T-Cell Responses Are Associated with Survival in Acute Melioidosis Patients

Background

Melioidosis is an increasingly recognised cause of sepsis and death across South East Asia and Northern Australia, caused by the bacterium Burkholderia pseudomallei. Risk factors include diabetes, alcoholism and renal disease, and a vaccine targeting at-risk populations is urgently required. A better understanding of the protective immune response in naturally infected patients is essential for vaccine design.

Methods

We conducted a longitudinal clinical and immunological study of 200 patients with melioidosis on admission, 12 weeks (n = 113) and 52 weeks (n = 65) later. Responses to whole killed B. pseudomallei were measured in peripheral blood mononuclear cells (PBMC) by interferon-gamma (IFN-γ) ELIspot assay and flow cytometry and compared to those of control subjects in the region with diabetes (n = 45) and without diabetes (n = 43).

Results

We demonstrated strong CD4+ and CD8+ responses to B. pseudomallei during acute disease, 12 weeks and 52 weeks later. 28-day mortality was 26% for melioidosis patients, and B. pseudomallei-specific cellular responses in fatal cases (mean 98 IFN-γ cells per million PBMC) were significantly lower than those in the survivors (mean 142 IFN-γ cells per million PBMC) in a multivariable logistic regression model (P = 0.01). A J-shaped curve association between circulating neutrophil count and mortality was seen with an optimal count of 4000 to 8000 neutrophils/μl.Melioidosis patients with known diabetes had poor diabetic control (median glycated haemoglobin HbA_1c 10.2%, interquartile range 9.2–13.1) and showed a stunted B. pseudomallei-specific cellular response during acute illness compared to those without diabetes.

Conclusions

The results demonstrate the role of both CD4+ and CD8+ T-cells in protection against melioidosis, and an interaction between diabetes and cellular responses. This supports development of vaccine strategies that induce strong T-cell responses for the control of intracellular pathogens such as B. pseudomallei.     

Transcriptional profiling of Caulobacter crescentus during growth on complex and minimal media

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.     

Isolation and characterization of an alpha-amylase gene in cassava (Manihot esculenta).

The roots of cassava plants (Manihot esculenta Crantz) accumulate starch as their major form of carbohydrate reserve. Starch accumulation and properties are determined by a balance between starch biosynthesis and degradation processes. Alpha-amylases (EC 3.2.1.1) are alpha-1,4 endoglycolytic enzymes, responsible for the mobilization of stored carbohydrate reserves by initiating the degradation process. Alpha-amylase genes have been shown to be differentially expressed at various developmental stages and environmental conditions through the action of plant hormones such as abscisic acid (ABA) and gibberellic acid (GA). In this study, we isolated an alpha-amylase gene from cassava tuberous roots (designated as MEamy2, GenBank accession number DQ011041). The deduced product of MEamy2 is 407 amino acid residues in length, with a calculated molecular mass of 46.7 kDa and an isoelectric point of 8.66. Southern blot analysis showed that the MEamy2 is present as a single copy in cassava genome. It shares the highest homology with AMY8 from apple fruit. The predicted structural model of MEamy2 contains three domains, active sites and starch-binding domain that are common with other plant alpha-amylases. RT-PCR analysis showed that the MEamy2 gene expression was induced in cassava roots within 2 hours after treatment with GA, but not ABA.     

Phytoremediation of arsenic in submerged soil by wetland plants

Wetland aquatic plants including Canna glauca L., Colocasia esculenta L. Schott, Cyperus papyrus L. and Typha angustifolia L. were used in the phytoremediation of submerged soil polluted by arsenic (As). Cyperus papyrus L. was noticed as the largest biomass producer which has arsenic accumulation capacity of 130-172 mg As/kg plant. In terms of arsenic removal rate, however, Colocasia esculenta L. was recognized as the largest and fastest arsenic remover in this study. Its arsenic removal rate was 68 mg As/m2/day while those rates of Canna glauca L., Cyperus papyrus L. and Typha angustifolia L. were 61 mg As/m2/day, 56 mg As/m2/day, and 56 mg As/m2/day, respectively. Although the 4 aquatic plants were inferior in arsenic accumulation, their high arsenic removal rates were observed. Phytostabilization should be probable for the application of these plants.     

Feasibility of Modified Surviving Sepsis Campaign Guidelines in a Resource-Restricted Setting Based on a Cohort Study of Severe S. Aureus Sepsis

Background

The Surviving Sepsis Campaign (SSC) guidelines describe best practice for the management of severe sepsis and septic shock in developed countries, but most deaths from sepsis occur where healthcare is not sufficiently resourced to implement them. Our objective was to define the feasibility and basis for modified guidelines in a resource-restricted setting.

Methods and Findings

We undertook a detailed assessment of sepsis management in a prospective cohort of patients with severe sepsis caused by a single pathogen in a 1,100-bed hospital in lower-middle income Thailand. We compared their management with the SSC guidelines to identify care bundles based on existing capabilities or additional activities that could be undertaken at zero or low cost. We identified 72 patients with severe sepsis or septic shock associated with S. aureus bacteraemia, 38 (53%) of who died within 28 days. One third of patients were treated in intensive care units (ICUs). Numerous interventions described by the SSC guidelines fell within existing capabilities, but their implementation was highly variable. Care available to patients on general wards covered the fundamental principles of sepsis management, including non-invasive patient monitoring, antimicrobial administration and intravenous fluid resuscitation. We described two additive care bundles, one for general wards and the second for ICUs, that if consistently performed would be predicted to improve outcome from severe sepsis.

Conclusion

It is feasible to implement modified sepsis guidelines that are scaled to resource availability, and that could save lives prior to the publication of international guidelines for developing countries.     

Staphylococcus aureus Bacteraemia in a Tropical Setting: Patient Outcome and Impact of Antibiotic Resistance

Background

Most information on invasive Staphylococcus aureus infections comes from temperate countries. There are considerable knowledge gaps in epidemiology, treatment, drug resistance and outcome of invasive S. aureus infection in the tropics.

Methods

A prospective, observational study of S. aureus bacteraemia was conducted in a 1000-bed regional hospital in northeast Thailand over 1 year. Detailed clinical data were collected and final outcomes determined at 12 weeks, and correlated with antimicrobial susceptibility profiles of infecting isolates.

Principal Findings

Ninety-eight patients with S. aureus bacteraemia were recruited. The range of clinical manifestations was similar to that reported from temperate countries. The prevalence of endocarditis was 14%. The disease burden was highest at both extremes of age, whilst mortality increased with age. The all-cause mortality rate was 52%, with a mortality attributable to S. aureus of 44%. Methicillin-resistant S. aureus (MRSA) was responsible for 28% of infections, all of which were healthcare-associated. Mortality rates for MRSA and methicillin-susceptible S. aureus (MSSA) were 67% (18/27) and 46% (33/71), respectively (p = 0.11). MRSA isolates were multidrug resistant. Only vancomycin or fusidic acid would be suitable as empirical treatment options for suspected MRSA infection.

Conclusions

S. aureus is a significant pathogen in northeast Thailand, with comparable clinical manifestations and a similar endocarditis prevalence but higher mortality than industrialised countries. S. aureus bacteraemia is frequently associated with exposure to healthcare settings with MRSA causing a considerable burden of disease. Further studies are required to define setting-specific strategies to reduce mortality from S. aureus bacteraemia, prevent MRSA transmission, and to define the burden of S. aureus disease and emergence of drug resistance throughout the developing world.     

Synthesis and SAR of novel benzoxaboroles as a new class of β-lactamase inhibitors

A new class of benzoxaborole β-lactamase inhibitors were designed and synthesized. 6-Aryloxy benzoxaborole 22 inhibited AmpC P99 and CMY-2 with K_i values in the low nanomolar range. Compound 22 restored antibacterial activity of ceftazidime against Enterobacter cloacae P99 expressing AmpC, a class C β-lactamase enzyme. The SAR around the arylbenzoxaboroles, which included the influence of linker and substitutions was also established.     

Public Awareness of Melioidosis in Thailand and Potential Use of Video Clips as Educational Tools

Background

Melioidosis causes more than 1,000 deaths in Thailand each year. Infection occurs via inoculation, ingestion or inhalation of the causative organism (Burkholderia pseuodmallei) present in soil and water. Here, we evaluated public awareness of melioidosis using a combination of population-based questionnaire, a public engagement campaign to obtain video clips made by the public, and viewpoints on these video clips as potential educational tools about the disease and its prevention.

Methods

A questionnaire was developed to evaluate public awareness of melioidosis, and knowledge about its prevention. From 1 March to 31 April 2012, the questionnaire was delivered to five randomly selected adults in each of 928 districts in Thailand. A video clip contest entitled “Melioidosis, an infectious disease that Thais must know” was run between May and October 2012. The best 12 video clips judged by a contest committee were shown to 71 people at risk from melioidosis (diabetics). Focus group interviews were used to evaluate their perceptions of the video clips.

Results

Of 4,203 Thais who completed our study questionnaire, 74% had never heard of melioidosis, and 19% had heard of the disease but had no further knowledge. Most participants in all focus group sessions felt that video clips were beneficial and could positively influence them to increase adherence to recommended preventive behaviours, including drinking boiled water and wearing protective gear if in contact with soil or environmental water. Participants suggested that video clips should be presented in the local dialect with simple words rather than medical terms, in a serious manner, with a doctor as the one presenting the facts, and having detailed pictures of each recommended prevention method.

Conclusions

In summary, public awareness of melioidosis in Thailand is very low, and video clips could serve as a useful medium to educate people and promote disease prevention.

Presented in Part

World Melioidosis Congress 2013, Bangkok, Thailand, 18–20 September 2013 (abstract OS VII-04).     

The Comparison of Fluid Dynamics Parameters in an Andersen Cascade Impactor Equipped With and Without a Preseparator

The fluid dynamic data in Andersen cascade impactor (ACI) are still lacking. Airflows and those affected parameters can be predicted in a preseparator and Andersen cascade impactor (ACI) by computational modeling. This study developed a validated computational fluid dynamic (CFD) model of an ACI and investigated the effects of the preseparator on the CFD parameters. Validation of the computational nozzle velocity for each of the stage 0 to stage 5 of the ACI stages was found to be within a 3.56% error. The flow field indicated that the preseparator accelerated the airflow velocity at the induction tube from 1.13 to 3.71 ± 0.09 m/s and 2.40 to 8.68 ± 0.16 m/s (at 28.3 and 60 L/min of flow rate, respectively). The preseparator produced a nozzle''s wall shear stress ranged from 0.08 to 0.34 Pa on a collection plate, while the ex-preseparator spread wall shear from the plate''s center was in a range of 0.11 to 0.37 Pa (at 28.3 L/min of flow rate). Moreover, the nozzle velocities increased along the distance from the middle of the collection plate to the periphery. The CFD explained the airflow of the preseparator equipped model by accelerating the airflow along the inlet port to maximize the trapping of desirable particles and the generation of a smooth wall shear stress at the collection plate to reduce the particle re-entrainment. While, the ex-preseparator generated an airflow that resulted in a higher wall shear stress occurring on the lower stages.Key words: ACI, flow field, preseparator, wall shear stress