BackgroundToll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.Methods and FindingsPatient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.ConclusionsPatients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.

Intestinal colonization by antibiotic-resistant Enterococcus faecium is the first step in a process that can lead to infections in hospitalized patients. By comparative genome analysis and subsequent polymerase chain reaction screening, we identified a locus that encodes a putative phosphotransferase system (PTS). The PTS locus was widespread in isolates from hospital outbreaks of infection (84.2%) and nonoutbreak clinical infections (66.0%) but absent from human commensal isolates. Deletion of pstD, which is predicted to encode the enzyme IID subunit of this PTS, significantly impaired the ability of E. faecium to colonize the murine intestinal tract during antibiotic treatment. This is the first description of a determinant that contributes to intestinal colonization in clinical E. faecium strains.

The incidence of infections with Enterococcus faecium is increasing worldwide. TLRs have been implicated in the recognition of pathogens and the initiation of an adequate innate immune response. We here sought to determine the roles of MyD88, the common adaptor protein involved in TLR signaling, TLR2, TLR4, and CD14 in host defense against E. faecium peritonitis. MyD88 knockout (KO) mice demonstrated an impaired early response to E. faecium peritonitis, as reflected by higher bacterial loads in peritoneal fluid and liver accompanied by a markedly attenuated neutrophil influx into the abdominal cavity. In vitro, not only MyD88 KO macrophages but also TLR2 KO and CD14 KO macrophages displayed a reduced responsiveness to E. faecium. In accordance, transfection of TLR2 rendered human embryonic kidney 293 cells responsive to E. faecium, which was enhanced by cotransfection of CD14. TLR2 KO mice showed higher bacterial loads in peritoneal fluid after in vivo infection with E. faecium and a diminished influx of neutrophils, whereas CD14 KO mice had an unaltered host response. E. faecium phagocytosis and killing were not affected by MyD88, TLR2, or CD14 deficiency. TLR4 did not play a role in the immune response to E. faecium in vitro or in vivo. These data suggest that MyD88 contributes to the effective clearance of E. faecium during peritonitis at least in part via TLR2 and by facilitating neutrophil recruitment to the site of the infection.