Comparative detection and recovery of Pseudomonas aeruginosa by membrane filtration and a Most Probable Number technique

Abstract

Two reference membrane filtration methods (Standard Methods: SM 9213E and U.S.EPA), the Most Probable Number method (Pseudalert®) and a membrane filtration method currently used by the Ontario Ministry of the Environment and Climate Change (MOECC) Laboratory Services Branch Etobicoke, Canada (Method E3371) were compared for the detection and recovery of Pseudomonas aeruginosa (P. aeruginosa) in sterile phosphate buffered dilution water (PBDW), un-spiked and spiked environmental water samples. The confirmation of presumptive-positive P. aeruginosa colonies was performed by API®20NE and on Milk Agar.Detection and recoveries were compared by determining the sensitivity and specificity of the methods and the performance of the methods was evaluated for statistical differences using ANOVA.The recovery of P. aeruginosa from PBDW spiked at a level of 100CFU/100mL was significantly higher (p<0.05) with Pseudalert® compared to the other methods. However, there were no significant differences (p>0.5) between all four methods when spiked at 5 and 40CFU/100mL.In the analysis of 61 environmental surface water samples, the MPN method (Pseudalert®) demonstrated the highest sensitivity (100%) while the Standard Method-SM9213E had the lowest sensitivity (3%). The U.S.EPA method and SM9213E demonstrated the highest specificity (100%) while Pseudalert® had the lowest (71.0%). Pseudalert® was able to detect P. aeruginosa in samples with higher amount of suspended solids as compared to other MF methods.Analysis of 24 environmental surface water samples (previously found to be negative for P. aeruginosa), which were spiked with P. aeruginosa at 5CFU/100mL, showed different sensitivities in recovery including Pseudalert® (100%), MOECC E3371 (92%), U.S.EPA (33%) and SM 9213E (33%). The highest mean recovery was observed with Pseudalert® (5.3 MPN/100mL) and the least with the U.S.EPA method (0.4CFU/100mL).Pseudalert® demonstrated improved performance in the detection and recovery of P. aeruginosa over SM9213E, U.S.EPA and MOECC E3371 in terms of sensitivity. However, Pseudalert® reported the highest amount of false positive results compared to the other three MF methods. The addition of a confirmation step with Milk Agar eliminated the false positive results. Therefore, this confirmatory step is recommended in order to increase the specificity of the Pseudalert® method.