Abstract
United States Pharmacopeia (USP) General Chapter <60> for the detection of Burkholderia cepacia complex (Bcc) members in nonsterile products became official in December 2019. This isolation method requires confirmation of the identity of any growth found on Burkholderia cepacia Selective Agar (BCSA) by additional identification tests (refer to the Interpretation section). This article presents a singleplex polymerase chain reaction (PCR) method to rapidly confirm the membership of any microbial grown on BCSA (and other nutrient medium) in the Bcc group. This method is cost effective as it does not require expensive equipment or reagents; therefore, it can be easily adopted in the industry without an important investment. We validated this singleplex PCR Bcc identification method with previously published PCR primers with an expanded panel of 37 clinical and environmental Bcc isolates. The sources and repositories of these Bcc isolates include contaminated health products and medical devices, patients infected with cystic fibrosis, the National Microbiology Laboratory (NML) internal strain bank, and the American Type Culture Collection (ATCC). All 37 isolates that belong to the Bcc tested positive using our confirmatory identification method. Twenty-two negative controls including four isolates belonging to the genus Burkholderia tested negative as expected. Our work indicates that this singleplex PCR is an efficient confirmatory method for Bcc identification, and it can successfully supplement USP <60> for Bcc isolates identification found in pharmaceutical products.
Introduction
Burkholderia cepacia are Gram-negative bacilli bacteria that belong to the Betaproteobacteria class. These organisms are typically found in water and soil. Previously known as Pseudomonas cepacia, first described by Walter H. Burkholder as a causal agent of the onion skin rot plant disease, B. cepacia is also found to be pathogenic to humans (1). In 1973, the genus Pseudomonas was divided into five groups by Palleroni et al. (2) based on rRNA-DNA hybridizations. On the basis of this exercise, Pseudomonas sensu stricto has been reclassified as genera Burkholderia, Ralstonia, Pandoraea, Acidovorax, Comamonas, Delftia, Brevundimonas, and Stenotrophomonas (1, 2).
B. cepacia, as its name suggest, is a member of the B. cepacia complex (Bcc), a group of closely related bacteria species (3). The Bcc includes at least 23 published genomovars that are difficult to differentiate either by their biochemical properties or by genetic traits. Table I shows species included in the complex. Eberl et al. (4) showed the classification of Burkholderia species within Bcc in phylogenetic trees based on 16 s rRNA gene sequences.
In addition to their high resistance to antibiotics such as carboxypenicillins, cephalosporins, and polymyxins, Bcc bacteria can thrive in challenging environments due to their large genomic size and genomic plasticity. Bcc microorganisms are also resistant to antiseptics and disinfectants. One of the factors that contributes to their multidrug resistance is the high-level expression and activation of efflux pumps (5). Consequently, Bcc bacteria are opportunistic pathogens, especially for cystic fibrosis (CF) patients and chronic granulomatous disease (CGD) patients. Because Bcc bacteria have antimicrobial resistance and they can survive under stressful conditions such as nutrient-limited and water-based environments, colonization of the CF lung environment is a serious concern (3, 4).
Bcc members are also an occasional contaminant of medications and medicinal products and have been responsible for several outbreaks (3). In the United States, the Bcc is the most frequent contaminant in nonsterile products such as nasal sprays, lotions and oils, water-based products, mouthwash, cleansing washcloths, baby wipes, preoperative skin solutions, sanitizers, oral pharmaceuticals, and gas relief drops (3, 6). Since 2006 in Canada, B. cepacia contaminations causing recalls were mainly found in wipes and disinfectant products (https://healthycanadians.gc.ca/recall-alert-rappel-avis/index-eng.php). This is not surprising, because Bcc contamination in pharmaceutical water has been a long concern in the manufacturing industry (3, 7). Bcc is known for its growth in nutrient-scarce environments such as water environments, because it can form a structural matrix on surfaces through production of proteins and extracellular polymeric substance matrix, creating biofilms. Consequently, contaminated water supplies in manufacturing environments lead to biofilm formation on various materials such as plastic, metals, hospital equipment, and so forth. This indicates the seriousness of Bcc contamination to the industry (1).
Bcc are recognized as objectionable microorganisms by the Food and Drug Administration (FDA) and the industry. Undetected presence of Bcc in nonsterile products has been a concern for the FDA since 1981. Persistent Burkholderia contamination in nonsterile products is, in part, attributed to Good Manufacturing Practices (GMPs) deficiencies (cleaning procedure as well as water system control and design deficiencies, insufficient environmental controls, no tracking for environmental floral studies, and so forth). Consequently, companies are expected to develop and validate a method to detect those microorganisms (3, 8, 9).
In December 2019, the U.S. Pharmacopeial Convention published in USP <60> a culture method to determine the presence or absence of Bcc in nonsterile products. Similar to the method described in USP <62> “Tests for Specified Microorganisms”, the possible presence of Bcc is indicated by typical growth on selective media, Burkholderia cepacia selective agar (BCSA), and it needs to be confirmed by further/additional identification tests.
Identification Methods for Members of Bcc
Routine isolation of Bcc members can be achieved using selective media such as BCSA, media that is required in the USP <60>, Oxidative Fermentative Polymyxin B Bacitracin Lactose (OFPBL) agar, or Pseudomonas cepacia agar; recovery of the bacteria; however, can require an incubation time of up to 72 h (10). These media contain several antibiotics such as polymyxin B, ticarcillin, bacitracin, gentamicin, or vancomycin to induce a rapid recovery of Bcc bacteria while inhibiting the growth of other microorganisms (11). Although this isolation technique is easy and routinely used, it has its limitations such as low specificity. Indeed, the isolation of Bcc bacteria on selective media cannot fully discriminate Bcc members from nonmembers, and it allows growth of some organisms belonging to the other genera, particularly, Ralstonia picketti or Cupriavidus basilensis (11, 12). This is why isolation of Bcc on selective media should be followed by additional identification tests. A combination of phenotypic tests such as enzyme-based tests (oxidase, lysine decarboxylase, ornithine decarboxylase, oxidation of sucrose, adonitol or lactose, gelatin liquefaction, aesculin hydrolysis, ß-galactosidase activity, and ß-hemolysis) and growth at 42°C has proven useful for the differentiation of members of the complex (11, 12). Although these tests are easily feasible in the lab, they are time-consuming and often insufficient to achieve adequate specificity for Bcc bacteria. In addition, phenotypic variations, conflicting results, and misinterpretation make Bcc identification arduous. Commercially available tests such as API strips and VITEK 2 System require 24–48 h incubation and suffer the same issue of low specificity and sensitivity, requiring additional tests for confirmation (11, 12).
In 1997, Vandamme et al. (13) presented a taxonomic structure of Burkholderia cepacia and its relationship with other Burkholderia species using a whole-cell protein analysis technique on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). This technique appears to be suitable as an additional confirmation method; however, there are disadvantages to adopting this as a routine method. These include the need to build a large database, poor discrimination between different genomovars, and the requirement for highly trained personnel.
Alternatively, Vitek MS and Bruker Biotyper are matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) systems based on protein profiles of microorganisms, and they can rapidly identify and/or discriminate Bcc from non-Bcc members depending on the quality of the database. However, the high acquisition cost of these instruments as well as maintenance and frequent calibrations costs make these options less accessible.
Polymerase chain reaction (PCR)-based tools like amplified fragment length polymorphism (AFLP) can successfully distinguish the Bcc isolates from the other Burkholderia organisms with high reproducibility. Nonetheless, these methods are laborious, expensive, and require development of a database and are therefore unsuitable as routine diagnostic tools (11, 12, 14).
Another valuable and simple technique for Bcc bacteria identification is fatty acid analysis as it offers rapid identification at low cost. Nevertheless, it is considered inappropriate to discriminate Bcc members because it can identify only at the genus level (11).
Nucleic acid sequence-based technologies such as multilocus sequence typing (MLST) and 16S rRNA sequencing can be used to differentiate Bcc members, but their usefulness is hampered by the phylogenetic proximity of certain species (1, 15⇓–17). The need for a robust database, specialized instrumentation, and highly trained personnel make these techniques expensive and difficult to implement for routine analyses (1).
Currently, PCR is becoming more widely accepted as the most appropriate technique for distinguishing Bcc from related genera/species. Subsequently, several genes are being studied as candidates for Bcc complex markers. The markers that are reported to increase the discrimination of Bcc include 16S rRNA, recA, hisA, and rpsU (18).
The recombinase A gene, recA, is useful for bacterial classification (22) and one of the most commonly used targets for PCR identification. Present in all bacteria species, recA is essential for their survival and its protein, RecA, is conserved across bacterial organisms as it is involved in many crucial biological mechanisms such as homologous recombination, DNA repair, and SOS response. The changes in DNA are observed when the SOS response is activated (13, 19). The recA gene shows 94%–95% similarity between the different genomovars within the Bcc (11), and an identification method based on its sequence analysis was proposed for the entire Burkholderia genus (20).
Many scientists have designed PCR-based methods for discriminating Bcc bacteria based on the recA gene similarity. In 2000, Mahenthiralingam et al. (21) designed a set of specific PCR primers (BCR1/BCR2) to discriminate Bcc members from nonmembers by aligning the Burkholderia recA sequences. Jimenez et al. (23) tested a real-time PCR approach for pharmaceutical products artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium and was able to detect B. cepacia in 30 h. In 2016, Attia et al. (24) proposed a seminested PCR (SN-PCR) with BCR primers followed by BCR1/Mr primers for the direct detection of B. cepacia from aqueous pharmaceuticals products.
The objective of our work is to validate a rapid PCR method for the confirmation of Bcc membership, which could be used after the isolation of bacterial colonies following the USP <60> procedure. This method will be able to distinguish species/genomovars of Bcc from other Burkholderia species and related microorganisms. When these pathogens are isolated from a pharmaceutical product or a medical device, a risk assessment does not require an identification at a genomovar level; a Bcc identification is sufficient. This is because the risk assessment would depend on the level and type of exposure rather than a specific genomovar (Health Canada Therapeutic Products Directorate, personal communication, 2013 and 2020). In this study, singleplex PCR assays were performed on 37 Burkholderia isolates within the complex (inclusivity test), 4 Burkholderia outside of the complex, and 18 other negative controls (exclusivity test). All Bcc isolates DNA was extracted from colonies grown on BCSA to avoid the extra step of subculturing isolated colonies on nutrient media.
We believe that this easy singleplex PCR confirmation method will be useful for regulatory laboratories and the industry as it is quick, accurate, cost-effective, and efficient.
Materials and Method
Bacterial Isolates
The collection of Bcc bacteria used in this study includes 37 Burkholderia isolates within the complex, 4 Burkholderia isolates outside the complex, and 18 non-Burkholderia negative controls (Table II). Ten Bcc and 2 Burkholderia isolates were ATCC strains. Five Bcc and 1 Burkholderia isolate were provided by the National Microbiology Laboratory, Public Health Agency of Canada (NML, PHAC, Winnipeg, Canada). Thirteen Bcc isolates were collected from CF patients’ throats at Queen Elizabeth II Health Sciences Center (Halifax, Nova Scotia, Canada). Six Bcc isolates were recovered from medical instruments, 2 from consumer products (teething ring and hair conditioner), and 1 from a natural health product analyzed in the Health Canada Health Products Laboratories in Longueuil and Toronto (HPLL and HPLT), now known as ROEB Microbiology Laboratory. One Burkholderia isolate was found from a teething ring analyzed in HPLL. From the 18 isolates used as negative controls, 5 were ATCC strains, 12 were isolated from contaminated health products, and 1 was recovered from a food product.
All isolates from the collection were identified in our laboratory with Axcess System MALDI-TOF (Microflex LT/SH, Charles River Axcess, Charleston, SC, United States) except for one negative control Pseudomonas aeruginosa, which was identified with a VITEK 2 instrument (BioMérieux, Saint-Laurent, QC). Twelve isolates were also confirmed with 16S rRNA sequencing done by Charles River Accugenix AccuGENX-ID service (Newark, DE, United States). The six samples provided by the NML were identified using MultiLocus Sequence Typing (MLST) (Table II). Among the Burkholderia isolates identified by the MALDI-TOF, several isolates were identified as two different genomovars because this method could not distinguish between the two.
Bacteria cultures were grown on Tryptic Soy Agar (TSA) slants at 30°C–35°C and stored at 20°C–25°C. Prior to DNA extraction, isolates were regrown at 30°C–35°C on BCSA (Hardy Diagnostics, Santa Maria, CA, United States) or on TSA (BD diagnostics, Franklin Lakes, NJ, United States) when it was unable to grow on BCSA.
DNA Extraction
DNA extraction was performed by suspending isolated colonies in 20 µL of lysis buffer (2.5% SDS, 50 mM NaOH). The mix of colonies and lysis buffer was heated for 15 min at 100°C and then cooled on ice or in a cooling block for 5 min. After the addition of 180 µL of nanopure water (molecular biology grade), the samples were centrifuged at 9300 ×g, 4°C for 5 min. The supernatants were transferred into a 96-well plate and used for PCR. The 96-well plate was conserved at −20°C before use.
PCR Detection and Analysis
The PCR reaction mixture was composed of 12.5 µl 2X GoTaq G2 Hot Start Green Mastermix (Promega, Madison, WI, United States), 1 µM of each PCR primer, and 2 µl DNA in a final volume of 25 µl. For the Bcc identification, previously published primers that were specific to Bcc, BCR1 (5′-TGACCGCCGAGAAGAGCAA-3′) and BCR2 (5′-CTCTTCTTCGTCCATCGCCTC-3′), were used (21). As for an internal amplification control (IAC), universal 16S rDNA primers 8 F and 1510 R were used (25). The thermal cycling conditions were: 94°C for 2 min, followed by 30 cycles at 94°C for 30 sec, 58°C for 45 sec, 72°C for 1 min, then a final extension at 72°C for 10 min using an Eppendorf Mastercycler ep gradient (Eppendorf, AG, Hamburg, Germany). PCR products were confirmed by electrophoretic separation at 90 V for 45 min using a Bio-Rad electrophoresis system on 1.0% agarose gel with an Invitrogen 1 kb Plus DNA ladder (Invitrogen, Waltham, MA, United States). The gels were visualized and photographed using a Gel Doc XR Imager (Bio-Rad, Mississauga, ON, Canada).
Sequencing
The PCR amplification products for 26 isolates were confirmed by sequencing of the PCR products at Genome Québec Innovation Center by Sanger-type sequencing (Montreal, Canada). Both strands of BCR amplicons (PCR product from BCR1 and BCR2 primers) were sequenced using BCR1, BCR2, as well as recA sequencing primers BCR3 (5′-GTCGCAGGCGCTGCGCAA-3′) (3′ half of Bcc recA gene in combination with primer BCR2) and BCR4 (5′-GCGCAGCGCCTGCGACAT-3′) (5′ half of Bcc recA gene in combination with primer BCR1), as described by Mahenthiralingam et al. in 2000. Raw sequences from both strands of the PCR products were verified using the Basic Local Alignment Search Tool (BLAST®; https://blast.ncbi.nlm.nih.gov/Blast.cgi) to ensure that isolates matched at 100% identity to sequences belonging to the Bcc recA gene.
Results
Isolates Collection
As part of this study, 9 genomovars have been tested out of the 23 that comprise the Bcc (Table I). The isolates and their various sources are further described in Table II. Our collection contains numerous Burkholderia outside of the complex such as Burkholderia glathei, Burkholderia andropogonis, Burkholderia gladioli and Burkholderia tropica and several non-Burkholderia negative controls related to the Burkholderia genus.
PCR Amplification
As the BCR primers used here were first published in 2000, they could be considered outdated. In order to verify with the continuously updated new bacterial genomes, the primers were confirmed using BLAST. They matched with the sequences belonging to the Bcc recA gene at 100% identities.
The BCR primers were first tested on DNA isolated from Bcc bacteria colonies grown on TSA (26 Bcc isolates). All Bcc isolates yielded strong amplicon of the expected size (1043 bp) when visualized on 1% agarose gel. The amplicons were sequenced and their identity was verified using BLAST. All the sequences matched published sequences of the recA gene from Bcc isolates.
The BCR primers were then tested on all available Bcc isolates, using DNA extracted from bacteria colonies grown on BCSA; 37 Bcc isolates grown on BCSA yielded a PCR amplicon of the accurate size (inclusivity/sensitivity, 100%).
Four Burkholderia isolates, which are not part of the Bcc (B. glathei, B. andropogonis, B. gladioli and B. tropica) were used as negative controls. B. gladioli and B. glathei were grown on BCSA before DNA extraction, but B. andropogonis and B. tropica were grown on TSA along with 18 other negative controls due to their inability to grow on BCSA. These other controls were chosen among Burkholderia related genera (Pseudomonas, Stenotrophomonas, Delftia, Ralstonia, and Cupriavidus). The PCR target band was not observed for the negative controls (exclusivity/specificity 100%).
Nonspecific bands obtained from the following non-Bcc isolates could be distinguished by size from the Bcc target band. B. glathei, B. andropogonis, B. tropica, and Pseudomonas aeruginosa produced amplicons of around 550 bp; Delftia acidovorans, Cupriavidus basilensis, B. gladioli, and Ralstonia picketti produced amplicons of 1250 bp, 700 bp, 400 bp and 300 bp, respectively (Figure 1). This is particularly important because the strains that grow on the selective media, BCSA, such as B. glathei, D. acidovorans, and R. picketti, can be immediately discriminated from the Bcc bacteria due to an absence of the recA amplicon at 1043 bp.
To confirm the PCR reaction, primers that amplify part of the 16S rRNA gene were included as an IAC. An amplicon representing the IAC (1502 bp) was observed for all isolates (25). All isolates successfully showed a positive PCR reaction, resulting in a band of the expected size (1502 bp) on an agarose gel. Only D. acidovorans has shown a weak band.
Multiplex assays were attempted for 34 Bcc isolates and 20 negative control strains. Even after several trials of PCR condition optimization, the results were found to be unreliable (results not shown). Indeed, at least five Bcc isolates showed inconsistent results such as the presence or absence of a Bcc-specific band on a few different assays. Consequently, our study found the singleplex PCR was most effective for Bcc bacteria identification.
Discussion
The Bcc bacteria strains used in this study were isolated from diverse sources and are a good representation of reference strains as well as clinical and environmental isolates. These include ATCC strains and isolates from medical devices (defogger for mouth mirrors) and health (wound-cleaning saline) and consumer products (teething ring and hair conditioner). These are environmental strains most likely originated from manufacturing facilities. Thirteen isolates were recovered from CF patients’ sputum or throat cultures from the Queen Elizabeth Hospital, whereas six isolates were donated by the Antimicrobial Resistance and Nosocomial Infections Laboratory (NML). Those are interesting because they may have been exposed to selective pressures (antibiotic therapy, disinfectants, and production processes). Our results indicate that the singleplex PCR method for Bcc identification presented here works with high specificity for the isolates coming from diverse backgrounds and potentially mutated through exposure to selective agents. To our knowledge, the collection of bacteria studied in this article is the most extensive representation and coverage of existing Bcc bacteria strains to this date.
Previously, Attia et al. (24) showed a similar study of the direct detection of Burkholderia cepacia in pharmaceuticals products using a seminested PCR with BCR primers. According to their study, B. cepacia in two samples that had not been detected by the VITEK 2 system were successfully identified by the PCR-based method. Although the number of tested isolates belonging to Bcc was limited in their study, Attia et al. demonstrated that the PCR-based method is suitable for Bcc detection in pharmaceutical products.
Our collection of Bcc bacteria isolates covers an important range of Bcc genomovars (at least 9 out of 23, 39%) and includes the most common genomovars isolated from CF and non-CF patients (5, 18, 26) (∼15 isolates), B. multivorans (2 isolates) and B. vietnamiensis (1–2 isolates). Twelve out of the 13 isolates isolated from CF patients from the Queen Elizabeth II Health Science Center are identified as B. cenocepacia, which is consistent with findings in the literature (5, 18, 26). This result strongly suggests that the singleplex PCR-based Bcc identification method is highly effective with the most clinically relevant Bcc genomovars.
Negative controls were specifically chosen based on phylogenetic closeness to the Bcc and the Burkholderia genus; the Burkholderia, Cupriavidus, Delftia, and Ralstonia genera are all Gram-negative rods, previously part of the Pseudomonas genus, that are susceptible to inaccurate Bcc identifications. Isolates phylogenetically related to the Burkholderia genus were also included in our study to ensure that our suggested singleplex PCR method can correctly identify strains that are often tested as false-positives. Negative controls were isolates from Pseudomonas, Ralstonia, Stenotrophomonas, Cupriavidus, and Delftia genera (11). Ralstonia, Cupriavidus, Pandoraea, Achromobacter, Brevundimonas, Comamonas, and Delftia are the most common genera that cause Bcc misidentification due to genetic proximity to Bcc (18). The PCR-based Bcc identification method that we have proposed here is a confirmation method that allows the accurate classification of bacteria that belong to the Bcc group from other Burkholderia species as well as Cupriavidus basilensis, D. acidovorans, and R. picketti. As we observed in our laboratory, those isolates are able to grow on BCSA. Our results strongly indicate that this identification method can discriminate the bacterial strains that are genetically closely related to Bcc.
All of the PCR amplification results from the IAC confirmed the successful PCR reaction. The weak band from D. acidovorans may be attributed to the fact that it was grown in a selective media, BCSA. In fact, all the positive control amplifications from the bacterial strains that were grown on TSA consistently showed stronger bands than those of the strains grown on BCSA.
As shown by the results obtained from negative controls, the use of a selective media before this confirmation method is not mandatory. However, the results have shown that the use of the selective media BCSA does not interfere with the PCR amplification. DNA extraction from BCSA and TSA culture have been tested and have both demonstrated the same amplification results. Thus, this approach could be used as a rapid confirmation method complementary to USP <60> without the need for subculturing candidate colonies on nonselective media before the testing.
Although it is possible to distinguish nonspecific bands from the Bcc band using a DNA ladder, it would be a good practice to include a known Bcc strain as a positive control along with the candidate strains, for example, Burkholderia cepacia ATCC25416, Burkholderia cenocepacia BAA-245 or Burkholderia multivorans BAA-247 as suggested as positive controls in USP <60>. This would allow comparison of the candidate bands with a known Bcc band.
Selectivity of BCSA
The isolation method published in USP <60> was developed for detection of Burkholderia cepacia complex in nonsterile products. It is a culture method allowing the isolation of Bcc on BCSA selective media containing gentamicin, vancomycin, and polymyxin B. The growth of typical colonies on this medium indicates the possible presence of Bcc members. However, the USP requires that all growth must be confirmed by additional identification tests. This is particularly important because our results have shown that B. glathei, B. gladioli, C. basilensis, D. acidovorans, and R. picketti are able to grow on BCSA (not shown). Although the phenotypic characteristics are not identical when grown on BCSA, these microorganisms are difficult to differentiate on this media. For example, R. picketti and C. basilensis present gray colonies on fuchsia media similar to B. stabilis or B. anthina. Indeed, Coenye (27) reported in 2013 that the selective media such as fuchsia media also allow the growth of other related species, for example, Ralstonia and Cupriavidus species. This reinforces the need for a confirmation method, PCR, for a more accurate Bcc bacteria identification. Our study has confirmed that the singleplex PCR-based method is much less laborious and more cost-effective and time-efficient than the biochemical tests.
The DNA extraction method used is simple and quick. The PCR-based method that we propose here works with crude extracts without the use of a DNA extraction kit, purification, quality verification, or quantification. As shown in our results, PCR reactions from the DNA extracted from the Bcc strains grown in BCSA are all successful and not hindered by inhibitors that may exist in the selective medium. This makes it a rapid and accurate confirmation method following USP <60>.
A next step would be to develop a multiplex assay combining BCR primers with universal primers as an internal control.
Conclusion
Since USP <60> for the detection of Burkholderia cepacia complex (Bcc) members in nonsterile products became official in December 2019, a need has arisen for the development of a simple Bcc membership confirmation method. Our results have shown that the confirmation by a PCR-based method is the simplest and most accessible procedure, because this technique does not require specialized instruments or highly trained personnel. Our singleplex PCR method was developed based on group-specific primers published in 2000 by Mahenthiralingam et al. Our work tested the specificity and inclusivity of the primers using a collection of isolates belonging to and closely related to the Bcc group. Of the 37 isolates belonging to the complex, 100% tested positive for the confirmatory test and of the 22 negative controls including 4 isolates belonging to the genus Burkholderia, 100% tested negative. The results show that our suggested PCR method is highly accurate in Bcc bacteria identification with 100% sensitivity and 100% specificity.
The accurate identification of the collection of bacteria from various sources used in this article strongly indicates that the method proposed here is highly sensitive and efficient in Bcc identification confirmation in nonsterile products. From this collection of bacteria, at least 9 genomovars out of 23 published have been correctly identified as belonging to the Bcc group. Three Burkholderia species not belonging to the Bcc group and 19 isolates related to the Burkholderia species—such as Pseudomonas, Ralstonia, Cupriavidus, Delftia, and Stenotrophomonas—have not shown the target band of 1043 bp.
Included in this study are numerous environmental strains isolated from various pharmaceuticals products. These strains are representatives of potential isolates obtained from manufacturing facilities according to USP <60>. Our study demonstrated that the presented PCR identification technique is applicable to various strains that are members and nonmembers of Bcc.
Although a sequencing-based method such as a whole-genome sequencing (WGS) approach would offer the most accurate results at the genomovar level, as a routine diagnostic technique, a rapid, low cost, and accessible method should be prioritized.
The proposed PCR identification method is much more rapid and suitable for identification of suspected Bcc isolates than the traditional culture method either from selective or nonselective media. This method is also inexpensive, less labor intensive, and requires a minimal training.
We believe that the confirmatory approach based on PCR will facilitate the management of the colonies isolated according to USP <60> as well as product conformity assessment and health risk evaluation.
Conflict of Interest Declaration
The authors declare that they have no competing interests.
Acknowledgments
We are very grateful to Kathryn Bernard, formerly head of the Special Bacteriology Unit at the National Microbiology Laboratory (Public Health Agency of Canada) and her team for providing Bcc isolates and their expertise. Thanks to Dr. David Haldane from the Queen Elizabeth II Health Sciences Centre in Halifax, Nova Scotia, for graciously providing us with clinical isolates. Without their help, our microbial collection would not have been as diverse and significant. We thank Roxane Arsenault, Jamili Ahmarani, Roland Yang, Julio Bran Barrera, Guillaume Massicotte, and Nathalie Poulin for their help with isolates preparation, DNA extraction, and PCR. We would also like to thank Daniel Plante and Hayline Kim for critical review of the manuscript.
- © PDA, Inc. 2023
References
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