RESUMO
Salmonella is a common food-borne pathogen with Enteritidis and Typhimurium being among the most important serovars causing numerous outbreaks. A rapid method was investigated to identify these serovars using whole-cell MALDI-TOF MS coupled with multivariate analysis and artificial intelligence and 113 Salmonella strains, including 38 Enteritidis (SE), 38 Typhimurium (ST) and 37 strains from 32 other Salmonella serovars (SG). Datasets of ions (presence/absence) with high discriminative power were created using newly developed criteria and subject to multivariate analyses and eight artificial intelligence (AI) tools. Principal Component Analysis based on 55 or 88 selected ions separated SE, ST and SG without overlap on the first three principal components. Datasets were partitioned using five partitioning methods with 70% of samples for AI model training and 30% for validation. Of the eight AI models evaluated, high performance (HP) SVM and HP Neural were the top performers, identified three serovar groups 97% correctly on average (range 82%-100%) according to the validation results. Selection of serovar specific ions facilitated differentiation of serotypes using unsupervised model PCA and improved the accuracy of classification using AI significantly (p < 0.01). MALDI-TOF MS incorporated with advanced data processing and classification tools is a promising method to allow rapid identification of Salmonella serovars of concern in routine diagnostic laboratories.
Assuntos
Inteligência Artificial , Salmonella enteritidis , Sorogrupo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise Multivariada , Íons , LasersRESUMO
Accurate detection of all Salmonella serovars present in a sample is important in surveillance programs. Current detection protocols are limited to detection of a predominant serovar, missing identification of less abundant serovars in a sample. An alternative method, called CRISPR-SeroSeq, serotyping by sequencing of amplified CRISPR spacers, was employed to detect multiple serovars in a sample without the need of culture isolation. The CRISPR-SeroSeq method successfully detected 34 most frequently reported Salmonella serovars in pure cultures and target serovars at 104 CFU/mL in 27 Salmonella-negative environmental enrichment samples post-spiked with one of 15 different serovars, plus 2 additional serovars at 1 log CFU/mL higher abundance. When the method was applied to 442 naturally contaminated environmental samples collected from 192 poultry farms, 25 different serovars were detected from 430 of the samples. In 73.1% of the samples, 2 to 7 serovars were detected, with Salmonella Kiambu (55.7%), Salmonella Infantis (48.4%), Salmonella Kentucky (27.1%), Salmonella Livingstone (26.6%), and Salmonella Mbandaka/Montevideo (23.4%) being the most prevalent on the farms. Single isolates from 384 samples were also analyzed using a traditional serotyping method, and the same serovar identified by culture was detected by CRISPR-SeroSeq in 96.1% (369/384) of samples, with the former missing detection of additional and sometimes critical serovars. The surveillance data obtained via CRISPR-SeroSeq revealed a significant emergence of Salmonella Kiambu and Salmonella Rissen on poultry farms in Ontario. The results highlight the effectiveness of the CRISPR-SeroSeq approach in detecting multiple Salmonella serovars in poultry environmental samples under applied conditions, providing updated surveillance information on Salmonella serovars on poultry farms in Ontario. IMPORTANCE The CRISPR-SeroSeq method represents an alternative molecular tool to the traditional culture-based serotyping method that can detect multiple Salmonella serovars in a sample and provide rapid serovar results without the need of selective enrichment and culture isolation. The evaluation results can facilitate implementation of the method in routine Salmonella surveillance on poultry farms and in outbreak investigations. The application of the method can increase the accuracy of current serovar prevalence information. The results highlight the effectiveness of the validated method and the need for monitoring Salmonella serovars in poultry environments to improve current surveillance programs. The updated surveillance data provide timely information on emergence of different Salmonella serovars on poultry farms in Ontario and support on-farm risk assessment and risk management of Salmonella.
Assuntos
Aves Domésticas , Salmonelose Animal , Animais , Sorogrupo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ontário , Galinhas , Salmonella , Salmonelose Animal/epidemiologiaRESUMO
One of the human- and animal-pathogenic species in genus Yersinia is Yersinia enterocolitica, a food-borne zoonotic pathogen that causes enteric infections, mesenteric lymphadenitis, and sometimes sequelae such as reactive arthritis and erythema nodosum. Y. enterocolitica is able to proliferate at 4 C, making it dangerous if contaminated food products are stored under refrigeration. The most common source of Y. enterocolitica is raw pork meat. Microbiological detection of the bacteria from food products is hampered by its slow growth rate as other bacteria overgrow it. Bacteriophages can be exploited in several ways to increase food safety with regards to contamination by Y. enterocolitica. For example, Yersinia phages could be useful in keeping the contamination of food products under control, or, alternatively, the specificity of the phages could be exploited in developing rapid and sensitive diagnostic tools for the identification of the bacteria in food products. In this review, we will discuss the present state of the research on these topics.
Assuntos
Bacteriófagos/fisiologia , Microbiologia de Alimentos , Inocuidade dos Alimentos , Yersiniose/microbiologia , Yersinia enterocolitica/virologia , Animais , Humanos , Yersinia enterocolitica/isolamento & purificaçãoRESUMO
Raw chia and flax seeds are increasingly associated with Salmonella contamination. However, intervention technologies for these seeds that maintain them in a raw state, without causing clumping because of mucilage production upon moisture exposure, are limited. In this study, a commercial ethanol and paracetic acid sanitizing solution meeting these criteria was evaluated for efficacy against Salmonella and Enterococcus faecium NRRL B-2354, a known Salmonella surrogate for thermal intervention technologies. Samples (100 g each) of chia and flax seeds ( n = 5) were inoculated with either a cocktail of Salmonella Newport, Senftenberg, Oranienburg, Saintpaul, Typhimurium DT104, and Cubana or E. faecium NRRL B-2354. After overnight acclimatization, samples were treated with 4 mL of sanitizing solution per sample and then held at ambient temperature (20 to 25°C) for 1 h before bacterial enumeration. Separate 1-kg-treated batches were evaluated for germination ability (4 replicates of 100-g samples), as well as nutrient content and rancidity ( n = 3), compared with untreated control. Following the posttreatment holding time, these batches were dried back to original moisture content at 70°C to evaporate residual sanitizing solution, thereby stopping treatment. The sanitizing solution was found to be an effective intervention method for chia and flax seeds, reducing Salmonella to below the level of detection by more than 4 and more than 5 average log CFU/g, respectively. Germination was not significantly affected ( P ≥ 0.05) for chia seed. For both seeds, nutrition and rancidity were not significantly affected ( P ≥ 0.05). Furthermore, E. faecium NRRL B-2354 was found to be an appropriate Salmonella surrogate for treatment of chia and flax seeds with this sanitizing solution, showing comparable but higher resistance to treatment with the sanitizing solution than the Salmonella cocktail.
Assuntos
Anti-Infecciosos , Enterococcus faecium , Linho/microbiologia , Ácido Peracético/farmacologia , Salvia/microbiologia , Anti-Infecciosos/farmacologia , Contagem de Colônia Microbiana , Descontaminação , Dessecação , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/crescimento & desenvolvimento , Microbiologia de Alimentos , Salmonella , Sementes/microbiologiaRESUMO
The genomes of two strains of Salmonella enterica subsp. enterica serovar Cubana and serovar Muenchen, isolated from dry hazelnuts and chia seeds, respectively, were sequenced using the Illumina MiSeq platform, assembled de novo using the overlap-layout-consensus method, and aligned to their respective most identical sequence genome scaffolds using MUMMER and BLAST searches.
RESUMO
Bacillus cereus is a pathogenic adulterant of raw milk and can persist as spores and grow in pasteurized milk. The objective of this study was to determine the prevalence of B. cereus and its enterotoxins in pasteurized milk at its best-before date when stored at 4, 7, and 10°C. More than 5.5% of moderately temperature-abused products (stored at 7°C) were found to contain >105 CFU/mL B. cereus , and about 4% of them contained enterotoxins at a level that may result in foodborne illness; in addition, more than 31% of the products contained >105 CFU/mL B. cereus and associated enterotoxins when stored at 10°C. Results from a growth kinetic study demonstrated that enterotoxin production by B. cereus in pasteurized milk can occur in as short as 7 to 8 days of storage at 7°C. The higher B. cereus counts were associated with products containing higher butterfat content or with those produced using the conventional high-temperature, short-time pasteurization process. Traditional indicators, aerobic colony counts and psychrotrophic counts, were found to have no correlation with level of B. cereus in milk. The characterization of 17 representative B. cereus isolates from pasteurized milk revealed five toxigenic gene patterns, with all the strains carrying genes encoding for diarrheal toxins but not for an emetic toxin, and with one strain containing all four diarrheal enterotoxin genes (nheA, entFM, hblC, and cytK). The results of this study demonstrate the risks associated even with moderately temperature-abused pasteurized milk and the necessity of a controlled cold chain throughout the shelf life of fluid milk to enhance product safety and minimize foodborne illness.
Assuntos
Bacillus cereus/isolamento & purificação , Enterotoxinas/análise , Contaminação de Alimentos/análise , Leite/microbiologia , Animais , Microbiologia de Alimentos , PrevalênciaRESUMO
UNLABELLED: Bacteriophages present huge potential both as a resource for developing novel tools for bacterial diagnostics and for use in phage therapy. This potential is also valid for bacteriophages specific for Yersinia enterocolitica To increase our knowledge of Y. enterocolitica-specific phages, we characterized two novel yersiniophages. The genomes of the bacteriophages vB_YenM_TG1 (TG1) and vB_YenM_ÏR1-RT (ÏR1-RT), isolated from pig manure in Canada and from sewage in Finland, consist of linear double-stranded DNA of 162,101 and 168,809 bp, respectively. Their genomes comprise 262 putative coding sequences and 4 tRNA genes and share 91% overall nucleotide identity. Based on phylogenetic analyses of their whole-genome sequences and large terminase subunit protein sequences, a genus named Tg1virus within the family Myoviridae is proposed, with TG1 and ÏR1-RT (R1RT in the ICTV database) as member species. These bacteriophages exhibit a host range restricted to Y. enterocolitica and display lytic activity against the epidemiologically significant serotypes O:3, O:5,27, and O:9 at and below 25°C. Adsorption analyses of lipopolysaccharide (LPS) and OmpF mutants demonstrate that these phages use both the LPS inner core heptosyl residues and the outer membrane protein OmpF as phage receptors. Based on RNA sequencing and quantitative proteomics, we also demonstrate that temperature-dependent infection is due to strong repression of OmpF at 37°C. In addition, ÏR1-RT was shown to be able to enter into a pseudolysogenic state. Together, this work provides further insight into phage-host cell interactions by highlighting the importance of understanding underlying factors which may affect the abundance of phage host receptors on the cell surface. IMPORTANCE: Only a small number of bacteriophages infecting Y. enterocolitica, the predominant causative agent of yersiniosis, have been previously described. Here, two newly isolated Y. enterocolitica phages were studied in detail, with the aim of elucidating the host cell receptors required for infection. Our research further expands the repertoire of phages available for consideration as potential antimicrobial agents or as diagnostic tools for this important bacterial pathogen.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Especificidade de Hospedeiro , Porinas/metabolismo , Receptores Virais/metabolismo , Yersinia enterocolitica/virologia , Proteínas de Bactérias/genética , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Genoma Viral , Humanos , Filogenia , Porinas/genética , Receptores Virais/genética , Temperatura , Replicação Viral , Yersiniose/microbiologia , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismoRESUMO
The efficient production of a high concentration of bacteriophage in large volumes has been a limiting factor in the exploration of the true potential of these organisms for biotechnology, agriculture and medicine. Traditional methods focus on generating small volumes of highly concentrated samples as the end product of extensive mechanical and osmotic processing. To function at an industrial scale mandates extensive investment in infrastructure and input materials not feasible for many smaller facilities. To address this, we developed a novel, scalable, generic method for producing significantly higher titer psychrophilic phage (P < 2.0 × 10(-6)), 2- to 4-fold faster than traditional methods. We generate renewable high yields from single source cultures by propagating phage under refrigeration conditions in which Listeria, Yersinia and their phages grow in equilibrium. Diverse Yersinia and Listeria phages tested yielded averages of 3.49 × 10(8) to 3.36 × 10(12) PFU/ml/day compared to averages of 1.28 × 10(5) to 1.30 × 10(10) PFU/ml/day by traditional methods. Host growth and death kinetics made this method ineffective for extended propagation of mesophilic phages.
Assuntos
Bacteriófagos/crescimento & desenvolvimento , Temperatura Baixa , Listeria/virologia , Cultura de Vírus/métodos , Carga Viral , Ensaio de Placa Viral , Yersinia/virologiaRESUMO
The Thermo Scientific™ SureTect™ Listeria species assay is a new real-time PCR assay for the detection of all species of Listeria in food and environmental samples. The assay was originally certified as Performance Tested Methods(SM) (PTM) 071304 in 2013. This report details the method modification study undertaken to extend the performance claims of the assay for matrixes of raw ground turkey, raw ground pork, bagged lettuce, raw pork sausages, pasteurized 2% fat milk, raw cod, pasteurized brie cheese, and ice cream. The method modification study was conducted using the AOAC Research Institute (RI) PTM program to validate the SureTect PCR assay in comparison to the reference method detailed in ISO 11290-1:1996 including amendment 1:2004. All matrixes were tested by Thermo Fisher Scientific (Basingstoke, United Kingdom). In addition, three matrixes (raw cod, bagged lettuce, and pasteurized brie cheese) were analyzed independently as part of the AOAC RI-controlled independent laboratory study by the University of Guelph, Canada. Using probability of detection statistical analysis, there was no significant difference in the performance between the SureTect assay and the International Organization for Standardization reference method for any of the matrixes analyzed in this study.
Assuntos
DNA Bacteriano/genética , Análise de Alimentos , Microbiologia de Alimentos , Listeria/classificação , Listeria/isolamento & purificação , Temperatura , Animais , Listeria/genética , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de ReferênciaRESUMO
The Thermo Scientific™ SureTect™ Listeria monocytogenes assay is a real-time PCR assay for the detection of Listeria monocytogenes in food and environmental samples, which was certified during 2013 by the AOAC Research Institute (RI) as Performance Tested Method(SM) (PTM) 061302 for a representative range of key food matrixes and production surfaces. This report details the method modification study, which was conducted during 2014, using the AOAC-RI PTM program to extend the validated matrix claims of the assay in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996, including Amendment 1:2004, to gain certification for raw ground turkey, raw ground pork, pasteurized 2% milk, raw pork sausages, raw cod, pasteurized brie cheese, cooked sliced ham, and bagged lettuce. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, brie cheese, bagged lettuce, and raw cod were analyzed independently by the University of Guelph, Canada, during the AOAC-RI controlled independent laboratory study. Using probability of detection (POD) statistical analysis, a significant difference was demonstrated between the candidate and reference methods for the high spiking level with raw ground pork and brie cheese. For all other matrixes and the low spiked levels for raw ground pork and brie cheese, no significant difference by POD was seen between the two methods during the study.
Assuntos
Laticínios/microbiologia , Listeria monocytogenes/genética , Carne/análise , Alimentos Crus/análise , Reação em Cadeia da Polimerase em Tempo Real/normas , Alimentos Marinhos/microbiologia , Animais , Bovinos , Análise de Alimentos , Contaminação de Alimentos/análise , Humanos , Alimentos Crus/microbiologia , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Bacteriophage vB_YenP_AP5 is a lytic bacteriophage capable of infecting Yersinia enterocolitica strains of serotype O:3, an epidemiologically significant serotype within this bacterial species that causes yersiniosis in humans. This work describes the complete genome sequence of this phage. RESULTS: The genome consists of linear double-stranded DNA of 38,646 bp, with direct terminal repeats of 235 bp in length, and a GC content of 50.7%. There are 45 open reading frames which occupy 89.9% of the genome. Most of the proteins encoded by this virus exhibit sequence similarity to Yersinia phage φYeO3-12 and Salmonella phage φSG-JL2 proteins. CONCLUSIONS: Genomic and morphological analyses place the bacteriophage vB_YenP_AP5 in the T7likevirus genus of the subfamily Autographivirinae within the family Podoviridae.
Assuntos
Bacteriófagos/genética , Genoma Viral , Podoviridae/genética , Esgotos/virologia , Yersinia enterocolitica/virologia , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Sequência de Bases , Especificidade de Hospedeiro , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Podoviridae/classificação , Podoviridae/isolamento & purificação , Sorotipagem , Yersinia enterocolitica/classificação , Yersinia enterocolitica/isolamento & purificaçãoRESUMO
The Thermo Scientific SureTect Salmonella species Assay is a new real-time PCR assay for the detection of Salmonellae in food and environmental samples. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested Methods program to validate the SureTect Salmonella species Assay in comparison to the reference method detailed in International Organization for Standardization 6579:2002 in a variety of food matrixes, namely, raw ground beef, raw chicken breast, raw ground pork, fresh bagged lettuce, pork frankfurters, nonfat dried milk powder, cooked peeled shrimp, pasteurized liquid whole egg, ready-to-eat meal containing beef, and stainless steel surface samples. With the exception of liquid whole egg and fresh bagged lettuce, which were tested in-house, all matrixes were tested by Marshfield Food Safety, Marshfield, WI, on behalf of Thermo Fisher Scientific. In addition, three matrixes (pork frankfurters, lettuce, and stainless steel surface samples) were analyzed independently as part of the AOAC-RI-controlled laboratory study by the University of Guelph, Canada. No significant difference by probability of detection or McNemars Chi-squared statistical analysis was found between the candidate or reference methods for any of the food matrixes or environmental surface samples tested during the validation study. Inclusivity and exclusivity testing was conducted with 117 and 36 isolates, respectively, which demonstrated that the SureTect Salmonella species Assay was able to detect all the major groups of Salmonella enterica subspecies enterica (e.g., Typhimurium) and the less common subspecies of S. enterica (e.g., arizoniae) and the rarely encountered S. bongori. None of the exclusivity isolates analyzed were detected by the SureTect Salmonella species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation (enrichment time and temperature, and lysis temperature), which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the assay shelf life.
Assuntos
Microbiologia de Alimentos/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Salmonella/classificação , Animais , Ovos/microbiologia , Microbiologia de Alimentos/normas , Carne/microbiologia , Leite/microbiologia , Padrões de Referência , Sensibilidade e Especificidade , Especificidade da Espécie , Aço Inoxidável , Verduras/microbiologiaRESUMO
The Thermo Scientific SureTect Listeria species Assay is a new real-time PCR assay for the detection of all species of Listeria in food and environmental samples. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested Methods program to validate the SureTect Listeria species Assay in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996 including amendment 1:2004 in a variety of foods plus plastic and stainless steel. The food matrixes validated were smoked salmon, processed cheese, fresh bagged spinach, cantaloupe, cooked prawns, cooked sliced turkey meat, cooked sliced ham, salami, pork frankfurters, and raw ground beef. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, three matrixes (pork frankfurters, fresh bagged spinach, and stainless steel surface samples) were analyzed independently as part of the AOAC-RI-controlled independent laboratory study by the University ofGuelph, Canada. Using probability of detection statistical analysis, a significant difference in favour of the SureTect assay was demonstrated between the SureTect and reference method for high level spiked samples of pork frankfurters, smoked salmon, cooked prawns, stainless steel, and low-spiked samples of salami. For all other matrixes, no significant difference was seen between the two methods during the study. Inclusivity testing was conducted with 68 different isolates of Listeria species, all of which were detected by the SureTect Listeria species Assay. None of the 33 exclusivity isolates were detected by the SureTect Listeria species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation, which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the assay shelf life.
Assuntos
Técnicas Bacteriológicas/métodos , Microbiologia de Alimentos/métodos , Listeria/isolamento & purificação , Animais , Técnicas Bacteriológicas/normas , Queijo/microbiologia , DNA Bacteriano/genética , Microbiologia Ambiental , Microbiologia de Alimentos/normas , Listeria/genética , Carne/microbiologia , Plásticos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Aço Inoxidável , Verduras/microbiologiaRESUMO
The Thermo Scientific SureTect Listeria monocytogenes Assay is a new real-time PCR assay for the detection of Listeria monocytogenes in food and environmental samples. This assay was validated using the AOAC Research Institute (AOAC-RI) Performance Tested Methods program in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996, including Amendment 1:2004 with the following foods and food contact surfaces: smoked salmon, processed cheese, fresh bagged spinach, fresh cantaloupe, cooked prawns (chilled product), cooked sliced turkey meat (chilled product), ice cream, pork frankfurters, salami, ground raw beef meat (12% fat), plastic, and stainless steel. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, three matrixes (pork frankfurters, bagged lettuce, and stainless steel) were analyzed independently as part of the AOAC-RI controlled laboratory study by the University of Guelph, Canada. Using probability of detection (POD) statistical analysis, a significant difference was demonstrated between the candidate and reference methods for salami, cooked sliced turkey and ice cream in favor of the SureTect assay. For all other matrixes, no significant difference by POD was seen between the two methods during the study. Inclusivity and exclusivity testing was also conducted with 53 and 30 isolates, respectively, which demonstrated that the SureTect assay was able to detect all serotypes of L. monocytogenes. None of the exclusivity isolates analyzed were detected by the SureTect assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside the recommended parameters open to variation, i.e., enrichment time and temperature and lysis temperature, which demonstrated that the assay gave reliable performance. Accelerated stability testing was also conducted, validating the assay shelf life.
Assuntos
Técnicas Bacteriológicas/métodos , Microbiologia Ambiental , Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Técnicas Bacteriológicas/instrumentação , Cucumis melo/microbiologia , Laticínios/microbiologia , Carne/microbiologia , Plásticos , Spinacia oleracea/microbiologia , Aço InoxidávelRESUMO
The Thermo Scientific™ SureTect™ Salmonella species Assay is a new real-time PCR assay for the detection of Salmonellae in food and environmental samples. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested MethodsSM program to validate the SureTect Salmonella species Assay in comparison to the reference method detailed in International Organization for Standardization 6579:2002 in a variety of food matrixes, namely, raw ground beef, raw chicken breast, raw ground pork, fresh bagged lettuce, pork frankfurters, nonfat dried milk powder, cooked peeled shrimp, pasteurized liquid whole egg, ready-to-eat meal containing beef, and stainless steel surface samples. With the exception of liquid whole egg and fresh bagged lettuce, which were tested in-house, all matrixes were tested by Marshfield Food Safety, Marshfield, WI, on behalf of Thermo Fisher Scientific. In addition, three matrixes (pork frankfurters, lettuce, and stainless steel surface samples) were analyzed independently as part of the AOAC-RI-controlled laboratory study by the University of Guelph, Canada. No significant difference by probability of detection or McNemars Chi-squared statistical analysis was found between the candidate or reference methods for any of the food matrixes or environmental surface samples tested during the validation study. Inclusivity and exclusivity testing was conducted with 117 and 36 isolates, respectively, which demonstrated that the SureTect Salmonella species Assay was able to detect all the major groups of Salmonella enterica subspecies enterica (e.g., Typhimurium) and the less common subspecies of S. enterica (e.g., arizoniae) and the rarely encountered S. bongori. None of the exclusivity isolates analyzed were detected by the SureTect Salmonella species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation (enrichment time and temperature, and lysis temperature), which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the assay shelf life.
RESUMO
A study was conducted to evaluate the performance of the ALOA (chromogenic media) in combination with immunomagnetic separation (IMS) for the detection of Listeria monocytogenes in ready-to-eat food products. IMS-ALOA method was found to be equivalent to Health Canada's reference culture method as well as comparable to BAX-PCR method in terms of the sensitivity of the methods for the detection of L. monocytogenes in ready-to-eat foods such as turkey roast, beef roast, mixed vegetable salads, potato and egg salad, soft cheese and smoked salmon. The IMS-ALOA method gave 100% sensitivity in the inclusivity tests with 42 pure L. monocytogenes strains. Exclusivity testing with five other species of Listeria genus and 29 pure non-L. monocytogenes strains from 21 different genera showed 97% specificity. The method was able to detect L. monocytogenes at levels near or below 1cfu/25g regulatory limit in ready-to-eat food matrices after 24h enrichment, with a turnaround time of 3days compared to 7-8days for culture method. IMS-ALOA method is a valuable alternate test method for the screening of L. monocytogenes in a variety of foods especially ready-to-eat foods.
Assuntos
Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Microbiologia de Alimentos , Separação Imunomagnética/métodos , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Ágar , Canadá , Sensibilidade e Especificidade , Fatores de TempoRESUMO
An automated immunomagnetic separation (IMS) and enzyme immunoassay (EIA) was applied to the detection of Salmonella enterica subspecies enterica serotypes from poultry environmental samples. The analytical sensitivity and specificity of the IMS-EIA for 46 S. enterica serotypes and 33 non-salmonellae isolates belonging to 21 different genera were 91.3% and 90.9%, respectively. From post enrichment S. enterica cultures, the limit of detection of the assay was estimated at 10(4)-10(6) CFU/mL. Application of IMS-EIA on 850 naturally contaminated poultry environmental samples achieved 98.4% sensitivity and 96.8% specificity, as compared with a standard culture reference method performed concurrently on the same set of samples. The IMS-EIA described, allows for the identification of suspect positive samples within 48 h of testing versus 4-6 days required by standard culture methods while significantly reducing the materials and labour required for the detection of S. enterica serotypes in poultry environmental samples.
Assuntos
Técnicas Bacteriológicas/métodos , Microbiologia Ambiental , Técnicas Imunoenzimáticas/métodos , Separação Imunomagnética/métodos , Salmonella enterica/isolamento & purificação , Animais , Automação , Aves Domésticas , Sensibilidade e EspecificidadeRESUMO
The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.
Assuntos
Técnicas Bacteriológicas/métodos , Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação , Ágar , Compostos Cromogênicos/química , Meios de Cultura , Listeriose/microbiologia , Listeriose/prevenção & controle , Sensibilidade e EspecificidadeRESUMO
Automated immunomagnetic separation (AIMS), using Dynabeads anti-Salmonella (Dynal, Oslo), was evaluated for its ability to detect Salmonella spp. in poultry environmental samples in comparison with standard, culture-based method (Health Canada, Health Protection Branch, MFHPB-20). AIMS was found to be more reliable in detecting Salmonella from artificially inoculated enrichment broths at low levels and exhibited a 15.5% higher sensitivity value than the culture method.
Assuntos
Técnicas Bacteriológicas , Microbiologia de Alimentos , Separação Imunomagnética/métodos , Aves Domésticas/microbiologia , Salmonella/isolamento & purificação , Animais , Técnicas Bacteriológicas/estatística & dados numéricos , Humanos , Separação Imunomagnética/estatística & dados numéricos , Salmonella/patogenicidade , Intoxicação Alimentar por Salmonella/prevenção & controle , Sensibilidade e EspecificidadeRESUMO
An increase in the prevalence of Salmonella enterica serotype Typhimurium DT104 has been reported worldwide. This study examined the prevalence of this microorganism in poultry environmental samples from commercial layer flocks and pullet environments as well as the sensitivity and specificity of a PCR-based method, and multiple antibiotic resistance profile of Salmonella serogroup B isolates in relation to the serotype and phagetype reference method for the identification of Salmonella Typhimurium DT104. A total of 435 Salmonella isolates were obtained from poultry house environmental samples tested during a 20-month period representing a prevalence of 5.5%. Of these, 313 (72%) isolates were identified as Salmonella serogroup B isolates. These isolates were tested by a PCR-based assay, and for resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) for the rapid identification of Salmonella Typhimurium DT104. Upon comparing the antibiotic resistance and PCR results with serotype and phage type data, the sensitivity and specificity for the identification of Salmonella Typhimurium DT104 of both methods were found to be 100%, and 99.6%, respectively. Both methods can be completed within 24 h after obtaining an isolate, while serotyping and phagetyping required more than 5 days to complete.