Antibacterial Activity of Essential Oil of Aeollanthus pubescens on Multidrug Resistant Strains of Salmonella and Escherichia coli Isolated from Laying Hens Farming in Benin

Infections due to Escherichia coli and Salmonella are of the major constraints for the laying hen’s industry as they cause mortality and serious economic losses. The use of conventional antibiotics to control bacterial has shown limits because it allows multidrug-resistance. The main objective of this study was to assess the antibacterial activity of essential oil from Aeollanthus pubescens on multidrug resistant strains of Salmonella and Escherichia coli isolated from laying hens faming in the department of Atlantique in Benin. Altogether, 11 strains of Salmonella and 16 strains of Escherichia coli have been isolated from 101 samples of different organs including liver, spleen, lung, feces and yolk according to standardized methods and their biochemical profile using API 20E gallery. Test of sensitivity was carried out on 11 antibiotics of six different families on identified strains in order to determine their resistance profile. A sensitivity test was carried out on multi-drug resistant strains with Aeollanthus pubescens essential oil to determine their sensitivity with regard to this essential oil. The results showed that the majority of Salmonella strains presented resistance to Tetracyclines (72.7%) and Sulfonamides (63.6%) and all Escherichia coli strains are resistant to Sulfonamides (100%) followed by Tetracycline (93.75%) and Ampicillin (75%). Aeollanthus pubescens essential oil was active on all the multi-drug resistant strains investigated with Minimal inhibitory concentration varying from 0.41 ± 0 mg/ml to 0.83 ± 0 mg/ml for Salmonella and from 0.41 ± 0 mg/ml to 1.66 ± 0 mg/ml for EscheHow to cite this paper: Nestor, A.O., Cyrille, B.K., Philippe, S., Mahudro, Y., Gwladys, K.S., Yannick, A., Alain, A.G., Felicien, A., Souaïbou, F. and Dominique, S.K.C. (2019) Antibacterial Activity of Essential Oil of Aeollanthus pubescens on Multidrug Resistant Strains of Salmonella and Escherichia coli Isolated from Laying Hens Farming in Benin. Advances in Microbiology, 9, 804-823. https://doi.org/10.4236/aim.2019.99049 Received: July 26, 2019 Accepted: September 7, 2019 Published: September 10, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Poultry farming is very important for food and economy of third world countries and is a booming sector in West Africa. It occupies a prominent place in development and poverty reduction strategies in these countries. In Benin, poultry production continues to increase and there are two types of poultry farming: Traditional poultry farming and commercial poultry farming [1]. The poultry livestock is estimated in 2017 at 19,830,000 traditional poultry and 813,000 improved poultry [2]. This livestock has increased to the present day due to the boom in peri-urban poultry farming and the increase need for animal protein [3]. However, the current production methods, introduced many risk factors considerably and the poultry industry performance is hampered by several health obstacles, among which the avian pathologies of which the dominant ones are: Newcastle disease, Gumboro disease, chronic respiratory disease, coccidiosis, colibacillosis, and salmonellosis [4]. The latter two are the main bacterial pathologies affecting laying hens farming and are one of mortality causes and economic losses in the poultry industry [5]. Faced with these pathologies, the means of the struggle of farmers rely essentially on antibiotic therapy which consists of the use synthetic antibiotics. However, the excessive use of Antibiotics has led to the emergence of multi-resistant strains of Salmonella and Escherichia coli in poultry farms [6] [7]. This multidrug resistance phenomenon is becoming a public health problem because previous studies indicate that among pathogenic microorganisms most commonly found in food, poultry products (eggs, chicken meat, etc.) include Salmonella and Escherichia coli strains [8] [9].
Thus, these strains gradually acquired the major antibiotic resistance genes used both in veterinary and human medicine, leaving the prospect of a therapeutic impasse for the most severe infections [10]. As a result, antimicrobial resistance has become a major concern worldwide and it's important to find new molecules who can ensure satisfactory substitution of synthetic antibiotics. Thus, medicinal plants represent a significant source of new drugs, especially since they have fewer side effects [11]. The present study aim is to evaluate the antibacterial activity of the essential oil of Aeollanthus pubescens on multi-resistant strains of

Study Field
This study was conducted from July to December 2018 in the department of Atlantique located in southern Benin. Department of Atlantique is located between 6˚66'0" North Latitude and 2˚22'0" East Longitude and covers an area of 3.233 km 2 . It stretches nearly 100 km from the coast to the interior of the country. Limited to the North by Zou, south by the Atlantic Ocean to the west by the Mono and Couffo and east by the department of Ouémé, this department has 8 towns: Ouidah, Abomey, Allada, Kpomassè, Toffo, Tori-bossito, Zè and Sô-Ava ( Figure 1).

Survey Material
As part of the field work, a survey form was developed. It mainly includes information relating to the identification of the farm, its status on the dominant bacterial pathologies and therapeutic practices implemented on farms.

Essential Oil Used
The essential oil of Aeollanthus pubescens used in this study in previously extracted by hydro distillation and analyzed chemically using GC and GC-MS by Alitonou et al. [12].

Methodology
This study was conducted in three phases namely survey, sampling and laboratory.

Survey
A retrospective study was conducted in 26 laying hens farming in the department of Atlantique to identify the dominant bacterial diseases and highlight antibiotics used by farmers.

Sampling
86 corpses freshly dead chickens were collected randomly from 30 poultry farms where mortality cases have been observed and reported during the study period.
The collected samples were immediately transported to the laboratory of Research Unit on Communicable Diseases in Benin for bacteriological analysis.

Laboratory Tests
Laboratory analysis consisted firstly to autopsy of corpses collected in order to organ harvesting for the isolation and identification of Salmonella and Escherichia coli strains and sensitivity test of isolated strains bolt-to-bolt synthetic antibiotics, in order to determine their resistance profile and finally to test the action of Aeollanthus pubescens essential oil on antibiotic-resistant strains tested.

1) Autopsy and Sampling
Autopsy was performed on the collected corps (86 dead) in laying hens farming visited by the conventional procedure poultry autopsy. Indeed, it was to the external examination of the corps, skin incisions, open abdominal and thoracic cavities and evisceration. These steps were followed by gross examination itself of tissues and organs in order to detect any lesion changes. Of the 86 corps collected 50 corpses had lesions that can be investigated. Thus, samples such as the liver, spleen, lungs, caeca and yolk were collected from damaged organs and tissues. A total of 101 samples were investigated for the isolation and identification of Escherichia coli and Salmonella strains.

2) Bacteriological Analysis
The analyses were performed according to standard isolation techniques, identification and antibiotic susceptibility.
Research and identification of Salmonella The Salmonella research was carried out in four stages, following the ISO 6579 Isolation and identification of Escherichia coli For the isolation of E. coli, a frank incision samples was made and the contents were then inoculated directly and simultaneously on blood agar and Mac-Conkey agar. All plates were incubated at 37˚C for 24 hours. After 24 hours of incubation, characteristic colonies were transplanted on nutrient agar for pure colonies. The identification has been confirmed by API 20E gallery.

3) Determination of Sensitivity and Resistance Profile of Strains to the Antibiotics
After biochemical identification, all Escherichia coli and Salmonella isolates identified were selected for carrying out antibiogram to determine their resistance profile. Thus, 11 antibiotics belonging to 6 families were tested: Ampicillin (10 μg), Cefotaxime (30 μg), Cephalotine (30 μg), Cefoxitin (30 μg), Aztreonam (30 μg), Amoxicillin + Clavulanic acid (20/10) μg), Chloramphenicol (30 μg), Ciprofloxacin (5 μg), Gentamicin (10 μg), Sulfonamide (300 μg) and Tetracycline (30 μg). The method used is agar diffusion on Muller-Hinton agar medium of antibiotic discs according to Clinical and Laboratory Standards Institute [13]. In fact, a colony of 24 hours pure strain was inoculated in 5 ml of Mueller Hinton Broth and incubated for 2 hours to obtain an exponential growth phase suspension (0.5 on the scale McFarland, approximately 1.5 × 10 8 cells/ml). 0.1 ml of this suspension was inoculated by spreading on the surface of the MHA agar previously poured into Petri dishes. Tested antibiotic disks were deposited on the surface of the inoculated plates using sterile forceps and all plates were incu-  [17]. Thus, all the strains for which an inhibition diameter greater than or equal to 14 mm was observed were retained for the determination of MIC, CMB and antibiotical power.

5) Determination of Minimum Inhibitory Concentration (MIC), Minimal
Bactericidal Concentration (CMB) and the Antibiotic Power of Aeollanthus pubescens Essential Oil against Strains Investigated MIC, CMB and antibiotic power (PA) of Aeollathus pubescens essential oil has been made following the technique of dilution in liquid medium on microplate  [18] and reported by Sessou et al. [7] For this, a solution was obtained by mixing 40 μl of the essential oil with 2 ml of Mueller Hinton broth (MHB) and a drop of Tween 80 to emulsify the mixture. Then, 100 μl of the Mueller Hinton broth (MHB) were distributed into the wells of a microplate and 100 μl of the oil extract (suspension prepared from 40 μl of the pure essential oil diluted in 2000 μl of MHB) were added to each of the wells in the first column. Successive dilutions of reason 2, wells by well until to the last well in each row were made and 100 μl of the last well were discarded. All the wells were inoculated except those serving as a negative control with 100 μl of the bacterial suspension at 10 6 germs/ml (density equal to the scale 2 of MCFarland). The negative control consisted of the oil-based Mueller Hinton broth and the positive control consisted of inoculum and Muller Hinton broth. The microplates were covered with parafilm and incubated at 37˚C for approximately 24 hours. On reading, the well corresponding to the lowest concentration of essential oil extract for which we did not observe turbidity or visible growth with the naked eye was taken as the minimum inhibitory concentration (MIC) oil on the strains tested. The CMB, which is the lowest concentration for which we do not have germs growth, was determined following the MIC by dissolving dilutions with a concentration greater than or equal to the MIC on the Mueller Hinton Agar medium. poured into sterile petri dishes and the antibiotic power (pa) of the oil was calculated by the formula: p.a = CMB/CMI. When p.a is less than 4, it is said that the oil tested has an antibiotical power.

Statistical Analysis
The data were coded and stored in the database designed Excel software. For data relating to the investigation, frequencies and confidence intervals were calculated with respect to the status of the dominant bacterial diseases and therapeutic practices. Then, these frequencies were compared with each other by the bilateral Z test (Test Z). For the results of microbiological analysis, descriptive analysis was made, and the means and standard deviations were calculated.
These averages were compared with each other by an ANOVA one way analysis.
In addition, the Turkey test was conducted for structuring averages. All analysis were performed with R3.4.4. Software

Farms Status on Dominant Bacterial Pathologies and Therapeutic Practices
The counting of the 26 survey sheets made it possible to gather various information from poultry farmers in department of Atlantique, particularly in town of Abomey-Calavi, Allada, Ouidah, Tori-bossito and Zè (

Bacteriological Analysis
After the bacteriological analysis of 101 samples, 11 Salmonella strains or 10.89% and 16 Escherichia coli strains or 15.84% were identified (Table 3). Figure 2 and Figure 3 shows respectively the sensitivity and resistance profile   of Salmonella and Escherichia coli strains to the antibiotics tested. All Salmonella strains are more sensitive to Ampicillin (100%), Amoxicillin (100%), Aztreonam (100%), Cefoxitin (100%) and Cephalotin (100%); however, these strains resist the action of Tetracycline (72.7%) and Sulfonamides (63.6%). Escherichia coli strains are particularly sensitive to Amoxicillin (100%), Cefoxitin (100%), Aztreonam (81.25%), Cefotaxime (75%) and are more resistant to Sulfonamides (100%) followed by Tetracycline (93.75%) and Ampicillin (75%). It appears that the two categories of identified strains have developed more resistance to tetracycline and Sulfonamides belonging respectively to the family of Tetracyclines and sulfonamide. Furthermore, Table 4 and Table 5 show respectively the average inhibition diameter of strains identified bolt-to-bolt antibiotics tested. The analysis of these tables shows that, in both Salmonella and Escherichia coli strains, the sensitivity and resistance profiles for the different antibiotics vary from one strain to another. It also appears from these tables that of the 11 strains of Salmonella identified, five (SB1, SB2, SC1, SC2, SC3) exhibited at least one pattern of resistance to three antibiotics of different families (Table 4). With regard to the Escherichia coli strains, almost all (EF1, EF2, EG1, EH1, EH2, EI1, EI2, EJ, EK, EL1, EL2, EL3, EL4, EM1, EM2) presented a resistance profile to at least three antibiotics of different families (Table 5).

Sensitivity of Strains to the Essential Oil of Aeollanthus pubescens
The sensitivity test to the essential oil of Aeollanthus pubescens considered only the Salmonella and Escherichia coli strains qualified as multidrug-resistant.
Thus, the evaluation of the sensitivity of these different strains to the essential oil revealed that the strains were sensitive to this oil. Table 6 and Table 7     antibiotics, they showed a sensitivity towards the Aeollanthus pubescens essential oil with a significant difference (p < 0.001).

Minimal Inhibitory Concentration (MIC), Bactericidal Concentration (CMB) and Antibiotic Power of A. pubescens Essential Oil
The determination of minimal inhibitory and bactericidal concentrations allowed us not only to confirm the activity of A. pubescens oil, but also to evaluate its antibiotic power on the different strains investigated. The results of the MIC, MBC and the antibiotic power of this essential oil on the Salmonella and Escherichia coli strains are presented respectively in Tables 8-13. Upon reading these results, we note that, the MIC of oil on Salmonella strains range from 0.41 mg/ml to 0.83 mg/ml with a non-significant difference between strains. For Escherichia coli strains, MIC ranged from 0.41 mg/ml to 1.66 mg/ml with a significant difference between strains (P < 0.001). It appears that the lowest concentrations of MIC are those obtained on Salmonella strains (Table 8 and Table   9). With regard to CMB, the same observations were made where the lowest concentrations are those obtained on Salmonella strains and range from 0.83 mg/ml to 1.66 mg/ml against 0.83 mg/ml to 5 mg/ml on Escherichia coli strains.
There is no significant difference between the strains. (Table 10 and Table 11).
With respect to the antibiotic power of A. pubescens oil, the calculation of the         (Table 12 and Table 13). Indeed, according to Joubert et al. [19] reported by Yovo [20], when the CMB/MIC ratio is less than or equal to 4, the extract is described as bactericidal and when it is greater than 4, the extract is said to be bacteriostatic. In view of these different results, we can say that the essential oil of Aeollanthus pubescens has a great antibiotic power that deserves to be valued.

Discussion
The survey carried out in some towns in Department of Atlantique allowed us to identify some information on the pathological status of the various farms visited, particularly on the therapeutic practice based on antibiotics using. The choice of the study area is justified by the fact that this department has a favorable climate for poultry farming. In addition, this department has several peri-urban areas with a significant concentration of poultry farming and we meet different categories of livestock. Of the 26 farms visited, the dominant bacterial diseases commonly encountered by her farmers were Chronic Respiratory Disease followed by salmonellosis and colibacillosis. The same observation was made by Sid Nassim et al. [21], in Algeria who took stock of avian pathologies in some poultry farms. The frequency of occurrence of these pathologies in poultry farming could be explained by the health practices of farmers on these farms based on the non-compliance with hygienic and sanitary rules. According to the study by Boko et al. [3] on poultry farming practices in southern Benin, the manure storage location is often in the open air and is not always far enough from poultry houses or it can be a contamination source by air. This study also reported that most farmers use a lot of antibiotics, which could promote the resistance of the various germs responsible for these diseases, hence their persistence in these farms. Regarding antibiotics, the most molecules used by these farmers are: oxytetracycline, colistin, enrofloxacin, tylosin, norfloxacin and the sulfadiazinetrimethoprim combination. The same molecules were identified by Fofana [22] in a survey of 23 broiler farming in Senegal.
For bacteriological analysis, samples were taken from the liver (n = 34), lungs (n = 20), caeca (n = 25), spleen (n = 12) and yolk (n = 10) from cadavers of chickens and chicks autopsied with lesions such as generalized congestion, congestion and hypertrophy, hypertrophy and inflammation. In total, 11 Salmonella strains and 16 Escherichia coli strains were identified in these different samples. Escherichia coli outside the caeca was isolated from the liver (n = 07), spleen (n = 03), lung (n = 04) and yolk (n = 02) and Salmonella was isolated from the liver (n = 04) and caeca (n = 07). Our results outside the Salmonella strains are close to those obtained by Rahmatallah et al. [6], who also isolated and identified Escherichia coli strains in the liver, spleen, lungs, yolk sac, but also in the bone marrow and heart on chickens and chicks in Morocco. Al Hassane [23], in Senegal, in his study on "chicken-flesh colibacillosis: anatomy-clinical study and circumstances of emergence in the peri-urban area of Dakar" also isolated and identified these Escherichia coli strains in the liver, spleen but also in the heart and intestine. Our study did not consider the removal from the heart and intestines although these strains can be isolated from its places in case of infection with Escherichia coli and Salmonella, can be explained by the absence of characteristic lesions may attract our attention for possible samples. Moreover, the presence of these different strains in the elements collected already poses a problem and could be explained by a superinfection of the subjects by these  [20] in Benin (0.27 mg/ml and 0.54 mg/ml respectively). For MIC and CMB). The same observation was made for the results of Sessou et al. [7] in Benin (0.81 ± 0.38 mg/ml and 1.62 ± 0.76 mg/ml respectively for CMI and CMB). These differences observed between our results and those of these authors could be explained by the fact that in our study, the investigated strains were multiresistants strains. In contrast to these authors, we determined the resistance profile of these strains and it is after the results obtained that we tested the action of this essential oil on these different strains. In addition, the essential oil of Aeollanthus pubescens showed a bactericidal effect on the various strains investigated, which is in accordance with the results obtained by Sessou et al. [7] It is important to emphasize that the work of these authors did not consider the investigation of Salmonella in their studies. The action of this essential oil of A. pubescens is due to its high concentration of thymol, α-terpinene, carvacrol and borneol [12] [26]. Indeed, these compounds are already recognized for their antibacterial activity, particularly thymol and carvacrol, which are the most bactericidal [27]. According to Zayyad et al. [26] reported by Yovo [20], borneol is a compound with high antimicrobial potency due to its high solubility in water, which gives it a high ability to cross bacterial cell membranes. This study showed, on the one hand, the importance of the antimicrobial resistance of Salmonella and Escherichia coli strains in our laying hens farming and, on the other hand, the effectiveness of the oil of Aeollanthus pubescens which deserves to be valued. Regarding the high antimicrobial activity of this oil, it is necessary to develop some strategies that will immensely boost Aellonthus pubescens plant production and therefore a large scale production of its essential oil in order to overcome this important bacterial problem which is a big regulator of economy in several countries.

Conclusion
In order to make our contribution to the endogenous fight against the bacterial resistance which is observed more and more in our farming and which constitutes a real public health problem these days, this study was interested in the activity antimicrobial effect of Aeollanthus pubescens essential oil on Salmonella and Escherichia coli multidrug-resistant strains isolated from laying hens farming in Department of Atlantique. At the end of this study, 11 Salmonella strains and 16 Escherichia coli strains were isolated and identified from liver, spleen, lungs, yolk and caeca collected from chickens and chicks corps collected in some laying hens farming in this department. The resistance profile of these isolated strains was determined and showed that almost all of them had a high resistance to tetracyclines, sulfonamides and ampicillin belonging to three different families of antibiotics (Tetracycline, Sulfamides and β-lactam), which are among the antibiotics commonly used in poultry farming. Faced with this resistance noted, this study has used the essential oil of Aeollanthus pubescens to propose an alternative therapeutic solution. Thus, this oil proved effective against the different A. O. Nestor et al.