Background-Improvement in lung function following macrolide antibiotic therapy has been attributed to reduction in bronchial infection due to specific bacteria. However, the airway may be populated by a more diverse microbiota, and clinical features of asthma may be associated with characteristics of the airway microbiota present.

Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective “early” and “late” colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations.

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

PAI-1 concentrations in bBALs correlated with mortality in ventilated patients with positive cultures for P. aeruginosa. Elevated bBAL PAI-1 concentrations also correlated with the secretion of type III exotoxins by P. aeruginosa.

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 posttranslational control, specifically governing type III cytotoxin secretion, exists in this species.

Background Type 2 diabetes (diabetes) and its complications can sometimes be prevented, if identified and treated early. One fifth of diabetics in the U.S. remain undiagnosed. Commonly used screening guidelines are inconsistent. Purpose To examine the optimal age cut-point for opportunistic universal screening, compared to targeted screening, which is recommended by U.S. Preventive Services Task Force (USPSTF) and American Diabetes Association (ADA) guidelines. Methods Cross-sectional analysis of a nationally representative sample from the National Health and Nutrition Examination Survey, 2007–2010. Number of people needed to screen (NNS) to obtain one positive test result was calculated for different guidelines. Sampling weights were applied to construct national estimates. The 2010 Medicare fee schedule was used for cost estimation. Analysis was conducted in January 2014. Results NNS, under universal screening, drops sharply at age 35 years, from 80 (30–34-year-olds) to 31 (35–39-year-olds). Opportunistic universal screening of eligible people aged ≥35 years would yield a NNS of 15, translating to $66 per positive test. Among people aged 35–44 years (who are not recommended for universal screening by ADA), most (71%) were overweight or obese and all had at least one other ADA risk factor. Only 34% of individuals aged ≥35 years met USPSTF criteria. Strictly enforcing USPSTF guidelines would have resulted in a majority (61%) of potential positive tests cases being missed (5,508,164 cases nationwide). Conclusions Opportunistic universal screening among individuals aged ≥35 years could greatly reduce the national prevalence of undiagnosed pre-diabetes or diabetes at relatively low cost.

The type III secretion system of P. 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.

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