In vitro Assessment of the Safety and Potential Probiotic Characteristics of Three Bacillus Strains Isolated From the Intestine of Hybrid Grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂)

Probiotics serving as an alternative to the criticized antibiotics mainly focus on improving animal's growth and health. After realizing the dangers posed by diseases that have led to lots of economic losses, aquaculture scientists have sought the usage of probiotics. However, most probiotics are ineffective in eliciting aquatic animals' preferred effects, since they are from non-fish sources. Again, there are even a few marine aquatic probiotics. Given this, a study was conducted to investigate the probiotic potential of the bacteria species isolated from the digestive tract of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂). Based on the morphological, biochemical, 16S rRNA sequencing analysis and evolutionary relationships, the isolated species were identified as Bacillus tequilensis GPSAK2 (MW548630), Bacillus velezensis GPSAK4 (MW548635), and Bacillus subtilis GPSAK9 (MW548634), which were designated as GPSAK2, GPSAK4, and GPSAK9 strains, respectively. Their probiotic potentials including their ability to tolerate high bile salt concentration, low pH, high temperatures, adhesion ability (auto-aggregation and cell-surface hydrophobicity), antimicrobial activity and biosafety test, compatibility test, hemolytic activity, and antibiotic susceptibility test were evaluated. While GPSAK2 and GPSAK9 strains were γ-hemolytic, that of GPSAK4 was α-hemolytic. All the isolates were resistant to low pH (1) and higher bile salt concentration (0.5%), showed higher viability ability after higher temperature exposure (80, 90, and 100°C), as well as higher cell-surface percentage hydrophobicity and auto-aggregation. All isolates exhibited positive compatibility with each other, signifying their ability to be used as multispecies. The three strains were susceptible to ampicillin (except GPSAK9, which was resistant), penicillin, kanamycin, ceftriaxone, chloramphenicol, erythromycin, clindamycin, furazolidone (except GPSAK2 and GPSAK9, which were moderately susceptible and resistant, respectively), polymyxin B, vancomycin (except GPSAK9, which was resistant), sulfamethoxazole (except GPSAK9, which was moderately susceptible), amikacin, minocycline, ofloxacin, norfloxacin, doxycycline, neomycin, gentamicin, tetracycline, carbenicillin, midecamycin (except GPSAK9, which was moderately susceptible), ciprofloxacin, piperacillin, and cefoperazone. All isolates demonstrated good antimicrobial activity against four pathogens, viz. Streptococcus agalactiae, Streptococcus iniae, Vibrio harveyi, and Vibrio alginolyticus. The results collectively suggest that Bacillus strains GPSAK2, GPSAK4, and GPSAK9 could serve as potential probiotic candidates that can be used to improve the growth and health status of aquatic animals, especially grouper.

Resistant gastric environment of Lactobacillus crispatus from stomach inhibits Helicobacter pylori colonization and attenuates gastric inflammation

Background Recent studies have shown that gastric-derived Lactobacillus can inhibit the colonization of H. pylori and attenuate gastric inflammation in conventional animals, but the resistant of Lactobacillus to gastric environment is still unknown. Here, we aimed to screen the candidate Lactobacillus that could adapt to the harsh gastric environment and inhibit the colonization of H. pylori. Results In vitro, the growth rate of seven Lactobacillus strains in different pH and bile salt concentration were tested, the size of inhibition zone and adhesion rate of H. pylori when Lactobacillus exist were measured. In gnotobiotic mice models, we examined the amount of colonization of L. crispatus and H. pylori by qRT-PCR and evaluated the inflammation in the gastric tissue by the content of MPO and H&E stain. In vitro experiments showed L. crispatus had a better growth rate than other six Lactobacilluses in pH 2.5 to 4.5; under the 0.2% bile salt concentration, other bacteria did not grow except for L. crispatus; L. crispatus yielded 24.2 mm of mean inhibitory zone diameters; the adhesion rate of H. pylori only reached 41.3% in H. pylori-L. crispatus group(HLG). In vivo, the amount of colonization of H. pylori in HLG is fifteen times less than that in H. pylori group (HG) (p < 0.05); the MPO value of HG was 1.4 times that of HLG; the gastric tissue inflammation of HLG was obviously lighter than HG. L. crispatus may be an adjunctive therapy for treating H. pylori- associated disease in clinic. Conclusions L. crispatus has resistance to low acid and high bile salts environment and it inhibits the growth of H. pylori and the subsequent inflammation H. pylori caused in gnotobiotic Kunming mice model, which suggest the potential of developing L. crispatus as clinical agents.

Uji Potensi Perlekatan Bakteri Asam Laktat Isolat TLA-15 Dan TLA-20 Pada Sel Epitel Usus Tikus (Rattus norvegicus)

In Vitro probiotic potential test that had been conducted before, which consist of probiotic bacteria test resistance toward the gastro-intestinal track pH and the bile salt concentration shows that the strain bacteria TLA-15 and TLA-20 had potential as probiotic bacteria. An advance test are required to fullfill the requirement as the probiotic bacteria. So that, the objective of this research are to study the viability of lactic acid bacteria strain TLA-15 and TLA-20 at the Rattus norvegicus GI track. At the first step of this research was added 4 106 cell/ml lactic acid bacteria TLA-15 and TLA-20 given orally.

Very high aquaporin-1 facilitated water permeability in mouse gallbladder

Water transport across gallbladder epithelium is driven by osmotic gradients generated from active salt absorption and secretion. Aquaporin (AQP) water channels have been proposed to facilitate transepithelial water transport in gallbladder and to modulate bile composition. We found strong AQP1 immunofluorescence at the apical membrane of mouse gallbladder epithelium. Transepithelial osmotic water permeability (Pf) was measured in freshly isolated gallbladder sacs from the kinetics of luminal calcein self-quenching in response to an osmotic gradient. Pf was very high (0.12 cm/s) in gallbladders from wild-type mice, cAMP independent, and independent of osmotic gradient size and direction. Although gallbladders from AQP1 knockout mice had similar size and morphology to those from wild-type mice, their Pf was reduced by ∼10-fold. Apical plasma membrane water permeability was greatly reduced in AQP1-deficient gallbladders, as measured by cytoplasmic calcein quenching in perfluorocarbon-filled, inverted gallbladder sacs. However, neither bile osmolality nor bile salt concentration differed in gallbladders from wild-type vs. AQP1 knockout mice. Our data indicate constitutively high water permeability in mouse gallbladder epithelium involving transcellular water transport through AQP1. The similar bile salt concentration in gallbladders from AQP1 knockout mice argues against a physiologically important role for AQP1 in mouse gallbladder.