Volume 2 Issue 1 - 2015
Burkholderia cepacia: A problem that does not go away!
Luis Jimenez*, Elizabeth Kulko, Elyssa Barron and Thomas Flannery
Department of Biology and Horticulture, Bergen Community College, USA
*Corresponding Author: Luis Jimenez, Biology and Horticulture Department, Bergen Community College, 400 Paramus, New Jersey, 07652, USA.
Received: June 17, 2015; Published: July 13, 2015
Citation: Luis Jimenez., et al. “Burkholderia cepacia: A problem that does not go away!”. EC Microbiology 2.1 (2015): 205-210.
Because of the genetic and metabolic capabilities to overcome environmental stresses during processing and manufacturing, Burkholderia cepacia is still the number one bacterial species found in contaminated pharmaceutical products. Nucleic acid based technologies are available to detect, identify, and quantify the numbers of B. cepacia cells in environmental samples such as pharmaceutical water, raw materials, and finished products. However, development and application of these technologies to pharmaceutical quality control have been rather slow. Rapid PCR detection of B. cepacia in pharmaceutical products contaminated with a mixed bacterial culture allowed faster detection times and higher resolution than standard microbiological methods that require time consuming and multiple procedures.
Keywords: Burkholderia cepacia; Pharmaceutical products; Biocides; PCR; Recalls
Microbial contamination is still a major reason for product recall in the United States. Several publications have reported the frequency of microbial contamination in non-sterile and sterile products from 1998 to 2011 [1,2] . When microbial contamination was found the number one species isolated were Burkholderia cepacia [1,2]. Looking at FDA recall data from a year after the last publication was reported, B. cepacia was still the number one microbial contaminant in non-sterile products (Table 1) [3]. Thirty nine percent of bacteria isolated from contaminated samples were identified as B. cepacia (Figure 1). The contaminated samples were sanitizers, oral pharmaceuticals, and gas relief drops.
Furthermore, a recent outbreak of B. cepacia complex (Bcc) pseudo bacteremia was associated to contaminated antiseptic formulations [4]. During that outbreak B. cepacia was isolated from blood cultures of 40 patients and antiseptic formulations. The outbreak investigation determined that the formulation was misused as a skin antiseptic during blood culture. The contaminated product was discarded and the staff retrained. Another outbreak was reported at a private hospital where 13 cancer patients undergoing chemotherapy developed B. cepacia bacteremia due to a contaminated antiemetic drug [5]. The outbreak lasted 2 months and was controlled when hospital personnel was properly educated to optimize daily aseptic practices. Opened and unopened vials of the antiemetic drug grew B. cepacia. Environmental samples from water, surfaces, equipment, air, disinfectants, and antiseptics did not show the presence of B. cepacia.
The persistence of B. cepacia in pharmaceutical products can be explained by the lack of proper good manufacturing practices (GMP) and the use of compendial methods that do not provide the sensitivity and resolution to detect B. cepacia in pharmaceutical water, raw materials, and finished products [1,2,6]. Most companies relied on traditional cultivation and phenotypic methods to isolate and identify microbial contamination. These methods are time consuming (5-7 days) and laborious. They relied upon the growth of microorganisms on the specific substrates in the media. However, some bacteria do not grow on those substrates or grow extremely slow to be detected by the incubation times currently used. In some cases microbial cells undergo a physiological state by reducing metabolic reactions rates and cell size which are affecting the protein and enzymatic profiles used to identify environmental isolates [1]. These microbial cell changes are triggered by physical processes and environmental systems implemented to reduce or eliminate microorganisms during manufacturing. If the processes and systems are not validated or properly implemented, microorganisms contaminated products, raw materials, and equipment [1,6]. B. cepacia genetic and metabolic diversity are severely underestimated by industrial operators. B. cepacia genome consists of more than one chromosome containing a wide variety of metabolic genes, which appear to be acquired by horizontal gene transfer [7]. B. cepacia is capable of growing on nitro aromatic and aromatic compounds by the action of different enzymes such as mono oxygenases and di oxygenases [8]. These enzymesnot only oxidize aromatic structures but also breakdown halogenated compounds [9]. Therefore is not only the health hazard to patients that makes B. cepacia a real nightmare for quality control microbiologists but product stability and purity are compromised by the degradation of active ingredients and excipients resulting in sub potent formulations. Nitro aromatic compounds are major components of many pharmaceutical drugs [10]. For instance, antipsychotic and analgesic drugs are based upon aromatic structures sensitive to biodegradation attack by mono and di oxygenases from microbial contaminants.
B. cepacia populations grow and survive in biocides, pharmaceutical products, active ingredients, and excipients leading to the development of resistance to preservative systems used to protect formulations [11-13]. They have also intrinsic antibiotic resistance by the release of b-lactamases, the impermeability of the outer envelope to antimicrobial agents, and specific efflux pumps. Biofilm formation is another adaptation that provided physiological resistance to antimicrobial treatments. Recent studies showed that a wide variety of commercial products contain biocides concentrations that are insufficient to kill B. cepacia and other Bcc species [13]. Based upon their analysis, investigators calculated that Bcc bacteria might need more than 25 times more biocides over the MIC value to achieve killing. No correlation was found between susceptibility to biocides, susceptibility to antibiotics, and biofilm formation. Some strains showing very high biocide resistance exhibited vigorous biofilm formation.
Other studies showed that after exposure to biocides, biofilm populations of B. cenocepacia expressed different genes related to membrane proteins, regulatory proteins, efflux pumps, oxidative stress response, and chemo taxis [14]. The transcriptional response to biocide treatment was ascertained by microarray analysis and real-time quantitative PCR. A wide variety of efflux pump systems were more predominant in biofilm-grown cells while plank tonic cells relied on other resistance mechanisms. Many genes encoding for transport-related proteins were down regulated as a consequence of biocide treatment. All these studies indicate the need to improve and develop rapid detection methods for B. cepacia to implement control practices leading to the reduction of microbial contamination and morbidity reports.
However, the polymerase chain reaction (PCR) has been shown to detect and identify B. cepacia in artificially contaminated pharmaceutical samples within 27 hours [15]. The assay was validated using DNA primers targeting a specific and highly conserved 209 base pair (bp) 16S rRNA gene fragment. Adding 10 µl l of the product suspension to 200 µl of the lysis buffer provided enough material for the PCR reaction to be performed [15]. The lysis buffer was based upon mild chemical ingredients. The samples were incubated at 37°C for 20 minutes in Tris-EDTA buffer and Tween 20 followed by a Proteinase K treatment for 10 minutes at 95°C. The DNA extraction procedure was basically a two-step protocol that was easy to perform with minimal sample manipulation and no hazardous chemicals. The DNA primers targeting the 209 bp fragment were added to different aliquots of the extracted microbial DNA containing Ready-To-Go PCR beads. The beads provided all the reagents needed for the PCR reaction in a convenient, ambient-temperature-stable form. They contain buffer, nucleotides, and Taq polymerase. The use of the beads minimized sample handling and cross contamination. None of the tested products or raw materials inhibited the PCR reaction. No amplified DNA fragment was obtained with uninoculated samples. All contaminated samples, e.g., 10 products and raw materials, showed the presence of the fragment indicating a positive detection and identification [15].
However, microbial contamination of pharmaceutical products is not only caused by pure cultures of specific microorganisms. For example, Table 1 shows that microbial contamination can be a result of mixed cultures of microorganisms. PCR detection of a target bacterial species present in pharmaceutical samples contaminated with a mixed bacterial culture was previously reported [16]. A recent study showed that PCR was used to detect Burkholderia species in high purity water systems despite the high background with mixed microbial contamination [17]. After water samples were concentrated on membrane filters, microbial DNA was extracted and amplified by a Quantitative Real-Time PCR assay. Quantitative detection and identification were completed in less than 5 hours.
In our laboratory, we spiked two pharmaceutical products, an over the counter antihistamine medication (product A) and nausea medication (product B) with low levels, < 100 colony forming units (CFU), of B. cepacia, Escherichia coli, Bacillus sp., and Dietzia sp. Figure 2 shows the counts for the B. cepacia culture used during the study. E. coli is one of the indicators in the Microbial Limits testing of pharmaceutical products. Different types of Bacillus species are commonly found in environmental samples during pharmaceutical operations [1]. For instance, B. cereus was found in 17% of the recalled products (Table 1) (Figure 1). Dietzia is a member of the Actinobacteria phylum with slow growth rates in environmental samples.
The contaminated samples were incubated at 37°C for 24 hours with shaking, e.g., 200 rpm. After incubation, DNA was extracted as previously described [15]. Other DNA extraction protocols were analyzed but none of them provided enough material to optimize the PCR reaction. For optimal PCR detection of B. cepacia in pharmaceutical products contaminated with mixed bacterial cultures, an aliquot of 50 µl was necessary to obtain a positive reaction (Figure 3). Lanes 3 and 4 showed the presence of the 209 bp 16S rRNA fragment. Lanes 5 and 6 contained 10 µl aliquots of the extracted microbial DNA that evidently did not show the same band intensity.
Rapid detection of B. cepacia was completed within 27 hours despite the presence of the other microorganisms in the product suspensions. Our laboratory is currently working on a Real Time Quantitative PCR protocol to detect, identify, and quantify the numbers of B. cepacia in pure and/or mixed cultures of contaminated pharmaceutical materials with a minimal incubation time, e.g., 3-5 hours. Rapid detection of B. cepacia in pharmaceutical products contaminated with mixed bacterial cultures demonstrated that nucleic acid based technologies can improve detection times leading to process optimization by early detection of potential problems with the possibility of rapid implementation of corrective actions. Pharmaceutical quality control is greatly improved when systems are developed to provide critical assessment of product quality and process control. Pharmaceutical manufacturing is a highly technical and detailed operation that requires specialized personnel and complex processes to control the continuity, reproducibility, and stability of the different systems put in place to guarantee the compliance to GMP regulations and provide safe, efficacious, and stable products. B. cepacia persistent presence in contaminated pharmaceutical samples indicates the inadequate application of control strategies and detection methods to optimize quality and process control.
Recall Number Non Sterile Reason
1 Alcohol Free Sanitizer B. cepacia
2 Alcohol Free Sanitizer B. cepacia
3 Oral pharmaceuticals B. cepacia
4 Alcohols pads Bacillus cereus
5 Alcohols pads Bacillus cereus
6 Gelatin capsules Microbial contamination
7 Alcohols pads Bacillus cereus
8 Pharmaceutical cream Enterobacter gergoviae and Pseudomonas monteilii/plecoglossicida.
9 Povidone iodine solution Elizabethkingia meningoseptica
10 Pharmaceutical gel Microbial contamination
11 Baby lotion Microbial contamination, Staphylococcus
12 Pepto bismol Microbial contamination
13 Gas relief drops (Simethicone) Microbial contamination, B. cepacia
14 Gas relief drops (Simethicone) Microbial contamination, B. cepacia
15 Pharmaceutical solution LSA Antimicrobial preservative failure
16 Pharmaceutical solution LSA Antimicrobial preservative failure
17 Hand sanitizer B. cepacia
18 Hand sanitizer B. cepacia
Table 1: Pharmaceutical product recall data from January 2012 to July 2012.
Figure 1: Percentage of bacterial species isolated in pharmaceutical products recalled during the first 6 months of 2012. N = 18.
Figure 2: Inoculum counts for B. cepacia culture spiked into pharmaceutical product suspensions contaminated with a mixed bacterial culture.
Figure 3: Detection of B. cepacia in pharmaceutical products contaminated with a mixed bacterial culture. After microbial DNA was extracted from product suspensions, 50 μl and 10 μl aliquots were added to 3 Ready-To-Go PCR beads, sterile water, and B. cepacia DNA primers.
Lane 1. Molecular Weight Markers: 4000, 2000, 1250, 800, 500, 300, 200, 100 bp.
Lane 2. B. cepacia DNA, positive control
Lane 3. 50 μl of DNA from product A.
Lane 4. 50 μl of DNA from product B.
Lane 5. 10 μl of DNA from product A.
Lane 6. 10 μl of DNA from product B.
  1. Jimenez L. “Microbial diversity in pharmaceutical product recalls and environments”. PDA Journal of Pharmaceutical Science and Technology 61.5 (2007): 383-399.
  2. Sutton SW and L Jimenez. “A Review of Reported Recalls Involving Microbiological Control 2004-2011 with Emphasis on FDA Considerations of ‘Objectionable Organisms’”.American Pharmaceutical Review15.2 (2012): 42-57.
  3. http://www.fda.gov/Safety/Recalls/
  4. Ko S., et al. “An outbreak of Burkholderiacepacia complex pseudo bacteremia associated with intrinsically contaminated commercial 0.5% chlorhexidine solution”. American Journal of Infection Control 43.3 (2015): 266-268.
  5. Singhal T., et al. “Outbreak of Burkholderia cepacia complex bacteremia in a chemotherapy day care un1it due to intrinsic contamination of an antiemetic drug”. Indian Journal of Medical Microbiology 33.1 (2015): 117-119.
  6. Torbeck L., et al.Burkholderia cepacia, the decision is overdue”. PDA Journal of Pharmaceutical Sciences and Technology 65 (2011): 535-543.
  7. Juhas M., et al. “High confidence prediction of essential genes in Burkholderia cenocepacia”. PloS ONE 7.6 (2012): e40064.
  8. Lessie TG., et al. “Genomic complexity and plasticity of Burkholderia cepacia”. FEMS Microbiology Letters 144.2-3 (1996): 117-128.
  9. Wang JW and SL Doty. “Cometabolic degradation of trichloroethylene by Burkholderia cepacia G4 with poplar leaf homogenate”. Canadian Journal of Microbiology 60.7 (2014): 487-890.
  10. Kou-San J and RE Parales. “Nitro aromatic compounds from synthesis to biodegradation”. Microbiology and Molecular Biology Reviews 74.2 (2010): 250-274.
  11. Kim A., et al. “Survival and susceptibility of Burkholderia cepacia complex in chlorhexidine gluconate and benzalkonium chloride”. Journal of Industrial Microbiology and Biotechnology 42.6 (2015): 905-913.
  12. Zani F., et al. “Evaluation of preservative effectiveness in pharmaceutical products: the use of a wild strain of Pseudomonas cepacia”. Journal of Applied Microbiology 83.3 (1997): 322-326.
  13. Rose H., et al. “Biocide susceptibility of the Burkholderia cepacia complex”. Journal of Antimicrobial Chemotherapy 63.3 (2009): 502-510.
  14. Coenye T., et al. “Molecular mechanisms of chlorhexidine tolerance in Burkholderia cenocepacia biofilms”. Antimicrobial Agents and Chemotherapy 55.5 (2011): 1912-1919.
  15. Jimenez L and S Smalls. “Molecular detection of Burkholderia cepacia in toiletry, cosmetic and pharmaceutical raw materials and finished products”. Journal of AOAC International 83.4 (2000): 963-966.
  16. Jimenez L., et al. “PCR detection of Salmonella typhimurium in pharmaceutical raw materials and products contaminated with a mixed bacterial culture using the BAXTM system”. PDA Journal of Pharmaceutical Science and Technology 55 (2001): 286-289.
  17. Minogue E., et al. “A rapid culture independent methodology to quantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water”. BMC Biotechnology 15.6 (2015).
Copyright: © 2015 Luis Jimenez., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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