Conclusions Our work demonstrates a novel, real-time monitoring system for Salmonella enterica serotypes that is stable and has potential use for in in vivo and in vitro trials.
Our results show the efficiency of plasmid pBEN276 to confer bioluminescence to eleven wild-type Salmonella enterica isolates by inserting the luxCDABE operon into the attTn7 site on the chromosome. Chromosomal insertion of the gene is significant SN-38 in that external antibiotic pressure is not required for perpetuation of the luxCDABE cassette. This system has the potential to eventually be utilized for the evaluation of potential pathogen mitigation strategies upon Salmonella under different environmental conditions over extended time courses, which was not previously www.selleckchem.com/products/AZD8931.html possible due to limitations of plasmid-based reporter systems. Detection was successful following metabolic inactivity due to refrigeration temperatures and results provide support for application of our model in trials simulating
processing plant environmental conditions. Future experiments are planned using this system to evaluate the efficacy of various AMCs. We expect this research may provide a foundation for future work to understand the mechanism of attachment of Salmonella to chicken skin and its ability to persist during the poultry processing continuum. Methods Bacterial serotypes and growth media As part of a previous study, Salmonella enterica isolates from five different sites along the broiler production continuum (day one placement, end of growout, arrival at the plant, pre-chill tank, and post-chill tank) were cataloged [25]. In the current study, 11 Salmonella enterica serotypes (S. Alachua, S. Braenderup, S. Enteritidis, S. Heidelberg, S. Kentucky, S. Mbandaka,
S. Montevideo, S. Newport, S. Schwarzengrund, S. Seftenberg, S. Typhimurium) were selected. Salmonella enterica serotypes were cultured using Luria-Bertani broth and agar plates at 37°C. Ampicillin (100 μg mL-1) and was used for selection Cepharanthine and 0.1% arabinose was used for transposition induction. Construction of plasmid pBEN276 The luxCDABE operon was amplified from the genome of Photorhabdus luminescens using primers PG131 (GATGCTACCTCGAGGTACAACCAGTTTGCAAGATG) and PG132 (TACGCTCAGGATCCGAATTCACTCCCTTGCCATC) and cloned in pCR2.1 (Invitrogen) to yield plasmid pBEN139. Primers PG131 and PG132 were added to include XhoI and BamHI restriction sites. A XhoI-BamHI restriction fragment from plasmid pBEN139 carrying luxCDABE was subcloned into plasmid pBEN129, a derivative of plasmid pACYC184 [26] containing XhoI and BamHI sites, yielding plasmid pBEN135. A XhoI-NotI fragment from plasmid pBEN135 carrying the luxCDABE operon was subcloned into plasmid pGRG25 [20] to give plasmid pBEN275. The promoter of the housekeeping gene frr [27] was amplified from the E.