Staphylococcus aureus has reemerged as an important human pathogen in recent decades. Although many infections caused by this microbial species persist through a biofilm mode of growth, little is known about how the host's adaptive immune system responds to these biofilm infections. In this study, S. aureus cells adhered to pins in culture and were subsequently inserted into the tibiae of C57BL/6 mice, with an infecting dose of 2 ؋

There is currently intensive research on the design of novel human immunodeficiency virus type 1 (HIV-1) vaccine immunogens that can elicit potent neutralizing antibodies. A prerequisite for comparing and optimizing these strategies is the ability to precisely measure neutralizing antibody responses. To this end, we sought to develop an assay that directly quantifies single-round HIV

In vivo bioluminescent imaging permits the visualization of bacteria in live animals, allowing researchers to monitor, both temporally and spatially, the progression of infection in each animal. We sought to engineer stably luminescent clinical strains of Staphylococcus aureus, with the goal of using such strains in mouse models. The gram-positive shuttle vector pMAD was used as the backbone for an integration plasmid. A chloramphenicol resistance gene, a modified lux operon from Photorhabdus luminescens, and approximately 650 bp of homology to the chromosome of the USA300 S. aureus strain NRS384 were added, generating plasmid pRP1195. Electroporation into strain RN4220 followed by temperature shift led to integration of pRP1195 into the chromosome. The integrated plasmid was transferred to clinical strains by phage transduction. Luminescent strains displayed no in vitro growth defects. Moreover, luminescence was stable in vitro after three rounds of subculture over 48 hours of growth in the absence of antibiotics. Mice were infected with a luminescent strain of NRS384 in skin and intravenous models. In a mouse skin model, luminescent bacteria were present in lesions that formed and cleared over the course of several days, and in an intravenous model, bacteria inoculated in the mouse tail vein were observed spreading to multiple tissues. No statistically significant difference in virulence was observed between NRS384 and the luminescent strain in either infection model. These preliminary data suggest that this luminescent USA300 strain is suitable for use in mouse models. Similar strains were engineered using other sequenced clinical strains. Because these strains are stably luminescent, they should prove useful in animal models of infection.

Staphylococcus aureus is a common cause of prosthetic implant infections, which can become chronic due to the ability of S. aureus to grow as a biofilm. Little is known about adaptive immune responses to these infections in vivo. We hypothesized that S. aureus elicits inflammatory Th1/Th17 responses, associated with biofilm formation, instead of protective Th2/Treg responses. We used an adapted mouse model of biofilm-mediated prosthetic implant infection to determine chronic infection rates, Treg cell frequencies, and local cytokine levels in Th1-biased C57BL/6 and Th2-biased BALB/c mice. All C57BL/6 mice developed chronic S. aureus implant infection at all time points tested. However, over 75% of BALB/c mice spontaneously cleared the infection without adjunctive therapy and demonstrated higher levels of Th2 cytokines and anti-inflammatory Treg cells. When chronic infection rates in mice deficient in the Th2 cytokine interleukin-4 (IL-4) via STAT6 mutation in a BALB/c background were assessed, the mice were unable to clear the S. aureus implant infection. Additionally, BALB/c mice depleted of Treg cells via an anti-CD25 monoclonal antibody (MAb) were also unable to clear the infection. In contrast, the C57BL/6 mice that were susceptible to infection were able to eliminate S. aureus biofilm populations on infected intramedullary pins once the Th1 and Th17 responses were diminished by MAb treatment with anti-IL-12 p40. Together, these results indicate that Th2/Treg responses are mechanisms of protection against chronic S. aureus implant infection, as opposed to Th1/Th17 responses, which may play a role in the development of chronic infection.