A Pilot Study on the Effect of Thyme Microemulsion Compared with Antibiotic as Treatment of Salmonella Enteritidis in Broiler

Multidrug resistance poses a global threat to the poultry industry and public health, so the direction towards eliminating the use of antibiotics and finding alternatives is a vital step to solve this problem. Thyme microemulsion (10% oil/water) had nanodrop size 28.65 ± 0.89 nm, with a polydispersity index (PDI) of 0.28 with greater homogeneity. It showed IC50 > 100 ug/ml on cytotoxicity assay and 14 active components by GC-Mass. The study was carried out using 210 Cobb chicks divided into fourteen groups. The infected groups were challenged using two Salmonella Enteritidis multidrug resistance (MDR) and Salmonella Enteritidis sensitive strains to the sulpha-trimethoprim antibiotic. The challenged inoculum was 1 × 109 CFU of Salmonella Enteritidis by oral route. The MIC treatments doses were 1 ml/liter water for thyme oil and thyme microemulsion and 33.34 mg/kg b.wt sulfadiazine for 5 days. The results showed that both thymol oil (0.1%) and microemulsion (0.01%) are able to decrease the count of Salmonella Enteritidis in cecal content and fecal dropping and the mortality rates after five days of treatment. In addition, thyme oil and microemulsion had no pathological alteration on chickens' tissues that were collected two weeks after giving the treatment. By the robust HPLC method, the SDZ and TMP residues in tissues of infected groups treated with Cotrimazine® + thyme oil microemulsion had a slight significant economic impact (P < 0.05) compared to Cotrimazine® alone. In conclusion, thymol oil and microemulsion could be an alternative economic choice for multidrug resistance Salmonella Enteritidis treatment in poultry farms.


Introduction
Poultry is an important source of animal protein for human. Salmonella is considered one of the most important zoonotic bacteria which causes food-borne diseases to human, through the consumption of poultry and poultry byproducts [1][2][3]. On the other hand Salmonella causes high economic losses in poultry production due to a decrease in productivity, low egg production, and premature deaths, in addition to the high cost of Salmonella infection treatment. So control of Salmonella infection in poultry is considered very important goal for decreasing human infection and economic losses [4,5].
Salmonella Enteritidis (S. Enteritidis) serotype is one of the most important members of 2500 Salmonella serovars which can be transmitted from poultry and poultry byproducts to human causing food poisoning diseases [6]; furthermore, S. Enteritidis became highly resistant, so prevention and control of S. Enteritidis infection of poultry is very important for human health and the poultry industry [7,8].
e critical task of poultry producers is to stop the uncontrolled usage of some antibiotics as growth promoters in poultry feed [9,10]. Unfortunately the misuse of antibiotics leads to the development of multidrug resistant bacterial strains especially in Salmonella species [11,12].
Development of products rather than antibiotics is done to treat or at least stop the growth of bacteria such as essential oils (EOs) and organic acids (EOA) which have antimicrobial and antioxidant effects, improve animal intestinal health, and promote the absorption of nutrients. Nowadays these products used as antibiotic substitutes [13,14]. e action of EOs depends on their chemical composition; generally EOs can inhibit bacterial growth, alter the permeability of cell membrane, and render the synthesis of some proteins and ATP [15]. Adding of EOs in poultry feed increases feed Conversion Ratio and improve growth performance in poultry [16].
ymol oil is one of the major components of thyme. It represents about 47-59% of thyme and has a great effect on Salmonella Typhimurium [17]. ymol oil has an effect on the synthesis of ATP leading to increase in the concentration of ATP inside the bacterial cell and lead to destruction and death of the bacterial cell, also can affect the production of protein which is involved in energy metabolism, and also changes other proteins which adversely affect survival of bacteria in adverse conditions, so the permeability of cell membrane of Salmonella Typhimurium is affected by thymol oil. ymol-benzoic acid has a bacteriostatic effect on Salmonella Enteritidis [15,18,19]. e combination of the sulfadiazine (SDZ) and trimethoprim (TMP) formulation at a 5 : 1 ratio is widely used in poultry industry against different microorganisms [20].
is may lead to residues in poultry tissues with possible hazards like development of antimicrobial resistance and toxic and allergic effect in addition to diminished effect on gut microflora [21].
Microemulsions are colloidal dispersions made up of small oil droplets (20-200 nm) suspended in water. e microemulsions must be properly constructed to ensure that they remain physically stable during storage and use [22]. Antimicrobial microemulsions are emulsified oil, water, and detergent combinations that have been demonstrated to have wide activity against enveloped viruses, bacteria, and fungus [23], at harmless quantities in animals. Microemulsions' antibacterial activity is unlike antibiotics', nonspecific, allowing for broad-range action while decreasing the potential for resistance progress. When microemulsions bind to lipid bilayers in cell membranes, the energy contained in the oil-and-detergent emulsion is released, destabilizing the bacteria's lipid membrane; therefore, their antibacterial action developed [24].
Infection of chicken with Salmonella Typhimurium leads to severe pathological changes in cecal wall like sever inflammation and degeneration in cecal wall treatment with thymol oil and thymol microemulsion leading to high improvement of histopathological picture of cecal wall of infected chickens [25].
Aim of this study is trying to use thymol oil and thymol microemulsion for controlling of Salmonella Enteritidis infection with/without the sulpha-trimethoprim antibiotics in broilers and show its residual and histopathological effect in broiler tissues in comparison with the sulpha-trimethoprim antibiotics. (1) Preparation of ymol Microemulsion. ymol oil was purchased from Alamal for natural oil, Gharbia, Tween 80, deionized water, propylene glycol, and sodium chloride were obtained from Sigma-Aldrich Co. Preparation of thymol oil microemulsion (10% oil in water) was done in nanomaterial research and synthesis unit by using the method according to [26].

Materials and Methods
(2) Characterization of ymol Microemulsion. Characterization of thymol microemulsion was done using Zetasizer Malvern Instrument (Corp, Malvern, UK) used to measure droplet size, surface charge (zeta potential), size distribution (polydispersity indexes, PDI), and electrical conductivity of the microemulsion. High-resolution transmission electron microscopy (HRTEM) observations were performed with a JEM 1400F HRTEM at a beam energy of 300 keV. ymus oil and microemulsion components were analyzed using GC-MS at Central Laboratory in Faculty of Agriculture, Cairo University.
(3) Cytotoxicity Assay. Cell viability was assessed by SRB assay with different concentrations (0.01, 0.1, 1, 10, and 100 ug/ml) according to [27] using Vero cell (Green monkey cell line) obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Cells were maintained in DMEM media supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% of heat-inactivated fetal bovine serum in humidified 5% (v/v) CO 2 atmosphere at 37°C.  (35 days). Chickens were fed a commercial starter diet (23% crude protein and 3000 kcal ME/kg diet) during the first two weeks of age and then a commercial grower diet (22% crude protein and 3150 kcal ME/kg diet). No antibiotics were added to the ration. No vaccination program was supported to the trial. Feed and water were available ad libitum. Colonies of Salmonella grew to approximately 1 × 10 9 cfu/mL on average and were diluted according to McFarland standard in normal saline to inoculate 1 ml/chick once orally [29] at 15-day-old chicks.

Drug Used in
(2) Antimicrobial Activity of Essential Oil and the Nanoproduct (In Vitro Challenge). e disk diffusion method was applied for the determination of the antibacterial activity of the thyme oil as an essential oil and thyme microemulsion product according to [30]. e result measured according to the bacterial growth inhibition was the diameter of the zones (mm) without bacterial growth. For the interpretation of the results, four groups of inhibitory zones were examined: (i) no inhibition, (ii) <12 mm (weak activity zone), (iii) 12 mm ≥ inhibition zone <20 mm (intermediate activity), and (iv) inhibition zone ≥20 mm (strong activity).

Experimental Design.
Two hundred and fifteen oneday-old Cobb broiler chicks were kept for 49 days. At arrival we examined the paper lining of all chicks' boxes and organs (liver, yolk, and cecum) separately of five sacrificed chicks which are humanly euthanized and cultured to confirm their freedom from Salmonella strains. Chicks were randomly divided into fourteen groups (1 to 14). Each group consists of fifteen chicks as shown in (Table 1).
Chicks in groups one to twelve were orally inoculated with single dose of 1 × 10 9 cfu/1 ml of Salmonella Enteritidis strains on day fifteen. On the eighteen day, the sulpha-trimethoprim was orally administered in a dose of 10 mg/kg b.wt/5 days while thymol oil and microemulsion were given in the drinking water in a dose of 1 ml/1 liter water/5 days after inoculation of both Salmonella Enteritidis strains by three days.
is challenge was done in period from December 2020 to January 2021.

Laboratory Examination
(1) Enumeration of Salmonella Enteritidis. Five cecal contents and fecal dropping from each group at 35, 42, and 49 days of age were examined for Salmonella Enteritidis enumeration [31]. Decimal dilutions in BPW were prepared and 0.1 mL of each dilution was inoculated by spread plate to XLD in duplicate. ese plates were incubated for 24 h at 37°C and pink colonies with dark center were counted as Salmonella.
(2) Sample Extraction for Detection of the Antibiotic Residue.
e extraction was done according to [32]. Tissues were homogenized and accurately weighed (3 g of muscle, 0.5 g of liver, and 0.25 g of kidney) in a 50 mL plastic centrifuge tube. irty mL of dichloromethane were added and the samples were homogenized with an Ultra Turrax for 1 min and centrifuged at 3000 g for 10 min. Supernatant was filtered through filter paper and collected. Ten mL of this liquid was transferred to a 15 mL glass centrifuge tube; 1 mL of hydrochloric acid 3 N was added, vortexed for 15 sec, and centrifuged for 5 min at 3000 g. From the upper aqueous layer, 250 μL was transferred to a 15 mL glass centrifuge tube and another 250 μL of 3.8 M sodium acetate solution was added and vortexed for 15 sec. Finally, a 100 μL aliquot was injected into the LC system.

(3) Reagents, Equipment and Chromatographic Conditions for
Detection of the Antibiotic Residues. All chemicals and reagents were of analytical grade. Analytical standards of SDZ and TMP were from Sigma Co. Liquid Chromatography (LC) Agilent Series 1200 was used. e chromatographic column was a reversed phase Venusil XBP C8 column (Agela Technologies, 4.6 mm i.d., 250 mm, 5 μm) ( Table 2).
(4) Histopathological Examination. Tissue specimens (heart, liver, spleen, proventriculus, and cecum) were collected from three chickens at second weak after treatment with antibiotics, thyme oil, and microemulsion. All organs were fixed in 10% neutral buffered formalin and processed by paraffin embedding technique for histopathological examination according to [33].

Statistical Analysis.
Statistical analysis was done using SPSS IBM 21 software and data were analyzed using statistical analysis system software (One-Way ANOVA). Results of all tests used in this study were considered significant at P value ≤ 0.05.

3.1.
Characterization of ymol Microemulsion. Microemulsion characterization of the nanodroplet was mainly determined by TEM whose size is 28.65 ± 0.89 nm with a narrow size distribution (polydispersity index: 0.28) which indicated that greater homogeneity can be realized (Figure 1(a)). e zeta potential is an indicator that stable suspensions are generally taken by using dynamic light scattering (DLS) of a −2.6 ± 8.32 mV and conductivity 0.089 ms/cm.

In Vitro: Antibacterial Activity of
ymol Oil and Microemulsion. Antimicrobial activity of thymol oil and microemulsion to Salmonella Enteritidis strain was done by measuring the diameter of inhibitory zone of bacterial growth surrounding the disc of three tested concentrations (0.5, 1.0, and 5.0 ml) according to [29] which was 15, 25, and 50 millimeters, respectively, for thymol oil while microemulsion results were 17, 32, and 60 ml. In the in vivo Negative control GP14 Only antibiotic

Mortality Rate
Results. High mortality rates were detected in group one (which was only inoculated with Salmonella Enteritidis MDR strain), group two (inoculated with Salmonella Enteritidis strain sensitive to sulpha-trimethoprim group), and group three (inoculated with MDR Salmonella Enteritidis strain and treated with sulpha-trimethoprim antibiotics) to be 40%, 26.7%, and 20%, respectively; meanwhile, the other groups show a significant decrease in the mortality rate reaching 6.7% after treatment with thymol oil dose (0.1%) and microemulsion dose (0.01%) and sulpha-trimethoprim, as shown in Table 3.

Results of Salmonella Enteritidis Count during
Experiment. Results of the fourteen groups showed high count of Salmonella Enteritidis in positive groups. Sulphatrimethoprim antibiotic has great effect on group inoculated with Salmonella sensitive strain while it has no effect on the group which was inoculated with Salmonella Enteritidis MDR strain. Other groups which were treated with thymol oil and thymol microemulsion with or without antibiotics showed lower count as shown in Table 4.
(1) Statistical Analysis of Total Salmonella Enteritidis Count in Cecal Content. e statistical analysis was done for total Salmonella Enteritidis count of cecal content of three chickens, from each group which were infected with Salmonella MDR strain, and other groups which were infected with Salmonella Enteritidis sensitive to sulpha-trimethoprim (sxt). e results of the count of Salmonella Enteritidis sensitive strain from both the second and fourth weeks showed a highly significant difference between groups which marked by ( * ) at the same column at P value ≤ 0.05, as shown in Tables 5 and 6.

Measuring Sulfadiazine and Trimethoprim Residuals in Different Boiler Tissues.
e distributions of sulfadiazine and trimethoprim in different boiler tissues after administration of 33.34 and 6.67 mg/kg b.wt for 5 days, respectively, are illustrated in Table 7 for sulfadiazine and in Table 8 for trimethoprim.
e results showed that both compounds were rapidly distributed in all examined tissues.

Histopathological Results.
Microscopic examination of negative control as well as treated groups with microemulsion dose (0.01%) and thyme oil dose (0.1%) revealed normal histological architecture of examined organs. Meanwhile, variable grades of pathological alterations were recorded in examined organs from infected/and or treated groups described in Tables 9 and 10 and Figures 2 and 3.

Discussion
Salmonella infection is one of the major bacterial diseases in poultry, which economically has an adverse effect on cost of the treatment especially in case of multidrug resistance strains [34]. is study aimed to investigate, in vitro and in vivo, the effect of antibiotics compared with thymol and microemulsion on antibiotic-resistant bacteria. ymol was found to have a positive effect on significantly reducing the number of antibiotic-resistant bacteria, which may make it a good alternative to antibiotics. is natural product could be an efficient alternative that would have positive effect to fight antimicrobial resistance that is observed in human which originates from the administering of antibiotics to farm animals [10,35,36]. e fabrication of microemulsion with lesser droplet size in the presence of double bonds in the nonpolar chain of nonionic surfactants obtained results in agreement with different results [37]. e conductivity of the microemulsions increased by increasing essential oil concentration which demonstrated that   Veterinary Medicine International water is the continuous phase (P < 0.05). is is due to the fact that conductivity of the solutions is directly proportional to the amount of ions and increases by increasing the ions. e cell viability reached 88-90% for microemulsions containing thyme and rosemary oil, respectively, which showed that prepared microemulsions are safe and nontoxic [38]. Kumari et al. [39] reported that in the stability study for the characterization of thymol based microemulsions they had spherical droplets size (293 ± 2.7 nm), PDI (0.15), and zeta potential (−32 mV) in 50 min sonicated as compared to other microemulsions. e results are strengthened by the fact that greater sonic energy flows to emulation through greater surfactant adsorption on hydrophobic droplet surfaces and helps to reduce the droplet dimensions and the spread of small droplet. Diffusion or movement of solubilized oil molecules from small droplets to large droplets through dispersed droplets causes dispersion of droplets to grow with reduced size (Ostwald ripening method). e optimized 0.82% of thymus oil microemulsions was pale yellow to amber transparent microemulsion with a globule size of 14.23 ± 0.3 nm, zeta potential of −0.69 mV, and PDI value of 0.00143 indicating a stable microemulsion [40].

In Vitro Experiment.
In our study, disc diffusion method is used for determination of doses and concentration of thyme oil and nanomaterial emulsion which affected the Salmonella strains used in our experimental design which is the same as [30,41], while minimum inhibitory concentration method (MIC) is used in the detection of the effective concentration which affects Salmonella strain in this study.
"In in vivo challenge in this study, the mortality rates were 40% in the positive control while other groups which were treated with the antibiotic and thymol oil and the microemulsion show lower percentage of 6.7% which is in accordance with [43] who reported that, after challenge with Salmonella and treatment with thymol oil and its microemulsion, the mortality rate among chicken treated with thymol oil and its microemulsion was 3%." Paper [44] did not find any significant difference in mortality rates of groups which were treated with essential oil and positive control.
In the present study, there was a little Salmonella count in cecal content when adding thymol oil in the water of chickens; these results agreed with [19,44]. On the other hand, [42] did not find any significant decrease in the Salmonella count in cecal content when treated with thymol oil. In our study, we found the effect of thymol microemulsion was higher than thymol oil on the percentage of reduction in the count of Salmonella in cecal content, which is similar to [25] who found better effect of 0.5% concentration of thymol microemulsion than 1% of thymol oil on reduction of Salmonella count in cecal content.
As a result of threats of drug residues in foods industry of animal origin, limits for these residues have been set by the  Veterinary Medicine International competent authorities and bodies as the European Union has set MRL of residues of both SDZ and TMP in different poultry tissues, 100 and 50 ppb, respectively [45,46]. After administration of SDZ and TMP at a doses of 33.34 and 6.67 mg/kg b.wt, respectively, for 5 days, SDZ residues were still detected till the 2nd day in muscle and till the 5th day in liver and kidneys, but TMP residues were still detected till the 2nd day in muscle, the 4th day in liver, and the 5th day in kidneys after cessation of treatment in healthy broiler chickens; these findings agree with that detected in broilers by [47], but are not in the same line with that noted by [48] who revealed that TMP residues were still detected only till the 2nd day in muscle, liver, and kidney in healthy broiler chickens and this may rely on using different dose at 33.4 g/l for just 3 consecutive days. Infection with MDR and SS strains in the other four groups significantly decreases the SDZ concentrations in the muscle at the 2nd day and in liver and kidneys at 4th day and significantly decreases the TMP concentrations in analyzed tissues to be detected in the muscle at the 2nd day and in liver and kidneys at 3rd day after last oral dose. e obtained data revealed that the recommended withdrawal time of SDZ and TMP in healthy broilers according to the accepted guidelines is 5 days and this agrees with [49] who found that the withdrawal period for such an oral formulation of SDZ and TMPt in healthy pigs should not be less than 5 days using different animal species and this also agrees with [48] who revealed that residue concentrations of SQX and TMP were lower than their maximum residual limits (MRLs) in all tissues at 5 days after the treatment.
In our study we found no histopathological effects of thymol oil and thymol microemulsion in noninfected groups while the infected groups which were treated with thyme oil show lower histopathological changes than those treated with thymol microemulsion and antibiotic and these results were similar to [35]. On the other hand our results disagree with what is mentioned by [25] who found better histopathological improvement in cecal wall of infected chicken with Salmonella after treatment with the thymol microemulsion than after treatment with thymol oil.

Conclusion
ymol oil and thymol microemulsion have antibacterial effect, which makes reduction in Salmonella count in broiler cecum after administration of thymol and its microemulsion as a treatment to the infected chicken with Salmonella Enteritidis even with the multidrug resistance strain. Treatment with thymol oil makes better improvement in histopathological picture of infected chicken than the thymol microemulsion. By robust HPLC method, the SDZ and TMP residues in tissues of infected groups treated with

Recommendations
A further study with increasing the thymol oil and thymol microemulsion concentration dose and/or increasing treatment period is recommended. e recommended withdrawal time of SDZ and TMP in healthy broilers is 5 days and in infected broilers with MDR and SS strains alone and in presence of thyme oil is four days to be safe for human consumption.

Data Availability
All data are included within the manuscript.

Ethical Approval
e experiment was carried out according to the national regulations on animal welfare and approved by Institutional Animal Ethical Committee. e present study was carried out at the Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP), Animal Health Research Institute.