Inclusivity and Exclusivity Strains
The objective of this study was to ensure that the LFIA test kit had the ability to selectively detect a wide range of E. coli O157 strains (including H7 and non-motile), while excluding non-target organisms closely related to E. coli O157 and/or commonly found in raw meat products. The inclusivity panel was assembled considering the variability within E. coli
O157 strains and the relevance of other O157:non-H7 E. coli as causes of human illness (27,154,223). A total of 50 E. coli O157 strains (including H7 and non-motile) was selected from different sources. Only 39 were O157:H7 serotype, 5 were O157:NM, and 6 of them were O157:non-H7 serotypes (H45, H42, H29, H25, H19, H12). In addition, 27 strains were from bovine origin isolated from different sources such as feces and meat (ground and salami). Around 18 strains were from human origin; 16 mainly from clinical isolation and 2 from feces. However, four strains did not have an origin specified, but their original depositor was stated by the contributor. Only one strain did not have any information regarding its history. All strains were biochemically characterized and confirmed as E. coli
O157 prior to testing (Appendix A).
For exclusivity, 37 non-E. coli O157 strains were selected from different sources that reflected the variability of organisms that can be present in the food matrices chosen for this study. Most of these (33) were Gram negative and belonged to the Enterobacteriaceae
family, with the exception of Aeromonas hydrophila. From those Enterobacteria, 18 were
E. coli non-O157, while the rest were bacteria frequently found in meat and closely related to E. coli O157. In addition, four Gram positive strains that can be found in meat were included in the panel (Bacillus subtilis, Enterococcus faecalis, and Listeria monocytogenes
4a and 1/2c). Strains obtained from the Public Health Agency of Canada (PHAC) in Guelph, ON, Canada, were verified using the Vitek® microbial identification system before they were provided (Appendix A).
Upon receipt, all 87 strains were initially grown in 5 ml of BBLTM TrypticaseTM Soy Broth [TSB; Becton, Dickinson and Company (BD), Sparks, MD, USA] or BactoTM Brain Heart Infusion (BHI) medium (BD, USA) incubated overnight at 37°C to reach late-exponential
phase (108–109 CFU/ml). A sub-culture was prepared by transferring 50 µl of the initial culture into 5 ml of TSB or BHI and incubated as previously stated. Cells were harvested by centrifugation (Eppendorf Centrifuge 5430, Rotor FA-45-30-11, Eppendorf AG, Hamburg, Germany) at 5000 ×g and re-suspended using TSB or BHI with 20% (v/v) glycerol as cryoprotectant and stored at -80°C for long-term storage. Furthermore, working cultures were prepared from frozen stocks by loop inoculation of 5 ml of TSB incubated overnight at 37°C to reach late-exponential phase (108–109 CFU/ml).
Bacterial Culture Enumeration
Bacterial enumerations or plate counts were performed by plating 50 µl on TrypticaseTM Soy Agar (TSA; BD, USA) and/or the E. coli O157:H7 selective agar, cefixime rhamnose sorbitol MacConkey (CR-SMAC; Oxoid Limited, Basingstoke, Hampshire, UK) using the Spiral Plate Method (224) (Eddy Jet Spiral Plater, E Mode; Neu-tec Group Inc., Farmingdale, NY, USA) then incubating the plates at 37°C for 18-24 h. Two serial dilutions were normally plated per sample. Colonies on the agar plates were counted using the spiral plate counting grid and following the “rule of 20 colonies”. In brief, a wedge from the grid was selected and colonies were counted from the outer edge towards the center until more than 20 colonies had been reached. A similar area in the opposite side to the wedge selected was also counted, added up and divided by the sample volume deposited in those two areas as shown in the formula below:
!"#$% 1 + !"#$% 2
*"+#,- .-/"01%-. =
!34 ,+
Results were reported as CFU/ml of sample plated and recorded for further use in dilution calculations or estimation of cell concentration in samples (CFU/g or ml).
Preparation of Stressed E. coli O157 Cells.
The protocol selected was adopted from a previous study done by Jasson et al. (225), which consisted of mimicking inherent factors normally present in food. Working cultures of E. coli O157 DSM17076, EC20060233, EC20001018, EC19970515, and EC20040339 were prepared as described in Section 3.1.1. In addition, a “food” broth was formulated using
TSB supplemented with 0.6% yeast extract (Sigma-Aldrich, St. Louis, MO, USA) (TSBYE) and acidified with HCl until a pH of 4.9 was reached. In addition, 130 g/L NaCl was added to simulate the salt concentration of a fermented sausage. The broth was filter sterilized and kept at 4°C. To determine the initial cell concentration, cultures were plated on TSA supplemented with 0.6% yeast extract (TSAYE) and CR-SMAC agar plates, which were considered non-selective and selective respectively. Serial dilutions were performed using the “food” broth up to a level of 106 CFU/ml. The inoculated broths were kept at 4°C for 10 d after which aliquots were plated on TSAYE and CR-SMAC plates for enumeration. The percentage of sub-lethal injury was determined by comparing the number of colonies in non-selective and selective agars using the formula below:
% 0#6 − +-%ℎ9+ 1$:#;< = $"$0-+-=%1>- − 0-+-=%1>-
$"$0-+-=%1>- ? 100
For each strain, at least three different experiments were undertaken and the percentage of sub-lethal injury was expressed as the average ± standard error of the mean. The % of sub- lethal injury caused solely by the stress treatment was confirmed by determining if significant difference between selective and non-selective media performance existed using a two-sided t-test withα= 0.05.
Artificial Inoculation of Food Samples
Fresh retail lean ground beef or trimmed beef was obtained from local supermarkets one day before starting the experiments (Loblaws, London, ON, Canada) and aseptically divided into 25 g samples using Stomacher® bags. In order to mimic real scenarios, samples were inoculated either with healthy or stressed E. coli O157 cells prepared as described in Section 3.1.3. Both cultures were serially 10-fold diluted using DifcoTM Buffered Peptone Water (BPW; BD, USA) in order to achieve three inoculation levels: low (1-10 CFU/25 g), medium (102 CFU/25 g) and/or high (103 CFU/25 g), as per the objective of each experiment. Cell concentrations of inocula were confirmed by plate counts on TSA and CR-SMAC. In addition, an uninoculated 25 g sample was included as a negative control. After manual homogenization, samples were stored at 4°C for 48 h for equilibration before further use.
Determination of Aerobic Plate Count (APC) of Food Samples
APC, also known as total viable count (TVC), was also determined to establish the initial level of contamination in the food samples. A 25 g sample, from the same batch used in each experiment, was weighed into a Stomacher® bag and mixed with 225 ml (1:10) of BPW. The bag was loosely closed and stomached using a lab blender (BagMixer® 400P, Interscience Laboratories Inc., Woburn, MA, USA) for 1 min. A 10-fold dilution was prepared using BPW and an aliquot of both was plated on TSA. Plates were incubated at 37°C for 48 h for enumeration. Cell concentrations were expressed as CFU/g of food sample.
Evaluation of Enrichment Conditions
Table 4 summarizes the commercially available enrichment broths used throughout the
development process. The first screening involved using pure cultures. Briefly, a working culture of E. coli O157 DSM 17076 was prepared as previously described and serially diluted in TSB to a final concentration of 102 CFU/ml. Initial inoculation levels were confirmed by plate counts on TSA and CR-SMAC. Cultures were incubated at 42°C for a maximum of 24 h. When necessary, bacterial growth was monitored by plating 50 µl every 2 h on TSA and CR-SMAC.
The final selection of the enrichment broth was based on the following: 1) the ability to recover E. coli O157 from artificially inoculated food samples prepared as per Section
3.1.4; and 2) its interaction with the LFIA Test Kit. Therefore, after equilibration,
Stomacher® bags containing the inoculated samples were filled with 225 ml of the appropriate enrichment broth, stomached for 1 min and regenerated 40 min at RT (room temperature). Food samples were incubated at 42°C for a maximum of 24 h. When necessary, a time-course study was performed with sampling every 2 h starting at 16 h. For enumeration, samples were plated on TSA and CR-SMAC and, if needed, they were previously serially diluted using BPW.
Cloning and Expression Bacterial Strains
E. coli XL1-Blue competent cells (Stratagene) were used for plasmid expression and E. coli BL21 (DE3) competent cells (Novagen) were used to express the recombinant protein.
Table 4. Enrichment broths and their selective agents. Broth Selectivity Selective
Agents
Supplier
Modified Tryptone Soya Broth (mTSB)
Selective Bile salts No. 3 Oxoid Limited (Basingstoke, Hampshire, UK)
RapidCultTME. coli Selective Sodium thioglycolate
EMD Chemicals (Darmstadt, Germany) BBLTM TripticaseTM Soy
Broth+ Novobiocin (TSBN)
Selective Novobiocin Becton, Dickinson and Company (Sparks, MD, USA)/ Sigma-Aldrich Co. LLC (St. Louis, MO, USA)
BBLTM TripticaseTM Soy Broth (TSB)
Non- selective
N/A Becton, Dickinson and Company (Sparks, MD, USA)
Both strains were grown aerobically at either 37°C or RT in Luria Broth (LB) (BD, USA) or on LB with 1.5% agar (BioShop, Burlington, ON, Canada). To maintain plasmid selectivity, the LB broth or agar was supplemented with ampicillin (20 µg/ml) and/or chloramphenicol (10 µg/ml), both from Sigma-Aldrich, USA. For long-term storage, bacterial strains were frozen in LB broth with 20% glycerol and kept at -80°C. Table 5
summarizes the main characteristics of the two strains used within this part of the study.