Antibacterial and Antibiofilm Activities of Crude Extract of Lasiodiplodia pseudotheobromae IBRL OS-64 against Foodborne Bacterium, Yersinia enterocolitica

The antibacterial and antibiofilm activities of crude extract of Lasiodiplodia pseudotheobromaeIBRL OS-64 was studied and tested against a foodborne pathogenic bacterium, Yersinia enterocolitica. The ethylacetate extract exhibited favorable antibacterial activity with the zone of inhibition was 20.3±0.6 mm compared to dichloromethane (15.0±0.3 mm) and butanol (9.0±0.3 mm) extracts. Minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) values of the extract were 125 and 250 μg/mL, respectively. Structural degeneration studies through scanning electron microscopy (SEM) and transmission electron microscope (TEM) micrographs exhibited major abnormalities that occurred on thebacterial cells after exposureto the extract were complete alterations in their morphology and collapsed of the cells beyond repair. The findings showed that the extract possesses antibiofilm activity against the initial and preformed biofilm of Y. enterocoliticawith the highest inhibition value of 69.12% and 58.70%, respectively The results also revealed the initial biofilm was more susceptible to the extract as compared to pre-formed biofilm. The light microscopy (LM) and SEM photomicrographs proved that thefungal extract significantly eliminates extracellular polysaccharide (EPS) matrices and hinder the attachment of the bacterial cells for biofilm formation. Therefore, the current study suggested the ethyl acetate crude extract from an endophytic fungus, L. pseudotheobromae IBRL OS-64 may be an effective antibacterial and anti-biofilm agent to treat foodborne pathogens

Effect of ethyl acetate crude extract of Lasiodiplodia pseudotheobromae IBRL OS-64 against oral cavity bacteria with emphasis on Streptococcus mutans

Streptococcus mutans and Streptococcus agalactiae have been reported in human oral diseases including cariesand periodontitis. Thus, the present study was performed to investigate the effect of ethyl acetate crude extract ofLasiodiplodia pseudotheobromae IBRL OS-64, an endophytic fungus isolated from Ocimum sanctum leaf againstS. mutans and S. agalactiae. Disk diffusion and broth microdilution assays were used to determine the antimicrobialactivities with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values.Meanwhile, the microscopic analysis was used to study the structural degeneration of bacterial cells. The fungal crudeextracts demonstrated favorable antibacterial activity toward both test bacteria and produced an inhibition zone rangingfrom 16.0 to 21.2 mm. The MIC and MBC values of the fungal crude extract toward S. mutans and S. agalactiae weredetermined and the results showed that the MIC and MBC values were in the range of 125–500 µg/ml and 125–1,000µg/ml, respectively. The time-kill study suggested that the ethyl acetate crude extract possessed bactericidal effectwith concentration and time-dependent. Structural degeneration studies revealed the major abnormalities occurredto S. mutans cells after treated with the fungal crude extract where completed alterations of their morphology withthe formation of cavities and collapsed cells beyond repair occurred. Therefore, the current study suggested the ethylacetate crude extract of L. pseudotheobromae IBRL OS-64 could be an effective antibacterial agent to treat oral cavitybacteria.

Antibacterial activity of an endophytic fungus Lasiodiplodia pseudotheobromae IBRL OS-64 residing in leaves of a medicinal herb, Ocimum sanctum Linn.

The study was carried out to investigate the antibacterial activity of an endophytic fungal isolate, Lasiodiplodiapseudotheobromae IBRL OS-64 residing in leaves of a medicinal herb, Ocimum sanctum Linn. Qualitative screeningof the antimicrobial activity was done using an agar plug assay, and the results showed that the fungal isolate wasable to inhibit all the 13 test bacteria. Three Gram-positive bacteria (methicillin-resistant Staphylococcus aureus[MRSA] ATCC 33591, Staphylococcus aureus, and Streptococcus mutans) were the most susceptible species withthe inhibition zones of ≥21 mm. The other three (Bacillus cereus ATCC 10876, Bacillus subtilis IBRL A3, andStreptococcus agalactiae) showed the inhibition zones of 11–≤20 mm of diameter. As for Gram-negative bacteria,Yersinia enterocolitica was the most susceptible to the fungal isolate with the size of inhibition zone of ≥21 mm,followed by Klebsiella pneumoniae ATCC 13883, Salmonella typhimurium, and Shigella boydii ATCC 9207 withthe inhibition zones of 11–≤20 mm, whereas Escherichia coli IBRL 0157, Proteus mirabilis, and Pseudomonasaeruginosa ATCC 27883 were the least susceptible with the inhibition zones of ≤10 mm. Quantitative screeningusing disc diffusion assay showed that the fungal ethyl acetate extract prepared from the fermentative broth(extracellular) exhibited better antibacterial activity compared to the methanolic extract prepared from the fungalbiomass (intracellular). The results showed that the ethyl acetate extract exhibited antibacterial activity against all the13 test bacteria with the inhibition zone sizes of 20.0 ± 0.3–31.3 ± 1.2 mm in diameter for Gram-positive bacteria and10.31 ± 0.6–20.1 ± 0.6 mm in diameter for Gram-negative bacteria. On the other hand, the methanolic extract onlyinhibited three Gram-positive bacteria (MRSA ATCC 33591, S. aureus, and S. mutans) with the inhibition zones of9.0 ± 0.6–11.0 ± 0.3 mm in diameter, whereas only one Gram-negative (S. typhimurium) with the inhibition zone sizeof 13.3 ± 1.5 mm diameter. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration(MBC) values of the ethyl acetate extract on Gram-positive bacteria were in the range of 62.50–125.00 and 62.50–500.00 µg/mL, respectively, whereas for the Gram-negative bacteria, the MIC and MBC values were 125.00–250.00and 250.0–1000.00 µg/mL, respectively. On the other hand, the MIC and MBC values for methanolic extract againstGram-positive bacteria were 250.00–500.00 µg/mL and against Gram-negative bacteria were 1000.00 µg/mL,respectively. Both of the extracts exhibited bactericidal effects on test bacteria with the MBC/MIC ratio ≤4. Further,detail of the effects of the ethyl acetate extract on the bacterial cells was observed from the scanning electronmicroscopy photomicrographs which revealed the severity of the morphological deterioration experienced by theextract-treated cells were beyond repair, and the most possible mode of actions were by interrupting the cell wallbiosynthesis and cell membrane permeability