Abstract
Antibiotic-resistant microorganisms are becoming a global concern. There are several antibiotic-resistant strains of microbes of clinical significance. Infections from antibiotic-resistant microbes adds significant costs to the healthcare system. These costs are due to prolonged and/or more expensive treatments, extended hospital stays and the like. (CDC, 2013) Rao (2017) identities ten clinical organisms of significant concern relative to antibiotic resistance. Burkholderia cepacia is identified as the second organism on his list, following Methicillin-resistant Staphylococcus aureus. See Figure 1.
Recently, regulatory scrutiny has increased due to BCC. BCC includes Burkholderia cepacia and several other species of organisms. The exact number of species changes depending upon the reference, but at least 20 organisms are included. BCC has been the reason for many recalls of contaminated non-sterile products.
On May 22, 2017, new regulatory guidance was provided that affected the manufacturers of non-sterile aqueous pharmaceutical products. (FDA, 2017) This guidance requires: establishment of a test to show absence of BCC in the final product and validation of the BCC absence test method. This requirement is more difficult to implement as currently no compendial test method for BCC exists today.
BCC Defined
“BCC can survive or multiply in a variety of non-sterile and waterbased products because it is resistant to certain preservatives and antimicrobial agents,” FDA said in a statement. “Detecting BCC bacteria is also a challenge and requires validated testing methods that take into consideration the unique characteristics of different BCC strains.” According to FDA, people exposed to BCC are at an increased risk for illness or infection, especially patients with compromised immune systems. (FDA, 2017a)
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!
There are at least 18 different species of BCC. The typical source of these organisms is water, although some have been found in soil, with prolonged survival in moist environments, and in biofilm formation. This organism complex is an opportunistic pathogen, and is associated with endocarditis, wound infections, IV bacteremia, foot infections and respiratory infections. As such, this organism is a concern for nonsterile aqueous products. It tends to show a resistance/persistence for organic solvents, antiseptics, disinfectants and low nutrient states. It has also shown resistance to efflux pumps. (Ensor, 2017)
It was believed that BCC was only a concern for patients with cystic fibrosis or other immune-compromised individuals. However, in the 2016-time period, numerous “healthy” personnel were affected by the presence of Burkholderia cepacia in non-sterile products. The Centers for Disease Control (CDC) claim that the bacteria are known to be resistant to many common antibiotics, making infection more difficult to treat. (Mezher, 2016)
Regulatory Recalls Due to BCC
In recent years (2016 and 2017) a variety of product recalls occurred that were mandated by either FDA or the affected companies. These recalls were due to Burkholderia cepacia complex (BCC) contamination. BCC contamination is frequently due to water-borne contamination with opportunistic pathogens. (Brennan, 2017)
Case Study 1: Is It BCC?
There are a variety of water-borne microorganisms, many of which have been reclassified in recent years. A manufacturer of a non-sterile mouthwash had a contamination issue with a confirmed BCC organism. The root cause of the contamination was found and eradicated. Several months later, another contamination was detected. The organism was identified as Cupriavidus pauculus. While not a member of the BCC, there was concern at the site, due to the previous BCC contamination.
Upon close examination of the characteristics of Cupriavidus pauculus it was found that: Cupriavidus pauculus is a type of Gram-negative, non-fermentative, motile bacterium, from the genus of Cupriavidus and the family of Burkholderiaceae. It has been isolated from water, the water in hospital ultrafiltration systems, as well as bottled mineral water. (Aspinall and Graham, 1989; Balada-Llasat, et al., 2010; Clark, et al., 1998; Ori, et al., 1998; Manara, et al., 1990; Anderson, et al., 1997)
This microorganism was previously classified as Ralstonia paucula (Vandamme, et al., 1999) and Wautersia paucula (Vaneechoutte, et al., 2004). Ralstonia paucula is described as a Gram-negative environmental bacterium. It is reported to be found in pool water, ground water, bottled mineral water and clinical specimens. This organism was also associated with serious nosocomial infections. (Moissenet, et al., 2001) Ralstonia spp. has been implicated in various patients with cystic fibrosis (like BCC does. Vandamme, et al. (1999) describes Wautersia paucula as being identical to the Ralstonia paucula.
Understanding the Health-Risks Associated with These Organisms
Cuprividus pauculus is rarely found as a human pathogen. When found it can cause significant disease especially in immune-compromised patients. (Balada-Llasat, et al. 2010) Tasbakan, et al. (2010) reported 19 cases of peritonitis and one case of tenosynovitis associated with Cupriavidus pauculus. (Tasbakan, et al., 2010) Cupriavidus pauculus has also been isolated as the causative agent in airway infections of individuals with cystic fibrosis. (Kalka-Moli, et al. 2009)
Since this organism is not routinely isolated as a human pathogen, no data could be found identifying the concentration of microorganism that is required to cause a pathogenic action.
Conclusion
While the Cupriavidus pauculus Is not officially part of the BCC grouping, it causes the same types of health risks as BCC and the route of administration for the oral product would be a considered a high risk for a BCC product, the company decided to discard the product.
Case Study 2: Water-based Issues with BCC
Since BCC are typically water-borne organisms, it is not unusual to find BCC contamination in water systems or areas of high-water content. Several different companies have found BCC in their facility and investigated the source of the contaminants. BCC sources have been found in the following environments:
Water Storage Tanks
In some cases, large water storage tanks are used as part of the water distribution system. It was found that when the tanks are routinely only partially-filled, it is not unusual to find biofilm organisms above the water line. In some facilities, the BCC contaminant was found in this biofilm.
Hoses
The uses of hoses from point of use (POU) sites on the water distribution system can be problematic. It is critical to ensure that the hoses are properly used to prevent water sitting in the hose and developing biofilm. At one site, the hoses were used on an ambient line. The hose connected to the point-of-use and was hanging into a sink. The bottom of the hose was sitting on the base of the sink. Biofilm was confirmed in the hose and BCC was found in the biofilm.
Ambient Distribution Lines
At one company, an ambient water distribution loop was used to transfer ambient water to different points-of-use. While the site had a policy to hot water sanitize the loop, the method used was described as sanitizing with hot water whenever the POU was not in use. Further investigation showed that the sanitization did not occur unless “All” POUs on the line were not in use. It could go many hours without sanitization occurring. Biofilm was detected and contained the BCC organisms.
Conclusion
In all these occurrences, ozonated water (at least 5 ppm) was used to sanitize or sterilize the contaminated systems. (Some companies chose to sterilize the line with ozonated water for two reasons: verifying the biological indicator kill was easier to qualify, and ozonated water is very effective at removing biofilm. In some cases, the companies also added preventative sanitizations at designated periods. The product dispositions varied, depending upon whether the process water was used in the product.
Case Study 3: Non-Sterile Aqueous Product
The facility manufactures several non-sterile drug products (both aqueous and non-aqueous). There were no complaints or product failures on record for the past year. The FDA initiated inspections at several companies who manufactured non-sterile products due to a multi-state contamination event where “healthy” patients became infected with BCC organisms.
BCC was not found in any of the product release test results. The products at this site were released based upon USP <61> and <62>, the tests for microbial limits and objectionable organisms. The site had performed method suitability testing using the USP panel of organisms with each of their products prior to the FDA inspection. During the inspection, the site was advised that the method suitability tests needed to be updated with testing of “healthy” and “injured” Burkholderia cepacia. Since these organisms were not part of an environmental monitoring library, the site had to develop a method to create injured BCC microorganisms, using a paper by Gray, et al. (2010). The existing methodology was evaluated to determine whether “injured” BCC could be detected. They found that the methodology used was not suitable for recovery of injured BCC. Refinements were made to the method and suitability was shown for the USP organisms and Burkholderia cepacia (both healthy and injured).
Testing of product bioburden, using the revised method suitability testing showed the presence of Burkholderia cepacia. An intensive investigation was conducted to determine the source of the BCC.
Extensive microbiological sampling was conducted throughout the site and for the water system. No presence of BCC was found in the point-of-use samples or the sampling ports. BCC was found in a filling line used for two products. It was also found in the drains in the warehouse. Further investigation into the filling line, showed that during cleaning of the fill line, a dead leg was created, and it was hypothesized that a biofilm was present in this fill line.
The site purchased an ozonated water cart and sanitization/sterilization of the filling lines. The cart was designed to generate ozonated water at a concentration of 5-8ppm ozone. Using a half-cycle approach per USP 1229.6, a sterilization cycle was validated. Following sterilization and periodic sanitization cycles no further BCC isolates were found. The same cycle duration was used in the drains to eradicate BCC. Samples taken after this rinse showed no BCC.
Conclusion
The affected products were quarantined and had not been released at the time the contamination occurred. No new BCC events have occurred since sterilizing the system and implementing a routine sanitization schedule with ozonated water.
Case Study: Sterile Aqueous Product
A company which manufactures sterile parenteral and oral products was audited. During this inspection, the auditor identified an increasing trend of Burkholderia cepacia and other related species over the past three years. All the contaminants were found in the pre-filtration bioburden samples for the product and recommended shutting down the operation.
There was some confusion in the facility, as there is no requirement for absence of BCC in sterile product manufacturing facilities. The only “objectionable organism” claims were designated for non-sterile, aqueous formulations and in the finished “sterile” product.
The data showed that all the BCC was isolated in four different product formulations. All the contaminated solutions were in a Grade B laminar air flow area which was part of the Aseptic Filling Suite. In total, ten different products are manufactured in this same suite. Four different strains of Burkholderia had been isolated. None of these organisms had been isolated in routine environmental monitoring. To date, no sterility failures or product complaints of contamination had taken place.
It was important to know whether the data generated could be believed. As such, method suitability was repeated using healthy and injured BCC to show that the methods could detect BCC. Studies were also conducted to show that the disinfectants would eradicate BCC. First, however, a method to injure BCC was developed using the methods in Gray, et al. (2010).
Numerous systems were reviewed and evaluated as a source of BCC. No link could be found to systems or equipment. Ingredients of the products were evaluated to determine a potential link to the contamination, but none could be found.
Additional studies were conducted to determine whether the existing media and incubation conditions would recover BCC. Testing included R2A, TSA, SDA and FTM. A selective media BCSA was also evaluated. Testing was conducted both at 20°C – 25°C and 30°C – 35°C using both healthy and injured microorganisms. These tests indicated that R2A and TSA yielded the best recovery for injured BCC at 30°C – 35°C. As such, it was determined that it was not necessary to utilize BCSA for routine monitoring. This showed the water testing data showing no growth of BCC could be believed.
The sterility test method suitability tests (B&F) were updated to include testing of healthy and injured BCC organisms. This provided confirmation that the sterility test could detect BCC organisms, within the normal abilities of the sterility test.
Another concern was possible masking of BCC presence, due to the kinetics of growth. Studies were conducted to ensure that this did not take place. (Metcalfe, 2017) No inhibition of growth was found.
After exhausting many other tests, the pH buffers were tested for microbial content. The pH 7 calibration buffer was found to be grossly contaminated with Burkholderia cepacia. The stock solution of buffer (unopened containers) was also tested and found to be contaminated. These stock solutions had been in place for the entire three years where the contamination event occurred. While this product was used for all ten products, further investigation showed that the way pH testing was done protected six products from contamination but not the other four. Samples of the isolates from the buffer were sent for genetic analysis and comparison to the original bioburden contaminants.
Conclusion
No product was deemed at risk due to the heat treatment and sterile filtration of the product. Both processes were validated.
Acknowledgements
This article was based upon a presentation given at the PDA European Pharmaceutical Conference in Berlin, 2018. This topic is discussed in additional detail in Contamination Control Volume 5, published by PDA/DHI in 2018. It is edited by Russell Madsen and Jeanne Moldenhauer.
References
- Anderson, R.R., Warnick, P. and Scheckenberger, P.C. (1997) Recurrent CDC Group IVc-2 bacteremia in a human with AIDS J. Clin. Microbiol.(Mar) 35(3):780-2.
- Aspinall, S., Graham, R. (1989) Two Sources of Contamination of a Hydrotherapy Pool by Environmental Organisms. J.Hosp. Infect. 14:285-292.
- Balada-Llasat, J-M, Elkins, C., Swyers, L., Bannerman, T., and Pancholi,P. (2010) Pseudo-Outbreak of Cupriavidus pauculus Infection at an Outpatient Clinic Related to Rinsing Culturette Swabs in Tap Water. Journal of Clinical Microbiology. 48(7): 2645-2647
- CDC (2013) Antibiotic Resistance Threats in the United States, 2013. U.S. Department of Health and Human Services. Centers for Disease Control and Prevention. Downloaded from: file:///C:/DocLib/Literature/Antibiotic%20Resistant%20Bugs/CDC%20Antibiotic%20Resistant-threats-2013-508.pdf on October 29, 2018.
- Clark, W., et al. (1984) Identification of Unusual Pathogenic Gram Negative Aerobic and Facultatively Anaerobic Bacteria. CDC, Atlanta, Georgia.
- Ensor, L.A. (2017) Microbiological Quality Considerations in Non-sterile Pharmaceutical Product Manufacture. Presentation given at the PDA European Pharmaceutical Microbiology Conference held in Porto, Portugal, on February 14-15, 2017.
- FDA (2017a) FDA advises drug manufacturers that Burkholderia cepacia complex poses a contamination risk in non-sterile, water-based drug products. Downloaded from: https://www.fda.gov/Drugs/DrugSafety/ucm559508.htm?source=govdelivery&utm_medium=email&utm_source=govdelivery on January 2, 2017.
- Gray, J.C.; Staerk, A.; Berchtold, M.; Hecker, W.; Neuhaus, G.; Wirth, A. (2010) Growth promoting Properties of Different Solid Nutrient Media Evaluated with Stressed and Unstressed Micro-organisms: Pre-study for the Validation of a Rapid Sterility Test. PDA Journal of Pharmaceutical Science and Technology. 64(3): 249-263.
- Kalka-Moli, W.M., LiPuma, J.J., Accurso, F.J., Plum, G., van Koningsbruggen, S., and Vandamme, P. (2009) Airway Infection with a Novel Cupriavidus Species in Persons with Cystic Fibrosis. J.Clinc. Microbiol. (September) 47(9): 3026-3028.
- Manara, C.M., et al. (1990) Heterotrophic Plate Counts and the Isolation of Bacteria from Mineral Waters on Selective and Enrichment Media. J. Appl. Bacteriol. 69:871-876.
- Mezher, M. (2016) Florida CMO Recalls All Lots of Liquid Drugs Due to Bacteria Outbreak. RAPS. Downloaded from: http://www.raps.org/Regulatory-Focus/News/2016/08/10/25563/Florida-CMO-Recalls-All-Lots-of-Liquid-Drugs-Due-to-Bacteria-Outbreak/ on January 2, 2017.
- Metcalfe, J. (2017) CDER Case Study: A Microbial Investigation of Contamination by Burkholderia multivorans. PDA European Pharmaceutical Microbiology Conference. Porto, Portugal.
- Microbewiki (2017) Burkholderia cepacia complex. Downloaded from: www.microbewiki.kenyon.edu on January 2,2018.
- Moissenet, D.,Bidet, P., Garbarg-Chenon, A., Arlet, G., and Vu-Thien, H. (2001) Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum). J. Clin. Microbiol. (January) 39(1): 381-384.
- Ori, S., et al. (1998) Microbial Contamination of Sterile Water in Japanese Hospitals. J. Hosp. Infection 38:61-65.
- Rao, T.V. (2017) Facts on Ten Most Dangerous Antibiotic Resistant Bacteria. Downloaded from: https://www.slideshare.net/doctortvrao/most-dangerous-antibiotic-resistantbacteria on October 29, 2018.
- Tasbakan, M.S., Yamazhan, T., Aydemir, S., and Bacakoğlu, F. (2010) A case of Ventilator associated Pneumonia Caused by Cupriavidus pauculus. Microbiyol. Bul. (January) 44(1): 127 – 131.
- USP <61>/<62> Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests and Tests for Specified Microorganisms,
- Vandamme, P., Goris, J., Coenye, T., Hoste, B., Janssens, D., Kersters, K., De Vos, P. & Falsen, E. (1999) Assignment of Centers for Disease Control group IVc-2 to the genus Ralstonia as Ralstonia paucula sp. nov. Int. J. Syst. Bacteriol. 49, 663–669.
- Vaneechoutte, M., Kämpfer, P., De Baere, T., Falsen, E. & Verschraegen, G. (2004). Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov. Int. J. Syst. Evol. Microbiol. 54: 317–327.
- Wikipedia (2017) Burkholderia cepacia complex. Downloaded from: https://en.wikipedia.org/wiki/Burkholderia_cepacia_complex on January 2,2018,