The Pseudomonas aeruginosa type III translocon is required for biofilm formation at the epithelial barrier.

Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection.

Management of pediatric multidrug-resistant tuberculosis and latent tuberculosis infections in New York City from 1995 to 2003.

BACKGROUND: Few studies have assessed the management and outcomes of multidrug-resistant tuberculosis (MDR-TB) in the pediatric population. Treatment of children with second-line TB drugs is complicated by potential toxicities of these agents. METHODS: We performed a retrospective study of children <15 years of age treated for MDR-TB or MDR-latent TB infection (LTBI) from 1995 to 2003. We reviewed the New York City Department of Health and Mental Hygiene (DOHMH) computerized TB registry to characterize demographic characteristics, clinical presentations, treatment, and outcomes of the study subjects. RESULTS: Twenty subjects with MDR-TB (mean age 2.7 years) and 51 with MDR-LTBI (mean age 9.8 years) were studied. The most commonly used second-line TB drugs were cycloserine, quinolone agents, and ethionamide, which were used in 70%, 69%, and 54% of subjects, respectively. Sixteen (80%) of 20 MDR-TB and 38 (75%) of 51 MDR-LTBI cases completed treatment. A greater proportion of subjects receiving care at a DOH clinic completed treatment for LTBI (36/41, 88%), when compared with subjects treated at non-DOH sites [(2/9, 22%) P < 0.001]. Review of the TB registry indicated that no subjects had recurrent disease or progression of LTBI to active disease during the study period and for 2 years thereafter. CONCLUSIONS: Children with MDR-TB and LTBI were best cared for in public health settings. A multicenter registry for pediatric MDR-TB and MDR-LTBI would be desirable to obtain accurate rates of toxicity and cure.

Host cell polarity proteins participate in innate immunity to Pseudomonas aeruginosa infection.

The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to external pathogens. The apical surface of the epithelium constitutes the first point of contact between mucosal pathogens, such as Pseudomonas aeruginosa, and their host. We observed that binding of P. aeruginosa aggregates to the apical surface of polarized cells led to the striking formation of an actin-rich membrane protrusion with inverted polarity, containing basolateral lipids and membrane components. Such protrusions were associated with a spatially localized host immune response to P. aeruginosa aggregates that required bacterial flagella and a type III secretion system apparatus. Host protrusions formed de novo underneath bacterial aggregates and involved the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NF-κB response. Our data reveal a role for spatiotemporal epithelial polarity changes in the activation of innate immune responses.