Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.

Mycobacteria in Minas cheese commercialized in open fairs in São Paulo, Brazil / Mycobacteria em queijos tipo Minas comercializados em feiras-livres de São Paulo, Brasil

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects dairy herds throughout the Brazilian territory, constituting a neglected zoonosis transmitted by raw milk and its derivatives. In this study, we evaluated the presence of M. bovis and other mycobacteria in Minas cheese obtained from open fairs in the city of São Paulo between 2012 and 2013. Samples (n = 133) were decontaminated using hexa-cetylpyridinium chloride and seeded on Stonebrink­Leslie medium. The isolates were submitted to molecular identification by TB Multiplex PCR targeting the 16S rRNA gene and amplicon nucleotide sequencing. From 16 cheese samples (12%), we obtained 26 putative colonies of Mycobacterium spp, none of which belonged to any of the Mycobacterium tuberculosis, Mycobacterium avium, or Mycobacterium intracellulare complexes. Phylogenetic analysis showed that sample sequences were grouped in a clade that includes only non-tuberculous mycobacteria with proximity to sequences obtained from Mycobacterium novocastrense (3 sequences), Mycobacterium holsaticum (1 sequence), and Mycobacterium elephantis (2 sequences). Although no epidemiological evidence was found regarding the importance of oral transmission of mycobacteria in healthy people, their importance in the immunosuppressed population remains uncertain.(AU)

Mycobacterium bovis é o agente da tuberculose bovina, doença que acomete o rebanho em todo território brasileiro e é uma negligenciada zoonose transmitida pelo leite e seus derivados. Este trabalho avaliou a presença de M. bovise outras micobactérias, em queijo minas meia-cura, obtidos em feiras-livres na cidade de São Paulo, entre os anos de 2012 e 2013. As amostras (n = 133) foram descontaminadas pelo método HPC (hexa-cetyl-pyridinium chloride) e semeadas em meio Stonebrink Leslie. Os isolados foram submetidos à identificação molecular por PCR TB multiplex, pesquisando-se o gene 16S rRNA, e ao sequenciamento nucleotídico. Dezesseis amostras (12%) possuiam 26 colônias sugestivas de Mycobacterium spp, mas nenhuma delas pertencia aos complexos Mycobacterium tuberculosis, Mycobacterium avium e Mycobacterium intracellulare. A análise filogenética mostrou que todas as amostras estavam agrupadas em clados que incluem apenas micobactérias não tuberculosas (MNT), sendo que algumas possuiam proximidade com sequências obtidas de Mycobacterium novocastrense (3 sequências), Mycobacterium hosaticum(1 sequência) e Mycobacterium elephantis (2 sequências). Embora no momento não haja evidência epidemiológica da importância da transmissão oral das micobactérias pra indivíduos saudáveis, sua importância na população imunossuprimida ainda é incerta.(AU)

Assessment of viral and bacterial contamination of fresh and ripened semi-hard cheeses.

This study aimed to evaluate viral and bacterial contamination from typical Brazilian cheeses, such as Minas (fresh) and Prato (ripened), commercially obtained in the Greater Metropolitan Region of the State of Rio de Janeiro, Brazil. Minas [30], Prato [30] and sliced Prato [30] cheese samples were investigated for norovirus genogroup I and II (NoV GI-II) and human adenovirus (HAdV) by direct nucleic acid extraction using TRIzol and amplification by TaqMan based quantitative polymerase chain reaction. Listeria monocytogenes, Salmonella spp., coagulase-positive staphylococci (CPS) and fecal coliforms were also assessed by using standard counting methods. NoV GI and GII were detected in one sample (1.1%) each and HAdV in nine samples (10.0%) while bacteriological analysis revealed five samples (5.5%) contaminated with L. monocytogenes, 27 (30.0%) with fecal coliforms and 10 (11.1%) with CPS. Salmonella spp. was not detected in any sample. Viruses were detected in 11 samples (12.2%), of which 9 met the microbiological criteria used to evaluate the microbiological quality of the cheeses, stressing the importance of considering virological parameters for monitoring this food matrix.

Vaccinia virus detection in dairy products made with milk from experimentally infected cows.

Vaccinia virus (VACV) is the agent of bovine vaccinia (BV), an emerging zoonosis that causes exanthematic lesions on the teats of dairy cows and on the hands of milkers. The virus has been detected in the milk of naturally infected cows. The objective of this study was to investigate and quantify VACV DNA as well as the presence of infectious virus particles in samples of cheese curd, cheese whey and pasteurized milk produced using milk from cows experimentally inoculated with VACV-GP2, a Brazilian isolate of VACV (VACV-BR). VACV DNA was detected in samples of cheese and pasteurized milk at different time points, even after the resolution of the typical lesions caused by VACV, which occurred after 22 days post-infection (dpi), on average. Moreover, it was possible to detect infectious viral particles in cheese samples on alternate days until 27 dpi. The presence of both VACV DNA and infectious viral particles in cheese samples throughout the clinical course of BV and even after the disappearance of the typical clinical signs of disease draws attention to the risk associated with consumption of the cheese. Furthermore, VACV-contaminated milk and cheese may represent an occupational risk to cheesemakers who often manipulate milk and cheese curd without wearing gloves.

Short communication: Survival of Vaccinia virus in inoculated cheeses during 60-day ripening.

Bovine vaccinia is a neglected zoonosis caused by Vaccinia virus (VACV) and has a major economic and public health effect in Brazil. Previous studies showed infectious VACV particles in milk from either experimentally or naturally infected cows and in fresh cheeses prepared with experimentally contaminated milk. Ripening is a process that leads to major changes in the physical and chemical characteristics of cheese, reducing contamination by spoilage, pathogenic microorganisms, or both. However, it is not known if VACV infectious particles persist after the ripening process. To investigate this issue, viral infectivity at different ripening times was studied in cheeses manufactured with milk experimentally contaminated with VACV strain Guarani P2 (GP2). Cheeses were analyzed at 1, 7, 14, 21, 45, and 60 d of ripening at 25°C. Viral DNA was quantified by real-time PCR, and VACV isolation and titration were performed in Vero cells. The whole experiment was repeated 4 times. Analysis of the mean viral DNA quantification and infectivity indicated a reduction of approximately 2 logs along the ripening process; however, infectious viral particles (1.7 × 102 pfu/mL) could still be recovered at d 60 of ripening. These findings indicate that the ripening process reduces VACV infectivity, but it was not able to inactivate completely the viral particles after 60 d.

Phage-host interactions in Streptococcus thermophilus: Genome analysis of phages isolated in Uruguay and ectopic spacer acquisition in CRISPR array.

Three cos-type virulent Streptococcus thermophilus phages were isolated from failed mozzarella production in Uruguay. Genome analyses showed that these phages are similar to those isolated elsewhere around the world. The CRISPR1 and CRISPR3 arrays of the three S. thermophilus host strains from Uruguay were also characterized and similarities were noted with previously described model strains SMQ-301, LMD-9 and DGCC7710. Spontaneous bacteriophage-insensitive S. thermophilus mutants (BIMs) were obtained after challenging the phage-sensitive wild-type strain Uy02 with the phage 128 and their CRISPR content was analyzed. Analysis of 23 BIMs indicated that all of them had acquired at least one new spacer in their CRISPR1 array. While 14 BIMs had acquired spacer at the 5'-end of the array, 9 other BIMs acquired a spacer within the array. Comparison of the leader sequence in strains Uy02 and DGCC7710 showed a nucleotide deletion at position -1 in Uy02, which may be responsible for the observed ectopic spacer acquisition. Analysis of the spacer sequences upstream the newly acquired ectopic spacer indicated presence of a conserved adenine residue at position -2. This study indicates that natural strains of S. thermophilus can also acquire spacers within a CRISPR array.

Control of Listeria monocytogenes growth in soft cheeses by bacteriophage P100.

The purpose of this study was to determine the effect of bacteriophage P100 on strains of Listeria monocytogenes in artificially inoculated soft cheeses. A mix of L. monocytogenes 1/2a and Scott A was inoculated in Minas Frescal and Coalho cheeses (approximately 10(5) cfu/g) with the bacteriophage added thereafter (8.3 × 10(7) PFU/g). Samples were analyzed immediately, and then stored at 10 °C for seven days. At time zero, 30 min post-infection, the bacteriophage P100 reduced L. monocytogenes counts by 2.3 log units in Minas Frescal cheese and by 2.1 log units in Coalho cheese, compared to controls without bacteriophage. However, in samples stored under refrigeration for seven days, the bacteriophage P100 was only weakly antilisterial, with the lowest decimal reduction (DR) for the cheeses: 1.0 log unit for Minas Frescal and 0.8 log units for Coalho cheese. The treatment produced a statistically significant decrease in the counts of viable cells (p < 0.05) and in all assays performed, we observed an increase of approximately one log cycle in the number of viable cells of L. monocytogenes in the samples under refrigeration for seven days. Moreover, a smaller effect of phages was observed. These results, along with other published data, indicate that the effectiveness of the phage treatment depends on the initial concentration of L. monocytogenes, and that a high concentration of phages per unit area is required to ensure sustained inactivation of target pathogens on food surfaces.

Leuconostoc bacteriophages from blue cheese manufacture: long-term survival, resistance to thermal treatments, high pressure homogenization and chemical biocides of industrial application.

Nine Leuconostoc mesenteroides phages were isolated during blue cheese manufacture yielding faulty products with reduced eye formation. Their morphologies, restriction profiles, host ranges and long-term survival rates (25°C, 8°C, -20°C and -80°C) were analysed. Based on restriction analysis, six of them were further examined regarding resistance to physical (heat and high pressure homogenization, HPH) and chemical treatments (ethanol, sodium hypochlorite, peracetic acid, biocides A, C, E and F). According to their morphology, L. mesenteroides phages studied in the present work belonged to the Caudovirales order and Siphoviridae family. Six distinct restriction patterns were obtained with EcoRV, HindIII, ClaI and XhoI enzymes, revealing interesting phage diversity in the dairy environment. No significant reductions in phage counts were observed after ten months of storage at -20°C and -80°C, while slightly and moderate decrease in phage numbers were noticed at 8°C and 25°C, respectively. The phages subjected to heat treatments generally showed high resistance at 63°C and moderate resistance at 72°C. However, 80°C for 30 min and 90°C for 2 min led to complete inactivation of viral particles. In general, the best ethanol concentration tested was 75%, as complete inactivation for most Leuconostoc phages within 30 min of incubation was achieved. Peracetic acid, and biocides A, C, E and F were highly effective when used at the same or at a moderately lower concentration as recommended by the producer. Usually, moderate or high concentrations (600-1,600 ppm) of sodium hypochlorite were necessary to completely inactivate phage particles. Leuconostoc phages were partially inactivated by HPH treatments as remaining viral particles were found even after 8 passes at 100 MPa. This is the first report of L. mesenteroides phages isolated from an Argentinean dairy cheese plant. The results of this work could be useful for establishing the most effective physical and chemical treatments for inactivating phages in industrial plants and laboratory environments.

Control of Listeria monocytogenes growth in soft cheeses by bacteriophage P100

The purpose of this study was to determine the effect of bacteriophage P100 on strains of Listeria monocytogenes in artificially inoculated soft cheeses. A mix of L. monocytogenes 1/2a and Scott A was inoculated in Minas Frescal and Coalho cheeses (approximately 10(5) cfu/g) with the bacteriophage added thereafter (8.3 x 10(7) PFU/g). Samples were analyzed immediately, and then stored at 10 ºC for seven days. At time zero, 30 min post-infection, the bacteriophage P100 reduced L. monocytogenes counts by 2.3 log units in Minas Frescal cheese and by 2.1 log units in Coalho cheese, compared to controls without bacteriophage. However, in samples stored under refrigeration for seven days, the bacteriophage P100 was only weakly antilisterial, with the lowest decimal reduction (DR) for the cheeses: 1.0 log unit for Minas Frescal and 0.8 log units for Coalho cheese. The treatment produced a statistically significant decrease in the counts of viable cells (p < 0.05) and in all assays performed, we observed an increase of approximately one log cycle in the number of viable cells of L. monocytogenes in the samples under refrigeration for seven days. Moreover, a smaller effect of phages was observed. These results, along with other published data, indicate that the effectiveness of the phage treatment depends on the initial concentration of L. monocytogenes, and that a high concentration of phages per unit area is required to ensure sustained inactivation of target pathogens on food surfaces.(AU)

Control of Listeria monocytogenes growth in soft cheeses by bacteriophage P100

The purpose of this study was to determine the effect of bacteriophage P100 on strains of Listeria monocytogenes in artificially inoculated soft cheeses. A mix of L. monocytogenes 1/2a and Scott A was inoculated in Minas Frescal and Coalho cheeses (approximately 10(5) cfu/g) with the bacteriophage added thereafter (8.3 x 10(7) PFU/g). Samples were analyzed immediately, and then stored at 10 ºC for seven days. At time zero, 30 min post-infection, the bacteriophage P100 reduced L. monocytogenes counts by 2.3 log units in Minas Frescal cheese and by 2.1 log units in Coalho cheese, compared to controls without bacteriophage. However, in samples stored under refrigeration for seven days, the bacteriophage P100 was only weakly antilisterial, with the lowest decimal reduction (DR) for the cheeses: 1.0 log unit for Minas Frescal and 0.8 log units for Coalho cheese. The treatment produced a statistically significant decrease in the counts of viable cells (p < 0.05) and in all assays performed, we observed an increase of approximately one log cycle in the number of viable cells of L. monocytogenes in the samples under refrigeration for seven days. Moreover, a smaller effect of phages was observed. These results, along with other published data, indicate that the effectiveness of the phage treatment depends on the initial concentration of L. monocytogenes, and that a high concentration of phages per unit area is required to ensure sustained inactivation of target pathogens on food surfaces.

Vaccinia virus is not inactivated after thermal treatment and cheese production using experimentally contaminated milk.

Bovine vaccinia is an emergent zoonosis caused by the Vaccinia virus (VACV). The disease is characterized by the appearance of exanthematic lesions that occur in humans and dairy cows. Previous studies have revealed the presence of infectious viral particles in milk samples during an outbreak of bovine vaccinia in Brazil, indicating the possibility of disease transmission through raw milk. To assess the viability of the virus in milk after thermal treatment and processing procedures, milk samples were experimentally contaminated with 10(3) plaque forming units (PFU)/mL (group I) and 10(5) PFU/mL (group II) VACV Guarani P2 virus, and the third group was not contaminated and served as a control. The samples were submitted to storage temperatures in a cold chamber, freezer for 48 hours, and to low temperature long-time treatment. Moreover, the viral viability was evaluated in cheese produced with contaminated milk using 10(4) PFU/mL VACV Guarani P2. Notably, the virus remained viable in milk after storage for 48 hours in both the cold chamber and the freezer, with a reduction in viral titer of 14.49% and 25.86%, respectively. Group II showed a viral reduction in titer of 61.88% and 75.98%, respectively. Thermal treatment 65°C for 30 minutes showed a reduction of viral titer of 94.83% and 99.99%, respectively, in group I and group II, but still showed remaining viable virus particles. In addition, it was possible to recover infectious viral particles from both the solid curds and the whey of the cheese produced with experimentally contaminated milk. The cheese shows a reduction in viral titer of 84.87% after storage at 4°C for 24 hours. The presence of viable viral particles in milk after both thermal treatment and cheese production indicates a potential public health risk.

A rapid procedure for detecting noroviruses from cheese and fresh lettuce.

Noroviruses (NoVs) are recognized as the most common agents of outbreaks of food-borne viral gastroenteritis and the efficiency of different methods for detection of NoVs from food matrices have been tested in several laboratories worldwide. The aim of this study was to develop a rapid and sensitive method for recovery of NoVs by using a filtration concentration method followed by PCR amplification for detection of NoVs from cheese and fresh lettuce. Experimentally, a fecal suspension containing different number of NoVs copies was spiked in the food surface and extracted by a direct elution using a Stomacher apparatus. An Ozone-Safe solvent Vertrel XF treatment was included for cheese samples for removing particulate matter. The watery phase was collected and the viral concentration was performed by the adsorption-elution method using negatively charged membranes with inorganic solvents in a Stericup and afterwards ultrafiltered using a Centriprep Concentrator 50 to obtain a final volume of 2ml. RNA isolation was carried out with the QIAamp Viral RNA Mini Kit available commercially and reverse transcription was carried out with a Pd(N6) random primer. Real time quantitative PCR (TaqMan) and qualitative PCR were used for molecular detection of NoVs. The recovery rate of NoVs ranged from 5.2 to 72.3% in lettuce and from 6 to 56.3% in cheese. The results indicate that this method is suitable for detection of NoVs contamination in food and will help establish the cause and source of NoVs outbreaks of food-borne illness.

Frecuencia de Listeria spp., en quesos colombianos costeños / Frequency of Listeria spp. in coastal Colombian cheeses

Objetivo. Determinar la frecuencia de Listeria spp., en quesos frescos costeños, distribuidos en plazas de mercado populares de las ciudades de Montería y Cereté. Materiales y métodos. Teniendo en cuenta la importancia económica de esta zona ganadera para Colombia se tomaron 217 muestras entre Junio y Agosto de 2005, los aislamientos obtenidos fueron identificados por pruebas bioquímicas presuntivas, PCRMúltiple (L1-U1/LF-LR) y pruebas bioquímicas para confirmación de especie. Adicionalmente, se determinó la frecuencia de las especies del género y se caracterizó la resistencia antimicrobiana de las cepas de mayor frecuencia. Resultados. Las pruebas bioquímicas y la PCR detectaron 49 aislamientos positivos para Listeria (22.58%), de los cuales 16.33% (8/49) correspondieron a Montería y 24.40% (41/168) a Cereté. La frecuencia por especies fue 14.75% para L. ivanovii, 2.30% para L. innocua, 1.84% para L. welshimeri y 1.38% para L. seeligeri, no se detectó L. monocytogenes. Sólo 3/32 cepas de L. ivanovvi (9.38%) mostraron resistencia a penicilina, estreptomicina y eritromicina respectivamente. Conclusiones. Los resultados confirman que los quesos costeños están frecuentemente contaminados con Listeria spp. La presencia de L. ivanovvi patógeno involucrado en algunos casos de infecciones oportunistas en humanos y L. innocua; microorganismo utilizado en muchas industrias de alimento como indicador del grado o calidad de sanitización; demuestra que las condiciones de producción y expendio no son adecuadas y que el consumo de queso costeño no es seguro. La resistencia antimicrobiana aunque baja muestra posibilidades para la transmisión horizontal y/o vertical de los genes de resistencia.

Diversity of Streptococcus thermophilus phages in a large-production cheese factory in Argentina.

Phage infections still represent a serious risk to the dairy industry, in which Streptococcus thermophilus is used in starter cultures for the manufacture of yogurt and cheese. The goal of the present study was to analyze the biodiversity of the virulent S. thermophilus phage population in one Argentinean cheese plant. Ten distinct S. thermophilus phages were isolated from cheese whey samples collected in a 2-mo survey. They were then characterized by their morphology, host range, and restriction patterns. These phages were also classified within the 2 main groups of S. thermophilus phages (cos- and pac-type) using a newly adapted multiplex PCR method. Six phages were classified as cos-type phages, whereas the 4 others belonged to the pac-type group. This study illustrates the phage diversity that can be found in one factory that rotates several cultures of S. thermophilus. Limiting the number of starter cultures is likely to reduce phage biodiversity within a fermentation facility.

Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries.

Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml. The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant.