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Ratio 1:100 Ratio 100:1 0 2 4 6 8 10 Log 10 CFU/mL Ratio 1:1 0 2 4 6 8 10 Log 10 CFU/mL 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Time (hours) OD 600nm Afia Boumail 1 , Anne Helmer 1 , Marie-Goreth Nicizanye 1 , Anna Yattara 1 , Michael Giuffre 2 and Sergiy Olishevskyy 1 1 FoodChek Laboratories Inc., Ste-Julie, Quebec, Canada 2 FoodChek Systems Inc., Calgary, Alberta, Canada One-Step Enrichment Broth for the Simultaneous Recovery of Salmonella enterica and Cronobacter sakazakii in Powdered Infant Formula Salmonella enterica and Cronobacter sakazakii are categorized as dangerous contaminants of powdered infant formula that represent a serious health risk for newborn infants. Conventional culture methods are time consuming and not user-friendly for detecting both pathogens. Therefore, the development and implantation of rapid and cost- effective methods allowing for the simultaneous detection of these pathogens remain important. The objective of this study was to develop a novel enrichment broth, Actero Simultaneous Recovery Broth (ASRB), that allows the one-step simultaneous recovery of S. enterica and C. sakazakii followed by both RT-PCR and culture detection. Introduction Conclusions In vitro Simultaneous Culture Studies Method Principle Infant Formula Samples Studies Fig. 1. Growth of Salmonella Agona and Cronobacter sakazakii in Actero Simultaneous Recovery Broth One Step Enrichment Sampling and Dilution DNA Isolation DNA Amplification Result Analysis 18H As short as Time-to-Result RT-PCR Detection MAX 40H Time-to-Result One Step Enrichment Sampling Plating Incubation Result Analysis Culture Detection Table 1. Growth Kinetics Data Strain Kinetics Parameter ASRB S. Agona Lag Phase Duration 5.912 ± 0.469 Growth Rate 0.195 ± 0.002 C. sakazakii Lag Phase Duration 4.985 ± 0.609 Growth Rate 0.298 ± 0.013 Notes : N – Number of experiments. Culture Conditions: • Initial Inoculum – 20 CFU/well • Medium Volume – 200 μL • Temperature – 35°C • Time – 16 hours A total of 170 artificially contaminated infant formula samples were examined to evaluate performance of the ASRB alternative method in comparison with the conventional method using culture detection. Actero Simultaneous Recovery Broth has a strong ability to provide an ideal growing environment for low numbers of sublethally injured Salmonella enterica and C. sakazakii in powdered infant formula. Single-step enrichment with Actero Simultaneous Recovery Broth reduces presumptive reporting time to as short as 18 hours without the loss of sensitivity and reliability of methods used for detection of Salmonella enterica and C. sakazakii in powdered infant formula. No significant differences were observed in the growth values of C. sakazakii and Salmonella in both monoculture and co-culture using different ratios of each bacterium. The simultaneous recovery of Cronobacter and Salmonella was comparable between the two methods. Among 170 tested samples, no false positives or false negatives were detected by direct plating using the alternative method. Detection by RT-PCR showed no false positives or false negatives using the alternative method. Fig. 2. Growth of Cronobacter sakazakii and Salmonella Agona Co-cultured in Actero Simultaneous Recovery Broth for 16 hours at 35°C Cronobacter Salmonella Monoculture Co-culture of Salmonella/Cronobacter Cronobacter Salmonella Table 2. Experimental Design Matrix Powdered Infant Formula (25 g) Strain S. Agona C. sakazakii Stress Drying Mortality 90% 85% Inoculum Level (CFU/sample) 5.5 2.7 Table 3. Recovery Rates of S. Agona and C. sakazakii in Powdered Infant Formula Detection Method N S. Agona C. sakazakii S. Agona C. sakazakii No Recovery ASRB 16 35/170 45/170 62/170 28/170 Conventional 16 34/170 34/170 63/170 39/170 The performance of RT-PCR detection method following the enrichment with ASRB was assessed using 30 artificially contaminated powdered infant formula samples. 1 Conventional Method 25g + 225 mL BPW 20-24h at 35°C Salmonella RV Broth 1 24h a t 42°C HE/XLD 24h a t 35°C Biochemical Tests C. sakazakii mLST+Vancomycin 2 24h a t 44°C ESIA 24h a t 44°C TSA 44-48h a t 25°C Biochemical Tests Concentration (centrifugation) 3 Biochemical Tests ASRB Method 25g + 225 mL ASRB 16h at 35°C Salmonella •RT-PCR •HE/XLD, 24h at 35°C C. sakazakii •RT-PCR •Rapid’Sakazakii, 24h a t 44°C Total Time = 90 - 140 hrs Total Time = 68 hrs Total Time = 19 - 40 hrs •PCR •DFI + R&PCRF Chromogenic agar Day 3 Day 2 Day 1 Day 4 Day 6 In vitro Culture Studies Matrix Study Design Both strains showed short lag-phases and comparable growth rates in ASRB. Notes: P – positive, N – negative, FP – false positive, FN – false negative. Table 4. Performance Parameters for Culture Detection in Powdered Infant Formula Using ASRB Strain Time (h) Total Tested P N FP FN Relative Sensitivity, % Relative Specificity, % FP Rate, % FN Rate, % Test Efficacy, % S. Agona 16 170 97 73 0 0 100 100 0.0 0.0 100 C. sakazakii 16 170 107 63 0 0 100 100 0.0 0.0 100 Notes: P – positive, N – negative, FP – false positive, FN – false negative. Table 5. Performance Parameters for RT-PCR Detection in Powdered Infant Formula Using ASRB Strain Time (h) Total Tested P N FP FN Relative Sensitivity, % Relative Specificity, % FP Rate, % FN Rate, % Test Efficacy, % S. Agona 16 30 25 5 0 0 100 100 0.0 0.0 100 C. sakazakii 16 30 21 9 0 0 100 100 0.0 0.0 100 1. ISO6579-1:2017 2. ISO/TS 22964 (2006) 3. BAM Ch. 29 BPW – Buffered Peptone Water Notes:
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