The pentose phosphate pathway leads to enhanced succinic acid flux in biofilms of wild-type Actinobacillus succinogenes.

Increased pentose phosphate pathway flux, relative to total substrate uptake flux, is shown to enhance succinic acid (SA) yields under continuous, non-growth conditions of Actinobacillus succinogenes biofilms. Separate fermentations of glucose and xylose were conducted in a custom, continuous biofilm reactor at four different dilution rates. Glucose-6-phosphate dehydrogenase assays were performed on cell extracts derived from in situ removal of biofilm at each steady state. The results of the assays were coupled to a kinetic model that revealed an increase in oxidative pentose phosphate pathway (OPPP) flux relative to total substrate flux with increasing SA titre, for both substrates. Furthermore, applying metabolite concentration data to metabolic flux models that include the OPPP revealed similar flux relationships to those observed in the experimental kinetic analysis. A relative increase in OPPP flux produces additional reduction power that enables increased flux through the reductive branch of the TCA cycle, leading to increased SA yields, reduced by-product formation and complete closure of the overall redox balance.

Succinic acid production with Actinobacillus succinogenes: rate and yield analysis of chemostat and biofilm cultures.

BACKGROUND: Succinic acid is well established as bio-based platform chemical with production quantities expecting to increase exponentially within the next decade. Actinobacillus succinogenes is by far the most studied wild organism for producing succinic acid and is known for high yield and titre during production on various sugars in batch culture. At low shear conditions continuous fermentation with A. succinogenes results in biofilm formation. In this study, a novel shear controlled fermenter was developed that enabled: 1) chemostat operation where self-immobilisation was opposed by high shear rates and, 2) in-situ removal of biofilm by increasing shear rates and subsequent analysis thereof. RESULTS: The volumetric productivity of the biofilm fermentations were an order of magnitude more than the chemostat runs. In addition the biofilm runs obtained substantially higher yields. Succinic acid to acetic acid ratios for chemostat runs were 1.28±0.2 g.g(-1), while the ratios for biofilm runs started at 2.4 g.g(-1) and increased up to 3.3 g.g(-1) as glucose consumption increased. This corresponded to an overall yield on glucose of 0.48±0.05 g.g(-1) for chemostat runs, while the yields varied between 0.63 g.g(-1) and 0.74 g.g(-1) for biofilm runs. Specific growth rates (µ) were shown to be severely inhibited by the formation of organic acids, with µ only 12% of µ(max) at a succinic acid titre of 7 g.L(-1). Maintenance production of succinic acid was shown to be dominant for the biofilm runs with cell based production rates (extracellular polymeric substance removed) decreasing as SA titre increases. CONCLUSIONS: The novel fermenter allowed for an in-depth bioreaction analysis of A. succinogenes. Biofilm cells achieve higher SA yields than suspended cells and allow for operation at higher succinic acid titre. Both growth and maintenance rates were shown to drastically decrease with succinic acid titre. The A. succinogenes biofilm process has vast potential, where self-induced high cell densities result in higher succinic acid productivity and yield.

Succinic acid-producing biofilms of Actinobacillus succinogenes: reproducibility, stability and productivity.

Continuous anaerobic fermentations were performed in a biofilm reactor packed with Poraver® beads. Dilution rates (D) varied between 0.054 and 0.72 h(-1), and D-glucose and CO2 gas were used as carbon substrates. Steady-state conditions were shown to be repeatable and independent of the operational history. Production stability was achieved over periods exceeding 80 h at values of D below 0.32 h(-1). In these situations, steady-state variation (expressed as fluctuations in NaOH neutralisation flow rates) exhibited a standard deviation of less than 5 % while no indication of biofilm deactivation was detected. The total biomass amount was found to be independent of the dilution rate with an average dry concentration of 23.8 ± 2.9 g L(-1) obtained for all runs. This suggests that the attachment area controls the extent of biofilm accumulation. Specific succinic acid (SA) productivities, based on the total biomass amount, exhibited a substantial decrease with decreasing D. An SA volumetric productivity of 10.8 g L(-1) h(-1) was obtained at D = 0.7 h(-1)-the highest value reported to date in Actinobacillus succinogenes fermentations. SA yields on glucose increased with decreasing D, with a yield of 0.90 ± 0.01 g g(-1) obtained at a D of 0.054 h(-1). Production of formic acid approached zero with decreasing D, while the succinic to acetic acid ratio increased with decreasing D, resulting in an increasing SA yield on glucose.

Organization and biology of the porcine serum amyloid A (SAA) gene cluster: isoform specific responses to bacterial infection.

Serum amyloid A (SAA) is a prominent acute phase protein. Although its biological functions are debated, the wide species distribution of highly homologous SAA proteins and their uniform behavior in response to injury or inflammation in itself suggests a significant role for this protein. The pig is increasingly being used as a model for the study of inflammatory reactions, yet only little is known about how specific SAA genes are regulated in the pig during acute phase responses and other responses induced by pro-inflammatory host mediators. We designed SAA gene specific primers and quantified the gene expression of porcine SAA1, SAA2, SAA3, and SAA4 by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in liver, spleen, and lung tissue from pigs experimentally infected with the Gram-negative swine specific bacterium Actinobacillus pleuropneumoniae, as well as from pigs experimentally infected with the Gram-positive bacterium Staphylococcus aureus. Our results show that: 1) SAA1 may be a pseudogene in pigs; 2) we were able to detect two previously uncharacterized SAA transcripts, namely SAA2 and SAA4, of which the SAA2 transcript is primarily induced in the liver during acute infection and presumably contributes to circulating SAA in pigs; 3) Porcine SAA3 transcription is induced both hepatically and extrahepatically during acute infection, and may be correlated to local organ affection; 4) Hepatic transcription of SAA4 is markedly induced in pigs infected with A. pleuropneumoniae, but only weakly in pigs infected with S. aureus. These results for the first time establish the infection response patterns of the four porcine SAA genes which will be of importance for the use of the pig as a model for human inflammatory responses, e.g. within sepsis, cancer, and obesity research.

An atypical case of respiratory actinobacillosis in a cow.

A not pregnant 4-year-old Jersey cow was presented with the sudden appearance of respiratory noise, nasal discharge and moderate respiratory difficulty. Upon physical examination a snoring-like noise, extended head and neck position, exaggerated abdominal effort, bilateral nasal discharge and left prescapular lymph node enlargement were noted. Sub-occlusion of the initial portion of the respiratory tract was suspected. Radiographic and endoscopic examinations revealed a pedunculate mass on the dorsal aspect of the rhinopharynx, which was removed with endoscopically assisted electrosurgery. Histologic examination revealed a chronic pyogranulomatous inflammation with eosinophilic club-like bodies surrounding small colonies of rod-shaped bacteria. Results of histochemical staining were consistent with Actinobacillus-like bacteria and a diagnosis of respiratory actinobacillosis was reached. Surgery and antibiotic therapy were resolutive, as demonstated by an endoscopic check at the second month after surgery, even without the association of the traditional iodine cure, which is regarded as the treatment of choice for actinobacillosis.

An atypical case of respiratory actinobacillosis in a cow

A not pregnant 4-year-old Jersey cow was presented with the sudden appearance of respiratory noise, nasal discharge and moderate respiratory difficulty. Upon physical examination a snoring-like noise, extended head and neck position, exaggerated abdominal effort, bilateral nasal discharge and left prescapular lymph node enlargement were noted. Sub-occlusion of the initial portion of the respiratory tract was suspected. Radiographic and endoscopic examinations revealed a pedunculate mass on the dorsal aspect of the rhinopharynx, which was removed with endoscopically assisted electrosurgery. Histologic examination revealed a chronic pyogranulomatous inflammation with eosinophilic club-like bodies surrounding small colonies of rod-shaped bacteria. Results of histochemical staining were consistent with Actinobacillus-like bacteria and a diagnosis of respiratory actinobacillosis was reached. Surgery and antibiotic therapy were resolutive, as demonstated by an endoscopic check at the second month after surgery, even without the association of the traditional iodine cure, which is regarded as the treatment of choice for actinobacillosis.

Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membrane-driven fumarate reduction and energy conservation.

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.

Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen.

Strain 130ZT was isolated from the bovine rumen. It is a facultatively anaerobic, pleomorphic, Gram-negative rod. It exhibits a 'Morse code' form of morphology, which is characteristic of the genus Actinobacillus. Strain 130ZT is a capnophilic, osmotolerant succinogen that utilizes a broad range of sugars. It accumulates high concentrations of succinic acid (> 70 g l-1). Strain 130ZT is positive for catalase, oxidase, alkaline phosphatase and beta-galactosidase, but does not produce indole or urease. Acid but no gas is produced from D-glucose and D-fructose. 16S rRNA sequence analysis places strain 130ZT within the family Pasteurellaceae; the most closely related members of the family Pasteurellaceae have 16S rRNA similarities of 95.5% or less with strain 130ZT. Strain 130ZT was compared with Actinobacillus lignieresii and the related Bisgaard Taxa 6 and 10. Based upon morphological and biochemical properties, strain 130ZT is most similar to members of the genus Actinobacillus within the family Pasteurellaceae. It is proposed that strain 130ZT be classified as a new species, Actinobacillus succinogenes. The type strain of Actinobacillus succinogenes sp. nov. is ATCC 55618T.

Synergistic effects of Actinobacillus suis cells in combination with 5-fluorouracil on mice bearing murine L1210 leukemia cells.

The mean survival rates of female BDF1 mice transplanted intravenously (i.v.) with murine L1210 leukemia cells were significantly prolonged by intraperitoneal (i.p.) pretreatment (before i.v. transplantation) or by i.p. pre- and post-treatment (before and after the i.v. transplantation) with heat-killed Actinobacillus suis cells ATCC 15557 (AS 15557) alone, as compared with untreated L1210-leukemia-cell-bearing control mice. However, significant prolongation of the mean survival rates was not elicited by the i.p. post-treatment with AS 15557 alone. When 5-fluorouracil (5-FU) was applied i.p. to mice receiving post-treatment with AS 15557 alone, the mean survival rates of the L1210-leukemia-cell-bearing mice were significantly prolonged. The antileukemic action of AS 15557, alone or in combination with 5-FU, against L1210 leukemia was superior to that of a streptococcal preparation (OK-432) and was almost the same as of bacillus Calmette-Guérin with or without 5-FU. The results suggest the possibility that the synergism of AS 15557 in combination with 5-FU may be dependent on the relationship between the indirect immunological function of AS 15557 and the direct cytotoxic action of 5-FU on L1210 leukemia cells.

Ecology and significance of Pasteurellaceae in man--an update.

Within the last decade new knowledge has emerged concerning the significance of Pasteurellaceae in man; the classification has undergone some changes, and new taxa were described. Haemophilus influenzae serotype b was shown to have a clonal distribution that is related to demographic patterns of the human host. Brazilian purpuric fever is caused by a special clone of Haemophilus aegyptius. H. influenzae biotype IV seems to be a genital pathogen, and may deserve species rank. New Pasteurella species have been described, that occur in well known pathological foci in man, e.g. bite wounds. Toxigenic P. multocida may occur in man also; the significance of toxigenicity in man is not known. The real actinobacilli of man, A. ureae and A. hominis are still very rarely reported. In order to avoid wrong epidemiological conclusions, correct diagnosis is emphasized.

Ecology and significance of Pasteurellaceae in animals.

The reservoir of eighty-one taxa/groups classified with the family Pasteurellaceae Pohl 1981 is reviewed based upon published data and own investigations. With the exception of certain strains of P. multocida, A. pleuropneumoniae and [H.] paragallinarum organisms belonging to this family are usually regarded as opportunistic, secondary invaders which under normal conditions coexist peacefully with the animal host on mucosal membranes of the upper respiratory- and lower genital tracts. Very little is known about factors that govern the ecological preferences that certain members of this family show for specific surfaces and hosts. Mechanisms of colonization, survival and multiplication, invasion and pathogenic action are incompletely understood. The significance of Pasteurellaceae in animals and man has recently been reviewed. Subsequent publications have underlined the significance of biovars 2 of P. canis and P. avium and ornithine negative P. multocida in pneumonia in cattle. In addition, differences in pathogenicity have been demonstrated for different serovars of [H.] parasuis. The disease potential of many taxa/groups is only incompletely known.

Actinobacillus suis-like organisms and evidence of hemolytic strains of Actinobacillus lignieresii in horses.

Thirty-seven local isolates of Actinobacillus suis-like organisms from diseased and clinically normal horses and 1 llama were compared with reference strains of A suis, A lignieresii, A equuli, A capsulatus, A hominis, A (Pasteurella) ureae, and equine A suis-like organisms (ASLO) previously described in literature. Comparison was by cultural characteristics, carbohydrate fermentation, enzyme profiles, and whole-cell protein polyacrylamide gel electrophoresis. Carbohydrate fermentation, determined by API-CH gallery, divided 36 equine ASLO isolates into 6 API-CH biotypes. The llama isolate was an additional distinct biotype. The biochemical comparisons between A suis and ASLO did not reveal remarkable and consistent differences. Enzyme analysis revealed 5 API-ZYM biotypes, one of which included the same strains as one of the API-CH biotypes and consisted in both instances of 4 esculin-negative ASLO cultures and the reference strain of A lignieresii. We conclude that the 4 strains were hemolytic variants of A lignieresii. Protein electrophoresis disclosed 15 banding patterns, 10 of which represented equine ASLO strains. The reference strains of A suis shared the pattern predominant among equine ASLO. Four of the remaining reference strains of Actinobacillus species each had a unique profile, whereas the type strain of A capsulatus and the llama isolate had similar profiles. The groupings of cultures resulting from the different testing methods had little relation to each other and to the anatomic source of the strains except the strains comprising API-CH biotype II, which originated in the equine respiratory tract, and the A lignieressi cluster.

Biochemical typing of Actinobacillus pleuropneumoniae.

A study was conducted to evaluate the possibility of using biochemical differences among strains of a given serotype of Actinobacillus pleuropneumoniae as epidemiological markers, to rapidly identify the source of infection in herds affected with swine pleuropneumonia. Out of 38 different biochemical and physiological tests performed on a total of 67 strains belonging to serotypes 1 and 5 of A. pleuropneumoniae, three fermentation tests, glycerol, lactose and raffinose, allowed the classification of serotype 1 strains into 6 phenotypic groups and serotype 5 strains into 4 of these groups. Groups II and III were exclusively composed of serotype 1 strains, whereas the majority of strains in groups I and IV belonged to serotypes 1 and 5 respectively, the latter comprising almost all the serotype 5 studied.

Endothelial cytotoxicity of Actinobacillus pleuropneumoniae.

The cytotoxicity of Actinobacillus pleuropneumoniae serotype 1 strain CM5 for porcine and bovine endothelial cells in vitro, was dose-dependent. This strain and its attenuated and avirulent substrain CM5A were equally cytotoxic. The cytotoxicity observed during five hours of exposure of endothelial cells to bacterial products was abolished if the bacteria were inactivated by heat or sonication. Exposure of the endothelial cells for five hours to 100 and 200 micrograms of purified lipopolysaccharide resulted in a partial cytotoxicity only, which was not enhanced in the presence of fresh guinea pig serum. The cytotoxicity of viable bacteria could be neutralised by a polyclonal rabbit antiserum to the purified 104kD haemolysin. A bacteria-free supernate of a culture of strain CM5 had both haemolytic and cytotoxic activity. The haemolytic activity could be neutralised completely by the anti-serum to the 104kD haemolysin, whereas the cytotoxic activity was only partially neutralisable. Hence A pleuropneumoniae is cytotoxic for endothelial cells and this cytotoxicity is possibly mediated by the 104kD haemolysin.

Immune suppression induced by Actinobacillus actinomycetemcomitans: effects on immunoglobulin production by human B cells.

Actinobacillus actinomycetemcomitans produces an immunosuppressive factor (ISF) which has been shown to suppress mitogen- and antigen-induced DNA, RNA, and protein synthesis in human T lymphocytes. In this study, we examined purified A. actinomycetemcomitans ISF for its ability to alter immunoglobulin production by human B cells. The ISF caused a dose-dependent inhibition of pokeweed mitogen (PWM)-induced immunoglobulin G (IgG) and IgM production. Preexposure to ISF was not required to achieve maximal inhibition of immunoglobulin synthesis, as previously observed for its effect on T-cell activation. Nevertheless, the ISF appeared to act by irreversibly affecting the early stages of cell activation. While PWM-induced immunoglobulin production is under the influence of T-regulatory circuits, it appears that the ISF interacts directly with B cells. First, ISF failed to alter either the synthesis of interleukin-2 (IL-2) or the expression of IL-2 receptors on T cells. Second, experiments in which individual purified populations of cells were exposed to ISF, washed, and placed back into tissue culture indicated that when all cells (i.e., T cells, B cells, and monocytes) were exposed to ISF, significant suppression was observed. However, when only one cell population was treated with ISF, suppression of both IgG and IgM synthesis was observed only when the B-cell-enriched population was exposed to ISF. These results in conjunction with our earlier findings suggest that the ISF functions via the activation of a regulatory subpopulation of B lymphocytes, which in turn either directly or indirectly (via suppressor T cells) downregulate both B- and T-cell responsiveness. Furthermore, it is hypothesized that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. This suppression may enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism.

Actinobacillus actinomycetemcomitans and Bacteroides intermedius in human periodontitis: age relationship and mutual association.

This study examined age relationships and mutual interrelationships between cultivable Actinobacillus actinomycetemcomitans and Bacteroides intermedius in 1624 periodontitis patients, 15 to 89 years of age. Each subject contributed a pooled subgingival sample, obtained from 3 deep periodontal pockets with paper points. A. actinomycetemcomitans occurred with higher prevalence (74%) and mean recovery (7% in culture-positive patients) in patients less than 25 years old than in adult and geriatric patients (prevalence about 31%; mean recovery about 1%). The organism was detected in 85% of localized juvenile periodontitis patients. B. intermedius was recovered from 45% of the study subjects, averaged about 7% of total isolates in positive patients, and showed no predilection for any age group. As determined from predicted and observed values for A. actinomycetemcomitans and B. intermedius, occurring alone and in combination, no synergistic or antagonistic relationships between the organisms could be delineated with respect to subgingival colonization. The therapeutic implication of these findings is discussed.

Morphological compositions of subgingival microbiota in Actinobacillus actinomycetemcomitans-associated periodontitis.

Infrequent occurrence of spirochetes and rather low proportions of these organisms have been reported in localized juvenile periodontitis, where periodontal lesions often harbour large numbers of Actinobacillus actinomycetemcomitans, a suspected principal periopathogen strongly implicated in the pathogenesis of this and other forms of chronic periodontitis. We studied the association of subgingival A. actinomycetemcomitans with the morphological composition of the subgingival microbiota in a large population of patients suffering from advanced periodontitis. Subgingival plaque from the deepest pockets of every quadrant of their dentitions was sampled and pooled in 70 patients between 14 and 63 years of age, and analysed morphologically by phase-contrast microscopy. A minimum % similarity index was employed to define 4 clusters with different morphological composition of the floras. The actual proportion of A. actinomycetemcomitans was determined at 2 sites with deep periodontal pockets. All clusters harboured patients infected with A. actinomycetemcomitans. If present, in clusters predominated by motile rods or medium-sized spirochetes, the organism was found in rather low proportions (median 5.3% and 3.4%, respectively). However, the cluster with a pooled flora mainly consisting of coccoid cells revealed periodontal sites with A. actinomycetemcomitans in proportions of more than 53% (median), if the organism was present (p less than 0.01). We found a positive correlation between proportions of A. actinomycetemcomitans and cocci (R = 0.65) and negative correlations with spirochetes and motile rods (R = 0.61, R = -0.59, respectively). Cautious interpretation of subgingival plaque predominated by coccoid cells is recommended, if deep periodontal pockets and obvious signs of inflammation are present, since these pockets were found to be often infected with large numbers of A. actinomycetemcomitans.

Identification of the rhamnose-sensitive adhesin of Capnocytophaga ochracea ATCC 33596.

The coaggregation of the Gram-negative microorganism Capnocytophaga ochracea ATCC 33596 with several Gram-positive bacteria, including streptococci, actinomyces and rothia, is mediated by rhamnose-sensitive adhesin(s). In the present study, MAbs against the C. ochracea adhesin(s) were prepared. These antibodies inhibited all the rhamnose-sensitive interactions, indicating that they recognize epitopes at or near the rhamnose binding site of the adhesin. The monoclonals served as probes in immunoblot analysis and recognized a polypeptide of Mr 155 K present in the wild-type organism but absent in a coaggregation defective mutant. It is concluded that the rhamnose-sensitive interactions of C. ochracea are mediated by a 155 K Mr polypeptide present on its outer surface.

Actinobacillus actinomycetemcomitans serotypes and leukotoxicity in Korean localized juvenile periodontitis.

Actinobacillus actinomycetemcomitans is though to play an important role in the pathogenesis of localized juvenile periodontitis (LJP). Preliminary data suggested that the serotype distribution of A. actinomycetemcomitans in Korea and the United States differ. This study evaluated A. actinomycetemcomitans prevalence, serotype distribution, and leukotoxicity in Korean LJP patients by culture, enzyme-linked immunosorbent assay, indirect immunofluorescence, and lactate dehydrogenase release from polymorphonuclear leukocytes exposed to A. actinomycetemcomitans. A. actinomycetemcomitans occurred in 75% of LJP lesions and 6% of normal sites with approximately equal distribution of serotype a, b, and c. Single serotypes were isolated from nine patients while three patients harbored two serotypes either in the same or different disease sites. A. actinomycetemcomitans leukotoxicity occurred in 22% isolates with a 69% prevalence. Individual sites harbored both leukotoxic and non-leukotoxic strains with no serotype association. The distribution of serotypes and leukotoxic strains of A. actinomycetemcomitans in Korean LJP patients differed from those reported in the United States. This suggests that serotype b may not be more important in the pathogenesis of LJP.

Identification of a hemolysin from Actinobacillus pleuropneumoniae and characterization of its channel properties in planar phospholipid bilayers.

A proteinaceous hemolysin secreted by strain 4074 of serotype 1 of Actinobacillus pleuropneumoniae was purified by diafiltration and ion exchange chromatographic techniques. The hemolytic activity is associated with a 107-kDa band as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and confirmed by Western blotting and immunoprecipitation. This hemolysin produces pores in membranes as demonstrated by osmotic protection studies using red blood cells and carbohydrate compounds of various molecular weights. These assays suggest a pore diameter in the order of 2 nm. Phospholipid bilayers composed of 1:1 w/w phosphotidylserine:phosphotidylethanolamine exposed to this toxin display discrete current flow events typical of transmembrane channels and consistent with the interpretation that this toxin acts by forming pores in phospholipid membranes. The linear relationship of current amplitude to holding potential when examined over the -60 to +60 mV range indicates that this pore has a constant mean single channel conductance level of 350-400 pS.