Impact of metabolite accumulation on the structure, viability and development of succinic acid-producing biofilms of Actinobacillus succinogenes.

Biofilms of Actinobacillus succinogenes have demonstrated exceptional capabilities as biocatalysts for high productivity, titre and yield production of succinic acid (SA). The paper presents a microscopic analysis of A. succinogenes biofilms developed under varied fermenter conditions. The concentration of excretion metabolites is controlled by operating the fermenter in a continuous mode where the liquid throughput is adjusted. It is clearly illustrated how the accumulation of excreted metabolites (concomitant with the sodium build-up due to base dosing) has a severe effect on the biofilm structure and physiology. Under high accumulation (HA) conditions, some cells exhibit severe elongation while maintaining a cross-sectional diameter like the rod/cocci-shaped cells predominantly found in low accumulation (LA) conditions. The elongated cells formed at high accumulation conditions were found to be more viable than the clusters of rod/cocci-shaped cells and appear to form connections between the clusters. The global microscopic structure of the HA biofilms also differed significantly from the LA biofilms. Although both exhibited shedding after 4 days of growth, the LA biofilms were more homogenous (less patchy), thicker and with high viability throughout the biofilm depth. The viability of the HA biofilms was threefold lower than the corresponding LA biofilms towards the end of the fermentation. Visual observations were supported by quantitative analysis of multiple biofilm samples and strengthened the main observations. The work presents valuable insights on the effect of metabolite accumulation on biofilm structure and growth.

The Influence of Crowding Conditions on the Thermodynamic Feasibility of Metabolic Pathways.

Intracellular reactions are carried out in a crowded medium where the macromolecules occupy ∼40% of the total volume. This decrease in the available volume affects the activity of the reactants. Scaled particle theory is used for the estimation of the activity coefficients of the metabolites, and thereby for the assessment of the impact of the presence of background molecules, on the estimation of the Gibbs free energy change (ΔrG) of the reactions. The lactic acid pathway and the central carbon metabolism of Actinobacillus succinogenes for the production of succinic acid from glycerol have been used as illustrative case studies. Results suggest the importance of maintaining intracellular crowded regions to favor the feasibility of a pathway that in other circumstances would be infeasible. Moreover, the crowding conditions may change the directionality of reactions and can modify the feasible range of fluxes estimated for a metabolic system compared with those obtained at standard biological conditions.

Immobilization of Actinobacillus succinogenes by adhesion or entrapment for the production of succinic acid.

The production of succinic acid was studied with entrapped and adsorbed Actinobacillus succinogenes. The adsorption of fermentation products (organic acids in the concentration range of 1-20 g/L) on different supports was evaluated. It was found that succinic acid was adsorbed in small quantities on diatomite and zeolite (12.6 mg/g support). The highest production of succinic acid was achieved with A. succinogenes entrapped in agar beads. Batch fermentations with immobilized cells were carried out with glucose concentrations ranging from 20 to 80 g/L. Succinic acid (43.4 g/L) was obtained from 78.3g/L glucose, and a high productivity (2.83 g/Lh) was obtained with a glucose concentration of 37.6g/L. For repeated batch fermentations (5 cycles in 72 h) with immobilized cells in agar, the total glucose consumed was 147.55 g/L, while the production of succinic acid was 107 g/L. Immobilized cells reduced significantly the fermentation time, yield, productivity and final concentration of succinic acid.

Uptake pathways of anionic and cationic photosensitizers into bacteria.

The effect of divalent cations (calcium and magnesium) and a permeabilizing agent (EDTA) on the uptake of a cationic photosensitizer (PS), methylene blue (MB), and two anionic PSs, rose bengal (RB) and indocyanine green (ICG), by Gram-positive Enterococcus faecalis and Gram-negative Actinobacillus actinomycetemcomitans was examined. The possible roles of multidrug efflux pumps and protein transporters in photosensitizer uptake were assessed in E. faecalis cells by studies using an efflux pump inhibitor (verapamil) and trypsin treatment respectively. Divalent cations enhanced the uptake and photodynamic inactivation potential of both RB and ICG in E. faecalis and A. actinomycetemcomitans, while they decreased the uptake and bacterial killing by MB. Verapamil increased the uptake of RB (possibly due to efflux pump inhibition), whereas trypsin treatment resulted in significant decrease in RB and ICG uptake. The results suggested that the uptake of anionic PSs by bacterial cells may be mediated through a combination of electrostatic charge interaction and by protein transporters, while the uptake of cationic PSs, as previously reported, is mediated by electrostatic interactions and self promoted uptake pathways.

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes.

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l(-1). In batch fermentation, 45.5 g l(-1) succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l(-1) of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l(-1) at a rate of 1.21 g l(-1) h(-1) after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.

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.

Invasive growth of Actinobacillus actinomycetemcomitans on solid medium (TSBV).

When grown on agar, most Actinobacillus actinomycetemcomitans form circular and convex colonies with an internal star-shaped morphology. Such colonies adhere firmly to the agar, and when removed, a star-shaped imprint similar to that of the intact colony remains. This study was undertaken to determine the nature of this in vitro growth. Stereo-microscopy and scanning and transmission electron microscopy showed that the star-shaped imprint of A. actinomycetemcomitans colonies reflected pseudopod-like extensions of the bacterial colony which penetrated deep into the TSBV agar. The center of the colonies consisted primarily of ghost-like cells, while the dense border of each colony, including the pseudopods, expressed characteristics of vital cells. The latter were embedded in vast amounts of extracellular vesicles, and the outer aspect of the pseudopods was lined with a border of such vesicles.

Colonial variation and fimbriation of Actinobacillus actinomycetemcomitans.

Three colonial variants of Actinobacillus actinomycetemcomitans, which formed transparent rough (TR)-, transparent smooth (TS)-, and opaque smooth (OS)-surfaced colonies, were described in relation to their fimbriation. TR- and TS-cells were adhesive to agar and glass surfaces but not the OS-cells. The examination by electron microscopy revealed that TR-cells were highly fimbriated but not TS- and OS-cells. Thus, TS-cells seemed to be an intermediate type. The fimbriae were isolated from TR-cells by suspending in 0.15 M ethanolamine-HCl buffer (pH 10.5) and purified by dissolving non-fimbrial components in 0.5% deoxycholate and 0.7% n-octyl-beta-D-glucopyranoside. The relative molecular mass of the fimbrial subunit protein was 54,000.

Microbiological study of localized juvenile periodontitis in Panama.

The occurrence of subgingival Actinobacillus actinomycetemcomitans and Capnocytophaga in 12 localized juvenile periodontitis and 10 gingivitis patients from Panama was determined using selective culture techniques. A actinomycetemcomitans was present in all localized juvenile periodontitis lesions studied and was, on average, recovered in hundred-fold-higher numbers from localized juvenile periodontitis lesions than from gingivitis lesions. Capnocytophaga was only recovered in approximately threefold-higher numbers from localized juvenile periodontitis than from gingivitis. The study confirms and extends previous data indicating a close relationship between A actinomycetemcomitans and localized juvenile periodontitis. It is proposed that identification of A actinomycetemcomitans may be a valuable adjunct in the diagnosis of localized juvenile periodontitis.

Suppression of penicillin-resistant oral Actinobacillus actinomycetemcomitans with tetracycline. Considerations in endocarditis prophylaxis.

Actinobacillus actinomycetemcomitans is an oral bacterium which is being encountered with increasing frequency in infective endocarditis. This organism occurs in high numbers in periodontitis lesions of patients with localized juvenile periodontitis (periodontosis). It is present infrequently, and only in low numbers in most other individuals. Its common resistance to penicillin, erythromycin and vancomycin represents a clinical problem in patients at risk of developing endocarditis after dental treatment. However, the high activity of tetracyclines against A. actinomycetemcomitans may be useful in prophylactic endocarditis considerations by allowing a suppression of the organism prior to the institution of recommended prophylactic protocols. In this study, we determined the effect of systemic tetracycline-HCl therapy (1 gm/day) on the oral A. actinomycetemcomitans population in five localized juvenile periodontitis patients who were heavily infected with the organism. A. actinomycetemcomitans could not be detected in samples of subgingival and supragingival dental plaque and cheek mucosal surfaces following 14 days of administration of systemic tetracycline. The organism was still undetectable 3 weeks after therapy but it reappeared at a few oral sites at week 8 post-treatment. On the basis of this data, it is proposed that the prophylactic endocarditis therapy of patients with high numbers of penicillin-resistant A. actinomycetemcomitans include a two-stage approach: first, the systemic administration of tetracycline for 14 days, and second, institution of a conventional prophylactic protocol during the time of dental treatment.

Salient Biochemical Characters of Actinobacillus actinomycetemcomitans.

A total of 136 strains of Actinobacillus actinomycetemcomitans were studied for 135 features. All isolates were small nonmotile capnophilic gram-negative rods which grew with no requirement of X or V growth factors. They all decomposed hydrogen peroxide, were oxidase-negative and benzidine-positive, reduced nitrate, produced strong alkaline and acid phosphatases, and fermented fructose, glucose and mannose. Variable fermentation results were obtained with dextrin, maltose, mannitol and xylose. Some isolates produced small amounts of gas. Representative strains of Haemophilus aphrophilus were morphologically and biochemically quite similar to A. actinomycetemcomitans. Characters which should prove to be useful to identify and distinguish these two species include catalase reaction. fermentation of lactose, starch, sucrose and trehalose, and resistance to sodium fluoride. This information allows a rapid diagnosis by species and may be helpful in studies of infections involving these organisms.

Morphology and ultrastructure of oral strains of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells.

The effects of fluoride on the percentage bacterial composition of dental plaque, on caries incidence, and on the in vitro growth of Streptococcus mutans, Actinomyces viscosus, and Actinobacillus sp.

Administration of 250 mug/ml NaF in drinking water to rats reduced both caries incidence (P less than 0.001) and the percentage of S mutans (0.01 greater than P greater than 0.001) although the percentage of Actinobacillus sp. was increased (P less than 0.001). An Actinobacillus sp. and S mutans FA1 both proliferated in NaF broth only if the NaF greater than 10 mug/ml, which was associated with the inhibition of acid production.