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m» MICROBIOLOGY ))
Recovery of Stressed (Acclimated) Burkholderia cepacia Complex Organisms
16 I Rb"Vrew I April 2014 vol 17
Introduction The FDA has adopted the position that all new product submissions for
non-sterile drugs must address recovery of Burkholderia cepacia [1,2).
The rationale for this requirement from the review section of the Center
for Drug Evaluation and Research (CDER) was published late in 2012 in
the trade literature [3]. Both the published article and the regulatory
requests have noted the disturbing ability of the Bee (Burkholderia
cepacia complex) group to proliferate in normally well-preserved
products and their ability to cause serious complications in susceptible
populations [4]. The Agency has expressed concern that "acclimated"
Bee organisms may not be recovered by standard microbiological
methods and so evade detection [2]. The potential failure of these
methods is of special concern as Bee organisms have been implicated
in a series of FDA recalls for both sterile and non-sterile products. The
product types included eyewash, nasal spray, mouthwash, anti-cavity
rinse, skin cream, baby and adult washcloths, surgical prep solution,
electrolyte solution, and radio-opaque preparations [5). B. cepacia
complex organisms have also been implicated in a series of outbreaks
in hospital settings and have earned their reputation as objectionable
organisms in specific product categories [6].
This study investigates the concern that compendia! methods
(especially the use of rich nutrient recovery agar) may not be capable
of recovering Bee microorganisms that had been acclimated to an
environment of USP Purified Water under refrigeration (2-8°() for an
extended period of time (up to 42 days). This acclimation method is
one suggested specifically for 8. cepacia in a pharma environment [2,7).
The Burkholderia cepacia Complex
Members of the Burkholderia cepacia complex are gram-negative
bacteria of the 13-proteobacteria subdivision and include plant, animal,
and human pathogens, with a widespread distribution in natural
and man-made habitats [8]. These bacteria exhibit an extraordinary
metabolic versatility, allowing their adaptation to a wide range of
environments including nutritionally limited ones [9). Burkho/deria
cepacia was first described by Burkholder as an agent causing bacterial
soft rot in onions [1 OJ.
.,,. MICROBIOLOGY ))
The genus Burkholderia currently comprises more than 60 species. This
genus was proposed in 1992 to accommodate the former rRNA group
II pseudomonads [1 OJ. Taxonomy of the entire Burkholderia genus
has changed rapidly: for instance, 8. cepacia has gone from a single
species to being a complex comprising 17 closely related species, or
genomovars (see Table 1 ), which can only be correctly classified by
using a combination of multiple molecular diagnostic procedures.
The literature published prior, and sometimes after, the definition
of the cepacia complex identified all the Bee species as Burkholderia
cepacia (or Pseudomonas cepacia), leading to some confusion. Several
Bee strains have developed beneficial interactions with their plant
hosts and have proven to be very efficient biocontrol, bioremediation,
or plant-growth promoting agents [12-14]. Refer to Table 1 for an
overview of the Burkholderia cepacia complex.
In the past two decades, Burkholderia cepacia has also emerged as
an opportunistic human pathogen causing numerous outbreaks,
particularly among cystic fibrosis (CF) and other immunocompromised
patients. One highly transmissible strain has spread across North
America and Britain, and another between hospitalized CF and
non-CF patients [15]. In addition, Burkholderia cepacia is inherently
resistant to multiple antibiotics and molecular epidemiology, and
phylogenetic studies demonstrate that highly transmissible strains
emerge randomly; the organism has a capacity for rapid mutation and
adaptation (facilitated by numerous insertion sequences) and a large,
complex genome divided into separate chromosomes.
An interesting side note on 8. cepacia physiology was recently
described by Vial et al. [7], who described experiments showing
that Bee can survive and grow within the vacuoles of both amoeba
and mammalian macrophages and monocytes. Nasal mucosa has
been known to carry amoeba and "consequently could represent an
important natural reservoir for Bee strains and act as a Trojan horse
Table 1. Overview of the Burkholderia cepacia Complex*
Species Natural Environment Clinical Environment
8. cepacia Rhizosphere, soil, water Cystic fibrosis (CF), medical solution
8. multivorans Rhizosphere, soil, water CF, CGD, non-CF -----
8. cenocepacia Rhizosphere, plant, soil, water, animal CF, non-CF
B. stabilis Rhizosphere CF, (rare) hospital equipment
8. vietnamiensis Rhizosphere, plant, soil, water, animal CF
B. dolosa Maize rhlzosphere, plant CF
8. ambifaria Rhizosphere, soil CF (rare)
B.anthina Rhizosphere, so_il CF(rare)
8. pyrrocinia Rhizosphere, soil, water, plant CF, non-CF (rare)
8. ubonensis Soil Nosocornial infection --- - ---8. letens No environmental strain reported CF
8.diffusa Soil, water CF, hospital equipment, non-CF
8. abroris Rhizosphere, soil, water CF, non-CF
8. seminalis Rice rhizosphere CF, nosocornial Infection
8.metallica No environmental strain reported CF
8. contaminans Soll, water, animal CF, hospital equipment, non-CF
8.lata Soil, water, flower CF, non-CF
*Based on Ref. 11.
18 I Tu:..'i\TIE:.,'>\V I April 2014
allowing bacteria to access the respiratory tract" [7]. These authors
also demonstrated the growth of Bee in the glucose-rich nutrient
medium following the cultivation of the eucaroytic cells. Therefore,
this medium type (ATCC Medium 712 PYG) was included in the studies
to recover Bee after growth of amoeba in the medium.
.~~.~~.?.~?..~.?.&Y ...................................................................... . Organism Preparation
Three distinct types of organisms from the Burkholderia cepacia
complex were obtained from the American Type Culture Collection
(ATCC a): Burkholderia cepacia (Be) ATCC 25608, Burkholderia
cenocepacia (Bceno) ATCC BAA-245, and Burkholderia multivorans (8m)
ATCC BAA-247 [16]. Organisms were chosen based on discussions with
FDA, availability from ATCC, source, and nomenclature history. The
organisms were reconstituted as per ATCC instructions, cultured, and
then frozen and stored at -70°C using an internal seed lot technique.
Two cryovials per organism type were defrosted and transferred onto
two trypticase soy agar (TSA) slants for this study. After 48 hours of
incubation at 30-35°C, the slants were rinsed with sterile phosphate
buffer (SPB) and combined per each organism type. Each slurry of
organism was diluted and added to 500ml sterile USP Purified Water
to yield 103-104 organisms per ml. Seven bottles were prepared per
each organism (one bottle for each time point of testing). Inoculated
bottles were kept overnight at room temperature and then transferred
into a refrigerator (2-8°C) for the rest of the study to create a low
nutrient/low-temperature environment.
Materials
Both liquid and solid media were employed during this test (see Table
2). The selection of media was based on compendia! test methods (USP
<61 >, Microbiological Examination of Nonsterile Products Microbial
Enumeration Tests, and USP<62> Microbiological Examination of
Nonsterile Products: Tests for Specified Microorganisms), commonly
used environmental test methodology media, and media that were
documented to be used for isolation recovery of Burkholderia cepacia.
Acanthamoeba castellanii and Amoeba-Enriched Medium (ABM)
Acanthamoeba castellanii (AQ ATCC 30234 was reconstituted as per
manufacturer instructions and transferred into a 16 x 125mm plastic
test tube with 5ml ATCC medium 712 PYG. The culture was incubated at
25°C at approximately 15° horizontal slant. To maintain culture, 0.25ml
was transferred into 5ml fresh ATCC medium 712 PYG every 10-11 days
of incubation (multiple tubes were created at each transfer). Amoeba
Enriched Medium (AEM) was prepared as follows: AC was grown
for 4 days at 25°C in ATCC Medium 712 PYG (ATCC b). After 4 days of
incubation AC was removed by centrifugation and the medium was
filter-sterilized using a 0.22-micron sterile filter. At this point the medium
712 PVC was denoted as Amoeba-Enriched Medium (AEM). This AEM
was then evaluated for its ability to support growth of acclimated Bee.
-~~P..~.~-~~~~~~~ .. !.?.~~-~~~ ................................................. . Acclimation (Stress) of Organisms in Cold Environment
Organisms were acclimated by storage at 2-8°C for a total of six weeks
in Sterile USP Purified Water as described above. Time zero was defined
Type Reference
R2A Broth and 10% R2A Broth* 17
Tryptic Soy Broth with Lecithin and Tween (TSB+LT) and 18
10% (TSB+LT)*
Burkholderia cepacia Selective Broth (BCSB) 19 (without Agar)
TB Broth Enrichment Medium (TBEM) 20
Minimal Defined Broth (MOB) with two sources of carbon 21
ATCC Medium 712 PYG 22,23
Amoeba-Enriched Medium (AEM) ATCC Medium 712 PYG-after growth of amoeba (see Media section)
(( MICROBIOLOGY ~
as no more than 24 hours after the inoculated bottles were refrigerated.
The organism's suspensions were tested at weekly intervals marked as
day 7, 14, 21, 28, 35, and 42 during the acclimation period. Each interval
was tested to determine recoverable organisms by the following
methods (for media types refer to the previous section and Table 2):
1. Burkholderia cepacia Selective Agar (BCSA) Count: A 1 :1 O
dilution from each acclimation bottle was spread in duplicate
on BCSA without supplement for count confirmation.
Type I Reference
OFPBL Agar 19
Tryptlc Soy Agar 18
Total Count Agar Strips N/ A**
R2AAgar 17
Cetrimide Agar 18,24
Burkholderia cepacia Agar (BCSA) with and without 8. cepacia 19 selective supplement
*Low nutrient 10% broths were used as alternative under assumption that acclimated Bee organisms will have difficulties growing in high-nutrient versions.
**Common media used in recovery of environmental samples.
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., MICROBIOLOGY »
6.0
s.o
,4.0 .s 3.0
t s 2.0
1.0
0.0
-.-BC
,.,,.,.Jlce"'1_ ..... Bm
14 21 28 35 42
z.ac st.,.... !In ays)
Figure 1. Burkholderia cepacia complex organisms recovery on Burkholderia cepacia Agar w/o supplements
4.5
4.0
3 .5
i 3.0
i ~ :: ~ 1.5 -
1.0
0.5
0 .0
: : -Sc live cells logtO
.-.eceno llw cells Lcs10
-Bm Live Cells LoslO
0 14 21 28
2-BC Sto111•• In Deya
35 42
Figure 2. Live Cells Count by Epifluorescent Method for Burkholderia cepacia complex organisms
-- -
2. Epiflourescence Dye Count: 30ml from each bottle was
filtered through 25mm diameter stainless steel filter
holders pre-loaded with filter for live/dead epifluorescence
test method using a bacterial viability kit. A 95%
confidence level was applied to the test results [25,26].
The purpose of this treatment is to provide a culture
independent estimate of the number of dead vs living
bacterial cells in the sample.
3. Liquid Media Enhancement Comparison: A 1:10 dilution
of each acclimation bottle was made into liquid media
type (see Table 2 for listing) in duplicate. One set of tubes
was placed into a 30-35°C incubator and the second set
was placed into a 20-25°C incubator for total of 48 hours.
5.0
4.5
4.0
::: 3.5
.! 3.0
'i 2.5
i 2.0 ::; 1.5
1.0
0.5
0.0 0
-+-Be Dead cells Log10
_._Beene Dead Cells log10
..... Bm Dead Cells loglO
14 21 28
2« Storage In D•ys
35 42
Figure 3. Dead Cells Count by Epifluorescent Method for Burkho/deria cepacia complex organisms
All tubes were then streaked onto each of five solid media
types (Cetrimide, TSA, oxidation-fermentation polymyxin
bacitracin lactose [OFPBLJ, and Burkholderia cepacia
Selective Agar [BCSAJ with and without supplement) after
both 24 and 48 hours of incubation (unless growth was
confirmed within 24 hours). Streaked plates were incubated
at 30-35°C for up to 48 hours. Growth was checked after 24
hours and 48 hours. If growth was observed after 24 hours
further testing was discontinued. The liquid media used are
listed in in Table 2.
4. Membrane Filtration Enhancement Study: 1 OOmL from
each inoculated bottle was filtered in triplicate through
0.22-micron (nominal pore size) filters. Filters were
placed into 1 OOmL bottles with TSB+LT, R2A Broth, and
BCSB that were incubated at 30-35°C for a total of 48
hours. Bottles with filters were streaked onto each of five
solid media types (Cetrimide, TSA, OFPBL, and BCSA with
and without supplement) after both 24 and 48 hours
of incubation (unless growth was confirmed after 24
hours). Streaked plates were incubated at 30-35°C for up
to 48 hours.
5. Direct Plating: 0.1 ml from each inoculated bottle was
spread on Total Count Agar Strips using the spread
Table 3. Evaluation of Microbial Growth from TSB+LT incubated at 30-35°C
Media type/ Incubation: TSB+LT / 30-35°( after 48 hours of incubation
Solid Recovery Medium
Storage at OFPBL Cetrimide TSA BCSAw/ BCSAw/o
2-8°C (Days) Be Bceno Bm Be Bceno Bm Be Bceno Bm Be Bceno Bm Be Bee no Bm
1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
14 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
21 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
28 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
35 4 ~ 4 4 4 4 4 4 4 4 4 4 4 4 4 4
42 4 4 4 4 4 4 4 4 4 1 4 4 4 4 4 4
20 l Rev-iew I April 2014
I I
J
f
« MICROBIOLOGY
Table 4. Evaluation of Microbial Growth from Minimal Def!ned Broth incubated at 20-25°(
Media type / Incubation: Minimal Defined Broth 1 / 20-25°C after 48 hours of incubation
Solid Recovery Medium
OFPBL Cetrimide TSA BCSAw/ BCSA Storage at Bceno I Bm 2-8°C (Days) Be Bee no Bm Be Bee no Bm Be Be Bee no Bm Be Bee no Bm
1
7
14
21
28
35
42
No Data
4 1 4 4 2 4
4 2 4 4 2 4
4 0 4 4 2 4
4 0 4 4 1 4
4 0 4 4 0 4
4 1 2 4 1 2
plate method and incubated at 30-35°( for up to 3 days.
These strips were used to represent a common media
presentation used in environmental monitoring of air.
4
4
4
4
4
4
6. R2A Filter Recovery Study: 1 ml and 1 OOmL of each
organism were filtered through 0.22-micron filter onto R2A
Agar and incubated at 30-35°( for up to 5 days. This study
was performed to mimic standard water testing using
minimal media.
_2 ______ i 4 4 1 4 4 2 4
2
+~----4 2 4 4 2 4
1 4 1 4 4 1 1 4
2 1 4 4 2 4 4 2 4
1 1 4 4 1 4 4 1 4
1 1 4 4 1 4 4 1 4
Results
Study #1 (as described above) was performed to count live Bee
microorganisms surviving in a low-nutrient, low-temperature
environment (USP Purified Water at 2-8°C) by culturing. The
Epiflourescence test methodology (Study #2) was able to enumerate
viable microorganisms even ifthe bacteria were unable to be cultured.
Counts obtained by spread plate method on BSCA without addition
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MICROBIOLOGY »
· ·- ~ -- - ~-:-_ --:::--~T~~l~.-5! Eyj~~atf~~ ~f Micrc:>~i~f G~~':l from Minimal Defined Broth incubated at 30-35°(
Media type / Incubation: Minimal Defined Broth 1 / 30-35°C after 48 hours of incubation
Solid Recovery Medium
Storage at OFPBL Cetrimide TSA BCSAw/ BCSA
2-8°C (Days) Be Bee no Bm Be Bee no Bm Be Bceno Bm Be Bceno Bm Be Bee no Bm
1 4 4 4 4 4 1 4 4 4 4 4 4 4 4 4 4
7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -·---·
14 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
21 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
28 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
35 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
42 4 2 4 4 2 2 4 4 4 4 4 4 4 4 4
.. Table 6. Evaluation of Microbial ~rowth from Tryptan Blue Broth incubated at 20-25°( I
Media type / Incubation: Tryptan Blue Broth I 20-25°C after 48 hours of incubation
Solid Recovery Medium
Storage at OFPBL Cetrimide TSA BCSAw/ BCSA
2-8°C (Days) Be Bee no Bm Be Bceno I-Bm ____ Be Bee no Bm Be Bceno Bm Be Bee no Bm
1 No Data
7 4 0 0 4 __ o ---t~--- 4 0 0 4 0 0 4 0 0
--14 2 0 0 0 o I o 2 0 0 0 0 0 0 0 0
21 2 * * 1 . I • 4 * . 2 * . 2 * *
28 4 * * 4 * l • 4 * * 4 * . 4 . * I 35 4 * * 2 . I • 4 . * 4 * . 4 * *
42 1 0 0 0 0 I o 0 0 0 0 0 0 0 0 0
•Test was not performed if no recovery was observed on two consecutive time points. All tests were performed on last time point at 42 day of acclimatization.
Table 7. Ev~luation of_M~rol>.ial Growth from Tryptan Blue Broth incubated at 30-35°C
Media type / Incubation: Tryptan Blue Broth / 30-35°( after 48 hours of incubation
Solid Recovery Medium
Storage at OFPBL Cetrimide TSA BCSAw/ BCSA
2-8°C (Days) Be Bee no Bm Be Bee no Bm Be Bee no Bm Be Bceno Bm Be Bceno Bm
1 4 0 4 4 0 0 4 0 4 4 0 4 4 0 4
7 4 0 0 4 0 0 4 0 0 4 0 0 4 0 0
14 4 0 0 4 0 0 4 0 0 4 0 0 4 0 0
21 4 . * 4 . . 4 * . 4 * . 4 I • *
28 4 . . 4 * I • i
4 * * 4 * * 4 . . 35 4 " . 4 . . 4 . . 4 . . 4 I • *
42 4 0 0 4 0 l o 4 0 0 4 0 0 4 0 0
*lest was not performed if no recovery was observed on two consecutive time points. Ali tests were performed on last time point at 42 day of acclimatization.
of supplement (see Figure 1) were compared to counts obtained by counts for live cells vs counts for dead cells over time. This might be
epitluorescent live cell count (Figure 2}. due to the formation of a biofilm structure or to inherent variability
It was noted by both counting methods that counts for Burkholderia
cepacia increased after several weeks at 2-8°(, while counts for Burkholderia
cenocepacia and Burkholderia multivorans declined moderately.
Epitluorescence counts of live and dead cells were also compared
(Figures 2 and 3}. There was no direct correlation observed between
22 I &:view I April 2014
in the test.
Study #3 evaluated the ability of broth enrichment media to recover
Bee organisms. This was measured by streaking onto solid agar medium
(Test #3, see above). This ability was measured semi-quantitatively
using the key listed below. The recovery was grouped by medium
type and incubation temperature of the broth_ Table 3 provides these
data where the recovery was recorded as follows: 0 = no recovery,
1 = little growth to no growth, 2 = little growth, 3 = little growth/
sufficient growth, and 4= sufficient growth. Successful recovery was
considered when "sufficient growth" was observed on the solid media
after 48 hours of enrichment broth incubation and 48 hours of solid
media incubation: Bc-Burkholderia cepacia, Bceno-Burkholderia
cenocepacia, Bm-Burkholderia multivorans.
It was of interest in Study #3 that variations were evident between
liquid media recovery of Bee when incubated at different temperatures.
An example of these differences is shown in Tables 4 and 5 when the
recovery rate is compared between 30-35°( and 20-25°( incubation of
Minimal Defined Broth 1 for 48 hours.
The least successful liquid media to recover selected organisms was
Tryptan Blue Broth; refer to Tables 6 and 7.
It also was observed that through the 42-day acclimation period
evaluated, Burkholderia cepacia was the most consistently recovered
from all liquid media types after 24 hours of incubation enhancement
and plating on all solid agar types with "sufficient growth" (data not
shown). The least consistently recovered organism was Burkholderia
cenocepacia (see Tables 2 and 3 ).
All three organisms were resuscitated with "sufficient growth" when
1 OOmL were filtered through 0.2-micron filter and placed into BCB,
TSB, and R2A Broth (Test #4).
Acceptable recovery of all three organisms
was also observed when 0.1 ml was spread
« MICROBIOLOGY ~
throughout the study, they were still detectable by spread plate
method and by the epitluorescent live/dead test method. This
allows us to conclude that acclimated Burkholderia cepacia complex
organisms could be recovered when broth is enriched for 24-48
hours and then streaked for confirmation onto TSA or even more
selective media, such as OFPBL or BCSA.
We showed in this study that even very glucose-rich broth (ATCC
Medium 712 PYG as well as Amoeba-Enriched Medium) could
also successfully recover all three organisms after organisms
were acclimated to a low-temperature/low-nutrient environment.
However, it should be noted that preparation of these media types is
very complex and not necessary for routine testing performed in the
quality control microbiology laboratory.
Acknowledgments ·············································································································· The authors want to thank Steve Steinberger and Linda Contiliano of
Perritt Laboratories, Inc., for the ordering and preparation of all the
media used in the study, and Stephen Carpenter, Ph.D., from Pacific
Analytical Laboratory for providing its laboratory support with
epitluorescent testing on short notice.
over Total Count Agar strips (Test #5) and
after filtration of 1 ml and 1 OOmL through
0.22-micron filters that were placed on
R2A Agar (Test #6).
CONFIDENCE COMES WITH A HIGHER CALIBER OF DATA.
• \ \
Discussion/ Conclusions This study was designed to directly
address concerns about the ability of
standard microbiological test methods
to recover cold-water acclimated Bee
organisms. While it is well accepted
that the "Absence of Pseudomonas
aeruginosa" test in USP is not optimal
for recovery of Bee [27], this study has
shown conclusively that acclimated
species of Burkholderia cepacia complex
(representing different genomovars)
could be recovered using a non-specific
enrichment step with most compendia!
media as well as other common media
after 48 hours of incubation at 30-
350C. We also show that although
counts of Burkholderia cenocepacia and
Burkholderia multivorans were declining
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www.americanpharmaceuticalreview.com I Review I 23
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~ MICROBIOLOGY »
Author Biographies
Julie Barlasov has been the Laboratory Manager at Perritt
Laboratories in Hightstown, New Jersey for the past five years. Julie
manages a team of microbiologists responsible for testing products,
materials, water, and environmental samples for various clients
(mostly non-sterile pharmaceutical manufacturers). Julie has an MBA
degree in Pharmaceutical Industry from the University of Sciences in
Philadelphia and B.S. in Life Science from the Open University of Israel.
Julie has been working in the' Quality Control Microbiology field for
over 7 4 years. She can be reached at [email protected].
Scott Sutton has over 25 years of experience in the
the pharmaceutical, medical device, cosmetics, and
personal products industries with extensive publications
and presentations. Consulting and training in GMP,
contamination control, investigations of MOD (OOS), laboratory
management, and microbiology-related project management are
areas of special interest. His clients have included startups, generics,
established Fortune 500 companies, law firms, and investment broker
houses. Scott has owned and operated The Microbiology Network
(http://www.microbiol.org) since 7996. This company provides
consulting and training services to industry.
Rick Jakober is the Vice President, Laboratory Services, at
Perritt Laboratories, Inc. He earned a B.S. in Environmental
Sciences at Cook College, Rutgers University. Mr. Jakober
is a seasoned researcher with wide ranging experience
in microbiological analyses including in-depth knowledge of
FDA and compendia/ cGMP requirements combined with broad
knowledge and experience in USP, Ph. Eur., JP, ISO, ASTM, EPA, and
AOAC methodologies. In addition, he has over 30 years' experience
in pharmaceutical microbiology, specializing in non-sterile products.
This includes Microbial Content Testing, Antimicrobial Effectiveness
Testing, Water Systems, Environmental Monitoring, and Inorganic
Chemical Analyses. Mr. Jakober has been with Perritt Laboratories
since 7 987 and previously was with Carter-Wallace and Ayerst
Laboratories. Rick may be reached at [email protected].
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