Sepsis induced by Staphylococcus aureus: participation of biomarkers in a murine model

Abstract

Background: This study aimed to evaluate the role of biomarkers in the pathophysiological process induced by a Staphylococcus aureus strain obtained in a hospital environment. For this, we intraperitoneally inoculated groups of male BALB/c mice with S. aureus, using a clinical isolate (CI) of S. aureus.

Material/methods: Mice were divided into groups according to time of euthanasia (24, 48, 72, 96, 120, 144, and 168 hours of infection). After being euthanized, blood samples were collected for quantification of microorganisms and leukocytes, as well as measurement of biomarkers of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), C-reactive protein (CRP), and Procalcitonin (PCT) by ELISA. Heart, kidneys, and lungs were removed for histopathological analysis, assessment of biomarkers of tissue expression by RT-PCR (polymerase chain reaction with reverse transcriptase), and quantification of microorganisms by real-time quantitative PCR (real-time PCR).

Results: The animals infected at between 120 hours and 168 hours had the highest blood levels of S. aureus. We observed that infection promoted increases in the levels of circulating neutrophils and monocytes. However, there was a reduction of circulating neutrophils and monocytes after 96 hours of infection. The infected mice also had increased levels of blood lymphocytes. In this model of infection with S. aureus, IL-6, CRP, and PCT demonstrated greater fidelity as markers of infection, since serum levels were elevated and lowered along with the number of circulating neutrophils and monocytes after resolution of the infection. The lungs showed hyperemia, with enlargement of the alveolar septa. On the other hand, infection with S. aureus did not promote visible change in histological tissue in the heart and kidneys.

Conclusions: In this model of infection with S. aureus, IL-6, CRP, and PCT demonstrated greater fidelity as markers of infection, since serum levels were elevated and lowered along with the number of circulating neutrophils and monocytes after resolution of the infection. We believe our results may provide a better understanding of the pathophysiology, as well as aid in the search for a more reliable method of diagnosis.

Figure 1

Quantification in blood using qPCR of Staphylococcus aureus in a sepsis model induced by clinical strain in Balb/c mice infected for 7 days. Infected animals showed higher systemic levels of S. aureus within 120 hours and 168 hours of infection. *** p<0.05 vs. Saline.

Figure 2

Blood leukocytes of BALB/c mice infected for 7 days with strain clinical isolate of S. aureus. The animals infected showed significant increases in blood levels of both neutrophils (A) and monocytes (B), peaking at between 96 and 120 hours. *** p<0.05 vs. Saline. The lymphocytes (C) showed considerable reduction after 72 hours of infection. *** p<0.05 vs. 96, 120, and 144 hours.

Figure 3

Plasma levels of biomarkers in BALB/c mice infected for 7 days with clinical isolate strain of S. aureus. The increase in plasma was more significant with respect to IL-6 (A) and PCT (D) compared to the control group. A borderline significant relation was observed in TNF-α (B) and CRP (C) dosages. *** p<0.05 vs. saline.

Figure 4

Gene expression of IL-6 in BALB/c mice infected for 7 days with clinical isolate strain of S. aureus in heart (A), kidneys (B) and lungs (C). Infected animals showed an increased expression of IL-6 in the heart within 48 hours. In the kidneys the increase was more evident at 96 hours. In the lungs there was a more pronounced increase of IL-6 at 72 hours of infection. Electrophoresis represents the gene expression profile obtained from lung tissue. *** p<0.05 vs. saline.

Figure 5

Gene expression of PCT in BALB/c mice infected for 7 days with clinical isolate strain of S. aureus in heart (A), kidneys (B), and lungs (C). There was a significant increase in the expression of PCT, more evident within 96 hours of infection in the heart, kidneys and lungs. Electrophoresis represents the gene expression profile obtained from lung tissue. *** p<0.05 vs. saline.

Figure 6

Gene expression of CRP in BALB/c mice infected for 7 days with clinical isolate strain of S. aureus in heart (A), kidneys (B), and lungs (C). Infected animals showed significant increases in gene expression of CRP in the heart within 24 hours of infection, and 48 hours in the kidneys and lungs. Electrophoresis represents the gene expression profile obtained from lung tissue. *** p<0.05 vs. saline.

Figure 7

Gene expression of TNF-α in BALB/c mice infected for 7 days with clinical isolate strain of S. aureus in heart (A), kidneys (B), and lungs (C). Infected animals showed an increased gene expression of TNF-α between 48 and 72 hours of infection in the heart, kidneys, and lungs. Electrophoresis represents the gene expression profile obtained from lung tissue. *** p<0.05 vs. saline.

Figure 8

Representative photographs of lung tissue sections from male BALB/c mice infected with S. aureus. In the control group and 24 hours after infection, we observed clear alveoli without cellular infiltration. However, there was a marked hyperemia with more pronounced enlargement of the alveolar septa between 48 and 96 hours. It was less pronounced, but still present, at 144 hours and 168 hours. Arrows indicate enlarged alveolar septa.