Microbial Spoilage, Infection Risk and Contamination Control Notes

Introduction

• Pharmaceutical products must meet GPMP requirements for quality, safety, and efficacy.

• Products must be stable and acceptable to patients, meeting high microbiological specifications.

• Rogue products with unacceptable contamination levels can escape quality assurance, leading to spoilage, financial problems, litigation, and health hazards.

• Contamination with opportunist pathogens (e.g., Pseudomonas spp.) can cause nosocomial infections in compromised patients.

• Low levels of pathogenic organisms (e.g., Salmonella) or toxic microbial metabolites (e.g., mycotoxins) can also cause significant problems.

Spoilage – Chemical and Physicochemical Deterioration of Pharmaceuticals

• Microorganisms recycle biological matter and degrade substances under mild conditions.

• Mixed microbial communities are more effective at biodeterioration than individual species.

• Pharmaceutical formulations are specialized microenvironments susceptible to microbial attack.

• Naturally occurring ingredients are sensitive to attack, and synthetic components are designed to be readily degraded.

• Crude vegetable and animal drug extracts contain microbial nutrients, increasing the potential for microbial attack.

Pharmaceutical Ingredients Susceptible to Microbial Attack

• Therapeutic agents: Active drug constituents may be metabolized to less potent or inactive forms.

  • Microorganisms can metabolize alkaloids (morphine, strychnine, atropine), analgesics (aspirin, paracetamol), thalidomide, barbiturates, steroid esters, and mandelic acid.

  • Microorganisms are used to transform steroid molecules for commercial production of therapeutic steroidal agents (see Chapter 26).

  • Examples of drug destruction include atropine metabolism in eye drops by fungi, penicillin inactivation by β-lactamase-producing bacteria (see Chapters 11 and 13), steroid metabolism in damp tablets/creams by fungi, aspirin hydrolysis in suspension by esterase-producing bacteria, and chloramphenicol deactivation by a contaminant.

• Surface-active agents

  • Anionic surfactants: Alkali metal and amine soaps of fatty acids are generally stable due to alkaline pH but degrade upon dilution into sewage. Alkyl and alkylbenzene sulfonates and sulfate esters are metabolized by ω-oxidation and β-oxidation. Degradation decreases with increasing chain length and branching.

  • Non-ionic surfactants: Alkylpolyoxyethylene alcohol emulsifiers are readily metabolized; degradation decreases with increasing chain length and branching. Alkylphenol polyoxyethylene alcohols are more resistant. Lipolytic cleavage of fatty acids from sorbitan esters, polysorbates, and sucrose esters produces molecules for microbial growth. Ampholytic surfactants (phosphatides, betaines, alkylamino-substituted amino acids) are reasonably biodegradable. Cationic surfactants (antiseptics/preservatives) degrade slowly at high dilution. Pseudomonads can grow in quaternary ammonium antiseptic solutions, metabolizing other ingredients and some surfactant.

• Organic polymers: Thickening and suspending agents are subject to microbial depolymerization by extracellular enzymes.

  • Examples: amylases (starches), pectinases (pectins), cellulases (carboxymethylcelluloses), uronidases (polyuronides like tragacanth/acacia), dextranases (dextrans), and proteases (proteins).

  • Agar is a relatively inert polymer used as a support for solidifying microbiological culture media.

  • Low molecular weight polyethylene glycols degrade via sequential oxidation of the hydrocarbon chain; larger congeners are more recalcitrant. Synthetic packaging polymers like nylon, polystyrene, and polyester are resistant, while cellophane is susceptible under humid conditions.

• Humectants: Low molecular weight materials like glycerol and sorbitol reduce water loss and may be readily metabolized unless in high concentrations (see section 2.3.3).

• Fats and oils: These are attacked when dispersed in aqueous formulations like oil-in-water emulsions, aided by high oxygen solubility in oils. Fungal attack occurs in condensed moisture films on oil surfaces or in water droplets contaminating the bulk oil phase. Lipolytic rupture of triglycerides releases glycerol and fatty acids, which undergo β-oxidation, producing odiferous ketones. Microbial metabolism of pharmaceutical hydrocarbon oils is rarely reported but is a problem in engineering and fuel technology when water accumulates in oil storage tanks, leading to fungal colonization and corrosion.

• Sweetening, flavoring, and coloring agents: Many sugars and sweetening agents used in pharmacy are ready substrates for microbial growth. Some are used in high concentrations to reduce water activity and inhibit microbial attack (see section 2.3.3). Stock solutions of coloring (e.g., tartrazine, amaranth) and flavoring agents (e.g., peppermint water) once supported Pseudomonas spp. growth; these should be preserved or freshly made from alcoholic solutions.

• Preservatives and disinfectants: Many preservatives and disinfectants can be metabolized by Gram-negative bacteria, commonly at concentrations below effective use levels. Pseudomonads can grow in stock solutions of quaternary ammonium antiseptics and chlorhexidine, resulting in patient infections. Pseudomonas spp. have metabolized 4-hydroxybenzoate (parabens) ester preservatives in eye drops, causing serious eye infections, and preservatives in oral suspensions and solutions. Detailed knowledge of agent properties, susceptibility to contamination, and limitations is essential for formulation.

Observable Effects of Microbial Attack on Pharmaceutical Products

• Microbial contaminants attack formulation ingredients to create substrates needed for biosynthesis and energy production before they can replicate to levels where obvious spoilage becomes apparent. 10^6 microbes will have a degradative effect 10^6 times faster than one cell.

• Early indications of spoilage are organoleptic, with unpleasant smells and tastes like sour fatty acids, fishy amines, bad eggs, or sickly tastes/smells. Products may become discolored by microbial pigments.

• Thickening and suspending agents like tragacanth, acacia, or carboxymethylcellulose can be depolymerized, reducing viscosity and causing sedimentation. Microbial polymerization of sugars and surfactant molecules can produce slimy, viscous masses in syrups, shampoos, and creams; fungal growth in creams has produced gritty textures.

• Changes in product pH can occur, permitting secondary attack by microbes previously inhibited by the initial pH. Gaseous metabolites may be seen as trapped bubbles in viscous formulations.

• When oil-in-water emulsions are attacked:

  • Metabolism of surfactants reduces stability and accelerates creaming of oil globules.

  • Lipolytic release of fatty acids lowers pH, encouraging coalescence of oil globules and cracking of the emulsion.

  • Fatty acids and their oxidation products provide a sour taste and unpleasant smell.

  • Bubbles of gaseous metabolites may be visible, and pigments may discolor the product.

Factors Affecting Microbial Spoilage of Pharmaceutical Products

• Understanding environmental parameters' influence on microorganisms enables manipulation of formulations to create unfavourable conditions for growth and spoilage, while considering patient acceptability and therapeutic efficacy. Formulation characteristics indicate susceptibility to various microbial classes.

• Successful formulation against microbial attack involves prediction of product use and challenges. Knowledge of microbial ecology and contaminant identification can track defective steps in design or production.

• Low levels of contaminants may not cause spoilage if unable to replicate. Unexpected surges in contaminant bioburden may challenge formulations. Raw material contamination, lapses in cleaning protocols, biofilm detachment from pipework, or product misuse during administration can cause unexpected surges.

• Inoculum size is unreliable as an indicator of spoilage potential. Aggressive pseudomonads in weakly preserved solutions pose a greater risk than tablets with high numbers of fungal and bacterial spores.

• Aggressive microorganisms contaminating medicine may have a lag period before significant spoilage, decreasing disproportionately with increasing contaminant loading. This can occur between manufacture and administration.

• Isolation of microorganisms from spoiled products does not necessarily mean it initiated the attack; it could be a secondary opportunist.

Nutritional Factors

• Spoilage microorganisms can use many formulation components as substrates for biosynthesis and growth due to their simple nutritional requirements and metabolic adaptability. Crude vegetable or animal products in a formulation provide a nutritious environment. Even demineralized water prepared by ion-exchange methods contains nutrients for significant growth of waterborne Gram-negative bacteria such as Pseudomonas spp.

• Failure of such contaminants to survive is unlikely from nutrient limitation but from non-supportive physicochemical or toxic properties.

• Acute pathogens require specific growth factors normally associated with the tissues they infect, often absent in pharmaceutical formulations. They are unlikely to multiply but may remain viable and infective.

Moisture Content: Water Activity ($A_w$)

• Microorganisms need readily accessible water to grow. $A_w$ measures uncomplexed water available in formulation to support microbial growth.

• Formula: $A_w = \frac{\text{vapour pressure of formulation}}{\text{vapour pressure of water under similar conditions}}$

• Greater solute concentration lowers water activity. Most microorganisms grow best in dilute solutions (high $A<em>w$). As solute concentration rises (lowering $A</em>w$), growth rates decline until a minimal growth-inhibitory $A_w$ is reached.

  • Limiting $A<em>w$ values: 0.95 for Gram-negative rods; 0.9 for staphylococci, micrococci, and lactobacilli; 0.88 for most yeasts. Syrup-fermenting osmotolerant yeasts can spoil products with $A</em>w$ levels as low as 0.73, while some filamentous fungi such as Aspergillus glaucus can grow at 0.61.

• $A<em>w$ can be lowered by adding high concentrations of sugars or polyethylene glycols. Syrup BP (67% sucrose; $A</em>w$ = 0.86) has occasionally failed to inhibit osmotolerant yeasts, requiring additional preservation. Alternative solutes not encouraging dental caries, such as sorbitol and fructose, have been investigated. $A_w$ can also be reduced by drying, but dry, hygroscopic medicines (tablets, capsules, powders, vitreous glasses) require suitable packaging to prevent water resorption and microbial growth.

• Tablet film coatings reduce water vapour uptake during storage, increasing microbial stability in humid climates, though foil strip packing may be more effective but expensive.

• Condensed water films can accumulate on the surface of 'dry' products like tablets or bulk oils following storage in damp atmospheres with fluctuating temperatures, creating sufficiently high localized $A_w$ to initiate fungal growth. Condensation formed on the surface of viscous products like syrups and creams, or exuded by syneresis from hydrogels, may allow surface yeast and fungal spoilage.

Redox Potential

• Microbial growth is influenced by oxidation-reduction balance (redox potential), as they require compatible terminal electron acceptors for respiratory pathways to function. Redox potential in viscous emulsions may be high due to oxygen solubility in fats and oils.

Storage Temperature

• Spoilage can occur from −20 °C to 60 °C, though less likely at extremes. Storage temperature selectively determines microorganisms involved in spoilage. Deep freeze at −20 °C or lower used for long-term storage of materials and short-term storage of TPN feeds. Reconstituted syrups and multidose eye drop packs are dispensed with instructions to store in a cool place (2–8 °C). Water for Injections (EP) should be held at 80 °C or above after distillation and before sterilization to prevent regrowth of Gram-negative bacteria and endotoxin release.

pH

• Extremes of pH prevent microbial attack. Bacterial spoilage is more likely around neutrality, with pseudomonads and related Gram-negative bacteria growing in antacid mixtures, mouthwashes, and distilled/demineralized water. Spoilage is rare above pH 8 (e.g., soap-based emulsions). Mold or yeast attack is more likely in products with low pH levels (e.g., fruit-juice-flavored syrups with pH 3–4). Yeasts can metabolize organic acids and raise pH to levels where secondary bacterial growth can occur. Low pH adjustment to preserve foodstuffs is well-established but not practical for medicines.

Packaging Design

• Packaging influences microbial stability by controlling entry of contaminants during storage and use. Containers are designed to prevent ingress of contaminants into parenteral medicines. Self-sealing rubber wads prevent microbial entry into multidose injection containers. Wide-mouthed cream jars are replaced by narrow nozzles and flexible tubes to reduce operator-introduced contamination. Hand creams, previously in glass jars, are now in closed, disposable dispensers. Medicines relying on low $A_w$ for spoilage prevention require water-vapour-proof packaging with efficient seals. Cardboard outer packaging and labels can become substrates for microbial attack in humid conditions, so preservatives are often included to reduce damage.

Protection of Microorganisms Within Pharmaceutical Products

• Microorganism survival is influenced by inert materials. Microbes can be more resistant to heat or desiccation in the presence of polymers like starch, acacia, or gelatin. Adsorption onto particulate material may aid establishment and survival. Suspended particles like kaolin, magnesium trisilicate, or aluminum hydroxide gel may influence contaminant longevity. Surfactants, suspending agents, and proteins can increase microorganism resistance to preservatives, beyond their direct inactivating effect on the preservative itself.

Hazard to Health

• Inadvertent use of contaminated products poses a health hazard. Outbreaks of medicament-related infections have been reported since the early 20th century, with increased understanding in the 1960s and 1970s.

• True pathogens such as Salmonella spp. and associated infections attract attention. Common saprophytic and non-fastidious opportunist contaminants with limited pathogenicity may present a challenge to compromised patients.

• Gram-negative contaminants, particularly Pseudomonas spp., multiply in aqueous products and cause numerous outbreaks. Intact cornea is resistant to infection but offers little resistance to pseudomonads when scratched. Loss of sight has occurred following the use of poorly designed ophthalmic solutions contaminated by Pseudomonas aeruginosa, which supports its growth. Pseudomonas-contaminated antiseptic solutions have infected the skin of badly burnt patients, resulting in skin graft failure and death from Gram-negative septicaemia. Infections of eczematous skin and respiratory infections in neonates have been traced to ointments and creams contaminated with Gram-negative bacteria. Oral mixtures and antacid suspensions can support the growth of Gram-negative bacteria and serious consequences have resulted following their inadvertent administration to patients who were immunocompromised.

Microbial Toxins

• Gram-negative bacteria contain lipopolysaccharides (endotoxins) in their outer cell membranes (Chapter 22); these remain active after cell death and some survive moist heat sterilization. Endotoxins induce physiological effects if they enter the bloodstream via infusion fluids or diffusion across membranes, even in nanogram quantities. Such effects include fever, cytokine activation, endothelial cell damage, leading to septic and fatal febrile shock.

• Acute bacterial toxins associated with food poisoning are not commonly reported in pharmaceutical products, although aflatoxin-producing aspergilli have been detected in some vegetable/herbal ingredients. Metabolites of microbial deterioration have unpleasant tastes and smells, deterring patients from using the medicine.

Sources and Control of Contamination

In Manufacture

• The microbiological quality of the finished product depends on formulation components, manufacturing environment, and the manufacturing process itself.

  • Raw materials, especially water and those of natural origin, must be of high microbiological standard.

  • All processing equipment should undergo planned preventive maintenance and be properly cleaned after use to prevent cross-contamination.

  • Cleaning equipment should be appropriate and maintained properly.

  • Manufacture should occur in suitable premises with filtered air, varying according to the product type.

  • Staff should have good health and knowledge of personal/production hygiene.

  • The end-product requires suitable packaging to protect against contamination and be free from contamination.

Hospital Manufacture

• Raises certain additional problems with regard to contamination control.

Water

• Mains water in hospitals is frequently stored in large roof tanks, some of which may be relatively inaccessible and poorly maintained. Water for pharmaceutical manufacture requires further treatment, usually by distillation, reverse osmosis or deionization or a combination of these, depending on the intended use of water. Storage of water requires particular care, as some 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 the distribution system at a fl ow rate of 1 – 2 m/s to prevent the build - up of bacterial biofi lms in the piping.

Environment

• The microbial flora of the hospital pharmacy environment is a reflection of the general hospital environment and the activities undertaken there. Cleaning equipment, such as mops, buckets, cloths and scrubbing machines, may be responsible for distributing these organisms around the pharmacy; if stored wet they provide a convenient niche for microbial growth, resulting in heavy contamination of equipment.

Packaging

• Sacking, cardboard, card liners, corks and paper are unsuitable for packaging pharmaceuticals, as they are heavily contaminated, for example with bacterial or fungal spores. These have now been replaced by non - biodegradable plastic materials.

In Use

• Pharmaceutical manufacturers' responsibility ends with the supply of a well - preserved product of high microbiological standard in a suitable pack and that the subsequent use, or indeed abuse, of the product is of little concern to them. Although much less is known about how products become contaminated during use, their continued use in a contaminated state is clearly undesirable, particularly in hospitals where it could result in the spread of cross - infection.

Human Sources

• During normal usage, patients may contaminate their medicine with their own microbial flora; subsequent use of such products may or may not result in self - infection. Topical products are considered to be most at risk, as the product will probably be applied by hand, thus introducing contaminants from the resident skin flora.

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.

Equipment Sources

• Patients and nursing staff may use a range of applicators (pads, sponges, brushes and spatulas) during medicament administration, particularly for topical products. If reused, these easily become contaminated. In hospitals today a wide variety of complex equipment is used in the course of patient treatment. Chemical disinfectants used for this purpose have in the past, through misuse, become contaminated with opportunist pathogens, such as Ps. aeru- ginosa , and ironically have contributed to, rather than reduced, the spread of cross - infection in hospital patients.

The Extent of Microbial Contamination

In Manufacture

• Investigations carried out by the Swedish National Board of Health in 1965 revealed some startling findings on the overall microbiological quality of non - sterile products immediately after manufacture.

  • Two hundred patients were involved in an outbreak of salmonellosis, caused by thyroid tablets contaminated with Salmonella.

  • Eight patients had severe eye infections following the use of a hydrocortisone eye ointment contaminated with Pseudomonas aeruginosa.

• Hospital manufacturing operations were later rationalized and Competitive purchasing from industry produced cheaper alternatives, and small - scale manufacturing was largely discouraged.

• Removal of Crown immunity from the NHS in 1991 meant that manufacturing operations in hospitals were then subject to the full licensing provisions of the Medicines Act 1968

In Use

• Higher rates of contamination are invariably seen in products after opening and use and, among these, medicines used in hospitals are more likely to be contaminated than those used in the general community.

Factors Determining the Outcome of a Medicament-borne Infection

Type and degree of microbial contamination

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

Route of administration

• As stated previously, 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.

Resistance of the patient

• A patient’s resistance is crucial 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, elderly people, diabetics and patients traumatized by surgery or accident may have impaired defense mechanisms.

Preservation of Medicines Using Antimicrobial Agents: Basic Principles

Introduction

• An antimicrobial 'preservative' may be included in a formulation to minimize the risk of spoilage and preferably to kill low levels of contaminants introduced during storage or repeated use of a multidose container. However, where there is a low risk of contamination, as with tablets, capsules and dry powders, the inclusion of a preservative may be unnecessary.

Effect of preservative concentration, temperature and size of inoculum

• For example, halving the concentration of phenol ($\eta = 6$) gives a 64-fold ($2^6$) reduction in killing activity, whereas a similar dilution for chlorhexidine ($\eta = 2$) reduces the activity by only four-fold ($2^2$).

Factors Affecting the ‘Availability’ of Preservatives

Effect of product pH

• In the weakly acidic preservatives, activity resides primarily in the unionized molecules and they only have significant efficacy at pH values where ionization is low.

  • e.g. benzoic and sorbic acids ($pKa = 4.2$ and $4.75$, respectively) have limited preservative usefulness above pH 5

Efficacy in multiphase systems

• In a multiphase formulation, such as an oil-in-water emulsion, preservative molecules will distribute themselves in an unstable equilibrium between the bulk aqueous phase and the oil phase by partition.

Effect of Container or Packaging

• Preservative availability may be appreciably reduced by interaction with packaging materials. Phenolics, for example, will permeate the rubber wads and teats of multidose injection or eye drop containers and also interact with flexible nylon tubes for creams.

Quality Assurance and the Control of Microbial Risk in Medicines

Introduction

• Manufacturers of medicinal products must comply with the requirements of their marketing authorization (product license) and ensure that their products are fit for their intended use in terms of safety, quality and efficacy.

Quality assurance in formulation design and development

• The risk of microbial infection and spoilage arising from microbial contamination during manufacture, storage and use could be eliminated by presenting all medicines in sterile, impervious, single - dosage units.

Good pharmaceutical manufacturing practice (GPMP)

• GPMP is concerned with the manufacture of medicines, and includes control of ingredients, plant construction, process validation, production and cleaning
  (see also
  Chapter 23 ). Current GPMP (cGPMP) requirements are found in the Medicines and Healthcare Products
  Regulatory Agency (MHRA) Rules and Guidance for Pharmaceutical Manufacturers and Distributors, known
  as the Orange Guide (Anon 2007), and its 20 annexes.

Quality control procedures

• While there is general agreement on the need to control total microbial levels in non - sterile medicines and to exclude certain species that have previously proved troublesome, the precision and accuracy of current methods for counting or even detecting some microbes in complex products are poor. Pathogens, present in low numbers, and often damaged by processing, can be very difficult to isolate.

Postmarket surveillance

• Despite extensive development and a rigorous adherence to procedures, it is impossible to guarantee that a medi- cine will never fail under the harsh abuses of real - life conditions.