A Neonatal Model of Intravenous Staphylococcus epidermidis Infection in Mice <24 h Old Enables Characterization of Early Innate Immune Responses

Abstract

Staphylococcus epidermidis (SE) causes late onset sepsis and significant morbidity in catheterized preterm newborns. Animal models of SE infection are useful in characterizing disease mechanisms and are an important approach to developing improved diagnostics and therapeutics. Current murine models of neonatal bacterial infection employ intraperitoneal or subcutaneous routes at several days of age, and may, therefore, not accurately reflect distinct features of innate immune responses to bacteremia. In this study we developed, validated, and characterized a murine model of intravenous (IV) infection in neonatal mice <24 hours (h) old to describe the early innate immune response to SE. C57BL/6 mice <24 h old were injected IV with 106, 107, 108 colony-forming units (CFU) of SE 1457, a clinical isolate from a central catheter infection. A prospective injection scoring system was developed and validated, with only high quality injections analyzed. Newborn mice were euthanized between 2 and 48 h post-injection and spleen, liver, and blood collected to assess bacterial viability, gene expression, and cytokine production. High quality IV injections demonstrated inoculum-dependent infection of spleen, liver and blood. Within 2 h of injection, SE induced selective transcription of TLR2 and MyD88 in the liver, and increased systemic production of plasma IL-6 and TNF-α. Despite clearance of bacteremia and solid organ infection within 48 h, inoculum-dependent impairment in weight gain was noted. We conclude that a model of IV SE infection in neonatal mice <24 h old is feasible, demonstrating inoculum-dependent infection of solid organs and a pattern of bacteremia, rapid and selective innate immune activation, and impairment of weight gain typical of infected human neonates. This novel model can now be used to characterize immune ontogeny, evaluate infection biomarkers, and assess preventative and therapeutic modalities.

Staphylococcus epidermidisBacteremia Induces Brain Injury in Neonatal Mice via Toll-like Receptor 2-Dependent and -Independent Pathways

“…For the preparation of inocula, 500 mL of bacterial stock was added to 30 mL of trypticase soy broth in a 125 mL baffled flask. Bacteria were grown for 16-20 hours in an incubator/shaker at 37°C and 240 rpm (MaxQ™ 4450 Benchtop Orbital Shakers -Thermo Scientific, Stockholm, Sweden), as previously described [20]. For murine injections, 1 × 10 8 S. epidermidis were resuspended in 1 mL of sterile, pyrogen-free saline, and mice were injected with 5 × 10 6 S. epidermidis in 50 μL saline.…”

“…PND1 mice (≤24 hours of age), similar to preterm human infants with respect to CNS development [22], were injected via the intrajugular vein with 50 μL of either saline (control) or 5 × 10 6 S. epidermidis as previously described [20].…”

“…After a brief intracardial perfusion with saline, the brain, spleen, and liver were collected as described previously [20]. Tissue specimens were homogenized and were spread onto trypticase soy agar plates (TSA II; BD Diagnostics; Franklin Lakes, New Jersey) with 5% sheep blood and incubated at 37°C for 24 hours to enumerate colony-forming units (CFUs), as described previously [20].…”

“…A Mouse TLR Signaling Pathway PCR Array (PAMM-018A; Qiagen) containing primers for 84 genes of interest and 12 controls was used as described elsewhere [20].…”

“…To assess the possible link between S. epidermidis bacteremia and early life brain injury, we characterized the impact of S. epidermidis bacteremia and the potential role of TLR2 using an established neonatal mouse model [20]. We found that S. epidermidis bacteremia at postnatal day 1 (PND1), even in the absence of bacteria in the cerebrospinal fluid (CSF), induces both systemic and brain inflammation and associated TLR2-dependent early brain caspase-3 activation (6 hours after injection on PND1) and TLR2-independent late white matter injury (PND14) providing fresh insights into the potential role of bacteremia in preterm brain injury.…”