Abstract

1. Abstract In this study, water samples were collected from agricultural areas of Bilecik Karaköy, Pazaryeri, Söğüt; Eskişehir Alpu and İnönü in Turkey. Water samples were investigated to detect contamination of Listeria. FDA with MOX and MMLA was employed to isolate Listeria from the samples. Listeria sp. could not been isolated on MMLA. However, 55 water isolates were isolated using MOX. In this study, cold enrichment technique at 30 ºC for 48 hours was found more efficient than enrichment technique at 4 ºC for 4 weeks. Fifty fiveListeria strains isolated from water samples. Among those strains, 24 of them were L. monocytogenes, 15 strains were L. denitrificans, 7 strains were L. welshineri and 9 of the strains were L. innocua. As a result, L. monocytogenes were recorded from 43.63 % of water samples contaminated by Listeria. Keywords: Listeria spp., irrigation water, modified Oxford medium IntroductionListeria monocytogenes, a Gram positive, facultative anaerobic and non-acid fast bacterium, is a non-spore forming rod that expresses a typical tumbling motility at 20-25 ºC. It is widely distributed throughout the environment and is considered hardy bacteria because of their ability to growth over a wide range of temperatures (0.5-45 ºC), pH (4.7-9.2) and osmotic pressures, which allows them to survive longer under adverse conditions. Listeria have been isolated from various environments including soil, sewage, vegetables and various foods (Ryser and Marth, 1991). L monocytogenes has been isolated from the surface water samples obtained from canals and lakes in Northern Holland (Dijkstra, 1982) and in Northern Greece (Arvanitidou et al., 1997). Vegetables can become contaminated from the soil and water or from manure used as fertilizer. The bacterium has been found in a variety of raw foods, such as uncooked meats and vegetables, as well as in processed foods that become contaminated after processing, such as soft cheese and cold cuts at the delicounter. Listeria monocytogenes(figure 1) is widespread pathogen of concern to public health and the food industry. This microorganism has been associated with outbreaks of listeriosis (Fleming et al., 1985; Farber and Peterkin, 1991) with a mortality rate near 30%. Figure 1Listeria monocytogenes scanning EM (http://textbookofbacteriology.net) L. monocytogenes is known to be present on vegetation (Welshimer, 1968) and in silage prepared from a variety of grasses (Irvin, 1969). Thus, the organism may exist on raw vegetable and fruits at the time of consumption. In addition, L. monocytogenes apparently is tolerant to acid pH commonly associated with anaerobically fermanted vegetation (Beuchat, 1986). In this study, water samples were collected from agricultural areas of Bilecik Karaköy, Pazaryeri, Söğüt; Eskişehir Alpu and İnönü in Turkey. Water samples were investigated to detect contamination of Listeria. Material and methods Collection of irrigation water samples Water samples were collected from agricultural areas of Bilecik Karaköy, Pazaryeri, Söğüt; Eskişehir Alpu and İnönü in Turkey, were taken for a period 6 months. Then they were brought by using 500 ml steril glass bottles and were immediately kept under ice-cold conditions and microbiological analyses were performed within 3 h of collection. Isolation and identification of the Listeria A25 ml of irrigation water sample was measured into each of a series of cap to which were added 225 ml of tryptose broth (added Listeria supplement Oxoid SR+141E). After 2 min of mixture, contents were incubated at 30 ºC, for 48 h and for 4 weeks at 4 ºC (Lovett et al., 1987). For the Food and Drug Administration (FDA) method (Lovett et al., 1987), enrichment broth culture was streaked, with and without KOH pre-treatment, to modified Oxford medium (MOX) and modified McBride Agar (MMLA) after 48 h of incubation at 30 ºC, plates were incubated for 48 h at 35 ºC. Up to 10 colonies that had the appearance typical of L.monocytogenes on MMLA and MOX were picked from each plate, using 45º incident-transmitted light and a dissecting microscope and stab-transferred into TSA–5% sheep blood plates. The plates were incubated at 35 ºC for 24 h. Test used to confirm these isolates included the examination of cellular morphology and tumbling motility is wet mounts, Gram reaction, urease production, H2Sproduction, nitrate reduction, urease production, Voges-proskauer reaction, haemolytic activity on blood agar, umbrella-shaped growth in motility test medium and ability to produce acid from the fermentation of glucose, mannitol, maltose, rhamnose and xylose. Detection of Listeria spp. in irrigation waterM. Kıvanç, E. Çakır and N. ÖzbaşaranAnadolu University, Science Faculty, Department of Biology, Eskişehir, Turkey(E-mail: mkivanc@anadolu.edu.tr; ecakir@anadolu.edu.tr) The isolation of L. monocytogenes greatly depended on the procedure used. No isolates were made by direct plating of the irrigation water before cold enrichment and at 30 ºC for 48 h enrichment. All isolates were detected after enrichment at 4 ºC for at 4 weeks and at 37 ºC for 48 hours. Direct plating after 4 week of cold enrichment at 4 ºC yielded 26 isolates. However direct plating after 48 h at 30 ºC enrichment obtained 29 isolates. In two earlier studies (Al-Ghazali and Al-Azawi, 1986, 1988), using the cold enrichment procedure, higher prevalence rates (100%) were reported. Table 2 Isolation of Listeria species in irrigation water samples at different temperature, month and site. FDA with MOX and MMLA was employed to isolate Listeria from the samples. Listeria sp. could not been isolated on MMLA. However, 55 Listeria isolates were isolated using MOX. In this study, cold enrichment technique at 30ºC for 48 hours was found more efficient than enrichment technique at 4oC for 4 weeks ( Table 3). FDA in USA uses an enrichment at 30 ºC in its most recent methods. However, Bernagozzi (1995) reported the best medium for recovering Listeria species from natural water samples was a Palcam agar. In this study the best procedure for recovering Listeria species was extended enrichment at 30 ºC for 48h in the selective broth, and plating on MOX agar. The most effective isolation media was MOX, as 100% of the positive samples were detected by this medium, while no Listeria were recovered on the other selective medium (MMLA). The advantage of the direct technique is the shorter time required for isolation. The number of isolation of L. monocytogenes weresimilar after 24 and 48h incubation, with or without KOH treatment,Lovett et al. (1987), also found that with low levels of Listeria , recoveries were not significantly different after 24 or 48 h. Table 3 Isolation ofListeria species in irrigation water samples on two media Results and Discussion Fifty five Listeria strains were isolated from the irrigation water samples, these strains were identified by biochemical tests. Of the 55 Listeria strains at 30 ºC for 48 h, 29 (52.73 %) strains were positive for Listeria spp.,L. monocytogenes was isolated from 10 (18.18%) water samples, L. denitrificans from 9 (16.36%) water samples, L. welshimeri from 5 (9.09%) water samples, L. innocua from 5 (9.09%) water samples and of the 55 Listeria strains at 4 ºC for 4 weeks, 26 (47.27 %) were positive for Listeria sp.,L. monocytogenes was isolated from 14 (25.45%) water samples, L. denitrificans from 6 (10.91%) water samples, L. welshimeri from 2 (3.64%) water samples, L. innocua from 4 (7.27%) water samples (Table1). Vegetables often have considerable amounts of soil adhering to them, manure fertilization and irrigation of fields in which vegatables are grown in close association with animals and their manure could account for sources of L. monocytogenes. Table 1 Isolation of Listeria species in the irrigation waters surveyed after enrichment technique at 30 oC for 48 h and cold enrichment technique at 4 oC for 4 weeks. L. monocytogenes presence in raw vegetable, even in low numbers, is a potential risk for the consumers because it would lead to intestinal colonization and in certain situations such as immuno-suppressive illnesses or therapies, to the development of a clinical listeriosis. References Al-Ghazali M.R. and Al-Azawi S.K. (1986). Detection and enumeration of Listeria monocytogenes in a sewage treatment plant in Iraq. J. Appl. Bacteriol., 60, 251-254. Al-Ghazali M.R. and Al-Azawi S.K. (1988). Effects of sewage treatment on the removal of Listeria monocytogenes. J. Appl. Bacteriol., 65, 203-208. Arvanitidou M., Papa A., Constantinidis T.C., Danielides V., Katsouyannopoulos V. (1997). The occurrence of Listeria spp. and Salmonella spp. in surface waters. Microbiol. Res ., 152, 395-397. Bernagozzi M., Bianucci F., Sacchetti R. and Bisbini P. (1994). Study of the prevalence of Listeria spp. in surface water. Zentralbl Hyg Umweltmed., 196(3), 237-244. Bernagozzi M., Sacchetti R., Polenta L. and Bisbini P. (1995). Comparison of different selective methods for detection of Listeria species in surface water. Zentralbl Hyg Umweltmed., 198(2), 124-137. Beuchat L.R., Bracket D.Y., Hao Y. and Conner D.E. (1986). Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice. Can. J. Microbiol., 32, 791-795. Ceballos B.S.O., Soares N.E., Moraes M.R., Catão R.M.R. and Konig A. (2003). Microbiological aspects of an urban river used for unrestricted irrigation in the semi-arid region of north-east Brazil. Wat. Sci. Tech., 47(3), 51-57. Combarro M.P., González M., Araujo M., Amezaga A.C., Sueiro R.A. and Garrido M.J. (1997). Listeria species incidence and characterisation in a river receiving town sewage treatment plant. Wat. Sci. Tech., 35(11-12), 201-204. Dijkstra R.G. (1982). The occurrence of Listeria monocytogenes in surface water of canals and lakes, in ditches of one big polder and in effluents of canals of a sewage treatment plant. Zentralbl Backteriol. Hyg Abt. 1 Orig B, 170, 202-205. Farber J.M. and Peterkin P.I. (1991). Listeria monocytogenes a foodborne pathogen. Microbiol. Rev., 55, 476-511. Fleming D.W., Cochi S.L. and MacDonald K.L. (1985). Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New England J. Medicine, 312, 404-407. Frances N., Hornby H. and Hunter P.R. (1991). The isolation of Listeria species from fresh water sites in Cheshire and North Wales. Epidemiol. Infect., 107, 235-238. Garrec N., Picard-Bonnaud F. and Sutra L. (2003). Occurrence of Listeria sp. and Listeria monocytogenes, in sewage sludge used for land application: effect of dewatering, limiting and storage in tank on survival of Listeria species. FEMS Immunol. Med. Microbiol., 35, 275-283. Geuenich H.H., Mueller H.E., Schretten-Brunner A. and Seeliger H.P.R. (1985). The occurrence of different Listeria species in municipal waste water. Zbl. Bakteriol. Mikrobiol. Hyg Abt. B., 81, 563-565. Irwin A.D. (1969). The inhibition of Listeria monocytogenes by anorganism resembling Bacillus mycoides, present in normal silage. Res. Vet. Sci., 10, 106-108. Lowett J., Francis D.W. and Hunt J.M. (1987). Listeria monocytogenes in raw milk detection incidence and pathogenicity. J. Food Prot., 50, 182-192. Paillard D., Dubois V., Thiebaut R., Nathier F., Hoogland E., Caumette P. and Quentin C. (2005). Occurrence of Listeria spp. in effluents of French urban wastewater treatment plants. Appl. Environ. Microbiol., 71(11), 7562-7566. Rodriguez L.D., Fernandez J.F., Vazquez J.A., Rodriguez E. and Suarez G. (1985). Isolation de microorganisms du genre Listeria a partir de lait cru destine a la consommation humaine. Can. J. Microbiol., 31, 938-941. Roszak D.B. and Colwell R.R. (1987). Survival strategies of bacteria in natural environment. Microbiol. Rev., 51, 365-379. Ryser E.T. and Marth E.H. (1991). Listeria, listeriosis and food safety. Marcel Dekker Inc., New York U.S.A. Welshimer H.J.(1968). Isolation of Listeria monocytogenes from vegetation. J. Bacteriol., 95, 300-303. L. monocytogenes was the main species isolated andL. denitrificans secondary. L. monocytogenes was generally found to be predominant. The prevalence of L. monocytogenes in water was also observed by Geuenich et al. (1985), Bernagozzi et al. (1994) and Combarro et al. (1997) who referred to the identification of this species in 92.5%, 72.4% and 92% of the isolates respectively (Ceballos et al. 2003). In the latest study (Paillard et al. 2005), treated waterswhich appears to be similar to our water, showed prevalence of Listeria spp. L. seeligeri were not detected in any of water samples.Frances et al. (1991), only detected low concentration of L. seeligeri and to a lesser extent of L. innocua and L. welshimeri in waters which they regarded as being indicative of the absence of faecal contaminations of these waters (Combarro et al. 1997).Dominguez Rodriguez et al. (1985) found L. grayi predominant, followed by L. monocytogenes, with L. innocua third most common species. Only L. monocytogenes is pathogenic for human and L. denitrificans and L. innocua are pathogenic for mice. L. monocytogenes was found most frequently and in greatest number during autumn (Table 2). Lower prevalence and densities of Listeria spp were observed in water in Summer in contraction with previously studies (Al-Ghazali and Al-Azawi, 1986; Garrec et al. 2003). Paillard et al. (2005) reported to be similar to our data. This seasonal for difference could be due to a variety of reasons such as different levels of available nutrients and predation by other organisms (Roszak and Colwell, 1987). Although significant differences were found in the counts of Listeria isolates between sites, a notable increase of these microorganisms was observed in Karaköy. This water is used for unrestricted irrigation which increases the possibility of human and vegetable contamination. Karaköy water receives diffusion and discharges of domestic sewage. Of the 55 samples analyzed , 29 (52.73 %) and 26 (47.27 %) contained Listeria spp., including 2 (3.63% ) and 4 (7.27% ) that provided a single strain and 27 and 22 were mixtures of two or more strains, in 30 ºC and 4 ºC respectively.