Brief report
Novel bioassay using Bacillus megaterium to detect tetracycline in milk
Novedoso bioensayo con Bacillus megaterium para detectar tetraciclina en leche
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Calles 1, 2 y 3: muestras de ADN de los aislamientos correspondientes a las cabras 188, 200 y 911 de la majada MJ2 (Colonia Km 503). Calles 4 a 8: muestras de ADN de leche de las cabras 105,184 (negativas), 188, 200 y 911 (infectadas). Calle 9: cepa de referencia de <span class="elsevierStyleItalic">B. melitensis</span> bv. 1. Calle 10: control negativo. Calle 11: marcador molecular de 100 pb (Invitrogen, EE. UU.).</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Ana M. Russo, Orlando A. Mancebo, Carlos M. Monzón, Juan J. Gait, Rubén D. Casco, Susana M. Torioni de Echaide" "autores" => array:6 [ 0 => array:2 [ "nombre" => "Ana M." "apellidos" => "Russo" ] 1 => array:2 [ "nombre" => "Orlando A." "apellidos" => "Mancebo" ] 2 => array:2 [ "nombre" => "Carlos M." "apellidos" => "Monzón" ] 3 => array:2 [ "nombre" => "Juan J." "apellidos" => "Gait" ] 4 => array:2 [ "nombre" => "Rubén D." "apellidos" => "Casco" ] 5 => array:2 [ "nombre" => "Susana M." "apellidos" => "Torioni de Echaide" ] ] ] ] ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754115001595?idApp=UINPBA00004N" "url" => "/03257541/0000004800000002/v1_201607030039/S0325754115001595/v1_201607030039/es/main.assets" ] "itemAnterior" => array:19 [ "pii" => "S0325754116300013" "issn" => "03257541" "doi" => "10.1016/j.ram.2016.03.003" "estado" => "S300" "fechaPublicacion" => "2016-04-01" "aid" => "95" "copyright" => "Asociación Argentina de Microbiología" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2016;48:137-42" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 1196 "formatos" => array:3 [ "EPUB" => 27 "HTML" => 797 "PDF" => 372 ] ] "en" => array:12 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Brief reports</span>" "titulo" => "Amino acid substitution in <span class="elsevierStyleItalic">Cryptococcus neoformans</span> lanosterol 14-α-demethylase involved in fluconazole resistance in clinical isolates" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "137" "paginaFinal" => "142" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Sustitución aminoacídica en la enzima lanosterol 14 α-demetilasa de <span class="elsevierStyleItalic">Cryptococcus neoformans</span> involucrada en la resistencia al fluconazol de aislamientos clínicos" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "María E. Bosco-Borgeat, Mariana Mazza, Constanza G. Taverna, Susana Córdoba, Omar A. Murisengo, Walter Vivot, Graciela Davel" "autores" => array:7 [ 0 => array:2 [ "nombre" => "María E." "apellidos" => "Bosco-Borgeat" ] 1 => array:2 [ "nombre" => "Mariana" "apellidos" => "Mazza" ] 2 => array:2 [ "nombre" => "Constanza G." "apellidos" => "Taverna" ] 3 => array:2 [ "nombre" => "Susana" "apellidos" => "Córdoba" ] 4 => array:2 [ "nombre" => "Omar A." "apellidos" => "Murisengo" ] 5 => array:2 [ "nombre" => "Walter" "apellidos" => "Vivot" ] 6 => array:2 [ "nombre" => "Graciela" "apellidos" => "Davel" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754116300013?idApp=UINPBA00004N" "url" => "/03257541/0000004800000002/v1_201607030039/S0325754116300013/v1_201607030039/en/main.assets" ] "en" => array:20 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Brief report</span>" "titulo" => "Novel bioassay using <span class="elsevierStyleItalic">Bacillus megaterium</span> to detect tetracycline in milk" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "143" "paginaFinal" => "146" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Melisa Tumini, Orlando G. Nagel, Pilar Molina, Rafael L. Althaus" "autores" => array:4 [ 0 => array:3 [ "nombre" => "Melisa" "apellidos" => "Tumini" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 1 => array:3 [ "nombre" => "Orlando G." "apellidos" => "Nagel" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "Pilar" "apellidos" => "Molina" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 3 => array:4 [ "nombre" => "Rafael L." "apellidos" => "Althaus" "email" => array:1 [ 0 => "ralthaus@fcv.unl.edu.ar" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:2 [ 0 => array:3 [ "entidad" => "Cátedra de Biofísica, Departamento de Ciencias Básicas, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, R.P.L. Kreder 2804, 3080 Esperanza, Argentina" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Instituto de Ciencia y Tecnología Animal, Universidad Politécnica de Valencia, Camino de Vera 14, 46071 Valencia, Spain" "etiqueta" => "b" "identificador" => "aff0010" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Novedoso bioensayo con <span class="elsevierStyleItalic">Bacillus megaterium</span> para detectar tetraciclina en leche" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2575 "Ancho" => 1474 "Tamanyo" => 284650 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Tetracyclines dose–response curves for different chloramphenicol concentrations (□ CAP: 0<span class="elsevierStyleHsp" style=""></span>μg/l; ○ CAP: 1000<span class="elsevierStyleHsp" style=""></span>μg/l; ▵ CAP: 1500<span class="elsevierStyleHsp" style=""></span>μg/l; × CAP: 2000<span class="elsevierStyleHsp" style=""></span>μg/l).</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall">Tetracyclines (TCs) are antibiotics used for the prevention and control of a variety of infectious diseases. These compounds are active against both gram-negative and gram-positive bacteria<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">11</span></a>. In dairy cattle, TCs are used for the treatment of bacterial enteritis, infectious metritis, colibacillary mastitis and keratoconjunctivitis.</p><p id="par0010" class="elsevierStylePara elsevierViewall">Cows metabolize about 25–50%<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">13</span></a> of tetracyclines administered, and an appreciable amount of these drugs can be excreted into milk. TC residues can cause effects on consumers, such as allergic reactions, liver damage, yellowing of teeth and gastrointestinal disorders<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">5</span></a>. In the dairy industry, TC residues produce changes in the organoleptic characteristics of fermented products<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">10</span></a>.</p><p id="par0015" class="elsevierStylePara elsevierViewall">For this reason, control authorities such as the European Union<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">4</span></a> and Codex Alimentarius<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">3</span></a> have recommended a Maximum Residue Level (MRL) of 100<span class="elsevierStyleHsp" style=""></span>μg/l for chlortetracycline, oxytetracycline and tetracycline in milk.</p><p id="par0020" class="elsevierStylePara elsevierViewall">Antibiotics in milk are widely evaluated using microbiological inhibition methods. Some authors propose the use of <span class="elsevierStyleItalic">Bacillus cereus</span> ATCC 11778 in a Petri dish to detect TC residues in milk<a class="elsevierStyleCrossRefs" href="#bib0085"><span class="elsevierStyleSup">2,6,9,12</span></a>. These microbiological methods are highly sensitive to TCs but require trained personnel and a prolonged incubation time to measure their response (18–24<span class="elsevierStyleHsp" style=""></span>h).</p><p id="par0025" class="elsevierStylePara elsevierViewall">In order to decrease the response time of these microbiological methods, Nagel et al.<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">8</span></a> and Tumini et al.<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">15</span></a> recommend the use of bioassays in microtiter plates containing <span class="elsevierStyleItalic">B. cereus</span> and <span class="elsevierStyleItalic">Bacillus pumilus</span> spores, which reduces the response time (5–6<span class="elsevierStyleHsp" style=""></span>h). However, it should be noted that <span class="elsevierStyleItalic">B. cereus</span> spores present risks for operators because they produce toxins that cause gastrointestinal disturbances<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">1</span></a>. Furthermore, the bioassay developed by Tumini et al.<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">15</span></a> requires the use of a photometric reader to interpret the results.</p><p id="par0030" class="elsevierStylePara elsevierViewall">Therefore, the aim of this work was to design a microbiological inhibition bioassay in microtiter plates using <span class="elsevierStyleItalic">Bacillus megaterium</span> with a dichotomous response (positive–negative) indicated by a change in the color of the redox indicator present in the culture medium. This bioassay is economical and easy to implement in a laboratory for the control of residues in milk.</p><p id="par0035" class="elsevierStylePara elsevierViewall">For the bioassay elaboration, Mueller Hinton Agar culture medium (38<span class="elsevierStyleHsp" style=""></span>g/l, Biokar<span class="elsevierStyleSup">®</span>, Ref. 10272, France) was fortified with glucose (10<span class="elsevierStyleHsp" style=""></span>g/l, Sigma Aldrich<span class="elsevierStyleSup">®</span>, Ref. G8270, St. Louis, MO, USA), brilliant black (200<span class="elsevierStyleHsp" style=""></span>μg/l Sigma Aldrich<span class="elsevierStyleSup">®</span>, Ref. 211842, St. Louis, MO, USA) and toluidine blue (10<span class="elsevierStyleHsp" style=""></span>μg/l of Sigma Aldrich<span class="elsevierStyleSup">®</span>, Ref. 89640, St. Louis, MO, USA) indicators<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">15</span></a> and <span class="elsevierStyleItalic">B. megaterium</span> ATCC 9885 spores (2.8<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">8</span><span class="elsevierStyleHsp" style=""></span>spores/ml) at pH 8.5<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>0.1. These concentrations were obtained by diluting a stock spore suspension of <span class="elsevierStyleItalic">B. megaterium</span> (5.6<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">10</span> spores/ml) determined by counting with Petrifilm ™ plates (3M, St Paul, MN, USA). The media was fractionated into four aliquots and a chloramphenicol (CAP) solution was added to obtain concentrations of 0, 1000, 1500 and 2000<span class="elsevierStyleHsp" style=""></span>μg CAP/l in the culture medium. Subsequently, 100<span class="elsevierStyleHsp" style=""></span>μl of the preparation was added to each microplate well using an electronic dispenser (Eppendorf Research<span class="elsevierStyleSup">®</span> Pro, Hamburg, Germany). Bioassay plates were sealed and conserved at 4<span class="elsevierStyleHsp" style=""></span>°C until use. Next, sixteen replicates of twelve concentrations of chlortetracycline (CTC, Sigma C-4881), oxytetracycline (OTC, Sigma O-5750) and tetracycline (TC, Sigma T-3258) were analyzed (0, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 500<span class="elsevierStyleHsp" style=""></span>μg/l), with the aim of obtaining at least two negative results in the lowest concentrations and two positive results at the highest levels. Subsequently, 50<span class="elsevierStyleHsp" style=""></span>μl of solution containing milk and the corresponding antibiotic concentration was added to each microplate well and left to diffuse into the agar medium for 1<span class="elsevierStyleHsp" style=""></span>h. The microplate was washed several times with distilled water and incubated in a water floating bath (Dalvo, Santa Fe, Argentina) at 45<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>1<span class="elsevierStyleHsp" style=""></span>°C until the color of the negative controls changed (from black to yellow). The visual interpretation was carried out by 3 qualified people, and the test results were evaluated as “negative” or “positive”. “Ambiguous” qualifications were considered “positive”. Since the visual evaluation of the bioassay is an ordinal variable with two dichotomous responses (“negative” and “positive”), it is appropriate to use a logistic model to evaluate the data. The results were analyzed using stepwise logistic regression in SAS<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">14</span></a>. The logistic regression model used was the following:<elsevierMultimedia ident="eq0005"></elsevierMultimedia>where <span class="elsevierStyleItalic">L</span><span class="elsevierStyleInf"><span class="elsevierStyleItalic">ijk</span></span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>the dependent or response variable of the linear logistic model; [Pijk]=[Pp/(1−Pp)] or the ratio of the probability of a “positive” response/the probability of a “negative” response; [TCs]<span class="elsevierStyleInf"><span class="elsevierStyleItalic">i</span></span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>effect of tetracycline concentration (<span class="elsevierStyleItalic">i</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>1, 2, …12 levels), [CAP]<span class="elsevierStyleInf"><span class="elsevierStyleItalic">j</span></span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>effect of chloramphenicol concentrations (<span class="elsevierStyleItalic">j</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0, 1000, 1500 or 2000<span class="elsevierStyleHsp" style=""></span>μg/l), ([TCs]*[CAP])<span class="elsevierStyleInf"><span class="elsevierStyleItalic">ij</span></span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>effect of interaction between tetracycline and chloramphenicol concentrations; <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">0</span>, <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1</span>, <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">2</span>, and <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">12</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>coefficients estimated for intercept terms, tetracycline, chloramphenicol and interaction between tetracycline and chloramphenicol, respectively; and <span class="elsevierStyleItalic">¿</span><span class="elsevierStyleInf"><span class="elsevierStyleItalic">ijk</span></span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>residual error. The detection limits of the bioassay were calculated as the concentration of antibiotic that produces 95% of the positive frequency<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">7</span></a>.</p><p id="par0040" class="elsevierStylePara elsevierViewall">The results show that the [CAP] and [TCs] terms were significant for the TCs analyzed (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05); however, their interaction [CAP]*[TCs] was not significant (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>><span class="elsevierStyleHsp" style=""></span>0.05), indicating that CAP produces an antimicrobial effect in the bioassay<a class="elsevierStyleCrossRefs" href="#bib0115"><span class="elsevierStyleSup">8,15</span></a>. High “<span class="elsevierStyleItalic">χ</span><span class="elsevierStyleSup">2</span>” values for CAP (χCTC2=199.02;   χOTC2=204.68;   χTC2=134.23) showed that CAP incorporation into the culture medium improves bioassay sensitivity for detecting TCs in milk. The coefficients calculated for the factors found to be statistically significant using the logistics regression model are reported in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>. Concordance percentages were adequate (CTC<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>88.5%; OTC<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>93.3%; TC<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>89.8%) and showed good fit to the model. The “<span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1</span>” coefficient indicates that the increase in the frequency of positive results rise with the TC concentration in milk. These coefficients showed that <span class="elsevierStyleItalic">B. megaterium</span> has similar sensitivity to all three antibiotics in milk, since their “<span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1</span>” values were equivalent (<span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1CTC</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.0534; <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1OTC</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.0730; <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1TC</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.0570). The “<span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1</span>” coefficients evidence the antimicrobial effect of CAP; the values obtained were similar (<span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">2CTC</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.0049; <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">2OTC</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.0058; <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">2TC</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.0037), indicating that the CAP's antimicrobial activity acted in a similar manner. <a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a> represents the dose–response curves elaborated with the coefficients calculated by the logistic regression model (<span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">0</span>, <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1</span> and <span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">2</span>). It depicts the effect of [TC] and [CAP] on the relative frequency of positive results in this bioassay. The frequency of positive results increases as the concentration of antibiotics in the milk increases. The addition of CAP to the culture medium displaces dose–response curves to a lower detection level<a class="elsevierStyleCrossRefs" href="#bib0115"><span class="elsevierStyleSup">8,15</span></a>. The detection limits of the bioassay for each tetracycline and different CAP levels (<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>) were calculated by applying the logistic regression model, using the 95% relative frequency of positive results. Additionally, <a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a> shows the MRLs established by the European Union. Chloramphenicol incorporation into the culture medium (0–2000<span class="elsevierStyleHsp" style=""></span>μg/l) decreases the TC detection limits of the bioassay (CTC: from 290 to 105<span class="elsevierStyleHsp" style=""></span>μg/l; OTC: from 260 to 100<span class="elsevierStyleHsp" style=""></span>μg/l; TC: from 268 to 134<span class="elsevierStyleHsp" style=""></span>μg/l). The levels obtained are similar to the MRLs established by the previously mentioned legislation (100<span class="elsevierStyleHsp" style=""></span>μg/l). The traditional microbiological methods developed in Petri dishes require an incubation period of between 18 and 24<span class="elsevierStyleHsp" style=""></span>h. Using these methods, Nouws et al.<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">9</span></a> report sensitivities of 100<span class="elsevierStyleHsp" style=""></span>μg/l of TC, 100<span class="elsevierStyleHsp" style=""></span>μg/l of OTC and 15<span class="elsevierStyleHsp" style=""></span>μg/l of OTC when using <span class="elsevierStyleItalic">B. cereus</span>. In a similar study, Raspor Lainscek et al.<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">12</span></a> determine 100<span class="elsevierStyleHsp" style=""></span>μg/l for tetracycline, 100<span class="elsevierStyleHsp" style=""></span>μg/l for oxytetracycline, 80<span class="elsevierStyleHsp" style=""></span>μg/l for chlortetracycline in milk when using <span class="elsevierStyleItalic">B. cereus</span> ATCC 11778 in the STAR protocol. In addition, Gaudin et al.<a class="elsevierStyleCrossRef" href="#bib0105"><span class="elsevierStyleSup">6</span></a> detected higher concentrations for OTC (250<span class="elsevierStyleHsp" style=""></span>μg/l) and TC (250<span class="elsevierStyleHsp" style=""></span>μg/l) and good sensitivity for CTC (50<span class="elsevierStyleHsp" style=""></span>μg/l). In sheep milk, Althaus et al.<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">2</span></a> obtained low detection limits of tetracycline residues in a Petri dish when using <span class="elsevierStyleItalic">B. cereus</span> (DL<span class="elsevierStyleInf">CTC</span>: 25<span class="elsevierStyleHsp" style=""></span>μg/l; DL<span class="elsevierStyleInf">OTC</span>: 75<span class="elsevierStyleHsp" style=""></span>μg/l; DL<span class="elsevierStyleInf">TC</span>: 85<span class="elsevierStyleHsp" style=""></span>μg/l). Subsequently, Nagel et al.<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">8</span></a> optimized a bioassay in microtiter plates using the same bacteria test with 470<span class="elsevierStyleHsp" style=""></span>μg CAP/l. These authors detected 100<span class="elsevierStyleHsp" style=""></span>μg/l of OTC and 109<span class="elsevierStyleHsp" style=""></span>μg/l of TC, but did not detect levels close to the MRL of CTC (300<span class="elsevierStyleHsp" style=""></span>μg/l). In contrast, the bioassay using <span class="elsevierStyleItalic">B. megaterium</span> developed in this work has better sensitivity for the detection of chlortetracycline residues in milk (105<span class="elsevierStyleHsp" style=""></span>μg/l). Additionally, the detection limits calculated using visual readings of the bioassay developed in this work (105<span class="elsevierStyleHsp" style=""></span>μg/l of CTC, 100<span class="elsevierStyleHsp" style=""></span>μg/l of OTC and 134<span class="elsevierStyleHsp" style=""></span>μg/l of TC) are similar to those calculated by Tumini et al.<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">15</span></a> when using a photometric reader to interpret the results of a bioassay in microtiter plates using <span class="elsevierStyleItalic">B. pumilus</span> spores (DL<span class="elsevierStyleInf">CTC</span>: 117<span class="elsevierStyleHsp" style=""></span>μg/l; DL<span class="elsevierStyleInf">OTC</span>: 142<span class="elsevierStyleHsp" style=""></span>μg/l; DL<span class="elsevierStyleInf">TC</span>: 105<span class="elsevierStyleHsp" style=""></span>μg/l). This microbiological inhibition bioassay using <span class="elsevierStyleItalic">B. megaterium</span> spores and 2000<span class="elsevierStyleHsp" style=""></span>μg/l of chloranphenicol detects adequate levels of tetracycline residues in milk with a 5<span class="elsevierStyleHsp" style=""></span>h response time. Furthermore, this method provides a dichotomous response that facilitates interpretation of the results. Moreover, this bioassay can be incorporated into a microbiological multi-residue system for the identification of tetracyclines in milk in order to select samples for subsequent unequivocal confirmation of these molecules in high resolution chromatographic techniques such as HPLC-MS–MS.</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="tbl0010"></elsevierMultimedia><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Ethical disclosures</span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Protection of human and animal subjects</span><p id="par0045" class="elsevierStylePara elsevierViewall">The authors declare that no experiments were performed on humans or animals for this study.</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Confidentiality of data</span><p id="par0050" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Right to privacy and informed consent</span><p id="par0055" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article.</p></span></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Conflict of interest</span><p id="par0060" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:8 [ 0 => array:3 [ "identificador" => "xres680264" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec686116" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres680265" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec686115" "titulo" => "Palabras clave" ] 4 => array:3 [ "identificador" => "sec0005" "titulo" => "Ethical disclosures" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0010" "titulo" => "Protection of human and animal subjects" ] 1 => array:2 [ "identificador" => "sec0015" "titulo" => "Confidentiality of data" ] 2 => array:2 [ "identificador" => "sec0020" "titulo" => "Right to privacy and informed consent" ] ] ] 5 => array:2 [ "identificador" => "sec0025" "titulo" => "Conflict of interest" ] 6 => array:2 [ "identificador" => "xack229396" "titulo" => "Acknowledgements" ] 7 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2015-11-23" "fechaAceptado" => "2016-02-23" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec686116" "palabras" => array:6 [ 0 => "Tetracyclines" 1 => "Milk" 2 => "<span class="elsevierStyleItalic">Bacillus megaterium</span>" 3 => "Antibiotics" 4 => "Detection" 5 => "Bioassay" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec686115" "palabras" => array:6 [ 0 => "Tetraciclinas" 1 => "Leche" 2 => "<span class="elsevierStyleItalic">Bacillus megaterium</span>" 3 => "Antibióticos" 4 => "Detección" 5 => "Bioensayo" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Tetracyclines are used for the prevention and control of dairy cattle diseases. Residues of these drugs can be excreted into milk. Thus, the aim of this study was to develop a microbiological method using <span class="elsevierStyleItalic">Bacillus megaterium</span> to detect tetracyclines (chlortetracycline, oxytetracycline and tetracycline) in milk. In order to approximate the limits of detection of the bioassay to the Maximum Residue Limit (100<span class="elsevierStyleHsp" style=""></span>μg/l) for milk tetracycline, different concentrations of chloramphenicol (0, 1000, 1500 and 2000<span class="elsevierStyleHsp" style=""></span>μg/l) were tested. The detection limits calculated were similar to the Maximum Residue Limits when a bioassay using <span class="elsevierStyleItalic">B. megaterium</span> ATCC 9885 spores (2.8<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">8</span><span class="elsevierStyleHsp" style=""></span>spores/ml) and chloramphenicol (2000<span class="elsevierStyleHsp" style=""></span>μg/l) was utilized. This bioassay detects 105<span class="elsevierStyleHsp" style=""></span>μg/l of chlortetracycline, 100<span class="elsevierStyleHsp" style=""></span>μg/l of oxytetracycline and 134<span class="elsevierStyleHsp" style=""></span>μg/l of tetracycline in 5<span class="elsevierStyleHsp" style=""></span>h. Therefore, this method is suitable to be incorporated into a microbiological multi-residue system for the identification of tetracyclines in milk.</p></span>" ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Las tetraciclinas son utilizadas para la prevención y el control de las enfermedades del ganado lechero; los residuos de estos medicamentos pueden ser excretados en la leche. El objetivo de este estudio fue desarrollar un método microbiológico con esporas de <span class="elsevierStyleItalic">Bacillus megaterium</span> para detectar las tetraciclinas en la leche. Con el propósito de aproximar los límites de detección del bioensayo al límite máximo de residuo permitido para tetraciclinas en leche (100<span class="elsevierStyleHsp" style=""></span>μg/l), se analizaron diferentes concentraciones de cloranfenicol (0, 1.000, 1.500 y 2.000<span class="elsevierStyleHsp" style=""></span>μg/l). Los límites de detección son similares a sus respectivos límites máximos de residuos cuando se utiliza un bioensayo con esporas de <span class="elsevierStyleItalic">Bacillus megaterium</span> ATCC 9885 (2,8 x 10<span class="elsevierStyleSup">8</span> esporas/ml) y cloranfenicol (2.000<span class="elsevierStyleHsp" style=""></span>μg/l). Este bioensayo detectó 105<span class="elsevierStyleHsp" style=""></span>μg/l de clortetraciclina, 100<span class="elsevierStyleHsp" style=""></span>μg/l de oxitetraciclina y 134<span class="elsevierStyleHsp" style=""></span>μg/l de tetraciclina en 5<span class="elsevierStyleHsp" style=""></span>h. Por lo tanto, este método es adecuado para ser incorporado en un sistema microbiológico multirresiduo para la identificación de tetraciclinas en leche.</p></span>" ] ] "multimedia" => array:4 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2575 "Ancho" => 1474 "Tamanyo" => 284650 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Tetracyclines dose–response curves for different chloramphenicol concentrations (□ CAP: 0<span class="elsevierStyleHsp" style=""></span>μg/l; ○ CAP: 1000<span class="elsevierStyleHsp" style=""></span>μg/l; ▵ CAP: 1500<span class="elsevierStyleHsp" style=""></span>μg/l; × CAP: 2000<span class="elsevierStyleHsp" style=""></span>μg/l).</p>" ] ] 1 => array:8 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at1" "detalle" => "Table " "rol" => "short" ] ] "tabla" => array:2 [ "leyenda" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">TCs: tetracyclines; CAP: chloramphenicol; <span class="elsevierStyleItalic">C</span>%: concordance correlation coefficient.</p>" "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">TCs \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">L</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>log[<span class="elsevierStyleItalic">P</span>]<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">0</span><span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">1</span>[TCs]<span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">β</span><span class="elsevierStyleInf">2</span>[CAP] \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="center" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">C</span>% \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Chlortetracycline \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top"><span class="elsevierStyleItalic">L</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>−12.436<span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span>0.0534*[CTC]<span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span>0.0049*[CAP] \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">88.5 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Oxytetracycline \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top"><span class="elsevierStyleItalic">L</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>−16.111<span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span>0.0730*[OTC]<span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span>0.0058*[CAP] \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">93.3 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Tetracycline \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top"><span class="elsevierStyleItalic">L</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>−12.137<span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span>0.0570*[TC]<span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span>0.0037*[CAP] \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">90.8 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1116586.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Logistic regression models representing TC and CAP effects on the bioassay response</p>" ] ] 2 => array:8 [ "identificador" => "tbl0010" "etiqueta" => "Table 2" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at2" "detalle" => "Table " "rol" => "short" ] ] "tabla" => array:2 [ "leyenda" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">CAP: chloramphenicol; MRLs: Maximum Residue Limits (μg/l).</p>" "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td-with-role" title="table-head ; entry_with_role_rowhead " align="left" valign="top" scope="col">Tetracyclines \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " colspan="4" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Concentration CAP (μg/l)</th><th class="td" title="table-head " align="left" valign="top" scope="col">MRLs \t\t\t\t\t\t\n \t\t\t\t</th></tr><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">0 \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">1000 \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">1500 \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">2000 \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Chlortetracycline \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">290 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">198 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">154 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">105 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">100 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Oxytetracycline \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">260 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">182 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">140 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">100 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">100 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Tetracycline \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">268 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">199 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">167 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">134 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">100 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1116587.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Effect of chloramphenicol on the detection limits (μg/l) of tetracyclines in milk</p>" ] ] 3 => array:6 [ "identificador" => "eq0005" "etiqueta" => "(1)" "tipo" => "MULTIMEDIAFORMULA" "mostrarFloat" => false "mostrarDisplay" => true "Formula" => array:5 [ "Matematica" => "Lijk=Logit[Pijk]=β0+β1[TCs]i+β2[CAP]j+β12([TCs]∗[CAP])ij+εijk" "Fichero" => "STRIPIN_si1.jpeg" "Tamanyo" => 3928 "Alto" => 44 "Ancho" => 279 ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:15 [ 0 => array:3 [ "identificador" => "bib0080" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Germination and outgrowth of spores of <span class="elsevierStyleItalic">Bacillus cereus</span> group members: diversity and role of germinant receptors" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "T. 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N° 140/12, Agencia Nacional de Promoción Científica y Tecnológica).</p>" "vista" => "all" ] ] ] "idiomaDefecto" => "en" "url" => "/03257541/0000004800000002/v1_201607030039/S0325754116000067/v1_201607030039/en/main.assets" "Apartado" => array:4 [ "identificador" => "41740" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Microbiología de alimentos" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/03257541/0000004800000002/v1_201607030039/S0325754116000067/v1_201607030039/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754116000067?idApp=UINPBA00004N" ]
| Year/Month | Html | Total | |
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| 2018 August | 2 | 18 | 20 |
| 2018 July | 4 | 0 | 4 |
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| 2018 May | 3 | 8 | 11 |
| 2018 April | 2 | 2 | 4 |
| 2018 March | 6 | 0 | 6 |
| 2018 February | 6 | 2 | 8 |
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| 2017 November | 5 | 2 | 7 |
| 2017 October | 6 | 4 | 10 |
| 2017 September | 3 | 9 | 12 |
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| 2017 June | 16 | 29 | 45 |
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| 2017 April | 15 | 8 | 23 |
| 2017 March | 10 | 49 | 59 |
| 2017 February | 13 | 5 | 18 |
| 2017 January | 13 | 1 | 14 |
| 2016 December | 20 | 8 | 28 |
| 2016 November | 26 | 9 | 35 |
| 2016 October | 29 | 10 | 39 |
| 2016 September | 68 | 13 | 81 |
| 2016 August | 52 | 10 | 62 |
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