D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei.

Melioidosis is a severe disease caused by Burkholderia pseudomallei. The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC50 values of the peptide and antibiotic and caused also disruption of biofilms of B. pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.

Characterization and functional analysis of novel circulating NK cell sub-populations.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.

Ocular involvement in melioidosis: a 23-year retrospective review.

BACKGROUND: Ocular involvement in melioidosis is rare and has devastating outcomes. Although there have been few reports on the condition, Khon Kaen, a city in northeast Thailand, has been called the "capital of melioidosis" due to the high prevalence of the condition in the region. We retrospectively reviewed all admitted cases of melioidosis with ocular involvement from the two largest hospitals in Khon Kaen. We reviewed cases from Srinagarind Hospital (a university hospital) of patients admitted between 1993 and 2016 and from Khon Kaen Hospital (a provincial hospital) of patients who presented from 2012 to 2016. RESULTS: We identified 16 cases of ocular involvement. Eight of these cases were proven from positive culture, and the remaining eight were implied from high melioidosis titer. The prevalence was estimated as being from 0.49 to 1.02%. Most patients had underlying diseases (14, 88%), of which diabetes mellitus was the most prevalent (12, 75%). Nine cases (56%) were part of disseminated septicemia. Patients suffered from blindness in 11 (73%) of the 15 cases in which visual acuity was recorded. Orbital cellulitis was the most common manifestation (7, 44%) followed by endophthalmitis (4, 25%). Interestingly, all patients with necrotizing fasciitis (100%) developed septic shock as a consequence. In most of the cases, patients underwent surgery (13, 81%) including incision and drainage, debridement, and pars plana vitrectomy. Despite appropriate management, the visual outcomes were disappointing (9, 64%). CONCLUSION: To summarize, ocular melioidosis is a highly destructive disease. Early detection and prompt surgical management may reduce morbidity and mortality from septic shock.

Characterization of a novel two-component system response regulator involved in biofilm formation and a low-iron response of Burkholderia pseudomallei.

Two-component systems (TCSs) regulate an adaptive response to environmental conditions, leading to changes in bacterial cellular processes. In this study, we identified a novel TCS response regulator gene, designated as bfmR (biofilm formation-associated regulator) that regulates biofilm formation by Burkholderia pseudomallei (Bp). An insertion mutant of the Bp bfmR gene resulted in a significant decrease in expression of fimbriae chaperone-usher assembly genes (BPSL2O28 and BPSL22 7), leading to suppression of assembly of fimbriae on the cell surface and reduced biofilm formation. The defective phenotypes of the mutant strain were restored by introducing a complementing plasmid having an intact bfmR gene. Using RT-PCR analyses, we found that bfmR gene expression was upregulated under low-iron growth conditions. In addition, the bfmR mutant strain showed retarded growth in low-iron medium and in phagocytic cells compared to the wild-type strain. These results indicate that bfmR is a novel positive regulator for controlling assembly of fimbriae and biofilm formation, and is upregulated under low-iron conditions.

Cationic liposomes extend the immunostimulatory effect of CpG oligodeoxynucleotide against Burkholderia pseudomallei infection in BALB/c mice.

Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Previously we showed that pretreatment of mice with CpG oligodeoxynucleotide (CpG ODN) 2 to 10 days prior to B. pseudomallei challenge conferred as high as 90% protection, but this window of protection was rather short. In the present study, we therefore aimed to prolong this protective window and to gain further insight into the mechanisms underlying the protection induced by CpG ODN against B. pseudomallei infection. It was found that the CpG ODN incorporated with cationic liposomes (DOTAP) but not zwitterionic liposomes (DOPC) provided complete protection against bacterial challenge. Although marked elevation of gamma interferon (IFN-γ) was found in the infected animals 2 days postinfection, it was significantly lowered by the DOTAP-plus-CpG ODN pretreatment. When appropriately activated, the phagocytic index and oxidative burst responses of neutrophils appeared not to be elevated. However, macrophages from stimulated mice showed higher levels of nitric oxide production and exhibited higher levels of antimicrobial activities, judging from lower numbers of viable intracellular bacteria. Taken together, our results demonstrate that DOTAP can enhance the protective window period of CpG ODN to at least 30 days and provide 100% protection against B. pseudomallei infection. The protective effect of DOTAP plus CpG ODN could provide an alternative approach to preventing this lethal infection, for which no vaccine is yet available.

Comparative in vivo and in vitro analyses of putative virulence factors of Burkholderia pseudomallei using lipopolysaccharide, capsule and flagellin mutants.

Burkholderia pseudomallei is a gram-negative bacillus that is the causative agent of melioidosis. We evaluated host-pathogen interaction at different levels using three separate B. pseudomallei mutants generated by insertional inactivation. One of these mutants is defective in the production of the polysaccharide side chains associated with lipopolysaccharide; one does not produce the capsular polysaccharide with the structure -3)-2-O-acetyl-6-deoxy-beta-d-manno-heptopyranose-(1-; and the third mutant does not produce flagellin. We compared the in vivo virulence in BALB/c mice, the in vitro fate of intracellular survival inside human polymorphonuclear cells (PMNs) and macrophages (Mphis) and the susceptibility to killing by 30% normal human serum, reactive nitrogen and oxygen intermediates and antimicrobial peptides with that of their wild-type counterpart. The lipopolysaccharide and capsule mutants demonstrated a marked reduction in virulence for BALB/c mice, but the flagellin mutant was only slightly less virulent than the parent strain. The results from the BALB/c mice experiments correlated with survival in Mphis. The lipopolysaccharide and capsule mutants were also more susceptible to killing by antimicrobial agents. All bacteria were equally susceptible to killing by PMNs. Altogether, the data suggest that lipopolysaccharide and capsule and, to a much lesser extent, flagella, are most likely associated with the virulence of this bacterium and highlight the importance of intracellular killing by PMNs and Mphis in disease pathogenesis.

Analyses of vaccination protocols for Leptospira interrogans serovar autumnalis in hamsters.

Leptospirosis, caused by Leptospira spp., is a zoonotic disease found worldwide. Killed whole cell leptospiral vaccines have been used as effective vaccines to elicit specific antibodies for protection. However, the involvement of cytokine responses after vaccination is not well characterized. Hamsters were immunized with killed L. interrogans serovar Autumnalis before challenge to study cytokine mRNA expression levels (interferon [IFN]-gamma, tumor necrosis factor [TNF]-alpha, interleukin [IL]-10, and IL-4). Vaccinated groups showed 92-100% survival rates, whereas control hamsters died within 6-10 days. However, live organisms were detected in vaccinated groups, and mild to moderate pathology was observed early in infection. IFN-gamma and TNF-alpha mRNA expression levels correlated with the severity of infection and lung pathology, whereas IL-4 and IL-10 expression levels were significantly higher in vaccinated groups. In summary, commonly used vaccines changed the cytokine profiles and protected hamsters from death but failed to stimulate sterile immunity and were unable to prevent the occurrence of pathology.

Differential response of cytokines induced by Leptospira interrogans, serogroup Pomona, serovar Pomona, in mouse and human cell lines.

Leptospira interrogans, the causative agent of leptospirosis, is an important zoonotic bacterium. The mechanisms and roles of cytokine induction in both humans and animals remain unclear. Therefore, the IFN-gamma, IL-6, IL-10 and IL-12 levels were measured by enzyme-linked immunosorbent assay (ELISA) in human THP-1 and mouse RAW 264.7 monocyte cell lines following stimulation with heat-killed Leptospira interrogans serogroup Pomona serovar Pomona, L. biflexa, E. coli or Salmonella group B. The production of IL-6 and IL-12 were higher and rose more rapidly in the RAW 264.7 cells with all bacteria. The IL-10 was not detected in the RAW 264.7 cells when induced by leptospires. The IFN-gamma level in human peripheral blood mononuclear cells (PBMCs) induced by leptospires was also significantly lower than with other bacteria. When IL-10 and IL-12 mRNA expressions were detected in hamster's spleen, their patterns were similar to what was observed in THP-1 in that IL-12 was only slightly increased while IL-10 expression was high. Moreover, the IFN-gamma expression could not be detected in hamsters. The more potent cytokine responses in the RAW 264.7 cells may indirectly reflect the disease outcome in mice which render them to be a good reservoir of leptospirosis. Whether these cytokines have contributed to immunoprotection during the L. interrogans infection remains to be further investigated.

Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei.

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

Immunostimulatory CpG oligodeoxynucleotide confers protection in a murine model of infection with Burkholderia pseudomallei.

Although CpG oligodeoxynucleotides (CpG ODNs) are known to enhance resistance against infection in a number of animal models, little is known about the CpG-induced protection against acute fatal sepsis such as that associated with the highly virulent bacterium Burkholderia pseudomallei. We previously demonstrated in an in vitro study that immunostimulatory CpG ODN 1826 enhances phagocytosis of B. pseudomallei and induces nitric oxide synthase and nitric oxide production by mouse macrophages. In the present study, CpG ODN 1826 given intramuscularly to BALB/c mice 2 to 10 days prior to B. pseudomallei challenge conferred better than 90% protection. CpG ODN 1826 given 2 days before the bacterial challenge rapidly enhanced the innate immunity of these animals, judging from the elevated serum levels of interleukin-12 (IL-12)p70 and gamma interferon (IFN-gamma) over the baseline values. No bacteremia was detected on day 2 in 85 to 90% of the CpG-treated animals, whereas more than 80% of the untreated animals exhibited heavy bacterial loads. Although marked elevation of IFN-gamma was found consistently in the infected animals 2 days after the bacterial challenge, it was ameliorated by the CpG ODN 1826 pretreatment (P = 0.0002). Taken together, the kinetics of bacteremia and cytokine profiles presented are compatible with the possibility that protection by CpG ODN 1826 against acute fatal septicemic melioidosis in this animal model is associated with a reduction of bacterial load and interference with the potential detrimental effect of the robust production of proinflammatory cytokines associated with B. pseudomallei multiplication.