6 5 4 3 2 1 0 -1 0 -2 -3 1 qPCR R2 = 0.84 y a r r a o r c i M -2 -1 2 3 4 qVFuiagaliundtariteiaot1niveofrmeailctriomaerrRayT-dPifCfeRre(nqtPiaCl Rge)ne expression data by Validation of microarray differential gene expression data by quantitative real time RT-PCR (qPCR). Ratio of treated vs. control were calculated for microarray data (ordinate) and qPCR data (abscissa). A total of six genes that were up or down regulated with amoxicillin, chlortetracy- cline or enrofloxacin were compared. The correlation coeffi- cient r2 was 0.84. target is often not affected, bacteria typically have a "sig- nature" gene expression response to specific antibiotics. Indeed, bacterial transcription profiles are now some- times used to obtain initial indications of the mechanism of action for new compounds [17,18]. Common trends in response to AMX, CTC and ENR Despite differences in their mechanism of action, the majority of genes with significantly altered expression (282) were common to all three antibiotics (Figure 2). Beyond the effects on specific target gene expression, anti- biotics are known to cause secondary effects on genes involved in general physiology as part of the adaptive response to antibiotic stress. A hallmark of recently described antibiotic mediated bacterial cell death com- mon to bactericidal antibiotics in E. coli is the generation of hydroxyl radicals with subsequent induction of SOS response [19,20]. These effects were noted for MIC as well as sub-MIC concentrations of the antibiotics. There is increasing appreciation for the fact that fine tuning of these responses is unique to each organism and needs to be evaluated as such to facilitate identification of targets that potentiate the bactericidal activity of antibiotics. The P. multocida genome is known to contain genes necessary for antibiotic mediated SOS response [6] (for example, sulA, which is necessary for beta-lactam mediated SOS response). Antibiotic stress in P. multocida resulted in increased ATP synthesis (Additional file 2, supplemental table 2). While this effect has been described for gyrase inhibitors [21], in P. multocida this response was common to all three antibi- otics. In particular, all three antibiotics influenced gene BMC Genomics 2009, 10(Suppl 2):S4 http://www.biomedcentral.com/1471-2164/10/S2/S4 CTC 3 280 84 42 67 AMX 7 42 ENR aVFneigtniubnirodetiia2cgsram of gene expression in response to sub-MIC Venn diagram of gene expression in response to sub- MIC antibiotics. The data intersections between significant changes in gene expression of P. multocida in response to sub- MIC amoxicillin (AMX), chlortetracycline (CTC) and enro- floxacin (ENR). expression involved in de novo nucleotide biosynthesis. The overall effects on expression of purM (increase), purK (decrease), and purF (decrease) could reduce the intracel- lular concentrations of IMP, while increased expression of pyrG and pyrF with ENR could result in increased concen- trations of CTP and UDP. Both AMX and CTC impaired the expression of hsp90, thus affecting proper protein folding under stress condi- tions in P. multocida. All three classes of antibiotics adversely affected putrescine (potE), ribose (rbscA and rbscC), and molybdate (modC) transport systems. Altera- tions to modC expression could have wide ranging effects on bacterial physiology due to effects on the synthesis of molybdoenzymes (Additional file 2, supplemental table 2). Reduced levels of polyamines like putrescine could have profound negative effects on protein translation and impair the ability to cope with oxidative stress [22]. Expression of 121 genes annotated as coding for hypo- thetical proteins were altered in response to the antibiot- ics (Additional file 1, supplemental table 1). Lack of functional information for these genes hampers our understanding of their role in adaptation of P. multocida to antibiotic stress and demonstrates the need for contin- uous functional annotation of genes beyond the initial annotation that is described with the genome sequence. One of the demonstrated outcomes of antibiotic stress in E. coli is alterations in iron homeostasis; namely, expres- sion of genes involved in siderophore mediated iron transport are affected (for example, fecC) [19]. In P. multo- Page 4 of 10 (page number not for citation purposes)