Elsevier

Burns

Volume 37, Issue 7, November 2011, Pages 1125-1133
Burns

Validation of the Zürich burn-biofilm model

https://doi.org/10.1016/j.burns.2011.05.017Get rights and content

Abstract

Background

Despite advances in the use of topical and parenteral antimicrobial therapy and the practice of early tangential burn-wound excision, bacterial infection remains a major problem in the management of burn victims today. The purpose of this study was to design and evaluate a polyspecies biofilm model with bacteria known to cause severe infections in burn patients. The model is simple to prepare, maintain and analyse, and allows for short-term exposure to antimicrobials.

Results

Initial experiments showed that it was impossible to establish balanced polyspecies biofilms with an inoculum of Gram-positive and -negative bacteria. After 64.5 h of incubation, the Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) had suppressed the Gram-positives (Enterococcus faecalis, Staphylococcus aureus and Streptococcus intermedius). However, adding the Gram-negative bacteria after 41.5 h to an established biofilm of Gram-positives resulted in a balanced microbial consortium. After 64.5 h, all species were present in high numbers (107 to 108 colony forming units (CFU) per biofilm). Multiple repetitions showed high reproducibility of biofilm formation without significant differences between and within experiments. Combined fluorescence in situ hybridisation/confocal laser scanning microscopy (FISH/CLSM) analyses, for which biofilms had to be grown on a different non-flexible substrate (hydroxy apatite), revealed that, by 41.5 h, the biofilm consisted of an almost confluent layer of bacteria firmly adherent to the substratum. After 64.5 h (22 h after the addition of the Gram negatives), the biofilm consisted of a confluent mixture of single cells, an abundance of galaxies of bacteria with small lacunae and large amounts of extracellular matrix polysaccharides.

Conclusions

The polyspecies biofilm model contains the most prevalent burn-associated Gram-positive and Gram-negative bacterial pathogens and mimics the Gram-negative shift observed in vivo. It shows excellent reproducibility. It should allow adaptation to the bacterial spectrum prevalent in different burn centres and lead to a much more reliable investigation of the efficiency of topical antimicrobial agents than models operating with planktonic bacteria. The experiments further open up the perspective to create an in vivo model using these biofilms as infectious agents.

Section snippets

Background

Despite advances in the use of topical and parenteral antimicrobial therapy and the practice of early tangential burn-wound excision to reduce bacterial load, bacterial infection remains a major problem in the management of burn victims today. Critically ill thermally injured patients have a propensity to develop nosocomial infections for several reasons. Rates of ventilator-associated pneumonia, bloodstream infection and wound infection have all been shown to be higher than those for other

Bacteria used, preparation of inoculum

Enterococcus faecalis OMZ 422 (ATCC 29212), Escherichia coli OMZ 56 (ATCC 25922), Streptococcus intermedius OMZ 871 (SK57, ATCC 9895), Pseudomonas aeruginosa OMZ 154 (ATCC 27853) and Staphylococcus aureus OMZ 1122 (ATCC 25923) were obtained from the culture collection of the Institute of Oral Biology, University of Zürich. Strains were maintained by aerobic incubation at 37 °C for 36 h on plates of Columbia blood agar base (CBA, Oxoid, Basingstoke, Hamps., UK) supplemented with 5% (v/v)

Biofilm formation, growth and composition

The experimental set-up for biofilm formation is given in Fig. 1. The biofilms were formed in two steps, the 1st consisting of the establishment of a Gram-positive biofilm with S. aureus, S. intermedius and E. faecalis (0–41.5 h) and the 2nd bringing the addition and the establishment of the Gram-negatives P. aeruginosa and E. coli (41.5–64.5 h).

Aliquots of cell suspensions recovered from different biofilms and time points were examined for viability by fluorescence microscopy and were found to

Biofilm model mimicking the bacterial colonisation of burn wounds

The aim of our study was to establish and validate a burn-biofilm model, in particular in terms of repeatability. Whereas the clinical significance of bacterial biofilms has been increasingly recognised lately, our model represents, to the best of our knowledge, the first dedicated polyspecies burn-biofilm model. Based on methods developed in our laboratories in past years [11], [12], [13], [14], we have dubbed it ‘the Zürich burn-biofilm model’.

We have composed the polyspecies biofilm

Conclusions

Polyspecies burn-biofilm models are very important to study the efficiency of topical agents. These results should be more accurate in terms of use of these agents in vivo as compared with results obtained with planktonic cultures or monospecies biofilms. The Zürich burn-biofilm model represents a polyspecies-biofilm model that not only contains some of the most prevalent burn-wounds-associated Gram-positive and Gram-negative bacterial pathogens but also mimics the Gram-negative shift observed

Authors’ contributions

MG and BG designed the study, evaluated the data and wrote the article, TT performed the CLSM analyses, RG designed probes and edited the article, while PG provided editorial assistance. All authors read and approved the final manuscript.

Conflict of interest statement

The authors declare no conflicts of interest.

Acknowledgements

The authors are grateful to André Meier and Bärbel Sauer for excellent assistance in carrying out these biofilm experiments.

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