Melioidosis, infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is a common cause of sepsis in northeast Thailand. In white North Americans, common functional genetic variation in TLR1 is associated with organ failure and death from sepsis. We hypothesized that TLR1 variants would be associated with outcomes in Thais with melioidosis. We collated the global frequencies of three TLR1 variants that are common in white North American populations: rs5743551 (-7202A/G), rs4833095 (742A/G), and rs5743618 (1804G/T). We noted a reversal of the minor allele from white North American subjects to Asian populations that was particularly pronounced for rs5743618. In the Utah residents of European ancestry, the frequency of the rs5743618 T allele was 17% whereas in Vietnamese subjects the frequency was >99%. We conducted a genetic association study in 427 patients with melioidosis to determine the association of TLR1 variation with organ failure or death. We genotyped rs5743551 and rs4833095. The variants were in high linkage disequilibrium but neither variant was associated with organ failure or in-hospital death. In 300 healthy Thai individuals we further tested the association of TLR1 variation with ex vivo blood responses to Pam3CSK4, a TLR1 agonist. Neither variant was robustly associated with blood cytokine responses induced by Pam3CSK4. We identified additional common variation in TLR1 by searching public databases and the published literature and screened three additional TLR1 variants for associations with Pam3CSK4-induced responses but found none. We conclude that the genetic architecture of TLR1 variation differs substantially in southeast Asians compared to other populations and common variation in TLR1 in Thais is not associated with outcome from melioidosis or with altered blood responses to Pam3CSK4. Our findings highlight the need for additional studies of TLR1 and other innate immune genetic modulators of the inflammatory host response and determinants of sepsis in southeast Asian populations.
B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.
Several factors contribute to the high mortality attributed to severe infections in resource-limited settings. While improvements in survival and processes of care have been made in high-income settings among patients with severe conditions, such as sepsis, guidelines necessary for achieving these improvements may lack applicability or have not been tested in resource-limited settings. The World Health Organization’s recent publication of the Integrated Management of Adolescent and Adult Illness District Clinician Manual provides details on how to optimize management of severely ill, hospitalized patients in such settings, including specific guidance on the management of patients with septic shock and respiratory failure without shock. This manuscript provides the context, process and underpinnings of these sepsis guidelines. In light of the current deficits in care and the limitations associated with these guidelines, the authors propose implementing these standardized best practice guidelines while using them as a foundation for sepsis research undertaken in, and directly relevant to, resource-limited settings.
Sepsis; Resource-limited setting; Severe illness; Guidelines
Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR51174C>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney–293 cells transfected with TLR5 and found that B. pseudomallei induced TLR51174C- but not TLR51174T-dependent activation of NF-κB. We tested the association of TLR51174C>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR51174C>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08–0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19–0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR51174C>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR51174T compared with carriers of TLR51174C. B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR51174T. We conclude that the hypofunctional genetic variant TLR51174C>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis.
Melioidosis is a tropical infection caused by the Gram-negative soil saprophyte Burkholderia pseudomallei. Despite broad exposure of northeast Thais, disease develops in only a small proportion of individuals. Although diabetes is a risk factor, the mechanisms of host susceptibility to melioidosis are still poorly understood. We postulated that Toll-like receptors (TLRs) regulate host susceptibility to disease, and that genetic variation in TLRs is associated with melioidosis. We analyzed the frequency of eight previously described TLR pathway polymorphisms in 490 cases compared to 950 non-hospitalized controls or 458 hospitalized controls. Based on these results, we then analyzed the frequency of additional TLR4 or TLR6-1-10 region polymorphisms in cases and controls. We found that the TLR41196C>T variant was associated with protection from melioidosis when compared to non-hospitalized controls. The TLR1742A>G and TLR1−7202A>G variants were associated with melioidosis when compared to hospitalized controls. In further analyses, we found that two additional TLR4 region polymorphisms were associated with disease. In diabetics, three other TLR6-1-10 region polymorphisms were associated with disease when compared to hospitalized controls. We conclude that TLR genetic variants may modulate host susceptibility to melioidosis. Confirmation of these findings and further investigation of the mechanisms is required.
melioidosis; Burkholderia pseudomallei; infection; Toll-like receptor; innate immunity; genetic variation
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.
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.
In their article, Baelani and colleagues surveyed anesthesia providers from African low- and middle-income countries (LMICs) to evaluate whether or not the current Surviving Sepsis Campaign (SSC) guidelines are feasible in such resource-constrained settings. The authors report that an alarmingly low percentage of hospitals have the capacity to implement the SSC guidelines in their entirety but a higher percentage are able to implement the majority of SSC guidelines and grade 1 recommendations. In reality, the probability of adherence to SSC guidelines for septic management is even lower than reported, given that the majority of sepsis management in African LMICs is likely performed by non-intensivists outside of intensive care units. Efforts to address the challenges of managing severely ill patients in LMICs have recently been taken on by the World Health Organization. After reviewing available evidence for sepsis management predominantly from high-income countries, a panel of experts developed a consensus-based strategy tailored for resource-limited settings. However, more research that can evaluate the challenges specific to sepsis management in LMICs and not currently addressed by the SSC guidelines is needed. Comprehensive, evidence-based guidelines combined with innovative approaches to sepsis management in LMICs are required to make a meaningful impact on worldwide sepsis survival.
Melioidosis is a frequently lethal tropical infection caused by the environmental saprophyte Burkholderia pseudomallei. Although transcutaneous inoculation and inhalation are considered the primary routes of infection, suggestive clinical evidence implicates ingestion as a possible alternative route. We show that in BALB/c and C57BL/6 mice, direct gastric inoculation of high doses of B. pseudomallei causes systemic infection that may be lethal or cause chronic disseminated infection. Mice may shed bacteria in the stool for weeks after infection, and high titers of B. pseudomallei-specific IgG are detectable. This report of enteric murine melioidosis supports further consideration of this route of infection.
Francisella tularensis is a Gram-negative intracellular pathogen that causes the zoonosis tularemia. Because F. tularensis LPS causes weak TLR4 activation, we hypothesized that administration of a synthetic TLR4 agonist, aminoalkyl glucosaminide phosphate (AGP), would boost the innate immune system and compensate for reduced TLR4 stimulation. Intranasal administration of AGPs induced intrapulmonary production of proinflammatory cytokines and chemokines. Mice treated with AGPs before and after inhalation of Francisella novicida exhibited augmented cytokine and inflammatory responses to infection; reduced bacterial replication in lung, liver, and spleen; and increased survival, whereas all PBS-treated control mice died within 4 days of infection, all AGP-treated mice showed prolonged time-to-death, and 30–60% of AGP-treated mice survived. The protective effect of AGP was lost in mice lacking IFN-γ. Long-term survivors developed specific Th1 splenocyte responses and specific Abs dominated by IgG2 isotypes. Survivors were fully protected from rechallenge with aerosolized F. novicida. Thus, preventive administration of AGP successfully modulated innate immune responses to aerosolized F. novicida, leading to protective immunity to pneumonic tularemia. This is the first report of the protective effect of a TLR ligand on resistance to F. novicida-induced pneumonic tularemia.
Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans—leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.
Many bacteria encounter both eukaryotic cells and other bacterial species as a part of their lifestyles. In order to compete and survive, these bacteria have evolved specialized pathways that target these distinct cell types. Type VI secretion systems (T6SSs) are bacterial protein export machines postulated to puncture targeted cells using an apparatus that shares structural similarity to bacteriophage. We investigated the role of the five T6SSs of Burkholderia thailandensis in the defense of the organism against other bacteria and higher organisms. B. thailandensis is a relatively avirulent soil saprophyte that is closely related to the human pathogen B. pseudomallei. Our work uncovered roles for two B. thailandensis T6SSs with specialized functions either in the survival of the organism in a murine host, or against another bacterial cell. We also found that B. thailandensis lacking the bacterial-targeting T6SS could not persist in a mixed biofilm with a competing bacterium. Based on the evolutionary relationship of T6SSs, and our findings that B. thailandensis engages other bacterial species in a T6S-dependent manner, we speculate that this pathway is of general significance to interbacterial interactions in polymicrobial human diseases and the environment.
Melioidosis is a tropical disease endemic in southeast Asia and northern Australia caused by the gram-negative soil saprophyte Burkholderia pseudomallei. Although infection is often systemic, the lung is frequently involved. B. thailandensis is a closely related organism that at high doses causes lethal pneumonia in mice. We examined the role of Toll-like receptors (TLRs), essential components of innate immunity, in vitro and in vivo during murine B. thailandensis pneumonia. TLR2, TLR4, and TLR5 mediate NF-κB activation by B. thailandensis in transfected HEK293 or CHO cells. In macrophages, TLR4 and the adaptor molecule MyD88, but not TLR2 or TLR5, are required for tumor necrosis factor alpha production induced by B. thailandensis. In low-dose airborne infection, TLR4 is needed for early, but not late, bacterial containment, and MyD88 is essential for control of infection and host survival. TLR2 and TLR5 are not necessary to contain low-dose infection. In high-dose airborne infection, TLR2 deficiency confers a slight survival advantage. Lung and systemic inflammatory responses are induced by low-dose inhaled B. thailandensis independently of individual TLRs or MyD88. These findings suggest that redundancy in TLR signaling or other MyD88-dependent pathways may be important in pneumonic B. thailandensis infection but that MyD88-independent mechanisms of inflammation are also activated. TLR signaling in B. thailandensis infection is substantially comparable to signaling induced by virulent B. pseudomallei. These studies provide additional insights into the host-pathogen interaction in pneumonic Burkholderia infection.
Acyl-homoserine lactone (acyl-HSL) quorum-sensing signaling is common to many Proteobacteria. Acyl-HSLs are synthesized by the LuxI family of synthases, and the signal response is mediated by members of the LuxR family of transcriptional regulators. Burkholderia thailandensis is a member of a closely related cluster of three species, including the animal pathogens Burkholderia mallei and Burkholderia pseudomallei. Members of this group have similar luxI and luxR homologs, and these genes contribute to B. pseudomallei and B. mallei virulence. B. thailandensis possesses three pairs of luxI-luxR homologs. One of these pairs, BtaI2-BtaR2, has been shown to produce and respond to 3OHC10-HSL and to control the synthesis of an antibiotic. By using a markerless-exhange method, we constructed an assortment of B. thailandensis quorum-sensing mutants, and we used these mutants to show that BtaI1 is responsible for C8-HSL production and BtaI3 is responsible for 3OHC8-HSL production. We also show that a strain incapable of acyl-HSL production is capable of growth on the same assortment of carbon and nitrogen sources as the wild type. Furthermore, this mutant shows no loss of virulence compared to the wild type in mice. However, the wild type self-aggregates in minimal medium, whereas the quorum-sensing mutant does not. The wild-type aggregation phenotype is recovered by addition of the BtaI1-R1 HSL signal C8-HSL. We propose that the key function of the BtaR1-BtaI1 quorum-sensing system is to cause cells to gather into aggregates once a sufficient population has been established.
Francisella tularensis causes the zoonosis tularemia in humans, and inhaled F. tularensis ssp. novicida induces lethal murine tularemia. Transcription of virulence factors in F. novicida is regulated by macrophage growth locus A (mglA), a global regulator required for bacterial replication in macrophages in vitro. We examined the infectivity and immunogenicity of attenuated F. novicida ΔmglA in the lung in vivo. Aerosolized ΔmglA caused replicative pulmonary infection that peaked at 7 days and was cleared thereafter, without clinical evidence of disease. In contrast, inhalation of wild type F. novicida resulted in more rapid bacterial replication and dissemination leading to death within 96 hours. Early containment of ΔmglA infection was partially dependent on myeloid differentiation factor 88 and interferon-γ but did not require B or T cells. However, lymphocytes were necessary for subsequent bacterial clearance. Infection with ΔmglA elicited specific IgG1-predominant antibodies and variable interferon-γ recall responses to wild type F. novicida. Inoculation of mice with aerosolized ΔmglA afforded no protection against a subsequent low-dose aerosol challenge with wild type F. novicida. These findings establish that inhalation of F. novicida ΔmglA results in replicative infection that elicits innate and adaptive immune responses but not protective immunity against invasive pneumonic tularemia.
Francisella tularensis; Tularemia; Pneumonia; Immunology
Burkholderia pseudomallei is a bacterial pathogen that causes a broad spectrum of clinical symptoms collectively known as melioidosis. Since it can be acquired by inhalation and is difficult to eradicate due to its resistance to a wide group of antibiotics and capacity for latency, work with B. pseudomallei requires a biosafety level 3 (BSL-3) containment facility. The bsa (Burkholderia secretion apparatus)-encoded type III secretion system (TTSS) has been shown to be required for its full virulence in a number of animal models. TTSSs are export devices found in a variety of gram-negative bacteria that translocate bacterial effector proteins across host cell membranes into the cytoplasm of host cells. Although the Bsa TTSS has been shown to play an important role in the ability of B. pseudomallei to survive and replicate in mammalian cells, escape from the endocytic vacuole, and spread from cell to cell, little is known about its effectors mediating these functions. Using bioinformatics, we identified homologs of several known TTSS effectors from other bacteria in the B. pseudomallei genome. In addition, we show that orthologs of these putative effectors exist in the genome of B. thailandensis, a closely related bacterium that is rarely pathogenic to humans. By generating a Bsa TTSS mutant B. thailandensis strain, we also demonstrated that the Bsa TTSS has similar functions in the two species. Therefore, we propose B. thailandensis as a useful BSL-1 model system to study the role of the Bsa TTSS during Burkholderia infection of mammalian cells and animals.
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.
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.
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.
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.
Melioidosis, a lethal tropical infection that is endemic in southeast Asia and northern Australia, is caused by the saprophytic Gram-negative bacterium Burkholderia pseudomallei. Overall mortality approaches 40% yet little is known about mechanisms of host defense. Toll-like receptors (TLRs) are host transmembrane receptors that recognize conserved pathogen molecular patterns and induce an inflammatory response. The lipopolysaccharide (LPS) of Gram-negative bacteria is a potent inducer of the host innate immune system. TLR4, in association with MD-2, is the archetype receptor for LPS although B. pseudomallei LPS has been previously identified as a TLR2 agonist. We examined TLR signaling induced by B. pseudomallei, B. pseudomallei LPS, and B. pseudomallei lipid A using gain-of-function transfection assays of NF-κB activation and studies of TLR-deficient macrophages.
In HEK293 cells transfected with murine or human TLRs, CD14, and MD-2, heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6) and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B. pseudomallei augmented TNF-α and MIP-2 production whereas in TLR4-/- cells, TNF-α, MIP-2, and IL-10 production was reduced. Cytokine production by macrophages stimulated with B. pseudomallei LPS or lipid A was entirely dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule MyD88 strongly regulated TNF-α production in response to heat-killed B. pseudomallei.
B. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B. pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or lipid A, cytokine production is augmented. Other MyD88-dependent signaling pathways may also be important in the host response to B. pseudomallei infection. These findings provide new insights into critical mechanisms of host defense in melioidosis.
Sharon Peacock and colleagues discuss management of adult patients with sepsis in low- and middle-income settings, with a particular emphasis on tropical regions.