Investigation of the possibility of detection of fluoroquinolones in carp kidney by microbiological diffusion method

Abstract

Fish is one of the most valuable food products used in human nutrition, due to the content of proteins, fat, minerals, vitamins, essential n -3 polyunsaturated fatty acids (PUFAs) and cholesterol. To meet the growing needs of the population for this kind of food, fish are increasingly grown in aquaculture. Intensive fish production, due to increased stock density, is favoring the occurrence of bacterial diseases. As a consequence, there is increased morbidity and mortality, reduced growth and reduced leasing sockets materials, which pose a serious problem for the aquaculture and lead to massive use of chemotherapeutics. Antibiotics are the common practice for the treatment of bacterial infections in fish ponds, and flurohinolones are used as antibiotics of choice. Fluoroquinolones are a group of antibiotics that have a broad spectrum of activity, low toxicity and only a few side effects in the treated fish. However, residues of antibiotic in fish tissues represent a real risk to human health. Consumption of fish containing residues of antibiotics can cause allergic, toxic, carcinogenic, mutagenic and teratogenic effects. Therefore, it is necessary to establish the residual amounts of antibiotics in fish tissues, that can be achieved by using reliable laboratory methods and techniques. Because of this, the goal of the work was set to investigate the possibility of identification and quantification of fluoroquinolones in the kidneys of carp by microbiological diffusion method, using the test organism E. coli ATCC 11303. The investigations revealed that microbiological diffusion method enables detection of all five fluoroquinolones (oxolinic acid, enrofloxacin, sarafloxacin, difloxacin and flumequine) in kidney of carp at different maximum residue levels, MRL (100 mg/kg, 100 mg/kg, 30 mg/kg, 300 mg/kg and 600mg/kg, respectively). Identification and quantification of fluoroquinolones at MRL levels was only achieved for enrofloxacin, flumequine and difloksacin. These fluoroquinolones can be detected and quantified at the level below the MRL, i.e. at the level of 1/4 MRL. Contrary to these fluoroquinolones, sarafloxacin can be detected only at the level of 2MRL and oxolinic acid can be detected at the level of 4MRL, as well. EU regulations provide that a screening method can be applicable only if a compound can be detected in the amount of at least at the MRL, and the ½ MRL is recommended. This means that the microbiological diffusion method can be used in routine analytical practice for the identification and quantification of enrofloxacin, flumequine and difloxacin in kidney tissues of carp.

Riba je, zbog sadržaja i količina proteina, masti, minerala, vitamina, esencijalnih n-3 polinezasićenih masnih kiselina (PNMK) i holesterola, jedna od nutritivno najvrednijih namirnica koja se koristi u ishrani ljudi. Da bi se zadovoljile rastuće potrebe stanovništva za ovom vrstom namirnice, riba se sve više gaji u akvakulturi. Intenzivna proizvodnja ribe, zbog povećane gustine nasada, pogoduje nastanku bakterijskih oboljenja. Kao posledica toga, javlja se povećan morbiditet i mortalitet, smanjen prirast i smanjenje nasadnog materjala, što predstavlja ozbiljan problem za akvakulturu i dovodi do masovne upotrebe hemioterapeutika u terapijske svrhe. Za lečenje bakterijskih infekcija riba u ribnjacima koriste se antibiotici. Kao antibiotik izbora u uzgojnom ribarstvu se koriste flurohinoloni. Fluorohinoloni su grupa antibiotika koji imaju širok spektar delovanja, nisku toksičnost i mali broj neželjenih dejstava na tretiranu ribu. Međutim, ostaci antibiotika u tkivima riba predstavljaju realan rizik za zdravlje ljudi. Ishrana ribom koja sadrži ostatke antibiotika može da dovede do pojave alergijskih, toksičnih, karcinogenih, mutagenih i teratogenih efekata. Stoga je neophodno da se ustanove rezidualne količine antibiotika u tkivima riba, što se može postići korišćenjem pouzdanih laboratorijskih metoda i tehnika kojima se ispituju ostaci antibiotika u mesu riba. Zbog navedenog, kao cilj rada definisano je da se ispita mogućnost identifikacije i kvantifikacije fluorohinolona u bubrezima šarana mikrobiološkom difuzionom metodom uz pomoć test mikroorganizma, E. coli ATCC 11303. Ispitivanjima je ustanovljeno da mikrobiološka difuziona metoda omogućava detekciju svih pet fluorohinolona (oksolinska kiselina, enrofloksacin, sarafloksacin, difloksacin i flumekvin) u tkivima bubrega šarana na različitim nivoima maksimalno dozvoljenih količina, MDK (100 μg/kg, 100 μg/kg, 30 μg/kg, 300 μg/kg i 600 μg/kg, respektivno). Identifikacija i kvantifikacija fluorohinolona na nivou MDK moguće je samo za enrofloksacin, difloksacin i flumekvin. Ovi fluorohinoloni mogu da se detektuju i kvantifikuju i na nivou ispod MDK, odnosno na nivou od 1/4 MDK. Za razliku od navedenih fluorohinolona, sarafloksacin se može detektovati samo na nivou od 2 MDK. Oksolinska kiselina se može detektovati na nivou od 4 MDK. Propisi EU predviđaju da trijažna (screening) metoda može biti primenjiva samo ukoliko se neko jedinjenje može detektovati u visini MDK, a preporučljivo je do 1/2 MDK. To znači da se mikrobiološka difuziona metoda može koristiti u rutinskoj analitičkoj praksi za identifikaciju i kvantifikaciju enrofloksacina, difloksacina i flumekvina u tkivima bubrega šarana.