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

Sobrepoblación bacteriana del intestino delgado y diarrea crónica / Chronic diarrhea and small bowell bacterial overgrowth

Se estudiaron de modo prospectivo 40 pacientes adultos atendidos en el Consultorio Externo del Servicio de Gastroenterología del Hospital Cayetano Heredia (Lima), catalogados como casos de Diarrea Crónica (DC), por presentar diarrea en un período mayor de 3 semanas. Los pacientes fueron sometidos a exámenes de laboratorio: hematológicos, bioquímicos, coproparasitológicos y coprocultivos. Exámenes endoscópico digestivo y radiografías de tórax y transito intestinal. En 36 casos se practicó el cultivo del contenido duodenal mediante la técnica del Enterotest, con el objeto de determinar la existencia de Sobrepoblación Bacteriana del Intestino Delgado Alto (SOBIA); por el hallazgo de cuentas totales mayor de 10000 gérmenes por ml, y/o coliformes mayor de 1000 gérmenes por ml. Los resultados de los 36 casos mostraron 13 casos (36.1 por ciento) con diagnóstico finales únicos y en 23 casos (63.8 por ciento) éstos eran mixtos, indicando la multifactoriedad de causas de DC. Los diagnósticos principales, fueron Trastornos psicoemocionales 15 casos (41.6 por ciento), SOBIA 11 casos (30.5 por ciento), Parasitosis 6 casos (16.6 por ciento), 2 casos de Diabetes Mellitus (5.5 por ciento), un caso (2.7 por ciento) con Tuberculosis Intestinal y un caso (2.7 por ciento) Desnutrición. El hallazgo el 30.5 por ciento de Sobrepoblación Bacteriana del Intestino Delgado Alto, correspondería a una cifra mayor de la población adulta sana de Lima, y ocupa el segunda lugar en frecuencia de causas de Diarrea Crónica en el presente estudio, confirmando esta importante asociación ya antes señalada por otros investigadores. Orienta a ampliar estos estudios, más aún al tratarse de pacientes que pertenecían a sectores socio-económicos bajos y con limitadas condiciones de saneamiento ambiental

Rahnella aquatilis, a potential contaminant in lager beer breweries.

The species Rahnella aquatilis has been isolated mostly from water, soil, and, in a few cases, from human clinical specimens; little is known about its ecological role. The application of polyacrylamide gel electrophoresis of soluble proteins, DNA-DNA hybridizations and API 20 E systems has shown that Rahnella aquatilis might also be encountered as a contaminant in lager beer breweries.

A conserved domain on enterobacterial porin protein analysed by monoclonal antibody.

Broadly cross-reactive monoclonal antibodies (MAbs) against enterobacterial outer membrane (OM) porin (Po) protein were isolated after immunization of BALB/c mice with whole cells of E. coli 055:B5. MAbs (n = 6) of the IgG class but of four different isotypes were studied. Based on a competition ELISA, all of the MAbs were directed against one and the same Po protein domain (Po I). The MAbs cross-reacted with 72 of 74 strains from 10 different genera of the Enterobacteriaceae. One Morganella and one Salmonella strain showed no cross-reactivity. Also, nine strains of various Neisseria spp. cross-reacted while 21 strains of various other nonenteric Gram-negative bacteria showed no cross-reactivity. The Po I sites were inaccessible in intact homologous bacteria but partially accessible in the OM. Digestion of OM with lysozyme or lysostaphin affected the accessibility of the Po I sites in OMs of various enterobacteria. Lysostaphin strongly enhanced the immunoaccessibility, whereas lysozyme had lesser effects. The enzymes also affected the binding by Neisseria OMs of the anti-Po I MAb. The Po I site was immunogenic both in humans and rabbits. The data indicate that Po I is an important Po protein domain, and that the effects of peptidoglycan-degrading enzymes must be considered in studies of Po protein domains.

Location and identification of substituents with free amino group in lipopolysaccharides of gram-negative bacteria with the use of chromophore labelling.

Interaction of ten different lipopolysaccharides (LPS) with 2,4-dinitrofluorobenzene yielded quantitatively yellow dinitrophenyl derivatives (DNP-LPS) to show the presence of substituents with free amino group. The DNP-LPS samples were degraded with 1% acetic acid, and after removal of lipid A precipitates the supernatants were separated on a Sephadex G-25 column to give coloured polysaccharide, oligosaccharide and monomeric fractions monitored at lambda DNP = 365 nm. The coloured materials, including DNP-derivative of lipid A, were dephosphorylated with hydrofluoric acid followed by identification of the released DNP-amines by thin layer chromatography (TLC) on silica gel. Subsequently, the dephosphorylated materials were hydrolysed with hydrochloric acid followed by TLC analysis. The approach allowed to detect, locate and identify the substituents with free amino group within the LPS molecules. Moreover, two types of core structures within LPS preparation from one strain were discovered for five microorganisms.

Identification of enterobactin and linear dihydroxybenzoylserine compounds by HPLC and ion spray mass spectrometry (LC/MS and MS/MS).

The production of catecholate siderophores was studied in some selected species of Enterobacter (Enterobacteriaceae). The extracted catecholates were separated as iron-free compounds by HPLC on a C18 reversed-phase column using methanol/0.1% phosphoric acid or methanol/0.1% formic acid as a solvent system and identified by ion spray mass spectrometry (LC/MS, MS/MS). Five catecholate compounds were identified which include 2,3-dihydroxybenzoylserine, its linear dimer and trimer, the cyclic enterobactin and an unidentified isomer of enterobactin. In addition, a new large-scale method for the isolation of catecholate siderphores is described which is based on adsorption on XAD-2 and subsequent purification on Sephadex LH20.

[Analysis of cellular fatty acids of Enterobacteria species by gas chromatography-mass spectrometry].

Cellular fatty acid compositions of 15 Enterobacteria were analyzed by gas chromatography-mass spectrometry(GC-MS). About 30 fatty acids were detected in chromatograms, and 13 of them were chemically identified, e.i. C11:0, C12:0, C13:0, C14:0, C15:0, 2OH-C14:0, 3OH-C14:0, C16:1, C16:0, aC17:0, delta C17:0, C18:1 and C18:0. The major cellular fatty acids in all fifteen species were C16:0, C18:1, C15:0, C14:0, C16:1, C13:0, 3OH-C14:0 and C18:0. The cellular fatty acids of Enterobacteria species were characterized by normal straight-chain saturated acids and monounsaturated acids, of which the most abundant fatty acid was C16:0. 3OH-C14:0 was found in all strains of Enterobacteria, whereas 2OH-C14:0 was only found in strains of Serratia species. Other unknown compositions also would have been certain characteristics for bacteria. The present paper would provide some of useful reference data for chemotaxonomy and molecular microbiology of Enterobacteria.

Protein profile typing--a new method of typing Morganella morganii strains.

A new, simple and stable method for typing Morganella morganii strains is described. The 150 strains examined, principally from faeces, contained haemolytic and non-haemolytic representatives of diverse O serogroup, bacteriocin type and biotype. Among the biotypes were some trehalose-fermenting, tetracycline-resistant strains and some non-motile, tetracycline-sensitive, glycerol fermenters. After analysis of cell lysates by sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis, strains could be differentiated into 21 types on the basis of outer membrane proteins (OMP) of 35-40 Kda. The OMP profile was not altered by culture on various common media and was unrelated to either O antigen or morganocin p-type. The finest strain recognition in M. morganii can be achieved by application of all three distinct typing methods.

Numerical analysis of electrophoretic protein patterns of Morganella morganii strains from faeces, wound, urine and other clinical sources.

Sixty-five strains of Morganella morganii (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 13 were from stools (including one from a toucan), 13 from wounds, 11 from urine, five from blood (including one from a snake), five from the respiratory tract (four sputum, one lung), 12 from miscellaneous sources and six from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of 67 M. morganii cultures plus those of the type strains of seven Proteus and Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the M. morganii strains formed 21 clusters at the 91% S level. In the second analysis, in which the principal protein bands (in the 31.6-43.2 kDa range) were excluded, the 67 M. morganii cultures formed a single cluster at the 80% S level distinct from the seven Proteus and Providencia reference strains. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of M. morganii. Reference strains of each of the 21 PAGE types identified are available from NCTC for inclusion in future studies.

Numerical analysis of electrophoretic protein patterns of Providencia stuartii strains from urine, wound and other clinical sources.

Eighty-six strains of Providencia stuartii (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 52 were from urine, 11 from wounds, five from blood (one of these also from urine), four from ear infections, two each from faeces and sputum, one from 'alimentation' and nine from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of 46 Prov. stuartii strains (selected to represent the full range of protein pattern diversity) plus those of the type strains of the four other Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the Prov. stuartii strains formed 13 clusters at the 88% S level. In the second analysis, in which the principal protein bands (in the 33.8-40.7 kDa range) were excluded, 45 of the 46 Prov. stuartii strains formed a single cluster at the 82% S level, whilst the four Providencia reference strains remained unclustered. The 40 strains of Prov. stuartii not included in the cluster analysis were assigned to a protein type by calculating their similarity with the strains in the database used for the cluster analysis. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. stuartii. Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.

Determinants of OmpF porin antigenicity and structure.

Sixty-six murine hybridomas raised to Escherichia coli B/r porin were used to identify and differentiate the epitopes of this outer membrane protein. Anti-porin monoclonal antibodies (mAb) were raised against outer membrane fragments, purified native trimeric porin (trimer), and purified sodium dodecyl sulfate-denatured monomeric porin (monomer). Immunochemical and flow cytometric methods identified five distinct cell surface-exposed determinants on OmpF. The peptide composition of porin epitopes was determined by analysis of mAb reactivity with cyanogen bromide-generated peptide fragments. Four of 43 anti-monomer mAb reacted with surface exposed sites on OmpF, defining epitopes that consist of residues within CNBr peptides d2, d3, and B. The anti-porin mAb panel was also used to evaluate changes in porin antigenic structure in strains with short ompF deletions. Flow cytometric experiments indicated that despite changes in porin permeability, little if any alteration of surface epitopes occurred in these strains. Western immunoblot analysis of the mutant porins showed loss of reactivity with numerous mAb, which was caused by changes in three spatially distinct epitopes at residues 108-111, 118-123, and 124-129. Our findings indicate that in these ompF mutants the residues responsible for altering porin permeability are not exposed on the cell surface, but are buried within the tertiary structure of the protein. One of these regions, which is apparently involved in the determination of channel permeability characteristics, is conserved among 15 of 16 different porin molecules which were screened with the anti-OmpF mAb panel.

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the O-specific polysaccharide from Enterobacter cloacae strain N.C.T.C. 11579 (serogroup O10).

The O-specific polysaccharide from the reference strain (N.C.T.C. 11579) for Enterobacter cloacae serogroup O10 has been isolated and characterised. By means of n.m.r. spectroscopy and methylation analysis, and by studies of the products obtained by Smith degradation or by N-deacetylation-deamination, the repeating unit of the polysaccharide could be allocated the structure shown. The polysaccharides from two cross-reacting serogroups (O9 and O11) have the same monosaccharide composition. (Formula: see text)

Hafnia alvei lipopolysaccharides: isolation, sugar composition and SDS-PAGE analysis.

Lipopolysaccharides (LPS) of 33 strains of Hafnia alvei were isolated and purified. LPS content of the dry bacterial mass ranged from 1.2 to 4.5%. All examined lipopolysaccharides contained glucose, glucosamine, heptose, 3-deoxy-octulosonic acid and often galactose. Rhamnose, mannose, galactosamine, mannosamine and unidentified amino sugars were found in some H. alvei strains. Sialic acid was present in LPS of one strain. D-3-Hydroxybutyryl groups also were identified in lipopolysaccharides of 5 strains of this genus. SDS-PAGE of the lipopolysaccharides was presented in the paper. According to these results two core types exist in H. alvei.

Chemical characterization of lipopolysaccharide from Edwardsiella ictaluri, a fish pathogen.

The chemical components of lipopolysaccharide (LPS) from the fish pathogen Edwardsiella ictaluri (Ed. ictaluri) were analyzed by SDS-PAGE, gas chromatography, and spectrophotometry, and compared with those of Salmonella typhimurium and Escherichia coli 0111:B4. Only four to five low molecular weight species of LPS from Ed. ictaluri were detected by silver staining after separation by polyacrylamide gel electrophoresis. The low molecular weight species, as well as a low sugar content, indicate that the LPS from Ed. ictaluri was of the rough type, compared with that of S. typhimurium and E. coli which were both of the smooth type LPS. Quantitatively, mannose was not a major sugar component in Ed. ictaluri, unlike S. typhimurium. Palmitic, palmitoleic, and cis-9,10-methylene-hexadecanoic acids were predominant fatty acids among the total cellular lipids of Ed. ictaluri. C14 fatty acids comprised 78% of the total in the LPS of this bacterium, with beta-hydroxy-myristate representing 55%. The results of this study suggest that the lipid A segment of the LPS molecule of Ed. ictaluri is similar to S. typhimurium and E. coli, at least with respect to fatty acid content; however, the core polysaccharide of E. ictaluri differs in that it has twice the heptose content.

A large family of bacterial activator proteins.

At least nine different bacterial proteins belong to the LysR family. The gene sequence for one of these proteins is presented here. Six others (Escherichia coli LysR, IlvY, CysB; Salmonella typhimurium MetR; Rhizobium NodD; and Enterobacter cloacae AmpR) are known to activate other genes. Based on sequence alignments, each member of this family is predicted to have a helix-turn-helix DNA binding motif near its amino terminus. The combined evidence indicates that all nine proteins are related by common ancestry, are similarly folded, and are not detectably related to other known bacterial regulatory proteins. The DNA database searching procedure and other methods used in this study should be useful in detecting other groups of related proteins.

Multitest system for biochemical identification of Salmonella, Escherichia coli, and other Enterobacteriaceae isolated from foods: collaborative study.

Nine laboratories evaluated the ability of the MICRO-ID test system to correctly identify members of the Enterobacteriaceae. A total of 78 isolates representing 11 genera of enterics that had been previously isolated from foods were used in the collaborative study. The collaborators streaked each isolate on plate count agar and incubated the plates overnight at 35 degrees C to check purity of the isolates. Then they proceeded with the method in which an isolated colony is transferred to a plate count agar slant and is incubated 18-24 h at 35 degrees C. Growth from the slant is emulsified in 3.5 mL physiological saline to a density comparable to a McFarland No. 2 tube and is then used to inoculate the test (MICRO-ID) strip. The strip is incubated 4 h at 35 degrees C and the reactions are read and recorded. Isolates are identified by using an octal code and the test kit manual. The system correctly identified 98.8% of the Salmonella isolates, 97.7% of the E. coli isolates, and 84.6% of the other 9 enteric genera tested. The system has been approved interim official first action as an alternative to conventional biochemical tests (1) for presumptive generic identification of food-borne Salmonella and for screening and elimination of non-Salmonella isolates; (2) for identification of E. coli from foods; and (3) for presumptive generic identification of other Enterobacteriaceae isolated from foods.

Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC.

The NTRC protein (ntrC product) of enteric bacteria activates transcription of nitrogen-regulated genes by a holoenzyme form of RNA polymerase that contains the ntrA product (sigma 54) as sigma factor. Although unmodified NTRC will bind to DNA, it must be phosphorylated to activate transcription. Both phosphorylation and dephosphorylation of NTRC occur in the presence of the NTRB protein (ntrB product). We here demonstrate rigorously that it is the NTRB protein that is a protein kinase by showing that NTRB can phosphorylate itself, whereas NTRC cannot. Phosphorylated NTRC (NTRC-P) is capable of autodephosphorylation with a first-order rate constant of 0.14-0.19 min-1 (t 1/2 of 5.0-3.6 min) at 37 degrees C. In addition, there is regulated dephosphorylation of NTRC-P. By contrast to the autophosphatase activity, regulated dephosphorylation requires three components in addition to NTRC-P: the PII regulatory protein, NTRB, and ATP. NTRC is phosphorylated within its amino-terminal domain, which is conserved in one partner of a number of two-component regulatory systems in a wide variety of eubacteria. A purified amino-terminal fragment of NTRC (approximately equal to 12.5 kDa) is sufficient for recognition by NTRB and is autodephosphorylated at the same rate as the native protein.