A fluorogenic micrococcal nuclease-based probe for fast detection and optical imaging of Staphylococcus aureus in prosthetic joint and fracture-related infections

Abstract

Purpose: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the 'smart-activatable' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus.

Methods: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria.

Results: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min.

Conclusion: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials.

Keywords: Staphylococcus aureus; Fracture-related infection; Infection imaging; Micrococcal nuclease; Optical probe; Orthopedic infection; Periprosthetic joint infection.

Fig. 1

Nuclease-mediated activation of the AttoPolyT probe. The AttoPolyT probe is specifically cleaved by the secreted S. aureus micrococcal nuclease (MN) enzyme. This leads to a separation of the quencher from the fluorophore and, consequently, emission of fluorescence (515–535 nm, red arrows) upon excitation in the 490 nm Cy2 range (yellow arrow)

Fig. 2

Formula applied to calculate the Target-to-Background (T/B) ratio for micrococcal nuclease activity in synovial fluid using the AttoPolyT probe assay

Fig. 3

Fluorescence target-to-background ratios of nuclease assays with the AttoPolyT probe on synovial fluids from patients with suspected (prosthetic) joint infections. A significant difference was observed in the optical intensities measured in T/B ratios for synovial fluid samples containing S. aureus (n = 13) or other Gram-positive bacterial pathogens (n = 13; ****, p < 0.0001), as well as samples containing Gram-negative bacterial pathogens (n = 4; **, p < 0.01), or non-infected samples (“no growth”; n = 8, p < 0.01)

Fig. 4

Imaging of in vitro-grown S. aureus biofilms on glass coverslips using the AttoPolyT probe. After incubation with or without the probe for 1 h, fluorescence was recorded using an Amersham™ Typhoon 5 Biomolecular Imager using the Cy2 filter. A–C Glass slides with S. aureus biofilm. D–F Glass slides without adherent bacteria. A, D Incubation with reaction buffer containing 3.9 µM AttoPolyT probe. B, E Incubation with reaction buffer alone. C, F Incubation with PBS. A clear difference is observed in the intensity of the emitted fluorescence between the S. aureus biofilms incubated with the A AttoPolyT probe and B–F the control groups. White-light imaging after parallel incubation of the coverslips with Congo Red confirms the G presence or H absence of S. aureus biofilms on the coverslips

Fig. 5

Ex vivo multispectral imaging of biofilms on patient-derived osteosynthesis materials (OSMs) with vanco-800CW and the AttoPolyT probe. OSMs that had tested either culture-positive or culture-negative were incubated with the AttoPolyT and vanco-800CW probes. Fluorescence images were recorded with an Amersham™ Typhoon 5 Biomolecular Imager using the Cy2 and IRLong filters to detect fluorescence of the AttoPolyT probe and vanco-800CW, respectively. Fluorescence signal was observed in both the Cy2 and IRLong range for OSMs from patients 1 and 2, who presented S. aureus-positive OSM cultures. In contrast, only fluorescence signal in the IRLong range was observed for OSMs from patients 3 and 4, who presented Gram-positive bacterial OSM cultures that did not include S. aureus. There were no fluorescence signals detectable for OSMs from patients 5 and 6, which had tested culture-negative

Fig. 6

Ex vivo imaging of S. aureus biofilms on patient-derived osteosynthesis materials (OSMs) with the AttoPolyT probe. OSMs that had tested either culture-positive for S. aureus or culture-negative were incubated with the AttoPolyT probe. Fluorescence images were recorded with an Amersham™ Typhoon 5 Biomolecular Imager using the Cy2 filters. Fluorescence signal was observed in the Cy2 range for OSM from patient 7, who presented S. aureus-positive OSM cultures. There were no fluorescence signals detectable for OSMs from patient 8, who had tested culture-negative