Association with the healthcare environment now has little predictive value for distinguishing patients with infection due to multidrug resistant MRSA isolates from those infected by CA-MRSA isolates, that is, isolates that are clindamycin-susceptible, PVL+, ST8, and/or contain SCCmec type IV. Defining CA-MRSA by the absence of risk factors for healthcare exposure greatly underestimates the burden of epidemic CA-MRSA disease.

The number of infections due to resistant gram-positive bacteria, including penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, and vancomycinresistant Enterococcus faecium, continues to increase (8,10,12,17). The development of effective antimicrobial agents to treat these infections is an area of intense research. Oxazolidinone antimicrobial agents represent a promising new class of compounds which act by inhibiting protein synthesis and have demonstrated potency against these emerging pathogens (13, 16; R. N. Jones, M. A. Pfaller, M. E. Erwin, and M. L. Beach, Abstr. 37th Infect. Dis. Soc. Am. Annu. Meet., abstr. 97, 1999).Linezolid is a new oxazolidinone that has demonstrated both in vitro activity and in vivo efficacy in animal models and clinical trials (2, 13, 15; Jones et al., Abstr. 37th Infect. Dis. Soc. Am. Annu. Meet.). The goals of our studies were (i) to characterize the in vivo time course of antimicrobial activity of linezolid against susceptible and resistant S. pneumoniae and S. aureus strains and (ii) to determine the PK/PD parameter and the magnitude of the PK/PD parameter predictive of efficacy.(Part of this work was presented at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., September 1998.) MATERIALS AND METHODSBacteria, media, and antibiotic. Eight strains of S. pneumoniae (one penicillinsusceptible, two penicillin-intermediate, and five penicillin-resistant S. pneumoniae strains) and four strains of S. aureus (two methicillin-susceptible and two methicillin-resistant S. aureus strains) were used for these experiments. S. aureus and S. pneumoniae organisms were grown, subcultured, and quantified in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.), on Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.), and on sheep blood agar plates (Remel, Milwaukee, Wis.). Linezolid was supplied by Pharmacia & Upjohn Company, Kalamazoo, Mich.In vitro susceptibility studies. The MICs of linezolid, penicillin, and methicillin for the various isolates were determined by using standard National Committee for Clinical Laboratory Standards microdilution methods.Murine infection model. Six-week-old, specific-pathogen-free, female ICR/ Swiss mice weighing 23 to 27 g (Harlan Sprague-Dawley, Madison, Wis.) were used for all studies. All animal studies were approved by the Animal Research Committee of the William S. Middleton Memorial VA Hospital. Mice were rendered neutropenic (neutrophils at Ͻ100/mm 3 ) by injecting cyclophosphamide (Mead Johnson Pharmaceuticals, Evansville, Ind.) intraperitoneally 4 days (150 mg/kg) and 1 day (100 mg/kg) before experimental infection. Previous studies have shown that this regimen produces neutropenia in this model for 5 days (1). Broth cultures of freshly plated bacteria were grown to logarithmic phase overnight to an absorbance of 0.3 at 580 nm (Spectronic 88; Bausch and Lomb, Rochester, N.Y.). After a 1:10 dilution into fresh Mueller-Hinton broth, bacterial counts of the ...

Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic pathogen capable of causing meningitis in humans. The mechanisms that enable pathogens to traverse the blood-brain barrier (BBB) are incompletely understood. Here, we investigated the role of a newly identified Fic domain-containing protein, BifA, in SEZ virulence. BifA was required for SEZ to cross the BBB and to cause meningitis in mice. BifA also enhanced SEZ translocation across human Brain Microvascular Endothelial Cell (hBMEC) monolayers. Purified BifA or its Fic domain-containing C-terminus alone were able to enter into hBMECs, leading to disruption of monolayer barrier integrity. A SILAC-based proteomic screen revealed that BifA binds moesin. BifA’s Fic domain was required for its binding to this regulator of host cell cytoskeletal processes. BifA treatment of hBMECs led to moesin phosphorylation and downstream RhoA activation. Inhibition of moesin activation or moesin depletion in hBMEC monolayers abrogated BifA-mediated increases in barrier permeability and SEZ’s capacity to translocate across monolayers. Thus, BifA activation of moesin appears to constitute a key mechanism by which SEZ disrupts endothelial monolayer integrity to penetrate the BBB.

A novel antibiotic 210-A, named as (6S,8aS,9S,11S,12aR)-6-hydroxy-9,10-dimethyldecahydrobenzo[d]azecine-2,4,12(3H)-trione, was isolated from the fermentation broth of Streptomyces noursei Da07210, its structure was unambiguously established by spectral analyses and chemical comparison with related cycloheximide. Experiments demonstrated that 210-A bore strong activity against Fusarium oxysporum f. sp. cubense race four (Foc race four), which also showed antitumor activity against SMMC-7721 human hepatocarcinoma cells and S180 murine sarcoma, and the IC(50) values were 0.77 and 0.74 microg/ml, respectively.

Streptococcus equi ssp. zooepidemicus (SEZ) causes meningitis in both humans and animals. Some dissociative proteins of SEZ are cytotoxic to mouse brain microvascular endothelial cells (mBMECs) and may contribute to the penetration of SEZ across the blood-brain barrier (BBB). In this study, the ability of SEZ to penetrate across an in vitro BBB model was confirmed. We used stable isotope labeling with amino acids in cell culture (SILAC) to label SEZ proteins with heavy or light isotope-tagged amino acids, along with LC-MS/MS to determine which SEZ proteins were involved in interactions with mBMECs. The efficiency of SEZ protein isotope labeling was 94.7 %, which was sufficient for further analysis. Forty-nine labeled peptides were identified as binding to mBMECs, which matched to 25 SEZ proteins. Bioinformatic analysis indicated that most of these proteins were cytoplasmic. These proteins may have functions in breaching the host BBB, and some of them are known virulence factors in other bacteria. Indirect immunofluorescence results indicated that SEZ enolase had binding activity toward mBMECs. Protective test results showed that enolase was a protective antigen against SEZ infection. This research is the first application of SILAC combined with LC-MS/MS to identify SEZ proteins that may contribute to the infection of mBMECs and potentially show functions related to breaching the BBB. The outcomes provide many future avenues for research into the mechanism of SEZ-induced meningitis.

Swine streptococcosis is a significant threat to the Chinese pig industry, and Streptococcus equi ssp. zooepidemicus (SEZ) is one of the major pathogens. SEZ ATCC35246 is a classical virulent strain, while SEZ ST171 is a Chinese attenuated vaccine strain. In this study, we employed stable isotope labeling by amino acids in cell culture and liquid chromatography-mass spectrometry (LC-MS) to determine the differential response of macrophages to infection by these two strains. Eighty-seven upregulated proteins and 135 downregulated proteins were identified. The proteomic results were verified by real-time polymerase chain reaction for 10 chosen genes and Western blotting for three proteins. All differentially abundant proteins were analyzed for their Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations. Certain downregulated proteins were associated with immunity functions, and the upregulated proteins were related to cytomembrane and cytoskeleton regulation. The phagocytosis rate and cytokine genes transcription in Raw264.7 cells during SEZ ATCC35246 and ST171 infection were detected to confirm the bioinformatics results. These results showed that different effects on macrophage phagocytosis and cytokine expression might explain the different phenotypes of SEZ ATCC35246 and ST171 infection. This research provided clues to the mechanisms of host immunity responses to SEZ ST171and SEZ ATCC35246, which could identify potential therapy and vaccine development targets.

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