Combined Effect of DNase I and Proteinase K on Dual-Species Biofilm of Campylobacter jejuni and Acinetobacter baumannii

Abstract

In food-associated environments, foodborne bacteria can form biofilms that are likely to exist as multiple species and are potentially a food safety concern. In this study, we focused on the effects of DNase I and proteinase K on dual-species biofilms containing Campylobacter jejuni and bacterial isolates from food-associated environments. The viable cell counts of C. jejuni differed significantly depending on the counterpart strain in the biofilms. In mature monospecies biofilms, both C. jejuni and Acinetobacter baumannii were susceptible to both enzymes. Acinetobacter baylyi was susceptible only to DNase I, while Staphylococcus epidermidis was susceptible only to proteinase K. Analysis of confocal laser scanning microscopy images of A. baumannii biofilm showed that the protein distribution was consistent with that of the biofilm-embedded cells, whereas it was distinct from the polysaccharide distribution. Among the dual-species biofilms, that of C. jejuni and A. baumannii was the only biofilm susceptible to both enzymes. Combined treatment using DNase I followed by proteinase K was far more effective than DNase I monotherapy against both A. baumannii mono- and dual-species biofilms. Our study suggests that proteins could be a primary target for inactivating biofilm-embedded cells in A. baumannii, and the use of multiple enzymes could be an efficient strategy for biofilm removal.

Keywords: Campylobacter; DNase I; biofilm; dual-species; proteinase K; reduction.

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Figure 2

Quantification of surface‐attached mono‐ or dual‐species biofilms comprising C. jejuni and contaminating bacteria isolated from a cafeteria kitchen before or after DNase I treatment. Biofilms were formed on a polystyrene 96‐well microtiter plate at 37°C under microaerobic conditions for 48 h, treated with DNase I at 37°C for 1 h, and quantified using a crystal violet assay. The data are based on three independent experiments in triplicate. Asterisk (∗) denotes a significant difference between untreated and DNase‐I treated samples (p < 0.05).

Figure 3

Quantification of surface‐attached biofilms of mono‐ or dual‐species biofilms comprising C. jejuni and contaminating bacteria isolated from a cafeteria kitchen after proteinase K treatment. Biofilms were formed on a polystyrene 96‐well microtiter plate at 37°C under microaerobic conditions for 48 h, treated with proteinase K at 37°C for 1 h, and quantified using crystal violet assay. The data are based on three independent experiments in triplicates. Asterisk (∗) denotes a significant difference between non‐treated and proteinase K treated samples (p < 0.05).

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Figure 5

Effect of the combined biofilm treatment using DNase I and proteinase K. DNase I was applied, and the plate was then incubated at 37°C for 1 h. Following incubation, the wells were washed once with 150 μL of MHB per well. Subsequently, proteinase K was added and the plate was incubated for an additional hour at 37°C.

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