Performance of current microbial tests for screening antibiotics in sheep and goat milk

Documen downloaded om: This pape mus be ci ed as: The inal publica ion is a ailable a Copy igh Addi ional In o ma ion h p://doi.o g/10.1016/j.idai yj.2014.09.007 h p://hdl.handle.ne /10251/80581 Else ie Bel án Ma ínez, MC.; Be uga Fe nandez, MI.; Molina Casano a, A.; Lisand o Al haus, R.; Molina Pons, MP. (2015). Pe o mance o cu en mic obial es s o sc eening an ibio ics in sheep and goa milk. In e na ional Dai y Jou nal. 41:13-15. doi:10.1016/j.idai yj.2014.09.007. 1 Pe o mance o cu en mic obial es s o sc eening an ibio ics in sheep and 1 goa milk 2 Bel án M.Ca., Be uga M.Ib., Molina Ab., Al haus R.Lc., Molina M.Pa. 3 aIns i u o de Ciencia y Tecnología Animal, Uni e si a Poli ècnica de València, Camino de 4 Ve a, s/n, 46022 Valencia, Spain. [email p o ec ed]; [email p o ec ed] 5 bDepa amen o de Ciencia y Tecnología Ag o o es al, ETSIA-IDR, Uni e sidad de Cas illa- 6 La Mancha, 02071 Albace e, Spain. [email p o ec ed]; [email p o ec ed] 7 cCá ed a de Bio ísica, Facul ad de Ciencias Ve e ina ias, Uni e sidad Nacional del Li o al, 8 R.P.L., K ede , 3080 Espe anza, A gen ina. [email p o ec ed] 9 10 11 12 13 14 15 16 17 Co esponding au ho : Ma ia Ca men Bel án Ma ínez 18 Ins i u o de Ciencia y Tecnología Animal 19 Uni e si a Poli ècnica de València 20 Camino de Ve a, s/n 21 46022 Valencia, Spain 22 Tel.: + 34 963877727; Fax: +34 963877439 23 [email p o ec ed] 24 2 ABSTRACT 25 The de ec ion capabili y (CCβ) o some mic obial sc eening es s (BRT MRL, Del o es 26 MCS SP-NT, Del o es MCS Accele a o and Eclipse 100) cu en ly a ailable was 27 calcula ed in acco dance wi h Commission Decision 657/2002/EC. The CCβ was a o 28 below he maximum esidue limi (MRL) o mos β-lac ams assessed and o he non-β- 29 lac am d ugs such as neomycin, ylosin, sul adiazine and sul adime hoxine. Howe e , 30 he es s we e less sensi i e in he de ec ion o mos non-β-lac am d ugs such as 31 quinolones and e acyclines a sa e y le els. When indi idual sheep milk samples ee 32 o an ibio ics we e analysed, an ele a ed soma ic cell coun (SCC) was ela ed o he 33 occu ence o non-complian esul s in all he me hods assessed. In o de o gua an ee 34 he sa e y o milk and dai y p oduc s om small uminan s, he pe iodical 35 implemen a ion o sc eening es s mo e sensi i e owa ds non-β-lac am d ugs would be 36 con enien . 37 1. In oduc ion 38 Mic obial inhibi o es s a e ou inely applied o sc eening an ibio ics in aw milk as 39 hey a e ela i ely inexpensi e, use - iendly, and ha e a high sample h oughpu . 40 Mos cu en mic obial sc eening es s we e ini ially de eloped o de ec β-lac ams in 41 cow milk and a e based on he inhibi ion o Geobacillus s ea o e mophilus a . 42 calidolac is being highly sensi i e o hese subs ances. Se e al s udies on he mic obial 43 es sensi i i y using sheep milk ha e been ca ied ou in he las wo decades (Al haus, 44 To es, Mon e o, Balasch & Molina, 2003a; Molina, Al haus, Molina & Fe nández, 45 2003), while e y ew s udies ha e been unde aken in goa milk (Sie a e al., 2009a,b), 46 demons a ing ha hese es s a e able o de ec β-lac ams a o below he Maximum 47 Residue Limi s (MRLs) es ablished by Eu opean legisla ion (EC, 2010), bu canno 48 sui ably de ec o he e e ina y d ugs. 49 3 The e o e, modi ica ions such as he addi ion o chela ing agen s in o he cul u e 50 medium o enhance he de ec ion o e acyclines, espec i ely, o an i ola es o imp o e 51 sulphonamide de ec ion ha e been p oposed (Lange eld, Beuke s, Bommele, & S a k, 52 2005), and consequen ly manu ac u e s ha e imp o ed some pe o mance 53 cha ac e is ics o mic obial sc eening es s in new e sions now a ailable. 54 Mo eo e , sheep and goa milk is cha ac e ised by a highe a and p o ein con en han 55 cow milk (Pa k, Jua ez, Ramos & Haenlein, 2007), an ele a ed na u al inhibi o con en 56 (e.g. immunoglobulins, lac o e in, o lysozyme) (C osson, Thomas, & Rossi, 2010) and 57 a highe soma ic cell coun (SCC) e en in he absence o in a-mamma y in ec ions 58 (Medhid, Díaz, Ma í, Vidal & Pe is, 2013), po en ially in e e ing in he mic obial 59 inhibi o es esponse. 60 Sheep and goa milk p oduc ion is mainly des ined o he elabo a ion o dai y p oduc s. 61 An ibio ic esidues in milk may pa ially o o ally inhibi e men a ion p ocesses in 62 cheese and yogu p oduc ion (Packham, B oome, Limsow in, & Roginski, 2001). 63 Mo eo e , consume sa e y may be comp omised by he p esence o hese esidues in 64 dai y p oduc s (Oli e , Mu inda & Ja ayao, 2011). Thus, he aim o his s udy was o 65 assess he pe o mance o new e sions o some mic obial sc eening es s o de ec 66 an imic obial esidues in sheep and goa milk acco ding o Eu opean Commission 67 Decision nº 657/2002 (EC, 2002). 68 2. Ma e ial and me hods 69 2.1. Mic obial inhibi o es s: The sc eening es s used we e he BRT MRL (Analy ik in 70 Milch P oduk ions-und Ve iebs-GmbH. Munich, Ge many), Del o es MCS SP-NT 71 (DSM Food Special ies. Del , he Ne he lands), Del o es MCS Accele a o (DSM 72 Food Special ies), and Eclipse 100 (Zeu-Inmuno ec. Za agoza, Spain). All es s we e 73 conduc ed acco ding o each manu ac u e ’s ins uc ions. 74 4 2.2. Milk samples: An ibio ic- ee milk samples we e ob ained om he expe imen al 75 locks o Manchega sheep o Uni e sidad de Cas illa-La Mancha (Albace e, Spain), and 76 Mu ciano-G anadina goa s o Uni e si a Poli ècnica de València (Valencia, Spain). 77 Animals had a good heal h s a us and had no ecei ed any e e ina y d ugs, nei he 78 be o e no du ing he expe imen al pe iod, no was medica ed eed used in hei die . All 79 milk samples we e analysed o g oss composi ion (MilkoScan 6000, Foss. Hille d, 80 Denma k), soma ic cell coun (Fossoma ic 5000, Foss), o al bac e ial coun (Bac oscan 81 FC, Foss), and pH alue (pHme e , C ison, Ba celona, Spain). 82 2.3. An imic obials and spiked milk samples: A o al o 37 subs ances was in es iga ed: 83 amoxicillin, ampicillin, benzylpenicillin, cloxacillin, dicloxacillin, na cillin, oxacillin, 84 ce alonium, ce api in, ce azolin, ce ope azone, ce quinome, ce io u , cephalexin, 85 chlo e acycline, cip o loxacin, colis in, en o loxacin, e y h omycin, gen amicin, 86 lincomicin, ma bo loxacin, neomycin, oxy e acycline, s ep omycin, sul adiazine, 87 sul adime hoxine, sul ame azine, e acycline, ime hop im, and ylosin we e p o ided 88 by Sigma-Ald ich Química, S.A. (Mad id, Spain). Des u oylce io u was supplied by 89 To on o Resea ch Chemicals, Inc. (To on o, Canada) and he 4-epime s o e acyclines 90 we e u nished by Ac os O ganics (Geel, Belgium). Finally, desace ylce api in and 91 ce ace ile, no comme cially a ailable, we e kindly p o ided by Fa o S.p.A. (Bologna, 92 I aly) and ACS Dob a , S.p.A. (Milan, I aly), espec i ely. Spiked milk samples we e 93 p epa ed ollowing In e na ional Dai y Fede a ion ecommenda ions (ISO/IDF, 2003), 94 and es ed by he mic obial sc eening es s immedia ely a e spiking. 95 2.4. De ec ion capabili y (CCβ): Tes de ec ion capabili y (CCβ) was in es iga ed 96 ollowing he “Guidelines o he alida ion o sc eening me hods o esidues o 97 e e ina y medicines” p oposed by Communi y Re e ence Labo a o ies o esidues 98 (CRLs, 2010), supplemen ing Commission Decision nº 657/2002 (EC, 2002). 99 5 An imic obial- ee milk samples we e spiked indi idually wi h di e en subs ances a 100 0.5·MRL, 0.75·MRL, and 1·MRL equi alen d ug concen a ion, and analysed 20, 40 o 101 60 imes, espec i ely, by he di e en mic obial es s. 102 2.5. In e e ences ela ed o milk ma ix cons i uen s: An ibio ic- ee milk samples we e 103 ob ained on a wo-week basis along he en i e milking pe iod (sheep: n=250, sampling 104 days 30 o 180 pos -pa um; goa s: n=350, sampling days 15 o 200 pos -pa um) and 105 analysed simul aneously, in h ee eplica es, by he ou es s. The e ec o he milk 106 ma ix cons i uen s on he es esponse was in es iga ed using he s epwise op ion o 107 he logis ic p ocedu e o he SAS so wa e, acco ding o he ollowing model: 108 Lij= logi [Pi] = β0 + β1[SL] + β2[pH] + β3[F] + β4[P] + β5[L] + β6[TS] + β7[logSCC] + 109 β8[logBC] + εij (Eq. 1) 110 whe e: Lij= logis ic model; [Pi]= p obabili y o he esponse ca ego y (posi i e/nega i e); 111 β0= in e cep ; βi= es ima e pa ame e s o he model; [SL]= lac a ion s age e ec (day); 112 [pH]= pH e ec ; [F]= a con en e ec ; [P]= p o ein con en e ec ; [L]= lac ose con en 113 e ec ; [TS]= o al solids con en e ec ; [logSCC]= soma ic cell coun e ec ; [logBC]= 114 bac e ial coun e ec ; εij= esidual e o . 115 3. Resul s and discussion 116 3.1. De ec ion capabili y (CCβ): The CCβ o he BRT MRL, Del o es SP-NT, 117 Del o es DA, and Eclipse 100 es s in sheep and goa milk we e shown in Table 1. In 118 gene al, mic obiological es s de ec ed high equencies o β-lac am an ibio ics in milk 119 om sheep (70.6 %: BRT MRL, and 88.2 %: Del o es SP-NT, Del o es DA, and 120 Eclipse 100) and goa s (76.4 %: BRT MRL, and 82.3 %: Del o es SP-NT; Del o es 121 DA, and Eclipse 100). Only ce quinome and ce ope azone could no be de ec ed by any 122 es a MRL equi alen an ibio ic concen a ion. 123 6 Fo non-β-lac am d ugs, he mic obiological es s could no de ec ed esidues o 124 quinolones, e acyclines, s ep omycin, lincomicin, sul ame azine, colis in and 125 ime hop im a sa e y le els. Con e sely, neomycin, ylosin sul adiazine and 126 sul adime oxine we e de ec ed a o below egula o y limi s (Table 1). Mo eo e , he 127 BRT MRL es was also able o de ec gen amicin and e y h omycin a hei MRLs. 128 3.2. In e e ences ela ed o he milk ma ix e ec 129 Indi idual milk, ee o an imic obials, p esen ed a wide ange o a ia ion o all milk 130 quali y pa ame e s conside ed. When indi idual sheep milk samples we e analysed an 131 ele a e pe cen age o non-complian esul s we e ob ained (BRT MRL: 4.8 %, 132 Del o es SP-NT: 8.0 %, Del o es DA: 10 % and Eclipse 100: 9.6 %). Applying 133 logis ic eg ession analysis, an inc ease in SCC was associa ed wi h an ele a ion in he 134 p edic ed likelihood o posi i e ou comes in all cases (Figu e 1), he BRT MRL es 135 esponse being he less a ec ed by his pa ame e . These esul s we e in ag eemen wi h 136 hose ob ained by Cullo e al. (1992) and Al haus e al. (2003b) using indi idual milk 137 samples om cows and sheep, espec i ely. Ins ead, he pe cen age o non-complian 138 esul s in goa milk we e lowe in all cases (BRT MRL: 1.4 %, Del o es SP-NT: 4.3 %, 139 Del o es DA: 3.1 % and Eclipse 100: 0.6 %), and he e o e, logis ic p ocedu e was no 140 pe o med. 141 4. Conclusions 142 Mic obial inhibi o es s a e e icien o de ec β-lac ams and o he non-be a-lac am 143 d ugs such as neomycin, ylosin, sul adiazine and sul adime hoxine in aw milk om 144 small uminan s. Howe e , in spi e o he imp o emen s made in hese es s in he las 145 decade, hey con inue o be ine icien o he de ec ion o o he d ugs, such as 146 quinolones and e acyclines, a sa e y le els. The e o e, he pe iodic use o mo e 147 sensi i e es s owa ds hese subs ances would be con enien o widen he de ec ion 148 7 ange in sc eening and gua an ee he quali y o milk and dai y p oduc s om small 149 uminan s. 150 5. Acknowledgemen s 151 This esea ch o ms pa o he P ojec AGL2009-11524 inanced by he Minis e io de 152 Ciencia e Inno ación (Mad id, Spain). 153 6. Re e ences 154 Al haus, R.L., To es, A., Mon e o, A., Balasch, S., & Molina, M.P. (2003a). 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