Burkholderia Cepacia Complex Organisms Recovery on Burkholderia Cepacia Agar W/O Supplements Figure 3

Recovery of Introduction The FDA has adopted the position that all new product submissions for Stressed (Acclimated) 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 Burkholderia cepacia 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 Complex Organisms 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.

16 I Rb"Vrew I April 2014 vol 17 .,,. MICROBIOLOGY ))

The genus Burkholderia currently comprises more than 60 species. This allowing bacteria to access the respiratory tract" [7]. These authors genus was proposed in 1992 to accommodate the former rRNA group also demonstrated the growth of Bee in the glucose-rich nutrient II pseudomonads [1 OJ. Taxonomy of the entire Burkholderia genus medium following the cultivation of the eucaroytic cells. Therefore, has changed rapidly: for instance, 8. cepacia has gone from a single this medium type (ATCC Medium 712 PYG) was included in the studies species to being a complex comprising 17 closely related species, or to recover Bee after growth of amoeba in the medium. 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 .~~. ~~. ?.~?..~.?.&Y ...... cepacia (or Pseudomonas cepacia), leading to some confusion. Several Bee strains have developed beneficial interactions with their plant Organism Preparation

hosts and have proven to be very efficient biocontrol, bioremediation, Three distinct types of organisms from the Burkholderia cepacia or plant-growth promoting agents [12-14]. Refer to Table 1 for an complex were obtained from the American Type Culture Collection overview of the Burkholderia cepacia complex. (ATCC a): Burkholderia cepacia (Be) ATCC 25608, Burkholderia In the past two decades, Burkholderia cepacia has also emerged as cenocepacia (Bceno) ATCC BAA-245, and Burkholderia multivorans (8m) an opportunistic human pathogen causing numerous outbreaks, ATCC BAA-247 [16]. Organisms were chosen based on discussions with particularly among cystic fibrosis (CF) and other immunocompromised FDA, availability from ATCC, source, and nomenclature history. The patients. One highly transmissible strain has spread across North organisms were reconstituted as per ATCC instructions, cultured, and America and Britain, and another between hospitalized CF and then frozen and stored at -70°C using an internal seed lot technique. non-CF patients [15]. In addition, Burkholderia cepacia is inherently Two cryovials per organism type were defrosted and transferred onto resistant to multiple antibiotics and molecular epidemiology, and 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 phylogenetic studies demonstrate that highly transmissible strains buffer (SPB) and combined per each organism type. Each slurry of emerge randomly; the organism has a capacity for rapid mutation and organism was diluted and added to 500ml sterile USP Purified Water adaptation (facilitated by numerous insertion sequences) and a large, to yield 103-104 organisms per ml. Seven bottles were prepared per complex genome divided into separate chromosomes. each organism (one bottle for each time point of testing). Inoculated An interesting side note on 8. cepacia physiology was recently bottles were kept overnight at room temperature and then transferred described by Vial et al. [7], who described experiments showing into a refrigerator (2-8°C) for the rest of the study to create a low that Bee can survive and grow within the vacuoles of both amoeba nutrient/low-temperature environment. and mammalian macrophages and monocytes. Nasal mucosa has been known to carry amoeba and "consequently could represent an Materials important natural reservoir for Bee strains and act as a Trojan horse Both liquid and solid media were employed during this test (see Table 2). The selection of media was based on compendia! test methods (USP Table 1. Overview of the Burkholderia cepacia Complex* <61 >, Microbiological Examination of Nonsterile Products Microbial Species Natural Environment Clinical Environment Enumeration Tests, and USP<62> Microbiological Examination of 8. cepacia Rhizosphere, soil, water Cystic fibrosis (CF), medical solution Nonsterile Products: Tests for Specified Microorganisms), commonly Rhizosphere, soil, water CF, CGD, non-CF 8. multivorans ----- used environmental test methodology media, and media that were 8. cenocepacia Rhizosphere, plant, soil, water, animal CF, non-CF documented to be used for isolation recovery of Burkholderia cepacia. B. stabilis Rhizosphere CF, (rare) hospital equipment 8. vietnamiensis Rhizosphere, plant, soil, water, animal CF Acanthamoeba castellanii and Amoeba-Enriched B. dolosa Maize rhlzosphere, plant CF Medium (ABM) 8. ambifaria Rhizosphere, soil CF (rare)

B.anthina Rhizosphere, so_il CF(rare) Acanthamoeba castellanii (AQ ATCC 30234 was reconstituted as per

8. pyrrocinia Rhizosphere, soil, water, plant CF, non-CF (rare) manufacturer instructions and transferred into a 16 x 125mm plastic test tube with 5ml ATCC medium 712 PYG. The culture was incubated at 8. ubonensis Soil Nosocornial infection ------25°C at approximately 15° horizontal slant. To maintain culture, 0.25ml 8. letens No environmental strain reported CF was transferred into 5ml fresh ATCC medium 712 PYG every 10-11 days 8.diffusa Soil, water CF, hospital equipment, non-CF of incubation (multiple tubes were created at each transfer). Amoeba 8. abroris Rhizosphere, soil, water CF, non-CF Enriched Medium (AEM) was prepared as follows: AC was grown 8. seminalis Rice rhizosphere CF, nosocornial Infection for 4 days at 25°C in ATCC Medium 712 PYG (ATCC b). After 4 days of 8.metallica No environmental strain reported CF incubation AC was removed by centrifugation and the medium was 8. contaminans Soll, water, animal CF, hospital equipment, non-CF filter-sterilized using a 0.22-micron sterile filter. At this point the medium 8.lata Soil, water, flower CF, non-CF 712 PVC was denoted as Amoeba-Enriched Medium (AEM). This AEM *Based on Ref. 11. was then evaluated for its ability to support growth of acclimated Bee.

18 I Tu:..'i\TIE:.,'>\V I April 2014 (( MICROBIOLOGY ~

as no more than 24 hours after the inoculated bottles were refrigerated. The organism's suspensions were tested at weekly intervals marked as -~~P..~.~ -~~~~~~~ .. !.?. ~~ -~~~...... day 7, 14, 21, 28, 35, and 42 during the acclimation period. Each interval was tested to determine recoverable organisms by the following Acclimation (Stress) of Organisms in Cold methods (for media types refer to the previous section and Table 2):

Environment 1. Burkholderia cepacia Selective Agar (BCSA) Count: A 1:1 O Organisms were acclimated by storage at 2-8°C for a total of six weeks dilution from each acclimation bottle was spread in duplicate in Sterile USP Purified Water as described above. Time zero was defined on BCSA without supplement for count confirmation.

Type Reference Type I Reference R2A Broth and 10% R2A Broth* 17 OFPBL Agar 19

Tryptic Soy Broth with Lecithin and Tween (TSB+LT) and 18 Tryptlc Soy Agar 18 10% (TSB+LT)*

Burkholderia cepacia Selective Broth (BCSB) 19 (without Agar) Total Count Agar Strips N/ A**

TB Broth Enrichment Medium (TBEM) 20 R2AAgar 17

Minimal Defined Broth (MOB) with two sources of carbon 21 Cetrimide Agar 18,24

ATCC Medium 712 PYG 22,23 Burkholderia cepacia Agar (BCSA) with and without 8. cepacia 19 selective supplement

Amoeba-Enriched Medium (AEM) ATCC Medium 712 PYG-after growth of amoeba (see Media section)

*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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5.0 6.0 4.5 s.o 4.0 ,4.0 ::: 3.5 .! 3.0 .s 3.0 t 'i 2.5 s2 .0 -.-BC i 2.0 -+-Be Dead cells Log10 1.0 ,.,,.,.Jlce"'1_ ::; 1.5 .....Bm 1.0 _._Beene Dead Cells log10 0.0 0.5 14 21 28 35 42 ..... Bm Dead Cells loglO z.ac st.,.... !In ays) 0.0 0 14 21 28 35 42 2« Storage In D•ys Figure 1. Burkholderia cepacia complex organisms recovery on Burkholderia cepacia Agar w/o supplements Figure 3. Dead Cells Count by Epifluorescent Method for Burkho/deria cepacia complex organisms

4.5 4.0 3.5 i 3.0 : : All tubes were then streaked onto each of five solid media i ~ : : types (Cetrimide, TSA, oxidation-fermentation polymyxin ~ 1.5 - -Sc live cells logtO bacitracin lactose [OFPBLJ, and Burkholderia cepacia 1.0 .-.eceno llw cells Lcs10 0.5 -Bm Live Cells LoslO Selective Agar [BCSAJ with and without supplement) after 0 .0 0 14 21 28 35 42 both 24 and 48 hours of incubation (unless growth was 2-BC Sto111•• In Deya confirmed within 24 hours). Streaked plates were incubated at 30-35°C for up to 48 hours. Growth was checked after 24 Figure 2. Live Cells Count by Epifluorescent Method for Burkholderia cepacia complex organisms 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.

2. Epiflourescence Dye Count: 30ml from each bottle was 4. Membrane Filtration Enhancement Study: 1OOmL from filtered through 25mm diameter stainless steel filter each inoculated bottle was filtered in triplicate through holders pre-loaded with filter for live/dead epifluorescence 0.22-micron (nominal pore size) filters. Filters were test method using a bacterial viability kit. A 95% placed into 1OOmL bottles with TSB+LT, R2A Broth, and confidence level was applied to the test results [25,26]. BCSB that were incubated at 30-35°C for a total of 48 The purpose of this treatment is to provide a culture hours. Bottles with filters were streaked onto each of five independent estimate of the number of dead vs living solid media types (Cetrimide, TSA, OFPBL, and BCSA with bacterial cells in the sample. and without supplement) after both 24 and 48 hours 3. Liquid Media Enhancement Comparison: A 1:10 dilution of incubation (unless growth was confirmed after 24 of each acclimation bottle was made into liquid media hours). Streaked plates were incubated at 30-35°C for up type (see Table 2 for listing) in duplicate. One set of tubes to 48 hours. was placed into a 30-35°C incubator and the second set 5. Direct Plating: 0.1 ml from each inoculated bottle was was placed into a 20-25°C incubator for total of 48 hours. spread on Total Count Agar Strips using the spread

I Table 3. Evaluation of Microbial Growth from TSB+LT incubated at 30-35°C I Media type/ Incubation: TSB+LT / 30-35°( after 48 hours of incubation Solid Recovery Medium OFPBL Cetrimide TSA BCSAw/ BCSAw/o Storage at 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 J

42 4 4 4 4 4 4 4 4 4 1 4 4 4 4 4 4 f

20 l Rev-iew I April 2014 « 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/ Storage at BCSA 2-8°C (Days) Be Bee no Bm Be Bee no Bm Be Bceno I Bm Be Bee no Bm Be Bee no Bm 1 No Data 7 4 1 4 4 2 4 4 _2 ______i 4 4 1 4 4 2 4 14 4 2 4 4 2 4 4 2 4 2 4 4 2 4 21 4 0 4 4 2 4 4 1 +~---- 4 1 4 4 1 1 4 28 4 0 4 4 1 4 4 2 1 4 4 2 4 4 2 4

35 4 0 4 4 0 4 4 1 1 4 4 1 4 4 1 4

42 4 1 2 4 1 2 4 1 1 4 4 1 4 4 1 4

plate method and incubated at 30-35°( for up to 3 days. These strips were used to represent a common media Results presentation used in environmental monitoring of air. Study #1 (as described above) was performed to count live Bee 6. R2A Filter Recovery Study: 1ml and 1OOmL of each microorganisms surviving in a low-nutrient, low-temperature organism were filtered through 0.22-micron filter onto R2A environment (USP Purified Water at 2-8°C) by culturing. The Agar and incubated at 30-35°( for up to 5 days. This study Epiflourescence test methodology (Study #2) was able to enumerate was performed to mimic standard water testing using viable microorganisms even ifthe bacteria were unable to be cultured. minimal media. Counts obtained by spread plate method on BSCA without addition

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· ·- ~ -- - ~-:-_ --:::--~T~~l~.-5! Eyj~~atf~~ ~f Micrc:>~i~fG ~~ ':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 OFPBL Cetrimide TSA BCSAw/ BCSA Storage at 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

.. I Table 6. Evaluation of Microbial ~rowth from Tryptan Blue Broth incubated at 20-25°( Media type / Incubation: Tryptan Blue Broth I 20-25°C after 48 hours of incubation Solid Recovery Medium OFPBL Cetrimide TSA BCSAw/ BCSA Storage at Bceno Bm____ 2-8°C (Days) Be Bee no Bm Be I- Be Bee no Bm Be Bceno Bm Be Bee no Bm

1 No Data

7 4 0 0 4 4 0 0 4 0 0 4 0 0 -- __o ---t ~--- 14 2 0 0 0 o I o 2 0 0 0 0 0 0 0 0 21 2 * * 1 . I • 4 * . 2 * . 2 * * l • 28 4 * * 4 * I 4 * * 4 * . 4 . * 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 OFPBL Cetrimide TSA BCSAw/ BCSA Storage at 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 • 4 * * 4 * * 4 . . i 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 in the test. It was noted by both counting methods that counts for Burkholderia Study #3 evaluated the ability of broth enrichment media to recover cepacia increased after several weeks at 2-8°(, while counts for Burkholderia Bee organisms. This was measured by streaking onto solid agar medium cenocepacia and Burkholderia multivorans declined moderately. (Test #3, see above). This ability was measured semi-quantitatively Epitluorescence counts of live and dead cells were also compared using the key listed below. The recovery was grouped by medium (Figures 2 and 3}. There was no direct correlation observed between type and incubation temperature of the broth_ Table 3 provides these

22 I &:view I April 2014 « MICROBIOLOGY ~

data where the recovery was recorded as follows: 0 = no recovery, throughout the study, they were still detectable by spread plate 1 = little growth to no growth, 2 = little growth, 3 = little growth/ method and by the epitluorescent live/dead test method. This sufficient growth, and 4= sufficient growth. Successful recovery was allows us to conclude that acclimated Burkholderia cepacia complex considered when "sufficient growth" was observed on the solid media organisms could be recovered when broth is enriched for 24-48 after 48 hours of enrichment broth incubation and 48 hours of solid hours and then streaked for confirmation onto TSA or even more media incubation: Bc-Burkholderia cepacia, Bceno-Burkholderia selective media, such as OFPBL or BCSA. cenocepacia, Bm-Burkholderia multivorans. We showed in this study that even very glucose-rich broth (ATCC It was of interest in Study #3 that variations were evident between Medium 712 PYG as well as Amoeba-Enriched Medium) could liquid media recovery of Bee when incubated at different temperatures. also successfully recover all three organisms after organisms An example of these differences is shown in Tables 4 and 5 when the were acclimated to a low-temperature/low-nutrient environment. recovery rate is compared between 30-35°( and 20-25°( incubation of However, it should be noted that preparation of these media types is Minimal Defined Broth 1 for 48 hours. very complex and not necessary for routine testing performed in the The least successful liquid media to recover selected organisms was quality control microbiology laboratory. 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 Acknowledgments from all liquid media types after 24 hours of incubation enhancement ·············································································································· and plating on all solid agar types with "sufficient growth" (data not The authors want to thank Steve Steinberger and Linda Contiliano of shown). The least consistently recovered organism was Burkholderia Perritt Laboratories, Inc., for the ordering and preparation of all the cenocepacia (see Tables 2 and 3 ). media used in the study, and Stephen Carpenter, Ph.D., from Pacific All three organisms were resuscitated with "sufficient growth" when Analytical Laboratory for providing its laboratory support with 1OOmL were filtered through 0.2-micron filter and placed into BCB, epitluorescent testing on short notice. TSB, and R2A Broth (Test #4).

Acceptable recovery of all three organisms was also observed when 0.1 ml was spread over Total Count Agar strips (Test #5) and CONFIDENCE COMES • after filtration of 1ml and 1OOmL through \ 0.22-micron filters that were placed on WITH A HIGHER \ R2A Agar (Test #6). CALIBER OF DATA. Introducing The BioTrak® Discussion/ Real-Time Viable Particle Counter Conclusions An accurate real-time assessment of environmental • microbial conditions. It's the dividing line between This study was designed to directly knowing and hoping that your pharmaceutical address concerns about the ability of microbial levels are under control. standard microbiological test methods The BioTrak® Particle Counters feature patented, to recover cold-water acclimated Bee field proven viable measurement technology for organisms. While it is well accepted the most discrminating data available. that the "Absence of Pseudomonas The end results are real-time microbial information, aeruginosa" test in USP is not optimal cost savings, and improved process control. for recovery of Bee [27], this study has Understand more at tsi.com/biotrak 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- .,~· UNDERSTANDING, ACCELERATED 350C. We also show that although counts of Burkholderia cenocepacia and *Patents: 5,701,012; 5.895,922; 6,831,279 Burkholderia multivorans were declining

www.americanpharmaceuticalreview.com I Review I 23 I

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5. S. Sutton and L. Jimenez. A review of reported recalls involving microbiological control Author Biographies 2004-2011 with emphasis on FDA considerations of "objectionable organisms." Amer. Pharm. Rev. 2072; 15(1):42-57.

Julie Barlasov has been the Laboratory Manager at Perritt 6. S. Sutton. What is an objectionable organism? Amer. Pharm. Rev. 2012; 15(6):36-48.

Laboratories in Hightstown, New Jersey for the past five years. Julie 7. L. Vial, A. Chapalain, M.C. Groleau, E. Deziel. Minireview-the various lifestyles of the manages a team of microbiologists responsible for testing products, Burkholderia cepacia complex species: a tribute to adaptation. Envir. Microbial. 2071; materials, water, and environmental samples for various clients 13(1): 1-12. (mostly non-sterile pharmaceutical manufacturers). Julie has an MBA 8. E. Mahenthiralingam, T.A. Urban, and J.B. Goldberg. The multifarious, multireplicon degree in Pharmaceutical Industry from the University of Sciences in Burkholderia cepacia complex. Nature Reviews Microbial. 2005; 3(2) : 144-156. Philadelphia and B.S. in Life Science from the Open University of Israel. 9. W Beckman and T. G. Lessie. Response of Pseudomonas cepacia to p-lactam antibiotics: Julie has been working in the' Quality Control Microbiology field for utilization of penicillin Gas the carbon source. J. Bacterial. 19 79; 140: 1126-1128. over 74 years. She can be reached at [email protected]. 10. W Burkholder. Sour skin, a bacterial rot of onion bulbs. Phytopathology 1950; 40: 715-8. 11. L. Chiarini, et al. Burkholderia cepacia complex species: health hazards and Scott Sutton has over 25 years of experience in the biotechnological potential. Trends Microbial. 2006; 14(6):277-286.

the pharmaceutical, medical device, cosmetics, and 72. E. Yabuuchi, Y. Kosako, H. Oyaizu, I. Yono, H. Hatta, Y. Hashimoto, et al. Proposal of personal products industries with extensive publications Burkholderia gen. nov. and transfer of 7 species of the genus Pseudomonas homology and presentations. Consulting and training in GMP, group-I/ to the new genus, with the type species Burkholderia cepacia. Microbial. contamination control, investigations of MOD (OOS), laboratory lmmunol. 1992; 36(12): 1251-1275. management, and microbiology-related project management are 13. J.L. Parke, D. Gurian-Sherman. Diversity of the Burkholderia cepacia complex and areas of special interest. His clients have included startups, generics, implications for risk assessment of biological control strains. Ann. Rev. Phytopatho/. 2001; 39: 225-58. established Fortune 500 companies, law firms, and investment broker houses. Scott has owned and operated The Microbiology Network 14. T. Coen ye and P. Vandamme. Diversity and significance ofBurkholderia species occupying diverse ecological niches. Environ. Microbial. 2003; 5(9):719-29. (http://www.microbiol.org) since 7996. This company provides 15. A. Holmes, J. Govan, and R. Goldstein. Agricultural use of Burkholderia (Pseudomonas) consulting and training services to industry. cepacia: a threat to human health? Emerging Infectious Dis. 1998; 4(2): 221-228. Rick Jakober is the Vice President, Laboratory Services, at 16. ATCC a. Product Sheet for BAA-247 (Burkholderia multivorans), BAA-245 (Burkholderia Perritt Laboratories, Inc. He earned a B.S. in Environmental cenocepacia), and 25416 (Burkholderia cepacia). Sciences at Cook College, Rutgers University. Mr. Jakober 17. D.J. Reasoner and E.E. Geldreich. A new medium for the enumeration and subculture of is a seasoned researcher with wide ranging experience bacteria from potable water. Appl. Environ. Microbial. 1985; 49(1):7-7. in microbiological analyses including in-depth knowledge of 78. USP. 2073 < 61 > Microbiological Examination of Nonsterile Products: Microbial FDA and compendia/ cGMP requirements combined with broad Enumeration Tests, and <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. USP36/NF31 vol. 1, The United States Pharmacopeial knowledge and experience in USP, Ph. Eur., JP, ISO, ASTM, EPA, and Convention. AOAC methodologies. In addition, he has over 30 years' experience 19. M.B. Glass, C.A. Beesley, P.P. Wilkins, and A.R. Hoffmaster. Comparison of four selective in pharmaceutical microbiology, specializing in non-sterile products. media for the isolation of Burkholderia ma/lei and Burkholderia pseudomal/ei. Am. J. This includes Microbial Content Testing, Antimicrobial Effectiveness Trap. Med. Hyg. 2009; 80(6): 7023-1028. Testing, Water Systems, Environmental Monitoring, and Inorganic 20. C. Hagedorn, WD. Gould, T.R. Bardinel/i, and D.R. Gustavson. A selective medium for Chemical Analyses. Mr. Jakober has been with Perritt Laboratories enumeration and recovery of Pseudomonas cepacia biotypes from soil. Appl. Envir. since 7987 and previously was with Carter-Wallace and Ayerst Microbial. 1987; 53(9): 2265-2268.

Laboratories. Rick may be reached at [email protected]. 21 . K. Vermis, M. Brachkova, P. Vandamme, and H. Neils. Isolation of Burkholderia cepacia complex genomovars from waters. Systematic and Applied Microbial. 2003; 26:595- 600.

22. ATCC b. Collection of Protists-Production Information Sheet for ATCC 30234 References (Acanthamoeba castellanii).

1. FDA. 2011 Product Quality Microbiology Review NDA: 202-245 Codeine Sulfate 23. F.L. Schuster. Cultivation of pathogenic and opportunistic free-living ameobas. Clin. Oral Solution (30 mg/5 ml); ttp://www.accessdata.fda.gov/ drugsatfda_ docs/ Microbial. Rev. 2002; 15(3):342-354. nda/2011/2022450rig1 sOOOMicroR.pdf (accessed 12/ 19/13). 24. E.O. King, et al. Two simple media for the demonstration of pyocyanin and fluorescein. J. 2. J.W Metcalf. Regulatory Review Perspectives for Non-Sterile Drug Products, 2013. Lab. Clin. Med. 1954; 44:301-307. Presented at the 2013 PDA Microbiology Conference in October, 2013. 25. L. Boulos, L. Live/Dead Badight: application of a new rapid staining method for direct 3. L.D. Torbeck, D. Racassi, D. Guilfoyle, R.L. Friesdman, and D. Hussong. Burkholderia enumeration of viable and total bacteria in drinking water. J. Microbial. Meth. 7999; cepacia: this decision is overdue. PDA Journal of Pharm. Sci. and Technology 2011; 37:77-86. 65(5): 535-543. 26. Molecular Probes lnvitrogen Detection Technologies, 2004. Live/Dead BacLight Bacteria/ 4. S. Sutton . Letter to the Editor, 2072, in response to L. Torbeck, et al., Burkholderia Viability Kits, Product Information MP07007. cepacia: This Decision Is Overdue. PDA Journal of Pharm. Sci. and Technol. 2012, 66(2): 27. USP. 1982. Microbial Contamination of Sterile and Non-Sterile Articles, with Special 91-95. Reference to Pseudomonas cepacia. Pharm. Forum 1982; 8(4):2239.

24 I Revrew I April 2014