Document Text Contents
Page 2
Rapid Microbiological Methods in the
Pharmaceutical Industry
Edited by
Martin C.Easter
Boca Raton London New York Washington, D.C.
Page 98
Parameters observed to influence RAPD profiles and reproducibility References citing confirmatory observations
[DNA] Gao et al. (1996), MacPherson et al. (1993), Meunier and
Grimont (l993)
Preparation Khandka et al. (1997), Micheli et al. (1994), Hilton et al.
(1997)
Secondary structure Caetano-Anolles (1993)
[Primer] Khandka et al. (1997), MacPherson et al. (1993), Meunier
and Grimont (1993)
Length MacPherson et al. (1993), Williams et al. (1990)
G+C content MacPherson et al. (1993), Williams et al. (1990)
Importance of primer selection Berg et al. (1994), He et al. (1994), Welsh and McClelland
(1990)
Ratio Primer/Template Caetano-Anolles (1993), del Tufo and Tingey (1994),
Ellsworth et al. (1993), MacPherson et al. (1993),
Template/DNA polymerase Khandka et al. (1997)
Reaction buffer and pH Hilton et al. (1997)
Thermocycler -Annealing temperature Ellsworth et al. (1993), Meunier and Grimont (1993),
Welsh and McClelland (l990),
-Cycle number and time del Tufo and Tingey (1994), MacPherson et al. (1993),
Meunier and Grimont (1993)
-Extension time Van Leuven (199l), Wilson (1994)
-Ramping Cocconcelli et al. (1995)
-Model del Tufo and Tingey (1994), MacPherson et al. (1993),
Meunier and Grimont (1993), Penner et al. (1993)
-Temperature across block Van Leuven (1991)
RNA contamination Ellsworth et al. (1993), Micheli et al. (1994),
Type of gel Berg et al. (1994)
Interlaboratory -due to Taq Meunier and Grimont (1993) Schierwater and Ender (1993)
-due to thermocycler MacPherson et al. (1993), Meunier and Grimont (1993),
Penner et al. (1993)
Shown not to affect profiles
Reaction buffer components and pH (including additives—gelatin, DMSO) Gao et al. (1996), Meunier and Grimont (1993)
Subculturing Gao et al. (1996)
Plasmid content Elaichouni et al. (1994)
Note: Items in bold represent those factors cited most often as contributing to variability.
Adapted from Tyler et al. 1997. Journal of Clinical Microbiology, 35. (Used with permission of American Society of Microbiobgy,
Washington, D.C.)
serotype (Jersek et al. 1999). In addition, the method has been extended as a means of identifying particular species of
eucaryotic microorganisms (Judd et al. 1993; van Belkum et al. 1992). The rep-PCR technique is quite reproducible and has
moderate discriminatory power.
PCR—restriction fragment length polymorphism (RFLP) is a genotypic method that, in contrast to standard southern
hybridizations, is rapid and only involves individual genes or gene clus ters (Arbeit 1995; Swaminathan and Matar 1993).
Popular targets of PCR-RFLP are virulence genes (Samadpour 1995), because they are commonly associated with only
pathogenic strains of a particular species. This technique incorporates the principles of PCR and REA in one rapid method.
Initially, a target gene is amplified using known primers to a particular virulence region. The resulting amplicon (usually 1 to
2 kb) is then digested with a suitable restriction endonuclease, and the fragments are run out onto an agarose gel and stained with
ethidium bromide to detect polymorphisms in the gene. Theoretically, any known region of interest can be amplified by PCR
and then examined by means of enzyme restriction (e.g., genes coding for flagella [Madden et al. 1998]). Even though prior
knowledge of the DNA region of interest is needed and the discriminatory power of the method tends to vary substantially
depending on the different species, loci, and restriction enzyme, this technique has the advantage of speed, simplicity, and
reproducibility. Investigators have shown the utility of this method by using it not only for typing, but also for strain
identification when applied to the ribosomal RNA region or some species-specific gene sequence. Organisms such as
Mycobacterium spp. (Taylor et al. 1997), S. aureus (Schmitz et al. 1998), Helicobacter pylori (Dzierzanowska et al. 1996),
90 RAPID MICROBIOLOGICAL METHODS
Page 99
Campylobacter (Madden et al. 1998), and L. monocytogenes (Vaneechoutte et al. 1998), have all been typed with this
method.
PCR-ribotyping, a relatively new genotypic typing method, is also based on the PCR reaction (Jensen et al. 1993; Kostman
et al. 1992). The method takes advantage of the heterogeneity found within the spacer regions that exist between the rRNA
subunits (16S, 23S, and 5S rRNA) of prokaryotic microorganisms (Campbell et al. 1993; Loughney et al. 1982), most notably
the 16S-23S intergenic spacer region. The spacer regions separate the rRNA subunits and may show a large degree of
sequence and length variation at both the genus and species level (Campbell et al. 1993; Loughney et al. 1982). Because most
bacterial genera contain multiple copies of the rRNA operon, multiple bands of varying lengths may be obtained from a
particular strain after amplification with primers designed to target the conserved regions flanking the spacer sequences in
different ribosomal operons. As discussed under ribotyping, a distinct advantage of involving the highly conserved rRNA
gene as a target in amplification is the fact that universal primers may be designed that will work across several genera of
bacteria (Jensen et al. 1993). However, as with traditional PCR, one must have some knowledge of the conserved nature of
the rRNA sequence of the particular organism one is trying to type in order to know if primers are adequate. This technique
has been used to type organisms such as S. aureus, Enterococcus faecium, E. coli, Enterobacter spp., and L. monocytogenes
(Kostman et al. 1995; Sontakke and Farber 1995).
Although it has been shown that the greatest variation lies within the 16S-23S spacer region and considerably less in the
23S–5S region, researchers have found that increased discrimination can be obtained in some organisms by employing
primers that span the entire 16S—5S region, so that the spacer region between the 23S and 5S rRNA is also amplified
(Sontakke and Farber 1995). Another approach has been to use restriction endonucleases to cut the amplicons arising from the
initial amplification (Kostman et al. 1992) in order to reveal hidden polymorphisms and thereby increase the discriminating
ability of PCR-ribotyping. In this respect PCR-ribotyping can be thought of as a form of PCR-RFLP utilizing the rRNA gene.
Although not as discriminatory as PFGE and possibly RAPD typing, PCR-ribotyping has the advantage of producing stable,
easily detectable amplification patterns in a rapid manner and has the potential to be widely useful in molecular epidemiology
(Kostman et al. 1992).
Insertion sequence (IS)—PCR takes advantage of the insertion sequence elements that have been discovered in several
bacteria. These elements are mobile genetic units that can vary in physical location within the chromosome of different
isolates and therefore make an appealing target for molecular typing. Primers can be designed to flank the ends of the IS
element and face outward to amplify the intervening space. If two IS elements are close enough, then the PCR operates
efficiently and a DNA fragment is amplified. This approach has been taken with organisms such as E. coli (Thompson et al.
1998), Mycobacterium (Devallois and Rastogi 1997), and S. aureus (Deplano et al. 1997).
Amplified restriction fragment length polymorphism (AFLP) is a DNA fingerprinting technique involving RFLP and PCR
(Janssen et al. 1996; Vos et al. 1995) (see Figure 8.1). Whole genomic DNA is isolated, then digested with a specific combination
of two restriction endonucleases (REs). The first must be a frequently cutting RE such as H MseI , HindIII, or TaqI that
recognizes a specific sequence of 4 bases. This generates numerous small DNA fragments that are in the optimal size range for
amplification and separation. A second infrequent or rare cutting RE is also incorporated into the digestion to further reduce
the number of potential amplicons, because the system is set up to amplify only those fragments having a different restriction
site at each end. This latter feat is accomplished through the use of specially designed double-stranded (ds) adapter sequences
that will ligate to the overhanging ends of the restricted fragments and then be amplified by primers that recognize the adapter
sequence. Two adapters are required, one specific for each of the restriction sites generated. Adapters consist of a core
sequence of 11 to 14 homologous ds bases that are arbitrarily selected, followed by the specific addition of bases at the 5’ end
to provide an overhang suitable for ligation to one of the restriction sites (i.e., an enzyme specific sequence). A convenient
omission that does not affect the results is that adapters are not phosphorylated, and thus only one strand of adapter will ligate
to the restricted fragment. Ligation will, however, occur in several different ways to the various combinations of restriction
fragments generated by the two REs, but only those that ligate in the proper orientation and in the desired combination will be
amplified, because of the design of the primer set. Each single-stranded primer consists of a core sequence that is homologous
to the majority of bases in the core sequence of the respective adapter molecule, plus the 5’ overhanging enzyme specific
sequence. In addition, 1 to 3 arbitrarily selected nucleotides are added to the 3’ end of the primer. Each addition of a selective
base to the AFLP primer reduces the number of amplicons approximately 4-fold and results in a subset of the original
fingerprint. Usually one primer (usually the rare cutter) is radioactively labeled, and primers are annealed at high temperatures
to ensure stringency. In this way, the system will amplify only the specific subset of properly ligated adapter-fragment
hybrids that are homologous for the selective bases added to the 3’ end of the primer. This addition of selective bases
provides a further specificity to the primer and in so doing reduces again the number of potential amplicons. This technique
still results in a DNA fingerprint pattern of approximately 50–100 fragments of varying sizes and thus most typically requires
a denaturing polyacrylamide (sequencing) gel for adequate resolution. Gels are visualized by autoradiography or
phosphoimaging; DNA analysis software is recommended to properly interpret the likelihood of two strains being related
because of the quantity of bands.
TRADITIONAL AND AUTOMATED RAPID METHODS FOR SPECIES IDENTIFICATION AND TYPING 91
Page 196
testing, 42–46.
See also validation biocidal activity, 110–113
clean-in-place (CIP), 95
component, 227
drug, 198
environmental, 95
finished product, 44, 50, 51, 52, 55
food, 180, 181, 182, 183, 233
hygiene, 51
in-process, 44, 95, 198, 224
market, size of, 2
minimum inhibitory concentration (MIC), 76, 110, 111
nonsterile product, 42, 46, 108
preparatory, 200
preservative efficacy, 50, 53, 108, 111
protocols, 216
raw material, 50
release, 44
sterility, 47, 48, 51, 113, 179–185
susceptibility, 125, 143
tests
antimicrobial effectiveness, 35–36, 227
bacterial endotoxin, 196, 199–202
colorimetric, 193
in vitro pyrogen, 197
Inhibition or Enhancement, 200–201
Limulus Amebocyte Lysate (see under Limulus Amebocyte
Lysate (LAL) test)
mandatory (USP) requirements for, 223
Microbial Limits Test (MLT), 3, 250
Most Probable Number (MPN), 15, 16, 52, 67
presence or absence, 163, 180, 250
rabbit pyrogen, 15, 188–189, 190, 196, 203
Sterility of Liquids, 179
tetanus, 11, 12
tetrathionate broths, 27, 28
thermal conductivity detector (TCD), 147
thermal hydrolysis and methylation (THM), 139, 143
thermophilic microorganisms, 24
thioglycolate broth, 26
thioglycollate medium, 113, 117
threshold pyrogenic dose, 190
thyroid-stimulating hormone (TSH), 238, 240
time of flight (TOF), 140
time to detection (TDD), 50, 102, 104
tissue culture media, 182
TMA (transcription mediated amplification), 138, 163, 166–168
TMAO (trimethylamine-N-oxide), 114–115, 116
total viable count, 32, 47, 75, 108, 109
toxic metabolic products, 25
toxicity, selective, 117–118
toxin genes, 130, 163
Toxinometer®, 194, 204
toxins.
See also endotoxin aflatoxin, 62
food-borne, 161
screening for, 49
Streptococcus, 49
traceability, 47
trailing endpoints, 76
training records, 217
transcription mediated amplification (TMA), 138, 163, 166–168
transfusion-transmitted sepsis, 181
TRANSIA, 49
Trichinella, 62
tricolsan, 90
trimethylamine-N-oxide (TMAO), 114–115, 116
trimethylphenylammonium hydroxide (TMAH), 143
triphenyl-tetrazolium chloride (TTC), 65
triple sugar iron agar, 28
tryptone soya bean broth, 113
tryptones, 25
TTC (triphenyl-tetrazolium chloride), 65
tuberculosis, 12
tumors, 117
turbidimetric assay, 193, 204, 205, 206
two-photon emission, 237
Tyler, Kevin, 125
Tyndall, John, 12
Type I Amendment, 93, 219
Type I variation, 219, 229–230
Type II variation, 219, 229, 230
typhoid bacillus, 11, 15
UK Medicines Control Agency (MCA), 46, 52, 93, 227
ultramicrobacteria, 20
universal media, 25, 27
universal primers, 135
updates, 214
urinary tract infections, 144, 189, 206
U.S. Environmental Protection Agency (EPA), 30
U.S. Food and Drug Administration (FDA), 30, 182, 230
bacterial endotoxin preparations, 189–190, 203
Center for Biologics Evaluation and Research (CBER), 190, 221,
222, 227
Center for Drug Evaluation and Research (CDER), 222, 227
draft guideline for validation of Limulus Amebocyte Lysate
(LAL) test, 196
FDA Bacteriological Analytical Manual, 14
Guidance Documents, 222–223, 226, 228–229
guidelines for Limulus Amebocyte Lysate (LAL) test, 197–199
Inspection Guides, 223, 224–226
product licensing, 219
recognition and acceptance, 221–223
U.S. Pharmacopeia, 14, 212, 221
bacterial endotoxin test, 195, 199–202, 203
Endotoxin Reference Standard, 199
General Chapters, 225
microbial limits, 30, 225
monographs, 199, 225
nutrient media of, 26, 27–29
objectionable microorganisms, 30
revision process, 227
standard methods, 31–36
standards, 223
sterility testing, 179, 180, 182, 184
validation, 33, 43, 52, 213
U.S. Standard Endotoxin, 190, 191
USP. See U.S. Pharmacopeia
UV treatment of surfaces, 30
188 RAPID MICROBIOLOGICAL METHODS
Page 197
vaccination, 10
validation, 2, 57, 100, 118, 225–226.
See also ICH Barr decision, 226, 230
cGMP amendments, importance to, 224
criteria, 213–214, 216
definition of, 211–212
of media, 48
of methods, 33, 35, 42–43, 45, 52
Parenteral Drug Association Technical Report No. 33, 6, 45, 52,
213, 221, 250
plan, 214–215
protocol, 215–216, 218
of recovery, 32
summary, 217
support, 45, 47, 53
vancomycin-resistant enterococci (VRE), 132
variations, 219, 229–230
verification, 212, 216
viability staining, 53
viable cells, 36, 108
viable count, 32, 47, 75, 108, 109, 110
viable nonculturable state, 30
Vibrio cholera, 11, 13
Vibrio parahaemolyticus, 64
Vibrio vulnifcus, 136
Vida, 50
video cameras, 72
VIP, 49
virulence genes, 132, 135
viruses, 146, 196, 242
Vitek, 14, 56
Vogel-Johnson agar, 29
von Behring, Emil, 11
VRB counts, 66
VRE (vancomycin-resistant enterococci), 132
WASP, 69–70
Wasserman, August, 11
waste products, 24
water
activity, 22, 113
contamination, 34–35
drinking, 21
as excipient, 32
for injection, 47, 52, 54
microbiology, 35
pharmaceutical, 14, 52, 54
purified, 30, 47, 54, 88
quality, 51, 52
testing, 53, 95, 227
treatment compounds, 30
water systems
bioburden in, 30–31
hemodialysis, 31
hospital, 31
pharmaceutical, 184
purified, 19–20, 32
sampling of, 47
validation of, 32
Watson, James, 16
Weigert, Carl, 13
Welch, William, 11
western blotting, 150
Whitley Impedance Broth®, 102, 116
WHO (World Health Organization), 190, 203
whole-cell protein profile (WCPP), 128, 149
Wilkins and Chalgrens anaerobe broth, 114
Wills, Kirsty, 85, 211
Winogradsky, Serge, 14
World Health Organization. See WHO
xylose-lysine-deoxycholate (XLD) agar, 26, 28
yeast, 12, 34, 47, 249
ATP bioluminescence, 86
detection with specialized techniques, 55, 64, 90–91, 95, 102
extract, 25
identification, 56, 143, 146
Petrifilm™ yeast and mold count method, 66–67
temperature conditions for growth, 90–91
Yersinia enterocolitica, 62, 64
Yersinia, FTIR identification, 146
Yersinia O9, 66
Zinsser Microbiology, 14
Zwittergent®, 197
Zymate robot, 206
INDEX 189
Rapid Microbiological Methods in the
Pharmaceutical Industry
Edited by
Martin C.Easter
Boca Raton London New York Washington, D.C.
Page 98
Parameters observed to influence RAPD profiles and reproducibility References citing confirmatory observations
[DNA] Gao et al. (1996), MacPherson et al. (1993), Meunier and
Grimont (l993)
Preparation Khandka et al. (1997), Micheli et al. (1994), Hilton et al.
(1997)
Secondary structure Caetano-Anolles (1993)
[Primer] Khandka et al. (1997), MacPherson et al. (1993), Meunier
and Grimont (1993)
Length MacPherson et al. (1993), Williams et al. (1990)
G+C content MacPherson et al. (1993), Williams et al. (1990)
Importance of primer selection Berg et al. (1994), He et al. (1994), Welsh and McClelland
(1990)
Ratio Primer/Template Caetano-Anolles (1993), del Tufo and Tingey (1994),
Ellsworth et al. (1993), MacPherson et al. (1993),
Template/DNA polymerase Khandka et al. (1997)
Reaction buffer and pH Hilton et al. (1997)
Thermocycler -Annealing temperature Ellsworth et al. (1993), Meunier and Grimont (1993),
Welsh and McClelland (l990),
-Cycle number and time del Tufo and Tingey (1994), MacPherson et al. (1993),
Meunier and Grimont (1993)
-Extension time Van Leuven (199l), Wilson (1994)
-Ramping Cocconcelli et al. (1995)
-Model del Tufo and Tingey (1994), MacPherson et al. (1993),
Meunier and Grimont (1993), Penner et al. (1993)
-Temperature across block Van Leuven (1991)
RNA contamination Ellsworth et al. (1993), Micheli et al. (1994),
Type of gel Berg et al. (1994)
Interlaboratory -due to Taq Meunier and Grimont (1993) Schierwater and Ender (1993)
-due to thermocycler MacPherson et al. (1993), Meunier and Grimont (1993),
Penner et al. (1993)
Shown not to affect profiles
Reaction buffer components and pH (including additives—gelatin, DMSO) Gao et al. (1996), Meunier and Grimont (1993)
Subculturing Gao et al. (1996)
Plasmid content Elaichouni et al. (1994)
Note: Items in bold represent those factors cited most often as contributing to variability.
Adapted from Tyler et al. 1997. Journal of Clinical Microbiology, 35. (Used with permission of American Society of Microbiobgy,
Washington, D.C.)
serotype (Jersek et al. 1999). In addition, the method has been extended as a means of identifying particular species of
eucaryotic microorganisms (Judd et al. 1993; van Belkum et al. 1992). The rep-PCR technique is quite reproducible and has
moderate discriminatory power.
PCR—restriction fragment length polymorphism (RFLP) is a genotypic method that, in contrast to standard southern
hybridizations, is rapid and only involves individual genes or gene clus ters (Arbeit 1995; Swaminathan and Matar 1993).
Popular targets of PCR-RFLP are virulence genes (Samadpour 1995), because they are commonly associated with only
pathogenic strains of a particular species. This technique incorporates the principles of PCR and REA in one rapid method.
Initially, a target gene is amplified using known primers to a particular virulence region. The resulting amplicon (usually 1 to
2 kb) is then digested with a suitable restriction endonuclease, and the fragments are run out onto an agarose gel and stained with
ethidium bromide to detect polymorphisms in the gene. Theoretically, any known region of interest can be amplified by PCR
and then examined by means of enzyme restriction (e.g., genes coding for flagella [Madden et al. 1998]). Even though prior
knowledge of the DNA region of interest is needed and the discriminatory power of the method tends to vary substantially
depending on the different species, loci, and restriction enzyme, this technique has the advantage of speed, simplicity, and
reproducibility. Investigators have shown the utility of this method by using it not only for typing, but also for strain
identification when applied to the ribosomal RNA region or some species-specific gene sequence. Organisms such as
Mycobacterium spp. (Taylor et al. 1997), S. aureus (Schmitz et al. 1998), Helicobacter pylori (Dzierzanowska et al. 1996),
90 RAPID MICROBIOLOGICAL METHODS
Page 99
Campylobacter (Madden et al. 1998), and L. monocytogenes (Vaneechoutte et al. 1998), have all been typed with this
method.
PCR-ribotyping, a relatively new genotypic typing method, is also based on the PCR reaction (Jensen et al. 1993; Kostman
et al. 1992). The method takes advantage of the heterogeneity found within the spacer regions that exist between the rRNA
subunits (16S, 23S, and 5S rRNA) of prokaryotic microorganisms (Campbell et al. 1993; Loughney et al. 1982), most notably
the 16S-23S intergenic spacer region. The spacer regions separate the rRNA subunits and may show a large degree of
sequence and length variation at both the genus and species level (Campbell et al. 1993; Loughney et al. 1982). Because most
bacterial genera contain multiple copies of the rRNA operon, multiple bands of varying lengths may be obtained from a
particular strain after amplification with primers designed to target the conserved regions flanking the spacer sequences in
different ribosomal operons. As discussed under ribotyping, a distinct advantage of involving the highly conserved rRNA
gene as a target in amplification is the fact that universal primers may be designed that will work across several genera of
bacteria (Jensen et al. 1993). However, as with traditional PCR, one must have some knowledge of the conserved nature of
the rRNA sequence of the particular organism one is trying to type in order to know if primers are adequate. This technique
has been used to type organisms such as S. aureus, Enterococcus faecium, E. coli, Enterobacter spp., and L. monocytogenes
(Kostman et al. 1995; Sontakke and Farber 1995).
Although it has been shown that the greatest variation lies within the 16S-23S spacer region and considerably less in the
23S–5S region, researchers have found that increased discrimination can be obtained in some organisms by employing
primers that span the entire 16S—5S region, so that the spacer region between the 23S and 5S rRNA is also amplified
(Sontakke and Farber 1995). Another approach has been to use restriction endonucleases to cut the amplicons arising from the
initial amplification (Kostman et al. 1992) in order to reveal hidden polymorphisms and thereby increase the discriminating
ability of PCR-ribotyping. In this respect PCR-ribotyping can be thought of as a form of PCR-RFLP utilizing the rRNA gene.
Although not as discriminatory as PFGE and possibly RAPD typing, PCR-ribotyping has the advantage of producing stable,
easily detectable amplification patterns in a rapid manner and has the potential to be widely useful in molecular epidemiology
(Kostman et al. 1992).
Insertion sequence (IS)—PCR takes advantage of the insertion sequence elements that have been discovered in several
bacteria. These elements are mobile genetic units that can vary in physical location within the chromosome of different
isolates and therefore make an appealing target for molecular typing. Primers can be designed to flank the ends of the IS
element and face outward to amplify the intervening space. If two IS elements are close enough, then the PCR operates
efficiently and a DNA fragment is amplified. This approach has been taken with organisms such as E. coli (Thompson et al.
1998), Mycobacterium (Devallois and Rastogi 1997), and S. aureus (Deplano et al. 1997).
Amplified restriction fragment length polymorphism (AFLP) is a DNA fingerprinting technique involving RFLP and PCR
(Janssen et al. 1996; Vos et al. 1995) (see Figure 8.1). Whole genomic DNA is isolated, then digested with a specific combination
of two restriction endonucleases (REs). The first must be a frequently cutting RE such as H MseI , HindIII, or TaqI that
recognizes a specific sequence of 4 bases. This generates numerous small DNA fragments that are in the optimal size range for
amplification and separation. A second infrequent or rare cutting RE is also incorporated into the digestion to further reduce
the number of potential amplicons, because the system is set up to amplify only those fragments having a different restriction
site at each end. This latter feat is accomplished through the use of specially designed double-stranded (ds) adapter sequences
that will ligate to the overhanging ends of the restricted fragments and then be amplified by primers that recognize the adapter
sequence. Two adapters are required, one specific for each of the restriction sites generated. Adapters consist of a core
sequence of 11 to 14 homologous ds bases that are arbitrarily selected, followed by the specific addition of bases at the 5’ end
to provide an overhang suitable for ligation to one of the restriction sites (i.e., an enzyme specific sequence). A convenient
omission that does not affect the results is that adapters are not phosphorylated, and thus only one strand of adapter will ligate
to the restricted fragment. Ligation will, however, occur in several different ways to the various combinations of restriction
fragments generated by the two REs, but only those that ligate in the proper orientation and in the desired combination will be
amplified, because of the design of the primer set. Each single-stranded primer consists of a core sequence that is homologous
to the majority of bases in the core sequence of the respective adapter molecule, plus the 5’ overhanging enzyme specific
sequence. In addition, 1 to 3 arbitrarily selected nucleotides are added to the 3’ end of the primer. Each addition of a selective
base to the AFLP primer reduces the number of amplicons approximately 4-fold and results in a subset of the original
fingerprint. Usually one primer (usually the rare cutter) is radioactively labeled, and primers are annealed at high temperatures
to ensure stringency. In this way, the system will amplify only the specific subset of properly ligated adapter-fragment
hybrids that are homologous for the selective bases added to the 3’ end of the primer. This addition of selective bases
provides a further specificity to the primer and in so doing reduces again the number of potential amplicons. This technique
still results in a DNA fingerprint pattern of approximately 50–100 fragments of varying sizes and thus most typically requires
a denaturing polyacrylamide (sequencing) gel for adequate resolution. Gels are visualized by autoradiography or
phosphoimaging; DNA analysis software is recommended to properly interpret the likelihood of two strains being related
because of the quantity of bands.
TRADITIONAL AND AUTOMATED RAPID METHODS FOR SPECIES IDENTIFICATION AND TYPING 91
Page 196
testing, 42–46.
See also validation biocidal activity, 110–113
clean-in-place (CIP), 95
component, 227
drug, 198
environmental, 95
finished product, 44, 50, 51, 52, 55
food, 180, 181, 182, 183, 233
hygiene, 51
in-process, 44, 95, 198, 224
market, size of, 2
minimum inhibitory concentration (MIC), 76, 110, 111
nonsterile product, 42, 46, 108
preparatory, 200
preservative efficacy, 50, 53, 108, 111
protocols, 216
raw material, 50
release, 44
sterility, 47, 48, 51, 113, 179–185
susceptibility, 125, 143
tests
antimicrobial effectiveness, 35–36, 227
bacterial endotoxin, 196, 199–202
colorimetric, 193
in vitro pyrogen, 197
Inhibition or Enhancement, 200–201
Limulus Amebocyte Lysate (see under Limulus Amebocyte
Lysate (LAL) test)
mandatory (USP) requirements for, 223
Microbial Limits Test (MLT), 3, 250
Most Probable Number (MPN), 15, 16, 52, 67
presence or absence, 163, 180, 250
rabbit pyrogen, 15, 188–189, 190, 196, 203
Sterility of Liquids, 179
tetanus, 11, 12
tetrathionate broths, 27, 28
thermal conductivity detector (TCD), 147
thermal hydrolysis and methylation (THM), 139, 143
thermophilic microorganisms, 24
thioglycolate broth, 26
thioglycollate medium, 113, 117
threshold pyrogenic dose, 190
thyroid-stimulating hormone (TSH), 238, 240
time of flight (TOF), 140
time to detection (TDD), 50, 102, 104
tissue culture media, 182
TMA (transcription mediated amplification), 138, 163, 166–168
TMAO (trimethylamine-N-oxide), 114–115, 116
total viable count, 32, 47, 75, 108, 109
toxic metabolic products, 25
toxicity, selective, 117–118
toxin genes, 130, 163
Toxinometer®, 194, 204
toxins.
See also endotoxin aflatoxin, 62
food-borne, 161
screening for, 49
Streptococcus, 49
traceability, 47
trailing endpoints, 76
training records, 217
transcription mediated amplification (TMA), 138, 163, 166–168
transfusion-transmitted sepsis, 181
TRANSIA, 49
Trichinella, 62
tricolsan, 90
trimethylamine-N-oxide (TMAO), 114–115, 116
trimethylphenylammonium hydroxide (TMAH), 143
triphenyl-tetrazolium chloride (TTC), 65
triple sugar iron agar, 28
tryptone soya bean broth, 113
tryptones, 25
TTC (triphenyl-tetrazolium chloride), 65
tuberculosis, 12
tumors, 117
turbidimetric assay, 193, 204, 205, 206
two-photon emission, 237
Tyler, Kevin, 125
Tyndall, John, 12
Type I Amendment, 93, 219
Type I variation, 219, 229–230
Type II variation, 219, 229, 230
typhoid bacillus, 11, 15
UK Medicines Control Agency (MCA), 46, 52, 93, 227
ultramicrobacteria, 20
universal media, 25, 27
universal primers, 135
updates, 214
urinary tract infections, 144, 189, 206
U.S. Environmental Protection Agency (EPA), 30
U.S. Food and Drug Administration (FDA), 30, 182, 230
bacterial endotoxin preparations, 189–190, 203
Center for Biologics Evaluation and Research (CBER), 190, 221,
222, 227
Center for Drug Evaluation and Research (CDER), 222, 227
draft guideline for validation of Limulus Amebocyte Lysate
(LAL) test, 196
FDA Bacteriological Analytical Manual, 14
Guidance Documents, 222–223, 226, 228–229
guidelines for Limulus Amebocyte Lysate (LAL) test, 197–199
Inspection Guides, 223, 224–226
product licensing, 219
recognition and acceptance, 221–223
U.S. Pharmacopeia, 14, 212, 221
bacterial endotoxin test, 195, 199–202, 203
Endotoxin Reference Standard, 199
General Chapters, 225
microbial limits, 30, 225
monographs, 199, 225
nutrient media of, 26, 27–29
objectionable microorganisms, 30
revision process, 227
standard methods, 31–36
standards, 223
sterility testing, 179, 180, 182, 184
validation, 33, 43, 52, 213
U.S. Standard Endotoxin, 190, 191
USP. See U.S. Pharmacopeia
UV treatment of surfaces, 30
188 RAPID MICROBIOLOGICAL METHODS
Page 197
vaccination, 10
validation, 2, 57, 100, 118, 225–226.
See also ICH Barr decision, 226, 230
cGMP amendments, importance to, 224
criteria, 213–214, 216
definition of, 211–212
of media, 48
of methods, 33, 35, 42–43, 45, 52
Parenteral Drug Association Technical Report No. 33, 6, 45, 52,
213, 221, 250
plan, 214–215
protocol, 215–216, 218
of recovery, 32
summary, 217
support, 45, 47, 53
vancomycin-resistant enterococci (VRE), 132
variations, 219, 229–230
verification, 212, 216
viability staining, 53
viable cells, 36, 108
viable count, 32, 47, 75, 108, 109, 110
viable nonculturable state, 30
Vibrio cholera, 11, 13
Vibrio parahaemolyticus, 64
Vibrio vulnifcus, 136
Vida, 50
video cameras, 72
VIP, 49
virulence genes, 132, 135
viruses, 146, 196, 242
Vitek, 14, 56
Vogel-Johnson agar, 29
von Behring, Emil, 11
VRB counts, 66
VRE (vancomycin-resistant enterococci), 132
WASP, 69–70
Wasserman, August, 11
waste products, 24
water
activity, 22, 113
contamination, 34–35
drinking, 21
as excipient, 32
for injection, 47, 52, 54
microbiology, 35
pharmaceutical, 14, 52, 54
purified, 30, 47, 54, 88
quality, 51, 52
testing, 53, 95, 227
treatment compounds, 30
water systems
bioburden in, 30–31
hemodialysis, 31
hospital, 31
pharmaceutical, 184
purified, 19–20, 32
sampling of, 47
validation of, 32
Watson, James, 16
Weigert, Carl, 13
Welch, William, 11
western blotting, 150
Whitley Impedance Broth®, 102, 116
WHO (World Health Organization), 190, 203
whole-cell protein profile (WCPP), 128, 149
Wilkins and Chalgrens anaerobe broth, 114
Wills, Kirsty, 85, 211
Winogradsky, Serge, 14
World Health Organization. See WHO
xylose-lysine-deoxycholate (XLD) agar, 26, 28
yeast, 12, 34, 47, 249
ATP bioluminescence, 86
detection with specialized techniques, 55, 64, 90–91, 95, 102
extract, 25
identification, 56, 143, 146
Petrifilm™ yeast and mold count method, 66–67
temperature conditions for growth, 90–91
Yersinia enterocolitica, 62, 64
Yersinia, FTIR identification, 146
Yersinia O9, 66
Zinsser Microbiology, 14
Zwittergent®, 197
Zymate robot, 206
INDEX 189
