| Summary: | Infectious diseases caused by bacteria are at risk of spreading and prolonging due to antimicrobial resistance. It is, therefore, urgently necessary to develop a more effective antibiotic alternative strategy to control pathogen spread. In general, probiotics have been recommended as a substitute for antibiotics that inhibit pathogens. This study was isolated and probiotic characteristics and antibacterial bacterial efficiency against various infection-causing pathogens were determined by different in vitro methods. A 16S rRNA sequence confirmed that the isolated strains belonged to a species of <i>Leuconostoc citreum</i>. <i>L. citreum</i> KCC-57 and KCC-58 produced various extracellular enzymes and fermented different carbohydrates. There was significant tolerance for both strains under the harsh conditions of the gastrointestinal tract (GIT). In addition, <i>L. citreum</i> KCC-57 and <i>L. citreum</i> KCC-58 showed significant auto-aggregations and hydrophobicity properties that varied with incubation time. Moreover, the cell-free secondary supernatant (CFS) of <i>L. citreum</i> KCC-57 and <i>L. citreum</i> KCC-58 inhibited growth of <i>Enterococcus faecalis</i>, <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus.</i> According to a co-culture study, <i>L. citreum</i> KCC-57 and <i>L. citreum</i> KCC-58 were highly competitive for pathogen growth. <i>L. citreum</i> KCC-57 and <i>L. citreum</i> KCC-58 showed significant probiotic potential and strong antibacterial activities against different pathogens, suggesting that these strains could be used instead of antibiotics to control infectious pathogens.
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