Bull Vet Inst Pulawy 56, 563-568, 2012 DOI: 10.2478/v10213-012-0099-9
MICROBIOLOGICAL AND MARINE BIOTOXINS CONTAMINATION OF RAW BIVALVE MOLLUSCS COMMERCIALLY AVAILABLE IN POLAND REMIGIUSZ POMYKAŁA, MIROSŁAW MICHALSKI, ARTUR JÓŹWIK1, AND JACEK OSEK Department of Hygiene of Food of Animal Origin National Veterinary Research Institute, 24-100 Pulawy, Poland 1 Institute of Genetics and Animal Breeding, Jastrzebiec, 05-552 Magdalenka, Poland
[email protected] Received: September 18, 2012
Accepted: November 30, 2012
Abstract The study was carried out on live bivalve molluscs available on Polish market. Microbiological tests were performed for the presence of Salmonella sp., Vibrio parahaemolyticus, spore-forming anaerobe bacteria, and coagulase-positive Staphylococcus sp., and for the enumeration of Escherichia coli. ELISA was used for the determination of marine biotoxins, paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), and diarrhoeic shellfish poisoning (DSP). Microbiological examinations were performed according to ISO and Polish Standards. Salmonella sp. was not detected in any sample tested. Coagulase-positive staphylococci were identified in 9.0% of the samples. V. parahaemolyticus was isolated from 17.0% of mussels. Shellfish were highly contaminated by anaerobes, which were isolated from 68.0% of the samples. The number of E. coli ranged from 1.8 x 104 MPN/100 g. The majority of mussels were free from the marine biotoxins tested or contained them bellow the permitted level. The analysis of microbiological and toxicological status of raw bivalve molluscs available on Polish market indicates that they are generally safe for the consumers.
Key words: raw bivalve molluscs, microbiological contamination, marine biotoxins. Polish consumers show an increasing interest in “frutti di mare”, including bivalve molluscs, which may be a potential source of foodborne diseases and poisonings. This food is easily digested, free of additives and minimally processed when consumed raw or only cooked. These features of bivalves make them a food product completely fulfilling the requirements of the consumers. Consequently, there is a global increase in the demand of shellfish products (18). Bivalves, as filter-feeder organisms, accumulate different contaminants such as bacteria, viruses, parasites, biotoxins, heavy metals, pesticides, or drug residues (7, 30). Bacterial pathogens found in shellfish and involved in foodborne diseases in humans include those associated with faecal contamination (Salmonella sp., Shigella sp., Campylobacter sp., Yersinia sp., Clostridium sp., Staphylococcus sp., and Escherichia coli) and naturally occurring belonging mainly to the genus of Vibrio (V. parahaemolyticus, V. vulnificus, and V. cholerae) (9, 18). Consumption of bivalves contaminated by these pathogens may lead to severe and potentially fatal foodborne diseases where gastroenteritis is the most frequent clinical syndrome. Biotoxins constitute another serious health problem for the consumers. There are several illnesses, caused by marine biotoxins, which are connected with
the consumption of contaminated shellfish. They include paralytic shellfish poisoning (PSP), caused by a saxitoxin group, diarrhoeic shellfish poisoning (DSP), caused by okadaic acid, and amnesic shellfish poisoning (ASP), caused by domoic acid (14, 15, 16, 17). Heat resistance of the toxins cause that even well cooked bivalves might still constitute a risk for human health (4). The occurrence of symptoms of intoxications such as nausea, vomiting, weakness, dysphasia, dysphonia, respiratory paralysis, diarrhoea, abdominal pain, memory loss, disorientation, coma, with varying severity, depends on the individual sensitivity of human, and type and quantity of consumed toxins (10, 17, 24). The requirements for the production, harvesting, storage conditions, and use in food processing industry of bivalve shellfish are described in the EU food acts (25, 26). Due to the epidemiological relation between the occurrence of diseases and consumption of raw molluscs, microbiological and toxicological criteria for these food products have been established (3, 26). Testing for other bacterial pathogens, especially for V. parahaemolyticus, is recommended for shellfish growing and harvesting from waters where these bacteria are present at high level (9). The objective of the study was to evaluate
564 microbiological and toxicological contamination of live bivalves available for human consumption in Poland.
Material and methods Bivalve molluscs. The study was conducted on live bivalve molluscs listed in Table 1. They were obtained from the wholesale companies and markets in Poland. Samples were collected in original packaging and were from different countries of Europe. A total of 100 samples collected in 2011 were used for microbiological analyses and 40 out of them for marine biotoxins determination. After collection, the samples were immediately delivered to the laboratory at refrigerated temperature. The time from harvesting to the beginning of analyses did not exceed 48-72 h. Microbiological analyses. To ensure the representativeness, at least six individual organisms (together with flesh and intervalve water) were used for the preparation of laboratory samples. The molluscs were homogenised in a blender for 2 min and subjected to specific analyses. The microbiological tests were conducted according to ISO and Polish Standards: EN ISO 6579 for detection of Salmonella sp., ISO/TS 21827-1 for detection of V. parahaemolyticus, EN ISO 6888-3 for detection of coagulase-positive Staphylococcus sp., PN-A-82055-12 for detection of spore-forming anaerobe bacteria, and ISO/TS 16649-3 for enumeration of E. coli (18-20, 25, 26). Marine biotoxins. For determination of PSP toxins the RIDACSREEN® FAST PSP SC test was used with the detection limit 50 µg/kg of shellfish meat. For the determination of ASP toxins the Biosense ASP
DIRECT ELISA test was used. The limit of detection was 0.01 mg/kg of shellfish. For determination of DSP toxins the TOXILINE DSP test was used. The limit of detection was 63 μg/kg of shellfish. Preparation of samples and tests were performed according to the test procedure described by the producers.
Results Microbiological analyses. As it is shown in Table 2, none of the 100 samples tested was contaminated with Salmonella sp. V. parahaemolyticus was isolated from 17 (17.0%) samples of all types of molluscs tested, except of razor clams. The highest prevalence of V. parahaemolyticus was noted for manila clams (41.2%). Coagulase-positive staphylococci were detected in nine samples (9.0%) of which the highest number of positive samples was found among manila clams (23.5%). No staphylococci were found in Japanese carpet shell molluscs. Spore-forming anaerobe bacteria were isolated from 68 samples (68.0%) and the presence of these bacteria was found in all types of bivalve molluscs examined. The number of E. coli determined in samples varied between 1.8 x 104 MPN/100 g (MPN, most probable number) and the majority of them (90.0%) contained E. coli in quantities not exceeding the acceptable limit of 230 MPN/100 g. However, E. coli was present in number higher than 230 MPN/100 g in two (7.1%) samples of blue mussels, also in two samples of Japanese carpet shell molluscs, and six (35.3%) samples of manila clams.
Table 1 Samples used for microbiological examinations Species of bivalve mollusc
Country of origin
Pacific oyster (Crassostrea gigas) Great scallop (Pecten maximus)
The Netherlands France
Number of samples 22 4
Norway
15
Norway The Netherlands Spain Italy The Netherlands The Netherlands France
15 11 2 14 3 5 3
The Netherlands
6
Blue mussel (Mytilus edulis) Manila clam (Tapes semidecussatus) Japanese carpet shell (Tapes philippinarum) Razor clam (Ensis directus)
Total number of samples 26 15 28
17 8 6
Table 2 Results of microbiological analyses of live bivalve molluscs
Number (%) of positive samples / number of samples tested Species
Salmonella sp. in 25 g
V. parahaemolyticus in 25 g
Coagulase-positive Staphylococcus sp. in 1 g
Spore-forming anaerobe bacteria in 1 g
Escherichia coli/ out of limit (230 MPN/100 g)
Pacific oyster
0/26
4/26 (15.4)
1/26 (4.0)
16/26 (61.5)
0/26 (0.0)
Great scallop
0/15
2/15 (13.3)
1/15 (6.7)
6/15 (40.0)
0/15 (0.0)
Blue mussel
0/28
3/28 (10.7)
1/28 (3.6)
19/28 (67.9)
2/28 (7.1)
Manila clam
0/17
7/17 (41.2)
4/17 (23.5)
15/17 (88.2)
6/17 (35.3)
Japanese carpet shell
0/8
1/8
0/8
7/8
2/8
Razor clam
0/6
0/6
2/6
4/6
0/6
Total number of positive samples/ Total number (%) of samples tested
0/100
17/100 (17.0)
9/100 (9.0)
68/100 (68.0)
10/100 (10.0)
565
566 Table 3 Presence of PSP, ASP, and DSP in bivalve molluscs Species
Number of samples
PSP (µg/kg)
ASP mg/kg
DSP (µg/kg)
Pacific oyster
10
bld – 263.29
bld
bld – 88.0
Great scallop
7
71.42 – 255.91
1.1 – 6.3
bld – 80.0
Blue mussel
11
bld – 428.51
bld
bld – 76.0
Manila clam
7
bld – 83.87
bld
bld – 85.0
Japanese carpet shell
3
73.63 – 89.53
bld
bld
Razor clam
2
56.36 – 109.1
bld
bld
Number of samples with biotoxins
32
8
8
Limit of detection for used tests
50 µg/kg
0.01 mg/kg
30 µg/kg
Maximum legal limit
800 µg/kg, as saxitoxine
20 mg/kg, as domoic acid
160 µg/kg, as okadaic acid
bld – below limit of detection; PSP - paralytic shellfish poisoning; ASP - amnesic shellfish poisoning; DSP - diarrhoeic shellfish poisoning Marine biotoxin analyses. The results of analysis of the biotoxins in shellfish samples, with division into individual species, are shown in Table 3. The maximum amount of PSP biotoxins, (428.51 µg/kg) were found in blue mussels. Of 40 tested samples, only in eight samples saxitoxin and its isomers were not detected. PSP was found in all species of shellfish analysed. ASP biotoxins were detected in eight samples (one blue mussel and seven great scallops). The maximum amount of ASP toxin (6.3 mg/kg) was found in great scallops. In other five species of molluscs, ASP was not detected. Biotoxins from the DSP group were detected in eight samples: pacific oysters (four samples), blue mussel (one sample), manila clam (two samples) and great scallop (one sample), with a maximum content of 88 µg/kg (in oyster). Only in Japanese carpet shell and razor clams no DSP toxin was identified.
Discussion It is widely known that bivalve molluscs may be a source of human disease agents and there are serious safety concerns connected with the consumption of raw or cooked shellfish due to the presence of biological and toxicological hazards (2, 9). According to the EFSA report, in 2010 in the EU countries crustaceans, shellfish, and molluscs were the source of 8.5% among noted foodborne outbreaks, whereas bacteria and marine biotoxins were the causative agents in 11.9% and 18.6%, respectively (5). Due to the global increase in the production and consumption of shellfish, there is a need for ensuring consumer’s health. For this reason, studies on the presence of pathogenic bacteria
and marine biotoxins in bivalve molluscs are performed in many countries. In the study, no Salmonella sp. was found in any of the examined samples. Other investigations concerning live bivalve molluscs confirmed that Salmonella sp. can tolerate coastal water salinity. The bacteria may be found in 0%-2.5% of live shellfish and consumption of these foods may lead to salmonellosis, characterised by enteric fever along with gastroenteritis and diarrhoea (1, 18). The presence of Salmonella sp. in water depends on many factors, including human and animal faecal pollutions (7). However, epidemiological data indicate that prevalence of Salmonella strains in molluscs may vary from 3% to even 34% in areas with warm sea water such as Vietnam or India (1). In Europe, according to the data published by EFSA, Salmonella sp. remains the main causative agent, responsible for 30.5% of noted outbreaks associated with the consumption of food, where shellfish and molluscs were the source in 6.8% (5). The study showed that the vast majority of live bivalve molluscs met the microbiological criteria for E. coli listed in Commission Regulation (EC) No. 2073/2005 (4). These bacteria are used as an indicator of faecal contamination of water designated for culture of molluscs and it seems that the occurrence of the bacteria in the levels below the limit (230 MPN/100 g) in molluscs does not constitute a high risk to the consumer’s health. Nevertheless, disease outbreaks associated with the consumption of shellfish contaminated by E. coli are reported. Foodborne diseases caused by V. parahaemolyticus are most commonly found in countries where the temperature of water is high and
567 molluscs are consumed raw. In Europe, the main types of bivalve molluscs eaten raw are oysters and mussels, which constitute a significant risk to the consumers. V. parahaemolyticus can be isolated from estuarine environments suitable for growth of a variety of shellfish. Bacteria were detected in the marine water of several countries in Europe, including Great Britain, France, Spain, Italy, and Greece (2, 9, 18). In the study, 17 (17.0%) samples were positive for V. parahaemolyticus and these results are similar to those reported by other authors where the prevalence of the vibrios in shellfish varied from few to as high as 35.0% (19). Ingestion of these bacteria with contaminated molluscs may lead to the development of acute gastroenteritis due to bacterial toxins: thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH). Correlation between pathogenicity of V. parahaemolyticus and its ability to form these toxins is well recognised although not much is known about the prevalence of TDH- and TRH-producing V. parahaemolyticus in marine water in Europe (8, 9). A microbiological criterion for raw bivalve molluscs in relation to V. parahaemolyticus has not been established but testing for the presence of these microorganisms is recommended for molluscs harvested from water suspected to be harboured by Vibrio sp. (9, 19). Several authors reported a relationship between the prevalence of Vibrio sp. and E. coli count in bivalve molluscs (8, 18, 19). The results of the study revealed that there was no significant correlation between the presence of E. coli and Vibrio sp. However, it was found that the most shellfish harboured by V. parahaemolyticus and high numbers of E. coli were manila clam molluscs. The ubiquitous nature of Vibrio sp. in marine and estuarine environments makes impossible to obtain seafood free of these bacteria and the significance for public health mostly depends on the health status of the consumer as well as on the prevalence of virulence factors of the pathogen (8). Coagulase-positive Staphylococcus sp. were detected in eight (9.0%) samples of the examined bivalve molluscs and the contamination rate was similar to those reported by other authors (19, 27). Existing microbiological criteria relating to staphylococci focus only on cooked shellfish and there is no obligation to examine raw bivalve molluscs for the presence of these bacteria. Therefore, reports on isolations of staphylococci from shellfish are limited. Nevertheless, enterotoxigenic coagulase-positive staphylococci are well recognised causative agents of food poisoning and may constitute a serious risk to human health if present in consumed raw bivalve molluscs (30). The high prevalence (68.0%) of anaerobe bacteria, mainly presumed Clostridium sp., may be related to the faecal contamination of marine environments where spores of the microorganisms permit their persistence (7). Clostridia are ubiquitous in aquatic environments and they were isolated from water, sediments, fish, and shellfish. These bacteria have been proposed as an alternative indicator of faecal contamination of marine environments and molluscs but
the idea has not found a universal acceptance due to their high prevalence in marine water (7, 18, 20). The presence of biotoxins in consumed bivalve molluscs exceeding a permissible by law content causes a lot of food poisoning. Serious intoxications occur mainly when shellfish contain a high amount of biotoxins far in excess of permissible concentrations. For the marine shellfish biotoxins, the same maximum levels apply within the EU and in Poland. The legal limit for PSP content in meat of mussels is 800 μg/kg, for ASP - 20 mg/kg, and for DSP - 160 μg/kg of clam meat. In the countries where the mussels are commonly consumed, a lot of people are affected, even with fatal poisoning (6, 11-14, 24, 31). The study showed that the shellfish available on Polish markets are generally safe for the consumers. However, safety of this kind of seafood can be guaranteed mainly by preventive measures and application of appropriate procedures such as a suitable selection of harvesting areas, programmes for monitoring of water quality, final product inspections, and hygiene control for food business operators involved in whole food chain. As a conclusion, to reduce the outbreaks of foodborne diseases due to the consumption of raw bivalve molluscs, it would be reasonable to carry out the investigation of pathogens that constitute a potential threat to public health, such as V. parahaemolyticus, and the consumers should be provided with a clear information on possible health hazards associated with the consumption of these products.
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