Body fat modulated activity of Gallus gallus domesticus Linnaeus (1758) and Meleagris gallopavo Linnaeus (1758) in association with antibiotics against bacteria of veterinary interest

Microbial Pathogenesis Available online 22 August 2018 In Press, Accepted ManuscriptWhat are Accepted Manuscript articles? Author links open overlay panelDiógenesde Queiroz DiasaDébora LimaSalesaJacqueline CosmoAndradeaAna RaquelPereira da SilvabSaulo RelisonTintinobCíceraDatiane de Morais Oliveira-TintinobGyllyandesonde Araújo DelmondesbMarcos FábioGadelha RochacJosé GalbertoMartins da CostabRômuloRomeu da Nóbrega AlvesdFelipe SilvaFerreiraeHenrique DouglasMelo CoutinhobWaltéciode Oliveira Almeidab a Universidade Federal Rural de Pernambuco – UFRPE, Recife, PE, Brazil b Universidade Regional do Cariri – URCA, Crato, CE, Brazil c Universidade Estadual do Ceará – UECE, Fortaleza, CE, Brazil d Universidade Estadual da Paraíba – UEPB, Campina Grande, PB, Brazil e Universidade Federal do Vale do São Francisco – UNIVASF, Senhor do Bomfim, BA, Brazil Received 23 March 2018, Revised 15 August 2018, Accepted 18 August 2018, Available online 22 August 2018. Show less https://doi.org/10.1016/j.micpath.2018.08.029Get rights and content Highlights • The OFGG and OFMG did not present clinically relevant antibacterial activity. • The OFGG modulated synergistically the action of the antibiotics Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline; . • The OFMG modulated synergistically the action of Amikacin, Amoxicillin, and Norfloxacin. Abstract In the Northeast of Brazil, ethnoveterinary studies have shown that the body fat from Gallus gallus domesticus and Meleagris gallopavo are used for diseases that affect domestic animals. The objective of this study was to identify the chemical composition and to evaluate the antibacterial activity of the Gallus gallus domesticus (OFGG) and Meleagris gallopavo (OFMG) fixed oils in isolation and in association with antibiotics. The OFGG and OFMG from the poultry's body fat were extracted using hexane as a solvent in Soxhlet. Their composition was indirectly determined using fatty acid methyl esters. The OFGG and OFMG antibacterial and modulatory activities against standard and multi-resistant bacterial strains were performed through the broth microdilution test. In the OFGG chemical composition, 4 constituents were identified. The saturated fatty acid (AGS) and unsaturated fatty acid (AGI) percentages were 35.1% and 64.91% respectively, with linoleic acid being the major component. In the OFMG, 3 constituents were identified. The AGS percentage was 27.71% and 72.29% for AGI, with oleic acid as the most abundant component. The oils did not present antibacterial activity when tested in isolation, presenting Minimum Inhibitory Concentrations (MICs) > 512 μg/mL. However, when associated with antibiotics the OFGG showed synergistic activity with the antibiotics Amikacin, Amoxicillin, Norfloxacin and Oxytetracycline, while the OFMG promoted a synergistic action with the antibiotics Amikacin, Amoxicillin and Norfloxacin. Keywords EthnoveterinaryZootherapyFatty acidsAntibiotic modulating activity 1. Introduction There are many parallels between traditional medicine for human beings and traditional medicine for animals, encompassing not only health care and belief concepts, but also almost all modes of medical material administrations, skills, techniques, and behaviors [1]. According to Alves and Rosa [2], ethnomedical information may indicate the presence of biologically active constituents and may represent important sources for the discovery of new medications. The information from ethnoveterinary systems has led to the bio-prospection of natural products which can be used to treat parasitic [3] and bacterial diseases which affect domestic animals [4,5]. The concern with the increasing occurrence of bacterial strains resistant to several groups of antibiotics is one of the most worrying problems for research directed towards animal health [[6], [7], [8]]. Studies indicate that the increase in antibiotic-resistant bacterial strains coincide with the wide use of different antibiotic groups for the treatment of several clinical manifestations in human beings and animals, as well as in the food preservation and experimental medicine industries [9,10]. Several microorganisms are responsible for infectious diseases in domestic animals, for example, infectious mastitis in animals is caused by a wide variety of microorganisms with the Staphylococcus and Streptococcus genera being the main contagious microorganisms, in addition to environmental agents such as Enterobacteriaceae Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa [11]. The potential for the Enterobacteriaceae family to be zoonotic is also a concerning factor in addition to antibiotic resistance [12]. One of the alternatives to preventing antibiotic bacterial strain resistance occurs through the bio-prospection of natural products that may demonstrate antibacterial or antibiotic modulating activity [13]. Natural resources such as plants [14] and animals [15] are used in ethnoveterinary practice. Plant-derived materials represent the majority of ingredients used in traditional health systems around the world, however animal origin products, such as urine and fat, are also important elements in the ethnomedical sense [2]. In recent years, some studies have tried to analyze the antibacterial and antifungal potential of animals (named as zootherapy), such as in the study by Sales et al. [16], where they demonstrated that glandular secretions from the Rhinella Jimi frog potentiated aminoglycoside effects against bacteria. Tadesse et al. [17] reported that compounds from sponges and ascidians showed activity against bacteria and fungi. Although products from domestic animals are cited for the treatment of diseases that affect humans and other animals, studies that seek to validate these pharmacological activities are still rare [13,18]. In the Northeast of Brazil, ethnoveterinary studies have shown that fat is one of the main products used for the treatment of diseases affecting domestic animals [[18], [19], [20]]. For domestic animals, turkey (Meleagris gallopavo) and chicken (Gallus gallus domesticus) fats are cited for the treatment of blisters, and the chicken fat is also used to treat mastitis and dermal nodules [18,20]. However, studies that seek to validate the antibacterial and modulatory activity of these species' fats against bacteria that cause diseases in domestic animals were not found. In this study, the antibacterial and modulatory activity of the Gallus gallus domesticus and Meleagris gallopavo body fat against bacteria of veterinary interest were verified. 2. Materials and methods 2.1. Zoological bird materials The abdominal fat of eight male and seven female Meleagris gallopavo (Linnaeus, 1758) as well as fifteen male and fifteen female Gallus gallus (Linnaeus, 1758) were donated by the commercial slaughterhouse Adriano do Frango ME, located at Street F7, number 7, Santa Terezinha Village, in the city of Barbalha - Ceará (Brazil). All animals used for fat extraction were adults of undefined strains (UDS) from several non-commercial farms, raised extensively (free range) and without receiving specific rations. For each species, the total fat obtained was mixed and crushed before sending it for extraction of their respective fixed oils. This study was approved by the Commission of Experimentation and Use of animals of the Regional University of Cariri (CEUA - URCA), under protocol number: 0300/2015.1. 2.2. Obtaining the Meleagris gallopavo (OFMG) and Gallus gallus domesticus (OFGG) fixed oils The fixed oils were extracted from body fat located in the ventral region of the birds. The extraction was performed in a Soxhlet device for 4 h using hexane P.A. as a solvent. After the mixtures were filtered and decanted, the poultry oils were dried in a water bath at 70 °C for 2 h. Thereafter, they were stored in a freezer (- 4 °C) until testing them. 2.3. Fatty acid determination The Meleagris gallopavo and Gallus gallus domesticus fatty acids were indirectly determined using their corresponding methyl esters. Each species was weighed with 0.2 g of oil and saponified for 30 min under reflux with potassium hydroxide solution in methanol, following the methodology described by Hertman and Lago [21]. After appropriate treatment and pH adjustment, the free acids were methylated using methanol through acid catalysis to obtain the respective methyl esters. 2.4. Gas chromatography-mass spectrometry (GC-MS) GC-MS analyses were performed on a Shimadzu GC–MS QP2010 series fitted with a fused silica Rtx-5MS (30 m × 0.25 mm I.D.; 0.25 m film thickness) capillary column and temperature programmed as follow: 60–240 °C at 3 °C/min, then to 280 °C at 10 °C/minute, ending with 10 min at 280 °C. Helium was the carrier gas with a flow rate of 1.5 mL/min and a split mode ratio of 1:50. The injection port was set at 220 °C. Significant quadrupole MS operating parameters: interface temperature 240 °C; electron impact ionization at 70 eV with scan mass range of 40–350 m/z at a sampling rate of 1.0 scan/s. Injected volume: 1 μL of 5 μg/mL solution in dichloromethane. Constituents were identified by computer search using digital mass spectral data libraries (NIST 08) and by comparison of their authentic mass spectra [22]. The GC-MS analyses revealed peaks corresponding to the elution and molecular mass of saturated and unsaturated fatty components often found in fixed oils. 2.5. Microorganisms The experiments were carried out with Escherichia coli (EC06), Pseudomonas aeruginosa (PA24), Staphylococcus aureus (SA10), Multiresistant Staphylococcus epidermidis (SEMR01) and Proteus mirabilis (PM01) clinical isolates as well as a standard Staphylococcus epidermidis ATCC 12228 (SEATCC) bacterial strain. All strains were maintained on Heart Infusion Agar slants (HIA, Difco). Before the assays, the cells were cultured for 24 h at 37 °C in Brain Heart Infusion (BHI, Difco). The Escherichia coli (EC06), Pseudomonas aeruginosa (PA24) and Staphylococcus aureus (SA10) bacteria came from the Microbiology and Molecular Biology Laboratory of the Regional University of Cariri, (LMBM-URCA); while the Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis (MR01), and Proteus mirabilis (PM01) bacteria were obtained from the Bacteriology Laboratory of the Federal University of Ceará (LB - UFC) (Table 1). Table 1. Strains of bacterial clinical isolates used for testing with their antibiotic resistance and origin profile. Bactéria Origem Perfil de resistencia Escherichia coli 06 Urine culture Cephalothin, cephalexin, cefadroxil, ceftriaxone, cefepime, ampicilin-sulbactam Proteus mirabilis 01 Urine culture Colistin, nalidix acid, Nitrofurantoin, imipenem Pseudomonas aeruginosa 03 Urine culture Amikacin, imipenem, ciprofloxacin, levofloxacin, piperacilin-tazobactam, ceftazidime, merpenem, cefepime. Staphylococcus aureus 10 Rectal swab culture Cephalothin, cephalexin, cefadroxil, ceftriaxone, cefepime, ampicilin-sulbactam Staphylococcus epidermidis 01 Surgical wound Benzylpenicilin; ciprofloxacin; Moxifloxacin; sulfamethoxazole-trimethoprim; gentamicin; norfloxacin Source: Laboratório de Microbiologia e Biologia Molecular - LMBM - Universidade Regional do Cariri-URCA and Laboratório de Microbiologia LB – UFC – Universidade Federal do Ceará. 2.6. Drugs The antibiotics Amoxicillin (União Química Farmacêutica Nacional S/A, Brazil), Oxytetracycline/LA (Zoetis Manufacturing & Research Spain, SL), Amikacin (Sigma Co., St. Louis, USA) and Norfloxacin (Sigma Co., St. Louis, USA) were selected for testing. All of these components were dissolved in distilled water and sterilized before their use. The aforementioned antibiotics were chosen for use in this study because they were used in bacterial diseases affecting domestic animals, whose etiological agents are often the microorganisms mentioned above (item 2.5), and because there are bacterial resistance reports in veterinary medicine on these antibiotics when they are used in domestic animals [10] [23]. 2.7. Initial solution and test solution preparations The Gallus gallus domesticus and Meleagris gallopavo fixed oils were solubilized in 1 mL of dimethyl sulfoxide (DMSO-Merk, Darmstadt, Germany) in the starting solution preparation. Thereby, a concentration of 10 mg/mL was obtained. Then, these solutions were microdiluted in sterile distilled water, reaching a concentration of 1024 μg/mL, reducing the DMSO concentration to below 10% and thus avoiding its possibly toxic effect [24]. 2.8. Minimal Inhibitory Concentration The broth microdilution procedure was adopted [25], where solutions were prepared in eppendorf® type microtubes containing 1 mL of solution with 900 μL of 10% BHI and 100 μL of the bacterial suspension with 106 CFU according to the McFarland scale. The plate was filled numerically by adding 100 μL of this solution into each well in a total of 96 wells, followed by serial microdilution performed with a 100 μL solution of the oil or antibiotics, with varying concentrations from 512 to 8 μg/mL. The plates were then taken to an incubator for 24 h at 37 °C. To demonstrate the MIC of the samples, a resazurin sodium (Sigma) solution in sterile distilled water at the concentration of 0.01% (w/v) was prepared. After incubation, 20 μL of the indicator solution was added into each well and the plates were subjected to an incubation period of 1 h at room temperature. The staining change from blue to pink due to the reduction of the resazurin pH indicated the presence of bacterial growth. The MIC was determined as the lowest concentration in which no growth was observed, which was evidenced by the unaltered blue color [26]. All procedures were performed in quadruplicates. 2.9. Modulation of drug action To verify if the fixed oils could modify the action of the antibiotics against the tested strains, the methodology proposed by Coutinho et al. (2008) [27] was used, where the oil solutions were tested at sub-inhibitory concentrations (MIC/8). Eppendorf® microtubes containing 1.5 mL of 10% BHI solution, 150 μL of the bacterial suspension and 23 μL of the fixed oil were prepared. For the control, eppendorf® microtubes were prepared with 1.5 mL of a solution containing 1350 μL of BHI (10%) and 150 μL of microorganisms in suspension. The plate was filled alphabetically by adding 100 μL of this solution into each well. The antibiotic (100 μL) was added to the first well and serial microdilutions at a 1:1 ratio were performed up to the penultimate cavity. All procedures were performed in quadruplicates. The antibiotics Amikacin, Amoxicillin, Norfloxacin and Oxytetracycline L.A. were evaluated at concentrations ranging from 512 to 0.5 μg/mL. 2.10. Statistical analysis Statistical significance was assessed using a two-way ANOVA (Analysis of Variance) followed by Bonferroni's post hoc test (where p < 0.05 and p < 0.0001 are considered significant and p > 0.05 is not significant) using the software Graph Pad Prism 6.0. 3. Results GC/MS fixed oil analyses allowed the Gallus gallus domesticus and Meleagris gallopavo fatty acid methyl esters to be identified. In the OFGG, 4 chemical constituents were identified. The saturated and unsaturated methyl ester percentages found were of 35.1% and 64.91%, respectively, with linoleic acid (55.35%) as the major constituent. The OFMG had 3 constituents, with 72.29% of them being unsaturated and 27.71% saturated; oleic acid (56.83%) was its major constituent (Table 2). Table 2. Methyl esters identified in the fixed oils Gallus gallus (OFGG) and Meleagris gallopavo (OFMG) with their respective percentages. Componentes Estrutura química OFGG OFMG RIa (%) RIa (%) Hexadecanoic Acid Image 1 27.617 29.24 – – 9,12 – Octadecadienoic acid Image 2 31.018 9.56 31.023 15.46 9 – Octadecenoic acid Image 3 31.103 55.35 31.107 56.83 Tridecanoic acid Image 4 31.484 5.86 27.627 27.71 Satured Esters 35.10% 27.71% Insatured Esters 64.90% 72.29% Total 100% 100% The resazurin added to the bacterial culture wells established a MIC of >512 μg/mL for the OFGG and OFMG. The MIC results for both species demonstrated that although OFGG and OFMG are indicated in traditional veterinary medicine for the treatment of infections, they have not shown any clinically relevant antibacterial activity. In the antibiotic modulation assays (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6), when associated with Amikacin, the OFGG presented synergism for EC 06, SE ATCC, SE 01 and PM 01. When in association with Amoxicillin, synergism for EC 06 and SE 01 was observed. For Norfloxacin, synergism occurred with the OFGG against SE 01 and PA 24, as well as in association with Oxytetracycline against EC 06. Antagonistic effects occurred for the OFGG and Amoxicillin association against PA 24 and PM 01, as well as with Amikacin against PA 24 and Oxytetracycline against PM 01. Fig. 1 Download high-res image (258KB)Download full-size image Fig. 1. Effect of the modulation of fixed oils of Gallus gallus (OFGG) and Meleagris gallopavo (OFMG) in combination with antibiotics (Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline) against Escherichia coli (strains EC06). The columns represent the Minimal Inhibitory Concentration (MIC) expressed in Geometric Mean (MG) ± Mean Standard Error SEM, analyzed through the two-way ANOVA (Two-Way Analysis of Variance) followed by the Bonferroni test and multiple 't' post hoc. The significance level for rejection of the null hypothesis was p < 0.05 (* - p < 0.05; ** - p < 0.01; *** - p < 0.001; **** - p < 0.0001). Fig. 2 Download high-res image (226KB)Download full-size image Fig. 2. Effect of the modulation of the fixed oils of Gallus gallus (OFGG) and Meleagris gallopavo (OFMG) in association with antibiotics (Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline) against Pseudomonas aeruginosa strains (PA24). The columns represent the Minimal Inhibitory Concentration (MIC) expressed in Geometric Mean (MG) ± Mean Standard Error SEM, analyzed through the two-way ANOVA (Two-Way Analysis of Variance) followed by the Bonferroni test and multiple 't' post hoc. The significance level for rejection of the null hypothesis was p < 0.05 (* - p < 0.05; ** - p < 0.01; *** - p < 0.001; **** - p < 0.0001). Fig. 3 Download high-res image (254KB)Download full-size image Fig. 3. Effect of the modulation of fixed oils of Gallus gallus (OFGG) and Meleagris gallopavo (OFMG) in combination with antibiotics (Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline) against Proteus mirabilis strains (PM01). The columns represent the Minimal Inhibitory Concentration (MIC) expressed in Geometric Mean (MG) ± Mean Standard Error SEM, analyzed through the two-way ANOVA (Two-Way Analysis of Variance) followed by the Bonferroni test and multiple 't' post hoc. The significance level for rejection of the null hypothesis was p < 0.05 (* - p < 0.05; ** - p < 0.01; *** - p < 0.001; **** - p < 0.0001). Fig. 4 Download high-res image (245KB)Download full-size image Fig. 4. Effect of the modulation of fixed oils of Gallus gallus (OFGG) and Meleagris gallopavo (OFMG) in combination with antibiotics (Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline) against Staphylococcus aureus strains (SA10). The columns represent the Minimal Inhibitory Concentration (MIC) expressed in Geometric Mean (MG) ± Mean Standard Error SEM, analyzed through the two-way ANOVA (Two-Way Analysis of Variance) followed by the Bonferroni test and multiple 't' post hoc. The significance level for rejection of the null hypothesis was p < 0.05 (* - p < 0.05; ** - p < 0.01; *** - p < 0.001; **** - p < 0.0001). Fig. 5 Download high-res image (196KB)Download full-size image Fig. 5. Effect of modulation of fixed oils of Gallus gallus (OFGG) and Meleagris gallopavo (OFMG) in combination with antibiotics (Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline) against Staphylococcus epidermidis strains ATCC 12228 (SEATTC). The columns represent the Minimal Inhibitory Concentration (MIC) expressed in Geometric Mean (MG) ± Mean Standard Error SEM, analyzed through the two-way ANOVA (Two-Way Analysis of Variance) followed by the Bonferroni test and multiple 't' post hoc. The significance level for rejection of the null hypothesis was p < 0.05 (* - p < 0.05; ** - p < 0.01; *** - p < 0.001; **** - p < 0.0001). Fig. 6 Download high-res image (275KB)Download full-size image Fig. 6. Effect of the modulation of the fixed oils of Gallus gallus (OFGG) and Meleagris gallopavo (OFMG) in combination with antibiotics (Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline) against the Multiresistant Staphylococcus epidermis strains (SEMR01). The columns represent the Minimal Inhibitory Concentration (MIC) expressed in Geometric Mean (MG) ± Mean Standard Error SEM, analyzed through the two-way ANOVA (Two-Way Analysis of Variance) followed by the Bonferroni test and multiple 't' post hoc. The significance level for rejection of the null hypothesis was p < 0.05 (* - p < 0.05; ** - p < 0.01; *** - p < 0.001; **** - p < 0.0001). When associated with Amikacin, the OFMG presented synergism against SA 10, PM 01 and SE 01. Other synergistic effects still occurred in the association of the OFMG with Norfloxacin for EC 06 and SE 01 and with Amoxicillin against SE 01. Antagonistic effects occurred for this fixed oil when associated with: Amikacin against EC 06, PM 01, PA 24; Oxytetracycline against PM 01 (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6). 4. Discussion The fatty acids in OFGG and OFMG are similar. However, the proportion between saturated and unsaturated fatty acids is different, where the OFGG presents 35.1% of saturated fatty acids and 64.91% of unsaturated fatty acids, the OFMG presents 72.29% unsaturated and 27.71% saturated fatty acids. In animal fats, mainly saturated fatty acids are found [28]. A basic poultry diet is composed of vegetables, where today the supply of animal-derived ingredients is prohibited [29]. The percentage and types of fatty acids present in the poultry's body fat may vary according to the amount and type of lipid source provided in poultry feed [30]. The major components found in this study are different, with linoleic acid being the main constituent of OFGG and oleic acid of OFMG, where this variation may be due to a different lipid diet provided for the species evaluated here. Previous studies investigating the antibacterial activity of animal fats as well as the results of the present study demonstrate that the composition of saturated and unsaturated fatty acids may vary according to the species analyzed [13], [28]. Our data indicates that in the antibacterial activity analysis, the OFGG and OFMG when used in isolation, did not demonstrate any relevant activity against the strains tested here. The results indicate that there is no pharmacological basis for the use of OFMG and OFGG to treat bacterial diseases in domestic animals that have the bacterial strains used as etiological agents. The modulatory action of the fixed oils mentioned above when associated with antibiotics commonly used to treat domestic animals were also investigated. OFGG showed a synergistic effect when associated with the antibiotics Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline. In another study, Coutinho et al. (2014) [31] had already found that OFGG had a modulatory effect against Staphylococcus aureus when associated with aminoglycosides. However, due to the absence of the OFGG chemical composition in their study, it is impossible to associate our results with respect to the compounds. Similar to OFGG, OFMG also demonstrated a synergistic effect in association with the antibiotics Amikacin, Amoxicillin, and Norfloxacin. Several chemical compounds, whether synthetic or from natural sources, have direct activity against several bacterial species increasing the specific activity of the antibiotic, reverting the bacterial natural resistance to specific antibiotics, eliminating plasmids and inhibiting the active effluent of antibiotics through the plasma membrane. The potentiation of antibacterial activity or antibiotic resistance reversal permits the categorization of these compounds as modifiers of antibiotic activity [32,33]. Several mechanisms may be involved in bacterial growth inhibition by the fixed oil, where this inhibitory effect may be partly attributed to the inhibitory nature of some components. As a result, these components may exhibit greater interaction with the cell membrane lipid bilayer, affect the respiratory chain and energy production, or even leave the cell more permeable to antibiotics, thus providing interruption of cellular activity. Several components from the fixed oils can permeabilize cell membranes, increasing antibiotic penetration. Enzymatic action interference can also be considered a potential mechanism of action. All the mechanisms of action mentioned herein can be obtained by the associating of antibiotics with fixed oils at a sub-inhibitory concentration administered directly into the culture medium [34]. This study corroborates with previous studies where, even when a direct fixed oil antibacterial activity does not occur, the association between antibiotics and zootherapy fixed oils can inhibit bacterial action against certain strains [13,35,36]. Other reports on the association of fixed oils and antibiotics against bacteria were observed all indicating a potentiation of antibiotic activity due to a greater membrane permeability, which is why we conducted the birds' fixed oil modulation tests, although these did not present direct antibacterial action [34,37]. The fixed oil from the two bird species tested here were capable of modulating the action of essentially the same antibiotics. However, the response from each of the fixed oils against certain strains was different. The major fatty acids from the two bird species were unsaturated fatty acids. However, these were different components with an Omega-6 being the major component of the OFGG and an omega-9 of the OFMG, corroborating with the analysis by Huang et al. (2010) [38], where the authors demonstrated that certain fatty acids can present greater antibacterial activity over specific strains. Granowitz and Brown [39] and Ferreira et al. [13] report that antagonistic effects may also occur when we associate natural products with antibiotics, where this effect is attributed to the occurrence of a mutual chelation. Similar effects may be attributed here to antagonistic effects having occurred with the OFGG and OFMG. 5. Conclusions The results presented here indicate that OFGG and OFMG did not present clinically relevant antibacterial activity against the bacterial strains used when tested in isolation. However, when poultry fixed oils were tested in combination with antibiotics, OFGG modulated the action of the antibiotics Amikacin, Amoxicillin, Norfloxacin, and Oxytetracycline, while OFMG modulated the action of Amikacin, Amoxicillin, and Norfloxacin. This study examined the effects of the OFGG and OFMG in vitro, thus additional studies with living organisms should be performed to verify systemic efficacy and elucidate additional information related to clinical issues which occur in veterinary medicine. Conflicts of interest The authors declare that they have no competing interests regarding the publication of this article. Acknowledgment The authors would like to thank the Cearense Foundation for Scientific and Technological Development Support (FUNCAP) for Diógenes de Queiroz Dias doctoral scholarship and the National Research Council (CNPq) for the research productivity subsidy of Waltécio de Oliveira Almeida, process: 302429/2015-8 and Débora Castelo Branco de Souza Colares Maia of the LB-UFC for giving the strains of S. epidermidis and Proteus mirabilis. Appendix A. Supplementary data The following is the supplementary data related to this article: Research data for this article Data not available / No data was used for the research described in the article About research data References [1] C.M. McCorkle An introduction to ethnoveterinary research and development J. Ethnobiol., 6 (1) (1986), pp. 129-149 View Record in Scopus [2] R.R.N. Alves, I.L. Rosa Why study the use of animal products in traditional medicines? J. Ethnobiol. Ethnomed., 1 (5) (2005), pp. 1-5 View Record in Scopus [3] M.V.B. 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