#34: What’s lurking in your water system? Bacteria, design, control and sampling

A working knowledge of the microbial ecology of water is of importance to those working in the pharmaceutical and healthcare fields. Water is the most widely used raw material - as a constituent of many products. It is also a cleaning agent, a temporary suspending agent, diluent, or used for cooling processes.

As outlined in this week’s article, to deliver pharmaceutical grade water of the right quality, attention needs to be made to design and control, supported by a sound sampling regime.

Water and microorganisms

Water is both a potential source of contamination; a life source for microorganisms and provision of optimal conditions for many microorganisms; and it may also act as a vector for transmitting contamination and encouraging the proliferation of contaminants.

The quality of the raw water, its treatment, storage and distribution all have an influence on its microbial quality. The types of aquatic bacteria recovered from pharmaceutical grade water systems will be either:

Autochthonous

• Originating within the water system.
• Consequence of poor design: mains to purified water to Water for Injections.

Allochthonous

• Introduced from a different environment.
• Consequence of poor maintenance or repairs. Plus impact of shutdowns and subsequent recovery.

Natural inhabitants of freshwater include Pseudomonas spp., Alcaligenes spp., Flavobacterium spp., Chromobacterium spp., and Serratia spp., and others, with some variations due to geographical locales and methods of treatment. Examinations of water supplies tend to indicate that 90 to 98% of the contaminants are Gram-negative bacteria, with additional implications for pyrogen presence. Other organisms isolated include Micrococcus spp., and Cytophaga spp.

Based on sampling, water testing regimes are biased towards heterotrophic bacteria (heterotrophs  need an external source of organic carbon for growth) as a result of the common culture media and incubation conditions (such as plate count agar or R2A).

Many of these bacteria may be considered as opportunistic pathogens (that is, pathogenic if provided with the right opportunity). The main concern with these organisms is that they have simple nutritional requirements and often have a relatively low optimum growth temperature. This enables them to not only survive but also multiply using traces of organic matter present in treated water, such that in some situations they might be present in high numbers, perhaps in excess of 10^6 CFU / ml at room temperature.

As indicated above, bacteria may also be introduced to water by indirect means, for example soil erosion resulting from heavy rainfall, and decaying plant matter might lead to an increase in the number of Bacillus species and Enterobacter species, as examples. Similarly, contamination by sewage results in the presence of Proteus spp., Escherichia coli and other enterobacteria. However, in many cases microorganisms that are derived from animal or plant debris do not survive for long due to unfavorable growth (nutrient) conditions.

Water systems contain higher numbers of bacteria that not culturable using standard methodologies. These are the so-termed “viable but non-culturable” organisms..

This level of contamination means that water supplied to pharmaceutical facilities requires processing. The water reaching the facility must be of potable water standard.

As water goes through purification, the organisms recovered will be mainly non-fermenting Gram-negative bacteria, with a high level of taxonomic biodiversity. There should be very few Gram-positive bacteria are isolated; those that are identified are typically Bacillus spp. The numbers and sizes of the bacteria will vary according to the supply of nutrients and other aspects of the microenvironment, such as pH, water flow, opportunities for surface attachment and so on.

Potable water

Potable water generally has a microbiological specification of no more than 500 CFU / mL (or as per national water standards) and the absence of Enterobacteriaceae. The quality of water will vary both from time to time and from place to place. Also of significance is where and when a sample is taken.

Often sampling is easier through a break tank; however, this represents a significant source of potential contamination because on prolonged storage microorganisms either settle out or attach themselves to the storage vessel surfaces and grow as biofilm communities. This results in the so called ‘bottle effect’, whereby bacterial growth and activity are substantially enhanced through growth as a biofilm (due primarily to increased nutrient trapping and concentration) as opposed to a free floating (planktonic) lifestyle.

Pharmaceutical grade water

There are many grades of water used in the pharmaceutical industry. Water for manufacturing may be water purified by ion-exchange, reverse osmosis or distillation. Water used for parenteral products must be apyrogenic and is usually produced in a specially designed still (although reverse osmosis is permissible).

Purified water

Water purification begins with deionization. Purified water is used extensively in the manufacture of tablets, syrups, suspensions, creams, lotions and for washing of all manufacturing equipment.

Purified water is prepared by passing potable water through anion and cation exchange resin beds to remove the ions. Any bacteria present in the mains water will therefore be present in the deionized water. Deionization beds are prone to contamination because they must be protected from the corrosive potential of chlorine which acts as a bacteriostat in potable water. Those beds that are not regenerated frequently with strong acids or alkali are often heavily contaminated. Consequently, there is a lot of emphasis on the development of new resins that are able to resist microbial contamination.

The common process for completing the purification is with the use of reverse osmosis. The process of producing water by reverse osmosis involves forcing water by an osmotic pressure through a semi-permeable membrane which acts as a molecular filter. Soluble materials dissolved in the water are impeded and those with a molecular weight in excess of 250 do not diffuse at all. In this manner microorganisms, and pyrogens, are removed, resulting in water of suitable purity being produced.

Contamination may, however, occur in the storage vessel on the distribution system if they are not kept free from microorganisms. Care must also be taken to disinfect the membrane at regular intervals, otherwise an opportunity is provided for biofilm formation. This interval will be determined by the results of regular sampling but will probably be of the order of once per month, depending on use.

The microbiological limit applying to purified water is normally in the region of no more than 100 CFU / ml.

Water for Injections

Water for Injections can be prepared by reverse osmosis or by distillation. With distillation, the still needs to be designed to prevent the entrainment of water droplets. As it leaves the still, distilled water is free from microorganisms, but its microbiological quality can deteriorate quickly as a result of a fault in the cooling system, the distribution system or incorrect storage conditions.

Water prepared in this way can either be used immediately for the preparation of injections, provided they are sterilized within 4 hours of water collection or stored at a temperature of 80°C to prevent bacterial growth and consequent pyrogen production.

Although cold water systems for WFI exist, optimally water should be stored at temperatures in excess of 65°C (usually 80°C) and circulated in the distribution system at a flow rate of 1-2 meters per second to prevent build-up of bacterial biofilm in the piping.

Owing to its high manufacturing cost, distilled water is usually used only for parenteral manufacture either as an ingredient or as a pyrogen-free rinsing agent for product contact surfaces. It can on occasion be used in the formulation of oral and topical pharmaceutical products where a low bacterial count is acceptable.

Ongoing controls

The microbiota of contaminated pharmaceutical water is usually Gram-negative bacteria (commonly Pseudomonas spp.). When water systems become contaminated, the use of the appropriate method depends on what is causing the microbial deterioration, the source of the problem, the quality of the water required and the volume to be treated and the type of distribution system.

The design of a system is influential on the size of the microbial populations and the ability of the user to remove them. Dead-legs, long pipework runs to taps, un-drainable pipes and U-bends all create microbiological problems once installed.

Three methods are routinely used for treating water: heat, chemicals, filtration or UV light. Chemical treatment (e.g. sodium hypochlorite, chlorine dioxide, ozone) is applicable to raw, mains water, but can also be used to treat distribution systems of water produced by distillation, deionization and reverse osmosis. The concentration of the chemical used will vary depending upon the location of the water in the distribution system.

Membrane filtration, using a 0.22µm porosity-filter, is useful where the usage is moderate, and a continuous circulation of water can be maintained. Thus, with the exception of that drawn off for use, water is continually being returned to the storage tank and re-filtered. In principle, filtration works well but is relatively expensive for high throughputs because the filters may need regular changing to prevent blockages and ‘grow through’. For this reason, use of 0.22µm filters as a means of controlling contamination in waters used directly for product manufacture is frowned upon. In essence, filters should only be used prior to the distribution process.

Ultraviolet radiation (254nm) is used for the disinfection of water of good optical clarity and works particularly well in a re-circulating system where water flows over a multiple lamp system. Caveats are that penetration of UV light into water is small, and any dead bacteria present in the system will further hinder penetration.

The most effective form of control, and remediation, is the use of heat.

Drains

The most heavily contaminated water in pharmaceutical manufacturing facilities is in the drains, the main habitat for Gram-negative microorganisms. Since these may be transferred throughout the facility on the feet and garments of operators who work close to the drains, the locations should be minimized and the drains designed to cope with the expected volumes of water without fear of backflow. Any unused drain should be capped.

Cooling systems

Water of poor microbiological quality may also be used as a coolant on, for example, stirrers. As such, great care should be taken to ensure that all seals are in place and intact, and that no leaks occur.

What does all this mean?

The above factors indicate why water system, design and control is important and why regular monitoring is necessary in order to demonstrate the maintenance of control.

Dr. Tim Sandle is a pharmaceutical microbiologist, please visit the Pharmaceutical Microbiology Resources website for a companion video to this article.

References

• Kulakov, L.A., McAlister, M.B., Ogden, K.L. Larkin, M.J. and O'Hanlon, J.F. ‘Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems’, Appl Environ Microbiol, 2002, 68(4): 1548–1555
• McAlister MB, Kulakov LA, O'Hanlon JF, Larkin MJ, Ogden KL. Survival and nutritional requirements of three bacteria isolated from ultrapure water, J Ind Microbiol Biotechnol. 2002, 29(2):75-82
• Patterson, M. K., G. R. Husted, A. Rutkowski, and D. C. Mayette. Isolation, identification and microscopic properties of biofilms in high-purity water distribution systems. Ultrapure Water, 1991, 8:18-23
• Penna, V.T.C., Martins, S.A.M., Mazzola, P.G. Identification of Bacteria in Drinking and Purified Water during the Monitoring of a Typical Purification System, BMC Public Health, 2002, 2 (13): 1-11
• Sande, T. Characterizing the Microbiota of a Pharmaceutical Water System-A Metadata Study, SOJ Microbiology & Infectious Diseases, 3 92): 1-8
• Thereza V.,  Penna, C., Martins, S.A.M.,  and Mazzola, P.G. Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system, BMC Public Health, 2002, 2 (13): 1471-2458