Resumo O controle higiênico e sanitário nas Unidades de Alimentação e Nutrição (UAN) é considerado um procedimento padrão para produzir refeições adequadas e reduzir o risco de doenças transmitidas pelos alimentos e infecções hospitalares. Este estudo teve como objetivo isolar e identificar bactérias de equipamentos e superfícies de contato com alimentos em uma UAN hospitalar, bem como avaliar a condição sanitária. Do mesmo modo, analisou-se a adesão dos micro-organismos em tábuas de corte de polietileno. A presença de micro-organismos aeróbios mesófilos, leveduras, fungos, Sthapylococcus coagulase-positivos, coliformes, coliformes fecais e Escherichia coli foi analisadas na superfície de mesas do refeitório, superfícies de bancada e tábuas de corte usadas para manuseio de carne ou vegetais e, em equipamentos como micro-ondas e refrigeradores. A identificação molecular foi feita pelo sequenciamento do gene 16S rRNA. A adesão dos micro-organismos (formação de biofilmes) em tábuas de corte de carne e de vegetais também foi avaliada por microscopia eletrônica de varredura. Os resultados mostraram elevada contagem para todos os micro-organismos analisados, exceto para E. coli, a qual não foi observada nas amostras. A análise molecular identificou espécies da família Enterobacteriaceae e Pseudomonadaceae. A análise de microscopia eletrônica de varredura revelaram adesão bacteriana nas superfícies das placsa de corte. Os resultados obtidos neste estudo indicaram que as condições higiênicas das superfícies e de equipamentos nesta UAN hospitalar estavam inadequadas. A aplicação de procedimentos operacionais padrão poderia auxiliar positivamente na padronização do controle higiênico-sanitário, reduzindo a contaminação microbiana e fornecendo um alimento seguro para pacientes hospitalizados.

Abstract The hygienic and sanitary control in Food and Nutrition Units (FNU) is considered a standard procedure to produce adequate meals and reduce the risk of foodborne diseases and hospital infections. This study aimed to evaluate the isolation and identification of bacteria from equipment and food contact surfaces in a hospital FNU as well as to evaluate the sanitary condition. Likewise, it was analyzed the adhesion of the microorganisms on polyethylene cutting boards. The presence of aerobic mesophilic microorganisms, yeasts, molds, coagulase-positive staphylococci, coliform and fecal coliform, and Escherichia coli were analyzed on eating tables, countertop surfaces and cutting boards used for meat or vegetable handling, and equipment such as microwaves and refrigerators. The molecular identification it was done by 16S rRNA gene sequencing. The adhesion of the microorganisms (biofilm formation) on meat and vegetable cutting boards was also evaluated by scanning electron microscopy. The results showed high numbers of all microorganisms, except for E. coli , which was not observed in the samples. The molecular analysis identified species of the Enterobacteriaceae family and species of the Pseudomonadaceae family. Scanning electron microscopy analyses revealed bacterial adhesion on the cutting board surfaces. The results obtained in this study indicated that the hygienic conditions of surfaces like plastic cutting boards and equipment in this hospital FNU were inadequate. The achievement and application of standard operating procedures could positively help in the standardization of sanitary control, reducing the microbial contamination and providing a safe food to hospitalized patients.

ABSTRACT The adhesion ability of bacteria to abiotic surfaces has important implications in food industries, because these organisms can survive for long periods through the biofilm formation. They can be transferred from one place to another in the industry causing contamination of the food processing environment. In this study, the antibacterial and antibiofilm activities of the antimicrobial peptide P34, characterized as a bacteriocin-like substance (BLS P34) were tested against planktonic and sessile cells of Staphylococcus aureus and Enterococcus faecalis isolated from foods. The BLS P34 showed inhibitory effect against all planktonic cells of E. faecalis. The inhibition of biofilm formation and the eradication of pre-formed biofilm were evaluated with the crystal violet assay and with the reduction of 3-bromide [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. The BLS P34 promoted a reduction of percentage of adhered microbial cells on the surface, not being able to perform the complete elimination of biofilm formation. The metabolic activity of S. aureus biofilms decreased considerably between 41-95%. However, E. faecalis cells showed up metabolically stimulated. The BLS P34 has the potential antibiofilm for the species S. aureus. Studies suggest more detailed approaches to a better understanding of the interactions between the antimicrobial and bacterial cells within the biofilm structure.

RESUMO P34 é um peptídeo antimicrobiano produzido pelo Bacillus sp. P34, isolado do conteúdo intestinal de um peixe na Bacia Amazônica. Esse peptídeo demonstrou propriedades antibacterianas contra bactérias Gram-positivas e Gram-negativas e foi caracterizado como uma substância do tipo bacteriocina. Foi demonstrado que o peptídeo P34 exibiu atividade antiviral contra o herpesvírus felino tipo 1 in vitro. O objetivo deste trabalho foi avaliar o P34 in vitro, em cultivo de células RK 13, contra o vírus da arterite equina, uma vez que não há tratamento específico e uma variável proporção de garanhões pode permanecer persistentemente infectada. Os resultados obtidos mostram que o peptídeo exerce atividade antiviral e virucida contra o vírus da arterite equina, agindo provavelmente no envelope viral. Os ensaios antivirais realizados demonstraram que o P34 reduz significativamente os títulos do vírus nas células infectadas e tratadas. O mecanismo de ação do P34 parece ser tempo/temperatura dependente. Esse peptídeo tende a ser um antiviral promissor para o tratamento e a prevenção das infecções por arterivírus, tendo em vista que ele possui um alto índice terapêutico. Contudo, estudos mais detalhados devem ser realizados para precisar a etapa exata da infecção viral em que o P34 age, para que ele possa ser usado como antiviral in vivo no futuro.

ABSTRACT P34 is an antimicrobial peptide produced by Bacillus sp. P34, isolated from the intestinal contents of a fish from the Amazon basin. This peptide showed antibacterial properties against Gram-positive and Gram-negative bacteria and was characterized as a bacteriocin like substance. It was demonstrated that the peptide P34 exhibited antiviral activity against feline herpesvirus type 1 in vitro. The aim of this work was to evaluate P34 for its antiviral properties in vitro, using RK 13 cells, against the equine arteritis virus, since it has no specific treatment and a variable proportion of stallions may become persistently infected. The results obtained show that P34 exerts antiviral and virucidal activities against equine arteritis virus, probably in the viral envelope. The antiviral assays performed showed that P34 reduces significantly the viral titers of treated cell cultures. The mechanism of action of P34 seems to be time/temperature-dependent. This peptide tends to be a promising antiviral compound for the prevention and treatment of arteriviral infections since it has a high therapeutic index. However, more detailed studies must be performed to address the exact step of viral infection where P34 acts, in order to use this peptide as an antiviral drug in vivo in the future.

Resumo As bactérias ácido láticas (BAL) têm um papel importante em uma grande variedade de alimentos fermentados. Em adição à sua contribuição para as características sensoriais, estes microorganismos melhoram a conservação de alimentos e podem ser utilizados como probióticos. Neste estudo, as atividades antimicrobiana e antioxidante do sobrenadante e dos extratos livres de células de 16 isolados de LAB de carne e produtos lácteos foram investigadas. Os isolados foram identificados pelo sequenciamento da região 16S do rRNA. Após a comparação das sequências obtidas com aquelas disponíveis na base de dados GenBank, observou-e que todos os isolados foram pertencentes à espécie Enterococcus faecium. A atividade antimicrobiana contra o microrganismo indicador (Listeria monocytogenes) foi observada no sobrenadante das culturas em 11 isolados, e nos extratos livres de células por 4 isolados. A sensibilidade da cultura sobrenadante foi avaliada pela proteinase K e tripsina e observou-se que a atividade da substância antimicrobiana foi completamente perdida após o tratamento com as enzimas proteolíticas. Todos os isolados apresentaram atividade antioxidante, como determinado pelo método do ácido tiobarbitúrico de substâncias reativas (TBARS) com ambos os tipos de extratos. Quando a capacidade antioxidante foi investigada usando o método do ABTS (2,2 azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) e o método de DPPH (2,2-diphenyl-1-picrylhydrazyl) observou-se que apenas os sobrenadantes das culturas demonstraram capacidade antioxidante. Estas bactérias poderiam particularmente ajudar a reduzir ou inibir microorganismos patogênicos, bem como a deterioração oxidativa em alimentos e rações.

Abstract Lactic acid bacteria (LAB) have an important role in a great variety of fermented foods. In addition to their contribution to sensory characteristics, they enhance food preservation and can be used as probiotics. In this study, the antimicrobial and antioxidant activities of culture supernatants and cell free extracts of 16 LAB isolated from meat and dairy products were investigated. The bacterial were identified by 16S rRNA sequencing. GenBank BLAST analysis revealed that all the isolates belong to Enterococcus faecium species. Antimicrobial activity against the indicator microorganism (Listeria monocytogenes) was observed at 11 culture supernatants and 4 cell free extracts. The sensibility of culture supernatant was evaluated by proteinase K and trypsin and it was observed that activity of antimicrobial substance was completely lost after the treatment. All of the isolates showed antioxidant activity as determined by the Thiobarbituric Acid Reactive Substances (TBARS) method with both types of extracts. When the antioxidant capacity was investigated using ABTS•+ method (2,2 azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH method (2,2-diphenyl-1-picrylhydrazyl) it was observed that only culture supernatants showed antioxidant capacity. These bacteria could particularly help to reduce or inhibit pathogenic microorganisms as well as oxidative spoilage in foods and feed.

This report demonstrates the usefulness of PCR for the genes spaS and sboA as a means of identifying Bacillus strains with a potential to produce subtilin and subtilosin A. One collection strain and five Bacillus spp. isolated from aquatic environments in the Amazon basin were screened by PCR using primers for sboA and spaS designed specifically for this study. The sequences of the PCR products showed elevated homology with previously described spaS and sboA genes. Antimicrobial peptides were isolated from culture supernatants and analyzed by mass spectrometry. For all samples, the mass spectra revealed clusters with peaks at m/z 3300-3500 Da, corresponding to subtilosin A, subtilin and isoforms of these peptides. These results suggest that the antimicrobial activity of these strains may be associated with the production of subtilosin A and/or subtilin. The PCR used here was efficient in identifying novel Bacillus strains with the essential genes for producing subtilosin A and subtilin.

The mode of action of cerein 8A, a bacteriocin produced by the soil bacterium Bacillus cereus 8A, was investigated. The effect of cerein 8A was tested against Listeria monocytogenes and a bactericidal effect at 400 arbitrary units (AU)/ml was observed. In addition, cerein 8A was bactericidal against Bacillus cereus at 200 AU/ml, and inhibited the growth of Escherichia coli and Salmonella Enteritidis. Stronger inhibition of these gram-negative bacteria was achieved when the chelating agent EDTA was added together with bacteriocin. The effect of cerein 8A on B. cereus and L. monocytogenes was also investigated by Fourier transform infrared spectroscopy (FTIR). Treated cells had an important frequency increase at 2920 cm-1 and a decrease at 1400 cm-1, corresponding to assignments of fatty acids. Transmission electron microscopy showed damaged cell walls and loss of protoplasmic material. These results suggest that the mode of action of cerein 8A is to interfere with cell membranes and the cell wall.

O modo de ação da cereina 8A, uma bacteriocina produzida pela bactéria do solo Bacillus cereus 8A, foi investigado. O efeito da cereina 8A foi testado contra Listeria monocytogenes, resultando em um efeito bactericida com uma concentração de 400 unidades arbitrárias (UA)/ml. A cereina 8A também foi bactericida contra Bacillus cereus à 200 UA/ml. A bacteriocina inibiu o crescimento de Escherichia coli e Salmonella Enteritidis. Uma maior inibição contra estas bactérias gram-negativas foi alcançada quando o agente quelante EDTA foi adicionado junto com a bacteriocina. O efeito da cereina 8A sobre B. cereus e L. monocytogenes também foi investigado por espectroscopia de infravermelho com transformada de Fourier (FTIR). As células tratadas mostraram um importante aumento de frequência em 2920 cm-1 e uma diminuição na banda de 1400 cm-1, correspondendo a designações de ácidos graxos. A microscopia eletrônica de transmissão mostrou que as células apresentaram danos na parede celular com perda de protoplasma. Estes resultados sugerem que o modo de ação da cereina 8A é interferindo nas membranas celulares e parede celular.

Se investigó el modo de acción de la cereína 8A, una bacteriocina producida por la bacteria del suelo Bacillus cereus 8A. El efecto de la cereína 8A fue probado contra Listeria monocytogenes, obteniendo un efecto bactericida a concentraciones de 400 unidades arbitrarias (AU)/ml. La cereína 8A también tuvo un efecto bactericida contra Bacillus cereus a una concentración de 200 AU/ml. La bacteriocina inhibió el crecimiento de Escherichia coli y Salmonella Enteritidis. Mayor inhibición contra estas bacterias gram-negativas se consiguió cuando a la bacteriocina se le añadió el agente quelante EDTA. El efecto de la cereína 8A sobre B. cereus y L. monocytogenes también fue investigado por espectroscopía de infrarrojos de transformación de Fourier (FTIR). Las células tratadas mostraron un importante crecimiento en frecuencia de 2920 cm-1 y un decrecimiento de 1400 cm-1 de banda, correspondiéndose con la asignación de los ácidos grasos. La microscopía electrónica de transmisión mostró que las células habían padecido daños en la pared celular, con pérdida de material protoplásmico. Los resultados sugieren que el modo de acción de la cereína 8A se produce mediante su intervención en las membranas celulares y en la pared celular.

A levedura Kluyveromyces marxianus CBS 6556 foi cultivada em soro de queijo para produzir extrato celular rico em nucleotídeos. O soro foi pré-tratado com Alcalase TM . K. marxianus foi inoculada e cultivada em batelada sob agitação e aeração de 7,0 L ar/min por 20 h em biorreator de 2 L de volume. A biomassa resultante foi submetida a diferentes tratamentos para obter os extratos enriquecidos. Tratamento térmico das células a 35ºC ou 50ºC por 15-30h resultou em extratos contendo cerca de 20 g/L de proteína, mas apenas com o segundo tratamento apresentando pequenas quantidades de RNA, medido por eletroforese de gel e espectrofotometria. Em contraste, tratamento autolítico em solução tamponada foi o único a resultar na liberação de importantes quantidades de RNA intracelular, sendo portanto, o mais adequado para a produção de 5'-nucleotídeos a partir de extratos celulares de K. marxianus crescidos em soro de queijo.

The yeast Kluyveromyces marxianus CBS 6556 was grown on whey to produce nucleotide-rich yeast extracts. Thermal treatments of cells at 35 or 50ºC for 15-30h resulted in yeast extracts containing about 20 g/L protein, with only the second treatment resulting in the presence of small amounts of RNA. In contrast, autolysis in buffered solution was the unique treatment that resulted in release of high amounts of intracellular RNA, being, therefore, the better procedure to produce 5'-nucletide rich extract with K. marxianus.

The antibacterial activity of a series of 1,4-naphthoquinones was demonstrated. Disk diffusion tests were carried out against several Gram-positive and Gram-negative bacteria. The compound 5-amino-8-hydroxy-1,4-naphthoquinone was the most effective, presenting inhibition zones measuring 20 mm against staphylococci, streptococci and bacilli at 50 µg/ml. Methicillin-resistant Staphylococcus aureus and several clinical isolates of this bacterium were also inhibited. Naphthazarin, 5-acetamido-8-hydroxy-1,4-naphthoquinone, and 2,3-diamino-1,4-naphthoquinone were the next most active compounds. The minimal inhibitory concentration of the active compounds was determined against S. aureus, ranging from 30 to 125 µg/ml. All compounds presented a minimal bactericidal concentration higher than 500 µg/ml, indicating that their effect was bacteriostatic. The EC50, defined as the drug concentration that produces 50% of maximal effect, was 8 µg/ml for 5-amino-8-hydroxy-1,4-naphthoquinone against S. aureus, S. intermedius, and S. epidermidis. These results indicate an effective in vitro activity of 5-amino-8-hydroxy-1,4-naphthoquinone and encourage further studies for its application in antibiotic therapy.

A presença de bactérias do gênero Aeromonas nos alimentos tem sido demonstrada. Normalmente destacam-se os alimentos que durante sua industrialização entraram em contato com a água, a qual é tida como habitat natural das diversas espécies e principal fonte de contaminação. Objetivando determinar a ocorrência de espécies de Aeromonas, bem como aspectos relacionados a sua virulência como capacidade de hemólise e hemoaglutinação, foram analisadas 70 amostras de água divididas entre água de abastecimento e de escoamento de lavagem das carcaças. De acordo com os resultados obtidos verificou-se a ocorrência de Aeromonas spp. em 21,4% das amostras. A. hydrophila obteve o maior índice de isolamento, 11,4% em água de abastecimento e 25,7% em águas de escoamento, já A. sobria foi isolada somente em água de abastecimento num percentual de 5,7%. O maior número de amostras positivas foram de águas de escoamento, porém com uma única espécie, isto sugere que a fonte de contaminação ainda é a água de abastecimento. Em relação ao teste de sensibilidade, a espécie A. hydrophila, apresentou uma maior resistência aos antibióticos testados do que a A. sobria. Foram observadas reações positivas de hemólise e hemoaglutinação para ambas espécies. Estes resultados indicam que Aeromonas spp. estão presentes no sistema de abastecimento de água, sendo potenciais contaminantes das carcassas e dos produtos derivados.

The presence of Aeromonas spp. in food has been demonstrated. They are often introduced from water, which is the natural habitat of many species and is thought to be the main source of contamination. The occurrence of Aeromonas spp. was investigated in 70 water samples of a bovine abattoir. Aeromonas spp. were present in 21.4% of water samples. A. hydrophila was isolated from 11.4% of supplying water samples and from 25.7% of the water drained from carcasses, whereas A. sobria was sole isolated from 5.7% of supplying water samples. Although greater number of positive samples were from water drained from carcasses, only A. hydrophila isolates were present. This suggests that the supplying water is the source of contamination. The antibiotic susceptibility testing revealed all strains were resistant to b-lactam antibiotics. However, the susceptibility to other antimicrobials was variable, being A. hydrophila more resistant than A. sobria strains. The investigation for virulence factors revealed positive reactions of hemolysis and hemagglutination. The results indicate that Aeromonas spp. are present in the supplying water system. These microorganisms may be a potential contaminant of carcasses and widespread in derived food.

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40% by PF from controls (PFc) and 50% by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60%) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control, NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11%) and PFe/PFi groups (17%), and the ionophore-induced AR was higher for PFi (33%) than PFc/PFe (25%). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR.

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface