Neutrophil extracellular traps (NETs) are released, as neutrophils die in vitro, in a process requiring hours, leaving a temporal gap for invasive microbes to exploit. Functional neutrophils undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live PMN in vivo rapidly releasing NETs, which prevented bacterial dissemination. NETosis occurred during crawling thereby casting large areas of NETs. NET-releasing PMN developed diffuse decondensed nuclei ultimately becoming devoid of DNA. Cells with abnormal nuclei displayed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A combined requirement of Tlr2 and complement mediated opsonization tightly regulated NET release. Additionally live human PMN developed decondensed nuclei and formed NETS in vivo and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection, non-cell death NETosis occurs in vivo during Gram-positive infection in mice and humans.

The nematode Caenorhabditis elegans has recently been developed as a host model for the study of Staphylococcus aureus virulence and pathogenesis. Here, the toxicity and virulence of representative clinical isolates of our methicillin-resistant S. aureus (MRSA) epidemic strains were studied using this model. The strains USA300 (associated with community infection outbreaks), USA400 (associated with sporadic community infections) and CMRSA2 (associated with both hospital and community infections), as well as the nematocidal reference strain NCTC8325, showed high nematocidal activity, both by killing the majority of the nematodes (> 90%) over 9 days, and by inhibiting second-generation nematode growth. By contrast, the typical hospital-associated MRSA strain CMRSA6, the colonization strain M92, and the non-pathogenic Staphylococcus epidermidis control strain ATCC12228 were non-toxic to the nematode, which behaved normally. The absence of nematocidal activity does not reflect lack of growth or reduced growth of the bacterial inoculum. The two non-nematocidal strains share similar genomic backgrounds, bacterial growth curve patterns and virulence gene profiles. However, the nematocidal strains each showed the same low maximum density growth curve patterns, but possessed distinct genetic profiles; no common virulence gene patterns or specific genes have been elucidated. Our findings demonstrate that community-associated MRSA strains are more pathogenic than hospital-associated MRSA in the C. elegans model and support the use of this model for studying the virulence of S. aureus strains.

BackgroundStaphylococcus aureus strains with distinct genetic backgrounds have shown different virulence in animal models as well as associations with different clinical outcomes, such as causing infection in the hospital or the community. With S. aureus strains carrying diverse genetic backgrounds that have been demonstrated by gene typing and genomic sequences, it is difficult to compare these strains using mammalian models. Invertebrate host models provide a useful alternative approach for studying bacterial pathogenesis in mammals since they have conserved innate immune systems of biological defense. Here, we employed Drosophila melanogaster as a host model for studying the virulence of S. aureus strains.ResultsCommunity-associated methicillin-resistant S. aureus (CA-MRSA) strains USA300, USA400 and CMRSA2 were more virulent than a hospital-associated (HA)-MRSA strain (CMRSA6) and a colonization strain (M92) in the D. melanogaster model. These results correlate with bacterial virulence in the Caenorhabditis elegans host model as well as human clinical data. Moreover, MRSA killing activities in the D. melanogaster model are associated with bacterial replication within the flies. Different MRSA strains induced similar host responses in D. melanogaster, but demonstrated differential expression of common bacterial virulence factors, which may account for the different killing activities in the model. In addition, hemolysin α, an important virulence factor produced by S. aureus in human infections is postulated to play a role in the fly killing.ConclusionsOur results demonstrate that the D. melanogaster model is potentially useful for studying S. aureus pathogenicity. Different MRSA strains demonstrated diverse virulence in the D. melanogaster model, which may be the result of differing expression of bacterial virulence factors in vivo.