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.
References
[1]
Levy O (2007) Innate immunity of the newborn: basic mechanisms and clinical correlates. Nature reviews Immunology 7: 379–390.
[2]
Angelone DF, Wessels MR, Coughlin M, Suter EE, Valentini P, et al. (2006) Innate immunity of the human newborn is polarized toward a high ratio of IL-6/TNF-alpha production in vitro and in vivo. Pediatr Res 60: 205–209.
[3]
Adkins B, Leclerc C, Marshall-Clarke S (2004) Neonatal adaptive immunity comes of age. Nature Reviews Immunology 4: 553–564.
[4]
Strunk T, Currie A, Richmond P, Simmer K, Burgner D (2011) Innate immunity in human newborn infants: prematurity means more than immaturity. J matern Fetal Neonatal Med 24: 25–31.
[5]
Wynn JL, Levy O (2010) Role of innate host defenses in susceptibility to early-onset neonatal sepsis. Clin Perinatol 37: 307–337.
[6]
Kaufman D, Fairchild KD (2004) Clinical microbiology of bacterial and fungal sepsis in very-low-birth-weight infants. Clinical Microbiology Reviews 17: 638–680.
[7]
Mohamed MA, Cunningham-Rundles S, Dean CR, Hammad TA, Nesin M (2007) Levels of pro-inflammatory cytokines produced from cord blood in-vitro are pathogen dependent and increased in comparison to adult controls. Cytokine 39: 171–177.
[8]
Schultz C, Rott C, Temming P, Schlenke P, Moller JC, et al. (2002) Enhanced interleukin-6 and interleukin-8 synthesis in term and preterm infants. Pediatr Res 51: 317–322.
Strunk T, Prosser A, Levy O, Philbin V, Simmer K, et al. (2012) Human monocyte responsiveness to the commensal bacterium Staphylococcus epidermidis develops late in gestation. Peds Research 27: 10–18.
[11]
Belderbos ME, Levy O, Stalpers F, Kimpen JL, Meyaard L, et al. (2012) Neonatal Plasma Polarizes TLR4-Mediated Cytokine Responses towards Low IL-12p70 and High IL-10 Production via Distinct Factors. PLoS One 7: e33419.
[12]
Kollmann TR, Crabtree J, Rein-Weston A, Blimkie d, Thommai F, et al. (2011) Neonatal Innate TLR-mediated responses are distinct from those of adults. J Immunol 183: 7150–7160.
[13]
Burl S, Townsend J, Njie-Jobe J, Cox M, Adetifa UJ, et al. (2011) Age-dependent maturation of Toll-like receptor-mediated cytokine responses in Gambian infants. PLoS One 13: e18185.
[14]
Cheung GY, Otto M (2010) Understanding the significance of Staphylococcus epidermidis bacteremia in babies and children. Curr Opin Infect Dis 23: 208–216.
[15]
Hartel C, Osthues I, Rupp J, Haase B, Roder K, et al. (2008) Characterisation of the host inflammatory response to Staphylococcus epidermidis in neonatal whole blood. Arch Dis Child Fetal Neonatal Ed 93: F140–145.
[16]
Isaacs D (2003) A ten year, multicentre study of coagulase negative staphylococcal infections in Australasian neonatal units. Arch Dis Child Fetal Neonatal Ed 88: F89–93.
[17]
Klingenberg C, Ronnestad A, Anderson AS, Abrahamsen TG, Zorman J, et al. (2007) Persistent strains of coagulase-negative staphylococci in a neonatal intensive care unit: virulence factors and invasiveness. Clin Microbiol Infect 13: 1100–1111.
[18]
Widerstrom M, Wistrom J, Sjostedt A, Monsen T (2012) Coagulase-negative staphylococci: update on the molecular epidemiology and clinical presentation, with a focus on Staphylococcus epidermidis and Staphylococcus saprophyticus. Eur J Clin Microbiol Infect Dis 31: 7–20.
[19]
Otto M (2009) Staphylococcus epidermidis–the ‘accidental’ pathogen. Nat Rev Microbiol 7: 555–567.
[20]
Strunk T, Power Coombs MR, Currie AJ, Richmond P, Golenbock DT, et al. (2010) TLR2 mediates recognition of live Staphylococcus epidermidis and clearance of bacteremia. PLoS One 5: e10111.
[21]
Vuong C, Otto M (2002) Staphylococcus epidermidis infections. Microbes Infect 4: 481–489.
[22]
Healy C, Palazzi D, Edwards M, Campbell J, Baker C (2004) Features of Invasive Staphylococcal Disease in Neonates. Pediatrics 114: 953–961.
[23]
Wynn J, Cornell TT, Wong HR, Shanley TP, Wheeler DS (2010) The host response to sepsis and developmental impact. Pediatrics 125: 1031–1041.
[24]
Chau V, Brant R, Poskitt KJ, Tam EWY, Synnes A, et al. (2012) Postnatal infection is associated with widespread abnormalities of brain development in premature newborns. Pediatric Research 274–279.
[25]
Schlapbach LJ, Aebischer M, Adams M, Natalucci G, Bonhoeffer J, et al. (2011) Impact of sepsis on neurodevelopmental outcome in a Swiss National Cohort of extremely premature infants. Pediatrics 128: 348–357.
[26]
Wang X, Mallard C, Levy O (2012) Potential Role of Coagulase-negative Staphylococcus Infection in Preterm Brain Injury. Advances in Neuroimmune Biology 3: 41–48.
[27]
Srinivasan L, Harris MC (2012) New technologies for the rapid diagnosis of neonatal sepsis. Curr Opin Pediatr 24: 165–171.
[28]
Radbruch A, Isaacs J (2009) Animal models in infection and inflammation - chance and necessity. Eur J Immunol 39: 1991–1993.
[29]
Gallimore B, Gagnon RF, Subang R, Richards GK (1991) Natural history of chronic staphylococcus epidermidis foreign body infection in a mouse model. Journal of Infectious Diseases 164: 1220–1223.
[30]
Kinsman OS, Arbuthnott J (1980) Experimental staphylococcal infection in newborn mice: inhibition of weight gain as an index of virulence. Journal of Medical Microbiology 13: 281–290.
[31]
Krause P, Kristie J, Wang W, Kreutzer D (1987) Pentoxifylline enhancement of defective neutrophil function and host defense in neonatal mice. The American Journal of Pathology 129: 217–222.
[32]
Maderazo EG, Breaux S, Woronick CL, Krause PJ (1990) Efficacy, toxicity and pharmacokinetics of pentoxifylline and its analogs in experimental Staphylococcus aureus infections. Antimicrobial Agents and Chemotherapy 34: 1100–1106.
[33]
McKay SE, Arbuthnott J (1979) Age-related Susceptibility of Mice to Staphylococcal Infection. Journal of Medical Microbiology 12: 99–106.
[34]
Placencia FX, Kong L, Weisman LE (2009) Treatment of methicillin-resistant Staphylococcus aureus in neonatal mice: lysostaphin versus vancomycin. Pediatric Research 65: 420–424.
[35]
Wynn J, Scumpia PO, Winfield RD, Delano MJ, Kelly-Scumpia K, et al. (2008) Defective innate immunnity predisposes murine neonates to poor sepsis outcome but is reversed by TLR agonists. Blood 112: 1750–1758.
[36]
Sun C, Fiette L, Tanguy M, Leclerc C, Lo-Man R (2003) Ontongeny an innate properties of neonatal dendritic cells. Blood 102: 585–591.
[37]
Zhou S, Kurt-Jones EA, Fitzgerald KA, Wang JP, Cerny AM, et al. (2007) Role of MyD88 in route-dependent susceptibility to vesicular stomatitis virus infection. Journal of Immunology 178: 5173–5181.
[38]
Major AS, Cuff CF (1996) Effects of the route of infection on immunoglobulin G subclasses and specificity of the reovirus-specific humoral immune response. J Virol 70: 5968–5974.
[39]
Gunn BA (1989) Comparative virulence of human isolates of coagulase-negative staphylococci tested in infant mouse weight retardation model. Journal of Clinical Microbiology 27: 507–511.
[40]
Mrozek JD, Georgieff MK, Blazar BR, Mammel MC, Schwarzenberg SJ (2000) Effect of Sepsis Syndrome on Neonatal Protein and Energy Metabolism. Journal of Perinatology 2: 96–100.
[41]
Mack D, Fischer W, Hartmann R, Egge H, Laufs R (1996) The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis. J Bacteriol 178: 175–183.
[42]
Mack D, Sabottke A, Dobinsky S, Rohde H, Horstkotte MA, et al. (2002) Differential expression of methicillin resistance by different boifilm-negative Staphylococcus epidermidis transposon mutant classes. Antimicrobials Agents and Chemotherapy 46: 178–183.
[43]
JAX Mice and services (2009) Mice Pup Appearance by Age. jaxmice.jax.org/literature/factsheet/LT0？001_Pups.pdf.
[44]
Rodewald AK, Onderdonk AB, Warren HB, Kasper DL (1992) Neonatal mouse model of group B streptococcal infection. Journal of Infectious Diseases 166: 635–639.
[45]
Kienstra KA, Freysdottir D, Gonzales NM, Hirschi KK (2007) Murine neonatal intravascular injections: modeling newborn disease. Journal of the American Association for Laboratory Animal Science. JAALAS 46: 50–54.
[46]
Hoff J (2000) Methods of Blood Collection in the Mouse. Lab Animal 29: 47–53.
[47]
Le Rouzic V, Corona J, Zhou H (2010) Postnatal Development of Hepatic Innate Immune Reponse. Inflammation 34: 576–584.
[48]
Sweeney T, Sulima H, Hollingsworth J, Welty-Wolf K, Piantadosi C (2011) A Toll-like receptor 2 pathway regulates the Ppargc1a/b metabolic co-activators in mice with Staphylococcal aureus sepsis. PLoS One 6: e25249.
[49]
Mancuso G, Midiri A, Beninati C, Biondo C, Galbo R, et al. (2004) Dual role of TLR2 and myeloid differentiation factor 88 in a mouse model of invasive group B streptococcal disease. J Immunol 172: 6324–6329.
[50]
Pietrocola G, Arciola C, Rindi S, DiPoto A, Missineo A, et al. (2011) Toll-like receptors (TLRs) in innate immune defense against Staphylococcus aureus. Int J Artif Organs 34: 799–810.
[51]
Knuefermann P, Sakata Y, Baker J, Huang C, Sekiguchi K, et al. (2004) Toll-like receptor 2 mediates Staphylococcus aureus-induced myocardial dysfunction and cytokine production in the heart. Circulation 110: 3693–3698.
[52]
Takeda K, Akira S (2004) TLR signaling pathways. Seminars in Immunology 16: 3–9.
[53]
Zhang JP, Yang Y, Levy O, Chen C (2010) Human neonatal peripheral blood leukocytes demonstrate pathogen-specific coordinate expression of TLR2, TLR4/MD2, and MyD88 during bacterial infection in vivo. Pediatric Research 68: 479–483.
[54]
Santos-Sierra S, Golenbock D, Henneke P (2006) Tol-like receptor-dependent discrimination of streptococci. J Endotoxin Res 12: 307–312.
[55]
Du X, Fleiss B, Hongfu L, D'Angelo B, Sun Y, et al. (2010) Systemic Stimulation of TLR2 Impairs Neonatal Mouse Brain Development. PLoS One 6 (5) e19583.
[56]
Mallard C, Wang X (2012) Infection-induced Vulnerability of Perinatal Brain Injury. Neurol Res Int 2012: 102153.
