Effect of challenge temperature and solute type on heat tolerance of Salmonella serovars at low water activity

Salmonella spp. are reported to have an increased heat tolerance at low water activity (a(w); measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of Salmonella enterica serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80 degrees C) and a(w) (rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = -bt(n)) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different Salmonella strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce a(w), or six low-a(w) foods artificially contaminated with Salmonella in place of the sugar broths. The data demonstrate that, at temperatures of > or =70 degrees C, Salmonella cells at low a(w) were more heat tolerant than those at a higher a(w) but below 65 degrees C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce a(w), but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-a(w) foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.     

Toxic effects of linear alkylbenzene sulfonate on metabolic activity, growth rate, and microcolony formation of Nitrosomonas and Nitrosospira strains

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC(50)], 6 to 38 mg liter(-1)) was affected much less by LAS than the growth rate and viability (EC(50), 3 to 14 mg liter(-1)) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter(-1)); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH(4)(+) oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.     

Epithelium-associated bacteria in the gastrointestinal tract of Arctic charr (Salvelinus alpinus L.). An electron microscopical study

AIMS: The primary aim was to use transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to define the location of epithelium-associated bacteria in the digestive tract of the salmonid fish, Arctic charr (Salvelinus alpinus). METHODS AND RESULTS: TEM and SEM examination of the gastrointestinal tract demonstrated substantial numbers of ovoid and rod-shaped bacterial cells associated with the microvillous brush borders of enterocytes. Bacteria were found at the tips of microvilli as well as between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in two regions (pyloric caeca and midgut). CONCLUSION: Electron microscope examination of the gut is an important tool for evaluating the microbial ecology of the fish digestive tract ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the current study clearly demonstrate that the intestine is involved in bacterial endocytosis.     

Bone bank service in Odense, Denmark. Audit of the first ten years with bone banking at the Department of Orthopaedics, Odense University Hospital

There has been an increase in the demand for allograft bone in recentyears. The Odense University Hospital bone bank has been in function since1990,and this paper outlines our results during the 10 year period 1990–1999.Potential donors were screened by contemporary banking techniques which includea social history, donor serum tests for HIV, hepatitis B and C, and graftmicrobiology. The bones were stored at –80 °C. No typeofsecondary sterilisation was made. 423 femoral heads were approved and donatedto300 patients,1–6 heads/operation. The allografts have been used mainly toreconstruct defects at revision hip arthroplasty (34%), and for fracturesurgery(24%). 7 % of all transplanted patients were reoperated because of infection.Inthe hip revision group the infection rate was 4 %. There were no cases ofdisease transmission. During the 10 year period there was a change in theclinical use of the allografts. In the first years the allografts were mainlyused for spinal fusion surgery, but today the majority are used in hip revisionand fracture surgery. The clinical results correspond to those reported inlarger international series.     

Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. or oryzae and tobacco

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-alpha-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.     

Overexpression of Bud5p can suppress mutations in the Gsp1p guanine nucleotide exchange factor Prp20p in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The gene product Prp20p, which is located in the nucleus, serves as the nucleotide exchange factor (GEF) for the small nuclear G protein Gsp1p in Saccharomyces cerevisiae, and catalyses the replacement of Gsp1-bound GDP by GTP. These proteins are involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules, cell cycle progression, DNA replication and maintenance of chromosome structure/stability. It is believed that in order to complete a full GDP/GTP cycle, Gsp1p has to shuttle between the nucleus and the cytoplasm, where its GTPase Activating Protein (GAP) Rna1p is located. Here, we report on the ability of Bud5p, the exchange factor for Rsr1p, to suppress conditional prp20 mutants when an extra copy of GSP1 is present. This suppression by BUD5 can be reversed by simultaneous overexpression of RNA1, and is not Rsr1p-dependent, nor allele-specific. We also show that Bud5p can physically interact with Gsplp, both in vitro and in vivo. These,findings raise the possibility that Bud5p could act as a cytoplasmic exchange factor for Gsp1p and, therefore, that a complete GDP/GTP cycle could take place in the cytoplasm.     

Cloning of maltase gene from a methylotrophic yeast, Hansenula polymorpha

The Hansenula polymorpha maltase structural gene (HPMAL1) was isolated from a genomic library by hybridization of the library clones with maltase-specific gene probe. An open reading frame of 1695 nt encoding a 564 amino-acid protein with calculated molecular weight of 65.3 kD was characterized in the genomic DNA insert of the plasmid p51. The protein sequence deduced from the HPMAL1 exhibited 58 and 47% identity with maltases from Candida albicans and Saccharomyces carlsbergesis encoded by CAMAL2 and MAL62, respectively, and 44% identity with oligo-alpha-1,6-glucosidase from Bacillus cereus. The recombinant Hansenula polymorpha maltase produced in Escherichia coli hydrolyzed p-nitrophenyl-alpha-D-glucopyranoside (PNPG), sucrose, maltose and alpha-methylglucoside and did not act on melibiose, cellobiose, trehalose and o-nitrophenyl-beta-D-galactopyranoside (ONPG). The affinity of the recombinant enzyme for its substrates increased in the order maltose 相似文献   

Two mutants of human heparin binding protein (CAP37): toward the understanding of the nature of lipid A/LPS and BPTI binding

Heparin binding protein (HBP) is an inactive serine protease homologue with important implications in host defense during infections and inflammations. Two mutants of human HBP, [R23S,F25E]HBP and [G175Q]HBP, have been produced to investigate structure-function relationships of residues in the putative lipid A/lipopolysaccharide (LPS) binding site and BPTI (bovine pancreatic trypsin inhibitor) binding site. The X-ray structures have been determined at 1.9 A resolution for [G175Q]HBP and at 2.5 A resolution for the [R23S,F25E]HBP mutant, and the structures have been fully refined to R-factors of 18.2 % and 20.7 %, respectively. The G175Q mutation does not alter the overall structure of the protein, but the ability to bind BPTI has been eliminated, and the mutant mediates only a limited stimulation of the LPS-induced cytokine release from human monocytes. The lipid A/LPS binding property of [G175Q]HBP is comparable with that of native HBP. The R23S,F25E mutations do not affect the binding of lipid A/LPS and BPTI or the LPS-induced cytokine release from human monocytes. This shows that two diverse ligands, lipid A/LPS and BPTI, do not share binding sites. Previously, there was convincing evidence for the proposed lipid A/LPS binding site of HBP. Unexpectedly, the extensive structural changes introduced by mutation of Arg23 and Phe25 do not affect the binding of lipid A/LPS, indicating that another not yet identified site on HBP is involved in the binding of lipid A/LPS.