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Salina E. Salamah, Michael Kovach, Rachida Bouhenni; L -2-amino-4-methoxy-trans-3-butenoic acid Production by the Keratitis Associated Pseudomonasaeruginosa. Invest. Ophthalmol. Vis. Sci. 2018;59(9):503.
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We have previously reported elevated protein levels of non-ribosomal peptide synthetases (NRPS) in an ocular strain of Pseudomonas aeruginosa (P. aeruginosa) isolated from an active corneal infection (E3) compared to a lab strain ATCC10145 that lacked the proteins. NRPSs are responsible for the production of the toxin L -2-amino-4-methoxy-trans-3-butenoic acid (AMB). In this study, we sought to investigate AMB production by E3 compared to an NRPS mutant (ambE, PA2302) where P. aeruginosa PAO1 was used as the positive control and ATCC10145 as the non-pathogenic, non AMB producer negative control.
AMB production was assessed using a previously described assay based on the growth inhibition of E. coli K-12. P. aeruginosa strains (E3, PAO1, PA2302 and ATCC10145, n=5/strain) were grown in 1X Vogel-Bonner Minimal Media (MME) supplemented with succinate and with or without iron overnight at 37°C with shaking. The supernatant was filtered through a 0.22 µm polyvinylidene difluoride (PVDF) membrane and 75 µls were added to the disks. Disks were placed on 1X MME/succinate agar plates inoculated with E. coli ATCC 27325 (W3110 K12). Spot plate assay (antagonism assay) was performed in a similar way with and without methionine. One-way analysis of variance (ANOVA) was used for statistical analysis to determine significance in differences between strains.
Using the disk inhibition assay, E3 exhibited a significantly larger zone of inhibition than that seen in PAO1 and PA2302 (27.6 ±1.6 mm vs. 16.1±2.5 mm and 13.0±0.0 mm respectively, P<0.01). Addition of methionine to the medium significantly reversed the growth inhibition of E. coli ATCC 27325 by E3 (4.2±0.2 mm with methionine vs. 7.2±0.4 mm without methionine zone of inhibition to colony size ratio, P<0.01). No significant inhibition was noticed with PAO1 or PA2302.
Results from this study confirm that the keratitis associated P. aeruginosa strain inhibits E. coli K12 growth most likely due to AMB production. This study will add more insight into the pathogenesis of pseudomonas associated keratitis and will help develop novel antimicrobial agents for the treatment of ocular infections caused by P. aeruginosa such as those that result from the wear of contact lenses.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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