Lacticaseibacillus spp.; Probiotic candidates from Palmyra palm sugar possesses antimicrobial and anti-biofilm activities against methicillin-resistant Staphylococcus aureus

Abstract

Background and aim: Probiotics are beneficial microorganisms that play important roles by adhering to the gut and producing antimicrobial substances to inhibit pathogens. The objective of this study was to isolate and characterize the probiotic lactic acid bacteria (LAB) from Palmyra palm sugar, which can produce antimicrobial compounds against methicillin-resistant Staphylococcus aureus (MRSA), a new zoonotic and food-borne pathogens.

Materials and methods: Twenty-six LAB isolates were isolated from 30 Palmyra palm sugar samples. Three selected LAB were further characterized as probiotics. In addition, the antibacterial and anti-biofilm-forming activities of the probiotics' culture supernatants against MRSA and food-borne pathogens were investigated. Finally, the selected probiotics were identified by aligning 16S rRNA sequences.

Results: The three confirmed probiotics, WU 0904, WU 2302, and WU 2503, showed strong antibacterial activities against S. aureus, MRSA, Escherichia coli O157:H7, and Listeria monocytogenes, as measured by a broth microdilution assay. Among the LAB isolates, 82.22-86.58%, 91.83-96.06%, and 64.35-74.93% exhibited resistance to low pH, pancreatin treatment, and bile salts, respectively. It was found that 59.46% and 83.33% auto-aggregation was observed in 2 and 24 h, respectively. Moreover, 50.25-57.24% adhesion was detected after the incubation of the bacterial cells to Caco-2 cells.. Biofilm inhibition (82.81-87.24%) was detected after the treatment of MRSA with the culture supernatants, when compared with that to the control. By the alignment of 16S rRNA sequences, the isolate WU 2302 was identified as Lacticaseibacillus spp. with 98.82% homology when compared to the GenBank database.

Conclusion: This study indicates that isolated probiotics can produce antimicrobial compounds against MRSA and food-borne pathogens. The obtained results strongly suggest that these probiotics are promising candidates for pharmaceutical products.

Keywords: Lacticaseibacillus spp; Palmyra palm sugar; antibacterial activity; methicillin-resistant Staphylococcus aureus; probiotics.

Figure-1

Auto-aggregation (%) of the selected lactic acid bacteria isolated from Palmyra palm sugar. The suspension of the bacteria was incubated at 37°C for 0, 2, and 24 h. Auto-aggregation at different time points was determined by the optical density of the aqueous phase at 600 nm. The data are presented as mean ± SD.

Figure-2

Percentage of hydrophobicity (a) and adhesion (b) of the selected lactic acid bacteria isolated from Palmyra palm sugar to Caco-2 cells. The data are presented as mean ± SD.

Figure-3

Scanning electron microscope (SEM) analysis of Caco-2 cells where the probiotic isolate adheres to the surface of Caco-2 cells. a; Untreated Caco-2 cells as control, b; adhesion of the probiotics to Caco-2 cells. Magnification: a = 2500×, b = 10,000×

Figure-4

Effects of probiotic supernatants on biofilm formation of Staphylococcus aureus ATCC25923 and MRSA. The organism was treated with the supernatants, incubated at 37°C for 24 h. The inhibitory activity was carried out using crystal violet assay. The culture medium was used as a negative control. The relative percentage of the biofilm inhibition (%)=100–[(570treatment/A570control)×100], (*significant difference; p<0.05).

Figure-5

Neighbor-joining phylogenetic tree showing the phylogenetic position of Lacticaseibacillus spp. WU 2502, WU 0904, and WU2302 based on 16S rRNA gene sequences. The scale bar indicates 1 nucleotide substitution per 100 nucleotides. Numbers in parentheses indicate accession numbers of 16S rRNA genes from type strains. Bootstrap values over 50% (based on 1000 replications) are shown at the nodes.