In vitro inhibitory effect of lactic acid bacteria and their supernatants on flavobacterium columnare biofilm

Abstract

Flavobacterium columnare is an opportunistic bacterium causing columnaris disease to more than 37 fish species. It is believed that the bacteria form biofilm on the fish skin/surfaces before the bacteria in the biofilm produce and release enzymes that degrade connective and other tissues resulting in lesions on the infected fish. Treatments to inhibit the bacterial biofilm without interfering with its planktonic cell growth can be an alternative method to control columnaris disease. This study aimed to evaluate the efficacy of lactic acid bacteria (LAB) isolated from white leg shrimp gut (Penaeus vannamei) and the root of Jerusalem artichoke (Helianthus tuberosus) in biofilm inhibition and modulation of biofilm-associated genes of F. columnare. Cell-free supernatants (CFSs) of five different LAB isolates were first screened for their abilities to inhibit biofilm formation and growth in broth cultures of three highly pathogenic F. columnare isolates; 15, CUVET1359, and CUVET1365. Then, optimal culture conditions that resulted in the LAB cells and CFSs with the most potent F. columnare biofilm inhibiting activities were determined for two LAB isolates (WS2021 and SC1) with the most potent F. columnare biofilm inhibiting activities. The two LAB isolates were cultured for 48 h, at 0, 3, 6, 9, 12, 24, 36, and 48 h timepoints, aliquots of each culture were collected, and their cells and CFSs were separated and tested for their abilities to inhibit the biofilm and growth of F. columnare isolate 15. The results showed that cells and CFSs from 48 h incubating timepoints inhibited F. columnare biofilm strongly. To determine if LAB cells and CFSs treatment affect or modulate the expression of seven biofilm-associated genes in F. columnare, a quantitative real-time PCR method was used. After co-incubation at three different time points (24, 48, and 72 h) with either the CFSs or cells of WS2021 and SC1 obtained from 48 h, iron acquisition, type IX secretion system (T9SS), and quorum sensing (luxR) genes in F. columnare biofilm was predominantly downregulated, especially at 48 h and 72 h co-incubation. Based on 16S rDNA sequence, WS2021 and SC1 isolates were identified as Enterococcus gallinarum and Leuconostoc pseudomesenteroides, respectively. In summary, both CFSs or cells of these two bacteria isolates possess F. columnare biofilm inhibiting activity which could be due to the downregulation of iron acquisition (alcB, rhbC, and tonB), T9SS (porV, gldL, and sprA), and luxR genes.