Diarrheal diseases, the third leading cause of death in the world, after respiratory diseases and AIDS (OMS, 2015) continue to be one of the leading causes of morbidity and mortality. Globally, they constitute the second leading cause of infant mortality in children less than five years (Bhutta  et  al.,  2013),  with   about   2.5   billion  diarrhea episodes and 2.2 million deaths all years (Bahmani et al., 2015). Of the 2.2 million annual deaths, 37% of cases occur in sub-Saharan Africa (OMS, 2016). In Côte d'Ivoire, diarrhea represents 14% of the reasons for consultation in maternal and child health centers (Kouakou, 2012) and 15% of deaths of children less than 5 years (Liu et al., 2014). The main agents involved are viruses, protozoa and bacteria among which Escherichia coli is one of the most important etiologic agents (Asadi et al., 2010).

Given the magnitude of this situation, World Health Organization (WHO) has adopted a treatment based on oral rehydration (OR) and oral rehydration solution (OSR). However, the annual rates of use of OR and OSR, respectively of 0.39 and 1.02% are still very low (Birger et al., 2006). In addition to this program, antibiotics, although used, are becoming increasingly ineffective because of microbial resistance and toxicity problems. They are also still beyond the reach of population grants in developing countries because of their high cost. In this situation, it is necessary and urgent to offer to disadvantaged populations more accessible new therapeutic solutions taking into account their culture and their purchasing power.

To meet this challenge, exploring the rich Ivorian medicinal flora with nearly 800 species of medicinal plants (Aké-Assi, 1991) could offer alternative therapeutic solutions. It is in this context, that the present study focused on Anogeissus leiocarpa (DC) Guill. & Perr, a plant of the Ivorian medicinal flora. Phytochemical studies carried out on A. leiocarpa root barks revealed the presence of phenolic compounds (flavonoids, tannins, leucoanthocyanins and polyphenols), saponins and sterols (Moronkola and Kunle, 2014; Gbadamosi and Ogunsuyi, 2014). These compounds are known to have antimicrobial activity (Mann et al., 2014; Adamu et al., 2017).

This study focused on the in vitro and in vivo investigation of the antibacterial potential of Combretaceae, a selected medicinal plant of Côte d’Ivoire flora by using infected rats with E. coli extended-spectrum β-lactamases (ESBL).

 

diameter (IZD) greater than 7 mm were reported in results

Minimum inhibitory concentration (MIC)

The incorporation of the plant extracts into Muller-Hinton (MH) agar was done using the double dilution method in agar slant tubes as described by Ouattara et al. (2013). Nine experimental tubes whose concentration varies to double dilution from 50 to 0.195 mg/ml and 2 control tubes, the growth control tube (TC) and the sterility control tube (TS) are prepared. The slope of the experimental tubes and that of the TC tube was seeded. The tubes were incubated at 37°C for 24 h. The MIC was the concentration of the first tube from which no microbial visible growth (Khaleel et al., 2016).

Minimum bactericidal concentration (MBC)

MBC is the lowest concentration of substance that leaves at most 0.01% of surviving germs. Using a loop calibrated at 2 μl, the contents of the tubes in which no haze was observed were seeded on MH (Box B) in parallel streaks 5 cm in length at the surface, starting with by the MIC tube. After 24 h incubation in an oven at 37°C, the numbers of colonies on the streaks of Box B with those of Box A were compared. In practice, the CMB corresponds to the concentration of the experimental tube whose number of colonies present on the streak is less than or equal to the number of colonies present on the streak of the dilution 10^-4.

In vivo antibacterial activity

Experimental design

Forty-two rats of both sexes previously deparasized through oral administration of 10 mg/kg body weight of tetracycline for 3 days (Ricicová et al., 2010) were used. They were divided into 7 Lots of six rats each. Rats from 6 Lots received, orally, 2 ml of the infective dose (Eman et al., 2008) evaluated at 2 × 108 CFU/ml with a sterile disposable needle-less syringe. The 7th batch is uninfected. Treatment started 48 h after the induction of diarrhea following the appearance of diarrheal faeces. For treatment, the infected rats received orally concentrations of the ethanolic fraction of A. leocarpa and ciprofloxacin (the reference antibiotic) according to the prescription of Venkatesan et al. (2005). Thus, daily and for 14 days, Lot 1 (uninfected and untreated) received 1 ml of distilled water; Lot 2 (infected and untreated) received 1 ml of distilled water; Lot 3 (infected and treated) received 2 ml of ETHA at dose of 69.44 mg/kg body weight corresponding to 1 × MIC; Lot 4 (infected and treated) received 2 ml at a dose of 271.87 mg/kg body weight (5 × CMI); Lot 4 (infected and treated)  received 2 ml of ETHA at a dose of 631.3 mg/kg body weight (10 × CMI) ; Lot 6 (infected and treated) received 2 ml of ETHA at a dose of 2222.22 mg/kg body weight (40 × CMI) and Lot 7 (infected and treated) received 1 ml of ciprofloxacin at dose of 5 mg/kg body weight w.

Enumeration of faecal E. coli

Rats faeces were collected in sterile polyethylene sterile bags of 500 ml (LMR®, France) placed under the cage of the rats, twice daily for 2 weeks. One (1) g of faeces removed with a spatula was transferred to 9 ml of BMH, then vortexed for 10 s and incubated at 37°C for 3 to 4 h to obtain dilution inoculum10°. From this dilution 10°, dilutions ranging from 10-1 to 10-5 were prepared. This serial dilution were cultured (0.1 ml) in duplicate on Mac Conkey agar (two plates per dilution). After incubation at 37°C for 18 to 24 h, typical smooth pink color colonies were counted on two  successive 

dilutions. Plates inoculated with a sample dilution that yields between 10 and 300 colonies per plate were read (ISO 7218, 2007). The E. coli faeces load (UFC per g of faeces) was calculated according to the formula proposed by ISO 7218 (2007):

where ΣC: sum of colonies counted on the two Petri dishes retained, V: volume of seeded inoculum on each Petri dish, and d: dilution corresponding to the first retained Petri dish, with the least diluted inoculum.

Evaluation of the water content of faeces

Fresh faeces was collected after 6 h and weighed individually to determine the wet weight (WW). Faecal samples were dried in a conventional oven at a temperature of 70°C. After 24 h, dried faeces were weighed again for determining their dry weight (DW) (Navarro et al., 2006). The difference between the WW and the DW helped to determine the water content of faeces (Navarro et al., 2006) expressed as a percentage, by the following relation:

Mortality rate and pathological manifestations

The mortality rate of the rats in the different groups is calculated as the number of dead rats during the experiment compared to the total number of rats used in each Lot (Eman and Hoda, 2008). Faeces were checked continuously for color and consistency, the development of diarrhea in infected Lots, as well as any change in activity and behavior were recorded weekly throughout the experiment.

Statistical analysis

All data during this experience were expressed in three replicates. The results are expressed as mean ± standard error of means. They were determined by Dunnett’s test using GraphPad Prism 7.0 Statistic Software and were considered significant at p-value less than 0.05.

Ethical approval

The experiments were conducted according to the ethical guidelines of Ethics Committee of Pasteur Institute of Côte d’Ivoire (Charter of Ethics of the Pasteur Institute, Text of September, 2012).

 

 

Antimicrobial activity of aqueous extract and organic fractions of A. leiocarpa

The effects of total aqueous extract and organic A. leiocarpa fractions obtained by agar well diffusion method on bacterial growth are shown in Table 1. The total aqueous extract and the dichloromethane fraction showed no  activity  on all the bacteria tested with inhibition zones diameters less than 8 mm. 

 

 

Both ethanol (ETHA) and ethylacetate (EAA) fractions were active against all infectious bacteria tested with an IZD values ranging from 8 to 16 mm and 8 to 15 mm, respectively. Diameter of zone of inhibition decreased when reducing gradually the extract or fraction concentration excepted for EDMA and ETAA. On a general note ETHA exhibited higher sensitivity than EAA. Only S. aureus Meti-R and Streptococcus spp. displayed the highest sensitivity with an IZD up to 16.64 at 250 and 500 mg/ml, highest zone of inhibition observed with ETHA compared to EAA. With the exception of E. coli ESBL which is the main resistant strain at 125 mg/ml, the IZD of most bacteria tested with ETHA are larger than those of EAA. In addition, EAA at the concentration of 125 mg/ml revealed 3 resistant strains which are E. coli ATCC, E. coli ESBL and P. aeruginosa with an IZD below 8 mm. The inhibition  zone  of    total aqueous   extract   and dichloromethane fraction at any concentration is below 8 mm. For standard drugs, extreme sensitivity was observed with an IZD that ranged from 10 to 30 mm.

Antibacterial parameters of the total aqueous extract and organic fractions of A. leiocarpa

MIC, MBC and MBC/MIC ration values are shown in Table 2. The MICs values of all strains studied with both ETAA and EDMA extracts were above 50 mg/ml, thus their MBC and MBC/MIC ratio were not determined. The MICs of A. leiocarpa ranged from 6.25 to 12.5 mg/ml in ETHA fraction. For EAA fraction, it was 6.25 mg/ml for S. Typhi 43PI16 and S. Typhi 1586, but was up to 25 mg/ml for E. Coli ATCC, ESBL, and P. aeruginosa but it was up to 50 mg/ml for Shigella dysenteriae and K. oxytoca. The MBC/MIC ratio of EAA and ETHA fractions observed were strictly inferior to  4 corroborating the bactericidal effect against all bacteria tested.

 

 

In vivo antibacterial activity

Infection rate, mortality in rats and faeces appearance

The antibacterial activity in vivo was evaluated by increasing the concentration of ETHA shown in Table 3.

 

 

Effect of ETHA on E. coli enumeration in rats diarrheal faeces

The effect of ethanolic fraction on bacterial load in diarrheic faeces is as shown in Figure 1. It can be seen that after the 2 days of rats infection, the E. coli colony forming units per g of faeces (UFC) counted decrease from Lots 3 to 7 when the concentration of ETHA was increased progressively. Outstandingly, the UFC of Lot 6 is close to that of the Lot 7, the standard drug that drops rapidly to zero after 6 days of treatment but the UFC of Lot 2 remained higher than those with treatment during the 13 days of study.

 

 

Effect of ETHA on water content

From the Figure 2 that shows the percent of facael water content, a significant decrease of water lost during the ETHA treatment was observed and it is strongly correlated to the diminishing feacal E. coli load in Figure 1. Only the rats treated with ciprofloxacin or cefotaxime showed no statistically significant difference (p > 0.05) in water content when compared with Lot 1 which represent not uninfected and untreated rats.

 

 

Effect of ETHA on the volume of diarrheal faeces

To gain additional information on the antibacterial activity of ETHA, the effect of ETHA on the volume of diarrheic faeces investigation was perfomed on both volume and weight of the rats’ faeces (Figures 3 and 4). There was a significant difference (p < 0.01) on both volume and weight of diarrheic faeces. Also, there was no significant (p > 0.05) decrease in infected and treated rats from 69.99 to 2222.22 mg/kg body weight when compared with uninfected and untreated rats. Notably, it is observed that the weight and volume estimated for the Lot 6 is almost similar to that of the drug control (Lot 7).

 

 

 

 

 

 

Mortality rate of 50% was observed in the untreated and infected Lot but no death was noted for both Lots treated with Ciprofloxacin and the ETHA fraction of 631.3 and 2222.22 mg/kg body weight, respectively. In fact, a significant reduction (p < 0.001 and p < 0.01) of rats’ death was observed from 30 to 0% when increasing the dose of ETHA and a decrease of the rats weight and volume of diarrheal faeces indicating a progressive eradication of the E. coli inducted infection compared to infected and untreated Lots. These results illustrated the therapeutic action of the ETHA fraction.

These observation was corroborated by an important decreasing of faecal E. coli ESBL load during the experiment that may be ascribed to the healing  power  of the extract. After the treatment, the bacterial load of the infected and treated Lots decreased significantly (p < 0.001) as during the treatment and dose-dependent manner compared to the untreated infected Lot. Similar results were obtained by Niehaus et al. (2011) and Ibrahim and Sarhan (2015) when enumerating E. coli from diarrheal rat faeces. These results highlighted the therapeutic potentialities of the ethanolic fraction of A. leiocapa.

The antibacterial effect of A. leiocarpa extracts could be attributed to the bioactive compounds present in the extracts such as phenolic compounds (flavonoids, tannins, leucoanthocyanins and polyphenols), and saponins and sterols (Moronkola and Kunle, 2014). It has been suggested that bioactive compounds act through two main mechanisms of action. The first is related to their hydrophobic property, which facilitates their adhesion to the surface of bacteria, causing their instability (Jongbloed et al., 2007). The second was the inactivation of different bacterial molecules such as enzymes or receptors, following the binding of bioactive compounds (Pandey and  Kumar,  2013).  Some of these compounds could lead to cell membrane perturbations and exert a β-lactam action on the transpeptidation of the cell wall. Other compounds could interact with the lipid bilayers in cell membranes, leading to the separation of the two membranes, thus leading to cellular swelling and cell death (Tshingani et al., 2017).

The reduction in the bacterial burden of faeces in infected and untreated rats may be explained by the protective action of their immune system against the pathogens (Lunga et al., 2014). The in vitro and in vivo analyses of antibacterial activity revealed the antibacterial and therapeutic potential of A. leiocarpa.

 

 

This study showed that root barks of A. leiocarpa extracts possessed antibacterial activity by inhibiting in vitro bacterial growth. Its therapeutic activity was carried out against E. coli in experimentally infected rats by the significant reduction of defecation frequency, water content of faeces, weight and volume of diarrheic faeces, and E. coli load of faeces. The present results demonstrated that A. leiocarpa should be used in the traditional therapeutic arsenal against resistant infectious germs.

 

The authors have not declared any conflict of interests.