Endophytic fungi from tropical ethnoveterinary plants and their antibacterial efficacy against Pasteurella multocida Capsular Type A strain / Hongos endófitos de plantas tropicales etnoveterinarias y su eficacia antibacteriana contra cepas capsulares de Pasteurella multocida tipo A

AbstractPasteurella multocida is an important veterinary pathogen causing infections in animals and birds. Nowadays, different reports have described the severity of infections, increasing resistance of micro-organisms to antibiotics, and the contribution of ethnoveterinary practices towards the treatment of various ailments of animals. The aim of the present study was to investigate the antibacterial efficacy of the ethanolic extracts of endophytic fungi against P. multocida Capsular Type A strains. A total of six endophytic fungi were isolated from two tropical ethnoveterinary plants: Garcinia xanthochymus H. and Polygonum chinense L. The ethanolic extracts of the endophytic fungi were subjected to in vitro antimicrobial activity by the well diffusion method. Besides, we evaluated the treatment of mice with the potent fungal extract and observed the effects in different organs under electron microscopy. Our results showed that four fungi had antimicrobial activity against the selected pathogen. The best antibacterial activity was showed by the extract of the endophytic fungi, Glomerella magna isolated from G. xanthochymus, with a minimum inhibitory concentration of 46.9 µg/mL and minimum bactericidal concentration of 750 µg/mL. Treatment of mice with the potent fungal extract caused a considerable inhibitory effect on the pathogen growth in vital organs, results that was also confirmed by histopathological studies made by scanning electron microscopy. The present findings indicated that the endophytic fungi G. magna has the potential to provide an effective treatment against infections caused by Pasteurella multocida. However, the isolation of bioactive components needs further investigation. Rev. Biol. Trop. 64 (2): 733-745. Epub 2016 June 01.

ResumenPasteurella multocida es un importante patógeno veterinario que causa infecciones en animales y aves. Hoy en día, diferentes informes han descrito la gravedad de las infecciones, aumentando la resistencia de los microorganismos a los antibióticos, y la contribución de las prácticas etnoveterinarias hacia el tratamiento de diversas enfermedades de los animales. El objetivo del presente estudio fue investigar la eficacia antibacteriana de los extractos etanólicos de hongos endófitos contra cepas de P. multocida tipo capsular A. Un total de seis hongos endófitos fueron aisladas de dos plantas etnoveterinarias tropicales: Garcinia xanthochymus H. y Polygonum chinense L. Los extractos etanólicos de los hongos endófitos fueron sometidos a la actividad antimicrobiana in vitro por el método de difusión. Además, se evaluó el tratamiento de ratones con el extracto de hongos potente y observamos los efectos en diferentes órganos bajo el microscopio electrónico. Nuestros resultados mostraron que cuatro hongos tenían actividad antimicrobiana contra el patógeno seleccionado. La mejor actividad antibacteriana la mostró el extracto de los hongos endófitos, Glomerella magna aislado de G. xanthochymus, con una concentración inhibitoria mínima de 46.9 mg/ml y la concentración bactericida mínima de 750 mg/ml. El tratamiento de ratones con el extracto de hongos potente causó un considerable efecto inhibidor sobre el crecimiento de patógenos en órganos vitales, resultados que también fueron confirmados por estudios histopatológicos realizados por microscopía electrónica de barrido. Los presentes hallazgos indican que el hongos endófitos G. magna tienen el potencial de proporcionar un tratamiento eficaz contra las infecciones causadas por Pasteurella multocida. Sin embargo, el aislamiento de componentes bioactivos necesita más investigación.

Endophytic fungi from tropical ethnoveterinary plants and their antibacterial efficacy against Pasteurella multocida Capsular Type A strain.

Pasteurella multocida is an important veterinary pathogen causing infections in animals and birds. Nowadays, different reports have described the severity of infections, increasing resistance of micro-organisms to antibiotics, and the contribution of ethnoveterinary practices towards the treatment of various ailments of animals. The aim of the present study was to investigate the antibacterial efficacy of the ethanolic extracts of endophytic fungi against P. multocida Capsular Type A strains. A total of six endophytic fungi were isolated from two tropical ethnoveterinary plants: Garcinia xanthochymus H. and Polygonum chinense L. The ethanolic extracts of the endophytic fungi were subjected to in vitro antimicrobial activity by the well diffusion method. Besides, we evaluated the treatment of mice with the potent fungal extract and observed the effects in different organs under electron microscopy. Our results showed that four fungi had antimicrobial activity against the selected pathogen. The best antibacterial activity was showed by the extract of the endophytic fungi, Glomerella magna isolated from G. xanthochymus, with a minimum inhibitory concentration of 46.9 µg/mL and minimum bactericidal concentration of 750 µg/mL. Treatment of mice with the potent fungal extract caused a considerable inhibitory effect on the pathogen growth in vital organs, results that was also confirmed by histopathological studies made by scanning electron microscopy. The present findings indicated that the endophytic fungi G. magna has the potential to provide an effective treatment against infections caused by Pasteurella multocida. However, the isolation of bioactive components needs further investigation.

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits.

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.

Interaction of Pasteurella multocida with free-living amoebae.

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.

Characterization of a 39kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies.

The role of a 39kDa protein of avian Pasteurella multocida in pathogenesis of fowl cholera was investigated using monoclonal antibodies (Mabs). Mabs were prepared by immunization of BALB/c mice with a crude capsular extract (CCE) of P. multocida strain P-1059 (serovar A:3). Totally eight hybridomas producing Mab were obtained. Immunoblot analysis of the hybridomas revealed that all the Mabs recognized a 39kDa protein of CCE. Treatment of CCE antigen with proteinase K or periodic acid indicated that the epitope recognized was proteinaceous. The Mabs reacted with a major 39kDa protein of CCE from encapsulated strains but not with any protein of non-capsulated strains indicating that a direct correlation between encapsulation and the 39kDa protein. Immunoelectron microscopy on strain P-1059 and the non-capsulated derivative P-1059B (serovar -:3) reacting with the Mabs and gold-labeled anti-mouse IgG indicated that the protein is associated with the capsule. The Mabs significantly inhibited the adherence of encapsulated P. multocida strains to chicken embryo fibroblast cells, but only slightly that of non-capsulated strains. Mice passively immunized with the Mabs were protected from lethal challenge with virulent strains P-1059 and X-73 (serovar A:1). Thus the capsular 39kDa protein was determined to be an adherence factor and a cross-protective antigen of avian P. multocida type A strains.

Capsule thickness and amounts of a 39 kDa capsular protein of avian Pasteurella multocida type A strains correlate with their pathogenicity for chickens.

Capsule thickness of avian Pasteurella multocida type A strains was determined by transmission electron microscopy after labeling with polycationic ferritin and compared with their pathogenicity for chickens. The capsule thickness of P. multocida strains Pm-18 and X-73 was 81.4 and 50.1 nm on average, respectively. These strains were highly virulent for chicken, whereas the less virulent strains Pm-1 and Pm-3 had a thin and irregular capsule, 21.0 and 29.8 nm on average, respectively. However, the thickest capsule was observed in strain P-1059, 101.2 nm on average, and the strain revealed moderate virulence. The noncapsulated variant P-1059B, which was derived from strain P-1059, revealed low virulence. The six P. multocida strains were examined with regard to protein content on the capsule of organisms. Amounts of total proteins of crude capsular extract (CCE) from capsulated strains were approximately twice those of the noncapsulated strains. The amount of an antigenic 39 kDa protein in the CCE were found to correlate with the capsule thickness, since heavily capsulated strains exhibited the greatest amount, whereas noncapsulated strains including noncapsulated and low virulent variant P-1059B possessed little 39 kDa protein. The results demonstrated that the capsule thickness and the quantity of a 39 kDa capsular protein of avian P. multocida type A strains correlated with their pathogenicity for chickens.

Pasteurella multocida- and Pasteurella haemolytica-ghosts: new vaccine candidates.

Pasteurella multocida is an important animal pathogen. Bacterial ghosts produced by the expression of phage PhiX174 lysis gene E are empty cells devoid of cytoplasmic and genomic material. Lysis of P. multocida 7A and P. haemolytica A1 carrying Pasteurella-specific lysis vectors (pSR2 and pSON2) occurred 140 min after induction of gene E expression induced by temperature upshift. The E-mediated cell lysis and killing activity was the same in both Pasteurella species and no viable cells could be detected after lysis of P. multocida and P. haemolytica. Pasteurella ghosts were used for immunization of rabbits and mice. Rabbits immunized subcutaneously with either P. multocida- or P. haemolytica-ghosts developed antibodies reacting with the immunizating strain, as well as with other Pasteurella strains. The number of proteins in whole cell protein extracts recognized by the sera constantly increased during the observation period of 51 days. In addition, dose-dependent protection against homologous challenge was observed in mice immunized with P. multocida-ghosts. Animals which received 1.15 x 10(8) ghosts and a challenge dose of up to 60 cfu (LD90), showed 100% protection. According to these results, we suggest ghosts of P. multocida and P. haemolytica as new vaccine candidates.

Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A.

We have constructed a defined acapsular mutant in Pasteurella multocida X-73 (serogroup A:1) by disrupting the hexA gene through the insertion of a tetracycline resistance cassette. The genotype of the hexA::tet(M) strain was confirmed by PCR and Southern hybridization, and the acapsular phenotype of this strain was confirmed by electron microscopy. The hexA::tet(M) strain was attenuated in both mice and chickens. Complementation of the mutant with an intact hexAB fragment restored lethality in mice but not in chickens. In contrast to the results described previously for P. multocida serogroup B (J. D. Boyce and B. Adler, Infect. Immun. 68:3463-3468, 2000), the hexA::tet(M) strain was sensitive to the bactericidal action of chicken serum, whereas the wild-type and complemented strains were both resistant. Following inoculation into chicken muscle, the bacterial count of the hexA::tet(M) strain decreased significantly, while the wild-type and complemented strains both grew rapidly over 4 h. The capsule is thus an essential virulence determinant in the pathogenesis of fowl cholera.

Ultrastructural location of the cross-protection factor on in vivo and in vitro grown Pasteurella multocida.

Pasteurella multocida from infected turkey tissues expresses a unique immunogen called cross-protection factor (CPF) that induces immunity to challenge by both homologous and heterologous serotypes. In this study, we used a monoclonal antibody (AMP MAb) to CPF and protein A-colloidal gold (PACG) to locate CPF on P. multocida. After incubation with AMP MAb and PACG, CPF was detected at the bacterial surface and cell periphery of P. multocida in infected turkey liver and P. multocida isolated from infected turkey blood. CPF was not detected on P. multocida incubated with control monoclonal antibody. Pasteurella multocida isolated from infected turkey blood and cultivated in the peptone-based medium did not express CPF consistently, and some cells contained more CPF than others. The location of CPF also varied, and CPF was detected both intracellular and extracellular on the cell surface. In the latter cells, CPF was heavily concentrated to a specific lateral site or detected sloughing from the cell surface. These results correlate with laboratory observations that CPF detected on P. multocida from infected turkey tissues, P. multocida isolated from infected turkey blood, and P. multocida cultivated in peptone-based medium is associated with outer membrane fractions.

Adaptive acquisition of novobiocin resistance in Pasteurella multocida strains of avian origin.

Naturally occurring strains of Pasteurella multocida are atypically susceptible to hydrophobic antibiotics such as novobiocin, despite their Gram-negative cell envelope ultrastructure. Four strains adaptively resistant to 1000 micrograms/ml of novobiocin were obtained by sequentially subculturing cell surface hydrophobic variants of avian origin in the presence of increasing antibiotic concentrations. Adaptive novobiocin resistance was accompanied in all cases by the concomitant acquisition of resistance to coumermycin, a hydrophobic antibiotic possessing the same mechanism of action, but not to the functionally disparate hydrophobic antibiotic rifamycin. The acquisition of resistance was not accompanied by alterations in the lipid composition of the cell envelope. Subsequent growth of adaptively resistant strains in the absence of novobiocin did not result in the restoration of susceptibility to either novobiocin or coumermycin. Acquisition of adaptive resistance in encapsulated parental strains resulted in an inability to synthesize capsular material and enhanced cell surface hydrophobicity; however, parental encapsulation and decreased cell surface hydrophobicity were restored upon removal of novobiocin. These data suggest that acquisition of adaptive resistance to novobiocin conferred in this manner is the result of a stable genetic event affecting the mechanistic target of both novobiocin and coumermycin rather than a physiological adaptation involving outer membrane impermeability.

Pasteurella multocida infection in the chicken embryo.

Pasteurella multocida infection in embryonated chicken eggs was studied by chorio-allantoic membrane inoculation. Strain differences were demonstrated in terms of lesion severity and time to death, especially during the first 24 h post-inoculation. A strain of low virulence gave a clear dose response but more virulent strains did not. Comparable results were obtained by infecting 6-week-old chickens. The main lesions in inoculated embryos appeared as severe vascular involvement of the entire embryo and feather tracts, thickening of the chorio-allantoic membrane, and enlargement and congestion of the yolk sac. The bacteria were demonstrated by transmission electron microscopy, either extracellularly or multiplying intracellularly in hepatocytes, heart tissue, and in the hyperplastic layer of the chorio-allantoic membrane, with resulting damage to the cellular organelles, and severe tissue changes.

Pasteurella multocida enters polarized epithelial cells by interacting with host F-actin.

We investigated the interaction of an avian strain of Pasteurella multocida with the cytoskeleton of MDCK cells, which formed a polarized epithelium when grown on type I collagen coated filters. Bacteria were incubated with MDCK cells for 30 min. 2, 4 and 6 hours and their location and association with the cell cytoskeleton determined by double-label immunofluorescence confocal microscopy. Cells were stained with a polyclonal antiserum to the outer-membrane proteins of P. multocida and with rhodamine phalloidin which specifically binds filamentous (F) actin. Confocal microscopy revealed that bacteria entered the cells by 30 min, and that by 6 hours there was a marked alteration in the actin cytoskeleton in which long filaments were reorganized to discrete foci of short actin filaments, within which were one or more bacteria. Electron microscopy demonstrated that by 2 hours, each bacterium was associated with many short 5-6 nm filaments. Treatment of MDCK cells with cytochalasin D for either 30 minutes or 24 hours prior to infection disrupted the actin cytoskeleton and inhibited entry of P. multocida.

Pili of Pasteurella multocida of porcine origin.

Using electron microscopy, pili with at least two distinct morphologies were observed on strains of Pasteurella multocida isolated from pigs with atrophic rhinitis. Rigid pili were found on 60-80% of all cells observed. These pili had a strong tendency to lie flat along the side of the outer cell membrane of P. multocida and as a result frequently were difficult to see. After growth in vitro, piliated P. multocida cells produced few pili (approx. 3-5 per cell). Heavily piliated cells were occasionally observed. The second type of pili were curly and also were difficult to visualize. Cells from cultures containing piliated cells failed to attach to red blood cells and to immobilized mucus.

Effect of polymyxin B nonapeptide on daptomycin permeability and cell surface properties in Pseudomonas aeruginosa, Escherichia coli, and Pasteurella multocida.

The present study was carried out to determine if sensitization of Gram-negative bacteria to the polyanionic antibiotic daptomycin by cationic molecules can be explained on the basis of decreased cell surface charge in order to better understand intrinsic resistance. Turbidimetric assessments of batch cultural growth kinetics revealed the outer membrane permeabilizer polymyxin B nonapeptide sensitized Pseudomonas aeruginosa and Escherichia coli to the hydrophobic probe novobiocin, whereas little or no sensitization was observed for two surface hydrophobicity variants of Pasteurella multocida. Polymyxin B nonapeptide and daptomycin synergistically inhibited growth of P. aeruginosa only. A hydrocarbon adherence assay revealed permeabilizing concentrations of polymyxin B nonapeptide increased cell surface hydrophobicity of P. aeruginosa and the hydrophobic P. multocida variant, while E. coli and the hydrophilic P. multocida variant remained unaffected. Measurement of cellular electrophoretic mobility showed polymyxin B nonapeptide permeabilization of P. aeruginosa to daptomycin occurred concomitantly with a significant decrease in cell surface charge, while no such sensitization occurred in organisms which failed to undergo polymyxin B nonapeptide-induced surface charge decreases. These data suggest that sensitization of Gram-negative bacteria to polyanionic lipopeptides by growth in the presence of polycationic outer membrane permeabilizers such as polymyxin B nonapeptide is dependent on diminution of overall cell surface charge and polarity, thereby allowing outer cell envelope permeation.

Virulence of capsulated and noncapsulated isolates of Pasteurella multocida and their adherence to porcine respiratory tract cells and mucus.

The virulence and the adherence to porcine respiratory tract cells and mucus of three toxigenic, capsular type D Pasteurella multocida isolates and their noncapsulated variants were evaluated in the present study. Loss of capsule by P. multocida, verified by transmission electron microscopy after polycationic ferritin labeling, was associated with a massive reduction in virulence of the organisms in mice. Specific-pathogen-free piglets inoculated intranasally with one of the capsulated isolates or its noncapsulated variant developed turbinate lesions characterized by bone resorption and by an inflammation of the mucosa associated with hyperplasia and squamous metaplasia of the epithelium. Infection with the capsulated isolate led to more severe lesions and atrophy of turbinates. The interactions of these P. multocida isolates with porcine respiratory tract cells and mucus were studied in vitro. The presence of capsule resulted in a decrease in binding of respiratory tract mucus were studied in vitro. The presence of capsule resulted in a decrease in binding of respiratory tract mucus to P. multocida isolates as determined by a dot blot assay. The presence of capsule also resulted in a significant decrease in adherence to porcine tracheal rings maintained in culture. The capsule seemed to mask outer membrane components which are involved in adherence. One of these components might be lipopolysaccharide since purified lipopolysaccharide bound respiratory tract mucus and blocked adherence of this microorganism to porcine tracheal rings. Our data indicate that capsular material does not seem to be involved in adherence of P. multocida to respiratory tract cells and mucus, but capsulated isolates are more virulent in mice and also in piglets.

[The localization of toxin in the cells of Pasteurella multocida]. / Die Lokalisation des Toxins in Zellen von Pasteurella multocida.

The application of the degradation procedure for Gram-negative bacteria according to Bewick and Lo to Pasteurella multocida indicates that the obvious localization of the toxin is in the periplasm. The stability of the outer membrane and of the substances adhering to it is essential for the release of the toxin. The production of the toxin clearly depend on the media used.

Enhanced adherence of Pasteurella multocida to porcine tracheal rings preinfected with Bordetella bronchiseptica.

Adherence of 25 isolates of Pasteurella multocida to porcine tracheal rings was evaluated. Results indicated that adherence was not related to the isolate's origin, capsular or somatic types, dermonecrotoxin production or hemagglutination activity. The effect of a preinfection with Bordetella bronchiseptica on the colonization by P. multocida was then studied. On rings infected with P. multocida alone, bacteria initially adhered to the epithelium, but within a few hours, the level of colonization decreased progressively. On rings preinfected with B. bronchiseptica, or pretreated with a cell-free B. bronchiseptica culture supernate (or filtrate), a high level of P. multocida colonization was maintained for at least 24 hours. Results indicate that B. bronchiseptica appears to facilitate upper respiratory tract colonization by P. multocida by a process which involves a low molecular weight (less than or equal to 1000) heat-stable substance, possibly the tracheal cytotoxin.

Cell surface characteristics and virulence in mice of Pasteurella multocida.

The virulence of three avian strains of Pasteurella multocida was evaluated in mice. Strains P-1059I (serotype A:3) and its uncapsulated variant P-1059B and strain 2723 (serotype A:16) were compared. Capsular material thickness after polycationic ferritin labelling of dextrose starch agar (DSA)-grown P. multocida was shown to vary with the strain and was not always related to virulence. Addition of alpha,alpha' bipyridyl (BIP) (160 microM) to the culture medium did not affect capsule production but increased virulence of strains P-1059B and 2723. None of the strains tested showed dermonecrotic activity. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), outer membrane protein (OMP) profiles indicated for strains P-1059I and P-1059B three proteins of 30, 35, and 38 KDa with the 30 KDa protein being the major one. Strain 2723 showed the same OMP profile but the 38 KDa protein was the major one. DSA + BIP-grown strains showed the same OMP profiles. Whole cell profiles were similar for all strains tested. However, addition of BIP to the culture media increased the virulence of strains P-1059B and 2723 and for all strains a 39 KDa protein was induced by the iron chelator. The results indicate that encapsulation may be important for virulence, but other surface components such as OMPs may be required as well.

Effects of antibiotics on the growth and morphology of Pasteurella multocida.

The effects of subminimal inhibitory concentrations (subMICs) of certain antibiotics, namely penicillin G, tetracycline and trimethoprim/sulphamethoxazole, on the growth and morphology of Pasteurella multocida were evaluated. SubMICs of penicillin markedly reduced the growth of P. multocida. Tetracycline and trimethoprim/sulphamethoxazole had no effect on its growth. SubMICs of penicillin greatly affected the morphology of P. multocida. At the highest concentrations tested (1/2 and 1/4 MIC) cells were acapsulate, and long filamentous cells (4-6 microns) were observed with some isolates. There was no correlation between the observed differences in the penicillin-binding proteins of the P. multocida isolates, and the extent of cell filamentation induced by penicillin G. SubMICs of tetracycline and trimethoprim/sulphamethoxazole did not seem to affect capsule production although filamentation was observed. Our results indicate that subMICs of penicillin can reduce growth of P. multocida. Furthermore, results also indicate that subMICs of antibiotics can affect the production of capsular material and the morphology of P. multocida.

Resistance of Pasteurella multocida A:3,4 to phagocytosis by turkey macrophages and heterophils.

A virulent field isolate and 2 vaccine strains of Pasteurella multocida A:3,4 were compared for resistance to phagocytosis by turkey macrophages and heterophils, using in vitro assays. The least virulent vaccine strain (M-9) was phagocytosed to a greater degree than was the field isolate or the other vaccine strain (Clemson University). All 3 bacteria differed significantly from each other in the amount of capsular material present as measured by polycationic ferritin labeling and electron microscopy. Removal of the capsule with hyaluronidase resulted in a significant increase in phagocytosis of the field isolate.