Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas Suffredini Elisabetta, Mioni Renzo, Mazzette Rina, Paola Bordin, Serratore Patrizia, Federica Fois, Annamaria Piano, Cozzi Loredana, Croci Luciana PII: DOI: Reference:
S0168-1605(14)00192-5 doi: 10.1016/j.ijfoodmicro.2014.04.016 FOOD 6513
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
3 December 2013 9 April 2014 11 April 2014
Please cite this article as: Elisabetta, Suffredini, Renzo, Mioni, Rina, Mazzette, Bordin, Paola, Patrizia, Serratore, Fois, Federica, Piano, Annamaria, Loredana, Cozzi, Luciana, Croci, Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas, International Journal of Food Microbiology (2014), doi: 10.1016/j.ijfoodmicro.2014.04.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT DETECTION
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SHELLFISH FROM ITALIAN PRODUCTION AREAS
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Suffredini Elisabetta1*, Mioni Renzo2, Mazzette Rina3, Paola Bordin2, Serratore Patrizia4,
1
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Federica Fois3, Annamaria Piano4, Cozzi Loredana1 and Croci Luciana1
Istituto Superiore di Sanità, Department of Veterinary Public Health and Food Safety, viale
2
MA
Regina Elena 299, 00161, Rome, Italy
Istituto Zooprofilattico Sperimentale delle Venezie, viale Università 10 , 35020 Legnaro (PD),
University of Sassari, Department of Veterinary Medicine, via Vienna 2, 07100 Sassari, Italy
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Italy
4 University of Bologna, Department of Veterinary Medical Sciences, viale Vespucci 2, 47042
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Cesenatico (FC), Italy
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Elisabetta Suffredini, PhD Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare Istituto Superiore di Sanità Viale Regina Elena, 299 00161 Rome Italy tel +39 (0)6 49903656 fax +39 (0)6 49902045 e-mail: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
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V. parahaemolyticus is a marine microorganism, recognised as an important cause of foodborne
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illness particularly in Asia, South America and United States. Outbreak are rarely reported in
SC R
Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e carrying the tdh and/or the trh genes) but currently there is a lack of
NU
occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member
MA
States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian Regions (Sardinia and Veneto) were analysed for E. coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic Vibrio parahaemolyticus
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D
strains using a new DNA colony hybridisation method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with
CE P
the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus was 84 CFU/g and 73
AC
CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7x103 CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120 CFU/g and most of the positive samples (n=17) showing values equal or below 20 CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency. The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs.
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Keywords: Vibrio parahaemolyticus, colony hybridization, quantitative analysis, tdh, trh, shellfish
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ACCEPTED MANUSCRIPT 1. INTRODUCTION
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Vibrio parahaemolyticus is a halophilic Gram negative microrganism, naturally present in marine
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and estuarine environment of tropical and temperate areas. Currently, strains carrying the tdh gene,
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encoding thermostable direct haemolysin (TDH) and/or the trh gene, encoding TDH-related haemolysin (TRH) are considered pathogenic to human (Nishibuchi and Kaper, 1995; Zhang and Austin, 2005). The tdh gene can be subdivided in five subtype (tdh1-tdh5), sharing 96 to 98%
NU
similarity level (Kamruzzaman et al., 2008; Nishibuchi and Kaper, 1990), whereas the trh genes,
MA
that present a higher sequence variation, can be clustered into two main subgroups (trh1 and trh2), which share 84% identity (Kishishita et al., 1992).
Epidemiological data show that V. parahaemolyticus is an important cause of foodborne illness in
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D
Asia (Chiou et al., 2000; Chowdhury et al., 2013; Kubota et al., 2008; Ma et al., 2014; Okuda et al., 1997; Tuyet et al., 2002; Vuddhakul et al., 2006), South America (Cabanillas-Beltran et al., 2006;
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Cordova et al., 2002; Fuenzalida et al., 2007; Garcia et al., 2009; Gil et al., 2007; Leal et al., 2008), United States (Haendiges et al., 2014; Sims et al., 2011), while V. parahaemolyticus isolation has
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been reported in some African countries (Ansaruzzaman et al., 2008; Soumare et al., 2007; Utsalo et al., 1992). In Europe only a few outbreaks or sporadic cases were reported in the last decade as a consequence of the consumption of local or imported seafood (Martinez-Urtaza et al., 2005; Ottaviani et al., 2010b; Ottaviani et al., 2008; Ottaviani et al., 2012; Quilici et al., 2005; Sala et al., 2009), but previous experiences showed that outbreaks can occur unexpectedly, in relation, among other reasons, to the spread of highly virulent strains, as demonstrated by the pandemic strain O3:K6 (Martinez-Urtaza et al., 2004; Okuda et al., 1997) or by the Pacific Nortwest strain O4:K12 / O4:KUT (Martinez-Urtaza et al., 2013). Consumption of raw or undercooked shellfish is considered a major risk for Vibrio parahaemolyticus infection (Potasman et al., 2002), however, according to current European Legislation (EC Reg. n. 2073/2005 and subsequent amendments), the evaluation of shellfish safety 4
ACCEPTED MANUSCRIPT is based entirely on the use of E. coli as an indicator of faecal contamination, that is not related to the presence of seawater autochthonous bacteria, as V. parahaemolyticus (Joseph et al., 1982; Koh
T
et al., 1994). Furthermore, while studies would be needed to fill the data gap on V.
IP
parahaemolyticus levels in shellfish production areas in the EU Member States, several authors
SC R
report that, in environmental and seafood samples, pathogenic V. parahaemolyticus (i.e. either carrying tdh or trh genes) range from
S0168-1605(14)00192-5 doi: 10.1016/j.ijfoodmicro.2014.04.016 FOOD 6513
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
3 December 2013 9 April 2014 11 April 2014
Please cite this article as: Elisabetta, Suffredini, Renzo, Mioni, Rina, Mazzette, Bordin, Paola, Patrizia, Serratore, Fois, Federica, Piano, Annamaria, Loredana, Cozzi, Luciana, Croci, Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas, International Journal of Food Microbiology (2014), doi: 10.1016/j.ijfoodmicro.2014.04.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT DETECTION
AND
QUANTIFICATION
OF
VIBRIO
PARAHAEMOLYTICUS
IN
IP
T
SHELLFISH FROM ITALIAN PRODUCTION AREAS
SC R
Suffredini Elisabetta1*, Mioni Renzo2, Mazzette Rina3, Paola Bordin2, Serratore Patrizia4,
1
NU
Federica Fois3, Annamaria Piano4, Cozzi Loredana1 and Croci Luciana1
Istituto Superiore di Sanità, Department of Veterinary Public Health and Food Safety, viale
2
MA
Regina Elena 299, 00161, Rome, Italy
Istituto Zooprofilattico Sperimentale delle Venezie, viale Università 10 , 35020 Legnaro (PD),
University of Sassari, Department of Veterinary Medicine, via Vienna 2, 07100 Sassari, Italy
TE
3
D
Italy
4 University of Bologna, Department of Veterinary Medical Sciences, viale Vespucci 2, 47042
AC
CE P
Cesenatico (FC), Italy
*
Elisabetta Suffredini, PhD Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare Istituto Superiore di Sanità Viale Regina Elena, 299 00161 Rome Italy tel +39 (0)6 49903656 fax +39 (0)6 49902045 e-mail: [email protected]
1
ACCEPTED MANUSCRIPT ABSTRACT
T
V. parahaemolyticus is a marine microorganism, recognised as an important cause of foodborne
IP
illness particularly in Asia, South America and United States. Outbreak are rarely reported in
SC R
Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e carrying the tdh and/or the trh genes) but currently there is a lack of
NU
occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member
MA
States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian Regions (Sardinia and Veneto) were analysed for E. coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic Vibrio parahaemolyticus
TE
D
strains using a new DNA colony hybridisation method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with
CE P
the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus was 84 CFU/g and 73
AC
CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7x103 CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120 CFU/g and most of the positive samples (n=17) showing values equal or below 20 CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency. The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs.
2
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
Keywords: Vibrio parahaemolyticus, colony hybridization, quantitative analysis, tdh, trh, shellfish
3
ACCEPTED MANUSCRIPT 1. INTRODUCTION
T
Vibrio parahaemolyticus is a halophilic Gram negative microrganism, naturally present in marine
IP
and estuarine environment of tropical and temperate areas. Currently, strains carrying the tdh gene,
SC R
encoding thermostable direct haemolysin (TDH) and/or the trh gene, encoding TDH-related haemolysin (TRH) are considered pathogenic to human (Nishibuchi and Kaper, 1995; Zhang and Austin, 2005). The tdh gene can be subdivided in five subtype (tdh1-tdh5), sharing 96 to 98%
NU
similarity level (Kamruzzaman et al., 2008; Nishibuchi and Kaper, 1990), whereas the trh genes,
MA
that present a higher sequence variation, can be clustered into two main subgroups (trh1 and trh2), which share 84% identity (Kishishita et al., 1992).
Epidemiological data show that V. parahaemolyticus is an important cause of foodborne illness in
TE
D
Asia (Chiou et al., 2000; Chowdhury et al., 2013; Kubota et al., 2008; Ma et al., 2014; Okuda et al., 1997; Tuyet et al., 2002; Vuddhakul et al., 2006), South America (Cabanillas-Beltran et al., 2006;
CE P
Cordova et al., 2002; Fuenzalida et al., 2007; Garcia et al., 2009; Gil et al., 2007; Leal et al., 2008), United States (Haendiges et al., 2014; Sims et al., 2011), while V. parahaemolyticus isolation has
AC
been reported in some African countries (Ansaruzzaman et al., 2008; Soumare et al., 2007; Utsalo et al., 1992). In Europe only a few outbreaks or sporadic cases were reported in the last decade as a consequence of the consumption of local or imported seafood (Martinez-Urtaza et al., 2005; Ottaviani et al., 2010b; Ottaviani et al., 2008; Ottaviani et al., 2012; Quilici et al., 2005; Sala et al., 2009), but previous experiences showed that outbreaks can occur unexpectedly, in relation, among other reasons, to the spread of highly virulent strains, as demonstrated by the pandemic strain O3:K6 (Martinez-Urtaza et al., 2004; Okuda et al., 1997) or by the Pacific Nortwest strain O4:K12 / O4:KUT (Martinez-Urtaza et al., 2013). Consumption of raw or undercooked shellfish is considered a major risk for Vibrio parahaemolyticus infection (Potasman et al., 2002), however, according to current European Legislation (EC Reg. n. 2073/2005 and subsequent amendments), the evaluation of shellfish safety 4
ACCEPTED MANUSCRIPT is based entirely on the use of E. coli as an indicator of faecal contamination, that is not related to the presence of seawater autochthonous bacteria, as V. parahaemolyticus (Joseph et al., 1982; Koh
T
et al., 1994). Furthermore, while studies would be needed to fill the data gap on V.
IP
parahaemolyticus levels in shellfish production areas in the EU Member States, several authors
SC R
report that, in environmental and seafood samples, pathogenic V. parahaemolyticus (i.e. either carrying tdh or trh genes) range from
