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 standardized laboratory detection methods for these organisms have not been established. Aim: This study 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 laboratories and sent to the Glasgow Royal Infirmary laboratory 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 that the methodology could identify organisms from a mixed culture. Volumes of 100 mL were filtered under negative pressure on to culture media and incubated at 30 °C and 37 °C. The incubation time was 7 days, with plates read on days 2, 5 and 7. Further identification of colonies was undertaken using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Findings: Optimal recovery of organisms was obtained by culturing water samples on tryptic soy agar, chocolate bacitracin agar and pseudomonas selective agar. The optimal temperature for isolation was 30 °C. The optimal incubation time was 5 days, and MALDI-TOF MS identified all test species reliably. Conclusion: The methodology described was able to detect the six tested waterborne pathogens reliably, and can be utilized by laboratories involved in testing water samples during outbreak investigations.