Cundell Et Al PDA J Pharm Sci Tech March 2010 (Full Article) Sfs
Cundell Et Al PDA J Pharm Sci Tech March 2010 (Full Article) Sfs
RESEARCH
ABSTRACT: The pharmacopoeias list a number of microorganisms to be used in the compendial microbiological tests for
confirming the growth-promoting, indicative, and inhibitory properties of the media and demonstrating the suitability of the
test for a specific test article. Major national culture collections are specified as the sources for these test strains based on
their history of deposition and maintenance and use in the compendial tests. Using these microorganisms, it has long been
assumed that these strains are interchangeable and that sourcing the strains from different culture collections has no impact
on the result of the media quality control and method qualification tests. In order to evaluate whether this assumption is
correct and to add more certainty to the procedures, we investigated whether there are detectable differences among isolates
of the same strain sourced from different culture collections. Using various phenotypic and genotypic identification and
strain typing methods, nine major pharmacopoeial species were analyzed. As expected, most of the species showed very
uniform patterns across the isolates, indicating that the strains were indeed identical. Surprisingly, the strains of Salmonella
enterica subsp. enterica serotype abony showed distinct differences at both the genotypic and the phenotypic level,
suggesting that the strains sourced from the different culture collections were not identical strains, or that they have
undergone detectable genetic shift from the time they were derived from the original depositor. Irrespective of the level of
genotypic or phenotypic homology identified here, there are no practical consequences on their performance in compendial
assays. It is concluded that the compendial strains investigated in this study are indeed equivalent and will perform
identically in compendial tests, making it safe to base pharmaceutical quality control procedures on the strains sourced from
any of the recognized national culture collections.
KEYWORDS: Microbial identification, Quality control, Strain typing, Pharmacopoeia, MALDI-TOF mass spectrom-
etry, Rep-PCR, Ribotyping, 16S rRNA gene sequencing, Serotyping
Introduction the compendial tests are strictly referee tests that would
be used to assess the microbiological quality of a phar-
The three compendia, the United States, European, and maceutical article, they are typically referenced in regu-
Japanese Pharmacopeia (USP, Ph. Eur. and JP, respec- latory filings, hence becoming product release tests. The
tively), are most widely used as standard-setting organi- tests used in the pharmaceutical industry need to comply
zations to guide pharmaceutical quality control (QC). with the compendia of the country the product is to be
Each of them is recognized by the respective authorities sold in, often resulting in slightly different test needs.
as the official compendium, e.g., in the US by the Federal After many years of effort, the chapters in the compendia
Food, Drug, and Cosmetic (FDC) Act. The compendial for microbial testing of nonsterile and sterile products
standards are used to determine the identity, strength, have been successfully harmonized and are in the pro-
quality, and purity of pharmaceutical articles. Although cess of implementation (1–3). Only the USP official tests
are referenced as representative of the tripartite harmo-
nized tests.
* Corresponding authors: [email protected],
Tel:⫹1908-820-6966;[email protected].
In order to confirm method suitability, different
com, Tel: ⫹61 28877 9150
species of bacteria, yeasts, and molds as well as
TABLE I
USP-Listed Cultures for the Official Compendial Microbiological Test Methods
suitable strains are specified. Moreover, the com- the culture collection that at some point in time the
pendia list a limited number of national culture strains were derived from each other or a common
collections as sources for these strains as equivalent source. There are other well recognized culture col-
(see Table I), with the assumption that the strains lections that make the same strains available and
are indeed identical (i.e., monoclonal) and could be can be identified using the bioportal of the Univer-
used interchangeably in the compendial tests. There sity of Gent (www.straininfo.ugent.be). Further-
is, however, no formal information in the literature more, these stains can be purchased as semi-
to confirm this belief (4). The equivalency is solely quantitative or quantitative prepared inocula from
based on the culture history and the affirmation of commercial suppliers.
This study investigates whether there are detectable than 30 min. The card, placed on the tray and applied
differences of the same strains sourced from different to the VITEK 2 system, was automatically processed
culture collections and if so whether this has an impact in a vacuum chamber, incubated at 35.5 °C, and au-
on the outcome of the compendial tests. To achieve tomatically subjected to a colorimetric measurement
this, multiple methods generally aimed at the identi- by use of an optical reading head every 15 min for a
fication at the species level (VITEK威 2, Biolog™, maximum incubation period of 8 –18 h depending on
API威, MALDI-TOF MS, and MicroSeq威, Applera the VITEK 2 card used. Data were analyzed using
Corporation) and for typing at subspecies level (Di- VITEK 2 database version 4.01.
versiLab威, riboprinting, and serotyping) were em-
ployed. This study, however, did not attempt to compare API Microbial Identification
the merits and suitability of the different methods avail-
able for species identification and strain typing. In the Some of the isolates were also tested on API strips
case of Salmonella enterica subsp. enterica serotype (bioMérieux) according to the manufacturer’s instruc-
abony where appreciable differences could be detected, tions. ID32E was used for Gram-negative bacteria,
the impact on the suitability of the strains in compendial Candida albicans isolates were tested on ID32C strips,
tests such as the growth promotion test was evaluated. Staphylococcus aureus was tested on ID32 Staph, and
Clostridium sporogenes was checked on ID32A.
Materials and Methods
Biolog Microbial Identification
Strains Evaluated
A pure culture, isolated on soybean casein digest agar,
Bacterial and fungal strains (see Table II) were pur- was subcultured onto Biolog BUG agar with 5% sheep
chased from the Australian Collection of Microorgan- blood and incubated at 35 °C for 16 –18 h. From the
isms (ACM, Australia), the American Type Culture Col- subculture, a uniform cell suspension was prepared to
lection (ATCC威, USA), CAB International (CABI, UK), a density of 61 ⫾ 2% light transmittance in Biolog
the Collection de l’Institut Pasteur (CIP, France), the GN/GP-IF (Gram-negative/Gram-positive inoculating
Deutsche Sammlung von Mikroorganismen und Zellkul- fluid) containing 5 mM sodium thioglycolate. A 96-
turen (DSMZ, Germany), the National Biological Re- well microplate was then inoculated with 150 L/well
source Center (NBRC, Japan), the National Collections of the cell suspension. The microplate was incubated
of Industrial and Marine Bacteria (NCIMB, Scotland), at 35 °C for 16 –24 h with readings being taken during
the National Collection of Pathogenic Fungi (NCPF, this time interval.
UK), and the National Collection of Type Cultures
(NCTC™, Health Protection Agency, UK) and were Matrix-Assisted Laser-Desorption/Ionization
subcultured no more than five times prior to testing. Time-of-Flight Mass Spectrometry (MALDI-TOF MS)
Please note that since the Japanese Institute for Fermen-
tation (IFO) joined the NBRC, all IFO numbers are Sample preparation for MALDI-TOF MS protein anal-
identical to their respective NBRC numbers. ysis was carried out according to the ethanol/formic
acid extraction protocol recommended by Bruker Dal-
VITEK 2 Microbial Identification tonik (Bremen, Germany) as described previously (7).
First, about 10 mg biomass from agar cultures was
The frozen strains were subcultured twice on Colum- resuspended in 300 L water by careful mixing. Then,
bia agar with 5% sheep blood or Sabouraud dextrose the suspension was mixed with 900 L ethanol. The
agar. All strains but Aspergillus isolates and Clostrid- biomass was collected by centrifugation and the pellet
ium strains were identified by the colorimetric VITEK was resuspended in 50 L 70% formic acid. The
2 cards (BCL, GN, GP, YST cards; bioMérieux, suspension was mixed carefully with 50 L acetoni-
Durham, NC) according to the instructions of the trile. After centrifugation, the supernatant was re-
manufacturer (McFarland standard of 0.5–2 depending moved immediately and aliquots of 1.5 L were
on the card used). A bacterial suspension made in placed on each spot of a stainless steel target plate.
0.45% aqueous NaCl was adjusted to the recom- After air-drying, 1.5 L matrix solution (saturated
mended McFarland standard with a VITEK 2 Den- solution of alpha-cyanohydroxycinnaminic acid in
siChek™ instrument. The time period from prepara- 50% aqueous acetonitrile containing 2.5% trifluoro-
tion of the inoculum to loading of the card was less acetic acid) per spot was applied. MALDI-TOF MS
140
History of Deposition of Compendial Cultures in Six Major Culture Collections
National Culture Collectionsa
Origin: CDC
Isolation: tissue animal
(pools of heart & liver
from 4 wk old chickens)
S. enterica subsp. enterica not available CIP 80.39 4 Inst. Pasteur 4 F. DSM 4224 4 CIP IFO100797 4 CIP 80.39 not available NCTC 6017 4 F.
serotype abony Kaufmann, K 103 80.39 Kaufmann, K 103
Origin: F. Kaufmann.
Denmark
Isolation: Unknown
S. aureus ATCC 6538 4 FDA 209P CIP 4.83 4 ATCC 6538 DSM 799 4 ATCC IFO 13276 4 ATCC 6538 NCIMB 9518 4 Diversey (UK) NCTC 10788 4
4 Walter Reed AMC 6538 Ltd 4 Diversey Corp. ATCC 6538
Origin: Walter Reed AMC Chicago 4 ATCC 6538
Isolation: human lesion
Strain numbers in bold were analyzed in this study.
a
ATCC ⫽ American Type Culture Collection (USA), NCIMB ⫽ National Collections of Industrial and Marine Bacteria (Scotland), CIP ⫽ Collection de l’Institut Pasteur (France), NBRC ⫽ National
Biological Resource Center (Japan), NCTC ⫽ National Collection of Type Cultures (UK), DSM ⫽ Deutsche Sammlung von Mikroorganismen (Germany), IFO ⫽ Institute for Fermentation (Japan).
b
was conducted using a Microflex威 L20 mass spec- priate for the microorganism (Bacterial Barcodes, Inc.,
trometer (Bruker Daltonik) equipped with an N2 laser. Athens, GA) following the manufacturer’s instruc-
All spectra were recorded in linear, positive ion mode. tions. The amplicons were then analyzed using the
The acceleration voltage was 20 kV. Spectra were DiversiLab system including a microfluidics chip
collected as a sum of 500 shots across a spot. A mass (Bacterial Barcodes). Data analysis was performed
range of 2000 –20,000 m/z was used for analysis. The with the Web-based DiversiLab software version 3.3
spectra were analyzed by using the BioTyper software, using the Pearson Correlation coefficient and UPGMA
Version 1.1 (Bruker Daltonik). to automatically compare the rep-PCR-based DNA
fingerprints of unknown isolates.
16S rRNA Base Sequencing
Inter Simple Sequence Repeats (ISSR) and Variable
Sequencing of the 16S rRNA gene was carried out Number Tandem Repeats (VNTR)
either as a partial 5⬘ sequence of 500 bp or the full-
length 1500 bp using the MicroSeq 16S rDNA proto- ISSR and VNTR analyses were performed by CABI,
col (Applied Biosystems, Foster City, CA) and was as described by Grünig et al. (8) and Bridge et al. (9),
conducted at the Australian Genome Research Facility respectively, except that the annealing temperature in
in Melbourne, Australia. the VNTR assays was 45 °C for Aspergillus niger and
48 °C for C. albicans.
Ribotyping
Serotyping
Automated ribotyping was performed according to the
Strains of S. enterica subsp. enterica serotype abony
instructions of the manufacturer of the Riboprinter
were subjected to serological typing according to the
microbial characterization system (DuPont Qualicon,
Kauffman-White scheme (6) at the Institute of Clinical
Wilmington, DE) as described previously (5). The
Pathology and Medical Research in Sydney, Australia.
automated process included bacterial cell lysis and
digestion of the DNA by using either the restriction
Growth Promotion Testing
enzyme EcoRI or PvuII. DNA fragments were sepa-
rated by size using agarose gel electrophoresis. The Freeze-dried inocula containing a precise number of
DNA fragments were hybridized with a labeled rRNA viable cells of S. enterica subsp. enterica serotype
operon probe derived from Escherichia coli, and the abony strains ACM 5080 and CIP 80.39 were prepared
bands were detected by a chemiluminescent substrate. using the proprietary BioBall technology (BTF bio-
The image was recorded by using a customized charge- Mérieux, Sydney, Australia) (10). Briefly, cultures
coupled device camera. Data were normalized to a stan- were grown in a modified 2YT medium and counted
dard marker set. The band patterns were compared using and sorted using flow cytometry. Droplets were snap-
the BioNumerics威 software (Applied Maths NV, St- frozen in liquid nitrogen and freeze-dried. The freeze-
Martens-Latem, Belgium) and clustering was carried out dried BioBall was rehydrated directly on xylose lysine
by the unweighted-pair group method with the arithmetic deoxocholate agar (XLD), soybean-casein digest agar
mean (UPGMA) based on Pearson’s correlation coeffi- (SCDA) ⫽ trypticase soy agar (TSA), or nutrient agar
cient (optimization coefficient, 1.2%). (NA) in 100 L 0.9% NaCl solution. For experiments
utilizing non-freeze-dried cultures, the strains were
Repetitive Sequence-Based Polymerase Chain Reaction cultured in 2YT into mid-log phase, counted and
(Rep-PCR) sorted as above, and dropped directly onto the agar
plates. After spreading and drying, the plate was in-
The bacterial isolates were cultured on plates of Co- cubated at 30 –35 °C for the time indicated and the
lumbia 5% sheep blood agar with the exception of C. colonies were counted or their diameter measured.
sporogenes, which was cultured in brain heart infusion
broth. Yeasts and molds were subcultured on Sab- Results and Discussion
ouraud dextrose agar plates. The DNA from each
culture was extracted from a 10-L loop of colony Strain Culture History
mass using the UltraClean microbial DNA isolation kit
(Mo Bio Laboratories, Solana Beach, CA). DNA (30 In the past, culture collections would extensively ex-
ng/L) was amplified using the DiversiLab kit appro- change strain isolates deposited at only one culture
collection with others in order to make the strains storage and subcultivation. In order to gain more res-
widely accessible to the research and industry com- olution power, two commercially available strain typ-
munities. In every culture collection, the same strain ing methods were employed, ribotyping (Riboprinter,
assumes a new identity code, and the dependencies DuPont Qualicon) and rep-PCR (DiversiLab, Bacterial
become less apparent. Table II lists the culture history Barcodes). Both methods rely on differences in the
of the deposition of compendial cultures in six major genomic DNA. Although both methods return results
culture collections. For the purpose of this study we that look very similar, there are fundamental differ-
follow the definition of Brenner and coworkers and ences as to how the banding patterns are generated and
define a strain as the descendant of a single isolation in how much information they bear. Briefly, for ribotyp-
pure culture stored in a culture (strain) collection, ing, the extracted genomic DNA is digested with a
typically from a single colony isolated on a plate, that specific restriction enzyme (typically EcoRI) and the
is, monoclonal (11). One strain of a species is desig- resulting fragments are separated by gel electrophore-
nated as the type culture due to its early history of sis and transferred onto a membrane. On this mem-
deposition in the culture collection and its taxonomic brane, the DNA is subjected to Southern hybridization
status, with all other strains sufficiently similar in both using a probe derived from the rRNA operon of E.
phenotype and genotype to be considered within the coli. Hybridization highlights the fragments of
species. For example, Pseudomonas aeruginosa genomic DNA with sufficient homology to the probe
ATTC 10145 is the type strain for both the genus and to create a specific banding pattern. The length of the
species while P. aeruginosa ATCC 9027 isolated from detected fragments depends on the location of the
an inner ear infection was designated as a compendial target sites for the restriction endonuclease used.
QC strain in USP 具61典, 具62典, and 具71典.
Rep-PCR, on the other hand, is not limited to the
Strain Identification Confirmation at Species Level conserved rRNA coding sequences on the genomic
DNA but utilizes repetitive DNA sequences dispersed
In order to confirm the species identity, three commer- throughout the genome. In a PCR reaction utilizing
cially available phenotypic identification methods primers complementary to those repetitive DNA ele-
were employed. API, Biolog, and VITEK 2 identifi- ments, fragments of different lengths are generated.
cation reagents are based on biochemical methods The amplified fragments are separated and detected by
mainly measuring carbon source utilization and enzy- microfluidics-based electrophoresis. Both methods re-
matic activities. While both API strips and VITEK 2 sult in a pattern of DNA fragments, the origin of which
cards contain biochemical substrates, their nature, however is fundamentally different. Comprehensive
concentration, and number in the manual API strip and descriptions of both technologies can be found in the
the automated VITEK 2 card are different, leading to literature (5, 8, 12, 13). As expected, ribotyping the
a large menu of claimed species. As expected, culture strains of Bacillus subtilis, E. coli, P. aeruginosa, and
collection strains were correctly identified and no dif- S. aureus specified in the USP and EP Ph. Eur. re-
ference based on the source of the isolate was noted. sulted in very similar banding patterns, regardless of
The BIOLOG results were not always definitive, as the culture collection from which they were sourced
some strains were identified incorrectly as phenotyp- (Figure 1). This set of ribotyping experiments was
ically related bacteria. Salmonella typhimurium ATCC performed with EcoRI, the restriction enzyme most
14028 was identified as Salmonella gp 1 (cholerae- commonly used for ribotyping. For comparison, an
suis) or Citrobacter sedlakii while S. typhimurium unrelated strain of the same species was chosen ran-
NCTC 12023 was identified as Salmonella gp 1 (chol- domly and subjected to the same ribotyping and
eraesuis). Salmonella abony CIP 80.39, ACM 5080, showed marked differences in the banding pattern.
and NCTC 6017 were identified as Salmonella gp 1 Pearson correlation analysis documented for each spe-
(choleraesuis). cies a very close clustering of the isolates of the same
strain from different culture collections, whereas a
Genetic Typing Discriminates Strains much lower correlation percentage of the unrelated
strain to that cluster was observed. In the case of P.
Despite confirming the species in each case, the phe- aeruginosa, and to a lesser extent S. aureus, two
notypic identification methods did not uncover possi- unrelated strains appeared to be more similar to each
ble subtle differences among strains of the same spe- other than to the other strain cluster (Figure 1, C and
cies or changes that might have occurred during D). It should be noted here that this close clustering in
Figure 1
of the banding pattern, PvuII was unable to discrimi- be detected (Figure 3). For each strain cluster, the
nate the unrelated strain DSM 1563 from the compen- overall similarity was always above 96%. As the band-
dial strain cluster, whereas EcoRI produced distinct ing pattern is dependent on the location of repetitive
fragment patterns clearly distinguishing these strains elements dispersed throughout the genome and is not
(compare Figures 1B and 2A). In order to gain insight focused on a conserved region, the rep-PCR analysis
into the repeatability of the method, the same isolate offers strain discrimination information distinct from
of P. aeruginosa (NCTC 12924) was ribotyped inde- the ribotyping results. In the rep-PCR results there was
pendently 10 times (Figure 2B). The resulting patterns no evidence of a shift of the banding patterns as seen
were very similar with slight variations in the migra- in ribotyping. The authors contribute this observation
tion distances (Figure 2B). to the different methods used to separate the DNA
fragments in the different systems, as well as the
When the same set of compendial strains was analyzed normalization of the data by the software. In each
using rep-PCR, again no appreciable differences could rep-PCR analysis, at least one strain was included in
duplicate to facilitate the discrimination of true ge-
netic differences from method-inherent variability.
For example, the parallel analysis of two samples of B.
subtilis ATCC 6633 taken from the exact same culture
did not cluster next to each other, whereas the two
samples of B. subtilis NCTC 10400 did (Figure 3A).
The algorithm calculating the similarity takes both the
position and the intensity of the bands into account
and picks up minor differences in band intensities,
which are hard to quantify with the naked eye. This
repeat analysis was expanded using P. aeruginosa as
an example (Figure 4A). Ten independent repeat rep-
PCR analyses resulted in indistinguishable patterns
with only minor differences in band intensity. A com-
plete absence or reappearance of a prominent band
was never observed. While all samples clustered to-
gether, these small method-inherent variations in in-
tensity, which occur in both ribotyping and rep-PCR,
are likely to be the reason why the order of the
Figure 3
Figure 8
Figure 11
Full sequence alignment of the 16S rRNA of Salmonella abony strains. Most prominently, a fragment of 310 bp in length
is deleted in CIP 80.39 when aligned with NCTC 6017 and ACM 5080. Furthermore, multiple single nucleotide changes
were detected. Two changes at positions 972 and 973 resulted in the appearance of an additional Eco R1 restriction site,
which could be the reason for a specific banding pattern difference in the Riboprinting.
150 PDA Journal of Pharmaceutical Science and Technology
Downloaded from journal.pda.org on August 23, 2010
Figure 11 (Continued)
Figure 12
Growth promotion testing of Salmonella abony strains. Colony numbers of (A) freeze-dried and (B) fresh
cultures. (C) Growth kinetics on different media. The graphs show colony number mean and standard
deviation of triplicate plates. For the colony size, images of the plates were taken at the indicated time points.
The size of all colonies of one representative plate each was measured on calibrated printouts. The graph
represents mean and standard deviation of about 30 colonies each.
For ACM 5080 and CIP 80.39, the batch mean was differences in growth properties might have been ob-
29.0 and 29.2 cfu per BioBall, respectively, measured scured by freezing and freeze-drying, the growth pro-
by plating on nutrient agar. Similar counts were ob- motion assay was repeated, omitting the freezing in
served when plated on SCDA (Figure 12A). Direct liquid nitrogen and freeze-drying (Figure 12B). This
spreading of the BioBall on XLD agar resulted in a time, the droplets containing the flow-cytometric
reduction of colonies for both strains, with CIP 80.39 counted and sorted bacteria were captured directly
recovering slightly fewer colonies than ACM 5080. onto the agar plates, spread, and the plates incubated
The difference in cfu between ACM 5080 and CIP as before. Again, there was no significant difference
80.39, however, was not significant when tested by between the colony numbers recovered from NA,
paired Student’s t-test. In order to investigate whether SCDA, or XLD plates. Finally, the growth kinetics on
the different media determined by measuring colony This study was limited to the analysis of strains of the
sizes did not differ significantly over a time period of major national culture collections. Subsequent work
72 h on any of the media tested (Figure 12C). In should include strains sourced from other culture col-
summary, growth promotion assays of strains of the lections as well as other microorganisms used in com-
two genetically differentiable S. enterica subsp. en- pendial tests, for example, microbial assay of antibi-
terica serotype abony “lineages” did not reveal signif- otics and vitamins. Moreover, the managers of the
icant differences in growth characteristics. The au- culture collections might be inspired to compare type
thors suspect that the differences will not affect strains held in their collections, which are taxonomi-
suitability testing with specific test articles. cally more important than the QC strains used in the
pharmaceutical industry.
Conclusions
Acknowledgments
The compendia require standardized stable suspen-
sions of test strains to be used for growth promotion We are grateful to Barry Holmes for detailed informa-
and inhibitory properties testing as well as for method tion on strain culture history, Scott Sutton for valuable
validation. For each test, the compendia suggest a comments throughout the study, and Mimi Healy,
suitable strain for each species and name a number of Mary Jane Hilbert, Danielle Dennis, and Nick Herman
culture collections as sources. Additionally, the same
for excellent technical work and review of the manu-
strains can be sourced from a number of national
script.
culture collections. The compendia require that the
cultures should be removed no more than five passages
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