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Direct Detection of Burkholderia cepacia in Susceptible Pharmaceutical Products Using Semi-Nested PCR
Mohamed A Attia1, Amal E Ali2, Tamer M Essam3
1Biotechnology Center, Faculty of Pharmacy, Cairo University, Cairo, Egypt;
Abstract:
Burkholderia cepaciahas recently received a considerable attention as one of the major risks in susceptible pharmaceutical products. This microorganism can easily propagate and cause vast and severe contamination, especially to the water supplies for pharmaceutical companies. Moreover, it proliferates within the products and can cause severe infections for humans. Therefore, fast and sensitive detection of these bacteria is of a great demand. The present study introduces improved application of a polymerase chain reaction assay with relatively high sensitivity and specificity for the direct detection ofB. cepaciafrom the aqueous pharmaceutical products. A semi-nested polymerase chain reaction approach using the primer set BCR1/BCR2 followed by BCR1/Mr yielding a 465 bp fragment of the recA gene was applied and tested using both crude lysate from isolated colonies and DNA directly extracted from artificially prepared and spiked reference syrup. The polymerase chain reaction assay showed no interference with other bacterial reference and environmental strains tested, includingStaphylococcus aureusATCC® 6538,Pseudomonas aeruginosaATCC® 9027,Escherichia coliATCC® 8739,Salmonella abonyNCTC® 6017,Bacillus subtilisATCC® 6633,Micrococcus luteus, Staphylococcus warneri, Pseudomonas fluorescens, Pseudomonas putida, andRalstonia pickettii Moreover, this semi-nested assay showed a detection limit of around 10 colony-forming units per sample and could detectB. cepaciastrains isolated from a municipal pre-treated potable water tank. Comparing the results for detection ofB. cepaciain 100 randomly collected commercial syrup preparations using both conventional standard method and polymerase chain reaction assay revealed thatB. cepaciawas detected in two samples using polymerase chain reaction assay while all samples showed negative results by conventional culturing and biochemical methods. These results highlight the advantage of using this polymerase chain reaction assay to detectB. cepaciain contaminated pharmaceutical products and even water for pharmaceutical purposes, without the need of culturing or pre-enrichment, where it may give false-negative results and may be misidentified when biochemically tested.
Insights
A new polymerase chain reaction (PCR) assay offers sensitive and specific detection of Burkholderia cepacia in pharmaceutical products. This method surpasses traditional culturing, identifying contamination missed by standard techniques.
Area of Science:
- Microbiology
- Molecular Biology
- Pharmaceutical Science
Background:
- Burkholderia cepacia poses a significant risk to pharmaceutical products and patient safety due to its ability to contaminate water supplies and proliferate in medications.
- Rapid and sensitive detection methods for B. cepacia are crucial for ensuring pharmaceutical product integrity and public health.
- Conventional culturing and biochemical methods can be slow and may yield false-negative or misidentified results for B. cepacia.
Purpose of the Study:
- To develop and validate an improved polymerase chain reaction (PCR) assay for the direct, sensitive, and specific detection of Burkholderia cepacia in pharmaceutical products.
- To evaluate the performance of the developed PCR assay against conventional methods for detecting B. cepacia in commercial pharmaceutical samples and water.
Main Methods:
- A semi-nested PCR approach was employed, targeting a 465 bp fragment of the recA gene using primer sets BCR1/BCR2 and BCR1/Mr.
- The assay was tested using both crude bacterial lysates and DNA extracted directly from spiked pharmaceutical samples (syrup).
- Specificity was confirmed against a panel of relevant bacterial strains, and the limit of detection was determined.
Main Results:
- The semi-nested PCR assay demonstrated high specificity, showing no interference with other tested bacterial strains.
- A detection limit of approximately 10 colony-forming units per sample was achieved.
- In a survey of 100 commercial syrup preparations, the PCR assay detected B. cepacia in two samples that were negative by conventional culturing and biochemical methods.
Conclusions:
- The developed semi-nested PCR assay provides a sensitive, specific, and rapid method for detecting Burkholderia cepacia in pharmaceutical products and associated water supplies.
- This PCR method offers a significant advantage over traditional techniques, enabling detection without the need for culturing or pre-enrichment, thus avoiding potential false-negative results.
- The assay's ability to detect B. cepacia in samples missed by conventional methods underscores its utility for quality control in the pharmaceutical industry.
