Direct Detection of Burkholderia cepacia in Susceptible Pharmaceutical Products Using Semi-Nested PCR

2.1. PCR-Based Detection Methods

2.2. Test Samples

Five liters from an aqueous syrup preparation was prepared as a reference, sterilized using 0.22 μm membrane filters, and stored and refrigerated to simulate commercial aqueous syrup samples within the whole study. The reference preparation composed of the following: purified water, Quinoline Yellow (D&C Yellow NO10), ethyl alcohol, methyl paraben, Plasdone K25 (PVP25) (Povidone K25), propyl paraben base, sorbitol 70/70% solution, sucrose, and a flavor (Orange Flavour E9904557). Further, 100 randomly collected commercial aqueous preparations were included in order to be used during the application of the testing procedures. The commercial preparations were labeled from P1 to P100.

2.3. Conventional Detection Methods

Conventional method suitability testing was carried out according to the procedures of the recovery in liquid medium by the membrane filtration technique in the USP (16⇓–18). Two dilutions were prepared from the reference preparation and from each of the commercial products (1:10 and 1:100) using USP phosphate-buffered solution (PBS) + 4% Tween® 80 as a diluent.

The microorganisms used in conventional method suitability testing were as follows: Staphylococcus aureus ATCC® 6538, Pseudomonas aeruginosa ATCC® 9027, Bacillus subtilis ATCC® 6633, Candida albicans ATCC® 10231, Aspergillus brasiliensis ATCC® 16404, and B. cepacia ATCC® 25416 to ensure that the used methods are highly reliable in the recovery of various microorganisms.

Three groups each of 200 mL from the reference preparation and from each of the commercial products were pooled in sterilized bottles.

For each preparation, a 10 mL sample from each of the 1:10 and 1:100 dilutions was inoculated with 0.1 mL containing <100 cfu of each microorganism. Then samples were then filtered through membrane filters of 0.45 μm pore size, rinsed once with 100 mL PBS 4%. The membrane filters were aseptically transferred to tryptic soy agar (TSA) plates for recovery of bacteria and Saboraud dextrose agar (SDA) plates for recovery of Candida albicans and Aspergillus brasiliensis. A negative control was carried out for each dilution from each bottle used from the reference preparation or commercial products. The microbial recovery of each dilution for each microorganism used was compared to a positive control containing the microorganism and the media without the product. The lowest dilution factor showing a microbial count greater than or equal to 50% of the positive control of each microorganism was the accepted dilution. For recovery of B. cepacia, another membrane filter was aseptically transferred to 100 mL tryptic soy both supplemented with 4% Tween® 80 (TSB 4%), incubated at 30–35 °C for 18 h, and then 0.1 mL from the incubated TSB 4% bottles was streaked on TSA, cetrimide, and BBL™ oxidation/fermentation-polymyexin-bacitracin-lactose (OFPBL). After incubating the TSA and cetrimide plates at 30–35 °C for 18 h and OFPBL media at 30–35 °C for 4 days, growth was recorded and the lowest dilution showing growth and a microbial count greater than or equal to 50% of the positive control was chosen as the accepted dilution.

Each of the reference and the commercial samples was tested later using the accepted dilution.

2.4. Testing of Filterable Pharmaceutical Preparations via SN-PCR

One hundred milliliters of the reference syrup were spiked with B. cepacia ATCC 25416 (on triplicate basis) and with the environmental isolates that were identified as B. cepacia either biochemically or by SN-PCR at an inoculum size of 10⁷ cfu/100 mL. Bacterial DNA was extracted by filtering the whole spiked dilution through a membrane filter. The membrane filter was soaked in 5 mL saline for 30 min, and then bacterial genomic DNA was extracted from the saline solution using a QIAamp® DNA Mini kit, following the manufacturer's instructions, and was used as a template in the first round of the SN-PCR for identification of B. cepacia.

2.5. Sensitivity of SN-PCR versus Conventional Methods

B. cepacia ATCC 25416 was serially diluted to 1000 ± 200 cfu/0.1 mL, 100 ± 20 cfu/0.1 mL, and 50 ± 10 cfu/0.1 mL. An aliquot of 0.1 mL from each serial dilution was used to spike 10 of the accepted dilutions identified by the conventional method suitability testing of the prepared syrup. Each of the spiked dilutions was tested by membrane filtering of the whole volume, transferring the membrane filters on TSB 4%, incubated at 30 - 35 °C for 24 h, and then streaking 0.1 mL on TSA, cetrimide agar, and OFPBL agar.

Ten groups each of 100 mL of the prepared syrup (without being diluted) were spiked with 0.1 mL containing around 10 cfu (10–15 cfu) of B. cepacia ATCC 25416 as counted on OFPBL agar, then the bacterial DNA was extracted and purified directly from the product, as previously described, with the following modifications of using 5 mL Qiagen® tissue lysis (ATL) buffer for soaking the membrane filters, using Qiagen® proteinase K instead of Qiagen® protease, incubating the sample with ATL buffer and proteinase K at 56 °C for 3 h before adding Qiagen® lysis (AL) buffer, warming Qiagen® elution (AE) buffer to 60 °C, and following the steps of purification of DNA from whole blood using the QIAamp® Blood Midi kit (Spin Protocol). During the elution phase, 100 μL of the warmed AE buffer was added to the QIAamp® Midi column, left for 10 min before starting elution centrifugation, re-elution with the same eluate, and finally elution with another 100 μL of the AE buffer. The final eluate was evaporated to around 50 μL using a Thermoscientific® heat block at 60 °C with frequent vortexing to increase the DNA concentration in the eluate, and then SN-PCR method was applied as previously described.

2.6. Testing Commercial Products by SN-PCR and Conventional Methods

One hundred aqueous commercial products collected randomly from the market were tested by both conventional methods and SN-PCR for the detection of B. cepacia. One hundred milliliters of each product was used, half of the product's volume was diluted to the suitable dilution factor and was membrane-filtered, and the membrane was rinsed with 100 mL 4% PBS. The membrane filter was then aseptically transferred to TSB 4% and incubated at 30–35 °C for 24 h. One hundred microliters of the incubated TSB 4% was then streaked on TSA, cetrimide agar, and OFPBL agar. All recovered colonies were identified via biochemical reactions using a Vitek 2 compact system. The other half of each product was filtered undiluted, and the same steps of SN-PCR in sensitivity tests were followed.