A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.

PCR and probe-PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.) ovarian fluid and kidney tissue for presence of DNA of the fish pathogen Renibacterium salmoninarum.

A simple, rapid PCR assay for the identification of Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.) tissues detected DNA extracted from between 4 and 40 bacterial cells. PCR was at least as sensitive as culture when it was used to identify subclinically infected fish experimentally challenged with R. salmoninarum. However, PCR identified much higher numbers of kidney tissue and ovarian fluid samples from commercially reared broodstock fish to be positive for R. salmoninarum than did culture. This difference may be due to the antibiotic chemotherapy of broodstock fish used by the industry in 1994 to control the vertical transmission of R. salmoninarum. A much closer relationship between PCR and culture results was observed for ovarian fluid samples collected from broodstock fish in 1993. Also, PCR scored a much higher percentage of kidney tissue samples than ovarian fluid samples from 1994 broodstock fish positive for R. salmoninarum, which may reflect the uneven distribution of the pathogen in different fish tissues. Inclusion of a nested probe to identify the PCR-positive 1994 ovarian fluid samples increased the sensitivity of detection to between one and four cells and the number of samples that scored positive by almost threefold. These data indicate that many infected ovarian fluid samples contained very low numbers of R. salmoninarum cells and, because almost all these samples were culture negative, that PCR may have detected dead or otherwise unculturable bacterial cells.

Effects of phentolamine or yohimbine on naloxone's actions during endotoxin shock in rats.

Endogenous opioids are known to mediate some of the cardiovascular sequelae of sepsis. Inhibition of adrenergic action has been implicated as a physiological path by which endogenous opioids cause deleterious changes in cardiovascular function during endotoxin shock, but where and to what extent this accounts for changes in regional vascular resistance remains unclear. In this study, we addressed this question by examining the role of alpha-adrenergic actions in cardiovascular performance and the regional perfusion changes caused by naloxone during endotoxin shock. Rats had catheters inserted into the tail artery, left cardiac ventricle, and jugular vein. Twenty-four hours later, rats received saline or endotoxin (2 mg/kg) challenge intravenously over 30 min, followed at 40 min by intravenous naloxone (or saline) treatment (4 mg/kg + 2 mg/kg x h) in the presence or absence of phentolamine (100 micrograms/kg + 600 micrograms/kg x h) or yohimbine (40 micrograms/kg + 4 micrograms/kg x h). Radiolabeled microspheres were used to determine cardiac outputs and blood flows at 0, 30, 60, and 120 min after beginning endotoxin infusion. Naloxone attenuated the endotoxin-induced decline in mean arterial pressure (MAP) and cardiac output (CO), but had no effect on increased systemic vascular resistance (SVR). Phentolamine blocked naloxone's ability to increase MAP and CO, but permitted an increase in SVR by naloxone. In the presence of yohimbine, naloxone still increased MAP, but not CO nor SVR. Regional vascular responses varied, with naloxone demonstrating a vasoconstrictive effect despite alpha-adrenergic receptor blockade in some beds, and no effect in others. The response of individual organs in the hepatosplanchnic circulation was heterogenous as well. These data suggest that some effects of endogenous opioids during endotoxin shock are mediated via inhibition of alpha-adrenergic effects, but that some cardiovascular effects of endogenous opioids are independent of adrenergic control during endotoxin shock.

Further characterization of Renibacterium salmoninarum extracellular products.

Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrations of extracellular protein in tissues of infected fish. The extracellular protein consists almost entirely of a 57-kDa protein and derivatives of degradation and aggregation of the same molecule. The 57-kDa protein and its derivatives were fractionated into defined ranges of molecular mass. Separated fractions continued to produce degradation and aggregation products. One-dimensional electrophoretic separation of extracellular protein revealed a number of proteolytically active bands from > 100 to approximately 18 kDa associated with various 57-kDa protein derivatives in the different molecular mass fractions. Two-dimensional separation of extracellular protein showed that continued degradation and aggregation, similar both in location and behavior to some of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after their separation. Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mechanism for the proteolytically active polypeptides characteristic of a thiol proteinase. The results suggested that the 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically active polypeptide(s) of at least 18 kDa. Soluble polysaccharide-like material also was detected in extracellular products and tissue from infected fish. Antiserum to the polysaccharide-like material cross-reacted with O-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity between these polysaccharides. The polysaccharide and the proteolytic activity associated with the 57-kDa protein derivatives should be investigated with respect to the pathogenesis of R. salmoninarum infections.

Beta-adrenergic-dependent and -independent actions of naloxone on perfusion during endotoxin shock.

Naloxone, an opioid antagonist, has been shown to improve cardiovascular status during endotoxin shock, including splanchnic perfusion. Enhancement of adrenergic action has been implicated as a physiological path by which naloxone effects changes in cardiac function during endotoxin shock, but the mechanism for changes in various splanchnic vascular beds has not been examined. In this study, we examined the role of beta-adrenergic actions in cardiovascular performance and the splanchnic perfusion changes caused by naloxone during endotoxin shock. Rats were instrumented with catheters in the tail artery, left cardiac ventricle, and jugular vein. Twenty-four hours later, rats received saline or endotoxin (2 mg/kg) challenge intravenously over 30 min, followed at 40 min by i.v. naloxone (or saline) treatment (4 mg/kg + 2 mg/kg.hr) in the presence or absence of propranolol (1 mg/kg + 1 mg/kg.hr). Radiolabelled microspheres were used to determine cardiac outputs and blood flows at 0, 30, 60, and 120 min after beginning endotoxin infusion. Blood pressure was not affected by endotoxin challenge, but cardiac output and most organ blood flows fell over time. beta-Adrenergic blockade did not alter this response. Naloxone improved cardiac output and blood flow to the stomach, small intestine, colon, and spleen but not to other splanchnic organs. Naloxone also increased renal and coronary blood flows. The improvements in cardiac output with naloxone were ablated in the presence of propranolol, as were the increases in gastric, colonic, splenic, coronary, and renal blood flows. However, the beneficial effect of naloxone on small bowel blood flow was not diminished by blockage of beta receptors. These results suggest that the effects of opioid antagonism are mediated, in part, by enhancing endogenous beta-adrenergic actions in vivo. Improvements in the splanchnic circulation are selectively altered by naloxone during endotoxin shock, some independent of beta-adrenergic actions. Understanding this phenomenon can lead to the appropriate use of opioid antagonism, should it prove clinically useful in the treatment of septic shock.

Characterization of Aeromonas salmonicida variants with altered cell surfaces and their use in studying surface protein assembly.

Aeromonas salmonicida variants were characterized for alterations in their cell surface structure and used to examine reconstitution of the surface protein layer (A-layer). Variants lacking outer membrane O-polysaccharide were devoid of A-layer and excreted stainable floret-like material of the surface protein (A-protein). One variant, showing partial loss of O-polysaccharide, was associated with a disrupted A-layer and excretion of some A-protein. Variants lacking A-protein but possessing O-polysaccharide rapidly absorbed and concentrated sufficient excreted A-protein at the cell surface to coat the cells with a single confluent layer. Although differences in electrophoretic mobilities of A-proteins and O-polysaccharides from "typical" and "atypical" strains were evident, the different A-proteins and A-protein-deficient variants were interchangeable for reconstitution of a surface protein layer. No association of A-protein with cell surfaces of unrelated gram-negative bacteria was observed.

Characterization of Aeromonas salmonicida mutants with low-level resistance to multiple antibiotics.

Aeromonas salmonicida mutants selected for low-level resistance to small-molecular-mass antibiotics occur at frequencies that suggest point mutations and exhibit pleiotropic effects such as a multiple low-level antibiotic resistance, changes in outer membrane protein profiles, and loss of major exoprotease activity. Multiple low-level resistance appeared as the result of decreased outer membrane permeability associated with a change from a 38.5- to a 37-kilodalton (kDa) outer membrane protein. This decreased outer membrane permeability was determined by rates of nitrocefin hydrolysis by periplasmic beta-lactamase activity. The findings described above were supported by isolation of revertant strains selected for regained exoprotease activity, which also lost multiple low-level resistance and possessed outer membrane protein profiles indistinguishable from those of the original parent strains. Exoprotease from parent and revertant strains was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major extracellular protein of approximately 69 kDa. No accumulation of a protein in this molecular mass range was observed in extracellular or periplasmic fractions from the mutants. The results suggested that exoprotease loss is not simply the result of an inability to export protease from the periplasm because of outer membrane protein changes, as has been reported for certain mutants of some other gram-negative bacteria. Also, several growth conditions were used, including some that have been reported to influence outer membrane protein expression and permeability in other enteric gram-negative bacteria. Although exoprotease expression in A. salmonicida was influenced by these conditions, no major outer membrane protein changes which would correspond to changes observed in the mutants were observed in parent strains.

Effects of polymyxin B nonapeptide on Aeromonas salmonicida.

In contrast to polymyxin B-susceptible gram-negative bacteria of human origin, the fish pathogen Aeromonas salmonicida was resistant to sensitization by polymyxin B nonapeptide (PMBN) to hydrophobic antibiotics. Similarly, sensitization of A. salmonicida strains by PMBN to the bactericidal action of brook trout (Salvelinus fontinalis) serum complement was less pronounced than the similar effect of PMBN against other gram-negative bacteria in certain mammalian sera. The surface array protein (A layer), overlying the outer membrane of virulent A. salmonicida strains, did not appear to cause resistance to PMBN sensitization since an A-layer-deficient mutant showed similar responses to PMBN as its parent strain. Electron micrographs of PMBN-treated A. salmonicida cells revealed very little visible outer membrane disruption when compared with the extensive blebbing on the outer membrane surface caused by PMBN in certain enteric bacteria, including Escherichia coli K-37. However, small extracellular vesiclelike components, which may have been derived from the outer membrane, were numerous around PMBN-treated A. salmonicida cells. In this connection, PMBN caused disruption of the A layer in the form of bulges, breaks, and detached fragments which appeared to be associated with the accumulation of these vesicles underneath the A layer of wild-type A. salmonicida strains.

Multiple low-level antibiotic resistance in Aeromonas salmonicida.

Mutants with multiple low-level antibiotic resistance were isolated from virulent wild-type Aeromonas salmonicida strains exposed to a low concentration of any one of several low-molecular-mass (approximately 635 daltons or less) antibiotics. Multiple resistance was toward beta-lactam compounds (penicillin G, ampicillin, cloxacillin), quinolones (flumequine, oxolinic acid, nalidixic acid), tetracyclines, chloramphenicol, and novobiocin. Susceptibilities of the mutants toward several higher-molecular-mass (greater than 700 daltons) hydrophobic or polycationic antibiotics such as rifampin, erythromycin, polymyxin B, and streptomycin sulfate were not affected. The mutants were obtained at frequencies suggesting point mutations. Outer membrane protein profiles, examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that all multiple low-level resistant mutants were deficient in a major protein of approximately 38.5 kilodaltons and contained a major protein of approximately 37 kilodaltons which was not present in significant amounts in the wild-type strains. In addition, these mutants lacked exoprotease activity. Furthermore, mutants isolated as deficient in exoprotease were found, with the exception of one avirulent strain, to exhibit multiple low-level antibiotic resistance and the outer membrane protein changes.

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens.

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.

The role of some membrane dehydrogenases in Pseudomonas fluorescens.

The membrane-associated oxidative enzymes glucose, gluconate, and malate dehydrogenases were examined in psychrotrophic Pseudomonas fluorescens. The function and activity of these enzymes was determined by measuring extracellular product formation by washed cell suspensions. Membrane dehydrogenase activities and corresponding transport activities for the substrates glucose, gluconate and malate were compared in batch cultures grown with these substrates at 30 and 5 degrees C. These activities correlated with the production or lack of extracellular oxidation products. in chemostat cultures grown at 30 and 5 degrees C, the membrane enzymes and production of their extracellular oxidation products appeared to be regulated by the available carbon concentration. The enzymes were induced and high concentrations of extracellular oxidation products were produced under conditions of nitrogen limitation (carbon excess) but not carbon limitation. The lower affinities of the three membrane enzymes for their respective substrates, when compared with the transport systems utilizing the same substrates, correlated with the observed major function of these enzymes in carbon-excess environments. The primary role of the membrane-associated oxidative enzymes, in carbon dissimilation by this psychrotrophic microorganism at low temperatures in carbon-excess environments, was strongly implicated.

Effect of temperature on Pseudomonas fluorescens chemotaxis.

The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 5 degrees C) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 5 degrees C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 0 degree C and appeared to be functional over a broad temperature range of 3 to 35 degrees C.

Effect of temperature on diauxic growth with glucose and organic acids in Pseudomonas fluorescens.

Growth of Pseudomonas fluorescens in batch culture with glucose and organic acids resulted in typical diauxic responses at 30 degrees C but no detectable diauxic lag at 5 degrees C. At 30 degrees C, organic acids were preferentially utilized during the first growth phase. Glucose utilization was delayed until onset of the second growth phase. Systems involved in direct uptake and catabolism of glucose responded in a manner compatible with repression by malate and/or its metabolites and induction by glucose and/or its metabolites. The oxidative non-phosphorylated pathway, through gluconate and 2-ketogluconate (2-KG) as intermediates, was not induced during either growth phase. At 5 degrees C, growth with glucose and organic acids was biphasic but without diauxic lag. Organic acids were preferentially utilized during the first growth phase. Although carbon from glucose was not fully catabolized until onset of the second growth phase, glucose was oxidized to and accumulated extracellularly as gluconate and 2-KG during the first growth phase. No significant repression of glucose-catabolizing enzymes was observed during growth with organic acids in the presence of glucose. However, uptake activities for gluconate and 2-KG did not increase significantly until onset of the second growth phase. Thus, at low temperatures, psychotrophic P. fluorescens oxidized glucose to extracellular 2-KG, while growing on preferred carbon sources. The 2-KG was then catabolized after depletion of the organic acid.

Effect of temperature on the activity and synthesis of glucose-catabolizing enzymes in Pseudomonas fluorescens.

The activity of the enzymes of the oxidative non-phosphorylated pathway, glucose and gluconate dehydrogenases, were not significantly affected by changes in the assay temperature. Both enzymes demonstrated only a threefold difference in activity when compared at assay temperatures of 30 degrees C and 5 degrees C. In contrast, the enzymes involved in the direct phosphorylation and catabolism of glucose or its oxidation products, gluconate and 2-ketogluconate, exhibited a more pronounced response to decreasing assay temperatures. At least one enzyme in each pathway, involved in the direct phosphorylation and catabolism of glucose or 2-ketogluconate (2KG), demonstrated an eightfold decrease in activity with a decrease in assay temperature from 30 degrees C to 5 degrees C. A similar decrease in assay temperature resulted in a fivefold decrease in activity of the enzymes involved in the direct phosphorylation and catabolism of gluconate. The observed differential effect of temperature on the activity of the enzymes of glucose catabolism and on the accumulation of direct oxidation products during growth with glucose in P. fluorescens E-20 is discussed. Growth with glucose at 5 or 20 degrees C resulted in high induced levels of all glucose-catabolizing enzymes examined when compared with the levels of these same enzymes in pyruvate-grown cells. However, only low levels of glucose dehydrogenase were detected during growth at 30 degrees C with glucose, gluconate, or 2-KG. Similarly, only low levels of gluconate dehydrogenase were detected during growth with glucose at 30 degrees C, although a weak induction was observed during growth with gluconate or 2-KG at 30 degrees C. The levels of 2-KG kinase plus KPG reductase during growth at 30 degrees C were undetectable with glucose, weakly induced with gluconate, and fully induced with 2-KG. High induced levels of glucose dehydrogenase, gluconate dehydrogenase, and 2-KG kinase plus KPG reductase were present during growth at 20 degrees C with glucose or 2-KG. The low levels of glucose and gluconate dehydrogenases present at a growth temperature of 30 degrees C was not due to heat lability of the enzymes at this temperature. The low amounts of these two enzymes during growth with glucose at 30 degrees C probably prevented sufficient inducer(s) formation from glucose to allow induction of enzymes of 2-KG catabolism. The results demonstrated that temperature may regulate the pathways of glucose dissimilation by regulating, either directly or indirectly, the activity and synthesis of the enzymes involved in these pathways.

Effect of growth temperature on the accumulation of glucose-oxidation products in Pseudomonas fluorescens.

The effect of the growth temperature, on the accumulation of glucose-oxidation products, was examined in aerated cultures with carbon excess in two strains of Pseudomonas fluorescens. At low growth temperatures (0 and 5 degrees C), 2-ketogluconate (KG) a-cumulated in the medium as the major oxidation product of glucose (up to 70%) before further metabolism. As the growth temperature was increased, the amount of 2-KG found to accumulate in the medium from glucose oxidation decreased. At a growth temperature of 20 degrees C, up to 25% of the glucose originally added accumulated in the medium as 2-KG. At the optimum growth temperature of 30 degrees C or above, no 2-KG was detected at any time during growth with glucose. Similar results were obtained when gluconate was used as the sole carbon and energy source. The results demonstrated a differential effect of growth temperature on the accumulation of oxidation products from glucose and gluconate. At low growth temperatures the major route for the catabolism of glucose and gluconate was the direct oxidative non-phosphorylated pathway.