Antibacterial activity of amoxicillin in vitro and its oral bioavailability in broiler chickens under the influence of 3 water sanitizers

Antibacterial activity of amoxicillin in vitro and its oral bioavailability in broiler chickens... ABSTRACT The interaction of 3 water sanitizers (sodium hypochlorite, iodine-polyvinylpyrrolidone, and citrate) utilized in poultry production on antibacterial activity and bioavailability of amoxicillin trihydrate (AMX) were studied. Sanitizers were mixed with AMX in prepared water, the resulting substances were regarded as amoxicillin-sanitizer products (ASP). First, the in vitro antibacterial activity of each ASP was compared to that of AMX. Then, pharmacokinetics (PK) of ASP and AMX diluted in prepared water, were carried out in broiler-chickens. Amoxicillin or ASP (20 mg/kg) from different concentrations of sanitizers was directly placed into the chicken's crop and blood samples were taken. Basic PK parameters were obtained. Serum activity/concentrations of AMX were assessed by agar diffusion and corroborated with high performance liquid chromatography. Results show that ASP of AMX/sodium hypochlorite decrease both, the antimicrobial activity of in vitro AMX and its relative bioavailability (Fr) assessed with the maximum serum concentration (Cmax), the area under the concentration-time curve, and the mean residence time (MRT) (3.80 μg/mL, 2.70 μg/mL·h, and 0.59 h, respectively), compared to the AMX administered alone (12.54 μg/mL, 44.02 μg/mL·h, and MRT 2.78 h). ASP from amoxicillin/ionophore, reduced the Cmax (10.62 μg/mL), Fr (94.67%), and MRT (2.07 h), at the highest tested concentrations. In contrast, the 2 highest concentrations of the citrate sanitizer increased the Cmax (15.07 and 15.47 μg/mL), Fr (119 and 132%), and MRT (3.32 and 4.06 h) and their in vitro antimicrobial activity. Interactions between the tested water sanitizers and AMX modify the Cmax, Fr, MRT of the latter, altering the PK/pharmacodymanic ratios for a time-dependent antibiotic. Results also reveal that the use of amoxicillin trihydrate administered through the drinking water does not meet the required PK/pharmacodymanic ratios. Thus, it is here postulated that this antibiotic should be administered at least twice a day and that its interaction with water sanitizers should be considered. INTRODUCTION Rational use of antibacterial drugs has been highlighted and considered a worldwide priority (Kathleen and Van Dijr, 2011). Therefore, antimicrobial dosing schedules and methods should be revised in order to comply with desired pharmacokinetics/pharmacodynamic (PK/PD) ratios appointed for veterinary medicine (McKellar et al., 2004). In poultry medicine, well planned dosing protocols and adequate equipment are followed to attempt the accurate delivery of antibacterial drugs to the flock, and eventually to each chicken (Vermeulen, 2002). The preferred manner for administering antibacterial drugs is through their drinking water. Besides its easy administration, this route and vehicle are chosen because it is believed that for most antibacterial drugs, better bioavailability (F) is obtained (Esmail, 1996). Even so, considerable variations in F among flocks and even among individuals should be expected. Among other causes, drinking habits in the flock of each chicken, are accountable for these variations (Bailey, 1999; Ribeiro et al., 2004; Ziaei et al., 2011). Also, quality of the drinking water has been shown to modify F of antibacterial drugs in chicken (Bell and Weaver, 2002; Sumano et al., 2004; Fairchild et al., 2006) as it also happens with inadequate plumbing and the way drinkers are positioned in the chicken coop (NRC, 1994; May et al., 1997; Quilumba et al., 2015). One further aspect that may also induce variations in F of antibacterial drugs, when they are given to poultry through drinking water, is the concurrent use of water sanitizers (Vermeulen, 2002). The possible interaction of a sanitizer added to drinking water with a given antibacterial drug has not been addressed in formal literature. The efficacy of most water-sanitizing agents is based on their high reactivity with chemical and organic entities (Kahrs, 1995); therefore it is reasonable to think that they can react with a given antibacterial drug when concurrently added to the water source, and such interactions may consequently modify F (Esmail, 1996). Iodine-polyvinylpyrrolidone, sodium hypochlorite preparations, and a citrate-based sanitizer are often used in poultry medicine (Acero et al., 2010; Hua et al., 2015). However, the consequences of the interaction between these chemicals and many antimicrobial drugs, in terms of loss or increase of in vitro antibacterial activity and F, await characterization. Amoxicillin (AMX) is a semisynthetic β-lactam antibiotic belonging to the aminopenicillin group. It possesses a broad antimicrobial spectrum, a reasonably good absorption rate, and considerable tissue penetration (Anadón et al., 1996). Additionally, it exhibits low toxicity compared to other antimicrobials (Krasucka et al., 2015) and has a low cost (Sun et al., 2016). It is used in poultry farming (Sun et al., 2016) and is commercially available in México (SAGARPA, 2012), Latin America (SENASA, 2015; ICA, 2017; MIDA, 2017; SAG, 2017), Canada (Agunos et al., 2012; Sun et al., 2016), and the European Union (Table 1). According to the sixth European Surveillance of Veterinary Antimicrobial Consumption, penicillins are the second most used group of antimicrobials in production animals (25.5%), only surpassed by tetracyclines (33.4%); and from these, AMX and ampicillin are the most consumed and are primarily administered orally (EMA, 2016). However, like all beta-lactam antibacterials, the amoxicillin molecule is highly susceptible to undergo chemical reactions (Acero et al., 2010). Therefore, the aim of this study was to assess whether or not the interaction of AMX with sodium hypochlorite, iodine-polyvinylpyrrolidone, and a citrate-based disinfectant results in the modification of the antibacterial activity of the drug in vitro, and whether the concurrent administration of these water sanitizers with AMX alters its relative bioavailability (Fr) in broiler chickens. Table 1. Examples of amoxicillin trihydrate preparations available for administration through drinking water for poultry medicine in different parts of the world. Country/ No. of region approved products Source Argentina 6 Servicio Nacional de Sanidad y calidad agroalimentaria: Servicio Nacional de Sanidad y calidad agroalimentaria: http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales Canada 3 Health Canada: https://cal.naccvp.com/search/main?query=amoxicillin Chile 2 Ministerio de agricultura: http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp Colombia 5 Instituto Colombiano Agropecuario: http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx Costa Rica 2 Servicio Nacional de Salud Animal, Costa Rica: Servicio Nacional de Salud Animal, Costa Rica: http://www.senasa.go.cr/medivet/busque_avanzada.aspx Germany 7 Pharmanet in cooperation with the Federal ministry of health (Bundesministerium für Gesundheit): https://www.pharmnet-bund.de/static/de/suche/?q=amoxicillin#&query=amoxicillin&sortdir=asc Italy 15 Ministero della Salute:https://www.vetinfo.sanita.it/j6_prontuario/farmaci/public/prodottomd/;jsessionid=95E50FE72628918695E06827886D9A8E-n1.tomcatprod2 Mexico 9 SAGARPA: SAGARPA: http://dev.sagarpa.gob.mx/tramitesyServicios/Lists/Direccin%20General%20de%20Salud%20Animal/Attachments/13/SENASICA%2001-024.pdf Spain 22 Agencia Española de Medicamentos y Productos Sanitarios: https://cimavet.aemps.es/cimavet/medicamentos.do UK 12 Veterinary Medicines Directorate service: http://www.vmd.defra.gov.uk/ProductInformationDatabase/Default.aspx Country/ No. of region approved products Source Argentina 6 Servicio Nacional de Sanidad y calidad agroalimentaria: Servicio Nacional de Sanidad y calidad agroalimentaria: http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales Canada 3 Health Canada: https://cal.naccvp.com/search/main?query=amoxicillin Chile 2 Ministerio de agricultura: http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp Colombia 5 Instituto Colombiano Agropecuario: http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx Costa Rica 2 Servicio Nacional de Salud Animal, Costa Rica: Servicio Nacional de Salud Animal, Costa Rica: http://www.senasa.go.cr/medivet/busque_avanzada.aspx Germany 7 Pharmanet in cooperation with the Federal ministry of health (Bundesministerium für Gesundheit): https://www.pharmnet-bund.de/static/de/suche/?q=amoxicillin#&query=amoxicillin&sortdir=asc Italy 15 Ministero della Salute:https://www.vetinfo.sanita.it/j6_prontuario/farmaci/public/prodottomd/;jsessionid=95E50FE72628918695E06827886D9A8E-n1.tomcatprod2 Mexico 9 SAGARPA: SAGARPA: http://dev.sagarpa.gob.mx/tramitesyServicios/Lists/Direccin%20General%20de%20Salud%20Animal/Attachments/13/SENASICA%2001-024.pdf Spain 22 Agencia Española de Medicamentos y Productos Sanitarios: https://cimavet.aemps.es/cimavet/medicamentos.do UK 12 Veterinary Medicines Directorate service: http://www.vmd.defra.gov.uk/ProductInformationDatabase/Default.aspx View Large Table 1. Examples of amoxicillin trihydrate preparations available for administration through drinking water for poultry medicine in different parts of the world. Country/ No. of region approved products Source Argentina 6 Servicio Nacional de Sanidad y calidad agroalimentaria: Servicio Nacional de Sanidad y calidad agroalimentaria: http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales Canada 3 Health Canada: https://cal.naccvp.com/search/main?query=amoxicillin Chile 2 Ministerio de agricultura: http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp Colombia 5 Instituto Colombiano Agropecuario: http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx Costa Rica 2 Servicio Nacional de Salud Animal, Costa Rica: Servicio Nacional de Salud Animal, Costa Rica: http://www.senasa.go.cr/medivet/busque_avanzada.aspx Germany 7 Pharmanet in cooperation with the Federal ministry of health (Bundesministerium für Gesundheit): https://www.pharmnet-bund.de/static/de/suche/?q=amoxicillin#&query=amoxicillin&sortdir=asc Italy 15 Ministero della Salute:https://www.vetinfo.sanita.it/j6_prontuario/farmaci/public/prodottomd/;jsessionid=95E50FE72628918695E06827886D9A8E-n1.tomcatprod2 Mexico 9 SAGARPA: SAGARPA: http://dev.sagarpa.gob.mx/tramitesyServicios/Lists/Direccin%20General%20de%20Salud%20Animal/Attachments/13/SENASICA%2001-024.pdf Spain 22 Agencia Española de Medicamentos y Productos Sanitarios: https://cimavet.aemps.es/cimavet/medicamentos.do UK 12 Veterinary Medicines Directorate service: http://www.vmd.defra.gov.uk/ProductInformationDatabase/Default.aspx Country/ No. of region approved products Source Argentina 6 Servicio Nacional de Sanidad y calidad agroalimentaria: Servicio Nacional de Sanidad y calidad agroalimentaria: http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales Canada 3 Health Canada: https://cal.naccvp.com/search/main?query=amoxicillin Chile 2 Ministerio de agricultura: http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp Colombia 5 Instituto Colombiano Agropecuario: http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx Costa Rica 2 Servicio Nacional de Salud Animal, Costa Rica: Servicio Nacional de Salud Animal, Costa Rica: http://www.senasa.go.cr/medivet/busque_avanzada.aspx Germany 7 Pharmanet in cooperation with the Federal ministry of health (Bundesministerium für Gesundheit): https://www.pharmnet-bund.de/static/de/suche/?q=amoxicillin#&query=amoxicillin&sortdir=asc Italy 15 Ministero della Salute:https://www.vetinfo.sanita.it/j6_prontuario/farmaci/public/prodottomd/;jsessionid=95E50FE72628918695E06827886D9A8E-n1.tomcatprod2 Mexico 9 SAGARPA: SAGARPA: http://dev.sagarpa.gob.mx/tramitesyServicios/Lists/Direccin%20General%20de%20Salud%20Animal/Attachments/13/SENASICA%2001-024.pdf Spain 22 Agencia Española de Medicamentos y Productos Sanitarios: https://cimavet.aemps.es/cimavet/medicamentos.do UK 12 Veterinary Medicines Directorate service: http://www.vmd.defra.gov.uk/ProductInformationDatabase/Default.aspx View Large MATERIAL AND METHODS This study was conducted in 2 phases. The first phase (antibacterial activity) was carried out in order to identify possible modifications of the in vitro antimicrobial activity of AMX in the presence of a sanitizer; whereas, the second phase (bioavailability) was designed to identify if the variations in Fr, maximum serum concentration (Cmax), mean residence time (MRT), and time in which the serum activity/concentrations were at or above the minimum inhibitory concentration (MIC) value (T ≥ MIC), could be observed in chickens dosed with various amoxicillin-sanitizer products (ASP). Antibacterial Activity Water sanitizers were mixed with AMX in prepared water, characterized by originating from any type of water supply (including municipal water); and additionally, subjected to any treatment that could modify the original water in order to comply with the chemical and microbiological safety requirements for pre-packaged water and commercialized as purified or drinking water (WHO, 1999; Sumano and Gutiérrez, 2008). It has a pH 8 and a resistivity of 200 Ω•m, as analyzed with an Oaklon multiparametric apparatus (Testr 35 series, Vernon Hills, USA) at 20°C. In this study, the bacterial charge was always smaller than 50 UFC/mL E. coli. After 30 min, the resulting substances were regarded as ASP. This ASP was inoculated into 80 agar wells prepared in a large plate, as suggested by Pillai et al. (2005) and based on directions of CLSI (2012). Serial dilutions from 0.05 to 50 μg/mL were prepared to interact with free chlorine (5 to 40 μg/mL) from a commercially available preparation of Na-hypochlorite (pH 11) (Hipoclorito de sodio al 13%; Organizacion Química de Calidad, Morelos, México), with free iodine (8 to 64 μg/mL) from iodine-polyvinylpyrrolidone (pH 3.5) (Iodosol 50; Loeffler, CDMX, México) and with a Citrex sanitizer (Citrex, Inc., USA), manufactured with an undisclosed mixture of orange and grapefruit seed extracts and citric acid (2 to 16 mg/mL) (pH 2.3). The tested ranges covered the maximum concentrations recommended by manufacturers and were approximately 8 times more concentrated. Control wells included AMX alone (pH 5.67) (Amoxi-40; FIORI, Querétaro, México) and the corresponding sanitizer, repeating the same serial concentrations as above. The tested bacteria were Bacillus subtilis ATCC 6633. Bioavailability This study phase complied with the Mexican regulations for use of experimental animals, as laid out by the Universidad Nacional Autónoma de Mexico (UNAM) and Mexican prescripts in NOM-062-ZOO-1999. Written permission was granted on 2015 January 13. A total of 1,365 healthy, 6 wk old, Cobb 500 female chickens, weighing 2.5 kg ± 20 g SD were included in this trial. Cobb chickens were managed using the recommended guidelines for this genetic-line (Cobb-Vantress Inc., 2005), having a population density of 30 kg of biomass/m2, nipple-type drinkers (10 chickens/nipple), and manual hopper feeders (50 chickens/feeder). They were placed in an experimental chicken coop with concrete floor (8 wide × 14.5 m long × 4 m high), covered with clean corn-straw bedding, in groups of 35; each group had 3 replicates (105 chickens per group), separated by a wire mesh. The room temperature was kept at 21°C and a mechanic negative-pressure ventilation system was used. The birds were fed with a balanced diet, based on National Research Council requirements (1994). Four Fr studies were carried out for each ASP with 3 replicates per group (see Table 2). Also an Fr study with 3 replicates was performed, administering only AMX in free-sanitizer drinking water that can be described as prepared water. Table 2. Description of groups in which bioavailability of amoxicillin was assessed after oral dosing the product(s) of the interaction of amoxicillin plus and sanitizer. Description Inclusion percentage of Group (mg/kg body weight) amoxicillin and disinfectant in water A Amoxicillin 20 Amoxicillin 5 ACl+ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.002 chlorine 0.00051 ACl++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.004 chlorine 0.0010 ACl+++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.008 chlorine 0.0020 ACl++++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.016 chlorine 0.0040 AI+ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0032 iodine 0.00081 AI++ Amoxicillin 20 plus Amoxicillin 5 plus Iodine 0.0064 iodine 0.0016 AI+++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0128 iodine 0.0032 AI++++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0256 iodine 0.0064 AC+ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 0.8 citric-based 0.21 AC++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 1.6 citric-based 0.4 AC+++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 3.2 citric-based 0.8 AC++++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 6.4 citric-based 1.6 Description Inclusion percentage of Group (mg/kg body weight) amoxicillin and disinfectant in water A Amoxicillin 20 Amoxicillin 5 ACl+ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.002 chlorine 0.00051 ACl++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.004 chlorine 0.0010 ACl+++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.008 chlorine 0.0020 ACl++++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.016 chlorine 0.0040 AI+ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0032 iodine 0.00081 AI++ Amoxicillin 20 plus Amoxicillin 5 plus Iodine 0.0064 iodine 0.0016 AI+++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0128 iodine 0.0032 AI++++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0256 iodine 0.0064 AC+ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 0.8 citric-based 0.21 AC++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 1.6 citric-based 0.4 AC+++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 3.2 citric-based 0.8 AC++++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 6.4 citric-based 1.6 1Highest recommended concentration as suggested by the manufacturer View Large Table 2. Description of groups in which bioavailability of amoxicillin was assessed after oral dosing the product(s) of the interaction of amoxicillin plus and sanitizer. Description Inclusion percentage of Group (mg/kg body weight) amoxicillin and disinfectant in water A Amoxicillin 20 Amoxicillin 5 ACl+ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.002 chlorine 0.00051 ACl++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.004 chlorine 0.0010 ACl+++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.008 chlorine 0.0020 ACl++++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.016 chlorine 0.0040 AI+ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0032 iodine 0.00081 AI++ Amoxicillin 20 plus Amoxicillin 5 plus Iodine 0.0064 iodine 0.0016 AI+++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0128 iodine 0.0032 AI++++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0256 iodine 0.0064 AC+ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 0.8 citric-based 0.21 AC++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 1.6 citric-based 0.4 AC+++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 3.2 citric-based 0.8 AC++++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 6.4 citric-based 1.6 Description Inclusion percentage of Group (mg/kg body weight) amoxicillin and disinfectant in water A Amoxicillin 20 Amoxicillin 5 ACl+ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.002 chlorine 0.00051 ACl++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.004 chlorine 0.0010 ACl+++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.008 chlorine 0.0020 ACl++++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.016 chlorine 0.0040 AI+ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0032 iodine 0.00081 AI++ Amoxicillin 20 plus Amoxicillin 5 plus Iodine 0.0064 iodine 0.0016 AI+++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0128 iodine 0.0032 AI++++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0256 iodine 0.0064 AC+ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 0.8 citric-based 0.21 AC++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 1.6 citric-based 0.4 AC+++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 3.2 citric-based 0.8 AC++++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 6.4 citric-based 1.6 1Highest recommended concentration as suggested by the manufacturer View Large Each chicken was individually weighed and fasted for 2 h before receiving 1 of the ASP or AMX, as a single oral bolus dose by means of a plastic cannula (BD Insyte; USA) (1.7 mm by 7 cm) attached to a syringe, directed into the crop. Once the cannula was ensured to be properly placed, the experimental solution was slowly delivered. In all cases, the dose was 20 mg/kg of AMX alone or as ASP, and the volume was adjusted to deliver 1 mL of AMX or ASP per chicken. After treatment, blood samplings were taken at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h, using 5 randomly chosen chickens per sampling time and drawing 3 mL of blood per chicken. Each bird was bled only twice. In order to achieve a close timing interval between the drug administration and blood sampling from the wing or jugular vein, technical assistance and clock-watch timing were ensured. Thus, differences between the targeted and the actual blood sampling times were never more than 5 min. Blood samples were immediately centrifuged and approximately 1.0 mL of serum was recovered, identified, and frozen until analyzed. Serum AMX activity/concentrations were determined through the modified agar diffusion analysis, described by Bennet et al. (1966) using Bacillus subtilis (ATCC 6633) as the test microorganism, and then corroborated by high performance liquid chromatography as described by Krasucka et al. (2015). For the modified agar diffusion analysis, the intra-assay variance coefficient was <6.6 and the inter-assay error was <7.4. The analytical assay was linear over a concentration range of 0.05 to 10 μg/mL, with a recovery percentage of 90 ± 2 and a correlation coefficient (r) of 0.96. The detection limit was 0.05 μg/mL, whereas the quantification limit was 0.01 μg/mL. For the high performance liquid chromatography analysis, the intra-assay variance coefficient was <1.7 and the inter-assay error was <1.6. The analytic assay was linear over a concentration range of 0.01 to 15 μg/mL. The mean ± 1 SD recovery was 94 ± 3% (r = 0.98). The detection limit was 0.003 μg/mL, whereas the quantification limit was 0.01 μg/mL. Compliance between both methods for determining AMX serum concentrations was carried out using the amoxicillin-spiked poultry serum samples processed by the 2 analytical techniques. Subtraction of the recover percentages revealed an error of no more than 12.4%. The serum concentrations of AMX vs. time relationships were analyzed using compartmental PK through the software from PKAnalyst (MicroMath, PKAnalyst for Windows. 1995. Version 1.1. MicroMath Inc., St. Louis, MO, USA). Best fit was obtained in model 5 (r < 0.95) with the following general formula: \begin{eqnarray*} {\rm{concentration}} \ ({\rm{time}}) \ &=& \ \frac{{{\rm{Dose}} \ * \ {{\rm{K}}_{{\rm{AE}}}} \ * \ {\rm{time}}}}{{{\rm{Volume}}}}\,{{\rm{e}}^{ - {{\rm{K}}_{{\rm{AE}}}}}}\nonumber\\ && * \, {\rm{time}} \end{eqnarray*} The following PK parameters were obtained: elimination half-life (T½β); Cmax; time to reach Cmax (Tmax): area under the serum concentrations vs. time curve (AUC); area under the moment curve (AUMC); and mean residence time (MRT). Statistical Analysis Antibacterial Activity The obtained data were processed through a generalized linear model (GzLM) (Garson, 2013) for a continuous variable, expressed as follows: \begin{equation*} {{\rm{\eta }}_{\rm{I}}} = {{\rm{\beta }}_0} + {\beta _1}{X_i}_1 + {\beta _2}{X_{j2}} + {\beta _3}{X_1}{X_2} \end{equation*} where ηi = linear predictor of inhibition zone; β0 = intersection; β1 = regression coefficient for the antibiotic X1; β2 = regression coefficient for sanitizer X2; β3 = regression coefficient of the interaction between the different concentrations of antibiotic and sanitizer X1X2. Then, this was analyzed by the maximum likelihood method. Marginal means of interaction were calculated between the different levels of interaction of the antibiotic and each sanitizer (X1X2). Using these multiple comparisons, a Bonferroni procedure was performed. The accepted significance level was P < 0.05. All data analyses were performed using the SPSS statistical package (IBM SPSS, Statistics for Windows. 2011. Version 20.0. IBM Corp. Armonk, NY). Bioavailability Mean value of AMX serum concentrations vs. time from all groups were analyzed by means of a Shapiro–Wilk test (Shapiro and Wilk, 1965), in order to test for normal distribution; and PK parameters with normal distribution using a general linear model as follows: \begin{equation*} Yij = \mu + Di + eij \end{equation*} where Yij = individual pharmacokinetic parameter value in the ith dilution of sanitizer; μ = general mean; Di = dilution of the sanitizer (i = 1, 2, 3, 4, 5); eij = random standard error N (μ, σe2). Bonferroni multiple comparison tests for marginal means and standard errors were adjusted for the considered model; these were performed with a significance level P < 0.05. The model was analyzed by means of least squares, using the SPSS statistical package. RESULTS Antibacterial Activity Table 3 shows the in vitro antibacterial concentration/activity as measured by the inhibition zones, when allowing the interaction of AMX with chlorine, citric-based sanitizer, or iodine. As chlorine sanitizer concentrations increased, the antibacterial action of the ASP diminished in a linear manner (YIJ = 4.35 + 0.047b1−0.035b2); where YIJ: inhibition halos; b1: AMX; b2: chlorine; with an r2 = 0.73, F2, 79 = 105.94; P = 0.0001. The ASP of the AMX-citric-based sanitizer, showed an increase in the antibacterial activity with the highest sanitizer concentrations (50 and 5 μg/mL) (P < 0.05 in both cases). In contrast, the highest concentration of the iodine-based sanitizer (64 μg/mL), diminished the antibacterial activity of the ASP when AMX was tested at 50 and 5 μg/mL (P < 0.05 in both cases). Table 3. Antibacterial in vitro activity of different amoxicillin and water sanitizer combinations. Arithmetic mean ± SD of inhibition zones (mm). Amoxicillin Disinfectant 50 μg/mL 5 μg/mL 0.5 μg/mL 0.05 μg/mL 0 μg/mL Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Chlorine 0.0 μg/mL 36.47a ± 0.68 28.29a ± 0.30 22.77a ± 0.89 15.98a ± 0.26 0.00a ± 0.00 5.0 μg/mL 6.96b ± 0.35 4.78b ± 0.69 4.60b ± 0.68 4.50b ± 0.72 10.06b ± 0.80 10.0 μg/mL 6.32b,c ± 0.42 4.33b–d ± 0.42 3.59b–d ± 0.34 3.05c–e ± 0.51 14.11c ± 0.42 20.0 μg/mL 5.71c ± 0.35 4.02b,d ±0.25 3.41c ± 0.54 2.51d,e ± 0.46 17.42d ± 0.84 40.0 μg/mL 4.41d ± 0.29 3.45 c,d ± 0.46 2.97 c,d ± 0.07 2.49e ± 0.67 22.11e ± 0.49 Citrate-based sanitizer 0.0 mg/mL 35.23a ± 0.28 28.29a ± 0.44 22.77a ± 0.76 15.98a ± 1.07 0.00a ± 0.00 2.0 mg/mL 36.08b ± 0.69 28.96a,d ± 0.65 21.61a ± 0.69 14.83a–c ± 0.47 8.74b ± 0.51 4.0 mg/mL 37.29c ± 0.45 29.26a,d ± 0.75 21.93a ± 0.64 14.95a,b ± 0.16 13.65c ± 0.61 8.0 mg/mL 37.65c ± 0.49 29.32b,d ± 0.39 22.04a ± 0.47 15.69a–c ± 0.64 15.22d ± 0.35 16.0 mg/mL 38.46c ± 0.55 30.85c ± 0.66 22.71a ± 0.57 16.75a ± 0.63 17.14e ± 0.44 Iodine 0.0 μg/mL 35.23a ± 0.56 28.29a ± 0.83 22.77a ± 0.54 15.98a ± 0.21 0.00a ± 0.00 8.0 μg/mL 35.25a ± 0.29 28.35a ± 0.35 23.55a ± 0.42 16.12a ± 0.31 14.25b ± 0.28 16.0 μg/mL 35.18a ± 0.66 28.31a ± 0.22 23.11a ± 0.57 15.83a ± 0.56 15.54c ± 0.68 32.0 μg/mL 34.99a ± 0.65 28.40a ± 0.41 22.68a ± 0.47 15.72a ± 0.48 16.92d ± 0.41 64.0 μg/mL 33.78b ± 0.44 26.96b ± 0.51 22.46a ± 0.32 15.66a ± 0.73 19.20e ± 0.38 Amoxicillin Disinfectant 50 μg/mL 5 μg/mL 0.5 μg/mL 0.05 μg/mL 0 μg/mL Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Chlorine 0.0 μg/mL 36.47a ± 0.68 28.29a ± 0.30 22.77a ± 0.89 15.98a ± 0.26 0.00a ± 0.00 5.0 μg/mL 6.96b ± 0.35 4.78b ± 0.69 4.60b ± 0.68 4.50b ± 0.72 10.06b ± 0.80 10.0 μg/mL 6.32b,c ± 0.42 4.33b–d ± 0.42 3.59b–d ± 0.34 3.05c–e ± 0.51 14.11c ± 0.42 20.0 μg/mL 5.71c ± 0.35 4.02b,d ±0.25 3.41c ± 0.54 2.51d,e ± 0.46 17.42d ± 0.84 40.0 μg/mL 4.41d ± 0.29 3.45 c,d ± 0.46 2.97 c,d ± 0.07 2.49e ± 0.67 22.11e ± 0.49 Citrate-based sanitizer 0.0 mg/mL 35.23a ± 0.28 28.29a ± 0.44 22.77a ± 0.76 15.98a ± 1.07 0.00a ± 0.00 2.0 mg/mL 36.08b ± 0.69 28.96a,d ± 0.65 21.61a ± 0.69 14.83a–c ± 0.47 8.74b ± 0.51 4.0 mg/mL 37.29c ± 0.45 29.26a,d ± 0.75 21.93a ± 0.64 14.95a,b ± 0.16 13.65c ± 0.61 8.0 mg/mL 37.65c ± 0.49 29.32b,d ± 0.39 22.04a ± 0.47 15.69a–c ± 0.64 15.22d ± 0.35 16.0 mg/mL 38.46c ± 0.55 30.85c ± 0.66 22.71a ± 0.57 16.75a ± 0.63 17.14e ± 0.44 Iodine 0.0 μg/mL 35.23a ± 0.56 28.29a ± 0.83 22.77a ± 0.54 15.98a ± 0.21 0.00a ± 0.00 8.0 μg/mL 35.25a ± 0.29 28.35a ± 0.35 23.55a ± 0.42 16.12a ± 0.31 14.25b ± 0.28 16.0 μg/mL 35.18a ± 0.66 28.31a ± 0.22 23.11a ± 0.57 15.83a ± 0.56 15.54c ± 0.68 32.0 μg/mL 34.99a ± 0.65 28.40a ± 0.41 22.68a ± 0.47 15.72a ± 0.48 16.92d ± 0.41 64.0 μg/mL 33.78b ± 0.44 26.96b ± 0.51 22.46a ± 0.32 15.66a ± 0.73 19.20e ± 0.38 a–eDifferent letters in each column indicate a statistically significant difference (P ≤ 0.05). Note: Multiple comparisons by means of Bonferroni tests were carried out, using marginal means and standard error values as adjusted by the statistical model. View Large Table 3. Antibacterial in vitro activity of different amoxicillin and water sanitizer combinations. Arithmetic mean ± SD of inhibition zones (mm). Amoxicillin Disinfectant 50 μg/mL 5 μg/mL 0.5 μg/mL 0.05 μg/mL 0 μg/mL Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Chlorine 0.0 μg/mL 36.47a ± 0.68 28.29a ± 0.30 22.77a ± 0.89 15.98a ± 0.26 0.00a ± 0.00 5.0 μg/mL 6.96b ± 0.35 4.78b ± 0.69 4.60b ± 0.68 4.50b ± 0.72 10.06b ± 0.80 10.0 μg/mL 6.32b,c ± 0.42 4.33b–d ± 0.42 3.59b–d ± 0.34 3.05c–e ± 0.51 14.11c ± 0.42 20.0 μg/mL 5.71c ± 0.35 4.02b,d ±0.25 3.41c ± 0.54 2.51d,e ± 0.46 17.42d ± 0.84 40.0 μg/mL 4.41d ± 0.29 3.45 c,d ± 0.46 2.97 c,d ± 0.07 2.49e ± 0.67 22.11e ± 0.49 Citrate-based sanitizer 0.0 mg/mL 35.23a ± 0.28 28.29a ± 0.44 22.77a ± 0.76 15.98a ± 1.07 0.00a ± 0.00 2.0 mg/mL 36.08b ± 0.69 28.96a,d ± 0.65 21.61a ± 0.69 14.83a–c ± 0.47 8.74b ± 0.51 4.0 mg/mL 37.29c ± 0.45 29.26a,d ± 0.75 21.93a ± 0.64 14.95a,b ± 0.16 13.65c ± 0.61 8.0 mg/mL 37.65c ± 0.49 29.32b,d ± 0.39 22.04a ± 0.47 15.69a–c ± 0.64 15.22d ± 0.35 16.0 mg/mL 38.46c ± 0.55 30.85c ± 0.66 22.71a ± 0.57 16.75a ± 0.63 17.14e ± 0.44 Iodine 0.0 μg/mL 35.23a ± 0.56 28.29a ± 0.83 22.77a ± 0.54 15.98a ± 0.21 0.00a ± 0.00 8.0 μg/mL 35.25a ± 0.29 28.35a ± 0.35 23.55a ± 0.42 16.12a ± 0.31 14.25b ± 0.28 16.0 μg/mL 35.18a ± 0.66 28.31a ± 0.22 23.11a ± 0.57 15.83a ± 0.56 15.54c ± 0.68 32.0 μg/mL 34.99a ± 0.65 28.40a ± 0.41 22.68a ± 0.47 15.72a ± 0.48 16.92d ± 0.41 64.0 μg/mL 33.78b ± 0.44 26.96b ± 0.51 22.46a ± 0.32 15.66a ± 0.73 19.20e ± 0.38 Amoxicillin Disinfectant 50 μg/mL 5 μg/mL 0.5 μg/mL 0.05 μg/mL 0 μg/mL Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Chlorine 0.0 μg/mL 36.47a ± 0.68 28.29a ± 0.30 22.77a ± 0.89 15.98a ± 0.26 0.00a ± 0.00 5.0 μg/mL 6.96b ± 0.35 4.78b ± 0.69 4.60b ± 0.68 4.50b ± 0.72 10.06b ± 0.80 10.0 μg/mL 6.32b,c ± 0.42 4.33b–d ± 0.42 3.59b–d ± 0.34 3.05c–e ± 0.51 14.11c ± 0.42 20.0 μg/mL 5.71c ± 0.35 4.02b,d ±0.25 3.41c ± 0.54 2.51d,e ± 0.46 17.42d ± 0.84 40.0 μg/mL 4.41d ± 0.29 3.45 c,d ± 0.46 2.97 c,d ± 0.07 2.49e ± 0.67 22.11e ± 0.49 Citrate-based sanitizer 0.0 mg/mL 35.23a ± 0.28 28.29a ± 0.44 22.77a ± 0.76 15.98a ± 1.07 0.00a ± 0.00 2.0 mg/mL 36.08b ± 0.69 28.96a,d ± 0.65 21.61a ± 0.69 14.83a–c ± 0.47 8.74b ± 0.51 4.0 mg/mL 37.29c ± 0.45 29.26a,d ± 0.75 21.93a ± 0.64 14.95a,b ± 0.16 13.65c ± 0.61 8.0 mg/mL 37.65c ± 0.49 29.32b,d ± 0.39 22.04a ± 0.47 15.69a–c ± 0.64 15.22d ± 0.35 16.0 mg/mL 38.46c ± 0.55 30.85c ± 0.66 22.71a ± 0.57 16.75a ± 0.63 17.14e ± 0.44 Iodine 0.0 μg/mL 35.23a ± 0.56 28.29a ± 0.83 22.77a ± 0.54 15.98a ± 0.21 0.00a ± 0.00 8.0 μg/mL 35.25a ± 0.29 28.35a ± 0.35 23.55a ± 0.42 16.12a ± 0.31 14.25b ± 0.28 16.0 μg/mL 35.18a ± 0.66 28.31a ± 0.22 23.11a ± 0.57 15.83a ± 0.56 15.54c ± 0.68 32.0 μg/mL 34.99a ± 0.65 28.40a ± 0.41 22.68a ± 0.47 15.72a ± 0.48 16.92d ± 0.41 64.0 μg/mL 33.78b ± 0.44 26.96b ± 0.51 22.46a ± 0.32 15.66a ± 0.73 19.20e ± 0.38 a–eDifferent letters in each column indicate a statistically significant difference (P ≤ 0.05). Note: Multiple comparisons by means of Bonferroni tests were carried out, using marginal means and standard error values as adjusted by the statistical model. View Large Bioavailability Figure 1 shows the AMX serum profiles from ASP that differ from the values obtained for the reference group (A), in a statistically significant manner (P < 0.05) and resulted in higher Cmax, MRT, AUC, and T ≥ MIC values. Figure 2 depicts the opposite: lower Cmax, AUC, and T ≥ MIC values of ASP, compared to the reference values. These figures consider the breakpoint for resistant Pasteurella sp. (1 μg/mL) as reference microorganism (CLSI, 2012). Table 4 presents the PK parameters for AMX derived from ASP and the reference ones. Two groups presented higher Cmax, MRT, and T ≥ MIC values than the A group: AC+++ and AC++++, which belong to ASP from the 2 highest concentrations of ASP derived from the citrate-based sanitizer combined with AMX (P < 0.05 in both cases). The T ≥ MIC obtained for the AC+++ group was 7.44 h; that is, 24% higher than the control one. This same parameter was 7.75 h for the AC++++ group (29% higher than the control group). All groups of the chlorine series caused the ASP to have statistically inferior Cmax, values compared to the reference value (P < 0.05 in all cases). Other variations in PK parameters were also noted, namely a decrease in AUC, MRT, and obviously an inferior T ≥ MIC (Table 4). Figure 1. View largeDownload slide Mean ± SD of amoxicillin serum concentrations after a single bolus administration of 20 mg/kg delivered directly into the proventriculus (group A), and amoxicillin serum concentrations derived from administering amoxicillin plus different concentrations of water sanitizers, as follows: AC+++ group: amoxicillin 20 mg/kg plus citrate-based sanitizer 3.2 mg/kg and AC++++ group: amoxicillin 20 mg/kg plus citrate-based sanitizer 6. 4 mg/kg. Figure 1. View largeDownload slide Mean ± SD of amoxicillin serum concentrations after a single bolus administration of 20 mg/kg delivered directly into the proventriculus (group A), and amoxicillin serum concentrations derived from administering amoxicillin plus different concentrations of water sanitizers, as follows: AC+++ group: amoxicillin 20 mg/kg plus citrate-based sanitizer 3.2 mg/kg and AC++++ group: amoxicillin 20 mg/kg plus citrate-based sanitizer 6. 4 mg/kg. Figure 2. View largeDownload slide Mean ± SD of serum concentrations of amoxicillin after a single bolus administration of 20 mg/kg delivered into the proventriculus (group A), and serum concentrations of amoxicillin derived from administering amoxicillin plus different concentration of water sanitizers as follows: ACL+ group: amoxicillin 20 mg/kg plus chlorine 0.002 mg/kg (from sodium hypochlorite), ACL++ group: amoxicillin 20 mg/kg plus chlorine 0.004 mg/kg, ACL+++ group: amoxicillin 20 mg/kg plus chlorine 0.008 mg/kg, ACL++++ group: amoxicillin 20 mg/kg plus chlorine 0.016 mg/kg and AI++++ group: amoxicillin 20 mg/kg plus iodine 0.0256 mg/kg. Figure 2. View largeDownload slide Mean ± SD of serum concentrations of amoxicillin after a single bolus administration of 20 mg/kg delivered into the proventriculus (group A), and serum concentrations of amoxicillin derived from administering amoxicillin plus different concentration of water sanitizers as follows: ACL+ group: amoxicillin 20 mg/kg plus chlorine 0.002 mg/kg (from sodium hypochlorite), ACL++ group: amoxicillin 20 mg/kg plus chlorine 0.004 mg/kg, ACL+++ group: amoxicillin 20 mg/kg plus chlorine 0.008 mg/kg, ACL++++ group: amoxicillin 20 mg/kg plus chlorine 0.016 mg/kg and AI++++ group: amoxicillin 20 mg/kg plus iodine 0.0256 mg/kg. Table 4. Mean ± SD of pharmacokinetic1 variables for amoxicillin administered at 20 mg/kg in broiler chickens after dosing them orally with the interaction products of amoxicillin plus chlorine (from sodium hypochlorite), or a citrate-based or iodine-based water sanitizer. Variable Group T½β2 (h) Tmax3 (h) Cmax4 (μg/mL) AUC5 (μg/mL·h) MRT6 (h) Fr7 (%) T ≥ MIC8 (h) A 0.96a ± 0.10 1.39a ± 0.12 12.54a ± 0.80 44.02a ± 0.61 2.78a ± 0.04 100a ± 0.00 6.00a ± 0.01 ACl+ 0.78b ± 0.06 1.14b ± 0.04 5.78b ± 1.36 12.14b ± 0.99 2.30b ± 0.14 27.58b ± 2.24 4.40b ± 0.06 ACl++ 0.24c ± 0.09 0.35c ± 0.03 4.12c ± 0.28 5.22c ± 0.08 0.72c ± 0.03 11.85c ± 0.19 2.44c ± 0.02 ACl+++ 0.19c ± 0.08 0.29c ± 0.04 3.80b,c ± 1.20 2.70d ± 0.06 0.59c ± 0.12 6.14d ± 0.13 0.85d ± 0.03 ACl++++ — 0.54d ± 0.09 1.67d ± 0.60 — — — 0.62e ± 0.01 AI+ 0.96a ± 0.03 1.49a ± 0.05 12.28a ± 0.46 44.04a ± 1.31 2.79a ± 0.11 100.04a ± 2.97 6.03a ± 0.02 AI++ 0.98a ± 0.04 1.50a ± 0.10 12.45a ± 0.66 44.00a ± 2.26 2.79a ± 0.12 99.96a ± 5.14 6.02a ± 0.01 AI+++ 0.94a ± 0.03 1.46a ± 0.06 11.91a ± 0.74 44.00a ± 2.66 2.78a ± 0.09 99.96a ± 6.06 5.99a ± 0.03 AI++++ 1.17b ± 0.09 1.66b ± 0.08 10.62b ± 0.34 41.67a ± 1.16 2.07b ± 0.11 94.67b ± 2.64 4.02b ± 0.01 AC+ 0.94a,b ± 0.14 1.30a,b ± 0.16 12.63a ± 0.39 44.67a ± 3.39 2.71a ± 0.17 101.49a ± 7.70 6.02a ± 0.02 AC++ 0.90a,b ± 0.09 1.28a,b ± 0.22 13.72b ± 0.09 48.64a,b ± 3.67 2.55a ± 0.10 110.51b ± 8.33 6.02a ± 0.01 AC+++ 0.87a,b ± 0.05 1.24a,b ± 0.09 15.07c ± 0.63 52.82b ± 1.45 3.32b ± 0.17 119.99b ± 3.29 7.44b ± 0.06 AC++++ 0.77b ± 0.06 1.13b ± 0.07 15.47c ± 0.26 58.28c ± 4.43 4.06c ± 0.27 132.39c ± 10.08 7.75c ± 0.02 Variable Group T½β2 (h) Tmax3 (h) Cmax4 (μg/mL) AUC5 (μg/mL·h) MRT6 (h) Fr7 (%) T ≥ MIC8 (h) A 0.96a ± 0.10 1.39a ± 0.12 12.54a ± 0.80 44.02a ± 0.61 2.78a ± 0.04 100a ± 0.00 6.00a ± 0.01 ACl+ 0.78b ± 0.06 1.14b ± 0.04 5.78b ± 1.36 12.14b ± 0.99 2.30b ± 0.14 27.58b ± 2.24 4.40b ± 0.06 ACl++ 0.24c ± 0.09 0.35c ± 0.03 4.12c ± 0.28 5.22c ± 0.08 0.72c ± 0.03 11.85c ± 0.19 2.44c ± 0.02 ACl+++ 0.19c ± 0.08 0.29c ± 0.04 3.80b,c ± 1.20 2.70d ± 0.06 0.59c ± 0.12 6.14d ± 0.13 0.85d ± 0.03 ACl++++ — 0.54d ± 0.09 1.67d ± 0.60 — — — 0.62e ± 0.01 AI+ 0.96a ± 0.03 1.49a ± 0.05 12.28a ± 0.46 44.04a ± 1.31 2.79a ± 0.11 100.04a ± 2.97 6.03a ± 0.02 AI++ 0.98a ± 0.04 1.50a ± 0.10 12.45a ± 0.66 44.00a ± 2.26 2.79a ± 0.12 99.96a ± 5.14 6.02a ± 0.01 AI+++ 0.94a ± 0.03 1.46a ± 0.06 11.91a ± 0.74 44.00a ± 2.66 2.78a ± 0.09 99.96a ± 6.06 5.99a ± 0.03 AI++++ 1.17b ± 0.09 1.66b ± 0.08 10.62b ± 0.34 41.67a ± 1.16 2.07b ± 0.11 94.67b ± 2.64 4.02b ± 0.01 AC+ 0.94a,b ± 0.14 1.30a,b ± 0.16 12.63a ± 0.39 44.67a ± 3.39 2.71a ± 0.17 101.49a ± 7.70 6.02a ± 0.02 AC++ 0.90a,b ± 0.09 1.28a,b ± 0.22 13.72b ± 0.09 48.64a,b ± 3.67 2.55a ± 0.10 110.51b ± 8.33 6.02a ± 0.01 AC+++ 0.87a,b ± 0.05 1.24a,b ± 0.09 15.07c ± 0.63 52.82b ± 1.45 3.32b ± 0.17 119.99b ± 3.29 7.44b ± 0.06 AC++++ 0.77b ± 0.06 1.13b ± 0.07 15.47c ± 0.26 58.28c ± 4.43 4.06c ± 0.27 132.39c ± 10.08 7.75c ± 0.02 a–d a–dDifferent letters within each column indicate a statistically significant difference within a given group (P ≤ 0.05). 1 1Pharmacokinetic parameters were calculated with non-transformed data considering that Shapiro–Wilk test indicated a normal distribution of the data. 1 2T½β = elimination half-life. 2 3Tmax = time to reach Cmax. 3 4Cmax = maximum serum concentration. 4 5AUC = area under the serum concentrations vs. time curve. 5 6MRT = mean residence time. 6 7Fr = relative bioavailability (AUCinteraction/AUCE) * 100. 8 8T ≥ MIC = time in which serum activity/concentrations were at or above the MIC value. View Large Table 4. Mean ± SD of pharmacokinetic1 variables for amoxicillin administered at 20 mg/kg in broiler chickens after dosing them orally with the interaction products of amoxicillin plus chlorine (from sodium hypochlorite), or a citrate-based or iodine-based water sanitizer. Variable Group T½β2 (h) Tmax3 (h) Cmax4 (μg/mL) AUC5 (μg/mL·h) MRT6 (h) Fr7 (%) T ≥ MIC8 (h) A 0.96a ± 0.10 1.39a ± 0.12 12.54a ± 0.80 44.02a ± 0.61 2.78a ± 0.04 100a ± 0.00 6.00a ± 0.01 ACl+ 0.78b ± 0.06 1.14b ± 0.04 5.78b ± 1.36 12.14b ± 0.99 2.30b ± 0.14 27.58b ± 2.24 4.40b ± 0.06 ACl++ 0.24c ± 0.09 0.35c ± 0.03 4.12c ± 0.28 5.22c ± 0.08 0.72c ± 0.03 11.85c ± 0.19 2.44c ± 0.02 ACl+++ 0.19c ± 0.08 0.29c ± 0.04 3.80b,c ± 1.20 2.70d ± 0.06 0.59c ± 0.12 6.14d ± 0.13 0.85d ± 0.03 ACl++++ — 0.54d ± 0.09 1.67d ± 0.60 — — — 0.62e ± 0.01 AI+ 0.96a ± 0.03 1.49a ± 0.05 12.28a ± 0.46 44.04a ± 1.31 2.79a ± 0.11 100.04a ± 2.97 6.03a ± 0.02 AI++ 0.98a ± 0.04 1.50a ± 0.10 12.45a ± 0.66 44.00a ± 2.26 2.79a ± 0.12 99.96a ± 5.14 6.02a ± 0.01 AI+++ 0.94a ± 0.03 1.46a ± 0.06 11.91a ± 0.74 44.00a ± 2.66 2.78a ± 0.09 99.96a ± 6.06 5.99a ± 0.03 AI++++ 1.17b ± 0.09 1.66b ± 0.08 10.62b ± 0.34 41.67a ± 1.16 2.07b ± 0.11 94.67b ± 2.64 4.02b ± 0.01 AC+ 0.94a,b ± 0.14 1.30a,b ± 0.16 12.63a ± 0.39 44.67a ± 3.39 2.71a ± 0.17 101.49a ± 7.70 6.02a ± 0.02 AC++ 0.90a,b ± 0.09 1.28a,b ± 0.22 13.72b ± 0.09 48.64a,b ± 3.67 2.55a ± 0.10 110.51b ± 8.33 6.02a ± 0.01 AC+++ 0.87a,b ± 0.05 1.24a,b ± 0.09 15.07c ± 0.63 52.82b ± 1.45 3.32b ± 0.17 119.99b ± 3.29 7.44b ± 0.06 AC++++ 0.77b ± 0.06 1.13b ± 0.07 15.47c ± 0.26 58.28c ± 4.43 4.06c ± 0.27 132.39c ± 10.08 7.75c ± 0.02 Variable Group T½β2 (h) Tmax3 (h) Cmax4 (μg/mL) AUC5 (μg/mL·h) MRT6 (h) Fr7 (%) T ≥ MIC8 (h) A 0.96a ± 0.10 1.39a ± 0.12 12.54a ± 0.80 44.02a ± 0.61 2.78a ± 0.04 100a ± 0.00 6.00a ± 0.01 ACl+ 0.78b ± 0.06 1.14b ± 0.04 5.78b ± 1.36 12.14b ± 0.99 2.30b ± 0.14 27.58b ± 2.24 4.40b ± 0.06 ACl++ 0.24c ± 0.09 0.35c ± 0.03 4.12c ± 0.28 5.22c ± 0.08 0.72c ± 0.03 11.85c ± 0.19 2.44c ± 0.02 ACl+++ 0.19c ± 0.08 0.29c ± 0.04 3.80b,c ± 1.20 2.70d ± 0.06 0.59c ± 0.12 6.14d ± 0.13 0.85d ± 0.03 ACl++++ — 0.54d ± 0.09 1.67d ± 0.60 — — — 0.62e ± 0.01 AI+ 0.96a ± 0.03 1.49a ± 0.05 12.28a ± 0.46 44.04a ± 1.31 2.79a ± 0.11 100.04a ± 2.97 6.03a ± 0.02 AI++ 0.98a ± 0.04 1.50a ± 0.10 12.45a ± 0.66 44.00a ± 2.26 2.79a ± 0.12 99.96a ± 5.14 6.02a ± 0.01 AI+++ 0.94a ± 0.03 1.46a ± 0.06 11.91a ± 0.74 44.00a ± 2.66 2.78a ± 0.09 99.96a ± 6.06 5.99a ± 0.03 AI++++ 1.17b ± 0.09 1.66b ± 0.08 10.62b ± 0.34 41.67a ± 1.16 2.07b ± 0.11 94.67b ± 2.64 4.02b ± 0.01 AC+ 0.94a,b ± 0.14 1.30a,b ± 0.16 12.63a ± 0.39 44.67a ± 3.39 2.71a ± 0.17 101.49a ± 7.70 6.02a ± 0.02 AC++ 0.90a,b ± 0.09 1.28a,b ± 0.22 13.72b ± 0.09 48.64a,b ± 3.67 2.55a ± 0.10 110.51b ± 8.33 6.02a ± 0.01 AC+++ 0.87a,b ± 0.05 1.24a,b ± 0.09 15.07c ± 0.63 52.82b ± 1.45 3.32b ± 0.17 119.99b ± 3.29 7.44b ± 0.06 AC++++ 0.77b ± 0.06 1.13b ± 0.07 15.47c ± 0.26 58.28c ± 4.43 4.06c ± 0.27 132.39c ± 10.08 7.75c ± 0.02 a–d a–dDifferent letters within each column indicate a statistically significant difference within a given group (P ≤ 0.05). 1 1Pharmacokinetic parameters were calculated with non-transformed data considering that Shapiro–Wilk test indicated a normal distribution of the data. 1 2T½β = elimination half-life. 2 3Tmax = time to reach Cmax. 3 4Cmax = maximum serum concentration. 4 5AUC = area under the serum concentrations vs. time curve. 5 6MRT = mean residence time. 6 7Fr = relative bioavailability (AUCinteraction/AUCE) * 100. 8 8T ≥ MIC = time in which serum activity/concentrations were at or above the MIC value. View Large High iodine concentrations produced a decrease in Cmax for the EI++++ group (64 μg/mL of free iodine) (P < 0.05). Additionally, a decrease of MRT, Fr, and T ≥ MIC was also obtained for this group (P < 0.05 in all cases). As referred before, the ASP from 2 of the highest concentrations of citrate-based sanitizer showed statistically higher Cmax, MRT, Fr, and T ≥ MIC (P < 0.05 in all cases). DISCUSSION The results derived from the interaction of water sanitizers with in vitro AMX and on the bioavailability of AMX in broiler chickens, reveal 3 different outcomes. If the interaction is with sodium hypochlorite, there is an almost linear progressive reduction of the in vitro antibacterial activity of AMX and this is somehow replicated in the observed Cmax, MRT, Fr, and T ≥ MIC values. Based on their oxidizing ability, halogenated disinfectants (iodine and chlorine) are commonly used as drinking water sanitizers (McDonnell and Russel, 1999; Sumano et al., 2015), and it is precisely this oxidative capacity that may react and alter the chemical structure of amoxicillin or modify its antimicrobial effects. For example, it has been stated that the aromatic ring and the amino group of AMX are susceptible to chlorine oxidation (Acero et al., 2010; Wang et al., 2010). In contrast and despite being also a halogenated compound, the iodine-based sanitizer used (iodine-polyvinylpyrrolidone, pH 3.5) was unable to interact sufficiently with the AMX and induce tangible changes in either the in vitro antibacterial activity or in the Fr patterns. Yet, reduction in the Cmax antibacterial activity, Fr, and MRT was only observed with the highest concentration of iodine. Iodine-polyvinylpyrrolidone is a slow iodine release formulation and this may partly explain the in vitro and in vivo antibacterial activity/concentration patterns. It is possible that if a longer AMX–iodine interaction time was allowed, a greater deterioration of AMX’s antibacterial activity could have been obtained. This must be further studied, because the AMX prevalence in water tanks could last much longer than 30 min; which was the AMX–water sanitizer interaction-time allowed during this trial. The citrate-based compound enhanced the in vitro AMX antibacterial activity. Relative bioavailability followed a similar pattern, where the Cmax, Fr, and MRT were significantly enhanced (P <0.05) with the 2 highest concentrations of the citrate-based water sanitizer (8 and 16 mg/mL) (see Table 2). This type of water sanitizers are usually made from grapefruit extracts and other citric seeds (Bevilacqua et al., 2013). It has been reported that grapefruit seed extracts contain furanocoumarins, which in turn can reduce or inhibit the gastrointestinal glycoprotein G activity, allowing a better bioavailability of some drugs (Dahan and Altman, 2004; Bailey, 2010; Ahmed et al., 2015). Additionally, it has been shown that grapefruit extracts also inhibit the P-450 (CYP) enzyme activity at the gastrointestinal epithelial level (Bailey, et al., 1998; Kane and Lipsky, 2000; Giorgi et al., 2003), contributing to a better absorption of some drugs. Consequently, it is likely to think that the active principles of the citrate-based sanitizer contribute to increase the Cmax, MRT, and Fr of AMX; such as what was observed in the results obtained during this experimental model (Kalpana et al., 2015). In this work, the chlorine, iodine, and citrate-based studied sanitizers included the maximum recommended concentrations found in the literature, and even they were 8 times more concentrated (CES, 1998; Sumano et al., 2015). It is not uncommon that the drinking water in poultry coops contain high concentrations of iodine, chlorine, or citrate products as a result of accidental accumulation or due to an inadequate dosing (Maharjan et al., 2016). For example, in the case of iodine, it has been used at high doses to treat outbreaks caused by adenovirus: 1.6 times the maximum recommended dose of iodine as water sanitizer (Abdul-Aziz and Hasan, 1996). Alterations of either, in vitro antibacterial activity or ASP bioavailability at lower concentrations, were generally much more discrete. Further work is needed to define changes in these parameters when longer interactions occurs within the water tank, considering that under field conditions the total consumption of the water source may take much longer than 30 min. However, a conclusion that can be reached from this study is that, if it has been decided to administer AMX to chickens through their drinking water, the water sanitizer of choice is the citrate-based one. The rational use of antimicrobials has been worldwide prioritized due to the increase of microbial resistance to these agents and the lack of new families of antimicrobial agents destined to poultry medicine (Saga and Yamaguchi, 2009; Bjork et al., 2015; Limayem et al., 2015). One of the obvious strategies that emerge from this situation is the use of antibacterial drugs based on PK/PD ratios (Errecalde, 2004). Amoxicillin being a time-dependent antimicrobial drug, the most important PK/PD ratio is T ≥ MIC. In order to obtain optimal clinical results, it has been set that depending on the pathogen, the dosage interval of T ≥ MIC can range from 40 to 100% (McKellar et al., 2004; MacGowan, 2011). In spite of the above, Cmax has also been highlighted as important and has been set at 1–5 times the MIC value (McKellar et al., 2004). It is unlikely that commercial preparations of AMX comply with the referred ratios, because AMX preparations for poultry that are administered through drinking water are usually prescribed with a 24-h interval. For example, based on the MRT and Cmax obtained for AMX during this trial (2.78 h ± 0.04 and 12.54 μg/mL ± 0.80, respectively) and considering a theoretical MIC of 1 μg/mL for Pasteurella sp. (Huang et al., 2009), it can be observed that the referred PK/PD ratios were not met using AMX alone. The results obtained in this trial, coincide with those of Krasucka and Kowalski (2010). By analyzing their reported serum concentrations of AMX vs. time relationships in poultry, they obtained a T ≥ MIC of only 8.33% after dosing broiler chickens with 20 mg/kg and considering the same Pasteurella sp. MIC values. They also reported a Cmax value that was only equivalent to the MIC for this pathogen. Similarly, Abo et al. (2004) showed that a dose of 10 mg/kg administered orally induced a T ≥ MIC of only 2.91 to 12.5%, also considering Pasteurella sp. Similarly, by analyzing data from Kandeel (2015), a dose of 10 mg/kg PO offered a T ≥ MIC that ranged from 8.3 to 25%. Also, the Cmax failed to reach 4–5 times the MIC value. In contrast, the same analysis performed on an earlier study by Anadón et al. (1996), offered AMX serum concentrations that complied well with the referred PK/PD ratios. Differences in the analytical precision may account for discrepancies among these researchers. It has been shown that the so-called post-antibiotic effect is either minimum or nonexistent for beta-lactamic antibacterial drugs (Cars, 1997; Papich, 2014). Thus, dosing intervals should ideally be established at the time when serum concentrations of the beta-lactamic drug fall below the MIC value. This is not possible for poultry medicine. However, in order to comply better with T ≥ MIC, the AMX dosing should at least be set twice a day. A specialized pharmaceutical design of AMX for poultry should then be investigated in order to provide protection to this drug when it has been diluted in drinking water; particularly, if it contains chlorine-based water sanitizers. The rapid and significant degradation of AMX in water has been demonstrated (Jerzsele and Nagy, 2009). Also, other variables should be considered, such as water temperature, bacterial load, water hardness, among others (Sumano et al., 2004; Fairchild et al., 2006). FUNDING Part of this work was supported by the Support Program for Research Projects and Technological Innovation (PAPIIT) of the National Autonomous University of Mexico (UNAM) No. IN212815. REFERENCES Abdul-Aziz T. A. , Hasan S. Y. . 1996 . Preliminary observations on the efficacy of an iodophor in reducing the mortality in chickens experimentally affected by the “hydropericardium syndrome.” Vet. Res. Commun. 20 : 191 – 194 . Google Scholar CrossRef Search ADS PubMed Abo E. K. , Al-Tarazi Y. H. , Al-Bataineh M. M. . 2004 . Comparative pharmacokinetics and bioavailability of amoxycillin in chickens after intravenous, intramuscular and oral administrations . Vet. Res. Commun. 28 : 599 – 607 . Google Scholar CrossRef Search ADS PubMed Acero J. L. , Benitez F. J. , Real F. J. , Roldan G. . 2010 . Kinetics of aqueous chlorination of some pharmaceuticals and their elimination from water matrices . Water Res . 44 : 4158 – 4170 . Google Scholar CrossRef Search ADS PubMed Agunos A. , Léger D. , Carson C. . 2012 . Review of antimicrobial therapy of selected bacterial diseases in broiler chickens in Canada . Can. Vet. J . 53 : 1289 – 1300 . Google Scholar PubMed Ahmed I. S. , Hassan M. A. , Kondo T. . 2015 . Effect of lyophilized grapefruit juice on P-glycoprotein-mediated drug transport in-vitro and in-vivo . Drug Dev. Ind. Pharm . 41 : 375 – 381 . Google Scholar CrossRef Search ADS PubMed Anadón A. , Martinez M. R. , Larrañaga , Diaz M. J. , Bringas P. , Fernandez M. C. , Martinez M. A. , Fernandez M. L. , Cruz . 1996 . Pharmacokinetics of amoxicillin in broiler chickens . Avian Pathol. 25 : 449 – 458 . Google Scholar CrossRef Search ADS PubMed Bailey M. 1999 . The water requirements of poultry . 342 in Developments in Poultry Nutrition 2 . Wiseman J. , Garnsworthy P. C. eds. Nottingham University Press , United Kindgom . Google Scholar CrossRef Search ADS Bailey D. 2010 . Fruit juice inhibition of uptake transport: a new type of food-drug interaction . Br. J. Clin. Pharmacol . 70 : 645 – 655 . Google Scholar CrossRef Search ADS PubMed Bailey D. , Malcolm J. , Arnold O. , Spence D. . 1998 . Grapefruit juice-drug interactions . Br. J. Clin. Pharmacol. 46 : 101 – 110 . Google Scholar CrossRef Search ADS PubMed Bell D. , Weaver W. . 2002 . Consumption and quality of water . Pages 411 – 430 in Commercial Chicken Meat and Egg Production . 5th ed . Bell D. , Weaver W. eds. Kluwer Academic Publishers , Norwell, MA . Google Scholar CrossRef Search ADS Bennet J. , Brodie J. L. , Benner E. J. , Kirby W. . 1966 . Simplified accurate method for antibiotic assay of clinical specimens . Am. Soc. Microbiol . 14 : 170 – 177 . Bevilacqua A. , Campaniello D. , Speranza B. , Sinigaglia M. , Corbo M. R. . 2013 . Control of Alicyclobacillus acidoterrestris in apple juice by citrus extracts and a mild heat-treatment . Food Control 31 : 553 – 559 . Google Scholar CrossRef Search ADS Bjork K. E. , Kopral C. A. , Wagner B. A. , Dargatz D. A. . 2015 . Comparison of mixed effects models of antimicrobial resistance metrics of livestock and poultry Salmonella isolates from a national monitoring system . Prev. Vet. Med . 122 : 265 – 272 . Google Scholar CrossRef Search ADS PubMed Cars O. 1997 . Efficacy of beta-lactam antibiotics: integration of pharmacokinetics and pharmacodynamics . Diagn. Microbiol. Infect. Dis. 27 : 29 – 33 . Google Scholar CrossRef Search ADS PubMed CLSI . 2012 . Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard . Clinical and Laboratory Standards Institute , Wayne, PA . Cobb-Vantress Inc . 2005 . COBB Guia de Manejo de Pollo de Engorde . Cobb-Vantress Inc. , Siloam Springs, Arkansas . Cooperative Extension Service (CES) . 1998 . Sanitizing poultry drinking water . 1 in The Disaster Handbook . National Edition Institute of Food and Agricultural Sciences . University of Florida, USA . PubMed PubMed Dahan A. , Altman H. . 2004 . Food-drug interaction: grapefruit juice augments drug bioavailability-mechanism, extent and relevance . Eur J Clin Nutr . 58 : 1 – 9 . Google Scholar CrossRef Search ADS PubMed EMA . 2016 . Sales of Veterinary Antimicrobial Agents in 29 European Countries in 2014. Sixth ESVAC Report . European Medicines Agency , London . Errecalde, J. O. 2004 . Uso de antimicrobianos en animale de consumo, incidencia del desarrollo de resistencias en salud pública . FAO , Roma, Itallia . Esmail S. 1996 . Water: the vital nutrient . Poult. Int . 15 : 72 – 76 . Fairchild B. , Batal A. , Ritz C. , Vendrell P. . 2006 . Effect of drinking water iron concentration on broiler performance . J. Appl. Poult. Res . 15 : 511 – 517 . Google Scholar CrossRef Search ADS Garson G. D. 2013 . Generalized Linear Models / Generalized Estimating Equations . Statistical Associates Publishers , Asheboro, NC . Giorgi M. , Meucci V. , Vaccaro E. , Mengozzi G. , Giusiani M. , Soldani G. . 2003 . Effects of liquid and freeze-dried grapefruit juice on the pharmacokinetics of praziquantel and its metabolite 4΄-hydroxy praziquantel in beagle dogs . Pharmacol. Res. 47 : 87 – 92 . Google Scholar CrossRef Search ADS PubMed Hua P. , Vasyukova E. , Uhl W. . 2015 . A variable reaction rate model for chlorine decay in drinking water due to the reaction with dissolved organic matter . Water Res. 75 : 109 – 122 . Google Scholar CrossRef Search ADS PubMed Huang T. M. , Lin. T. L. , Wu C. C. . 2009 . Antimicrobial susceptibility and resistance of chicken Escherichia coli, Salmonella spp., and Pasteurella multocida isolates . Avian Dis. 53 : 89 – 93 . Google Scholar CrossRef Search ADS PubMed ICA . 2017 . Productos medicamentos veterinarios . Accessed Feb. 2017. http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx . Jerzsele Á. , Nagy G. . 2009 . The stability of amoxicillin trihydrate and potassium clavulanate combination in aqueous solutions . Acta Vet. Hung. 57 : 485 – 493 . Google Scholar CrossRef Search ADS PubMed Kahrs R. F. 1995 . Principios generales de la desinfección . Rev. sci. tech. Off. int. Epiz . 14 : 143 – 163 . Google Scholar CrossRef Search ADS Kalpana S. , Srinivasa Rao G. , Malik J. K. . 2015 . Impact of aflatoxin B1 on the pharmacokinetic disposition of enrofloxacin in broiler chickens . Environ. Toxicol. Pharmacol. 40 : 645 – 649 . Google Scholar CrossRef Search ADS PubMed Kandeel M. 2015 . Pharmacokinetics and oral bioavailability of amoxicillin in chicken infected with caecal coccidiosis . J. vet. Pharmacol. Therap. 38 : 504 – 507 . Google Scholar CrossRef Search ADS Kane G. C. , Lipsky J. J. . 2000 . Drug-grapefruit juice interactions . Mayo Clin. Proc. 75 : 933 – 942 . Google Scholar CrossRef Search ADS PubMed Kathleen H. , Van Dijr L. . 2011 . The World Medicines Situation 2011, Rational Use of Medicines . 3rd ed . WHO , Geneva, Switzerland . Krasucka D. , Kowalski C. J. . 2010 . Pharmacokinetic parameters of amoxicillin in pigs and poultry . Acta Pol. Pharm. - Drug Res . 67 : 729 – 732 . Krasucka D. , Kowalski C. , Osypiuk M. , Opielak G. . 2015 . Determination of amoxicillin in poultry plasma by high-performance liquid chromatography after formaldehyde derivation . Acta Chromatogr. 27 : 55 – 65 . Google Scholar CrossRef Search ADS Limayem A. , Donofrio R. S. , Zhang C. , Haller E. , Johnson M. G. . 2015 . Studies on the drug resistance profile of Enterococcus faecium distributed from poultry retailers to hospitals . J. Environ. Sci. Heal. Part B 50 : 827 – 832 . Google Scholar CrossRef Search ADS MacGowan A. 2011 . Revisiting beta-lactams - PK/PD improves dosing of old antibiotics . Curr. Opin. Pharmacol. 11 : 470 – 476 . Google Scholar CrossRef Search ADS PubMed Maharjan P. , Clark T. , Kuenzel C. , Foy M. K. , Watkins S. . 2016 . On farm monitoring of the impact of water system sanitation on microbial levels in broiler house water supplies . J. Appl. Poult. Res. 25 : 266 – 271 . Google Scholar CrossRef Search ADS May J. , Loot B. , Simmons J. . 1997 . Water consumption by broilers in high cyclic temperatures: bell versus nipple waterers . Poult. Sci. 76 : 944 – 947 . Google Scholar CrossRef Search ADS PubMed McDonnell G. , Russel D. A. . 1999 . Antiseptics and disinfectants activity, action, and resistance . Clin. Microbiol. Rev . 12 : 147 – 179 . Google Scholar PubMed McKellar Q. A. , Sanchez Bruni S. F. , Jones D. G. . 2004 . Pharmacokinetic/pharmacodynamic relationships of antimicrobial drugs used in veterinary medicine . J. Vet. Pharmacol. Ther . 27 : 503 – 514 . Google Scholar CrossRef Search ADS PubMed MIDA . 2017 . Registros farmacéuticos.Panama . Accessed Feb. 2017 http://www.mida.gob.pa/upload/documentos/registrosfarmaceuticosoct-15(1).pdf . National Research Council (NRC) . 1994 . Nutrient Requirements of Poultry . 9th rev. ed . Natl. Acad. Press , Washington, DC . Papich M. G. 2014 . Pharmacokinetic-pharmacodynamic (PK-PD) modeling and the rational selection of dosage regimes for the prudent use of antimicrobial drugs . Vet. Microbiol. 171 : 480 – 486 . Google Scholar CrossRef Search ADS PubMed Pillai K. , Eliopoulos G. , Moellering C. . 2005 . Antimicrobial combinations . 365 – 409 in Antibiotics in Laboratory Medicine . V. , Lorian , ed. 5th ed. Lippincott Williams and Wilkins , Philadelphia, USA . Quilumba C. , Quijia E. , Gernat A. , Murillo G. , Grimes J. . 2015 . Evaluation of different water flow rates of nipple drinkers on broiler productivity . J. Appl. Poult. Res . 24 : 58 – 65 . Google Scholar CrossRef Search ADS Ribeiro A. , Krabbe E. , Penz J. A. , Renz S. , Gomez H. . 2004 . Effect of chick weight, geometric mean diameter and sodium level in prestarter diets (1 to 7 days) on broiler perfomance up to 21 days of age . Poult. Sci . 6 : 225 – 230 . SAG . 2017 . Sistema Medicamentos Veterinarios . Accessed Feb. 2017. http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp . Saga T. , Yamaguchi K. . 2009 . History of antimicrobial agents and resistant bacteria . Japan Med. Assoc. J . 52 : 103 – 108 . SAGARPA . 2012 . Acuerdo por el que se modifica el diverso por el que se establece la clasificación y prescripción de los productos farmacéuticos veterinarios por el nivel de riesgo de sus ingredientes activos . Diario Oficial , Mexico . SENASA . 2015 . Listados oficiales de productos veterinarios . Accessed Feb. 2017. http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales . Shapiro S. , Wilk M. . 1965 . An analysis of variance test for normality (complete samples) . Biometrika 52 : 591 – 611 . Google Scholar CrossRef Search ADS Sumano H. S. , Gutiérrez L. . 2008 . Farmacología clínica en aves . 3rd ed . McGraw Hill/Interamericana , Mexico City, Mexico . Sumano L. H. , Gutierrez O. L. , Aguilera R. , Rosiles M. R. , Bernard B. M. J. , Gracia M. J. . 2004 . Influence of hard water on the bioavailability of enrofloxacin in broilers . Poult. Sci. 83 : 726 – 731 . Google Scholar CrossRef Search ADS PubMed Sumano L. H. , Ocampo C. L. , Gutiérrez O. L. . 2015 . Farmacología Veterinaria . 4th ed . Diseño e Impresiones Aranda SA de CV , México . Sun L. , Jia L. , Xie X. , Xie K. , Wang J. , Liu J. , Cui L. , Zhang G. , Dai G. , Wang J. . 2016 . Quantitative analysis of amoxicillin, its major metabolites and ampicillin in eggs by liquid chromatography combined with electrospray ionization tandem mass spectrometry . Food Chem. 192 : 313 – 318 . Google Scholar CrossRef Search ADS PubMed Vermeulen B. 2002 . Drug administration to poultry . Adv. Drug. Deliv. Rev. 54 : 795 – 803 . Google Scholar CrossRef Search ADS PubMed Wang P. , He Y. L. , Huang C. H. . 2010 . Oxidation of fluoroquinolone antibiotics and structurally related amines by chlorine dioxide: reaction kinetics, product and pathway evaluation . Water Res. 44 : 5989 – 5998 . Google Scholar CrossRef Search ADS PubMed WHO . 1999 . Codex Alimentarius Commission . 23rd ed . WHO , Rome . Ziaei N. , Kermanshahi H. , Pilevar M. . 2011 . Effects of dietary crude protein and calcium / phosphorus content on growth, nitrogen and mineral retention in broiler chickens . Afr. J. Biotechnol . 10 : 13342 – 13350 . © 2018 Poultry Science Association Inc. This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Antibacterial activity of amoxicillin in vitro and its oral bioavailability in broiler chickens under the influence of 3 water sanitizers

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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10.3382/ps/pey114
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Abstract

ABSTRACT The interaction of 3 water sanitizers (sodium hypochlorite, iodine-polyvinylpyrrolidone, and citrate) utilized in poultry production on antibacterial activity and bioavailability of amoxicillin trihydrate (AMX) were studied. Sanitizers were mixed with AMX in prepared water, the resulting substances were regarded as amoxicillin-sanitizer products (ASP). First, the in vitro antibacterial activity of each ASP was compared to that of AMX. Then, pharmacokinetics (PK) of ASP and AMX diluted in prepared water, were carried out in broiler-chickens. Amoxicillin or ASP (20 mg/kg) from different concentrations of sanitizers was directly placed into the chicken's crop and blood samples were taken. Basic PK parameters were obtained. Serum activity/concentrations of AMX were assessed by agar diffusion and corroborated with high performance liquid chromatography. Results show that ASP of AMX/sodium hypochlorite decrease both, the antimicrobial activity of in vitro AMX and its relative bioavailability (Fr) assessed with the maximum serum concentration (Cmax), the area under the concentration-time curve, and the mean residence time (MRT) (3.80 μg/mL, 2.70 μg/mL·h, and 0.59 h, respectively), compared to the AMX administered alone (12.54 μg/mL, 44.02 μg/mL·h, and MRT 2.78 h). ASP from amoxicillin/ionophore, reduced the Cmax (10.62 μg/mL), Fr (94.67%), and MRT (2.07 h), at the highest tested concentrations. In contrast, the 2 highest concentrations of the citrate sanitizer increased the Cmax (15.07 and 15.47 μg/mL), Fr (119 and 132%), and MRT (3.32 and 4.06 h) and their in vitro antimicrobial activity. Interactions between the tested water sanitizers and AMX modify the Cmax, Fr, MRT of the latter, altering the PK/pharmacodymanic ratios for a time-dependent antibiotic. Results also reveal that the use of amoxicillin trihydrate administered through the drinking water does not meet the required PK/pharmacodymanic ratios. Thus, it is here postulated that this antibiotic should be administered at least twice a day and that its interaction with water sanitizers should be considered. INTRODUCTION Rational use of antibacterial drugs has been highlighted and considered a worldwide priority (Kathleen and Van Dijr, 2011). Therefore, antimicrobial dosing schedules and methods should be revised in order to comply with desired pharmacokinetics/pharmacodynamic (PK/PD) ratios appointed for veterinary medicine (McKellar et al., 2004). In poultry medicine, well planned dosing protocols and adequate equipment are followed to attempt the accurate delivery of antibacterial drugs to the flock, and eventually to each chicken (Vermeulen, 2002). The preferred manner for administering antibacterial drugs is through their drinking water. Besides its easy administration, this route and vehicle are chosen because it is believed that for most antibacterial drugs, better bioavailability (F) is obtained (Esmail, 1996). Even so, considerable variations in F among flocks and even among individuals should be expected. Among other causes, drinking habits in the flock of each chicken, are accountable for these variations (Bailey, 1999; Ribeiro et al., 2004; Ziaei et al., 2011). Also, quality of the drinking water has been shown to modify F of antibacterial drugs in chicken (Bell and Weaver, 2002; Sumano et al., 2004; Fairchild et al., 2006) as it also happens with inadequate plumbing and the way drinkers are positioned in the chicken coop (NRC, 1994; May et al., 1997; Quilumba et al., 2015). One further aspect that may also induce variations in F of antibacterial drugs, when they are given to poultry through drinking water, is the concurrent use of water sanitizers (Vermeulen, 2002). The possible interaction of a sanitizer added to drinking water with a given antibacterial drug has not been addressed in formal literature. The efficacy of most water-sanitizing agents is based on their high reactivity with chemical and organic entities (Kahrs, 1995); therefore it is reasonable to think that they can react with a given antibacterial drug when concurrently added to the water source, and such interactions may consequently modify F (Esmail, 1996). Iodine-polyvinylpyrrolidone, sodium hypochlorite preparations, and a citrate-based sanitizer are often used in poultry medicine (Acero et al., 2010; Hua et al., 2015). However, the consequences of the interaction between these chemicals and many antimicrobial drugs, in terms of loss or increase of in vitro antibacterial activity and F, await characterization. Amoxicillin (AMX) is a semisynthetic β-lactam antibiotic belonging to the aminopenicillin group. It possesses a broad antimicrobial spectrum, a reasonably good absorption rate, and considerable tissue penetration (Anadón et al., 1996). Additionally, it exhibits low toxicity compared to other antimicrobials (Krasucka et al., 2015) and has a low cost (Sun et al., 2016). It is used in poultry farming (Sun et al., 2016) and is commercially available in México (SAGARPA, 2012), Latin America (SENASA, 2015; ICA, 2017; MIDA, 2017; SAG, 2017), Canada (Agunos et al., 2012; Sun et al., 2016), and the European Union (Table 1). According to the sixth European Surveillance of Veterinary Antimicrobial Consumption, penicillins are the second most used group of antimicrobials in production animals (25.5%), only surpassed by tetracyclines (33.4%); and from these, AMX and ampicillin are the most consumed and are primarily administered orally (EMA, 2016). However, like all beta-lactam antibacterials, the amoxicillin molecule is highly susceptible to undergo chemical reactions (Acero et al., 2010). Therefore, the aim of this study was to assess whether or not the interaction of AMX with sodium hypochlorite, iodine-polyvinylpyrrolidone, and a citrate-based disinfectant results in the modification of the antibacterial activity of the drug in vitro, and whether the concurrent administration of these water sanitizers with AMX alters its relative bioavailability (Fr) in broiler chickens. Table 1. Examples of amoxicillin trihydrate preparations available for administration through drinking water for poultry medicine in different parts of the world. Country/ No. of region approved products Source Argentina 6 Servicio Nacional de Sanidad y calidad agroalimentaria: Servicio Nacional de Sanidad y calidad agroalimentaria: http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales Canada 3 Health Canada: https://cal.naccvp.com/search/main?query=amoxicillin Chile 2 Ministerio de agricultura: http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp Colombia 5 Instituto Colombiano Agropecuario: http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx Costa Rica 2 Servicio Nacional de Salud Animal, Costa Rica: Servicio Nacional de Salud Animal, Costa Rica: http://www.senasa.go.cr/medivet/busque_avanzada.aspx Germany 7 Pharmanet in cooperation with the Federal ministry of health (Bundesministerium für Gesundheit): https://www.pharmnet-bund.de/static/de/suche/?q=amoxicillin#&query=amoxicillin&sortdir=asc Italy 15 Ministero della Salute:https://www.vetinfo.sanita.it/j6_prontuario/farmaci/public/prodottomd/;jsessionid=95E50FE72628918695E06827886D9A8E-n1.tomcatprod2 Mexico 9 SAGARPA: SAGARPA: http://dev.sagarpa.gob.mx/tramitesyServicios/Lists/Direccin%20General%20de%20Salud%20Animal/Attachments/13/SENASICA%2001-024.pdf Spain 22 Agencia Española de Medicamentos y Productos Sanitarios: https://cimavet.aemps.es/cimavet/medicamentos.do UK 12 Veterinary Medicines Directorate service: http://www.vmd.defra.gov.uk/ProductInformationDatabase/Default.aspx Country/ No. of region approved products Source Argentina 6 Servicio Nacional de Sanidad y calidad agroalimentaria: Servicio Nacional de Sanidad y calidad agroalimentaria: http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales Canada 3 Health Canada: https://cal.naccvp.com/search/main?query=amoxicillin Chile 2 Ministerio de agricultura: http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp Colombia 5 Instituto Colombiano Agropecuario: http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx Costa Rica 2 Servicio Nacional de Salud Animal, Costa Rica: Servicio Nacional de Salud Animal, Costa Rica: http://www.senasa.go.cr/medivet/busque_avanzada.aspx Germany 7 Pharmanet in cooperation with the Federal ministry of health (Bundesministerium für Gesundheit): https://www.pharmnet-bund.de/static/de/suche/?q=amoxicillin#&query=amoxicillin&sortdir=asc Italy 15 Ministero della Salute:https://www.vetinfo.sanita.it/j6_prontuario/farmaci/public/prodottomd/;jsessionid=95E50FE72628918695E06827886D9A8E-n1.tomcatprod2 Mexico 9 SAGARPA: SAGARPA: http://dev.sagarpa.gob.mx/tramitesyServicios/Lists/Direccin%20General%20de%20Salud%20Animal/Attachments/13/SENASICA%2001-024.pdf Spain 22 Agencia Española de Medicamentos y Productos Sanitarios: https://cimavet.aemps.es/cimavet/medicamentos.do UK 12 Veterinary Medicines Directorate service: http://www.vmd.defra.gov.uk/ProductInformationDatabase/Default.aspx View Large Table 1. Examples of amoxicillin trihydrate preparations available for administration through drinking water for poultry medicine in different parts of the world. Country/ No. of region approved products Source Argentina 6 Servicio Nacional de Sanidad y calidad agroalimentaria: Servicio Nacional de Sanidad y calidad agroalimentaria: http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales Canada 3 Health Canada: https://cal.naccvp.com/search/main?query=amoxicillin Chile 2 Ministerio de agricultura: http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp Colombia 5 Instituto Colombiano Agropecuario: http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx Costa Rica 2 Servicio Nacional de Salud Animal, Costa Rica: Servicio Nacional de Salud Animal, Costa Rica: http://www.senasa.go.cr/medivet/busque_avanzada.aspx Germany 7 Pharmanet in cooperation with the Federal ministry of health (Bundesministerium für Gesundheit): https://www.pharmnet-bund.de/static/de/suche/?q=amoxicillin#&query=amoxicillin&sortdir=asc Italy 15 Ministero della Salute:https://www.vetinfo.sanita.it/j6_prontuario/farmaci/public/prodottomd/;jsessionid=95E50FE72628918695E06827886D9A8E-n1.tomcatprod2 Mexico 9 SAGARPA: SAGARPA: http://dev.sagarpa.gob.mx/tramitesyServicios/Lists/Direccin%20General%20de%20Salud%20Animal/Attachments/13/SENASICA%2001-024.pdf Spain 22 Agencia Española de Medicamentos y Productos Sanitarios: https://cimavet.aemps.es/cimavet/medicamentos.do UK 12 Veterinary Medicines Directorate service: http://www.vmd.defra.gov.uk/ProductInformationDatabase/Default.aspx Country/ No. of region approved products Source Argentina 6 Servicio Nacional de Sanidad y calidad agroalimentaria: Servicio Nacional de Sanidad y calidad agroalimentaria: http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales Canada 3 Health Canada: https://cal.naccvp.com/search/main?query=amoxicillin Chile 2 Ministerio de agricultura: http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp Colombia 5 Instituto Colombiano Agropecuario: http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx Costa Rica 2 Servicio Nacional de Salud Animal, Costa Rica: Servicio Nacional de Salud Animal, Costa Rica: http://www.senasa.go.cr/medivet/busque_avanzada.aspx Germany 7 Pharmanet in cooperation with the Federal ministry of health (Bundesministerium für Gesundheit): https://www.pharmnet-bund.de/static/de/suche/?q=amoxicillin#&query=amoxicillin&sortdir=asc Italy 15 Ministero della Salute:https://www.vetinfo.sanita.it/j6_prontuario/farmaci/public/prodottomd/;jsessionid=95E50FE72628918695E06827886D9A8E-n1.tomcatprod2 Mexico 9 SAGARPA: SAGARPA: http://dev.sagarpa.gob.mx/tramitesyServicios/Lists/Direccin%20General%20de%20Salud%20Animal/Attachments/13/SENASICA%2001-024.pdf Spain 22 Agencia Española de Medicamentos y Productos Sanitarios: https://cimavet.aemps.es/cimavet/medicamentos.do UK 12 Veterinary Medicines Directorate service: http://www.vmd.defra.gov.uk/ProductInformationDatabase/Default.aspx View Large MATERIAL AND METHODS This study was conducted in 2 phases. The first phase (antibacterial activity) was carried out in order to identify possible modifications of the in vitro antimicrobial activity of AMX in the presence of a sanitizer; whereas, the second phase (bioavailability) was designed to identify if the variations in Fr, maximum serum concentration (Cmax), mean residence time (MRT), and time in which the serum activity/concentrations were at or above the minimum inhibitory concentration (MIC) value (T ≥ MIC), could be observed in chickens dosed with various amoxicillin-sanitizer products (ASP). Antibacterial Activity Water sanitizers were mixed with AMX in prepared water, characterized by originating from any type of water supply (including municipal water); and additionally, subjected to any treatment that could modify the original water in order to comply with the chemical and microbiological safety requirements for pre-packaged water and commercialized as purified or drinking water (WHO, 1999; Sumano and Gutiérrez, 2008). It has a pH 8 and a resistivity of 200 Ω•m, as analyzed with an Oaklon multiparametric apparatus (Testr 35 series, Vernon Hills, USA) at 20°C. In this study, the bacterial charge was always smaller than 50 UFC/mL E. coli. After 30 min, the resulting substances were regarded as ASP. This ASP was inoculated into 80 agar wells prepared in a large plate, as suggested by Pillai et al. (2005) and based on directions of CLSI (2012). Serial dilutions from 0.05 to 50 μg/mL were prepared to interact with free chlorine (5 to 40 μg/mL) from a commercially available preparation of Na-hypochlorite (pH 11) (Hipoclorito de sodio al 13%; Organizacion Química de Calidad, Morelos, México), with free iodine (8 to 64 μg/mL) from iodine-polyvinylpyrrolidone (pH 3.5) (Iodosol 50; Loeffler, CDMX, México) and with a Citrex sanitizer (Citrex, Inc., USA), manufactured with an undisclosed mixture of orange and grapefruit seed extracts and citric acid (2 to 16 mg/mL) (pH 2.3). The tested ranges covered the maximum concentrations recommended by manufacturers and were approximately 8 times more concentrated. Control wells included AMX alone (pH 5.67) (Amoxi-40; FIORI, Querétaro, México) and the corresponding sanitizer, repeating the same serial concentrations as above. The tested bacteria were Bacillus subtilis ATCC 6633. Bioavailability This study phase complied with the Mexican regulations for use of experimental animals, as laid out by the Universidad Nacional Autónoma de Mexico (UNAM) and Mexican prescripts in NOM-062-ZOO-1999. Written permission was granted on 2015 January 13. A total of 1,365 healthy, 6 wk old, Cobb 500 female chickens, weighing 2.5 kg ± 20 g SD were included in this trial. Cobb chickens were managed using the recommended guidelines for this genetic-line (Cobb-Vantress Inc., 2005), having a population density of 30 kg of biomass/m2, nipple-type drinkers (10 chickens/nipple), and manual hopper feeders (50 chickens/feeder). They were placed in an experimental chicken coop with concrete floor (8 wide × 14.5 m long × 4 m high), covered with clean corn-straw bedding, in groups of 35; each group had 3 replicates (105 chickens per group), separated by a wire mesh. The room temperature was kept at 21°C and a mechanic negative-pressure ventilation system was used. The birds were fed with a balanced diet, based on National Research Council requirements (1994). Four Fr studies were carried out for each ASP with 3 replicates per group (see Table 2). Also an Fr study with 3 replicates was performed, administering only AMX in free-sanitizer drinking water that can be described as prepared water. Table 2. Description of groups in which bioavailability of amoxicillin was assessed after oral dosing the product(s) of the interaction of amoxicillin plus and sanitizer. Description Inclusion percentage of Group (mg/kg body weight) amoxicillin and disinfectant in water A Amoxicillin 20 Amoxicillin 5 ACl+ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.002 chlorine 0.00051 ACl++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.004 chlorine 0.0010 ACl+++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.008 chlorine 0.0020 ACl++++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.016 chlorine 0.0040 AI+ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0032 iodine 0.00081 AI++ Amoxicillin 20 plus Amoxicillin 5 plus Iodine 0.0064 iodine 0.0016 AI+++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0128 iodine 0.0032 AI++++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0256 iodine 0.0064 AC+ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 0.8 citric-based 0.21 AC++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 1.6 citric-based 0.4 AC+++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 3.2 citric-based 0.8 AC++++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 6.4 citric-based 1.6 Description Inclusion percentage of Group (mg/kg body weight) amoxicillin and disinfectant in water A Amoxicillin 20 Amoxicillin 5 ACl+ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.002 chlorine 0.00051 ACl++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.004 chlorine 0.0010 ACl+++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.008 chlorine 0.0020 ACl++++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.016 chlorine 0.0040 AI+ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0032 iodine 0.00081 AI++ Amoxicillin 20 plus Amoxicillin 5 plus Iodine 0.0064 iodine 0.0016 AI+++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0128 iodine 0.0032 AI++++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0256 iodine 0.0064 AC+ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 0.8 citric-based 0.21 AC++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 1.6 citric-based 0.4 AC+++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 3.2 citric-based 0.8 AC++++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 6.4 citric-based 1.6 1Highest recommended concentration as suggested by the manufacturer View Large Table 2. Description of groups in which bioavailability of amoxicillin was assessed after oral dosing the product(s) of the interaction of amoxicillin plus and sanitizer. Description Inclusion percentage of Group (mg/kg body weight) amoxicillin and disinfectant in water A Amoxicillin 20 Amoxicillin 5 ACl+ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.002 chlorine 0.00051 ACl++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.004 chlorine 0.0010 ACl+++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.008 chlorine 0.0020 ACl++++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.016 chlorine 0.0040 AI+ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0032 iodine 0.00081 AI++ Amoxicillin 20 plus Amoxicillin 5 plus Iodine 0.0064 iodine 0.0016 AI+++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0128 iodine 0.0032 AI++++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0256 iodine 0.0064 AC+ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 0.8 citric-based 0.21 AC++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 1.6 citric-based 0.4 AC+++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 3.2 citric-based 0.8 AC++++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 6.4 citric-based 1.6 Description Inclusion percentage of Group (mg/kg body weight) amoxicillin and disinfectant in water A Amoxicillin 20 Amoxicillin 5 ACl+ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.002 chlorine 0.00051 ACl++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.004 chlorine 0.0010 ACl+++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.008 chlorine 0.0020 ACl++++ Amoxicillin 20 plus Amoxicillin 5 plus chlorine 0.016 chlorine 0.0040 AI+ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0032 iodine 0.00081 AI++ Amoxicillin 20 plus Amoxicillin 5 plus Iodine 0.0064 iodine 0.0016 AI+++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0128 iodine 0.0032 AI++++ Amoxicillin 20 plus Amoxicillin 5 plus iodine 0.0256 iodine 0.0064 AC+ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 0.8 citric-based 0.21 AC++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 1.6 citric-based 0.4 AC+++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 3.2 citric-based 0.8 AC++++ Amoxicillin 20 plus Amoxicillin 5 plus citric-based 6.4 citric-based 1.6 1Highest recommended concentration as suggested by the manufacturer View Large Each chicken was individually weighed and fasted for 2 h before receiving 1 of the ASP or AMX, as a single oral bolus dose by means of a plastic cannula (BD Insyte; USA) (1.7 mm by 7 cm) attached to a syringe, directed into the crop. Once the cannula was ensured to be properly placed, the experimental solution was slowly delivered. In all cases, the dose was 20 mg/kg of AMX alone or as ASP, and the volume was adjusted to deliver 1 mL of AMX or ASP per chicken. After treatment, blood samplings were taken at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h, using 5 randomly chosen chickens per sampling time and drawing 3 mL of blood per chicken. Each bird was bled only twice. In order to achieve a close timing interval between the drug administration and blood sampling from the wing or jugular vein, technical assistance and clock-watch timing were ensured. Thus, differences between the targeted and the actual blood sampling times were never more than 5 min. Blood samples were immediately centrifuged and approximately 1.0 mL of serum was recovered, identified, and frozen until analyzed. Serum AMX activity/concentrations were determined through the modified agar diffusion analysis, described by Bennet et al. (1966) using Bacillus subtilis (ATCC 6633) as the test microorganism, and then corroborated by high performance liquid chromatography as described by Krasucka et al. (2015). For the modified agar diffusion analysis, the intra-assay variance coefficient was <6.6 and the inter-assay error was <7.4. The analytical assay was linear over a concentration range of 0.05 to 10 μg/mL, with a recovery percentage of 90 ± 2 and a correlation coefficient (r) of 0.96. The detection limit was 0.05 μg/mL, whereas the quantification limit was 0.01 μg/mL. For the high performance liquid chromatography analysis, the intra-assay variance coefficient was <1.7 and the inter-assay error was <1.6. The analytic assay was linear over a concentration range of 0.01 to 15 μg/mL. The mean ± 1 SD recovery was 94 ± 3% (r = 0.98). The detection limit was 0.003 μg/mL, whereas the quantification limit was 0.01 μg/mL. Compliance between both methods for determining AMX serum concentrations was carried out using the amoxicillin-spiked poultry serum samples processed by the 2 analytical techniques. Subtraction of the recover percentages revealed an error of no more than 12.4%. The serum concentrations of AMX vs. time relationships were analyzed using compartmental PK through the software from PKAnalyst (MicroMath, PKAnalyst for Windows. 1995. Version 1.1. MicroMath Inc., St. Louis, MO, USA). Best fit was obtained in model 5 (r < 0.95) with the following general formula: \begin{eqnarray*} {\rm{concentration}} \ ({\rm{time}}) \ &=& \ \frac{{{\rm{Dose}} \ * \ {{\rm{K}}_{{\rm{AE}}}} \ * \ {\rm{time}}}}{{{\rm{Volume}}}}\,{{\rm{e}}^{ - {{\rm{K}}_{{\rm{AE}}}}}}\nonumber\\ && * \, {\rm{time}} \end{eqnarray*} The following PK parameters were obtained: elimination half-life (T½β); Cmax; time to reach Cmax (Tmax): area under the serum concentrations vs. time curve (AUC); area under the moment curve (AUMC); and mean residence time (MRT). Statistical Analysis Antibacterial Activity The obtained data were processed through a generalized linear model (GzLM) (Garson, 2013) for a continuous variable, expressed as follows: \begin{equation*} {{\rm{\eta }}_{\rm{I}}} = {{\rm{\beta }}_0} + {\beta _1}{X_i}_1 + {\beta _2}{X_{j2}} + {\beta _3}{X_1}{X_2} \end{equation*} where ηi = linear predictor of inhibition zone; β0 = intersection; β1 = regression coefficient for the antibiotic X1; β2 = regression coefficient for sanitizer X2; β3 = regression coefficient of the interaction between the different concentrations of antibiotic and sanitizer X1X2. Then, this was analyzed by the maximum likelihood method. Marginal means of interaction were calculated between the different levels of interaction of the antibiotic and each sanitizer (X1X2). Using these multiple comparisons, a Bonferroni procedure was performed. The accepted significance level was P < 0.05. All data analyses were performed using the SPSS statistical package (IBM SPSS, Statistics for Windows. 2011. Version 20.0. IBM Corp. Armonk, NY). Bioavailability Mean value of AMX serum concentrations vs. time from all groups were analyzed by means of a Shapiro–Wilk test (Shapiro and Wilk, 1965), in order to test for normal distribution; and PK parameters with normal distribution using a general linear model as follows: \begin{equation*} Yij = \mu + Di + eij \end{equation*} where Yij = individual pharmacokinetic parameter value in the ith dilution of sanitizer; μ = general mean; Di = dilution of the sanitizer (i = 1, 2, 3, 4, 5); eij = random standard error N (μ, σe2). Bonferroni multiple comparison tests for marginal means and standard errors were adjusted for the considered model; these were performed with a significance level P < 0.05. The model was analyzed by means of least squares, using the SPSS statistical package. RESULTS Antibacterial Activity Table 3 shows the in vitro antibacterial concentration/activity as measured by the inhibition zones, when allowing the interaction of AMX with chlorine, citric-based sanitizer, or iodine. As chlorine sanitizer concentrations increased, the antibacterial action of the ASP diminished in a linear manner (YIJ = 4.35 + 0.047b1−0.035b2); where YIJ: inhibition halos; b1: AMX; b2: chlorine; with an r2 = 0.73, F2, 79 = 105.94; P = 0.0001. The ASP of the AMX-citric-based sanitizer, showed an increase in the antibacterial activity with the highest sanitizer concentrations (50 and 5 μg/mL) (P < 0.05 in both cases). In contrast, the highest concentration of the iodine-based sanitizer (64 μg/mL), diminished the antibacterial activity of the ASP when AMX was tested at 50 and 5 μg/mL (P < 0.05 in both cases). Table 3. Antibacterial in vitro activity of different amoxicillin and water sanitizer combinations. Arithmetic mean ± SD of inhibition zones (mm). Amoxicillin Disinfectant 50 μg/mL 5 μg/mL 0.5 μg/mL 0.05 μg/mL 0 μg/mL Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Chlorine 0.0 μg/mL 36.47a ± 0.68 28.29a ± 0.30 22.77a ± 0.89 15.98a ± 0.26 0.00a ± 0.00 5.0 μg/mL 6.96b ± 0.35 4.78b ± 0.69 4.60b ± 0.68 4.50b ± 0.72 10.06b ± 0.80 10.0 μg/mL 6.32b,c ± 0.42 4.33b–d ± 0.42 3.59b–d ± 0.34 3.05c–e ± 0.51 14.11c ± 0.42 20.0 μg/mL 5.71c ± 0.35 4.02b,d ±0.25 3.41c ± 0.54 2.51d,e ± 0.46 17.42d ± 0.84 40.0 μg/mL 4.41d ± 0.29 3.45 c,d ± 0.46 2.97 c,d ± 0.07 2.49e ± 0.67 22.11e ± 0.49 Citrate-based sanitizer 0.0 mg/mL 35.23a ± 0.28 28.29a ± 0.44 22.77a ± 0.76 15.98a ± 1.07 0.00a ± 0.00 2.0 mg/mL 36.08b ± 0.69 28.96a,d ± 0.65 21.61a ± 0.69 14.83a–c ± 0.47 8.74b ± 0.51 4.0 mg/mL 37.29c ± 0.45 29.26a,d ± 0.75 21.93a ± 0.64 14.95a,b ± 0.16 13.65c ± 0.61 8.0 mg/mL 37.65c ± 0.49 29.32b,d ± 0.39 22.04a ± 0.47 15.69a–c ± 0.64 15.22d ± 0.35 16.0 mg/mL 38.46c ± 0.55 30.85c ± 0.66 22.71a ± 0.57 16.75a ± 0.63 17.14e ± 0.44 Iodine 0.0 μg/mL 35.23a ± 0.56 28.29a ± 0.83 22.77a ± 0.54 15.98a ± 0.21 0.00a ± 0.00 8.0 μg/mL 35.25a ± 0.29 28.35a ± 0.35 23.55a ± 0.42 16.12a ± 0.31 14.25b ± 0.28 16.0 μg/mL 35.18a ± 0.66 28.31a ± 0.22 23.11a ± 0.57 15.83a ± 0.56 15.54c ± 0.68 32.0 μg/mL 34.99a ± 0.65 28.40a ± 0.41 22.68a ± 0.47 15.72a ± 0.48 16.92d ± 0.41 64.0 μg/mL 33.78b ± 0.44 26.96b ± 0.51 22.46a ± 0.32 15.66a ± 0.73 19.20e ± 0.38 Amoxicillin Disinfectant 50 μg/mL 5 μg/mL 0.5 μg/mL 0.05 μg/mL 0 μg/mL Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Chlorine 0.0 μg/mL 36.47a ± 0.68 28.29a ± 0.30 22.77a ± 0.89 15.98a ± 0.26 0.00a ± 0.00 5.0 μg/mL 6.96b ± 0.35 4.78b ± 0.69 4.60b ± 0.68 4.50b ± 0.72 10.06b ± 0.80 10.0 μg/mL 6.32b,c ± 0.42 4.33b–d ± 0.42 3.59b–d ± 0.34 3.05c–e ± 0.51 14.11c ± 0.42 20.0 μg/mL 5.71c ± 0.35 4.02b,d ±0.25 3.41c ± 0.54 2.51d,e ± 0.46 17.42d ± 0.84 40.0 μg/mL 4.41d ± 0.29 3.45 c,d ± 0.46 2.97 c,d ± 0.07 2.49e ± 0.67 22.11e ± 0.49 Citrate-based sanitizer 0.0 mg/mL 35.23a ± 0.28 28.29a ± 0.44 22.77a ± 0.76 15.98a ± 1.07 0.00a ± 0.00 2.0 mg/mL 36.08b ± 0.69 28.96a,d ± 0.65 21.61a ± 0.69 14.83a–c ± 0.47 8.74b ± 0.51 4.0 mg/mL 37.29c ± 0.45 29.26a,d ± 0.75 21.93a ± 0.64 14.95a,b ± 0.16 13.65c ± 0.61 8.0 mg/mL 37.65c ± 0.49 29.32b,d ± 0.39 22.04a ± 0.47 15.69a–c ± 0.64 15.22d ± 0.35 16.0 mg/mL 38.46c ± 0.55 30.85c ± 0.66 22.71a ± 0.57 16.75a ± 0.63 17.14e ± 0.44 Iodine 0.0 μg/mL 35.23a ± 0.56 28.29a ± 0.83 22.77a ± 0.54 15.98a ± 0.21 0.00a ± 0.00 8.0 μg/mL 35.25a ± 0.29 28.35a ± 0.35 23.55a ± 0.42 16.12a ± 0.31 14.25b ± 0.28 16.0 μg/mL 35.18a ± 0.66 28.31a ± 0.22 23.11a ± 0.57 15.83a ± 0.56 15.54c ± 0.68 32.0 μg/mL 34.99a ± 0.65 28.40a ± 0.41 22.68a ± 0.47 15.72a ± 0.48 16.92d ± 0.41 64.0 μg/mL 33.78b ± 0.44 26.96b ± 0.51 22.46a ± 0.32 15.66a ± 0.73 19.20e ± 0.38 a–eDifferent letters in each column indicate a statistically significant difference (P ≤ 0.05). Note: Multiple comparisons by means of Bonferroni tests were carried out, using marginal means and standard error values as adjusted by the statistical model. View Large Table 3. Antibacterial in vitro activity of different amoxicillin and water sanitizer combinations. Arithmetic mean ± SD of inhibition zones (mm). Amoxicillin Disinfectant 50 μg/mL 5 μg/mL 0.5 μg/mL 0.05 μg/mL 0 μg/mL Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Chlorine 0.0 μg/mL 36.47a ± 0.68 28.29a ± 0.30 22.77a ± 0.89 15.98a ± 0.26 0.00a ± 0.00 5.0 μg/mL 6.96b ± 0.35 4.78b ± 0.69 4.60b ± 0.68 4.50b ± 0.72 10.06b ± 0.80 10.0 μg/mL 6.32b,c ± 0.42 4.33b–d ± 0.42 3.59b–d ± 0.34 3.05c–e ± 0.51 14.11c ± 0.42 20.0 μg/mL 5.71c ± 0.35 4.02b,d ±0.25 3.41c ± 0.54 2.51d,e ± 0.46 17.42d ± 0.84 40.0 μg/mL 4.41d ± 0.29 3.45 c,d ± 0.46 2.97 c,d ± 0.07 2.49e ± 0.67 22.11e ± 0.49 Citrate-based sanitizer 0.0 mg/mL 35.23a ± 0.28 28.29a ± 0.44 22.77a ± 0.76 15.98a ± 1.07 0.00a ± 0.00 2.0 mg/mL 36.08b ± 0.69 28.96a,d ± 0.65 21.61a ± 0.69 14.83a–c ± 0.47 8.74b ± 0.51 4.0 mg/mL 37.29c ± 0.45 29.26a,d ± 0.75 21.93a ± 0.64 14.95a,b ± 0.16 13.65c ± 0.61 8.0 mg/mL 37.65c ± 0.49 29.32b,d ± 0.39 22.04a ± 0.47 15.69a–c ± 0.64 15.22d ± 0.35 16.0 mg/mL 38.46c ± 0.55 30.85c ± 0.66 22.71a ± 0.57 16.75a ± 0.63 17.14e ± 0.44 Iodine 0.0 μg/mL 35.23a ± 0.56 28.29a ± 0.83 22.77a ± 0.54 15.98a ± 0.21 0.00a ± 0.00 8.0 μg/mL 35.25a ± 0.29 28.35a ± 0.35 23.55a ± 0.42 16.12a ± 0.31 14.25b ± 0.28 16.0 μg/mL 35.18a ± 0.66 28.31a ± 0.22 23.11a ± 0.57 15.83a ± 0.56 15.54c ± 0.68 32.0 μg/mL 34.99a ± 0.65 28.40a ± 0.41 22.68a ± 0.47 15.72a ± 0.48 16.92d ± 0.41 64.0 μg/mL 33.78b ± 0.44 26.96b ± 0.51 22.46a ± 0.32 15.66a ± 0.73 19.20e ± 0.38 Amoxicillin Disinfectant 50 μg/mL 5 μg/mL 0.5 μg/mL 0.05 μg/mL 0 μg/mL Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Chlorine 0.0 μg/mL 36.47a ± 0.68 28.29a ± 0.30 22.77a ± 0.89 15.98a ± 0.26 0.00a ± 0.00 5.0 μg/mL 6.96b ± 0.35 4.78b ± 0.69 4.60b ± 0.68 4.50b ± 0.72 10.06b ± 0.80 10.0 μg/mL 6.32b,c ± 0.42 4.33b–d ± 0.42 3.59b–d ± 0.34 3.05c–e ± 0.51 14.11c ± 0.42 20.0 μg/mL 5.71c ± 0.35 4.02b,d ±0.25 3.41c ± 0.54 2.51d,e ± 0.46 17.42d ± 0.84 40.0 μg/mL 4.41d ± 0.29 3.45 c,d ± 0.46 2.97 c,d ± 0.07 2.49e ± 0.67 22.11e ± 0.49 Citrate-based sanitizer 0.0 mg/mL 35.23a ± 0.28 28.29a ± 0.44 22.77a ± 0.76 15.98a ± 1.07 0.00a ± 0.00 2.0 mg/mL 36.08b ± 0.69 28.96a,d ± 0.65 21.61a ± 0.69 14.83a–c ± 0.47 8.74b ± 0.51 4.0 mg/mL 37.29c ± 0.45 29.26a,d ± 0.75 21.93a ± 0.64 14.95a,b ± 0.16 13.65c ± 0.61 8.0 mg/mL 37.65c ± 0.49 29.32b,d ± 0.39 22.04a ± 0.47 15.69a–c ± 0.64 15.22d ± 0.35 16.0 mg/mL 38.46c ± 0.55 30.85c ± 0.66 22.71a ± 0.57 16.75a ± 0.63 17.14e ± 0.44 Iodine 0.0 μg/mL 35.23a ± 0.56 28.29a ± 0.83 22.77a ± 0.54 15.98a ± 0.21 0.00a ± 0.00 8.0 μg/mL 35.25a ± 0.29 28.35a ± 0.35 23.55a ± 0.42 16.12a ± 0.31 14.25b ± 0.28 16.0 μg/mL 35.18a ± 0.66 28.31a ± 0.22 23.11a ± 0.57 15.83a ± 0.56 15.54c ± 0.68 32.0 μg/mL 34.99a ± 0.65 28.40a ± 0.41 22.68a ± 0.47 15.72a ± 0.48 16.92d ± 0.41 64.0 μg/mL 33.78b ± 0.44 26.96b ± 0.51 22.46a ± 0.32 15.66a ± 0.73 19.20e ± 0.38 a–eDifferent letters in each column indicate a statistically significant difference (P ≤ 0.05). Note: Multiple comparisons by means of Bonferroni tests were carried out, using marginal means and standard error values as adjusted by the statistical model. View Large Bioavailability Figure 1 shows the AMX serum profiles from ASP that differ from the values obtained for the reference group (A), in a statistically significant manner (P < 0.05) and resulted in higher Cmax, MRT, AUC, and T ≥ MIC values. Figure 2 depicts the opposite: lower Cmax, AUC, and T ≥ MIC values of ASP, compared to the reference values. These figures consider the breakpoint for resistant Pasteurella sp. (1 μg/mL) as reference microorganism (CLSI, 2012). Table 4 presents the PK parameters for AMX derived from ASP and the reference ones. Two groups presented higher Cmax, MRT, and T ≥ MIC values than the A group: AC+++ and AC++++, which belong to ASP from the 2 highest concentrations of ASP derived from the citrate-based sanitizer combined with AMX (P < 0.05 in both cases). The T ≥ MIC obtained for the AC+++ group was 7.44 h; that is, 24% higher than the control one. This same parameter was 7.75 h for the AC++++ group (29% higher than the control group). All groups of the chlorine series caused the ASP to have statistically inferior Cmax, values compared to the reference value (P < 0.05 in all cases). Other variations in PK parameters were also noted, namely a decrease in AUC, MRT, and obviously an inferior T ≥ MIC (Table 4). Figure 1. View largeDownload slide Mean ± SD of amoxicillin serum concentrations after a single bolus administration of 20 mg/kg delivered directly into the proventriculus (group A), and amoxicillin serum concentrations derived from administering amoxicillin plus different concentrations of water sanitizers, as follows: AC+++ group: amoxicillin 20 mg/kg plus citrate-based sanitizer 3.2 mg/kg and AC++++ group: amoxicillin 20 mg/kg plus citrate-based sanitizer 6. 4 mg/kg. Figure 1. View largeDownload slide Mean ± SD of amoxicillin serum concentrations after a single bolus administration of 20 mg/kg delivered directly into the proventriculus (group A), and amoxicillin serum concentrations derived from administering amoxicillin plus different concentrations of water sanitizers, as follows: AC+++ group: amoxicillin 20 mg/kg plus citrate-based sanitizer 3.2 mg/kg and AC++++ group: amoxicillin 20 mg/kg plus citrate-based sanitizer 6. 4 mg/kg. Figure 2. View largeDownload slide Mean ± SD of serum concentrations of amoxicillin after a single bolus administration of 20 mg/kg delivered into the proventriculus (group A), and serum concentrations of amoxicillin derived from administering amoxicillin plus different concentration of water sanitizers as follows: ACL+ group: amoxicillin 20 mg/kg plus chlorine 0.002 mg/kg (from sodium hypochlorite), ACL++ group: amoxicillin 20 mg/kg plus chlorine 0.004 mg/kg, ACL+++ group: amoxicillin 20 mg/kg plus chlorine 0.008 mg/kg, ACL++++ group: amoxicillin 20 mg/kg plus chlorine 0.016 mg/kg and AI++++ group: amoxicillin 20 mg/kg plus iodine 0.0256 mg/kg. Figure 2. View largeDownload slide Mean ± SD of serum concentrations of amoxicillin after a single bolus administration of 20 mg/kg delivered into the proventriculus (group A), and serum concentrations of amoxicillin derived from administering amoxicillin plus different concentration of water sanitizers as follows: ACL+ group: amoxicillin 20 mg/kg plus chlorine 0.002 mg/kg (from sodium hypochlorite), ACL++ group: amoxicillin 20 mg/kg plus chlorine 0.004 mg/kg, ACL+++ group: amoxicillin 20 mg/kg plus chlorine 0.008 mg/kg, ACL++++ group: amoxicillin 20 mg/kg plus chlorine 0.016 mg/kg and AI++++ group: amoxicillin 20 mg/kg plus iodine 0.0256 mg/kg. Table 4. Mean ± SD of pharmacokinetic1 variables for amoxicillin administered at 20 mg/kg in broiler chickens after dosing them orally with the interaction products of amoxicillin plus chlorine (from sodium hypochlorite), or a citrate-based or iodine-based water sanitizer. Variable Group T½β2 (h) Tmax3 (h) Cmax4 (μg/mL) AUC5 (μg/mL·h) MRT6 (h) Fr7 (%) T ≥ MIC8 (h) A 0.96a ± 0.10 1.39a ± 0.12 12.54a ± 0.80 44.02a ± 0.61 2.78a ± 0.04 100a ± 0.00 6.00a ± 0.01 ACl+ 0.78b ± 0.06 1.14b ± 0.04 5.78b ± 1.36 12.14b ± 0.99 2.30b ± 0.14 27.58b ± 2.24 4.40b ± 0.06 ACl++ 0.24c ± 0.09 0.35c ± 0.03 4.12c ± 0.28 5.22c ± 0.08 0.72c ± 0.03 11.85c ± 0.19 2.44c ± 0.02 ACl+++ 0.19c ± 0.08 0.29c ± 0.04 3.80b,c ± 1.20 2.70d ± 0.06 0.59c ± 0.12 6.14d ± 0.13 0.85d ± 0.03 ACl++++ — 0.54d ± 0.09 1.67d ± 0.60 — — — 0.62e ± 0.01 AI+ 0.96a ± 0.03 1.49a ± 0.05 12.28a ± 0.46 44.04a ± 1.31 2.79a ± 0.11 100.04a ± 2.97 6.03a ± 0.02 AI++ 0.98a ± 0.04 1.50a ± 0.10 12.45a ± 0.66 44.00a ± 2.26 2.79a ± 0.12 99.96a ± 5.14 6.02a ± 0.01 AI+++ 0.94a ± 0.03 1.46a ± 0.06 11.91a ± 0.74 44.00a ± 2.66 2.78a ± 0.09 99.96a ± 6.06 5.99a ± 0.03 AI++++ 1.17b ± 0.09 1.66b ± 0.08 10.62b ± 0.34 41.67a ± 1.16 2.07b ± 0.11 94.67b ± 2.64 4.02b ± 0.01 AC+ 0.94a,b ± 0.14 1.30a,b ± 0.16 12.63a ± 0.39 44.67a ± 3.39 2.71a ± 0.17 101.49a ± 7.70 6.02a ± 0.02 AC++ 0.90a,b ± 0.09 1.28a,b ± 0.22 13.72b ± 0.09 48.64a,b ± 3.67 2.55a ± 0.10 110.51b ± 8.33 6.02a ± 0.01 AC+++ 0.87a,b ± 0.05 1.24a,b ± 0.09 15.07c ± 0.63 52.82b ± 1.45 3.32b ± 0.17 119.99b ± 3.29 7.44b ± 0.06 AC++++ 0.77b ± 0.06 1.13b ± 0.07 15.47c ± 0.26 58.28c ± 4.43 4.06c ± 0.27 132.39c ± 10.08 7.75c ± 0.02 Variable Group T½β2 (h) Tmax3 (h) Cmax4 (μg/mL) AUC5 (μg/mL·h) MRT6 (h) Fr7 (%) T ≥ MIC8 (h) A 0.96a ± 0.10 1.39a ± 0.12 12.54a ± 0.80 44.02a ± 0.61 2.78a ± 0.04 100a ± 0.00 6.00a ± 0.01 ACl+ 0.78b ± 0.06 1.14b ± 0.04 5.78b ± 1.36 12.14b ± 0.99 2.30b ± 0.14 27.58b ± 2.24 4.40b ± 0.06 ACl++ 0.24c ± 0.09 0.35c ± 0.03 4.12c ± 0.28 5.22c ± 0.08 0.72c ± 0.03 11.85c ± 0.19 2.44c ± 0.02 ACl+++ 0.19c ± 0.08 0.29c ± 0.04 3.80b,c ± 1.20 2.70d ± 0.06 0.59c ± 0.12 6.14d ± 0.13 0.85d ± 0.03 ACl++++ — 0.54d ± 0.09 1.67d ± 0.60 — — — 0.62e ± 0.01 AI+ 0.96a ± 0.03 1.49a ± 0.05 12.28a ± 0.46 44.04a ± 1.31 2.79a ± 0.11 100.04a ± 2.97 6.03a ± 0.02 AI++ 0.98a ± 0.04 1.50a ± 0.10 12.45a ± 0.66 44.00a ± 2.26 2.79a ± 0.12 99.96a ± 5.14 6.02a ± 0.01 AI+++ 0.94a ± 0.03 1.46a ± 0.06 11.91a ± 0.74 44.00a ± 2.66 2.78a ± 0.09 99.96a ± 6.06 5.99a ± 0.03 AI++++ 1.17b ± 0.09 1.66b ± 0.08 10.62b ± 0.34 41.67a ± 1.16 2.07b ± 0.11 94.67b ± 2.64 4.02b ± 0.01 AC+ 0.94a,b ± 0.14 1.30a,b ± 0.16 12.63a ± 0.39 44.67a ± 3.39 2.71a ± 0.17 101.49a ± 7.70 6.02a ± 0.02 AC++ 0.90a,b ± 0.09 1.28a,b ± 0.22 13.72b ± 0.09 48.64a,b ± 3.67 2.55a ± 0.10 110.51b ± 8.33 6.02a ± 0.01 AC+++ 0.87a,b ± 0.05 1.24a,b ± 0.09 15.07c ± 0.63 52.82b ± 1.45 3.32b ± 0.17 119.99b ± 3.29 7.44b ± 0.06 AC++++ 0.77b ± 0.06 1.13b ± 0.07 15.47c ± 0.26 58.28c ± 4.43 4.06c ± 0.27 132.39c ± 10.08 7.75c ± 0.02 a–d a–dDifferent letters within each column indicate a statistically significant difference within a given group (P ≤ 0.05). 1 1Pharmacokinetic parameters were calculated with non-transformed data considering that Shapiro–Wilk test indicated a normal distribution of the data. 1 2T½β = elimination half-life. 2 3Tmax = time to reach Cmax. 3 4Cmax = maximum serum concentration. 4 5AUC = area under the serum concentrations vs. time curve. 5 6MRT = mean residence time. 6 7Fr = relative bioavailability (AUCinteraction/AUCE) * 100. 8 8T ≥ MIC = time in which serum activity/concentrations were at or above the MIC value. View Large Table 4. Mean ± SD of pharmacokinetic1 variables for amoxicillin administered at 20 mg/kg in broiler chickens after dosing them orally with the interaction products of amoxicillin plus chlorine (from sodium hypochlorite), or a citrate-based or iodine-based water sanitizer. Variable Group T½β2 (h) Tmax3 (h) Cmax4 (μg/mL) AUC5 (μg/mL·h) MRT6 (h) Fr7 (%) T ≥ MIC8 (h) A 0.96a ± 0.10 1.39a ± 0.12 12.54a ± 0.80 44.02a ± 0.61 2.78a ± 0.04 100a ± 0.00 6.00a ± 0.01 ACl+ 0.78b ± 0.06 1.14b ± 0.04 5.78b ± 1.36 12.14b ± 0.99 2.30b ± 0.14 27.58b ± 2.24 4.40b ± 0.06 ACl++ 0.24c ± 0.09 0.35c ± 0.03 4.12c ± 0.28 5.22c ± 0.08 0.72c ± 0.03 11.85c ± 0.19 2.44c ± 0.02 ACl+++ 0.19c ± 0.08 0.29c ± 0.04 3.80b,c ± 1.20 2.70d ± 0.06 0.59c ± 0.12 6.14d ± 0.13 0.85d ± 0.03 ACl++++ — 0.54d ± 0.09 1.67d ± 0.60 — — — 0.62e ± 0.01 AI+ 0.96a ± 0.03 1.49a ± 0.05 12.28a ± 0.46 44.04a ± 1.31 2.79a ± 0.11 100.04a ± 2.97 6.03a ± 0.02 AI++ 0.98a ± 0.04 1.50a ± 0.10 12.45a ± 0.66 44.00a ± 2.26 2.79a ± 0.12 99.96a ± 5.14 6.02a ± 0.01 AI+++ 0.94a ± 0.03 1.46a ± 0.06 11.91a ± 0.74 44.00a ± 2.66 2.78a ± 0.09 99.96a ± 6.06 5.99a ± 0.03 AI++++ 1.17b ± 0.09 1.66b ± 0.08 10.62b ± 0.34 41.67a ± 1.16 2.07b ± 0.11 94.67b ± 2.64 4.02b ± 0.01 AC+ 0.94a,b ± 0.14 1.30a,b ± 0.16 12.63a ± 0.39 44.67a ± 3.39 2.71a ± 0.17 101.49a ± 7.70 6.02a ± 0.02 AC++ 0.90a,b ± 0.09 1.28a,b ± 0.22 13.72b ± 0.09 48.64a,b ± 3.67 2.55a ± 0.10 110.51b ± 8.33 6.02a ± 0.01 AC+++ 0.87a,b ± 0.05 1.24a,b ± 0.09 15.07c ± 0.63 52.82b ± 1.45 3.32b ± 0.17 119.99b ± 3.29 7.44b ± 0.06 AC++++ 0.77b ± 0.06 1.13b ± 0.07 15.47c ± 0.26 58.28c ± 4.43 4.06c ± 0.27 132.39c ± 10.08 7.75c ± 0.02 Variable Group T½β2 (h) Tmax3 (h) Cmax4 (μg/mL) AUC5 (μg/mL·h) MRT6 (h) Fr7 (%) T ≥ MIC8 (h) A 0.96a ± 0.10 1.39a ± 0.12 12.54a ± 0.80 44.02a ± 0.61 2.78a ± 0.04 100a ± 0.00 6.00a ± 0.01 ACl+ 0.78b ± 0.06 1.14b ± 0.04 5.78b ± 1.36 12.14b ± 0.99 2.30b ± 0.14 27.58b ± 2.24 4.40b ± 0.06 ACl++ 0.24c ± 0.09 0.35c ± 0.03 4.12c ± 0.28 5.22c ± 0.08 0.72c ± 0.03 11.85c ± 0.19 2.44c ± 0.02 ACl+++ 0.19c ± 0.08 0.29c ± 0.04 3.80b,c ± 1.20 2.70d ± 0.06 0.59c ± 0.12 6.14d ± 0.13 0.85d ± 0.03 ACl++++ — 0.54d ± 0.09 1.67d ± 0.60 — — — 0.62e ± 0.01 AI+ 0.96a ± 0.03 1.49a ± 0.05 12.28a ± 0.46 44.04a ± 1.31 2.79a ± 0.11 100.04a ± 2.97 6.03a ± 0.02 AI++ 0.98a ± 0.04 1.50a ± 0.10 12.45a ± 0.66 44.00a ± 2.26 2.79a ± 0.12 99.96a ± 5.14 6.02a ± 0.01 AI+++ 0.94a ± 0.03 1.46a ± 0.06 11.91a ± 0.74 44.00a ± 2.66 2.78a ± 0.09 99.96a ± 6.06 5.99a ± 0.03 AI++++ 1.17b ± 0.09 1.66b ± 0.08 10.62b ± 0.34 41.67a ± 1.16 2.07b ± 0.11 94.67b ± 2.64 4.02b ± 0.01 AC+ 0.94a,b ± 0.14 1.30a,b ± 0.16 12.63a ± 0.39 44.67a ± 3.39 2.71a ± 0.17 101.49a ± 7.70 6.02a ± 0.02 AC++ 0.90a,b ± 0.09 1.28a,b ± 0.22 13.72b ± 0.09 48.64a,b ± 3.67 2.55a ± 0.10 110.51b ± 8.33 6.02a ± 0.01 AC+++ 0.87a,b ± 0.05 1.24a,b ± 0.09 15.07c ± 0.63 52.82b ± 1.45 3.32b ± 0.17 119.99b ± 3.29 7.44b ± 0.06 AC++++ 0.77b ± 0.06 1.13b ± 0.07 15.47c ± 0.26 58.28c ± 4.43 4.06c ± 0.27 132.39c ± 10.08 7.75c ± 0.02 a–d a–dDifferent letters within each column indicate a statistically significant difference within a given group (P ≤ 0.05). 1 1Pharmacokinetic parameters were calculated with non-transformed data considering that Shapiro–Wilk test indicated a normal distribution of the data. 1 2T½β = elimination half-life. 2 3Tmax = time to reach Cmax. 3 4Cmax = maximum serum concentration. 4 5AUC = area under the serum concentrations vs. time curve. 5 6MRT = mean residence time. 6 7Fr = relative bioavailability (AUCinteraction/AUCE) * 100. 8 8T ≥ MIC = time in which serum activity/concentrations were at or above the MIC value. View Large High iodine concentrations produced a decrease in Cmax for the EI++++ group (64 μg/mL of free iodine) (P < 0.05). Additionally, a decrease of MRT, Fr, and T ≥ MIC was also obtained for this group (P < 0.05 in all cases). As referred before, the ASP from 2 of the highest concentrations of citrate-based sanitizer showed statistically higher Cmax, MRT, Fr, and T ≥ MIC (P < 0.05 in all cases). DISCUSSION The results derived from the interaction of water sanitizers with in vitro AMX and on the bioavailability of AMX in broiler chickens, reveal 3 different outcomes. If the interaction is with sodium hypochlorite, there is an almost linear progressive reduction of the in vitro antibacterial activity of AMX and this is somehow replicated in the observed Cmax, MRT, Fr, and T ≥ MIC values. Based on their oxidizing ability, halogenated disinfectants (iodine and chlorine) are commonly used as drinking water sanitizers (McDonnell and Russel, 1999; Sumano et al., 2015), and it is precisely this oxidative capacity that may react and alter the chemical structure of amoxicillin or modify its antimicrobial effects. For example, it has been stated that the aromatic ring and the amino group of AMX are susceptible to chlorine oxidation (Acero et al., 2010; Wang et al., 2010). In contrast and despite being also a halogenated compound, the iodine-based sanitizer used (iodine-polyvinylpyrrolidone, pH 3.5) was unable to interact sufficiently with the AMX and induce tangible changes in either the in vitro antibacterial activity or in the Fr patterns. Yet, reduction in the Cmax antibacterial activity, Fr, and MRT was only observed with the highest concentration of iodine. Iodine-polyvinylpyrrolidone is a slow iodine release formulation and this may partly explain the in vitro and in vivo antibacterial activity/concentration patterns. It is possible that if a longer AMX–iodine interaction time was allowed, a greater deterioration of AMX’s antibacterial activity could have been obtained. This must be further studied, because the AMX prevalence in water tanks could last much longer than 30 min; which was the AMX–water sanitizer interaction-time allowed during this trial. The citrate-based compound enhanced the in vitro AMX antibacterial activity. Relative bioavailability followed a similar pattern, where the Cmax, Fr, and MRT were significantly enhanced (P <0.05) with the 2 highest concentrations of the citrate-based water sanitizer (8 and 16 mg/mL) (see Table 2). This type of water sanitizers are usually made from grapefruit extracts and other citric seeds (Bevilacqua et al., 2013). It has been reported that grapefruit seed extracts contain furanocoumarins, which in turn can reduce or inhibit the gastrointestinal glycoprotein G activity, allowing a better bioavailability of some drugs (Dahan and Altman, 2004; Bailey, 2010; Ahmed et al., 2015). Additionally, it has been shown that grapefruit extracts also inhibit the P-450 (CYP) enzyme activity at the gastrointestinal epithelial level (Bailey, et al., 1998; Kane and Lipsky, 2000; Giorgi et al., 2003), contributing to a better absorption of some drugs. Consequently, it is likely to think that the active principles of the citrate-based sanitizer contribute to increase the Cmax, MRT, and Fr of AMX; such as what was observed in the results obtained during this experimental model (Kalpana et al., 2015). In this work, the chlorine, iodine, and citrate-based studied sanitizers included the maximum recommended concentrations found in the literature, and even they were 8 times more concentrated (CES, 1998; Sumano et al., 2015). It is not uncommon that the drinking water in poultry coops contain high concentrations of iodine, chlorine, or citrate products as a result of accidental accumulation or due to an inadequate dosing (Maharjan et al., 2016). For example, in the case of iodine, it has been used at high doses to treat outbreaks caused by adenovirus: 1.6 times the maximum recommended dose of iodine as water sanitizer (Abdul-Aziz and Hasan, 1996). Alterations of either, in vitro antibacterial activity or ASP bioavailability at lower concentrations, were generally much more discrete. Further work is needed to define changes in these parameters when longer interactions occurs within the water tank, considering that under field conditions the total consumption of the water source may take much longer than 30 min. However, a conclusion that can be reached from this study is that, if it has been decided to administer AMX to chickens through their drinking water, the water sanitizer of choice is the citrate-based one. The rational use of antimicrobials has been worldwide prioritized due to the increase of microbial resistance to these agents and the lack of new families of antimicrobial agents destined to poultry medicine (Saga and Yamaguchi, 2009; Bjork et al., 2015; Limayem et al., 2015). One of the obvious strategies that emerge from this situation is the use of antibacterial drugs based on PK/PD ratios (Errecalde, 2004). Amoxicillin being a time-dependent antimicrobial drug, the most important PK/PD ratio is T ≥ MIC. In order to obtain optimal clinical results, it has been set that depending on the pathogen, the dosage interval of T ≥ MIC can range from 40 to 100% (McKellar et al., 2004; MacGowan, 2011). In spite of the above, Cmax has also been highlighted as important and has been set at 1–5 times the MIC value (McKellar et al., 2004). It is unlikely that commercial preparations of AMX comply with the referred ratios, because AMX preparations for poultry that are administered through drinking water are usually prescribed with a 24-h interval. For example, based on the MRT and Cmax obtained for AMX during this trial (2.78 h ± 0.04 and 12.54 μg/mL ± 0.80, respectively) and considering a theoretical MIC of 1 μg/mL for Pasteurella sp. (Huang et al., 2009), it can be observed that the referred PK/PD ratios were not met using AMX alone. The results obtained in this trial, coincide with those of Krasucka and Kowalski (2010). By analyzing their reported serum concentrations of AMX vs. time relationships in poultry, they obtained a T ≥ MIC of only 8.33% after dosing broiler chickens with 20 mg/kg and considering the same Pasteurella sp. MIC values. They also reported a Cmax value that was only equivalent to the MIC for this pathogen. Similarly, Abo et al. (2004) showed that a dose of 10 mg/kg administered orally induced a T ≥ MIC of only 2.91 to 12.5%, also considering Pasteurella sp. Similarly, by analyzing data from Kandeel (2015), a dose of 10 mg/kg PO offered a T ≥ MIC that ranged from 8.3 to 25%. Also, the Cmax failed to reach 4–5 times the MIC value. In contrast, the same analysis performed on an earlier study by Anadón et al. (1996), offered AMX serum concentrations that complied well with the referred PK/PD ratios. Differences in the analytical precision may account for discrepancies among these researchers. It has been shown that the so-called post-antibiotic effect is either minimum or nonexistent for beta-lactamic antibacterial drugs (Cars, 1997; Papich, 2014). Thus, dosing intervals should ideally be established at the time when serum concentrations of the beta-lactamic drug fall below the MIC value. This is not possible for poultry medicine. However, in order to comply better with T ≥ MIC, the AMX dosing should at least be set twice a day. A specialized pharmaceutical design of AMX for poultry should then be investigated in order to provide protection to this drug when it has been diluted in drinking water; particularly, if it contains chlorine-based water sanitizers. The rapid and significant degradation of AMX in water has been demonstrated (Jerzsele and Nagy, 2009). Also, other variables should be considered, such as water temperature, bacterial load, water hardness, among others (Sumano et al., 2004; Fairchild et al., 2006). FUNDING Part of this work was supported by the Support Program for Research Projects and Technological Innovation (PAPIIT) of the National Autonomous University of Mexico (UNAM) No. IN212815. REFERENCES Abdul-Aziz T. A. , Hasan S. Y. . 1996 . Preliminary observations on the efficacy of an iodophor in reducing the mortality in chickens experimentally affected by the “hydropericardium syndrome.” Vet. Res. Commun. 20 : 191 – 194 . Google Scholar CrossRef Search ADS PubMed Abo E. K. , Al-Tarazi Y. H. , Al-Bataineh M. M. . 2004 . Comparative pharmacokinetics and bioavailability of amoxycillin in chickens after intravenous, intramuscular and oral administrations . Vet. Res. Commun. 28 : 599 – 607 . Google Scholar CrossRef Search ADS PubMed Acero J. L. , Benitez F. J. , Real F. J. , Roldan G. . 2010 . Kinetics of aqueous chlorination of some pharmaceuticals and their elimination from water matrices . Water Res . 44 : 4158 – 4170 . Google Scholar CrossRef Search ADS PubMed Agunos A. , Léger D. , Carson C. . 2012 . Review of antimicrobial therapy of selected bacterial diseases in broiler chickens in Canada . Can. Vet. J . 53 : 1289 – 1300 . Google Scholar PubMed Ahmed I. S. , Hassan M. A. , Kondo T. . 2015 . Effect of lyophilized grapefruit juice on P-glycoprotein-mediated drug transport in-vitro and in-vivo . Drug Dev. Ind. Pharm . 41 : 375 – 381 . Google Scholar CrossRef Search ADS PubMed Anadón A. , Martinez M. R. , Larrañaga , Diaz M. J. , Bringas P. , Fernandez M. C. , Martinez M. A. , Fernandez M. L. , Cruz . 1996 . Pharmacokinetics of amoxicillin in broiler chickens . Avian Pathol. 25 : 449 – 458 . Google Scholar CrossRef Search ADS PubMed Bailey M. 1999 . The water requirements of poultry . 342 in Developments in Poultry Nutrition 2 . Wiseman J. , Garnsworthy P. C. eds. Nottingham University Press , United Kindgom . Google Scholar CrossRef Search ADS Bailey D. 2010 . Fruit juice inhibition of uptake transport: a new type of food-drug interaction . Br. J. Clin. Pharmacol . 70 : 645 – 655 . Google Scholar CrossRef Search ADS PubMed Bailey D. , Malcolm J. , Arnold O. , Spence D. . 1998 . Grapefruit juice-drug interactions . Br. J. Clin. Pharmacol. 46 : 101 – 110 . Google Scholar CrossRef Search ADS PubMed Bell D. , Weaver W. . 2002 . Consumption and quality of water . Pages 411 – 430 in Commercial Chicken Meat and Egg Production . 5th ed . Bell D. , Weaver W. eds. Kluwer Academic Publishers , Norwell, MA . Google Scholar CrossRef Search ADS Bennet J. , Brodie J. L. , Benner E. J. , Kirby W. . 1966 . Simplified accurate method for antibiotic assay of clinical specimens . Am. Soc. Microbiol . 14 : 170 – 177 . Bevilacqua A. , Campaniello D. , Speranza B. , Sinigaglia M. , Corbo M. R. . 2013 . Control of Alicyclobacillus acidoterrestris in apple juice by citrus extracts and a mild heat-treatment . Food Control 31 : 553 – 559 . Google Scholar CrossRef Search ADS Bjork K. E. , Kopral C. A. , Wagner B. A. , Dargatz D. A. . 2015 . Comparison of mixed effects models of antimicrobial resistance metrics of livestock and poultry Salmonella isolates from a national monitoring system . Prev. Vet. Med . 122 : 265 – 272 . Google Scholar CrossRef Search ADS PubMed Cars O. 1997 . Efficacy of beta-lactam antibiotics: integration of pharmacokinetics and pharmacodynamics . Diagn. Microbiol. Infect. Dis. 27 : 29 – 33 . Google Scholar CrossRef Search ADS PubMed CLSI . 2012 . Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard . Clinical and Laboratory Standards Institute , Wayne, PA . Cobb-Vantress Inc . 2005 . COBB Guia de Manejo de Pollo de Engorde . Cobb-Vantress Inc. , Siloam Springs, Arkansas . Cooperative Extension Service (CES) . 1998 . Sanitizing poultry drinking water . 1 in The Disaster Handbook . National Edition Institute of Food and Agricultural Sciences . University of Florida, USA . PubMed PubMed Dahan A. , Altman H. . 2004 . Food-drug interaction: grapefruit juice augments drug bioavailability-mechanism, extent and relevance . Eur J Clin Nutr . 58 : 1 – 9 . Google Scholar CrossRef Search ADS PubMed EMA . 2016 . Sales of Veterinary Antimicrobial Agents in 29 European Countries in 2014. Sixth ESVAC Report . European Medicines Agency , London . Errecalde, J. O. 2004 . Uso de antimicrobianos en animale de consumo, incidencia del desarrollo de resistencias en salud pública . FAO , Roma, Itallia . Esmail S. 1996 . Water: the vital nutrient . Poult. Int . 15 : 72 – 76 . Fairchild B. , Batal A. , Ritz C. , Vendrell P. . 2006 . Effect of drinking water iron concentration on broiler performance . J. Appl. Poult. Res . 15 : 511 – 517 . Google Scholar CrossRef Search ADS Garson G. D. 2013 . Generalized Linear Models / Generalized Estimating Equations . Statistical Associates Publishers , Asheboro, NC . Giorgi M. , Meucci V. , Vaccaro E. , Mengozzi G. , Giusiani M. , Soldani G. . 2003 . Effects of liquid and freeze-dried grapefruit juice on the pharmacokinetics of praziquantel and its metabolite 4΄-hydroxy praziquantel in beagle dogs . Pharmacol. Res. 47 : 87 – 92 . Google Scholar CrossRef Search ADS PubMed Hua P. , Vasyukova E. , Uhl W. . 2015 . A variable reaction rate model for chlorine decay in drinking water due to the reaction with dissolved organic matter . Water Res. 75 : 109 – 122 . Google Scholar CrossRef Search ADS PubMed Huang T. M. , Lin. T. L. , Wu C. C. . 2009 . Antimicrobial susceptibility and resistance of chicken Escherichia coli, Salmonella spp., and Pasteurella multocida isolates . Avian Dis. 53 : 89 – 93 . Google Scholar CrossRef Search ADS PubMed ICA . 2017 . Productos medicamentos veterinarios . Accessed Feb. 2017. http://www.ica.gov.co/Areas/Pecuaria/Servicios/Regulacion-y-Control-de-Medicamentos-Veterinarios/Medicamentos/VADEMECUM-MV-Feb-2017-WEB.aspx . Jerzsele Á. , Nagy G. . 2009 . The stability of amoxicillin trihydrate and potassium clavulanate combination in aqueous solutions . Acta Vet. Hung. 57 : 485 – 493 . Google Scholar CrossRef Search ADS PubMed Kahrs R. F. 1995 . Principios generales de la desinfección . Rev. sci. tech. Off. int. Epiz . 14 : 143 – 163 . Google Scholar CrossRef Search ADS Kalpana S. , Srinivasa Rao G. , Malik J. K. . 2015 . Impact of aflatoxin B1 on the pharmacokinetic disposition of enrofloxacin in broiler chickens . Environ. Toxicol. Pharmacol. 40 : 645 – 649 . Google Scholar CrossRef Search ADS PubMed Kandeel M. 2015 . Pharmacokinetics and oral bioavailability of amoxicillin in chicken infected with caecal coccidiosis . J. vet. Pharmacol. Therap. 38 : 504 – 507 . Google Scholar CrossRef Search ADS Kane G. C. , Lipsky J. J. . 2000 . Drug-grapefruit juice interactions . Mayo Clin. Proc. 75 : 933 – 942 . Google Scholar CrossRef Search ADS PubMed Kathleen H. , Van Dijr L. . 2011 . The World Medicines Situation 2011, Rational Use of Medicines . 3rd ed . WHO , Geneva, Switzerland . Krasucka D. , Kowalski C. J. . 2010 . Pharmacokinetic parameters of amoxicillin in pigs and poultry . Acta Pol. Pharm. - Drug Res . 67 : 729 – 732 . Krasucka D. , Kowalski C. , Osypiuk M. , Opielak G. . 2015 . Determination of amoxicillin in poultry plasma by high-performance liquid chromatography after formaldehyde derivation . Acta Chromatogr. 27 : 55 – 65 . Google Scholar CrossRef Search ADS Limayem A. , Donofrio R. S. , Zhang C. , Haller E. , Johnson M. G. . 2015 . Studies on the drug resistance profile of Enterococcus faecium distributed from poultry retailers to hospitals . J. Environ. Sci. Heal. Part B 50 : 827 – 832 . Google Scholar CrossRef Search ADS MacGowan A. 2011 . Revisiting beta-lactams - PK/PD improves dosing of old antibiotics . Curr. Opin. Pharmacol. 11 : 470 – 476 . Google Scholar CrossRef Search ADS PubMed Maharjan P. , Clark T. , Kuenzel C. , Foy M. K. , Watkins S. . 2016 . On farm monitoring of the impact of water system sanitation on microbial levels in broiler house water supplies . J. Appl. Poult. Res. 25 : 266 – 271 . Google Scholar CrossRef Search ADS May J. , Loot B. , Simmons J. . 1997 . Water consumption by broilers in high cyclic temperatures: bell versus nipple waterers . Poult. Sci. 76 : 944 – 947 . Google Scholar CrossRef Search ADS PubMed McDonnell G. , Russel D. A. . 1999 . Antiseptics and disinfectants activity, action, and resistance . Clin. Microbiol. Rev . 12 : 147 – 179 . Google Scholar PubMed McKellar Q. A. , Sanchez Bruni S. F. , Jones D. G. . 2004 . Pharmacokinetic/pharmacodynamic relationships of antimicrobial drugs used in veterinary medicine . J. Vet. Pharmacol. Ther . 27 : 503 – 514 . Google Scholar CrossRef Search ADS PubMed MIDA . 2017 . Registros farmacéuticos.Panama . Accessed Feb. 2017 http://www.mida.gob.pa/upload/documentos/registrosfarmaceuticosoct-15(1).pdf . National Research Council (NRC) . 1994 . Nutrient Requirements of Poultry . 9th rev. ed . Natl. Acad. Press , Washington, DC . Papich M. G. 2014 . Pharmacokinetic-pharmacodynamic (PK-PD) modeling and the rational selection of dosage regimes for the prudent use of antimicrobial drugs . Vet. Microbiol. 171 : 480 – 486 . Google Scholar CrossRef Search ADS PubMed Pillai K. , Eliopoulos G. , Moellering C. . 2005 . Antimicrobial combinations . 365 – 409 in Antibiotics in Laboratory Medicine . V. , Lorian , ed. 5th ed. Lippincott Williams and Wilkins , Philadelphia, USA . Quilumba C. , Quijia E. , Gernat A. , Murillo G. , Grimes J. . 2015 . Evaluation of different water flow rates of nipple drinkers on broiler productivity . J. Appl. Poult. Res . 24 : 58 – 65 . Google Scholar CrossRef Search ADS Ribeiro A. , Krabbe E. , Penz J. A. , Renz S. , Gomez H. . 2004 . Effect of chick weight, geometric mean diameter and sodium level in prestarter diets (1 to 7 days) on broiler perfomance up to 21 days of age . Poult. Sci . 6 : 225 – 230 . SAG . 2017 . Sistema Medicamentos Veterinarios . Accessed Feb. 2017. http://medicamentos.sag.gob.cl/ConsultaUsrPublico/BusquedaMedicamentos_1.asp . Saga T. , Yamaguchi K. . 2009 . History of antimicrobial agents and resistant bacteria . Japan Med. Assoc. J . 52 : 103 – 108 . SAGARPA . 2012 . Acuerdo por el que se modifica el diverso por el que se establece la clasificación y prescripción de los productos farmacéuticos veterinarios por el nivel de riesgo de sus ingredientes activos . Diario Oficial , Mexico . SENASA . 2015 . Listados oficiales de productos veterinarios . Accessed Feb. 2017. http://www.senasa.gov.ar/informacion/prod-vet-fito-y-fertilizantes/productos-veterinarios/listados-oficiales . Shapiro S. , Wilk M. . 1965 . An analysis of variance test for normality (complete samples) . Biometrika 52 : 591 – 611 . Google Scholar CrossRef Search ADS Sumano H. S. , Gutiérrez L. . 2008 . Farmacología clínica en aves . 3rd ed . McGraw Hill/Interamericana , Mexico City, Mexico . Sumano L. H. , Gutierrez O. L. , Aguilera R. , Rosiles M. R. , Bernard B. M. J. , Gracia M. J. . 2004 . Influence of hard water on the bioavailability of enrofloxacin in broilers . Poult. Sci. 83 : 726 – 731 . Google Scholar CrossRef Search ADS PubMed Sumano L. H. , Ocampo C. L. , Gutiérrez O. L. . 2015 . Farmacología Veterinaria . 4th ed . Diseño e Impresiones Aranda SA de CV , México . Sun L. , Jia L. , Xie X. , Xie K. , Wang J. , Liu J. , Cui L. , Zhang G. , Dai G. , Wang J. . 2016 . Quantitative analysis of amoxicillin, its major metabolites and ampicillin in eggs by liquid chromatography combined with electrospray ionization tandem mass spectrometry . Food Chem. 192 : 313 – 318 . Google Scholar CrossRef Search ADS PubMed Vermeulen B. 2002 . Drug administration to poultry . Adv. Drug. Deliv. Rev. 54 : 795 – 803 . Google Scholar CrossRef Search ADS PubMed Wang P. , He Y. L. , Huang C. H. . 2010 . Oxidation of fluoroquinolone antibiotics and structurally related amines by chlorine dioxide: reaction kinetics, product and pathway evaluation . Water Res. 44 : 5989 – 5998 . Google Scholar CrossRef Search ADS PubMed WHO . 1999 . Codex Alimentarius Commission . 23rd ed . WHO , Rome . Ziaei N. , Kermanshahi H. , Pilevar M. . 2011 . Effects of dietary crude protein and calcium / phosphorus content on growth, nitrogen and mineral retention in broiler chickens . Afr. J. Biotechnol . 10 : 13342 – 13350 . © 2018 Poultry Science Association Inc. This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Poultry ScienceOxford University Press

Published: Jun 22, 2018

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