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Achromobacter xylosoxidans and Pseudomonas aeruginosa biofilms can develop in ophthalmic products and accessories such as contact lens cases, leading to the development of ocular infections. This study evaluated the efficacy of the antimicrobials polyaminopropyl biguanide (PAPB) and benzalkonium chloride (BAK) against A. xylosoxidans and P. aeruginosa biofilms.Biofilms of A. xylosoxidans and P. aeruginosa used as a comparative control were formed by incubating the bacteria on contact lens cases and on coverslips in phosphate-buffered saline. The biofilms were then exposed to PAPB and BAK for 5 minutes and 4 hours. After exposure, alginate swabs were used to remove the biofilms from the lens cases and the bacteria were plated on tryptic soy agar for determination of survivors. Also, after exposure to these disinfectants, the A. xylosoxidans and P. aeruginosa biofilms were stained with SYTO 9 and propidium iodide. Using a confocal microscope with a 488-nm laser, the number of cells with damaged cell membranes was determined.After 5 minutes of exposure to BAK or PAPB, A. xylosoxidans biofilms were more resistant to the antimicrobial effects of these disinfectants than P. aeruginosa biofilms. After 4 hours, both organisms were reduced by more than 3 logs after exposure to either BAK or PAPB. Confocal microscopy studies revealed that BAK was more effective at damaging A. xylosoxidans and P. aeruginosa cell membranes than PAPB at the concentrations used in ophthalmic products.Biofilms of the emerging pathogen A. xylosoxidans were more resistant to the disinfectants PAPB and BAK than biofilms of P. aeruginosa. Because of the emergence of A. xylosoxidans and the demonstrated greater resistance to the common ophthalmic preservatives BAK and PAPB than the standard Gram-negative organism P. aeruginosa, A. xylosoxidans biofilms should be assessed in antimicrobial challenge tests to assure the safety of multiuse ophthalmic products.