Microbial Spoilage

Microbial Spoilage

Contents

• Spoilage of pharmaceutical products

• Pharmaceutical ingredients which are susceptible to microbial attack

• Factors affecting microbial spoilage

• Sources and control of contamination

• The extent of microbial contamination

• Factors determining the extent of microbial contamination

Intended Learning objectives

At the end of this lecture the student will be able to:

• Describe Spoilage of pharmaceutical products and ways to minimize them

• Discuss different pharmaceutical ingredients which are susceptible to microbial attack

• Discuss factors affecting microbial spoilage

• Explain various sources and control of contamination

• Describe effects of microbial contamination in pharmaceutical product

• Summarise factors determining the extent of microbial contamination

Spoilage

• Spoilage is a process where the product gets deteriorated to such an extent that it is considered as unfit for the human consumption

• It is a complex event, which may be combination of microbial and biochemical activities

• Spoilage process led to the addition of preservatives into the product to extend its shelf-life

Microbial Spoilage

• Microbial Spoilage include the contamination of pharmaceutical products with the microbes which lead to spoilage of the product affecting drug safety and quality

• Microorganisms have the ability to degrade compounds at mild physicochemical conditions

• Mixture of microbial species are effective biodeteriogens

• The rate of degradation of materials varies from product to product

• The overall rate of deterioration of a pharmaceutical compound depends on:

– Its molecular structure

– The physicochemical properties of a particular environment

– Type & quantity of microbes present

– Whether the metabolites produced can serve as source of usable energy & precursor for biosynthesis

Pharmaceutical ingredients susceptible to microbial attack are

• Therapeutic agents

• Surface active agents

• Organic polymer: Starch, CMC, pectin -Agar –Polystyrene

• Humectant: Glycerin, sorbitol

• Oil & Fats

• Organoleptic additives

• Preservatives, disinfectants

– When used in concentration less than the effective concentration, Gram negative organisms can attack

Factors affecting microbial spoilage

1. Types and size of contaminant inoculum

2. Nutritional factors

3. Moisture content: water activity (Aw)

4. Redox potential

5. Storage temperature

6. pH

7. Packaging design

8. Protection of microorganism’s products within pharmaceutical

1. Types and size of contaminant inoculum

• While designing formulation, the formulator should consider the environment and usage to which the product is likely to be subjected

• Very low levels of contaminants which are unable to replicate in a product might not cause appreciable spoilage

• Inoculum size alone is not always a reliable indicator of spoilage

• For example, a very low level of, aggressive pseudomonas in a weakly preserved solution may suggest a greater risk than tablets containing fairly high numbers of fungal and bacterial spores

• When an aggressive contaminant enters a medicine, there may be a lag period before significant spoilage begins

• Since there is usually a long delay between manufacture and administration of medicines, growth and attack could start during this period unless additional steps are taken to prevent it like addition of preservation

2. Nutritional factors

• Many common spoilage microorganisms have simple nutritional requirements and metabolic adaptability which enable them to utilize many of the components of medicines as substrates for biosynthesis and growth

• The use of crude vegetable or animal products in a formulation provides an additionally nutritious environment

• Even demineralized water prepared by good ion-exchange methods normally contains sufficient nutrients to allow significant growth of many water-borne Gram-negative bacteria such as Pseudomonas species

3. Moisture content: water activity

• Microorganisms require 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

• The greater the solute concentration, the lower is the water activity

• Hygroscopic medicines (tablets, capsules, powders) will require suitable packaging to prevent resorption of water and consequent microbial growth

• Dilute aqueous films formed on the surface of viscous products such as syrups and creams can lead to surface yeast and fungal spoilage

4. Redox potential

• The ability of microbes to grow in an environment is influenced by its oxidation-reduction balance

• The redox potential in viscous emulsion may be quite high due to the high solubility of oxygen in most fats & oils

5. Storage temperature

• Spoilage of pharmaceuticals could occur over the range of about -20° to 60°C

• Storage in a deep freeze at -20°C or lower is used for long- term storage of foodstuffs and some pharmaceutical raw materials, to even further minimize the risk of growth of any contaminants

• Reconstituted suspensions and multi-dose eye drop packs are sometimes dispensed with the instruction to 'store in a cool place' (8°-12°C), or “store in refrigerator”

• Pharmacopoeial Water for Injections is recommended to be held at 80°C or above

6. pH

• Extremes of pH prevent microbial attack

• Around neutrality bacterial spoilage is more likely, with reports of pseudomonas and related Gram-negative bacteria growing in antacid mixtures, flavoured mouth washes and in distilled or demineralised water

• Above pH 8, e.g with soap-based emulsions, spoilage is rare

• For products with low pH levels such as the fruit juice-flavoured syrups (pH 3-4) mould or yeast attack is more

• 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 (pickles, yoghurt) but not suitable for pharmaceutical products

7. 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 of administration

• Self-sealing rubber closures must be used to prevent microbial entry into multi-dose injection containers following withdrawals with a hypodermic needle

• 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 are potential means microbial attack in humid conditions

8. Protection of microorganisms within pharmaceutical products

• The survival of microorganisms in particular environments is influenced by the presence of various relatively inert materials

• Microbes can be more resistant to heat or desiccation in the presence of some polymers such as starch, acacia or gelatin

• Presence of suspended particles such as kaolin, magnesium trisilicate or aluminium hydroxide gel may influence contaminant longevity in medicines

• Presence of some surfactants, suspending agents and proteins can increase the resistance of microorganisms to preservatives, over and above their direct inactivating effect on the agents

Sources and control of contamination

During manufacturing

– Hospital manufacture

– Water

– Environment

– Packaging

In use

– Human sources

– Environmental sources

– Equipment sources

During manufacturing

Microbiological quality of the finished product will be determined by

– The formulation components used,

– The environment in which they are manufactured and

– The manufacturing process itself

Quality should not be only inspected at the end of manufacturing but must be built into the product at all stages of the process

• Raw materials, like water and ingredients of natural origin

• All processing equipment should be subject to planned preventive maintenance and should be properly cleaned after use to prevent cross-contamination between batches

• Cleaning equipment should be appropriate for the task in hand and should be thoroughly cleaned and properly maintained

• Manufacture should take place in suitable premises, supplied with filtered air, for which the environmental requirements vary according to the type of product being made

• Staff involved in manufacture should not only have good health but also a good knowledge of the importance of personal and production hygiene

• The end-product requires suitable packaging which will protect it from contamination during its shelf-life and is itself free from contamination

Hospital manufacture

• Manufacture in hospital premises increases contamination issues

Environment

• Wet areas are sources of microorganisms

• Cleaning equipment, such as mops, buckets, cloths and scrubbing machines, may be responsible for distributing these organisms around the pharmacy

• Good manufacturing practices minimises contamination

Packaging

• Cardboard, card liners, corks and papers are unsuitable for packaging pharmaceuticals, as they are heavily contaminated with bacterial or fungal spores

• Re-usable containers must be thoroughly washed and dried

• Common practice in hospitals is the repackaging of products purchased in bulk into smaller containers increasing the chances of contamination

Water

• Water for pharmaceutical manufacture requires some treatment such as distillation, reverse osmosis, deionization or a combination of these

• Gram-negative opportunist pathogens can survive on traces of organic matter present in treated water and will readily multiply to high numbers at room temperature

• Water should therefore be stored at a temperature in excess of 80°C and circulated in at a flow rate of 1–2 m/s to prevent the build-up of bacterial biofilms in the piping

In use

• All multi-dose products are vulnerable to contamination during use

• The risk of contamination during product use has been much reduced in recent years due to improvements in packaging and changes in nursing practices

Human sources

• During normal usage, patients may contaminate their medicine with their own microbial flora

• Topical products are considered to be most at risk, as the product will probably be applied by hand thus introducing contaminants from the skin

• In hospitals, multi-dose products, once contaminated, may serve as a vehicle of cross contamination or cross-infection between patients

• These contaminations can be minimised by effective hand

Environmental sources

• Small numbers of airborne contaminants may settle in products left open to the atmosphere

• Larger numbers of waterborne contaminants may be accidentally introduced into topical products by wet hands or by a ‘splash-back mechanism’ if left at the side of a basin

• When to hospitals, there are fewer opportunities for contamination in the home, as patients are generally issued

Equipment sources

• Disposable applicators or swabs should be used

• Reused applicators such as sponges, brushes, spatulas are main reasons of contaminations

• Humidifiers, incubators, ventilators, resuscitators and other apparatus require proper maintenance and decontamination after use

• Chemical disinfectants minimises the spread of cross-infection among hospital patients

Assessment of microbial contamination and spoilage

Observable effects of microbial attack on pharmaceutical products are –

• Early indications of spoilage are organoleptic (smell & taste) Example: sour, fishy, bad eggs, bitter, earthy taste and smells

• Discoloration to products by microbial pigments

• Loss of viscosity due to depolymerization of thickening & suspending agent like acacia, carboxy methyl cellulose (CMC) resulting in sedimentation of suspended ingredients

• Microbial polymerization of sugars and surfactant molecules can produce slimy, viscous, masses in syrups, shampoos and creams, fungal growth in creams produce 'gritty’ textures

• Acidic or basic microbial metabolites change pH of the formulation & enhance microbial growth

• Gaseous metabolites may be seen as trapped bubbles within viscous formulations

• When emulsions are attacked, it reduces the stability and accelerates creaming, coalescence and cracking of emulsion

The extent of microbial contamination

Medicament-borne contamination have become very common these days

• A wide range of non-streile products were found to be contaminated with Bacillus subtilis, Staph. albus, yeasts and moulds, and large numbers of coliforms soon after manufacture

• Under Medicines Act 1968, pharmaceutical products made in industry were expected to conform microbiological and chemical quality specifications

• Higher rates of contamination are seen in products after opening and using

• Medicines used in hospitals are more likely to be contaminated than those used in the general community

• Medicines used in the home are not only less often contaminated but also contain lower levels of contaminants and fewer pathogenic organisms

Factors determining the extent of microbial contamination

• Type and degree of microbial contamination

• Resistance of the patient

• The route of administration

Type and degree of microbial contamination

• Microorganisms that contaminate medicines and cause disease in patients may be classified as true pathogens or opportunist pathogens

• Pathogenic organisms like Clostridium tetani and Salmonella spp. rarely occur in products, but when present cause serious problems

• Opportunist pathogens like Ps. aeruginosa, Klebsiella, Serratia require simple nutritional requirements when enable them to survive in a wide range of pharmaceuticals

• Opportunist pathogens can survive in disinfectants and antiseptic solutions that are normally used in the control of hospital cross-infection

Resistance of the patient

• A patient’s resistance is important in determining the outcome of a medicament-borne infection

• Hospital patients are more exposed and susceptible to infection than those treated in the general community

• Neonates, the elderly, diabetics and patients traumatized by surgery or accident may have impaired defence mechanisms

• People suffering from leukaemia and those treated with immunosuppressants are most vulnerable to infection

• The critical dose of microorganisms that will initiate an infection is largely unknown and varies between species but also within a species.

• Animal and human volunteer studies have indicated that the infecting dose reduces significantly in the presence of trauma or foreign bodies or if accompanied by a drug having a local vaso-constrictive action.

The route of administration

• Contaminated products injected directly into the bloodstream or instilled into the eye cause the most serious problems

• Injectable and ophthalmic solutions are often simple solutions and provide Gram-negative opportunist pathogens with sufficient nutrients to multiply during storage

• Contaminants in topical products may cause harm when deposited on broken skin

• Patients treated with topical steroids are also prone to local infections due to contaminations

• Eye when damaged through the improper use of contact lenses or scratched by fingernails or cosmetic applicators, infections due to ophthalmic preparations

• The acidity of the stomach provides a good barrier for contaminants ingested orally in medicines

Summary