Functional Activities of O-Polysaccharide and Hemolysin Coregulated Protein 1 Specific Antibodies Isolated from Melioidosis Patients.

Melioidosis is a fatal tropical disease caused by the environmental Gram-negative bacterium, Burkholderia pseudomallei. This bacterium is intrinsically resistant to several antibiotics and treatment of melioidosis requires prolonged antibiotic administration. To date, there are no vaccines available for melioidosis. Previous studies have shown that humoral immunity is critical for surviving melioidosis and that O-polysaccharide (OPS) and hemolysin coregulated protein 1 (Hcp1) are important protective antigens in animal models of melioidosis. Our previous studies revealed that melioidosis patients had high levels of OPS- and Hcp1-specific antibodies and that IgG against OPS (IgG-OPS) and Hcp1 (IgG-Hcp1) were associated with patient survival. In this study, we characterized the potential function(s) of IgG-OPS and IgG-Hcp1 from melioidosis patients. IgG-OPS and IgG-Hcp1 were purified from pooled serum obtained from melioidosis patients using immuno-affinity chromatography. Antibody-dependent cellular phagocytosis assays were performed with pooled serum from melioidosis patients and compared with serum obtained from healthy controls. Serum from melioidosis patients significantly enhanced B. pseudomallei uptake into the human monocytic cell line THP-1 compared with pooled serum from healthy donors. Enhanced opsonization was observed with IgG-OPS and IgG-Hcp1 in a dose-dependent manner. Antibody-dependent complement deposition assays were performed with IgG-OPS and IgG-Hcp1 using flow cytometry and showed that there was enhanced C3b deposition on the surface of B. pseudomallei treated with IgG-OPS but to a lesser degree with IgG-Hcp1. This study provides insight into the function of IgG-OPS and IgG-Hcp1 in human melioidosis and supports that OPS and Hcp1 are potential vaccine antigens for immunization against melioidosis.

Effect of temperature on Burkholderia pseudomallei growth, proteomic changes, motility and resistance to stress environments.

Burkholderia pseudomallei is a flagellated, gram-negative environmental bacterium that causes melioidosis, a severe infectious disease of humans and animals in tropical areas. We hypothesised that B. pseudomallei may undergo phenotypic adaptation in response to an increase in growth temperature. We analysed the growth curves of B. pseudomallei strain 153 cultured in Luria-Bertani broth at five different temperatures (25 °C-42 °C) and compared the proteomes of bacteria cultured at 37 °C and 42 °C. B. pseudomallei exhibited the highest growth rate at 37 °C with modest reductions at 30 °C, 40 °C and 42 °C but a more marked delay at 25 °C. Proteome analysis revealed 34 differentially expressed protein spots between bacterial cultures at 42 °C versus 37 °C. These were identified as chaperones (7 spots), metabolic enzymes (12 spots), antioxidants (10 spots), motility proteins (2 spots), structural proteins (2 spots) and hypothetical proteins (1 spot). Of the 22 down-regulated proteins at 42 °C, redundancy in motility and antioxidant proteins was observed. qRT-PCR confirmed decreased expression of fliC and katE. Experiments on three B. pseudomallei strains demonstrated that these had the highest motility, greatest resistance to H2O2 and greatest tolerance to salt stress at 37 °C. Our data suggest that temperature affects B. pseudomallei motility and resistance to stress.

Comprehensive analysis of clinical Burkholderia pseudomallei isolates demonstrates conservation of unique lipid A structure and TLR4-dependent innate immune activation.

Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a major community-acquired infection in tropical regions. Melioidosis presents with a range of clinical symptoms, is often characterized by a robust inflammatory response, may relapse after treatment, and results in high mortality rates. Lipopolysaccharide (LPS) of B. pseudomallei is a potent immunostimulatory molecule comprised of lipid A, core, and O-polysaccharide (OPS) components. Four B. pseudomallei LPS types have been described based on SDS-PAGE patterns that represent the difference of OPS-type A, type B, type B2 and rough LPS. The majority of B. pseudomallei isolates are type A. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) followed by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) and gas chromatography to characterize the lipid A of B. pseudomallei within LPS type A isolates. We determined that B. pseudomallei lipid A is represented by penta- and tetra-acylated species modified with 4-amino-4-deoxy-arabinose (Ara4N). The MALDI-TOF profiles from 171 clinical B. pseudomallei isolates, including 68 paired primary and relapse isolates and 35 within-host isolates were similar. We did not observe lipid A structural changes when the bacteria were cultured in different growth conditions. Dose-dependent NF-κB activation in HEK cells expressing TLR4 was observed using multiple heat-killed B. pseudomallei isolates and corresponding purified LPS. We demonstrated that TLR4-dependent NF-κB activation induced by heat-killed bacteria or LPS prepared from OPS deficient mutant was significantly greater than those induced by wild type B. pseudomallei. These findings suggest that the structure of B. pseudomallei lipid A is highly conserved in a wide variety of clinical and environmental circumstances but that the presence of OPS may modulate LPS-driven innate immune responses in melioidosis.

Effective Aeromonas specific monoclonal antibody for immunodiagnosis

Objective:To identify the monoclonal antibody specific to Aeromonas spp.,a Gram negative bacteria causing gastroenteritis and wound infection.Methods:The monoclone,namely 88F2-3F4,was produced from hybridoma technology.The specificity of antibody secreted from 88F2-3F4 was tested against other Gram negative bacteria frequently found in gastrointestinal tract.Then the antibody was used for searching Aeromonas antigens in artificial seeded rectal swab cultures by dot-blot enzyme linked immunosorbent assay.Results:88F2-3F4 produced an antibody that recognized an antigen with a molecular mass of 8.5 kDa in all 123 isolates of the seven Aeromonas species tested,but recognized no epitope of any other Gram-negative bacterium typically found in the gastrointestinal tract.A dot-blot enzyme linked immunosorbent assay based on this antibody showed 86.49％ sensitivity and 92.13％ specificity.Conclusions:88F2-3F4 monoclonal antibody could react with all Aeromonas isolates,but not other Gram negative bacteria,therefore it should be a useful tool for the detection of Aeromonas antigen in clinical and environmental samples.

Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand.

BACKGROUND: Tackling multidrug-resistant Escherichia coli requires evidence from One Health studies that capture numerous potential reservoirs in circumscribed geographic areas. METHODS: We conducted a survey of extended ß-lactamase (ESBL)-producing E. coli isolated from patients, canals and livestock wastewater in eastern Thailand between 2014 and 2015, and analyzed isolates using whole genome sequencing. RESULTS: The bacterial collection of 149 isolates consisted of 84 isolates from a single hospital and 65 from the hospital sewer, canals and farm wastewater within a 20 km radius. E. coli ST131 predominated the clinical collection (28.6%), but was uncommon in the environment. Genome-based comparison of E. coli from infected patients and their immediate environment indicated low genetic similarity overall between the two, although three clinical-environmental isolate pairs differed by ≤ 5 single nucleotide polymorphisms. Thai E. coli isolates were dispersed throughout a phylogenetic tree containing a global E. coli collection. All Thai ESBL-positive E. coli isolates were multidrug resistant, including high rates of resistance to tobramycin (77.2%), gentamicin (77.2%), ciprofloxacin (67.8%) and trimethoprim (68.5%). ESBL was encoded by six different CTX-M elements and SHV-12. Three isolates from clinical samples (n = 2) or a hospital sewer (n = 1) were resistant to the carbapenem drugs (encoded by NDM-1, NDM-5 or GES-5), and three isolates (clinical (n = 1) and canal water (n = 2)) were resistant to colistin (encoded by mcr-1); no isolates were resistant to both carbapenems and colistin. CONCLUSIONS: Tackling ESBL-producing E. coli in this setting will be challenging based on widespread distribution, but the low prevalence of resistance to carbapenems and colistin suggests that efforts are now required to prevent these from becoming ubiquitous.

Susceptibility of Clinical Isolates of Burkholderia pseudomallei to a Lipid A Biosynthesis Inhibitor.

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infection associated with high mortality and relapse. Current antimicrobial therapy using ceftazidime (CAZ) is often ineffective. Inhibitors of LpxC, the enzyme responsible for lipid A biosynthesis, have potential antimicrobial activity against several Gram-negative bacteria in vivo, but their activity against B. pseudomallei is unclear. Herein, we investigated the susceptibility of B. pseudomallei clinical isolates to LpxC-4, an LpxC inhibitor, and LpxC-4 in combination with CAZ. Time-kill assays for bactericidal activity were conducted for B. pseudomallei K96243, revealing growth inhibition and bactericidal effect at LpxC-4 concentrations of 2 µg/mL and 4 µg/mL, respectively. No significant synergistic effect was observed with the combination of LpxC-4 and CAZ. LpxC-4 susceptibility was tested on three groups of clinical isolates:1) CAZ- and trimethoprim-sulfamethoxazole (SXT)-susceptible (N = 71), 2) CAZ-resistant (N = 14), and 3) SXT-resistant (N = 23) isolates, by broth microdilution. The minimum concentration of LpxC-4 required to inhibit the growth of 90% of organisms was 2 µg/mL for all isolates. The median minimum inhibitory concentration of both the CAZ/SXT-susceptible and CAZ-resistant groups was 1 µg/mL (interquartile range [IQR] = 1-2 µg/mL), compared with 2 µg/mL (IQR = 2-4 µg/mL) for the SXT-resistant group. Cell morphology was observed after drug exposure by immunofluorescent staining, and a change from rod-shaped to cell wall-defective spherical cells was observed in surviving bacteria. LpxC-4 is a potent bactericidal agent against B. pseudomallei and warrants further testing as a new antibiotic to treat melioidosis.

Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a gram-negative bacterial species capable of occupying a broad range of environmental and clinical habitats. Known as an opportunistic pathogen, it has recently become a major causative agent of clinical infections worldwide. Despite growing knowledge about the highly diverse population of K. pneumoniae, the evolution and clinical significance of environmental K. pneumoniae, as well as the relationship between clinical and environmental K. pneumoniae, are poorly defined. METHODS: We isolated and sequenced K. pneumoniae from in-patients in a single hospital in Thailand, as well as hospital sewage, and surrounding canals and farms within a 20-km radius. RESULTS: Phylogenetic analysis of 77 K. pneumoniae (48 clinical and 29 non-clinical isolates) demonstrated that the two groups were intermixed throughout the tree and in some cases resided in the same clade, suggesting recent divergence from a common ancestor. Phylogenetic comparison of the 77 Thai genomes with 286 K. pneumoniae from a global collection showed that Thai isolates were closely related to the clinical sub-population of the global collection, indicating that Thai clinical isolates belonged to globally circulating lineages. Dating of four Thai K. pneumoniae clades indicated that they emerged between 50 and 150 years ago. Despite their phylogenetic relatedness, virulence factors and ß-lactamase resistance genes were more numerous in clinical than in environmental isolates. Our results indicate that clinical and environmental K. pneumoniae are closely related, but that hospitals may select for isolates with a more resistant and virulent genotype. CONCLUSIONS: These findings highlight the clinical relevance of environmental K. pneumoniae isolates.

Validation of a monoclonal antibody-based immunofluorescent assay to detect Burkholderia pseudomallei in blood cultures.

BACKGROUND: Identification of Burkholderia pseudomallei, the cause of melioidosis, using routine methods takes several days. Use of a monoclonal antibody-based immunofluorescent assay (IFA) on positive blood cultures may speed diagnosis. METHODS: We tested the diagnostic accuracy of the IFA on 545 blood cultures positive for Gram-negative organisms at Udon Thani Hospital, Thailand, between June 2015 and August 2016. RESULTS: Sensitivity of the IFA was 100% and specificity was 99.6%. The median decrease in time to pathogen identification between the IFA result and routine methods was 28 h (IQR 25-51), p<0.0001. CONCLUSIONS: The IFA accurately expedites the diagnosis of melioidosis.

Plasma resistin, insulin concentration in non-diabetic and diabetic, overweight/obese Thai.

This study investigated levels of fasting plasma glucose (FBS), homeostasis model of the assessment of the insulin resistance (HOMA), lipid profile, insulin, and resistin hormones in 202 individuals, divided into four groups. Two groups had type II diabetes mellitus (DM): one group had been overnourished (DM/OB) (body mass index: BMI equal or above 25) and the other had not (DM/nOB). Two additional groups not suffering from diabetes were either overnourished (nDM/OB) or of normal nutritional status (nDM/nOB). Only the DM/OB group had insulin levels elevated above the other three groups. Resistin levels had been lowest in the nDM/nOB group. When participants of the two nOB groups were pooled into one group and the subjects of the two OB groups were combined into another group, the median plasma resistin levels of the OB groups were significantly higher compared with the nOB groups. Likewise the DM groups had higher resistin levels than the nDM groups. A significant correlation of plasma resistin with BMI, waist circumference, waist-to-hip ratio, FBS, and HOMA score had been observed. The result suggests that plasma resistin has a role in linking central obesity and obesity-related insulin resistance to type II diabetes mellitus.

Relationship between soluble leptin receptor, leptin, lipid profiles and anthropometric parameters in overweight and obese Thai subjects.

Median, range and 95% confidence interval (CI) for median of age, anthropometric variables, soluble leptin receptor, serum leptin and lipid profile levels of 48 overweight (Body mass index (BMI) = 25.00-29.99 kg/m2) and obese (BMI > or = 30. 00 kg/m2) Thai males and 166 overweight and obese Thai females, compared with 26 males and 81 females in a control group (BMI = 18.50-24.99 kg/m2), were determined The study subjects were persons who turned up regularly for physical check-ups at the Out-patient Department, General Practice Section, Ratchawithi Hospital, Bangkok, aged between 18-60 years. Serum leptin, triglyceride and low density lipoprotein cholesterol/high density lipoprotein cholesterol ratios (LDL-C/ HDL-C ratio) were significantly higher in the overweight and obese males and females. Soluble leptin receptor and HDL-C were significantly lower in the overweight and obese males and females. Cholesterol and LDL-C were significantly higher in the overweight and obese females, but there was no significant difference in the overweight and obese males when compared with the control males. Low soluble leptin receptor levels were found in 38.1% (8/21) of the overweight and obese males, while 31.5% (29/92) were found in the overweight and obese females. Elevated leptin levels were found in 66.7% (32/48) and 89.8% (149/166) of the overweight and obese males and females, respectively. Both low soluble leptin receptor levels and elevated leptin levels were found in 9.5% (2/21) and 29.4% (27/92) of the overweight and obese males and females, respectively. A significant positive correlation was found between soluble leptin receptor and cholesterol, and between weight, BMI, waist, hip and HDL-C, with leptin. Serum soluble leptin receptor levels were significantly negatively correlated with leptin and BMI. The results can elucidate the causes and consequences of obesity, and are expected to aid the provision of care for overweight and obese Thai people.