Bioassay-Guided Isolation of Antimicrobial Components and LC/QToF Profile of Plumeria obtusa: Potential for the Treatment of Antimicrobial Resistance

Abstract

The methanolic fraction (M-F) of the total extract (TE) of Plumeria obtusa L. aerial parts showed promising antibacterial effects against the MDR (multidrug-resistant) gram-negative pathogens Klebsiella pneumoniae and Escherichia coli O157:H7 [Shiga toxin-producing E. coli (STEC)]. In addition, M-F had a synergistic effect (in combination with vancomycin) against the MDR gram-positive strains MRSA (methicillin-resistant Staphylococcus aureus) and Bacillus cereus. After treating the K. pneumoniae- and STEC-infected mice with M-F (25 mg/kg, i.p.), the level of IgM and TNF-α was decreased and the severity of pathological lesions were reduced better than that observed after administration of gentamycin (33 mg/kg, i.p.). Thirty-seven compounds including 10 plumeria-type iridoids and 18 phenolics, 7 quinoline derivatives, 1 amino acid, and 1 fatty acid were identified in TE using LC/ESI-QToF. Furthermore, five compounds; kaempferol 3-O-rutinoside (M1), quercetin 3-O-rutinoside (M2), glochiflavanoside B (M3), plumieride (M4), and 13-O-caffeoylplumieride (M5) were isolated from M-F. M5 was active against K. pneumoniae (MIC of 64 μg/mL) and STEC (MIC of 32 μg/mL). These findings suggested that M-F and M5 are promising antimicrobial natural products for combating MDR K. pneumoniae and STEC nosocomial infections.

Figure 1

LC-DAD-QToF chromatograms of P. obtusa aerial parts: (A, B) QToF-MS base peak chromatograms in negative and positive modes and (C–E) DAD chromatograms at 254, 280, and 330 nm, respectively.

Figure 2

Chemical structure of compounds isolated (M1–M5) from the 100% methanolic fraction (M-F) of P. obtusa L. total extract.

Figure 3

Highest zone diameter observed against E. coli O157:H7 after combination of 100% methanolic fraction (M-F) of P. obtusa and gentamycin (GEN).

Figure 4

Tumor necrosis factor-alpha (TNF-α, pg/mL) levels in model of mice infected by MDR K. pneumoniae (A) and E. coli O157:H7 (B) and then treated with 100% methanolic fraction (M-F) of P. obtusa and gentamycin (GEN). Results are expressed as mean ± SD. Statistical analysis was carried out by one-way ANOVA. ^aSignificant difference from normal control group at p < 0.05. ^bSignificant difference from positive infected group at p < 0.05. ^abSignificant difference from normal control group and positive infected group at p < 0.05.

Figure 5

Immunoglobulin M (IgM, ng/mL) levels in model of mice infected by MDR K. pneumoniae (A) and E. coli O157:H7 (B) and then treated with 100% methanolic fraction (M-F) of P. obtusa and gentamycin (GEN). Results are expressed as mean ± SD. Statistical analysis was carried out by one-way ANOVA. ^aSignificant difference from normal control group at p < 0.05. ^bSignificant difference from positive infected group at p < 0.05. ^abSignificant difference from the normal control group and positive infected group at p < 0.05.

Figure 6

Histological sections of lung stained with H & E and scoring of pulmonary lesions in model mice infected with MDR K. pneumoniae. (a) Negative control (X 400), (b) mice infected with K. pneumoniae (X 200), (c) mice infected with K. pneumoniae and then treated by gentamycin (GEN) (X 200), and (d) mice infected with K. pneumoniae and then treated with 100% methanolic fraction of P. obtusa (M-F) (X 400).

Figure 7

Histological sections of kidney stained with H&E and scoring of renal lesions in model mice infected with E. coli O157:H7. (a) Normal control (X 400), (b) Mice infected with E. coli O157:H7 (X 400), (c) mice infected with E. coli O157:H7 and then treated with gentamycin (GEN) (X 200), and (d) mice infected with E. coli O157:H7 and then treated with 100% methanolic fraction of P. obtusa (M-F) (X 200).