Valéria de Mello Medeiros

Virus recovering from strawberries: Evaluation of a skimmed milk organic flocculation method for assessment of microbiological contamination

Skimmed milk organic flocculation method was adapted, optimized and compared with polyethylene glycol (PEG) precipitation and filtration methods for recovering viruses from a strawberry matrix. Spiking experiments with norovirus genogroup II genotype 4 (NoV GII.4) and murine norovirus 1 (MNV-1) demonstrated that the organic flocculation method associated with a glycine elution buffer, filter bag and cetyltrimethylammonium bromide (CTAB) showed a recovery percentage of 2.5 and 32 times higher than PEG precipitation and filtration methodologies for NoV recovering. Furthermore, this method was used for investigating NoV and human adenoviruses (HAdVs) in 90 samples of fresh strawberries commercialized in Rio de Janeiro markets. NoV GI and GII were not detected in those samples and MNV-1, used as internal process control (IPC), was recovered in 95.5% (86) of them. HAdVs were detected in 18 (20.0%) samples and characterized by nucleotide sequencing as Human Mastadenovirus specie F and as type specie HAdV-2. Bacterial analysis did not detect Salmonella spp. and Listeria monocytogenes, however, 3.3% of fecal coliforms were detected in those samples. These results indicate the organic flocculation method as an alternative for recovering enteric viruses from strawberries, emphasizing a need for virus surveillance in food matrices.

Isolation, molecular and phenotypic characterization of Cronobacter spp. in ready-to-eat salads and foods from Japanese cuisine commercialized in Brazil

The aim of this study was to detect Cronobacter from 30 samples of ready-to-eat (RTE) salads and 30 foods from Japanese cuisine as commercially available in Brazil. The detection of Cronobacter was as according to the ISO standard 22964:2017. The isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile was determined using the standardized agar disc diffusion method. Molecular characterization was accomplished by real-time PCR targeting dnaG gene, multiplex-PCR targeting cgcA gene, and fusA allele sequencing. Twenty-seven samples (45.0%) contained Cronobacter, 14 (23.3%) samples of foods from Japanese cuisine and 13 (21.7%) samples of RTE salads. Twenty-nine unique Cronobacter isolates were selected from the 27 positive samples and were identified as C. sakazakii (n = 18), C. malonaticus (n = 8), and C. dublinensis (n = 3). A high genetic diversity was observed, with 29 Cronobacter strains being assigned to 11 different fusA alleles, a ratio of 2.6 strains by fusA allele was found. The cgcA multiplex-PCR failed to identify many of the Cronobacter isolates at the species level. Four (13.8%) Cronobacter isolates were resistant to one or more antibiotics tested (n = 12). The presence of Cronobacter in RTE foods could be a potential threat to human health and highlights the need for high levels of hygiene, particularly when preparing food for elderly, immunosuppressed persons or adults with prior underlying pathology. Epidemiological surveillance agencies should be aware of the risk that these RTE foods may represent, for these groups.

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.

Evaluation of skimmed milk flocculation method for virus recovery from tomatoes

This study aimed to evaluate the elution-concentration methodology based on skimmed milk flocculation from three varieties of tomatoes (Solanum lycopersicum L. [globe], Solanum lycopersicum var. cerasiforme [cherry] and hybrid cocktail [grape tomato]) for further monitoring of field samples. Spiking experiments were performed to determine the success rate and efficiency recovery of human norovirus (NoV) genogroup II, norovirus murine-1 (MNV-1) used as sample process control virus and human adenovirus (HAdV). Mean values of 18.8%, 2.8% and 44.0% were observed for NoV GII, MNV-1 and HAdV, respectively with differences according to the types of tomatoes, with lower efficiency for cherry tomatoes. Analysis of 90 samples, obtained at commercial establishments in the metropolitan region of Rio de Janeiro State, revealed 4.5% positivity for HAdV. Bacterial analysis was also performed with no detection of Salmonella spp., L. monocytogenes and fecal coliforms. Data demonstrated that the skimmed milk flocculation method is suitable for recovering HAdV from tomatoes and highlights the need for considering investigation in order to improve food safety.