Conclusions In this study we have shown that SPI-1 and SPI-2 pathogeniCity islands are central to the virulence of S. Enteritidis for chickens. The presence of either of these two pathogeniCity islands resulted in
a significant increase in the liver and spleen colonisation by S. Enteritidis. The remaining three major pathogeniCity islands (SPI-3, SPI-4 and SPI-5) influenced S. Enteritidis virulence for day-old chickens collectively but not individually. Methods Bacterial strains and culture buy Geneticin VE822 conditions S. Enteritidis strain 147 was used throughout the study . A clone spontaneously resistant to nalidixic acid was propagated in LB broth supplemented with ampicillin, chloramphenicol or kanamycin if necessary. Construction and characterisation of SPI deletion mutants SPI-5 was removed from the S. Enteritidis genome using the λ Red recombination as described . For the construction of the remaining SPI mutants, a modified procedure of λ Red recombination was used. The modification was used because we had failed to remove a sequence greater than 10 kb by a single-step procedure in Tideglusib mw S. Enteritidis 147. We therefore first introduced the chloramphenicol gene cassette at the left end of the sequence to
be removed by the standard protocol and in the next step, a kanamycin gene cassette was inserted at the right end of the sequence to be removed. In the case of SPI-1 removal, the chloramphenicol gene cassette was used for the replacement of the avrA gene and then the kanamycin gene cassette was used for the replacement of the invH gene. The intermediate avrA::Cm invH::Kan mutant was transformed with pCP20 and any sequence in between the frt sequences was removed by pCP20-encoded flipase. Originally we expected to obtain two constructs, ΔSPI1 and SPI1::Cm (or Erastin clinical trial SPI1::Kan), the latter being suitable for transduction. However, since all the mutants
recovered were ΔSPI-1, free of any antibiotic resistance marker, to obtain SPI1::Cm (or SPI1::Kan) mutation suitable for transduction, we inserted chloramphenicol or kanamycin resistance gene cassettes into the ΔSPI1 mutant once more using a PCR product resulting from the amplification of pKD3 or pKD4 plasmid template with avrA44For and invH44Rev primers. Using this protocol, we constructed strains in which SPI-1, SPI-2, SPI-3, SPI-4 or SPI-5 were replaced with either chloramphenicol or kanamycin resistance gene cassettes. All the primers used for SPI removal are listed in Table 2. Table 2 List of primers used for the generation and verifications of SPI mutants in S. Enteritidis.