily contaminated.
These have now been replaced by non-biodegradable
plastic materials.

13. Prevention and Control of
Contamination
In manufacture the principles of good
manufacturing practice must be
observed, and control measures must be
built in at all stages.
The manufacture of liquid or semi-solid
preparations for either oral or topical use
requires a clean environment for both
the production and filling stages.

14. Spoilage
Spoilage

15. Microbial spoilage may include:
 Survival of low levels of acutely pathogenic
microorganisms, or higher levels of opportunist
pathogens.
 The presence of toxic microbial metabolites.
 Microbial growth and initiation of chemical and
physicochemical deterioration of the formulation.

16. Types of Spoilage
1. Infection induced by contaminated medicines
Although infrequently reported as pharmaceutical
contaminants, acute human pathogens attract
considerable attention when they are present.
For example Pseudomonads contaminating 'antiseptic'
solutions have infected the skin of badly burnt patients.

17. Cont.…
2. Chemical and physicochemical deterioration of
pharmaceutical products
The overall rate of deterioration of a chemical will
depend upon:
 Its chemical structure;
 The physico-chemical properties of a particular
environment;
 The type and quantity of microbes present.

18. 3. Pharmaceutical
ingredients susceptible
to microbial attack
API
Preservatives,
disinfectants
Humectants Oil & Fats
Surface
Active Agents
Organic
Polymer

19. Fungal growth on a tablet which has become damp during
storage under humid conditions.

20. Factors affecting microbial spoilage of
pharmaceutical products
Types and size
of contaminant
inoculum
Nutritional
factors
Moisture
content:
water activity
Storage
temperature
pH
Packaging
design

21. Types and size of contaminant
inoculum
This might arise if:
1. raw materials were unusually contaminated
2. a problem of the plant-cleaning protocol occurred
3. biofilm detached itself from within supplying
pipework
4. there was demolition or maintenance work in the
vicinity of the manufacturing site
5. gross misuse of the product during administration

22. Cont.…
Inoculum size alone is not always a reliable indicator of
spoilage potential.
For example, a very low level of, aggressive
pseudomonads in a weakly preserved solution may
suggest a greater risk than tablets containing fairly high
numbers of fungal and bacterial spores.

23. Nutritional factors
 The simple nutritional requirements and metabolic
adaptability of many common spoilage microorganisms
enable them to utilize many formulation components as
substrates for biosynthesis and growth.
 A formulation containing crude vegetable or animal
products provide additional nutritious environment.
 Even demineralized water prepared by ion exchange method
normally contains sufficient nutrients to allow growth of some
Pseudomonas spp.
 Acute pathogens require specific growth factors which are
often absent in pharmaceutical formulations so they do not
multiply but remain viable and infective for an appreciable time.

24. Moisture content: water activity (Aw )
 Microorganisms require readily accessible water in
appreciable quantities for growth.
 Although some solute-rich medicines such as syrups
appear to be 'wet', microbial growth in them may be
difficult since the microbes have to compete for water
molecules with the large numbers of sugar and other
molecules of the formulation which also interact with
water via hydrogen bonding.

25.  An estimate of the proportion of the uncomplexed
water in a formulation available to equilibrate with
any microbial contaminants and facilitate growth can
be obtained by measuring its water activity (Aw).
Aw = vapour pressure of formulation ÷
vapour pressure of water under similar conditions

26. Cont.…
The greater the solute concentration, the lower is the
water activity.
With the exception of halophilic bacteria, it grows best in
dilute solutions (high Aw) and as solute conc. rises
(lowering Aw), growth rates decline until a minimal,
growth-inhibitory Aw is reached.
Limiting Aw values are of the order of:
Gram-negative rods 0.95
staphylococci, micrococci and lactobacilli 0.9
most yeasts 0.88

27. Prevention
 The Aw of aqueous formulations can be lowered to
increase resistance to microbial attack by the addition
of high concentrations of sugars or polyethylene
glycols. However, even Syrup BP (67% sucrose; Aw =
0.86) has been reported to fail occasionally to inhibit
osmotolerant yeasts and additional preservation may
be necessary.
 Aw can also be reduced by drying, although the dry,
often hygroscopic medicines (tablets, capsules,
powders) will require suitable packaging to prevent
resorption of water and consequent microbial growth.

28. Prevention
 Some tablet film coatings are now available which
greatly reduce water vapour uptake during storage.
These might contribute to increased microbial stability
during storage in particularly humid climates, although
suitable foil strip packing may be more effective, but
also more expensive.

29. Storage temperature
 Spoilage of pharmaceuticals could occur over the range
of about 20° to 60°C, although much less likely at the
extremes.
 The actual storage temperature may determine the
spoilage by particular types of microorganisms.

30. Prevention of different dosage forms
Temp. Dosage form Consequences
Deep freeze at
-20°C or lower
Foodstuffs and some
pharmaceutical raw materials
Long-term storage
-20°c Total parenteral nutrition
(TPN) feeds
Minimize the risk of growth of any
contaminants which might have
been introduced during their
aseptic compounding.
'Store in a cool
place'
(8°-12°C
Reconstituted suspensions
and multi-dose eye drop
packs
Partly to reduce the risk of in-use
contamination growing before
The expiry date
80°C or above
after
distillation
Water for injections Prevent possible regrowth of gram-
negative Bacteria, and the release
of endotoxins.

31. pH
 Extremes of pH prevent microbial attack (although
slight mould growth can occur in dilute hydrochloric
acid).
 Around neutrality bacterial spoilage is more likely,
with reports of pseudomonads and related Gram-
negative bacteria growing
 Antacid mixtures
 Flavored mouth washes
 distilled or demineralized water

32. Cont.…
 Above pH 8, e.g with soap-based emulsions, spoilage
is rare.
 For products with low pH levels such as the fruit
juice-flavored syrups (pH 3-4) mould or yeast attack
is more likely.
 Yeasts can metabolize organic acids and raise the pH
to levels where secondary bacterial growth can occur.
 In food industry low pH adjustment can be made to
preserve foodstuffs (pickling, yoghurt), but this is not
practical for medicines.

33. Packaging design
 Packaging should be made in a way to control the
entry of contaminants during both storage and use.
 The most important dosage form to be protected are
the parenteral drugs because of the high risks of
infection by this route.
 Self-sealing rubber closures must be used to prevent
microbial entry into multi-dose injection containers
following withdrawals with a hypodermic needle.

34. Cont.…
 Wide-mouthed cream jars are now replaced with
narrow nozzle and flexible screw capped tubes to
remove the likelihood of operator-introduced
contamination during use.
 For medicines which rely on their low Aw to prevent
spoilage, packaging such as strip foils must be of
water vapour-proof materials with fully efficient
seals.
 Cardboard outer packaging and labels themselves
(especially the gluing material) can become substrates
for microbial attack under humid conditions.

35. Articles
Effect of water activity (aw) on microbial growth
and toxins production
compared the effects of water content and water
activity (aw) values of cellulose substrate on the
growth of a filamentous fungus Penicillium roqueforti.
o Four water activity values (0.94, 0.96, 0.97, 0.99)
o four water content values: 0.4, 0.6, 0.8, 1 (g/g dry
matter) have been tested in a cross experiment.

36. Articles
Results:
The effect of water activity has been shown to be
highly significant while the water content level did not
significantly modify the development of the fungus.
Conclusion:
Thus it appeared clear that a decrease of aw
drastically decreased the development of the
fungus, whatever the water content level.

37. ArticlesEffect of temperature on microbial growth and
toxins production
1) Schindler et al. studied the production of two
aflatoxins isolated from Aspergillus flavus grown for
5 days on work media at 2, 7, 13, 18, 24, 29, 35, 41,
46, and 52oC.
Results:
They found that maximal growth for the two
isolates occurred at 29 and 35oC and the optimal
temperature for growth of both Aspergillus flavus
isolates was higher than optimal temperatures for
aflatoxin production.

38. Articles
2) Laciaková et al. studied devitalization of
Penicillium glabrum and Aspergillus niger germs (as an
example of Aspergillus and Penicillium spp.) and other
microorganisms at different temperatures of 22, 37,
60oC.
Result:
All of the tested strains withstood the temperature
22oC during the period of 42 days in Sabouraud agar.
At the temperature of 37oC the devitalization of
Penicillium glabrum occurred after 21 days and
Aspergillus niger devitalization occured after 35 days.
Temperature of 60oC devitalized all tested strains of
microorganisms within 5 hours.

39. ArticlesEffect of pH on microbial growth and toxins
production
Kathryn et al. determined the effect of pH on the growth
rates of 61 isolates belonging to 13 important
toxigenic fungi derived from Aspergillus spp.,
Fusarium spp., Penicillium spp., over the pH range 2 to
11 at 25, 30 and 37°C.
Results:
Nearly all species studied were able to grow over the
entire range examined on a laboratory agar medium.
However, in general, Aspergillus spp. was more
tolerant on alkaline pH while Penicillium spp.
appeared to be more tolerant on acidic pH.

40. ArticlesCombined effects of water activity (aw), pH and
antimicrobial agent on microbial growth
The combined effects of water activity (0.99 or 0.95),
pH (4.5 or 3.5) and antimicrobial agent (potassium
sorbate, sodium benzoate, sodium bisulfate, carvacrol,
citral, eugenol, thymol, or vanillin) concentration (0,
100, 200 up to 1800 ppm) on the growth of Aspergillus
flavus were evaluated by López-Malo et al. in potato
dextrose agar (PDA).
Results:
Mold spore germination time and radial growth rates
(RGR) were significantly (p<0.05) affected by the
variables.

41. Articles
 Germination time increased as antimicrobial agent
concentration increased and when (aw) and pH
decreased.
 Important antimicrobial differences were observed,
being, in general, the natural antimicrobials less
pH-dependent than chemical preservatives.
 Aspergillus flavus exhibited higher sensitivity to
thymol, eugenol, carvacrol, potassium sorbate,
sodium bisulfate, and sodium benzoate (at pH 3.5)
than to vanillin or citral.

42. ArticlesMicrobiological stability of tablets stored under
tropical conditions
The effect of storage under tropical conditions on the
behaviour of microbial contamination of tablets was
studied.
Materials
The tablet ingredients used were:
 lactose monohydrate 200 mesh
 Mg stearate
 potato starch
 rice starch
 potassium sorbate
 Tapioca starch
 sodium methylhydroxybenzoate

43. ArticlesPreparation of spore suspensions
1. Aspergilh niger (A. niger) was grown on Trypton
Soya Agar at 30°C. After 5 days the spores were
harvested by washing the plates with a 0.1%
polysorbate 80 solution. The spore suspension was
diluted to contain: 5 X lo7 spores/ml.
2. Bacillus brevis (3. brevis) was grown on Lab Lemco
Agar, containing 0.88 mg/l manganese chloride, at
30o C. After 7 days the spores were harvested by
washing the plates with a 0.1% polysorbate 80
solution. The spore suspension was diluted to
contain: 5 X lo7 spores/ml.

44. ArticlesTablet composition
Control
tablets
(mg)
0.1% w/w
preservative
(mg)
1% w/w
preservative
(mg)
Lactose monohydrate 245 245 242.5
Potato starch, rice starch
or tapioca starch.
245 245 242.5
Sodium
methylhydroxybenzoate
or potassium sorbate.
- 0.5 5
5% starch a paste 163 163 163
Magnesium stearate 2.5 2.5 2.5

45. ArticlesResults:
 The investigation of the microbiological quality of
the starting materials showed that rice and tapioca
starch had a higher level of natural contamination
than potato starch.
 Lactose/potato starch tablets without preservatives,
inoculated with Aspergillus niger spores, spoiled due
to mould growth when stored under tropical
conditions (31° C and 95% RH). Reason (Under
these conditions tablets prepared with lactose and
rice or tapioca starch spoiled due to growth of natural
contaminants)

46. Articles
 No growth of bacterial cells (Bacillus breuis) was
observed during storage under these conditions.
 When the tablets were stored under more moderate
conditions (31° C, 75% RH) they were not at risk of
microbiological spoilage.
Effect of preservative:
 Sodium methylhydroxybenzoate and potassium
sorbate were evaluated for then efficacy against
microbiological spoilage of tablets.
 A concentration of 1% w/w of either preservative
prevented growth of Aspergiliw niger on
lactose/potato starch tablets stored at 31° and 95%
relative humidity.
 A 0.1% w/w concentration level of preservative was
not as effective.

47. Articles
 Adding a preservative to lactose/potato starch tablets
contaminated with Bacillus breuis spores did not affect
the viability of these bacterial spores.
 The addition of preservatives to tablets prepared with
lactose and rice or tapioca starch and stored under
tropical conditions prevented microbiological spoilage
caused by growth of natural contaminants.

48. Hazards

49. An inherent chemical or physical characteristic that
has potential for causing damage to people,
property or the environment.
Definition

50. Causes of hazards
Infection due to bacterial contamination
Lesions or cuts caused by handling of
damaged glass ware.
Workers are exposed to UV radiation when
operating in sterile area.
Severe skin and eye irritations.

51. Prevention of hazards
Use of laminar flow extraction hoods in order to
reduce product contamination and/or operator contact.
Protective face masks and gloves should be worn.
Use of cut-resistant gloves, operator information and
training.
Work surfaces must be clean and disinfected at all
times.

52. Prevention of hazards
Skin must be adequately protected by appropriate
clothing.
Protective eye wears should be used.
Access to microbiology activity areas shall be
restricted by duly authorized personnel.