Marcelo Luiz Lima Brandão

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.

Desenvolvimento de material de referência para microbiologia de alimentos contendo estafilococos coagulase positiva em matriz queijo

O uso de materiais de referencia (MR) e uma das principais ferramentas utilizadas para garantia e controle da qualidade de laboratorios de microbiologia de alimentos. No Brasil, a RDC n.o 12/01 da Anvisa preve como um dos parâmetros para a avaliacao da qualidade de queijos a enumeracao de estafilococos coagulase positiva (ECP). O grande desafio na producao de MR destinados a ensaios microbiologicos e a instabilidade natural dos micro-organismos, o que dificulta o desenvolvimento e a manutencao desses MR. O objetivo deste estudo foi produzir um MR quantitativo destinado ao ensaio de enumeracao de ECP em matriz queijo. Uma amostra de queijo ultrafiltrado com contagem de ECP <10 UFC/g e numero de aerobios viaveis de 1,22 × 103 UFC/g foi utilizada como matriz para producao do MR. A matriz foi distribuida em frascos, contaminada com a bacteria alvo em concentracao especifica e submetida a liofilizacao. Como crioprotetor, foi utilizada sacarose. O MR produzido foi considerado homogeneo e estavel a temperatura < -70 oC durante todo o periodo estudado (dez meses). O material apresentou estabilidade a 4, 25, 30 e 35 oC durante quatro dias; contudo, os resultados indicam que, a 35 oC, ocorre um decrescimo na concentracao celular. A -20 oC, o MR apresentou-se estavel durante 48 dias. Conclui-se que o material apresentou todos os requisitos necessarios de um MR de qualidade e poderia ser transportado aos laboratorios participantes de um ensaio de proficiencia a temperaturas de ate 35 oC por ate quatro dias, uma vez que os resultados indicaram a manutencao da concentracao celular neste periodo. Esse foi o primeiro trabalho a descrever uma metodologia de producao de MR contendo ECP em matriz queijo.

Desenvolvimento de material de referência para microbiologia de alimentos contendo Listeria monocytogenes em matriz queijo

The standard most used in testing laboratories, ISO/IEC 17025:2005, describes the participation of laboratories in periodic proficiency testing (PT), as a criteria for quality assurance of analytical results. The analyses used in PT are reference materials (MR) from the same lot, and must have characteristics of homogeneity and stability. This study aimed to produce a qualitative RM for detection of Listeria monocytogenes assays in cheese matrix. For the production of RM, Minas Frescal cheese (MFQ) was used as matrix. The matrix was distributed in flasks, contaminated with the target bacteria and submitted to freeze-drying. Sucrose was used as cryoprotector. The RM produced was considered homogeneous and stable at -70°C during the entire period of study (10 months). The material showed stability at 4, 25, 30 and 35°C for 4 days and at -20°C the RM showed stability for 48 days, and the statistical results indicate a tendency to maintain stability. It was concluded that the material showed all the requirements of an RM quality and could be transported to the laboratory participants of a PT at 35°C up to 4 days, since the results indicate the maintenance of cell concentration during this period. This is the first study to describe a methodology for producing MR containing L. monocytogenes in cheese matrix.

Phenotypic characterization of Cronobacter spp. strains isolated from foods and clinical specimens in Brazil

Several Cronobacter species are opportunistic pathogens that cause infections in humans. This study evaluated the phenotypic characteristics of 57 Cronobacter strains (C. sakazakii n=41, C. malonaticus n=10, C. dublinensis n=4, and C. muytjensii n=2) isolated from food (n=54) and clinical specimens (n=3) in Brazil. These strains included sequence types (ST): ST395-ST398, ST402, ST413 and ST433-ST439, isolated from food samples, and three C. malonaticus clinical strains previous isolated from an outbreak which were ST394 (n=1) and ST440 (n=2). Strains were tested for capsule production, biofilm formation, protease activity, hemolytic activity, cell-cell aggregation, and desiccation resistance. Capsule formation was observed with all Cronobacter strains. Forty-four (77.2%) strains showed proteolytic activity on milk agar. All strains showed β-hemolysis against erythrocytes from guinea pig, horse and rabbit. Using erythrocytes from sheep, the majority of strains (53/57; 92.9%) showed α-hemolysis and the remaining, β-hemolysis. All Cronobacter strains produced weak biofilms in microtiters polystyrene plates, which were independent of temperature (4, 25 and 37°C) and/or growth conditions. In glass tubes, formation of either a moderate or strong biofilm was observed in 15/57 (26.3%), 19/57 (33.3%) and 27/57 (47.4%), at 4, 25 and 37°C, respectively. Desiccation treatment decreased Cronobacter viability by 1.55 to >3.87Log10CFU/mL. Cell-cell aggregation was observed in 17 (29.8%) strains. This study showed that the Cronobacter species evaluated showed differing phenotypes, independent of their origin (clinical or not) and ST. Further studies are necessary to elucidate the factors affecting phenotype expression. This may identify novel bacterial targets that could be useful in the development of strategies to control Cronobacter in food chain and to prevent cases of infections.

Pesquisa de Cronobacter spp. em fórmulas infantis desidratadas

Cronobacter spp. is an opportunistic bacterium that is associated with infection outbreaks in neonates and infants due to the consumption of powdered infant formulas (PIF). In this context, the official regulator sector has created the specific standards for the industries to control these pathogens in the PIF. The present study aimed at evaluating the occurrence of Cronobacter spp. in 60 samples of PIF commercialized in Rio de Janeiro, Brazil. Thirty samples of infant formulas (0-6 months) and 30 samples of follow-up infant formulas (> 6 months) were analyzed according to the conventional methodologies described in the Bacteriological Analytical Manual Online-FDA (2012). The identification of characteristic colonies was performed using kits ID32E, API20E andVitek 2.0 system and polymerase chain reaction (PCR) targeting the gluA gene. No sample showed contamination by Cronobacter spp. The occurrence of Cronobacter spp. in PIF seems to be low, indicating that the producers are complying with the provisions of the Brazilian standards in force to avoid the product contamination by this microorganism.

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.

Fatal Cronobacter sakazakii Sequence Type 494 Meningitis in a Newborn, Brazil

We describe a case of infection with Cronobacter sakazakii sequence type 494 causing bacteremia and meningitis in a hospitalized late premature infant in Brazil. We conducted microbiological analyses on samples of powdered infant formula from the same batch as formula ingested by the infant but could not identify the source of contamination.

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.

Cronobacter spp.: infecções, ocorrência e regulação em alimentos ‒ uma revisão no Brasil

Cronobacter is an opportunistic bacterium associated with infections in newborns due to the consumption of powdered infant formulae. It is now known that Cronobacter can cause infections in individuals of any age with a higher prevalence in the elderly, urinary infections being the most common syndrome in this group. The identification of powdered infant formulae as the contamination vehicle in cases of meningitis caused by Cronobacter in neonates led the regulatory agencies to establish specific measures to control such pathogens in these products. Thus a literature review was carried out concerning the epidemiology of Cronobacter infections, the occurrence of the pathogen and food regulations in Brazil. It was found that cases of the infection were reported in Brazil from 1997-2013, with a higher occurrence in neonates. The precise number of cases could not be determined, and in some reports, identification of the microorganism was questionable. In most of the cases reported the contamination vehicle was not investigated or could not be determined. There have been reports of contamination by Cronobacter in samples of powdered infant formulae commercialized in Brazil, but their occurrence seems to have reduced after the adoption of stricter sanitary measures by the regulatory agencies. Cronobacter has also been isolated from spices and herbs, flours, cheese and from cereal mixes destined for children; and from these samples, four species were identified: C. sakazakii, C. malonaticus, C. dublinensis and C. muytjensii. The presence of the pathogen Marcelo Luiz Lima Brandão1*, Natália Scudeller Umeda1, Ivano de Filippis1 1 Instituto Nacional de Controle de Qualidade em Saúde, Departamento de Microbiologia, Rio de Janeiro/RJ Brasil *Corresponding Author Marcelo Luiz Lima Brandão, Instituto Nacional de Controle de Qualidade em Saúde, Departamento de Microbiologia, Av. Brasil, 4365, Manguinhos, CEP: 21040-900, Rio de Janeiro/RJ Brasil, e-mail: marcelollb8@gmail.com Cite as: Cronobacter spp.: infections, occurrence and food regulations – a review in Brazil. Braz. J. Food Technol., v. 21, e2017074, 2018. Received: May 19, 2017; Accepted: Sept. 22, 2017 http://bjft.ital.sp.gov.br 2 Braz. J. Food Technol., Campinas, v. 21, e2017074, 2018 Cronobacter spp.: infecções, ocorrência e regulação em alimentos – uma revisão no Brasil Brandão, M. L. L. et al.