Figure

Figure. Increased susceptibility of collagen graft to biofilm formation compared with gelatin graft. Graft patches were inoculated with indicated bacterial strains for 72 h and analyzed quantitatively and qualitatively. A) Biofilm formation...

For the in vitro experiments, we dissected the grafts into 5 × 5 mm square pieces and inoculated them with bacterial strains representing pathogens implicated in thoracic PVGI in our patient cohort. These were derived from either Vascular Graft Cohort Study (VASGRA) patients or laboratory strains (Appendix Table 1, Figure 1) and maintained on Columbia blood agar plates (bioMérieux SA, https://www.biomerieux.com) and in tryptic soy broth (Becton Dickinson, https://www.bd.com) at 37°C. Graft patches were incubated with bacteria in tryptic soy broth–glucose solution (glucose concentration 8 mmol/L) at 37°C for 72 h, and medium was exchanged every 24 h. The patches were washed with phosphate-buffered saline (PBS), sonicated at 44 khz, and the resulting optical density at a wavelength of 600 was measured in a microplate reader. All bacteria, apart from Pseudomonas aeruginosa strain 2, showed increased biofilm formation on the collagen graft compared with the gelatin graft patches (Figure, panel A).

Biofilms of selected strains were stained with SYTO 9 of the LIVE/DEAD BacLight Bacterial Viability Kit (ThermoFisher Scientific, https://www.thermofisher.com) according to the manufacturer’s instructions. The graft patches were placed in 8-well microslides (ibidi, https://ibidi.com) and visualized by confocal laser scanning microscopy with a Leica TCS SP8 inverted microscope (https://www.leica-microsystems.com) under a 63×/1.4 oil immersion objective. We selected 2 representative spots per graft patch, providing a stack of horizontally acquired images (512 × 512 pixels representing an area of 244.8 μm × 244.8 μm) with a z-step size of 0.12 μm. We processed the obtained stacks by using Imaris 9.2.0 software (Bitplane; Oxford Instruments, https://imaris.oxinst.com/support/imaris-release-notes/9-2-0). Biofilm height and volume were determined as previously described (11). This approach illustrated the increased biofilm formation on collagen graft patches (Appendix Figure 2). Quantitative analysis from the obtained confocal laser scanning microscopy images corroborated the initial findings because biofilm grown on collagen graft patches displayed increased total biofilm mass volume as well as maximal biofilm height (Figure, panel B).

One possible explanation for the increased susceptibility to biofilm formation could be the distinct coatings of the grafts. Hence, we coated well plates overnight at 4°C with either rat tail collagen 1 (10 μg/mL; ThermoFisher Scientific) or type B gelatin solution (10 μg/mL; Sigma-Aldrich, https://www.sigmaaldrich.com). The plates were incubated with bacteria at 37°C for 30 min. Bacteria were washed, stained with 0.1% crystal violet (Fluka; Sigma Aldrich, https://www.sigmaaldrich.com), solubilized in 95% ethanol, and the resulting optical density at 570 nm was measured. The tested strains adhered substantially better to collagen (Figure, panel C). Our findings are supported by studies demonstrating the potential of gram-positive bacteria to adhere to collagen, whereas only minor affinity was observed for gelatin (12,13).

To assess the effects of these findings in vivo, we analyzed 412 prospective participants with woven polyester grafts: 28 VASGRA participants in whom intracavitary thoracic PVGI developed and 384 contemporaneous open-chest cardiac surgery patients in whom PVGI did not develop (controls) (Table). Data and strains isolated from patients were used in accordance with Cantonal Ethics Committee approval KEK-ZH-Nr. 2012-0583. For statistical analyses with GraphPad Prism 8 (GraphPad Software, https://www.graphpad.com), we used nonparametric tests (Fisher exact or Wilcoxon rank-sum, as appropriate). When normalized to the total number of control patients (n = 384 who had undergone the cardiac surgery but had no PVGI), the calculated percentage of intracavitary thoracic PVGI (n = 28 VASGRA patients who had undergone the cardiac surgery and had PVGI) was higher for patients in the collagen-graft (10.8%) versus the gelatin-graft group (3.52%; p<0.005).

We found more biofilm formation on collagen-coated polyester vascular grafts than on gelatin-coated grafts, possibly because the tested strains adhered substantially better to collagen than to gelatin. When we analyzed prospective patients with PVGI and contemporary controls, the percentage of PVGI associated with collagen-coated grafts was also higher.

The strengths of our study include use of patient-derived strains of bacteria and the prospective collection of patients with incident PVGIs and controls. Our study has some limitations. First, it was a single-center study, resulting in low patient numbers, and the role of the graft material as a risk factor for PVGI is difficult to prove because of the rarity of the infection. Furthermore, data for patients with PVGI have to be interpreted with caution because some patients have additional foreign material in the heart. Second, publicly available information on the exact type of coating as well as the application procedures used is lacking. In addition, the experiments were performed in a static experimental setup instead of a flow chamber. However, because we were interested in direct bacterial adherence to the graft material, simulating a scenario in which infection would occur as consequence of unintentional inoculation during the perioperative period, we believe that the setup used is adequate. We did not reproduce a gram-positive and gram-negative mixed biofilm formation experiment because a monomicrobial setup enabled us to determine which material and coating was more susceptible to bacterial adherence in a more controllable fashion.

In conclusion, biofilm formation was increased on collagen-coated vascular grafts compared with gelatin-coated grafts in vitro. As opposed to another risk factor analysis from the VASGRA study (3), in our study, the graft material was associated with the PVGI rate. Parameters such as vascularization potential, secure pseudointima growth, and reduced thrombogenicity are perceived as affecting successful integration and functionality of prosthetic vascular grafts (14). Further parameters should be considered in the future design and development of vascular prostheses to reduce the emerging trend of PVGI.