John Bodner, PhD
Demonstration of the Robustness of a Hermetically Sealed and Permanently Locked Detection Tube for Pathogen Assays in a Food Production Environment
CERTUS Food Safety, June 2018
The current study was undertaken to validate that biocontainment, a key feature of the CERTUS rapid environmental monitoring system delivers the benefit of safe use within a food production facility. The CERTUS BIO-LOCK™ Sampling Swab and detection tube allows for the simultaneous enrichment and detection of the Listeria bacterial pathogen in a closed system using the CERTUS EL Detection Kit. The system design eliminates accidental environmental or human exposure to bacterial pathogens while undergoing enrichment. A polypropylene detection tube was engineered to allow placement of a proprietary sampling swab with a BIO-LOCK™ Sampling Swab into a reagent containing enrichment media. Once inserted the cap design prevents removal of the swab from the detection tube. Positive internal pressure testing of the detection tube – cap device has shown it can withstand internal pressure build up that could incur through introduction of CO2 producing microorganisms that may be present in a food producer site sample. The current embodiment of the tube and cap assembly has been shown to be leak proof up to 60 psi. On board instrument leak testing of the detection tube – cap system during actual Listeria runs where the tube experiences horizontal agitation cycles did not show loss of media through gravimetric measurement. A more stringent test to directly look for release of enriched Listeria was done by swabbing of the exterior of the tube and streaking on non-selective and selective agar. Plate results were negative. The CERTUS BIO-LOCK™ Sampling Swab and detection tube when used with the CERTUS Detection Unit provides a bio-contained rapid pathogen detection system that allows in- plant pathogen testing that is simple and safe.
On-site testing for food & environmental pathogens enables early and focused remediation, reduces cost, minimizes risk, and shortens the time to product release. Handling environmental samples with potentially harmful bacterial pathogens in proximity to food production is a concern. If a completely bio-contained detection system (tube/cap/swab) can be developed where pathogen enrichment and detection is completed simultaneously and where once the cap/swab is engaged and locked prevents re-opening; then the goal of safe on-site testing is reached. The current study objectives were: 1) To empirically determine what level of pressure could be expected if microorganisms were brought into the detection tube during environmental swab sampling. This was achieved using known CO2 producing microorganisms. 2) To show that it is achievable to engineer a permanently lockable detection tube-cap system, that is capable of withstanding potential internal pressure build up during pathogen enrichment and would not allow leaking of the enrichment media. The innovative hermetically sealed and locked CERTUS BIO-LOCK™ Sampling Swab and detection tube, provides this solution, for in-house environmental sampling. The cap and tube design protect the environment and operator from accidental contamination. Once the sample swab is placed in the detection tube and the cap is locked, the tube is placed in the CERTUS Detection Unit where enrichment and detection proceed simultaneously without the need for further sample manipulation.
Materials and Methods
A. Pressure measurement of CO2 microbial producers
An E. coli cocktail (ATCC 25922, 35218, 11229 and 8739) was inoculated at a concentration of 103 to 104 CFU/mL into Escherichia coli broth with 4-methylumbelliferyl-ß-D-glucuronide (EC-MUG). The yeast cocktail, consisting of Saccharomyces cerevisiae, Candida albicans and Zygosaccharomyces bailli was also inoculated at a final concentration of 103 to 104 CFU/mL into Yeast Mold (YM) broth. Lactic acid bacteria were inoculated at a concentration of 103 to 104 CFU/mL into Lactobacilli MRS Broth.
ii. Pressure measurement
Pressure vessels used were the PRV Footed Starter Set, 3 oz. from Andrews Glass Co. Vineland, NJ. Pressure gauges used were the MediaGaugeTM Model MGA-9V from McMaster-Carr which had a pressure range of 0-30 psi and a resolution of 0.01 PSI. The volume of broth added to the pressure vessels was adjusted to approximate the headspace of the CERTUS BIO-LOCK™ system after media broth is added to the detection tube and the BIO-LOCK™ Sampling Swab is engaged.
The pressure vessels were placed in an incubator at the optimal temperature for the respective group of organisms and incubated for 30 minutes to allow the temperature and pressure to stabilize. The gauges were then reset to begin the study. At each time point, the static pressures were read and recorded first. The vessels were then gently shaken by hand for one minute to swirl the contents (75-100 RPM). Following shaking, the vessels were allowed to stabilize before reading the pressure.
Bacterial growth was also monitored independently by turbidity using McFarland standards. Once the McFarland score was recorded the vessels were returned to the appropriate incubator until the next time point.
B. Internal Pressure Tolerance Studies of the CERTUS BIO-LOCK™ Sampling Swab and detection tube
i. The CERTUS BIO-LOCK™ Sampling Swab and detection tubeThe assembly is comprised of a polypropylene tube (detection tube) that has locking tabs designed into the sides and a BIO-LOCK™ Sampling Swab comprised of a polypropylene material has integrated receiving ports. Once the cap is engaged with the locking tabs a leak proof and permanently locked detection tube results in which the CERTUS food pathogen assays can be run. See Figure 1 showing the tube / cap assembly interface.
Figure 1: BIO-LOCK™ Sampling Swab and detection tube with cap attached to the top of the detection tube
ii. Internal Pressure Measurement Tool
The CERTUS BIO-LOCK™ Sampling Swab and detection tube are manufactured by injection molding. The manufactured parts underwent dimensional measurements and were subjected to a pressure test that pumped air into the assembled detection tube and BIO-LOCK™ Sampling Swab cap containing a fixed volume of water as a substitute for media broth that would nominally be present in the actual assay run. See Figure 2.
Figure 2: In-house pressure testing apparatus. The tubing is connected to the top of the cap through a machined port and then the cap is locked onto the tube
Each design revision was tested on this system. Pressure was slowly increased and held for a set period while leakage of water around the detection tube and BIO-LOCK™ Sampling Swab cap was observed. A failure was recorded when water was observed to be escaping at a given pressure.
A. On-Board Instrument Studies
i. Mechanical agitation Profile
To determine if the optimal detection tube and BIO-LOCK™ Sampling Swab design would prevent leakage of fluid out of the system during an actual assay run, a series of experiments were designed. The instrument used was a breadboard instrument that has the same mixing profile and cycle times, that are present in the on market CERTUS Detection Unit. See Table 1 below for the respective motion profile.
ii. Gravimetric Determination of Fluid Loss
One approach to examine the robustness of the CERTUS BIO-LOCK™ Sampling Swab and detection tube cap was through gravimetric measurement of the filled tube pre- and post-mechanical agitation.
Ten tubes were filled with 15 mL of colored water, the BIO-LOCK™ Sampling Swab was placed on top of the tube and pressed down to fully engage the cap with the tube. The prepared tubes were measured gravimetrically. The tubes were then inserted into the Breadboard unit within a 30°C incubator, where the programed mixing profile listed in Table 1 was initiated and the run was set for 72 h. After the 72-h run the tubes were re-measured gravimetrically.
iii. Gravimetric and culture recovery of Listeria spiked tubes
Ten tubes were filled with 15 mL of CERTUS proprietary media broth to which 111 CFU of Listeria monocytogenes 4b LMG 21264 in 100µl of Maximum Recovery Diluent was added. The BIO-LOCK™ Sampling cap was placed on top of the detection tube and pressed down to fully engage the cap with the tube. The prepared tubes were measured gravimetrically. The tubes were then inserted horizontally into individual holders attached to the mixing plates onboard the R&D instrument that is housed within a 30°C incubator. The programed mixing profile listed in Table 1 was initiated and the run was set for 116 h. After the 116-h run the tubes were re-measured gravimetrically.
A second approach to complement the gravimetric measurement studies was to test for leakage of viable Listeria on the outside of tube. In this experiment a sterile cotton tipped wood sampling swab was pre- wet with Maximum Recovery Diluent and used to swab and collect any potential expelled Listeria from around the joint of the cap and tube.
The swab head was snapped off into 10 mL non- selective Brain Heart Infusion Broth (BHI) and grown at 30°C for 24 h. Post 24 h BHI enrichment, 0.1 mL from each BHI broth was transferred to 10 mL selective Fraser Broth and incubated at 37°C for 24 h.
In parallel, a 10µl loop from both the BHI primary and secondary selective Fraser broths were transferred to non-selective Tryptone Soya Agar and incubated at 37°C for 24 h. Also, 10µl were respectively transferred to Listeria selective chromogenic agars, OA and Palcam and incubated at 37°C for 24-48 h
A. Pressure measurement of CO2 microbial producers
Table 2 provides both turbidity (McFarland) and observed pressure (psi) readings for E. coli, yeast, and the lactic acid bacteria.
Table 2. Pressure and turbidity measurements over time
The increase in measured pressure followed other growth indicators, including turbidity and gas production (E. Coli Durham Tube). Each method exhibited a lag phase of around 8 h, followed by increased growth between 8 to 24 h. This growth was indicated by the rise in pressure, turbidity, and gas production with the increase in growth continuing through 36 h. In all cases shaking of the culture resulted in a higher pressure reading. This may be the result of trapped CO2 in the broth which becomes released by simple agitation of the vessel. Previous studies have demonstrated the same increasing pressure with bacterial growth over time. (Wilkins, Applied Microbiology, January 1974).
Theoretical calculation for the expected rise in pressure under the growth conditions above showed the maximum CO2 production expected from each media as follows: TSB + EC-MUG – 37.4 psi, MRS – 40.9 psi, and YM broth – 5.1 psi (calculations not shown). This assumed 100% fermentation of the sugar in the media.
It should be noted the CERTUS Listeria media has selective reagents in it that during enrichment should preferentially allow the growth of Listeria over other background microflora. If background microflora does grow it will be at significantly slower rates than any Listeria present. Subsequent inoculation of the yeast cocktail, E. coli cocktail and lactic acid bacteria at a final concentration of 104 CFU/mL into CERTUS proprietary media failed to result in growth and media turbidity after a 24-enrichment period. This also correlated with a lack of positive detection on the R&D instrument run using the programmed mixing profile. This adds additional assurance that minimal gas builds up will occur during the Listeria assay. If pressure was to build through CO2 generation the CERTUS BIO-LOCK™ Sampling swab and detection tube is capable of handling high internal pressures without leaking. (see section B, below)
B. Internal Pressure Tolerance Studies of the CERTUS BIO-LOCK™ System
During development of the tube and cap multiple attributes were evaluated. This included dimensional parameters, locking mechanism, ease of use, material resin, and maximum pressure achievable before failure by leakage out of the tube from visual observations. The base resin for the tube and cap was polypropylene (PP) ± additives as noted. In one case Acrylonitrile Butadiene Styrene ABS was used for the cap but was found not to be acceptable for ease of closing. Table 3 shows the early characterization work on tube and cap materials.
Table 3: Summary Status of Material for CERTUS Bio Lock™ Sampling Swab Cap and Detection Tube
Depending on the combination of tube and cap material tested various attributes were scored differently. For example, POM Cap and PP + 10% Vistamax additive or PP alone tubes achieved good pressure holding, however, the POM cap was subject to fracturing. ABS cap and PP + 10% Vistamax additive showed good pressure holding, however, closing strength and locking feel were not acceptable plus cost was high and lead time for resin long. Based on optimizing for the various tube cap attributes, a decision was made to move forward with PP tube and PP cap with no additives. Table 4 shows pressure testing work on the final optimized tube and cap design.
Table 4: Pressure testing of final optimal tube and cap design
On-Board Instrument Studies
i. Gravimetric Determination of Fluid Loss
There were two gravimetric studies to look at fluid lost from the optimal designed BIO-LOCK™ Sampling Swab and detection tube when run in the standard mechanical profile at 30°C. A preliminary test was done using colored water before running live Listeria cultures. Table 5 shows the colored water results.
Table 5: Gravimetric record of colored water volume retained in the tube after 72 h
Within the sensitivity limits of the analytical balance used in this study, there was no observed change in the gravimetric measurement recorded in the pre- and post-instrument run.
The second gravimetric experiment used CERTUS proprietary single enrichment selective media broth that was designed for maximum recovery of Listeria while suppressing the growth of cross-reactors. The media broth was spiked with 111 cfu Listeria monocytogenes 4b LMG 21264 prior to the instrument being run in the standard programed mixing profile for 116 h at 30°C. Table 6 shows the live Listeria culture results.
Table 6: Gravimetric record of CERTUS proprietary broth volume retained in the tube after 116 h
As what was observed with the colored water, within analytical balance sensitivity limits there was no change in the gravimetric measurement recorded in the pre- and post-instrument run. Visual inspection of the tubes after 116 h of agitation did not indicate loss of broth as shown in Figure 3 below.
Figure 3. Visual Comparison Media Level of the 10 Tubes Post 116 h of Mixing on board the R&D Instrument
Though gravimetric measurement and visual inspection for fluid loss are good indicators for evaluating whether leakage has occurred during the instrument run we wanted to determine through microbiology testing if a biofilm was building up on the outside of the tube which would be most prevalent at the cap and tube interface. So, culture recovery was carried out as described under the material and methods sections.
The results of adding the swab head into 10 mL non- selective Brain Heart Infusion Broth (BHI) and grown at 30°C for 24 h are shown in Figure 4 below.
Figure 4: 10 mL BHI cultures containing swab, taken after 24 h at 30°C incubation
No turbidity was recorded from any of the ten BHI broths, indicating that no bacteria has leaked from the BIO-LOCK™ Sampling Swab cap and detection tube.
Furthermore, 0.1 mL from each BHI broth was transferred to 10 mL selective Fraser Broth and incubated at 37°C for 24 h. Results are shown in Figure 5 below.
Figure 5: 10 mL Fraser Broth, taken after 24 h at 37°C incubation
Note: Blackening of the media is presumptive evidence of Listeria presence. The picture shows only the positive control has presumptive Listeria present. All the others resemble the aseptic control vial (far right), further confirming that no Listeria has leaked from the tube.
Despite the observation of no turbidity, 10µl loopful was transferred to non-selective Tryptone Soya Agar to check for any sign of growth, incubated at 37°C for 24 h. 10µl was also transferred to Listeria chromogenic agars, OA and Palcam and incubated at 37°C for 24-48 h.
No growth was recorded from the non-selective and selective agar plates indicating no bacteria had leaked from the tube-cap system and thus no growth in the BHI cultures. This was further confirmed by no sign of growth in the Fraser Broth and on any of the Listeria selective chromogenic agars.
These observations together with the negligible loss recorded gravimetrically, conclude that the BIO-LOCK™ Sampling Swab and detection tube as a sealed unit are fit for purpose.
Development of the CERTUS BIO-LOCK™ Sampling Swab and detection tube was undertaken to provide customers with a robust, simple, and safe detection tube that accommodates the CERTUS BIO-LOCK™ Sampling Swab for environmental sampling which remains in the tube during the assay run. In addition, the materials used to manufacture the detection tube were required to be compatible with signal acquisition using Raman resonance. The materials tested are listed in Table 3 as well as the attributes evaluated. Hardness, closing strength, locking feedback feel, tendency to crack, cost, and pressure. These attributes were balanced against the critical attribute pressure, i.e., the capability to withstand a rise in the internal pressure when the cap and tube is engaged. The internal pressure increase could be a mechanism by which media can leak out of the tube and therefore pathogen release could occur. An additional design requirement is that once the cap and tube are seated the cap is locked to the tube preventing removal of the BIO-LOCK™ Sampling Swab making it tamper resistant. Table 4 shows the test results of the critical attribute, pressure, for the optimal tube and cap design. Across five samples the internal pressure was continuously increased from 28 psi to 60 psi. At predetermined pressure points the pressure was held for increments of 4 minutes. The cumulative time for each tube and cap assembly held under continuous pressure was 22 minutes and the maximum pressure recorded for each tube and cap was 60 psi.
As part of the evaluation to demonstrate the tube and cap design does not allow leakage of the contents an independent study was done to look at known microbial CO2 producers found in food plants to estimate a range of pressure rise that could occur during fermentation.
Listeria fermentative growth does produce acid as a by-product it does not generate CO2. (Bergey’s Manual® of Systematic Bacteriology Second Edition Volume Three). However, in food facilities there are many microorganisms that both produce acid and CO2 as by-products of fermentation of sugar. This raises a concern that if these organisms can come along on the swab used to collect Listeria samples used for collecting environmental samples. If this occurs and they can grow in the CERTUS proprietary media there could be a build of pressure in the tube that could force the seal between the CERTUS detection tube and BIO-LOCK™ Sampling Swab to be compromised allowing leakage of food pathogens from the detection tube.
An experiment was designed to estimate the type of pressures that could result from the growth of known CO2 producers that could be encountered in a food production facility. Table 2 shows the observed results in pressure rise across several CO2 microbial producers grown under their respective optimal growth conditions. The pressure rise observed was from 2 to 5 psi (shaking pressure) in 36 hours which is 12 hours more than what is required to run the CERTUS EL assay. To approximate a worst-case scenario a theoretical calculation was done to estimate the pressure rise attributed to CO2 production during fermentation. The calculation assumed 100% conversion of the sugar present in the media used to grow the microorganism in Table 2. The highest pressure estimated was for lactic acid bacteria grown in MRS broth which was calculated to be 40.9 psi.
As shown on Table 4, the design of the tube and cap allowed pressure to build to 60 psi without leakage failure. This is 12 times the maximum pressure recorded in Table 2 and approximately 20 psi above the highest theoretically calculated pressure assuming 100% conversion of the sugar present in the media.
To directly test if the optimal tube and cap design is fit for purpose a series of experiments were performed using an R&D instrument that approximates the commercial CERTUS Detection Unit. The R&D instrument allows for horizontal placement of the tube as does the commercial instrument. In addition, the programed mixing profile shown in Table 1 and maintenance of a 30°C temperature during the assay run is the same in both the R&D instrument and the commercial unit.
Two gravimetric experiments were performed. The first used color water to look for major failures. There was no difference in the gravimetric weight recorded pre- and post-running the tubes for 72 h on the instrument, Table 5. The next experiment was to grow Listeria on the R&D instrument and measure the gravimetric weight pre- and post-running the tubes for 116 h. Again, no difference in the gravimetric weight was recorded pre- and post-running of the tubes on the instrument, Table 6 and Figure 3.
A more stringent test to directly look for release of enriched Listeria was done by swabbing of the exterior of ten tubes and incubating the individual swab heads in BHI broth for 24 h, Figure 4. No turbidity was observed. Then a sample from each of the 10 BHI broths were transferred to selective Fraser broth and incubated for 24 h. No turbidity was observed, Figure 5. Finally, from the Fraser broth samples were streaked onto Listeria selective agar plates and non-selective agar plates. No growth was recorded on either the selective or the non-selective agar plates.
Handling environmental samples for testing of potentially harmful bacterial pathogens in proximity to food production is a concern. This has curtailed the adoption of bringing food pathogen testing on site at food production facilities, particularly in small-medium sized production facilities lacking microbiological laboratory.
The development of the innovative hermetically sealed and locked detection tube, the CERTUS BIO-LOCK™ Sampling Swab and detection tube, opens the possibility to bring on site testing to realization. The cap and tube design protect the environment and operator from accidental contamination. The CERTUS BIO-LOCK™ Sampling Swab and detection tube when used with the CERTUS Detection Unit provides a bio-contained rapid pathogen detection system that allows for in-plant pathogen testing that is simple and safe.
The technical contributions on the content of this paper by Nevin Perera, PhD and Holly Urquhart, of Solus Scientific Solutions, Ltd. East Kilbride Scotland and Heidi Wright, PhD and Ninalynn Daquigan of Aemtek, Inc. Fremont California, and Takuya Kurimoto and Yoshi Yamaki Toho Technology Inazawa-City, Japan are greatly appreciated.