Biological material is made of chicken corps and freshly collected dead chicks on farms of laying hens farming visited.

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].

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.

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 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 (2002) standard adapted to our studies.

Pre-Enrichment

One gram (1 g) of each sample was removed aseptically and added to 9 ml of sterile buffered peptone water (EPT). The whole was incubated at 37˚C for 24 hours.

Enrichment

After 24 hours of incubation, 0.1 ml of the pre-enriched was placed in 10 ml of Rappaport-Vassiliadis broth (RV) for selective enrichment and then incubated at 41.5˚C for 24 hours. Also, 1 ml of the medium was added to 10 ml of Muller Kauffman broth with tetrathionate and novobiocin always for selective enrichment.

Isolation

A loopful of the RV-enriched was seeded on XLD (Xylose-Lysine-Desoxicholate) and Hecktoen agar and incubated at 37˚C for 24 hours. Characteristic colonies on XLD agar were removed and subcultured on nutrient agar to obtain pure colonies. Incubation was performed at 37˚C for 24 h.

Identification

Gram stain, catalase and oxidase tests were firstly performed, and the identification of Salmonella strains was confirmed using API 20E gallery. In fact, 24-hour colonies obtained on nutritive agar were used to prepare the microbial suspension (a colony in 5 ml of 0.85% NaCl solution). The API 20E plate was then inoculated according to the manufacturer’s instructions and incubated at 37˚C for 24 hours. The reading and the interpretation were made by referring to the API 20E analytical catalog and from Abis online software.

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 MacConkey 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 × 10⁸ 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 incubated at 37˚C for 24 hours. After 24 hours incubation, the inhibition diameters in Table 1 observed were measured using calipers and the values obtained were compared to the standards specified by CLSI [13] and EUCAST [14]. Escherichia coli and Salmonella strains showing resistance patterns were selected for the sensitivity test to the essential oil of Aeollanthus pubescens.

4) Strain Sensitivity Test with Essential Oil of Aeollanthus pubescens

Strain susceptibility testing of this essential oil was performed by the disk diffusion method on Muller-Hinton Agar medium according to National Committee for Clinical Laboratory Standards [16] reported by Lakhdar [17] and by Sessou et al. [7] Thus, sterile disks of 6 mm diameter impregnated with 5 μl of Aeollanthus pubescens essential oil were deposited on the MHA agar previously inoculated with a microbial suspension of approximately 1.5 × 10⁸ cells/ml. All plates were then incubated at 37˚C for 24 hours. After 24 hours incubation, inhibitions diameters observed were measured using a vernier caliper. Indeed, the germ sensitivity is zero for a diameter less than or equal to 8 mm. It is limited to a diameter between 8 and 14 mm, and average diameters between 14 and 20 mm. To a diameter greater than or equal to 20 mm the germ is sensitive [7] [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

AMP: ampicillin; AMC: Amoxicillin + clavulanic acid; ATM: Aztreonam; CTX: Cefotaxime; FOX: Cefoxitine; CEF: Cephalotin; GMN: Gentamicin; TET: Tetracycline; SSS: Sulfonamide.

96 wells coupled to the plating on solid medium as described by Chabert et al. [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 10⁶ 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.

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

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 (Table 2). At the end of this survey, all the farms visited are private. The dominant bacterial diseases commonly encountered by its farmers were Chronic Respiratory Disease (46.14%) followed by Salmonellosis (38.46%) and Colibacillosis (15.37%). All farmers

NS: not significant (P > 0.05); *: P < 0.05; **: P < 0.01; ***: P < 0.001.

(100%) have a prophylaxis plan, but 76.92% strictly respect it against 23.08% with a significant difference (p < 0.05). In addition, 57.69% of farmers vaccinate against salmonellosis and 42.31% do not. For vaccination against colibacillosis, only 19.23% of farmers do it. In case of pathology, only 23.07% of farmers confirm their diagnosis by autopsy and laboratory analysis against 53.8% who confirm their diagnosis by only performing the autopsy. Regarding drugs, the most used molecules by these farmers were: oxytetracycline, colistin, enrofloxacin, tylosin, norfloxacin and the sulfadiazine-trimethoprim combination. Most of these farmers use these curative antibiotics (76.92%) and often obtain the expected result (69.23%) with a significant difference (p < 0.05).

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).

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 respectively show the diameters of inhibition of Salmonella and Escherichia coli multiresistants strains in contact with the essential oil of Aeollanthus pubescens. The

***: P < 0.001; the average of the same line followed by different letters, differ significantly at the threshold of 10/00.

***: P < 0.001; the average of the same line followed by different letters, differ significantly at the threshold of 10/00.

analysis of these tables shows that the essential oil of A. pubescens had an important activity on all the strains investigated. In fact, at 5 μl of the essential oil, the Salmonella strains (SB2, SC1 and SC2) showed great sensitivity with respective inhibition diameters of 22.5 ± 0.70 mm, 25.5 ± 0.70 mm. and 24.5 ± 0.70 mm while strains (SB1 and SC3) exhibited average sensitivity with respective inhibition diameters of 15 ± 0 mm and 19 ± 0 mm (Table 6). As for the Escherichia coli strains, with 5 μl of the essential oil, strains (EG1, EH1, EH2, EI1, EI2, EJ, EK, EL1, EL2, EL3, EL4 and EM1 also presented a high sensitivity to oil with respective inhibition diameters of 32 ± 00 mm, 27.5 ± 0.70 mm, 30 ± 00 mm, 20 ± 00 mm, 20 ± 00 mm, 22.5 ± 0.70 mm, 23.5 ± 0.70 mm, 23 ± 00 mm, 22.5 ± 00 mm, 29 ± 00 mm, 34.5 ± 0.70 mm and 22 ± 00 mm and only the strains (EF1, EF2 and EM2) showed average sensitivity with respective inhibition diameters of 16 ± These results show that, despite the resistance of these different strains to antibiotics, they showed a sensitivity towards the Aeollanthus pubescens essential oil with a significant difference (p < 0.001).

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

***: P < 0.001; The averages of the same line followed by different letters, differ significantly at the threshold of 10/00.

***: P<0.001; The averages of the same line followed by different letters, differ significantly at the threshold of 10/00.

CMB/MIC ratio revealed that it has a bactericidal effect on Salmonella and Escherichia coli strains (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.