Basal-Level Effects of (p)ppGpp in the Absence of Branched-Chain Amino Acids in Actinobacillus pleuropneumoniae.

The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory mechanism in bacterial pathogens, enabling adaptation to adverse environments, and is linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs of pigs, its only known natural host. Pig lungs are known to have a low concentration of free branched-chain amino acids (BCAAs) compared to the level in plasma. We had investigated the role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae Now, we sought to determine whether (p)ppGpp was a trigger signal for the SR in A. pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic analyses of the wild type (WT) and an relA spoT double mutant [which does not produce (p)ppGpp], we found that (p)ppGpp could repress de novo purine biosynthesis and activate antioxidant pathways. There was a positive correlation between GTP and endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology, and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the biosynthesis of BCAAs were constitutively upregulated, regardless of the existence of BCAAs, without accumulation of (p)ppGpp beyond a basal level. Collectively, our study shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. pleuropneumoniae (p)ppGpp-mediated regulation in A. pleuropneumoniae is different from that described for the model organism Escherichia coli Further work will establish whether the (p)ppGpp-dependent SR mechanism in A. pleuropneumoniae is conserved among other veterinary pathogens, especially those in the Pasteurellaceae family.IMPORTANCE (p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional challenges. Here, we present transcriptional and phenotypic differences of A. pleuropneumoniae grown in different chemically defined media in the absence of (p)ppGpp. We show that the deprivation of branched-chain amino acids (BCAAs) does not elicit a change in the basal-level (p)ppGpp, but this level is sufficient to regulate the expression of BCAA biosynthesis. The mechanism found in A. pleuropneumoniae is different from that of the model organism Escherichia coli but similar to that found in some Gram-positive bacteria. This study not only broadens the research scope of (p)ppGpp but also further validates the complexity and multiplicity of (p)ppGpp regulation in microorganisms that occupy different biological niches.

The roles of flp1 and tadD in Actinobacillus pleuropneumoniae pilus biosynthesis and pathogenicity.

Pili have been demonstrated to contribute to the pathogenicity of many bacterial pathogens. Flp pilus encoded by the tad locus belongs to the type IVb pilus. Our previous study has revealed that the intact tad locus is essential for Flp pilus formation in Actinobacillus pleuropneumoniae, a very important porcine respiratory pathogen. To further investigate the functions of Flp pilus in A. pleuropneumoniae pathogenesis, the flp1 and tadD single deletion mutants were constructed by homologous recombination. Both of the mutant strains lost pilus on their cell surfaces. The abilities of biofilm formation, cell adhesion, resistance to phagocytosis, survival in swine whole blood, and in vivo colonization of the two mutants were significantly reduced compared with those of the parental strain. The corresponding complemented strains recovered the phenotypes. These results demonstrated that flp1 and tadD were essential for the biosynthesis of Flp pilus and that the pilus played important roles during infection of A. pleuropneumoniae.

Pharmacokinetic/pharmacodynamic integration and modelling of oxytetracycline for the porcine pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida.

Pharmacokinetic-pharmacodynamic (PK/PD) integration and modelling were used to predict dosage schedules of oxytetracycline for two pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined in broth and porcine serum. PK/PD integration established ratios of average concentration over 48 h (Cav0-48 h )/MIC of 5.87 and 0.27 µg/mL (P. multocida) and 0.70 and 0.85 µg/mL (A. pleuropneumoniae) for broth and serum MICs, respectively. PK/PD modelling of in vitro time-kill curves established broth and serum breakpoint values for area under curve (AUC0-24 h )/MIC for three levels of inhibition of growth, bacteriostasis and 3 and 4 log10 reductions in bacterial count. Doses were then predicted for each pathogen, based on Monte Carlo simulations, for: (i) bacteriostatic and bactericidal levels of kill; (ii) 50% and 90% target attainment rates (TAR); and (iii) single dosing and daily dosing at steady-state. For 90% TAR, predicted daily doses at steady-state for bactericidal actions were 1123 mg/kg (P. multocida) and 43 mg/kg (A. pleuropneumoniae) based on serum MICs. Lower TARs were predicted from broth MIC data; corresponding dose estimates were 95 mg/kg (P. multocida) and 34 mg/kg (A. pleuropneumoniae).

Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

BACKGROUND: Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. RESULTS: For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. CONCLUSIONS: In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.

Effect of bovine apo-lactoferrin on the growth and virulence of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes porcine pleuropneumonia, leading to economic losses in the swine industry. Due to bacterial resistance to antibiotics, new treatments for this disease are currently being sought. Lactoferrin (Lf) is an innate immune system glycoprotein of mammals that is microbiostatic and microbicidal and affects several bacterial virulence factors. The aim of this study was to investigate whether bovine iron-free Lf (BapoLf) has an effect on the growth and virulence of App. Two serotype 1 strains (reference strain S4074 and the isolate BC52) and a serotype 7 reference strain (WF83) were analyzed. First, the ability of App to grow in iron-charged BLf was discarded because in vivo, BapoLf sequesters iron and could be a potential source of this element favoring the infection. The minimum inhibitory concentration of BapoLf was 14.62, 11.78 and 10.56 µM for the strain BC52, S4074 and WF83, respectively. A subinhibitory concentration (0.8 µM) was tested by assessing App adhesion to porcine buccal epithelial cells, biofilm production, and the secretion and function of toxins and proteases. Decrease in adhesion (24-42 %) was found in the serotype 1 strains. Biofilm production decreased (27 %) for only the strain 4074 of serotype 1. Interestingly, biofilm was decreased (60-70 %) in the three strains by BholoLf. Hemolysis of erythrocytes and toxicity towards HeLa cells were not affected by BapoLf. In contrast, proteolytic activity in all strains was suppressed in the presence of BapoLf. Finally, oxytetracycline produced synergistic effect with BapoLf against App. Our results suggest that BapoLf affects the growth and several of the virulence factors in App.

Actinobacillus pleuropneumoniae genes expression in biofilms cultured under static conditions and in a drip-flow apparatus.

BACKGROUND: Actinobacillus pleuropneumoniae is the Gram-negative bacterium responsible for porcine pleuropneumonia. This respiratory infection is highly contagious and characterized by high morbidity and mortality. The objectives of our study were to study the transcriptome of A. pleuropneumoniae biofilms at different stages and to develop a protocol to grow an A. pleuropneumoniae biofilm in a drip-flow apparatus. This biofilm reactor is a system with an air-liquid interface modeling lung-like environment. Bacteria attached to a surface (biofilm) and free floating bacteria (plankton) were harvested for RNA isolation. Labelled cDNA was hybridized to a microarray to compare the expression profiles of planktonic cells and biofilm cells. RESULTS: It was observed that 47 genes were differentially expressed (22 up, 25 down) in a 4 h-static growing/maturing biofilm and 117 genes were differentially expressed (49 up, 68 down) in a 6h-static dispersing biofilm. The transcriptomes of a 4 h biofilm and a 6 h biofilm were also compared and 456 genes (235 up, 221 down) were identified as differently expressed. Among the genes identified in the 4 h vs 6h biofilm experiment, several regulators of stress response were down-regulated and energy metabolism associated genes were up-regulated. Biofilm bacteria cultured using the drip-flow apparatus differentially expressed 161 genes (68 up, 93 down) compared to the effluent bacteria. Cross-referencing of differentially transcribed genes in the different assays revealed that drip-flow biofilms shared few differentially expressed genes with static biofilms (4 h or 6 h) but shared several differentially expressed genes with natural or experimental infections in pigs. CONCLUSION: The formation of a static biofilm by A. pleuropneumoniae strain S4074 is a rapid process and transcriptional analysis indicated that dispersal observed at 6 h is driven by nutritional stresses. Furthermore, A. pleuropneumoniae can form a biofilm under low-shear force in a drip-flow apparatus and analyses indicated that the formation of a biofilm under low-shear force requires a different sub-set of genes than a biofilm grown under static conditions. The drip-flow apparatus may represent the better in vitro model to investigate biofilm formation of A. pleuropneumoniae.

Method to grow Actinobacillus pleuropneumoniae biofilm on a biotic surface.

BACKGROUND: Actinobacillus pleuropneumoniae is a Gram-negative bacterium and a member of the Pasteurellaceae family. This bacterium is the causative agent of porcine pleuropneumonia, which is a highly contagious respiratory disease causing important economical losses to the worldwide pig industry. It has been shown that A. pleuropneumoniae can form biofilms on abiotic surfaces (plastic and glass). Although in vitro models are extremely useful to gain information on biofilm formation, these models may not be representative of the conditions found at the mucosal surface of the host, which is the natural niche of A. pleuropneumoniae. RESULTS: In this paper, we describe a method to grow A. pleuropneumoniae biofilms on the SJPL cell line, which represents a biotic surface. A non-hemolytic, non-cytotoxic mutant of A. pleuropneumoniae was used in our assays and this allowed the SJPL cell monolayers to be exposed to A. pleuropneumoniae for longer periods. This resulted in the formation of biofilms on the cell monolayer after incubations of 24 and 48 h. The biofilms can be stained with fluorescent probes, such as a lectin against the polymer of N-acetyl-D-glucosamine present in the biofilm matrix, and easily observed by confocal laser scanning microscopy. CONCLUSIONS: This is the first protocol that describes the formation of an A. pleuropneumoniae biofilm on a biotic surface. The advantage of this protocol is that it can be used to study biofilm formation in a context of host-pathogen interactions. The protocol could also be adapted to evaluate biofilm inhibitors or the efficacy of antibiotics in the presence of biofilms.

Regulation of pga operon expression and biofilm formation in Actinobacillus pleuropneumoniae by sigmaE and H-NS.

Clinical isolates of the porcine pathogen Actinobacillus pleuropneumoniae often form adherent colonies on agar plates due to expression of an operon, pgaABCD, encoding a poly-beta-1,6-N-acetyl-D-glucosamine (PGA) extracellular matrix. The adherent colony phenotype, which correlates with the ability to form biofilms on the surfaces of polystyrene plates, is lost following serial passage in broth culture, and repeated passage of the nonadherent variants on solid media does not result in reversion to the adherent colony phenotype. In order to investigate the regulation of PGA expression and biofilm formation in A. pleuropneumoniae, we screened a bank of transposon mutants of the nonadherent serovar 1 strain S4074(T) and identified mutations in two genes, rseA and hns, which resulted in the formation of the adherent colony phenotype. In other bacteria, including the Enterobacteriaceae, H-NS acts as a global gene regulator, and RseA is a negative regulator of the extracytoplasmic stress response sigma factor sigma(E). Transcription profiling of A. pleuropneumoniae rseA and hns mutants revealed that both sigma(E) and H-NS independently regulate expression of the pga operon. Transcription of the pga operon is initiated from a sigma(E) promoter site in the absence of H-NS, and upregulation of sigma(E) is sufficient to displace H-NS, allowing transcription to proceed. In A. pleuropneumoniae, H-NS does not act as a global gene regulator but rather specifically regulates biofilm formation via repression of the pga operon. Positive regulation of the pga operon by sigma(E) indicates that biofilm formation is part of the extracytoplasmic stress response in A. pleuropneumoniae.

Transcriptional profiling of Actinobacillus pleuropneumoniae during the acute phase of a natural infection in pigs.

BACKGROUND: Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. Many virulence factors are involved in the pathogenesis, namely capsular polysaccharides, RTX toxins, LPS and many iron acquisition systems. In order to identify genes that are expressed in vivo during a natural infection, we undertook transcript profiling experiments with an A. pleuropneumoniae DNA microarray, after recovery of bacterial mRNAs from serotype 5b-infected porcine lungs. AppChip2 contains 2033 PCR amplicons based on the genomic sequence of App serotype 5b strain L20, representing more than 95% of ORFs greater than 160 bp in length. RESULTS: Transcriptional profiling of A. pleuropneumoniae recovered from the lung of a pig suffering from a natural infection or following growth of the bacterial isolate in BHI medium was performed. An RNA extraction protocol combining beadbeating and hot-acid-phenol was developed in order to maximize bacterial mRNA yields and quality following total RNA extraction from lung lesions. Nearly all A. pleuropneumoniae transcripts could be detected on our microarrays, and 150 genes were deemed differentially expressed in vivo during the acute phase of the infection. Our results indicate that, for example, gene apxIVA from an operon coding for RTX toxin ApxIV is highly up-regulated in vivo, and that two genes from the operon coding for type IV fimbriae (APL_0878 and APL_0879) were also up-regulated. These transcriptional profiling data, combined with previous comparative genomic hybridizations performed by our group, revealed that 66 out of the 72 up-regulated genes are conserved amongst all serotypes and that 3 of them code for products that are predicted outer membrane proteins (genes irp and APL_0959, predicted to code for a TonB-dependent receptor and a filamentous hemagglutinin/adhesin respectively) or lipoproteins (gene APL_0920). Only 4 of 72 up-regulated genes had previously been identified in controled experimental infections. CONCLUSIONS: These genes that we have identified as up-regulated in vivo, conserved across serotypes and coding for potential outer membrane proteins represent potential candidates for the development of a cross-protective vaccine against porcine pleuropneumonia.

Comparative profiling of the transcriptional response to iron restriction in six serotypes of Actinobacillus pleuropneumoniae with different virulence potential.

BACKGROUND: Comparative analysis of gene expression among serotypes within a species can provide valuable information on important differences between related genomes. For the pig lung pathogen Actinobacillus pleuropneumoniae, 15 serotypes with a considerable variation in virulence potential and immunogenicity have been identified. This serotypic diversity can only partly be explained by amount of capsule and differences in the RTX toxin genes in their genomes. Iron acquisition in vivo is an important bacterial function and in pathogenic bacteria, iron-limitation is often a signal for the induction of virulence genes. We used a pan-genomic microarray to study the transcriptional response to iron restriction in vitro in six serotypes of A. pleuropneumoniae (1, 2, 3, 5b, 6, and 7), representing at least two levels of virulence. RESULTS: In total, 45 genes were significantly (p < 0.0001) up-regulated and 67 genes significantly down-regulated in response to iron limitation. Not previously observed in A. pleuropneumoniae was the up-regulation of a putative cirA-like siderophore in all six serotypes. Three genes, recently described in A. pleuropneumoniae as possibly coding for haemoglobin-haptoglobin binding proteins, displayed significant serotype related up-regulation to iron limitation. For all three genes, the expression appeared at its lowest in serotype 3, which is generally considered one of the least virulent serotypes of A. pleuropneumoniae. The three genes share homology with the hmbR haemoglobin receptor of Neisseria meningitidis, a possible virulence factor which contributes to bacterial survival in rats. CONCLUSIONS: By comparative analysis of gene expression among 6 different serotypes of A. pleuropneumoniae we identified a common set of presumably essential core genes, involved in iron regulation. The results support and expand previous observations concerning the identification of new potential iron acquisition systems in A. pleuropneumoniae, showing that this bacterium has evolved several strategies for scavenging the limited iron resources of the host. The combined effect of iron-depletion and serotype proved to be modest, indicating that serotypes of both moderate and high virulence at least in vitro are reacting almost identical to iron restriction. One notable exception, however, is the haemoglobin-haptoglobin binding protein cluster which merits further investigation.

Enhancement of Apx Toxin Production in Actinobacillus pleuropneumoniae Serotypes 1, 2, and 5 by Optimizing Culture Condition.

Actinobacillus pleuropneumoniae (APP) is a causative agent of porcine pleuropneumonia. Therefore, the development of an effective vaccine for APP is necessary. Here, we optimized the culture medium and conditions to enhance the production yields of Apx toxins in APP serotype 1, 2, and 5 cultures. The use of Mycoplasma Broth Base (PPLO) medium improved both the quantity and quality of the harvested Apx toxins compared with Columbia Broth medium. Calcium chloride (CaCl2) was first demonstrated as a stimulation factor for the production of Apx toxins in APP serotype 2 cultures. Cultivation of APP serotype 2 in PPLO medium supplemented with 10 µg/ml of nicotinamide adenine dinucleotide (NAD) and 20 mM CaCl2 yielded the highest levels of Apx toxins. These findings suggest that the optimization of the culture medium and conditions increases the concentration of Apx toxins in the supernatants of APP serotype 1, 2, and 5 cultures and may be applied for the development of vaccines against APP infection.

Comparison of metabolic adaptation and biofilm formation of Actinobacillus pleuropneumoniae field isolates from the upper and lower respiratory tract of swine with respiratory disease.

Most outbreaks of disease due to infection with Actinobacillus (A.) pleuropneumoniae are caused by pigs already pre-colonised in tonsillar tissue, where the pathogen is protected from exposure to antibiotic substances administered for treatment. As it has been shown recently under experimental conditions, A. pleuropneumoniae displays host tissue-specific metabolic adaptation. In this study, pairs of A. pleuropneumoniae field isolates were recovered from lung as well as from tonsillar and nasal tissue from 20 pigs suffering from acute clinical signs of pleuropneumonia and showing characteristic pathological lung alterations. Metabolic adaptation to the porcine lower and upper respiratory tract of 32 A. pleuropneumoniae serotype 2 field isolates was examined using Fourier transform infrared (FTIR) spectroscopy as a high resolution metabolic fingerprinting method. All strains showed metabolic adaptations to organ tissue reflected by hierarchical cluster analysis of FTIR spectra similar to those previously observed under experimental conditions. Notably, differences in antimicrobial resistance patterns and minimal inhibitory concentrations of isolates from different tissues in the same animal, but not in biofilm production capability in a microtiter plate assay were found. Overall, biofilm formation was observed for 71 % of the isolates, confirming that A. pleuropneumoniae field isolates are generally able to form biofilms, although rather in a serotype-specific than in an organ-specific manner. A. pleuropneumoniae serotype 6 isolates formed significantly more biofilm than the other serotypes. Furthermore, biofilm production was negatively correlated to the lung lesion scores and tonsillar isolates tended to be more susceptible to antimicrobial substances with high bioavailability than lung isolates.

Analysis of the Actinobacillus pleuropneumoniae HlyX (FNR) regulon and identification of iron-regulated protein B as an essential virulence factor.

The Gram-negative rod Actinobacillus pleuropneumoniae is a facultative anaerobic pathogen of the porcine respiratory tract, and HlyX, the A. pleuropneumoniae homologue of fumarate and nitrate reduction regulator (FNR), has been shown to be important for persistence. An A. pleuropneumoniae hlyX deletion mutant has a decreased generation time but highly prolonged survival in comparison to its wild type parent strain when grown anaerobically in glucose-supplemented medium. Applying a combination of proteomic and transcriptomic approaches as well as in silico analyses, we identified 23 different proteins and 418 genes to be modulated by HlyX (> or = twofold up- or down-regulated). A putative HlyX-box was identified upstream of 54 of these genes implying direct control by HlyX. Consistent with its role as a strong positive regulator, HlyX induced the expression of genes for anaerobic metabolism encoding alternative terminal reductases and hydrogenases. In addition, expression of virulence-associated genes encoding iron uptake systems, a putative DNA adenine modification system, and an autotransporter serine protease were induced by HlyX under anaerobic growth conditions. With respect to virulence-associated genes, we focused on the iron-regulated protein B (FrpB) as it is the outer membrane protein most strongly up-regulated by HlyX. An frpB deletion mutant of A. pleuropneumoniae had the same growth characteristics as wild type grown aerobically and anaerobically. In contrast, A. pleuropneumoniae DeltafrpB did not cause any disease and could not be re-isolated from experimentally infected pigs, thereby identifying FrpB as a previously unknown virulence factor.

malT knockout mutation invokes a stringent type gene-expression profile in Actinobacillus pleuropneumoniae in bronchoalveolar fluid.

BACKGROUND: Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components found in the lungs, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF for 30 min. RESULTS: In reverse transcription PCR differential display (RT-PCR DD) experiments, A. pleuropneumoniae CM5 exposed to BALF up-regulated, among other genes, a gene predicted to encode LamB, an outer-membrane transport protein of the maltose regulon. To determine the role of the lamB and other genes of the maltose regulon in the pathogenesis of A. pleuropneumoniae, knockout mutations were created in the lamB and malT genes, the latter being the positive transcriptional regulator of the maltose regulon. Relative to the lamB mutant and the wild type, the malT mutant had a significant (P < 0.05) decrease in growth rate and an increased sensitivity to fresh porcine serum and high concentrations (more than 0.5 M) of sodium chloride. In DNA microarray experiments, the BALF-exposed malT mutant exhibited a gene-expression profile resembling that of a stringent type gene-expression profile seen in bacteria facing amino acid or carbon starvation. Genes encoding proteins for protein synthesis, energy metabolism, and DNA replication were down-regulated, while genes involved in stringent response (e.g., relA), amino acid and nucleotide biosynthesis, biofilm formation, DNA transformation, and stress response were up-regulated. CONCLUSION: These results suggest that MalT may be involved in protection against some stressors and in the transport of one or more essential nutrients in BALF. Moreover, if MalT is directly or indirectly linked to the stringent response, an important global mechanism of bacterial persistence and virulence in many bacterial pathogens, it might play a role in A. pleuropneumoniae pathogenesis.

Degraded neutrophil extracellular traps promote the growth of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (A.pp) causes severe pneumonia associated with enormous economic loss in pigs. Peracute diseased pigs die in <24 h with pneumonia. Neutrophils are the prominent innate immune cell in this infection that massively infiltrate the infected lung. Here we show that neutrophils release neutrophil extracellular traps (NETs) as response to A.pp infection. Numerous NET-markers were identified in bronchoalveolar lavage fluid (BALF) of A.pp-infected piglets in vivo, however, most NET fibers are degraded. Importantly, A.pp is able to enhance its growth rate in the presence of NETs that have been degraded by nucleases efficiently. A.pp itself releases no nuclease, but we identified host nucleases as sources that degrade NETs after A.pp infection. Furthermore, the nucleases of co-infecting pathogens like Streptococcus suis increase growth of A.pp in presence of porcine NETs. Thus, A.pp is not only evading the antimicrobial activity of NETs, A.pp is rather additionally using parts of NETs as growth factor thereby taking advantage of host nucleases as DNase1 or nucleases of co-infecting bacteria, which degrade NETs. This effect can be diminished by inhibiting the bacterial adenosine synthase indicating that degraded NETs serve as a source for NAD, which is required by A.pp for its growth. A similar phenotype was found for the human pathogen Haemophilus (H.) influenzae and its growth in the presence of human neutrophils. H. influenzae benefits from host nucleases in the presence of neutrophils. These data shed light on the detrimental effects of NETs during host immune response against certain bacterial species that require and/or efficiently take advantage of degraded DNA material, which has been provided by host nuclease or nucleases of other co-infecting bacteria, as growth source.

An Actinobacillus pleuropneumoniae arcA deletion mutant is attenuated and deficient in biofilm formation.

Actinobacillus pleuropneumoniae is a facultative anaerobic pathogen of the porcine respiratory tract requiring anaerobic metabolic activity for persistence on lung epithelium. The ArcAB two-component system facilitating metabolic adaptation to anaerobicity was investigated with regard to its impact on virulence and colonization of the porcine respiratory tract. Using pig infection experiments we demonstrate that deletion of arcA renders A. pleuropneumoniae significantly attenuated in acute infection and reduced long-term survival on unaltered lung epithelium as well as in sequesters. Contrary to its role in enterobacteria, the deletion of arcA in A. pleuropneumoniae does not affect growth and survival under anaerobic conditions. Instead, other than the parent strain A. pleuropneumoniae DeltaarcA does not show autoaggregation under anaerobic conditions and is deficient in biofilm formation. It is hypothesized that the lack of these functions is, at least in part, responsible for the reduction of virulence.

The CpxA/CpxR Two-Component System Affects Biofilm Formation and Virulence in Actinobacillus pleuropneumoniae.

Gram-negative bacteria have evolved numerous two-component systems (TCSs) to cope with external environmental changes. The CpxA/CpxR TCS consisting of the kinase CpxA and the regulator CpxR, is known to be involved in the biofilm formation and virulence of Escherichia coli. However, the role of CpxA/CpxR remained unclear in Actinobacillus pleuropneumoniae, a bacterial pathogen that can cause porcine contagious pleuropneumonia (PCP). In this report, we show that CpxA/CpxR contributes to the biofilm formation ability of A. pleuropneumoniae. Furthermore, we demonstrate that CpxA/CpxR plays an important role in the expression of several biofilm-related genes in A. pleuropneumoniae, such as rpoE and pgaC. Furthermore, The results of electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analysis demonstrate that CpxR-P can regulate the expression of the pgaABCD operon through rpoE. In an experimental infection of mice, the animals infected with a cpxA/cpxR mutant exhibited delayed mortality and lower bacterial loads in the lung than those infected with the wildtype bacteria. In conclusion, these results indicate that the CpxA/CpxR TCS plays a contributing role in the biofilm formation and virulence of A. pleuropneumoniae.

Transcriptional profiling of Actinobacillus pleuropneumoniae under iron-restricted conditions.

BACKGROUND: To better understand effects of iron restriction on Actinobacillus pleuropneumoniae and to identify new potential vaccine targets, we conducted transcript profiling studies using a DNA microarray containing all 2025 ORFs of the genome of A. pleuropneumoniae serotype 5b strain L20. This is the first study involving the use of microarray technology to monitor the transcriptome of A. pleuropneumoniae grown under iron restriction. RESULTS: Upon comparing growth of this pathogen in iron-sufficient versus iron-depleted medium, 210 genes were identified as being differentially expressed. Some genes (92) were identified as being up-regulated; many have confirmed or putative roles in iron acquisition, such as the genes coding for two TonB energy-transducing proteins and the hemoglobin receptor HgbA. Transcript profiling also led to identification of some new iron acquisition systems of A. pleuropneumoniae. Genes coding for a possible Yfe system (yfeABCD), implicated in the acquisition of chelated iron, were detected, as well as genes coding for a putative enterobactin-type siderophore receptor system. ORFs for homologs of the HmbR system of Neisseria meningitidis involved in iron acquisition from hemoglobin were significantly up-regulated. Down-regulated genes included many that encode proteins containing Fe-S clusters or that use heme as a cofactor. Supplementation of the culture medium with exogenous iron re-established the expression level of these genes. CONCLUSION: We have used transcriptional profiling to generate a list of genes showing differential expression during iron restriction. This strategy enabled us to gain a better understanding of the metabolic changes occurring in response to this stress. Many new potential iron acquisition systems were identified, and further studies will have to be conducted to establish their role during iron restriction.

Susceptibility of bacteria isolated from pigs to tiamulin and enrofloxacin metabolites.

Susceptibilities to metabolites of tiamulin (TIA) and enrofloxacin (ENR) were tested using selected bacteria with previously defined minimal inhibitory concentrations (MIC). The TIA metabolites tested were: N-deethyl-tiamulin (DTIA), 2beta-hydroxy-tiamulin (2beta-HTIA) and 8alpha-hydroxy-tiamulin (8alpha-HTIA), and the ENR metabolites were: ciprofloxacin (CIP) and enrofloxacin N-oxide (ENR-N). Bacteria, all of porcine origin, were selected as representatives of bacterial infections (Staphylococcus hyicus and Actinobacillus pleuropneumoniae), zoonotic bacteria (Campylobacter coli) and indicator bacteria (Escherichia coli and enterococci). Furthermore the effects of these compounds were tested on the microbial community of active sludge to test any negative effect on colony forming units (CFU). DTIA had a potency of 12.5-50% of the potency of TIA. 2beta-HTIA and 8alpha-HTIA had potencies less than 1% of the potency of TIA. ENR-N had a potency of 0.75-1.5% of the potency of ENR, while CIP and ENR had similar potencies. Results obtained here indicate that CIP and DTIA could contribute to the selective pressure for upholding antimicrobial resistant bacteria in animals under ENR or TIA treatment. The most potent metabolites CIP and DTIA showed considerable potencies against activated sludge bacteria compared to the parent compounds. EC(50) (microg/ml) for ENR, CIP, TIA and DTIA were 0.018 [95% CI: 0.028-0.149], 0.064 [95% CI: 0.007-0.046], 6.0 [95% CI: 3.6-9.8], and 9.7 [95% CI: 5.8-16.3], respectively. This indicates that the compounds can change the bacterial population in the sludge, and hereby alter the properties of the sludge.

Pharmacokinetic and Pharmacodynamic Evaluation of Marbofloxacin in Pig against Korean Local Isolates of Actinobacillus pleuropneumoniae.

The pharmacokinetics of marbofloxacin in pigs after intravenous (i.v.), intramuscular (i.m.), and peroral (p.o.) administration and pharmacokinetic/pharmacodynamic indices of this drug against Korean local isolates of Actinobacillus pleuropneumoniae were determined in this study. Marbofloxacin (2.50 mg/kg of body weight) was administered, and blood samples were collected with designated time intervals. Plasma-extracted marbofloxacin was injected into the LC-MS/MS system. The in vitro and ex vivo antibacterial activities of marbofloxacin were evaluated against 20 isolates of A. pleuropneumoniae. The mean peak plasma concentrations (Cmax) after i.v., i.m., and p.o administration were 2.60 ± 0.10, 2.59 ± 0.12, and 2.34 ± 0.12 µg/mL at 0.25 ± 0.00, 0.44 ± 0.10, and 1.58 ± 0.40 h, respectively. The area under the plasma concentration-time curves (AUC0-24) and elimination half-lives were 24.80 ± 0.90, 25.80 ± 1.40, and 23.40 ± 5.00 h·µg/mL and 8.60 ± 0.30, 12.80 ± 1.10, and 8.60 ± 0.00 h, for i.v., i.m., and p.o. administration, correspondingly. The AUC0-24/MICs of marbofloxacin after i.v., i.m., and p.o. administration were 253.86 ± 179.91, 264.1 ± 187.16, and 239.53 ± 169.75 h, respectively. The Cmax/MIC values were 26.58 ± 18.84, 26.48 ± 18.77, and 23.94 ± 16.97, and T>MICs were 42.80 ± 1.01, 36.40 ± 1.24, and 38.60 ± 1.18 h, after i.v., i.m., and p.o. administration, respectively. Thus, marbofloxacin dosage of 2.50 mg/kg of body weight by i.v., i.m., and p.o. administration with 24 h dosing interval will provide effective treatment for the infection of pig by A. pleuropneumonia.