Perioperative thrombocytopenia: evidence, evaluation, and emerging therapies.

Thrombocytopenia is a common perioperative clinical problem. While global haemostasis is influenced by many patient- and procedure-related factors, the contribution of thrombocytopenia to bleeding risk is difficult to predict, as platelet count does not linearly correlate with likelihood of bleeding. Thus, the widely used definition of thrombocytopenia and grading of its severity have limited clinical utility. We present a summary and analysis of the current recommendations for invasive procedures in thrombocytopenic patients, although the platelet count at which any given procedure may safely proceed is unknown. The benefits and risks of preoperative platelet transfusions should be assessed on a patient-by-patient basis, and alternatives to platelet transfusion should be considered. In non-emergent surgeries or in postoperative thrombocytopenic patients, haematology consultation should be considered to guide diagnostics and management. We present a pragmatic approach to the evaluation of perioperative thrombocytopenia.

Rearrangement of a large novel Pseudomonas aeruginosa gene island in strains isolated from a patient developing ventilator-associated pneumonia.

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative ß-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNA(lys) recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting.

Differences in biofilm formation and antimicrobial resistance of Pseudomonas aeruginosa isolated from airways of mechanically ventilated patients and cystic fibrosis patients.

Pseudomonas aeruginosa biofilms exhibit increased antimicrobial resistance compared with planktonic isolates and are implicated in the pathogenesis of both acute and chronic lung infections. Whilst antibiotic choices for both infections are based on planktonic antibiotic susceptibility results, differences in biofilm-forming ability between the two diseases have not previously been explored. The aim of this study was to compare differences in biofilm formation and antibiotic resistance of P. aeruginosa isolated from intubated patients and from patients with chronic pulmonary disease associated with cystic fibrosis (CF). The temporal evolution of antibiotic resistance in clonal P. aeruginosa strains isolated from CF patients during periods of chronic infection and acute pulmonary exacerbation was also evaluated. Biofilm formation and biofilm antibiotic susceptibilities were determined using a modified microtitre plate assay and were compared with antibiotic susceptibility results obtained using traditional planktonic culture. Clonality was confirmed using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Pseudomonas aeruginosa isolates collected from intubated patients produced substantially more biofilms compared with CF isolates. There was considerable heterogeneity in biofilm-forming ability amongst the CF isolates and this was unrelated to pulmonary status. Biofilm antibiotic resistance developed rapidly amongst clonal CF isolates over time, whilst traditional antibiotic resistance determined using planktonic cultures remained stable. There was a significant positive correlation between imipenem/cilastatin and ceftazidime resistance and biofilm-forming ability. The variability in biofilm-forming ability in P. aeruginosa and the rapid evolution of biofilm resistance may require consideration when choosing antibiotic therapy for newly intubated patients and CF patients.

Pseudomonas aeruginosa: acute lung injury or ventilator-associated pneumonia?

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative, aerobic, rod-shaped and polar-flagella bacterium with unipolar motility. Furthermore, it is an opportunistic pathogen responsible for ventilator-acquired pneumonia (VAP). VAP due to P. aeruginosa is usually multidrug-resistant and associated with severe infection and increased mortality. The goals of this review are as follows: (I) to present selected recent epidemiological literature characterizing the population at risk of P. aeruginosa pneumonia, (II) to describe some of the virulence factors of P. aeruginosa that are related to infection, and (III) to illustrate clinical outcomes of ventilated patients with large burdens of P. aeruginosa as well as the mechanism by which this bacterium may evade the host immune response.

Secretion of Pseudomonas aeruginosa type III cytotoxins is dependent on pseudomonas quinolone signal concentration.

Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent post-translational control, specifically governing type III cytotoxin secretion, exists in this species.

A prospective study of ventilator-associated pneumonia in children.

OBJECTIVE: We conducted a prospective, observational study in a tertiary care pediatric center to determine risk factors for the development of and outcomes from ventilator-associated pneumonia. METHODS: From November 2004 to June 2005, all NICU and PICU patients mechanically ventilated for >24 hours were eligible for enrollment after parental consent. The primary outcome measure was the development of ventilator-associated pneumonia, which was defined by both Centers for Disease Control and Prevention/National Nosocomial Infections Surveillance criteria and clinician diagnosis. Secondary outcome measures were length of mechanical ventilation, hospital and ICU length of stay, hospital cost, and death. RESULTS: Fifty-eight patients were enrolled. The median age was 6 months, and 57% were boys. The most common ventilator-associated pneumonia organisms identified were Gram-negative bacteria (42%), Staphylococcus aureus (22%), and Haemophilus influenzae (11%). On multivariate analysis, female gender, postsurgical admission diagnosis, presence of enteral feeds, and use of narcotic medications were associated with ventilator-associated pneumonia. Patients with ventilator-associated pneumonia had greater need for mechanical ventilation (12 vs 22 median ventilator-free days), longer ICU length of stay (6 vs 13 median ICU-free days), higher total median hospital costs ($308,534 vs $252,652), and increased absolute hospital mortality (10.5% vs 2.4%) than those without ventilator-associated pneumonia. CONCLUSIONS: In mechanically ventilated, critically ill children, those with ventilator-associated pneumonia had a prolonged need for mechanical ventilation, a longer ICU stay, and a higher mortality rate. Female gender, postsurgical diagnosis, the use of narcotics, and the use of enteral feeds were associated with an increased risk of developing ventilator-associated pneumonia in these patients.

Comparison of type III secretion system virulence among fluoroquinolone-susceptible and -resistant clinical isolates of Pseudomonas aeruginosa.

Fluoroquinolone resistance and type III secretion system (TTSS) virulence are independently associated in Pseudomonas aeruginosa infections with poor patient outcomes. In the present study, the virulence of fluoroquinolone-susceptible and -resistant isolates of P. aeruginosa was compared, focusing on TTSS virulence. Clinical isolates (n = 45) exhibiting a broad range of susceptibilities to fluoroquinolones, with differing mechanisms of resistance and associated with varying disease sites, were selected for the study. PCR, Southern blot and western immunoblot analyses were performed to determine the presence of TTSS-encoding genes and secretion of gene products. The cytotoxicity of the clinical isolates towards human lung epithelial cells was also determined. Clinical isolates encoding only the exoS cytotoxin gene occurred more frequently than those encoding only exoU (62% vs. 27%; p 0.0007). Compared with exoS(+) isolates, exoU(+) isolates were more likely to be fluoroquinolone-resistant (92% vs. 61%, p 0.05) and to exhibit both a gyrA mutation and the efflux pump over-expressed (EPO) phenotype (91% vs. 59%; p 0.06). Almost all exoU(+) strains secreted ExoU and exhibited increased cytotoxicity compared with ExoS-secreting strains (7% vs. 92.5%, relative to a PA103 reference strain control). These data suggest that exoU(+) and fluoroquinolone resistance may be co-selected traits that result in highly virulent and resistant strains. Adverse outcomes associated with infections caused by fluoroquinolone-resistant strains may, in part, be attributable to this co-association, which warrants further clinical investigation.

Loss of bacterial diversity during antibiotic treatment of intubated patients colonized with Pseudomonas aeruginosa.

Management of airway infections caused by Pseudomonas aeruginosa is a serious clinical challenge, but little is known about the microbial ecology of airway infections in intubated patients. We analyzed bacterial diversity in endotracheal aspirates obtained from intubated patients colonized by P. aeruginosa by using 16S rRNA clone libraries and microarrays (PhyloChip) to determine changes in bacterial community compositions during antibiotic treatment. Bacterial 16S rRNA genes were absent from aspirates obtained from patients briefly intubated for elective surgery but were detected by PCR in samples from all patients intubated for longer periods. Sequencing of 16S rRNA clone libraries demonstrated the presence of many orally, nasally, and gastrointestinally associated bacteria, including known pathogens, in the lungs of patients colonized with P. aeruginosa. PhyloChip analysis detected the same organisms and many additional bacterial groups present at low abundance that were not detected in clone libraries. For each patient, both culture-independent methods showed that bacterial diversity decreased following the administration of antibiotics, and communities became dominated by a pulmonary pathogen. P. aeruginosa became the dominant species in six of seven patients studied, despite treatment of five of these six with antibiotics to which it was sensitive in vitro. Our data demonstrate that the loss of bacterial diversity under antibiotic selection is highly associated with the development of pneumonia in ventilated patients colonized with P. aeruginosa. Interestingly, PhyloChip analysis demonstrated reciprocal changes in abundance between P. aeruginosa and the class Bacilli, suggesting that these groups may compete for a similar ecological niche and suggesting possible mechanisms through which the loss of microbial diversity may directly contribute to pathogen selection and persistence.

Effect of anti-PcrV antibody in a murine chronic airway Pseudomonas aeruginosa infection model.

Pseudomonas aeruginosa is one of the most important pathogens in patients with chronic airway conditions, such as cystic fibrosis and diffuse panbronchiolitis. Type III secretion system-mediated virulence factors contribute to the lung damage in chronic P. aeruginosa infection. The effects of the anti-PcrV immunoglobulin (Ig)G, which blocks the type III secretion system, were evaluated in a mouse model of chronic P. aeruginosa infection. On bacteriological examination, anti-PcrV IgG showed no bactericidal effects. On bronchoalveolar lavage fluid (BALF) analysis, total cell number and neutrophil ratios in the anti-PcrV IgG-treated groups were lower than those in the control group. In addition, macrophage inflammatory protein-2, tumour necrosis factor-alpha, and interleukin-beta concentrations in BALF were lower in the anti-PcrV IgG-treated groups when compared with controls. Plasma anti-PcrV IgG titre was elevated after administration of anti-PcrV IgG. Although plasma titre decreased gradually, a significant concentration was maintained during the experimental period. These data suggest that anti-PcrV immunoglobulin G reduces the inflammatory reaction caused by chronic Pseudomonas aeruginosa respiratory infection and may be useful in treating respiratory diseases.

Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.

Targeting mechanisms of Pseudomonas aeruginosa pathogenesis.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for ventilator-acquired pneumonia, acute lower respiratory tract infections in immunocompromised patients and chronic respiratory infections in cystic fibrosis patients. High incidence, infection severity and increasing resistance characterize P. aeruginosa infections, highlighting the need for new therapeutic options. One such option is to target the many pathogenic mechanisms conferred to P. aeruginosa by its large genome encoding many different virulence factors. This article reviews the pathogenic mechanisms and potential therapies targeting these mechanisms in P. aeruginosa respiratory infections.

Therapeutic administration of anti-PcrV F(ab')(2) in sepsis associated with Pseudomonas aeruginosa.

The effects of rabbit-derived polyclonal Ab against PcrV, a protein involved in the translocation of type III secreted toxins of Pseudomonas aeruginosa, was investigated in two animal models of P. aeruginosa sepsis. In a mouse survival study, the i.v. administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P. aeruginosa resulted in the complete survival of the animals. In a rabbit model of septic shock associated with Pseudomonas-induced lung injury, animals treated with anti-PcrV IgG intratracheally or i.v. had significant decreases in lung injury, bacteremia, and plasma TNF-alpha and significant improvement in the hemodynamic parameters associated with shock compared with animals treated in a similar manner with nonspecific control IgG. The administration of anti-PcrV F(ab')(2) showed protective effects comparable to those of whole anti-PcrV IgG. These results document that the therapeutic administration of anti-PcrV IgG blocks the type III secretion system-mediated virulence of P. aeruginosa and prevents septic shock and death, and that these protective effects are largely Fc independent. We conclude that Ab therapy neutralizing the type III secretion system has significant potential against lethal P. aeruginosa infections.

A randomized, controlled trial of advanced care planning discussions during preoperative evaluations.

BACKGROUND: Although many patients and physicians support the concept of advance care planning, only a small percentage of patients actually have the necessary discussion with health care providers. Hospital-based physicians other than primary care providers often are needed to increase physician, patient, and proxy communication about advanced directives. This study evaluated the effectiveness of a 5-10-min discussion designed to foster dialogue between patients and their proxies in a preoperative evaluation clinic. The discussions were lead by anesthesiologists. METHODS: A randomized controlled trial was conducted from September 1998 through May 1999 in a preoperative evaluation clinic at University of California, San Francisco, a tertiary care center. English-speaking patients aged 65 yr or older who were scheduled for elective surgery were randomized to receive a short information session stressing the importance of communication about end-of-life care between the patients and their proxies. Patients randomized to the control group received the standard preoperative anesthesia screening. An admitting counselor questioned all patients (control and intervention) about whether they have an advanced directive as part of the registration process before their arrival in clinic. RESULTS: The intervention significantly increased discussions about end-of-life care between patients and their proxies. Eighty seven percent of patients reported having discussions with their proxies as compared with only 66% of control patients (P = 0.001). The intervention also increased durable power of attorney completion rate to 27% as compared with 10% completion rate by controls. CONCLUSIONS: The preoperative evaluation period can be an opportunity to encourage patient and proxy communication about end-of-life care.

Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR.

The development of gene targeting strategies for specific modification of genomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologous replacement (SFHR), directly targets and modifies specific genomic sequences with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach has been effective for the in vitro modification of exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in human airway epithelial cells. As another step in the development of SFHR for gene therapy, studies were carried out to target and modify specific genomic sequences in exon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), homologous to mCFTR except for a 3-bp deletion (DeltaF508) and a silent mutation which introduces a unique restriction site (KpnI), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAMINE, DDAB, SuperFect). Successful modification was determined by PCR amplification of DNA or mRNA-derived cDNA followed by KpnI digestion. The results of these studies showed that SFHR can be used as a gene therapy to introduce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence.

Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections.

The ability of Pseudomonas aeruginosa to secrete specific toxins using the type III-mediated pathway has been reported. To determine the association of this phenotype with human illness, immunoblot analysis was used to detect expression of type III secretory proteins in P. aeruginosa isolates from respiratory tract or blood cultures of 108 consecutive patients. Relative risk of mortality was 6-fold greater with expression of the type III secretory proteins ExoS, ExoT, ExoU, or PcrV. Phenotype was independently correlated with toxicity in cellular and murine models. Prevalence of this phenotype was significantly higher in acutely infected patients than in chronically infected patients with cystic fibrosis. These results suggest that the type III protein secretion system is integral to increased P. aeruginosa virulence. A positive phenotype is a predictor of poor clinical outcome. In the future, such analyses may help distinguish potentially lethal infection from colonization and help determine appropriate therapy for critically ill patients.

Effects of antibiotic therapy on Pseudomonas aeruginosa-induced lung injury in a rat model.

The effect of antibiotics on the acute lung injury induced by virulent Pseudomonas aeruginosa PA103 was quantitatively analyzed in a rat model. Lung injury was induced by the instillation of PA103 directly into the right lower lobes of the lungs of anesthetized rats. The alveolar epithelial injury, extravascular lung water, and total plasma equivalents were measured as separate, independent parameters of acute lung injury. Four hours after the instillation of PA103, all the parameters were increased linearly depending on the dose of P. aeruginosa. Next, we examined the effects of intravenously administered antibiotics on the parameters of acute lung injury in D-galactosamine-sensitized rats. One hour after the rats received 10(7) CFU of PA103, an intravenous bolus injection of aztreonam (60 mg/kg) or imipenem-cilastatin (30 mg/kg) was administered. Despite an MIC indicating resistance, imipenem-cilastatin improved all the measurements of lung injury; in contrast, aztreonam, which had an MIC indicating sensitivity, did not improve any of the lung injury parameters. The antibiotics did not generate different quantities of plasma endotoxin; therefore, endotoxin did not appear to explain the differences in lung injury. This in vivo model is useful to quantitatively compare the efficacies of parenteral antibiotic administration on Pseudomonas airspace infections.

Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia.

The pathogenesis of septic shock occurring after Pseudomonas aeruginosa pneumonia was studied in a rabbit model. The airspace instillation of the cytotoxic P. aeruginosa strain PA103 into the rabbit caused a consistent alveolar epithelial injury, progressive bacteremia, and septic shock. The lung instillation of a noncytotoxic, isogenic mutant strain (PA103DeltaUT), which is defective for production of type III secreted toxins, did not cause either systemic inflammatory response or septic shock, despite a potent inflammatory response in the lung. The intravenous injection of PA103 did not cause shock or an increase in TNF-alpha, despite the fact that the animals were bacteremic. The systemic administration of either anti-TNF-alpha serum or recombinant human IL-10 improved both septic shock and bacteremia in the animals that were instilled with PA103. Radiolabeled TNF-alpha instilled in the lung significantly leaked into the circulation only in the presence of alveolar epithelial injury. We conclude that injury to the alveolar epithelium allows the release of proinflammatory mediators into the circulation that are primarily responsible for septic shock. Our results demonstrate the importance of compartmentalization of inflammatory mediators in the lung, and the crucial role of bacterial cytotoxins in causing alveolar epithelial damage in the pathogenesis of acute septic shock in P. aeruginosa pneumonia.

The effects of two antiinflammatory pretreatments on bacterial-induced lung injury.

BACKGROUND: Two antiinflammatory therapies that have been effective in preventing acid-induced lung injury were evaluated. Specifically, their effects on a subsequent bacterial-airspace challenge were compared. Bacteria were instilled 24 h after acid-induced lung injury. Pseudomonas aeruginosa PAO-1 was used as the bacteria, because its effects in healthy lungs was documented previously. METHODS: New Zealand white rabbits were anesthetized and three pretreatments were administered: (1) pentoxifylline pretreatment (a 20-mg/kg bolus dose and then 6 mg x kg(-1) x h(-1) given intravenously), (2) 1 ml anti-tumor necrosis factor alpha antiserum given intravenously, or (3) normal saline given intravenously. The pretreatment doses were shown previously to prevent acid-induced lung injury. Then 1.2 ml/kg hydrochloric acid (HCl), pH 1.25, was instilled into the rabbits' right lungs. All the animals underwent mechanical ventilation for 8 h. Twenty-four hours after the acid instillation, the rabbits were anesthetized again and 2 ml/kg (10(9) colony forming units/ml) PAO-1 was instilled into their left lungs. The rabbits' breathing was aided by mechanical ventilation for another 8 h, and then they were killed and exsanguinated. RESULTS: Both pretreatments attenuated the acid-induced lung injury of the noninstilled left lungs. Arterial oxygen tension and the lung edema of pretreated, acid-exposed animals were significantly and almost equally improved (compared with no pretreatments) by either of the pretreatments. However, when the bacteria were instilled into the left lungs 24 h after the acid injury, the pentoxifylline pretreatment but not the anti-tumor necrosis factor alpha pretreatment prevented much of the bacteria-induced lung injury. Pentoxifylline pretreatment significantly improved the measurements of left lung edema and epithelial and endothelial permeability. There was also a trend for improved oxygenation in the pentoxifylline-pretreated and infected animals. In contrast, the anti-tumor necrosis factor alpha pretreatment did not prevent the bacteria-induced lung injury and increased some of the measurements of lung injury. CONCLUSIONS: Two antiinflammatory therapies that prevented acid-induced lung injury to the noninstilled left lungs had significantly different effects on a subsequent bacteria-induced lung injury to the left lungs. The therapies differed in their mechanism of tumor necrosis factor alpha blockade, and this may have affected the bacteria-induced injury to the lungs.

Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Damage to the lung epithelium is associated with the expression of toxins that are directly injected into eukaryotic cells through a type Ill-mediated secretion and translocation mechanism. Here we show that the P. aeruginosa homolog of the Yersinia V antigen, PcrV, is involved in the translocation of type III toxins. Vaccination against PcrV ensured the survival of challenged mice and decreased lung inflammation and injury. Antibodies to PcrV inhibited the translocation of type III toxins.

Evaluation of antimicrobial and lipopolysaccharide-neutralizing effects of a synthetic CAP18 fragment against Pseudomonas aeruginosa in a mouse model.

CAP18 (cationic antimicrobial protein; 18 kDa) is a neutrophil-derived protein that can bind to and inhibit various activities of lipopolysaccharide (LPS). The 37 C-terminal amino acids of CAP18 make up the LPS-binding domain. A truncated 32-amino-acid C-terminal fragment of CAP18 had potent activity against Pseudomonas aeruginosa in vitro. We studied the antimicrobial and LPS-neutralizing effects of this synthetic truncated CAP18 peptide (CAP18106-137) on lung injury in mice infected with cytotoxic P. aeruginosa. To determine its maximal effect, the CAP18106-137 peptide was mixed with bacteria just prior to tracheal instillation, and lung injury was evaluated by determining the amount of leakage of an alveolar protein tracer (125I-albumin) into the circulation and by the quantification of lung edema. The lung injury caused by the instillation of 5 x 10(5) CFU of P. aeruginosa was significantly reduced by the concomitant instillation of CAP18106-137. However, the administration of CAP18106-137 alone, without bacteria, induced lung edema, suggesting that it has some toxicity. Also, the peptide did not significantly reduce the number of bacteria that had been simultaneously instilled, nor did it significantly improve the survival of the infected mice. The addition of CAP18106-137 to aztreonam along with the bacteria did decrease the level of antibiotic-induced release of inflammatory mediators including tumor necrosis factor alpha, interleukin-6, and nitric oxide and also improved the survival of the mice. Therefore, more investigations are needed to confirm the toxicities and the therapeutic benefits of CAP18106-137 as an adjunctive therapy to antibiotics in the treatment of infections caused by gram-negative bacteria.