Virological Quality of Urban Rivers and Hospitals Wastewaters in Addis Ababa, Ethiopia
Tesfaye L. Bedada1, *, Teshome B. Eshete2, Samson G. Gebre1, Firehiwot A. Dera1, Waktole G. Sima1, Tigist Y. Negassi1, Rahel F. Maheder1, Shiferaw Teklu1, Kaleab Awoke1, Tatek K. Feto1, Kassu D. Tullu3
Identifiers and Pagination:Year: 2019
First Page: 164
Last Page: 170
Publisher Id: TOMICROJ-13-164
Article History:Received Date: 23/02/2019
Revision Received Date: 15/05/2019
Acceptance Date: 22/05/2019
Electronic publication date: 30/06/2019
Collection year: 2019
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Polluted rivers and hospital wastewater become a greater concern because of their public health and environmental hazards with high tendency to result in epidemics.
Methods and Materials:
The current study investigated 84 samples of Urban rivers and 30 samples of hospitals wastewaters in Addis Ababa, Ethiopia between February and April, 2017. The simultaneous detection of male-specific and somatic Coliphages from the samples was carried out using Escherichia coli CB390 as the host according to the single agar layer plaque assay at public health microbiology laboratory of Ethiopian Public Health Institute.
Of the total 114 samples tested, coliphages were detected in 44 (52.4%) and 3 (10%) samples of urban rivers and hospital waste waters, respect ively. Total coliphages enumerations ranged from <1pfu/100ml to 5.2×103pfu/100ml for urban rivers and <1pfu/100ml to 4.92×103pfu/100ml for hospitals wastewaters.
The detection of total coliphages in our study settings warrants the possibility that the pollution of urban rivers and hospital wastewaters may be a source for pathogenic viral infections. Unless coliphages, viral and fecal indicators are also examined in the waters by public health agencies, waterborne infections cause a major risk to public health.
Quality of river water is essential to humans, animals and for a good environment . However, river waters are getting polluted by indiscriminate disposal of sewerage, industrial waste and excess of human activities, which affect water quality globally . The degradation of river water depends mainly on wastewater quality. The harmful effects of these effluents on the quality of river waters are diverse and rely on the volume of the discharge and microbiological load of the effluents .
In under developed countries, insufficient wastewater management systems can lead to increase waterborne diseases . The sanitary related risk for humans linked to the presence of pathogens is associated with the use of the water such as for drinking, recreational activities, bathing, irrigation and shellfish harvesting .
Wastewater generated mainly from hospital may also pose serious health risks to human beings and animals when contaminated with these wastes . Hospital wastewater where individuals with enteric illnesses are hospitalized is a principal problem during diarrheal illnesses outbreaks . The problems faced related to the management of wastewater are due to high population growth, urbanized societies and industrialization .
In Addis Ababa, Ethiopia due to insufficient and inefficient solid and liquid waste management services, pollutants including some of the clinical wastes are discharged directly or indirectly to the nearby rivers . These polluted waters are used by downstream residents to grow vegetables, which are sold and consumed in the city . Such polluted waters can also contaminate water supplies .
Fecal pollution in water sources is a public health risk and surrogates of fecal contamination are utilized widely to regulate water quality . The monitoring of river water and wastewater for fecal pollution is becoming increasingly significant as the world's population has become more urbanized. To reduce the risk of diseases in public, good fecal indicators are required. Conventionally, bacterial indicators have been used as microbial indicators to monitor fecal pollution in waters. Nevertheless, it has been recommended that these bacterial indicators are not good for predicting enteric viruses . The detection of all pathogenic microbes potentially present in water bodies is very difficult due to the large diversity of pathogens, low abundance of each species and the absence of standardized for their detection .
No information concerning the occurrence of phages in urban rivers and hospitals’ wastewaters is available for most countries including Ethiopia in general and in Addis Ababa in particular. The objective of this study was to assess the contamination level of urban rivers and hospitals wastewaters in Addis Ababa, Ethiopia using coliphages.
2. MATERIALS AND METHODS
A cross sectional study was carried out on a total of 114 urban rivers and hospital wastewaters samples at Public Health Microbiology Research Team Laboratory in Ethiopian Public Health Institute between February and April, 2017.
Thirty selected urban rivers and streams (R1-R30) that flow through nine sub cities (SC 1-SC 9) of Addis Ababa and four hospitals wastewaters in the city were used for collecting samples for the current study. Eighty-four samples were collected using a grab sampling technique from the rivers that have large water flow. Three discrete samples from the first sampling point, 100 meter downstream and 200 meter downstream, were collected from 11 rivers (R1, R3, R8, R9, R11, R12, R16, R17, R28, R29 and R30) in the first round and processed independently. Two discrete samples from the first sampling point and 100 meter downstream were collected in the second round except for three rivers. The samples were processed independently. A single discrete sample was collected from 15 rivers and streams (R2, R4, R5, R6, R10, R13, R14, R15, R18 - R21, R24, R25 and R27) in the first and second round and for the remaining four rivers (R7, R22, R23 and R26) only in the first round (Table 1).The second round samples were collected at 15 day intervals. For hospital waste waters, a total of 30 untreated samples were collected from various cafeterias, laundries, Wards and medical laboratories units of four government hospitals. For all the samples, about 150 ml samples were collected using sterilized glass bottles and transported on ice to the laboratory. The samples were maintained at 4 °C. Microbiological screenings were performed within 24 hours after collection. All the samples were tested for total coliphages using standard EPA method [14, 15].
|Rivers and streams in nine sub-cities of Addis Ababa||No. of Samples|
|R7, R22, R23 and R26||1|
|R2, R4, R5, R6, R10, R13, R14, R15, R18, R19, R20, R21, R24, R25 and R27||2|
|R8 and R30||3|
|R1, R3, R9, R11, R12, R16, R17 and R29||5|
2.1. Detection of Coliphages
Simultaneously, both types of male-specific and somatic Coliphages from polluted rivers, streams and hospitals wastewaters samples were detected using Escherichia coli CB390 [obtained from University of North Carolina, Chapel Hill] as the host bacterium according to the single agar layer plaque assay. The Host log phase containing 0.15% ampicillin was applied with magnesium chloride in double strength tryptic soy agar [Difco]. All the plaques, 1to 10 mm diameter lysis zone formation in the lawn of host bacteria, were counted after 16 to 24 hours of incubation at 37 °C per plates from a single sample for total coliphages positive; no circular zone of clearing or an intact lawn of bacteria identical to the background lawn of bacteria was found for coliphage negative. The coliphage enumerated was computed per 100 mL of the sample [14, 15].
Of 84 river and streams water samples tested between February and April 2017 in Addis Ababa, Ethiopia, total coliphages were observed in 44 (52.4%) samples ranging from <1pfu/100ml to 5.2×103pfu/100ml. All the nine sub-cities included in the study contained coliphages in one or more of their river water samples. Out of 30 rivers and streams in the sub-cities, 24 (80%) rivers had coliphages. The distributions of total coliphages detected in the river water samples in the sub-cities SC-1 to SC- 9 were 50% (4), 25% (2), 83.3% (10) 10% (1), 37.5% (3), 54.5% (6), 62.5% (5), 85.7% (6) and 87.5% (7), respectively (Table 2).
The maximum recovery of the phages in the urban river water samples was 5200 pfu/100ml (Table 3). Of 30 waste water samples tested from four hospitals in Addis Ababa, Ethiopia, total coliphages were detected in 3 (10%) samples ranging from <1pfu/100ml to 4.92×103 pfu/100ml. In the hospital wastewaters, total coliphages were detected in wastewater from the ward, medical laboratory and laundry facilities units with the concentrations of 4.92× 103, 4.2×102 and 2.7×102, respectively. P- value for coliphages using the nonparametric, Kruskal-Wallis test for the samples by the sample type (urban rivers and hospitals wastewaters) was 0.003.
|Coliphages [pfu/100ml]||Addis Ababa Sub-cities’ Rivers and Streams Water Samples|
|SC- 1||SC- 2||SC-3||SC-4||SC-5||SC-6||SC-7||SC-8||SC-9||Total|
|Rivers||Sub-Cities||Phage +ve Samples||Phage -ve Samples||Min. [pfu/100ml]||Max. [pfu/100ml]|
|Hospital||Hospital`s Units||No. of Negative Samples||No. of Positive Samples||Total Number of Samples|
This study was intended to investigate the incidence of total coliphages in polluted urban rivers and hospitals waste water. The coliphages were monitored using suitable E. coli strain CB 390 by the single agar layer plaque assay. The detection of coliphages in environmental water samples can be carried out by plaque assay using single-layer agar methods . Even if the two main types of coliphage are examined separately on different E. coli host bacteria; with the right choice of E. coli host, it can be possible to measure both somatic andmale-specific coliphages together on a single E. coli host. This can reduce the cost and work load in detecting and quantifying total coliphages in waste water. E. coli CB390 is the only E. coli host for simultaneous detection of total coliphages that give similar concentrations as the sum of coliphages detected by the individual somatic (CN13) and male-specific [Famp] E. coli hosts .
The occurrence of total coliphages in 52.4% of the urban rivers and 10% hospitals’ wastewater sources could indicate fecal pollution and hence the presence of enteric viruses and possibly also other pathogens [18, 19]. The excellent indicators of pathogenic viruses in wastewater and fecally contaminated water are because of compositional similarity, structural similarity, site of replication, size similarity, morphological similarity, their resistance to environmental changes  and different water treatments similarities .
Contamination by pathogenic viruses can be predicted by specifically detecting the viruses or by assessing the level of fecal contamination using some indicators . However, specific detection of enteric viruses is not adapted to routine analysis. Culturing, which is the reference method for the detection of environmental viruses, is time-consuming and does not allow the detection of all viral serotypes and no information is achieved regarding viral infections using molecular techniques . Seventy percent of sequences obtained from the environment had no match with any database ; therefore a suitable and cheap technique is needed .
As indicated by different researchers, total coliphages concentrations are correlated with pathogens . In somatic and male-specific coliphages in polluted rivers, viruses and protozoan parasites were detected in one study . In another study, the presence of coliphages and pathogenic viruses at different locations was observed . Quality of river water samples positive for enteric viruses enhances with increasing concentration of coliphages . The review of the EPA report shows that coliphages exist when fecal pathogens are present, but are likely to be absent in non-fecally contaminated water. They are a better surrogate for viruses than enterococci orE. coli in the effluent. In most cases, these organisms are present in greater numbers than the pathogen in this case, human viruses .
Nowadays, viral infections being the communicable diseases caused by bacteria and parasites are at the forefront . The extremely small size of the enteric viruses allows them to infiltrate soils and reaching aquifers to contaminate groundwater after being shed in large quantities in feces of infected individuals . They are commonly more resistant to treatment, more infectious; and do not need to be large in number to cause a disease than most of the other pathogens . These pathogenic viruses and also protozoa are major threats to human health in all freshwater supplies  and wastewaters .
Fecal pollution is highly problematic to both developing and developed countries, though the levels of pollution and contamination type vary among nations. Micro-organisms of fecal origins largely transfer to the water bodies through industrial and domestic wastewater discharges . This fecal pollutant contamination of water resources may pose signifi cant health risks to human beings and animals since many pathogens are frequently associated with feces .
The recovery of total coliphages in 52.4% of river water samples collected from Addis Ababa, Ethiopia was nearly in agreement with the study conducted in Malaysia by Foong who analyzed some coliphages in various rivers heavily polluted with sewage and animal fecal matter from residential areas . The detection rate of total coliphages in more than half of urban rivers water samples was lower than the study conduct ed in South Africa found at least one coliphage in 97.2% of the rivers samples in all rivers with maximum recovery of 2×104pfu/100ml .
The contamination of urban rivers waters in Addis Ababa, Ethiopia with coliphages (52.4%) in all of the nine sub-cities was higher as compared to hospitals wastewater (10%) with higher maximum recovery. This may indicate the degree of pollution of urban river waters. The pollution of the rivers can be due to damaged septic tanks, runoff from agricultural lands  and wastes discharged directly or indirectly to the nearby rivers because of insufficient and inefficient solid and liquid waste management services 
The detection of total coliphages in the hospitals especially in the medical laboratories showed pathogenic microbes that can pose a serious health risk for patients in the healthcare units, health professionals, community, and the environment . Hospital wastewater pollutants including viruses can simply reach the water resources in the environment causing aquatic pollution and human health crisis .
The detection of coliphages in the laundry facilities may indicate the impurity of healthcare textiles with enteric viruses or other microbes. Healthcare textiles such as gowns, bed sheets, towels, blankets, personal clothing, patient apparel and uniforms can be contaminated with large numbers of microbes from stool .
All the wastewater samples collected in the study were discharged to the environments without any pretreatment. Discharge of infectious agents in hospital wastewater to the groundwater and other environments might pose a high risk for hospital personnel and the nearby communities . Therefore, medical waste management is of greater significance because of its potential public health risks and environmental hazards with high tendency to result in epidemics 
The wastewater effluents areresponsible for the degradation of the water bodies, rivers or streams . Wastewater is used as a source of irrigation for farming in many urban areas in the world . As a result of easy access or no alternative source, this water is causing severe negative impacts on health [45, 46]. The water pollution is leading to cause large scale deaths of humans across the world . Furthermore, consumption of crops irrigated with wastewater causes 99.7% of deaths as the the consumption of such contaminated crops leads to diarrhea in developing countries and 90% of the deaths occur in children . These wastewaters contain numerous pathogenic microbes that may get eroded into drinking water supplies or receiving water bodies . Majorly, pathogenic viruses are extensively dispersed in waters and the environment and are consumed by humans and animals via drinking water , water used in food irrigation, and shellfish production [50-52].
The nonparametric Kruskal-Wallis test revealed that total quantity of coliphages differed statistically in different the sample types i.e urban rivers and hospitals wastewaters, (p values 0.003). The maximum recoveries of total coliphages in the three sub-cities were higher than other sub-cities.
The detection of coliphages in all the sub-cities and one-half of the hospitals’ samples might be a source for pathogenic viral infections. Unless the level of contamination of the rivers and hospital wastewaters is not properly monitored for coliphages by the scientific community, poor quality of the water will continue to cause a major health risk and will result in more number of deaths and also will affect the aquatic life, production of different crops, other sources, and drinking water.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.
CONSENT FOR PUBLICATION
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings of this research are available within the article.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
We thank Ethiopian Public Health Institute for all the support and Lydia S Abebe from the University of North Carolina, Chapel Hill for providing coliphage and bacterial strains.
|||Caraco NF, Cole JJ, Likens GE, Lovett GM, Weathers KC. Variation in NO3 export from flowing waters of vastly different sizes: does one model fit all? Ecosystems 2003; 6: 344-52.
|||Koshy M, Nayar TV. Water quality aspects of river Pamba. Pollut Res 1999; 18: 501-10.|
|||Owuli MA. Assesment of impact of sewage effluents on coastal water quality in Hafnarfjordur, Iceland. The United Nations Fishery Training Program, Final Report 2003.|
|||Gantzer C, Gillerman L, Kuznetsov M, Oron G. Adsorption and survival of faecal coliforms, somatic coliphages and F-specific RNA phages in soil irrigated with wastewater. Water Sci Technol 2001; 43(12): 117-24.
|||Servais P, Billen G, Goncalves GA, Garcia-Armisen T. Hydrology and earth system sciences modelling microbiological water quality in the seine river drainage network: Past, present and future situations. Hydrol Earth Syst Sci 2007; 11: 1581-92.
|||Akter N. Medical Waste Management: A 14. Rheinheimer G aquatic microbiology review.Asian Institute of Technology 2000 4 edn. 1991; 25-363.|
|||Emmanuel E, Perrodin Y, Keck G, Blanchard JM, Vermande P. Ecotoxicological risk assessment of hospital wastewater: A proposed framework for raw effluents discharging into urban sewer network. J Hazard Mater 2005; 117(1): 1-11.
|||Mc Casland M, Trautmann N, Porter K, Wagenet R. Nitrate: Health effects in drinking water [Accessed 05/04/2008];http:// pmep.cee.comell.edu /facts.slides–self/facts/nit-heefgrw85.html2008|
|||Teshager G. Economic valuation of protecting urban river water pollution in Addis Ababa, Ethiopia: A choice experiment approach MSc thesis Addis Ababa University 2014.|
|||Mersha Z. Determination of the pollution level of Great and Little Akaki rivers and trend of pollution Addis Ababa Environmental Protection Authority 2012.|
|||Nisar H, Ejaz N, Naushad Z. Ali1 Z. Impacts of solid waste management in Pakistan: A case study of Rawalpindi city. WIT Trans Ecol Environ 2008; 109
|||Maier RM, Pepper IL, Gerba CP. Environmental Microbiology 2nd ed. Part VI2009;|
|||Garcı’a-Aljaro C, Mun˜oz-Berbel X. A. Toby A. Jenkins XAT, Anicet R. Blanch AR, Francesc Xavier Mun˜oz1 FX. Surface plasmon resonance assay for real-time monitoring of somatic coliphages in wastewaters. Appl Environ 2008; 74(13): 4054-8.
|||Price MT. Comparison of Single-Agar Layer and two-step enrichment spot plate methods in the detection of somatic and male-specific coliphages in NC type II reclaimed water samples Master’s thesis 2014.|
|||Method 1602: Male-specific [F+] and somatic coliphage in water by Single agar layer [SAL] procedure EPA document 821-R-01-029] 2001.|
|||Bitton G. Wastewater microbiology 3rd ed.|
|||Bailey ES, Price M, Casanova LM, Sobsey MD. E. coli CB390: An alternative E. coli host for simultaneous detection of somatic and F+ coliphage viruses in reclaimed and other waters. J Virol Methods 2017; 250: 25-8.
|||Guidelines for drinking water quality, recommendations 4th ed. 2011.|
|||Lucena F, Jofre J. Potential use of bacteriophages as indicators of water quality and wastewater treatment processesBacteriophages in the Control of Food and Waterborne Pathogen 2010.|
|||Jurzik L, Hamza IA, Puchert W, Überla K, Wilhelm M. Chemical and microbiological parameters as possible indicators for human enteric viruses in surface water. Int J Hyg Environ Health 2010; 213(3): 210-6.
|||Ashbolt NJ, Grabow WO, Snozzi M. “Indicators of Microbial Water Quality” Water Quality: Guidelines, Standards, and Health 2001; 289-316.|
|||Skraber S, Gassilloud B, Gantzer C. Comparison of coliforms and coliphages as tools for assessment of viral contamination in river water. Appl Environ Microbiol 2004; 70(6): 3644-9.
|||Gassilloud B, Schwartzbrod L, Gantzer C. Presence of viral genomes in mineral water: a sufficient condition to assume infectious risk? Appl Environ Microbiol 2003; 69(7): 3965-9.
|||Cantalupo PG, Calgua B, Zhao G, et al. Raw Sewage Harbors Diverse Viral Populations mBio 2011; 2(5)e00180-11|
|||Lin J, Ganesh A. Water quality indicators: bacteria, coliphages, enteric viruses. Int J Environ Health Res 2013; 23(6): 484-506.
|||Wu J, Long SC, Das D, Dorner SM. Are microbial indicators and pathogens correlated? A statistical analysis of 40 years of research. J Water Health 2011; 9(2): 265-78.
|||Payment P, Franco E. Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts. Appl Environ Microbiol 1993; 59(8): 2418-24.0099|
|||Jiang SC, Chu W, He JW. 2007; Seasonal detection of human viruses and coliphage in newport bay California. Appl Environ 2007; 73(20): 6468.|
|||EPA (US Environmental Protection Agency) Review of coliphages as possible indicators of fecal contamination for ambient water quality 820-R-15-0982015.|
|||Akin BS. Contaminant properties of hospital clinical laboratory wastewater: A physiochemical and microbiological assessment. J Environ Prot (Irvine Calif) 2016; 7: 635-42.
|||Borchardt MA, Bertz PD, Spencer SK, Battigelli DA. Incidence of enteric viruses in groundwater from household wells in Wisconsin. Appl Environ Microbiol 2003; 69(2): 1172-80.
|||Gomez M, Rua A, Garralon G, Plaza F, Hontoria E, Gomez MA. Urban wastewater disinfection by filteration technologies. Desalin 2006; 190: 16-28.
|||Schijven JF, Hassanizadeh MS. Removal of viruses by soil passage: Overview of modeling, processes and parameters. Crit Rev Environ Sci Technol 2000; 30(1): 49-127.
|||Tree JA, Adams MR, Lees DN. Chlorination of indicator bacteria and viruses in primary sewage effluent. Appl Environ Microbiol 2003; 69(4): 2038-43.
|||Stewart JR, Gast RJ, Fujioka RS, et al. The coastal environment and human health: Microbial indicators, pathogens, sentinels and reservoirs. Environ Health 2008; 7(Suppl. 2): S3.
|||Reischer GH, Haider, Sommer JM, et al. Quantitative microbial faecal source tracking with samplingguided by hydrological catchment dynamics. Environ Microbiol 2008; 10: 2598-608.
|||Yee SY, Fong NY, Fong GT, Tak OJ, Hui GT, Su Ming Y. Male-specific RNA coliphages detected by plaque assay and RT-PCR in tropical river waters and animal fecal matter. Int J Environ Health Res 2006; 16(1): 59-68.
|||Bothma L. Bacteriophage levels and associated characteristics in selected temperate water systems 2017.|
|||Ribaudo MO. Regional estimates of off-site damages from soil erosion. In: Waddell TE, Ed. The off-site costs of soil erosion Proceedings of a symposium Conservation Foundation, Washington, D.C . 1985; p. 1985; 284.|
|||Ekhaise FO, Omavwoya BP. Influence of Hospital Wastewater Discharged from University of Benin Teaching Hospital [UBTH], Benin City on its Receiving Environment. Am-Eurasian J Agric Environ Sci 2008; 4(4): 484-8.|
|||Sehulster LM, Chinn RYW, Arduino MJ, et al. Guidelines for Environmental Infection Control in Health-Care Facilities Recommendations from CDC and the Healthcare Infection Control Practices Advisory Committee 2004. HICPAC|
|||Mesdaghinia AR, Naddafi K, Nabizadeh R, Saeedi R, Zamanzadeh M. Wastewater characteristics and appropriate method for wastewater management in the hospitals. Iran J Public Health 2009; 38(1): 34-40.|
|||Dehghani MH, Azam K, Changani F, Dehghani EF. Assessment of medical waste management in Educational Hospitals of Tehran University Medical Science. Iran J Environ Health Sci Eng 2008; 5(2): 131-6.|
|||Qadir M, Wichelns D, Raschid-Sally L, et al. The challenges of wastewater irrigation in developing countries. Agric Water Manage 2010; 97(4): 561-8.
|||Jimenez B. Irrigation in developing countries using wastewater. IRES 2006; 6(2): 229-50.|
|||Rutkowski TL, Raschid-Sally L, Buechler S. Wastewater irrigation in the developing world-two case studies from the Kathmandu Valley in Nepal. Agric Water Manage 2007; 88(1–3): 83-91.
|||Ashraf MA, Maah MJ, Yusoff I, Mehmood K. Effects of Polluted Water Irrigation on Environment and Health of People in Jamber, District Kasur, Pakistan. IJBAS 2010; 10(3): 37-57.|
|||Bos R, Carr R, Keraita B. Assessing and mitigating wastewater-related health risks in low-income countries: an introduction.Wastewater Irrigation and Health: Assessing and mitigating risk in low-income countries 2010; 29-37.|
|||Kris M. Wastewater pollution in China 2007.http: www.dbc.uci/wsu stain/suscoasts/krismin.html|
|||Bofill-Mas S, Albinana-Gimenez N, Clemente-Casares P, et al. Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices. Appl Environ Microbiol 2006; 72(12): 7894-6.
|||Bofill-Mas S, Rodriguez-Manzano J, Calgua B, Carratala A, Girones R. Newly described human polyomaviruses Merkel cell, KI and WU are present in urban sewage and may represent potential environmental contaminants. Virol J 2010; 7: 141.
|||Formiga-Cruz M, Tofiño-Quesada G, Bofill-Mas S, et al. Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom. Appl Environ Microbiol 2002; 68(12): 5990-8.