Evaluation of Contamination of Poultry Meat and Semi-finished Products with...

[Full title: Evaluation of Contamination of Poultry Meat and Semi-finished Products with Campylobacter Bacteria]

Asian Journal of Pharmaceutic s • Apr-Jun 2017 (Suppl) • 11 (2) | S 265 Evaluation of Contamination of Poultry Meat and Semi-finished Products with Campylobacter Bacteria Nikolay Khomenets 1 , Regina Gurina 1 , Nadiya Khairova 1 , Inga Nityaga 2 1 Agrarian Technological Institute, Peoples’ Friendship University of Russia (RUDN University), 8/2, Miklykho- Maklaya str., Moscow, Russia 117198, 2 FGBOU VPO Moscow State University of Food Production, 11, Volokolamsk Highway, Moscow, 125080, Russia Abstract Purpose: The scope of the paper was the assessment of the contamination (or monitoring) of poultry meat and semi-finished products with Campylobacter (“from the retail network and farms” may be added), using the culture identification technique and the polymerase chain reaction method, as well as the evaluation of the antibiotic resistance of strains isolated from poultry meat and semi-finished products. Materials and Methods: The experimental part of the work was carried out on the basis of the Institute of Veterinary and Sanitary Expertise, Biological and Food Safety of the Moscow State Institute of Food Production and the Federal Publicly Funded Institution of Science “FIC of Nutrition and Biotechnology” at the Nutriicrobiome Biosafety and Analysis Laboratory. The GOST ISO 10272-1-2013 “microbiology of food and animal feeds. Methods for the detection and counting of Campylobacter spp., Part 1. Detection method” was used in the work performed to isolate and identify the Campylobacter spp., Pure growths were identified per totality of cultural, morphological, and biochemical signs confirming the belonging of the isolated cultures to the bacteria of the Campylobacter genus. The belonging of Campylobacter spp., isolated cultures was also determined by polymerase chain reaction (PCR) with electrophoretic detection of amplification products in an agarose gel. The sensitivity to antibiotics was determined according to the clinical guidelines “determination of the sensitivity of microorganisms to antimicrobial agents” (IACMAC, 2014) by disc diffusion method using standard discs. Results: It was established that out of the 28 samples of poultry meat and semi-finished products, Campylobacter bacteria were found in 4 samples of the half-finished product “drumette” in the weights of 10 and 1.0 g. Conclusions: The results of the PCR analysis confirmed the belonging of the isolated cultures to Campylobacter Jejuni species. In turn, the PCR is a faster method than the culture technique and allows giving a definitive answer within a few hours Keywords: Antibiotic resistance, Campylobacter , contamination, microbiological evaluation, polymerase chain reaction, poultry meat Address for correspondence: Nikolay Khomenets, Agrarian Technological institute, Peoples’ Friendship University of Russia, 8/2, Miklykho-Maklaya str., Moscow, Russia - 117 198. E-mail: khomenets_ng@rudn.university Received: 28-12-2016 Revised: 06-04-2017 Accepted: 21-05-2017 INTRODUCTION I n Russia, the industrial poultry farming is one of the most important and rapidly developing sectors of agriculture. Poultry meat makes up more than 43% of the total volume of raw meat, and demand for it has been constantly growing. Thanks to the State Program of Development of Agriculture and Regulation of Agricultural Products, Raw Materials and Food Markets for 2008-2012, as well as the target program of the department “Development of Poultry Farming in the Russian Federation for 2010-2012,” the share of poultry meat in the total meat increased more than twice as compared with 1990. Thanks to government support and investment in the industry, in the near future the Russian poultry farming may become an export-oriented one [1] However, the proportion of nutritional diseases with the alimentary transmission has increased dramatically over the ORIGINAL AR TICLE

Khomenets, et al .: Contamination of poultry meat with Campylobacter Asian Journal of Pharmaceutic s • Apr-Jun 2017 (Suppl) • 11 (2) | S 266 past 10 years. These diseases cause both the physical damage to the human body, often leading to serious complications and even death, and the economic losses to the state. According to the WHO, the consumption of unsafe foods causes more than 200 diseases - from diarrhea to cancer. Every year, 600 million (approximately one in ten) people worldwide fall ill due to the consumption of substandard food containing pathogens. Out of them, 420 thousand die. This has resulted in the loss of 33 million years of healthy life to date. Children under 5 years old are subject to the highest risk: They make up 40% of all cases, and every year 125 thousand children die of nutritionrelated diseases . The diseases with lesions of the gastrointestinal tract constitute the greatest proportion: 550 million people annually, out of them 230 thousand with deaths [2,3] The nutritional diseases, whose etiologic factors are Salmonella spp., Campylobacter spp., and enterohemorrhagic Escherichia coli have acquired the highest prevalence in the recent years [4] Intestinal Campylobacter is widespread all over the world, and according to the WHO data for the period 2007-2015 it significantly exceeded the salmonellosis incidence [3] However, in contrast to salmonellosis, most often it is recorded in high-income countries [Figure 1] People aged 25-64 fall sick most often (57% of cases), and children under 5 years old account for 11% of cases, but in this group, the highest mortality is observed. The sex distribution is 50:50 (according to the WHO report for 2007-2015) Reservoir is represented by wild and domestic birds, in which Campylobacter spp., are the commensals of the gastrointestinal tract. There is the evidence of commensalism of some subspecies of Campylobacter in the gut of pigs [5] The main route of transmission is the nutritional one. The main transmission factors are products made from animal-based raw materials, as well as contaminated water [6] Poultry meat plays a leading role in the transmission of Campylobacter spp., - up to 78% of human cases of nutritional Campylobacter iosis originate from an infected chicken. According to Dawkins МS: People consume nearly 60 billion chickens yearly and, despite the intensive efforts to improve the biosecurity practice on farms over the past decade, 71.2% of chicken flocks in the EU sent for slaughter during 2008 were contaminated with Campylobacter spp., and the incidence among people continues to grow. Moreover, the highest concentration of pathogen is noted in the skin (over 1,000 of colony forming units/g) in the neck and the cloaca. Furthermore, the source of the pathogen may be the beef, pork, milk, and other foods [Figure 2] that have not been sufficiently treated by heat or are contaminated because of non-compliance with technological processes and conditions of storage and sale of finished products [7,8] According to the results of the audit conducted by the CDC in 2011, Campylobacter bacteria were detected in 47% of raw chicken samples sold in the retail network. According to the reports of EU member states for 2013, Campylobacter were isolated from 31.4% of raw chicken meat samples. In the period of 2014-2015, according to surveys conducted by the British Agency for Food Standards, 73.3% of the tested products contained Campylobacter , and the frequency of pathogen detection in the packaging swabs was 7% [3,9] MATERIALS AND METHODS The experimental part of the work was carried out at the Institute of Veterinary and Sanitary Examination, Biological and Food Safety FSBEU “Moscow State Institute of Food Production” and FSBUN “FIC of Food and Biotechnology” in the biosafety and nutrimicrobiom analysis laboratory A total of 28 samples were examined, including 12 - from the chicken carcass, 8 - from soup sets, consisting of the dorsal part and the skin, 4 - skin trimmings, and 4 - drumette. All Figure 1: Distribution of sickness cases by regions. AFR: African region, EMR: Central Asian region, AMR: North American region, EUR B: Western Europe, EUR C: Eastern Europe, including Russia Figure 2: The product distribution according to the degree of contamination

Khomenets, et al .: Contamination of poultry meat with Campylobacter Asian Journal of Pharmaceutic s • Apr-Jun 2017 (Suppl) • 11 (2) | S 267 samples were obtained from various farms of the Moscow and Tambov regions Defining by the culture method was performed according to GOST ISO 10272-1-2013 «Microbiology of food items and animal feed. Methods of detection and counting Campylobacter spp., bacteria. Part 1: Method of detection. Furthermore, the bioMerieux (France) API CAMPY diagnostic systems (strips) were used for the specific differentiation of Campylobacter isolated cultures. The species confirmation of the Campylobacter spp., isolated cultures was also performed using polymerase chain reaction (PCR) with the electrophoretic detection of amplification products in the agarose gel. Determination of sensitivity to antibiotics was performed according to clinical guidelines, “Determination of the sensitivity of microorganisms to antimicrobial agents” (IACMAC 2014) using the disk diffusion method with the standard disks After the cultivation time, the cups with inoculation were examined for signs of growth. Several types of colonies were detected. On coal agar: (1) Small white waxy opaque convex, the medium surface covered with white translucent bloom, the medium has a sharp, unpleasant smell, (2) small-dew translucent whitish flat, resembling drops of condensation. On Preston agar: (1) Small-dew flat with irregular shape, somewhat like spreading in the course of a stroke, (2) convex round waxy, yellow in the middle, and pale at the edges - translucent, the medium has a sharp odor and a hemolysis area, and (3) white waxy, with signs of creeping growth. Suspicious colonies were tested for oxidase and catalase The smears were prepared from the colonies similar with Campylobacter by cultural properties and giving a positive response to the oxidase and catalase and stained by Gram. A total of 64 smears were microscope, and in two of them, the multiple Gram-negative coccoid forms of thin and small single polymorphonuclear curved rods were found. Based on these data, a preliminary conclusion about the presence of Campylobacter spp., in two samples was made. To obtain pure cultures, the Columbia blood agar was reseeded to the surface On blood agar: Single, small-dew, translucent, spreading colonies, and a weak zone of hemolysis. In smears: Gramnegative small thin polymorphonuclear curved rods When cultured on TSA and in broth, there were no visible signs of growth for Brucella. That confirms the appurtenance of the isolated microorganisms to thermotolerant Campylobacter species The study of biochemical properties of the isolated strains was conducted using the bioMerieux api Campy diagnostic kit To confirm the appurtenance of the isolated species to Campylobacter jejuni , the PCR analysis was performed, as one of the highly specific molecular biological techniques available today RESULTS PCR results have confirmed that the isolated microorganisms are the C. jejuni [Table 1] In the study of antibiotic susceptibility, the multiple antibiotic resistance was evident in the isolated strains The cultures were resistant to penicillins, cephalosporins, and fluoroquinolones, which is consistent with the available literature data. Erythromycin - the drug of choice in the treatment of nutritional Campylobacter iosis - was ineffective against isolated strains. However, the cultures were sensitive to tetracyclines and aminoglycosides, although the available literature data indicate the stability of most strains to these drugs DISCUSSION OF THE STUDY RESULTS It was established that out of 28 samples of poultry and semi-finished products the Campylobacter bacteria had been detected in 4 semi-finished product samples “drumette” in 10 and 1.0 g weights. Based on the study of biochemical properties with the use of the bioMerieux api Campy diagnostic kit, it has been found that the isolated cultures belong to thermotolerant C. jejuni subsp. Jejuni . The results of the PCR analysis confirmed the appurtenance of the isolated cultures to C. jejuni form In its turn, the PCR technique is faster as compared to the culture method and allows giving a definitive answer within a few hours Isolates of Campylobacter spp., had a multiple antibiotic resistance and were sensitive to aminoglycosides, Table 1: PCR results April 28, 2016 Sample Result Phoresis Molecular weight marker Negative control of allocation Sample 1 + Sample 2 + Positive control + Molecular weight marker Negative control ‑ PCR: Polymerase chain reaction

Khomenets, et al .: Contamination of poultry meat with Campylobacter Asian Journal of Pharmaceutic s • Apr-Jun 2017 (Suppl) • 11 (2) | S 268 tetracyclines, and fenicoles. Among the macrolides, the azithromycin proved to be effective CONCLUSION Dynamic development of the poultry farming in Russia and the increased consumption of poultry by the population, the growing incidence of food Campylobacteriosis , often severe complications of the disease, high contamination of raw materials and the lack of sufficiently accurate and simple methodology for the detection of pathogens in food cause the relevance of obtaining microbiologically safe poultry products. For this purpose, it is proposed to carry out a constant microbiological control both in the procurement of raw materials and on the stages of production, storage and sale of food products REFERENCES 1. Buyarov VS, Buyarov AV, Kleymenov IS, Shalimova OA. Sostoianieiperspectivyrazvitiyamyasnogoptitsevodstva [Status and Prospects of development of the poultry meat farming]. Bull Orel State Agrarian Univ 2012;1:49-61 2. WHO. Estimates of the Global Burden of Foodborne Diseases. Foodborne Disease Burden Epidemiology Reference Group 2007-2015. Geneva: WHO; 2015. p. 83-84 3. WHO. Food Safety. Fact Sheet #399. Geneva: WHO; 2015. p. 2-3 4. Khomenets NG, Usha BV, Morozova EN, Khomenets NG. Improved control of Salmonella in meat and meat products by PCR in real time and robotics to select the template DNA. Bulletin of the Russian peoples’ friendship university. Ser Agric Cattle Breed 2016;1:61-5 5. Shurysheva ZN. Assessment of the Risk of Contamination of Food with Campylobacter Bacteria. Thesis of the Candidate of Medical Sciences. Moscow: SI “RAMS Research Institute of Nutrition”; 2007. p. 145 6. Efimochkina NR. Microbiology of Food and Modern Methods of Detecting Pathogens. Moscow: RAMS Publishing House; 2013. p. 518 7. Colles FM, Cain R, Nickson T, Smith A, Roberts SJ, Maiden MC, et al . Monitoring chicken flock behavior provides early warning of infection by human pathogen campylobacter. In: The Proceedings of the Royal Society; 2016. p. 57-8 8. Evans MR, Ribeiro CD, Salmon RL. Hazards of healthy living: Bottled water and salad vegetables as risk factors for campylobacter infection. Emerg Infect Dis 2003;10:1219-25 9. World Health Organization. WHO Estimates of the Global Burden of Foodborne Diseases. Foodborne Disease Burden Epidemiology Reference Group 2007- 2015. Geneva: WHO; 2015. p. 34 Source of Support: Nil. Conflict of Interest: None declared.

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