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.

A Florfenicol-Resistant Plasmid Shuttling Between Actinobacillus pleuropneumoniae and Glaesserella parasuis.

Porcine contagious pleuropneumonia, caused by Actinobacillus pleuropneumoniae, has resulted in significant economic losses to the swine industry. Although antibiotics are commonly employed to control this disease, their widespread use or misuse can lead to the development of antibiotic resistance in A. pleuropneumoniae. Consequently, it is crucial to conduct antimicrobial susceptibility testing on clinical isolates. In our study, we identified one strain of A. pleuropneumoniae with resistance to florfenicol and extracted a 5919 bp plasmid named pAPPJY, which confers florfenicol resistance. Sequence analysis revealed that the plasmid contains four open reading frames, namely rep, antioxin vbha family protein, floR, and a partial copy of lysr. Although a few variations in gene position were observed, the plasmid sequence exhibits a high degree of similarity to other florfenicol-resistant plasmids found in Glaesserella parasuis and A. pleuropneumoniae. Therefore, it is possible that the pAPPJY plasmid functions as a shuttle, facilitating the spread of florfenicol resistance between G. parasuis and A. pleuropneumoniae. In addition, partial recombination may occur during bacterial propagation. In conclusion, this study highlights the horizontal transmission of antibiotic resistance among different bacterial species through plasmids, underscoring the need for increased attention to antibiotic usage.

Actinobacillus ureae may be a critical pathogen in patients with predispositions: A case report and review of the literature.

RATIONALE: Actinobacillus ureae (A. ureae) is an unusual commensal of human respiratory flora, rarely causing human infection. The predisposing factors, identification, clinical features, and antibiotic therapy of A. ureae are seldomly reported. Herein, we present a case of 64-year-old man affected by A. ureae pneumonia after intracranial surgery. PATIENT CONCERNS AND DIAGNOSES: A 64-year-old male was admitted with vomiting, drowsiness, and a severe disturbance of consciousness and was later diagnosed with cerebral hemorrhage by computed tomography images. After a craniocerebral surgery, the patient suffered from intractable pneumonia, experiencing treatment failure with multiple anti-bacterial agents. Sputum culture yield pure colonies of A. ureae, confirmed by matrix-assisted laser desorption/ionization time of flight and 16S rRNA gene sequencing. INTERVENTIONS: Minocycline (100 mg p.o. per 12 hours) with a course of 15 days was administrated for this patient. OUTCOMES: The respiratory symptoms, presenting as intermittent coughing with purulent and yellowish sputum, were gone. A 3-month follow-up examination showed a complete resolution of radiological findings. LESSONS: Clinically, the actual incidence of A. ureae pneumonia may be higher than that we generally recognized, and clinicians should consider A. ureae as a possible etiologic agent in patients with predispositions. Currently, A. ureae may be susceptible to penicillin, ampicillin, and third-generation cephalosporins. Other antibacterial agents, such as tetracycline, amoxicillin/clavulanic acid, and aminoglycosides also respond well and can be a choice in the treatment of A. ureae 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.

Gamithromycin in swine: Pharmacokinetics and clinical evaluation against swine respiratory disease.

The pharmacokinetics of gamithromycin were evaluated in 26 male castrated and female crossbred swine administered gamithromycin 15% w/v (Zactran®, Boehringer Ingelheim) intravenously at 6 mg/kg bodyweight or intramuscularly at 3, 6 or 12 mg/kg bodyweight. Blood samples were collected up to Day 10 to establish the plasma profile of gamithromycin, bioavailability and dose proportionality. When administered by intramuscular injection at 6 mg/kg BWT, pharmacokinetic parameters were as follows: area under the curve until last quantifiable plasma concentration, 5.13 ± 0.957 µg*hours/ml; maximum plasma concentration, 960 ± 153 ng/ml at 5 to 15 min; terminal half-life of 94.1 ± 20.4 hr. Absolute bioavailability was 92.2%. Increase in systemic exposure was proportional to the gamithromycin dose level over the range 3-12 mg/kg BWT. No gender-related statistically significant difference in exposure was observed. For clinical evaluation of Zactran® against swine respiratory disease, 305 pigs from six commercial farms in three countries in Europe with signs associated with Actinobacillus pleuropneumoniae and/or Haemophilus parasuis and/or Pasteurella multocida and/or Bordetella bronchiseptica were used. At each site, animals were treated once in a 1:1 ratio with a single intramuscular dose of Zactran® (6 mg gamithromycin/kg bodyweight) or Zuprevo® (4% w/v tildipirosin at 4 mg/kg bodyweight; MSD Animal Health) at the recommended dose respectively. Animals were observed and scored daily for 10 consecutive days for signs of swine respiratory disease (depression, respiration and rectal temperature), and animals presenting signs of clinical swine respiratory disease (Depression Score 3 and/or Respiratory Score 3 associated with Rectal Temperature > 40.0°C) were removed from the study and considered as treatment failure. Animals which remained in the study were individually assessed for 'treatment success' or 'treatment failure' (Depression Score ≥ 1 and Rectal Temperature > 40.0°C or Respiratory Score ≥ 1 and Rectal Temperature > 40.0°C). Using a non-inferiority hypothesis test (non-inferiority margin = 0.10), the proportion of treatment successes in the Zactran® group (97%) was equivalent to or better than that in the Zuprevo® group (93%).

Exposure to benzylpenicillin after different dosage regimens in growing pigs.

BACKGROUND: Penicillin is important for treatment of pigs, but data on its absorption and disposition in pigs are sparse. This is reflected by the variation in recommended dosages in the literature. Inadequate dosage may lead to treatment failure and selection of resistant bacteria. To optimize treatment regimens, plasma exposure to benzylpenicillin for two sustained release formulations of procaine benzylpenicillin for intramuscular administration was studied in growing pigs by means of tandem mass spectrometry (UPLC-MS/MS). One formulation was an aqueous suspension, Ethacilin® vet (ETH), and the other an oily suspension, Ultrapen vet (UPA). Benzylpenicillin exposure after intravenous administration of potassium benzylpenicillin was also explored. Exposure profiles were first studied after single administrations of the approved dosages in healthy pigs and then after repeated administration of different dosages in pigs inoculated intranasally with an Actinobacillus pleuropneumoniae serotype 2 strain. RESULTS: After intravenous administration of benzylpenicillin (n = 6), maximum plasma concentration (Cmax), 1860-9318 µg/L, was observed after 15 min. At four h, plasma concentrations decreased to 15-76 µg/L. After intramuscular administration of ETH (n = 6) Cmax, 1000-4270 µg/L, was observed within one h (tmax) in 5 pigs but at four h in one pig. Cmax for UPA (n = 6), 910-3220 µg/L, was observed within one h in three pigs, but at four or 24 h in three pigs. For both ETH and UPA, the terminal phase was characterized by slow decline compared with intravenous administration. Repeated administration of different dosages of ETH and UPA in pigs inoculated with A. pleuropneumoniae (n = 54) showed that the approved dose for UPA (30 mg/kg, qd) but not for ETH (20 mg/kg, qd) gave adequate plasma exposure for bacteria with a penicillin MIC of 500 µg/L. However, more frequent dosing of ETH (bid) or increased dosage gave an adequate exposure. CONCLUSIONS: The approved dosage of ETH provided insufficient plasma exposure for adequate therapy of infections caused by A. pleuropneumoniae or other bacteria with a penicillin MIC of 500 µg/L. More frequent ETH dosing (bid) or an increased dosage would improve exposure. The approved dosage of UPA however provided adequate exposure.

Optimal regimens based on PK/PD cutoff evaluation of ceftiofur against Actinobacillus pleuropneumoniae in swine.

BACKGROUND: Actinobacillus pleuropneumoniae formerly known as Haemophilus pleuropneumoniae, can cause pleuropneumoniae in pigs, which lead to significant mortality. Ceftiofur was the first cephalosporin antibiotic used in animals, which was effective against gram-negative and gram-positive bacterium. This study aimed to formulate a rational dosage strategy and review the preceding recommended dosage based on PK/PD modeling and Establish Clinical breakpoint of ceftiofur against Actinobacillus pleuropneumoniae based on the pharmacodynamic-pharmacokinetic cutoff. RESULTS: The epidemiologic cutoff value was 0.125 µg/mL. The results of the pharmacodynamic study showed that the MICs of BW39 were 0.5 µg/mL and 1 µg/mL in vitro and ex-vivo, respectively. The minimal bactericidal concentrations (MBCs) under in vitro and ex vivo conditions were both 1 µg/mL. The time-killing profiles of ceftiofur against BW39 were time-dependent with a partly concentration-dependent pattern. Based on the inhibitory sigmoid Emax model, the AUC24 h/MIC values for the bacteriostatic, bactericidal, and elimination effects in serum were 45.73, 63.83, and 69.04 h for healthy pigs separately. According to the Monte Carlo simulation, the COPD was calculated as 2 µg/mL, and the optimized dosage regimen of ceftiofur against Actinobacillus pleuropneumoniae to achieve bacteriostatic, bactericidal, and elimination effects over 24 h was 2.13, 2.97, and 3.42 mg/kg for the 50% target attainment rate (TAR) and 2.47, 3.21, and 3.70 mg/kg for the 90% TAR respectively. CONCLUSIONS: In conclusion, we reveal the EOFF and PK/PD cutoff values of ceftiofur against A. pleuropneumoniae in piglets. However, with the paucity of clinical data for ceftiofur to establish a clinical cutoff against A. pleuropneumoniae, the PK/PD cutoff value of 2 µg/mL will be recommended as surrogate. According to the PK/PD data and the MIC distribution in China, the single bactericidal dose was 3.21 mg/kg for the 90% target, which would be more able to cure Actinobacillus pleuropneumoniae and avoid the emergence of resistance for clinical ceftiofur use in piglet.

The antimicrobial peptide MPX kills Actinobacillus pleuropneumoniae and reduces its pathogenicity in mice.

Actinobacillus pleuropneumoniae is the causative agent of highly contagious and fatal respiratory infections, causing substantial economic losses to the global pig industry. Due to increased antibiotic resistance, there is an urgent need to find new antibiotic alternatives for treating A. pleuropneumoniae infections. MPX is obtained from wasp venom and has a killing effect on various bacteria. This study found that MPX had a good killing effect on A. pleuropneumoniae and that the minimum inhibitory concentration (MIC) was 16 µg/mL. The bacterial density of A. pleuropneumoniae decreased 1000 times after MPX (1 × MIC) treatment for 1 h, and the antibacterial activity was not affected by pH or temperature. Fluorescence microscopy showed that MPX (1 × MIC) destroyed the bacterial cell membrane after treatment for 0.5 h, increasing membrane permeability and releasing bacterial proteins and Ca2+, Na+ and other cations. In addition, MPX (1 × MIC) treatment significantly reduced the formation of bacterial biofilms. Quantitative RT-PCR results showed that MPX treatment significantly upregulated the expression of the PurC virulence gene and downregulated that of ApxI, ApxII, and Apa1. In addition, the Sap A gene was found to play an important role in the tolerance of A. pleuropneumoniae to antimicrobial peptides. Therapeutic evaluation in a murine model showed that MPX protects mice from a lethal dose of A. pleuropneumoniae and relieves lung inflammation. This study reports the use of MPX to treat A. pleuropneumonia infections, laying the foundation for the development of new drugs for bacterial infections.

Caspase-1 inhibitor reduced the lung injury in a mouse model of pleuropneumonia caused by Actinobacillus pleuropneumoniae.

The pathogenesis of porcine contagious pleuropneumonia is poorly understood. In the present study, a mouse model of intranasal infection by Actinobacillus pleuropneumoniae (App) was used to examine lung inflammation. The pathogical results of lung tissues showed that App-infected mice showed dyspnea and anorexia, with severe damage by acute hemorrhage, and infiltration of eosinophils and lymphocytes, as well as increased expression of caspase-1 p20, interleukin (IL)-1ß, IL-6, IL-8, IL-18 and tumor necrosis factor (TNF)-α. Caspase-1 inhibitors reduced both lung tissue damage and the expression of caspase-1 p20, IL-1ß, IL-6, IL-8, TNF-α and IL-18 in infected mice. These findings suggest that the caspase-1 dependent pyroptosis involved in the pathogenesis of the mouse pleuropneumonia caused by App and the inhibition of caspase-1 reduced the lung injury of this pleuropneumonia.

Efficacy of enteric-coated tilmicosin granules in pigs artificially infected with Actinobacillus pleuropneumoniae serotype 2.

BACKGROUND: Porcine infectious pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one of the most serious infectious diseases in pigs and has brought huge economic losses to the world pig industry. The aim of this trial was to evaluate the effect of enteric-coated tilmicosin granule in the treatment and control of artificial infection of App. METHODS: Sixty Duroc and Yorkshire crossbred pigs (50 of which were artificially infected) were divided into six groups: BCG (Blank control group), ICG (Infection-only control group), HDG (High-dose enteric-coated tilmicosin granules), MDG (Medium-dose enteric-coated tilmicosin granules), LDG (Low-dose enteric-coated tilmicosin granules) and TPG (Tilmicosin premix drug control group). The cure rate, mortality, clinical respiratory score, body temperature score, weight gain, lung score and so on were recorded. RESULTS: The cure rate of HDG and MDG was as high as 90%, the mortality was 10%, and the clinical signs recovered quickly. CONCLUSION: The results showed that enteric-coated tilmicosin granules had obvious therapeutic effect on artificial infection, which could reduce the damage caused by the disease and reduce the mortality.

Comparison of pharmacokinetics of tilmicosin in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae.

Aim: To compare the pharmacokinetic profiles of tilmicosin, administered orally at a single dose of 20 mg/kg bodyweight, in healthy pigs and in pigs experimentally infected with Actinobacillus pleuropneumoniae. Methods: Twelve healthy crossbred pigs, aged approximately 8 weeks, were randomly assigned to uninfected and infected groups, with six pigs per group. Pigs in the infected group were inoculated intranasally with a bacterial suspension of A. pleuropneumoniae containing approximately 108 cfu. Each pig received a single oral dose of 20 mg/kg bodyweight of tilmicosin, given 3-4 hours after inoculation in infected pigs. Blood samples were collected before drug administration and up to 48 hours after tilmicosin administration. Concentrations of tilmicosin in plasma samples were determined by HPLC. Throughout the experimental period pigs were observed for signs of inappetence and clinical abnormalities. After sampling was complete pigs were subject to euthanasia and samples collected for gross and histopathology as well as microbiology. Results: Infected pigs showed signs of bradykinesia, nasal discharge dyspnoea, and coughing 1 hours after inoculation and A. pleuropneumoniae was cultured from the lungs of all infected pigs postmortem. Comparing pharmacokinetic parameters in uninfected and infected pigs, the maximum plasma concentration of tilmicosin was higher in uninfected pigs (1.17 (SD 0.17) vs. 0.96 (SD 0.17) µg/mL), the time to reach maximum concentration was shorter (1.53 (SD 0.23) vs. 2.40 (SD 0.37) hours), and the half-life of the absorption phase and half-life of the elimination phase were both shorter (0.66 (SD 0.08) vs. 1.00 (SD 0.27) hours) and (12.93 (SD 0.96) vs. 16.53 (SD 0.55) hours), respectively. The apparent volume of distribution was smaller in uninfected than infected pigs (1.91 (SD 0.22) vs. 2.16 (SD 0.21) L/kg). The relative bioavailability of tilmicosin in infected relative to uninfected pigs was 108.6 (SD 9.71)%. Conclusions and clinical relevance: The results of this study indicate that A. pleuropneumoniae infection significantly changed certain pharmacokinetic parameters of tilmicosin in pigs. In infected pigs tilmicosin exhibited a longer drug persistence and a better extent of absorption. These results indicate that it is necessary to monitor and adjust the dose of tilmicosin administration during the presence of pleuropneumonia. It is expected that this can optimise clinical efficacy and help avoid the development of resistance.

In Vivo Effectiveness of Injectable Antibiotics on the Recovery of Acute Actinobacillus pleuropneumoniae-Infected Pigs.

The aim of this trial was to evaluate the in vivo effectiveness of injectable antibiotics of one- or two-dose administration on recovery of acute App (Actinobacillus pleuropneumoniae)-infected pigs. Ninety pigs with moderate general clinical score (GCS) of a commercial farm, suffering from acute App infection, were divided in two groups: (a) T1: one administration of gamithromycin injectable solution and (b) T2: two administrations of florfenicol injectable solution. Morbidity/mortality, clinical scores (clinical appearance score index-CAS, clinical respiratory score-CRS, clinical cough score index [CCS], general respiratory clinical score-GCRS, and general clinical score-GCS), body temperature score (BTS), and posttreatment interval were recorded. The carcass weight and lung scoring were estimated, based on slaughterhouse pleurisy evaluation system score, lung lobes score, and pneumonia area. The results of this study indicated that the tested antibiotics are efficacious for the recovery of acute App-affected pigs. Quicker improvement of BTS in sick pigs (at day 1 and 2) and quicker recovery of clinical signs, based on the improvement of clinical parameters (CAS, CCS, GCRS, GCS on day 2 and 3, and CRS on day 2), were noticed in T1 group. In conclusion, the use of tested antibiotics in acute App-affected pigs is an effective strategy for the control of an acute outbreak.

Antimicrobial resistance, biofilm formation and virulence reveal Actinobacillus pleuropneumoniae strains' pathogenicity complexity.

Porcine pleuropneumonia is an important cause of lowered productivity and economic loss in the pig industry worldwide, associated primarily with Actinobacillus pleuropneumoniae infection. Its colonization and persistence within the upper respiratory tract of affected pigs depends upon interactions between a number of genetically controlled virulence factors, such as pore-forming repeats-in-toxin exoproteins, biofilm formation, and antimicrobial resistance. This study investigated correlations between biofilm-forming capacity, antimicrobial resistance, and virulence of A. pleuropneumoniae obtained from clinical outbreaks of disease, using a Galleria mellonella alternative infection model. Results suggest that virulence is diverse amongst the 21 strains of A. pleuropneumoniae examined and biofilm formation correlated with genetic control of antimicrobial resistance.

Pharmacokinetic/pharmacodynamic assessment of cefquinome against Actinobacillus Pleuropneumoniae in a piglet tissue cage infection model.

To evaluate the relationship between the pharmacokinetic/pharmacodynamic (PK/PD) parameters and the antibacterial effect of cefquinome against Actinobacillus pleuropneumoniae, a tissue cage infection model was established in piglets. In this model, an initial count of A. pleuropneumoniae of approximately 106 CFU/mL was exposed to different concentrations of cefquinome after multiple administration at dosages of 0.2, 0.4, 0.8, 1, 2, 4 mg/kg body weight once a day for 3 days. Concentration of cefquinome and bacterial numbers of A. pleuropneumoniae in the tissue-cage fluid (TCF) were monitered. An inhibitory form of sigmoid maximum effect (Emax) model was used to estimate the relationship between the antibacterial effect and PK/PD indices of cefquinome against A. pleuropneumoniae. The minimum inhibitory concentration of cefquinome against A. pleuropneumoniae was 0.016 µg/mL in TCF. The total maximum antibacterial effect was a 3.96 log10 (CFU/mL) reduction. In addition, the cumulative percentage of time over a 24 h period that the drug concentration exceeds the MIC (%T > MIC) was the pharmacokinetic-pharmacodynamic (PK-PD) index that best correlated with the antibacterial efficacy (R2 = 0.967). The estimated %T > MIC values were 11.59, 27.49, and 59.81% for a 1/3-log10 (CFU/mL) reduction, a 2/3-log10 (CFU/mL) reduction, and a 1-log10 (CFU/mL) reduction, respectively, during the 24h administration period of cefquinome. In conclusion, cefquinome exhibits excellent antibacterial activity and time-dependent characteristics against A. pleuropneumoniae in vivo. Furthermore, these data provide meaningful guidance to optimize regimens of cefquinome to treat respiratory tract infections caused by A. pleuropneumoniae.

In silico analysis of putative drug and vaccine targets of the metabolic pathways of Actinobacillus pleuropneumoniae using a subtractive/comparative genomics approach.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium that resides in the respiratory tract of pigs and causes porcine respiratory disease complex, which leads to significant losses in the pig industry worldwide. The incidence of drug resistance in this bacterium is increasing; thus, identifying new protein/gene targets for drug and vaccine development is critical. In this study, we used an in silico approach, utilizing several databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Database of Essential Genes (DEG), DrugBank, and Swiss-Prot to identify non-homologous essential genes and prioritize these proteins for their druggability. The results showed 20 metabolic pathways that were unique and contained 273 non-homologous proteins, of which 122 were essential. Of the 122 essential proteins, there were 95 cytoplasmic proteins and 11 transmembrane proteins, which are potentially suitable for drug and vaccine targets, respectively. Among these, 25 had at least one hit in DrugBank, and three had similarity to metabolic proteins from Mycoplasma hyopneumoniae, another pathogen causing porcine respiratory disease complex; thus, they could serve as common therapeutic targets. In conclusion, we identified glyoxylate and dicarboxylate pathways as potential targets for antimicrobial therapy and tetra-acyldisaccharide 4'-kinase and 3-deoxy-D-manno-octulosonic-acid transferase as vaccine candidates against A. pleuropneumoniae.

Thymol kills bacteria, reduces biofilm formation, and protects mice against a fatal infection of Actinobacillus pleuropneumoniae strain L20.

Actinobacillus pleuropneumoniae is the causative agent of the highly contagious and deadly respiratory infection porcine pleuropneumonia, resulting in serious losses to the pig industry worldwide. Alternative to antibiotics are urgently needed due to the serious increase in antimicrobial resistance. Thymol is a monoterpene phenol and efficiently kills a variety of bacteria. This study found that thymol has strong bactericidal effects on the A. pleuropneumoniae 5b serotype strain, an epidemic strain in China. Sterilization occurred rapidly, and the minimum inhibitory concentration (MIC) is 31.25µg/mL; the A. pleuropneumoniae density was reduced 1000 times within 10min following treatment with 1 MIC. Transmission electron microscopy (TEM) analysis revealed that thymol could rapidly disrupt the cell walls and cell membranes of A. pleuropneumoniae, causing leakage of cell contents and cell death. In addition, treatment with thymol at 0.5 MIC significantly reduced the biofilm formation of A. pleuropneumoniae. Quantitative RT-PCR results indicated that thymol treatment significantly increased the expression of the virulence genes purC, tbpB1 and clpP and down-regulated ApxI, ApxII and Apa1 expression in A. pleuropneumoniae. Therapeutic analysis of a murine model showed that thymol (20mg/kg) protected mice from a lethal dose of A. pleuropneumoniae, attenuated lung pathological lesions. This study is the first to report the use of thymol to treat A. pleuropneumoniae infection, establishing a foundation for the development of new antimicrobials.

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.

Antimicrobial resistance genes in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs.

OBJECTIVE: To identify genes associated with the observed antimicrobial resistance in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs. DESIGN: Isolates with known phenotypic resistance to ß-lactams, macrolides and tetracycline were screened for the presence of antimicrobial resistance genes. PROCEDURE: A total of 68 A. pleuropneumoniae, 62 H. parasuis and 20 P. multocida isolates exhibiting phenotypic antimicrobial resistance (A. pleuropneumoniae and P. multocida) or elevated minimal inhibitory concentrations (MICs) (H. parasuis) to any of the following antimicrobial agents - ampicillin, erythromycin, penicillin, tetracycline, tilmicosin and tulathromycin - were screened for a total of 19 associated antimicrobial resistance genes (ARGs) by PCR. RESULTS: The gene bla ROB-1 was found in all ampicillin- and penicillin-resistant isolates, but none harboured the bla TEM-1 gene. The tetB gene was found in 76% (74/97) of tetracycline-resistant isolates, 49/53 A. pleuropneumoniae, 17/30 H. parasuis and 8/14 P. multocida. One A. pleuropneumoniae isolate harboured the tetH gene, but none of the 97 isolates had tetA, tetC, tetD, tetE, tetL, tetM or tetO. A total of 92 isolates were screened for the presence of macrolide resistance genes. None was found to have ermA, ermB, ermC, erm42, mphE, mefA, msrA or msrE. CONCLUSION: The current study has provided a genetic explanation for the resistance or elevated MIC of the majority of isolates of Australian porcine respiratory pathogens to ampicillin, penicillin and tetracycline. However, the macrolide resistance observed by phenotypic testing remains genetically unexplained and further studies are required.