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 <2.0 x 101 up
to >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 <2.0 x 10 1
MPN/100 g and >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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