PURPOSE. Multi-species biofilms associated with contact lens cases and lenses can predispose individuals to contact lensrelated inflammatory complications. Our study used cultureindependent methods to assess the relationship between the severity of contact lens-related disease and bacteria residing in biofilms of contact lens cases and lenses.METHODS. Contact lens cases and lenses from 28 patients referred to the West Virginia University Eye Institute and diagnosed as having mild keratitis, keratitis with focal infiltrates, or corneal ulcers were processed and evaluated for bacterial composition based on 16S ribosomal RNA gene sequencing. Cases and lenses from nine asymptomatic contact lens wearers were processed in a manner similar to controls. Relationships between disease severity, bacterial types, and bacterial diversity were evaluated statistically.RESULTS. Disease severity and presenting visual acuity correlated with an increase in the diversity of bacterial types isolated from contact lens cases. A significant difference also was observed in the number of bacterial types associated with the three clinical groups. Achromobacter, Stenotrophomonas, and Delftia were prevalent in all disease groups, and Achromobacter and Stenotrophomonas were present in one asymptomatic control. Scanning electron microscopy revealed that Achromobacter and Stenotrophomonas formed a biofilm on the surface of contact lenses.CONCLUSIONS. Culture-independent methods identified an association between disease severity and bacterial diversity in biofilms isolated from cases and lenses of patients with contact lens-related corneal disease. Achromobacter, Stenotrophomonas, and Delftia were predominant bacteria identified in our study, drawing attention to their emerging role in contact lensrelated disease. (Invest Ophthalmol Vis Sci. 2012;53:3896-3905)

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Laboratory studies on a group of 20 patients from the Rio Negro Valley, Colombia selected for detailed study showed that 14 gave antibody reactions on immunoassay consistent with Trypanosoma cruzi or T. rangeli infections. Four were diagnosed as having T. rangeli infection, 4 had mixed infections and 6 were infected with T. cruzi alone. Immunoprecipitation analysis showed that sera from T. cruzi-infected patients recognized a similar range of trypomastigote-derived polypeptides as sera from patients in Brazil, and all of the Colombian sera reacted with the 160 kiloDalton (kDa) polypeptide associated with active infection. Although sera from patients with T. rangeli infection alone gave a positive immunofluorescence or ELISA reaction with T. rangeli, they failed to bind to parasite polypeptides by either immunoprecipitation or Western blotting. Intriguingly, sera from patients with mixed infections consistently gave a stronger, but qualitatively similar, binding reaction in immunoprecipitation and Western blotting compared to sera from patients infected with T. cruzi alone.

Due to increasing rates of invasive Salmonella enterica serovar Typhimurium infection, there is a need for an effective vaccine to prevent this disease. Previous studies showed that a mutation in the first gene of the Enterobacterial common antigen biosynthetic pathway, wecA, resulted in attenuation of S. Typhimurium in a murine model of salmonellosis. Furthermore, immunization with a wecA(-) strain protected against lethal challenge with the parental wild type S. Typhimurium strain. Herein, we examined whether the S. Typhimurium wecA(-) strain could also provide cross-protection against non-parental strains of S. Typhimurium and S. Enteritidis. We found that intraperitoneal immunization (IP) with S. Typhimurium SL1344 wecA(-) resulted in a significant increase in survival compared to control mice for all Salmonella challenge strains tested. Oral immunization with SL1344 wecA(-) also resulted in increased survival; however, protection was less significant than with intraperitoneal immunization. The increase in survival of SL1344 wecA(-) immunized mice was associated with a Salmonella-specific IgG antibody response. Furthermore, analysis of sera from IP and orally immunized animals revealed cross-reactive antibodies to numerous Salmonella isolates. Functional analysis of antibodies found within the sera from IP immunized animals revealed agglutination and opsonophagocytic activity against all tested O:4 Salmonella serovars. Together these results indicate that immunization with a S. Typhimurium wecA(-) strain confers protection against lethal challenge with wild type S. Typhimurium and S. Enteritidis and that immunization correlates with functional antibody production.

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration. INTRODUCTIONOriginally identified because of its role in Yersinia virulence (Cornelis et al., 1989), type III secretion (T3S) is now recognized to contribute to the pathogenesis of a large number of Gram-negative bacteria. T3S allows the direct translocation of 'effectors' from the bacterial cytosol into eukaryotic cells, enabling bacteria to manipulate host cells to establish infections while evading immune responses. The T3S system includes a bacterially formed 'injectisome' needle-like nanostructure that serves as a conduit for transferring bacterial effectors to eukaryotic cells. A bacterially formed 'translocon' channel is then believed to mediate effector translocation across host cell membranes. The mechanism underlying T3S translocon channel formation and host involvement in this process remain the least understood events in T3S. We have used the Abbreviations: CTB, cholera toxin B subunit; dsPFO, prepore locked PFO; HA, haemagglutinin; IF, immunofluorescent/immunofluorescence; IQGAP1, IQ-motif-containing GTPase-activating protein 1; LatB, latrunculin B; LE, leading edge; MbCD, methyl-beta-cyclodextrin; MT, microtubule; Pa-ExoS-HA, P. aeruginosa stra...