Medicinal plants have been presented as a valuable source of preservation of human health. In special, Stryphnodendron barbatiman has been employed due to its antimicrobial activity. This plant is rich in tannins and has been used in popular medicine for the treatment of gastrointestinal disorder, treatment of lesions, and also as anti-inflammatory microbicide. Citrobacter freundii is a member of the family Enterobacteriaceae and is one of the major causes of opportunistic infections. This microorganism is a bacterium (bacillus) aerobic gram-negative with a length in the range of 1 to 5 mm. C. freundii is commonly found in water, soil, food and occasionally in the gastrointestinal tract of animals and humans. In this paper, we have demonstrated the antibacterial activity of S. barbatiman by observing cellular death by using inhibition halo approach. Atomic force microscopy and FT-IR spectroscopy results suggested that interaction between the main active components of S. barbatiman with cellular wall of C. freundii gives rise to cellular wall damage, and then leads this microorganism to death.
Bacterial infections are usually treated using antibacterial drug therapies. The problem with this approach is the resistance developed by bacteria to these types of drugs. Natural products have emerged as a suitable way to the discovery of effective compounds with low toxicity. Several plants are used in the popular medicine including the species Stryphnodendron barbatiman. This one has, as main active, ingredient the called tannins, which are recognized as healing and antimicrobial agent against bacteria [
Bacteria used in this study were obtained from a scraping oropharyngeal sample and identified by an automated system Vitek/BioMérieux. S. barbatiman, popularly known as barbatimão, grows in Brazilian Cerrado and in Amazon region. Alcoholic extract was prepared using 5.0 g of fresh plant bark and dried with maceration in 96% ethanol (5.0 g/100.0 mL). Aqueous extract was obtained by cooking bark in purified water (5.0 g/100.0 mL). After filtering, the extract was stored in amber bottle. Suspension of the bacterium C. freundii was obtained from colony solubilisation in physiological solution (0.9% NaCl). For susceptibility test of C. freundii, the extracts of S. barbatiman were deposited in 10.0 mL droplets over the culture on the dish petri. Then, the plates were incubated at 36˚C for 24 h to monitor the formation of inhibition halo. Biofilms were prepared on glass substrates from the deposition of the suspension of C. freundii using the casting technique—which consisted in a deposition followed by solvent evaporation. Biofilms were left to stand for 20 min at 36˚C until complete solvent evaporation. Biofilms with extract of S. barbatiman were obtained by deposition of a combination of bacteria in suspension and extract after different contact times. Studies of the surface morphology of the biofilms were performed using an atomic force microscope (Nanosurf EasyScan II) in intermittent mode (512 × 512 pixels). Images were acquired in scanning windows of 5 mm × 5 mm. Roughnesses of biofilms were determined using the Nanosurf Instruments software and a spectrophotometer Perkin-Elmer Spectrum 100 FT-IR was used to obtain spectra of the extracts and bacteria.
It was observed that C. freundii is sensitive to extracts of S. barbatiman (aqueous or alcoholic) showing more stable results in inhibition halos for aqueous extracts of S. barbatiman (
Although the halo after 24 h for alcoholic extract is slightly larger than that of the aqueous extract, after 48 h is possible to observe the appearance of some colony in the region of the halo for alcoholic extract. This find was expected, since other studies have shown that S. barbatiman has antimicrobial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Candida krusei, Enterococcus faecalis, Klebsiella pneumoniae, Kocuria rhizophila, Neisseria gonorrhoeae, and Shigella flexneri [
As shown in
results support the hypothesis that cellular wall damage of C. freundii can be caused by chemical compounds like tannins, which may be found in S. barbatiman and are involved in antimicrobial activity.
FT-IR spectroscopy has been used to investigate the interaction between S. barbatiman and C. freundii, which could lead to death of these bacteria [
The most intense band of C. freundii includes the functional C=O group of lipid at 1741 cm−1. The amide I band at ~1630 cm−1 is associated with the C = O stretching of amide group of proteins. The region between 1300 and 900 cm−1 is characterized by the characteristic vibration of groups of nucleic acids, cell membrane and cell wall components [9-11]. The major band of this region includes stretching of the P = O groups (phosphodiester) at 1219 cm−1 in the backbone of nucleic acids and polysaccharide vibration (1200 - 900 cm−1) of the cell wall peptidoglycans and lipopolysaccharides.
The spectrum of C. freundii dispersed in S. barbatiman extract indicated significant variations in the region between 1300 and 1200 cm−1. This suggested a denaturation of nucleic acid and/or damages to the cellular walls [
The antimicrobial activity of S. barbatiman against C. freundii was demonstrated by susceptibility tests. FT-IR spectroscopy and AFM results suggested that S. barbatiman causes damages to the cellular wall of C. freundii bacteria and then leads them to death. Further studies are necessary to clear this point; however, possible therapeutic uses of the S. barbatiman against C. freundii should be explored in which FT-IR spectroscopy and AFM analyses can play an important role.
This work was supported by CNPq and Capes (Brazil). We thank M. Fátima Rodrigues Souza and Elpídio de Souza Filho for discussion about S. Barbatiman.