Development and Evaluation of Test Methods for the Detection and Enumeration of Opportunistic Waterborne Pathogens from the Hospital Environment.

BACKGROUND: Many Gram -negative bacteria other than Pseudomonas aeruginosa have been implicated in waterborne outbreaks but standardised laboratory detection methods for these organisms have not been established.

AIM: We aimed to establish laboratory testing methodologies for six waterborne pathogens; Acinetobacter spp, Burkholderia spp, Cupriavidus spp, Delftia acidovorans, Elizabethkingia spp and Stenotrophomonas maltophilia.

METHODS: Water samples were spiked by UK Health Security Agency labs and sent to the Glasgow Royal Infirmary lab for analysis. Water samples were spiked with either a pure culture of target organism or the target organism in water containing normal background flora, to ensure the methodology could identify organisms from a mixed culture. Volumes of 100 mL were filtered under negative pressure onto culture media and incubated at 30°C and 37°C. Incubation time was seven days with plates read on day two, day five and day seven. Further identification of colonies was undertaken using MALDI-TOF.

FINDINGS: Optimal recovery of organisms was obtained by culturing water samples on tryptic soy agar (TSA), Chocolate Bacitracin agar (BAC) and Pseudomonas selective agar (PSE). 30°C was the optimal temperature for isolation. Optimal incubation time was five days and MALDI-TOF identified all species tested reliably.

CONCLUSION: The methodology described can reliably detect the six waterborne pathogens tested and can be utilised by labs involved in testing water samples during outbreak investigations.