Formulation of Improved Traditional Drugs against virulent species of Salmonella spp in Benin: assessment of the properties from Uvaria chamae, Lantana camara and Phyllantus amarus in Benin, West Africa CURRENT STATUS: ACCEPTED

Background Uvaria chamae (Annonaceae), Phyllantus amarus (Phyllantaceae) and Lantana camara (Verbenaceae) are empirically alleged to be used as Beninese medicinal plants in the treatment of salmonellosis. This study aimed to produce scientific data on in vitro and in vivo efficacy of Uvaria chamae, Lantana camara and Phyllantus amarus on multiresistant Salmonella spp isolated in Benin. Results After in vitro tests on aqueous and ethanolic extracts of Uvaria chamae, Lantana camara and Phyllantus amarus , only the aqueous extract of Uvaria chamae (leaves) showed the best anti-Salmonella ’s activity. It has been used for the following experiments. The induction of salmonellosis revealed 9.0 10 8 CFU/ml was optimal concentration for triggering and maintaining the symptoms in chicks. This infective concentration has been used for in vivo assessment. 24 hours post inoculation later, the symptoms of salmonellosis (wet cloaca, diarrhea stool and somnolence) were observed in infected groups. After seven days of treatment, the rate of reduction of bacterial load at 100 mg / L, 200 mg / L, 400 mg / L of this extract was 85%, 52.38% and 98% respectively in the chicks groups infected with Salmonella Typhimurium ATCC 14028. About the groups infected with Salmonella spp (virulent strain), the rate of reduction of bacterial load at 100 mg / L, 200 mg / L, 400 mg / L of this extract was 0%, 98.66% and 99.33%. The toxicity tests did not show any significant effect of the Uvaria chamae ’s extract on the biochemical and hematological parameters of the chicks. The aqueous extract of Uvaria chamae is active in vitro and in vivo on multiresistant strains of Salmonella spp . This plant is a good candidate for the development of an improved traditional medicine for the management of salmonellosis. in 500 ml of distilled water or ethanol on a Stuart Bioblock Scientific Fisher stirrer for 72 hours at room temperature. Homogenate was filtered with hydrophilic cotton and Wattman No. 1 paper. This filtrate was then dried at 40 ° C for ethanolic extract and 50°C for aqueous extract in the Pasteur oven.

Background Salmonella spp is the most common foodborne pathogens habitually isolated from food-producing animals. This germ is responsible for zoonotic infections in humans and animal species including birds. Salmonella spp can be transmitted to humans along the farm-to-fork continuum, commonly through contaminated foods of animal origin, namely poultry and poultry-related products, pork, fish [1].
It is Gram negative, rod-shaped bacteria, and facultative anaerobes belonging to the Enterobacteriaceae's family and divided into three main species: Salmonella enterica, Salmonella bongori and Salmonella subterranean [2]. It isone of the top four causes of diarrheal diseases worldwide [3]. Recent studies estimated that there were approximately 94 million cases of nontyphoid Salmonella gastroenteritis resulting in 155,000 deaths globally each year [4]. Out of these cases, 80.3 million were estimated as foodborne origin [5]. Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg and Salmonella Newport are the epidemiologically important serotypes with poultry and have been associated with the majority of human salmonellosis burden worldwide [6][7][8][9].
Increasing prevalence of multidrug resistant Salmonella such as resistance towards clinically important antimicrobials like fluoroquinolones and third-generation cephalosporins has become an emerging problem worldwide [10][11][12][13]. As an alternative to antimicrobial resistance, exploration of medicinal plants with anti-Salmonella activity is becoming very common in West Africa. In vitro antibacterial activity data of medicinal plant extracts on Salmonella strains do exist [14] but there is a few data about in vivo efficacy of medicinal plants against this bacterium. The difficulty of choosing a suitable study model, the complexity of such research work, and the still limited data on the physiology of Salmonella strains could explain this situation. This study is then devoted to the experimental induction of salmonellosis in chicks as animals' models. These data can be a good starting point for in vivo efficacy testing of herbal extracts on Salmonella spp. Indeed, promoting medicinal plants used in the traditional treatment of salmonellosis involves a structured approach. At this time, an ethnopharmacological survey revealed 57 species of medicinal plants used in the treatment of salmonellosis in Benin [15]. Based on quotation frequency and literary data, Phyllantus amarus, Senna siamea, Uvaria chamae and Lantana camara have been selected. Toxicological, chemical and antibacterial (to ten enteropathogens) characterization have been done. This study showed interesting contents in polyphenols and flavonoids and an effective antibacterial activity at 100 mg/mL with MIC between 100 and 25 mg/mL and inhibition diameters between 7.5 and 21 mm [16].
With these results, it was found necessary to evaluate the in vitro and in vivo efficacy of extracts of  (leaves). The aqueous extract of Uvaria chamae (leaves)showed the best inhibition diameter (9.33 ± 2.08 mm) ( Figure 1). There is no significant difference between inhibition diameters (P = 0.6885).  In vivo anti-Salmonella activity of aqueous extracts (leaves) of Uvaria chamae using chick models Preliminary test This step aimed to choose the optimal effective concentration of inoculum for Salmonella's induction. 24 hours after infection, wet cloaca and diarrhea stools were detected in groups 1, 2 and 3 infected with inoculum concentrations 1, 2 and 3 respectively whereas these symptoms were absent in group 4, which received distilled water only. These symptoms were present until the 10th day of observation in the group 3 while they disappeared between the fifth and the 7 th day in the group 2 (Table 2). However, no deaths were recorded. Salmonella spp was investigated in faecal samples to support clinical observations. The strains were detected in chicks of group 2 and 3, three days after salmonellosis induction. They were present till the ninth day for group 3 whereas they disappeared from feces on the sixth day for group 2 (Table 3). On the ninth day, a bacterial count was made on the faeces samples to assess the bacterial load. The results are shown in Table 4.
Clinical observations suggest that only 9.0.10 8 UFC/ml could trigger and maintain the symptoms of salmonellosis.
In vivo efficacy of leaves aqueous extract of U. chamae on Salmonella Typhimurium ATCC 14028 (reference strain) and Salmonella spp P19 (Virulent strain) 24 hours after induction, symptoms were detected in all infected chicks. Diarrheal stools were abundant. The cloacae of the chicks were wet and somnolence was noticed in some chicks (Tables 5-10). During the 9 days of monitoring, including 7 days of treatment, these symptoms evolved considerably according to the groups. They remained persistent in infected and untreated chicks (group 2). However, diarrhea was slightly reduced at the last days of monitoring, indicating a progression of the disease to asymptomatic carriage (Tables 7 and 8). 3 days after infection with Salmonella Typhimurium, the bacterial load of Salmonella increased in all infected chicks, but differentially according to the groups. The bacterial load at day 3 was in the range 2000-30000 CFU/g. Treatment of the chicks with the extracts and colistin started at day 3 and continued until day 9. During treatment, the bacterial load gradually decreased in group 3 treated with colistin and disappeared on Day 9. In group 2 (infected and untreated), the bacterial load decreased slightly between the third and sixth day (11000 to 10000 CFU/g) before undergoing a considerable increase on the ninth day (10000 to 16000 CFU/g). In groups 4, 6 respectively treated with 100 and 400 mg/l of the extract, the bacterial load was first increased before falling down on day 9. In group 5 (treated with the extract at 200 mg/l), a gradual decline from the 3 rd to the 9 th day (21000 to 10.000 CFU/g) (figure 5) was observed.
Bacterial load reduction between the 3 rd and the 9 th day after the infection (7 days of treatment) was assessed globally and summarized in the figure 6. The inoculum increased by 45.45% in group 2 (infected and untreated). It was reduced by 100% in lot 3 (treated with colistin). Treatment with leaves aqueous extract of Uvaria chamae also showed remarkable efficacy (bacterial load reduction between 52.38% and 98%). During treatment, the bacterial load gradually decreased from 1500 to 1000 CFU/g in group 3 treated with colistin. In group 2 (infected and untreated), the bacterial load decreased slightly between the third and sixth day (9000 to 3000 CFU/g) before undergoing a considerable increase on the ninth day (3000 to 52,000 CFU/g). In groups 4, 6 respectively treated with 100 and 400 mg/l, the bacterial load was first increased before falling on day 9, whereas in group 5 treated with the extract at 200 mg / l, bacterial load were constant to 15000 CFU/g between day 3 and day 6 and decreased to 200 CFU/g at day 9.
Bacterial load reduction between the 3 rd and the 9 th day after the infection (7 days of treatment) was assessed globally and summarized in the figure 8. The inoculum increased by 477.77 % in group 2 (infected and untreated). It was reduced by 33.33 % in lot 3 (treated with colistin), 98.66% in group 5, 99.33 % in group 6. The extract at 100 mg / l did not allow a reduction of the bacterial load (group 4).

Effects of Leaves aqueous extract of Uvaria chamae on biochemical and hematological parameters
Effect of Leaves aqueous extract of Uvaria chamae on hematological and biochemical parameters was investigated to evaluate whether as a biologically active substance, this extract did not have a pathological effect on certain biochemical and hematological parameters. The results are shown in Uremia increased insignificantly (p>0.05) at day 4 in chicks which received 400 mg/l (4.66 g / l to 4.99 g / l) and 100 mg/l (4, 49 g / l at 5.27 g/l) of extract. As for those which received 200mg/l of extract, their uremia increased at day 7 (4.23g / l to 4.67g / l).The creatinine concentration at day 4 increased insignificantly in chicks having 100 mg of extract (0.17g / l to 0.21g / l) and those which received the antibiotic (0.16g / l to 0.21g / l). In chicks which received 200mg (0.16g / l to 0.18g / l) and 400mg (0.17g / l to 0.19g / l) of extract, their creatinine increased at day 7 (p>0.05). Same observation was done with AST and ALT (no significant variation) (figure 9). With hematological parameters, the same observations are made. There was no significant difference in all groups.
However, the number of blood cells increased from the 1 st to the 4th day and from the 4 th to the 7 th day ( figure 10).

Discussion
This study aimed to produce scientific data on in vitro and in vivo efficacy of extracts of Uvaria chamae, Lantana camara and Phyllantus amarus on multiresistant Salmonella spp isolated in Benin. In vitro anti-salmonella tests were used to assess the activity of the extracts of the three plants and to select the extract to be used for the in vivo tests.
The performance of the in vivo efficacy test was preceded by an experimental infection which made it possible to choose the optimal concentration of the inoculum.

In vitro anti-Salmonella activity of Uvaria chamae, Phyllantus amarus and Lantana camara
The aqueous and ethanolic extracts of the leaves and bark of U. chamae showed an inhibition of Salmonella spp with the exception of the ethanolic extract of U. chamae's roots. The aqueous extract of Uvaria chamae was active on 90% of the virulent Salmonella spp and on Salmonella Typhimurium ATCC 14028. These results can be compared to those obtained by Ogueke [17]. This author showed that at a concentration varying between 150-250 mg / ml, aqueous and ethanolic extracts of bark and the ethanolic extract of leaves of Uvaria chamae inhibited Salmonella Typhi.
Aqueous and ethanolic extracts of Phyllantus amarus inhibited Salmonella spp with maximal inhibition diameter of 12 mm. The activity of Phyllantus amarus extracts on Salmonella spp were reported in 2008. Using agar cup diffusion method, the authors showed that ethanolic extracts of P. amarus were active on Salmonella Typhi [18]. In our study, only leaves ethanolic extract of Phyllantus amarus inhibit Salmonella Typhimurium ATCC 14028 (9.33±1.53 mm). The inhibitory power of the extract on the reference strain was greater than those obtained in a previous study. For concentrations ranging from 200 to 1000 µg / ml, the inhibition diameters varied between 7 and 9 mm on Salmonella Typhimurium ATCC 6539 [19]. Firstly, Salmonella Typhimurium is known for his ability to infect birds, contaminate eggs [21] and be transmitted to humans. It is the main agent of salmonellosis in humans. Secondly, chicks have been used successfully for experimental infections of non-typhoid salmonellosis : for investigation of the invasiveness of Salmonella enterica in chickens [22] ; for investigation of the dynamics of egg contamination over an extended time course [23]; for investigation of pathogenicity of some avian Salmonella Serovars [24]. In this study, we choose three-day old chicks for various reasons. One had to choose an age of susceptibility, an age when the animal's immune system is not mature enough to prevent infection. Such an age guarantees the establishment of the infection. Also it is known that in poultry, the signs of the disease are rarely observed after the first two weeks of life [25]. These strategic choices appear to be optimal since, 24 hours after the infection, the animals showed signs of salmonellosis, particularly in groups 2 and 3, which received the concentrations of inoculum 2 and 3 respectively. It was observed wet cloaca, diarrhea stool and somnolence. Clinical signs have been  [27]. This study showed that the strain has 5 virulence genes: invA, spvR, SpvC, FimA and Stn. Spv genes are responsible for the systemic infection and multidrug resistance in humans and animals [28]. SpvC gene is able to inhibit the activation of macrophages [29]. Presence of fimA gene indicates the presence of fimbriae which is important for Salmonella spp to adhere to epithelial cells [27]. Stn gene is suspected to contribute to enterotoxigenic potency [30]. The presence of all these genes therefore guarantees the pathogenicity of Salmonella Typhimurium and its ability to infect chicks.
This virulence explains why salmonellosis symptoms were observed in infected animals. Three infective concentrations were chosen because we had no assurance of sufficient bacterial load to induce salmonellosis in chicks of this age. We had to expand the possibilities. The results showed that only concentrations 2 and 3 could trigger the symptoms of salmonellosis. These symptoms were present until the 10th day of observation in the group 3 chicks while they disappeared between the fifth and the 7th day for the group 2. Salmonella spp were investigated in faecal specimens to support clinical observations. Salmonella spp were detected in chicks of group 2 and 3, three days after infection. Chickens infected with concentration 3 of inoculum still host Salmonella.
By relating microbiological data to clinical observations, it seems obvious that only the infective 3 concentration was able to keep the 3-day-old chicks sicked for 10 days. This observation was reinforced by the count at day 9. The disappearance of Salmonella in group 2 could be explained by a positive reaction of the immune system of the birds.
In vivo anti-Salmonella activity of Uvaria chamae using chick model Ten multiresistant strains of Salmonella spp: they were isolated by Deguenon et al. [27]. The strains were multidrug-resistant to penicillins, first generation cephalosporins and some aminoglycosides. Each inoculum was seeded by swab on Petri dishes containing Mueller Hinton agar. Wells of 6 mm diameter were hollowed out using sterile Pasteur pipette tip. 50 μl of each extract was deposited in the wells. A well containing sterile distilled water served as a negative control. After 1 hour prediffusion at room temperature, the petri dish was incubated at 37 ° C in an oven for 24 hours. After incubation period, the dishes were examined for the measurements of the zones of inhibition. Oral inoculation was performed using 20-gauge feeding needle and disposal syringe. To confirm the viability of the strain used for inoculation, a sample of each inoculum (about 100 microliters) was taken before and after oral administration for culture at 37 ° C for 24-45 hours.

Production of aqueous and ethanolic extracts
The birds were observed for 10 days and the symptoms of salmonellosis were recorded. On days 3, 6, and 9, the feces from each lots of chicks were collected. Salmonella was sought using method described par Deguenon et al.[ [27]. Five (5)  Preliminary examination and inoculation were performed as previously but a single concentration of inoculum were used: 9.0 10 8 UFC/ml. The birds were observed for 9 days and the symptoms of salmonellosis were recorded. From the third day after infection, the chicks are subjected to oral treatment with the aqueous leaf extract of Uvaria chamae and colistin as reference antibiotic. The treatment was done for 7 days. On days 3, 6 and 9 after infection, faeces from each group were collected and Salmonella counts were performed [35].

Effects of Leaves aqueous extract of Uvaria chamae on biochemical and hematological parameters
In order to evaluate the toxicity of Uvaria chamae leaves aqueous extracts for chicks, the different concentrations of extracts tested and Colistin were administered for 7 days to three-week-old chicks.