Activities of selected medicinal plants against multi-drug resistant Gram-negative bacteria in Cameroon

Background: Medicinal plants are used worldwide for several human ailments including bacterial infections. The present work was designed to assess the in vitro antibacterial activities of some Cameroonian medicinal plants including Entada abyssinica, Entada africana, Pentaclethra macrophylla, Allexis cauliflora, Anthocleista leibrechtsiana, Carapa procera, Carica papaya and Persea americana against Gram-negative bacteria expressing multidrug resistant (MDR) phenotypes. Methods: The microbroth dilution was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the samples against eight bacterial strains belonging to four species, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Providencia stuartii. Results: The extracts displayed selective antibacterial activities with the minimal inhibitory concentrations (MIC) values ranges of 64 to 1024 μg/mL. The most active extract was that from Pentaclethra macrophylla (TPM) that showed inhibitory activities against five of the eight (62.5%) tested bacteria. The lowest MIC value (64 μg/mL) was recorded with the crude extract of Entada africana against E. coli AG100A whilst the best MBC (256 μg/mL) value was also obtained with methanol extract of Persea americana against this bacterial strain. Conclusion: The results of the present work provide baseline information on the possible use of Pentaclethra macrophylla, Entada africana and Entada abyssinica in the treatment of selected bacterial infections.


Introduction
The increase of bacterial resistance specifically leading to treatment failures is directly responsiblefor the current increase in morbidity and mortality associated with bacterial infections 1 .Among the known mechanisms of resistance, active efflux via resistance-nodulation-cell division (RND) pumps is one of the most occurring systems in Gram-negative bacteria 2 .This efflux system depends on membrane energy and efficiently expels structurally unrelated antibiotic molecules across the bacterial envelope via a tripartite complex (comprising an inner membrane pump, a periplasmic fusion protein, and an outer membrane channel) 3 .Today, the increase of resistance to antibiotics propels the search of new drugs to tada africana collected at Far Nord Region in February 2011; the bark of Pentaclethra macrophylla collected in November 2012 at Mfou division (Centre Region); the leaves of Allexis cauliflora and the bark of Carapa procera collected in August 2012 at Monkey mount, kribi division (South Region), Anthocleista leibrechtsiana collected in November 2012 in June 2012 at Pouma division (Littoral Region), the seeds of Carica papaya and stones of Persea americana collected in February 2013 at Mfoundi market (Centre Region).The botanical identification of these plants was done at the Cameroon National Her-barium in Yaounde by Mr Victor Nana, where voucher specimens were kept (Table 1).The powdered air-dried (under shade) sample from Allexis cauliflora, Anthocleista leibrechtsiana, Carapa procera, Carica papaya and Persea americana were extracted with methanol, that of Entada africana with the solvent mixture CH 2 Cl 2 /MeOH (1:1), those of Entada abyssinica with ethyl acetate and that of Pentaclethra macrophylla with CH 2 Cl 2 /MeOH (1:1) for 48 h at room temperature.The extract was then concentrated under reduced pressure under vacuum to give a residue that constituted the crude extract.They were then kept under 4 0 C until use.

Bacterial susceptibility determinations
The respective MICs of samples on the studied bacteria were determined by using rapid p-Iodonitrotetrazolium chloride (INT, Sigma-Aldrich, St. Quentin Fallavier, France) colorimetric assay 34 .Briefly, the test samples were first dissolved in dimethylsulfoxide (DMSO)/ MHB.The solution obtained then added to MHB, and serially diluted two fold (in a 96-well microplate).
One hundred microlitres (100 μL) of inoculum (1.5 × 10 6 CFU/mL) prepared in MHB was then added.The turbidity of the microbial suspension was adjusted with a densitometer to a McFarland standard of 0.5 that is equivalent to 1-5 x 10 8 CFU/mL.The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a shaker and incubated at 37•C for 18 hrs.The final concentration of DMSO was set at 2.5% (a concentration at which DMSO does not affect the microbial growth).Wells containing MHB and 100 μl of inoculums served as a negative control.Chloramphenicol (CHL) was used as reference antibiotic.The MICs of samples were detected after 18 h of incubation at 37•C, following addition (40 μL) of 0.2 mg/mL INT and incubation at 37•C for 30 min 5 .Viable bacteria reduced the yellow dye to pink.MIC was defined as the lowest sample concentration that exhibited complete inhibition of microbial growth and then prevented this change MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth.
For the determination of MBC, the microplates were filled by 150 µL of MHB without extract of plant; for wells not having received a INT (during the reading of the MIC), 50 µL of the contents of the wells corresponding to the concentrations higher or equal to the MIC was taken and introduced into these microplates.These were then incubated during 48 h à 37 0 C, followed by revelation with the INT.All the concentrations among which we did not observe pink coloring were taken as bactericidal and the lowest was noted as MBC.

Results
The data summarized in Table 3 shows the antibacterial activities of the tested strains.All extracts were active on at least one of the eight tested bacteria with the MIC values ranging from 64 to 1024 µg/mL.The most active extracts were those of P. macrophylla (TPM), E. africana (TM2), bark of young from plant E. abyssinica (TM1') and bark of old plant from E. abyssinica (TM1) with the respective inhibitory activities recorded against 62.5 %, 50%, 37.5% and 37.5%.The lowest MIC value (64 μg/mL) was obtained with E. africana (TM2) extract against E. coli AG100A.This strain was the most sensitive amongst the tested bacteria towards all the plant extracts whilst no activity was recorded against E. aerugenes CM64 at the tested concentrations.The MIC of chloramphenicol was lower compared to those of the tested extract on all bacteria used in this study.However they were still high and varied from 8 to above 256 µg/ mL.This confirms the high level of resistance of studied bacterial strains.The extracts of P. americana (AV), E. africana (TM2) and A. cauliflora (ACT) showed MBC values of 128; 256 and 512μg/mL respectively against Echerichia coli AG100A.

Discussion
Plants constitute a good source of anti-infective agents and were found to be effective in the fight against microbial infections 35 .A number of secondary metabolites derived from plants such alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, tannins, steroids and triterpenes have previously showed antibacterial activities 4,5,36 .The extracts are considered to possess significant activity when they have MIC below 100 μg/mL, moderate activity when their MICs vary between 100 and 625 μg/mL or weak activity their display MICs above 625 μg/mL 4 .Consequently, the activity of E. Africana (TM2) against E. coli AG100A (64 µg/mL) could be considered important.Nevertheless, the overall activity of the studied plants could be considered as selective and rather moderate or weak.To the best of our knowledge, the in vitro antibacterial activity of P. macrophylla is being reported for the first time.Nevertheless, the aqueous and ethanol leaf extracts of this plant were previously tested for their anti-diarrheal activity using experimental animal models.Diarrheal infections are also caused by pathogenic bacteria such as E. coli and other enterobacteriaceae.Besides, it was demonstrated several tannins, alkaloids, saponins, flavonoids, steroids and or terpenoids have antibacterial activities 37 .
Further detection of this class of chemical in this extracts will therefore provide better understanding on its antibacterial potential.The antimicrobial activities of plants of the genus Entada have also been demonstrated 13,15 .
Teke and al. 13 demonstrated that the methanol extract, fractions and compounds from the stem bark of E. abyssinica have moderate activities against bacteria and fungi.The weak activity observed in this work is therefore consistent with their studies.The weak antibacterial activities of the methanol extract of E. abyssinica stem bark have also been reported 38 , validating the low inhibitory potential of the plant as documented herein.
The presence of alkaloids, flavonoids, tannins, saponins and cardiac glycosides have been reported in E. abyssinica and E. africana 13,39 .
The activities recorded in this study may be due to the presence of such chemical classes chemicals in the tested extracts.Although the activity recorded with the methanol extract of P. americana was found weak, this plant is known to possess antimicrobial activities against sensitive bacteria and fungi 28 .The result of antibacterial activity obtained with the extract of the seeds of C. papaya is in accordance with those obtain by Ogunjobi and Ogunjobi 25 who previously demonstrated the antibacterial activity of ethanol and aqueous extract of the seeds of C. papaya on the various bacteria stains.
Ogunjobi and Ogunjobi 25 also revealed showed that the seeds of this plant contain reducing sugars, phenols, alkaloids and tannins which could be responsible for the inhibitory activities of this plant as observed against K. pneumoniae ATCC11296 and P. stuarti NAE16.The antibacterial activity of the ethanol extract of C. procera has also been demonstrated against S. aureus, E. coli, and P. aeruginosa strains 20 .The present study therefore brings additional information on the antibacterial activities of this plant against multi-resistant bacteria.our knowledge, the antibacterial activities of A. leibrechtsiana and A. cauliflora extracts are being reported here for the first time.The weak antibacterial activities of most of the studied plants could be due to the resistance features of the studied bacterial strains.However, their effects on at least one bacterial species could justify their use in African traditional medicine in the treatment of microbial infections as reported in Table 1.

Conclusions.
The present work provides a supportive information of the antibacterial activities of the tested medicinal plants and the possibility to use the extracts from Pentaclethra macrophylla, Entada africana, Entada abyssinica in the control of selected bacterial infections.