Construction and characterization of bioluminescent Salmonella Reading outbreak and non-outbreak strains

Abstract

Salmonella enterica serotype Reading has recently been identified as a significant foodborne pathogen from contaminated poultry products. There is a critical need for close monitoring of this newly emerged pathogen. This study developed bioluminescent strains of S. Reading for real-time pathogen tracking using bioluminescence imaging. Two strains of S. Reading were used: an outbreak strain and a non-outbreak strain. The chloramphenicol acetyltransferase gene was cloned into the plasmid pBS-slpGFPluxABCDE, which carries luxABCDE operon and an ampicillin resistance gene. The newly constructed plasmid was then transformed into the outbreak and non-outbreak strains of S. Reading by electroporation. The resulting colonies were confirmed by visualizing bioluminescence using an in vivo imaging system and by testing their resistance to chloramphenicol. These strains demonstrated a high bioluminescence level (10⁸-10⁹ Photons/s/cm²/sr) and were tested for growth and plasmid stability by daily subculturing in Luria-Bertani medium with and without antibiotics. The plasmid remained stable for 8 days under non-selective conditions, and growth rates were comparable to non-bioluminescent parent strains in antibiotic-free conditions. However, growth was notably different in the presence of chloramphenicol, indicating successful plasmid retention and function. This study successfully created stable bioluminescent S. Reading strains, marking a significant step forward in monitoring and potentially reducing the spread of this emergent foodborne pathogen in the poultry industry.

Importance: Salmonella enterica serotype Reading has recently become a significant foodborne pathogen linked to poultry products. To enhance pathogen monitoring, this study developed bioluminescent strains of S. Reading by inserting the chloramphenicol acetyltransferase gene into a plasmid containing a bioluminescence gene cluster. These modified strains were transformed into outbreak and non-outbreak bacterial strains via electroporation. The bioluminescent strains demonstrated stable plasmid retention and high bioluminescence levels. They also showed growth comparable to their parent strains, even in the absence of antibiotics. These bioluminescent strains could potentially facilitate real-time monitoring and control of S. Reading in poultry industries.

Keywords: Salmonella Reading; bioluminescence imaging; foodborne pathogen; outbreaks; transformation.

Fig 1

Representation of plasmid manipulation for gene insertion: (A) donor plasmid pMJH46 carrying the chloramphenicol resistance gene, (B) plasmid pBS-slpGFPluxABCDE with bioluminescence and ampicillin resistance genes, and (C) resulting cloned plasmid pBS-slpGFPlux-cat incorporating the chloramphenicol resistance gene.

Fig 2

Visualization of bioluminescent Salmonella Reading strains: (A) outbreak strain and (B) non-outbreak strain grown on LB with chloramphenicol. Both strains expressing high levels of bioluminescence of 10^8–9 photons/s/cm²/steradian quantified by an in vivo imaging system.

Fig 3

Comparative growth curves of the parent and bioluminescent Salmonella Reading outbreak (SRO) strains in LB media with and without addition of chloramphenicol. Differences between parent and bioluminescent strains in both media types were analyzed using Student’s t-test (significance level: P = 0.05).

Fig 4

Growth curve comparison of the parent and bioluminescent Salmonella Reading non-outbreak (SRN) strains in LB media with and without chloramphenicol. Growth differences between parent and bioluminescent strains under both conditions were performed using Student’s t-test (significance level: P = 0.05).

Fig 5

In vitro plasmid stability of the constructed bioluminescent Salmonella Reading strains without chloramphenicol. Both strain cultures were sub-cultured daily in LB media alone. The figure shows the percent proportion of Salmonella Reading that retained the plasmid at each day of passage.

Fig 6

In vitro plasmid stability in constructed bioluminescent Salmonella Reading (SR) strain cultures daily sub-cultured in LB media supplemented with 10 µg/mL chloramphenicol. The figure shows the reduction in CFU/mL determined in day 1–10 cultures through plating.

Fig 7

In vivo plasmid stability of the constructed bioluminescent Salmonella Reading strains in chicks. The graph displays the percent of fecal positivity for bioluminescent Salmonella Reading from eight cages per strain with six chicks per cage.