[show abstract][hide abstract] ABSTRACT: Airway diseases often feature persistent neutrophilic inflammation and infection. In cystic fibrosis bronchitis, for example, Pseudomonas aeruginosa is isolated frequently. Previously, this laboratory revealed that neutrophils become major sources of histamine in mice with tracheobronchitis caused by the wall-less bacterium Mycoplasma pulmonis. To test the hypothesis that more-broadly pathogenic P. aeruginosa (which expresses cell wall-associated LPS and novel toxins) has similar effects, we incubated naïve mouse neutrophils with two strains of P. aeruginosa. Strain PAO1 greatly increased neutrophil histamine content and secretion, whereas strain PA103 depressed histamine production by killing neutrophils. The histamine-stimulating capacity of PAO1, but not PA103-mediated toxicity, persisted in heat-killed organisms. In PAO1-infected mice, lung and neutrophil histamine content increased. However, PAO1 did not alter production by mast cells (classical histamine reservoirs), which also resisted PA103 toxicity. To explore mechanisms of neutrophil-selective induction, we measured changes in mRNA encoding histidine decarboxylase (rate-limiting for histamine synthesis), probed involvement of endotoxin-TLR pathways in Myd88-deficient neutrophils, and examined contributions of pyocyanin and exotoxins. Results revealed that PAO1 increased histamine production by up-regulating histidine decarboxylase mRNA via pathways largely independent of TLR, pyocyanin, and type III secretion system exotoxins. PAO1 also increased histidine decarboxylase mRNA in neutrophils purified from infected lung. Stimulation required direct contact with neutrophils and was blocked by phagocytosis inhibitor cytochalasin D. In summary, Pseudomonas-augmented histamine production by neutrophils is strain-dependent in vitro and likely mediated by up-regulation of histidine decarboxylase. These findings raise the possibility that Pseudomonas-stimulated neutrophils can enhance airway inflammation by producing histamine.
Journal of leukocyte biology 11/2011; 91(2):275-84. · 4.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Methicillin-resistant STAPHYLOCOCCUS AUREUS (MRSA) and PSEUDOMONAS AERUGINOSA are key pathogens in hospitals (particularly intensive care units), in long-term care facilities, and in outpatients with specific comorbidities and risk factors. Both MRSA and P. AERUGINOSA display resistance to a wide array of antibiotics. Further, both bacteria contain a variety of virulence products or systems that make it difficult to treat associated infections. Within the past several years, community-acquired MRSA containing virulence factors [particularly the Panton-Valentine leukocidin (PVL) gene] has emerged globally. Given the limited number of novel antibiotics to treat antibiotic-resistant organisms, there is growing interest in treating bacterial infections by targeting specific virulence products or systems. This article reviews potential therapeutic targets in the virulence systems of these two bacteria that are responsible for a large number of serious infections in critically ill patients.
Seminars in Respiratory and Critical Care Medicine 04/2011; 32(2):228-35. · 2.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International journal of antimicrobial agents 03/2011; 37(4):309-15. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Improvement in lung function after macrolide antibiotic therapy has been attributed to reduction in bronchial infection by specific bacteria. However, the airway might be populated by a more diverse microbiota, and clinical features of asthma might be associated with characteristics of the airway microbiota present.
We sought to determine whether relationships exist between the composition of the airway bacterial microbiota and clinical features of asthma using culture-independent tools capable of detecting the presence and relative abundance of most known bacteria.
In this pilot study bronchial epithelial brushings were collected from 65 adults with suboptimally controlled asthma participating in a multicenter study of the effects of clarithromycin on asthma control and 10 healthy control subjects. A combination of high-density 16S ribosomal RNA microarray and parallel clone library-sequencing analysis was used to profile the microbiota and examine relationships with clinical measurements.
Compared with control subjects, 16S ribosomal RNA amplicon concentrations (a proxy for bacterial burden) and bacterial diversity were significantly higher among asthmatic patients. In multivariate analyses airway microbiota composition and diversity were significantly correlated with bronchial hyperresponsiveness. Specifically, the relative abundance of particular phylotypes, including members of the Comamonadaceae, Sphingomonadaceae, Oxalobacteraceae, and other bacterial families were highly correlated with the degree of bronchial hyperresponsiveness. Conclusion: The composition of bronchial airway microbiota is associated with the degree of bronchial hyperresponsiveness among patients with suboptimally controlled asthma. These findings support the need for further functional studies to examine the potential contribution of members of the airway microbiota in asthma pathogenesis.
The Journal of allergy and clinical immunology 02/2011; 127(2):372-381.e1-3. · 9.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: To compare the ability of four biomarkers to distinguish between those with ventilator-associated pneumonia (VAP) vs. lower respiratory tract bacterial colonization in mechanically ventilated intensive care unit (ICU) pediatric patients.
Prospective, pilot cohort study.
Tertiary care children's hospital, pediatric ICU.
All pediatric ICU patients mechanically ventilated > 48 hrs were eligible for enrollment between April 2006 to May 2007. Thirty-three patients were consecutively screened and enrolled after institutional consent process.
VAP was defined by both Centers for Disease and Prevention/National Nosocomial Infections Surveillance criteria and clinician diagnosis; those not meeting the criteria were considered to be colonized. Plasminogen activation inhibitor (PAI-1), soluble triggering receptor expressed on myeloid cells, receptor for advanced glycation end-products, and surfactant protein D levels were measured in bronchoalveolar lavage samples on average within 24 hrs of suspicion for VAP, i.e., a positive screening endotracheal Gram stain. Sixteen patients were diagnosed with VAP and 17 met the criteria for colonization. PAI-1 was associated with VAP independent of age, sex, race, acute lung injury/acute respiratory distress syndrome, Pediatric Risk of Mortality 3 score, pediatric logistic organ dysfunction score, and duration of intubation. The receiver operating characteristics for PAI-1 showed good discrimination with an area under the curve of 0.82. PAI-1 levels of ≥ 2.8 ng/mL had a sensitivity of 81.3%, specificity of 76.5%, and positive likelihood ratio of 3.5. Levels of soluble triggering receptor expressed on myeloid cells, receptor for advanced glycation end-products, and surfactant protein D were not significantly associated with VAP.
In mechanically ventilated pediatric ICU patients, PAI-1 is independently associated with the diagnosis of VAP. Real-time measurement of PAI-1 levels in bronchoalveolar lavage fluid may be of benefit in the early diagnosis and subsequent treatment of VAP in ICU patients.
Pediatric Critical Care Medicine 01/2011; 12(1):21-7. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The determination of reliable biomarkers capable to predict clinical outcome of a trauma patient remains essential toward better therapeutic management of the patient in the intensive care unit. Assessment of global metabolic profiling using quantitative nuclear magnetic resonance (NMR)-based metabolomics offers an attractive modern methodology for fast and comprehensive determination of multiple circulating metabolites and for establishing metabolic phenotype of survivors versus nonsurvivors. Multivariate data analysis on 43 quantitative metabolic parameters identified three lipid metabolites, triacylglycerol, glycerol heads of phospholipids, and monounsaturated fatty acids, as being the most discriminative markers to separate survivors versus nonsurvivors at the time of admission. Glucose and glutamate were intermediate predictors, followed by lactate and hydroxybutyrate as two low-weight predictors. Ultimately, cellular and subcellular failure in nonsurviving trauma patients results in multiple systemic biochemical effects and in changes in circulating metabolites in the blood that are characteristic for decreased lipid synthesis and urea cycle activity in the liver, and for increased hyperglycemia, lactic, and ketoacidosis.
The Journal of trauma 07/2010; 69(1):31-40. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: The type III secretion system of Pseudomonas aeruginosa, responsible for acute infection, is composed of over twenty proteins that facilitate cytotoxin injection directly into host cells. Integral to this process is production and secretion of PcrV. Administration of a recently developed, anti-PcrV immunoglobulin, either as a therapeutic or prophylactic has previously demonstrated efficacy against laboratory strains of P. aeruginosa in a murine model. To determine if this therapy is universally applicable to a variety of P. aeruginosa clinical isolates, genetic heterogeneity of pcrV was analyzed among strains collected from three geographically distinct regions; United States, France and Japan. Sequence analysis of PcrV demonstrated limited variation among the clinical isolates examined. Strains were grouped according to the presence of non-synonymous single nucleotide polymorphisms. Representative isolates from each mutant group were examined for the ability of anti-PcrV to bind the protein secreted by these strains. The protective effect of anti-PcrV IgG against each strain was determined using an epithelial cell line cytotoxicity assay. The majority of strains tested demonstrated reduced cytotoxicity in the presence of anti-PcrV IgG. This study provides insights into the natural sequence variability of PcrV and an initial indication of the amino acid residues that appear to be conserved across strains. It also demonstrates the protective effect of anti-PcrV immunotherapy against a multitude of P. aeruginosa strains from diverse global regions with a variety of mutations in PcrV.
[show abstract][hide abstract] ABSTRACT: Acute exacerbations of chronic obstructive pulmonary disease (COPD) are a major source of morbidity and contribute significantly to healthcare costs. Although bacterial infections are implicated in nearly 50% of exacerbations, only a handful of pathogens have been consistently identified in COPD airways, primarily by culture-based methods, and the bacterial microbiota in acute exacerbations remains largely uncharacterized. The aim of this study was to comprehensively profile airway bacterial communities using a culture-independent microarray, the 16S rRNA PhyloChip, of a cohort of COPD patients requiring ventilatory support and antibiotic therapy for exacerbation-related respiratory failure. PhyloChip analysis revealed the presence of over 1,200 bacterial taxa representing 140 distinct families, many previously undetected in airway diseases; bacterial community composition was strongly influenced by the duration of intubation. A core community of 75 taxa was detected in all patients, many of which are known pathogens. Bacterial community diversity in COPD airways is substantially greater than previously recognized and includes a number of potential pathogens detected in the setting of antibiotic exposure. Comprehensive assessment of the COPD airway microbiota using high-throughput, culture-independent methods may prove key to understanding the relationships between airway bacterial colonization, acute exacerbation, and clinical outcomes in this and other chronic inflammatory airway diseases.
Omics: a journal of integrative biology 02/2010; 14(1):9-59. · 2.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mechanically ventilated trauma patients have a high risk for the development of ventilator-associated pneumonia (VAP). We have recently reported that reduced plasma protein C (PC) levels early after trauma/shock are associated with coagulopathy and mortality. Furthermore, trauma patients with tissue injury and shock are at higher risk for the development of VAP.
We hypothesized that low PC levels early after trauma are associated with an increased susceptibility to VAP in trauma patients.
Fifty-nine acutely injured, intubated trauma patients were admitted to the critical care unit. Serial blood samples were drawn and coagulation factors were measured. VAP was diagnosed by presence of bacteria on bronchial alveolar lavage specimen, bilateral infiltrates on chest roentgenogram, and fever or elevated white blood cell count.
There were no differences in demographic or injury characteristics between patients who developed VAP and those who did not. As expected, patients who developed VAP had more ventilator days, hospital days, intensive care unit days, and greater mortality (all p < 0.05). Patients in both groups had lower mean PC levels at 6 hours compared with baseline. Noninfected patients' PC subsequently returned to near baseline levels, whereas those patients who eventually acquired VAP had significantly lower PC levels at both 12 and 24 hours (12 hours: 79 vs. 96%, p = 0.05; 24 hours: 75 vs. 97% p = 0.02). Soluble endothelial PC receptor (sEPCR) levels were also lower at 24 hours (82 vs. 99% in the noninfected group, p = 0.04).
The activation of PC pathway early after trauma may protect the vascular endothelium by both its anticoagulant and cytoprotective effects. However, trauma patients who later developed VAP have significantly lower plasma levels of PC within 24 hours after injury, suggesting a possible consumption of this vitamin K-dependent protein and an inhibition of its activation by inflammatory mediators. EPCR is involved in the activation of PC and is also a mediator of its cytoprotective effects.
Critically ill trauma patients have an early activation of the PC pathway, associated with a rapid decrease in the plasma levels of this protein and increase in EPCR. Plasma levels of PC return to normal levels within 24 hours in most patients. However, patients who go on to acquire VAP have persistently low plasma levels of PC in the immediate period after trauma. Whether PC could play a mechanistic role in the host response against nosocomial lung infection warrants further study.
The Journal of trauma 12/2009; 67(6):1176-81. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: To consider the relevance to severe human lung infections of recently discovered virulence mechanisms of Staphylococcus aureus and Francisella tularensis.
S. aureus has long been considered an opportunistic pathogen. However, due to the emergence of community-acquired methicillin-resistant S. aureus (CA-MRSA) strains that can readily infect and kill normal hosts, S. aureus must now be considered a potentially virulent pathogen. The evolution of S. aureus from an organism associated with asymptomatic nasopharyngeal colonization to one associated with community-acquired lethal infections may reflect horizontal acquisition of bacterial genes that enable efficient spread, aggressive host invasion, and effective immune evasion. Alleviating the burden of staphylococcal disease will require better understanding of host susceptibility and of staphylococcal virulence and antibiotic resistance. In contrast to the rapidly evolving staphylococcal virulence strategy, recent genomic analysis of F. tularensis has revealed a small set of bacterial genes associated with the marked virulence of its North American subspecies. This suggests that a relatively stable strategy of immune evasion underlies this pathogen's ability to establish serious life-threatening lung infections from a very small inoculum.
Understanding bacterial pathogenesis will require additional research into both host susceptibility factors and bacterial virulence mechanisms, including horizontal gene transfer. Refinements in the molecular detection of bacteria in the clinical setting, as well as whole genome analysis of both pathogens and patients, are expected to aid in the understanding of bacterial-induced lung injury.
Current opinion in critical care 12/2009; 16(1):8-12. · 2.67 Impact Factor
[show abstract][hide abstract] ABSTRACT: Virulent P. aeruginosa strains express PcrV, one of the translocational components of the type III secretion system. PcrV has been reported to be a protective antigen against lethal P. aeruginosa infection. The PcrV region, which contributes to protective immunity against P. aeruginosa infection, was investigated by using genetically engineered, truncated PcrV proteins and affinity-purified anti-PcrV antibodies against the truncated PcrV proteins. The efficacy of active and passive immunization against PcrV was tested in mice with cyclophosphamide-induced immunosuppression by intraabdominal challenge of P. aeruginosa. Active immunization with either full-length PcrV1-294 or PcrV139-294 significantly improved the survival of mice infected with P. aeruginosa, while PcrV139-258, PcrV139-234, PcrV197-294, and PcrV261-294 were not protective. These results suggest that an effective PcrV vaccine needs to contain not only the Mab166 epitope (PcrV144-257) but also the carboxyl terminal tail of PcrV. In the case of passive immunization, administration of affinity-purified anti-PcrV IgG against either PcrV1-294 or PcrV139-258 showed significantly higher efficacy against lethal P. aeruginosa infection than did original anti-PcrV IgG and Mab166. The increased efficacy of affinity-purified anti-PcrV IgG implies that more potent anti-PcrV strategies are possible. The results of this study are crucial to the development of an effective PcrV vaccine for active immunization and to an appropriate blocking anti-PcrV antibody against P. aeruginosa infection in humans.
Microbiology and Immunology 11/2009; 53(11):587-94. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Higher plasma and pulmonary edema fluid levels of plasminogen activator inhibitor-1 (PAI-1) are associated with increased mortality in patients with pneumonia and acute lung injury. The 4G allele of the 4G/5G polymorphism of the PAI-1 gene is associated with higher PAI-1 levels and an increased incidence of hospitalizations for pneumonia. The authors hypothesized that the 4G allele would be associated with worse clinical outcomes (mortality and ventilator-free days) in patients with severe pneumonia.
The authors enrolled patients admitted with severe pneumonia in a prospective cohort. Patients were followed until hospital discharge. DNA was isolated from blood samples, and genotyping detection for the PAI-1 4G/5G polymorphism was carried out using Taqman-based allelic discrimination.
A total of 111 patients were available for analysis. Distribution of genotypes was 4G/4G 26 of 111 (23%), 4G/5G 59 of 111 (53%), and 5G/5G 26 of 111 (23%). Of 111 patients, 32 (29%) died before hospital discharge and 105 patients (94%) received mechanical ventilation. Patients with the 4G/4G and the 4G/5G genotypes had higher mortality (35% vs. 8%, P = 0.007) and fewer ventilator-free days (median 4 vs. 13, P = 0.04) compared to patients with the 5G/5G genotype.
The 4G allele of the 4G/5G polymorphism in the PAI-1 gene is associated with fewer ventilator-free days and increased mortality in hospitalized patients with severe pneumonia. These findings suggest that PAI-1 may have a role in pathogenesis and that the 4G/5G polymorphism may be an important biomarker of risk in patients with severe pneumonia.
[show abstract][hide abstract] ABSTRACT: In patients with ventilator-associated pneumonia (VAP), Pseudomonas aeruginosa type III (TTSS) secreting isolates have been linked to poor clinical outcomes. Differential expression of matrix metalloproteinases (MMPs) induced by type III effector proteins may herald an irreversible lung injury.
Serial bronchoalveolar lavage fluids collected from 41 patients with P. aeruginosa at onset of VAP, day 4, and day 8 after antibiotic therapy were assayed for MMP-8, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and alpha-2 macroglobulin levels.
At the onset of VAP, isolates secreting ExoU had the highest MMP-9 levels. The response to antimicrobial therapy showed a differential drop in MMPs with significant decrease in MMP-8 and MMP-9 levels on days 4 and 8 in patients with TTSS(-) compared to TTSS(+) phenotype. The ratio of MMP-9/TIMP-1 was significantly associated with alpha-2 macroglobulin at end of therapy (r=0.4, p=0.02). Patients who survived had a lower MMP-9/TIMP-1 ratio than those who died (p=0.003).
VAP linked to P. aeruginosa Type III phenotype portrays a divergent antibiotic treatment response in regards to the concentrations of metalloproteinases in the alveolar space. The imbalance between MMP-9 and TIMP-1 may determine the intensity of alveolocapillary damage and ultimate outcome of P. aeruginosa VAP.
The Journal of infection 06/2009; 59(1):49-55. · 4.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.
Infection and immunity 01/2009; 77(3):1083-90. · 4.21 Impact Factor