Estilos de aprendizaje

Healthy cells, prepared as previously described, were used when assessing the performance of the LFIA Test Kit under unprocessed raw meat conditions. However, when evaluating processed raw meat conditions, it was necessary to mimic realistic situations, where bacterial replication may be impaired due to exposure of meat to physical or chemical treatments (170,225). Therefore, a “food stress” treatment, which consisted of incubating an E. coli O157 culture for 10 d at 4°C with conditions similar to real processed raw meat samples (TSB, 0.6% yeast extract, pH 4.9, and 130 g/L NaCl), was chosen for preparing stressed cells that could be used for artificial inoculation (225). After treatment, E. coli O157 cells were plated in parallel on non-selective (TSAYE) and selective (CR-SMAC) media and the difference in growth was further compared to obtain the % of sub-lethal injury. Therefore, to ensure the proper performance of both TSAYE and CR-SMAC media, they were initially evaluated with a healthy E. coli O157 control. Plate counts were further compared using a two-sided t-test (α=0.05). The results showed that cells plated on

selective media (CR-SMAC) produced significantly lower CFUs compared with non- selective media (TSAYE) (p<0.05, Figure 12A). Hence, it was necessary to establish a % of sub-lethal injury, or threshold value, attributed to the media performance in order to distinguish from the true effect of the “food stress” treatment. The threshold value was estimated as one standard deviation above the mean of % of sub-lethal injury calculated for the eight healthy control samples assessed (225), which resulted in 21.7%. This threshold value was further used to assess whether the % of sub-lethal injury was truly caused by the “food stress” treatment or was simply an effect of the media performance. Furthermore, five different E. coli O157 strains were treated following the “food stress” protocol and the % of sub-lethal injury was compared with the threshold value previously obtained using a one-sample t-test (α=0.05). Two of them, EC20001018 and EC19970515, did not differ from the threshold value estimated (p=0.1426 and p=0.1385, respectively). Therefore, the % of sub-lethal injury for both of them was assumed to be due to the media performance rather than the effect of the “food stress” treatment. On the other hand, three strains showed a difference in % of sub-lethal injury (p<0.05), which was attributed to the effect of the “food stress” treatment (Figure 12B). These data demonstrated that although all strains evaluated belong to the same species, E. coli O157, there was an inherent cell- to-cell variation that could be reflected in the response to stress. Finally, despite the fact that there was an intrinsic effect caused by the performance of the media, three strains showed an adequate % of sub-lethal injury, which was optimal for their further use in the artificial inoculation of meat samples.

Optimization of the LFIA Device Blocking Conditions

In order to improve the visualization of the test line in the positive control while eliminating background signals, the first phase of the LFIA Test Kit development process focused on reducing the non-specific binding. Although different combinations of proteins and polymers were initially assessed, BSA, which is frequently reported to block non-specific binding in nitrocellulose strips (237–239), was selected as the best alternative for further optimization, based on our evaluation. In addition, Tween 20 was preferred over Triton X- 100 as a nonionic detergent for improvements in sample flow through wettability. Using a blocking buffer either as a sample diluent or pre-treatment decreased the red smear along

Figure 12. Preparation and assessment of stressed E. coli O157 cells. A) Bar graph shows the difference in CFUs between the non-selective (TSAYE) and selective (CR- SMAC) media performance using an E. coli O157 control culture. CFUs were significantly lower on CR-SMAC compared with TSAYE (1.41×109 ± 1.57×108 and 1.57×109 ± 1.77×108, respectively). The p-value is from a two-sided paired t-test (p= 0.0254, n=8). Values represent mean ± SEM. B) Bar graph representing the % of sub- lethal injury of 5 different E. coli O157 strains subjected to a “food stress” treatment. Each bar represents the mean value of % of sub-lethal injury ± SEM (n=3-5). A one sample t- test, using the threshold value (21.7%), was performed to determine whether the % of sub- lethal injury was truly caused by the “food stress” treatment or was mainly due to the performance of the media (*p ≤0.05)

TSA

YE

CR-SMA

C

0.0

5.0×10

1.0×10

1.5×10

2.0×10

CFU/ml

0.0254

p=

EC20

0602

33

EC20

0101

8

EC19

9705

15

EC20

0403

39

DSM1

7076

0

20

40

60

80

100

stressed E. coli O157 strains

% sub-lethal injury

*

*

*

B

A

the membrane and improved the flow of the sample through the nitrocellulose membrane, as indicated by a well-defined control line when compared with the device without blocking buffer (Table 10). In addition, positive samples showed a clearly defined test line within the 15 min window established for reading, while negative samples remained clear (Table 10). Despite the improved performance, the two approaches required additional steps, increasing the complexity of the assay. Thus, it was necessary to assess additional blocking strategies.