Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/26/2006
Publication Date: 6/30/2006
Citation: Killinger-Man, K., Dowd, S.E., Blanton, J.R., Brashears, M.M. 2006. Microarray analysis of a lactic acid stressed, multi-drug resistant mutant of Salmonella Typhimurium [abstract]. The 59th Annual Reciprocal Meat Conference, June 18-21, 2006, University of Illinois. Abstract No. 44P.
Technical Abstract: Salmonella Typhimurium is one of the most common Salmonella serovars associated with human foodborne infections and has been associated with multi-drug resistance properties. It is important to establish the ability of multi-drug resistant Salmonella to survive food processing interventions. Lactic acid washes are commonly used in the meat industry to reduce the prevalence of foodborne pathogens, such as Salmonella, on carcasses. A previous study indicated that a multi-drug resistant mutant of Salmonella Typhimurium (strain 2A) was more susceptible to lactic acid than the wild-type, parent strain (Salmonella Typhimurium ATCC 14028). The objective of this study was to identify differences in gene expression between the multi-drug resistant strain 2A and the wild-type strain after exposure to lactic acid. Strain 2A was resistant to 5 drugs: nalidixic acid, chloramphenicol, cefoxitin, sulfisoxazole and tetracycline, and the wild-type strain was sensitive to all antimicrobial drugs tested. For the microarray study, the wild-type strain and strain 2A were each exposed to 1% lactic acid in saline for 10 minutes, then RNA from each strain was isolated. RNA was converted to cDNA and labeled using a random primer based labeling kit. Salmonella microarray slides were prepared and contained 1152 oligonucleotide probes including 10 positive and 10 negative control elements. Labeled cDNA was hybridized to the slides using standard methods, and signal intensity was quantified using a microarray scanner. Significantly (P<0.05) upregulated or downregulated genes were identified using a t-test. A dye-swap experimental design was used to provide technical duplicate array analysis of each sample. Two biological replicates were performed. The microarray analysis indicated 38 genes were upregulated (P<0.05) and 22 genes were downregulated (P<0.05) in strain 2A compared to the wild-type control. In strain 2A, cyoD was upregulated (P<0.05) 3.4-fold. Upregulation of cyoD, which minimizes proton export, could contribute to the enhanced acid sensitivity observed in strain 2A compared to the wild-type strain. In strain 2A, several secretion system components were also upregulated (P<0.05): sseA, sseB, sseE, sseG, ssaC, ssaJ, ssaN, sscA. Most of these genes are associated with Salmonella type III secretion systems, which have been noted as virulence factors. Expression of rcsB was increased (P<0.05) in strain 2A, and rcsB is involved in a signal transduction cascade that can be triggered by loss of membrane integrity. Moreover, tolQ tended to be upregulated (P=0.09) in strain 2A; tolQ is believed to be controlled in part by rcsB. Additionally, tolB expression was also increased (P<0.05) in strain 2A. The tolB gene is believed to interact with outer membrane porins. The tol genes are associated with membrane stability and maintenance of membrane integrity. The sse, ssa and tol genes have been associated with both anti-microbial resistance as well as virulence. The acid sensitivity displayed by multi-drug resistant strain 2A may be explained by the expression of some genes that would limit the ability of strain 2A to remove protons from the cell, and the resultant damage to the cell may have induced other systems involved in cell envelope maintenance as well as virulence.