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Study of in vivo catheter biofilm infections using pediatric central venous catheter implanted in rat

Nature Protocols volume 11, pages 525–541 (2016)Cite this article

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Abstract

Venous access catheters used in clinics are prone to biofilm contamination, contributing to chronic and nosocomial infections. Although several animal models for studying device-associated biofilms were previously described, only a few detailed protocols are currently available. Here we provide a protocol using totally implantable venous access ports (TIVAPs) implanted in rats. This model recapitulates all phenomena observed in the clinic, and it allows bacterial biofilm development and physiology to be studied. After TIVAP implantation and inoculation with luminescent pathogens, in vivo biofilm formation can be monitored in situ, and biofilm biomass can be recovered from contaminated TIVAP and organs. We used this protocol to study host responses to biofilm infection, to evaluate preventive and curative antibiofilm strategies and to study fundamental biofilm properties. For this procedure, one should expect ∼3 h of hands-on time, including the implantation in one rat followed by in situ luminescence monitoring and bacterial load estimation.

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Figure 1: Schematic representation of the six different major stages of the in vivo model of TIVAP-related infection.
Figure 2: Preparation for surgery.
Figure 3: Shaving and disinfection procedures.
Figure 4: TIVAP surgical implantation (Steps 12–14).
Figure 5: TIVAP surgical implantation (Steps 15–17).
Figure 6: TIVAP surgical implantation (Steps 18–27).
Figure 7: Bacterial inoculation and monitoring of biofilm formation using bioluminescence.
Figure 8: Biofilm formation in TIVAPs was confirmed by scanning electron microscopy (SEM).

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Acknowledgements

We thank D. Lebeaux for critical reading of the manuscript. We thank R. Ramphal and P. Courvalin for providing strains. This work was supported by the French government's Investissement d'Avenir program, Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant ANR-10-LABX-62-IBEID) and the Fondation pour la 'Recherche Médicale grant' (Equipe FRM DEQ20140329508).

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Authors and Affiliations

  1. Département de Microbiologie, Institut Pasteur, Unité de Génétique des Biofilms, Paris, France

    Ashwini Chauhan, Jean-Marc Ghigo & Christophe Beloin

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  1. Ashwini Chauhan
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  3. Christophe Beloin
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Contributions

A.C. and C.B. initiated the development of the model. A.C., J.-M.G. and C.B. designed the experiments and wrote the manuscript. A.C. performed the experiments.

Corresponding author

Correspondence to Christophe Beloin.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Biofilm led to lethal infection in immunosuppressed rats.

(a) TIVAP implanted and cyclophosphamide-treated rats (see Supplementary Method 1 for detailed procedure) were injected with 102 CFU in 100µL of P. aeruginosa into the port of TIVAP at day +4 (4 days after TIVAP implantation) and photon emission was monitored for 3 days to evaluate biofilm formation and associated infection. (b) Bacterial load from different organs aseptically removed on day 3 post infection from dead animals was analyzed, organs were homogenized and were plated on LB agar for viable counts per mL. CFU results are means +/- standard deviations. Number of rats (n) used in the experiment, n = 4. Panel b is a modified version of Figure S5B and C from ref. 31 (Chauhan, A. et al., Antimicrob. Agents Chemother. 56, 6310–6318, 2012) and is adapted with permission from the American Society for Microbiology. Work on animals was performed in compliance with French and European regulations on care and protection of laboratory animals (European Commission directive 2010/63; French law 2013-118, 06th February, 2013). The protocols used in this study were approved by the ethics committee of “Paris Centre et Sud N°59” (reference 2012-0045).

Supplementary Figure 2 In vivo lock therapy against Staphylococcus aureus biofilm in the implanted TIVAP: 5-day regimen presented.

200 µL high dose antibiotics solution was instilled in TIVAP of rats (day +4) to treat methicillin sensitive S. aureus biofilm colonization (n = 5). Lock therapy was associated with systemic vancomycin for S. aureus. The lock was renewed every 24 h and 4 times (up to day +8), and its efficacy was monitored as photon emissions. See Supplementary Method 2 for detailed procedure. (a) Untreated control rats with 1X PBS lock. (b) 5 mg/mL gentamicin lock. (c) 30 mg/mL EDTA alone. (d) Combined gentamicin (5 mg/mL) and EDTA (30 mg/mL) lock. In panels (a) to (d), representative experiments are shown. (e-h) Rats were euthanized 7 days after the last lock instillation, (day +15) and TIVAP were harvested and monitored for photon emissions. (e) Untreated control 1X PBS lock, (f) 5 mg/mL gentamicin lock, (g) 30 mg/mL EDTA alone and (h) Combined gentamicin (5 mg/mL) and EDTA (30 mg/mL) lock. (i) Bacterial cells from TIVAP were harvested and plated on TSB agar for counts of CFU/mL. CFU results are means +/- standard deviations. One-way analysis of variance (ANOVA) with Graphpad Prism version 5.0c was used for statistical analysis. A P value of <0.05 was considered significant, **** P <0.0001. Panel i is a modified version of Figure 1G from ref. 31 (Chauhan, A. et al., Antimicrob. Agents Chemother. 56, 6310–6318, 2012) and is adapted with permission from the American Society for Microbiology, and is reprinted with authorization. Work on animals was performed in compliance with French and European regulations on care and protection of laboratory animals (European Commission directive 2010/63; French law 2013-118, 06th February, 2013). The protocols used in this study were approved by the ethics committee of “Paris Centre et Sud N°59” (reference 2012-0045).

Supplementary Figure 3 Modified anti-adhesive totally implantable venous access port (TIVAP).

(a) Commercially available pediatric TIVAP used in the study, and dismantled TIVAP (1, silicone catheter; 2 and 3, envelope of port; 4, sealing ring of port; 5, silicone septum; 6, titanium port) and anti-adhesive molecules used to modify TIVAP parts. The silicone catheter and septum were modified using methylcellulose (MeCe) derivative and the titanium port was modified using polyethylene glycol (PEG) derivative. (b) Rats with modified or unmodified implanted TIVAPs were inoculated with 106 colony-forming units (CFUs) of Staphylococcus aureus or 103 CFUs of Pseudomonas aeruginosa per 50μL of 1X phosphate-buffered saline. Bacteria were allowed to adhere to the TIVAP endoluminal surface for 3 hours (S. aureus) or 1.5 hours (P. aeruginosa) and biofilms were left to form for 5 days, and TIVAP was extracted to measure bacterial biofilm colonization. Viable bacteria were counted by plating on tryptic soy agar for S. aureus or lysogeny broth agar for P. aeruginosa. (Control: unmodified TIVAP. Si-Ti: TIVAP with silicone catheter and septum, and titanium modified parts as detailed in panel (a)). Statistical analysis was performed using 1-way analysis of variance (ANOVA) with GraphPad Prism software (version 5.0c). Differences were considered significant at P < 0.05. *P ≤ 0.01; **P ≤ 0.001. Panels (a) and (b) are reprinted from, respectively, Figure 1B and 3B from ref. 29 (Chauhan, A. et al., J. Infect. Dis. 210, 1347–1356, 2014) and is adapted with permission from Oxford University Press and the Infectious Diseases Society of America. Work on animals was performed in compliance with French and European regulations on care and protection of laboratory animals (European Commission directive 2010/63; French law 2013-118, 06th February, 2013). The protocols used in this study were approved by the ethics committee of “Paris Centre et Sud N°59” (reference 2012-0045).

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Chauhan, A., Ghigo, JM. & Beloin, C. Study of in vivo catheter biofilm infections using pediatric central venous catheter implanted in rat. Nat Protoc 11, 525–541 (2016). https://doi.org/10.1038/nprot.2016.033

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