Biocontrol Science, 2016, Vol. 21, No. 4, 193－201

Review Methods of Rapid Microbiological Assay and Their Application to Pharmaceutical and Medical Device Fabrication

HIDEHARU SHINTANI

Faculty of Science and Engineering, Chuo University, 1-13-27, Kasuga, Bunkyo, Tokyo 112-8551, Japan

Received 29 October, 2014/Accepted 26 January, 2016

There are several rapid microbiological methods becoming available that have useful applica- tions in pharmaceutical and medical devices. They are ATP bioluminescence, fluorescent labeling, electrical resistance, and nucleic acid probes. In choosing to employ rapid methods, the micro- biologist should examine their prospective performances against the specific requirements for that sector. Some methods may require expensive equipment and offer full automation, and others represent only a small investment. The regulatory view of these methods is changing and they still officially have not been approved in medical and pharmaceutical area, but it will still be up to the microbiologist to demonstrate that the method chosen is fit for the purpose intended.

Key words ： Rapid microbiological methods / Bioburden / Pharmaceuticals / Medical devices.

INTRODUCTION screening） and for microbiological quality assurance （MQA）. In the latter instance, the inevitable time delay Traditional microbiological methods of detection, enu- associated with incubation often determines that MQA meration, and identification using mostly culture meth- data are only of retrospective value. Pharmaceutical and ods are so often time-consuming and labor-intensive. medical device production can no longer accommodate These practical considerations often limit the extent to this delay（ Zuluaga et al., 2009; Akselband et al., 2005; which microbiological tests are routinely applied both at Dexter et al., 2003）. Considerable benefit would there- the formulation development stage（ i.e., preservative fore be gained from the introduction of suitable, more rapid methods of microbiological analysis to the phar- maceutical and medical device sector. For these pur- TABLE 1．Features of a rapid methods for application poses, this review article has been prepared. The typical to pharmaceutical and medical device fabrication requirements for such rapid methods are summarized in Rapid Table 1. Sensitive Broad spectrum detection 1. CURRENTLY AVAILABLE RAPID METHODS AND THEIR BRIEF EXPLANATION Potential for specificity Identification The development of rapid methods has been largely Viability assessment fed by the food, dairy, water, and medical diagnostics Simple industries and has resulted in a diverse range of meth- （ ） Potential for automation ods Table 2 . However, they are not always suited to pharmaceutical and medical devices due to the large Reproducible difference in the number of contaminating microorgan- Compatible with sample matrices isms. Their procedures of detection may be direct, in which individual or populations of or- ＊Corresponding author. Tel: +81-42-592-2336, Fax: +81-42- ganisms are directly observed, or indirect, whereby mi- 592-2336, E-mail : hshintan（i a）jcom.zaq.ne.jp crobial metabolism, metabolites, or components can be 194 H. SHINTANI

TABLE 2．A brief explanation of currently available rapid microbiological detection methods Method Detection Principle Direct Fluorescent labeling Stain microorganisms using a viability-indicating fluorophore; direct enumeration, usually after filter capture, by light excitation（ epifiuorescent microscopy or laser scanning） and image analysis

Indirect ATP bioluminescence Light emission from microbial ATP by luciferin/luciferase reaction. Amenable to amplification by intracellular adenylate kinase Carbon dioxide detection Monitoring of microbial metabolism using 14C-radiolabeld substrate to produce 14C-labeled

carbon dioxide. Infrared CO2 detection offers a more acceptable substitute Chromatographic analysis Detection of microbial metabolites and cellular components; gas chromatographic analysis of microbial fatty acid has been employed in identification Dye reduction Monitoring microbial metabolism of specified substrates by color changes in redox dyes; can form the basis of identification profile Electrical resistance Measurement of electrical changes（ conductance, impedance） in specialized media due to microbial growth; enumeration based on time to exceed a specified detection level Enzyme monitoring Detection of microbial enzymes. By using appropriate substrates can form the basis of identi- fication profiles Limulus amoebocyte lysate Detection of（ principally） Gram-negative bacterial lipopolysaccharide by gelation or colori- metric reaction Nucleic acid probes Labeled DNA or RNA probe hybridization to specific target sequences. Amplification of target by the polymerase chain reaction（ PCR） increases sensitivity; competitive quantitative PCR offers enumeration Phage-interaction technology Host-specific bacteriophage infects target cells leading to phage DNA replication. Detection by expression of new protein（ using recombinant phage） or cell lysis

monitored. Some methods may be highly developed labeling（ Pettipher, 1983; Hutcheson et al., 1988; with extensive equipment and information support, Rodrigues and Kroll, 1988; Rodrigues and Kroll, 1990; while others can still be considered to be at relatively Diaper and Edwards, 1994; Nebe-von Caron et al., 1998; early stages of research or currently developed for only Newby, 2000; Van Poucke and Nelis, 2000; DeCory et a narrow application range. A few studies appear able al., 2005）, electrical resistance（ Baynes et al., 1983; to meet the challenges of pharmaceutical and medical Firstenberg-Eden and Eden, 1984; Owens and Wacher- device （ Zuluaga et al., 2009; Akselband et Viveros, 1986; Silley and Forsythe, 1996; Newby, 2000）, al., 2005; Dexter et al., 2003; Newby, 2000）. It is also enzyme monitoring（ Kroll and Rodrigues, 1986; Watling important to remember that the term "rapid" is variously and Leech, 1996; Newby, 2000）, Limulus amoebocyte applied to techniques of 5 min to 24 h duration, the lysate（ Jorgensen and Alexander, 1981; Bussey and definition often reflecting the expectations of the user. Tsuji, 1984; Baines, 2000）, nucleic acid probes（ Bauters However, a method that may be deemed rapid in appli- et al., 1999; Jordan, 2000; Newby, 2000; Dunsmoor et cation with a high bioburden may require an extended al., 2001; Jimenez et al., 2001; Serin et al., 2005; Chaieb enrichment period in situations of lower or injured et al., 2007; Zuluaga et al., 2009）, phage-interaction bioburden. technology（ Wolber and Green, 1990; Turpin et al., Useful discussion and comparison of the principal 1993; Stewart et al., 1996; Stewart et al., 1998; Mole et methods can be found in the following works: general al., 1999; Wu et al., 2001）, and carbon dioxide radiom- aspects（ Jarvis and Easter, 1987; Balows et al., 1989; etry（ Cutler et al., 1989）. In addition to the methods Stannard et al., 1989; Blackburn, 1993; Watling and summarized in Table 2, other techniques have been in- Leech, 1996; Stewart, 1997; Geis, 2006）, adenosine vestigated but have received only modest development. triphosphate（ ATP） bioluminescence（ Jago et al., These include electrochemical methods（ Patchett et al., 1989; Stanley et al., 1989; Stewart et al., 1989; Stewart, 1989; Yang, 2008）, electronic particle counting 1990; Stewart, 1997; Dexter et al., 2003）, fluorescent （Kubitschek, 1969）, microcalorimetry（ Forrest, 1972; RAPID ASSAY AND APPLICATION 195

TABLE 3．Principal areas of application for rapid microbiological detection methods in pharmaceutical and medical device production Area Application Product quality assessment Microbial limit tests for raw materials and final nonsterile products（ includes total viable count and detection of pathogens） Sterility tests Process hygiene In-process samples Site hygiene Air quality Preservative efficacy Screening potential preservatives Examining the influence of formulation on preservative behavior Challenge testing Sterilizer testing Biological indicators

Beezer, 1980; Watling and Leech, 1996）, biophotome- 2.1 Product Quality Assessment try（ Thomas et al., 1985）, and flow cytometry The pharmaceutical and medical device industry has （Akselband et al., 2005）. Practical details of these tended to be conservative in its approach to rapid meth- methods potentially applicable to the examination of mi- ods for assessment of product quality. This is largely croorganisms attached to medical devices（ bioburden） because of the regulatory constraints imposed upon can be found done in Denyer et al.（ 1993）. these products. For this reason, much of the informa- We recognize that many different types of , tion accumulated in Table 4 is drawn from related indus- while remaining physiologically active, can enter periods tries, but using comparable products and environments. of nonculturability: in this form they are termed viable Table 4 clearly demonstrates the current low probability but nonculturable（ VBNC）（ Colwell, 1987; Kell et al., that any single method will satisfy the requirements for 1998; McDougald et al., 1998）. This may be an adap- all types of pharmaceutical and medical device applica- tive response to inimical environments; there is an evi- tion, although some manufacturers are now seeking dence that this characteristic may be the dominant form methods applicable to a collection of related products. in some environmental niches（ Bloomfield et al., 1998）. It is unlikely that any rapid method can be immediately VBNC microorganisms are theoretically capable of prod- applied in a wide range of situations without first under- uct spoilage and may be a potential infectious threat taking extensive protocol development. The sensitivity （Colwell et al., 1996; Rahman et al., 1996）. It is per- of all methods can be enhanced by sample enrichment haps reassuring therefore to discover that direct fluores- but this will lead to an inevitable increase in analysis cent staining（ labeling） techniques offer a suitable ap- time; additionally, contaminants grow at different rates proach to the detection of VBNC microorganisms（ Kawai and this may result in a substantially different microbial et al., 1999）. The ChemScanR process（ laser scanning flora from the original sampled product. In sterility test- cytometry） routinely shows water bioburdens in excess ing, where the bioburden is quite likely to be low, rapid of those determined by conventional culture, indicative methods generally require sample enrichment or ex- of an otherwise undisclosed VBNC population（ e.g., tended incubation period to reach the microbial levels Wallner et al., 1997）. required for detection, which significantly differs from rapid methods used in the food facilities. Food facilities 2. USE OF RAPID METHODS IN PHAR- have approved the use of rapid methods to detect mi- MACEUTICAL AND MEDICAL DEVICES croorganisms contaminating the food products, but pharmaceutical and medical device facilities have not From a pharmaceutical and medical device perspec- officially approved rapid detection methods yet. tive, the principal areas in which rapid methods may find application are given in Table 3. A method may be 2.2 Process Hygiene required to provide quantitative or qualitative evidence In general, the examination of in-process product of microbial presence（ survival of bioburden）, some samples can utilize the same methods of rapid analysis mechanism of contamination tracking, or to offer rapid as that for raw materials and final products（ Table 4）. confirmation of the absence of microorganisms. Few Surface hygiene assessment, using appropriate swab- methods show complete promise in their range and rel- bing or surface sampling techniques, may require an evance of reported applications（ Table 4）; some of the enrichment period if low counts are expected; similarly, practical implications of their use are considered by large volumes of water or air associated with the pro- Newby（ 2000）. cess may need to be sampled and concentrated by 196 H. SHINTANI

TABLE 4．Some examples of rapid methods applied to the detection of microorganisms Method Sensitivity（ CFU） Limitations Applications Reference ATP bioluminescence >102 , Presence of high levels of Cosmetics/toiletries 1 >102-103 bacteria, nonmicrobial ATP. reduced to 1-10 range with Interfering factors quenching Intravenous fluids 2 enrichment or an MPN-based light or adversely affecting approach. luciferase reaction. Medical devices 3 Packaging materials 4 Sterility testing 5 Surface hygiene 6 Water 7 Electrical resistance Threshold for detection Narrow spectrum of detectable Cosmetics 8 -106 /mL organisms without careful media selection; may be Preservative testing 9 overcome with indirect Sterility testing 10 impedance method Toiletries 11 Water 12 Fluorescent labeling Air, >105; liquid, generally Cannot be applied to highly Air 13 DE（ F） T 103-104/mL but down to viscous or particulate Intravenous fluids 14 25/mL ; liquid（ + enrichment）, materials. Direct correlation 6 organisms irrespective of with viable count not always Medical devices 15 sample volume possible. Preservative testing 16 Surface hygiene 17 Water 18 Laser scanning Liquid, to single organism Spores must be germinated. Cosmetics/toiletries 19 cytometry level; in flow cytometry, Viable nonculturable（ VNC） Sterility testing 20 -50/mL organisms may be detected Water 21 requiring reappraisal of limits. Nonfiltable products need to be tested by fiow cytometry.

Nucleic acid probes Better than 0.1 using PCR Interference from formulation Air 22 amplification excipients. Nonviable Blood products 23 organisms are also detected Contamination tracing 24 limiting utility. Current Water 25 development focused on specific organisms DE（ F） T: Direct epifluorescent（ filtration） technique Examples besides pharmaceutical and medical devices are also included. 1） Neilsen and van Dellen（ 1989）, Watling and Leech（ 1996）, Anonymous（ 1996）, 2） Bopp and Wachsmith（ 1981）, Anderson et al.（ 1986）, 3） Wassall et al.（ 1997）, 4） Senior et al.（ 1989）, 5） Bussey and Tsuji（ 1986）, 6） Blackburn et al.（ 1989）, Anonymous（ 2001）, 7） Webster（ 1986）, Woolridge（ 1989）, Tanaka et al.（ 1997）, Newby（ 2000）, 8） Kahn and Firstenberg- Eden（ 1984）, Kaiserman et al.（ 1989）, 9） Connolly et al.（ 1983, 1994）, 10） Dal Maso（ 1998）, 11） Watling and Leech（ 1996）, 12） Wilkins et al.（ 1980）, 13） Palmgren et al.（ 1986）, 14） Denyer and Ward（ 1983）, Denyer and Lynn（ 1987）, Denyer et al. （1989）, 15） Ladd et al.（ 1985）, Bridgett et al.（ 1993）, 16） Connolly et al.（ 1993）, 17） Holah et al.（ 1988）, 18） Mittelman et al.（ 1983, 1985）, Newby（ 1991）, Kawai et al.（ 1999）, 19） Newby（ 2000）, 20） Anonymous（ 1995）, 21） Wallner et al.（ 1997）, Gapp et al.（ 1999）, Reynolds and Fricker（ 1999）, Newby（ 2000）, 22） Alvarez et al.（ 1994）, 23） Jordan（ 2000）, 24） Newby （2000）, 25） Atlas（ 1991）, Maiwald et al.（ 1994） RAPID ASSAY AND APPLICATION 197

TABLE 5．Current performance of selected rapid methods Performance of Rapid Method Assay Feature ATP FL ER NAP Rapidity 30 min-24 h -2h 1-24h 4-5h Sensitivity（ CFU） 1-100 1-200 106-10 （with PCR） 0.1 Broad spectrum + + + - Specificity - - +/- - Viability assessment + + + - Simplicity + + + +/- Automation + +/- + +/- Sample compatibility +/- +/- + +/- ATP： ATP bioluminescence; FL： fluorescent labeling; ER： electrical resistance; NAP： nucleic acid probe +, satisfactory; +/-, moderate performance or potential; -, currently limited performance or potential.

filtration to ensure a sufficient microbial bioburden be- with biological indicators is necessary, which is required fore examination. in the sterilization validation and GMP. To ensure that every reasonable opportunity is given for the recovery of 2.3 Preservative Efficacy stressed and injured indicator spores, sometimes a lon- The official preservative efficacy test methods require ger incubation period, often in excess of two weeks, is challenge periods of up to 28 days and the introduction required before assurance of sterilizer efficacy can be of rapid methods in this situation would confer no mean- given; this provides little opportunity for early detection ingful benefit. Where rapid methodology can have an of partial sterilizer failure. A commercial detection sys- important role to play is in the rapid examination of sev- tem in which a spore enzyme, α-glucosidase（ reflective eral candidate preservative systems（ and their possible of spore viability）, converts a nonfluorescent substrate permutations of concentration and combination） for into a fluorescent product within an hour offers one so- use in new or developing formulations. Kinetic data from lution. Other approaches examined include ATP biolu- D-value determinations or estimation of growth-inhibito- minescence to detect spores surviving suboptimal ster- ry concentrations can quickly provide a useful indication ilization processes（ Webster et al., 1988） and in vivo of preservative or formulation incompatibilities and can bioluminescence as a reporter of recombinant spore vi- be used to compare the relative merits of potential pre- ability（ Stewart et al., 1989）. servative systems. Rapid methods have been applied to preservative evaluation（ Denyer, 1990; Connolly et al., 3. CONCLUSION 1993; Connolly et al., 1994）. It is important to distin- guish between those methods used primarily to explore There are several developed rapid microbiological bacteriostatic behavior and those able to examine bac- methods now becoming available that may have useful tericidal activity. In the latter instance, enrichment or ex- applications in pharmaceutical and medical devices; of tended incubation periods may be necessary to detect these, ATP bioluminescence, fluorescent labeling, elec- low numbers of survivors（ bioburden）, thereby extend- trical resistance, and nucleic acid probes appear among ing the overall detection time. the most promising（ Table 5）. Inevitably, no single In a product challenge designed to explore the capaci- method will satisfy easily all requirements, and further ty of a preservative system to withstand repeated mi- development will be needed to adapt them to the spe- crobial insults or to study the ability of spoilage microor- cific demands of the pharmaceutical and medical device ganisms to survive and grow, kinetic information is of situation. In this context, it is encouraging to see the less importance and the detection methods summa- developments in ATP bioluminescence offering increased rized in Table 4 are potentially applicable. sensitivity（ adenylate kinase amplification（ Corbitt et al., 2000））, potential specificity（ phage lysins（ Stewart, 2.4 Sterilizer Testing 1997）, phage lysis（ Wu et al., 2001））, and internal cali- Sterilization protocols require regular microbiological vali- bration against excipient effects（ caged ATP（ Calvert et dation; for some processes, continual efficacy monitoring al., 2000））, while proposed developments using 198 H. SHINTANI

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