Investigation of putative invasion determinants of Actinobacillus species using comparative genomics.

Actinobacillus spp. are Gram-negative bacteria associated with mucosal membranes. While some are commensals, others can cause important human and animal diseases. A. pleuropneumoniae causes severe fibrinous hemorrhagic pneumonia in swine but not systemic disease whereas other species invade resulting in septicemia and death. To understand the invasive phenotype of Actinobacillus spp., complete genomes of eight isolates were obtained and pseudogenomes of five isolates were assembled and annotated. Phylogenetically, A. suis isolates clustered by surface antigen type and were more closely related to the invasive A. ureae, A. equuli equuli, and A. capsulatus than to the other swine pathogen, A. pleuropneumoniae. Using the LS-BSR pipeline, 251 putative virulence genes associated with serum resistance and invasion were detected. To our knowledge, this is the first genome-wide study of the genus Actinobacillus and should contribute to a better understanding of host tropism and mechanisms of invasion of pathogenic Actinobacillus and related genera.

Identification of the pangenome and its components in 14 distinct Aggregatibacter actinomycetemcomitans strains by comparative genomic analysis.

BACKGROUND: Aggregatibacter actinomycetemcomitans is genetically heterogeneous and comprises distinct clonal lineages that may have different virulence potentials. However, limited information of the strain-to-strain genomic variations is available. METHODOLOGY/PRINCIPAL FINDINGS: The genome sequences of 11 A. actinomycetemcomitans strains (serotypes a-f) were generated de novo, annotated and combined with three previously sequenced genomes (serotypes a-c) for comparative genomic analysis. Two major groups were identified; serotypes a, d, e, and f, and serotypes b and c. A serotype e strain was found to be distinct from both groups. The size of the pangenome was 3,301 genes, which included 2,034 core genes and 1,267 flexible genes. The number of core genes is estimated to stabilize at 2,060, while the size of the pangenome is estimated to increase by 16 genes with every additional strain sequenced in the future. Within each strain 16.7-29.4% of the genome belonged to the flexible gene pool. Between any two strains 0.4-19.5% of the genomes were different. The genomic differences were occasionally greater for strains of the same serotypes than strains of different serotypes. Furthermore, 171 genomic islands were identified. Cumulatively, 777 strain-specific genes were found on these islands and represented 61% of the flexible gene pool. CONCLUSIONS/SIGNIFICANCE: Substantial genomic differences were detected among A. actinomycetemcomitans strains. Genomic islands account for more than half of the flexible genes. The phenotype and virulence of A. actinomycetemcomitans may not be defined by any single strain. Moreover, the genomic variation within each clonal lineage of A. actinomycetemcomitans (as defined by serotype grouping) may be greater than between clonal lineages. The large genomic data set in this study will be useful to further examine the molecular basis of variable virulence among A. actinomycetemcomitans strains.

Recurrent Actinobacillus peritonitis in an otherwise healthy thoroughbred horse.

A Thoroughbred gelding in North America was evaluated for Actinobacillus peritonitis on three different occasions over a 4-year period. At each presentation, peritoneal fluid had an elevated nucleated cell count (220,000-550,000 cells/µL) characterised by non-degenerate neutrophils, no visible bacteria, an elevated total protein (4.6-5.5 g/dL) and bacterial culture yielding Actinobacillus spp. Actinobacillus peritonitis appears to be a regional disease occurring in Australia and less commonly in New Zealand and North America. Recurrence, other than incomplete resolution, has not been previously reported. This case highlights the classical presentation, response to therapy and excellent prognosis despite the alarmingly abnormal peritoneal fluid characteristic of Actinobacillus peritonitis and questions the role of parasite migration in the pathogenesis. Finally, this case is remarkable because Actinobacillus peritonitis was recurrent over several years in an otherwise normal horse.

A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production.

BACKGROUND: Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process. RESULTS: Described here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogenes's potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae. CONCLUSIONS: Both A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogenes's natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes.

Modulation of gene expression in Actinobacillus pleuropneumoniae exposed to bronchoalveolar fluid.

BACKGROUND: Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia, is an important pathogen of swine throughout the world. It must rapidly overcome the innate pulmonary immune defenses of the pig to cause disease. To better understand this process, the objective of this study was to identify genes that are differentially expressed in a medium that mimics the lung environment early in the infection process. METHODS AND PRINCIPAL FINDINGS: Since bronchoalveolar lavage fluid (BALF) contains innate immune and other components found in the lungs, we examined gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after a 30 min exposure to BALF, using DNA microarrays and real-time PCR. The functional classes of genes found to be up-regulated most often in BALF were those encoding proteins involved in energy metabolism, especially anaerobic metabolism, and in cell envelope, DNA, and protein biosynthesis. Transcription of a number of known virulence genes including apxIVA and the gene for SapF, a protein which is involved in resistance to antimicrobial peptides, was also up-regulated in BALF. Seventy-nine percent of the genes that were up-regulated in BALF encoded a known protein product, and of these, 44% had been reported to be either expressed in vivo and/or involved in virulence. CONCLUSIONS: The results of this study suggest that in early stages of infection, A. pleuropneumoniae may modulate expression of genes involved in anaerobic energy generation and in the synthesis of proteins involved in cell wall biogenesis, as well as established virulence factors. Given that many of these genes are thought to be expressed in vivo or involved in virulence, incubation in BALF appears, at least partially, to simulate in vivo conditions and may provide a useful medium for the discovery of novel vaccine or therapeutic targets.

Granulomatous lymphadenitis and pneumonia associated with Actinobacillus porcitonsillarum in a slaughter pig.

Multiple coalescing granulomatous foci were detected in the pulmonary hilar and mediastinal lymph nodes and lung of a slaughtered pig aged 6 months. Haemolytic, Gram-negative bacilli were isolated from the lymph nodes. The isolate (strain TO17214) strongly cross-reacted with sera against Actinobacillus pleuropneumoniae serotype 12 in slide agglutination tests. Comparative 16S rDNA gene sequencing analysis identified strain TO17214 as Actinobacillus porcitonsillarum. Histologically, extensive inflammation took the form of large granulomas consisting of epithelioid cells and multinucleated giant cells in the lymph nodes and lung, and Gram-negative bacilli were discernible in the centres of the lesions. Immunohistochemically, the organisms cross-reacted with polyclonal antibodies against A. pleuropneumoniae serotypes 12 and 2. The results indicated that A. porcitonsillarum, previously considered non-pathogenic, can induce multifocal granulomatous lymphadenitis accompanied by pneumonia in the growing-finishing pig.

Diagnóstico por el laboratorio de las enfermedades periodontales y periimplantarias. Diagnóstico de la periodontitis / Diagnostic by laboratory in the periodontal and periimplantar disease. Diagnostic of the periodontitis

Desde el punto de vista epidemiológico la enfermedad periodontal tiene una enorme trascendencia. El diagnóstico del proceso es una necesidad para, con arreglo a él, realizar el tratamiento y la prevención mas apropiada. El enfoque del diagnóstico depende del concepto que sobre la patogenia se tiene en un momento dado. Desde esa perspectiva se hace un análisis de los distintos métodos de diagnóstico que existen. Se llega a la conclusión de que actualmente el método clínico, mediante el sondaje periodontal, es el mas apropiado para conocer el estado de gravedad del proceso y enfocar el tratamiento. Tras la revisión de los distintos procesos que estudian: los patógenos responsables, los mediadores de la inflamación, los parámetros de destrucción ósea y del conectivo, los factores genéticos y de riesgo ambiental y adquirido, se llega a la conclusión de que actualmente no existe ningún método capaz de identificar a la población de riesgo de padecer la enfermedad periodontal ni los períodos de actividad. Ello en gran medida se debe a que existen muchas incógnitas en la patogenia del proceso y que es preciso seguir la búsqueda de un parámetro eficaz que me ayude en la toma de decisiones a la hora de enfocar el tratamiento y la prevención del proceso (AU)

The periodontal disease is very important in the general health status of our patients. We need an adequate diagnosis of the process to treat and prevent successfully. The diagnosis depends on the concept about the illness is generally accepted nowadays. We revise all the diagnostic procedures we can use in the clinic. We conclude that the clinic diagnostic method with the periodontal probe is the best way to know the destructive bone process and treat it. The others methods that study the bacteria, inflammatory mediators, parameters of bone and collagen resorption, genetic tests cannot identify the high-risk patients and the period of activity. The reason could be the lack of a completely knowledge of the pathogenic process involved in the periodontal disease. We have to work in this field in the future to improve the treatment and prevention of the periodontitis (AU)

Non-pathogenic Actinobacillus isolates antigenically and biochemically similar to Actinobacillus pleuropneumoniae: a novel species?

Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae.

Host cell specific activity of RTX toxins from haemolytic Actinobacillus equuli and Actinobacillus suis.

We assessed and compared host cell specificity of the haemolytic and cytotoxic activity of the RTX toxins from Actinobacillus equuli, an equine pathogen, and Actinobacillus suis, which is pathogenic for pigs. The two bacterial species are closely related, phenotypically as well as phylogenetically, sharing the same 16S rRNA gene sequence. Both species contain specific protein toxins from the family of pore-forming RTX toxins, however, the two species differ in their RTX toxin profiles. Haemolytic A. equuli contains the operon for the Aqx toxin, whereas A. suis harbours genes for ApxI and ApxII. We tested the toxic activity of the corresponding proteins on erythrocytes as well as on lymphocytes isolated from horse and pig blood. The strength of the haemolytic activity for each of the toxins was independent of the origin of erythrocytes. When testing cytotoxic activity, the Aqx protein showed a higher toxic effect for horse lymphocytes than for porcine lymphocytes. On the other hand, ApxI and ApxII showed a strong cytotoxic effect on porcine lymphocytes and a reduced toxicity for horse lymphocytes; the toxicity of ApxII was generally much lower than ApxI. Our results indicate a host species specificity of the toxic activity of RTX toxins Aqx of A. equuli and ApxI and ApxII of A. suis.

Virulence properties of oral bacteria: impact of molecular biology.

Dental caries and periodontitis, although generally not life threatening, are nevertheless of significant importance. An understanding of the molecular nature of these diseases could aid the development of novel methods of prevention and control, and increase our knowledge of their etiology. The identification of virulence factors in oral bacteria could lead to the development of vaccines directed against these organisms, the design of inhibitors of biofilm formation, and the development of replacement therapy strategies.

Bacterial heat shock protein-60 increases epithelial cell proliferation through the ERK1/2 MAP kinases.

Heat shock proteins (hsp) have important roles in the regulation and protection of both prokaryotic and eukaryotic cells, especially during environmental stress. Hsps are also important bacterial virulence factors. We investigated whether bacterial hsp60 can alter epithelial cell mitogen-activated protein kinase (MAPK) signaling and cell proliferation. Human skin keratinocytes (HaCaT cell line) were cultured in the presence of hsp60 purified from Actinobacillus actinomycetemcomitans, an important oral pathogen. Protein kinases in the ERK1/2 and p38 MAPK signaling pathways were probed with kinase-specific and phosphorylation-site-specific antibodies on Western blots. In quiescent cultures, hsp60 increased ERK1/2 phosphorylation in a sustained manner and p38 phosphorylation transiently. Hsp60 also increased epithelial cell proliferation by about 30%. Inhibition of the ERK1/2 pathway by PD 98059 (a MEK1 inhibitor) reversed partially ERK1/2 phosphorylation and totally cell proliferation indicating that the ERK1/2 MAPK pathway is involved in the hsp60-induced cell growth. This was supported by findings that hsp60 stimulated phosphorylation of RSK1/2 and cyclic AMP response element-binding protein and increased expression of transcription factors c-Jun and c-Fos. Recombinant human hsp60 did not alter ERK1/2 or p38 phosphorylation and had no effect on epithelial cell proliferation. Inhibition of p38 MAPK pathway by SB 203580 increased both ERK1/2 phosphorylation and cell proliferation demonstrating that the inhibitor can either directly or indirectly activate the ERK1/2 MAPK pathway. The results show that exogenous bacterial hsp60 is able to activate ERK1/2 phosphorylation and thereby cause increased epithelial proliferation. In case of mucosal infection this effect may either lead to increased wound repair or participate in the pathological mechanism of some bacterial diseases that involve increased epithelial proliferation.

Pathogenicity of Actinobacillus minor, Actinobacillus indolicus and Actinobacillus porcinus strains for gnotobiotic piglets.

The purpose of the study was to evaluate the clinical significance of Actinobacillus minor, Actinobacillus porcinus and Actinobacillus indolicus strains in gnotobiotic piglets. Twenty-two 6-h-old Caesarean-delivered and colostrum-deprived piglets were intranasally and orally inoculated with 2 x 10(6) colony-forming units of an A. minor (group 2; n = 9), A. indolicus (group 3; n = 5), or A. porcinus (group 4; n = 8) strain. Six other piglets were inoculated in the same way with phosphate-buffered saline solution and used as controls (group 1). All pigs were observed for clinical signs and rectal temperatures were taken until euthanasia 7 days after inoculation. At necropsy, conchae, tonsils, lungs, brains, liver, spleen and kidneys were macroscopically examined for lesions and samples were taken for bacteriology. None of the pigs developed fever. Mild ataxia was observed in one pig from group 3 for 2 days. Clinical signs were not observed in the other animals. In none of the animals were macroscopic lesions detected at necropsy. NAD-dependent Pasteurellaceae were not isolated from control animals (group 1). The A. minor, A. indolicus and A. porcinus strains were isolated from the tonsils of one, two and one pigs, respectively. Actinobacillus porcinus was isolated from the brains of the pig with central nervous symptoms and from the conchae of another pig. The inoculation strains were not demonstrated in the other samples. It was concluded that, using these inoculation routes and dose, the A. minor, A. indolicus and A. porcinus strains had low capacity to colonize the upper respiratory tract of gnotobiotic piglets and demonstrated low or no pathogenicity in such animals.

Comparative pathogenicity of different Actinobacillus suis O/K serotypes.

The pathogenicity of Actinobacillus suis serotypes O1/K1 (strain SO4), O1/K2 (strain C84), and O2/K2 (strain H91-0380) was evaluated in specific-pathogen-free (SPF) piglets challenged by intraperitoneal inoculation with approximately 1 x 10(7) colony-forming units per mL. All 3 strains produced peritonitis, but differences were observed in the composite histopathologic scores (P = 0.001) and in their ability to spread (P = 0.008) at 7 h post challenge. The O2/K2 strain caused the most severe peritonitis and disseminated most widely to other tissues. Moderate lesions were seen with the O1/K2 strain while the O1/K1 strain caused mild lesions and remained largely localized to the peritoneum. In an attempt to explain the basis of observed differences, the serum sensitivity of 9 A. suis strains with different O and K types was assessed. Regardless of the O/K type, all of the isolates tested were serum resistant. Moreover, most A. suis isolates grew as well or better in complement-replete sera as they did in complement-depleted sera. These observations indicate that although 02 and K2 strains had a greater propensity to cause a disseminating septic inflammatory response in pigs, they were no more resistant to complement-mediated killing than O1 strains.

Actinobacillus species and their role in animal disease.

Actinobacillus species are Gram-negative bacteria responsible for several quite distinct disease conditions of animals. The natural habitat of the organisms is primarily the upper respiratory tract and oral cavity. A. lignieresii is the cause of actinomycosis (wooden tongue) in cattle: a sporadic, insidiously-developing granulomatous infection. In sharp contrast is A. pleuropneumoniae which is responsible for a rapidly spreading often fatal pneumonia, common among intensively reared pigs. Detailed investigation of this organism has provided a much clearer picture of the bacterial factors involved in causing disease. A. equuli similarly causes a potent septicaemia in the neonatal foal; growing apparently unrestricted once infection occurs. Other members of the genus induce characteristic pathogenesis in their preferred host, with one, A. actinomycetemcomitans, being a cause of human periodontal disease. This article reviews recent understanding of the taxonomy and bacteriology of the organisms, and the aetiology, pathogenicity, diagnosis and control of animal disease caused by Actinobacillus species.

Effects of Actinobacillus equuli culture supernatants on equine neutrophil functions and survival.

After exposure of equine granulocytes from both foals and adult horses to culture supernatants from clinical isolates of Actinobacillus equuli, phagocytic capacity and respiratory burst was examined by flow-cytometry and a chemiluminescence assay, respectively. One haemolytic isolate of an equine Actinobacillus was also included in the study. An average decrease of 22% in total number of granulocytes, in the flow cytometric assay (P < 0.01), and an average decrease of 26% in light emission, in the chemiluminescence assay (P < 0.001), was seen after exposure to bacterial culture supernatants of A. equuli, indicating that the supernatants contained leukotoxic bacterial products. Supernatants from the haemolytic isolate appeared to contain a higher amount or more potent leukotoxic metabolites when haemolysis was expressed, causing a decrease in total number of granulocytes of 44% (P < 0.01) and a decrease in light emission of 52% (P < 0.01). Evaluation of the stability of the methods used revealed that within-method variation was far less than the observed results. The leukotoxic effects of A. equuli culture supernatants were mainly reflected in the decreased survival of neutrophils and not in neutrophil functions.

Oral pathogens: from dental plaque to cardiac disease.

Oral bacteria exhibit highly specific adherence mechanisms and as a result they colonize and cause disease principally in the oral cavity. Oral pathogens, however, can produce systemic disease and are known causative agents of infective endocarditis. Recent studies have revealed that periodontal disease per se is also a statistically significant risk factor for cardiovascular disease. A link between the two diseases is the secretion and systemic appearance in periodontitis of pro-inflammatory cytokines capable of eliciting effects associated with atherosclerosis and coronary heart disease.