Mouse model of chronic post-arthroplasty infection: noninvasive in vivo bioluminescence imaging to monitor bacterial burden for long-term study

Abstract

Post-arthroplasty infections are a devastating problem in orthopaedic surgery. While acute infections can be treated with a single stage washout and liner exchange, chronic infections lead to multiple reoperations, prolonged antibiotic courses, extended disability, and worse clinical outcomes. Unlike previous mouse models that studied an acute infection, this work aimed to develop a model of a chronic post-arthroplasty infection. To achieve this, a stainless steel implant in the knee joints of mice was inoculated with a bioluminescent Staphylococcus aureus strain (1 × 10(2) -1 × 10(4) colony forming units, CFUs) and in vivo imaging was used to monitor the bacterial burden for 42 days. Four different S. aureus strains were compared in which the bioluminescent construct was integrated in an antibiotic selection plasmid (ALC2906), the bacterial chromosome (Xen29 and Xen40), or a stable plasmid (Xen36). ALC2906 had increased bioluminescent signals through day 10, after which the signals became undetectable. In contrast, Xen29, Xen40, and Xen36 had increased bioluminescent signals through 42 days with the highest signals observed with Xen36. ALC2906, Xen29, and Xen40 induced significantly more inflammation than Xen36 as measured by in vivo enhanced green fluorescence protein (EGFP)-neutrophil flourescence of LysEGFP mice. All four strains induced comparable biofilm formation as determined by variable-pressure scanning electron microscopy. Using a titanium implant, Xen36 had higher in vivo bioluminescence signals than Xen40 but had similar biofilm formation and adherent bacteria. In conclusion, Xen29, Xen40, and especially Xen36, which had stable bioluminescent constructs, are feasible for long-term in vivo monitoring of bacterial burden and biofilm formation to study chronic post-arthroplasty infections and potential antimicrobial interventions.

Figure 1. In vivo bioluminescence signals of the different S. aureus strains during a post-arthroplasty infection

1×10², 1×10³ and 1×10⁴ CFUs of the four S. aureus strains possessing the bioluminescent construct in a chloramphenicol selection plasmid (ALC2906), the bacterial chromosome (Xen29 and Xen40) and a stable plasmid (Xen36) or no bacteria (none) (n=4 to 8 mice per group) were inoculated into the right knee joints of mice in the presence of a stainless steel implant. (A, C, E, G) Bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux [photons/s/cm²/sr] ± sem (logarithmic scale). (B, D, F, H)Representative in vivo S. aureus bioluminescence on a color scale overlaid on top of a grayscale image of mice.*p<0.05, †p<0.01 ALC2906, Xen29, Xen40 or Xen36 versus none (Student’s t-test [one-tailed]).

Figure 2. In vivo EGFP-neutrophil fluorescence induced by the different S. aureus strains during a post-arthroplasty infection

1×10³ CFUs of each of the four S. aureus strains possessing the bioluminescent construct in a chloramphenicol selection plasmid (ALC2906), the bacterial chromosome (Xen29 and Xen40) and a stable plasmid (Xen36) (n=6 mice per group) were inoculated into the right knee joints of mice in the presence of a stainless steel implant. (A) Neutrophil infiltration (EGFP-neutrophil fluorescence) as measured by in vivo fluorescence (total radiant efficiency ([photons/s]/[μW/cm²]). (B) Representative neutrophil infiltration as measured by a color scale of fluorescence overlaid on top of a grayscale image of mice. *p<0.05, †p<0.01 ALC2906, Xen29 or Xen40 versus Xen36 (Student’s t-test [two-tailed]).

Figure 3. Biofilm formation induced by the different S. aureus strains on the implants

Implants were harvested from the mice at day 42 for all three inocula (1×10², 1×10³ and 1×10⁴ CFUs) of each bacterial strain possessing the bioluminescent construct in a chloramphenicol selection plasmid (ALC2906), the bacterial chromosome (Xen29 and Xen40) and a stable plasmid (Xen36). Representative VP-SEM images of biofilms on the cut ends of the stainless steel implants are shown (1 of 3 mice per group with similar results) with a low power magnification (120x) (left panels) and a higher magnification (600x) of the area boxed in red (right panels).

Figure 4. Comparison of Xen40 versus Xen36 using titanium implants

Xen40 or Xen36 (1×10³ CFUs) (n=8 per group) was inoculated into the right knee joints of mice in the presence of a titanium implant. (A) Bacterial counts measured by in vivo S. aureus bioluminescence (mean maximum flux [photons/s/cm²/sr] ± sem (logarithmic scale). The dotted line denotes the level of background bioluminescence. (B) VP-SEM images of implants on day 42 (1 of 4 mice per group with similar results) with a low power magnification (120x) (left panels) and a higher magnification (600x) of the area boxed in red (right panels). (C) Numbers of CFUs released from the implants (n=4 mice/group) on day 42. *p<0.05 Xen36 versus Xen40 (Student’s t-test [two-tailed]). n.s.= not significant.