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PurposePreviously, we showed that tear fluid protects corneal epithelial cells against Pseudomonas aeruginosa without suppressing bacterial viability. Here, we studied how tear fluid affects bacterial gene expression.MethodsRNA-sequencing was used to study the P. aeruginosa transcriptome after tear fluid exposure (5 h, 37 oC). Outcomes were further investigated by biochemical and physiological perturbations to tear fluid and tear-like fluid (TLF) and assessment of bacterial viability following tear/TLF pretreatment and antibiotic exposure.ResultsTear fluid deregulated ~180 P. aeruginosa genes ≥8 fold versus PBS including downregulating lasI, rhlI, qscR (quorum sensing/virulence), oprH, phoP, phoQ (antimicrobial resistance) and arnBCADTEF (polymyxin B resistance). Upregulated genes included algF (biofilm formation) and hemO (iron acquisition). qPCR confirmed tear down-regulation of oprH, phoP and phoQ. Tear fluid pre-treatment increased P. aeruginosa resistance to meropenem ~5-fold (4 μg/ml), but enhanced polymyxin B susceptibility ~180-fold (1 μg/ml), the latter activity reduced by dilution in PBS. Media containing a subset of tear components (TLF) also sensitized bacteria to polymyxin B, but only ~22.5-fold, correlating with TLF/tear fluid Ca2+ and Mg2+ concentrations. Accordingly, phoQ mutants were not sensitized by TLF or tear fluid. Superior activity of tear fluid versus TLF against wild-type P. aeruginosa was heat resistant but proteinase K sensitive.ConclusionP. aeruginosa responds to human tear fluid by upregulating genes associated with bacterial survival and adaptation. Meanwhile, tear fluid down-regulates multiple virulence-associated genes. Tears also utilize divalent cations and heat resistant/proteinase K sensitive component(s) to enhance P. aeruginosa sensitivity to polymyxin B.

The cornea of the eye differs from other mucosal surfaces in that it lacks a viable bacterial microbiome and by its unusually high density of sensory nerve endings. Here, we explored the role of corneal nerves in preventing bacterial adhesion. Pharmacological and genetic methods were used to inhibit the function of corneal sensory nerves or their associated transient receptor potential cation channels TRPA1 and TRPV1. Impacts on bacterial adhesion, resident immune cells, and epithelial integrity were examined using fluorescent labeling and quantitative confocal imaging. TRPA1/TRPV1 double gene-knockout mice were more susceptible to adhesion of environmental bacteria and to that of deliberately-inoculated Pseudomonas aeruginosa. Supporting the involvement of TRPA1/TRPV1-expressing corneal nerves, P. aeruginosa adhesion was also promoted by treatment with bupivacaine, or ablation of TRPA1/TRPV1-expressing nerves using RTX. Moreover, TRPA1/TRPV1-dependent defense was abolished by enucleation which severs corneal nerves. High-resolution imaging showed normal corneal ultrastructure and surface-labeling by wheat-germ agglutinin for TRPA1/TRPV1 knockout murine corneas, and intact barrier function by absence of fluorescein staining. P. aeruginosa adhering to corneas after perturbation of nerve or TRPA1/TRPV1 function failed to penetrate the surface. Single gene-knockout mice showed roles for both TRPA1 and TRPV1, with TRPA1-/- more susceptible to P. aeruginosa adhesion while TRPV1-/- corneas instead accumulated environmental bacteria. Corneal CD45+/CD11c+ cell responses to P. aeruginosa challenge, previously shown to counter bacterial adhesion, also depended on TRPA1/TRPV1 and sensory nerves. Together, these results demonstrate roles for corneal nerves and TRPA1/TRPV1 in corneal resistance to bacterial adhesion in vivo and suggest that the mechanisms involve resident immune cell populations.

BACKGROUND: The tortuosity of nerve fibers has been shown to be important for identifying and monitoring clinically relevant manifestations resulting from of a variety of ocular and systemic disease pathologies and disorders. However, quantifying tortuosity in dense neurite networks can prove challenging, as existing methods require manual scoring and/or complete segmentation of the neurite network. NEW METHOD: We measured neurite tortuosity by quantifying the degree of directional coherence in the Fourier transform of segmented neurite masks. This allowed for the analysis of neurite tortuosity without requiring complete segmentation of the neurite network. We were also able to adapt this method to measure tortuosity at different length and size scales. RESULTS: With this novel method, neurite tortuosity was accurately quantified in simulated data sets at multiple length scales and scale variant and scale invariant tortuosity was accurately distinguished. Use of this method on images of murine corneal neurites correctly distinguished known differences between neurite tortuosity in the peripheral and central cornea. COMPARISON WITH EXISTING METHOD(S): Other methods require complete segmentation of neurites, which can be prohibitive in dense and/or sparsely labeled neurite networks such as in the cornea. Additionally, other methods require manual curation, manual scoring, or generation of a curated training set, while our novel method directly measures tortuosity as an intrinsic property of the image. CONCLUSIONS: We report the use of Fourier transforms for quantification of neurite tortuosity at multiple length scales, and with an image input that contains incompletely segmented neurites. This new method does not require manual training or curation, allowing a direct and rapid measurement of neurite tortuosity, thereby enhancing the accuracy and utility of neurite tortuosity measurements for evaluation of ocular and systemic disease pathology.

Teachers are responsible for meeting the needs of increasingly diverse learners. Given their position as catalysts for educational change, teachers' positive attitudes towards inclusive education must be considered prerequisite to its success in Australian classrooms. This study investigated the extent to which pre-service training affects pre-service primary teachers' attitudes towards inclusive education. A survey was designed to examine such attitudes among primary pre-service teachers at all year levels of their Bachelor of Education (Primary). To reflect the increasingly broad definition of inclusion established in the literature, participants' attitudes towards gifted and talented students, those learning English as a second language or dialect and those with disabilities were considered. Using a sample of 56 primary pre-service teachers from three metropolitan universities in Australia, this study examined the nature of these attitudes according to child, teacher and environment related variables across the training years. Results showed that primary pre-service teachers' attitudes towards inclusion were generally positive and strengthened across the training years, though they varied according to demographic characteristics, constructs and areas of inclusion. The findings of this study have implications for teacher educators, teacher education institutions and future research.