Evaluation of Simultaneous Growth of Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes in Ground Beef Samples in Different Growth Media

Abstract

Several multiplex approaches for the simultaneous detection of pathogens in food have been developed in recent years, but the use of a single enrichment medium remains a problem. In this study, six enrichment broths (five non-selective media, tryptic soy broth (TSB), brain heart infusion broth (BHI), buffered peptone water (BPW), universal pre-enrichment broth (UPB), no. 17 broth, and a selective, Salmonella Escherichia Listeria broth (SEL)), were studied for the simultaneous detection of E. coli O157:H7, Salmonella spp., and L. monocytogenes, to validate the suitable enrichment broth to be used for the detection methods. Different ratios of E. coli O157:H7, Salmonella spp., and L. monocytogenes were used. Almost all non-selective broths evaluated in this study showed similar growth parameters and profiles among each other. The only selective enrichment broth under analysis (SEL) showed distinct growth features compared to the non-selective media, allowing for a slower but balanced growth of the three pathogens, which could be beneficial in preventing the overgrowth of fast-growing bacteria. In addition, when tested in ground beef samples, SEL broth seems to be the most distinctive medium with a balanced growth pattern observed for the three pathogens. Overall, this study is intended to provide the basis for the selection of suitable enrichment broths according to the technology detection to be used, the desired time of enrichment, and the expected balanced concentration of pathogens.

Keywords: culture media comparison; foodborne pathogens; growth media; pathogen detection.

Figure 1

Growth kinetics in single cultures. (A) Values of µ—Exponential Growth Rate (unit: h⁻¹). (B) DT—Doubling Time (unit: h) on single culture assays for the six enrichment media under analysis. ^a Indicates that there is a statistical difference in values of µ (p < 0.05) between TSB and BPW; TSB and SEL. ^b Indicates that there is a statistical difference in values of µ (p < 0.05) between BHI and BPW; BHI and SEL. ^c Indicates that there is a statistical difference in values of µ (p < 0.05) between UPB and BPW; UPB and SEL. ^d Indicates that there is a statistical difference in values of µ (p < 0.05) between BPW and no. 17. ^e Indicates that there is a statistical difference in values of µ (p < 0.05) between no. 17 and SEL. ^f Indicates that there is a statistical difference in values of DT (p < 0.05) between BPW and TSB; BPW and BHI; and BPW and UPB. Error bars represent standard deviation. Abbreviations: tryptic soy broth (TSB); brain heart infusion broth (BHI); buffered peptone water (BPW); universal pre-enrichment broth (UPB); no. 17 broth; and Salmonella Escherichia Listeria broth (SEL).

Figure 2

Growth curves for E. coli O157:H7, Salmonella spp., and L. monocytogenes co-cultured in SEL, at different pathogen ratios. E. coli O157:H7/Salmonella spp./L. monocytogenes. (A) Ratio of 1:1:1. (B) Ratio of 1:1000:10. (C) Ratio of 10:1:1000. (D) Ratio of 1000:10:1. ^a Indicates that there is a statistical difference in values of µ (p < 0.05) between E. coli O157:H7 and L. monocytogenes.

Figure 3

Growth kinetics in co-cultures. (A) Values of µ—Exponential Growth Rate (unit: log₁₀ CFUs mL⁻¹h⁻¹). (B) DT—Doubling Time (unit: h) (B) for E. coli O157:H7, Salmonella spp., and L. monocytogenes co-cultured in SEL, at different pathogen ratios. E. coli O157:H7/Salmonella spp./L. monocytogenes ratio of 1:1:1, 1:1000:10, 10:1:1000, and 1000:10:1. ^a Indicates that there is a statistical difference in values of µ (p < 0.05) between E. coli O157:H7 and L. monocytogenes. ^b Indicates that there is a statistical difference in values of µ (p < 0.05) between E. coli O157:H7 and Salmonella spp.

Figure 4

Morphology of some E. coli O157:H7 and Salmonella spp. colonies in McConkey agar plates at the early hours of SEL assays. (A) Regular morphology of E. coli O157:H7 and Salmonella spp. (B) Regular and irregular morphology of Salmonella spp. (C) Irregular morphology of E. coli O157:H7. The images were acquired by using the Stereo-Microscope SZ61TR.

Figure 5

Growth curves for E. coli O157:H7 CECT 4267, Salmonella serovar Typhimurium NCTC 12416, and L. monocytogenes 747 in an artificially contaminated ground beef sample. (A) Enrichment on TSB. (B) Enrichment on UPB. (C) Enrichment on no. 17 broth. (D) Enrichment on SEL. ^a Indicates that there is a statistical difference in values of µ (p < 0.05) between L. monocytogenes 747 and microbiota. ^b Indicates that there is a statistical difference in values of µ (p < 0.05) between Salmonella spp. and microbiota. ^c Indicates that there is a statistical difference in values of µ (p < 0.05) between E. coli O157:H7 and microbiota.