Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2016

Development of Fluorescence-Based Cell Assays Combined with in situ Hybridisation Enhanced Acoustic Flow Cytometry for Rapid Bacterial Detection in Patients with Bacteraemia (#222)

Xiao Xuan Huang 1 2 3 , Nadezda Urosevic 1 2 3 , Tim Inglis 1 2 3
  1. Department of Microbiology, PathWest Laboratory Medicine WA, Nedlands, WA, 6009
  2. School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, WA, 6009
  3. University of Western Australia, Western Australia

Background

Delayed antibiotic treatment in patients with bacteraemia can progress rapidly into sepsis with a mortality of 40-60%. The current standard procedure for bacterial identification takes at least 12h of blood culture incubation followed by protein and DNA-based molecular assays. Despite recent advances, these assays are incapable of identifying polymicrobial infections and differentiating like-organisms. Here we present an alternative method of rapid bacterial identification based on peptic nucleic acid (PNA) fluorescence in situ hybridization (FISH) and acoustic flow cytometry (aFLOW). This is the first time that PNA-FISH-aFLOW is used for bacterial detection in patients with bacteraemia.

 

Methods

We have used FITC-labelled 16S rRNA PNA probes for bacterial detection by FISH-aFLOW in simulated blood cultures (BC) spiked with K. pneumoniae (10 CFU/mL). Bacterial growth was monitored by plate count in BacTEC system. The sensitivity of PNA-FISH-aFLOW was determined by ten-fold serial dilutions of the spiked BC.

 

Results & Discussions

Time to positivity (TTP) was 12.5 hours in the BacTEC system, corresponding to 108 CFU/mL. Using PNA-FISH-aFLOW with FITC labelled 16S rRNA probe, the lowest bacterial load detected was 9.02x105 CFU/mL, equating to 7 hours of incubation with reduced TTP estimated as 5.5hrs. FITC was an inferior fluorophore due to photobleaching. AlexaFluor488 is recommended. This method provides a specific and rapid approach to bacterial detection in patients with bacteraemia.

 

Conclusions

Early bacterial identification can reduce mortality and improve treatment outcome in patients with bacteraemia. The PNA-FISH-aFLOW has a potential to greatly reduce turnaround time for bacteraemia diagnostics. Thanks to its unique molecular properties combined with the multi-parameter cell analyser (aFLOW), PNA-FISH provides rapid bacterial detection and identification in both mono/polymicrobial bacteraemia. The combination of PNA-FISH-aFLOW with other fluorescence-based cell proliferation assays (e.g. Click-iT) has the potential to replace current culture-dependent assays with a much faster approach to bacteraemia diagnostics.

  1. Papaparaskevas, J, Katsandri, A, Pantazatou, A, Stefanou, I, Avlamis, A, Legakis, NJ & Tsakris, A 2011, 'Epidemiological characteristics of infections caused by Bacteroides, Prevotella and Fusobacterium species: a prospective observational study', Anaerobe, vol. 17, no. 3, pp. 113-7
  2. Tang, P, Lee, C, Li, C, Li, M, Ko, W & Lee, N 2015, 'Time to positivity of blood culture: An independent prognostic factor of monomicrobial Pseudomonas aeruginosa bacteremia', Journal of Microbiology, Immunology and Infection, pp. 1-8.
  3. Bizzini, A, Durussel, C, Bille, J, Greub, G & Prod'hom, G 2010, 'Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Bacterial Strains Routinely Isolated in a Clinical Microbiology Laboratory', Journal of Clinical Microbiology, vol. 48, no. 5, pp. 1549-1554.