Virulence genes in Escherichia coli isolates from commercialized saltwater mussels Mytella guyanensis (Lamarck, 1819) / Presença de genes de virulência em isolados de Escherichia coli provenientes de sururu Mytella guyanensis (Lamarck, 1819) comercializado

Abstract The isolation of Escherichia coli from food is a major concern. Pathogenic strains of these bacteria cause diseases which range from diarrhea to hemolytic-uremic syndrome. Therefore the virulence genes in E. coli isolates from the mussel ( Mytella guyanensis) commercialized in Cachoeira, Bahia, Brazil were investigated. Samples were purchased from four vendors: two from supermarkets and two from fair outlets. They were conditioned into isothermal boxes with reusable ice and transported to the laboratory for analysis. E. coli strains were isolated in eosin methylene blue agar, preserved in brain-heart infusion medium with 15% glycerol and stored at -20 °C, after microbiological analysis. Virulence genes in the isolated strains were identified by specific primers, with Polymerase Chain Reaction. Twenty-four isolates were obtained, with a prevalence of elt gene, typical from enterotoxigenic infection, in 75% of the isolates. The stx and bfpA genes, prevalent in enterohemorragic and enteropathogenic E. coli, respectively, were not detected. The occurrence of elt virulence-related gene in the E. coli isolates of Mytella guyanensis reveals urgent improvement in food processing, including good handling practices, adequate storage and cooking before consumption, to ensure consumer's health.

Resumo O isolamento de Escherichia coli a partir de alimentos é uma grande preocupação, pois cepas patogênicas desta bactéria podem causar desde diarreia até síndrome hemolítico-urêmica. Diante do exposto, o objetivo do trabalho foi pesquisar genes de virulência em isolados de Escherichia coli provenientes do sururu Mytella guyanensis comercializado na cidade de Cachoeira, Bahia, Brasil. As amostras foram adquiridas de quatro comerciantes, sendo duas de mercados e duas em pontos de venda na feira livre da cidade de Cachoeira, acondicionadas em caixas isotérmicas com gelo reutilizável e transportadas até o laboratório para a análise. Após a análise microbiológica, as cepas de Escherichia coli foram isoladas em ágar Eosina Azul de Metileno e preservadas em caldo Brian Heart Infusion e glicerol a 15% e mantidas a - 20° C. A identificação dos genes de virulência nas cepas isoladas foi realizada utilizando primers específicos, por meio da Reação em Cadeia da Polimerase. Foram obtidos 24 isolados de Escherichia coli, destes a prevalência do gene elt , característico de Escherichia coli enterotoxigênica, foi de 75% dos isolados. Não houve a detecção dos genes stx e bfpA nos isolados, os quais são prevalentes nas cepas de Escherichia coli enterohemorrágica e Escherichia coli enteropatogênica, respectivamente. A presença do gene elt relacionado à virulência de Escherichia coli nos isolados de Mytella guyanensis revela a necessidade da melhoria no processamento, incluindo boas práticas de manipulação, armazenamento adequado e cocção previa ao consumo, visando a garantia da saúde do consumidor.

Aspectos da segurança alimentar no consumo de invertebrados marinhos do mercado informal / Aspects of food safety in the consumption of marine invertebrates from informal market

A atividade extrativa e o comércio informal de alguns organismos marinhos, tais como, do gastrópoda saquaritá (Stramonita haemastoma), pepinos-do-mar (Holothuria grisea), ouriços-do-mar (Echinometra lucunter) e mexilhões (Perna perna), é prática comum em áreas adjacentes aos costões rochosos na Baía de Santos-SP. O presente estudo objetivou diagnosticar o risco microbiológico no consumo de organismos marinhos extraídos e por vezes comercializados na região costeira da Baía de Santos-SP, utilizando métodos oficiais e limites normatizados para a discussão dos dados. Concluiu-se que o comércio informal de invertebrados marinhos na região pode colocar em risco a saúde dos consumidores e o meio ambiente, sendo imprescindível a informação quanto a origem do pescado para garantir a segurança no seu consumo e a sustentabilidade da sua produção.

In vitro resistance of Enterobacter cloacae isolated from fresh seafood to colistin

Abstract INTRODUCTION: Enterobacter cloacae is a clinically important bacterium from the Enterobacteriaceae family. This study evaluated resistance of E. cloacae strains from fish (n=14) and shrimp (n=9) to colistin. METHODS: Biochemical identification and antimicrobial susceptibility tests were carried out in an automated Vitek®2 instrument. RESULTS: Colistin resistance was observed in 21.4% and 66.7% of the strains from fish and shrimp, respectively. We observed minimum inhibitory concentrations of ≥16 mg/L and ≤5 mg/L in 8 and 15 of all strains, respectively. CONCLUSIONS: Fish and shrimp can carry drug-resistant enterobacteria, which can be of clinical interest.

Contaminación por mohos y aislamiento de cladosporium spp. y thamnidium spp. en cámaras frigoríficas de plantas procesadoras de alimentos marinos y empresas frigoríficas del sur de Chile / Mould contamination and isolation of cladosporium spp. and thamnidium spp. in frigorific chambers of sea-food processing plants and frigorific enterprises from South of Chile

La apertura de nuevos mercados para los productos hidrobiológicos chilenos ha impuesto el análisis del estatus sanitario de cámaras frigoríficas donde estos productos son almacenados. Para medir el estatus sanitario, la Unión Econó- mica Euroasiática (UEE) exige un recuento total de mohos e identificación de los géneros Cladosporium y Thamnidium en paredes y ambiente de las cámaras frigoríficas. Poco se sabe sobre los niveles de contaminación de mohos en cámaras frigoríficas de productos de exportación en Chile. Aquí, los resultados de 88 análisis de mohos en paredes y aire, realizados a 24 empresas diferentes que poseen cámaras frigoríficas conteniendo productos hidrobiológicos, son mostrados. El recuento total de mohos y el número de colonias de Cladosporium spp. y Thamnidium spp. fueron determinados. Además, los niveles de contaminación por mohos fueron comparados entre plantas procesadoras de alimentos y empresas frigoríficas. Thamnidium spp. no fue encontrado en ninguno de los análisis realizados. Los resultados indican que los niveles de contaminación por mohos son aceptables para la UEE. Este es el primer reporte de niveles de contaminación de mohos en cámaras frigoríficas de productos hidrobiológicos en el sur de Chile.

The opening of new markets for Chilean hydrobiological products had imposed the analysis of the sanitary status of frigorific chambers where those products are kept. To measure the sanitary status, the Euroasiatic Economical Union (EEU) requires a total mould count and identification of Cladosporium and Thamnidium in walls and air of the frigorific chambers. Little is known about contamination levels of moulds in frigorific chambers of export products in Chile. Here, the results of 88 mould analyses in walls and air, performed to 24 different enterprises that possess frigorific chambers containing hydrobiological products, are shown. Total mould count and Cladosporium spp. and Thamnidium spp. colony number were determined. Additionally the levels of moulds contamination were compared between food-processing plants and frigorific enterprises. Thamnidium spp. was not found in any of the analyses performed. The results indicate that levels of moulds contamination are acceptable to the EEU. This is the first report of mould contamination levels in frigorific chambers of hydrobiological products from south of Chile.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

Evaluación del riesgo microbiológico a Vibrio spp. en alimentos de origen marino en México / Microbial risk assessment of Vibrio spp. in seafood products in Mexico

Las enfermedades transmitidas por los alimentos son uno de los mayores problemas de salud pública que actualmente existen. La evaluación del riesgo microbiológico es un proceso utilizado para examinar los peligros ocultos en los alimentos, la probabilidad de exposición a éstos y su impacto en la salud pública. La evaluación del riesgo se realiza en cuatro fases: identificación del peligro, caracterización del peligro, evaluación de la exposición y caracterización del riesgo. De acuerdo con el proceso/resultado, las evaluaciones de riesgo microbiológico se clasifican en dos categorías: cualitativa y cuantitativa. La presente revisión pretende enmarcar la importancia de implementar estas evaluaciones en alimentos de origen marino que son consumidos crudos, fortaleciendo así el acceso a los alimentos inocuos y de buena calidad para beneficio del consumidor, y la necesidad de evaluaciones de riesgo microbiológico que hay en México.

Food-borne diseases are among the major public health problems that currently exist. Microbiological risk assessment is a process used to evaluate the hidden hazards in food, the likelihood of exposure to these hazards and their impact on public health. Risk assessment is performed in four steps: hazard identification, hazard characterization, assessment of exposure and risk characterization. According to the process/response microbial risk assessment is classified in two categories, qualitative and quantitative. The aim of this review is to underline the importance of implementing assessments in seafood that is usually consumed raw, strengthening access to good quality and safe food for the consumer's benefit and to stress the necessity of microbiological risks assessments in Mexico.

Prevalence of Vibrio cholerae and Other Vibrios from Environmental and Seafood Sources, Tamil Nadu, India.

Aims: The aim of this study was to investigate the prevalence of diarrhoea causing human pathogen V. cholerae and other vibrios from different environmental and seafood samples in Tamil Nadu, India. Place and Duration of Study: Laboratory of Clinical Microbiology, Department of Bio- Medical Science, School of Basic Medical Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India between 2012 and 2013. Methodology: Seafood, water and plankton samples were collected at different locations of Tamil Nadu, India. All the samples were primarily enriched with alkaline peptone water (APW). 2-3 loopful of overnight cultures were streaked onto Thiosulphate Citrate Bile salt Sucrose (TCBS) agar plates. Suspected Vibrio cholerae, V. parahaemolyticus and other vibrios were picked up and identified by using standard biochemical and serological characterization and also by molecular methods. Results: Among the various samples that includes freshwater, coastal water, plankton and various seafoods, only plankton and seafood samples were found to be harbored with V. cholerae, V. parahaemolyticus and V. fluvialis. The remaining samples were negative for vibrios. All V. cholerae, V. parahaemolyticus and V. fluvialis strains possessed outer membrane protein W (ompW), thermostable direct haemolysin (tdh) and toxin regulatory protein (toxR) gene respectively. Hemolytic activity of V. cholerae exihibited different reaction isolated from seafood and plankton. The median lethal dose (LD50) of some V. cholerae strains was generally high. Conclusion: The result of the study suggested that the seafoods may act as an important reservoir of pathogenic vibrios and pose threat to human health.

Vibrio vulnificus infection in Southern Brazil - Case report / Infecção por Vibrio vulnificus no sul do Brasil - Relato de caso

The genus Vibrio is a member of the family Vibrionaceae, and among their disease-causing species, Vibrio vulnificus, a lactose-positive gram-negative bacillus, is one of the most virulent pathogen of the noncholerae vibrios. We describe the case of a 39-year-old male patient, who was using immunosuppressive therapy, admitted to the hospital for liver transplantation. Twelve hours later, the patient presented high fever, myalgia, anuria and erythematous plaques on lower limbs, of rapid growth and proximal progression. The patient was treated with ceftriaxone, meropenem and oxacillin, however he expired within 30 hours. Blood cultures showed growth of a gram-negative bacillus, which was later identified as Vibrio vulnificus.

O gênero Vibrio é membro da família Vibrionaceae, e entre as espécies patogênicas, Vibrio vulnificus, bacilo gram negativo lactose positivo, tem sido frequentemente citado. Descrevemos o caso de um paciente masculino de 39 anos, em uso de medicação imunossupressora, admitido no hospital para transplante hepático. Doze horas após a internação, o paciente evoluiu com febre, mialgias, anúria e placas eritematosas em membros inferiores, com rápido crescimento e evolução proximal. O paciente foi tratado com ceftriaxona, meropenem e oxacilina sem melhora, evoluindo para óbito em 30 horas. Hemocultura mostrou crescimento de bacilo gram negativo posteriormente identificado como Vibrio vulnificus.

Serotyping & molecular characterization for study of genetic diversity among seafood associated nontyphoidal Salmonella serovars.

Background & objectives: Infections due to seafood associated Salmonella serovars are great risk to public health. Different phenotypic characteristics have been used previously for epidemiological investigation of Salmonella. Beyond the phenotypic characterization, a reliable genetic level discriminatory method is required. Therefore, this study was attempted to use different phenotypic and molecular fingerprinting methods for investigation of genetic diversity among seafood associated nontyphoidal Salmonella serovars. Methods: Fifty eight seafood associated Salmonella isolates were included in this study. All isolates were serotyped and epidemiological investigation was carried out using molecular fingerprinting methods, random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR) along with whole cell protein profiling using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in our study. Results: Among the 58 Salmonella isolates, S. Weltevreden was observed to be the most predominant serovar. Typing of Salmonella serovars using RAPD and ERIC-PCR suggested the existence of a genetic diversity. Though both PCR based techniques were found to have a good discriminatory index, a better discriminatory ability was observed when the results obtained by the two techniques were combined and taken for composite analysis. Protein profiling of whole cells using SDS-PAGE demonstrated the presence of several bands with two bands of sizes 38 kDa and 46 kDa common among all 58 isolates. Interpretation & conclusions: Our study shows that use of protein profiling in combination with established typing methods such as RAPD and ERIC-PCR may provide useful information in typing of non-typhoidal Salmonella isolates associated with seafood and to develop strategies to protect public from Salmonella infections.

Rapid detection and E-test antimicrobial susceptibility testing of Vibrio parahaemolyticus isolated from seafood and environmental sources in Malaysia

To find out the prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus in seafoods and environmental sources. The study was carried out at the Center of Excellence for Food Safety Research, University Putra Malaysia; University Kebangsaan Malaysia; Medical Molecular Biology Institute; and University Kebansaan Malaysia Hospital, Malaysia between January 2006 and August 2008. One hundred and forty-four isolates from 400 samples of seafood [122 isolates] and seawater sources [22 isolates] were investigated for the presence of thermostable direct hemolysin [tdh[+]] and TDH-related hemolysin [trh[+]] genes using the standard methods. The E-test method was used to test the antimicrobial susceptibility. The study indicates low occurrence of tdh[+] [0.69%] and trh[+] isolates [8.3%]. None of the isolates tested possess both virulence genes. High sensitivity was observed against tetracycline [98%]. The mean minimum inhibitory concentration [MIC] of the isolates toward ampicillin increased from 4 ug/ml in 2004 to 24 ug/ml in 2007. The current study demonstrates a low occurrence of pathogenic Vibrio parahaemolyticus in the marine environment and seafood. Nonetheless, the potential risk of vibrio infection due to consumption of Vibria parahaemolyticus contaminated seafood in Malaysia should not be neglected

PCR-based detection of enterotoxigenic isolates of Bacillus cereus from tropical seafood.

Background & objectives: Bacillus cereus is an important enterotoxigenic food borne pathogen. The present study was undertaken to assess the occurrence of B. cereus in tropical fish and evaluation of virulent gene specific PCR for differentiation of diarrhoeal enterotoxin producing isolates of B. cereus from non enterotoxigenic isolates. Methods: Selective plating on polymixin-pyruvate-egg yolk-mannitol-bromocresol purple agar (PEMPA) was used for isolation of B. cereus from finfish, prawn and clams. Enterotoxin producing ability of all 42 isolates obtained from the samples was judged by reverse passive latex agglutination (RPLA) test and the presence of different virulent genes i.e. hbla, bceT and entFM was screened by PCR. Results: B. cereus and enterotoxigenic B. cereus were found to be in 36.7 and 29.41 per cent of fish samples, respectively. All the diarrhoeal enterotoxin producing isolates showed the presence of hbla gene, but hbla gene was not present in any of the non-enterotoxigenic isolates tested in this study. Interpretation & conclusions: Our findings indicated that hbla gene specific PCR can be employed for differentiation of enterotoxigenic B. cereus isolates from non-enterotoxigenic isolates.

Primary Shewanella algae Bacteremia Mimicking Vibrio Septicemia

Shewanella algae infections are rare in humans. Previously reported cases of S. algae have mainly been associated with direct contact with seawater. We report a case of primary S. algae bacteremia occurring after the ingestion of raw seafood in a patient with liver cirrhosis that presented a fulminent course of necrotizing fasciitis.

Evaluación microbiológica y detección de enterotoxinas estafilococcicas en ensaladas de moluscos y vegetales / Microbiological evaluation and staphylococcus aureus enterotoxin detection in shellfish and vegetable salads

Un importante renglón de la pesca en el oriente de Venezuela es el de los moluscos. Las inadecuadas condiciones higiénicas cuando se preparan alimentos con estos productos y su venta en las playas señalan la importancia de ejercer un estricto control sanitario. El objetivo del trabajo fue evaluar la calidad microbiológica y detectar enterotoxinas de Staphylococcus aureus en ensaladas de moluscos con vegetales vendidas en las playas de la Isla de Margarita, Venezuela en los años 2004-2005. Se analizaron 63 unidades de muestra pertenecientes a 21 muestras. El 47,6 por ciento de las unidades superó los 10 NMP/g de E coli. El 79,3 por ciento tuvo recuentos mayores a 104 UFC/g de microorganismos acidúricos y el 57,1 por ciento mayor a 105 UFC/g de aerobios mesófilos. Se detectaron enterotoxinas estafilococcicas en el 8 por ciento de las muestras, (4,8 por ciento del tipo A y 3,2 por ciento del tipo B). En general, el 64,3 por ciento de las muestras no cumplió alguno de los requisitos internos del laboratorio de microbiología. Se halló relación significativa (r de Pearson) entre los recuentos de aerobios mesófilos y los de microorganismos acidúricos (r= 0,52; P<0,05), al igual que entre los coliformes fecales y S. aureus (r= 0,43; P<0,05), sugiriendo el incumplimiento de varias normas higiénicas, establecidas por organismos de la salud pública. Se concluye que estas ensaladas presentan un alto riesgo de generar problemas de salud a quienes las consumen.

Mollusks represent an important fishing product in Venezuela’s easthern region. Inadequate hygienic conditions applied in its preparation and sale at the beaches indicate the importance of a strict sanitary control. The objective of this research was to evaluate the microbiological quality and detect enterotoxins of Staphylococcus aureus in mollusks and vegetable salads sold at Margarita Island beaches during 2004-2005. Sixty three sample units were analyzed from to 21 samples, finding that 47.6% of them surpassed the 10 MPN/g of E coli. 79.3% had counts higher than 105CFU/g of aciduric microorganisms, and 57.1% higher than 104CFU/g of mesophilic aerobic. Staphylococcal enterotoxins were detected in 8% of the samples (of 4.8% type A and 3.2% of type B). In general, 64.3% of the samples did not fulfill some of the microbiological requirements. A significant relation (r of Pearson) exists between the counts of mesophilic aerobic and those of aciduric microorganisms (r= 0.52; P<0.05), as well as between fecal coliforms and S. aureus (r= 0.43; P<0.05). These findings suggest the breach of several hygienic norms. As a conclusion, these salads convey a high risk of generating health problems to those who consume them.

Effect of natural preservatives on the growth of histamine producing bacteria.

Present study deals with the hampering of the growth of histamine producing bacteria (HPB), by using NaCl and spices which are easily available and cheaper cost wise. For this experiment, four strains of HPB viz. Vibrio parahaemolyticus, Bacillus cereus, Pseudomonas aeruginosa and Proteus mirabilis were tested against 1 to 10% concentrations of NaCl and 1 to 5% concentrations of natural preservatives (turmeric, ginger and garlic) in a basal medium. HPB showed different growth rates at different concentrations of NaCl and natural preservatives. V. parahaemolyticus, B. cereus and Ps. aeruginosa showed no growth at 10% concentration. When the HPB growth was tested with garlic, turmeric and ginger extracts, growth of all the bacteria was inhibited by garlic and turmeric extracts at 5% concentration. In ginger, V. parahaemolyticus, B. cereus and P. mirabilis were totally inhibited at 5% concentration. But Ps. aeruginosa showed very less growth at this concentration.

Detection of tdh and trh genes in Vibrio parahaemolyticus isolated from Corbicula moltkiana prime in West Sumatera, Indonesia.

The occurrence of Vibrio parahaemolyticus in raw Corbicula moltkiana Prime from Lake Singkarak and Pasar Raya Padang market and in cooked samples in West Sumatera, Indonesia, was studied. Thirteen raw and seven cooked bivalve samples were positive using CHROMAgar Vibrio medium. All 47 V parahaemolyticus isolates were positive for toxR gene but negative for trh. However, 36% (17/47) of V parahaemolyticus strains were positive for tdh gene. Antibiotic profiling showed that 76% and 38% of isolates from raw and cooked bivalves respectively were resistant to ampicillin. Using RAPD-PCR analysis, most of the strains were clustered according to their source of isolation but some of the strains from raw and cooked samples were clustered together. These results indicate that pathogenic V parahaemolyticus isolates are present in Corbicula moltkiana Prime in West Sumatera, Indonesia, suggesting that V parahaemolyticus may also be present in seafood in other regions of Indonesia.

Monoclonal antibody-based dot-blot ELISA for the detection of Salmonella in foods.

Monoclonal antibody (MAb) produced to polysaccharides in the LPS molecule of salmonellae was used in a dot-blot ELISA for detecting Salmonella in 873 food samples, ie 100 fresh chicken, 261 frozen chicken, 78 pork, 84 beef, 100 hen eggs, 100 duck eggs, 50 sea-mussels, 50 shrimps and 50 squids in comparison with the conventional culture method. Salmonella culture from foods involved the following steps: pre-enrichment, enrichment in selective medium, isolation on selective and indicator media, followed by biochemical and serological identification of appropriate colonies, respectively. The whole culture procedure took 5 days. Food samples from the selective enrichment medium were also subjected to the MAb-based dot-blot ELISA. The whole procedure of dot-blot ELISA took less than 2 hours. Among 873 food samples, salmonellae could be recovered from 7.4% of the samples by the bacterial isolation method (16% of fresh chicken, 8.8% of frozen chicken, 24.4% of pork, 3.6% of beef and 2% each of hen eggs and duck eggs, respectively). Salmonella derby were predominant among pork samples while S.paratyphi B biovar java predominated in chicken. The MAb-based dot-blot ELISA were positive in 19.5% of the food samples, i.e. 30% of fresh chicken, 27.6% of frozen chicken, 34.6% of pork, 21.4% of beef, 20% of shrimp, 16% of sea-mussels, 2% of hen eggs and 4% of duck eggs. The sensitivity and specificity of the MAb-based dot-blot ELISA compared to the bacterial culture method were 81.5% and 85%, respectively. The discrepancy of the data between the culture method and the dot-blot ELISA might be due to the fact that the culture method could detect only living cells at numbers that gave at least one isolated colony on the selective/differential plate while the dot-blot ELISA detects any form of Salmonella antigen. The monoclonal antibody-based dot-blot ELISA offers several advantages over the conventional bacterial culture method when it is used for screening of Salmonella contamination in foods, especially export foods. These include rapidity, cost-effectiveness and simplicity (the dot-blot ELISA does not need highly trained personnel or equipment, in contrast to the culture method). The test can be performed in field conditions and the result can be read visually. It also offers multisample analysis at one time which renders more samples of food for screening possible, thus false negative results are fewer which, in turn, assures the quality of the export food in a cost-saving, short time frame.