Albanian j. agric. sci. ISSN: 2218-2020, (2012), (Special Edition) Copyright © Agricultural University of Tirana

EVALUATION OF ESCHERICHIA COLI AS AN INDICATOR OF POLLUTION AND VIBRIO PATHOGENS IN BIVALVE MUSSELS OF BUTRINTI LAGOON SONILA ÇOÇOLI1*, PRANVERA ÇABELI2, ELVIRA BELI1, TANA SHTYLLA2, RENIS MAÇI1 1 Food Safety and Veterinary Institute, Tirana, Albania 2 Agricultural University of Tirana, Faculty of Veterinary Medicine, Tiranë * Author of correspondence; Email: sonilacocoli@yahoo. com

Abstract: The aim of this study was to assess the contamination with Escherichia coli and detection of pathogens of genus Vibrio in mussels (Mytilus galloprovincialis), cultivated in Butrinti lagoon, and the variations of these indicators depending on the season. 84 samples were analyzed during a period of one year (April 2011-April 2012), sampled with a biweekly frequency. Overall the analyzed samples, the presence of E. coli was detected with a value greater than 230 MPN/100g in 19 samples (22, 7%) and the presence of Vibrio spp. in 4 samples (4, 8%). It is noticed that the highest values of E. coli appeared during the period November 2011-February 2012. The analytical results show that high levels of E. coli are obtained in winter season, related with the rainfalls presence and consequently the rinsing of polluted lands around the cultivation area and the drainage in the lagoon. Otherwise, the presence of Vibrio species (V. fluvialis and V. vulnificus) were detected during summer, as a result of increased temperature which favors their multiplication. According to the requirements of EU directive (Dir. 91/492/EEC), the mussels which exceed the levels of 230 MPN/100g of E. coli must necessarily undergo through the process of depuration to minimize these levels. Keywords: Escherichia coli; Vibrio fluvialis; Vibrio vulnificus; depuration.

1. Introduction Fishery products represent a very large category of food and are an important source of proteins in human food diet. Fish and mussels, result to be the second source of proteins for consumers behind meat products. However, all over the world have raised concerns about health risks derived from mussels contaminated with various pathogens [6]. Although most reports of outbreaks of food infections came from the United States, there are several reports from Europe, Australia and Asia. Since the end of 1800 there has been over 400 epidemic diseases from food origin and over 14 000 cases of gastroenteritis associated with the consumption of contaminated shellfish [2]. The aim of this study was to assess the contamination with Escherichia coli as an indicator of fecal contamination, as well as the detection of Vibrio spp. , as a very important pathogen causing gastroenteritis in humans [1]. Since gastroenteritis occurred at a higher frequency during the SummerAutumn it is also analyzed the variation of E. coli and the presence of Vibrio spp. depending on the seasons.

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2. Material and Methods This study was carried out on samples of bivalve mussels (Mytilus galloprovincialis) collected from Butrinti lagoon during the period April 2011 - April 2012, where it is analyzed a total of 84 samples. The samples were taken from 3 stations of the lagoon: North, South and West. All, the analysis were performed in the laboratories of microbiological control for aquatic products at Food Safety and Veterinary Institute. 1.1

The enumeration of Escherichia coli

It was carried out according to ISO / TS 166493/2005 [7]. For each sample, to prepare the initial homogenization, it was homogenized 10 g of sample in Blender using 90 ml of diluent Buffered Peptone Water. From the initial homogenate it was prepared subsequent dilutions 10-2, 10-3 which were inoculated into selective enrichment media Mineral Modified Glutamate Medium, 3 x 5 tubes and were incubated in the thermostat at 37 ± 1oC for 24 ± 2 h. After incubation, the MMGM tubes were checked for the production of acid and gas. Positive samples were

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inoculated in the second selective media Tryptone Bile Glucuronic agar (TBX) and incubated at 44 ± 1 ° C for 21 ± 3 h. The presence of green to blue colonies as a result of the action of β-glucuronidase enzyme in TBX media indicates the presence of Escherichia coli in MMGM tubes. Based on the positive tubes for each dilution the Most Probable Number (MPN) obtained is calculated on the basis of a table presented in ISO 7218 [9]. 1.2

Detection of Vibrio spp.

It is performed according to ISO / TS 218721:2007 [8]. To prepare initial dilution it is used the enrichment media Alkaline Saline Peptone Water. The representative sample (25 g) was homogenized with 225 ml ASPW. Further this homogenate is incubated at 41. 5 ° C for 6 h ± 1 h. After 24 hours the culture obtained above, was inoculated with a loop the surface of the plate with TCBS agar and Vibrio CROM agar, in such a way to allow the growth of well isolated colonies. The presence of opaque yellow colonies with a diameter of 2-3 mm in TCBS indicates the presence of Vibrio cholerae, Vibrio alginolyticus, Vibrio

fluvialis, Vibrio metschnikovii. While the growth of green colonies indicates the possibility of the presence of Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio harvey Vibrio mimicus. In Vibrio CHROM agar the presence of smooth colonies with blue color indicates the presence of Vibrio cholerae and smooth red colonies indicates the presence of Vibrio parahaemolyticus. At the end of incubation, suspected colonies were subject to the following tests for confirmation: determination of oxidative activity, planting in tryptophan broth for indole reaction, halotolerance test (inoculation in Peptone Water with different salt concentrations), and API 20E. 3. Results and Discussion From analyzes performed in 84 samples collected in Butrinti lagoon, 19 of them resulted contaminated with E. coli and 4 samples positive to Vibrio spp. (Table 1) (Figure 1). According to CE Reg. 853/2004 [10] these values (> 230MPN/100g), are not acceptable and bivalve mussels must necessarily pass the depuration process before issuing the market. During the study were isolated 2 species of Vibrio. (Table 2).

3. 1 Evaluation of Escherichia coli and detection of Vibrio spp. in bivalve mussels Table 1: E. coli values and presence of Vibrio spp. in bivalve mussels E. coli < 230 MPN/100g 65 cases 77, 3%

E. coli >230MPN/100g 19 cases 22, 7%

Vibrio spp. 4 cases 4, 8%

Table 2 Vibrio species identified in bivalve mussels of Butrinti lagoon Total number of samples analyzed for Vibrio spp, 84

Vibrio fluvialis 3

Runoff from agricultural lands and livestock farms is one of the main sources of fecal coliforms in surface waters [4], and E. coli concentrations tend to vary with the season due to seasonal variations, for this reason, these values tend to increase during winter, connected with the presence of numerous rainfalls (Figure 2). As shown in Figure 2 these values increase significantly in the period November-February (maximum value 16 000 MPN/100g).

Vibrio vulnificus 1

multiplication of Vibriones reach the maximum values [5]. E.coli  ≥230  22,7 %

Vibrio  spp. 4,8 %

E.coli <230 77,3 %

3. 2 Positive cases of Vibrio spp. All positive cases of Vibrio spp. (4. 8% of samples) are generally isolated in the summer months (July-September 2011), (Figure 3) during which the environmental factors such as air temperature and as result the water temperature necessary for the

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Figure 1: Graphic percentage of E.coli values and the presence of Vibrio spp.

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Evaluation of Escherichia coli as an indicator of pollution and Vibrio pathogens in bivalve mussels of Butrinti lagoon 20000.00 15000.00 10000.00 5000.00 15.03.2011 28.03.2011 27.04.2011 30.05.2011 13.06.2011 11.07.2011 09.08.2011 22.08.2011 20.09.2011 17.10.2011 31.10.2011 30.11.2011 26.12.2011 09.01.2012 06.02.2012 05.03.2012 19.03.2012

0.00

but must be carried out continuous monitoring of these species potentially pathogenic to humans. In many cases it is noticed that depuration has not been effective in eliminating Vibriones, otherwise their concentration has increased if the salinity was within the limits 10 to 30 ‰ and temperatures above 20 ° C [3]. For this reason, monitoring for Vibrio spp. should be carried out after the process of depuration. 5. References

Figure 2: E. coli values during different periods of the year

1. Arcangeli G: Il problema dei virus enterici e dei Vibrio. Eurofishmarket, 2005, (1): 4-10. 2. Blake PA, Weaver RW, Hollins DG: Diseases of humans (other than cholera) caused by vibrios. Annual Review of Microbiology, 1980, 34: 341– 367. 3. Croci L, Suffredini E, Cozzi L, Toti L: Effects of depuration of mollusks experimentally contaminated with Escherichia coli, Vibrio cholerae O1 and Vibrio parahaemolyticus. Journal of Applied Microbiology, 2002, 92: 460465. 4. Desmarchelier PM: Vibrio. In Encyclopedia of Food Microbiology: Robinson RC, Batt C, Patel P: 2000: 2237-2242.

Figure 3: The distribution of positive cases of Vibrio spp. during the year

4. Conclusions Regulations in force (Reg. CE 2073/05 [11] updated by Reg. CE 1441/2007 [12] defines as safety criteria E. coli, as an indicator of fecal contamination. Relatively safety parameters, only 22. 7% of the samples analyzed resulted with no acceptable values of E. coli (> 230 MPN/100g) [10]. For this reason, bivalve mussels in Butrinti lagoon must necessarily pass the depuration process until they reach minimum values. High values of E. coli are shown during the winter months (November 2011-February 2012), connected with numerous precipitation during this period. This indicates that the main sources of pollution of the lagoon come from the flood of waters from agricultural lands and livestock farms around the area, after the rainfalls. Although the number of positive samples (4. 8%) of Vibrio spp. is low compared with the total number of samples analyzed, this fact should not be overlooked International Conference 31 October 2012, Tirana

5. Hlady WG: Vibrio infections associates with raw oysters consumption in Florida, 1981-1994. Journal of Food Protection, 1997, 60: 353-357. 6. Rippey SR: Infectious diseases associated with mollusk shellfish consumption. Clinical Microbiology Reviews, 1994 (7): 419−425. 7. Technical Committee ISO/TC 34: ISO 16649-3 Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli -Part 3: Most probable number technique using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. Proceedings of the International for Standardization, 2005. 8. Technical Committee ISO/TC 34: ISO 21872-1 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of potentially enteropathogenic Vibrio spp. Proceedings of the International for Standardization, 2007. 9. Technical Committee ISO/TC 34: ISO 7218 Microbiology of food and animal feeding stuffs – General requirements and guidance for microbiological examinations. Proceedings of the International for Standardization, 2007.

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10. The European Parliament and the Council of European Union: Regulation (CE) n. 853/2004 which establishes specific hygienic standards for foods of animal origin, 2004. 11. The European Parliament and the Council of European Union: Regulation (CE) n. 2073/2005

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of the Commission on microbiological criteria applicable in foodstuffs, 2005. 12. The European Parliament and the Council of European Union: Regulation (CE) n. 1441/2007 which modifies the Commission Regulation (CE) n. 2073/2005 on microbiological criteria applicable in foodstuffs, December 5, 2007.

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