Listeria spp. were isolated from the tested samples on Fraser Broth Base supplemented with an antibiotic (Oxoid) by a membranous filtration. A 10-mL sample of water was filtered via membranous filters (0.45μl) (Millipore) in three repetitions and placed in a selective medium. 1 mL and 0.1 mL quantities of water were poured directly into the tubes. All samples were vortexed for 1 minute, and then incubated at 30°C for 72 hours. Positive cultures were reinoculated on Listeria Selective Agar Base supplemented with antibiotic (Oxoid) and incubated at 30°C for 72 h. Characteristic colonies were identified by APIŽLISTERIA (BioMerieux).

Selected samples underwent microbiological analysis according to the PN-77 C-04615 standard - Indication of coliforms by a tube fermentation method – including the ISO 7251:1993 standard – The general rules of indication of potential Escherichia coli- the most probable number method – on LPB medium (BTL).

The total microbial count was also performed on a nutrient agar (Difco) incubated at 20°C for 72 hours.

Statistical analysis, i.e. calculations of means values, standard deviations, as well as significance and correlation tests were performed with Statistica PL software.

RESULTS

Quantitative analysis revealed statistically insignificant (p<0.05) elimination of all groups of microorganisms tested during municipal sewage purification.

With reference to the untreated sewage a significant decrease (p<0.05) in the total number of microorganisms was observed for the sewage collected at the point of discharge to the river. At further stages of purification no significant differences in the total number of microorganisms were recorded (Table 2 and Figure 1).

An analysis aimed at detecting coliform presence displayed significant decrease (p<0.05) in their number in relation to the untreated sewage: two logarithms down in the industrial sewage after chemical purification, and three logarithms down in the treated one. No significant differences in the number of coliforms between the untreated sewage and the one at its further biological purification stages (Table 2 and Figure 1) were found.

A quantitative analysis of Listeria sp. in the industrial sewage showed significant decrease (p<0.05) in the MPN of Listeria after chemical treatment and in the aeration chamber. Sewage analyses performed in the secondary settler and at the point of treated sewage discharge to the environment revealed significant increase in the number of Listeria in comparison to the previous stages of the sewage treatment. At these stages of purification the level of Listeria spp. was similar to the one in the untreated sewage (p<0.05) (Table 2 and Figure 1).

Species analysis of Listeria spp. displayed Listeria monocytogenes as dominant (90%) in the untreated municipal sewage. Moreover, the presence of Listeria seeligeri and Listeria grayi was detected, 5% of each, whereas in the treated municipal sewage and in the industrial sewage at all the stages of its treatment (Table 3) solely the presence of Listeria monocytogenes was disclosed.

DISCUSSION

Industrialisation and urbanisation have negatively affected the quality of surface waters that provide drinking water to majority of Polish population. It has been reported that in Poland about 20% of sewage is discharged directly into the surface waters. Moreover, no analysis are officially required on sewage intended for agricultural utilisation, though it may be a cause of introducing to water not only allochtonic microflora but also pathogenic microorganisms. According to the regulation of the Minister for the Environmental Protection, Natural Resources and Forestry dated November 5^th, 1991, sewage introduced to surface waters cannot affect adversely the natural biocoenosis typical for such waters. Therefore, it should not contain pathogenic microorganisms and its coliform index should correspond to the quality class of water into which the sewage is discharged.

The index of sanitary contamination in the tested samples reveals that coliforms are mainly eliminated during chemical treatment of industrial sewage. The increase in the amount of coliforms observed during biological treatment may result from contamination of the secondary settler by birds or other animals. However, a significant decrease in the coliforms? index in the treated sewage may suggest utilisation of Escherichia sp. in an active sediment. Neither during mechanical nor biological treatment of domestic sewage a significant decrease in the number of coliforms was observed, and the quality of the treated sewage met the requirements for waters of the third class quality.

The studies revealed that in the process of mechanical and biological treatment a reduction in the number of classic microbiological indices fell to the level that compliant with the official standards. Nevertheless, the decrease was not correlated with the reduction of isolated Listeria spp.

Our work proved that though a significant decrease in the number of Listeria sp. was observed already during chemical treatment, the treatment of the municipal sewage was not efficient enough to eliminate pathogenic microorganisms. Even pH of 11 only reduced the number of the bacteria but not eliminated them completely. Hence, reports on efficient elimination of microorganisms due to high pH values have been disproved in this case [17]. Additionally, in the industrial sewage collected at the treated sewage discharge to the environment, a rise in the amount of Listeria monocytogenes was observed. It may result from the outdoor location of both the aeration chamber and the secondary settler, since birds are known to be a reservoir of this pathogen [35,18,34]. It is then very likely that contamination caused by their faeces increased the number of Listeria sp. Earlier studies also highlighted inefficiency of biological treatment in eliminating Listeria monocytogenes from sewage [22].

Usually research is focused on the presence of Listeria monocytogenes in domestic [6,28] and industrial [14,1,23] sewage. Occurrence of different pathogenic microorganisms in sewage, such as Candida albicans [16], Salmonella typhimurium [30], neuroviruses [25] and even parasite eggs Ascaris suum [17] have been also reported.

It is well known that waters quality depends on the quality of the sewage discharged into. Ferguson et al. [19] found the increased number of fecal bacteria, fecal coliforms, spores of Clostridium perfringens and Aeromonas correlated with the amount of sewage introduced into a river estuary. Similar results were presented by Contreras-Coll et al. [10] who confirmed occurrence of different groups of pathogenic sewage-borne microorganisms in various European recreational waters. We find it alarming that in the samples collected at the sewage discharge to the environment, Listeria monocytogenes constituted a 100% of all Listeria spp. Most likely it is the reason for a highly frequent (54,5%) contamination of Odra waters with this pathogen [12].

Still, even more alarming is the occurrence of Listeria monocytogenes detected not only in rivers [27,4], lakes [7,20], sea waters, but also in mountain surface and ground waters that very often serve as drinking water reservoirs for people and animals [32,4].

Quality evaluation of water meant for consumption is based mainly on application of fecal coliform and fecal streptococci counts. Nevertheless, it turns out that even water that meets the requirements defined in the official standards, may contain other, not specified in the regulations, dangerous pathogens. The supposition was confirmed by the results presented by El-Taweel and Shaban [16] who studied samples intended for consumption that were collected from sewage treatment plants. Studies of samples that underwent the entire treatment process showed that despite maintaining adequate levels of standard microbiological contamination indices, such as total number of bacteria, fecal coliforms, fecal streptococci, etc., the admissible limits for so-called ‘new indices’ were well exceeded. Namely, a total number of yeasts, Candida albicans, Aeromonas hydrophila, and streptococci were exceeded. Salmonella sp., Vibrio sp., Listeria sp. were also isolated from those samples.

Both the quoted and obtained in the presented study results highlight the problem of pathogenic Listeria distribution via waters. Our studies indicate inefficiency of sewage treatment plants with respect to eliminating the microorganisms in question. Even a multistage treatment process applied to municipal sewage cannot entirely eliminate this pathogen. Necessity and significance of introducing additional indices for correct and reliable evaluation of microbiological quality of waters is underlined.

CONCLUSIONS

1. Combined mechanical and biological treatment of domestic sewage does not eliminate Listeria monocytogenes.

2. Mechanical and biological treatment of sewage from a poultry processing industry does not eliminate Listeria monocytogenes.

3. A lack of correlation between Listeria sp. and MPN of fecal coliforms in municipal sewage was observed.

4. Irrespectively of the level of contamination evaluated in municipal sewage, Listeria monocytogenes was found to be dominant species of Listeria sp.

1. al-Ghazali M.R., al.-Azawi S.K., 1990. Listeria monocytogenes contamination of crops grown on soil treated with sewage sludge cake. Journal of Applied Bacteriology 69 (5), 642-647.

2. 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 (4), 395-397.

3. Baloda S.B., Christensen L., Trajcevska S., 2001. Persistence of a Salmonella enterica serovar Typhimurium DT12 clone in a piggery and in agricultural soil amended with Salmonella-contaminated slurry. Applied and Environmental Microbiology 67 (6), 2859-2862.

4. Bernagozzi M., Bianucci F., Sacchetti R., Bisbini P., 1994. Study of the prevalence of Listeria spp. in surface water. Zentralbl. Hyg. Umweltmed., 196 (3), 237-244.

5. Beumer R.R., te Giffel M.C., Kok M.T., Rombouts F.M., 1996. Confirmation and identification of Listeria spp. Lett Appl. Microbiol. 22 (6), 448-452.

6. Beumer R.R., te Giffel M.C., Spoorenberg E., Rombouts F.M., 1996. Listeria species in domestic environments. Epidemiol. Infect. 117 (3), 437-442.

7. Bogusławska-Wąs E., Czeszejko K., Dąbrowski W., 2002. Listeria spp. i mykocenozy na tle mikrobiologicznego wskaźnika zanieczyszczenia fekalnego w Jeziorach Pomorza Zachodniego [Listeria spp. presence and mycocoenoses diversity based on indicators of faecal contamination in the Western Pomeranian lakes]. Mat.konf. Mikroorganizmy w funkcjonowaniu ekosystemów wodnych, 2 Ogólnopolska Konferencja Hydromikrobiologiczna, Toruń-Ciechocinek. [in Polish]

8. Carducci A., Tozzi E., Rubulotta E., Casini B., Cantiani L., Rovini E., Muscillo M., Pacini R., 2000. Assessing airborne biological hazard from urban wastewater treatment Water Research 34 (4), 1173-1178.

9. Colburn K.G., Kaysner C.A., Abeyta C., Wekell M.M., 1990. Listeria species in a California Coast Estuarine Environment. Applied And Environmental Microbiology 56 (7), 2007-2011.

10. Contreras-Coll N., Lucena F., Mooijman K., Havelaar A., Pierzo V., Boque M., Gawler A., Höller C., Lambiri M., Mirolo G., Moreno B., Niemi M., Sommer R., Valentin B., Wiedenmann A., Young V., Jofre J., 2000. Occurrence and levels of indicator bacteriophages in bathing waters throughout Europe. Water Research 36 (20), 4963-4974.

11. Dąbrowski W., 1999. Listerie nowy problem w przemyśle rybnym [Listeria a new problem in fish industry], Magazyn Przemysłu Rybnego. [in Polish]

12. Dąbrowski W.,Czeszejko K., 2002. Listeria spp. jako zanieczyszczenie mikrobiologiczne surowca rybnego pochodzącego z Odry i Zalewu Szczecińskiego [Listeria spp. as contamination of raw fish material from Odra River and Bay of Szczecin]. Mat. Konf. Nauka o Żywności Osiągnięcia i perspektywy. 23 Sesja Naukowa Komitetu Technologii i Chemii Żywności PAN. [in Polish]

13. Dąbrowski W., Daczkowska -Kozon E., Koronkiewicz A., 2001. Incidence of Listeria spp. In selected raw fish, semi-processed and processed fish products. Folia Universitatis Agriculture Stetinensis Scienta Alimentaria (1), 25-28.

14. De Luca G., Zanetti F., Fateh-Moghadm P., Stampi S., 1998. Occurrence of Listeria monocytogenes in sewage sludge. Zentralbl. Hyg. Unweltmed 201 (3), 269-277.

15. Dz.U. 2001.115.1229 z dnia 11 października 2001 roku. Prawo wodne [Polish Acts Register dated November 11^th 2001. Water Law] [in Polish]

16. El-Taweel G.E., Shaban A.M., 2001. Microbiological quality of drinking water at eight water treatment plants. Int. J. Environ. Health Res. 11 (4), 285-290.

17. Eriksen L., Andreasen P., Ilsoe B., 1996. Inactivation of Ascaris suum eggs during storage in lime treated sewage sludge. Water Research 30 (4), 1026-1029.

18. Fenlon D.R., 1985. Wild birds and silage as reservoirs of Listeria in the agricultural environment. Journal Applied Bacteriology 59 (6), 537-543.

19. Ferguson C.M., Coote B.G., Ashbolt N.J., Stevenson I.M., 1996. Relationships between indicators, pathogens and water quality in an estuarine system. Water Research 30(9): 2045-2054.

20. Frances N., Hornby H., Hunter P.R., 1991. The isolation of Listeria species from fresh-water sites in Cheshire and North Wales. Epidemiol. Infect. 107 (1), 235-238.

21. Gantzer C., Gaspard P., Galves L., Huyard A., Dumouthier N., 2001. Monitoring of bacterial and parasitological contamination during various treatment of sludge. Water Research 35 (16), 3763-3770.

22. Geuenich H.H., Muller H.E., 1984. Isolation and germ count of Listeria monocytogenes in raw and biologically treated waste water. Zentralbl. Bakteriol. Mikrobiol. Hyg. 179 (3), 266-273.

23. Geuenich H.H., Muller H.E., Schretten-Brunner A., Seeliger H.P., 1985. The occurrence of different Listeria species in municipal waste water. Zentralbl. Bakteriol. Mikrobiol. Hyg. 181 (6), 563-565.

24. Hazen K.C.,1995. New and emerging yeast pathogens. Clin. Microbiol Rev. 8, 462-478.

25. Hitoshi Horie, Hiromu Yoshida, Kumiko Matsuura, Miwako Miyazawa, Yoshihiro Ota, Takashi Nakayama, Yutaka Doi, So Hashizume, 2001. Neurovirulence of type 1 Polioviruses isolated from sewage in Japan. Applied and Environmental Microbiology 68 (1), 138-142.

26. Kocwa-Haluch R., 1996. Comparison of the airborn spread of coliform and hemolytic bacteria around a sewage treatment plant. Ann. Agric. Environ. Med. 3, 13-17.

27. Luppi A., Bucci G., Maini P., Rocourt J., 1998. Ecological survey of Listeria in the Ferrara area (northern Italy). Zentralbl. Bakteriol. Mikrobiol. Hyg. 269 (2), 266-275.

28. MacGowan A.P., Bowker K., McLauhlin J., Bennett P.M., Reeves D.S., 1994. The occurrence and seasonal changes in the isolation of Listeria spp. in shop bought food stuffs, human faeces, sewage and soil from urban sources. Int. J. Food Microbiol. 21 (4), 325-334.

29. Melloul A., Amahmid O., Hassani L., Bouhoum K., 2002. Health effect of human wastes use in agriculture in El Azzouzia (the wastewater spreading area of Marrakesh city, Moroco). International Journal of Environmental Health Research 12 (1), 17-23.

30. Mezrioui N., Oufdou KH., 1996. Abundance and antibiotic resistance of non-O1 Vibrio cholerae strains in domestic wastewater before and after treatment in stabilization ponds in an arid region (Marrakesh, Morocco). FEMS Microbiology Ecology 21 (4), 277-284.

31. Monfort P., Piclet G., Plusquellec A., 2000. Listeria innocua and Salmonella panama in estuarine water sea water: a comparative study. Water Research 34 (3), 983-989.

32. Schaffer N., Parriaux A., 2002. Pathogenic-bacterial water contamination in mountainous catchments. Water Research, 36 (1),131-139.

33. Schwartz B., Pinner RW., Broome CV., 1990. Dietary risk factor for sporadic listeriosis: association with consumption uncooked hot dogs and undercooked chicken. In: Foodborn Listeriosis. Eds. A.J. Miller, J.L. Smith, G.A. Somkuti, Elsevier, Amsterdam-New York-Oxford 67.

34. Weber A., Potel J., Schäfer-Schmidt R., Prell A., Datzmann C., 1995. Studies on the occurrence of Listeria monocytogenes in fecal samples of domestic and companion animals. Zentralb Hyg Umweltmed. 198 (2), 117-123.

35. Weis J., Seeliger H.P., 1975. Incidence of Listeria monocytogenes in nature. Applied Microbiology 30 (1), 29-32.

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