Lacticaseibacillus casei ATCC 393 Cannot Colonize the Gastrointestinal Tract of Crucian Carp

Lactic acid bacteria (LAB) are commonly applied to fish as a means of growth promotion and disease prevention. However, evidence regarding whether LAB colonize the gastrointestinal (GI) tract of fish remains sparse and controversial. Here, we investigated whether Lacticaseibacillus casei ATCC 393 (Lc) can colonize the GI tract of crucian carp. Sterile feed irradiated with ⁶⁰Co was used to eliminate the influence of microbes, and 100% rearing water was renewed at 5-day intervals to reduce the fecal-oral circulation of microbes. The experiment lasted 47 days and was divided into three stages: the baseline period (21 days), the administration period (7 days: day -6 to 0) and the post-administration period (day 1 to 19). Control groups were fed a sterile basal diet during the whole experimental period, whereas treatment groups were fed with a mixed diet containing Lc (1 × 10⁷ cfu/g) and spore of Geobacillus stearothermophilus (Gs, 1 × 10⁷ cfu/g) during the administration period and a sterile basal diet during the baseline and post-administration periods. An improved and highly sensitive selective culture method (SCM) was employed in combination with a transit marker (a Gs spore) to monitor the elimination of Lc in the GI tract. The results showed that Lc (<2 cfu/gastrointestine) could not be detected in any of the fish sampled from the treatment group 7 days after the cessation of the mixed diet, whereas Gs could still be detected in seven out of nine fish at day 11 and could not be detected at all at day 15. Therefore, the elimination speed of Lc was faster than that of the transit marker. Furthermore, high-throughput sequencing analysis combined with SCM was used to reconfirm the elimination kinetics of Lc in the GI tract. The results show that the Lc in the crucian carp GI tract, despite being retained at low relative abundance from day 7 (0.11% ± 0.03%) to 21, was not viable. The experiments indicate that Lc ATCC 393 cannot colonize the GI tract of crucian carp, and the improved selective culture in combination with a transit marker represents a good method for studying LAB colonization of fish.