Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

RESUMO Este consenso de recomendações para a radioterapia (RT) hipofracionada de toda a mama foi organizado pela Sociedade Brasileira de Radioterapia (SBRT) considerando o cenário ideal para indicação e segurança na tecnologia aplicada. Questões de controvérsias e contraindicações (RT hipofracionada em pacientes submetidas à quimioterapia [QT], RT hipofracionada nas drenagens linfáticas, RT hipofracionada após mastectomia com ou sem reconstrução imediata, a realização de reforço de dose em leito cirúrgico [ou boost], RT hipofracionada em pacientes com idade menor que 50 anos, RT hipofracionada em mamas volumosas, RT hipofracionada em histologia de carcinoma in situ [CDIS]) foram discutidas em encontro presencial, sendo o consenso atingido quando existisse concordância de pelo menos 75% dos panelistas. O grau de recomendação foi também sugerido de acordo com o nível de evidência científico disponível, qualificado entre fraco, médio ou forte. Assim, este consenso deverá servir para auxiliar os especialistas da radioterapia brasileira em relação às indicações e particularidades dessa técnica, como uma alternativa segura e viável para a realidade nacional.

SUMMARY This recommendation consensus for hypofractionated whole-breast radiotherapy (RT) was organized by the Brazilian Society of Radiotherapy (SBRT) considering the optimal scenario for indication and safety in the technology applied. All controversies and contraindication matters (hypofractionated RT in patients who underwent chemotherapy [CT], hypofractionated RT in lymphatic drainage, hypofractionated RT after mastectomy with or without immediate reconstruction, boost during surgery, hypofractionated RT in patients under 50 years old, hypofractionated RT in large breasts, hypofractionated RT in histology of carcinoma in situ [DCIS]) was discussed during a meeting in person, and a consensus was reached when there was an agreement of at least 75% among panel members. The grade for recommendation was also suggested according to the level of scientific evidence available, qualified as weak, medium, or strong. Thus, this consensus will aid Brazilian radiotherapy experts regarding indications and particularities of this technique as a viable and safe alternative for the national reality.

Foi realizado o monitoramento epidemiológico molecular de estirpes de Staphylococcus aureus potencialmente toxigênicas isoladas no processo de produção do queijo Minas frescal em micro-usina do Estado de São Paulo. Para tanto, foram realizadas seis amostragens durante o período de junho de 2008 a julho de 2009, de modo a perfazer um total de 140 amostras. Essas amostras foram colhidas da superfície dos tanques de recepção e estocagem do leite cru, da superfície do tanque de equilíbrio do leite pasteurizado, da rede de abastecimento de água, das tubulações e equipamentos, das mãos do manipulador e de queijos embalados prontos para consumo. As colônias isoladas em Agar Baird-Parker confirmadas como cocos Gram positivos e que mostravam-se positivas às provas de catalase, coagulase e da produção de acetoína, foram submetidas à extração do DNA bacteriano através da utilização do Kit Invitek - Uniscience®. A confirmação molecular da espécie dos isolados e a presença de enterotoxinas SEA, SEB, SEC, SED e da toxina TSST-1 foi realizada a partir da amplificação dos fragmentos de DNA cromossômico específico. Entre as 74 estirpes de estafilococos coagulase positivos isoladas, somente 41 (55.4%) amostras foram confirmadas como sendo Staphylococcus aureus, das quais 25 (61,0%) mostraram-se positivas na pesquisa de toxinas estafilocócicas. A enterotoxina de maior frequência identificada foi a SEA. As estirpes de Staphylococcus aureus toxigênico foram mais isoladas nas mãos do manipulador (16,0%), no leite cru do tanque de recepção (12,0%), no leite pasteurizado para elaboração do queijo (12,0%) e no queijo Minas frescal pronto para consumo (12,0%).

We studied the molecular epidemiology of Staphylococcus aureus strains potentially toxigenic, isolated from the production process of Minas frescal cheese in a small dairy plant in the state of São Paulo. For this, samples were taken during the period from June 2008 to July 2009. Samples were collected from the surface of the receiving and storage tanks of raw milk, the surface of the balance tank of pasteurized milk, the water supply system, the pipes and equipments, the hands of the handler and from the packaged cheese, totaling 140 samples. The colonies isolated on Baird-Parker Agar confirmed as Gram positive and positive for catalase, coagulase and acetoin production, were submitted to extraction of bacterial DNA using the Invitek - Uniscience® kit. Confirmation of the isolated species and enterotoxins SEA, SEB, SEC, SED and TSST-1 toxin was carried out through the amplification of specific fragments of chromosomal DNA. Among the 74 strains of isolated coagulase-positive staphylococci, only 41 (55.4%) strains were confirmed as Staphylococcus aureus, of which 25 (61.0%) were positive to the presence of staphylococcal toxins. The most frequently identified enterotoxin was SEA. The toxigenic strains of Staphylococcus aureus were more frequently isolated from hands of the handler (16.0%), raw milk receiving tank (12.0%), pasteurized milk for cheese making (12.0%) and fresh white cheese ready for consumption (12.0%).

Biofilm formation is considered to be a selective advantage for Staphylococcus aureus mastitis isolates by facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers. The objective of this study was to determine the sensitivity and specificity of three techniques for the detection of S. aureus biofilm-positive strains. Two phenotypic tests, including growth on microtitre plates and Congo red agar, were compared with a PCR technique using 94 S. aureus strains obtained from cows with subclinical mastitis from two farms in the state of São Paulo. These strains were characterised by in vitro slime production on Congo red agar, biofilm formation on microtitre plates and the presence of the icaA and icaD genes. The results revealed that 85% of the isolates tested produced slime on the Congo red agar, 98.9% of the isolates produced biofilms in vitro by adhering to sterile 96-well "U" bottom polystyrene tissue culture plates, and 95.7% of the isolates carried the icaA and icaD genes. The results of the phenotypic tests for biofilm formation were compared with those of the molecular analysis, and the sensitivity and specificity of the Congo red agar test were 88.9% and 100%, respectively, while those of the microtitre plate test were 100% and 25%, respectively. When the phenotypic methods for the detection of biofilm producers, namely growth on microtitre plates and Congo red agar, were compared, the sensitivity and specificity were 86% and 100%, respectively. Therefore, growth on Congo red agar and the microtitre plate test are methods that could be used to determine whether an isolate has the potential for biofilm production.

The epidemiological relationships between isolated Staphylococcus aureus strains in milk samples of dairy cows, reagent to California Mastitis Test, individual and group milk was demonstrated in different sites of the production fluxogram, in 12 milk-producing farms in the Gameleira region, municipality of Sacramento MG Brazil, so that localization and transmission modes may be identified. Two hundred and forty-four strains out of 446 samples collected at several sites were isolated and bio-chemically characterized as coagulase-positive staphylococcus. Specific chromosome DNA fragment of the species Staphylococcus aureus was amplified to 106 strains and 103 underwent (PFGE). Samples' collection sites with the highest isolation frequency of Staphylococcus aureus strains comprised papillary ostia (31.1%), CMT-reagent cow milk (21.7%), mechanical milking machines' insufflators (21,7%), milk in milk pails (6.6%) and the milk in community bulk tanks (5.6%). Genetic heterogeneity existed among the isolated 103 Staphylococcus aureus strains, since 32 different pulse-types were identified. Pulse-type 1 had the highest similarity among the isolated strains within the different sites of the milk-production fluxogram. Highest occurrence of pulsetype 1 isolates of Staphylococcus aureus strains was reported in samples collected from the papillary ostia (10.6%), followed by milk samples from CMT-reagent dairy cows (5.8%) and mechanical milking machine insufflators (3.8%). The above shows the relevance of these sites in the agents' transmission mechanism within the context of the farms investigated.

OBJETIVO: Comparar a quantidade do glicosaminoglicano dermatam sulfato entre pacientes homens, portadores de hérnia inguinal tipo II de Nyhus e, indivíduos sem hérnia inguinal, com idade entre 20 e 40 anos. MÉTODOS: Foram constituídos dois grupos. Um de 15 pacientes do sexo masculino com hérnia inguinal tipo II de Nyhus e idade entre 20 e 40 anos, com risco ASA I e II, e um grupo controle com dez indivíduos, também do sexo masculino entre 20 e 40 anos, que morreram em período de até 24 h. Foram excluídos os pacientes do sexo feminino, diabéticos, portadores de doença do tecido conjuntivo, tabagistas e com risco cirúrgico ASA III e IV. Foi retirada uma amostra de 1cm² da fáscia transversal na parte intermediária do trígono inguinal, e 1cm² na bainha anterior do músculo reto abdominal na região inguinal correspondente e quantificados os glicosaminoglicanos dermatam sulfato por densitometria, após eletroforese em gel de agarose. RESULTADOS: A quantidade de dermatam sulfato não apresentou diferença estatisticamente significante entre os pacientes com hérnia inguinal e os indivíduos sem hérnia inguinal, tanto na fáscia transversal (p=0,108) quanto na bainha anterior do músculo reto abdominal (p=0,292). CONCLUSÃO: Não se encontrou diferença na quantidade do glicosaminoglicano dermatam sulfato entre os pacientes portadores de hérnia inguinal tipo II de Nyhus e indivíduos sem hérnia inguinal em homens adultos.

OBJECTIVE: To compare the amount of the dermatan sulfate glycosaminoglycan between male patients with Nyhus type II inguinal hernias and subjects without inguinal hernia, aged between 20 and 40 years. METHODS: Two groups were formed: One with 15 male patients with Nyhus type II inguinal hernia and aged between 20 and 40 years with ASA risk I and II, and a control group of ten individuals, also males between 20 and 40, who had died up to 24 h before. We excluded female patients, diabetic patients with connective tissue disease, smokers and surgical risk ASA III and IV. We resected a sample of 1 cm² of the transversalis fascia in the middle of the inguinal trigone, and 1 cm² of the anterior sheath of the rectus abdominis muscle in the groin for the quantification of dermatan sulfate glycosaminoglycans by densitometry after agarose gel electrophoresis. RESULTS: The amount of dermatan sulfate showed no statistically significant difference between patients with inguinal hernia and individuals without inguinal hernia in both the transverse fascia (p = 0.108) and anterior sheath of the rectus abdominis muscle (p = 0.292). CONCLUSION: There was no difference in the amount of the dermatan sulfate glycosaminoglycan among patients with Nyhus type II inguinal hernias and subjects without inguinal hernia in adult males.

The objective of the present study was to evaluate the occurrence of Staphylococcus aureus in milk produced in 37 farms located in the regions of Ribeirão Preto and São Carlos, state of São Paulo, Brazil. Two-hundred and eight samples of milk from individual cows showing subclinical mastitis, and 37 samples of bulk tank milk were analyzed. S. aureus strains were detected in 18 (7.3%) milk samples: 14 (6.7%) from samples of individual cows, and 4 (10.8%) from bulk tank milk. Two individual milk samples (14.3%) and two bulk milk samples contained enterotoxigenic S. aureus. PFGE analysis revealed the genetic heterogeneity of the strains isolated from raw milk, which presented to 13 S. aureus patterns. Results confirmed the potential transmission of staphylococcal food poisoning to consumers via milk of cows affected by subclinical mastitis, mainly when raw milk is ingested.

A total of 72 strains of Staphylococcus aureus were examined for the production of staphylococcal enterotoxins (SE) A, B, C, D and toxic shock syndrome toxin (TSST-1). The strains were isolated from milk samples from cows with mastitis in dairy herds of São Paulo State, Brazil. Off 72 isolates, 38 (52.8%) produced SEA, 38 (52.8%) SEB, 32 (44.4%) SED, 28 (38.9%) SEC and 27 (37.5%) TSST-1. From the 72 strains, 66 (91.7%) produced, at least, one or more toxin, including TSST-1.

Avaliou-se a relação custo-benefício do tratamento da mastite subclínica bovina causada por Staphylococcus aureus. Foram selecionados 270 quartos mamários com mastite subclínica e sadios, divididos em quatro grupos de acordo com o estádio de lactação e o tratamento. O grupo 1 foi formado por animais entre 10 e 60 dias da lactação e tratados contra mastites; o grupo 2 incluiu animais entre 61 dias da lactação e dois meses antes da secagem e tratados contra mastite; o grupo 3 foi formado por animais entre 10 e 60 dias da lactação, não tratados contra mastite; e o grupo 4 foi formado por animais entre 61 dias em lactação e dois meses antes da secagem, não tratados. O tratamento foi realizado pela infusão intramamária de 150mg de gentamicina, uma vez ao dia. A reavaliação foi efetuada após 30 dias. Para os cálculos dos custos com o tratamento, foram considerados uma prevalência de S. aureus de 5% e os gastos com medicamento, descarte do leite, antibiograma e mão-de-obra. Observou-se redução de 2% e 14% das receitas nos grupos 1 e 2, respectivamente, quando comparada com as receitas obtidas antes do tratamento, demonstrando ser economicamente inviável o tratamento da mastite subclínica bovina causada por S. aureus, durante a lactação.

Economic evaluation of the treatment bovine subclinical mastitis caused by S. aureus was evaluated. Two hundred and seventy udder quarters with or without subclinical mastitis were distributed into four groups, in conformity to lactational stage and treatments. Group 1 included animals treated between 10 and 60 days of lactation; group 2 included animals treated from 61 days of lactation to two months before drying; group 3 included animals no treated between 10 and 60 days of lactation; group 4 included animals no treated from 61 days of lactation to two months before drying. Treatment with gentamicin (150mg) was accomplished by intramammary doses, once a day, after performing sensitivity tests. The mammary quarters were re-evaluated after 30 days. The costs with the treatment were calculated considering a S. aureus prevalence of 5% as well as expenses with antibiotic, milk disposal, tests of drug sensitivity and workload. There was loss of income of 2% and 14% in the groups 1 and 2, respectively, when compared with the values before the treatment. In such case, the treatment of bovine subclinical mastitis caused by S. aureus in the lactation was economically unviable.

RESUMO Este trabalho teve por objetivo conhecer a sensibilidade antimicrobiana “in vitro” de 72 cepas de Staphylococcus aureus isoladas em amostras de leite de vacas com mastite clínica e subclínica, em 10 propriedades rurais do Estado de São Paulo. Os testes foram realizados pela da técnica de difusão em discos padronizada por Kirby-Bauer. Os principios ativos que apresentaram maior sensibilidade foram a gentamicina (98,6%) e a eritromicina (98,6%), seguidos pela estreptomicina (94,4%), oxacilina (84,7%), novobiocina (73,4%), vancomicina (72,2%), ampicilina (4,2%) e a penicilina (2,8%). Os resultados obtidos evidenciaram, ainda, que as 72 (100%) cepas estudadas apresentaram resistência a pelo menos 2 antibióticos ou quimioterápicos e que nenhum destes princípios ativos, agindo isoladamente, pode ser ativo contra qualquer das cepas experimentadas.

ABSTRACT The purpose of this study was to determine the in-vitro susceptibility pattern of 72 Staphylococcus aureus strains, isolated from bovine mastitic milk. The calves were reared on 10 dairy farms in the state of São Paulo, Brazil. These strains were tested by the standardized disc diffusion method. The highest susceptibility was to gentamicin (98.6%) and erytromycin (98.6%), followed by streptomycin (94.4%), oxacillin (84.7%), novobiocin (73.4%), vancomycin (72.2%), ampicillin (4.2%) and penicillin (2.8%). Results showed that 72 (100%) strains were resistant to at least 2 antibiotics and chemotherapeutics. None of the active principles, acting alone, was effective against any of the strains tested.

Relatamos o caso de uma mulher de 60 anos portadora da doença de von Willebrand tipo I, submetida a cirurgia da valva mitral. A paciente necessitou de cuidados especiais em razão da coagulopatia e foi necessária a utilização de concentrado de fator VIII (VIIIf) e fator de von Willebrand (vWf) antes, durante e depois da cirurgia. Não houve complicações durante e após a cirurgia. Nove meses depois, a paciente encontra-se assintomática. A correção para valores adequados de VIIIf e vWf permitiu a realização da cirurgia com segurança.

We report a case of a 60 year-old woman with von Willebrand disease type I that was submitted to a mitral valve repair. The patient needed special care due coagulopathy and needed VIII factor (VIIIf) and von Willebrand factor (vWf), before, during and after surgery. There was no complication during or after surgery. Patient is asymptomatic nine months postoperatively. The correction of VIIIf and vWf allowed the realization of a safety surgery.

Foram submetidas a PCR-Ribotipagem e aos testes de sensibilidade in vitro frente a 12 antimicrobianos 77 estirpes de Staphylococcus aureus isoladas em amostras de leite procedentes de 40 vacas da raça holandesa que apresentaram mastite subclínica, em uma propriedade rural localizada no Estado de São Paulo, Brasil. Os resultados obtidos revelaram quatro diferentes padrões de resistência a antimicrobianos, sendo observada a predominância de resistência à lincomicina entre 19 (24,7%) estirpes de S.aureus. As 58 (75,3%) estirpes restantes foram sensíveis aos 12 antimicrobianos testados. A PCR-ribotipagem revelou a ocorrência de nove padrões genotípicos distintos, além de apresentar uma capacidade discriminatória maior (D = 0,82) que a obtida nos antibiogramas (D = 0,42). Entre as 19 estirpes resistentes aos antimicrobianos, 14 (73,7%) foram agrupadas em três padrões de ribotipagem e, destas, 13 (92,9%) apresentaram resistência à eritromicina e à lincomicina, isoladamente ou em associação. O grande número de ribotipos e de padrões de resistência a antimicrobianos observados nesta propriedade demonstrou que há grande heterogeneidade genética em populações naturais de S. aureus, fato este que deve ser levado em consideração em programas de controle da mastite bovina.

Seventy-seven S. aureus strains, isolated in milk samples obtained from 40 Holstein cows suffering from subclinical mastitis in a dairy herd in the state of São Paulo - Brazil, were undergone to PCR-ribotyping and in vitro susceptibility testing to 12 antimicrobial drugs. PCR-ribotyping revealed nine different patterns and presented a higher discriminatory power (D = 0.82) in comparison with the in vitro antibiotic susceptibility test (D = 0.42). Moreover,, the results showed four antibiotic resistance patterns, with the resistance to lincomycin being the most predominant and comprising 19 (24.7%) S. aureus strains. The other 58 (75.3%) isolates were sensitive to all antibiotics tested. Of the 19 isolates which presented resistance to antibiotics, 14 (73.7%) were grouped in three ribotypes, and 13 (92.9%) of them were resistant to eritromycin and/or lincomycin. The large number of ribotypes and antibiotic resistance patterns found in this single dairy herd showed that one must be careful when considering the results of in vitro susceptibility tests obtained from a few S. aureus strains, because there is a high heterogeneity among naturally occurring populations of S. aureus, which must be taken into consideration in control programs against bovine mastitis.

RESUMO Com o objetivo de avaliar a eficiência da exposição à radiação solar na desinfecção da água, amostras de água de poços rasos foram acondicionadas em garrafas "PET" (Polietileno Teraftalato) com volume de 2.000 mL, e expostas ao sol por 2, 5 e 12h. Antes da exposição, após a exposição e em amostras que permaneceram 2, 5 e 12h protegidas do sol (controles) foram determinados os números mais prováveis de coliformes totais e Escherichia coli e os números de microrganismos mesófilos. Foi verificado ainda o recrescimento bacteriano após 72h da exposição ao sol em amostras protegidas da ação da luz solar e a quantidade de energia recebida pelas amostras após os três tempos de exposição à radiação solar. Os resultados obtidos evidenciaram que a radiação solar foi eficaz na desinfecção da água com reduções, após 12h de exposição, de 98,2%, 99,9% e 100% nos números de microrganismos mesófilos, coliformes totais e E. coli, respectivamente. Foi verificada a ausência de recrescimento de todos microrganismos pesquisados após 12h de exposição ao sol. Os resultados evidenciaram que a exposição à radiação solar da água de consumo humano, acondicionada em garrafas PET, pode ser utilizada para diminuir o risco de enfermidades de veiculação hídrica.

ABSTRACT This study evaluated the effictiveness of expusure to solar radiation for disinfecting water samples from shallow wells. Water sample were placed into 2-liter PET (Polyethylene Terephthalate) exposed to sun rays for 2, 5 and 12 hours. Probably numbers of total coliform bacteria, Escherichia coli and mesophyl microorganisms were determined before and after exposure to sun and in samples protected from the sun (controls) for 2, 5 and 12 hours. Bacterial re-growth after a 72h-exposure to the sun in samples which were protected from the sun rays and the amount of energy received by the samples after the three exposure periods to sun radiation were also evaluated. Results showed that sun radiation was effective for water disinfection with 98.2%, 99.9% and 100% reductions in mesophyl microorganisms, total coliform bacteria and E. coli respectively after a 12h-exposure to sun rays. Lack of re-growth of all microorganisms analyzed after a 12h exposure to sun rays was confirmed. Results showed that exposure of water placed in PET bottles for human consumption may be used to diminish water-transmitted disease risks.

RESUMO O presente trabalho teve por objetivo pesquisar diferenças na composição e na produção de leite ao serem estudadas as interações entre estágios de lactação, número de lactações e mastite subclínica. Quartos mamários foram distribuídos em 6 grupos: Grupo "A", animais com até 2 meses após o parto e até 5 lactações; Grupo "B", animais com até 2 meses após o parto e após a quinta lactação; Grupo "C", com vacas em meses intermediários do estágio de lactação e com até 5 lactações; Grupo "D", com animais em meses intermediários do estágio de lactação e após a quinta lactação; Grupo "E", com vacas nos 2 meses anteriores à secagem e até 5 lactações; e Grupo "F", composto por animais com até 2 meses antes da secagem e após a quinta lactação. Foram analisadas a produção láctea, a contagem de células somáticas (CCS), o teor de cloretos, a acidez titulável, a densidade, o extrato seco desengordurado (ESD) e a crioscopia. O estágio de lactação e o número de lactações influenciaram a produção láctea e o conteúdo de cloretos do leite de quartos sadios, devendo ser considerados quando estudada a influência da mastite subclínica sobre a composição e a produção de leite. Além disso, em conjunto com o período lactacional e o número de lactações, a etiologia infecciosa da mastite subclínica deve ser investigada pela possível variação do grau de acometimento da glândula mamária de acordo com os microrganismos envolvidos.

ABSTRACT This study was carried out to investigate differences in milk composition and production when followed the stages of lactation, parities and subclinical mastitis. The mammary quarters of cows were arranged in agreement to days in milk and parities: Group "A" (lactating cows until 2 months after parturition and until the fifth lactation); Group "B" (lactating cows until 2 months after parturition and after fifth lactation); Group "C" (animals in intermediary months of lactation stage and until the fifth lactation); Group"D" (animals in intermediary months of lactation stage and after fifth lactation); Group "E" (cows in corresponding period of 2 months before the end of lactation and until the fifth lactation); Group "F" (lactating cows within 2 months before the end of lactation and after fifth lactation). The milk characteristics studied were the milk production, somatic cell count (SCC), chloride levels, titratable acidity, density, non-fat solids and cryoscopy. The lactation stage and the parities changed the milk production and chloride levels and these factors should be considered when the influence of subclinical mastitis upon the milk composition and milk production is analyzed. The days in milk and the parities of lactating cows should be investigated in addition to the infective etiology of subclinical mastitis because variations of the effects can occur in agreement to the microorganisms.