Molecular identification and microbiological evaluation of isolates from equipments and food contact surfaces in a hospital Food and Nutrition Unit / Avaliação microbiológica e identificação molecular de isolados de equipamentos e superfícies de contato com alimentos em um hospital Unidade de Alimentação e Nutrição

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.

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.

Molecular identification and microbiological evaluation of isolates from equipments and food contact surfaces in a hospital Food and Nutrition Unit

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.

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.

Antimicrobial and antioxidant activities of Enterococcus species isolated from meat and dairy products.

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.

Antimicrobial and antioxidant activities of Enterococcus species isolated from meat and dairy products / Atividade antimicrobiana e antioxidante de espécies de Enterococcus isoladas de carne e produtos lácteos

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.

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.

Peptide-like substances as antimicrobial barriers to Corynebacterium sp. adhesion to silicone catheters.

AIMS: To show medical application of antimicrobial peptides such as Pep5 and epidermin in inhibiting adhesion of Corynebacterium spp. to silicone catheters. METHODS AND RESULTS: The inhibitory activity of crude preparations of Pep5 and epidermin was tested on Corynebacterium spp. isolated from catheter-related infections. The addition of these substances at 640 AU ml(-1) to a cell suspension of Corynebacterium sp. 633544 resulted in a decrease of 3 log cycles in the number of viable cells over a period of 12 h. When Pep5 and epidermin were added to in vitro catheter colonization experiments, there was a decrease of 1 log unit (P < 0.01) in the cell number of Corynebacterium spp. adhered to silicone catheters. Scanning electron microscopy revealed that antimicrobial-treated catheters presented zones with absence of adhered cells, and some parts of the catheter presented aggregates suggesting damaged cells. CONCLUSIONS: The crude preparations of Pep5 and epidermin were able to inhibit Corynebacterium sp. 633544 isolated from catheter-related infection. The capability of Pep5 and epidermin to inhibit catheter colonization may indicate their usefulness as a barrier to block or to reduce the bacteremia by Corynebacterium spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Peptide-like antimicrobial substances used to reduce bacterial attachment to medical devices may represent a novel strategy to control catheter-related infections.

Purification and partial chemical characterization of the antimicrobial peptide cerein 8A.

AIMS: To purify and to characterize the antimicrobial compound cerein 8A. METHODS AND RESULTS: Cerein 8A was isolated by ammonium sulfate precipitation, 1-butanol extraction and ion-exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. The purified substance corresponded to a 26 kDa peptide band. The native protein eluted at the void volume of Sephadex G-100, but within the included volume when a 1.5 mol l(-1) NaCl buffer was used, indicating that cerein 8A aggregates extracellularly. The antimicrobial activity was lost by treatment with proteases and heat. The ultraviolet spectrum was typical of a polypeptide and the infrared spectrum indicates that the peptide contains acyl group(s) in its structure. Intact Bacillus cereus spores were sensitive to cerein 8A at 1600 AU ml(-1). CONCLUSIONS: Cerein 8A show distinct properties from other antimicrobial peptides of B. cereus, and has a significant inhibitory effect on spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of a substance active against important pathogens addresses an important aspect of food safety.

In vitro antimicrobial activity of a new series of 1, 4-naphthoquinones

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 æg/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 æg/ml. All compounds presented a minimal bactericidal concentration higher than 500 æg/ml, indicating that their effect was bacteriostatic. The EC50, defined as the drug concentration that produces 50 percent of maximal effect, was 8 æg/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

Characterization of an antibacterial peptide produced by Brevibacterium linens.

AIMS: The aim of this research was to investigate the antimicrobial activity produced by Brevibacterium linens ATCC 9175. METHODS AND RESULTS: A bacteriocin produced by the red smear cheese bacterium B. linens ATCC 9175 was identified. The antimicrobial activity was first produced at the exponential growth phase. A crude bacteriocin obtained from the culture supernatant fluid was inhibitory to some indicator strains. It inhibited the growth of Listeria monocytogenes ATCC 7644, B. linens ATCC 9172 and Corynebacterium fimi NCTC 7547, but was inactive against the Gram-negative bacteria and yeast tested. The bacteriocin was stable at 30 degrees C but the activity was lost when the temperature reached 50 degrees C. It was sensitive to the proteolytic action of trypsin, papain and pronase E and was active between pH 6.0 and 9.0. The bacteriocin was bactericidal to L. monocytogenes at 40 AU ml(-1). Bacteriostasis was observed for a low dose of bacteriocin (20 AU ml(-1)). CONCLUSIONS: An antibacterial peptide produced by B. linens was characterized, presenting potential for use as a biopreservative in food systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of a novel bacteriocin active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage micro-organisms.

Hyperprolactinemia and luteal insufficiency in infertile patients with mild and minimal endometriosis.

The objective of the present paper was to assess the presence of hormonal alterations in infertile women with stage I or II endometriosis (Group III, n = 20) compared to fertile women without endometriosis (Group I, n = 14) and to fertile women with endometriosis (Group II, n = 7). Serum levels of FSH, LH, estradiol, TSH, and PRL were measured between days 1 and 5 of the early follicular phase; in the luteal phase, three serum samples were collected for progesterone measurement, and endometrial biopsies were performed. Serum estradiol levels were lower (p = 0.035) in infertile patients with endometriosis than in fertile patients without endometriosis. Six infertile patients with endometriosis presented prolactin levels above 20 ng/ml. This was not observed in the other groups. Luteal insufficiency was more frequent in infertile patients with endometriosis (78.9%) than in fertile patients with (42.9%) or without endometriosis (0%). In a multiple logistic regression analysis, only the presence of endometriosis and infertility was significantly associated with luteal insufficiency. The serum levels of LH, FSH, and TSH were not significantly different among the groups. Luteal insufficiency and altered prolactin secretion were associated with endometriosis, and could be important mechanisms causing infertility in this group of patients.

Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein

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 percent by PF from controls (PFc) and 50 percent 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 percent) 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 percent) and PFe/PFi groups (17 percent), and the ionophore-induced AR was higher for PFi (33 percent) than PFc/PFe (25 percent). 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

Proteolytic and partial characterization of human sperm-surface glycopeptides

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 treatmentmainly 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.

Characterization of beta-N-acetylglucosaminidase from human epididymis.

beta-N-acetylglucosaminidase (NAG) activity in human epididymal fluid was separated into two forms (I and II) after HPLC-hydrophobic interaction chromatography. Both forms exhibited maximal activity at a pH of around 4.5 and had a molecular weight of 125 kD when determined by Superose-HPLC. After incubation at 50 degrees C, form I retained only 30% of its activity while form II retained 90% activity. When analysed by non-denaturing electrophoresis, form I displayed higher electrophoretic mobility than did form II. These features indicate that the I and II isoforms found in the human epididymis are the A and B forms present in other tissues. NAG activity was measured in the fluid obtained form the different epididymal regions of 13 different samples. An average four-fold increase in activity between the proximal caput and distal corpus was found. The contribution of each isoform to the total activity was studied. The proximal caput found to be rich in the A isoform (59%), whereas the B form was predominant in the distal corpus (65%). Human spermatozoa contain membrane-associated NAG activity with an isoform distribution similar to that found in cauda epididymal fluid (CEP, 80% B). Finally, enzyme activity in CEP was two-fold greater than in seminal plasma. Taken together these results suggest that NAG may become associated with human spermatozoa during epididymal transit.