Host gene expression profiling and in vivo cytokine studies to characterize the role of linezolid and vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) murine sepsis model

Abstract

Linezolid (L), a potent antibiotic for Methicillin Resistant Staphylococcus aureus (MRSA), inhibits bacterial protein synthesis. By contrast, vancomycin (V) is a cell wall active agent. Here, we used a murine sepsis model to test the hypothesis that L treatment is associated with differences in bacterial and host characteristics as compared to V. Mice were injected with S. aureus USA300, and then intravenously treated with 25 mg/kg of either L or V at 2 hours post infection (hpi). In vivo alpha-hemolysin production was reduced in both L and V-treated mice compared to untreated mice but the reduction did not reach the statistical significance [P = 0.12 for L; P = 0.70 for V). PVL was significantly reduced in L-treated mice compared to untreated mice (P = 0.02). However the reduction of in vivo PVL did not reach the statistical significance in V- treated mice compared to untreated mice (P = 0.27). Both antibiotics significantly reduced IL-1β production [P = 0.001 for L; P = 0.006 for V]. IL-6 was significantly reduced with L but not V antibiotic treatment [P<0.001 for L; P = 0.11 for V]. Neither treatment significantly reduced production of TNF-α. Whole-blood gene expression profiling showed no significant effect of L and V on uninfected mice. In S. aureus-infected mice, L altered the expression of a greater number of genes than V (95 vs. 42; P = 0.001). Pathway analysis for the differentially expressed genes identified toll-like receptor signaling pathway to be common to each S. aureus-infected comparison. Expression of immunomodulatory genes like Cxcl9, Cxcl10, Il1r2, Cd14 and Nfkbia was different among the treatment groups. Glycerolipid metabolism pathway was uniquely associated with L treatment in S. aureus infection. This study demonstrates that, as compared to V, treatment with L is associated with reduced levels of toxin production, differences in host inflammatory response, and distinct host gene expression characteristics in MRSA sepsis.

Figure 1. Effect of antibiotics on bacterial load in A/J mice.

The bacterial loads in the (A) Kidney and (B) Blood from A/J mice after intraperitoneal infection with S. aureus (6×10⁶ CFU/g, strain USA300), and treatment with linezolid (L) or vancomycin (V) (25 mg/kg). Infected mice were sacrificed as follows: 2 hpi S. aureus; 24 hpi S. aureus; 24 hpi S. aureus with linezolid or vancomycin. Each symbol represents one mouse. P-values were obtained by F-test. The error bars correspond to the standard error of mean (SEM) and the dashed line correspond to the mean value. Values of P<0.05 were considered significant.

Figure 2. Effect of antibiotics on in vivo toxin production.

In vivo level of (A) Panton Valentine Leukocidin (PVL, µg) (B) and Alpha-toxin (ng) in the serum of A/J mice after 24 h of intraperitoneal (ip) infection with S. aureus (6×10⁶ CFU/g, strain USA300), and treatment with linezolid or vancomycin (25 mg/kg) at 2 h was measured using ELISA. P-value was obtained by unpaired t-test. The error bar corresponds to the standard error of mean (SEM). Values of P<0.05 were considered significant.

Figure 3. Effect of antibiotics on pro-inflammatory cytokines.

Level of (A) IL-1beta (pg/ml), (B) IL-6 (pg/ml), and (C)TNF-alpha (pg/ml) from the serum of A/J mice without infection, with 2 hours post infection (hpi) with S. aureus (6×10⁶ CFU/g, strain USA300), and 24 hpi with and without antibiotic treatment as labeled was measured using ELISA. P-value was obtained by unpaired t-test. The error bar corresponds to the standard error of mean (SEM). Values of P<0.05 were considered significant.

Figure 4. Unsupervised hierarchical clustering analysis of mouse blood microarray data.

Each colored row in the heat map represents the gene expression value for a probe and each column represents a sample. The color conventions are as follows: red indicates over-expressed transcripts, blue represents under-expressed transcripts. Time of analysis post-infection are marked as follows: green rectangles indicate 0 hpi, purple rectangles indicate 2 hpi, and orange rectangles indicate 24 hpi (with and without antibiotic treatment).

Figure 5. Comparisons of differentially expressed genes in mice with the appropriate treatment.

(A) Effect of linezolid and vancomycin on host, (B) Effect of linezolid and vancomycin on host with S. aureus infection, and (C) Effect of S. aureus infection on host immune response. Venn diagrams indicate the number of genes unique to each comparison pair and the number of genes common to each comparison pair (overlapped).