During the first countrywide outbreak of dengue hemorrhagic fever in Bangladesh, we conducted surveillance for dengue at a hospital in Dhaka. Of 176 patients, primarily adults, found positive for dengue, 60.2% had dengue fever, 39.2% dengue hemorrhagic fever, and 0.6% dengue shock syndrome. The Dengue virus 3 serotype was detected in eight patients.

Pneumolysin is a pore-forming cytolysin known as a major virulence determinant of Streptococcus pneumoniae. This protein toxin has also been shown to activate the Toll-like receptor 4 (TLR4) signaling pathway. In this study, a mutant S. pneumoniae strain deficient in pneumolysin (⌬ply) and a recombinant pneumolysin protein (rPLY) were constructed. Upon infection of macrophages in vitro, the ability to induce the production of interleukin-1␣ (IL-1␣), IL-1␤, and IL-18 was severely impaired in the ⌬ply mutant, whereas there was no marked difference in the induction of tumor necrosis factor alpha (TNF-␣) and IL-12p40 between the wild type and the ⌬ply mutant of S. pneumoniae. When macrophages were stimulated with rPLY, the production of IL-1␣, IL-1␤, and IL-18 was strongly induced in a TLR4-dependent manner, whereas lipopolysaccharide, a canonical TLR4 agonist, hardly induced these cytokines. In contrast, lipopolysaccharide was more potent than rPLY in inducing the production of TNF-␣, IL-6, and IL-12p40, the cytokines requiring no caspase activation. Activation of caspase-1 was observed in macrophages stimulated with rPLY but not in those stimulated with lipopolysaccharide, and the level of activation was higher in macrophages infected with wild-type S. pneumoniae than in those infected with the ⌬ply mutant. These results clearly indicate that pneumolysin plays a key role in the host response to S. pneumoniae, particularly in the induction of caspase-1-dependent cytokines.

Chronic pulmonary infections are the principal cause of morbidity and mortality in individuals with cystic fibrosis (CF). Due to the polymicrobial nature of these infections, the identification of the particular bacterial species responsible is an essential step in diagnosis and treatment. Current diagnostic procedures are time-consuming, and can also be expensive, invasive and unpleasant in the absence of spontaneously expectorated sputum. The development of a rapid, non-invasive methodology capable of diagnosing and monitoring early bacterial infection is desired. Future visions of real-time, in situ diagnosis via exhaled breath testing rely on the differentiation of bacteria based on their volatile metabolites. The objective of this proof-of-concept study was to investigate whether a range of CF-associated bacterial species (i.e. Pseudomonas aeruginosa, Burkholderia cenocepacia, Haemophilus influenzae, Stenotrophomonas maltophilia, Streptococcus pneumoniae and Streptococcus milleri) could be differentiated based on their in vitro volatile metabolomic profiles. Headspace samples were collected using solid phase microextraction (SPME), analyzed using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and evaluated using principal component analysis (PCA) in order to assess the multivariate structure of the data. Although it was not possible to effectively differentiate all six bacteria using this method, the results revealed that the presence of a particular pattern of VOCs (rather than a single VOC biomarker) is necessary for bacterial species identification. The particular pattern of VOCs was found to be dependent upon the bacterial growth phase (e.g. logarithmic versus stationary) and sample storage conditions (e.g. short-term versus long-term storage at  -18 °C). Future studies of CF-associated bacteria and exhaled breath condensate will benefit from the approaches presented in this study and further facilitate the production of diagnostic tools for the early detection of bacterial lung infections.

Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

Listeriolysin O (LLO), an hly-encoded cytolysin from Listeria monocytogenes, plays an essential role in the entry of this pathogen into the macrophage cytoplasm and is also a key factor in inducing the production of IFN-γ during the innate immune stage of infection. In this study, we examined the involvement of LLO in macrophage production of the IFN-γ-inducing cytokines IL-12 and IL-18. Significant levels of IL-12 and IL-18 were produced by macrophages upon infection with wild-type L. monocytogenes, whereas an LLO-deficient mutant (the L. monocytogenes Δhly) lacked the ability to induce IL-18 production. Complementation of Δhly with hly completely restored the ability. However, when Δhly was complemented with ilo encoding ivanolysin O (ILO), a cytolysin highly homologous with LLO, such a restoration was not observed, although ILO-expressing L. monocytogenes invaded and multiplied in the macrophage cytoplasm similarly as LLO-expressing L. monocytogenes. Induction of IL-18 was diminished when pretreated with a caspase-1 inhibitor or in macrophages from caspase-1-deficient mice, suggesting the activation of caspase-1 as a key event resulting in IL-18 production. Activation of caspase-1 was induced in macrophages infected with LLO-expressing L. monocytogenes but not in those with Δhly. A complete restoration of such an activity could not be observed even after complementation with the ILO gene. These results show that the LLO molecule is involved in the activation of caspase-1, which is essential for IL-18 production in infected macrophages, and suggest that some sequence unique to LLO is indispensable for some signaling event resulting in the caspase-1 activation induced by L. monocytogenes.

Since 1987, multidrug resistant (MDR) strains of Salmonella Typhi, resistant simultaneously to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole, have caused epidemics of severe typhoid fever in Asia and Africa. A retrospective analysis of blood culture results (1989-96) in a Diarrhoea Treatment Centre in Dhaka, Bangladesh detected MDR strains in 0.3% (8 of 2793) of samples in 1990. The isolation rate peaked to 3.2% (240 of 7501) in 1994 (P < 0.01) and decreased to 1.8% (165 of 9348) in 1995 and further to 1.0% (82 of 8587) in 1996 (P < 0.01 compared to 1994) indicating the emergence and decline of MDR typhoid epidemic. Ten of 15 MDR strains tested had a 176 kb conjugative R plasmid that mediates resistance to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole to Escherichia coli K12. Unlike MDR strains, the isolation rate (approximately 3.3%) of susceptible S. Typhi remained remarkably unchanged during the study. The significant decrease in isolation of MDR strains suggests that cheaper and effective first-line antibiotics may re-emerge as drugs of choice for the treatment of typhoid fever in Bangladesh.

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