Maniobras

We next tested the possibility that degradation of eDNA in biofilms would lead to biofilm disintegration. Previous work in P. aeruginosa biofilms has shown that eDNA is important in early stages of biofilm formation, however DNase I treatment has less impact on biofilm structure as the biofilm matures (Whitchurch et al., 2002). In order to assess if this was also the case in C. jejuni NCTC 11168 biofilms, DNase I was added at 12 hour intervals over the total 48 hour aerobic incubation period. The C. jejuni biofilm was degraded following addition of 4 U/ml DNase I regardless of the biofilms maturity, indicating that eDNA is an important ECM component throughout the entire C. jejuni biofilm life cycle (Figure 6-5).

Figure 6-5 DNase I is able to degrade C. jejuni NCTC 11168 biofilms regardless of their maturity DNase I was added at intervals to aerobically incubated NCTC 11168 cultures over a 48 hour static incubation and biofilm degradation assessed by crystal violet staining (A). At all time points a statistically significant reduction in biofilm biomass was observed, with DNase I treated samples being indistinguishable from a Brucella medium only control. Error bars show SE (N= 5), significance was measured using Bonferroni post-test following ANOVA analysis (** = P<0.01).

The rapidity of biofilm degradation was next assessed by treating mature (48 hour) biofilms with DNase I and then staining at intervals over a two hour period. Following only a five minute incubation with DNase I the majority of the biofilm was removed from the glass surface and showed no statistical difference in A590 value to a test tube containing only

Brucella medium (Figure 6-6). No further degradation occurred at later time points, suggesting that a five minute treatment is able to achieve maximal biofilm degradation.

Helen Louise Brown DNase I treatment of the biofilm

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Figure 6-6 DNase I degradation of mature C. jejuni NCTC 11168 biofilms occurs with a 5 minute incubation

Following a 48 hour static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 minutes before biofilm degradation was assessed. After a 5 minute incubation of the biofilm with DNase I there was a statistically significant decrease in biofilm biomass compared to an untreated control (yellow bar). No statistically significant further decrease in biofilm biomass, or regrowth of the biofilm was seen during a further two hour treatment. Error bars show SE (N = 5), significance was measured using Bonferroni post-test following ANOVA analysis (***=P<0.001).

Finally, the concentration of DNase I required to degrade the biofilm was also investigated. DNase I was diluted to concentrations, ranging from 5 to 0.01 U/ml, and added to biofilm cultures before incubating statically for 48 hours (Figure 4-8).

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Figure 6-7 Concentrations of DNase I as low as 0.01 U/ml are able to achieve biofilm degradation

The concentration of DNase I required for biofilm reduction was assessed by adding various concentrations of DNase I to biofilm cultures before incubating statically for 48 hours and assessing biofilm biomass by crystal violet staining. The graph shows aerobic data only since it is industrially relevant, microaerobic data was comparable to the aerobic data shown. All DNase I concentrations tested were able to prevent measurable C. jejuni NCTC 11168 attachment and biofilm formation, with all treated samples being indistinguishable statistically from the Brucella only control (black and white checked bar). Error bars show SE (N = 5), significance was measured using Bonferroni post-test following ANOVA analysis (** = P<0.01, ***=P<0.001).

All the tested concentrations significantly reduced the level of C. jejuni NCTC 11168 biofilm. As previously there was no statistically significant difference between DNase I treated test tubes and the negative control tube containing Brucella medium only. It is interesting to note that DNase I treatment had little impact on MPN, simply degrading the biofilm and releasing the attached cells in to suspension (Figure 6-7).

Helen Louise Brown DNase I treatment of the biofilm

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Figure 6-8 MPN of the C. jejuni NCTC 11168 planktonic phase is not effected by DNase I treatment.

Representative image (N = 5) of a spot plate containing serial 1:10 dilutions (highest dilution on the left of the image and lowest dilutions on the right) of C. jejuni NCTC 11168 cells incubated statically for 48 hours in aerobic conditions. The labels on the left of the image denote at what time during the incubation DNase I was added to the culture. There is little difference in MPN between those cultures incubated with DNase I (top four lines) and those without DNase I (bottom four lines), suggesting that although DNase I is able to effect the biofilm biomass, it does not significantly impact MPN. Control samples were as follows: DNase buffer only = biofilm suspension containing 4 µl of manufacturers DNase buffer, No treatment = biofilm suspension not containing any buffers or enzyme but physically handled in the same manner as the treatment samples, Incubation control = sample was incubated continuously throughout the experiment, rather than being removed for incubation for manipulations.

Since biofilms formed in both aerobic and microaerobic conditions showed the same pattern of disruption (Figure 6-8), it is suggested that the effects observed were not a response to atmospheric condition, but rather DNase I treatment. DNase I which had been heat inactivated (by heating to 95 °C for 10 min) lost the ability to inhibit the formation of C. jejuni NCTC 11168 biofilms (Figure 6-9).

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Figure 6-9 Heat inactivation of DNase I inhibits its ability to degrade C. jejuni biofilms

Biofilm formation by C. jejuni NCTC 11168 cultures treated with either DNase I (pale yellow bars), Heat treated DNase I (orange bars) or cultures with no DNase I added (yellow bars) were compared. DNase I enzyme which had been heated to 95 °C for 10 min was unable to statistically significantly inhibit C.

jejuni NCTC 11168 biofilm formation during a 48 hour static incubation at 37°C, unlike its un-treated

counterpart. Graph shows a representative replicate (with three internal, technical replicates). Error bars show SE of the three internal replicates.

1.1.1 Treatment of the biofilm with exogenous DNase I leads to irreversible biofilm