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Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols.

The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).

Alcohol Dehydrogenase↗

Rhamnolipid biosurfactants produced by Renibacterium salmoninarum 27BN during growth on n-hexadecane.

A new strain Renibacterium salmoninarum 27BN was isolated for its capacity to utilize n-hexadecane as sole substrate. Growth on n-hexadecane was accompanied with the production of glycolipid surface active substances detected by surface pressure lowering and emulsifying activity. Glycolipid detection by thin layer chromatography and infrared spectra analyses showed for the first time that Renibacterium salmoninarum 27BN secretes the two rhamnolipids RLL and RRLL typical for Pseudomonas aeruginosa. Growth of Renibacterium salmoninarum 27BN on n-hexadecane depended on the bioavailability of the substrate and the secreted rhamnolipids appeared to be efficient in increasing hexadecane availability for the cells.

Alkanes↗

In vitro activity of cefotaxime against clinically significant pathogens.

The present in vitro antibacterial activities of cefotaxime and 8 other cephalosporins (cefoperazone, cefmenoxime, cefpiramide, latamoxef, cefamandole, cefmetazole, cefotiam and cephazolin) were evaluated simultaneously in 384 strains of Gram-positive cocci, 595 strains of Enterobacteriaceae, 240 strains of non-fermenters and 143 strains of anaerobes and miscellaneous organisms. The results were expressed as minimum inhibitory concentration (MIC) range, MIC50 and MIC90. Of the beta-lactams, cefotaxime and latamoxef exhibited the highest activity against a wide variety of Gram-positive and Gram-negative bacteria. MIC90 of cefotaxime, however, for species of Pseudomonas aeruginosa, Xanthomonas maltophilia, enterococci, Bacteroides sp. and Clostridium difficile were more than 100 mg/L. Cefpiramide and cefoperazone were generally less active than these 2 agents. All strains were tested for beta-lactamase production by the cefinase disc method and the relationship of susceptibility to beta-lactams was evaluated in each species. The need was demonstrated for periodic susceptibility testing to be performed to better guide empirical antimicrobial therapy.

Cefamandole↗

Stomatococcus mucilaginosus septicemia in two bone marrow transplanted patients.

A 44-year-old man with chronic myeloid leukemia and a 10-year-old boy with adrenoleukodystrophy, both admitted for bone marrow transplantation in December 1992, developed clinical signs of septicemia within 2 weeks after transplantation. Three strains of Stomatococcus mucilaginosus were isolated from blood cultures. These were among the first cases of S. mucilaginosus infection diagnosed at our Laboratory and the first reported from Scandinavia. S. mucilaginosus is part of the endogenous oral flora. Both patients had signs of oral mucositis. All 3 strains were isolated earlier with the Laboratory's present blood culture system, compared with the one in use before spring 1992.

Adult↗

Immobilized cells in meat fermentation.

The immobilization of microbial cells can contribute to fermented meat technology at two basic levels. First, the solid/semisolid nature (low available water) of the substrate restricts the mobility of cells and results in spatial organizations based on "natural immobilization" within the fermentation matrix. The microniches formed influence the fermentation biochemistry through mass transfer limitations and the subsequent development and activity of the microflora. This form of immobilization controls the nature of competition between subpopulations within the microflora and ultimately exerts an effect on the ecological competence (ability to survive and compete) of the various cultures present. Second, immobilized cell technology (ICT) can be used to enhance the ecological competence of starter cultures added to initiate the fermentation. Immobilization matrices such as alginate can provide microniches or microenvironments that protect the culture during freezing or lyophilization, during subsequent rehydration, and when in competition with indigenous microflora. The regulated release of cells from the microenvironments can also contribute to competitive ability. The regulation of both immobilization processes can result in enhanced fermentation activity.

Fermentation↗

Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines.

Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.

Animals↗

Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes.

We report the application of a nucleic acid-based assay that enables direct detection and identification of bacterial pathogens in fish kidney tissue without the need for bacterial culture. The technique, known as terminal restriction fragment length polymorphism (T-RFLP), employs the polymerase chain reaction (PCR) using a primer pair that targets 2 highly conserved regions of the gene that encodes for the 16S small subunit of the bacterial ribosome. Each primer is 5' labeled with a different fluorescent dye, which results in each terminus of the resulting amplicon having a distinguishable fluorescent tag. The amplicon is then digested with a series of 6 restriction endonucleases, followed by size determination of the 2 labeled terminal fragments by capillary electrophoresis with laser-induced fluorescence detection. Comparison of the lengths of the full set of 12 terminal fragments with those predicted based on analyses of GenBank submissions of 16S sequences leads to presumptive identification of the pathogen to at least the genus, but more typically the species level. Results of T-RFLP analyses of genomic DNA from multiple strains of a number of fish bacterial pathogens are presented. The assay is further demonstrated on fish kidney tissue spiked with a known number of cells of Flavobacterium psychrophilum where a detection limit of ca. 30 CFU mg(-1) of tissue was estimated. A similar detection limit was observed for several other gram-negative pathogens. This procedure was also used to detect Aeromonas salmonicida and Renibacterium salmoninarum in the kidney tissue of 2 naturally infected salmonids.

Aeromonas↗

Relationship between antigen concentration and bacterial load in Pacific salmon with bacterial kidney disease.

Using data collected to test spawning female Pacific salmon (Oncorhynchus kisutch and O. tshawytscha for the presence and severity of bacterial kidney disease (BKD), a mathematical model of the relationship between bacterial load and antigen concentration in tissues and ovarian fluid is developed. Renibacterium salmoninarum, the causative agent of BKD, secretes large amounts of a 57 kDa protein ('p57'), its major soluble antigen, which eventually breaks down or is otherwise removed from free circulation. Bacterial load and soluble antigen concentration in tissues are strong indicators of fish health, while in ovarian fluid they are predictors of the success of offspring. Model results indicate either an exponentially increasing antigen removal rate or an exponentially decreasing per-bacterium antigen secretion rate with increasing antigen concentration. Possible mechanisms underlying the observed relationship include a nonlinear increasing autolytic rate of the 'p57' antigen and a bacterium-antigen interaction threshold which prevents bacterial antigen secretion.

Actinomycetales Infections↗

Renibacterium salmoninarum: effect of hypochlorite treatment, and survival in water.

The effect of different concentrations of sodium hypochlorite on Renibacterium salmoninarum and the survival of the bacterium in autoclaved river water and groundwater were examined. The disinfection trial was performed using R. salmoninarum ATCC 33209. The concentrations of free chlorine were 10, 50, 100 and 200 mg 1(-1), the contact times were 5, 15, and 30 min and 24 h, and the test suspensions were subcultured both on Kidney disease medium (KDM2) agar and in 3 parallel KDM2 broths, which were then subcultured on KDM2 and selective KDM (SKDM) agar. The survival of the bacterium in river water and groundwater was studied using 4 isolates of R. salmoninarum including ATCC 33209. Treatment with sodium hypochlorite effectively reduced the number of culturable cells of R. salmoninarum, but use of the recovery broth showed that small numbers of cells remained viable at all concentrations of free chlorine. The numbers of R. salmoninarum decreased to an undetectable level after 4 wk incubation in the survival trials, but low numbers of colonies were again found in the subculture after 5 wk incubation. Viable cells of R. salmoninarum were still detected in subcultures of all strains after 20 wk of incubation in river water.

Agar↗

Prevalence and diagnosis of bacterial kidney disease (BKD) in Scotland between 1990 and 2002.

Bacterial kidney disease (BKD) is a notifiable disease for salmonids under United Kingdom and European Union legislation. Within the UK, legislation and the control of infected fish with BKD has been operating for 25 yr. Infection by the bacterium Renibacterium salmoninarum results in a chronic, debilitating infection and mortality. Records of BKD outbreaks and the detection of R. salmoninarum were monitored from 1990 through to 2002 for Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss reared in Scottish waters. The test methods included ELISA, culture and light microscopy. New outbreaks of BKD in salmon in seawater declined during this period, but with year-to-year variation. Only 1 record of BKD has occurred in freshwater-reared salmon (prevalence 1.04). BKD in farmed rainbow trout in seawater is uncommon and was only identified in 1993 and between 1998 and 2000. The number of active designated area orders (DAOs) for outbreaks in salmon has fallen since 1990, but has remained relatively constant for trout over the period of study.

Actinomycetales Infections↗

A cohabitation challenge to compare the efficacies of vaccines for bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha.

The relative efficacies of 1 commercial and 5 experimental vaccines for bacterial kidney disease (BKD) were compared through a cohabitation waterborne challenge. Groups of juvenile chinook salmon Oncorhynchus tshawytscha were vaccinated with one of the following: (1) killed Renibacterium salmoninarum ATCC 33209 (Rs 33209) cells; (2) killed Rs 33209 cells which had been heated to 37 degrees C for 48 h, a process that destroys the p57 protein; (3) killed R. salmoninarum MT239 (Rs MT239) cells; (4) heated Rs MT239 cells; (5) a recombinant version of the p57 protein (r-p57) emulsified in Freund's incomplete adjuvant (FIA); (6) the commercial BKD vaccine Renogen; (7) phosphate-buffered saline (PBS) emulsified with an equal volume of FIA; or (8) PBS alone. Following injection, each fish was marked with a subcutaneous fluorescent latex tag denoting its treatment group and the vaccinated fish were combined into sham and disease challenge tanks. Two weeks after these fish were vaccinated, separate groups of fish were injected with either PBS or live R. salmoninarum GL64 and were placed inside coated-wire mesh cylinders (liveboxes) in the sham and disease challenge tanks, respectively. Mortalities in both tanks were recorded for 285 d. Any mortalities among the livebox fish were replaced with an appropriate cohort (infected with R. salmoninarum or healthy) fish. None of the bacterins evaluated in this study induced protective immunity against the R. salmoninarum shed from the infected livebox fish. The percentage survival within the test groups in the R. salmoninarum challenge tank ranged from 59% (heated Rs MT239 bacterin) to 81% (PBS emulsified with FIA). There were no differences in the percentage survival among the PBS-, PBS/FIA-, r-p57- and Renogen-injected groups. There also were no differences in survival among the bacterin groups, regardless of whether the bacterial cells had been heated or left untreated prior to injection.

Actinomycetales Infections↗

Immunosuppression in progeny of chinook salmon infected with Renibacterium salmoninarum: re-analysis of a brood stock segregation experiment.

Female spawner infection level and temperature variation through rearing are sufficient to explain in-hatchery mortality rates and infection levels and smolt to adult return ratios (SARs) of progeny of Renibacterium salmoninarum infected spring chinook salmon. Data from published reports and manuscripts regarding a 1988 brood stock segregation experiment that held progeny of highly infected female spring chinook salmon spawners separate from progeny of other spawners during 16 mo of hatchery rearing are analyzed to test the hypothesis that immunosuppression could account for differences in survival and infection levels between the 2 segregates. Immunosuppression, caused by the presence of the p57 antigen of R. salmoninarum in sufficient concentration within the salmon egg before spawning, can account for differences in infection levels, mortality rates, and SARs for each hatchery raceway in that study. This immunosuppression may be characterized by immunotolerance, or might only affect cell mediated immunity, which appears the most effective defense mechanism against R. salmoninarum infection, as antibody production can result in tissue damaging antibody-antigen complexes. Low-temperature mediated immunosuppression can account for the nearly identical trajectories of infection and mortality between the 2 segregates during the first 8 mo of hatchery rearing. There is no evidence of widespread vertical infection from spawner to progeny, nor is there evidence that brood stock segregation reduces overall mortality. Rather, the suppression of cell-mediated immune mechanisms may condemn progeny of highly infected female spawners to an almost certain eventual premature demise.

Actinomycetales Infections↗

Prevalence and analysis of Renibacterium salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha in North Puget Sound.

Renibacterium salmoninarum causes bacterial kidney disease (BKD), a chronic and sometimes fatal disease of salmon and trout that could lower fitness in populations with high prevalences of infection. Prevalence of R. salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha inhabiting neritic marine habitats in North Puget Sound, Washington, USA, was assessed in 2002 and 2003. Fish were collected by monthly surface trawl at 32 sites within 4 bays, and kidney infections were detected by a quantitative fluorescent antibody technique (qFAT). The sensitivity of the qFAT was within an order of magnitude of the quantitative real-time PCR (qPCR) sensitivity. Prevalence of infection was classified by fish origin (marked/hatchery vs. unmarked/likely natural spawn), month of capture, capture location and stock origin. The highest percentages of infected fish (63.5 to 63.8%) and the greatest infection severity were observed for fish collected in Bellingham Bay. The lowest percentages were found in Skagit Bay (11.4 to 13.5%); however, there was no difference in prevalence between marked and unmarked fish among the capture locations. The optimal logistic regression model of infection probabilities identified the capture location of Bellingham Bay as the strongest effect, and analysis of coded wire tagged (CWT) fish revealed that prevalence of infection was associated with the capture location and not with the originating stock. These results suggest that infections can occur during the early marine life stages of Chinook salmon that may be due to common reservoirs of infection or horizontal transmission among fish stocks.

Actinomycetales Infections↗

Correlation between bacterial population and axillary and plantar bromidrosis: study of 30 patients.

Although studies on the chemistry of odors are expanding to identify the chemical structures of odorous substances, there are no universal standards as yet to measure odor and intensity of bromidrosis. Clinical evaluation can be made on a subjective scoring from 0 to 3 prior to prescription of an antiseptic soap. In order to appreciate the correlation between the intensity of bromidrosis (BI) and bacterial activity, a study was carried out with both clinical and bacterial assessment in thirty patients with axillary or plantar BI. Odor intensity was evaluated by two physicians using a score from 0 to 3 (i.e. absent, minor, moderate, major), meanwhile bacterial composition and density were assessed before and after 10 days of hygiene using an antiseptic detergent (trichlocarbanilide) provided on the first visit. Baseline count of diphtheroids/cm2 was 35.104 and baseline micrococci average was 32.104/cm2. At the end of the study, the reduction of odor intensity was observed in 20 patients (67%) without any change in sweat production. The clinical improvement correlated with a reduction of both micrococci (70%) or diphtheroids (73%) as compared with initial data. In patients presenting persistant bromidrosis, the bacterial count/cm2 did not significantly decrease and remained above 104 diphtheroids/cm2. Thus, this study suggests that body odor may be at least indirectly correlated to microbia counts with a bacteria threshold of BI ranging around and above 104.

Anti-Infective Agents, Local↗

In vitro study of the influence of virginiamycin and spiramycin on the composition and biochemical activities of the gastrointestinal flora of piglets. I. influence on the composition of the flora.

In vitro fermentations with the gastro-intestinal flora of 18 treated piglets (Control, Virginiamycin, Spiramycin) were carried out to study the influence of Nutritional doses of Virginiamycin and Spiramycin on the composition of the microflora. Each total flora was used for three different incubations: Control, 50 ppm Virginiamycin, 50 ppm Spiramycin. To obtain comparable results with experiments in vivo, an artificial fermentation medium was made on the basis of several chemical analyses of intestinal contents. The treatments with nutritional doses in vivo had little influence on the different in vitro fermentations. Addition of Spiramycin during incubation was of little influence on the growth and the composition of the flora. Virginiamycin on the other hand caused marked changes in the gastro-intestinal microflora, such as a decrease of the total growth and a decrease in the number of Lactobacilli and Enterococci, combined with a slight increase of the Gram negative aerobic population.

Anaerobiosis↗

Magnetic susceptibility of microorganisms.

Total magnetic susceptibility of 13 species and varieties of bacteria was investigated using the relative method of Guy. It has been established that the index of magnetic susceptibility is a constant characteristic of bacteria. Total magnetic susceptibility ranged from --0.3295.10(-6) in Escherichia P678 to --0.4965.10(-6) in Proteus. It has also been established that magnetic susceptibility changes during long-term passages of bacteria in fluctuating +/- 0.1 Oe) magnetic field. This is suggestive of a low threshold of their magnetic susceptibility and permits a rough assessment of the importance of fluctuations of the geomagnetic field for the viability of microbes.

Alcaligenes↗