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

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Burkholderia Cepacia Complex Organisms Recovery on Burkholderia Cepacia Agar W/O Supplements Figure 3 m» MICROBIOLOGY )) 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
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