Antibacterial and antibiotic-potentiation activities of the methanol extract of some cameroonian spices against Gram-negative multi-drug resistant phenotypes

Background The present work was designed to evaluate the antibacterial properties of the methanol extracts of eleven selected Cameroonian spices on multi-drug resistant bacteria (MDR), and their ability to potentiate the effect of some common antibiotics used in therapy. Results The extract of Cinnamomum zeylanicum against Escherichia coli ATCC 8739 and AG100 strains showed the best activities, with the lowest minimal inhibitory concentration (MIC) of 64 μg/ml. The extract of Dorstenia psilurus was the most active when tested in the presence of an efflux pump inhibitor, phenylalanine Arginine-β- Naphtylamide (PAβN), a synergistic effect being observed in 56.25 % of the tested bacteria when it was combined with Erythromycin (ERY). Conclusion The present work evidently provides information on the role of some Cameroonian spices in the fight against multi-resistant bacteria.


Background
Infectious diseases are one of the leading causes of morbidity and mortality worldwide, especially in developing countries [1,3]. Following the massive use of antibiotics in human therapy, bacteria have developed several resistance mechanisms including the efflux of antibiotics [3]. Several Cameroonian spices are known to possess medicinal values [4]. In our previous report, we demonstared that several medicinal spices inhibited the growth of MDR bacteria and were also able to improve the activity of commonly used antibiotics [5]. In our continuous search of antimicrobial drugs from medicinal plant, we designed the present work to investigate the antibacterial potential against Gram-negative MDR bacteria of some of the commonly used medicinal spices in Cameroon

Plant materials and extraction
The eleven edible spices used in this work were purchased from Dschang local market, West Region of Cameroon in January 2010. The collected spices material were the fruits of Aframomum citratum, Aframomum melegueta, Scorodophloeus zenkeri, Tetrapleura tetraptera,  Table 1). The extracts were obtained by methanol (MeOH) maceration as previously described [5].

Preliminary phytochemical investigations
The major secondary metabolites classes were screened according to the common phytochemical methods described by Harborne [24].

Bacterial strains and culture media
The studied microorganisms included reference (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes and Enterobacter cloacae The bacterial strains and their features were previously reported [5]. The preliminary treatment of these organisms as well as the culture media were conducted as previously described [5].

Bacterial susceptibility determinations
The respective MICs of samples on the studied bacteria were determined using rapid INT colorimetric assay [25,26] with some modifications as previously reported [5]. The inoculum concentration used was 1.5 x10 6 CFU/ml and the samples were incubated at 37°C for      18 h [5]. The final concentration of DMSO was lower than 2.5 % and this concentration also served as negative control [5]. Chloramphenicol was used as reference antibiotic. The MICs of samples were detected after 18 h incubation at 37°C, following addition (40 μl) of 0.2 mg/ml INT and incubation at 37°C for 30 minutes [5]. MIC was defined as the lowest sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [27]. Samples were tested alone and then, in the presence of PAßN at 20 mg/L final concentration as previously reported [5]. Four of the best extracts, those from A. citratum, C. zeylanicum, D. psilurus and T. tetraptera were also tested in association [5] at the concentrations selected following a preliminary assay on P. aeruginosa PA124 (See Additional file 1: Table S1). All assays were performed in triplicate and repeated thrice. Fractional inhibitory concentration (FIC) [5] were calculated and the interpretations were made as follows: synergistic (<0.5), indifferent (0.5 to 4), or antagonistic (>4) [28] (The FIC values available in Additional file 1: Table S2 and S3).

Phytochemical composition of the spice extracts
The results of qualitative analysis showed that each plant contains various phytochemicals compounds such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, steroids, tannins and triterpenes as shown in Table 2.

Antibacterial activity of the spice extracts
The results summarized in Table 3 summarize the MIC of the extract tested alone or in combination with PAβN on the tested microorganisms. Its shows that all the studied extracts were active on at least one microbial strain. A. citratum showed the best activity, it inhibitory effect being recorded on 85% (24/28) of the tested bacteria. Other Table 7 Minimal inhibitory concentration (MIC) of antibiotics in absence and presence extracts Tetrapleura tetraptera (μg/ml)

Effects of the association of some spice extracts with antibiotics
A. citratum, C. zeylanicum, D. psilurus and T. tetraptera (Tables 4, 5, 6 and 7) were associated to antibiotics in view of evaluating the possible synergistic effect of these associations. A preliminary study using P. aeruginosa PA124 was carried out with ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP and NOR) to select the appropriate sub-inhibitory concentrations to be used. MIC/2.5 and MIC/5 were then selected as the sub-inhibitory concentrations (see Additional file 1: Table S1). All of these four extracts were then tested in association with antibiotics previously listed on strains of E. coli AG100A TET and AG102, E. aerogenes CM64, K. pneumonia KP63 and P. aeruginosa PA124. No antagonistic effect (FIC > 4) was observed between extracts and antibiotics meanwhile indifference was observe between T. tetraptera and antibiotics in most of the case (see Tables 5, 6, and 7, Additional file 1: S2, S3, S4 and S5). Significant increase of the activity was observed with the association of the extracts of A. citratum and D. psilurus on E. aerogenes CM64 and K. pneumoniae KP63, and with C. zeylanicum against K. pneumoniae KP63. A significant decrease (synergy effect) of MIC values was also observed when ERY was associated with various extracts, and when extracts of A. citratum and C. zeylanicum were each combined with aminoglycosides (KAN, STR), the best activity being noted against E. aerogenes CM64.

Phytochemical composition of the spice extracts
The phytochemical studies revealed the presence of secondary metabolite such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, sterols, tannins and triterpenes; several molecules belonging to these classes of secondary metabolites were found active on pathogenic microorganisms [29].

Antibacterial activity of the spice extract
Although this is the first time that plants used in this work are studied for their activities vis-à-vis multiresistant bacteria, plants belonging to some of the genus studied herein, like the Aframomum genus are well documented for their antimicrobial activity [6]. Some antibacterial compounds, such as acridone and chelerythrine have previously been isolated from the fruits of F. leprieurii [14,30]. The antimicrobial activity of P. brazzeana and S. zenkeri is mainly due to some sulfur compounds. In fact, sulfur compounds with antimicrobial properties have previously been isolated from the two plants [7,31]. Several alkaloids of the genus Piper proved to be responsible for the activity of P. guineense [32]. The detection of this class of secondary metabolites in the extract studied herein can explain the observed activities. According to Krishnaiah et al. [16], the antimicrobial activity of I. cylindricum can be due to the presence of tannins in this plant. However, tannins were not detected in the extract of I. cylindricum as found in the present work ( Table 2), suggesting that other classes of secondary metabolites might be responsible for the antibacterial activity of this plant.

Role of efflux pumps in susceptibility of gram negative bacteria to the tested spice extracts
The significant increase of the activity of the extract of D. psilurus in the presence of EPI, indicates that bioactive constituents of this plant extract are substrate of efflux pumps. Efflux through AcrAB-TolC pumps was reported as essential mode of resistance of several Gram-negative MDR bacteria to a number of flavonoids isolated from plants of the genus Dorstenia, such as isobavachalcone, kanzonol C, stipulin, etc. [4,15,[33][34][35]. This suggests that possible combination of the extract of D. psilurus with EPI can be envisaged to overcome MDR bacteria.

Effects of the association of extracts with antibiotics
The results obtained by combining the antibiotic with the extracts of A. citratum, C. zeylanicum, D. psilurus and T. tetraptera indicate that these extracts contain chemical compounds that can modulate the activity of antibiotics against bacteria expressing MDR phenotypes. The methanol extracts of A. citratum, C. zeylanicum and D. psilurus showed a synergistic effect with antibiotics inhibiting bacterial cell wall synthesis (AMP and CEF) on K. pneumoniae KP63. The intrinsic mode of action of the active extracts is to be investigated.

Conclusion
The present work evidently provides information in the role of some Cameroonian spices in the fight against multi-resistant bacteria. The study also highlights the potential of D. psilurus as a strong antibacterial agent when the extract is combined with efflux pump inhibitor and several antibiotics.