nes encoding proteins that moderate oxidative stress, virulence, and those involved in metabolism and capsule synthesis are generally upregulated, but the effects vary and some PBP genes are down-regulated after exposure for 30 minutes, and upregulated after exposure for 180 minutes [6]. In our previous experiments utilizing biotinylated ampicillin, betalactam binding to PBP 2 but not PBP 3 was significantly diminished after PMN exposure [4]. Utilizing a differing methodology by labeling with an anti-beta lactam monoclonal antibody [13], the reduction of beta-lactam binding to PBP-2 after PMN exposure was again demonstrated, and shown in Figure 4.

We sought to determine if cefotaxime, a third generation cephalosporin that primarily binds to PBP 1 and 2, has diminished activity in an abscess environment compared to the first generation cephalosporin cephalexin, which primarily binds to PBP 1 and 3, as would be predicted if PMN exposure alters the synthesis of PBP 2 or alters the ability of beta-lactams to bind to the PBP 2 target. Others have noted that strains of S. aureus, such as CDC-6, which has an altered PBP-2, is more resistant to cefotaxime as compared to antimicrobials that preferentially bind PBP-3 [14]. Similarly SC 12,700 is an organism with an altered PBP-3 and is more resistant to cephalexin, but is susceptible to cefotaxime [9]. In timekill curves against stationary phase S. aureus, cefotaxime was highly active when tested in MHB, and was more active than cephalexin when measured at 24 h. In abscess fluid that was obtained from neutropenic animals, cefotaxime remained highly active in killing stationary phase S. aureus as compared to cephalexin. However in abscess fluid obtained from non-neutropenic animals, cefotaxime had diminished activity in killing S. aureus, while cephalexin maintained activity. In the non-neutropenic abscess animal model, cephalexin was more active than cefotaxime after seven days of treatment.

In summary, cephalexin was more active against S. aureus exposed to PMNs or PMN products, while cefotaxime was more active against S. aureus in the absence of exposure to PMNs. A plausible explanation for these findings is that beta-lactam binding to PBP 2, which is an important target for the activity of cefotaxime but not cephalexin, is altered secondary to PMN exposure, which results in loss of activity of cefotaxime but not cephalexin. The present data suggest that the effect observed in vivo was secondary to the neutrophil.

Previous studies have suggested that the stress of increasing concentrations of antimicrobial may produce selection of altered PBPs with resultant loss of antimicrobial activity [7]. We and others [15] have found that PMN exposure also effects beta-lactam binding to PBPs. The in vivo significance of the altered beta-lactam binding to PBP has not been previously demonstrated. PMN induced alteration of antimicrobial binding proteins with resultant diminished antimicrobial activity defines a novel mechanism of in vivo antimicrobial resistance. Antimicrobial resistance to beta-lactams in stationary phase Streptococcus pyogenes has also been postulated to be secondary to altered PBP production [16]. The mechanisms by which bacterial persist in abscesses despite the presence of antimicrobials at concentrations above the MIC likely remain multifactorial, however included in the potential mechanisms could be alteration of targets of antibacterials by the PMN.

5. Acknowledgements

We gratefully acknowledge the contributions of Zarqa Imdad M.D., lab assistants Shena Latcham and Tahira Zufer, and medical students Christine Van Dillen and Nisha Mangalat.

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NOTES

*Corresponding author.

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