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1.
PLoS One ; 15(1): e0227864, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990915

RESUMO

The type VI secretion system (T6SS) is a nanomachine capable of killing adjacent microbial cells in a contact-dependent manner. Due to limited studies, relatively little is known about the range of marine bacteria that are susceptible to T6SS attack. Here, 15 diverse marine bacterial isolates from the phyla Bacteroidetes and Ɣ-Proteobacteria were challenged against the marine bacterium and human pathogen, Vibrio cholerae, which has a well described T6SS. V. cholerae killed several of the tested Ɣ-Proteobacteria, including members of the orders Vibrionales, Alteromonadales, Oceanospirillales, and Pseudomonadales. In contrast, V. cholerae co-existed with multiple Bacteroidetes and Ɣ-Proteobacteria isolates, but was killed by Vibrio coralliilyticus. Follow-up experiments revealed that five V. coralliilyticus strains, including known coral and shellfish pathogens survived the T6SS challenge and killed V. cholerae. By using predicted protein comparisons and mutagenesis, we conclude that V. coralliilyticus protected itself in the challenge by using its own T6SS to kill V. cholerae. This study provides valuable insight into the resilience and susceptibility of marine bacteria to the V. cholerae T6SS, and provides the first evidence for a functional T6SS in V. coralliilyticus, both of which have implications for human and ocean health.


Assuntos
Sistemas de Secreção Tipo VI/genética , Vibrio cholerae/patogenicidade , Vibrio/genética , Animais , Antozoários/microbiologia , Anticorpos Antibacterianos/genética , Proteínas de Bactérias/genética , Bacteroidetes/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteobactérias/genética , Frutos do Mar/microbiologia , Vibrio cholerae/genética
2.
Int J Food Microbiol ; 313: 108378, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31678817

RESUMO

Vibrio bacteria can accumulate in molluscan shellfish and cause human diseases. The United States (U.S.) has implemented Vibrio Control Plans to mitigate risks associated with these bacteria, which include time and temperature requirements for post-harvest processing and maintaining an unbroken cold chain. In this study, we tracked the performance of cold chains for U.S. farmed oysters distributed nationally and internationally using temperature sensors. Boxes and bags of oysters (n = 125) were shipped from farms in Washington State and the Chesapeake Bay to 143 unique businesses in 20 U.S. states, Washington D.C., and Hong Kong, China. Eighty-one percent of the temperature sensors were returned with usable data. The average product temperature among all participants was 4.4 ±â€¯2.7 °C (40 ±â€¯5 °F), which is 5.6 °C (10 °F) cooler than the 10 °C (50 °F) guidance criterium established by the U.S. government. There were spikes in temperature in some shipments: 18% of shipments (16/91) experienced oyster temperatures above 10 °C for one hour or more, and the median time spent out of temperature control was 2.5 h. We modeled V. parahaemolyticus abundance using temperature sensor data and 75% (68/91) of shipments had a net decrease in V. parahaemolyticus abundance in the cold chain. There are opportunities for improvements in cold chain performance in the shellfish industry and related businesses. In the discussion we provide recommendations for oyster producers related to product cooling, for businesses that handle shellfish, and for government and industry groups to develop guidance for shipping by air, among other issues.


Assuntos
Ostreidae/microbiologia , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/crescimento & desenvolvimento , Animais , China , Temperatura Baixa , Contagem de Colônia Microbiana , Fazendas , Contaminação de Alimentos/análise , Manipulação de Alimentos , Humanos , Ostreidae/química , Ostreidae/crescimento & desenvolvimento , Refrigeração , Frutos do Mar/análise , Temperatura , Estados Unidos
3.
Rev Sci Tech ; 38(2): 437-457, 2019 Sep.
Artigo em Inglês, Francês, Espanhol | MEDLINE | ID: mdl-31866683

RESUMO

The growth of aquaculture over the past 50 years has been accompanied by the emergence of aquatic animal diseases, many of which have spread to become pandemic in countries or continents. An analysis of 400 emerging disease events in aquatic animals that were logged by the Centre for Environment, Fisheries and Aquaculture Science between 2002 and 2017 revealed that more than half were caused by viruses. However, in molluscs, most events were parasitic. Categorising these events indicated that the key processes underpinning emergence were the movement of live animals and host switching. Profiles of key pathogens further illustrate the importance of wild aquatic animals as the source of new infections in farmed animals. It is also clear that the spread of new diseases through the largescale movement of aquatic animals for farming, for food and for the ornamental trade has allowed many to achieve pandemic status. Many viral pathogens of fish (e.g. infectious salmon anaemia, viral haemorrhagic septicaemia) and shrimp (e.g. white spot syndrome virus) affect a large proportion of the global production of key susceptible species. Wild aquatic animal populations have also been severely affected by pandemic diseases, best exemplified by Batrachochytrium dendrobatidis, a fungal infection of amphibians, whose emergence and spread were driven by the movement of animals for the ornamental trade. Batrachochytrium dendrobatidis is now widespread in the tropics and subtropics and has caused local extinctions of susceptible amphibian hosts. Given the rising demand for seafood, aquacultural production will continue to grow and diseases will continue to emerge. Some will inevitably achieve pandemic status, having significant impacts on production and trade, unless there are considerable changes in global monitoring and the response to aquatic animal diseases.


Assuntos
Anfíbios/microbiologia , Doenças dos Peixes/epidemiologia , Pandemias/veterinária , Frutos do Mar , Animais , Aquicultura , Quitridiomicetos , Micoses/microbiologia , Micoses/veterinária , Frutos do Mar/microbiologia , Frutos do Mar/parasitologia , Frutos do Mar/virologia
4.
Pol J Microbiol ; 68(4): 429-438, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880887

RESUMO

Penaeus monodon is highly susceptible to vibriosis disease. Aims of the study were to identify the pathogen causing vibriosis in P. monodon through molecular techniques and develop a biocontrol method of the disease by application of herbal extracts. Shrimp samples were collected aseptically from the infected farm and the bacteria were isolated from the infected region of those samples. Based on phenotypic identification, several isolates were identified as Vibrio sp. 16S rRNA gene sequences of the selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. An in vivo infection challenge test was performed by immersion method with V. alginolyticus where these isolates caused high mortality in juvenile shrimp with prominent symptoms of hepatopancreatic necrosis. Antibiogram profile of the isolates was determined against eleven commercial antibiotic discs whereas the isolates were found resistant to multiple antibiotics. A total of twenty-one herbal extracts were screened where Emblica officinalis, Allium sativum, and Syzygium aromaticum strongly inhibited the growth of V. alginolyticus in in vitro conditions. In in vivo conditions, the ethyl acetate extracts of E. officinalis and A. sativum successfully controlled the vibriosis disease in shrimp at a dose of 10 mg/g feed. This is the first report on molecular identification and biocontrol of V. alginolyticus in shrimp in Bangladesh.Penaeus monodon is highly susceptible to vibriosis disease. Aims of the study were to identify the pathogen causing vibriosis in P. monodon through molecular techniques and develop a biocontrol method of the disease by application of herbal extracts. Shrimp samples were collected aseptically from the infected farm and the bacteria were isolated from the infected region of those samples. Based on phenotypic identification, several isolates were identified as Vibrio sp. 16S rRNA gene sequences of the selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. An in vivo infection challenge test was performed by immersion method with V. alginolyticus where these isolates caused high mortality in juvenile shrimp with prominent symptoms of hepatopancreatic necrosis. Antibiogram profile of the isolates was determined against eleven commercial antibiotic discs whereas the isolates were found resistant to multiple antibiotics. A total of twenty-one herbal extracts were screened where Emblica officinalis, Allium sativum, and Syzygium aromaticum strongly inhibited the growth of V. alginolyticus in in vitro conditions. In in vivo conditions, the ethyl acetate extracts of E. officinalis and A. sativum successfully controlled the vibriosis disease in shrimp at a dose of 10 mg/g feed. This is the first report on molecular identification and biocontrol of V. alginolyticus in shrimp in Bangladesh.


Assuntos
Conservantes de Alimentos/farmacologia , Penaeidae/microbiologia , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Frutos do Mar/microbiologia , Vibrio alginolyticus/efeitos dos fármacos , Vibrio alginolyticus/genética , Animais , Antibacterianos/farmacologia , DNA Bacteriano/genética , Conservação de Alimentos , Testes de Sensibilidade Microbiana , Penaeidae/crescimento & desenvolvimento , RNA Ribossômico 16S/genética , Vibrio alginolyticus/crescimento & desenvolvimento , Vibrio alginolyticus/isolamento & purificação
6.
Mar Pollut Bull ; 149: 110559, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31543492

RESUMO

To reduce the outbreaks caused by the major pathogenic Vibrio species, V. parahaemolyticus, V. vulnificus, and V. cholerae, the distribution, antibiotic resistance, and virulence of these Vibrio strains were monitored in shellfish and seawater along the Korean coast. Among the Vibrio strains, V. parahaemolyticus was the most abundant species; during summer, this strain showed a substantial increase that correlated with the water temperature. Although >99.0% of the Vibrio species isolates were sensitive to seven antimicrobials recommended by the Center for Disease Control and Prevention for the treatment of Vibrio infections, multiple-antibiotic resistance to at least three antimicrobials was found in 14.3% to 50.0% of each Vibrio species. Among V. parahaemolyticus isolates, 14.3% were positive for the trh gene, whereas only 1% was positive for the tdh gene. These results should aid in implementing proper precautions to avoid potential human health risks associated with exposure to pathogenic Vibrio species.


Assuntos
Aquicultura , Monitoramento Ambiental , Alimentos Marinhos/microbiologia , Frutos do Mar/microbiologia , Vibrio/isolamento & purificação , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , República da Coreia , Água do Mar/microbiologia , Vibrio/efeitos dos fármacos , Vibrio/patogenicidade , Virulência
7.
Food Microbiol ; 84: 103258, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421777

RESUMO

The aim of the present study was to investigate the genetic variability of Vibrio parahaemolyticus strains isolated from naturally contaminated Mediterranean mussels (Mytilus galloprovincialis) and Grooved carpet shells (Ruditapes decussatus) from three harvesting areas of Sardinia (Italy) using a combination of different typing methods: traditional phenotypic systems and molecular techniques. Ninety-nine putative V. parahaemolyticus strains isolated from shellfish collected before and after purification were included in the study. Seventy-two isolates were confirmed as V. parahaemolyticus and were submitted to REP, ERIC and BOX PCRs. The combined dendrogram showed the similarity of the data set of the three typing methods and demonstrates how the different techniques grouped the strains in two clusters in accordance with each singular dendrogram. Several strains rendered a unique pattern regardless of the typing method, which indicates the high discriminatory power of the methods. Moreover, the use of multiple typing methods allowed a more accurate characterization of the genetic profiles of isolates and the identification of clones hardly revealed through the common techniques. The intraspecific typing of environmental V. parahaemolyticus can be of great interest in order to recognize clonal relationships between environmental contamination, foodborne disease, and geographical/temporal distribution of this pathogen. The comparative analysis focusing on the obtained genetic profiles supports the possibility for typing methods to discriminate strains with similar phenotypic profile, identifying the level of genetic correlation among the strains and the presence of genetic clones.


Assuntos
Variação Genética , Mytilus/microbiologia , Alimentos Marinhos/microbiologia , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/genética , Animais , Técnicas de Tipagem Bacteriana , Itália , Vibrio parahaemolyticus/patogenicidade
8.
Food Microbiol ; 84: 103270, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421783

RESUMO

Vibrio parahaemolyticus is the leading bacterial cause of seafood-associated gastroenteritis worldwide. Moreover, infections and outbreaks caused by V. parahaemolyticus has kept increasing over the last two decades. In this study, we investigated the genetic diversity, virulence factors and farm-to-table spread pattern of V. parahaemolyticus by analyzing 383 genomes of food-associated isolates. These strains were isolated from diverse sample types from six provinces of China in 2014, being classified into three tiers of the farm-to-table spread process: food production, circulation and consumption. The genetic diversity of V. parahaemolyticus in different classifications, including geographical location, sample type, source and spread tier, was similar, as the median number of pairwise SNPs within each classification was between 33,013 and 33,659. Specifically, there was no clear boundaries in genetic diversity of the isolates from inland vs. coastal provinces, as well as of those from freshwater vs. seawater products. Moreover, the virulence genes and genomic islands were only found in a small number of isolates, indicating a low disease risk of the food-associated isolates in this study. By further exploring 28 recently emerged clonal groups, we identified seven farm-to-table spread events, showing a common pattern of single-source radial spread accompanied with occasional gene gain/loss events. Generally speaking, our work highlighted the colonization of V. parahaemolyticus in inland provinces and freshwater environment, and provided a snapshot of the farm-to-table spread pattern of V. parahaemolyticus food-associated isolates. Our results showed the feasibility of tracking the farm-to-table spread of foodborne pathogen, which would help construct the whole genome sequencing-based molecular tracking network in the future.


Assuntos
Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Variação Genética , Vibrio parahaemolyticus/genética , Fatores de Virulência/genética , Animais , Técnicas de Tipagem Bacteriana , China , Surtos de Doenças , Ilhas Genômicas , Humanos , Tipagem de Sequências Multilocus , Filogenia , Frutos do Mar/microbiologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/isolamento & purificação , Virulência/genética
9.
Lett Appl Microbiol ; 69(4): 286-293, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392736

RESUMO

The prevalence of Listeria monocytogenes in the retail fish markets of the Kerala, India was investigated by screening 227 samples comprising of marine finfish (n = 97) shellfish (n = 19), ready-to-cook fish products (n = 47), ready-to-eat fish products (n = 10), dried fish (n = 11) and retail ice (n = 43). The prevalence of L. monocytogenes and L. innocua was 2·7% and 17·2% respectively. Sample category wise, prevalence of L. monocytogenes was higher in marine finfish (1·8%) and retail ice (0·9%). All the L. monocytogenes isolates carried virulent genes namely inlA, inlC, inlJ, hlyA, iap, plcA, prfA genes and majority (82%) belonged to 1/2a, 3a serogroups. L. monocytogenes isolates were multidrug-resistant and showed resistance to ampicillin, penicillin, erythromycin, tetracycline and clindamycin. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) delineated 58% genetic heterogeneity among the L. monocytogenes strains. The study reports that genetic similarities of the isolates were interlinked to their serogroup and sample origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of Listeria monocytogenes, in the retail fish markets of Kerala, India was low but their relatively higher presence in marine finfish and retail ice and virulent nature of the isolates signifies food safety concerns. Moreover, multidrug-resistant nature of these isolates may potentially lead to spread of antimicrobial resistance. This study identified retail ice as a vehicle for entry of L. monocytogenes in retail fish and hence, there is a need to ensure quality of retail ice used for maintaining the cold-chain.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Listeria monocytogenes/efeitos dos fármacos , Alimentos Marinhos/microbiologia , Frutos do Mar/microbiologia , Ampicilina/farmacologia , Animais , Clindamicina/farmacologia , Eritromicina/farmacologia , Pesqueiros , Peixes/microbiologia , Microbiologia de Alimentos , Inocuidade dos Alimentos , Heterogeneidade Genética , Gelo/análise , Índia , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Penicilinas/farmacologia , Prevalência , Sorogrupo , Tetraciclina/farmacologia
10.
Food Chem ; 298: 124980, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260963

RESUMO

Effect of phlorotannins (PT) treatments on the ployhenoloxidase (PPO) activity and quality changes of Pacific white shrimp during a 16-day period of storage in ice were studied. Among seaweeds, Sargassum tenerimum had the highest amount of PT (10.00 mg phloroglucinol/g), PPO inhibitory activity (71.94%) and therefore selected for PT extraction. The shrimp treated with 5% PT (w/v) showed the least melanosis score, pH, TVB-N values and lipid oxidation among all treatments throughout the iced storage. Lower counts of mesophilic and psychrotrophic bacteria (1-2 log CFU/g) were obtained with 5% PT treatment compared to the control at the last day of storage (P < 0.05). Sensory evaluation proved that 5% PT treatment could cause a 4-days increase in the shelf-life of shrimp compared to the control, PT1% and PT2% treatments. Therefore, 5% phlorotannins from S. tenerimum could be used as a safe melanosis inhibitor for the treatment of shrimp.


Assuntos
Armazenamento de Alimentos/métodos , Penaeidae , Floroglucinol/farmacologia , Frutos do Mar , Animais , Bactérias/efeitos dos fármacos , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/química , Catecol Oxidase/metabolismo , Microbiologia de Alimentos , Qualidade dos Alimentos , Gelo , Melanose , Oxirredução , Penaeidae/efeitos dos fármacos , Penaeidae/microbiologia , Sargassum/química , Frutos do Mar/microbiologia
11.
ISME J ; 13(10): 2578-2588, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31235840

RESUMO

Humans have profoundly affected the ocean environment but little is known about anthropogenic effects on the distribution of microbes. Vibrio parahaemolyticus is found in warm coastal waters and causes gastroenteritis in humans and economically significant disease in shrimps. Based on data from 1103 genomes of environmental and clinical isolates, we show that V. parahaemolyticus is divided into four diverse populations, VppUS1, VppUS2, VppX and VppAsia. The first two are largely restricted to the US and Northern Europe, while the others are found worldwide, with VppAsia making up the great majority of isolates in the seas around Asia. Patterns of diversity within and between the populations are consistent with them having arisen by progressive divergence via genetic drift during geographical isolation. However, we find that there is substantial overlap in their current distribution. These observations can be reconciled without requiring genetic barriers to exchange between populations if long-range dispersal has increased dramatically in the recent past. We found that VppAsia isolates from the US have an average of 1.01% more shared ancestry with VppUS1 and VppUS2 isolates than VppAsia isolates from Asia itself. Based on time calibrated trees of divergence within epidemic lineages, we estimate that recombination affects about 0.017% of the genome per year, implying that the genetic mixture has taken place within the last few decades. These results suggest that human activity, such as shipping, aquatic products trade and increased human migration between continents, are responsible for the change of distribution pattern of this species.


Assuntos
Vibrioses/microbiologia , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/isolamento & purificação , Variação Genética , Genoma Bacteriano , Humanos , Filogenia , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/genética
12.
Environ Sci Pollut Res Int ; 26(20): 21034-21043, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115814

RESUMO

Vibrio parahaemolyticus is the most common pathogen causing seafood-borne illnesses in Korea. The present study evaluated the occurrence, virulence, and antimicrobial resistance of V. parahaemolyticus in seawater and bivalves obtained in 2016 from the southern coast of Korea, an important region for commercial aquaculture industries, especially the Korean raw seafood culture. V. parahaemolyticus was detected in 87 of 160 (54.4%) bivalve samples and in 32 of 130 (24.5%) seawater samples. Especially high levels were detected during summer to early autumn. All the seawater and bivalves contained less than 2 and 5% of the tdh and trh genes of the isolates, respectively, and seawater isolates possessed two fewer genes than the bivalve isolates. Of 23 antimicrobials tested, three agents (ofloxacin, norfloxacin, and trimethoprim/sulfamethoxazole) effectively treated V. parahaemolyticus illness due to the sensitivity of the isolates. The isolates were highly resistant to ampicillin, however, excluding it as a treatment option. More than half of the isolates exhibited resistance to at least three antimicrobials. These findings indicate the importance of an integrated monitoring and surveillance program noting the occurrence, virulence, and antimicrobial resistance patterns of V. parahaemolyticus in various aquatic sources for preventing human health risks from seafood consumption.


Assuntos
Aquicultura/estatística & dados numéricos , Bivalves/microbiologia , Farmacorresistência Bacteriana , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/patogenicidade , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Humanos , República da Coreia , Estações do Ano , Água do Mar/microbiologia , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificação , Virulência/genética
13.
Food Microbiol ; 82: 209-217, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027776

RESUMO

Microorganisms harbored in oyster gills are potentially related to the spoilage and safety of oyster during storage. In this study, the microbial activities and pH changes of the gills of the two species, Crassostrea gigas and C. virginica, harvested from three different sites were determined and sensory evaluation was conducted during refrigerated storage. The bacteria in gills with an initial aerobic plate count (APC) of 3.1-4.5 log CFU/g rose remarkably to 7.8-8.8 log CFU/g after 8-days of storage. The APC of Enterobacteriaceae increased from 2.5 to 3.6 log CFU/g to 4.5-4.8 log CFU/g, and that of lactic acid bacteria (LAB) fluctuated in the range of 1.4-3.0 log CFU/g during the whole storage period. The results of sensory analysis indicated that the oysters had 8-days of shelf-life and that the gill presented the fastest deterioration rate. The pH of all samples showed a decrease in the early stages followed by an increased after 4-days of storage. The dynamic changes in microbial profiles were depicted to characterize gill spoilage by Illumina Miseq sequencing to characterize gill spoilage. The results revealed that oysters harvested at different sites showed common bacterial profiles containing Arcobacter, Spirochaeta, Pseudoalteromonas, Marinomonas, Fusobacterium, Psychrobacter, Psychromonas, and Oceanisphaera when spoiled, especially, among which Psychrobacter and Psychromonas (psychrotrophic genus) were represented as the most important gill spoiled bacteria during refrigerated storage, and Arcobacter with pathogenic potential was the dominated bacteria in all spoiled oysters. The consumption quality and safety of refrigerated oysters could be greatly improved by targeted control of bacteria in oyster gills according to the results the present study provided.


Assuntos
Bactérias/isolamento & purificação , Crassostrea/microbiologia , Microbiologia de Alimentos , Frutos do Mar/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Contagem de Colônia Microbiana , Crassostrea/química , Qualidade dos Alimentos , Armazenamento de Alimentos , Brânquias/química , Brânquias/microbiologia , Concentração de Íons de Hidrogênio , Microbiota/genética , Refrigeração
14.
Braz J Microbiol ; 50(3): 807-816, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31006836

RESUMO

Effective monitoring of Salmonella contamination in seafood processing to conform the requirements of HACCP is a great challenge today. Such challenges can be effectively addressed, if the conventional detection methods are replaced with DNA-based molecular methods. Accordingly, it was aimed to develop a robust PCR protocol for specific detection of Salmonella spp. Out of the different primers screened, one pair of primers developed in this study targeting invA gene demonstrated 100% inclusivity for a wide range of Salmonella serotypes and 100% exclusivity for wide range of non-target species. The in silico analysis of the nucleotide sequence obtained from the PCR product suggests its potential as a hybridization probe for genus specific detection of Salmonella spp. contamination. The PCR protocol was sensitive enough to detect 15 cells per reaction using crude DNA prepared within a short time directly from artificially contaminated shrimp tissue. The study demonstrated that the result of PCR reaction can come out on the same day of sample arrival. Incorporation of this pair of primers in a multiplex PCR designed for simultaneous detection of four common seafood-borne human pathogens yielded 147 bp, 302 bp, 403 bp, and 450 bp distinct DNA bands specifically targeting E. coli, toxigenic Vibrio cholerae, Salmonella spp., and V. parahaemolyticus, respectively in a single PCR tube. The PCR methods developed in this study has the potential to be used in the seafood processing plants for effective monitoring of CCPs required for implementation of HACCP-based quality assurance system.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Salmonella/isolamento & purificação , Frutos do Mar/microbiologia , Animais , Primers do DNA/genética , Contaminação de Alimentos/análise , Reação em Cadeia da Polimerase Multiplex/instrumentação , Palaemonidae/microbiologia , Salmonella/classificação , Salmonella/genética
15.
Int J Food Microbiol ; 298: 31-38, 2019 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-30903916

RESUMO

Marine bivalve shellfish are of public health interest because they can accumulate pollutants in their tissues. As they are usually consumed raw or lightly cooked, they are considered to be a possible source of foodborne infections, including giardiosis and cryptosporidiosis. Although data indicating contamination of shellfish with Giardia cysts and Cryptosporidium oocysts have been published, comparing results from different studies is difficult, as there is no standardized protocol for the detection and quantification of these parasites in mussels, and different researchers have used different analytical approaches. The aim of this study was to identify and characterize the most sensitive protocol for the detection of Giardia cysts and Cryptosporidium oocysts in shellfish. In an effort to test the sensitivity and the detection limits of the protocol, every step of the process was investigated, from initial preparation of the mussel matrix through detection of the parasites. Comparative studies were conducted, including several methods previously applied by other researchers, on commercial mussels Mytilus galloprovincialis spiked with a known number of (oo)cysts of both parasites. As preparation of the mussel matrix plays an important role in the sensitivity of the method, different techniques were tested. These included: (ia) removal of the coarse particles from the matrix with sieving, (ib) extraction of the lipids with diethyl ether, and (ic) artificial digestion of the matrix with pepsin digestion solution, and (ii) the use or not of immunomagnetic separation (IMS) for the concentration of the (oo)cysts. Pre-treatment of the mussel homogenate with pepsin digestion solution, followed by IMS, then detection with a direct immunofluorescence assay, achieved the highest sensitivity: 32.1% (SD: 21.1) of Giardia cysts and 61.4% (SD: 26.2) Cryptosporidium oocysts were recovered, with a detection limit of 10 (oo)cysts per g of mussel homogenate. The outcome of the current study was the standardization of a protocol, with defined detection limits, for the detection of these two protozoan transmission stages in mussels, in order to be used as a reference technique in future studies. Further advantages of this protocol are that it uses the whole mussel as a starting material and does not require difficult handling procedures. The method has potential to be applied in larger surveys and, potentially, to other species of shellfish for the detection of these parasites. However, the composition (lipid to protein ratio) may be of relevance for detection efficiency for some other species of shellfish.


Assuntos
Cryptosporidium/isolamento & purificação , Técnica Direta de Fluorescência para Anticorpo/normas , Microbiologia de Alimentos/métodos , Giardia/isolamento & purificação , Mytilus/microbiologia , Oocistos/isolamento & purificação , Frutos do Mar/microbiologia , Animais , Criptosporidiose/prevenção & controle , Giardíase/prevenção & controle , Separação Imunomagnética , Mar Mediterrâneo
16.
Food Environ Virol ; 11(2): 101-112, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30706411

RESUMO

Eastern oysters (Crassostrea virginica) from three locations along the Delaware Bay were surveyed monthly from May to October 2017 for levels of total Vibrio parahaemolyticus, pathogenic strains of V. parahaemolyticus and Vibrio vulnificus, and for strain-specific bacteriophages against vibrios (vibriophages). The objectives were to determine (a) whether vibriophages against known strains or serotypes of clinical and environmental vibrios were detectable in oysters from the Delaware Bay and (b) whether vibriophage presence or absence corresponded with Vibrio abundances in oysters. Host cells for phage assays included pathogenic V. parahaemolyticus serotypes O3:K6, O1:KUT (untypable) and O1:K1, as well as clinical and environmental strains of V. vulnificus. Vibriophages against some, but not all, pathogenic V. parahaemolyticus serotypes were readily detected in Delaware Bay oysters. In July, abundances of total and pathogenic V. parahaemolyticus at one site spiked to levels exceeding regulatory guidelines. Phages against three V. parahaemolyticus host serotypes were detected in these same oysters, but also in oysters with low V. parahaemolyticus levels. Serotype-specific vibriophage presence or absence did not correspond with abundances of total or pathogenic V. parahaemolyticus. Vibriophages were not detected against three V. vulnificus host strains, even though V. vulnificus were readily detectable in oyster tissues. Selected phage isolates against V. parahaemolyticus showed high host specificity. Transmission electron micrographs revealed that most isolates were ~ 60-nm diameter, non-tailed phages. In conclusion, vibriophages were detected against pandemic V. parahaemolyticus O3:K6 and O1:KUT, suggesting that phage monitoring in specific host cells may be a useful technique to assess public health risks from oyster consumption.


Assuntos
Bacteriófagos/fisiologia , Ostreidae/microbiologia , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/virologia , Animais , Delaware , Contaminação de Alimentos/análise , Vibrio parahaemolyticus/fisiologia , Vibrio vulnificus/fisiologia , Vibrio vulnificus/virologia
17.
J Food Prot ; 82(1): 30-38, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30702931

RESUMO

This article describes the identification and investigation of two extended outbreaks of listeriosis in which crabmeat was identified as the vehicle of infection. Comparing contemporary and retrospective typing data of Listeria monocytogenes isolates from clinical cases and from food and food processing environments allowed the detection of cases going back several years. This information, combined with the analysis of routinely collected enhanced surveillance data, helped to direct the investigation and identify the vehicle of infection. Retrospective whole genome sequencing (WGS) analysis of isolates provided robust microbiological evidence of links between cases, foods, and the environments in which they were produced and demonstrated that for some cases and foods, identified by fluorescent amplified fragment length polymorphism, the molecular typing method in routine use at the time, were not part of the outbreak. WGS analysis also showed that the strains causing illness had persisted in two food production environments for many years and in one producer had evolved into two strains over a period of around 8 years. This article demonstrates the value of reviewing L. monocytogenes typing data from clinical cases together with that from foods as a means of identifying potential vehicles and sources of infection in outbreaks of listeriosis. It illustrates the importance of reviewing retrospective L. monocytogenes typing alongside enhanced surveillance data to characterize extended outbreaks and inform control measures. Also, this article highlights the advantages of WGS analysis for strain discrimination and clarification of evolutionary relationships that refine outbreak investigations and improve our understanding of L. monocytogenes in the food chain.


Assuntos
Braquiúros/microbiologia , Listeria monocytogenes , Listeriose , Frutos do Mar/microbiologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Surtos de Doenças , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/epidemiologia , Genoma Bacteriano , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Listeriose/epidemiologia , Listeriose/microbiologia , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Estudos Retrospectivos , Sequenciamento Completo do Genoma
18.
Food Microbiol ; 79: 35-40, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30621873

RESUMO

This study investigated the influences of seawater to oyster ratio on depuration for decontaminating V. parahaemolyticus in raw oysters with a goal of identifying the proper ratio of oyster to seawater capable of improving the efficacy of the depuration process. The water to oyster ratios tested in this study ranged from 1.0 to 2.5 L of artificial seawater (ASW) per oyster (40 oysters in 40, 60, 80 and 100 L ASW). The depuration efficacy for purging V. parahaemolyticus from oysters was highest when we applied a 2:1, followed by 1.5:1, 2.5:1, and 1:1 L of ASW/oyster. Further studies of depuration with 2:1 L of ASW/oyster found that the concentration of V. parahaemolyticus in oysters decreased in a nonlinear manner. The depuration curve was fitted to a one phase decay model with a coefficient of determination (R2) of 0.933. The time for a 3 log reduction was 1.75 days with a 95% confidence interval from 1.65 to 1.85 days, which meets the FDA's requirement of larger than a 3.0 log (MPN/g) reduction as a post-harvest process for V. parahaemolyticus control. After 4 days levels in all trials were <100 MPN/g meeting performance standards established by Japan and Canada. Furthermore, the time for a 3.52 log reduction was 3.17 days with a 95% confidence interval from 2.92 to 3.54 days but it took 5 days to reduce levels to <30 MPN/g, which satisfies FDA's requirement as a post-harvest control process (>3.52 log MPN/g reduction) for the purpose of making safety added labeling claims for V. parahaemolyticus.


Assuntos
Crassostrea/microbiologia , Manipulação de Alimentos/métodos , Inocuidade dos Alimentos/métodos , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , Animais , Contagem de Colônia Microbiana , Contaminação de Alimentos/análise , Contaminação de Alimentos/prevenção & controle , Conservação de Alimentos , Humanos , Água do Mar , Frutos do Mar/normas , Temperatura
19.
Foodborne Pathog Dis ; 16(2): 137-143, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30457890

RESUMO

Shellfish is a highly valuated natural food product that is usually consumed minimally processed. Some foodborne pathogens have been associated to marine products and isolated from aquatic environments. Helicobacter pylori emerges as one of the most concerning human pathogens associated to water and, thereby, it could be present in raw and slightly treated marine food products. The present research work aimed to detect the presence of H. pylori in Spanish commercial samples of shellfish (mussels, clams, and cockles) by means of a quantitative real-time polymerase chain reaction (qPCR) approach based on the vacuolating cytotoxin A (vacA) gene specificity. Putative H. pylori amplicons were confirmed by sequencing. qPCR was positive for 12 out of the 100 samples, being 67% (8/12) from mussels, 25% (3/12) from clams, and only 8% (1/12) from cockles. After sequencing, three of the amplicons showed 97-99% homology with the H. pylori vacA gene. Quantitative results indicate that the levels of contamination remained below 102 log10 colony forming units per mL (CFU/mL). The present research shows for the first time the effectiveness of the optimized qPCR in the identification of potentially H. pylori contaminated shellfish products. Our results confirm the presence of H. pylori in shellfish from the Spanish western seacoast and verify the possible relationship between the presence of H. pylori in seawater and the role of contaminated seafoods as vehicles of H. pylori entrance into the food chain.


Assuntos
Proteínas de Bactérias/genética , Bivalves/microbiologia , Helicobacter pylori/isolamento & purificação , Frutos do Mar/microbiologia , Animais , DNA Bacteriano/análise , Corantes Fluorescentes , Helicobacter pylori/genética , Toxinas Marinhas/genética , Compostos Orgânicos , Reação em Cadeia da Polimerase em Tempo Real , Espanha
20.
Mar Pollut Bull ; 137: 360-369, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30503445

RESUMO

There is a substantial need for tools that effectively predict spatial and temporal fecal pollution patterns in estuarine waters. In this study, statistical models of exceedances of shellfish fecal coliform (FC) water quality criteria were developed using a 10-year dataset of FC levels and environmental data. Performance (sensitivity, specificity, and predictive capacity) of five different types of models was tested (MLR regression, Tobit (censored) regression, Firth's binary logistic regression (BLR), classification trees, and mixed-effects regression) for each of three conditionally managed shellfish-harvesting areas in Tillamook Bay, Oregon (USA). The most influential variables were related to precipitation and river stage height in the wet season and wind and tidal-stage in the dry season. Classification tree and Firth's BLR approaches better explained exceedances of shellfish water quality standards than the current closure thresholds. Findings demonstrate the utility of statistical modeling approaches for improved management of shellfish harvesting waters.


Assuntos
Enterobacteriaceae/isolamento & purificação , Fezes/microbiologia , Modelos Estatísticos , Frutos do Mar/microbiologia , Microbiologia da Água , Tomada de Decisões , Estuários , Modelos Logísticos , Qualidade da Água/normas
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