RESUMO
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.
Assuntos
Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/efeitos dos fármacos , Actinobacillus pleuropneumoniae/fisiologia , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana , Doenças dos Suínos/microbiologia , Infecções por Actinobacillus/tratamento farmacológico , Animais , Suínos , Doenças dos Suínos/tratamento farmacológico , VirulênciaRESUMO
Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 10(4) to 10(7) c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.
Assuntos
Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/fisiologia , Modelos Animais de Doenças , Mariposas/microbiologia , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Humanos , Larva/microbiologia , VirulênciaRESUMO
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.
Assuntos
Actinobacillus pleuropneumoniae/crescimento & desenvolvimento , Actinobacillus pleuropneumoniae/patogenicidade , Apoproteínas/fisiologia , Lactoferrina/fisiologia , Infecções por Actinobacillus/etiologia , Infecções por Actinobacillus/veterinária , Actinobacillus pleuropneumoniae/fisiologia , Animais , Antibacterianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/fisiologia , Apoproteínas/administração & dosagem , Apoproteínas/imunologia , Aderência Bacteriana , Toxinas Bacterianas/biossíntese , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Bovinos , Sinergismo Farmacológico , Células HeLa , Humanos , Ferro/metabolismo , Lactoferrina/administração & dosagem , Lactoferrina/imunologia , Oxitetraciclina/administração & dosagem , Pleuropneumonia/etiologia , Pleuropneumonia/veterinária , Suínos , Doenças dos Suínos/etiologia , VirulênciaRESUMO
Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia and is normally transmitted by aerosols and direct contact between animals. A. pleuropneumoniae has traditionally been considered an obligate pathogen of pigs and its presence in the environment has yet to be investigated. Here, the presence of A. pleuropneumoniae was detected in drinking water of pig farms in Mexico using a PCR specific for the RTX toxin gene, apxIV. The presence of A. pleuropneumoniae in farm drinking water was confirmed by indirect immunofluorescence using an A. pleuropneumoniae-specific polyclonal antibody and by fluorescent in situ hybridization. Viable bacteria from the farm drinking water were detected using the Live/Dead BacLight stain. Additionally, viable A. pleuropneumoniae was selected and isolated using the cAMP test and the identity of the isolated bacteria were confirmed by Gram staining, a specific polyclonal antibody and an A. pleuropneumoniae-specific PCR. Furthermore, biofilms were observed by scanning electron microscopy in A. pleuropneumoniae-positive samples. In conclusion, our data suggest that viable A. pleuropneumoniae is present in the drinking water of swine farms and may use biofilm as a strategy to survive in the environment.
Assuntos
Actinobacillus pleuropneumoniae/isolamento & purificação , Criação de Animais Domésticos , Água Potável/microbiologia , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/fisiologia , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Hibridização in Situ Fluorescente , México , Viabilidade Microbiana , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase , SuínosRESUMO
Actinobacillus pleuropneumoniae serotype 1 adhered to immobilized swine-lung collagen. Bacteria bound to collagen type I, III, IV and V. At 5 min incubation, 30 % of bacteria adhered to collagen, reaching saturation in around 90 min. Treatment of bacteria with divalent-metal chelators diminished their attachment to collagen, and Ca(2+) but not Mg(2+) increased it, suggesting Ca(2+) dependence for adherence. Proteolytic enzymes drastically reduced bacterial adherence to collagen, showing that binding involved bacterial surface proteins. Porcine fibrinogen, haemoglobin and gelatin partially reduced collagen adhesion. A 60 kDa outer-membrane protein of A. pleuropneumoniae recognized the swine collagens by overlay. This membrane protein was apparently involved in adhesion to collagen and fibrinogen, but not to fibronectin and laminin. Antibodies against the 60 kDa protein inhibited the adhesion to collagen by 70 %, whereas pig convalescent-phase antibodies inhibited it by only 40 %. Serotypes 1 and 7 were the most adherent to pig collagen (taken as 100 %); serotypes 6 and 11 were the lowest (approximately 50 %), and neither showed the 60 kDa adhesin to biotinylated collagens. By negative staining, cells were observed initially to associate with collagen fibres in a polar manner, and the adhesin was detected on the bacterial surface. The results suggest that swine-lung collagen is an important target for A. pleuropneumoniae colonization and spreading, and that the attachment to this protein could play a relevant role in pathogenesis.
Assuntos
Actinobacillus pleuropneumoniae/fisiologia , Aderência Bacteriana , Colágeno Tipo III/metabolismo , Pulmão/microbiologia , Suínos/microbiologia , Actinobacillus pleuropneumoniae/crescimento & desenvolvimento , Adesinas Bacterianas/isolamento & purificação , Adesinas Bacterianas/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Pulmão/químicaRESUMO
Actinobacillus pleuropneumoniae has been considered nonmotile and nonflagellate. In this work, it is demonstrated that A. pleuropneumoniae produces flagella composed of a 65-kDa protein with an N-terminal amino acid sequence that shows 100% identity with those of Escherichia coli, Salmonella, and Shigella flagellins. The DNA sequence obtained through PCR of the fliC gene in A. pleuropneumoniae showed considerable identity (93%) in its 5' and 3' ends with the DNA sequences of corresponding genes in E. coli, Salmonella enterica, and Shigella spp. The motility of A. pleuropneumoniae was observed in tryptic soy or brain heart infusion soft agar media, and it is influenced by temperature. Flagella and motility may be involved in the survival and pathogenesis of A. pleuropneumoniae in pigs.