Excluding Burkholderia cepacia complex from Aqueous, Non-Sterile Drug Products

• Microbiological Consulting, LLC

Introduction

In non-sterile drug product manufacturing, low microbial counts are tolerated and the final drug product does not necessarily need to be free of microorganisms to be released to the market. The recommended microbiological quality requirements for different dosage forms may found in USP <1111> Microbiological Examination Of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances For Pharmaceutical Use. The microbial limits and absence of specified microorganism requirements related to the invasiveness of the dosage form were established in USP <1111> and some microorganisms, if found in a particular product, are considered objectionable in that they can adversely affect the appearance, physicochemical attributes or therapeutic effects of a non-sterile drug product or due to their numbers and/or pathogenicity, may cause infection, allergic response or even toxemia in patients receiving the product. Recent U.S. recall surveys have found that the presence of objectionable microorganisms, and not microbial numbers, represent the vast majority of microbiologically related FDA recalls of non-sterile drug products.

This review article will discuss the definition of an objectionable microorganism, the prevalence of members of the Burkholderia cepacia complex (BCC) in U.S. product recall and nosocomial infection outbreaks, why BCC members are serious opportunistic pathogens, the screening and identification methods for members of the complex, and how the risk of microbial contamination of non-sterile drug products can be mitigated.

What is an Objectionable Microorganism?

It is notable that the U.S. Federal Good Manufacturing Regulations 21 CFR 211.113 Control of microbiological contamination requires that objectionable microorganisms be excluded from non-sterile drug products, but contains no actual definition of an objectionable microorganism and certainly does not provide a list of microorganisms to be excluded. However, FDA microbiologists attending industry meetings have stated that they plan to publish a guidance document in the next 1-2 years addressing non-sterile drug products. The assignment of the responsibility is to the pharmaceutical manufacturer who must develop a written program to exclude objectionable microorganisms from their drug products was, in the author’s opinion, the right decision. Not only does the manufacturer bear the responsibility for the safety of their products but alone has the complete range of knowledge of the pharmaceutical ingredients, formulation, manufacturing processes, product attributes, and intended patient population to make these critical judgments.

Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special offers
from American Pharmaceutical Review – all delivered right to your inbox! Sign up now!

So what is an objectionable microorganism? The PDA Technical Report No. 67 Exclusion of Objectionable Microorganisms from Non-sterile Pharmaceutical and OTC Drug Products, Medical Devices and Cosmetics defined objectionable microorganisms as follows:

1. Microorganisms that can proliferate in a product adversely affecting the chemical, physical, functional and therapeutic attributes of that pharmaceutical product.
2. Microorganisms that due to their numbers in the product and their pathogenicity can cause infection in the patient via the route of administration when treated with that pharmaceutical product.

The points to note in this definition are what is objectionable is drug product specific and the objectionable microorganism may either cause an infection in the patient or affect the functionality of the drug product.

Why are Members of the Burkholderia cepacia Complex Objectionable Microorganisms?

Why do we consider that the Gram-negative bacterium Burkholderia cepacia and many other members of the BCC are objectionable microorganisms? The reasons include:

• Prominence of B. cepacia in U.S. drug product recalls
• Recent high visibility in recent infection outbreaks for B. cepacia due to bacterial contamination of non-sterile drug products and consumer care products
• Role as an opportunistic pathogen in compromised hospital patients
• Unique ability to overcome antimicrobial preservative systems
• Resistance of BCC members to many widely used antibiotics

B. cepacia is an aerobic, Gram-negative, oxidase-positive, rod-shaped bacterium that is an opportunistic pathogen, with a reputation of overcoming antimicrobial preservative systems and antiseptics and growing in multiple-use preserved oral liquids, topical products and nasal sprays. It a member of a group of closely related species in the B. cepacia complex with a high metabolic versatility, wide environmental distribution, and variable virulence due in part to its large genomic size (7-8 mbp). Furthermore, members of B. cepacia complex have the ability to form biofilms in pharmaceutical water systems making it more difficult to eliminate from the system.

BCC members cause serious infections in individuals with cystitis fibrosis (CF) and chronic granulomatous disease. Two BCC members B. cenocepacia and B. multivorans account for greater than 85% of the CF infections (Reik et al, 2005). It is an opportunistic pathogen in mechanically ventilated patients, the immunosuppressed, infants, the elderly and those with serious underlying disease.

U.S. Drug Product Recalls

A 2012 survey analyzed 144 reported recalls of non-sterile pharmaceutical drug products (5%), over-the-counter drug products (42%), cosmetics (31%), medical devices (14%) and dietary supplements (8% of the total recalls) for microbiologically related issues for the 7-year period from 2004 through 2011. This represents an average of 20 recalls annually. The publication highlighted that the majority of these recalls (72%) were associated with objectionable microorganisms and not for exceeding microbial enumeration limits (Sutton and Jimenez, 2012).

The objectionable organisms associated with the recalls by microbial type were:

• Burkholderia cepacia – 34%
• Yeast/molds – 21%
• Other Pseudomonads – 15%
• Members of the family Enterobacteriaceae – 11%
• Bacillus cereus – 9%
• Chryseobacterium spp.– 5%
• Staphylococcus spp. – 4%
• Achromobacter spp. – 1%

A hierarchy of microbial infection risk can be established simply based solely on the invasiveness of the pharmaceutical dosage form. The increasing invasiveness would range from solid oral dosage forms to inhalation products. In addition, the medical status of the recipients Will influence the risk. For example, a recent survey published in a leading medical journal suggests 4% of U.S. adults are believed to be immunosuppressed (Harpaz et al, 2016). Note: Sterile injectable products have the greatest risk of microbial infection due to their parenteral administration.

Prominent Recall Case Histories

On May 2008, Hydrox Labs, Elgin, IL issued a voluntary recall of labeled alcohol-free mouthwash they manufacture. As a result of this recall, Cardinal Health initiated a voluntary recall of a lot of alcohol-free mouthwash. The unopened and used mouthwash containers were tested, and certain samples were found positive for B. cepacia strongly suggesting intrinsic microbial contamination. The product was distributed to hospitals, medical centers, and long-term care facilities nationwide but not for retail sale. Most troubling, the mouthwash may be found in certain Personal Hygiene Hospital Admission Kits distributed to patients.

It is widely recognized that B. cepacia poses little medical risk to healthy people. However, people who have certain health problems such as weakened immune systems or chronic lung diseases, particularly cystic fibrosis (CF), infants or the elderly or on mechanical ventilators, may be more susceptible to infections with B. cepacia. This pattern was observed in this outbreak, as the patient infection was limited to those ventilators in intensive care units and not the general patient population who also received the mouthwash in admission kits. Adding to the risk, B. cepacia is often resistant to common antibiotics, so they are more difficult to treat.

A recent review article (Abdallah et al, 2018) found by searching PubMed using the terms “Burkholderia”, “outbreak” and “intensive care unit” between January 1994 and December 2017 thirty outbreaks of BCC infection or colonization in non-CF patient in intensive care units with many outbreaks associated with contaminated medical products. Intrinsic contamination was associated with liquid laxative, skin antiseptics, alcohol-free mouthwash, ultrasound gel, and moisturizing body cream with resulting high mortality rates.

Another case history that is informative is the infection outbreak and the nationwide recall of an oral laxative (Marquez et al, 2017). On July 16, 2016 the FDA announced a voluntary nationwide recall of all non-expired lots of oral liquid docusate sodium manufactured by PharmaTech LLC, Davie, Florida in one pint (473 mL) bottles and distributed by Rugby Laboratories for contamination with B. cepacia and linked to a five state outbreak. Later laboratory evidence linked the B. cepacia to the company’s purified water system.

In an August 10, 2016 update, the CDC confirmed 60 cases from an eight state outbreak was caused by the same strain of B. cepacia using molecular typing and recommended that clinicians and their patients not use any brand of liquid docusate sodium as a stool softener or other medical reason. Liquid laxatives like Docusate™ are widely used to treat infants and the elderly that have difficulties swallowing and are typically higher risk groups for bacterial infection.

The FDA was sufficiently concerned in 2017 to issue an advisory notice of the dangers of BCC contamination of aqueous, non-sterile drug products (FDA, 2017)

What Defines Membership in the B. cepacia Complex?

The bacterium B. cepacia was originally viewed as a pseudomonad, first isolated from rotting onions. The genus Pseudomonas is a large and complex heterogeneous group of organisms belonging to the family Pseudomonadaceae that originally contained 211 validly described species but 56 of which have been reclassified to other genera. They are constantly undergoing continuous taxonomic revision due to improvements in methodologies of species classification. Organisms previously classified within the genus Pseudomonas (rRNA homology groups I-V) are now divided among the ten genera Pseudomonas, Burkholderia, Ralstonia, Comamonas, Acidovorax, Delftia, Hyrodenophaga, Brevundimonas, Stenotrophomonas and Xanthomonas.

The genus Burkholderia was created by Yabuuchi et al (1992) to accommodate the former rRNA Group II pseudomonads excluding P. pickettii and P. solonacearum, which were transferred to the genus Ralstonia. Traditionally, Burkholderia species are known as plant pathogens and soil bacteria with the exception of P. mallei and P. pseudomallei human and animal pathogens.

More recent research (Coenye et al, 2001) has resulted in a number of changes to the taxonomy of Burkholderia cepacia complex (BCC). The BCC currently comprises 17 species which exhibit a high degree of 16S rRNA (98–100%) and recA (94–95%) gene sequence similarity, and moderate levels of DNA–DNA hybridization (30–50%).

Characteristics that Make Members of B. cepacia Complex Opportunistic Pathogens

B. cepacia is a well-known nosocomial pathogen that is intrinsically resistant to aminoglycosides and first- and second-generation cephalosporins. Members of the BCC are widely found in wet locations in the environment and hospital settings. Their ecological and metabolic versatility and resistance to a wide range of antibiotics and antiseptics may be partly explained by their large genomic size (7-8 mbp). In December 1995, an outbreak involving B. cepacia in respiratory cultures from patients without cystic fibrosis was traced to intrinsically contaminated alcohol-free mouthwash (Bernstein et al, 1996). An investigation by the FDA suggested an association with the deionization procedure of the water used to prepare the product. Mechanically ventilated patients are vulnerable to pathogens in their mouths and upper airways because of their inability to maintain the mucociliary and cough mechanisms that normally protect the lower respiratory tract (Beck-Sague et al, 1996). These outbreaks of B. cepacia related to mouthwash highlight the increased risk for respiratory colonization and infection among patients on ventilators.

Testing for the Absence of B. cepacia Complex

The pharmaceutical industry needs a standardized test for screening non-sterile, aqueous, drug products for members of the BCC. In clinical microbiology, primary culture for BCC should be performed on a selective agar to avoid overgrowth by other bacteria and to readily identify the pathogen. Examples of selective solid media include Burkholderia cepacia selective agar (BCSA), Burkholderia cepacia agar (BCA) (formerly known as Pseudomonas cepacia agar or PCA), and Oxidation-Fermentation Polymyxin Bacitracin Lactose agar (OFPBL). Recent evaluations suggest that BCSA is more selective and grows BCC colonies more rapidly than the other media. All media contain multiple antibiotics to maintain selectively. B. cepacia selective agar (BCSA) contains 1% lactose and 1% sucrose in an enriched base of casein and yeast extract with 600 U of polymyxin B per ml, 10 μg of gentamicin per ml, and 2.5 μg of vancomycin per ml (Henry et al, 1997). Incubation at 30-35°C for 48-72 hours is recommended.

In response to stakeholder requests, a proposed USP test for the absence of Burkholderia cepacia complex was published as an inprocess revision in the September-October 2018 Pharmacopieal Forum. A 90-day comment period was available for stakeholders to review and comment on the proposed general test chapter <60> Microbiological Examination of Non-sterile Products: Tests for Burkholderia cepacia complex.

The proposed USP chapter briefing states: Because no standard method currently exists for the detection of Burkholderia cepacia complex (BCC), a new chapter is being proposed. BCC species are gram-negative, rod-shaped bacteria that include opportunistic pathogens. Many BCC have the potential for overcoming antimicrobial preservative systems and antiseptics, and grow in preserved aqueous oral liquids and topical products. BCC may cause serious infections in individuals with cystic fibrosis and chronic granulomatous disease, mechanically ventilated patients, immune-suppressed people, and those with serious underlying disease. This chapter proposal contains test procedures and media formulations for the detection of BCC.

To conduct the test, 10 g of drug product would be added to 90 mL of USP Fluid A and a 10 mL aliquot transferred to soybean-casein digest broth with Lecithin and Polysorbate 80 and incubated at 30-35°C for 48 to 72 hours. After the incubation period, the broth would be streaked out with a sterile inoculation loop onto BCSA and incubated at 30-35°C for 24-48 hours. BCC typically grows as yellow colonies with a pink to yellow zone in the medium. If no colonies are isolated or the colonies are not subsequently identified as a BCC member, the test material passes the test.

As with related tests for specified microorganisms as found in USP <62>, the BCC screening test is subjected to growth promotion and method suitability testing. Microorganisms used are: Growth- Promoting and Indicative - Burkholderia cepacia, Burkholderia cenocepacia, or Burkholderia multivorans and inhibitory - Pseudomonas aeruginosa. The three members of the BCC selected for suitability testing are the most significant in CF infection. More than one BBC member was selected as more representative of the 17 members of the complex.

Sandle (2018) has published a review article discussing the relative merits of the proposed USP <60> BCC screening test.

Identification of Members of the B. cepacia Complex

Basic commercial microbial identification systems may be limited in their ability to identify accurately non–fermenters and these organisms can be very time consuming to identify by phenotypic tests due to the necessity for subculture and low reactivity in key biochemical tests. Differentiation of species within the B. cepacia complex can be particularly problematic, even with an extended panel of biochemical tests, as they are phenotypically very similar and most commercial bacterial identification systems cannot reliably distinguish between them (See Manual of Clinical Microbiology. 10th ed. Washington DC: ASM Press; 2011).

Polymerase Chain Reaction (PCR) amplification and base sequencing is usually considered to be a good method for bacterial identification, as it is simple, rapid, sensitive and specific. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific gene sequences. However, it does have limitations. Although the 16S rRNA gene is generally targeted, identification is difficult when the 400-500 base sequences of the homologous genes have high similarity in BCC members.

The emerging identification technology Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MADLI-TOF MS), which analyzes the riboprotein composition of bacterial cells, is useful to identify member of the BCC. Despite the initial capital cost of the instrumentation, the advantage of MALDI-TOF MS is it can identify bacteria in minutes compared to days using traditional methods and the cost per test is less than a dollar. However, the database must include mass-charge fingerprints for all of the BCC members to be fully successful. The good news is that these databases are continuously being updated to include more microorganisms so they will improve over time.

Other molecular techniques include Pulsed Field Gel Electrophoresis (PFGE), Multi - Locus Sequence Typing (MLST), Multiple-Locus Variable-Number Tandem-Repeat Analysis (MVLA), repetitive extragenic palindromic sequence-based PCR (rep-PCR), and Whole Genome Sequencing (WGS) have been applied to microbial identification and strain typing especially in epidemiology studies in response to infection outbreaks. All of these approaches enable subtyping of related strains, but do so with different accuracy, discriminatory power, and reproducibility.

MLST has been successfully used with the closely related members of the B. cepacia complex. The technique uses seven housekeeping gene compared to the single target with 16S rRNA gene sequencing. The ability of this technique to carry out both strain differentiation and species identification in a single approach represents a major advance that should greatly aid the clinical diagnosis of B. cepacia complex infection (Baldwin et al, 2005). However, the equipment and expertise to conduct MLST is limited specialized clinical microbiology and CF clinics, contact identification laboratories and not pharmaceutical QC microbiology laboratories.

The BCC MLST schema uses fragments of these seven housekeeping genes:

• ATP synthase beta chain (atpD)
• Glutamate synthase large subunit (gltB)
• DNA gyrase subunit B (gyrB)
• Recombinase A (recA)
• GTP binding protein (lepA)
• Acetoacetyl-CoA reductase (phaC)
• Trptophan synthase subunit B (tryB)

At present, 17 current members of the BCC are recognized and include B. cepacia, B. multivorans, B. stabilis, B. vietnamiensis, B. ambifaria, B. athina, B. pyrrocinia, B. latens, B. diffusa, B. arboris, B. seminalis, B. cenocepacia, B. contaminans, B. dolosa, B. lata, B. ubonensis and B. metallica (Vandamme and Dawyndt, 2011). With the future application of WGS this number could change either up or down.

Risk Mitigation Steps to Exclude B. cepacia from Non-Sterile Drug Products

Risk mitigation steps to exclude B. cepacia complex include:

• Pharmaceutical ingredients selection
• Product formulation including robust antimicrobial preservative system
• Management of pharmaceutical water systems
• Equipment cleaning and sanitization
• Manufacturing processes
• Risk-based microbial testing programs

When selecting pharmaceutical ingredients the microbiological quality of these ingredients may be important depending on the dosage form. Keep in mind the quantity of the ingredient used in the product, the manufacturing process of the ingredient, physical attributes of the product especially water activity, the antimicrobial effectiveness of the formulation, and the intended use of the product. There is a hierarchy of risk for microbial contamination with chemically synthesized ingredients having the lowest risk and animal-derived ingredients the highest risk. The hierarchy is animal-derived > plantderived > mineral-derived > semi-synthetic > synthetic ingredients. This generalized sequence can be modified by the extent of processing during manufacturing (Cundell, 2005).

Multiple-use non-sterile products must be resistant to microbial contamination and growth due to their low water activity, i.e., <0.6, inherent antimicrobial activity or to the effectiveness of their antimicrobial preservative system. During product development, candidate formulations are assessed using the methods described in USP <51> Antimicrobial Effectiveness Testing. As recommended in the USP chapter, additional challenge organisms, including members of the BCC, can be added to the test to ensure the formulation has the most robust preservative system.

Investigations of BCC infection outbreaks have consistently identified the pharmaceutical-grade water system at the manufacturing site to be the probable cause of the product contamination. Manufacturers of aqueous non-sterile drug products should install a well-designed water system, and well maintain and manage the system aggressively.

Process equipment including tanks, pumps, and filling lines, especially their product contact surface must be adequately cleaned and stored dry to avoid product contamination. Microbial surface monitoring must be included within standard equipment cleaning protocols and periodic cleaning verification implemented.

For each dosage form, the unit manufacturing operational steps can be analyzed for potential microbial contamination risk. For example, for an oral liquid the manufacturing steps are ingredient procurement, equipment cleaning, weighing, blending, mixing, container-closure preparation, and filling. Ingredient water and processing equipment cleaning may be viewed as areas of greatest risk for microbial contamination. Another factor may be the order of addition of the preservative, as they must be present in the aqueous phase of an oil-water suspension. For a comprehensive discussion of microbial contamination of non-sterile drug products, the reader is referred to the USP General Informational Chapter <1115> Bioburden Control of Non-sterile Drug Substances and Products.

When USP <60> becomes official, whether a microbiological specification for the absence of B. cepacia complex will be added to any drug product monographs will remain to be seen. A risk-based microbial testing program would involve more frequent testing of aqueous drug products than non-aqueous products. For example, based on a comprehensive risk assessment and a testing history, a compressed tablet may not have a microbiological specification and would not be subjected to release testing, whereas each batch of a topical cream would be subject to full testing as recommended in USP <1111>.

Conclusions

Although microbial contamination of drug products results in few patient infections, the pharmaceutical industry must not be complacent working to eliminate this risk. The author hopes that this review article contributes to this goal through a timely discussion of the role that BCC plays in microbial contamination and how it can be detected.

References

1. Abdallah, M. et al, 2018 Burkholderia cepacia complex outbreaks amongst non-cystic fibrosis patients in the intensive care units: a review of adult and pediatric literature. Le Infez. in Med. 4:299-307
2. Baldwin, A., E. Mahenthiralingam et al 2005 Multi-locus sequence typing scheme that provides both species and strain differentiation for the Burkholderia cepacia complex. J. Clin. Microbiol. 43(9):4665-73.
3. Beck-Sague C.M., R. L. Sinkowitz et al. 1996 Risk factors for ventilator-associated pneumonia in surgical intensive care unit patients. Infect Control Hospital Epidemiol. 17:374-6.
4. Bernstein B, Dineen T, Kehl S, Wilson P, Sohnle P. 1996 Outbreak of Burkholderia cepacia colonization and infection related to contaminated oral mouthwash {Abstract}. In: Program and Abstracts of the 34th Annual Meeting of the Infectious Diseases Society of America, New Orleans, Louisiana, September 1996.
5. Coenye, T., P. Vandamme et al, 2001 Taxonomy and Identification of the Burkholderia cepacia Complex J. Clin. Microbiol. 39(10): 3427–3436
6. Cundell. A. M. 2005 Managing the Microbiological Quality of Pharmaceutical Excipients. PDA J. Pharm. Sci & Technol. 59(6): 381-395
7. FDA 2017 FDA advises drug manufacturers that Burkholderia cepacia complex poses a contamination risk in non-sterile, water-based drug products, U.S. Food and Drug Administration, U.S. Department of Health and Human Services, at: https://www.fda.gov/Drugs/DrugSafety/ucm559508.htm
8. Harpaz R, R.M. Dahl and K.L. Dooling 2016 Prevalence of Immunosuppression Among US Adults, 2013. JAMA. 316(23):2547-2548
9. Henry D. A., M.E. Campbell , J.J. LiPuma, and D.P. Speert 1997 Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium. J. Clin. Microbiol. 35:614–619
10. Marquez, L., K. N. Jones et al 2017 An outbreak of Burkholderia cepacia Complex infections associated with contaminated liquid Docusate. Infect. Control & Hosp. Epidemiol. 38(5): 567-573
11. Reik R, T. Spilker, and J.J. LiPuma 2005 Distribution of Burkholderia cepacia complex species among isolates recovered from persons with or without cystic fibrosis. J Clin Microbiol 43:2926–2928
12. Sandle, T. 2018 Burkholderia cepacia complex: Review of origins, risks and methodologies www.europeanpharmaceuticalreview.com/article/80557/burkholderia-cepaciacomplex-review-of-origins-risks-and-test-methodologies/
13. Sutton, S and L. Jimenez, 2012 A Review of Reported Recalls Involving Microbiological Control 2004-2011 with Emphasis on FDA Considerations of “Objectionable Organisms” Posted January 1 2012 Amer. Pharm. Rev.
14. USP <60> Microbiological Examination of Non-sterile Products: Tests for Burkholderia cepacia complex Pharmacopeial Forum 46(5) Sept.-Oct. 2018
15. Vandamme, P and P. Dawyndt, 2011 Classification and identification of the Burkholderia cepacia complex: Past, present and future Syst Appl Microbiol. 34(2):87-95
16. Yabuuchi, E. et al, 1992 Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol. Immunol. 36(12):1251-75.

Author Biography

Tony Cundell received a PhD in microbiology from Lincoln University, New Zealand and had a long career as a microbiologist working in quality and product development in the pharmaceutical industry. Currently he works as an independent consultant specializing in microbial testing, contamination risk assessment, and regulatory issues. He is a member of the 2015-2020 USP Microbiology Expert Committee. Telephone: 914 725-3947