Risks and critical points assessment of contamination by Enterococcus spp. and Bacillus cereus in the processing of ricota

Orientador: Arnaldo Yoshiteru KuayeDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: A ricota é um tipo de queijo fresco de origem italiana, obtido pela precipitação das proteínas do soro do queijo por acidificação associada ao calor. Por suas características nutricionais, físico-químicas e bioquímicas apresenta-se propícia ao desenvolvimento microbiano. No processamento deste produto destacam-se o Bacillus cereus, pela sua capacidade de esporular e ser um contaminante potencial do leite e do ambiente e as bactérias do gênero Enterococcus, pela característica ubíqua, habilidade de sobrevivência à condições diversas de pH, temperatura e salinidade e ocorrência em casos de infecções hospitalares. Os objetivos deste trabalho foram: a) verificar as possíveis fontes de contaminação de ricota por B. cereus e Enterococcus spp. ao longo do processamento; b) identificar as espécies de enterococos, avaliar o potencial de patogenicidade e o perfil de resistência destas espécies a antibióticos de uso clínico; e, c) avaliar a conformidade das amostras de ricota aos padrões microbiológicos legais. Amostras de leite cru e pasteurizado, soro de queijo, ricotas antes e após embalagem, superfícies diversas do ambiente e do ar obtidas em três coletas de laticínio da região Sul de Minas Gerais foram submetidas à determinação de B. cereus e Enterococcus spp. As contagens de B. cereus em leite cru, pasteurizado e soro de queijo, foram de 1,4 x104, 1,2 x103 e 1,0 x103 UFC/ml, respectivamente, e, apenas uma amostra de ricota final apresentou valor maior que 102 UFC/g. Dentre as 60 amostras ambientais, destaca-se a forma de moldagem da ricota, que apresentou contaminação persistente e contagem de até 1,7x107 UFC/unidade de B. cereus. Enterococcus spp. foram encontradas em todas as amostras de ricota, com contagens entre 103 e 107 UFC/g, e em todas de leite cru, com contagens de até 1,9 x106 UFC/ml. Nas superfícies de forma e tela, vassoura, parede e ralo foram encontrados valores superiores a 105 UFC/unidade; já para tanques, bancada da área de embalagem e caixa de recolhimento do soro os números foram superiores a 102 UFC/unidade. De um total de 136 isolados, confirmados para o gênero Enterococcus, 71,3% (97/136) foram confirmados para a espécie E. faecium e 20,6% (28/136) para E. faecalis, pela técnica de PCR. Os isolados (66) de E. faecium e E. faecalis das amostras de produto final submetidas aos testes fenotípicos resultaram em 89,4% (59/66) positivos para hemólise, nenhum para gelatinase (0/66) e 98,5% (65/66) positivos para termonuclease. A maioria dos isolados de E. faecium e E. faecalis mostrou resistência a pelo menos três dos 5 antimicrobianos testados, destacando-se que 100% deles apresentaram resistência à vancomicina. De 15 amostras de ricota avaliadas após 21 dias de armazenamento sob refrigeração, 13,3% (2/15) estavam em desacordo com o padrão legal para estafilococos coagulase positiva e em nenhuma delas foi detectada a presença de Salmonella, Listeria monocytogenes e coliformes termotolerantes. A natural e inevitável contaminação da matéria-prima e do ambiente de processamento de ricota por B. cereus e Enterococcus spp., bactérias estas potencialmente patogênicas, tem na eficiência dos programas de higienização um fator indispensável para o seu controleAbstract: The ricotta is a type of fresh cheese of Italian origin, obtained by precipitation of proteins from cheese whey by acidification associated with the heat. Because of its nutritional, physicochemical and biochemical characteristics it is conducive to microbial growth. On the processing of this product it can be emphasized the Bacillus cereus, due to its ability to sporulate and be a potential contaminant of milk and the environment, and the bacteria of the genus Enterococcus, due its ubiquitous characteristic, ability to survive the various conditions of pH, temperature and salinity and appearance in cases of hospital infections. The objectives of the present work were: (a) to verify the possible sources of ricotta contamination by B. cereus and Enterococcus spp. during processing; (b) to identify the species of enterococci, evaluate the pathogenic potential and the resistance profile of these species to antibiotics of clinical use; and (c) to assess the conformity of samples of ricotta under legal microbiological standards. Samples of raw and pasteurized milk, cheese whey, ricotta before and after packaging, various surfaces of the environment and of air obtained from three collections on a dairy industry located in southern Minas Gerais were subjected to the determination of B. cereus and Enterococcus spp. The counts of B. cereus in raw and pasteurized milk and in cheese whey were 1,4 x104, 1,2 x103 and 1,0 x103 CFU/ml, respectively, and only one sample of final ricotta had levels of B. cereus higher than 1,7x107 CFU/unity. Among the 60 environmental samples, it can be highlighted the mold where the ricotta is shaped, which showed persistent contamination and count up to 1,7x107 CFU/unity for B. cereus. All the samples of ricotta and raw milk showed the presence of Enterococcus spp. with counts between 103 and 107 CFU/g and up to 1,9 x106 CFU/ml, respectively. On the surfaces of the mold, mesh, broom, wall and drain it were found counts higher than 105 CFU/unity; for the tank, stand in the area of packaging and box for the collection of serum the counts were higher than 102 CFU/unity. Over 136 isolated of the genus Enterococcus, 71,3% (97/136) were confirmed for the species E. faecium and 20,6% (28/136) for E. faecalis by PCR technique. The isolates (66) of E. faecium and E. faecalis taken from the samples of the final product submitted to phenotypic tests resulted in 89,4% (59/66) positive for hemolysis, none for gelatinase (0 / 66) and 98,5% (65/66) positive for thermonuclease. Most of the isolates of E. faecium and E. faecalis showed resistance to at least three of the 5 antimicrobials, highlighting that 100% of them were resistant to vancomycin. From 15 samples of ricotta evaluated after 21 days of refrigerated storage, 13,3% (2/15) were in disagreement with the legal standard for coagulase-positive staphylococci and none were detected the presence of Salmonella, Listeria monocytogenes and thermotolerant coliforms. The natural and inevitable contamination of raw-materials and of the processing environment of ricotta by B. cereus and Enterococcus spp., which are potentially pathogenic bacteria, have in the efficiency of the hygiene programs a essential factor for its controlMestradoMestre em Tecnologia de Alimento

Quorum sensing em cepas de enterococcus faecium, enterococcus faecalis e bacillus cereus isoladas do processamento de ricota

The quorum sensing phenomenon is a process of intra- and inter-species microbial communication involving the production and detection of extracellular signaling molecules. The autoinducer AI-2 has been proposed to serve as a ‘universal signal’ for interspecies communication. This study aimed to evaluate the capability of Enterococcus faecium, Enterococcus faecalis, and Bacillus cereus strains isolated from ricotta processing to produce quorum sensing signalling molecules (AI-2). The strains were evaluated for the presence of the luxS gene using the polymerase chain reaction. AI-2 quorum sensing signalling molecules were measured in relative light units (RLUs) using a luminometer. A total of 74% of E. faecium, 91% of E. faecalis, and 95% of B. cereus isolates were positive for luxS gene. In addition, the induced bioluminescence in Vibrio harveyi BB170 was observed in all strains, indicating the presence of the AI-2 autoinducer482FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2010/10507-7O fenômeno quorum sensing corresponde a um processo de comunicação intra e interespécies microbianas e é mediado por sinais químicos extracelulares, denominados moléculas sinalizadoras ou auto indutoras (AI). A molécula AI2 está envolvida na comunicação interespécies, denominada sistema “universal” de comunicação. Este estudo teve como objetivo avaliar a capacidade de Enterococcus faecium, Enterococcus faecalis e Bacillus cereus isolados do processamento de ricota em produzir moléculas sinalizadoras de Quorum sensing (AI-2). Os isolados foram avaliados quanto à presença do gene luxS utilizando a reação em cadeia da polimerase (PCR). As moléculas sinalizadoras (AI-2) foram medidas em unidades relativas de luz (RLU) através de um luminômetro. Um total de 74% dos isolados de E. faecium, 91% de E. faecalis e 95% de B. cereus foram positivos para o gene luxS. Além disso, todos os isolados apresentaram capacidade de induzir o fenômeno de bioluminescência em Vibrio harveyi BB170, indicando a presença de auto indutores AI-

Biofilms Of Enterococcus Faecalis And Enterococcus Faecium Isolated From The Processing Of Ricotta And The Control Of These Pathogens Through Cleaning And Sanitization Procedures.

The biofilm formation of Enterococcus faecalis and Enterococcus faecium isolated from the processing of ricotta on stainless steel coupons was evaluated, and the effect of cleaning and sanitization procedures in the control of these biofilms was determined. The formation of biofilms was observed while varying the incubation temperature (7, 25 and 39°C) and time (0, 1, 2, 4, 6 and 8days). At 7°C, the counts of E. faecalis and E. faecium were below 2log10CFU/cm(2). For the temperatures of 25 and 39°C, after 1day, the counts of E. faecalis and E. faecium were 5.75 and 6.07log10CFU/cm(2), respectively, which is characteristic of biofilm formation. The tested sanitation procedures a) acid-anionic tensioactive cleaning, b) anionic tensioactive cleaning+sanitizer and c) acid-anionic tensioactive cleaning+sanitizer were effective in removing the biofilms, reducing the counts to levels below 0.4log10CFU/cm(2). The sanitizer biguanide was the least effective, and peracetic acid was the most effective. These studies revealed the ability of enterococci to form biofilms and the importance of the cleaning step and the type of sanitizer used in sanitation processes for the effective removal of biofilms.20097-10

Behavior of listeria monocytogenes in a multi-species biofilm with enterococcus faecalis and enterococcus faecium and control through sanitation procedures

The formation of mono-species biofilm (Listeria monocytogenes) and multi-species biofilms (Enterococcus faecium, Enterococcus faecalis, and L. monocytogenes) was evaluated. In addition, the effectiveness of sanitation procedures for the control of the multi-species biofilm also was evaluated. The biofilms were grown on stainless steel coupons at various incubation temperatures (7, 25 and 39 °C) and contact times (0, 1, 2, 4, 6 and 8 days). In all tests, at 7 °C, the microbial counts were below 0.4 log CFU/cm2 and not characteristic of biofilms. In mono-species biofilm, the counts of L. monocytogenes after 8 days of contact were 4.1 and 2.8 log CFU/cm2 at 25 and 39 °C, respectively. In the multi-species biofilms, Enterococcus spp. were present at counts of 8 log CFU/cm2 at 25 and 39 °C after 8 days of contact. However, the L. monocytogenes in multi-species biofilms was significantly affected by the presence of Enterococcus spp. and by temperature. At 25 °C, the growth of L. monocytogenes biofilms was favored in multi-species cultures, with counts above 6 log CFU/cm2 after 8 days of contact. In contrast, at 39 °C, a negative effect was observed for L. monocytogenes biofilm growth in mixed cultures, with a significant reduction in counts over time and values below 0.4 log CFU/cm2 starting at day 4. Anionic tensioactive cleaning complemented with another procedure (acid cleaning, disinfection or acid cleaning + disinfection) eliminated the multi-species biofilms under all conditions tested (counts of all micro-organisms < 0.4 log CFU/cm2). Peracetic acid was the most effective disinfectant, eliminating the multi-species biofilms under all tested conditions (counts of the all microorganisms < 0.4 log CFU/cm2). In contrast, biguanide was the least effective disinfectant, failing to eliminate biofilms under all the test conditions200512FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2010/10507-

Behavior Of Listeria Monocytogenes In A Multi-species Biofilm With Enterococcus Faecalis And Enterococcus Faecium And Control Through Sanitation Procedures.

The formation of mono-species biofilm (Listeria monocytogenes) and multi-species biofilms (Enterococcus faecium, Enterococcus faecalis, and L. monocytogenes) was evaluated. In addition, the effectiveness of sanitation procedures for the control of the multi-species biofilm also was evaluated. The biofilms were grown on stainless steel coupons at various incubation temperatures (7, 25 and 39°C) and contact times (0, 1, 2, 4, 6 and 8days). In all tests, at 7°C, the microbial counts were below 0.4 log CFU/cm(2) and not characteristic of biofilms. In mono-species biofilm, the counts of L. monocytogenes after 8days of contact were 4.1 and 2.8 log CFU/cm(2) at 25 and 39°C, respectively. In the multi-species biofilms, Enterococcus spp. were present at counts of 8 log CFU/cm(2) at 25 and 39°C after 8days of contact. However, the L. monocytogenes in multi-species biofilms was significantly affected by the presence of Enterococcus spp. and by temperature. At 25°C, the growth of L. monocytogenes biofilms was favored in multi-species cultures, with counts above 6 log CFU/cm(2) after 8days of contact. In contrast, at 39°C, a negative effect was observed for L. monocytogenes biofilm growth in mixed cultures, with a significant reduction in counts over time and values below 0.4 log CFU/cm(2) starting at day 4. Anionic tensioactive cleaning complemented with another procedure (acid cleaning, disinfection or acid cleaning+disinfection) eliminated the multi-species biofilms under all conditions tested (counts of all micro-organisms<0.4 log CFU/cm(2)). Peracetic acid was the most effective disinfectant, eliminating the multi-species biofilms under all tested conditions (counts of the all microorganisms <0.4 log CFU/cm(2)). In contrast, biguanide was the least effective disinfectant, failing to eliminate biofilms under all the test conditions.2005-1

Dissemination Of Enterococcus Faecalis And Enterococcus Faecium In A Ricotta Processing Plant And Evaluation Of Pathogenic And Antibiotic Resistance Profiles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)In this work, the sources of contamination by Enterococcus spp. in a ricotta processing line were evaluated. The isolated strains were tested for virulence genes (gelE, cylA,B, M, esp, agg, ace, efaA, vanB), expression of virulence factors (hemolysin and gelatinase), and the resistance to 10 different antibiotics. Enterococcus faecium and Enterococcus faecalis were subjected to discriminatory identification by intergenic spacer region (ITS)-polymerase chain reaction and sequencing of the ITS region. The results showed that Enterococcus spp. was detected in the raw materials, environment samples and the final product. None of the 107 Enterococcus isolates were completely free from all virulence genes considered. A fraction of 21.5% of the isolates containing all of the genes of the cylA, B, M operon also expressed -hemolysis. Most of the isolates showed the gelE gene, but only 9.3% were able to hydrolyze gelatin. In addition, 23.5% of the observed Enterococcus isolates had the vanB gene but were susceptible to vancomycin in vitro. The dissemination of antibiotic-resistant enterococci was revealed in this study: 19.3% of the E. faecium samples and 78.0% of the E. faecalis samples were resistant to at least one of the antibiotics tested. Sequencing of region discriminated 5 and 7 distinct groups among E. faecalis and E. faecium, respectively. Although some similarity was observed among some of the isolates, all E. faecalis and E. faecium isolates had genetic differences both in the ITS region and in the virulence profile, which makes them different from each other. Practical ApplicationEnterococcus spp. were found in raw materials, processing environment and ricotta. Mold was considered the major font of contamination in the processing ricotta line. The expression of hemolytic activity was greater for Enterococcus faecium isolates, while the expression of gelatinase and antibiotic resistance was greater for Enterococcus faecalis. Sequencing showed a wide diversity in the isolates of E. faecalis and E. faecium.804M765M775Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Apoio ao Ensino, a Pesquisa e a Extensao (FAEPEX), UNICAMPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2010/10507-7

Biofilm formation on stainless steel as a function of time and temperature and control through sanitizers

Enterococcus spp. contamination was screened from a Minas Frescal cheese processing line. Biofilm formation of Enterococcus faecium and Enterococcus faecalis isolates was evaluated and the effect of sanitization procedures in the control of these biofilms was investigated. Enterococcus spp. were detected in raw milk, milk machine, door handle, floor, drain, thermometer, and Minas Frescal cheese. Biofilm formation on stainless steel was modelled as a function of time (0, 1.2, 4, 6.8, and 8 days) and temperature (7, 13, 27, 41, and 47 °C) using response surface methodology. The model showed that E. faecium biofilms were formed from 1 to 8 days at 12–47 °C, while E. faecalis biofilms were formed from 1 to 8 days at 10–43 °C. None of the sanitizers (sodium hypochlorite 100 mg L−1, peracetic acid 300 mg L−1, and chlorhexidine digluconate 400 mg L−1) was able to completely eliminate the biofilms68916CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ140334/2009-

Biofilm formation of enterococcus faecium on stainless steel surfaces: modeling and control by disinfection agents

The objective of this study was to evaluate the biofilm formation by Enterococcus faecium, isolated from coalho cheese, on stainless steel and its control by disinfection agents. The biofilms formation on stainless steel 304#4 was modeled as a function of time (0, 1.2, 4, 6.8, and 8 days) and temperature (4.5, 10.5, 25.0, 39.5, and 45.5 °C) using response surface methodology. Analysis of variance revealed a significant (p  5 log cfu/cm2). E. faecium was not eliminated by the sodium hypochlorite at 100 mg/L and peracetic acid at 300 mg/L demonstrating the difficulty of disinfection stainless steel surfaces after the onset of biofilm formation413CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQSem informaçã