Microsoft Word - 13 VASILACHEart_FIA_full paper corect.doc Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 80 STUDIES REGARDING THE INCIDENCE OF BACTERIA FROM LISTERIA GENUS IN FISH MEAT *Violeta VASILACHE, Constantin CIOTAU Stefan cel Mare University of Suceava, Universitatii Street 13, Suceava, Romania e-mail adress: violetav@fia.usv.ro *Corresponding author Received 25 May 2012, accepted 15 June 2012 Abstract: Listeria is a bacterial genus whose species are Gram positive bacilli and it contains seven representatives. Joseph Lister was an English surgeon who was one of the first in the field of sterile surgery and the name Listeria is given in his honor. Sources of Listeria are soil, contaminated water, animals and vegetables. Because bacteria from Listeria monocytogenes specie are very dangerous for human health, with a mortality rate of 20% for infected patients, a serious attention has to be accorded in consumption of those foods able to transmit microorganisms. This article presents a study about the incidence of Listeria in two species of fish which were tested to prove presence or absence of Listeria monocytogenes, because fish meat is one of the foods susceptible to transmit bacteria and to induce grave intoxication. From fresh water fish Carp was selected and as a representative of ocean fish, Mackerel respectively. Different methods for detection were described and discussed. Those methods are legally accepted in European and Romanian normative for food consumption security as Food Microbiology and Specific Rules for Microbiological Analyzes. OXFORD, PALCAM and CAMP are test used for detection and confirmation of Listeria species of bacteria. Also API- Listeria tests were used for confirmation the presence or absence of different Listeria species in tested samples. Agar with sheep blood medium was inseminated with samples of fish meat because this is the proper medium for bacterial colonies development. The reason is the fact that bacteria colonies formed dark colored rings rounded by a black haloes, because in their metabolic process bacteria produce hydrolysis of aesculin (a glycoside contained in agar medium). The results of analyzes for those two species of fish meat do not confirm the presence of Listeria monocytogenes, but other species of Listeria (Listeria welshimeri and Listeria inocua), fortunately not pathogenic, were present. Keywords: pathogenicity, Listeria monocytogenes, fish meat, infection 1. Introduction Bacteria from Listeria genus and Listeria monocytogenes especially provide a high level of pathogenicity, being responsible by a lot of alimentary toxic- infections and other dangerous diseases, even female abortion. Many sources of Listeria monocytogenes exist in nature, like environment, animals, people [1-7]. From environment, Listeria monocytogenes was mainly isolated from soil, water and vegetables in decomposition. Weis detected Listeria monocytogenes in 21% of samples (from 779 samples of soil and plants). The high capacity of bacteria to survive in difficult conditions permits to explain infections which occur from sources contaminated long time before the reference moment. It was noted that in wastewater dispersed on farm land, bacteria remain active till eight weeks, what could explain human infections in New Scotland – Canada in 1981. The pathogenicity of Listeria monocytogenes is proved by the fact that Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 81 almost 20% of infected patients died. Even the reported cases of listeriosis diminished in developed countries, researchers found varied modes of transmission of bacteria through diverse foods [1,2,5,7,8]. 2. Materials and methods There were selected two species of fish, a carp representing a fresh water fish and a mackerel representing an ocean fish respectively. The samples were analyzed in The Laboratory for Food Safety of DSVSA Suceava. Figure 1. An image of laboratory with a carp fish and culture media for Listeria monocytogenes From fresh water fish Carp was selected and as a representative of ocean fish, Mackerel respectively. Different methods for detection were described and discussed. Those methods are legally accepted in European and Romanian normative for food consumption security as Food Microbiology and Specific Rules for Microbiological Analyzes [9-24]. Figure 2. An image of laboratory with a mackerel fish and culture media for Listeria monocytogenes Preparing of samples followed SR EN ISO 6887-4/2005 regarding Food Microbiology and Specific Rules for Microbiological Analyzes and initial suspensions and decimal dilutions preparing followed SR EN ISO 6887- 1/2002. Fish samples investigation was performed through the method of analyze for detection and confirmation of Listeria monocytogenes implemented and actualized in Romania by SR EN ISO 11290-1/2000 with amendment A1/2005 regarding isolation media and hemolysis test. [21-23]. Analyzes evolved following next stages: - Primary enrichment. In 225 mL liquid media with low concentration of selective agents of semi-Fraser bouillon, insemination of 25 mL of sample followed by 24 h incubation at 30°C was performed. - Secondary enrichment. From obtained culture 0.1 mL were transferred in each test tube with 10 mL Fraser bouillon, followed by incubation for 48 h at 35°C or 37°C. Corrugation and identification. From obtained cultures samples were passed in two isolation media (agar OXFORD and agar PALCAM), with 24 – 48 h incubation at 35°C. In parallel, plates inseminated with positive control tell-tale (reference stem of Listeria monocytogenes ATCC 13932) were incubated in similar conditions [3,5,8]. - Confirmation. 3. Results and discussions Examination of final obtained cultures confirmed that OXFORD and PALCAM media were highly selective for inhibition of associated flora [3,5,8]. On control tell-tale Listeria monocytogenes formed dark colored colonies, rounded by a black haloes, Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 82 because in their metabolic process bacteria produce hydrolysis of aesculin (contained in agar medium) (see figures 3 and 4). Figure 3. Aspect of colonies of Listeria monocytogenes from positive control tell-tale sample on PALCAM selective medium, after 24 hours in thermostatic conditions Figure 4. Aspect of colonies of Listeria monocytogenes on OXFORD selective medium, after 24 hours in thermostatic conditions Confirmation for Listeria monocytogenes was realized by hemolysis test and CAMP tests. Because Listeria generates hemolysis on agar with sheep blood, this could be used to confirm the presence or absence of bacteria. So, Listeria monocytogenes stem from control tell-tale sample produced small colonies rounded by small but clear zone of hemolysis, which is characteristic. Figure 5. Aspect of colonies of Listeria monocytogenes on an agar with sheep blood medium Another reaction, gentle or accentuated could be highlighted with a CAMP test. This test consists in insemination of agar surface with Staphylococcus aureus and Rhodococcus equi on vertical parallel lines (knurling). Then were knurled horizontal parallel lines with Listeria monocytogenes, Listeria innocua and Listeria ivanovii, but horizontal lines did not intersect vertical lines, a distance of 1 – 2 mm remaining. Incubation time was 24 h at 37°C. Image in figure 6 presents a positive reaction, consisting in an arrow form at virtual intersection of the lines. Figure 6. CAMP test – control tell-tale plate (+) The test results show tha absence of Listeria monocytogenes and Listeria ivanovii, but a possible contamination with Listeria inocua or other species of Listeria genus. Another confirmation was achieved using Api-Listeria tests, containing dehydrated substrates. Inseminated and incubated for 18 – 24 h at 35 – 37°C, the color change could signalize the presence or absence of bacteria. So, Listeria inocua and Listeria welshimeri respectively were confirmed for analyzed samples. Figure 7. Listeria monocytogenes identified on API-LISTERIA galleries Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 83 Figure 8. Listeria welshimeri identified on API- LISTERIA galleries Figure 9. Listeria inocua identified on API- LISTERIA galleries All results of tests are summarized in table 1. Table 1. Distribution of species of Listeria genus bacteria Examined product Num ber of sampl es Listeria monocyto genes Listeria inocua Listeria welshim eri Probe 1. Fish meat from Carp specie 5 absent absent 2 Probe 2. Fish meat from Mackerel specie 5 absent 2 1 4. Conclusions Analyzes of those two species of fish with five samples each infirm the presence of Listeria monocytogenes. Carp fish meat contain Listeria welshimeri at two samples from five and Mackerel fish meat contain Listeria inocua at two samples and Listeria welshimeri at one sample from five, respectively. Because Listeria inocua and Listeria welshimeri are not pathogenic, conclusion is that tested fish could be approved for consumption. 5. References [1]. BANU, C. & col., 2007, Food Treaty. Food Technology, Publisher ASAB, Bucureşti [2]. BERCEA I. & col., 1981, Infectious animal diseases, Pedagogical Publishing , Bucureşti [3]. BĂRZOI D., APOSTU S., 2002, Microbiology of food, Publisher RISOPRINT, Cluj Napoca [4]. BĂRZOI D. & col., 1999, Food Poisoning, Deacon Publishing House, Coresi, Bucureşti [5]. BONDOC I., ŞINDILAR E., 2002, Quality control of veterinary and food sanitation, Publishing House, Ion Ionescu de la Brad, Iaşi [6]. CIOTĂU C., 2010, Veterinary control of raw materials Alimentary, Suceava University Press [7]. ROTARU O., MIHAIU M., 2004, Veterinary hygiene of foodstuffs, vol.I., Publisher RISOPRINT, Cluj –Napoca. [8]. STĂNESCU V. APOSTU S., 2010, Hygiene, food safety inspection and animal.Vol.II. Fish and fishing products and avacultură, Publisher RISOPRINT, Cluj – Napoca. [9]. IOANCEA L., KATHREIN I., 1989, Condiţionarea şi valorificarea superioară a materiilor prime animale în scopuri alimentare, Editura Ceres, Bucureşti [10]. KOUBA M., 2002, Qualite des produits d,origine animale, INRA, Prod. Alim., p.161- 169 [11]. ROBIN L.T., CHURCHILL, HUNG LEE, J. CHRISTOPHER HALL, Detection of Listeria monocytogenes and the toxin listeriolysin in food, Journal of Microbiological Methods 64 (2006) 141–170 [12]. OKUTANI A., OKADA Y., YAMAMOTO S., IGIMI S., Overview of Listeria monocytogenes contamination in Japan, International Journal of Food Microbiology, 93 (2004) 131– 140 [13]. DUODU S., HOLST-JENSEN A., SKJERDAL T., CAPPELIER J-M., FRANCE PILET M., LONCAREVIC S., Influence of storage temperature on gene expression and virulence potential of Listeria Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 84 monocytogenes strains grown in a salmon matrix, Food Microbiology, 27 (2010) 795- 801 [14]. DE CESARE A., MIONI R., MANFREDA G., Prevalence of Listeria monocytogenes in fresh and fermented Italian sausages and ribotyping of contaminating strains, International Journal of Food Microbiology, 120 (2007) 124–130 [15]. ORSI R. H., DEN BAKKER H. C., WIEDMANN M., Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics, International Journal of Medical Microbiology, 301 (2011) 79–96 [16]. CORCORAN D., CLANCY D., O’MAHONY M. L., GRANT K., HYLAND E., SHANAGHY N., WHYTE P., MCLAUCHLIN J., MOLONEY A., SE´AMUS FANNING, Comparison of Listeria monocytogenes strain types in Irish smoked salmon and other foods, Int. J. Hyg. Environ.Health, 209 (2006) 527–534 [17]. CHANG Y., GU W., LYNNE MCLANDSBOROUGH, Low concentration of ethylenediaminetetraacetic acid (EDTA) affects biofilm formation of Listeria monocytogenes by inhibiting its initial adherence, Food Microbiology, 29 (2012) 10-17 [18]. ZUNABOVIC M., DOMIG J.K., KNEIFEL W., Practical relevance of methodologies for detecting and tracing of Listeriamonocytogenes in ready-to-eat foods and manufacture environments – A review, LWT- Food Science and Technology, 44 (2011) 351-362 [19]. RYSER E.T., Listeria Monocytogenes, Encyclopedia of Dairy Sciences, p.1650- 1655 [20]. CONTER M., PALUDI D., ZANARDI, GHIDINI E.S., VERGARA A., IANIERI A., Characterization of antimicrobial resistance of foodborne Listeria monocytogenes, International Journal of Food Microbiology, 128 (2009) 497–500 [21]. ***Order ANSVSA, no. 13/2005- Veterinary food safety standard that establishes rules for the sampling of animal products for laboratory examination. [22]. *** Regulation (EC) nr.2073/2005 the European Parliament and Council amended by Regulation (EC) nr.1441/2007 of on microbiological criteria for foods. [23]. ***SR EN ISO 11290-1/A 1:2005, Detection methods for Listeria monocytogenes. Amendment 1. Changing the isolation media and the haemolysis test and inclusion of precision.