Microbiota bacteriana no processamento industrial de frangos e sua influencia na vida util de carcacas refrigeradas / Bacterial flora in the industrial processing of chickens and their influence in shelf-life of refrigerated carcasses

Visando investigar qualitativa e quantitativamente a participacao da microbiota psicrotrofila e patogenica em carcacas de frango durante e apos o processamento industrial, foram examinadas cento e oitenta unidades de amostras coletadas ao longo do processo em uma industria do Estado de Santa Catarina (Brasil). Numa segunda etapa, foi estudado o comportamento de cinco sanificantes de uso comercial, objetivando verificar a eficiencia dos mesmos na extensao da vida util de carcacas de frango refrigeradas. As contagens medias de microrganismos aerobios em carcacas de frango no inicio do processamento apresentaram valores oscilando entre '10 POT.4'e '10 POT.6' ufc/'cm POT.2' com incubacao a 7 graus centigrados e '10 POT.5' e > '10 POT.6' ufc/'cm POT.2' quando incubadas a 20 graus centigrados e trinta e cinco graus centigrados, respectivamente. Estes valores nao foram muito diferentes daqueles obtidos nas analises do produto final

A resuscitation/selection system for rapid determination of Salmonella in foods.

A resuscitation medium was developed consisting of a trypticase soy broth base supplemented with 0.5% yeast extract, 0.25% sodium pyruvate, 0.01% sodium thioglycollate, and 0.1% chicken fat. After a resuscitation period of 4 h, the medium was made selective by addition of either sodium thiosulfate, bile salts and iodine, or sodium selenite and L-cystine. The now selective medium was incubated for 16 h. The presence or absence of Salmonella was determined by the Salmonella-Tek antibody-based detection kit. The present system was compared with a method of the Bacteriological Analytical Manual (BAM) for naturally contaminated foods. Nineteen egg products were screened; 3/19 were positive using the BAM method, 9/19 were positive using the present system. Seventeen chicken samples were assayed; 10/17 were positive using the BAM method; 13/17 were positive using the present system. Of 8 pepper samples, 4/8 were positive using the BAM method; 6/8 were positive using the present system. Of 8 spice samples, 6/8 were positive using the BAM method, 7/8 were positive using the present system. Of 6 onion products sampled, 5/6 were positive using the BAM method; 6/6 were positive using the present system.

Epidemias por salmonella y por shigella, importancia de la vigilancia epidemiologíca y control de la resistencia a los antibióticos / Epidemics by salmonella and shigella, importance of epidemiologic vigilance and control of the antibiotics resistence

El descubrimiento de los antibióticos y su aplicación clínica ha permitido el tratamiento y control de padecimientos infecciosos. Por otra parte, se ha descubierto que algunas enterobacterias desarrollan resistencia a las drogas antimocrobianas causada, principalmente por la presencia de elementos extracromosómicos denominados plasmidos. De entre estos microorganismos se han estudiado la salmonella y shigella con la finalidad de determinar las características de las mismas, los antibióticos a que se resisten y con cuales pueden ser tratados los padecimientos provocados por las mismas. La presencia de epidemias y las endemias en regiones de México así como en otras regiones del mundo, y consecuentemente el alto índice de morbilidad han inducido a los especialistas a investigar los procesos infecciosos provocados por shigella y la salmonella, se ha logrado establecer una clasificación de las mismas así como la sintomatología que producen las mismas. Para establecer un tratamiento determinado, se aisló al agente infeccioso, se le sometió a estudios para poder establecer a que drogas eran resistentes y a cuales no. De este modo se ha podido descrubir que algunos tipos de estas bacterias son resistentes, por ejemplo, al clorafenicol, la tetraciclina, la estreptomicina y a la ampicilina, en tanto que otras son suceptibles a los mismos, así mismo se ha observado que la región guarda cierta relación con el desarrollo de la resistencia

Determinación de Salmonella en aves en Ciudad de La Habana / Deterination of Salmonella in fowl in Havana City

Se sometieron a examen 200 canales de aves para la detección de Salmonella. De éstas 125 resultaron positivas a este microorganismo para el 62,5

. Se señala el medio de enriquecimiento Caldo Tetrationato Bierbrauer y el selectivo Agar Digralski como los medios más efectivos. Se establece la relación entre los serotipos presentes en aves y los serotipos que aparecen en personas enfermas en Ciudad de la Habana

Determinación de Salmonella en aves en Ciudad de La Habana / Determination of Salmonella in fowl in Havana City

Se sometieron a examen 200 canales de aves para la detección de Salmonella. De éstas 125 resultaron positivas a este microorganismo para el 62,5%. Se señala el medio de enriquecimiento Caldo Tetrationato Bierbrauer y el selectivo Agar Digralski como los medios más efectivos. Se establece la relación entre los serotipos presentes en aves y los serotipos que aparecen en personas enfermas en Ciudad de la Habana

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide.

We have reported here on the structural polymorphism of lipid A, the "endotoxic principle" of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg2+] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70 degrees C. These data were correlated with measurements of the beta----alpha phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges--in the absence of Mg2(+)-from 45 degrees C at high to 56 degrees C at low water content, and-at an equimolar content of Mg2(+)-from 52 degrees C at high to 59 degrees C at low water concentrations. In the gel phase-in which the lipid A acyl chains are more disordered than those from saturated phospholipids-cubic phases are adopted at high water content (greater than 60%) and at high [lipid A]:[Mg2+] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal HII state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg2+ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.

Synthesis of methyl 3-O-(alpha-D-glucopyranosyl)-7-O-(L-glycero-alpha-D- manno-heptopyranosyl)-L-glycero-alpha-D-manno-heptopyranoside.

The title trisaccharide glycoside, which is related to part of the core region of the lipopolysaccharide from Salmonella, and the disaccharide glycosides methyl 3-O-alpha-D-glucopyranosyl-L-glycero-alpha-D-manno-heptopyranoside and methyl 7-O-L-glycero-alpha-D-manno-heptopyranosyl-L-glycero-alpha-D-manno- heptopyranoside have been synthesised. Methyl 2,3,4-tri-O-benzyl-L-glycero-alpha-D-manno- heptopyranoside, obtained via a one-carbon elongation at C-6 of methyl 2,3,4-tri-O-benzyl-alpha-D-manno- hexodialdo-1,5-pyranoside, was used as precursor both for the heptosyl donor and acceptor.

Investigation into the fluidity of lipopolysaccharide and free lipid A membrane systems by Fourier-transform infrared spectroscopy and differential scanning calorimetry.

The phase behaviour, particularly the fluidity within each phase state and the transitions between them, of lipopolysaccharides and of their lipid moiety, free lipid A, of various species of Gram-negative bacteria, especially of Salmonella minnesota and Escherichia coli, has been investigated by applying mainly Fourier-transform infrared spectroscopy and differential scanning calorimetry. For enterobacterial strains, the transition temperatures of the gel----liquid crystalline (beta----alpha) phase transition of the hydrocarbon chains in dependence on the length of the sugar moiety are highest for free lipids A (around 45 degrees C) and lowest for deep rough mutant lipopolysaccharides (around 30 degrees C). Evaluating certain infrared active vibration bands of the hydrocarbon moiety, mainly the symmetric stretching vibration of the methylene groups around 2850 cm-1, it was found that, in the gel state, the acyl chains of lipopolysaccharides and free lipid A have a higher fluidity as compared with saturated and the same fluidity as compared with unsaturated phospholipids. This 'partial fluidization' of lipopolysaccharide below the transition temperature correlates with its reduced enthalpy change at that temperature compared to phospholipids with the same chain length. The fluidity depends strongly on ambient conditions, i.e. on the Mg2+ and H+ content: higher Mg2+ concentrations and low pH values make the acyl chains of free lipid A and lipopolysaccharide preparations significantly more rigid and also partially increase the transition temperature. The influence of Mg2+ is highest for free lipid A and decreases with increasing length of the sugar side chain within the lipopolysaccharide molecules, whereas the effect of a low pH is similar for all preparations. At basic pH, a fluidization of the lipopolysaccharide and lipid A acyl chains and a decrease in transition temperature take place. Free lipid A and all investigated rough mutant lipopolysaccharides exhibit an extremely strong lyotropic behaviour in the beta----alpha melting enthalpy but not in the value of the transition temperature. The phase transition is distinctly expressed only at water concentrations higher than 50-60%. A further increase of the water content still leads to an increase in the phase-transition enthalpy, particularly for lipopolysaccharides with a more complete sugar moiety. The fluidity of the hydrocarbon chains is shown to be an important parameter with respect to the expression of biological activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Architecture of bacterial lipid A in solution. A neutron small-angle scattering study.

The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram-negative bacteria, has been investigated by neutron small-angle scattering. The shape of the scattering curves obtained at different H2O/2H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40-45 degrees C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron-scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A.

Structural characterization of monophosphoryl lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide.

Sixteen monophosphoryl Lipid A (MLA) homologs obtained from the lipopolysaccharides of Salmonella minnesota Re595 were separated by preparative thin layer chromatography into eight fractions. The components of these fractions were analyzed directly (or as structural analogs) and characterized by mass spectrometry. Molecular weights were determined by negative and positive ion fast atom bombardment mass spectrometry and component structures were assigned following a study of fragmentation and metastable ion kinetic energy spectrometry. One fraction (TLC-8) contained a single heptaacyl MLA of Mr = 1,954, a structure previously elucidated (Qureshi, N., Mascagni, P., Ribi, E., and Takayama, K. (1985) J. Biol. Chem. 260, 5271-5278). The remaining seven fractions contained 15 additional MLAs with decreasing acylation. Two of these components have been previously reported in S. minnesota and Salmonella typhimurium. Three of the eight TLC fractions (TLC-8, -7, -6) were found to be biologically active toward human platelets inducing their aggregation and secretion of serotonin. All tested fractions induced varying degrees of phosphorylation of a platelet protein of Mr = 47,000 (P47) reflecting protein kinase C activation (Grabarek, J., Her, G. R., Reinhold, V. N., and Hawiger, J. J. (1990) J. Biol. Chem. 265, 8117-8121).

Endotoxic lipid A interaction with human platelets. Structure-function analysis of lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide.

We previously reported that human blood platelets are directly stimulated by endotoxic Lipid A via the protein kinase C pathway (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). To study the relationship between the molecular structure of Lipid A and its ability to activate human platelets, we used Lipid A homologs derived from Salmonella minnesota Re595 lipopolysaccharide. Preparations of Lipid A are heterogeneous in regard to the degree of substitution of fatty acids which result in multiple homologs. These were separated by thin-layer chromatography and characterized by fast atom bombardment spectroscopy and related techniques (Johnson R. S., Her, G.-R., Grabarek, J., Hawiger, J., and Reinhold, V. N. (1990) J. Biol. Chem. 265, 8108-8116). The homologs of monophosphoryl Lipid A (MLA) present in fractions TLC-8 (heptaacyl MLA ion, m/z 1953), TLC-7 (three hexaacyl species with predominant MLA ion m/z 1715), and TLC-6 (four pentaacyl homologs with predominant MLA ion, m/z 1505) induced secretion of [14C]serotonin and aggregation of platelets. Lipid A homologs in fractions TLC-5 (three tetraacyl MLA ions, m/z 1323, 1307, and 1279), TLC-4 (one major triacyl MLA ion, m/z 1097), TLC-3 (tetraacyl MLA ion, m/z 1278), TLC-2 (a diphosphoryl hexaacyl Lipid A ion, m/z 1795, and several ions of low abundance), and TLC-1 (two ions, m/z 1097 and 666) were not active in regard to human platelet aggregation and [14C]serotonin secretion. The most active homolog was heptaacyl MLA ion, m/z 1953, present in TLC-8, while homologs present in TLC-7 and TLC-6 were 5 and 10 times less active, respectively. Rapid phosphorylation of a human platelet protein of Mr 40,000-47,000 (P47), a substrate for protein kinase C activation, preceded secretion of serotonin when platelets were triggered by the most active heptaacyl MLA ion, m/z 1953. These events were time-dependent, with half-maximal response of phosphorylation of P47 at 30 s and [14C]serotonin secretion at 45 s. A marked difference in the degree of phosphorylation of P47 was observed with heptaacyl MLA homolog present in TLC-8 inducing complete phosphorylation (97%), whereas less acylated Lipid A homologs present in TLC-1 caused marginal phosphorylation (20%). These results indicate that the degree of acylation of monophosphoryl Lipid A determines its functional properties toward human platelets in regard to secretion of [14C]serotonin, aggregation, and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

Monomer sequence determination of carbohydrates using fast-atom bombardment mass spectrometry of periodate-oxidized acetate ester derivatives.

A derivatization method, adapted from that of Angel et al. (ref. 10), for sequencing sugar residues in partially degraded poly- and oligo-saccharides using positive-ion f.a.b.-m.s. is described. Derivative selection provides sequence information by directing fragmentation exclusively to both sides of glycosidic oxygen atoms and, in the case of opened rings, between glycosidic carbon and ring oxygen atoms. Polysaccharides or oligosaccharides are subjected to sequential periodate oxidation, borodeuteride reduction, and acetylation. The derivatized polysaccharides are then subjected to partial degradation, acetylation, and high-performance liquid chromatography (h.p.l.c.) purification. F.a.b.-m.s. data obtained on model compounds, using 3-nitrobenzyl alcohol as matrix for f.a.b.-m.s., demonstrated direction of fragmentation to both sides of the glycosidic oxygen atom in unoxidized residues, and to both sides of the acetal oxygen atoms in oxidized residues. Oligosaccharide linkage and sequence may thus be determined by observing fragmentation from both the reducing and non-reducing ends of the molecule. Two Salmonella lipopolysaccharides, derivatized by this procedure, were partially hydrolyzed and then acetylated. Analysis of the h.p.l.c.-purified oligosaccharide derivatives by f.a.b.-m.s. demonstrated the applicability of the technique for sequencing nmol quantities of branched structures.

Presencia de salmonella en ostiones: Cuba, 1985-1988 / Presence of salmonella in oyster: Cuba, 1985-1988

Se analizaron 1 177 muestras de ostiones para la determinación de Salmonella en 7 Centros Provinciales de Higiene y Epidemiología. Se obtuvieron valores promedio en el período que oscilaron desde 0 % de aislamiento en el año 1985 hasta el 4,7 % en el año 1987. Por provincias varió del 0 % en Granma al 10,7 % en Matanzas durante los años 1985-1988. Se valoraron los resultados y posibles causas de la contaminación. Se establecen conclusiones para mejorar la calidad del producto

Vigilancia y determinación de salmonella en dulces con crema / Surveillance and determination of salmonella in cream sweets

Se analizan 3 745 muestras para determinar la presencia de Salmonella en 6 Centros Provinciales de Higiene y Epidemiología en Pinar del Río, La Habana, Matanzas, Holguín, Granma y Santiago de Cuba. Se detectan valores superiores a otras provincias en Matanzas y La Habana. Se analizan los factores de contaminación y se sugieren medidas para evitar la misma en este producto

Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We utilized the recently described tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (TSDS-PAGE) system to study the lipooligosaccharides (LOS) and lipopolysaccharides (LPS) of gram negative bacteria. TSDS-PAGE resulted in a high degree of resolution of LOS and LPS in the 'mini-gel' format. TSDS-PAGE resulted in the LOS and LPS migrating as a function of their Mr during electrophoresis and allowed estimation of Mr from a protein standard. Several species of LOS were analyzed. The newly described procedure allowed a more rapid and accurate analysis of LOS and the core region of LPS.

Development of a new quantitative method for detection of endotoxin by fluorescence labeling of 3-hydroxy fatty acid.

New quantitative method for the detection of minute amounts of endotoxin has been developed using 3-hydroxytetradecanoic acid as a chemical marker. After converting 3-hydroxytetradecanoic acid to methyl ester, it was coupled with a fluorescent probe, anthracene-9-carboxyl chloride, obtained by chlorization of 9-anthroic acid with oxalyl chloride. The resulting ester was isolated by HPLC on silica column. The purified product, methyl-3-0-(9-carboxy-anthracenyl) tetradecanoate (M/Z 462), was highly responsive to a fluorescence spectrophotometer, showing maximum emission with excitation wavelength at 257 nm and emission wavelength at 458 nm in dichloromethane, the limit of detection being as little as 10 f mol. Using this method it is currently possible to detect Salmonella abortus equi endotoxin in aqueous solution at a level of 100 pg.

Reconstitution of the lipid matrix of the outer membrane of gram-negative bacteria as asymmetric planar bilayer.

This paper is a report on the reconstitution of the lipid matrix of the outer membrane of Gram-negative bacteria as an asymmetric planar bilayer. This is the first time that a planar membrane is described, which consists on one side of a phospholipid (PL) mixture and on the other side of lipopolysaccharide (LPS). Therefore, strong emphasis is placed on a physical characterization of this membrane via its electrical properties. The membranes were prepared from spread monolayers or from vesicle-derived monolayers. Contrary to observations for symmetric phospholipid membranes, specific capacitances of (0.67 +/- 0.02) mu F.cm-2, breakdown voltages between 200 and 400 mV and specific conductances between 10(-8) and 2 x 10(-7) S.cm-2 were obtained independent of the preparation method. The LPS-containing membranes were stable up to 3 hr if they were formed and kept at temperatures above the hydrocarbon chain melting temperature of the LPS. For the specific capacitance, a dependence on the aperture radius was observed. This is explained by assuming a toroidal transition zone at the rim of the aperture. First results on the action of the pore-forming alpha-toxin from Staphylococcus aureus on bilayers of different composition demonstrate particular characteristics of this asymmetric bilayer system. The pore-formation rate is highest in symmetric phospholipid bilayers, considerably lower in asymmetric PL/LPS systems and fully inhibited in LPS/LPS systems.

Structure of the lipopolysaccharide antigenic O-chain produced by Salmonella ohio (O:6,7).

Salmonella ohio, which belong to Group C1 (0:6,7) of the Kauffmann-White classification system, produces a smooth lipopolysaccharide which by glycose analysis, methylation, deamination, and 1H-n.m.r. studies was shown to have an O-polysaccharide chain composed of a repeating hexasaccharide unit having the structure [----2)-[alpha-D-Glcp-(1----3)]-alpha-D-Manp-(1----2)-alpha-D-M anp- (1----2)-beta-D-Manp-(1----3)-beta-D-GlcpNAc-(1----2)-beta-D-Ma np-(1----]n.

Characterization of the lipid A component of genuine smooth-form lipopolysaccharide.

The smooth-form lipopolysaccharide of Salmonella abortus equi had earlier been separated into three distinct fractions, a long-chain fraction with an O chain containing 20-50 repeating units, a short-chain fraction consisting of an R lipopolysaccharide and another with 1-6 repeating units, and an R fraction identical to the lipopolysaccharide synthesized by Ra.b-mutant bacteria [Galanos et al. (1988) J. Chromatogr. 440, 397-404]. In this paper, the corresponding lipid A from each fraction was prepared by a newly elaborated procedure based on hydrolysis of the fractions in calcium acetate buffer (pH 3.5) followed by separation of the resulting free lipid A from the polysaccharide on a Sephadex G-100 column. Chemical analysis revealed that lipid A of the R fraction contained the expected spectrum and amounts of fatty acids and it proved to be structurally identical to lipid A of previously studied Salmonella R mutants. In contrast, the lipid A of the long-chain fraction contained only about 60% fatty acids compared to that of the R fraction. The lipid A of the short-chain fraction also expressed a reduced substitution pattern of acyl residues.