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1.
Front Microbiol ; 8: 1174, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28702010

RESUMO

The demand for rapid methods for the quantification of pathogens is increasing. Among these methods, those based on nucleic acids amplification (quantitative PCRs) are the most widespread worldwide. Together with the qPCR, a new approach named digital PCR (dPCR), has rapidly gained importance. The aim of our study was to compare the results obtained using two different dPCR systems and one qPCR in the quantification of three different bacterial pathogens: Listeria monocytogenes, Francisella tularensis, and Mycobacterium avium subsp. paratuberculosis. For this purpose, three pre-existing qPCRs were used, while the same primers and probes, as well as PCR conditions, were transferred to two different dPCR systems: the QX200 (Bio-Rad) and the Quant Studio 3D (Applied Biosystems). The limits of detection and limits of quantification for all pathogens, and all PCR approaches applied, were determined using genomic pure DNAs. The quantification of unknown decimal suspensions of the three bacteria obtained by the three different PCR approaches was compared through the Linear Regression and Bland and Altman analyses. Our results suggest that, both dPCRs are able to quantify the same amount of bacteria, while the comparison among dPCRs and qPCRs, showed both over and under-estimation of the bacteria present in the unknown suspensions. Our results showed qPCR over-estimated the amount of M. avium subsp. paratuberculosis and F. tularensis cells. On the contrary, qPCR, compared to QX200 dPCR, under-estimated the amount of L. monocytogenes cells. However, the maximum difference among PCRs approaches was <0.5 Log10, while cultural methods underestimated the number of bacteria by one to two Log10 for Francisella tularensis and Mycobacterium avium subsp. paratuberculosis. On the other hand, cultural and PCRs methods quantified the same amount of bacteria for L. monocytogenes, suggesting for this last pathogen, PCRs approaches can be considered as a valid alternative to the cultural ones.

2.
Ital J Food Saf ; 5(3): 5848, 2016 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-27853714

RESUMO

In temperate climates, a seasonal trend was observed in the incidence of human campylobacteriosis cases, with peaks reported in spring and autumn in some countries, or in summer in others; a similar trend was observed in Campylobacter spp. dairy cattle faecal shedding, suggesting that cattle may play a role in the seasonal peak of human infection. The objectives of this study were to assess if a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk exists and to evaluate a possible relation between this and the increase of human campylobacteriosis incidence in summer months. The results showed a mean prevalence of 1.6% of milk samples positive for thermophilic Campylobacter spp. with a wide range (0.0-3.1%) in different months during the three years considered. The statistical analysis showed a significant difference (P<0.01) of the prevalence of positive samples for thermophilic Campylobacter spp. between warmer and cooler months (2.3 vs 0.6%). The evidence of a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk sold for direct consumption, with an increase of the prevalence in warmer months, may represent one of the possible links between seasonal trend in cattle faecal shedding and seasonal trend in human campylobacteriosis.

3.
Ital J Food Saf ; 4(3): 4585, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-27800408

RESUMO

The behaviour of Listeria monocytogenes and Escherichia coli O157:H7 was studied during the manufacture and ripening of two traditional Italian Alps cheeses. Each cheese type was manufactured in a pilot plan from raw cow milk (without the addition of starter cultures) artificially inoculated with L. monocytogenes and E. coli O157:H7 to a final concentration of about 4 log CFU/mL. The pathogens were enumerated throughout the cheese making and ripening processes to study their behaviour. When the milk was inoculated with 4 Log CFU/mL, the pathogens counts increased in the first time during the manufacturing process and then remained constant, until the end of ripening, or decreased significantly. Results indicate that the environment and nature of food borne pathogens affected the concentration of the bacteria during the manufacturing and ripening process. Thus, the presence of low cells numbers of L. monocytogenes and E. coli O157:H7 in milk destined for the production of raw milk cheeses characterized by a cooking of the curd less than 48°C can constitute a hazard for the consumer.

4.
Ital J Food Saf ; 4(2): 4587, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-27800394

RESUMO

Hepatitis E virus (HEV) is an important public health concern in many developing countries and it occurs in sporadic forms in industrialized areas. With the discovery of swine HEV in pigs, which is genetically closely related to human HEV, hepatitis E is considered to be a zoonotic disease. To investigate the circulation of HEV within a distinct area of Lombardy region (Northern Italy), 17 pig farms were subjected to monitoring study by collection of fresh stool samples each represented by ground-pooled specimens. In particular, three distinct types of breeding farms were focused, represented by farrow to weaning, farrow to finish and fattening farms, respectively. Epidemiological data confirm that in Europe the seroprevalence in pigs, more than 9 month of age, ranges from 51.4 to 75%, while in 3-9 months fatteners is about 38%. In France and Italy, the positivity among farms is respectively 30 and 97.4% and the seroprevalence in Italy is 50.2%. Since HEV viremia was typically observed in the early period of life in swine, faeces were collected in boxes containing weaning pigs. For the study, 183 stool samples were collected and amplifications were performed with universal primers specific for the ORF2 region of genome. Twentyeight samples resulted positive to HEV RNA and genotyping demonstrated that they were closely related to HEV strains belonging to genotype 3 and circulating in Europe. Comparison with reference strains from GenBank excluded their similarity to genotype 1, 2 or 4 confirming that genotype 3 strains are circulating in Europe. Since it was demonstrated that swine act as a reservoir for HEV, and since many strains into HEV genotype 3 share a strong molecular similarity to human HEV, it was important to detect the presence of HEV in a restricted area with a very high density of pigs.

5.
J Food Prot ; 78(1): 13-21, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25581173

RESUMO

Two quantitative risk assessment (RA) models were developed to describe the risk of salmonellosis and listeriosis linked to consumption of raw milk sold in vending machines in Italy. Exposure assessment considered the official microbiological records monitoring raw milk samples from vending machines performed by the regional veterinary authorities from 2008 to 2011, microbial growth during storage, destruction experiments, consumption frequency of raw milk, serving size, and consumption preference. Two separate RA models were developed: one for the consumption of boiled milk and the other for the consumption of raw milk. The RA models predicted no human listeriosis cases per year either in the best or worst storage conditions and with or without boiling raw milk, whereas the annual estimated cases of salmonellosis depend on the dose-response relationships used in the model, the milk storage conditions, and consumer behavior in relation to boiling raw milk or not. For example, the estimated salmonellosis cases ranged from no expected cases, assuming that the entire population boiled milk before consumption, to a maximum of 980,128 cases, assuming that the entire population drank raw milk without boiling, in the worst milk storage conditions, and with the lowest dose-response model. The findings of this study clearly show how consumer behavior could affect the probability and number of salmonellosis cases and in general, the risk of illness. Hence, the proposed RA models emphasize yet again that boiling milk before drinking is a simple yet effective tool to protect consumers against the risk of illness inherent in the consumption of raw milk. The models may also offer risk managers a useful tool to identify or implement appropriate measures to control the risk of acquiring foodborne pathogens. Quantification of the risks associated with raw milk consumption is necessary from a public health perspective.


Assuntos
Microbiologia de Alimentos/estatística & dados numéricos , Listeria monocytogenes/isolamento & purificação , Leite/microbiologia , Alimentos Crus/microbiologia , Salmonella/isolamento & purificação , Algoritmos , Animais , Distribuidores Automáticos de Alimentos/normas , Manipulação de Alimentos , Temperatura Alta , Humanos , Itália/epidemiologia , Listeriose/epidemiologia , Modelos Estatísticos , Distribuição Normal , Medição de Risco , Intoxicação Alimentar por Salmonella/epidemiologia
6.
Food Environ Virol ; 7(1): 76-85, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25344058

RESUMO

Consumption of raw or insufficiently cooked mussels contaminated with hepatitis A virus (HAV) is a major cause of infection to humans. The origin of mussels commonly used for the preparation of marinated seafood salads is often unknown, since different producers worldwide undergo a precooking treatment at the original collection site with methods and parameters not always indicated. These treatments could be insufficient for the inactivation of HAV, which is characterized by a high temperature resistance. Both high hydrostatic pressure (HHP) and marinade treatments have been shown to affect HAV vitality. In this study, two treatments (HHP and marinating) were combined in order to assess a potential synergistic effect on the virus vitality. A kinetic test was conducted by subjecting the experimentally-contaminated mussels (HAV titre: 10(6)/ml TCID50) to marinating, and to different HHP treatment (4,000; 5,000; and 6,000 bar for 1, 5, and 9 min). Virus post-treatment vitality was assessed by its ability to grow on cell cultures and by quantitative real-time RT-PCR to evaluate virus resistance under such conditions. Marinating treatment alone (final pH 4.3, and NaCl 2 %) did not inactivate the virus. On the other hand, the use of HHP treatment alone on non-marinated HAV-contaminated mussels was effective only above 5,000 bar for 5 min. The results of the present study elucidate the synergistic effect of a combination between marination and HHP treatments on the inactivation of the virus.


Assuntos
Bivalves/virologia , Conservação de Alimentos/métodos , Vírus da Hepatite A/química , Frutos do Mar/virologia , Animais , Contaminação de Alimentos/análise , Conservação de Alimentos/instrumentação , Vírus da Hepatite A/crescimento & desenvolvimento , Temperatura Alta , Pressão Hidrostática
7.
Food Environ Virol ; 6(3): 202-6, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24859055

RESUMO

Hepatitis A virus (HAV) was detected in two samples of mixed frozen berries linked to Italian hepatitis A outbreak in April and September 2013. Both viruses were fully sequenced by next-generation sequencing and the genomes clustered with HAV complete genomes of sub-genotype IA with nucleotide identities of 95-97%.


Assuntos
Contaminação de Alimentos/análise , Alimentos Congelados/virologia , Frutas/virologia , Vírus da Hepatite A/genética , Vírus da Hepatite A/isolamento & purificação , Genoma Viral , Vírus da Hepatite A/classificação , Itália , Dados de Sequência Molecular , Filogenia
8.
Ital J Food Saf ; 3(1): 2112, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-27800321

RESUMO

In the last years, consequently to EC Regulation no. 1924/2006 on nutrition and health claims made on foods, some Italian food businnes operators (FBOs) leaders in the meat sector, invested in research to develop innovative products such as low fat salami, containing up to 30% less fat than the traditional one. For FBOs it is essential to demonstrate for each production process whether the substrate allows the growth of L. monocytogenes and whether L. monocytogenes could reach or exceed the limit of 100 cfu g-1 at the end of the shelf life, as stated by EC Regulation no. 2073/2005. In the present study, the growth potential of L. monocytogenes during the shelf life of low fat salami packed in modified atmosphere was evaluated. The results show that the product is unable to support the growth of pathogen, even if the storage temperature is between 8 and 12°C.

9.
Ital J Food Saf ; 3(1): 2231, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-27800323

RESUMO

According to EC Regulation No 2073/2005, for food business operators that produce ready-to-eat (RTE) product, it is crucial to be able to demonstrate if the product supports the growth of Listeria monocytogenes. The objective of the study was therefore to evaluate the behaviour of L. monocytogenes in sliced RTE turkey bresaola (made by cured turkey breast 4.5% NaCl, 1% sodium lactate, sodium nitrite 150 ppm and flavouring) during the shelf life of the product, simulating a contamination during the slicing operation. Considering a shelf life of 90 days, as defined by manufacturer, the packages of sliced bresaola were stored at 5°C for 7 days and at 8°C for the remaining storage time (83 days). L. monocytogenes count decreased during storage test from 1.43/1.98 log cfu/g in the three batches tested to 1.03 log cfu/g in one batch and to undetectable levels in the other two batches. The results show that the investigated product is unable to support the growth of L. monocytogenes.

10.
Ital J Food Saf ; 3(1): 2243, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-27800324

RESUMO

Formagelle di capra is a raw goat cheese produced from whole chilled goat milk; traditional technology involving unpasteurised milk and indigenous lactic starter cultures is employed for its production in Italy. The purpose of this study was to assess the behaviour of Escherichia coli O157:H7 during the manufacturing and ripening of this raw goat milk cheese. Raw milk was experimentally inoculated with E. coli O157:H7 in a laboratory scale plant and the count was monitored during production and 30 days of ripening required for this cheese. Results showed that E. coli O157:H7 count increased to more than 1.5 Log cfu g-1 during cheese production and remained constant until the end of ripening. The evidence that E. coli O157:H7 is able to survive during the manufacturing and ripening process suggests that the 30-day ripening period alone is insufficient to eliminate levels of viable E. coli O157:H7 in Formaggelle di capra cheese and that the presence of low numbers of E. coli O157:H7 in milk destined for the production of raw goat milk cheeses could represent a potential source of infection for humans and a threat for consumers.

11.
Int J Food Microbiol ; 184: 134-8, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24513055

RESUMO

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.


Assuntos
Microbiologia de Alimentos/métodos , Carne/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Salmonella/isolamento & purificação , Animais , DNA Bacteriano/análise , Europa (Continente) , Salmonella/genética , Sensibilidade e Especificidade , Suínos
12.
Int J Food Microbiol ; 184: 128-33, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24468028

RESUMO

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.


Assuntos
Queijo/microbiologia , Microbiologia de Alimentos/métodos , Listeria monocytogenes/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Contagem de Colônia Microbiana , DNA Bacteriano/genética , Europa (Continente) , Listeria monocytogenes/genética , Sensibilidade e Especificidade
13.
New Microbiol ; 34(3): 287-90, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21811749

RESUMO

The study provides data on two cases of foodborne botulism caused by consumption of commercial vegetable products: artichoke preserve and cream of vegetable soup. By mouse bioassay, Clostridium botulinum toxin in both the suspected food samples was detected and identified as type B toxin. The detection of C. botulinum toxin in the artichoke preserve indicates an inadequate food production technology while the presence of C. botulinum toxin in the vegetable soup appeared to be related to wrong behavior on the part of the consumer and to faulty food preservation. The study confirms that an early identification and reporting of suspected botulism cases is vital in the prevention of accidental widespread outbreaks.


Assuntos
Botulismo , Animais , Clostridium botulinum , Itália
14.
Ann Ist Super Sanita ; 39(1): 97-104, 2003.
Artigo em Italiano | MEDLINE | ID: mdl-12820575

RESUMO

The "Istituti Zooprofilattici" are an important network whose main function is the monitoring of animal health as well as food. As a result of the recent improvements in aquaculture technology interest in the safety of seafood is increasing. Therefore, the purpose of this study was to set up diagnostic methods for the detection of virus contamination, as well as the use of in vitro techniques able to identify the different toxins. The results have allowed the development of molecular biology assays which, together with the isolation in cell cultures, can detect contaminations/infections by the hepatitis A virus and the most common enteroviruses. Moreover, specific selected cell lines have led to the detection of different toxins. These laboratory methods will be used in order to control seafood safety.


Assuntos
Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/prevenção & controle , Doenças Transmitidas por Alimentos/virologia , Alimentos Marinhos/virologia , Academias e Institutos , Animais , Células Cultivadas , Infecções por Enterovirus/prevenção & controle , Infecções por Enterovirus/virologia , Hepatite A/prevenção & controle , Hepatite A/virologia , Humanos , Itália , Biologia Marinha
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