Failure of an automated blood culture system to detect nonfermentative gram-negative bacteria.

During a 1-year study we observed that both aerobic and anaerobic blood culture bottles from patients were negative by the BacT/Alert system during a 7-day incubation period. However, upon subcultivation of negative bottles, growth of Pseudomonas aeruginosa was detectable. In an attempt to explain this observation, aerobic BacT/Alert Fan bottles were seeded with a defined inoculum (0.5 McFarland standard; 1 ml) of Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, P. aeruginosa, Stenotrophomonas maltophilia, or Acinetobacter baumannii. Half of the inoculated bottles were loaded into the BacT/Alert system immediately, and the remainder were preincubated for 4, 8, 16, and 24 h at 36 degrees C. With preincubation all bottles seeded with the Enterobacteriaceae signaled positive during the next 1.5 h. Organisms in bottles seeded with the nonfermentative species P. aeruginosa and A. baumannii remained undetected by the BacT/Alert system for 7 days. S. maltophilia was detected if the preincubation time was equal or less than 8 h. Without preincubation all bottles seeded with the Enterobacteriaceae or nonfermentative species signaled positive. Since nonfermentative species seem to enter a state of bacteriostasis within the preincubation period, we reasoned that an unknown factor is consumed. Accordingly, a smaller inoculum should allow the detection of nonfermentative species, even after preincubation, and serial dilutions of P. aeruginosa were detected in preincubated bottles. In this case preincubated bottles signaled positive faster than bottles without preincubation. We conclude that all bottles from clinical settings should be subcultured prior to loading to avoid false negatives. An alternative may be preincubation at room temperature.

Design of a potent novel endotoxin antagonist.

BACKGROUND: Bactericidal permeability increasing protein (BPI) binds to and neutralizes lipopolysaccharide (LPS, endotoxin). Small synthetic peptides based on the amino acid sequence of the LPS binding domain of BPI neutralize LPS, albeit inefficiently. Although the LPS binding domain of native BPI possesses a beta-turn secondary structure, this structure is not present in small derivative peptides. The purpose of this study was to determine whether the addition of a beta-turn to a BPI-derived peptide is associated with more potent endotoxin antagonism. METHODS: We generated a hybrid peptide (BU3) on the basis of (1) a portion of the LPS binding domain from BPI and (2) amino acids known to initiate a beta-turn. BU3 folds with a beta-turn, and we tested its effects on LPS neutralization and LPS-induced tumor necrosis factor-alpha secretion, comparing it with BPI-derived peptide BG22 that lacks a beta-turn and to an irrelevant peptide (BG16). RESULTS: Compared with BG22, BU3 demonstrated enhanced LPS neutralization and inhibition of LPS-induced tumor necrosis factor-alpha secretion in vitro and a similar diminution of endotoxemia and tumor necrosis factor-alpha secretion in a murine model of endotoxemia. CONCLUSIONS: These data demonstrate the potential for enhancing the biologic activity of a BPI-derived peptide endotoxin antagonist via manipulation of its conformational structure.

Candida albicans and Escherichia coli are synergistic pathogens during experimental microbial peritonitis.

Candida albicans has been isolated with increasing frequency during intraabdominal infection; yet its role as a pathogen or copathogen remains controversial. A recent experimental study of its effect during polymicrobial peritonitis indicated that it did not enhance mortality when added to an Escherichia coli challenge, but that study used fecal or mucin-based adjuvants which are known to markedly potentiate the lethality of intraperitoneal bacteria. Therefore, we sought to examine the hypothesis that C. albicans and E. coli are synergistic copathogens that act in concert to increase mortality rates in experimental models of polymicrobial peritonitis, irrespective of the presence of growth adjuvant. To test this hypothesis, we assessed the mortality rates of previously healthy Swiss-Webster mice (20 g) that were challenged intraperitoneally (i.p.) with E. coli, C. albicans, or both, in either the presence or the absence of hemoglobin-mucin. In the absence of hemoglobin-mucin, E. coli plus C. albicans resulted in 83.3% mortality (P < 0.02) compared to either E. coli (0%) or C. albicans (0%) alone. In the presence of hemoglobin-mucin, the synergistic effect was not observed, lower numbers of E. coli alone (62.5%), C. albicans alone (75%), or both organisms together (100%, P > 0.05) provoked high lethality. These data demonstrate that in the absence of adjuvant, E. coli plus C. albicans provoked synergistic lethality. However, in the presence of hemoglobin-mucin the synergistic effect was no longer observed. Therefore, this study provides support for the contention that C. albicans is capable of acting as a copathogen during experimental peritonitis, but that this effect may be obscured by the presence of an adjuvant substance that itself markedly potentiates microbial growth.

Immunization with antibodies that mimic LPS protects against gram negative bacterial sepsis.

We developed 9H1.B11, an anti-idiotypic anti-deep core/lipid A (DCLA), murine monoclonal antibody (mAb) that mimics the conserved DCLA region of lipopolysaccharide (LPS). It recognizes an epitope in the variable region of an DCLA mAb, binds to the murine macrophage cell surface, and inhibits LPS-induced macrophage cytokine secretion. We hypothesized that (1) active immunization with mAb 9H1.B11 would be associated with the development of anti-DCLA antibodies and (2) immunization would protect against subsequent gram negative bacterial challenge. Mice were immunized for 8 weeks before intraperitoneal (ip) challenge with Escherichia coli O111:B4 bacteria. Control animals were immunized with an irrelevant IgM antibody 8133 (negative control) or with LPS derived from Salmonella minnesota Re bacteria (positive control). Sera from immunized mice were collected, and titers against the core region of LPS (Re) and against LPS derived from the infecting E. coli strain were determined. Mice immunized with mAb 9H1.B11 developed measurable titers against S. minnesota Re LPS but not against the challenge strain of E. coli. However, immunization with 9H1.B11 on S. minnesota Re LPS protected against subsequent infection due to E. coli O111:B4 (100% survival). The group of mice immunized with IgM 8133 exhibited only 25% survival. The development of an anti-S. minnesota Re LPS titer after immunization with 9H1.B11 provides further evidence that a portion of 9H1.B11 mimics the conserved DCLA region of LPS. We believe that this approach holds considerable promise and plan further studies to define the mechanism by which protective capacity occurs.

Macrophages expressing a fusion protein derived from bactericidal/permeability-increasing protein and IgG are resistant to endotoxin.

OBJECTIVES: To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor alpha (TNF-alpha) secretion. DESIGN: A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-alpha protein secreted was measured and compared with that secreted by cells transfected with a control construct. SETTING: Basic science research laboratory. MAIN OUTCOME MEASURES: Secreted TNF-alpha protein concentration. RESULTS: After transfection, RAW 264.7-cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-alpha than did cells transfected with a control construct. CONCLUSIONS: The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-alpha secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients.

Differential sensitivity to Escherichia coli infection in mice lacking tumor necrosis factor p55 or interleukin-1 p80 receptors.

OBJECTIVE: To determine the effect of targeted disruption of the cellular receptors of either tumor necrosis factor alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta) during experimental gram-negative bacterial infection and endotoxemia. DESIGN: Transgenic (knockout [KO]) mice deficient in either the p55 TNF receptor (TNF RI) or the p80 IL-1 receptor (IL-1 RI) were challenged with intravenous lipopolysaccharide (endotoxin) or intraperitoneal live Escherichia coli 0111:B4. Mortality was assessed daily for 7 days. Serum endotoxin levels and quantitative blood cultures were monitored at multiple times during infection. SETTING: Surgical infectious disease research laboratory. MAIN OUTCOME MEASURES: Mortality, results of quantitative blood cultures, and serum endotoxin levels. RESULTS: Both TNF and IL-1 RI KO mice were resistant to endotoxin challenge (0% mortality for both groups) compared with control mice (100% mortality [P < .01]). In contrast, only the IL-1 RI KO mice were resistant to infection caused by viable gram-negative bacteria (43% mortality) compared with control mice (100% mortality [P < .01]). Infection led to 100% mortality in TNF RI KO mice. The IL-1 RI KO mice exhibited less bacteremia and diminished endotoxemia compared with control and TNF RI KO mice 18 and 24 hours after infection. CONCLUSION: The absence of either the TNF or the IL-1 RI receptor prevents cellular activation by each respective cytokine. Absence confers protection against intravenous endotoxin, which stimulates massive rapid release of cytokines into the systemic circulation. However, bacterial infection within the peritoneal cavity is known to cause more delayed cytokine release, and cytokines may act at the site of infection to enhance host defenses. We believe that IL-1 signaling may be more critical in provoking lethal systemic toxic effects than TNF signaling. However, TNF signaling may be an important component of host defense enhancement at the local site of infection.