It is a truism in ecology that as the complexity of ecosystems is reduced, the diversity of the fauna and flora is markedly reduced because of reduction in niches in the ecosystem. Because water for pharmaceutical purposes differs from potable water in seasonal variations in dissolved organic matter, tem- perature, and bacterial content, it is not unexpected that the microbial di- versity in purified water and water for injection is considerably less than potable water and water for injection (Table 14). As stated above, the bac- terial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments [15].
A major concern of water companies is the persistence of coliform bacteria in biofilms formed on the interior of pipes used to distribute the water that may be shed into the water distribution system. Occasional failures of coliform testing during the summer months (i.e., one total coliform in a 100- mL sample) have been attributed to seasonal biofilm formation and may have public health implications. Similarly, there is a potential for biofilm devel- opment in the distribution system for purified water. The temperature, low dissolved organic matter, aeration, absence of chlorine, uneven levels of de- mand, and recirculation all favor biofilm formation. Strategies to reduce biofilm formation include high turbulence, absence of doglegs, flushing of taps when drawing off water, and periodic sanitization by hot water, steam, or ozone treatment. Biofilms were the subject of a recent review article [21].
With the amount of dissolved organic matter in potable water up to three magnitudes higher than water for pharmaceutical purposes, the ability of these waters to support bacterial growth is probably limited. For example,
if the dry weight of water-borne bacterium is 10 13g, of which 50% is carbon, then water for injection containing 100 ppm of TOC (i.e., 10 9g/L) should be able to support up to 2 104bacteria. In general, water-borne bacteria adopt three different strategies to the nutrient level. When utilizable substrate is growth-limiting, then slow-growing bacteria with a high substrate affinity are favored, whereas when substrate is in excess, fast-growing bacteria are fa- vored [29]. The third strategy used is pharmaceutical water systems in biofilm formation on surfaces. Microbial habitats as diverse as oceanic waters and water for injection share the characteristic of extremely low utilizable sub- strate where bacteria with high substrate affinity may predominate.
A perennial question asked in pharmaceutical discussion groups is whether water for injection that is maintained at 80jC needs to be monitored for thermophilic bacteria. Thermophiles [16] are a diverse group of Archaea and bacteria that include photosynthetic bacteria, chemolithoautotrophic and heterotrophic aerobic Archaea and bacteria, and anaerobic Archaea and bacteria (Tables 15 and 16). In general, the nutritional requirements of thermophiles (e.g., rich nutrients, vitamins, light, electron acceptors, anaer- TABLE14 Parameters of Potable Water, Purified Water, and Water for Injection
Parameter Potable water Purified water
Water for injection Temperature Ambient temperature: 5–20jC Room temperature: 20–25jC >60jC Total organic carbon NMT 500 mg/L (total dissolved solids); typical ranges: 1–20 mg/L (surface water) and 0.1–2 mg/L (ground water) NMT 0.5 mg/L NMT 0.5 mg/L
Recirculation Demand-driven Recirculated Recirculated Residual chlorine Greater than 0.2 mg/L None None Microbial content NMT 500 cfu/mL NMT 100 cfu/mL NMT 1 cfu/ 100 mL
Total coliforms Zero in 100 mL NA NA
TABLE 15 Representative Thermophilic Archaea Class of thermophiles Representative organisms Temperature maximum/ temperature optimum pH optimum Methanogenic anaerobes Methanobacterium thermoautotrophicum 70–110jC/ 55–98jC 5.7–7.7 Aerobic thermoacidophiles Thermoplasma acidophilum 65–96jC/ 60–90jC 1.5–3.0 Sulfolobus acidocaldarius Acidianus inferus Anaerobic thermoacidophiles Thermococcus celer Pyrococcus woesei Thermoproteus neutrophiles 90–110jC 5.5–7.0
TABLE 16 Representative Thermophilic Bacteria
Class of thermophiles Representative organisms Temperature maximum/ temperature optimum pH optimum Aerobes Bacillus stearothermophilus 65–85jC/ 55–75jC 2.0–8.0 Thermus aquaticus Thermoleophilum album Anaerobes Clostridium stercorarium 65–90j C/ 60–75jC 5.7–8.0 Desulfovibrio thermophilus Thermotoga neapolitana
obic conditions, and elevated temperatures) make it highly unlikely that thermophiles will exist in hot water of injection. If they did persist in water for injection, they would not grow in the human body, which has a temperature around 37jC. Given this situation, monitoring pharmaceutical-grade waters for thermophiles is not recommended.
11. CONCLUSIONS
As stated earlier for pharmaceutical companies manufacturing drug products for the international market, a water monitoring strategy that accommodates both the USP and Ph. Eur. requirements must be developed. The author recommends that the USP-recommended methods (because of their 48- to 72- hr incubation time, ease of subculture of isolates, and ability to readily isolate fungi) be used for routine monitoring, whereas the Ph. Eur.-recommended methods with a 5-day incubation time be run periodically (i.e., monthly) so that a testing history is available to certify that, if tested, the water system will meet the Ph. Eur. requirements.
The methods are for purified water–pour plate or membrane filtration using plate count, R2A, or m-CPC agar, with a minimum sample size of 1 mL, incubated at 30–35jC for up to 48 hr, and for water for injection membrane filtration using plate count or R2A agar with a minimum sample size of 100 mL incubated at 30–35jC for up to 48 hr. The recommended membrane filters are 0.45-Am gridded membrane filters.
REFERENCES
1. Beuchat LR, Frandberg E, Deak T, Alzamora SM, Chen J, Guerrero S, Lopez- Malo A, Ohlsson I, Olsen M, Peinado JM, Schnurer J, de Siloniz MI, Tornai- Lehoczki J. Performance of mycological media in enumerating desiccated food spoilage yeasts: an interlaboratory study. Int J Food Microbiol 2001; 70(1– 2):89–96.
2. Brock TD, Koch R. A Life in Medicine and Bacteriology. Madison, WI: Sci- ence Tech Publishers, 1988.
3. Brozel VS, Cloete TE. Evaluation of nutrient agars for the enumeration of viable aerobic heterotrophs in cooling water. Water Res 1992; 28(8):111–1117. 4. Carter J. Evaluation of recovery filters for use in bacterial retention testing of
sterilizing-grade filters. PDA J Pharm Sci Technol 1996; 50(3):147–153. 5. Clark HF, Geldreich EE, Jeter HL, Karbler PW. The membrane filter in san-
itary bacteriology. Public Health Rep 1951; 66:951.
6. Colwell RR. Bacterial death revisited. In: Colwell RR, Grimes DJ, eds. Non- Culturable Microorganisms in the Environment. Washington, DC: ASM Press, 2000.
7. 40CFR Part 141, Monitoring Requirements for Public Drinking Water Supplies.
8. Geldreich EE. Microbial Quality of Water Supply in Distribution Systems. CRC Press, Inc., 1996:504.
9. Geldreich EE, Allen MJ, Taylor RH. Interferences to coliform detection in potable water supplies. In: Hendricks CW, ed. Evaluation of the Microbio- logical Standards for Drinking Water. Washington, DC: U.S. Environmental Protection Agency, 13–30.
10. Governal RA, Yahya MT, Gerba CP, Shadman F. Oligotrophic bacteria in ultra-pure water systems media selection and process component evaluations. J Ind Microbiol 1991; 8(4):223–228.
11. Guyomard S. Validation of a scanning laser system for microbiological quality control (QC) analysis. Pharm Technol Int 1997; 9(Sept):50, 52, 54.
12. Haas CN, Meyer MA, Paller MS. Analytical note: evaluation of the m-SPC method as a substitute for the standard plate count in water microbiology. J AWWA 1982; 74, 322.
13. Klein DA, Wu SA. Factors to be considered in heterotrophic microorganism enumeration from aquatic environments. Appl Microbiol 1974; 27:429–431. 14. Leclerc H, Mossel DA, Edberg SC, Stuijk CB. Advances in the bacteriology of
the coliform group: their suitability as markers of microbial water safety. Annu Rev Microbiol 2001; 55:201–234.
15. Kawai M, Matsutera E, Kanda H, Tani K, Yamaguchi N, Nasu M. Dominant bacteria in a viable but non-culturable state in pharmaceutical water. 2002: Abstracts of the General Meeting of the American Society for Microbiology. 2001; 101:639–640.
16. Kristjansson JK, Stetter KO. Thermophilic Bacteria. In: Kristjansson JK, ed. Thermophilic Bacteria. Boca Raton, FL: CRC Press, Inc., pp. 2–13.
17. Lillis, Bissonnette. Detection and characterization of filterable heterotrophic bacteria from rural groundwater supplies. Lett Appl Microbiol 2001; 32(4): 268–272.
18. Lombardo LR, West PR, Holbrook JL. A comparison of various media and incubation temperatures used in the Heterotrophic Plate Count analysis. Water Quality Technology Conference, AWWA, Denver, CO, 1985:251–270. 19. Maki JS, LaCroix SJ, Hopkins BH, Staley JT. Recovery and diversity of het-
erotrophic bacteria from chlorinated drinking waters. Appl Environ Microbiol 1986; 51:1047–1055.
20. Marino G, Maier C, Cundell AM. A comparison of the MicroCount Digital System to plate count and membrane filtration methods for enumeration of microorganisms in water for pharmaceutical purposes. PDA J Pharm Sci Technol, 1999.
21. Marshall KC. Starved and non-culturable microorganisms in biofilms. In: Colwell RR, Grimes DJ, eds. Non-Culturable Microorganisms in the Envi- ronment. Washington, DC: ASM Press, 2000.
22. Means EG, Hanami L, Ridway GF, Olson BH. Evaluating media and plating techniques for enumerating bacteria in water distribution systems. J AWWA 1981; 73:585.
23. Montgomery DC. Introduction to Statistical Quality Control. 4th ed. New York: John Wiley and Sons.
24. Muller G. Lactose–fushsin plate for detection of E. coli in drinking water. Z Hyg Infektionskr 1947; 127:187–190.
25. Pass T, Wright R, Sharp B, Harding GB. Culture of dialysis fluids on nutrient- rich media for short periods at elevated temperatures underestimate microbial contamination. Blood Purif 1996; 14(2):36–145.
26. Reasoner DJ, Geldreich EE. A new medium for the enumeration and subcul- ture of bacteria from potable water. Appl Environ Microbiol 1985; 39(10):1–7. 27. Reasoner DJ. Monitoring heterotrophic bacteria in potable water. In: McFeters GA, ed. Drinking Water Microbiology: Progress and Recent Developments. Berlin: Springer-Verlag, 1990:452–477.
28. Reynolds DT, Fricker CR. Application of LASER scanning for the rapid and automated detection of bacteria in water samples. J Appl Microbiol 1999; 86(5):785–795.
29. Schlegel HG, Jannasch HW. Prokaryotes and their habitats. In: Starr MP, Stolp H, Balows A, Truper HG, Dworkin M, Schlegel HG, eds. Prokaryotes. Berlin: Springer-Verlag, 1981:43–82.
30. Sharpe AN. Development and evaluation of membrane filtration techniques in microbial analysis. In: Patel PH, ed. Rapid Analysis Techniques in Food Mi- crobiology. Glasgow, Scotland: Blackie Academic and Professional, 29–60. 31. Shirley JJ, Bissonette GK. Detection and identification of groundwater bacteria
capable of escaping entrapment on 0.45-micron-pore-size membrane filters. Appl Environ Microbiol 1991; 57(8):2251–2254.
32. Standard Methods for the Examination of Dairy Products. 16th ed. Wash- ington, DC: American Public Health Association, 1992.
33. Standard Methods for the Examination of Water and Waste Water. 20th ed. Washington, DC: American Public Health Association, 1998.
34. Szewzyk U, Szewzyk W, Schleifer K-H. Microbiological safety of drinking water. Annu Rev Microbiol 2000; 54:81–127.
35. Wallner G, Tillman D, Haberer K. Evaluation of the ChemScan System for rapid microbiological analysis of pharmaceutical water. PDA J Pharm Sci Technol 1999; 53(2):70–74.
36. USP General Informational Chapter <1231> Water for Pharmaceutical Purposes.
37. Van der Linde BT, Lim JMM, Rondeel LPMT, Antonissen, De Jong GMT. Improved bacteriological surveillance of hemodialysis fluids: a comparison between Tryptic Soy Agar and Reasoner’s 2A media. Nephrol Dial Transplant 1999; 14(10):2433–2437.
38. www.microbeworld.org/cissues/wqual 39. www.epa.gov/safewater