25 l KEYWORDS Fluoroquinolones, Residues, Chicken, A ntibiotic resistance, Nigeria. PAGES 25 – 34 REFERENCES Vol. 1 No. 2 (2014) ARTICLE HISTORY Submitted: September 26, 2014 Revised: November 29, 2014 Accepted: December 02, 2014 Published: December 04, 2014 CORRESPONDING AUTHOR Omotoso Adekunbi B., Animal Products and Processing Unit, Department of Animal Science, University of Ibadan, Ibadan, Nigeria e-mail: kunbiadeshiyan@yahoo.com phone: +234 08033900828 JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Screening of fluoroquinolone residues in imported and locally produced broiler chicken meat in Ibadan, Nigeria. Omotoso Adekunbi B.1* and Omojola Andrew B.1 1 Animal Products and Processing Unit, Department of Animal Science, University of Ibadan, Ibadan, Nigeria ABSTRACT. The study was conducted to investigate residues of three fluoroquinolones (ciprofloxacin, norfloxacin and ofloxacin) in chicken meat sold in Ibadan, Nigeria. Two hundred and ninety-seven (297) samples of imported frozen (99) loc ally produced frozen (99) and freshly slaughtered (99) broiler chicken meat products were screened for antibiotic residues by microbiological as say using Escherichia coli as test organism. High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was used for the determination of ciprofloxacin, norfloxacin and ofloxacin in the positive samples. One hundred and sixty (160) samples, constituting more than half (53.87%) of total sample size tested positive for Escherichia coli–sensitive antibiotic residues. Positive samples were 54.55%, 56.57% and 52.53%, of freshly slaughtered, locally produced frozen chicken and imported frozen chic ken meat respectively. Residues of investigated fluoroquinolones occurred more frequently in locally produced frozen chic ken than in imported frozen chicken. The concentrations were however consistently higher in imported chicken. Among the three fluoroquinolones examined, the most abundant in imported frozen chicken was ciprofloxacin with mean 354.83±716.43µg/kg. Norfloxacin was the most abundant in freshly slaughtered and locally produced frozen chicken meat, having mean values of 107.70±138.36µg/kg and 120.96±162.83µg/kg respectively while ofloxacin was the lowest in all categories. M ost frozen chic ken products imported into Nigeria at the time of this study contain higher levels of residual fluoroquinolones than the locally produced chicken. In order to tackle fluoroquinolone resistance from a food safety perspective, proper usage and monitoring of fluoroquinolones in meat animals should be encouraged in developing countries. Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 25 - 34 26 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 1 Introduction The World Health Organization (WHO) recognises that while an tibiotics can play a critical role in food production, th ere is need to balan ce their use to ensure they remain a valuable tool for both human and animal health (WH O, 2011b). Antibiotics used in meat animal production do not pos e a health hazard provided they are used in accordance with th e recomm endations for th eir use, proper dosage, proper route of administration, proper species of animal and adequate withdrawal period before slaughter (Dipeolu, 2010). Quinolones are effective ch emotherapeutic agen ts with v ery good antibacterial activity that target DNA synth esis. Fluoroquinolones are derivatives of quinolon es which are typically fluorinated at C-6 or C-7 position of th e quinolon e ring. Norfloxacin, ciprofloxacin, ofloxacin, lomefloxacin, enrofloxacin are second generation quinolones, exhibiting a broader activity against gram negative and gram positive bacteria, less protein binding, higher drug tolerance, lower toxicity and longer half life than the first generation (Emami et al., 2010; Somasundaram and Manivannan, 2013). Drug residues in foods have been said to cause allergic reactions, toxicity, techn ological problems in fermented products and the developmen t of an tibiotic resistance in human pathogens. It has been documen ted that a major rou te of transmission of resistant micro- organisms from animals to humans is through the food chain (Hernandez -Serran o, 2005; Canada-Canada, 2012). Quite unfortunately, poultry production has been implicated in th e emerg ence of quinolon e resistan t bacteria. Resistance to fluoroquinolones in Escherichia coli and Salmo nella typhi is an increasing problem in Nigeria and many oth er countries (Daini et al., 2005). Researchers hav e demonstrated the prevalence of bacteria which are resistan t to various fluoroquinolon es in different parts of Nigeria, including Lagos (Aibinu et al., 2004), Benin City (Enabulele et al., 2006), Oshogbo (Olow e et al., 2008) and Ibadan (Makanjuola et al., 2012). Antibiotic resistance has long been recognised as a food safety issue and all stakeholders in food production are responsible for the preven tion and con trol of antibiotic resistance through th e food chain. The rising prevalen ce of an tibiotic resistance is a particularly importan t problem in developing countries whe re there is limited con trol of th e quality, distribution an d use of antibiotics in human medicin e, veterinary medicine and food animal ag ricultu re (Ok ek e et al., 1999). It is important for coun tries to monitor residues of antibiotics in their foods because the use of an tibiotics in on e sector, setting or coun try affects the spread of resistance in others. The WH O has noted that since animal products are traded w orldwide, th ey contribute to an tibiotic resistance in countries far from where the problem origin ates. Hen ce the need to monitor both imported and locally produced animal products for residues of antimicrobials, especially highest priority critically importan t an timicrobials such as th e fluoroquinolones (WH O, 2011a ). Ciprofloxacin and norfloxacin are am ong th e mos t commonly used fluoroquinolones in poultry production in Ibadan m etropolis and ofloxacin is the common drug for treatin g tuberculosis in Nigeria (Daniel et al., 2011). This study seeks to examine samples of locally produced and imported chicken products for residues of three fluoroquinolone compounds (ciprofloxacin, norfloxacin and ofloxacin) because of th eir im portance in both animal an d human medicine. Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 25 - 34 27 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 2 Materials and Methods 2.1 Study area The study was carried out in Ibadan, Oyo State, Nigeria. Samples were collected from small, medium and large scale farms as well as open frozen chick en market spread across th e eleven (11) local government areas that make up Ibadan, the capital of Oyo State, Nigeria . 2.2 Sample collection Sample size was two hundred and ninety seven (297) comprising 99 samples of freshly slaughtered chicken (from 3 small scale, 3 medium scale and 3 large scale farms in each of eleven local governmen t areas), 99 samples of locally produced frozen chick en and 99 samples of imported frozen chick en thigh muscles. The samples were collected on ice and kept frozen at -30C at the Meat Science Unit of the Department of Animal Scien ce, University of Ibadan, until th ey w ere further transported in padded coolers with ice packs to the Agulu Lab oratory of th e National Agency for Food and Drug Adminis tration and Control (NAFDAC) where th e analysis was done. The samples were screen ed for three fluoroquinolones, ciprofloxacin, norfloxacin and ofloxacin with structural formulae as shown in Figure 1. Figure 1. Structural formulae of ciprofloxacin, norfloxacin and ofloxacin. Source: http://www.newdruginfo.com/pharmacopeia/usp28/v28230/usp28nf23s0.htm Ciprofloxacin (b) Norfloxacin (c) Ofloxacin 2.3 Microbiological screening One Plate Test (OPT) as described by Alla et al. (2011) was adapted for this study. Microbiological screening using th e plate test depends on bacterial g rowth inhibition. The tes t organism was Escherichia coli (American Type Tissue Culture 11303 ). Escherichia coli has been indicated as a reliable test organism for th e detection of fluoroquinolon es in animal products. To prepare the sub-culture, wire loop ch rom e passed through Bunsen burner flam e was allowed to cool and used to s eed Escherichia coli in the nu trient broth which was then incubated at 370C for 24 hours. Th e culture m edium was prepared by suspending 34g of Mueller Hinton Agar (Merck KGaA, Damstadt, Germany) in 1 litre of demineralised water an d heating in a boiling water bath. It was th en autoclaved for 15 minu tes at 120 0C. The cultu re medium was sterilized in an autoclave at 121 0C for 20 minutes. javascript:modelesswin('imageViewer?doc='+parent.myTitle+'&img=uspnf/pub/images/v28230/g-1292.gif',500,500); Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 25 - 34 28 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 Sterilized stainless steel scissors, forceps and cork borers were used in handling meat samples. The work table was wiped down intermittently with 70% ethanol. 20ml of th e culture m edium was pou red in to each 90mm Petri dish and left on th e work - bench for abou t 15 minutes to solidify. With the aid of sterile swab sticks, Escherichia coli was seeded in th e plates. Holes were punched in to each aga r plate with a cork borer. Sterile forceps were used to place meat discs inside the hole. Th e plates w ere th en incubated a t 370C±2 for 24 hours. Zon e of inhibition was measured with a mm -graduated ruler. Samples with no clear zone or with clear zon es less than 1mm were taken as negative. Clear zon es betw een 1mm and 2mm were considered doubtful while zones from 2mm upwards were positive. 2.4 Confirmation and quantification of fluoroquinolones by HPLC with UV detection Negative samples w ere discarded while chick en samples that were either doubtful or positive for residues of an tibiotics were subjected to HPLC - UV for iden tification an d quantification. Residue extraction was done as described by (Ovando et al., 2004). 0.2g thigh muscle was homogenized with 2mL phosphate buffer (pH 7.2) prepared in the laboratory. 8mL Dicholorom ethan e (Sigma Aldrich, St. Louis, Mo, USA) was added to the homogenate, which was mixed on a Stuart Scien tific SA8 vortex mixer (Sigma Aldrich, St. Louis, Mo, USA) at 1000rpm for 1 minute and centrifuged at 4000 rpm for 20 minutes. The upper aqueous layer was discarded, the organic phase was transferred to a clean tube and the tissue was again extracted with 6mL of dichlorom ethane. Organic layers w ere combin ed and evaporated a t 300C under nitrog en stream. The extract was re-dissolved with 200μL of mobile phase an d 100μL was used for HPLC analysis. The HPLC equipmen t was Elite Lach rom VWR, Hitachi HPLC ch romatograph (Hitachi, Tokyo, Japan) comprising L2200 autosampler, L2130 pump and L2350 column oven, equipped with L2400 UV VIS detector and Ezch rom Elite software. Th e separating column was Elite C18 (250mmx4.6mmx5µm ) (Hitachi, Tokyo, Japan). Reference standards of ciprofloxacin (99.6%), norfloxacin (99.5%) and ofloxacin (99.9) were provided by NAFDAC. Buffer phosphate solution 0.1M, pH 7.2 was prepared in th e laboratory. Deionised water, passed through 0.45µm Whatman filter was used throughout. The solvents, dichlorom ethan e, acetonitrile and triethylamin e were HPLC grade. The mobile phase was water: acetonitrile: trieth ylamine (80:19:1), adjusted to pH 3.0 with phosphoric acid, filtered through 0.45µm nylon membrane. Samples and separate s tandard solu tions were identified using the HPLC ch romatograph with UV detector. Ciprofloxacin, norfloxacin and ofloxacin were identified individually by comparing the retention time, area and spectra of peaks of unknown substance with respective s tandard substan ces. The quan tity of iden tified substan ces was calculated using th e formula adapted from Naeem et al. (2006). Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 25 - 34 29 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 2.5 Statistical analysis Statistical analysis was accomplished by analysis of variance using SPSS (2005). Treatm en t means were separated by Duncan Multiple Range Test and statistical significance was set at a probability of P ≤ 0.05. 3 Results Results of microbiological screening show that out of a to tal sample size of 297 (tw o hundred and nin ety seven ), 162 (54.55%) w ere positive for at least one antibiotic to which Escherichia coli was susceptible. Samples were categorised as negative and positiv e based on the zon es of inhibition around m eat samples and confirmation with HPLC (Figure 2). Figure 2. Result of antibiotic residue screening in chicken meat pu rchased from differen t markets in Ibadan. Ciprofloxacin, norfloxacin and ofloxacin residues are present togeth er in most freshly slaughtered and locally produced frozen chicken samples. Fewer samples contain ed eith er ciprofloxacin alone or ofloxacin alone. How ever, norfloxacin residues did not exist in isolation in any of the samples. Distribution of identified fluoroquinolones in the three sample groups is shown in Table 1. Mean residual concen tration of ciprofloxacin in imported frozen chicken samples (354.83 ± 716.43 µg/kg) was significantly (P ≤ 0.05) higher than the con cen tration in locally produ ced frozen chick en (64.63 ± 120.77 µg/kg). Ofloxacin follows th e same trend bu t no significan t difference was obs erved in concen tration of n orfloxacin residue in th e th ree categories. 54,55 43,43 52,53 45,45 56,57 47,47 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Freshly slaughtered Locally produced frozen Imported frozen P e rc e n ta g e o f sa m p le s Class of chicken meat Antibiotic-negative Antibiotic-positive Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 25 - 34 30 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 Table 1: Occurrence of three fluoroquinolones in chicken m eat sold in Ibadan as confirm ed with HPLC Type of chicken meat Freshly slaughtered Locally produced frozen Imported frozen Samples with one of the fluoroqu inolones Ciprofloxacin only - - 5 Norfloxacin only - - - Ofloxacin only - 3 3 Samples with 2 of 3 fluoroquinolones Ciprofloxacin & norfloxacin 5 18 10 Ciprofloxacin & ofloxacin - - 5 Norfloxacin & ofloxacin 5 3 9 Samples with all three fluoroquinolones Ciprofloxacin, norfloxacin & ofloxacin 44 32 20 Total positive samples 54 56 52 Total no of samples 99 99 80 % of total positive with at least one fluoroquinolone 54.55 56.57 52.53 4 Discussion Investigating residues of fluoroquinolon es in broiler chicken is of g reat public h ealth concern (AL-Mustafa and Al-Ghamdi, 2000; WHO, 2011b). It has been argued that small doses of fluoroquinolones inges ted from consumption of m eat products with residues of fluoroquinolones weak ens their effectiveness leading to drug resistance by bacteria. The National Policy on Food H ygiene and Safety (NPFH S) seeks to ensure that all foods consumed in Nigeria, whether imported or locally produced, are wholesome, nutritious and free from contaminan ts (Omotayo and D enloye, 2002). There is curren tly a ban on th e importation of poultry products in Nig eria but im ported chick en parts are still freely sold in open markets, especially in the south western parts of th e country. Since the products are illegally imported, not much atten tion is given to their inspection and quality con trol (Dipeolu, 2010). Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 25 - 34 31 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 Table 2: Fluoroquinolone residues in chicken m eat pu rchased from different markets in Ibadan. Fluoroquinolones Mean concentration ± SD of fluoroquinolones (µg/ kg) P value Freshly slaughtered Locally produced frozen Imported frozen Ciprofloxacin 78.02 ± 202.38by 64.63 ± 120.77by 354.83 ± 716.43ax 0.000 Norfloxacin 107.70 ± 138.36x 120.96 ± 162.83x 127.99 ± 279.63y 0.931 Ofloxacin 15.18 ± 24.35bz 13.55 ± 22.46bz 42.33 ± 102.53ay 0.019 P value 0.007 0.000 0.049 a, b Means in the same row with different superscripts are signific antly different (P ≤ 0.05) x,y Means in the same column with different superscripts are significantly different (P ≤ 0.05) Table 3: Range of con centration of th ree flu oroquinolones in chicken meat purchased from different markets in Ibadan, Nigeria Fluoroquinolones Type of chicken meat Range of concentration in positive sampl es (µg/kg) % above 100 (µg/kg)* Ciprofloxacin Freshly slaughtered 2.45 to 888.90 15.15 Locally produced frozen 2.45 to 591.50 24.24 Imported frozen 14.00 to 2767.00 32.32 Norfloxacin Freshly slaughtered 3.98 to 393.17 40.40 Locally produced frozen 20.50 to 466.65 33.33 Imported frozen 6.18 to 1081.27 20.20 Ofloxacin Freshly slaughtered 4.36 to 77.51 0.00 Locally produced frozen 8.76 to 67.13 0.00 Imported frozen 14.53 to 431.66 0.00 * European Union MRL (Commission Regulation (EU) 37/2010) for the sum of enrofloxacin and c iprofloxacin in poultry muscle Maximum residue limits (MRL) hav e been set for different antimicrobials in differen t food matrices by different organizations and countries. If th e residue of a chemical exceeds th e MRL in meat, it is considered unsafe (Petrovic et al., 2006). MRL for antibiotics in foods of animal origin hav e not been s et in Nigeria. Thus the EU MRL for the sum of enrofloxacin an d ciprofloxacin is adopted for the pu rpose of this study. Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 25 - 34 32 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 Microbial inhibition assay was em ployed as a first qualitative screening step to sift ou t large number of compliant samples. Overall, one hundred and thirty seven (137) representin g 46.13% of total sample size con tained no residues of Escherichia coli - sensitive antimicrobials. Fluoroquinolone residues occu rred more frequ ently in locally produced frozen broiler chicken samples. Positive samples were 54.55%, 56.57% and 52.53%, of freshly slaughtered, locally produced frozen chicken and imported frozen chicken respectively. Norfloxacin residues are presen t in higher con centration in chicken produced within Nigeria and are thus of more concern than ciprofloxacin and ofloxacin. H owever, ciprofloxacin is more abundant than norfloxacin and ofloxacin in imported products (Table 2). In a similar experim ent, Al- Mustafa and Al-Ghamdi (2000) detected norfloxacin in raw chicken tissues from the eas tern province of Saudi Arabia at levels that were 2.7 to 34.3 folds higher than the MRL. Naeem et al. (2006) observed residues of ciprofloxacin, norfloxacin an d ofloxacin in liver samples purchased from various mark ets in Lahore, Pa kistan, to be 2.45 to 245.00 µg/kg, 2 20 to 31.00 µg/kg and 2.05 to 22 µg/kg respectively in summer. In the pres en t study, there is wide variation in concentration (Table 3) of flu oroquinolones in chicken samples. 5 Conclusions If antibiotic resistance must be tackled from a fo od safety perspective, chick en meat, whether imported or locally produced, must be monitored for residues of antimicrobials, especially those that have been classified as critically important. Th e present ban on importation of poultry m eat in Nigeria should be properly implemen ted to redu ce th e exposure of consumers to fluoroquinolon e residues. As a matter of public health, it is importan t to con trol the use of these antimicrobials in the local production of chicken so as to maintain their potency for use in human medicine. 6 Acknowledgements This study was technically supported by the National Agency for Food and Drug Administration and Control (NAFDAC), Nigeria. The authors are thankful for th e assistance of the staff of NAFDAC regional laboratory, Agulu. 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