Effects of neutrophils on cefazolin activity and penicillin-binding proteins in Staphylococcus aureus abscesses

Abstract

Bacteria survive within abscesses despite antimicrobial therapy, usually necessitating drainage. Our previous work showed that bacterial killing is diminished within the neutrophils of animals with abscesses. To further assess the role of neutrophils in Staphylococcus aureus survival and the poor activities of beta-lactams in abscesses, tissue cage abscess-bearing rats were given polymorphonuclear leukocyte (PMN)-depleting antibody prior to and several times following inoculation of the tissue cages with S. aureus. Cefazolin (300 mg/kg of body weight/day) was administered to all animals in appropriately divided doses. After 7 days of antimicrobial therapy, the 17 animals that received anti-PMN serum had significantly fewer abscess neutrophils than the 18 controls and fewer abscess bacteria (5.55 versus 3.79 log(10) CFU/ml [P = 0.04]) than the 18 controls. The data were consistent with the premise that cefazolin is more effective in abscesses depleted of neutrophils. To investigate further, S. aureus was incubated with rat peritoneal neutrophils; and bacterial cell membrane proteins were isolated, labeled with biotinylated ampicillin, separated by electrophoresis, blotted onto nitrocellulose, and stained for biotin reactivity. PBP 2 expression was consistently and significantly decreased after a brief, nonkilling PMN exposure. These experiments showed that PMN depletion enhanced the activity of cefazolin in the abscess milieu. Furthermore, altered bacterial cell wall cefazolin targets may be the mechanism by which the PMN diminishes antimicrobial activity, suggesting the importance of the staphylococcus-PMN interaction in the outcome of established infections.

FIG. 1.

Rat tissue cage model. A gas-sterilized table-tennis ball into which holes were drilled was implanted by sterile procedures intra-abdominally in large rats (one per rat). The balls were well tolerated when they were infected following 5 weeks of encapsulation. The model was developed so that reagents that were unavailable for the rabbit model could be used. The photograph was made at the time of necropsy of a rat with an 8-day-old S. aureus infection.

FIG. 2.

Circulating blood PMN counts (A) and abscess fluid PMN counts (B) during treatment with cefazolin in rats infected with S. aureus. Anti-PMN serum of rabbit origin (Accurate Chemical Corp.) was administered 1 day before infection and on days 2, 4, and 6 of infection (n = 17). Total cell counts and differential counts were obtained for cytospin samples made with abscess fluid sampled on days 2, 4, and 7 of cefazolin treatment. The sera of rats treated with anti-PMN serum demonstrated significant drops in PMN cell counts on all days on which the counts were measured compared to the counts in the sera of rats treated with control serum (n = 18). P values were determined by the Mann-Whitney U test. □, control treatment; ▧, treatment with anti-PMN serum; bars, maximum and minimum values; the symbols themselves delineate the values for 25 to 75% of the samples; ▪, median values.

FIG. 3.

S. aureus counts in abscesses. Abscess fluid was sampled on days 2, 4, and 7 of cefazolin treatment. The bacterial count at day 7 was significantly lower in the anti-PMN-treated group (n = 17) than in the control group (n = 18) (P = 0.043 by the Mann-Whitney U test). See the legend to Fig. 2 for definitions of the symbols.

FIG. 4.

Effect of incubation with PMNs on the S. aureus PBPs. Density values (means ± standard deviations for six runs) for PBPs isolated from bacteria incubated with PMNs (░⃞) or not incubated with PMNs (▧) were determined by differential centrifugation and identification with biotinylated ampicillin. PBPs were separated electrophoretically, blotted, and stained. Important PBPs were identified by molecular weight on the basis of two standards included in each run and were quantitated with imaging software (ImageQuaNT). After exposure to PMNs, a significant reduction in the level of PBP 2 expression was seen, as defined by a decrease in gel band density with equal amounts of protein (P < 0.01 by Student's t test). The levels of PBP 1 and PBP 3 expression were not significantly changed. The level of PBP 4 expression showed a significant increase in the biotinylated ampicillin series that was not seen by the methods that used fluorescent penicillin.

FIG. 5.

ImageQuaNT scans of S. aureus PBPs (the numbers above each peak). A representative run of viable S. aureus cells incubated or not incubated with viable neutrophils for 30 min is shown. Cell wall proteins were subsequently isolated; incubated with biotinylated ampicillin; and separated on a gel, blotted, and stained. The third scan at the bottom is a methods control: S. aureus membrane proteins were incubated with unlabeled ampicillin in excess for 30 min at 37°C before incubation with biotinylated ampicillin for 30 min at 37°C, followed by performance of the standard run.

FIG. 6.

Photo from the density program of S. aureus PBPs incubated without or with neutrophils before cell wall isolation and electrophoresis. The cell wall proteins were subsequently isolated, incubated with biotinylated ampicillin, and separated electrophoretically and were blotted and stained with streptavidin-alkaline phosphatase and then the BCIP-NBT substrate. The photo is from the software program used to determine band density. PBPs are indicated to the right of the gel.