Abstract

Medical care has provided for the use of chemo-selective prophylaxis for doxycycline for patients, health personnel and contacts persons who have no contraindications. Therapeutic failures and the rising costs of treating infections caused by resistant bacteria call for alternative care options. To asses the antibacterial activity of Combretum micranthum and Combretum adenogonium extracts on multi-resistant Vibrio cholerae strains resulting from the 2012-2019 epidemic in Benin. Strains of Vibrio cholerae from the 2012-2019 outbreak and three reference strains (V. cholerae O1 URF-ECMI U67, V. cholerae O1 URF-ECMI U27 and V. cholerae O1 URF-ECMI U14) has been re-isolated on nutrient agar. Strains were identified using standard bacteriology methods (culture, biochemistry, serogroup). The study of antibiotic sensitivity was carried out according to the Kirby-Bauer technique. Strains were sensitive to C. micranthum and C. adenogonium extracts by the agar diffusion method. A total of 84.38% of the Vibrio cholerae strains were recovered. The strains showed resistance (100%) to amoxicillin + clavulanic acid, ceftazidime and oxytetracycline while they are predominantly sensitive to gentamecine (88.46%) and ofloxacin (88.46%). Strains were resistant to Erythromycin (97.56%) and doxycycline (99%). The rate of resistance to Trimethoprim / sulfamethoxazole is 70% of the strains tested. Resistance to doxycycline is 99% while 16.67% of strains tested are resistant to all antibiotics. Strains resistant to antibiotics were sensitive to C. micranthum and C. adenogonium extracts. All are sensitive to the alcoholic extracts of C. adenogonium and 75% are sensitive to the alcoholic extract of C. micranthum. The minimum inhibitory concentrations of the extracts vary between 2.5 mg / ml and 10 mg / ml and the minimum bactericidal concentrations between 2.5 mg/ml and 10 m/ml. The high percentage of resistance to doxycycline and Trimethoprim-sulfamethoxazole insists on reviewing the patient care’s protocol. C. micranthum and C. adenogonium will be an alternative for treating cholera in the community.

1. Introduction

Cholera is an acute diarrheal infection caused by ingestion of food or water contaminated with the bacillus Vibrio cholerae. It is estimated that every year, there are roughly 1.3 to 4.0 million cases, and 21 000 to 143 000 deaths worldwide due to cholera ¹. Africa remains the most affected continent, with 46% of cholera-affected countries in sub-Saharan Africa ². Most of the Gulf of Guinea countries including Benin have an ecological potential favorable to the development of El Tor Vibrio cholera, responsible for this seventh pandemic according to Janny ³. This disease of uncleanliness and poor hygiene conditions occurs in communities with a low standard of living ⁴. Surveillance and response are part of the strategy for the control of epidemic-prone diseases including cholera proposed by WHO in the country ⁵. In Benin, this epidemic first appeared in 1970 and then became almost annual and usually occurs during the rainy season ⁶. Patient care is essentially based on parenteral rehydration. However, some authors argue that associated antibiotic therapy would accelerate healing and break the chain of transmission ⁷. The selection pressure exerted by the use of antibiotics has fostered the emergence of resistant Vibrio cholerae strains in several countries in Africa as well as in other countries of the world ^{8, 9, 10}. Thus, faced with therapeutic failures and the frequency of isolation of cholera strains in Benin, it becomes imperative to search for new antibacterial molecules; these must be, if possible, both effective, well tolerated and more economically accessible to the population. Plants, which have already provided major therapeutic molecules to the medicine, such as aspirin, morphine, quinine or taxol, offer a real potential for the search for molecules with antibacterial activity ¹¹. Combretaceae have several uses in Africa thanks to their metabolic activity ¹², anti-inflammatory, antifungal ¹³, antibacterial ¹⁴, analgesic ¹⁵ and antioxidant ¹⁶. The objective of this work is to assess the antibacterial activity of Combretum micranthum and Combretum adenogonium extracts on multi-resistant Vibrio cholerae strains resulting from the 2012-2019 epidemic in Benin.

2. Material and Methods

This is a cross-sectional study of strains of Vibrio cholerae isolated during the 2012 to 2019 outbreaks. The sensitivity of Vibrio cholerae O1 strains to conventional antibiotics and the sensitivity of strains to C. micranthum and C. adenogonium extracts was carried out on these strains.

The re-isolation of V. cholerae strains was done according to the method of Rodier et al. ¹⁷. To do this, three reference strains (V. cholerae O1 URF-ECMI U67, V. cholerae O1 URF-ECMI U27 and V. cholerae O1 URF-ECMI U14) and 64 strains were cultured in an alkaline nutrient broth (alkaline peptone water at 30g/l NaCl) for 24 h at 37 ° C. The veil developed on the surface of the medium was seeded on TCBS (Thiosulfate Citrate Bile Sucrose) medium. Thus, after 24 h of incubation at 37°C, the characteristic V. cholerae colonies were subcultured onto alkaline nutrient agar at 2% NaCl and incubated at 37°C to obtain pure strains. From suspicious colonies on GNA: fresh state, Gram stain, oxidase reaction, sugar fermentation and agglutination reaction were performed.

The agar slope in the Middle TSI (Triple Sugar/Iron/agar) was used to eliminate Pseudomonas spp. and some Enterobacteria. The reactions of V. cholerae on TSI medium which contains sucrose in addition to glucose and lactose gives type A / A reactions (alkaline slope, acid pellet), without gas, nor H₂S. Inoculate the slopes of TSI plates by pitting the pellet and streaking the surface of the medium. Incubate at 35-37 ° C and examine after 18-24 hours. The caps of each tube should not be tightly tightened during incubation, and this is especially important for TSI slopes. When the caps are too tight, anaerobic conditions appear in the TSI tube, and characteristic reactions of V. cholerae may not occur. This will result in an inaccurate reaction.

Place a colony on a strip of oxidase previously soaked with sterile distilled water placed on a sterile tear; after 30 seconds the positive reaction resulted in a dark purple color.

Serogroupage of Vibrio cholerae is carried out from isolated colonies on an alkaline GNA agar medium according to the principle of Bio-Rad. On one slide, 1 drop of saline water and next to a drop of antiserum. Then an öse from a pure and fresh culture of Vibrio cholerae was harvested. These bacteria were suspended in the drop of saline water taking care to make a homogeneous suspension by progressive addition of bacteria in the serum. Then a second step of the culture was suspended in the drop of antiserum taking the same precautions. The blade was shaken by a slight rotational movement. Observe the mixture with the naked eye over a dark surface or above a concave mirror. A positive reaction results in the appearance, with the antiserum, of agglutination in less than 30 seconds.

Fifty grams of extract powder was macerated in 500 ml of distilled water on a Bioblock Scientific Fisher Stuart shaker for 48 hours at room temperature. The homogenate obtained was filtered twice on hydrophilic cotton and once on Whatman No. 1 paper (Qualitative Circles 150 mm Cat No. 1001 150). This filtrate was then dried at 45°C in the oven and the residue thus obtained represents the total aqueous extract ready for use.

The extraction method used is an adaptation of the protocol used in the work of Sanogo et al. ¹⁸ and N'Guessan et al. ¹⁹. It has the advantage of putting the powder correctly in contact with the solvent through continuous agitation. A mass of 100 g of plant organ powder was macerated in one liter of ethanol 96 ° with continuous stirring for 48 hours. The mixture was filtered three times on hydrophilic cotton and then once on Whatman No. 1 filter paper. The 1/5 of the filtrate obtained with ethanol was evaporated at a temperature of 40°C in an oven until a dry mass was obtained which represents the ethanolic extract.

The antibiogram was performed for sensitivity tests with 54 strains of Vibrio cholerae O1 from the 2012 to 2019 epidemic in Benin. These strains were provided by the national laboratory of the Ministry of Health of Benin. Three reference strains (V. cholerae O1 URF-ECMI U67, V. cholerae O1 URF-ECMI U27 and V. cholerae O1 URF-EC14 U14) were used as controls.

For susceptibility testing, 3 reference strains (V. cholerae O1 URF-ECMI U67, V. cholerae O1 URF-ECMI U27 and V. cholerae O1 URF-ECMI U14) and 54 strains were cultured in a nutrient broth. alkaline (alkaline peptone water at 30 g/l NaCl) for 24h at 37°C. The veil developed on the surface of the medium was seeded on TCBS (Thiosulfate Citrate Bile Sucrose) medium. After incubation for 24 h at 37°C., the characteristic colonies of Vibrio cholerae were subcultured onto alkaline nutrient agar (GNA) at 2% NaCl and incubated at 37°C. to obtain pure strains. An antibiogram was systematically performed on these strains according to the Kirby-Bauer agar diffusion technique. The reading and the interpretation were made according to the criteria of the Antibiogram Committee of the French Society of Microbiology ²⁰. Eight antibiotic disks were tested: Amoxicillin / clavulanic acid (AMC, 30 μg), oxytetracycline (OT, 30 μg), sulfamethoxazole-trimethoprim (SXT, 25 μg), ceftazidime (CAZ, 30 μg), gentamicin (G, 10 μg), doxycycline, erythromycin (E, 15 μg); ofloxicin (OFX: 10 μg).

In addition to conventional antibiotics, aqueous and ethanolic extracts of C. micranthum and C. adenogonium leaves were tested by the well diffusion method Bauer et al ¹⁸. Pre-culture of V. cholerae was performed in Muller Hinton medium broth and incubated for 18h-24h at 37°C. One milliliter of the second decimal dilution of the 18-24 h pre-culture was flooded with a Petri dish containing the appropriate culture medium (MH). After seeding, the wells were thoroughly impregnated with 30 µl of plant extract of concentration 20 mg/ml. The impregnated cans were left for 15 to 30 min at room temperature (25°C±2°C) for pre-diffusion of the substances before being incubated at 37°C ²¹. The diameters of any zones of inhibition were measured using a graduated rule after an incubation time of 24 h to 48 h. For each extract, the experiment was performed in duplicate.

MICs were determined by the tube dilution method used by Dah-Nouvlessounon et al. ²². In a series of 10 test tubes numbered T1 to T10, 1ml of an extract solution at different concentrations ranging from 20-0.039 mg / ml respectively were introduced into the tubes ranging from T1-T10. To each tube containing 1 ml of extract was added 1 ml of inoculum whose turbidity was adjusted to 0.5 Mc Farland (10⁸ CFU / ml) and returned to 10⁶ CFU/ml in Mueller-Hinton broth twice concentrated. After 24h incubation at 37°C, the bacterial growth in each tube, which results in turbidity was examined. The MIC of an extract against a given strain corresponds to the smallest concentration showing no visible growth in the naked eye.

They were determined by the method of seeding on agar medium according to the method used by Dah-Nouvlessounon et al. ²². Referring to the MIC results, all non-growth tubes were inoculated aseptically on MH agar medium and incubated at 37°C for 24 h. The lowest concentration of extract showing no visible growth is considered Minimal Bactericidal Concentration.

The data entry and analysis was done in Excel Spreadsheet 2013. The percentage of resistance was calculated for each antibiotic by dividing the frequency of resistant bacteria by the number of bacteria tested. The data analyzes were done using the software.

3. Results

During re-isolation, 84.38% of Vibrio cholerae strains from the 2012-2019 outbreak were recovered.

The susceptibility of V. cholerae strains varies with antibiotics (Figure 1). All strains showed resistance (100%) to amoxicillin + clavulanic acid, ceftazidime and oxytetracycline while they are predominantly sensitive to gentamecine (88.46%) and ofloxacin (88.46%). Strains were also resistant to sulfamethoxazole-trimethoprim (70%), erythromycin (97.56%) and doxycycline (99%).

From these observations, we can draw five different profiles (Table 1) according to their capability to resist or not to the tested antibiotics.

Table 2 shows the diameters of inhibition of V. cholerae vis-à-vis strains of extracts. It appears that all extracts inhibit the proliferation of most V. cholerae without any persistence in time and the nature of the extract. All the reference strains are sensitive to the alcoholic extracts of C. adenogonium and 1/3 are sensitive to the alcoholic extract of C. micranthum.

Table 3 shows the inhibition diameters of the extracts with respect to Vibrio cholerae strains. It appears that all extracts inhibit the proliferation of most Vibrio cholerae without any persistence in time and the nature of the extract. It is noted that 100% of strains are sensitive to ethanolic extracts of C. adenogonium and 75% are sensitive to the ethanolic extract of C. micranthum. In addition, 95.83% that are sensitive to aqueous extracts of C. adenogonium and 75% are sensitive to the aqueous extract of C. micranthum. The strains of Profile 4 (W1, W5, W11, W14 etc.) are all sensitive to extracts.

The Minimum Inhibitory Concentrations obtained are variable depending on the types of extract and strains (Figure 2). The minimum inhibitory concentrations of the extracts vary between 2.5 mg / ml and 10 mg / ml. There is still a greater inhibitory concentration (2.5 ± 0 mg / ml) on the strains of profile 1, 3 and 4. In addition, a greater sensitivity is observed in the presence of the strains of profiles 1, 2 and 3 regardless of the type of extract.

Minimum Bactericidal Concentrations (MBC) vary with strain and type of extract (Figure 3). Minimum bactericidal concentrations between 2.5 mg / ml and 10 mg / ml. A higher sensitivity of tested Vibrio cholerae strains resulting from profile 1 (CMB = 2.5 mg / ml) independently of the type of extract used.

4. Discussion

Our objective was to evaluate the antibacterial activity of Combretum micranthum and Combretum adenogonium extracts against Vibrio cholerae strains from the 2012-2019 epidemic in Benin. During re-isolation, 84.38% of V. cholerae strains from the 2012-2019 outbreak were recovered. The death of some strains may be due to the culture medium of conservation, temperature and duration. We observed these phenomena in the strains which are from 2012 to 2014. We noted the loss of some strain of 2016. The death of these bacteria could be explained by the fact that V. cholerae strains were kept in skim milk. Skim milk contains 1 to 5 percent of a pure Lactobacillus acidophilus culture. These bacteria could make the environment when conditions are favorable which will cause the death of V. cholerae strains

Resistance rates of Vibrio cholerae O1 isolates varied according to conventional antibiotics (Figure 1). The resistance to doxycycline is 99% and the resistance to Trimethoprim/sulfamethoxazole is 70%. These molecules are widely used in the treatment of cholera. This could be due to the anarchic use and most often by self-medication of this molecule by the populations. Sulfametoxazole-trimethoprim resistance is lower than in other countries. It should be noted that high rates have been reported in Senegal from 2004-2006 (90.3%) ⁸, to Iran in 2009 (95%) 2009 ²³. In Ghana, the genotypic characterization of V. cholerae has revealed a transposon of SXT and a class-29 integron. These results show that this antibiotic should no longer be used in the treatment of cholera. Indeed, this molecule is often prescribed in the treatment of gastroenteritis and chemoprophylaxis in people infected with HIV. Studies have shown that 90% of subjects are asymptomatic carriers or have a common gastroenteritis. Taking this drug would participate in the selection and dissemination of emerging resistant strains in the population ¹⁰. In addition, the use of sulfonamide-based molecules in the treatment of malaria could be the cause of cross-resistance ²³. The resistance to doxicycline observed in our study was contrary to those obtained by Manga et al ⁹. In Senegal where all strains tested were sensitive to doxycline. This may be due to the chemo-selective prophylaxis for doxycycline that has been administered to all accompanying patients and to health personnel who have no contraindications in Benin. This resistance could also be explained by the inappropriate prescription of these antibiotics by health workers in health centers in health care settings without laboratory evidence. For erythromycin, resistance was 97%. Our results are superior to those obtained by Shah et al ²⁴ in Pakistan where they found a 15% resistance to erythromycin. This could be explained by the fact that the strains they used are from 1998-1999 but our strains are from 2012-2019. The resistance of Vibrio cholerae O1 to oxytetracycline (100%), Amoxicillin + clavulanic acid (100) was also observed. However, Vibrio cholerae O1 also showed variable susceptibility to C. micranthum and C. adenogonium extracts. The ethanolic extracts of C. micranthum and C. adenogonium show stronger antibacterial activity than the aqueous extract (Table 2). These results corroborate those of Hamid and Aiyelaagbe ²⁵ who showed that the Alafia barteri alcoholic extract better inhibits the in vitro growth of various bacterial strains (S. aureus, E. coli, Pseudomonas aeruginosa and Candida albicans) at the same concentrations ranging from 25-200mg / ml. The alcoholic extract is then more active and better concentrate the antibacterial active ingredients contained in the plant than the aqueous extracts. The antibacterial activity of this fraction is the most important of the two fractions of C. micranthum and C. adenogonium used in this study. This activity is observed both with the susceptible strains and the resistant and multi-resistant strains (Figure 1, Table 1, Table 2).

In Benin, the decoction or infusion of leaves, sometimes with the bark of these plants, are simply absorbed as beverages for treatment in fever and malaria. They are also used in the steam bath. Decoction of stem and root powder are easily treated. It is noted that antibiotic resistant strains are susceptible to the extract. All resistant strains are sensitive to the ethanoid extract of Combretum adenogonium. Therefore, these plant extracts were a healthy and sustainable alternative to overpriced conventional antibiotics and the misuse of antimicrobial resistance. In addition, according to Guevara et al ²⁶ in Peru, the decoction of Malus sativa and Cydenia oblonga showed a bactericidal effect for its acidity and avocado stone (Persea gratissima), a late bactericidal effect. The infusion of tea and the decoction of Punica granatum bark showed the best bactericidal effect. Thakurta et al ²⁷ also tested the susceptibility of V. cholerae to plant extracts. They found that serotypes O1, O139, non-O1 and non-O139 were inhibited by extracts of neem (Azadirachta indica). In addition, Akinsinde and Olukoya ²⁸ observed the effects of vibrios from medicinal plants in Nigeria and, in one study, the bactericidal in vitro of 14 plant species on V. cholerae, Guevara et al ²⁶ proposed Alternate of Punke granatum, on the Alternate for Chopera.

5. Conclusion

Since effective management of cholera is based primarily on replacement of fluid loss and antibiotic therapy, the circulation of multidrug resistant Vibrio cholerae strains can compromise the efficacy of probabilistic treatments and explain treatment failures. Therefore, it is important and urgent to set up community and prescriber awareness actions on the reasoned use of antimicrobials in order to mitigate the expansion of resistant strains in Benin. The aqueous and organic extracts of these plants had variable antimicrobial activities against the in vitro growth of clinical Vibrio cholerae strains tested. Our results will contribute to improving the living conditions of our populations and set up a validation of the use of C. adenogonium, C. micranthum, in traditional medicine.

Conflicts of Interest

The authors declare no conflict of interest.

References