Abstract: For many years, various drugs have been used for the treatment of infectious diseases but some bacterial microorganisms have induced resistance to several drugs. In the search for an alternative therapy for the infectious diseases, in this study the antibacterial activity of the steroid-brucine derivative against S. typhy, K. pneumoniae and E. coli was evaluated, using as control cefotaxime, gentamicin and ciprofloxacin. The evaluation of antimicrobial effect of the different compounds on the bacterial species was made by the method of microbial minimal inhibitory. The results indicate that bacterial growth of S. typhi was inhibited with cefotaxime, gentamicin, ciprofloxacin, brucine-ethylenediamine derivative and brucine-estradiol conjugate. Other results showed that bacterial growth of E. coli was inhibited with cefotaxime, gentamicin, ciprofloxacin, brucine-ethylenediamine and brucine-estradiol conjugate. Alternative experimental, indicate that bacterial growth of K. pneumoniae was inhibited with cefotaxime, gentamicin, ciprofloxacin, brucine-ethylenediamine derivative and brucine-estradiol conjugate. In conclusion, S. typhi, K. pneumoniae and E. coli were susceptible to gentamicin, cefotaxime, ciprofloxacin, brucine-ethylenediamine derivative and brucine-estradiol conjugate. These phenomenon, involves different molecular mechanism of brucine-ethylenediamine derivative and brucine-estradiol conjugate in comparison with the controls. In addition, the antibacterial activity induced by brucine-estradiol in comparison with the brucine-ethylenediamine derivative can depend of the degree of lipophilicity of these compounds.
INTRODUCTION
Infectious diseases still remain one of the most important causes of mortality worldwide (Pinner et al., 1996; Crossley and Peterson, 1996; Norman, 1999). There are different causal agents such as S. aureus (Henry, 2001), K. pneumoniae (Podschun and Ullman, 1998) and E. coli (Lautenbach et al., 2001) among others (Rothstein et al., 2003) that can induce various infectious diseases. It is important to mention, that for treatment of the infectious diseases is required many therapeutic agents (Wilson et al., 1995; Yoo et al., 2004; Killgore et al., 2004) unfortunately, prolonged antibiotic therapy may induce bacterial-resistance (Hackbarth and Chambers, 1989; Maguire et al., 1998), because some bacteria have developed ways to circumvent the effects of antibiotics (Peschel, 2002; Yeaman and Younth, 2003). For example, several studies indicate that β-lactam antibiotics may predispose to patients worldwide for acquisition of resistance to S. aureus (Mylotte et al., 1987; Lowy, 1998; Ayliffe, 1997; Merlino et al., 2002). Other reports showed that some antibiotic-resistant strains also have been emerging among Gram-negative bacilli such as K. pneumoniae (Podschun and Ullman, 1998) and E. colli (Prats et al., 2003). Therefore, antibiotic resistance can be considered a serious threat for the human health. In the search of alternative therapeutics for the treatment of infectious diseases, new drugs have been developed for control of bacterial resistance (Gordon et al., 1994; Schwab et al., 1999; Patch and Barron, 2003). In this sense, since several years ago, have been evaluated some steroid derivatives as potential therapeutic agents for infectious diseases (Li et al., 1999a). For example, there are data which indicate that the steroid derivative (dihydrotestosterone-ciprofloxacin) induce antibacterial activity against S. aureus and E. coli (Figueroa-Valverde et al., 2010). In addition, other steroid derivative (squalamine) has been used as antibacterial agent against a broad spectrum of microorganisms (Moore et al., 1993; Kikuchi et al., 1997). Other data indicate that a steroidal heterocyclic derivative showed antibacterial activity against gram positive and gram negative bacteria (Abdelhalim et al., 2007). All these experimental data indicate that several steroid derivatives can exert antibacterial activity on microorganism different. Therefore, in this study the antibacterial effect of the brucine-steroid derivative against S. typhi, K. pneumoniae and E. coli was evaluated using the method of microbial minimal inhibitory (Chiong and Betancourt, 1985). It is important to mention that the steroid-brucine derivative has a spacer arm in the D-ring of steroid whichis bound to the brucine fragment. In addition was used as biological tool a brucine derivative (brucine-ethylenediamine) to assess the molecular mechanism involved in the antibacterial activity of the brucine-steroid conjugate.
MATERIALS AND METHODS
General methods
Strains: The microorganisms in this study belonged to the strain
bank at the Departament of Pharmaco-chemistry at the Faculty of Chemical Biological
Sciences of the Universidad Autonóma de Campeche. The strains are certified
by Center for Disease Control in Atlanta and were as follows: S. tiphy
(ATCC 19430), K. pneumonia (ATCC 700603) and E. coli (ATCC 25922).
The strains are kept under refrigeration at 4°C in special gel (BBL).
| Fig. 1: | Chemical structure of brucine-ethylenediamine derivative (N1-(2,3-dimethoxystrychnidin-10-yliden)-ethane-1, 2-diamine) and brucine-steroid conjugate (N-[2-(2,3-dimethoxystrychnidin-10-ylideneamino)-ethyl]-succinamic acid 3-hydroxy-13-methyl-7,8,9,11,12,13,14, 15,16,17-decahydro-6 H-cyclo- penta[a]phenanthren-17-yl ester) |
Antimicrobial agents: Brucine-ethylenediamine [N1-(2,3-dimethoxystrychnidin-10-yliden)-ethane-1, 2-diamine], brucine-estradiol conjugate [N-[2-(2, 3-dimethoxystrychnidin-10-ylideneamino)-ethyl]-succinamic acid 3-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6 H-cyclopenta (a) phenanthren-17-yl ester] (Fig. 1) were synthesized by the previously method reported (Figueroa-Valverde et al., 2013). The compounds were dissolved in methanol and diluted with distilled water. Cefotaxime, gentamicinand ciprofloxacin were used as the standard drugs.
Antimicrobial activity: The evaluation of antimicrobial effect of the different compounds on the bacterial species was made by a method described previously (Chiong and Betancourt, 1985). The bacterial species were incubated on Mc Conkey (E. coli, S. typhi and K. pneumonia) agar for 24 h at 37°C. After such time, it was be determined whether growth had taken place or not. In addition, a series of tubes were prepared, the first of which contained 2 mL of culture medium (tripticase soye) at double concentration and the remainder (11 tubes), contained the same quantity of medium at single concentrations. From the first tube (double concentration) an aliquot of 2 mL of the studied compound (1 mg mL-1) was added and stirred, from this tube an aliquot of 2 mL was taken and added to the following tube (simple concentration) and the process was successively repeated until the last 2 mL of dissolution had been used up. After this process, each tube was inoculated with 0.1 mL of the bacterial suspension, whose concentration corresponded to Mc-Farland scale (9x108 cells mL-1) and all the tubes were incubated at 37°C for 24 h. Subsequently, a loop was taken from each of them and inoculated into the appropriate cultures for different bacterial organismsand were incubated for 24 h at 37°C. After such time, the Minimum Inhibitory Concentration (MIC) was evaluated to consider the antimicrobial effect of the different compounds. In order to discard the effect of methanol (solvent) on the bacterial species studied, a series of the same number of tubes was prepared in parallel, to which 2 mL of methanol at 60% was added to the first and corresponding successive dilutions were added in the same way as before. In addition a control series was also performed using distilled water to pH 7.0.
Statistical analysis: All the values obtained were expressed as Mean±SD error. The differences were considered significant when p-value was equal or smaller than 0.05.
RESULTS
The bacterial activity of brucine-estradiol conjugate was compared with the antibacterial effect of cefotoxime, gentamicinand ciprofloxacin (controls) in such bacterial microorganism studied. The results obtained (Fig. 2) indicate that bacterial growth of S. typhi was inhibited by cefotaxime (MIC = 5.23×10-4 mmol), gentamicin (MIC = 1.34×10-5 mmol) and ciprofloxacin (MIC = 3.01×10-3 mmol). In addition, the bacterial growth was significantly blocked (p = 0.05) in presence of the brucine-ethylenediamine derivative (MIC = 2.68×10-3 mmol) in comparison with the brucine-estradiol conjugate (MIC = 2.29×10-3 mmol).
On the other hand, alternative experimental were made in K. pneumoniae and E. coli using the same controls to evaluate the antibacterial effect of brucine-ethylenediamine derivative and brucine-estradiol conjugate. The results indicate that bacterial growth of E. coli was inhibited (Fig. 3) in presence of cefotaxime (MIC = 5.23×10-4 mmol), gentamicin (MIC = 1.34×10-5 mmol), ciprofloxacin (MIC = 3.01×10-3 mmol). Additionally, the growth of E. coli was significantly inhibited (p = 0.06) by brucine-estradiol conjugate (MIC = 2.68×10-3 mmol) in comparison with brucine-ethylenediamine derivative (MIC = 2.29×10-3 mmol).
| Fig. 2: | Antibacterial effects induced by brucine-estradiol derivative (B-S), brucine-ethylenediamine (B-ET) and controls (cefotaxime, CEFOT; gentamicin, GENT and ciprofloxacin, CIPROF) on S. typhi. The results showed that S. typhi was susceptibly to cefotaxime (MIC = 5.23×10-4 mmol), gentamicin (MIC = 1.34×10-5 mmol) and ciprofloxacin (MIC = 3.01×10-4 mmol). In addition, the bacterial growth in the presence of the brucine-ethylenediamine (MIC = 2.29×10-3 mmol) was significantly inhibited (p = 0.05) in comparison with the brucine-estradiol derivative (MIC = 2.68×10-3 mmol) |
| Fig. 3: | Antibacterial activity induced by brucine-estradiol derivative (B-S), brucine-ethylenediamine (B-ET) and controls (cefotaxime, CEFOT; gentamicin, GENT and ciprofloxacin, CIPROF) on E. coli. Experimental data showed that E. coli was susceptibly to CEFOT (MIC = 5.23×10-4 mmol), GENT (MIC = 1.34×10-5 mmol) and CIPROF (MIC = 3.01×10-3 mmol). Additionally, the growth bacterial was significantly blocked (p = 0.06) in the presence of B-S (MIC = 2.68×10-3 mmol) in comparison with B-ET (MIC = 2.29×10-3 mmol) |
| Fig. 4: | Effects induced by brucine-estradiol derivative (B-S), brucine-ethylenediamine (B-ET) and controls (cefotaxime, CEFOT; gentamicin, GENT and ciprofloxacin, CIPROF) on K. pneumoniae. The results showed that bacterial growth of K. pneumoniae in the presence of CEFOT (MIC = 2.61×10-4 mmol), GENT (MIC = 2.68×10-5 mmol), CIPROF (1.50×10-3 mmol) was inhibited. In addition, the growth of K. pneumoniae was significantly inhibited (p = 0.05) by the brucine-estradiol derivative (MIC = 2.68×10-3 mmol) and brucine-ethylenediamine (2.29×10-3 mmol) |
Other results obtained (Fig. 4) showed t hat bacterial growth of E. coli was significantly inhibited (p = 0.05) in presence brucine-ethylenediamine derivative (MIC = 2.29×10-3 mmol) in co mparison with the brucine-estradiol conjugate (MIC = 2.68×10-3 mmol). This results were compared with the antibacterial effect of controls, in this case, the cefotaxime showed a MIC of 2.61×10-4 mmol, the MIC for gentamicin was of 2.68×10-5 mmol and the MIC for ciprofloxacin was MIC = 1.50×10-3 mmol.
DISCUSSION
The experimental data obtained indicate that the brucine-estradiol conjugate had different antibacterial activity against S. typhi, K. pneumoniae and E. coli, in comparison with the controls (cefotaxime, gentamicin and ciprofloxacin). This phenomenon may be due mainly to the different molecular mechanisms involved in the antibacterial effect induced by the compounds studied. To evaluate the mechanism involved in the antibacterial activity induced by the brucine-estradiol conjugate against S. typhi, K. pneumoniae and E. coli, in this study the brucine-ethylenediamine compound was used as a biological tool. The result showed that bacterial growth of S. typhy, K. pneumoniae and E. coli in presence of brucine-ethylenediamine was blocked. It is important to mention that this compound contains in the chemical structure an arm with a free amine group. Therefore, the antibacterial activity of this compound may depend on the nature of the free amino group contained in their chemical structure (Fig. 1) which is a membrane-perturbing agent whose antibacterial activity can be possibly by the interaction with the positively charged phosphate groups contained in the Lipid A that are essential polymers that plays a vital role in the growth and development of the gram-negative bacteria (Fisher, 1990). It is important to mention, that some authors proposed a compelling model of complex formation involving ionic interactions between the phosphates on lipid A and the amine groups on polymyxin B. This phenomenon may increase the permeability of the outer membrane and induce bacterial growth inhibition on this gram-negative microorganism (Li et al., 1999b; Figueroa-Valverde et al., 2009). Nevertheless, also it is important to mention that when the brucine-ethylenediamine compound is bound with steroid to form the brucine-steroid conjugate, the antibacterial activity is increasing. This phenomenon, suggest that brucine-steroid conjugado requires of a hydrophobic region in order to interact with some components of bacterial cell, disturbing the bacterial growth and to cause cell death.
On the other hand, also possibly the antibacterial activity involve the intramolecular interaction of brucine-steroid with divalent cations (Mg2+ and Ca2+), involved in the membrane cell providing a substantial increase the permeability of the outer membrane of Gram-negative bacteria include bactericidal/permeability increasing protein. In conclusion, in this study the bacterial microorganism such as S. typhi, K. pneumoniae and E. coli were more susceptible to brucine-steroid in comparison with brucine ethylenediamine. Possibly the molecular mechanism could be by mimic the actions of several steroid-derivates with antibacterial characteristics (Ding et al., 2004). The molecular mechanism suggest that brucine-steroid conjugate can adopt cationic, facially amphiphilic conformations and involved the nature of functional groups contained in the chemical structure, which appears to be key requirement for antibacterial activity. This chemistry structure allows them to disrupt bacterial membranes at relatively different concentrations by the interaction with some factors in the bacterial membrane as effective targets of the brucine-steroid conjugate.
CONCLUSION
The antibacterial activity of the steroid-brucine derivative possibly involves, (1) Differences in the molecular mechanism involved in the antibacterial activity exerted by the brucine-ethylenediamine derivative and the brucine-estradiol conjugate in comparison with the controls and (2) The antibacterial activity induced by the brucine-estradiol derivative in comparison with the brucine-ethylenediamine conjugate can depend of the lipophilicity degree of this compound.
ACKNOWLEDGEMENTS
Lauro Figueroa is grateful to Angelica León Garcia for assistance valuable.