Biofilm growth and detachment of Actinobacillus actinomycetemcomitans

The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.     

Characterization of a novel riboswitch-regulated lysine transporter in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an opportunistic pathogen that resides primarily in the mammalian oral cavity. In this environment, A. actinomycetemcomitans faces numerous host- and microbe-derived stresses, including intense competition for nutrients and exposure to the host immune system. While it is clear that A. actinomycetemcomitans responds to precise cues that allow it to adapt and proliferate in the presence of these stresses, little is currently known about the regulatory mechanisms that underlie these responses. Many bacteria use noncoding regulatory RNAs (ncRNAs) to rapidly alter gene expression in response to environmental stresses. Although no ncRNAs have been reported in A. actinomycetemcomitans, we propose that they are likely important for colonization and persistence in the oral cavity. Using a bioinformatic and experimental approach, we identified three putative metabolite-sensing riboswitches and nine small regulatory RNAs (sRNAs) in A. actinomycetemcomitans during planktonic and biofilm growth. Molecular characterization of one of the riboswitches revealed that it is a lysine riboswitch and that its target gene, lysT, encodes a novel lysine-specific transporter. Finally, we demonstrated that lysT and the lysT lysine riboswitch are conserved in over 40 bacterial species, including the phylogenetically related pathogen Haemophilus influenzae.     

Actinobacillus actinomycetemcomitans serotype-specific genotypes and periodontal status in Brazilian subjects

Periodontitis is associated with members of the oral microbiota, such as Actinobacillus actinomycetemcomitans. To our knowledge, this is the first study to evaluate, by PCR, the occurrence of the six known bacterium serotypes that included subjects with and without periodontitis. Our group comprised 49 Brazilian subjects. We studied 146 bacterial isolates from 23 patients with aggressive or chronic periodontitis and 26 subgingival specimens from subjects with or without periodontitis, all originating in our collection. Serotypes b and c were observed in similar frequencies, and no subject harboured d, e, or f serotype strains. Around 78% subjects had single-serotype infection. Mixed infection was seen only in aggressive periodontitis patients. An association between serotype b and healthy periodontium and between serotype c and chronic periodontitis was observed. Our results diverge from those previously reported, which may be explained by specific distribution patterns in distinct populations. The association of different serotypes with the same periodontal status or conversely of a serotype with different periodontal conditions indicates that organism serotyping should not be used as a sole reliable marker for predicting the outcome of the infection. Evaluation of factors involved in human oral cavity colonization by subsets of A. actinomycetemcomitans is essential for elucidating organism-host-environment relationships.     

Rapid identification of oral isolates of Aggregatibacter actinomycetemcomitans obtained from humans and primates by an ultrafast super convection based polymerase chain reaction

Aggregatibacter actinomycetemcomitans is a Gram negative oral bacterium associated with localized aggressive periodontitis (LAP). Detection of A. actinomycetemcomitans in clinical samples is routinely done by PCR. Our aim was to develop a rapid and reliable PCR method that can be used as a chair-side tool to detect A. actinomycetemcomitans in clinical samples. Sensitivity and specificity assessment was performed on buccal and plaque samples obtained from 40 adolescents enrolled in an ongoing LAP study by comparing 20 A. actinomycetemcomitans-positive subjects and 20 who were negative. In a second study, A. actinomycetemcomitans presence was tested in oral samples from eighty-six primates that included rhesus monkeys, chimpanzees, marmosets, tamarins and baboons. All samples were processed for detection of A. actinomycetemcomitans by means of culture, conventional PCR (cPCR) and rapid PCR (rPCR) using a Super Convection based AmpXpress thermal cycler (AlphaHelix, Sweden). For human samples, culture, cPCR and rPCR showed perfect agreement. Using this method A. actinomycetemcomitans was detected in 27 of 32 rhesus monkeys, 4 of 8 chimpanzees and 1 of 34 marmosets. Rapidity of AmpXpress thermal cycler, combined with Ready-To-Go PCR beads (GE Life sciences), a quick DNA extraction kit (Epicentre Biotechnologies, Madison, Wisconsin, USA) and a bufferless fast agarose gel system, made it possible to obtain results on A. actinomycetemcomitans detection within 35 min. We conclude that AmpXpress fast PCR can be conveniently used as a chair-side tool for rapid detection of A. actinomycetemcomitans in clinical samples.     

The interaction between saliva and Actinobacillus actinomycetemcomitans influenced by the Zeta potential

The adhesion of Actinobacillus actinomycetemcomitans is a virulence factor in the aetiology of periodontitis and is determined by physico-chemical properties, e.g. surface charge and hydrophobicity, of the bacterial cell surface. Although oral surfaces are constantly coated with saliva, few studies have dealt with the binding of A. actinomycetemcomitans with saliva. In this report, the charge properties of A. actinomycetemcomitans have been studied through measurement of the zeta potential and the saliva-bacteria interaction investigated at different pH-values.At physiological conditions the zeta potential was negative, varying from -11 to -26 mV, for two laboratory and two fresh isolates of A. actinomycetemcomitans. Under these conditions, binding of the low-molecular-weight salivary mucin, lactoferrin, and S-IgA was confirmed using salivary samples and purified salivary fractions in liquid-phase and in ELISA. The iso-electric points of the laboratory and fresh clinical isolates of A. actinomycetemcomitans were determined at pH 4.6 and 3.8, respectively. At pH below the iso-electric point, giving positive values of the zeta potential, additional salivary protein species bound to A. actinomycetemcomitans, including the high-molecular-weight salivary mucin (MG1) and agglutinin. Binding of the low-molecular-weight salivary mucin (MG2), lactoferrin, and S-IgA, was hardly affected by this change in zeta potential. A salivary coating formed on the bacterium at pH 7 reduced the zeta potential of the laboratory strain Y4 greatly and an iso-electric point for the bacterium could not be determined. Overall, the study suggests that upon changes in environmental pH additional salivary attachment sites on the micro-organism are exposed.     

Lactate Metabolism by Veillonella parvula

A strain of Veillonella parvula M4, which grows readily in lactate broth without a requirement for carbon dioxide, has been isolated from the oral cavity. Anaerobic, washed cells of this organism fermented sodium lactate to the following products (moles/100 moles of lactate): propionate, 66; acetate, 40; carbon dioxide, 40; and hydrogen, 14. Cells grew readily in tryptone-yeast extract broth with pyruvate, oxaloacetate, malate, and fumarate, but poorly with succinate. The fermentation of pyruvate, oxaloacetate, or lactate plus oxaloacetate by washed cells resulted in the formation of propionate and acetate in ratios significantly lower than those observed with lactate as the sole carbon source. This was primarily due to increased acetate production. Cell-free extracts were unable to degrade lactate but metabolized lactate in the presence of oxaloacetate, indicating the presence of malic-lactic transhydrogenase in this organism. Lactic dehydrogenase activity was not observed. Evidence is presented for oxaloacetate decarboxylase and malic dehydrogenase activities in extracts.     

Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a K_m of approximately 150 µM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans.     

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.     

Prevalence and distribution of bacteriophage φAa DNA in strains of Actinobacillus actinomycetemcomitans

Abstract φAa is a bacteriophage that was originally isolated by induction of a lysogenic strain of the oral bacterium Actinobacillus actinomycetemcomitans . Since the discovery of phage φAa , additional phages infecting several other strains of A. actinomycetemcomitans have been identified. To determine the prevalence of φAa or φAa -related temperate phages in this species, a φAa -specific DNA probe was prepared to screen for homologous sequences among 42 strains of A. actinomycetemcomitans . Fourteen (33%) of the 42 strains examined contained DNA sequences that hybridized with the phage φAa probe. A bacteriophage designated φAa ₃₃₃₈₄ was isolated by induction from one of the strains (ATCC 33384) that contained a sequence that hybridized with the φAa probe. The φAa probe hybridized with the DNA extracted from bacteriophage φAa ₃₃₃₈₄. The distribution of the phage φAa sequence among A. actinomycetemcomitans serotypes was 5/13 (38%) of the serotype a strains, 0/16 (0%) of the serotype b strains, and 9/13 (69%) of the serotype c strains. The results of this investigation suggest that the target sequence prepared from the phage φAa genome is fairly common in the A. actinomycetemcomitans chromosome, and that the sequence is distributed among the A. actinomycetemcomitans serotypes in a seemingly nonrandom manner.     

Isolation of a novel Aggregatibacter actinomycetemcomitans serotype b bacteriophage capable of lysing bacteria within a biofilm

A bacteriophage specific for Aggregatibacter actinomycetemcomitans serotype b, able to kill the bacterium within a biofilm, was isolated. Random mutagenesis of this phage rendered a bacteriophage able to kill 99% of the bacteria within a biofilm. This is the first report of a biocontrol experiment against A. actinomycetemcomitans.     

Mixed carbon source utilization of meat-spoiling Pseudomonas fragi 72 in relation to oxygen limitation and carbon dioxide inhibition

The growth of meat-spoiling Pseudomonas fragi 72 was studied on a defined salt medium supplemented with L-aspartate, citrate, creatine, creatinine, D-glucose, L-glutamate, and L-lactate. The utilization of the different carbon sources was followed in batch and continuous culture and under the influence of oxygen limitation and carbon dioxide inhibition (50% CO2 in air). Under nonrestricted atmospheric conditions in batch culture, the organism showed a preference in the utilization of the carbon sources in the order glucose greater than lactate greater than citrate greater than aspartate-glutamate greater than creatine greater than creatinine. The first five sources were utilized simultaneously. The order of preference was changed in continuous culture to lactate-citrate-glutamate-aspartate greater than glucose greater than creatine greater than creatinine. All carbon sources were utilized at lower dilution rates, but as the rate was increased the concentration of the carbon sources started to increase in the effluent and the preference could be seen. Under conditions of oxygen limitation the preference for glucose was weakened, but for lactate it was slightly enhanced (batch and continuous culture). Under conditions of CO2 inhibition, the preference for glucose was enhanced. However, lactate and amino acids were still preferred to glucose in the continuous culture. The utilization of creatine and creatinine was blocked by CO2 in batch culture, and only a slight utilization of creatine was noticed in a chemostat at lower dilution rates.     

Mixed carbon source utilization of meat-spoiling Pseudomonas fragi 72 in relation to oxygen limitation and carbon dioxide inhibition.

The growth of meat-spoiling Pseudomonas fragi 72 was studied on a defined salt medium supplemented with L-aspartate, citrate, creatine, creatinine, D-glucose, L-glutamate, and L-lactate. The utilization of the different carbon sources was followed in batch and continuous culture and under the influence of oxygen limitation and carbon dioxide inhibition (50% CO2 in air). Under nonrestricted atmospheric conditions in batch culture, the organism showed a preference in the utilization of the carbon sources in the order glucose greater than lactate greater than citrate greater than aspartate-glutamate greater than creatine greater than creatinine. The first five sources were utilized simultaneously. The order of preference was changed in continuous culture to lactate-citrate-glutamate-aspartate greater than glucose greater than creatine greater than creatinine. All carbon sources were utilized at lower dilution rates, but as the rate was increased the concentration of the carbon sources started to increase in the effluent and the preference could be seen. Under conditions of oxygen limitation the preference for glucose was weakened, but for lactate it was slightly enhanced (batch and continuous culture). Under conditions of CO2 inhibition, the preference for glucose was enhanced. However, lactate and amino acids were still preferred to glucose in the continuous culture. The utilization of creatine and creatinine was blocked by CO2 in batch culture, and only a slight utilization of creatine was noticed in a chemostat at lower dilution rates.     

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.     

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium.

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.     

Genomic heterogeneity in Chlorobium limicola: chromosomic and plasmidic differences among strains

Abstract The purpose of this study is to certify the importance of the fimbriae as an attachment factor of Actinobacillus actinomycetemcomitans , a human periodontopathic bacterium, and the significance of anti-fimbrial antibody function as an attachment inhibitor. Fimbrial antigen was prepared from the A. actinomycetemcomitans 310-a strain. Oligopeptides were synthesized according to the amino acid sequence of the fimbrial protein. The peptide antigen was conjugated with branched lysine polymer resin beads. The peptide antigen was suspended in PBS emulsified with incomplete Freund's adjuvant and used to immunize rabbits. A rabbit antiserum reacted with an approximately 54 kDa protein of the fimbriae protein from A. actinomycetemcomitans 310-a and with those of other fimbriated strains. This antiserum strongly inhibited the attachment of fimbriated A. actinomycetemcomitans strains to saliva-coated hydroxyapatite beads, buccal epithelial cells, and a fibroblast cell line, Gin-1. Such a synthetic fimbrial peptide antigen may be effective in inducing antibodies which inhibit adhesion and subsequent colonization by A. actinomycetemcomitans .     

The highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans: evolutionary aspects, epidemiology and etiological role in aggressive periodontitis

For many years, attention has been given to the oral bacterium Aggregatibacter actinomycetemcomitans, as a species possibly implicated in the etiology of aggressive periodontitis in adolescents. One of the major virulence factors of A. actinomycetemcomitans is the leukotoxin which is able to kill important cells of the immune system. As demonstrated in population genetic analyses, the population structure of A. actinomycetemcomitans is mainly clonal with evolutionary lineages corresponding to the serotypes. A particular highly leukotoxic clone (JP2) of serotype b has been discovered. The JP2 clone, with an estimated origin some 2400 years ago, is found to be highly conserved, based on analyses of a collection of JP2 clone strains collected through more than 20 years from individuals of diverse origin and living geographically widespread. Despite demonstration of minor evolutionary changes within the genome of JP2 clone strains of A. actinomycetemcomitans, the JP2 clone strains constitute a unique clonal type, the characteristics of which include a 530 basepair deletion in the leukotoxin operon implicated in the enhanced leukotoxic activity of the clone. Mapping of the geographic occurrence of the JP2 clone of A. actinomycetemcomitans has revealed that its colonization is largely restricted to individuals of African descent. Characteristic mutations, which allow JP2 clone isolates from the Mediterranean region to be distinguished from isolates from West Africa, including the Cape Verde islands, suggest that the JP2 clone initially emerged as a distinct genotype in the Mediterranean region of Africa and subsequently spread to West Africa, from where it might have been transferred to the American continent during the transatlantic slave trade. The finding of a sustained selective colonization of individuals of African descent, despite geographical separation from the African continent for centuries, suggests that the JP2 clone might have a distinct host tropism. Further studies are needed to elucidate the reasons for the apparent selective colonization of the Mediterranean and Western African populations. The JP2 clone of A. actinomycetemcomitans appears to play a prominent role in the etiology of aggressive periodontitis compared to other clonal types of the species. While A. actinomycetemcomitans, in general, is considered an opportunistic pathogen of the resident oral microbiota, the JP2 clone has features similar to those of an exogenous pathogen. Clonal types other than JP2 can be isolated from healthy as well as periodontally diseased individuals, whereas the JP2 clone has been isolated primarily from periodontally diseased individuals. As demonstrated in a prospective cohort study in Morocco, where the JP2 clone is endemically present, the presence of this clone in dental plaque confers a remarkably increased risk for development of aggressive periodontitis, suggesting that the JP2 clone is an important etiological agent of aggressive periodontitis in adolescents. Support for association of clonal types other than JP2 of A. actinomycetemcomitans with aggressive periodontitis has also been provided, but the association is much weaker. Nearly half of the JP2 clone carriers were found to be persistently infected during a two-year follow-up period, which indicates a level of stability of colonization with the JP2 clone similar to that previously reported for non-JP2 clonal types of A. actinomycetemcomitans. The relative risk of aggressive periodontitis is highest for individuals with stable JP2 clone colonization. Although the method used is not quantitative, this finding adds to the evidence for a causal role of the JP2 clone in aggressive periodontitis. Longitudinal data shows that few individuals are colonized with the JP2 clone de novo after puberty. Patterns of parent-child carriage and shared colonization of JP2 clone strains among siblings have been demonstrated in other studies, altogether indicating that transmission of the JP2 clone, like other clonal types of A. actinomycetemcomitans, occurs vertically, and through close person to person contacts. In conclusion, a conserved and highly leukotoxic clone of A. actinomycetemcomitans with unique characteristics and with an apparent linkage to individuals of African descent has been identified. Being aware that the genetic constitution of the hosts has not been considered and that focus in our studies has been on A. actinomycetemcomitans only, and not on other members of the oral microbiota, we conclude that the JP2 clone of A. actinomycetemcomitans is a likely etiological agent of aggressive periodontitis in adolescents.     

Molecular phylogenetic analysis of tryptophanyl-tRNA synthetase of Actinobacillus actinomycetemcomitans

Aminoacyl-tRNA synthetase family enzymes are of particular interest for creating universal phylogenetic trees and understanding the gene flow as these enzymes perform the basic and analogous biochemical function of protein synthesis in all extant organisms. Among them, tryptophanyl-tRNA synthetase (Trp-RS) plays a foremost role in phylogeny owing to the close relationship with tyrosine-tRNA synthetase. In this study, the sequence of the gene Trp-RS was amplified using degenerated adenylation domain primers in the periodontal bacterium Actinobacillus actinomycetemcomitans. The sequence of the cloned PCR amplicon confirmed the adenylation domain sequence with glutamic acid residue, which is absent in five other oral bacteria used in this study as well as in a number of other bacteria described in the database. The Trp-RS sequence analysis prevailed the identify elements such as Rossmann-fold sequence and tRNA(Trp) binding domains including acceptor stem and anticodon. A theoretical model of Trp-RS of A. actinomycetemcomitans was generated. Guided docking of the ligand tryptophanyl-5'-AMP revealed a highly identical active site in comparison with the bacterial template. The phylogenetic positioning of Trp-RS among a group of oral bacterial species revealed that A. actinomycetemcomitans is closely related to Haemophilus influenzae, H. ducreyi and Pasteurella multocida.