Antibacterial activities of the methanol extracts and compounds from Erythrina sigmoidea against Gram-negative multi-drug resistant phenotypes

Background In the present study, the methanol extracts from the leaves, as well as compounds namely sigmoidin I (1), atalantoflavone (2), bidwillon A (3), neocyclomorusin (4), 6α-hydroxyphaseollidin (5) and neobavaisoflavone (6) (from the bark extract) were tested for their activities against a panel of Gram-negative bacteria including multi-drug resistant (MDR) phenotypes. Methods Broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) of the extracts as well as compounds 1–6. Results The MIC results indicated that the crude extracts from the leaves and bark of this plant were able to inhibit the growth of 96.3 % of the 27 tested bacteria. Compounds 2–6 displayed selective activities, their inhibitory effects being obtained on 8.3 %, 41.7 %, 58.3 %, 58.3 % and 66.7 % of tested bacteria respectively for 2, 3, 5, 6 and 4. The lowest MIC value of 8 μg/mL was obtained with 6 against Escherichia coli ATCC8739, Enterobacter cloacae ECCI69, Klebsiella pneumoniae KP55, Providencia stuartii NAE16 and Pseudomonas aeruginosa PA01. Conclusion The present study demonstrates that Erythrina sigmoidea is a potential source of antibacterial drugs to fight against MDR bacteria. Neobavaisoflavone (6) is the main antibacterial consituents of the bark crude extract.


Plant material and extraction
The leaves and bark of Erythrina sigmoidea (Fabaceae) were collected in April 2013 in Bangangté (West Region of Cameroon). The plant was identified by a botanist of the National Herbarium in Yaoundé, Cameroon and compared with voucher kept under the registration number N°24470/HNC.

INT colorimetric assay for MIC and MBC determinations
MIC determinations on the tested bacteria were conducted using rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay according to described methods [16] with some modifications [19,20]. The test samples and chloramphenicol were first of all dissolved in DMSO/ Mueller Hinton Broth (MHB) or DMSO/7H9 broth. The final concentration of DMSO was lower than 2.5 % and does not affect the microbial growth [21,22]. The 96-wells microplate were used and the inoculum concentration was 1.5 × 10 6 CFU/mL [19,20]. The plates were incubated at 37°C for 18 h. The assay was repeated thrice. Wells containing adequate broth, bacterial inoculum and DMSO to a final concentration of 2.5 % served as negative control. The MIC of samples was detected after 18 h incubation at 37°C, following addition (40 μL) of 0.2 mg/mL of INT and incubation at 37°C for 30 min. Viable bacteria reduced the yellow dye to a pink. MIC was defined as the sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [16]. The MBC was determined by adding 50 μL aliquots of the preparations, which did not show any growth after incubation during MIC assays, to 150 μL of  (2); bidwillon A (3); neocyclomorusin (4); 6α-hydroxyphaseollidin (5); neobavaisoflavone (6) adequate broth. These preparations were incubated at 37°C for 48 h. The MBC was regarded as the lowest concentration of extract, which did not produce a color change after addition of INT as mentioned above [19,20].

Results and discussion
Compounds tested in this study included five isoflavonoids: atalantoflavone (2), bidwillon A (3), neocyclomorusin (4), 6α-hydroxyphaseollidin (5), neobavaisoflavone (6) and one flavonoid: sigmoidin I (1) (Fig. 1). Their isolation and identification from the bark of Erythrina sigmoidea were previously reported [15]. These compounds as well as the crude extracts from the leaves and bark of Erythrina sigmoidea were tested for their antibacterial activities on a panel bacterial strains and the results are reported in Tables 2 and 3.
Results of the MIC determinations indicate that crude extracts from leaves and bark of this plants were able to Klebsiella pneumoniae ATCC11296  --na  --na  --na  ---na  --na   KP55  --na  --na  256  -na  256  512  2  512  -na  8  -na   Providencia stuartii   ATCC29916  --na  --na  256  -na  256  -na  512  -na  --na   NAE16  --na  --na  --na  256  -na  512  -na  8  -na Pseudomonas aeruginosa  [24][25][26]. However, a keen look of the MICs and MBCs of compounds indicated that they rather exerted bacteriostatic effects (MBC/MIC > 4) [24][25][26]. It should be noted that the antibacterial spectra of compounds were lower than that of the bark extract. This suggested that a possible synergistic effect between the constituents of this extract could be expected. It should also be noted that the bark extract was not active on the resistant P. aeruginosa PA124 strains contrary to the isolated compound 6. This can either be due to the fact that this active compound (6) is less concentrated in the initial crude extract or to the possible interactions with other constituent. Regarding the clinical involvement of MDR bacteria in treatment failures [11,12,27,28], the antibacterial activity of the crude extracts as well as that of compound 6 could be considered promising. Pseudomonas aeruginosa is an important nosocomial pathogen, highly resistant to clinically used antibiotics, leading to substantial morbidity and mortality [29]. MDR Enterobacteriaceae, including K. pneumoniae, E. aerogenes, E.cloacae and P. stuartii and E. coli have also been classified as antimicrobial-resistant organisms of concern in healthcare facilities [11,12,30].
To the best of our knowledge, the antibacterial activity of the crude extracts from the Erythrina sigmoidea as well as compounds 2-6 against MDR bacteria is being reported for the first time. However, the antibacterial activities of compounds belonging to the classes flavonoids and isoflavonoids are well known [31]. In addition, a preliminary antibacterial study of flavonoids from the stem bark of Erythrina burttii showed that bidwillon A was active against E. coli and Staphylococcus aureus [32]. Neobavaisoflavone also displayed antifungal activity against Aspergillus fumigatus and Cryptococcus neoformans [33]. The present study provides additional information on the antimicrobial potency of neobavaisoflavone (6).

Conclusions
The results of the present study are interesting, taking in account the medical importance of the studied microorganisms. These data provided evidence that the crude extracts from Erythrina sigmoidea as well as some of its constituents, and mostly neobavaisoflavone (6) could be potential antimicrobial drugs to fight MDR bacterial infections.