Rhein kills Actinobacillus pleuropneumoniae, reduces biofilm formation, and effectively treats bacterial lung infections in mice.

Background. Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae family, is known for its highly infectious nature and is the primary causative agent of infectious pleuropneumonia in pigs. This disease poses a considerable threat to the global pig industry and leads to substantial economic losses due to reduced productivity, increased mortality rates, and the need for extensive veterinary care and treatment. Due to the emergence of multi-drug-resistant strains, Chinese herbal medicine is considered one of the best alternatives to antibiotics due to its unique mechanism of action and other properties. As a type of Chinese herbal medicine, Rhein has the advantages of a wide antibacterial spectrum and is less likely to develop drug resistance, which can perfectly solve the limitations of current antibacterial treatments.Methods. The killing effect of Rhein on A. pleuropneumoniae was detected by fluorescence quantification of differential expression changes of key genes, and scanning electron microscopy was used to observe the changes in A. pleuropneumoniae status after Rhein treatment. Establishing a mouse model to observe the treatment of Rhein after A. pleuropneumoniae infection.Results. Here, in this study, we found that Rhein had a good killing effect on A. pleuropneumoniae and that the MIC was 25 µg ml-1. After 3 h of action, Rhein (4×MIC) completely kills A. pleuropneumoniae and Rhein has good stability. In addition, the treatment with Rhein (1×MIC) significantly reduced the formation of bacterial biofilms. Therapeutic evaluation in a murine model showed that Rhein protects mice from A. pleuropneumoniae and relieves lung inflammation. Quantitative RT-PCR (Quantitative reverse transcription polymerase chain reaction is a molecular biology technique that combines both reverse transcription and polymerase chain reaction methods to quantitatively detect the amount of a specific RNA molecule) results showed that Rhein treatment significantly downregulated the expression of the IL-18 (Interleukin refers to a class of cytokines produced by white blood cells), TNF-α, p65 and p38 genes. Along with the downregulation of genes such as IL-18, it means that Rhein has an inhibitory effect on the expression of these genes, thereby reducing the activation of inflammatory cells and the production of inflammatory mediators. This helps reduce inflammation and protects tissue from further damage.Conclusions. This study reports the activity of Rhein against A. pleuropneumoniae and its mechanism, and reveals the ability of Rhein to treat A. pleuropneumoniae infection in mice, laying the foundation for the development of new drugs for bacterial infections.

Tea Polyphenols Protects Tracheal Epithelial Tight Junctions in Lung during Actinobacillus pleuropneumoniae Infection via Suppressing TLR-4/MAPK/PKC-MLCK Signaling.

Actinobacillus pleuropneumoniae (APP) is the causative pathogen of porcine pleuropneumonia, a highly contagious respiratory disease in the pig industry. The increasingly severe antimicrobial resistance in APP urgently requires novel antibacterial alternatives for the treatment of APP infection. In this study, we investigated the effect of tea polyphenols (TP) against APP. MIC and MBC of TP showed significant inhibitory effects on bacteria growth and caused cellular damage to APP. Furthermore, TP decreased adherent activity of APP to the newborn pig tracheal epithelial cells (NPTr) and the destruction of the tight adherence junction proteins ß-catenin and occludin. Moreover, TP improved the survival rate of APP infected mice but also attenuated the release of the inflammation-related cytokines IL-6, IL-8, and TNF-α. TP inhibited activation of the TLR/MAPK/PKC-MLCK signaling for down-regulated TLR-2, TLR4, p-JNK, p-p38, p-PKC-α, and MLCK in cells triggered by APP. Collectively, our data suggest that TP represents a promising therapeutic agent in the treatment of APP infection.

Impact of growth matrix on pharmacodynamics of antimicrobial drugs for pig pneumonia pathogens.

BACKGROUND: The most widely used measure of potency of antimicrobial drugs is Minimum Inhibitory Concentration (MIC). MIC is usually determined under standardised conditions in broths formulated to optimise bacterial growth on a species-by-species basis. This ensures comparability of data between laboratories. However, differences in values of MIC may arise between broths of differing chemical composition and for some drug classes major differences occur between broths and biological fluids such as serum and inflammatory exudate. Such differences must be taken into account, when breakpoint PK/PD indices are derived and used to predict dosages for clinical use. There is therefore interest in comparing MIC values in several broths and, in particular, in comparing broth values with those generated in serum. For the pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, MICs were determined for three drugs, florfenicol, oxytetracycline and marbofloxacin, in five broths [Mueller Hinton Broth (MHB), cation-adjusted Mueller Hinton Broth (CAMHB), Columbia Broth supplemented with NAD (CB), Brain Heart Infusion Broth (BHI) and Tryptic Soy Broth (TSB)] and in pig serum. RESULTS: For each drug, similar MIC values were obtained in all broths, with one exception, marbofloxacin having similar MICs for three broths and 4-5-fold higher MICs for two broths. In contrast, for both organisms, quantitative differences between broth and pig serum MICs were obtained after correction of MICs for drug binding to serum protein (fu serum MIC). Potency was greater (fu serum MIC lower) in serum than in broths for marbofloxacin and florfenicol for both organisms. For oxytetracycline fu serum:broth MIC ratios were 6.30:1 (P. multocida) and 0.35:1 (A. pleuropneumoniae), so that potency of this drug was reduced for the former species and increased for the latter species. The chemical composition of pig serum and broths was compared; major matrix differences in 14 constituents did not account for MIC differences. Bacterial growth rates were compared in broths and pig serum in the absence of drugs; it was concluded that broth/serum MIC differences might be due to differing growth rates in some but not all instances. CONCLUSIONS: For all organisms and all drugs investigated in this study, it is suggested that broth MICs should be adjusted by an appropriate scaling factor when used to determine pharmacokinetic/pharmacodynamic breakpoints for dosage prediction.

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.

What is the true in vitro potency of oxytetracycline for the pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida?

The pharmacodynamics of oxytetracycline was determined for pig respiratory tract pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Indices of potency were determined for the following: (i) two matrices, broth and pig serum; (ii) five overlapping sets of twofold dilutions; and (iii) a high strength starting culture. For A. pleuropneumoniae, minimum inhibitory concentration (MIC) was similar for the two matrices, but for P. multocida, differences were marked and significantly different. MIC and minimum bactericidal concentration (MBC) serum: broth ratios for A. pleuropneumoniae were 0.83:1 and 1.22:1, respectively, and corresponding values for P. multocida were 22.0:1 and 7.34:1. For mutant prevention concentration (MPC) serum: broth ratios were 0.79:1 (A. pleuropneumoniae) and 20.9:1 (P. multocida). These ratios were corrected for serum protein binding to yield fraction unbound (fu) serum: broth MIC ratios of 0.24:1 (A. pleuropneumoniae) and 6.30:1 (P. multocida). Corresponding fu serum: broth ratios for MPC were almost identical, 0.23:1 and 6.08:1. These corrections for protein binding did not account for potency differences between serum and broth for either species; based on fu serum MICs, potency in serum was approximately fourfold greater than predicted for A. pleuropneumoniae and sixfold smaller than predicted for P. multocida. For both broth and serum and both bacterial species, MICs were also dependent on initial inoculum strength. The killing action of oxytetracycline had the characteristics of codependency for both A. pleuropneumoniae and P. multocida in both growth media. The in vitro potency of oxytetracycline in pig serum is likely to be closer to the in vivo plasma/serum concentration required for efficacy than potency estimated in broths.

Pharmacokinetic/pharmacodynamic evaluation of marbofloxacin in the treatment of Haemophilus parasuis and Actinobacillus pleuropneumoniae infections in nursery and fattener pigs using Monte Carlo simulations.

This study evaluated the theoretical clinical outcome of three marbofloxacin posology regimens in two groups of pigs (weaners and fatteners) for the treatment of Actinobacillus pleuropneumoniae (App) and Haemophilus parasuis (Hp) infection and the appearance of resistant bacteria due to the antibiotic treatment. The probability of target attainment (PTA) for pharmacokinetic/pharmacodynamics (PK/PD) ratios associated with clinical efficacy and with the appearance of antimicrobial resistance for fluoroquinolones at each minimum inhibitory concentration (MIC) or mutant prevention concentration (MPC) were calculated, respectively. The cumulative fraction of response (CFR) was calculated for the three posology regimens against App and they ranged from 91.12% to 96.37% in weaners and from 93% to 97.43% in fatteners, respectively. In the case of Hp, they ranged from 80.52% to 85.14% in weaners and from 82.01% to 88.49% in fatteners, respectively. Regarding the PTA of the PK/PD threshold associated with the appearance of antimicrobial resistance, results showed that marbofloxacin would prevent resistances in most of the animals up to the MPC value of 1 µg/mL.

In vitro efficacy of diallyl sulfides against the periodontopathogen Aggregatibacter actinomycetemcomitans.

The in vitro antibacterial effects of diallyl sulfide (DAS) against the Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans, the key etiologic agent of the severe form of localized aggressive periodontitis and other nonoral infections, were studied. A. actinomycetemcomitans was treated with garlic extract, allicin, or DAS, and the anti-A. actinomycetemcomitans effects of the treatment were evaluated. Garlic extract, allicin, and DAS significantly inhibited the growth of A. actinomycetemcomitans (greater than 3 log; P < 0.01) compared to control cells. Heat inactivation of the garlic extracts significantly reduced the protein concentration; however, the antimicrobial effect was retained. Purified proteins from garlic extract did not exhibit antimicrobial activity. Allicin lost all its antimicrobial effect when it was subjected to heat treatment, whereas DAS demonstrated an antimicrobial effect similar to that of the garlic extract, suggesting that the antimicrobial activity of garlic extract is mainly due to DAS. An A. actinomycetemcomitans biofilm-killing assay performed with DAS showed a significant reduction in biofilm cell numbers, as evidenced by both confocal microscopy and culture. Scanning electron microscopy (SEM) analysis of DAS-treated A. actinomycetemcomitans biofilms showed alterations of colony architecture indicating severe stress. Flow cytometry analysis of OBA9 cells did not demonstrate apoptosis or cell cycle arrest at therapeutic concentrations of DAS (0.01 and 0.1 µg/ml). DAS-treated A. actinomycetemcomitans cells demonstrated complete inhibition of glutathione (GSH) S-transferase (GST) activity. However, OBA9 cells, when exposed to DAS at similar concentrations, showed no significant differences in GST activity, suggesting that DAS-induced GST inhibition might be involved in A. actinomycetemcomitans cell death. These findings demonstrate that DAS exhibits significant antibacterial activity against A. actinomycetemcomitans and that this property might be utilized for exploring its therapeutic potential in treatment of A. actinomycetemcomitans-associated oral and nonoral infections.

Evaluation of a single dose versus a divided dose regimen of amoxycillin in treatment of Actinobacillus pleuropneumoniae infection in pigs.

The theory of a time-dependent effect of amoxycillin was examined in a model of porcine Actinobacillus pleuropneumoniae (Ap)-infection using clinically relevant dosage regimens. Twenty hours after infection of fourteen pigs, when clinical signs of pneumonia were present, one group of pigs received a single dose of amoxycillin (20 mg/kg, i.m.), whereas another group received four doses of 5 mg/kg injected at 8-h intervals. A similar AUC of the plasma amoxycillin concentration versus time curve was obtained in the two groups, whereas the maximum concentration was threefold higher using the single high dose. Plasma amoxycillin was above the MIC for twice as long using the fractionated dosage scheme. The condition of the animals was evaluated by clinical and haematological observations combined with quantification of biochemical infection markers: C-reactive protein, zinc and ascorbic acid. Within 48 h of treatment, the pigs in both treatment groups recovered clinically. No significant differences in the time-course of clinical observations or plasma concentrations of the biomarkers of infection were observed between the two treatments. In conclusion, the efficacy of these two dosage regimens of amoxycillin was not significantly different in treatment of acute Ap-infection in pigs.

Adjunctive antimicrobial therapy of periodontitis: long-term effects on disease progression and oral colonization.

BACKGROUND: This study assessed the long-term effects of adjunctive antimicrobial therapy on periodontal disease progression and oral colonization. METHODS: Patients with previously untreated chronic periodontitis and subgingival Actinobacillus actinomycetemcomitans and/or Porphyromonas gingivalis were randomly assigned to subgingival scaling without (control group) or with systemic amoxicillin plus metronidazole and CHX irrigation (test group). Relative attachment levels were determined and subgingival and mucosal plaque samples were taken at baseline, at 10 days (plaque only) and at 3, 6, 9, 12, 18, and 24 months following initial therapy. During maintenance therapy, patients received supragingival debridement only. RESULTS: After 24 months, the 18 test group patients showed at sites with initial probing depths (PD) > or =7 mm a significantly (P <0.05) higher frequency of 2 mm or more attachment gain (37.3% +/- 4.6%) and lower frequency of attachment loss (7.2% +/- 3.1%) compared to the 17 controls (8.2% +/- 3.9% and 19.1% +/- 13.1%, respectively). Compared to controls, the intraoral prevalence of A. actinomycetemcomitans (up to 18 months) and P. gingivalis (up to 3 months) decreased and that of Eikenella corrodens (at 10 days) increased in test patients (P <0.05). In both treatment groups, the detection frequency of Tannerella forsythensis decreased transiently, while an overall increase was recorded for Treponema ssp. CONCLUSIONS: Over the 24-month period, a single course of the administered adjunctive antimicrobial therapy led to a relative risk reduction of 62% for attachment loss at deep sites. However, with the exception of A. actinomycetemcomitans, it failed to induce long-term changes in the prevalence profiles of oral colonization.

Effect of oral enrofloxacin and florfenicol on pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 1.

This study was carried out to compare the efficacy of two oral anti-microbials as metaphylactic medication to pigs inoculated with Actinobacillus pleuropneumoniae serotype 1. Forty-two pigs with an average weight of 22.64 kg were randomly assigned to three treatment groups: group F was given doses of 40 ppm of florfenicol, group E received 150 ppm of enrofloxacin and group C received no medication. Groups F and E received medicated feed 12 h before being inoculated and for 7 days after inoculation. All the pigs were inoculated by aerosol, with 2 x 10(7) CFU/ml of A. pleuropneumoniae serotype 1 each. The average body temperature was higher in group C than in groups E and F, between 12 and 96 h after inoculation (P < 0.05). No differences were found between groups F and E in respiration pattern, nasal secretion and general condition (P > 0.05): however, differences were found in group C for respiration pattern and general condition (P < 0.05), 12 h after inoculation. There was no mortality in groups F and E, whereas a 50% mortality was recorded in group C during the first 48 h after inoculation (P < 0.05). Necropsies and bacterial cultures were performed 12 days after inoculation. Lesions were observed in five pigs of group F (35.71%) with an average damage of 1.16%; in four pigs of group E (28.57%) with 1.24%; and in 13 animals in group C (92.85%) with 34.5% of affected lung tissue (P < 0.05). The infective agent was cultured from various organs of animals in groups F and C, but not from those in group E.

Evaluation of a single dose versus a divided dose regimen of danofloxacin in treatment of Actinobacillus pleuropneumoniae infection in pigs.

A single versus a divided dose regimen of danofloxacin was evaluated in treatment of porcine Actinobacillus pleuropneumoniae infection using clinical observations combined with biochemical infection markers: C-reactive protein, zinc and ascorbic acid. Twenty hours after experimental infection, the 18 pigs received danofloxacin intravenously as a single dose of 2.5mg/kg or four doses of 0.6 mg/kg administered at 24h intervals. These dosage regimens resulted in similar AUCs of the plasma danofloxacin vs time curve. The maximum concentration was 3.5-fold higher using the single dose regimen, while the time with concentrations above the MIC was 2.5-fold longer using the fractionated regimen. Using the single dose regimen, temperature was normalised 32 h post-infection. In contrast, normalisation was delayed until 44 h post-infection using four low doses and a relapse with elevated temperatures at 52 and 68 h was observed. No other significant differences between the treatments were found, neither regarding clinical, haematological nor biochemical observations. The use of the more convenient single dose regimen was appropriate, as it was at least equivalent to the fractionated regimen.

Experimental infections with Actinobacillus pleuropneumoniae in pigs--I. Comparison of five different parenteral antibiotic treatments.

SPF pigs aged 10 weeks were infected intranasally with Actinobacillus pleuropneumoniae serotype 2. After the onset of clinical symptoms of respiratory disease, which occurred 20 h post-infection, parenteral treatment with ceftiofur, danofloxacin, enrofloxacin, penicillin or tiamulin was initiated (n = 8 per group). Untreated groups, of which one was infected, served as controls. The uninfected control group did not show any signs of disease, while the infected control group was severely affected by the infection and also expressed a decreased weight gain following the challenge. Based on clinical signs, the magnitude of pathological lesions in the respiratory tract found at necropsy performed 17 days post-infection and the number of reisolates of A. pleuropneumoniae made at necropsy, treatments with the quinolones (danofloxacin and enrofloxacin) and the cephalosporine (ceftiofur) were superior to those with penicillin and tiamulin. The latter groups also developed antibodies to A. pleuropneumoniae to a larger extent. Some of the pigs treated with ceftiofur and danofloxacin developed antibodies to A. pleuropneumoniae, and the microbe was reisolated from approximately 50% of these animals. In contrast, pigs treated with enrofloxacin did not develop antibodies to A. pleuropneumoniae, and the challenge strain was not found at necropsy. The performance with respect to daily weight gain and feed conversion corresponded well with the clinical signs developed and the findings made at necropsy. The decreased growth recorded during the acute phase of the disease was, to a large extent, caused by a reduced feed intake.

Experimental infections with Actinobacillus pleuropneumoniae in pigs--II. Comparison of antibiotics for oral strategic treatment.

The present study was aimed at scrutinizing the efficacy of oral antimicrobial treatments at experimental challenge using a strain of Actinobacillus pleuropneumoniae serotype 2 known to cause severe disease. SPF pigs aged 10 weeks were infected intranasally and the antimicrobial treatments were initiated 5 h prior to that exposure. Several antimicrobial drugs, as well as the length of the treatment period, were elucidated. The outcome of the challenge was monitored by registration of clinical symptoms, weight gains and the development of serum antibodies to A. pleuropneumoniae. At necropsy, the magnitude of pathological lesions in the respiratory tract and the rate of reisolation of the infective strain were recorded. Animals that became diseased displayed a decreased growth rate caused, to a large extent, by a reduced feed intake. The performance with respect to daily weight gain and feed conversion corresponded well with the clinical signs developed and serologic reactions, as well as with the findings made at necropsy. The results obtained among pigs treated with enrofloxacin, but also with florfenicol or chlortetracycline, were superior to those of pigs treated with penicillin, tiamulin or tilmicosin. A positive effect was obtained using a strategic in-feed medication against infection with A. pleuropneumoniae. Provided that the drug used is effective against the target microbe, initiating treatment prior to infection appeared to be more important than the length of the treatment. It should, however, be remembered that A. pleuropneumoniae was reisolated from all but one medicated group following an experimental challenge given after initiating the medication. Consequently medical treatment as described did not eradicate the microbe.

Susceptibility testing of Actinobacillus pleuropneumoniae in Denmark. Evaluation of three different media of MIC-determinations and tablet diffusion tests.

This study was conducted to compare the applicability of three different media in sensitivity testing of Actinobacillus pleuropneumoniae by means of MIC and tablet diffusion tests. The media used were: modified PPLO agar, chocolatized Mueller-Hinton-II and Columbia agar supplemented with NAD. Seven antimicrobial agents were tested: ceftiofur, enrofloxacin, penicillin, spectinomycin, tiamulin, trimethoprim + sulfadiazine and tylosin, against 40 randomly selected A. pleuropneumoniae isolates. In general, good agreement was found between results obtained with all combinations of media, most antimicrobials tested and the two-test systems. Some variations between media were observed for spectinomycin, tiamulin and tylosin. For ceftiofur and trimethoprim + sulfadiazine some isolates with low MIC-values were classified as resistant using tablet diffusion, indicating that the break points of resistance for these antimicrobials using the tablet diffusion tests need adjustment. Using current break points for resistance with MIC-determinations, all isolates tested susceptible to ceftiofur, enrofloxacin, penicillin, tiamulin and trimethoprim + sulfadiazine. A larger number of isolates tested resistant to spectinomycin and tylosin on all three media using both MIC determinations and tablet diffusion.

Effect of giving enrofloxacin in the diet to pigs experimentally infected with Actinobacillus pleuropneumoniae.

Three groups of 16 pigs were exposed individually when four weeks old to intranasal infection with 10(8.9) viable Actinobacillus pleuropneumoniae (serovar 3, strain 2/(10P2); a fourth group was kept in isolation from the others as uninfected controls. Seven days later the 62 surviving animals were killed and necropsied. The organism had caused typical, mainly subacute disease in 12 of the 16 unmedicated animals but in only two of the 16 which had had continuous access to a diet containing 150ppm of enrofloxacin from four hours before exposure to infection, and in six of the 16 given 32 ppm enrofloxacin. However, only 150 ppm enrofloxacin produced marked control of the infection in terms of reduced average severity of thoracic lesions and much reduced prevalence of the organism in the lung at necropsy, and the mean weight gain (1.55 kg) and feed conversion efficiency (2.08) of this infected group over seven days were similar to those of the unmedicated, uninfected controls (1.67 kg and 2.25). The infected but untreated group on average produced detectable antibody seven days after infection whereas in the infected and medicated groups a specific response against serovar 3 was absent.

Failure of vancomycin prophylaxis and treatment for Actinobacillus actinomycetemcomitans endocarditis.

Reported is a case of Actinobacillus actinomycetemcomitans endocarditis which developed after dental prophylaxis with vancomycin and erythromycin. In vitro results indicated that this isolate and 20 additional isolates were resistant to vancomycin and that potentially useful bactericidal antibiotics for the prophylaxis and treatment of A. actinomycetemcomitans infections included gentamicin, sulfamethoxazole-trimethoprim, cefotaxime, and ciprofloxacin.