Lactic acid bacteria and their inhibitory activity against L. gervieae

A review of, Perez-Sanchez, T., Balcazar, J.L., Garcia, Y., Halaihel, N., Vendrell, D., De Blas, I., Merrifield, D.L. & Ruiz-zarzuela, I., (2011), Identification and characterisation of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae, Journal of Fish Diseases, Vol: 34(7): 499-507

Lactococcus garvieae, a Gram-postiive pathogen is known to cause haemorrhagic septicaemia and meningoencephalitis in various fish species, as well as in mammals and has recently been isolated in humans, determining its potential as a zoonotic agent. Outbreaks of L, garvieae are common in aquaculture, where they are normally treated with antibiotics. However, this is often ineffective and has also led to antibiotic resistance.

Probiotics are now used to prevent pathogenic microorganisms such as L. garvieae causing disease in fish. Lactic acid bacteria (LAB), such as Lactococcus spp., Pediococcus spp. or Lactobacillus spp. are especially used as biological control agents in aquaculture.

This study aimed to identify and characterize the properties of the endogenous microbiota of rainbow trout, Oncorhynchus mykiss, against L. garvieae by studying their antibacterial activity, pH and bile tolerances, adherence characteristics and antibiotic resistance, in order to determine their potential as probiotics.

60 rainbow trout were collected from two fish farms. Sample mucus was obtained from the gills and intestines. Serial dilutions were then placed on tryptic soy agar, where 335 bacterial strains were isolated. They were screened for antagonistic activity against L.gervieae using agar spot assay. Then the strains were grouped using PCR and identified using 16s rRNA gene sequence analysis.

Eight of 198 isolated strains from the intestinal mucosa were antagonistic against L.gervieae. These strains were identified as Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides.

L. mesenteroides showed greater growth rate and doubling time compared to the other bacteria. They all showed a good hydrophobicity percentage, which acted as an indicator of adherence ability, with the highest in Lb. plantarum (31.59%) and significantly lower (P<0.05) in L. M=mesenteroides (11.42%) and L. lactis (24.99%). All strains showed relatively high resistance to bile, (resistant to 1.0% bile) with no significant difference in sensitivity between the bacteria. Similarly, they showed resistance to low pH, up to pH 2. L. lactis showed the highest levels at pH 1 whereas L. mesenteroides showed highest levels at pH 7.4.

The bacteria were also resistant to a majority of antibiotics tested, with little variation in susceptibility between strains. This is advantageous for establishing the beneficial microbiata in the intestines for a prolonged time, however it must be ensured that this resistance is not later passed on through plasmids.The antimicrobial substances exhibited by the three strains were also found to be sensitive to proteinase K. Moreover, after incubation of L.garvieae on the plates, measurable clearing zones were detected around the wells filled with supernatants from the strains, showing their antagonistic efficacy and that proteinaceous extracellular inhibitory compounds were at least partly responsible for the antagonism.

The clear antagonistic effects of these indigenous trout microbiota on the pathogen as shown by the results support previous studies which explain that establishing normal or protective microbiota in particular fish will constitute a key component of defensive barrier function against pathogens. This is also the first report to demonstrate clear and consistent antagonism of indigenous rainbow trout microbiota against L.garvieae, mediated in part at least, by extracellular antimicrobial peptides. Considering the good pH and bile tolerance, positive indications towards epithelial adherence and the ability to suppress pathogen growth under in vivo conditions, L.lactis, Lb, plantarum and L mesenteroides should be further studied in vivo for their potential use as probiotics for the control of L.garvieae, as it may provide an alternative to the current use of antimicrobial compounds.