scholarly journals KETAHANAN PANAS CEMARAN Escherichia coli, Staphylococcus aureus, Bacillus cereus dan BAKTERI PEMBENTUK SPORA YANG DIISOLASI DARI PROSES PEMBUATAN TAHU DI SUDAGARAN YOGYAKARTA

2015 ◽  
Vol 35 (03) ◽  
pp. 300
Author(s):  
Reny Mailia ◽  
Bara Yudhistira ◽  
Yudi Pranoto ◽  
Saiful Rochdyanto ◽  
Endang Sutriswati Rahayu
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Heat Resistance ◽  
Bacillus Cereus ◽  
Raw Materials ◽  
Raw Material ◽  
Isolation And Identification ◽  
Vegetative Cells ◽  
Protein Levels ◽  
Spore Forming Bacteria

Characteristics of tofu with higher a (0.89 to 0.90) and protein levels of 8% or more, made tofu to be a suitable medium for bacterial growth. This leads to out to be very easy to damage due to bacterial contamination. Contamination of bacteria is commonly found in the tofu because of contamination in the process making of tofu. Source of contamination can come out from the raw material, during the process of making tofu and hygienic sanitation level during processing. Generally, this study aimed to determine the level of contamination of Escherichia coli, Staphylococcus aureus, Bacillus cereus and spore-forming bacteria in the process of making tofu and study the properties of heat resistance of eachisolate. Phases of of the study started with the isolation and identification and then quantitative analysis of Escherichiawcoli, Staphylococcus aureus, Bacillus cereus and spore-forming bacteria in the tofu process from raw materials to end product, tofu, comprised from water and soybean, slurry, soymilk cooking, curd, whey and tofu. Isolates originating from the cooking process and the coagulation process was for testing the heat resistance (D value and Z value). D and Z values were calculated using linear regression. Escherichia coli found in the water, soybeans, soybean slurry, curd and tofu, the number 10 =4,83 min and the value of Z = 22.73°C. Staphylococcus aureus found in soybeans and curd, showed the number of 101-102 CFU/g. Escherichia coli GMP isolate had D60°C CFU/g. The Staphylococcus aureus GMP4 isolate, had D60°C 1=2.72 min and the value of Z = 18.87°C. The Staphylococcus aureus GMP 6 isolate, had D=2.54min and the value of Z = 18.18°C. Bacillus cereus found in the water, soybean, soybean slurry, soymilk cooking, curdand tofu, showed the number 102-103CFU/g. Bacillus cereus vegetative cells SK 2 had D=5.43 min and the value of Z = 22.72°C. Bacillus cereus vegetative cells SK 4 had D60°C 60°C =5.95 min and the value of Z = 22.22°C. Spore-forming bacteria found in water, soybean, soybean slurry from the grinding process, the process cooking of soymilk, the process of clotting, whey and tofu, showed the number of 102CFU/g.Keywords: Tofu, Escherichia coli, Staphylococcue aureus, Bacillus cereus, a spore forming bacteria, heat resistance 60°C ABSTRAKKarakteristik tahu dengan a0,89-0,90 dan kadar protein 8% atau lebih, menjadikan tahu sebagai media yang cocok bagipertumbuhan bakteri. Hal ini menyebabkan tahu menjadi sangat mudah rusak karena cemaran bakteri. Penelitian ini bertujuan untuk mengetahui tingkat cemaran Escherichia coli, Staphylococcus aureus, Bacillus cereus dan Bakteri pembentuk spora pada proses pembuatan tahu dan mempelajari sifat ketahanan panas dari masing-masing cemaran. Tahapan penelitian dimulai dari pengamatan proses pembuatan tahu, isolasi dan identifikasi dan analisa kuantitatif cemaran Escherichia coli, Staphylococcus aureus, Bacillus cereus dan bakteri pembentuk spora pada proses pembuatantahu. Isolat yang berasal dari proses pemasakan dan proses penggumpalan digunakan untuk pengujian ketahanan panasdengan melihat nilai D dan Z menggunakan regresi linier. Escherichia coli ditemukan pada air, kedelai, bubur kedelai, gumpalan tahu dan tahu, dengan jumlah 10w1-10CFU/g. Isolat Escherichia coli dari proses penggumpalan (GMP), nilaiD60°C 2=4,83 menit dan nilai Z=22,73°C. Staphylococcus aureus ditemukan pada kedelai, gumpalan tahu dan tahu, dengan jumlah 10=2,72 menit dan nilai Z =18,87°C. Untuk isolat Staphylococcus aureus GMP 6, nilai D1CFU/g.  Isolat Staphylococcus aureus GMP 4, memiliki nilai D60°C60°C =2,54 menit dan nilai Z =18,18°C. Bacillus cereus ditemukan pada air,kedelai, bubur kedelai, sari kedelai masak, gumpalan tahu dan tahu, dengan jumlah 102-10CFU/g. Sel vegetatif Bacilluscereus yang berasal dari sari kedelai (SK) 2, memiliki nilai D60°C3=5,43 menit dan nilai Z =22,72°C. Untuk sel vegetatif Bacillus cereus SK 4, memiliki nilai D60°C=5,95 menit dan nilai Z =22,22°C. Bakteri pembentuk spora ditemukan pada air, kedelai, bubur kedelai pada proses penggilingan, sari kedelai masak, gumpalan tahu, kecutan dan tahu, dengan jumlah 10CFU/g.Kata kunci: Tahu, Escherichia coli, Staphylococcue aureus, Bacillus cereus, bakteri pembentuk spora, ketahananpanas2    

Antibacterial Activities of Naturally Occurring Compounds against Antibiotic-Resistant Bacillus cereus Vegetative Cells and Spores, Escherichia coli, and Staphylococcus aureus

2004 ◽  
Vol 67 (8) ◽  
pp. 1774-1778 ◽  
Author(s):  
MENDEL FRIEDMAN ◽  
ROBERT BUICK ◽  
CHRISTOPHER T. ELLIOTT
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacillus Cereus ◽  
Resistant Bacteria ◽  
Vegetative Cells ◽  
Cinnamon Oil ◽  
Antibiotic Resistant ◽  
Thyme Oil ◽  
Oregano Oil ◽  
And Staphylococcus Aureus

After demonstrating the lack of effectiveness of standard antibiotics against the acquired antibiotic resistance of Bacillus cereus (NCTC 10989), Escherichia coli (NCTC 1186), and Staphylococcus aureus (ATCC 12715), we showed that the following natural substances were antibacterial against these resistant pathogens: cinnamon oil, oregano oil, thyme oil, carvacrol, (S)-perillaldehyde, 3,4-dihydroxybenzoic acid (β-resorcylic acid), and 3,4-dihydroxyphenethylamine (dopamine). Exposure of the three pathogens to a dilution series of the test compounds showed that oregano oil was the most active substance. The oils and pure compounds exhibited exceptional activity against B. cereus vegetative cells, with oregano oil being active at nanogram per milliliter levels. In contrast, activities against B. cereus spores were very low. Activities of the test compounds were in the following approximate order: oregano oil > thyme oil ≈ carvacrol > cinnamon oil > perillaldehyde > dopamine>β-resorcylic acid. The order of susceptibilities of the pathogens to inactivation was as follows: B. cereus (vegetative) ≫S. aureus ≈ E. coli ≫ B. cereus (spores). Some of the test substances may be effective against antibiotic-resistant bacteria in foods and feeds.

scholarly journals IDENTIFICATION OF FOOD PATHOGENS AND DETERMINATION OF THEIR DISTRIBUTION LEVEL IN UKRAINIAN FOOD PRODUCTS OF ANIMAL AND PLANT ORIGIN BY PCR METHOD

2019 ◽  
Vol 13 (4) ◽  
Author(s):  
O. Berhilevych ◽  
L. Pylypenko ◽  
V. Kasianchuk ◽  
A. Ilyeva ◽  
P. Shubin
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacillus Cereus ◽  
Clostridium Perfringens ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Raw Materials ◽  
Cow Milk ◽  
Plant Origin ◽  
Pcr Method

The foodborne pathogens cause serious public health problems in each country. In this regard, microbiological investigation is included in food safety management of the food chain. Molecular methods and mostly polymerase chain reaction (PCR) are considered highly sensitive, specific and rapid methods for pathogens detection from raw material and food. This study describes the using of specially designed and highly specific primers for PCR to identify 5 common and especially dangerous causeve agents of food poisoning and disease and to determine their level of distribution in food of animal and plant origin. The studies included the identification of methicillin-resistant Staphylococcus aureus (MRSA) and Cronobacter spp. (E. sakazakii) from raw milk, Shiga toxin-producing strains of Escherichia coli (STEC) from beef and swine carcasses, Bacillus cereus and Clostridium perfringens from various types of plant and animal raw materials and products of its processing - fruits, vegetables, berries, dried and preserved products, food concentrates, half-canned food. A total of 397 food samples were investigated to detect these pathogens using classical bacteriological methods and PCR. It was found that the distribution of foodborne pathogens in the studied products of animal and plant origin was as follows: Staphylococcus aureus (MRSA) and Cronobacterspp. (E. sakazakii) in raw cow milk in 6.5% and 19.4% of cases, respectively; shiga-toxin-producing Escherichia coli (STEC) from beef and pork carcasses in 8.1% and 5.7%; Bacillus cereus and Clostridium perfringens from different types of plant and animal raw materials and their processing products averages 27.5 % and 7.7 %, respectively. The advantages of molecular biological methods to which the PCR method relates, include their speed, as well as the specificity of identification of microorganisms by the features of genetic regions of genes that carry information about their pathogenicity factors. It has been found that the rate of detection of these pathogens when using the PCR method in comparison with classical methods increases at least 5-9 times. This data will be useful for assessing microbiological risk and will help authorities develop strategies to reduce consumer health risks.

scholarly journals Synthesis of CoFe 2 O 4 /BiOI Nanocomposite: Effects on Staphylococcus aureus, Escherichia coli, Pseudomonas Aeruginosa and Bacillus Cereus

2021 ◽  
Author(s):  
Davood Gheidari ◽  
Morteza Mehrdad ◽  
Saloomeh Maleki ◽  
Samanesadat Hosseini
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Bacillus Cereus ◽  
Antibacterial Properties ◽  
Limited Area ◽  
X Ray Diffraction ◽  
X Ray ◽  
Inhibition Concentration ◽  
Resolution Mapping

Abstract With the increase of general knowledge and the advancement of science and technology, antibacterial substances were used more than antibiotics. In our current study, the antibacterial virtues of CFO/BiOI nanocomposite were investigated due to its high importance on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus. MIC, MBC , Disk Diffusion and IC50 tests Cefalotin (CF), Amoxicillin (AMX), Gentamicin (GM), Trimethoprim-sulphamethoxazole (SXT) and Ceftriaxone (CRO) antibiotics in concentration 30W, 10i, 10t , 25h and 30 were used to find the antibacterial properties of the synthesized nanocomposite, respectively. For the synthesis of nanocomposites polyethylene glycol (PEG) and sulfonic acid was used as a solvent. It is noteworthy that the synthesis was performed by heat dissolution method without the presence of surfactant. Also, various techniques such as X-Ray Diffraction(XRD), Scanning Electron Microscope (SEM), High resolution mapping and Energy Dispersive X-ray spectroscopy (EDAX) have been used to determine the properties of produced nanocomposites. SEM test results showed that the formed nanoparticles were globular and their size was limited area of 22 to 34 nm. The results showed CFO / BiOI nanocomposite exhibits strong significant biological activity against Bacillus cereus. The results of MBC (Minimum Bactericidal Concentration) and MIC (Minimum Inhibition Concentration) tests for CFO/BiOI nanocomposites on bacteria were examined in the range of 0.12-0.48 mg/ml and 0.06 to 0.24 mg/ml respectively. According to the results, the minimum IC50 value was determined at a concentration of 0.061 mg/ml. On the other hand, the most resisting and susceptible bacteria in this method were Pseudomonas aeruginosa and Bacillus cereus, respectively. These findings are identical to those of a prior study on CoFe2O4 nanoparticles antibacterial properties. MBC of the nanocomposites, 50 µl from all the tubes that showed no obvious bacterial growth were distributed on BHI agar plates and incubated for 24 h at 37 ◦C. The MBC endpoint is defined as the lowest concentration which killed 98% of the bacterial population.

scholarly journals A New Prenylated Flavanoid with Antibacterial Activity from Propolis Collected in Egypt

2010 ◽  
Vol 5 (1) ◽  
pp. 1934578X1000500 ◽  
Author(s):  
Ashraf El-Bassuony ◽  
Sameh AbouZid
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Bacillus Cereus ◽  
Spectroscopic Methods ◽  
Gram Positive ◽  
Gram Negative

A novel prenylated flavanoid, isonymphaeol-D (1), together with two known compounds, isonymphaeol-B (2) and nymphaeol-B (3), were isolated from Egyptian propolis. The structures of the isolated compounds were determined by various spectroscopic methods. 1 exhibited antibacterial activity against Gram-positive (Bacillus cereus, Staphylococcus aureus) and Gram-negative strains (Serratia sp., Pseudomonos sp., Escherichia coli).

Fate of Pathogens Inoculated onto Vacuum-Packaged Sliced Hams to Simulate Contamination During Packaging1

1979 ◽  
Vol 42 (6) ◽  
pp. 464-469 ◽  
Author(s):  
M. E. STILES ◽  
L.-K. NG
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Salmonella Typhimurium ◽  
Bacillus Cereus ◽  
Clostridium Perfringens ◽  
Subsequent Treatment ◽  
Vacuum Packaging ◽  
E Coli ◽  
Survival And Growth ◽  
And Staphylococcus Aureus

Ham and chopped ham from two manufacturers were contaminated with five enteropathogens: Bacillus cereus, Clostridium perfringens, Escherichia coli, Salmonella typhimurium and Staphylococcus aureus, at time of slicing and vacuum-packaging, to simulate contamination by manufacturer. Subsequent treatment of the samples, representing sound and undesirable retail handling and consumer use conditions, indicated marked differences in the fate of the pathogens between these products and within product type between the two manufacturers. Greatest differences were observed between the chopped ham products. All pathogens, except C. perfringens, grew actively in fresh ham and chopped ham with abusive holding at 30 and 21 C. After storage at 4 or 10 C for 30 days, B. cereus and C. perfringens were no longer detected, even after subsequent holding at 30 or 21 C for 24 h. E. coli survival and growth was variable, S. typhimurium survived well and grew under some conditions and S. aureus was generally inhibited at high levels of competition.

scholarly journals Antibacterial Diterpenes from the Roots of Ceriops tagal

2008 ◽  
Vol 3 (1) ◽  
pp. 1934578X0800300 ◽  
Author(s):  
Musa Chacha ◽  
Renameditswe Mapitse ◽  
Anthony J. Afolayan ◽  
Runner R. T. Majinda
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Bacillus Cereus ◽  
Spectroscopic Data ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Bacterial Strains ◽  
Agar Dilution ◽  
Ceriops Tagal

Investigation of the roots of Ceriops tagal led to the isolation of a new isopimarane, together with the known diterpenes isopimar-8(14)-en-15,16-diol and erythroxyl-4(17),15(16)-dien-3-one. The structure of the new compound was identified as isopimar-8(14)-en-16-hydroxy-15-one. These structures were determined from extensive spectroscopic data analysis. The isolates were screened for antibacterial activity using the agar dilution method against ten test bacterial strains ( Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Micrococcus kristinae, Pseudomonas aeruginosa, Salmonella pooni, Serratia marcescens, Staphylococcus aureus, S. epidermidis and Streptococcus pyrogens). Isopimar-8(14)-en-16-hydroxy-15-one exhibited activity, with MIC values of 0.5 mg/mL against Streptococcus pyrogens; 0.25 mg/mL against Salmonella pooni and 0.1 mg/mL against Bacillus cereus, Staphylococcus aureus and Micrococcus kristinae.

Synthesis and Antibacterial Activities of N-Phenylpyrazolines from Veratraldehyde

2017 ◽  
Vol 901 ◽  
pp. 124-132
Author(s):  
Artania Adnin Tri Suma ◽  
Tutik Dwi Wahyuningsih ◽  
Deni Pranowo
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Bacillus Subtilis ◽  
Bacillus Cereus ◽  
Shigella Flexneri ◽  
Inhibition Zone ◽  
Antibacterial Activities ◽  
Diffusion Method ◽  
Agar Well Diffusion Method

Some novel N-phenylpyrazolines were synthesized and investigated for their antibacterial activitiy. Chalcones 2-4 which were prepared from acetophenone and veratraldehyde derivatives were reacted with phenylhydrazine to give N-phenylpyrazolines 5-7. All of the synthesized compounds were characterized using FTIR, GC-MS, and NMR spectrometers. Further, antibacterial activity of N-phenylpyrazolines were evaluated by agar well-diffusion method against Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Escherichia coli, and Shigella flexneri. The highest activity (highest inhibition zone) of compound 5 was 2.6 mm (at 1000 ppm) against B. subtillis, compound 6 was 7.25 mm (at 1000 ppm) against S. aureus, and compound 7 was 6.75 mm (at 500 ppm) against S. aureus. The results indicated that compound 6 and 7 exhibited promising antibacterial activity.

STUDIES OF ASPOROGENIC MUTANTS OF BACILLUS CEREUS: PRELIMINARY INVESTIGATIONS WITH CALCIUM AND ZINC

1962 ◽  
Vol 8 (6) ◽  
pp. 823-833 ◽  
Author(s):  
J. J. Cooney ◽  
D. G. Lundgren
Keyword(s):  
Heat Resistance ◽  
Bacillus Cereus ◽  
Trichloroacetic Acid ◽  
Minimal Medium ◽  
Soluble Fraction ◽  
Dipicolinic Acid ◽  
Insoluble Fraction ◽  
Temperature Sensitive ◽  
Vegetative Cells ◽  
Culture Cycle

The physiology of spore formation was studied in Bacillus cereus and a temperature-sensitive asporogenic mutant. The parent organism sporulates when cultured in a minimal medium at either 28 °C or 37 °C while the mutant sporulates only at 28 °C. The blocking of sporulation at 37 °C has been referred to as "abortive" sporulation. Uptake of calcium and zinc was followed during growth and sporulation or "abortive" sporulation. Calcium and dipicolinic acid (DPA) levels in sporogenic cultures increased as the medium calcium was increased. The asporogenic mutant took up less calcium and synthesized little DPA. Heat resistance of spores increased as the calcium and DPA increased. Over 99% of Ca45or Zn65were released from labelled spores when autoclaved to release DPA. Chemical fractionations were made of cells labelled with Zn65and Ca45and harvested at different times during the culture cycle. Smaller percentages of calcium than of zinc were located in the cold trichloroacetic acid soluble fraction. The alcohol-soluble, ether-insoluble fraction of spores contained a greater percentage of calcium than was found in vegetative cells. Cells which had undergone "abortive" sporulation contained the same percentage of calcium in this fraction as homologous vegetative cells.

scholarly journals Microbial Assessment of Bed Linens in Ekiti State University Students’ Hostels

2020 ◽  
pp. 43-52
Author(s):  
T. O. Olowomofe ◽  
J. O. Oluyege ◽  
A. O. Ogunlade ◽  
D. T. Makinde
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacillus Cereus ◽  
Proteus Mirabilis ◽  
Disease Transmission ◽  
Enterobacter Aerogenes ◽  
Antibiotics Resistance ◽  
Klebsiella Aerogenes ◽  
State University ◽  
Plasmid Profiling

Bed linen is clearly recognized as a potential reservoir for microorganisms and could be a vector of disease transmission. The present study was aimed at isolating and characterizing bacteria and fungi from different kinds of bed linen of student in Ekiti State University hostels. Pour plate method was used for the enumeration of total bacteria count from the posterior and anterior end of the bed linen. The average bacteria count for the anterior and posterior end was 7.46 and 7.16 Log10 CFU/ml respectively. The most dominant microbial species were bacteria and these were mostly found in the environment and on human skin. The bacteria isolated were Staphylococcus aureus, Staphylococcus epidermidis, Proteus mirabilis, Enterobacter aerogenes, Bacillus cereus, Escherichia coli and Klebsiella aerogenes. Bacillus cereus had the highest frequency of occurrence (25%), followed by Staphylococcus aureus (15%), Escherichia coli (15%), Klebsiella aerogenes (15%), Staphylococcus epidermis (15%), Enterobacter aerogenes (10%) and Proteus mirabilis (5%). The fungi isolates were Aspergillus sulphureus, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus and Fusarium spp. Antibiotics susceptibility test was carried out on the bacteria isolates with gram negative bacteria showing resistance to Cotrimoxazole and gram positive bacteria showing resistance to Amplicillin. Most of the bacteria isolates have multiple antibiotics resistance. The bacterial isolates were susceptible to Ciprofloxacin. Plasmid profiling was also done with Escherichia coli having three high molecular weight plasmids, Bacillus cereus and Enterobacter aerogenes do not possess plasmid. The identified species are suspected to be opportunistic pathogens for human, representing a risk for people with weakened immune system, especially in cases of super-infection.

scholarly journals Produção de itens de ensaio de proficiência contendo bactérias em matriz frango utilizando a técnica de liofilização

2021 ◽  
Vol 9 (2) ◽  
pp. 59-67
Author(s):  
Ingrid Camelo da Silva ◽  
◽  
Paula Vasconcelos Costa ◽  
Luiza Vasconcellos ◽  
Pablo Tavares Coimbra ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacillus Cereus ◽  
Salmonella Enteritidis ◽  
E Coli ◽  
Iso Guide 35

Introdução: A participação em ensaios de proficiência (EP) é utilizada para avaliar a competência de laboratórios de ensaio e calibração. Objetivo: Avaliar a viabilidade da técnica de liofilização na produção de seis lotes de itens de EP, dois contendo Escherichia coli, dois contendo Bacillus cereus e Staphylococcus aureus concomitantemente, e dois contendo Salmonella Enteritidis em matriz frango desfiado cozido. Método: Foram realizados testes de homogeneidade segundo o protocolo harmonizado e de estabilidade em longo prazo pelo modelo clássico e curto prazo pelo modelo isócrono segundo a ISO Guide 35. Resultados: Todos os lotes produzidos foram considerados suficientemente homogêneos. No estudo de estabilidade em longo prazo, todos os lotes se apresentaram suficientemente estáveis nas temperaturas de -80 ± 10ºC e -20 ± 4ºC, exceto o lote contendo B. cereus e S. aureus. Os outros lotes apresentaram estabilidade por pelo menos 126 dias a -80 ± 10ºC e 84 dias a -20 ± 4ºC. Na avaliação da estabilidade em curto prazo, foram analisados apenas os lotes suficientemente estáveis no estudo em longo prazo. Os lotes foram suficientemente estáveis nas temperaturas de 5 ± 3ºC e 35 ± 2ºC, com exceção do lote contendo Salmonella Enteritidis a 35 ± 2ºC, devido ao decréscimo significativo da concentração celular. Conclusões: A técnica de liofilização foi satisfatória para produção de itens de ensaio contendo E. coli e Salmonella Enteritidis em matriz frango viáveis para utilização em um EP, sendo que o lote contendo Salmonella Enteritidis deve ser transportado aos laboratórios participantes em temperatura ≤ 8ºC por até quatro dias. Lotes contendo S. aureus e B. cereus, simultaneamente, apresentaram estabilidade insuficiente, indicando que a produção de lotes individuais contendo cada bactéria individualmente é necessária.

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