Isolation and application of Bdellovibrio and like organisms (BALOs) for controlling Aeromonas hydrophila in the American eel (Anguilla rostrata) rearing water

Abstract

Bdellovibrio is a kind of fast-moving bacteria that mainly preys on Gram-negative bacteria. Aeromonas hydrophila is one of the most common pathogenic bacteria for many animals. However, the potential application of freshwater-derived Bdellovibrio in protecting eels from A. hydrophila infection is still unclear. In this study, Klebsiella was used as the host bacteria, and Bdellovibrio-and-like organisms (BALOs) were isolated by double-layer plate technique. 32 strains of pathogenic bacteria from different sources including: one Gram-positive pathogenic stain of Bacillus cereus and 31 Gram-negative pathogenic stains of A. hydrophila, Escherichia coli, Pantoea agglomerans, Providencia rettgeri, Pseudomonas aeruginosa, Salmonella Choleraesuis, Salmonella Typhimurium, Serratia odorifera, Shewanella Putrefaciens, Vibrio parahaemolyticus, to name just a few. Moreover, 5 strains of potential probiotics including Bacillus coagulans, Lactobacillus plantarum, and Lactobacillus salivarius were also used to select the Bdellovibrio strain with a relatively wide lysis spectrum and no adverse effects on potential probiotics. Transmission electron microscopy and BALO-specific polymerase chain reaction [PCR] techniques were employed to identify BALOs strains. The application of BALOs strains in controlling A. hydrophila infections of American eel (Anguilla rostrata) was also conducted. Results showed that 8 strains of BALOs, viz. FSBD1, FSBD2, FSBD3, FSBD4, FSBD5, FSBD6, ZZBD1 and ZZBD2 were isolated. Lysis experiments on 32 potentially pathogens showed that FSBD1, FSBD2 FSBD3, FSBD4, FSBD5, FSBD6, ZZBD1 and ZZBD2 lysed 65.6%6 % (21/32), 65.6% (21/32), 65.6 % (21/32), 65.6 %6 % (21/32), 87.5 % (28/32), 75.0 % (24/32), 84.4 % (27/32) and 71.9 % (23/32) pathogens, respectively. Among of them, FSBD5 had the highest lysis rate of 87.5 % (28/32). It showed great lysis ability to 100.0 % (4/4) Salmonella and 100.0 % (4/4) Aeromonas tested. However, its lysis rate of potential 5 probiotics strains was 0 % (0/5). Surprisingly, ZZBD1 lysed one Gram-positive pathogenic stain of B. cereus, with the Gram-positive lysis rate of 16.7 % (1/6). FSBD5 and ZZBD1 were identified as BALOs morphologically by transmission electron microscopy and partial 16S rDNA sequencing. Concerning the strain of FSBD5, compared with A. hydrophila infection group (AHI), adding the cultured FSBD5 to the rearing water of American eel significantly reduced the number of A. hydrophila in Bdellovibrio treatment group (BDT, from 6.19 ± 0.01 log CFU/mL to 2.84 ± 0.08 log CFU/mL, p < 0.05), and also significantly improved the survival rate of American eel (at the end of 7 days experiment, the survival rate of AHI and BDT group was 12.4 % and 64.3 %, correspondingly, p < 0.05). Our results firstly revealed that the BALOs strain FSBD5 could lyse a wide variety of potential pathogenic bacteria from different sources, but it couldn't lyse probiotics strains tested. FSBD5 also significantly reduced the risk of being infected by A. hydrophila in American eel rearing water. These results will provide a theoretical basis for the biological controlling of A. hydrophila infections by BALOs in food eel or its production process.