Effects of woody forages on biodiversity and bioactivity of aerobic culturable gut bacteria of tilapia (Oreochromis niloticus)

example, Pseudomonas fulva was identified in the guts of tilapia in the MOL group, and Pseu- domonas can produce ligninase and cellulase [68, 69]. In general, cellulose and lignin, as anti- nutritive factors, hinder the digestion and absorption of typical nutrient [70]. Fermenting Pseudomonas fulva to obtain some extracellular enzymes such as cellulase would be an excel- lent direction for future investigations. Rhodococcus sp. recovered in the guts of tilapia in the fermented MOL group may degrade steroid [71]. However, the fish guts are complex environ- ments, and the exact functions of Rhodococcus sp. in guts are unknown. As a probiotic, Bacillus megaterium was only isolated from the fermented FM group (Table 3). In a recent study, the application of Bacillus megaterium as a probiotic in feed could aid in maintaining the balance of intestinal microflora populations and increase the activity of digestive enzymes and the growth of catfish [72]. Therefore, whether Bacillus megaterium can also improve the growth of tilapia or even other fish is a question that is worth elucidating. In addition, P. mirabilis, a conditional pathogen [73], was also shown to be the most abundant isolate in the control group, while it was rarely observed in other woody forage groups, sug- gesting that the woody forage had a specific antibacterial activity [74, 75]. The present study showed that supplementation of forage modulated the adherent gut microbiota. In this respect a fundamental question arises: what effect does the gut microbiota

have on the pathogenic colonization? one can hypothesize that beneficial bacteria colonizing the gut may offer protection against invading fish pathogens [76]. In vitro growth inhibition of Streptococcus agalactiae, Streptococcus iniae, Micrococcus luteus and Vibrio parahemolyticus showed that Bacillus amyloliquefaciens and the Streptomyces rutgersensis isolated in the present study have broad and efficient agonistic activities. During the last 20 years, numerous papers have suggested that Streptomyces used as probiotics in aquatic animals can produce bacterio- cins [77], siderophores [78] and other antibacterial chemicals. The dietary supplementation of Bacillus amyloliquefaciens can be used to improve the health and growth rate of fish [79]. Therefore, in order to clarify whether supplementation of probiotic improves disease resis- tance, challenge studies have to be carried out. In addition, the remaining bacteria isolated in present study also have some antagonism to pathogenic bacteria, which was a valuable resource to be developed. Conclusions In summary, in this study, the diversity and bioactivity of aerobic culturable intestinal bacteria of tilapia in five diet groups (including four woody forages and the control group) was success- fully characterized. Woody forages had a great impact on the diversity of aerobic culturable bacteria in the guts of tilapia, and feeding different woody forages caused changes in the bacte- rial community in the gut of tilapia. In addition, a large number of bacterial resources that

have antagonistic effects on pathogenic bacteria were discovered by present study. These Table 6. The MIC value was determined by selecting strains with primary screen activity and bacteriostasis circle more than 1.5 cm. (Bacillus altitudinis, Bacillus amyloliquefaciens, Bacillus pumilus, Staphylococcus sp., Streptomyces rutgersensis). Bacterial isolates Antibacterial activity/ MIC (μg/mL) SA SI ML VP Bacillus altitudinis (MK281515) 12.5 25 6.2 - Bacillus amyloliquefaciens (MK281516) 6.2 3.1 12.5 25 Bacillus pumilus (MK281525) 25 50 6.2 Staphylococcus sp. (MK281545) - - - 12.5 Streptomyces rutgersensis (MK281548) 12.5 25 3.1 12.5 Control < 1.6 < 1.6 < 1.6 < 1.6 https://doi.org/10.1371/journal.pone.0235560.t006 PLOS ONE Effects of wood forages on gut bacterial biodiversity and bioactivity in tilapia PLOS ONE | https://doi.org/10.1371/journal.pone.0235560 July 2, 2020 13 / 17