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An evaluation of selective broths based on the bi-selenite ion and on hypertonic strontium chloride in Salmonellae detection in egg products.
Of the 104 isolations of Salmonella sp. from egg pulp, 97 were obtained from strontium chloride M broth, 42 from strontium selenite broth and 57 from strontium selenite A broth. The results suggest that the first medium may be used more successfully than bi-selenite based media for enrichment and subsequent detection of salmonellae in egg products; however, the growth of S. pullorum was not satisfactory in strontium chloride M broth.
Application of ozone for enhancing the microbiological safety and quality of foods: a review.
Ozone (O3) is a strong antimicrobial agent with numerous potential applications in the food industry. High reactivity, penetrability, and spontaneous decomposition to a nontoxic product (i.e., O2) make ozone a viable disinfectant for ensuring the microbiological safety of food products. Ozone has been used for decades in many countries and recently, the generally recognized as safe (GRAS) status of this gas has been reaffirmed in the United States. Ozone, in the gaseous or aqueous phases, is effective against the majority of microorganisms tested by numerous research groups. Relatively low concentrations of ozone and short contact time are sufficient to inactivate bacteria, molds, yeasts, parasites, and viruses. However, rates of inactivation are greater in ozone demand-free systems than when the medium contains oxidizable organic substances. Susceptibility of microorganisms to ozone also varies with the physiological state of the culture, pH of the medium, temperature, humidity, and presence of additives (e.g., acids, surfactants, and sugars). Ozone applications in the food industry are mostly related to decontamination of product surface and water treatment. Ozone has been used with mixed success to inactivate contaminant microflora on meat, poultry, eggs, fish, fruits, vegetables, and dry foods. The gas also is useful in detoxification and elimination of mycotoxins and pesticide residues from some agricultural products. Excessive use of ozone, however, may cause oxidation of some ingredients on food surface. This usually results in discoloration and deterioration of food flavor. Additional research is needed to elucidate the kinetics and mechanisms of microbial inactivation by ozone and to optimize its use in food applications.
The differential diagnosis of early gastric mucosa-associated lymphoma: polymerase chain reaction and paraffin section immunophenotyping.
The distinction between benign florid lymphoid hyperplasia and low-grade gastric mucosal-associated lymphoid tissue (MALT) lymphoma may be a challenge. The presence of monoclonal B cells in Helicobacter pylori-chronic active gastritis has suggested that polymerase chain reaction (PCR) data should be viewed with caution. We investigated the reliability of PCR versus immunophenotyping in diagnosing early gastric MALT lymphoma. We studied 1511 biopsies from eight patients with high-grade primary gastric lymphoma, 25 with low-grade MALT lymphoma, 32 with atypical lymphoid infiltrates, and 39 with Helicobacter pylori-chronic active gastritis. Paraffin sections from all cases were stained with antibodies to CD20, CD3, AE1/AE3, kappa and lambda. PCR was performed on paraffin sections using the primer set VH-FR3/J(H). Using histopathology as the gold standard in diagnosis, we confirmed monoclonality in 22 of 25 MALT lymphomas (88%); a clonal band was found in 38% (15 of 39) of patients with chronic active gastritis. An immunophenotype pattern with predominance of CD20-positive cells in lymphocytic infiltrates was associated with monoclonality in 92% of cases. The presence of an enlarged irregular mantle zone was found in both monoclonal and polyclonal areas. An equal prevalence of B and T cells in lymphocytic infiltrates was associated with a polyclonal pattern in 24 of 31 cases (77%). Immunostaining of sIg (kappa and lambda) was difficult in paraffin sections and convincing proof of monoclonality was not obtained. Lymphoepithelial lesions were infrequent in gastric biopsies and their presence was highlighted with keratin stains. Because monoclonal B cells are observed in Helicobacter pylori-associated gastritis, the correct interpretation of clonality by PCR remains unclear. Paraffin section IHC using CD20 and CD3 is especially useful to confirm the diagnosis of gastric MALT lymphoma.
Listeria monocytogenes ActA protein interacts with phosphatidylinositol 4,5-bisphosphate in vitro.
The N-terminal region of the Listeria monocytogenes ActA protein, in conjunction with host cell factors, is sufficient for actin polymerization at the bacterial surface. Previous data suggested that ActA could protect barbed ends from capping proteins. We tested this hypothesis by actin polymerization experiments in the presence of the ActA N-terminal fragment and capping protein. ActA does not protect barbed ends from capping protein. In contrast, this polypeptide prevents PIP(2) from inhibiting the capping activity of capping protein. Gel filtration and tryptophan fluorescence experiments showed that the purified ActA N-terminal fragment binds to PIP(2) and PIP, defining phosphoinositides as novels ligands for this functional domain of ActA. Phosphoinositide binding to the N-terminal region of ActA may induce conformational changes in ActA and/or facilitate binding of other cell components, important for ActA-induced actin polymerization.
Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru.
Twenty-nine epidemiological unrelated and mostly multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains from Peruvian patients.
To investigate the molecular genetics of MDR-TB strains recovered in a Latin American country.
Antimicrobial agent susceptibility testing, major genetic group designation, IS6110 fingerprinting, spoligotyping, and automated deoxyribonucleic acid sequencing of regions of the katG, rpoB, embB, gyrA, and pncA genes with mutations commonly associated with drug resistance.
Nineteen isolates were found to be multidrug-resistant by susceptibility testing. IS6110 typing showed that virtually all isolates were unique and therefore had independently acquired drug resistance. Seventy-nine percent of isoniazid-resistant strains had a Ser315Thr amino acid change in KatG. Ninety-five percent of rifampin-resistant isolates had amino acid replacements in the rifampin-resistance determining region of RpoB. Six of 11 ethambutol-resistant strains had EmbB alterations. Eleven pyrazinamide-resistant strains had distinct mutations in pncA.
Virtually all organisms evolved drug resistance independently. The types of drug resistance-associated mutations identified were very similar to changes occurring in isolates from other areas of the world. Nucleotide sequence-based strategies for rapid detection of drug resistance-conferring mutants will be applicable to organisms recovered in Peru, and potentially other areas of Latin America.
Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors.
Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.
Identification of a novel glycoprotein-binding activity in Streptococcus pyogenes regulated by the mga gene.
The interaction between Streptococcus pyogenes and the host cell surface is not completely understood. Characterization of the adhesion mechanisms of the bacterium to the host cell surface is needed in order to develop new vaccines and anti-adhesion drugs. The presence of glycoprotein-binding activities among streptococcal strains was investigated. An activity binding to thyroglobulin, fetuin, asialofetuin and mucin but not non-glycosylated proteins was found to be present in the majority of the S. pyogenes strains studied. Cross-inhibition experiments suggested that the glycoproteins share a common structure recognized by the bacteria. The glycoprotein-binding activity was found to be proteinaceous, tightly attached to the bacterial surface and it also mediated the adherence of bacteria to solid surfaces coated with glycoproteins. The activity was found by transposon mutagenesis and complementation to be regulated by the multiple-gene regulator Mga, which has been implicated as a regulator of S. pyogenes virulence factors.
Evaluation of three Brucella soluble antigens used in an indirect Elisa to discriminate S19 vaccinated from naturally infected cattle.
An O-polysaccharide (O-chain) and a hot-water extracted polysaccharide (PS), both obtained from Brucella abortus 1119-3, and a B. melitensis 16M native hapten (NH) were evaluated by indirect enzyme linked immunosorbent assay (ELISA) on three groups of cattle sera. The sera tested were: (a) 75 sera from cows naturally infected with B. abortus; (b) 130 sera from non-infected and non-vaccinated cattle; and (c) 61 sera from non-infected heifers recently vaccinated with B. abortus Strain 19 (S19). Sensitivity (Se), specificity (Sp) and the capability to discriminate vaccinated cattle (ADV) were determined. Using PS antigen, Se was 100% and the Sp was 97.7%, while the highest Sp was obtained by using the O-chain (99.2% ). For the NH antigen, Se was 94.7% and the Sp was 90.0%. The ADV of the three antigens was approximately 85%. Statistical analysis showed significant differences between O-chain/PS and O-chain/NH antigens. The agreement among antigens determined by kappa coefficient was 0.899 for O-chain/PS, 0.845 for O-chain/NH and 0.795 for PS/NH.
Genotyping by restriction endonuclease analysis compared to phenotyping by antibiogram for typing methicillin-resistant Staphylococcus aureus strains colonizing patients in a nursing home.
To assist in defining patterns of methicillin-resistant Staphylococcus aureus (MRSA) colonization in a skilled nursing facility (SNF), we compared genotyping by field-inversion gel electrophoresis (FIGE) restriction endonuclease digestion analysis (REA) with phenotyping by antibiogram for defining strain relatedness among MRSA isolates from SNF patients.
Prospective screening culture surveillance for MRSA among patients in a community SNF.
Nares and stool swab cultures were obtained from newly admitted patients and from all patients quarterly. MRSA were isolated by oxacillin screening agar. Antibiograms were determined by the disk-diffusion method, and genotyping was by FIGE REA.
It was shown that, among isolates with the same genotypes, many had different antibiograms; among isolates with the same antibiograms, many had different genotypes; and the discriminatory indices for isolates of MRSA by FIGE REA and by antibiogram were 0.56 and 0.78, respectively.
Our study demonstrated that, in patients from one SNF, genotyping by FIGE REA identified two prevalent REA DNA types, but with variability of antibiogram patterns within each DNA type; the antibiogram also identified prevalent patterns with variability of REA DNA type within each antibiogram pattern. The discriminatory index of antibiograms alone, or of genotypes alone as determined by FIGE REA, was poor for strains of MRSA isolated from the SNF patients in our study.
Immune response to infection with Salmonella typhimurium in mice.
Infection of mice with Salmonella typhimurium results in systemic infection and a disease similar to that seen in humans after infection with S. typhi. The innate immune system can restrict replication of S. typhimurium to a certain degree, but for effective control and eradication of bacteria, acquired immunity is essential. Salmonella infection induces the generation of specific CD4+ and CD8+ T cells, and both T cell populations are important for protection during primary and secondary responses, although the mechanisms underlying T cell-mediated protection are not yet completely understood. Infection with S. typhimurium also results in a strong antibody response to Salmonella antigens and, in contrast to most other intracellular bacteria, this antibody response participates in protection. In summary, the response to S. typhimurium involves both T and B cell-mediated immunity, and mechanisms mediated by both lymphocyte populations are important for control of primary infection and protection against secondary infection.
The role of Fcgamma receptor polymorphisms and C3 in the immune defence against Neisseria meningitidis in complement-deficient individuals.
Individuals with either a late (C5-9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti-meningococcal defence in both LCCD persons and properdin-deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcgammaRIIa (CD32) and FcgammaRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcgammaRIIa and FcgammaRIIIb allotypes was assessed in 15 LCCD and in 15 properdin-deficient patients with/without previous meningococcal disease, we found the combination of FcgammaRIIa-R/R131 with FcgammaRIIIb-NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13.9, Fisher's test P = 0.036). No such relation was observed in the properdin-deficient patients. The importance of FcgammaRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcgammaRIIa-R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0.05) less than PMN from FcgammaRIIa-H/H131 donors. When properdin-deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0.001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0.6568, P = 0. 01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcgammaR allotypes. Properdin-deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.
Symbiotic induction of pyruvate dehydrogenase genes from Sinorhizobium meliloti.
Genes coding for components of the pyruvate dehydrogenase (PDH) multienzyme complex (PDHc) from Sinorhizobium meliloti, the alfalfa symbiont, have been isolated on the basis of their high expression in symbiotic bacteria. The Elp component, PDH, is encoded by two genes, pdhAalpha (1,047 bp) and pdhAbeta (1,383 bp), a situation encountered in the alpha-proteobacteria Rickettsia prowazekii and Zymomonas mobilis as well as in some gram-positive bacteria and in mitochondria. pdhAalpha and pdhAbeta precede pdhB (1,344 bp), which encodes the E2p component, dihydrolipoamide acetyltransferase, of the PDHc. No gene encoding the E3 component, lipoamide dehydrogenase, was found in the immediate vicinity of pdhA and pdhB genes. pdhAalpha, pdhAbeta and pdhB likely constitute an operon. Here, we provide evidence that pdhA expression is induced in the symbiotic stage, compared with free-living conditions. We demonstrate that symbiotic expression of pdhA genes does not depend on the fix LJ regulatory cascade that regulates nitrogen fixation and respiration gene expression in symbiotic S. meliloti cells. Induction of pdhA expression could be obtained under free-living conditions upon the addition of pyruvate to the culture medium. Induction by pyruvate and symbiotic activation of pdh gene expression take place at the same promoter.
A new purification method for overproduced proteins sensitive to endogenous proteases.
Proteolysisis a major problem in purification of overproduced proteins for structural studies. We developed a new method to avoid proteolysis of the products even in cases where popular protease inhibitors do not work effectively. When we cloned FlgF, a flagellar rod protein, from Salmonella typhimurium and overproduced it in Escherichia coli, FlgF was highly susceptible to cleavage by endogenous proteases after cell disruption even in the presence of various protease inhibitors. However, FlgF was not digested when the cells were disrupted in the presence of urea, which allowed us to develop the following new purification procedure. After cell disruption in the presence of urea and removal of the cell debris, the supernatant was passed through tandem-connected cation- and anion-exchange columns. Proteases were trapped in the cation-exchange column, and protease-free FlgF was eluted from the disconnected anion-exchange column. This gave a stable full-length product suitable for crystallization trials. The key procedures are cell disruption in the presence of urea and linked ion-exchange chromatography to quickly remove proteases as well as urea. This fast and simple method can be applied to purification of other overproduced proteins that are very sensitive to proteolysis.
Effects of 2,2',5,5'-tetrachlorobiphenyl and biphenyl on cell membranes of Ralstonia eutropha H850.
The effects of 2,2',5,5'-tetrachlorobiphenyl (TeCB), a PCB congener, and biphenyl on the cytoplasmic membranes of Ralstonia eutropha H850 were investigated by measuring fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe, and determining the cellular fatty acid compositions. TeCB significantly affected the membrane of R. eutropha H850 cells grown on fructose by decreasing DPH fluorescence polarization. In contrast, the membrane of cells grown on biphenyl showed a considerably less significant effect of TeCB on membrane polarization than in fructose-grown cells. An increase in the ratio of total saturated to unsaturated fatty acids in cells grown on biphenyl suggested less of a fluidizing effect of TeCB on membranes in those cells. When biphenyl-grown cells were transferred back to a fructose medium, they required 25 generations for the membrane polarization and fatty acid compositions of these cells to revert back to those of the initial fructose-grown cells. The re-adaptation to a change in temperature required only five generations to return to normal. These results show that biphenyl affects cells in more ways than simply fluidizing the cytoplasmic membrane.
Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris.
Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter.
Cloning and expression analysis of Phytoplasma protein translocation genes.
Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.
Evaluation of two commercial methods for the detection of Listeria sp. and Listeria monocytogenes in a chicken nugget processing plant.
This study measures the detection performances of two rapid test systems (Listeria Rapid Test Clearview and Bax system) for the screening of Listeria sp. and Listeria monocytogenes, respectively. A total of 413 samples from different sources (product from (i) different stages of processing, (ii) different environments, and (iii) different food handlers), collected from a chicken nugget processing plant, were analysed by both rapid methods and a cultural method consisting of pre-enrichment, enrichment, and isolation onto selective agars (PALCAM, LPM, and HCLA). Overall, results showed an excellent correlation between data obtained using Clearview and the cultural method, with Clearview presenting an efficiency of 99%. Bax showed a lower correlation using the cultural method, with an efficiency of 71.1%. The type of sample did not affect the efficiency of Clearview, which varied from 98.1% for product samples to 100% for environmental and food handler samples, while for Bax it had a marked influence. Efficiency of Bax varied from as high as 100% for food handlers to 37.9% for product samples.
Long-term Helicobacter pylori infection and the development of atrophic gastritis and gastric cancer in Japan.
The incidence of gastric cancer and the prevalence of Helicobacter pylori are high in Japan, so it is an important issue whether long-term H. pylori infection leads to chronic atrophic gastritis, considered one of the precursors of gastric cancer. We have reported that the grade of atrophy was higher in H. pylori-positive subjects than in H. pylori-negative subjects. It has also been reported that the atrophy of gastric mucosa increased in H. pylori-infected monkeys compared with control monkeys in a 5-year follow-up study. Most H. pylori infections occur in children, and atrophy of the gastric mucosa progresses during aging. Long-term data show that H. pylori infection can lead to gastric atrophy and may play an important role in the development of gastric cancer. Interestingly, there was no difference in the prevalence of H. pylori between patients with chronic gastritis and gastric cancer in Japan, but the prevalence of H. pylori in young Japanese gastric cancer patients was significantly higher than in the control group. These data clearly show that H. pylori infection is one of the risk factors of gastric cancer in young Japanese people, There is no answer to whether curing H. pylori infection can reverse the atrophy of the gastric mucosa and decrease the risk of gastric cancer development. To clarify this issue, an intervention study must be done. A large clinical trial called the Japanese Intervention Trial of H. pylori is in progress.
Serotypes of Vibrio parahaemolyticus from clinical and environmental sources in Togo (West Africa).
Serological analysis of O and K antigens was performed on 343 strains of Vibro parahaemolyticus isolated from clinical and environmental sources in Togo. Only two strains were not typable by the available O antisera. K untypable strains were found in 4.8% of isolates from gastroenteritis patients, in 11% from healthy carriers, and in 47% and 46% of isolates, respectively, from water and fish samples. Thirteen serotypes identified in Togo are not considered in the Japanese antigenic scheme. The suitability of the Japanese typing scheme for geographic areas outside of Japan is discussed and its extension is suggested.
Emergence of resistance of vancomycin-resistant Enterococcus faecium in a thermal injury patient treated with quinupristin-dalfopristin and cultured epithelial autografts for wound closure.
Vancomycin-resistant Enterococcus faecium and faecalis (VRE) remains a major complication among critically ill patients. A 26-year-old patient with 65% total body surface area burns (TBSA) was infected with several E. faecium strains during his admission that were resistant to vancomycin. Because chloramphenicol was the standard treatment at this time, this drug was initiated until, the organism was identified as E. faecium and reported as susceptible to quinupristin-dalfopristin. Given these data, it was then decided to discontinue the chloramphenicol therapy. Quinupristin-dalfopristin therapy resulted in initial reduction of fever and white blood cell counts that continued over the next 5 days. However, on day 7 of quinupristin-dalfopristin therapy, a return of fever and elevation of the white blood cell count was noted and a repeated E. faecium blood culture demonstrated sudden resistance to quinupristin-dalfopristin (Bauer-Kirby zone size <14 mm). Chloramphenicol was restarted and the patient improved slowly over a period of 16 days. Our indigenous VRE had limited exposure to quinupristin-dalfopristin in the recent past; however, resistance emerged with the first commercial use of this agent in our burn treatment center. High-dose chloramphenicol treatment did not appear to impair engraftment of cultured epithelial autografts (CEA) in this patient.
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