Prevention of biofilm formation and removal of existing biofilms by extracellular DNases of Campylobacter jejuni

Abstract

The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.

Fig 1. Distribution of eDNase genes in C. jejuni genome sequences.

The Venn diagram shows the distribution of the eDNase genes dns (cje0256), cje0566 and cje1441 in the genome sequences of 1630 eDNase gene-positive C. jejuni strains (Table 2). Most genomes (1248 of 1630) only have a single eDNase gene, 357 genomes have two eDNase genes, while only 25 genomes, including C. jejuni RM1221, contain all three eDNase genes. The Venn diagram is encircled by the RM1221 chromosome showing open reading frames (blue), CJIE1–4 insertion elements (red), and the position of the three eDNase genes (black). Finally, the bottom part shows an amino acid sequence alignment of the Dns, CJE0566 and CJE1441 proteins, with the signal sequence and Pfam domains indicated. Signal sequences were predicted using PSORTb version 3.0.2.

Fig 2. Strain RM1221 is unable to form a monospecies biofilm but exhibits both swarming and autoagglutination (AAG).

Biofilm formation (A) of RM1221 (light grey bars) was measured by crystal violet staining and compared to NCTC 11168 (white bars), 81116 (dark grey bars), and a test tube containing only Brucella medium (black bar). Swarming ability (B) was calculated by measuring halo area on soft agar after 48 hours incubation in microaerobic conditions. Autoagglutination assessment (C) was carried out by observing the reduction in A₆₀₀ measurement over a 24 hour period. Both B and C show data for 11168 (white bars), RM1221 (light grey bars), 81116 (dark grey bars) and 11168 ΔflaAB (dark grey bars). Bars represent the median, error bars show range and significance was measured using Mann-Whitney tests (* = P<0.05).

Fig 3. Co-incubation of pre-formed biofilms with RM1221 leads to biofilm degradation.

Biofilms of NCTC 11168 (A and D) and 81116 (B) were allowed to form in static aerobic conditions for 24 hours before a further 24 hour treatment with RM1221 cell culture (A and B), or the cell free spent media of RM1221 (C). Graphs A, B and C show median A₅₉₀ values of each treatment. Bars represent the median, error bars show range and significance was measured using Mann-Whitney tests (* = P<0.05).

Fig 4. Inactivation of the cje1441 eDNase gene restores biofilm formation by C. jejuni strain RM1221.

(A) shows biofilm formation of NCTC 11168 (white bar), Δ1441 (dark grey bar), RM1221 (black bar) and a Brucella medium only control (light grey bar). The Δ1441 mutant shows similar levels of biofilm formation to NCTC 11168 and a significant increase in biofilm formation compared to the parent strain RM1221. (B) Shows that the biofilm produced by the Δ1441 mutant is susceptible to degradation by DNase I (white bar) and leads to levels of staining indistinguishable from the Brucella medium only control (black bars). (C) Shows biofilm formation of the Δ1441 mutant following secondary co-culture with strain NCTC 11168 (white bars), the Δ1441 mutant (dark grey bars), Brucella medium (black bars), or the RM1221 parent strain (light grey bars) showing that deletion of cje1441 inhibits the biofilm degrading ability of RM1221. Bars represent the median, error bars show range and significance was measured using Mann-Whitney tests (* = P<0.05).

Fig 5. C. jejuni RM1221 is able to degrade DNA in both static and shaking suspensions.

The ability of NCTC 11168 (A), RM1221 (B) and the Δ1441 mutant (C) to degrade NCTC 11168 genomic DNA was assessed by incubation of cell suspensions with genomic DNA at 37°C for three hours. Both NCTC 11168 and the Δ1441 mutant are unable to degrade the genomic DNA, with a band of genomic DNA of >10 kb remaining for the duration of the assay, while incubation with RM1221 results in degradation of genomic DNA (B), indicated by the ‘smearing’ shown as the time course progresses. RM1221 overnight suspensions were also shown to contain no eDNA when compared to NCTC 11168 and 81116 (D), again indicating that RM1221 is able to degrade its own exogenous DNA.