Effect of prodigiosin on the lysozyme activity in melioidosis]

Decreased lysozyme activity was observed under conditions of melioidosis intoxication in rats induced by intraperitoneal administration of an acetone-killed 3-day culture of the bacterial mass of the melioidosis causative agent. When prodigiozan was administered 48 hours by the 4th day which was indicative of prodigiozan activation of the factors of the microbial non-specific resistance.     

Phagocytability of Pseudomonas pseudomallei

Experiments were conducted on the cell culture of macrophages of animals significantly differing by the extent of resistance to melioidosis; the presence of correlation between the extent of the animal natural immunity and the intensity of dying of the microbes in the test system was demonstrated. The causative agent of melioidosis proved to be more resistant to phagocytosis with guinea pig macrophages than E. coli. Ps. aeruginosa and A. mallei. It was impossible to establish any relationship between the efficacy of phagocytosis by animal macrophages and the virulence or morphology of the colonies in the Ps. pseudomallei species.     

Neutrophil phagocytic activity of the peripheral blood in experimental keratoconjunctivitis in a sensitized macroorganism

The study of the characteristics of the phagocytic activity of peripheral blood neutrophils (the activity and intensity of phagocytosis, the index of its completeness) in the sensitized organism in experimental keratoconjunctivitis caused by Staphylococcus aureus and Escherichia coli has revealed a decrease in the phagocytic function of neutrophils. Still more pronounced suppression of the ingestive and digestive activity of leukocytes has been observed in cases of the combined action of bacterial allergens and benzylpenicillin potassium, which probably accounts for the ineffectiveness of the penicillin treatment of bacterial keratoconjunctivitis.     

Bordetella pertussis lipopolysaccharide resists the bactericidal effects of pulmonary surfactant protein A

Surfactant protein A (SP-A) plays an important role in the innate immune defense of the respiratory tract. SP-A binds to lipid A of bacterial LPS, induces aggregation, destabilizes bacterial membranes, and promotes phagocytosis by neutrophils and macrophages. In this study, SP-A interaction with wild-type and mutant LPS of Bordetella pertussis, the causative agent of whooping cough, was examined. B. pertussis LPS has a branched core structure with a nonrepeating trisaccharide, rather than a long-chain repeating O-Ag. SP-A did not bind, aggregate, nor permeabilize wild-type B. pertussis. LPS mutants lacking even one of the sugars in the terminal trisaccharide were bound and aggregated by SP-A. SP-A enhanced phagocytosis by human monocytes of LPS mutants that were able to bind SP-A, but not wild-type bacteria. SP-A enhanced phagocytosis by human neutrophils of LPS-mutant strains, but only in the absence of functional adenylate cyclase toxin, a B. pertussis toxin that has been shown to depress neutrophil activity. We conclude that the LPS of wild-type B. pertussis shields the bacteria from SP-A-mediated clearance, possibly by sterically limiting access to the lipid A region.     

Obtaining spheroplasts from the agents of glanders and melioidosis and separation of membrane structures from them]

Spheroplasts were obtained from the causative agents of glanders and melioidosis under the effect of lysozyme and antibiotics. In the capacity of an inducing agent lysozyme was effective in high concentration only (0.4%); preliminary washing and incubation in sucrose were necessary to obtain glanders spheroplasts. Of the antibiotics studied penicillin was more useful for obtaining melioidosis spheroplasts and ampicillin--for glanders spheroplasts. Membrane preparations were derived from the spheroplasts of glanders and melioidosis causative agents.     

Evaluation of recombinant antigens for diagnosis of melioidosis

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of the disease are diverse, ranging from chronic localized infection to acute septicaemia, with death occurring within 24-48 h after the onset of symptoms. Definitive diagnosis of melioidosis involves bacterial culture and identification, with results obtained within 3-4 days. This delayed diagnosis is a major contributing factor to high mortality rates. Rapid diagnosis is vital for successful management of the disease. This study describes the purification and evaluation of three recombinant antigenic proteins, BPSL0972, BipD and OmpA from B. pseudomallei 08, for their potential in the serodiagnosis of melioidosis using an indirect enzyme-linked immunosorbent assay (ELISA) method. The recombinant proteins were evaluated using 74 serum samples from culture-confirmed melioidosis patients from Malaysia, Thailand and Australia. In addition, 62 nonmelioidosis controls consisting of serum samples from clinically suspected melioidosis patients (n=20) and from healthy blood donors from an endemic region (n=18) and a nonendemic region (n=24) were included. The indirect ELISAs using BipD and BPSL0972 as antigens demonstrated poor to moderate sensitivities (42% and 51%, respectively) but good specificity (both 100%). In contrast, the indirect ELISA using OmpA as an antigen achieved 95% sensitivity and 98% specificity. These results highlight the potential for OmpA to be used in the serodiagnosis of melioidosis in an endemic area.     

Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei

Burkholderia pseudomallei is a potential bioterror agent and the causative agent of melioidosis, a severe disease that is endemic in areas of Southeast Asia and Northern Australia. Infection is often associated with bacterial dissemination to distant sites, and there are many possible disease manifestations, with melioidosis septic shock being the most severe. Eradication of the organism following infection is difficult, with a slow fever-clearance time, the need for prolonged antibiotic therapy and a high rate of relapse if therapy is not completed. Mortality from melioidosis septic shock remains high despite appropriate antimicrobial therapy. Prevention of disease and a reduction in mortality and the rate of relapse are priority areas for future research efforts. Studying how the disease is acquired and the host-pathogen interactions involved will underpin these efforts; this review presents an overview of current knowledge in these areas, highlighting key topics for evaluation.     

Phagocytosis of Burkholderia mallei as a criterion for immunogenicity evaluation of its capsular antigens

Test-system using index of phagocytosis of noncapsulated mutant loaded by one of the several capsular antigenic complexes was developed and used for screening for both immunogenic and protective capsular antigens of B. mallei. Direct correlation between index of phagocytosis, level of delayed-type hypersensivity, and protective effect of capsular antigens has been shown on the model of experimental melioidosis in susceptible white mice, guinea pigs and white rats. Obtained results let to use the developed test-system for initial selection of B. mallei protective capsular antigens and their further study as potential components of preparations for specific prophylaxis of glanders and melioidosis.     

Elevated anti-parasitic activity in peripheral blood monocytes and neutrophils of cattle infected with Babesia bovis

The innate immune response to bovine Babesia bovis infection in vivo has not previously been established. We used assays measuring phagocytosis and oxidative burst to investigate the immune response because they are indicative of the innate antimicrobial capacity of monocytes and neutrophils. Monocyte and neutrophil phagocytosis is thought to be non-specific in nature and so the phagocytosis of either opsonised Zymosan or Escherichia coli was used to indicate the non-specific phagocytic capacity of monocytes and neutrophils ex vivo. The kinetics of both phagocytic and oxidative burst activity in monocytes and neutrophils were followed twice weekly from pre-inoculation (day 0) through to 31 days after inoculation. Peripheral blood monocytes were found to display a pronounced oxidative burst, but a suppressed capacity to phagocytose during a primary infection. On the other hand, neutrophils exhibited an increased phagocytic capacity and reduced oxidative activity during a primary infection. These findings identified considerable antimicrobial activity evident in peripheral blood monocytes and neutrophils from cattle exposed to B. bovis as a primary exposure. This elevated antimicrobial activity was coincident with the time that parasite numbers peaked in the circulation and occurred prior to parasite clearance. These results suggest that peripheral blood monocytes and neutrophils are active mediators in the innate immune response to a primary B. bovis.     

G-CSF immunotherapy for treatment of acute disseminated murine melioidosis

Burkholderia pseudomallei, the causative agent of melioidosis, is an important pathogen in tropical regions of Australia and Southeast Asia. Antibiotic therapy can be ineffective in patients with acute septicaemic melioidosis. It has been proposed that adjunctive immunotherapy using granulocyte colony stimulating factor (G-CSF) combined with antibiotics may provide an alternative approach to antibiotics alone. We have developed a murine model for melioidosis that allows novel treatment approaches to be investigated. This study looked at the potential for murine G-CSF therapy both alone and as an adjunct in the treatment of acute disseminated B. pseudomallei infection in BALB/c mice. A number of therapeutic variables involving ceftazidime and recombinant murine G-CSF were studied. Surviving mice were sacrificed and splenic bacterial loads were determined. Combining recombinant murine G-CSF with ceftazidime offered no advantage over ceftazidime alone. Pre-treatment with recombinant murine G-CSF did not demonstrate a significant benefit. This would suggest that adjunct immunotherapy using G-CSF is of limited benefit.     

The effect of leukinferon on the activity of phagocytosing cells in human salmonellosis]

The functional activity of phagocytic cells in 52 salmonellosis patients was studied with regard to the following characteristics: percent share of phagocytosis, phagocytic index, nitro blue tetrazolium test results, digestive activity. In patients with the unfavorable course of salmonellosis (the formation of carrier state) disturbances in the bactericidal activity of neutrophils and monocytes were established. For 32 patients leukinferon was included in the complex of etiotropic and pathogenetic treatment. The preparation was introduced in 3 intramuscular injections of 10,000 I. U. at intervals of 48 hours (the course of treatment); 10 days after the last injection this course was repeated. The use of leukinferon restored the normal functioning of phagocytes and the number of T-lymphocytes.     

Formation of HCN by human phagocytosing neutrophils--1. Chlorination of Staphylococcus epidermidis as a source of HCN

Phagocytosis of Staphylococcus epidermidis by human neutrophils is accompanied by HCN liberation. The amount of HCN evolved is significantly higher when the bacteria are damaged by penicillin. One of the substrates yielding HCN during phagocytosis are N-dichloroglycyl residues of bacterial peptidoglycan formed by the chlorinating myeloperoxidase-H2O2-Cl- system of neutrophils. HCN deriving from the bacterial structures constitutes 6-12% of total HCN liberated during phagocytosis, which indicates that there are other substrates for HCN production. Chlorination process within phagocytosing neutrophils seems to be essential for formation of both, total HCN and that deriving from bacterial structures.     

Capsule Influences the Deposition of Critical Complement C3 Levels Required for the Killing of Burkholderia pseudomallei via NADPH-Oxidase Induction by Human Neutrophils

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD₅₀ via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.     

Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice

Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1). They were still 50-80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1). The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1). Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.     

Delayed phagocytosis and bacterial killing in Chediak-Higashi syndrome neutrophils detected by a fluorochrome assay. Ultrastructural aspects.

The few studies already published about phagocyte functions in Chediak-Higashi syndrome (CHS) has stated that neutrophils present slow rate of bacterial killing but normally ingest microorganisms. In the present study, both phagocytosis and killing of Staphylococcus aureus were verified to be delayed in neutrophils from two patients with CHS when these functions were simultaneously evaluated by a fluorochrome phagocytosis assay. Electron microscopic examination showed morphologic differences among neutrophils from CHS patients and normal neutrophils regarding the cytoplasmic structures and the aspects of the phagolysosomes. It was noteworthy the presence of giant phagolysosomes enclosing bacteria in active proliferation commonly observed in CHS neutrophils after 45 min of phagocytosis, which corresponded with the impaired bactericidal activity of these leukocytes. The present results suggest that phagocytosis may also be defective in CHS, and point out to the sensitivity of the fluorochrome phagocytosis assay and its application in clinical laboratories.     

Characterization of the capsular polysaccharide of Burkholderia (Pseudomonas) pseudomallei 304b.

Burkholderia (Pseudomonas) pseudomallei is the causative agent of melioidosis, a bacterial infection of considerable morbidity in areas of endemicity of Southeast Asia and northern Australia. Clinical isolates of B. pseudomallei have been demonstrated to produce a lipopolysaccharide (LPS) containing two separate and chemically distinct antigenic O polysaccharides against which infected patients produced antibodies. A putative capsular polysaccharide (CPS) has also been reported and is thought to be antigenically conserved based on results of serological studies with clinical B. pseudomallei isolates. In the present study, the CPS isolated from B. pseudomallei 304b from northeastern Thailand was found to have an [alpha]D of +99 degrees (water), was composed of D-galactose (D-Gal), 3-deoxy-D-manno-2-octulosonic acid (KDO), and O-acetyl 3:1:1), and was a linear unbranched polymer of repeating tetrasaccharide units having the following structure: -3)-2-O-Ac-beta-D-Galp-(1-4)-alpha-D-Galp-(1-3)-beta-D -Galp-(1-5)-beta-D-KDOp-(2-. Sera from 13 of 15 patients with different clinical manifestations of melioidosis but not normal controls recognize the CPS, which suggests that it is immunogenic and raises the possibility that it may have a role as a vaccine candidate and/or diagnostic agent.     

Pseudomonas pseudomallei and melioidosis, with special reference to the status in Thailand

Melioidosis is a long-known disease since 1912, but only quite recently we have obtained the knowledges about its actual clinical and epidemiological features. The disease is so unique in having a wide spectrum of disease course and clinical manifestation. The causative agent, P. pseudomallei, is free-living bacterium in the natural environments (soil and surface water) of tropical and subtropical areas. Just like legionnaires' disease, melioidosis is a good example of infectious disease in which pneumonia is produced by inhalation of contaminated soil dusts or water droplets. The infection becomes dormant for years, but with a chance of recrudescence under a variety of insults to the host resistance. The disease, may it be acute or chronic, will be symptomatically confused with malaria, typhoid fever, leptospirosis, septicemia caused by other gram-negative bacteria, tuberculosis and mycotic infections. Isolation of the causative agent from clinical specimens is the only reliable method for diagnosis. Because of the increasing clinical awareness and the development of diagnostic methods, the reported cases of melioidosis have numbered almost one thousand in Thailand during the past 20 years. This country has now the most ample clinical experiences on melioidosis. We have reviewed the history of melioidosis research from bacteriological, immunological, clinical and epidemiological viewpoints, especially including the recent reports in Thailand.     

Effect of Chemical Factors on Natural Biocontrol of the Melioidosis Agent by AMP1-Like Bacteriophages in Agricultural Ecosystems

Microbiology - The causative agent of melioidosis, Burkholderia pseudomallei, as well as closely related nonpathogenic bacteria of the B. pseudomallei/thailandensis complex of species, are found in...     

Concurrent measurement of antigen- and antibody-dependent oxidative burst and phagocytosis in monocytes and neutrophils

The current study aims to review flow cytometric (FCM) parameters for the quantification of phagocytosis. A limitation of existing methods is their difficulty with accurate quantification of the phagocytic index, i.e., number of beads per phagocyte, in individual cell lines in mixed cell suspensions. We have quantified phagocytosis and the oxidative burst simultaneously using fluorescent beads coated with meningococcal outer membrane vesicles (OMV beads) by the conversion of dihydrorhodamine 123 (DHR-123) to rhodamine 123 (R-123). Both these processes depend on specific serum opsonins. After the incubation, staining with a fluorescent anti-CD14 monoclonal antibody succeeded in discriminating phagocytosing monocytes from neutrophils. The spectral overlaps between OMV beads, R-123, and anti-CD14 could be completely compensated. Percentage of phagocytosis and the phagocytic index were similar in monocytes and neutrophils, but the oxidative burst behaved differently. Two monocyte subpopulations were observed. Both subpopulations spontaneously converted some DHR-123 into R-123, whereas the reaction was triggered by phagocytosis in neutrophils. The total oxidative response increased with increasing phagocytic index in both cell types, but the oxidative burst in monocytes was about twice that of neutrophils. The oxidative ratio (mean R-123 fluorescence value divided by the phagocytic index) declined with time in monocytes, but increased in neutrophils. Our results demonstrate the need for careful attention to technical details. This single-laser, three-color FCM method facilitates the comparative research of phagocytosis and the oxidative burst in monocytes and neutrophils and provides a basis for a number of applications in hematology, infectious medicine, and immunology.     

Evaluating the role of Burkholderia pseudomallei K96243 toxins BPSS0390, BPSS0395, and BPSS1584 in persistent infection

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with a mortality rate of up to 40% even with treatment. Despite the ability of certain antibiotics to control initial infection, relapse occurs in treated patients. The inability of antibiotics to clear this bacterial infection is in part due to persistence, an evasion mechanism against antibiotics and the effect of host defenses. Evaluation of antibiotic efficacy against B. pseudomallei revealed that up to 48% of in vitro grown populations can survive in a persister state. Toxin–antitoxin (TA) systems have been previously implicated in modulating bacterial persistence. We generated three isogenic TA mutants and found that loss of each toxin gene did not alter antibiotic persistence or macrophage survival. In response to macrophage‐induced persistence, all three toxin mutants demonstrated increased intracellular susceptibility to levofloxacin which in part was due to the inability of the mutants to induce persistence after nitric oxide or nutrient starvation. In an inhalational model of murine melioidosis, both ΔBPSS0395 and ΔBPSS1584 strains were attenuated, and treatment with levofloxacin led to significant reduction in lung colonisation and reduced splenic colonisation by ΔBPSS0395. Based on our findings, these toxins deserve additional evaluation as putative therapeutic targets.