B. Cepacia: A Case Study For Determining Objectionableness In Drug Manufacturing
By James E. Akers, Ph.D, James P. Agalloco, and Russell E. Madsen
After introducing the subject of colonization and opportunistic pathogenicity, we can now return to a discussion of B. cepacia. First, we should begin by discussing what B. cepacia is and what it isn’t. A critical place to start is the fact that B. cepacia sounds like a typical bacterial taxonomic construction consisting of a generic name “Burkholderia” and a species name “cepacia.” B. cepacia is a very different situation. We typically see B. cepacia described as a complex of organisms, which is typically abbreviated as “BCC.”. What then is meant by complex? In this case it means BCC describes a group that consists of at least 24 separate species.12 Therefore, when we say or write BCC we are talking about a group of relatively common human microbiome and human environmental microbiome organisms that are different enough to not be members of the same species.
A recent comment published (B. Friedman, https://eccwebinars.com/ecc-247/) stated that when the USP, Ph. Eur., and JP were harmonized in 2009, B. cepacia was excluded as a “specified” bacterium. The fact that B. cepacia is not listed as objectionable in any compendia is true, but it is also true that B. cepacia as we know it is not a single species of “bacterium.” Thus, the addition of B. cepacia would not be the addition or inclusion of a single species but rather the addition of about two dozen species. The “magnificent seven” would become the very strange 31 — strange because a great many of the 24 species most commonly included in the BCC are not associated with any human disease, and by definition those that don’t cause disease are neither opportunistic nor objectionable.
Further complicating matters are the following factors:
- The various species of the BCC group and entire genus Burkholderia can be very difficult to identify and doing so genetically depends very much on the database being used. Biochemical identification can be extremely tricky.
- Those listed in the red pathogen category in Figure 1 are not universally dangerous in humans; some have been found in as few as one patient and many of those patients have cystic fibrosis (CF) and some of the others have been reported to be opportunistic pathogens only in animals.
- Some Burkholderia species are primarily plant pathogens.
Most infections are caused by a handful of BCC members. B. mallei and B. pseudomallei cause glanders and meliodosis in horses and in man. Since it was first identified, B. mallei has been classified at one time or another as a member of the following genera:
The most common BCC organisms associated with disease in CF patients are B. cenocepacia and B. multivorans. Thus, the four species of the BCC group most commonly listed as opportunistic pathogens aren’t named B. cepacia. Only B. mallei among the 70 or so Burkholderia species appears to be a true pathogen.
Also, of interest is that between 1996 and 2012, when the FDA’s publication appeared in the PDA Journal, the frequency of BCC infections among cystic fibrosis patients had dropped from 3.6% to 3%. Cystic fibrosis is a low prevalence disease affecting about one out of 2,500 newborns annually.13,14 In the United States, rare diseases are defined by the Rare Disease Act of 2002 as those that affect fewer than 200,000 Americans, or about one out of each 5,000 people. This definition is essentially the same as the Orphan Drug act of 1983. In simple terms, the patients who are most likely to be impacted by a medicine containing one of the pathogenic Burkholderia is within a special treatment needs demographic.
But there is more to this story. An article published in 2011 by Couthinho, et al in Frontiers in Cellular and Infection Microbiology14 reported that 70% of the 41 CF patients they studied were already colonized with a member of the BCC. This study evaluated patients between the ages of 7 months and 9 years, so it appears that children are colonized quite early in life. They further reported that in a study of 500 patients in Europe, over a 16-year period five different BCC species were found to have colonized patients. They concluded from their investigation that the CF airway represents a constantly evolving ecosystem with “multiple phenotypic variants emerging from the clonal population and becoming established in the patient’s airway as a result of genetic adaptation.” Given this picture and the use of a broad panel of antibiotics to treat CF lung infections, it is not surprising that antibiotic resistance would appear, leading to the widespread reports that the BCC species are often resistant to antibiotics. Given the genetic adaptability of these species, this result is not surprising.
Antibiotic Resistance Of BCC
It is likely that the resistance of some BCC isolates to antibiotics is not an innate feature of genus Burkholderia but rather is an evolutionary adaptation resulting from colonizing humans and adapting to this ecological niche. This raises the question as to whether isolates from a manufacturing environment in which Burkholderia species may have as their source soil, water, or plant life are as likely to manifest antibiotic resistance. Certainly, aquatic Gram-negative organisms that are adapted to difficult nutritional conditions have physiological features that provide some inherent antimicrobial resistance; among these are efflux pumps and exclusion by cell membrane features, which is how Burkholderia may be resistant to polymixins. However, the resistance to third- and fourth-generation ß-lactam antibiotics (mostly cephalosporins) used clinically to treat these infections is primarily enzymatic and therefore acquired through genetic adaptation to the environment. The human microbiome studies are helping us better understand how colonization of humans with normally non-pathogenic or opportunistic organisms can lead to the spread of antibiotic resistance.15
Given that, it is unlikely that the use of non-sterile drugs would contribute to this emergence of antibiotic resistance in a meaningful way. Again, mathematics stands in the way. The numbers of organisms in drugs and in non-aqueous drugs and the inability of these organisms to proliferate in those drugs means that they are unlikely to develop or manifest antibiotic susceptibility. Also, given the fact that the most susceptible patients among us, those with CF, are commonly colonized with BCC or other Burkholderia species means that drug-carried organisms would have to displace through competition organisms already colonizing a human. It is not a simple task for the small number of organisms that may be present in orally consumed product to reach pulmonary tissues.
Physical Resistance Of BCC
Another worrisome characteristic feature of BCC often mentioned in regulatory presentations is the physical resistance of these species to environmental stress as well as sterilization and disinfection. First, the heat resistance of these organisms is unremarkable. A study done by Tano and Melhus16 found that drying hospital textiles in tumble cycles at 60 to 70 °C resulted in a 3- to 4-log kill of enterobacteria as well as Gram-negative rods. We know from our own experience in the pharmaceutical industry that circulating water at 75 to 80 °C is typically sterile and non-pyrogenic. A clinical manual on the properties of BCC published by Boston University reported that under dry conditions, BCC organisms cannot survive for more than a week; thus, it is unlikely that BCC would survive in a dry active pharmaceutical ingredient, inactive ingredient, tablet, or any other dry product. In fact, it seems unlikely in low water activity products that they’d survive long enough for a finished product to reach a patient.
BCC Resistance To Disinfection
Yet another feature of BCC often mentioned is its ability to resist disinfection. There are numerous clinical reports indicating the resistance of BCC to disinfections, even potent sporicides such as sodium hypochlorite at 0.05% concentrations, which is a common pharmaceutical industry disinfectant system. However, understanding the relevance of these data to a pharmaceutical manufacturing environment requires careful evaluation of the studies. Peeters and co-workers found that in biofilms such as hospital fomites containing blood or sputum, BCC could resist disinfection.17 However, in a planktonic state, which is to say in the absence of biofilm protection, the susceptibilities to disinfection are not significantly different from other Gram-negative bacteria. Peeters, et al further report that a 99.999% reduction of B. cenocepacia was observed using 0.3% H2O2 for 30 minutes, hot water at 70 °C for 1 minute, and chlorhexidine at 0.05%, and sodium hypochlorite at 0.05%, 0.1%, and 0.3%. So, yes, BCC in biological materials in a clinical setting, in biofilms on instruments, or in treatment areas can persist and can resist antimicrobials, including sporicides. But, significantly, in a manufacturing setting where blood and mucous are not present and cGMP procedures are followed, BCC will not manifest resistance that will challenge typical disinfection regimens.
When we examine the features of BCC in the pharmaceutical setting, these organisms’ superbug cape is removed, and they are found to have considerably less resistance to cleaning and disinfection procedures than the spore-forming organisms we generally use to access our processes. We also note that they are not any more able to survive or proliferate in dry conditions than any other bacteria or mold. Not surprisingly, the other human colonizer mentioned earlier in this article, S. aureus, presents much the same picture. It is far harder to kill when treated in infectious fomites containing blood, sputum, or mucous than it is when treated on clean surfaces. The conclusion to be drawn from this is that comparing clinical findings on the resistance of organisms in treatment facilities in which clinically ill people are being treated to the resistance of these same organisms in a cGMP drug manufacturing facility is a comparison of apples to broccoli. The conditions are simply not comparable.
While we understand why some FDA scientists in 2012 thought the decision was overdue with respect to what they called B. cepacia, we suggest that a deeper look into this issue is necessary. This is not a criticism of FDA, which has a tough job to do and should be concerned about emerging clinical findings. It must also be said that it had the misfortune of publishing its article just as the first human microbiome project studies were appearing and we have the advantage of working some nine or more years later when a far clearer picture of human colonization by opportunistic pathogens has emerged. We must also caution that the use of findings from clinical settings to project risk in a CGMP manufacturing setting is fraught with difficulty. These facilities have essentially nothing in common from a hygienic standpoint.
We suggest that the emergence of extensive data regarding human microbial ecology requires us to have another look at the property of “objectionableness” among microorganisms. It is apparent that in the case of non-aqueous products that are not administered as an inhalant, BCC isn’t objectionable. In fact, the diversity and complexity of this complex of organisms means that many of the organisms that could be considered part of this complex are generally harmless in humans and other mammals. It is also apparent that in aqueous products, the proliferation of BCC is objectionable, but we could also say that if there is one condition in pharmaceutical microbiological risk abatement that is always objectionable it is the proliferation of any microorganisms in anything, anywhere, and at any time in our manufacturing process.
USP <1115>18 presents a useful discussion of microbial risks and their management. Water activity, which is a critical component in any hazard analysis critical control point (HACCP) evaluation of microbial process risk, should be considered in any microbiological risk analysis. HACCP as commonly used in the food industry is far more effective as a microbial control risk analysis than risk studies based on FEMA or FEMCA. There are many good books on HACCP that are useful for study by both industry scientists and regulators.
It is reasonable to ask if the strategy that has been in force for microbial attribute quality control testing for decades has been rendered obsolete by our increasing knowledge of the human microbiome and, specifically, that study’s ecological/human colonization ramifications as well as simple mathematics. Specifically, how can organisms that colonize a substantial percentage of the healthy human population be “objectionable” in a broad sense? Also, it is an incontrovertible fact that humans harbor many hundreds of bacterial species among the 1014 organisms that live on and in us. Some of these organisms may rarely cause disease, and some of them colonize us and may evolve into opportunistic pathogens. However, is it possible for a small number of organisms that already live within us in vast numbers to be objectionable in a non-sterile product? To a layman, 2,000 organisms seem like a great many, but within our oral cavities, where so many of our drugs are administered, it doesn’t even amount to a drop in the bucket. Our current standards are reasonable, and we don’t need sterile oral drug products. All that would do is make those products far more costly to produce than they already are.
The USP wrote regarding chapter <61> Microbial Limits test in 1982 in PF1 in a stimulus to the revision process article: “The tests described in the Microbial Limits Tests <61> were not designed to be all-inclusive, i.e., to detect all potential pathogens. To accomplish this, an extensive text on laboratory detection of microorganisms would be required. The procedures in USP were designed to detect the presence of specific ‘index’ or ‘indicator’ organisms.” The authors went on to say, “nevertheless, the present chapter does not preclude the detection of P. cepacia – the organism requires subsequent differentiation.” The chapter does not provide specific methods for this, nor does it provide procedures for detecting the hundreds of other potentially opportunistically pathogenic species of microorganisms. Individual monographs include requirements for limits on total aerobic counts and/or absence of one or more of the four selected “indicator” organisms. The authors added this important statement, “the chapter on Microbial Limits Tests provides methods to assure that one may test for those microbial requirements in the individual monographs...”
Of course, the P. cepacia referred to in this passage was one of several taxonomic precursors of B. cepacia, which is now just one member of the BCC and a not particularly pathogenic one at that. We must also agree with our now deceased colleague Scott V.W. Sutton19, who wrote the following in 2012 in response to the FDA’s call for designation of the “species” B. cepacia as an objectionable organism and which neatly summarizes our conclusion:
“The main point for this analysis is that there are many organisms associated with clinical samples – most of them occur only rarely and cannot be tied to a disease state. Association with a disease state cannot be the only criterion for determination that a microorganism is “objectionable.” Even clear evidence of pathogenicity to a specific patient population by a specific route of administration should not be over-interpreted to the simplistic response of adding the organism to an “objectionable organism” list for all medications.”
Dr. Sutton also warned about using product recall data as a reason to designate objectionableness. He wrote this in that regard:
“The established method of risk analysis (USP <1111>) is a reasonable starting point to determine the actual risk from an organism that may be found in a raw material or finished product. We also must take into account the numbers of organisms seen. The recall (emphasis added) analysis is especially weak in this regard, as the magnitude of the contamination event is rarely listed in the enforcement report.”
There does need to be a discussion regarding “objectionableness” and what is really a “Bad Bug.” The FDA has a book called the Bad Bug Book, the second edition of which was issued in 2012. We should not be using this book or Bergey’s Manual in 2021 to decide whether either a recall or lot rejection is necessary. It’s just not that simple. We have left the era of black and white microbiology and we need to bring our industry into the 21st century. “The only good bug is a dead bug” school of microbiology is in our rearview mirrors. We need to finally recognize just how complex microbiology is, that “absence of” can’t be established, selective sterility doesn’t exist, and microbiology really is a logarithmic science. These are things current regulation, and too often standards as well, fail to consider at all. We believe pharmaceutical scientists can handle the truth.
Please note this is the full list of references from this article series (read Part 1 here).
- USP-NF 2021 Chapters <61> Microbial Enumeration Tests, <62> Tests for Specified Organisms, and <1111> Microbial Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use.
- A. Sawant, A.M. Cundell et al, 2014, PDA Technical Report Number 67 Exclusion of Objectionable Microorganisms from Nonsterile Pharmaceuticals, Medical Devices and Cosmetics.
- Transmission of SARS-CoV-2: Implications for infection and prevention precautions. WHO Scientific Brief July 9, 2020 https://www.who.int/publications/i/item/modes-of-transmission-of-virus-causing-covid-19-implications-for-ipc-precaution-recommendations.
- J. Cohen, W. Powderly, and Steven Opal. 2017 Opportunistic Infections 4th edition Vol. 1
- L. Torbeck, D. Raccasi, D. Guilfoyle, R. L Friedman and D. Hussong. 2011. Burkholderia cepacia: This Decision is Overdue. PDA J. Pharm Sci Technol. Sep-Oct 2011;65(5):535-543.
- USP-NF 2021 Chapter <60> Microbiological examination of nonsterile products- Test for Burkholderia cepacia complex (BCC).
- Kundrat, L, 2017. The Great Debate Over B. cepacia Testing: Your Opinions and FDA Recommendations. Microbiologics Blog https://blog.microbiologics.com/tag/burkholderia-cepacia-complex/
- Joanne S. Eglovitch. 2018. USP Sparks Debate Over Rapid Sterility Testing for Cell and Gene Therapies. Pink Sheet. Informa Publishing 31 Oct 2018 https://pink.pharmaintelligence.informa.com/PS124172/USP-Sparks-Debate-Over-Rapid-Sterility-Testing-For-Cell-And-Gene-Therapies
- A.A.T.M Bosch, G. Biesbroek, K. Trzcinski, E.A.M. Sanders and D. Bogeart. Viral and Bacterial Infections in the Upper Respiratory Tract. PLoS Pathogens. Published January 10. 2013. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1003057
- A.van Belkum et al 2009. Reclassification of S. aureus Nasal Carriage Types, J. of Infectious Disease: Vol. 199(12); 1820-1826.
- C. M. Bassis et al. Microbiome 2, Art. No. 27, 2014. Nasal Cavity microbiota of healthy adults.https://microbiomejournal.biomedcentral.com/articles/10.1186/2049-2618-2-27
- L. Eberl and P. Vandamme., 2016. Members of the genus Burkholderia: good and bad guys. F1000Research https://f1000research.com/articles/5-1007/v1
- V.C. Scoffone et al., 2017. Burkholderia cenocepacia Infecdtions in Cystic Fibrosis Patients Drug Resistance and Therapuetic Approaches. Front. Microbiol. Published online August 22, 2017; https://www.frontiersin.org/articles/10.3389/fmicb.2017.01592/full
- C.P. Coutinho et al. Long-term colonization of the cystic fibrosis lung by Burkholderia cepacia complex bacteria: epidemiology, clonal variation and genome-wide expression alterations. Front. Cell. Infect. Microbiol. Published Dec. 2, 2011 https://www.frontiersin.org/articles/10.3389/fcimb.2011.00012/full
- S. A. Baron, S.M. Diene, and J-M Rolain. 2018, Human Microbiomes and antibiotic resistance. Human Microbiome Journal, December 2018. Pages 43-52.
- E. Tano and A. Melhus. 2014 Level of decontamination after washing textiles and 60C or 70C followed by tumble drying. Infection Ecology and Epidemiology (4) :24314 https://www.tandfonline.com/doi/full/10.3402/iee.v4.24314
- E. Peeters, HJ Nelis and T Coenye, 2008. Evaluation of the efficacy of disinfecton procedures against Burkholderia cenocepacia biofilms. J. Hosp Infection 70(4) pages 361-368.
- USP-NF 2021 Chapter <1115> Bioburden Control of Nonsterile Drug Substances and Products.
- Scott V.W. Sutton. 2012. “Objectionable Organisms”- the Shifting Perspective. American Pharmaceutical Review. https://www.americanpharmaceuticalreview.com/Featured-Articles/122201-What-is-an-Objectionable-Organism-Objectionable-Organisms-The-Shifting-Perspective/
About The Authors:
Jim Akers, Ph.D., is owner and principal consultant of Akers Kennedy and Assoc. He has 39 years of experience in the pharmaceutical/biopharmaceutical industry and has held various management positions in quality, development, validation, and analytics. Akers is past president of PDA and spend 15 years on its Board of Directors. He has served the USP in several capacities since 1993, including chair of the Microbiology Expert Committee, where he continues to serve as a member. Akers has also served as a technical advisor to Shibuya Corporation since 1991. He has published 36 book chapters, edited two books on Isolator technology, published over 100 articles, and lectured widely on aseptic processing, analytical microbiology, sterilization, cell processing, and advanced aseptic processing.
James Agalloco, president of Agalloco & Associates (A&A), is a pharmaceutical manufacturing expert with more than 45 years of experience. He worked in organic synthesis, pharmaceutical formulation, pharmaceutical production, project/process engineering, and validation during his career at Merck, Pfizer, and Bristol-Myers Squibb. Since the formation of A&A in 1991, Agalloco has assisted more than 200 firms with validation, sterilization, aseptic processing, and compliance. He has edited/co-edited four texts, authored/coauthored 40+ chapters, published more than 150 papers, and lectured extensively on numerous subjects. He is a past president of PDA and a current member of USP’s Microbiology Expert Committee, and serves on the Editorial Advisory Boards of Pharmaceutical Technology and Pharmaceutical Manufacturing.
Russell E. Madsen is president of The Williamsburg Group, LLC, a consulting firm located in Gaithersburg, MD. Prior to forming The Williamsburg Group, he had served PDA as acting president and was senior VP of science and technology. Before joining PDA, he was employed by Bristol-Myers Squibb as director of technical services, providing technical and general consulting services to Bristol-Myers Squibb operations worldwide. He is vice chairman of ASTM Committee E55 on Manufacture of Pharmaceutical and Biopharmaceutical Products, a member of the USP Microbiology Expert Committee, a member of Pharmaceutical Technology’s Editorial Advisory Board, and an honorary member of PDA. He holds a BS degree from St. Lawrence University and a MS degree from Rensselaer Polytechnic Institute.