Rapid bacterial evaluation beyond the colony forming unit in osteomyelitis
- Centre for Orthopaedic & Trauma Research, Faculty of Health and Medical Sciences, University of Adelaide, Australia
- Department of Orthopaedics and Trauma, Royal Adelaide Hospital, Australia
Figures
Figure 1
Validation of DNA preparations from SaOS2-OY cells and S. aureus.
Digital droplet PCR (ddPCR) genome counting of SaOS2-OY (A), SK2 (B), and SK3 (C), comparing the Direct buffer approach (
) and a standard DNA kit (
) (four biological replicates with mean and standard errors were shown on graphs, ***p < 0.001 and ****p < 0.0001); demonstration of complete bacterial chromatin release from SK2 (D) and SK3 (E): correlation with CFU plating. Each data point represented the comparison of one colony-forming unit (CFU) recovery and one total DNA measurement; three independent experiments were carried out for each strain and similar results were achieved; representative results of one experiment are presented.
Figure 2
Measurement of intracellular S. aureus in SaOS2-OY cells in vitro.
Colony-forming unit (CFU) recovery of SK2 (A) and SK3 (B) from host SaOS2-OY cells, with haemolysis reactions (
) and numerous small colony variants (
) shown in SK2 group; quantification of SK2 and SK3 using CFU count (
, left Y) and digital droplet PCR (ddPCR) count (⬤, right Y) from low (C, E) and high (D, F) multiplicities of infection (MOI) groups; relative human genome copy (%) with CTRL (⬤) vs. INF (◼) quantified by ddPCR from low (G) and high (H) MOI groups (four biological replicates with mean and standard errors are sḩown; **p < 0.01, ***p < 0.001, ****p < 0.0001; ns = not significant).
Figure 3 with 2 supplements
Pathogen diagnosis in clinical PJI bone specimens.
Masson’s trichrome staining of bone tissue sections of an osteoarthritis (OA) subject (A) and culture-negative periprosthetic joint infection (PJI) subjects I–III (B–D). Pathogen profiling using total DNA of bone specimens from the above three PJI patients (E–G) by ONT sequencing for the readout of pathogen species and digital droplet PCR (ddPCR) to quantify bacterial load as a ratio of bacterial: human genomic copies (error bars shown are the combination of the standard error of the mean from two individual DNA preparations and the device generated error from each of the ddPCR runs, with over 15,000 droplets read per sample).
Figure 3—figure supplement 1
Polymerase chain reaction (PCR) analysis of bone samples from primary total hip replacement cases.
DNA isolated from five patient bone samples were analysed by PCR for the presence of human COL10A1 and bacterial tuf. The negative presence of bacterial tuf PCR product was confirmed by melt analysis post-quantitative PCR reactions using the DNA samples of five primary total hip replacement (non-infected) patients (coded with five different colours).
Figure 3—figure supplement 2
Multi-sequence alignment among S. aureus, S. epidermidis, S. haemolyticus, and S. hominis.
The green and blue boxes indicate the forward and reverse primer-binding areas, respectively; the region between the red brackets was the assaying sequence for analysis (A). A summary of the number of mismatched base pairs and % differences within the assayed regions among the above four species (B).
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Rapid bacterial evaluation beyond the colony forming unit in osteomyelitis
eLife 13:RP93698.
https://doi.org/10.7554/eLife.93698.3
