<i>Treponema denticola</i> Superoxide Reductase: In Vivo Role, in Vitro Reactivities, and a Novel [Fe(Cys)₄] Site

In vitro and in vivo results are presented demonstrating that superoxide reductase (SOR) from the air-sensitive oral spirochete, <i>Treponema denticola</i> (Td), is a principal enzymatic scavenger of superoxide in this organism. This SOR contains the characteristic non-heme [Fe­(His)₄Cys] active sites. No other metal-binding domain has been annotated for Td SOR. However, we found that Td SOR also accommodates a [Fe­(Cys)₄] site whose spectroscopic and redox properties resemble those in so-called 2Fe-SORs. Spectroscopic comparisons of the wild type and engineered Cys → Ser variants indicate that three of the Cys ligands correspond to those in [Fe­(Cys)₄] sites of “canonical” 2Fe-SORs, whereas the fourth Cys ligand residue has no counterpart in canonical 2Fe-SORs or in any other known [Fe­(Cys)₄] protein. Structural modeling is consistent with iron ligation of the “noncanonical” Cys residue across subunit interfaces of the Td SOR homodimer. The Td SOR was isolated with only a small percentage of [Fe­(Cys)₄] sites. However, quantitative formation of stable [Fe­(Cys)₄] sites was readily achieved by exposing the as-isolated protein to an iron salt, a disulfide reducing agent and air. The disulfide/dithiol status and iron occupancy of the Td SOR [Fe­(Cys)₄] sites could, thus, reflect intracellular redox status, particularly during periods of oxidative stress

In Vitro Pharmacodynamics of Vancomycin and Cefazolin Alone and in Combination against Methicillin-Resistant Staphylococcus aureus

Previous studies employing time-kill methods have observed synergistic effects against methicillin-resistant Staphylococcus aureus (MRSA) when a β-lactam is combined with vancomycin. However, these time-kill studies have neglected the importance of human-simulated exposures. We evaluated the effect of human simulated exposures of vancomycin at 1 g every 8 h (q8h) in combination with cefazolin at 1 g q8h against various MRSA isolates. Four clinical isolates (two MRSA isolates [vancomycin MICs, 0.5 and 2.0 μg/ml], a heterogeneous vancomycin-intermediate S. aureus [hVISA] isolate [MIC, 2.0 μg/ml], and a vancomycin-intermediate S. aureus [VISA] isolate [MIC, 8.0 μg/ml]) were evaluated in an in vitro pharmacodynamic model with a starting inoculum of 106 or 108 CFU/ml. Bacterial density was measured over 48 to 72 h. Time-kill curves were constructed, and the area under the bacterial killing and regrowth curve (AUBC) was calculated. During 106 CFU/ml studies, combination therapy achieved greater log10 CFU/ml changes than vancomycin alone at 12 h (−4.31 ± 0.58 versus −2.80 ± 0.59, P < 0.001), but not at 48 h. Combination therapy significantly reduced the AUBC from 0 to 48 h (122 ± 14) compared with vancomycin alone (148 ± 22, P = 0.017). Similar results were observed during 108 CFU/ml studies, where combination therapy achieved greater log10 CFU/ml changes at 12 h than vancomycin alone (−4.00 ± 0.20 versus −1.10 ± 0.04, P < 0.001) and significantly reduced the AUBC (275 ± 30 versus 429 ± 37, P < 0.001) after 72 h of incubation. In this study, the combination of vancomycin and cefazolin at human-simulated exposures improved the rate of kill against these MRSA isolates and resulted in greater overall antibacterial effect, but no differences in bacterial density were observed by the end of the experiments

Nationally Coordinated Program of Highway Research, Development and Technology: Annual Progress Report, Executive Summary, Fiscal Year 1989

Nationally Coordinated Program of Highway Research, Development and Technology: Annual Progress Report, Executive Summary, Fiscal Year 198