Bacillus cereus: Caracterização genômica na cadeia produtiva de leite e influência da pasteurização na expressão de genes relacionados a toxinas diarreicas
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Data
2017-12-06
Autores
Rossi, Gabriel Augusto Marques
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Universidade Estadual Paulista (Unesp)
Resumo
As bactérias pertencentes ao grupo do Bacillus cereus são importantes para indústrias processadoras de leite e produtos lácteos devido à capacidade de sobrevivência aos tratamentos térmicos utilizados durante os processamentos, e consequente deterioração dos produtos e risco à saúde pública. Assim, inicialmente, objetivou-se compreender a estrutura populacional de isolados pertencentes ao grupo do B. cereus na cadeia produtiva do leite e produtos lácteos. Por meio da utilização de amostragem estruturada e técnicas genômicas comparativas, investigou-se quais linhagens específicas e genes estão significativamente associados a estágios de produção específicos e produtos. Os genomas de 69 isolados obtidos de equipamentos, leite cru e produtos lácteos foram comparados com outros 193 disponíveis de diversas origens. A estrutura populacional incluiu os conhecidos grupos filogenéticos II, III, IV, V e VI, e quase todos os isolados dos produtos lácteos pertenceram ao grupo III. A investigação de genes específicos revelou um grande número de isolados carreando aqueles relacionados à produção de toxinas, como o cytK (53,62%), hblA (59,42%), hblC (44,93%), hblD (53,62%), nheA (84,06%), nheB (89,86%), nheC (84,06%), cesA (2,90%) e cesB (2,90%). Os isolados pertencentes aos grupos IV e V possuíram uma prevalência significativamente maior dos genes hblACD e o grupo IV do gene cytK. Os isolados obtidos de produtos lácteos tiveram uma prevalência significativamente menor dos genes cytK e hblACD comparados aos de equipamentos e leite cru/tanques de refrigeração. A análise genômica populacional demonstrou a diversidade dos isolados e a variedade de funções dentro do grupo do B. cereus da cadeia produtiva leiteira, com grande número de isolados potencialmente capazes de causar doenças alimentares. Posteriormente, objetivou-se avaliar a viabilidade do processo de tindalização de leite cru refrigerado e determinar se o processo de pasteurização do leite influencia na expressão dos genes relacionados à produção das toxinas diarreicas pelo B. cereus s.s. em leite experimentalmente contaminado. Para isso, realizou-se um processo de tindalização de leite cru refrigerado e então foi realizada a contaminação com um isolado potencialmente toxigênico, e, posteriormente, o mesmo foi pasteurizado, envasado e mantido em refrigeração durante 10 dias. Em momentos determinados, foram coletadas amostras do produto afim de avaliar a expressão de genes codificadores de toxinas (hblACD, nheABC e cytK). O protocolo de tindalização utilizado permitiu redução logarítmica da população de bactérias do grupo do B. cereus e foi possível detectar a expressão do gene hblA em amostras de leite 5 e 10 dias após a pasteurização. Conclui-se que o modelo proposto foi adequado e que a expressão do gene hblA não foi inibida após a realização da pasteurização do leite, demonstrando o potencial risco aos consumidores decorrentes do consumo de leite pasteurizado contaminado.
The bacteria belonging to Bacillus cereus group are important to dairy industries due their ability to survive to thermal treatments used during processing, and consequently cause food spoilage and risk to public health. Thus, firstly, this study aimed to better understand the population structure of B. cereus group isolates in dairy production chain. Using structured sampling of B. cereus in dairy production chain and comparative genomics techniques, we investigated if specific lineages and genes are significantly overrepresented in particular production stages. The genomes of 69 isolates obtained from equipments, raw milk and dairy products were compared with others 193 avaiable from several origins. The populational structure included the known phylogenetic groups II, III, IV, V and V, and almost all isolates from dairy products belonged to group III. The genes investigation showed a high number of isolates carrying genes related to toxins production, such as cytK (53.62%), hblA (59.42%), hblC (44.93%), hblD (53.62%), nheA (84.06%), nheB (89.86%), nheC (84.06%), cesA (2.90%) and cesB (2.90%). The isolates belonging to groups IV and V had a significant higher prevalence of genes hblACD an group VI of cytK. The isolates obtained from dairy products had a significant lower prevalence of genes cytK and hblACD compared to those from equipments and raw milk/bulk tanks. The populational genomic analyses showed the diversity of isolates and variability of functions in B. cereus group in dairy production chain, with a high number of isolates potentially able to cause foodborne disease. Posteriorly, this study aimed to evaluate the viability of tyndallizating raw milk and to establish if milk’s pasteurization influences on the expression of genes related to the production of diarrheal toxins by B. cereus s.s. using a milk contamined experimentally. For this purpose, the tyndallization of raw milk was performed and later it was contaminated with a potentially toxigenic isolate, and, posteriorly, it was pasteurized, packaged and kept under refrigeration during 10 days. In established periods, samples of this product were collected in order to evaluate the expression of genes related to toxins production (hblACD, nheABC and cytK). The protocol used for tyndallization allowed a logarithmic reduction of population of B. cereus group and the expression of hblA gene was detected in samples from milk after 5 and 10 days after pasteurization. It was concluded that the proposed model was appropriate and the expression of hblA gene was not inihibited after milk pasteurization, highlighting the potential risk for consumers through consumption of contamined pasteurized milk.
The bacteria belonging to Bacillus cereus group are important to dairy industries due their ability to survive to thermal treatments used during processing, and consequently cause food spoilage and risk to public health. Thus, firstly, this study aimed to better understand the population structure of B. cereus group isolates in dairy production chain. Using structured sampling of B. cereus in dairy production chain and comparative genomics techniques, we investigated if specific lineages and genes are significantly overrepresented in particular production stages. The genomes of 69 isolates obtained from equipments, raw milk and dairy products were compared with others 193 avaiable from several origins. The populational structure included the known phylogenetic groups II, III, IV, V and V, and almost all isolates from dairy products belonged to group III. The genes investigation showed a high number of isolates carrying genes related to toxins production, such as cytK (53.62%), hblA (59.42%), hblC (44.93%), hblD (53.62%), nheA (84.06%), nheB (89.86%), nheC (84.06%), cesA (2.90%) and cesB (2.90%). The isolates belonging to groups IV and V had a significant higher prevalence of genes hblACD an group VI of cytK. The isolates obtained from dairy products had a significant lower prevalence of genes cytK and hblACD compared to those from equipments and raw milk/bulk tanks. The populational genomic analyses showed the diversity of isolates and variability of functions in B. cereus group in dairy production chain, with a high number of isolates potentially able to cause foodborne disease. Posteriorly, this study aimed to evaluate the viability of tyndallizating raw milk and to establish if milk’s pasteurization influences on the expression of genes related to the production of diarrheal toxins by B. cereus s.s. using a milk contamined experimentally. For this purpose, the tyndallization of raw milk was performed and later it was contaminated with a potentially toxigenic isolate, and, posteriorly, it was pasteurized, packaged and kept under refrigeration during 10 days. In established periods, samples of this product were collected in order to evaluate the expression of genes related to toxins production (hblACD, nheABC and cytK). The protocol used for tyndallization allowed a logarithmic reduction of population of B. cereus group and the expression of hblA gene was detected in samples from milk after 5 and 10 days after pasteurization. It was concluded that the proposed model was appropriate and the expression of hblA gene was not inihibited after milk pasteurization, highlighting the potential risk for consumers through consumption of contamined pasteurized milk.
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Palavras-chave
doenças veiculadas por alimentos, genômica populacional, microbiologia, RT-PCR, sequenciamento de futura geração, foodborne diseases, microbiology, next generation sequecing, populational genomic