Cellular and Cytokine Responses to Salmonella enterica Serotype Typhi Proteins in Patients with Typhoid Fever in Bangladesh

We assessed interferon-gamma (IFN-γ) responses via enzyme-linked immunosorbent spot (ELISPOT) to a number of S. Typhi antigens in samples from humans with S. Typhi bacteremia and typhoid fever in Bangladesh. Compared with responses in healthy endemic zone controls, there were significantly increased IFN-γ responses at the time of clinical presentation (acute phase) and at convalescence 14–28 days later. The majority (80–90%) of IFN-γ expressing T cells were CD4+. We observed a significant increase in interleukin-17 (IL-17) positive CD4 + T cells at convalescent versus acute stage of infection using an intracellular cytokine staining assay. We also found that stimulated peripheral blood mononuclear cells (PBMCs) produced significantly increased levels of a number of cytokines at the convalescent versus acute phase of infection, including IFN-γ, MIP-1β, sCD40L, TNF-β, IL-13, and IL-9. These results suggest that S. Typhi antigens induce a predominantly Th1 response, but that elevations in other cytokines may be modulatory

Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen

Interferon-γ and Proliferation Responses to Salmonella enterica Serotype Typhi Proteins in Patients with S. Typhi Bacteremia in Dhaka, Bangladesh

Salmonella enterica serotype Typhi infection is a significant global public health problem and the cause of typhoid fever. Salmonella are intracellular pathogens, and cellular immune responses are required to control and clear Salmonella infections. Despite this, there are limited data on cellular immune responses during wild type S. Typhi infection in humans. Here we report the assessment of cellular immune responses in humans with S. Typhi bacteremia through a screening approach that permitted us to evaluate interferon-γ and proliferation responses to a number of S. Typhi antigens. We detected significant interferon-γ CD4 and CD8 responses, as well as proliferative responses, to a number of recombinantly purified S. Typhi proteins as well as membrane preparation in infected patients. Antigen-specific interferon-γ responses were present at the time of clinical presentation in patients and absent in healthy controls. These observations could assist in the development of interferon-γ-based diagnostic assays for typhoid fever

Analysis of Salmonella enterica Serotype Paratyphi A Gene Expression in the Blood of Bacteremic Patients in Bangladesh

Salmonella enterica serotype Paratyphi A is a significant and emerging global public health problem and accounts for one fifth of all cases of enteric fever in many areas of Asia. S. Paratyphi A only infects humans, and the lack of an appropriate animal model has limited the study of S. Paratyphi A infection. In this study, we report the application of an RNA analysis method, Selective Capture of Transcribed Sequences (SCOTS), to evaluate which S. Paratyphi A genes are expressed directly in the blood of infected humans. Our results provide insight into the bacterial adaptations and modifications that S. Paratyphi A may need to survive within infected humans and suggest that similar approaches may be applied to other pathogens in infected humans and animals

The Utility of Zebrafish to Study Bacterial Pathogenesis

Zebrafish <i>(Danio rerio) </i>are now established as an excellent model to study infections and their pathogenesis. Zebrafish have many advantages over other vertebrates in studying host immune responses to infectious diseases. Most importantly, the ease of manipulating host genes to generate transgenic fish lines, and the optical transparency of zebrafish that facilitates imaging of real-time interactions between host and pathogen with excellent resolution. In my PhD project, I optimized and applied a zebrafish infection model to study multiple virulence features of two major nosocomial pathogens, these being <i>Acinetobacter baumannii</i> and <i>Staphylococcus</i> <i>aureus. </i><br>     <br>    In the first part of my PhD, I used zebrafish as a substitute host to study <i>A. baumannii </i>infections and the genes required for its virulence. Bacterial mutants involving genes responsible for quorum-sensing and global virulence regulation <i>(gacSA)</i> were studied in zebrafish, and results corroborated with those observed in a murine model of infection. Neutrophil and macrophage migration responses were also studied to find the primary phagocyte responsible for initial killing of bacteria. These experiments led to an important finding related to bacterial-driven neutrophil chemoattraction. When zebrafish were infected with the <i>A. baumannii gacS</i> mutant, it induced prompt neutrophil swarming and dwelling at the site of infection, which was not observed with wild-type bacteria. To understand the mechanism of this neutrophil swarming, I then tested a number of mutants with deletion in genes controlled by the two-component global virulence regulator <i>(gacS)</i>. These experiments led me to find that a mutation in the phenylacetic acid metabolic pathway gene <i>(paaA)</i> was responsible for neutrophil dwelling at a localised site of infection. Investigation of metabolites in culture supernatant revealed that phenylactic acid was the chemoattractant for neutrophils, and this facilitated the survival of fish by clearing the infection more effectively. <br>     <br>    The second aim was focused on another problematic hospital-acquired pathogen; <i>Staphylococcus aureus</i>. More specifically, to understand the impact of resistance to a last-line antibiotic, known as daptomycin, on host-pathogen interactions. Daptomycin resistance in <i>S. aureus </i>is associated with changes in the bacterial membrane lipid composition. My work assessed how these changes in membrane lipid composition contributed to host immune evasion and persistent infection. Studying the lipid moieties present in the membrane of daptomycin-resistant clinical strains showed that there was an increase in cardiolipin content and a reduction in phosphatidylglycerol. Clinically relevant point mutations were generated in the <i>S. aureus</i> cardiolipin synthase <i>(cls2)</i> gene, which allowed a detailed assessment of the impact of bacterial membrane phospholipid changes on antibiotic resistance and interaction with the immune system. By using neutrophil migration assays in zebrafish, I showed that less neutrophils migrated to the site of infection caused by the point mutants compared to wild-type infection. To investigate the role of individual lipids in neutrophil migration, I used purified cardiolipin and phosphatidylglycerol isolated from wild-type bacteria and showed that phosphatidylglycerol was a neutrophil chemoattractant, whereas cardiolipin had no effect. These results indicated that gaining more cardiolipin and reducing phosphatidylglycerol, as was being observed in our clinical daptomycin-resistant strains, caused less neutrophil migration to the site of infection promoting bacterial survival and persistence. <br>     <br>    Overall, I used zebrafish to study the pathogenesis of two common hospital acquired organisms. The work has supported the importance of zebrafish as an infection model to study bacterial pathogenesis and has contributed novel findings related to drug resistance and bacterial driven neutrophil chemoattraction

Carbapenem resistance in Acinetobacter baumannii: laboratory challenges, mechanistic insights and therapeutic strategies

Unprecedented levels of antimicrobial resistance in bacterial isolates have prompted great concerns globally. In 2012 the WHO released a publication outlining the evolving threat of antimicrobial resistance in order to raise awareness and to stimulate coordinated international efforts. The carbapenem class of antibiotics is largely considered as an antibiotic of last-resort when treating infections. Now carbapenem resistance further limits treatment options. In this article the authors discuss carbapenem resistance in Acinetobacter baumannii, a bacterial isolate often implicated in nosocomial infections. Virulence factors, intrinsic and acquired resistance mechanisms, together with laboratory challenges in the detection and antibiotic susceptibility testing of A. baumannii make this a truly problematic isolate. Therapeutic options are exceedingly limited, relying on polymyxins in combinations with other antibiotics, with few, if any, new active agents in the pipeline

The Resistance to Host Antimicrobial Peptides in Infections Caused by Daptomycin-Resistant <i>Staphylococcus aureus</i>

Daptomycin is an important antibiotic for the treatment of infections caused by Staphylococcus aureus. The emergence of daptomycin resistance in S. aureus is associated with treatment failure and persistent infections with poor clinical outcomes. Here, we investigated host innate immune responses against clinically derived, daptomycin-resistant (DAP-R) and -susceptible S. aureus paired isolates using a zebrafish infection model. We showed that the control of DAP-R S. aureus infections was attenuated in vivo due to cross-resistance to host cationic antimicrobial peptides. These data provide mechanistic understanding into persistent infections caused by DAP-R S. aureus and provide crucial insights into the adaptive evolution of this troublesome pathogen

Assessment of disease specific immune responses in enteric diseases using dried blood spot (DBS).

BackgroundBlood collection, transportation and storage remain a problem in countries where infrastructure, laboratory facilities and skilled manpower are scarce. This limits evaluation of immune responses in natural infections and vaccination in field studies. We developed methods to measure antigen specific antibody responses using dried blood spot (DBS) in cholera, ETEC and typhoid fever patients as well as recipients of oral cholera vaccine (OCV).Methodology/principle findingsWe processed heparinized blood for preparing DBS and plasma specimens from patients with, cholera, ETEC and typhoid as well as OCV recipients. We optimized the conventional vibriocidal method to measure vibriocidal antibody response in DBS eluates. We measured responses in DBS samples and plasma (range of titer of 5 to 10240). Vibriocidal titer showed strong agreement between DBS eluates and plasma in cholera patients (ICC = 0.9) and in OCV recipients (ICC = 0.8) using the Bland-Altman analysis and a positive correlation was seen (r = 0.7, p = 0.02 and r = 0.6, p = 0.006, respectively). We observed a strong agreement of lipopolysaccharide (LPS) and cholera toxin B (CTB)-specific antibody responses between DBS eluates and plasma in cholera patients and OCV recipients. We also found agreement of heat labile toxin B (LTB) and membrane protein (MP)-specific antibody responses in DBS eluates and plasma specimen of ETEC and typhoid patients respectively.ConclusionOur results demonstrate that dried blood specimens can be used as an alternate method for preservation of samples to measure antibody responses in enteric diseases and vaccine trials and can be applied to assessment of responses in humanitarian crisis and other adverse field settings

Immunogenicity of a killed bivalent whole cell oral cholera vaccine in forcibly displaced Myanmar nationals in Cox's Bazar, Bangladesh.

After the large influx of Rohingya nationals (termed Forcibly Displaced Myanmar National; FDMN) from Rakhine State of Myanmar to Cox's Bazar in Bangladesh, it was apparent that outbreaks of cholera was very likely in this setting where people were living under adverse water and sanitation conditions. Large campaigns of oral cholera vaccine (OCV) were carried out as a preemptive measure to control cholera epidemics. The aim of the study was to evaluate the immune responses of healthy adults and children after administration of two doses of OCV at 14 days interval in FDMN population and compare with the response observed in Bangladeshi's vaccinated earlier. A cross-sectional immunogenicity study was conducted among FDMNs of three age cohort; in adults (18+years; n = 83), in older children (6-17 years; n = 63) and in younger children (1-5 years; n = 80). Capillary blood was collected at three time points to measure vibriocidal antibodies using either plasma or dried blood spot (DBS) specimens. There was a significant increase of responder frequency of vibriocidal antibody titer at day 14 in all groups for Vibrio cholerae O1 (Ogawa/Inaba: adults-64%/64%, older children-70%/89% and younger children-51%/75%). There was no overall difference of vibriocidal antibody titer between FDMN and Bangladeshi population at baseline (p = 0.07-0.08) and at day 14, day 28 in all age groups for both serotypes. The seroconversion rate and geometric mean titer (GMT) of either serotype were comparable using both plasma and DBS specimens. These results showed that OCV is capable of inducing robust immune responses in adults and children among the FDMN population which is comparable to that seen in Bangladeshi participants in different age groups or that reported from other cholera endemic countries. Our results also suggest that the displaced population were exposed to V. cholerae prior to seeking shelter in Bangladesh

Acinetobacter baumanniiphenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis

Innate cellular immune responses are a critical first-line defense against invading bacterial pathogens. Leukocyte migration from the bloodstream to a site of infection is mediated by chemotactic factors that are often host-derived. More recently, there has been a greater appreciation of the importance of bacterial factors driving neutrophil movement during infection. Here, we describe the development of a zebrafish infection model to study Acinetobacter baumannii pathogenesis. By using isogenic A. baumannii mutants lacking expression of virulence effector proteins, we demonstrated that bacterial drivers of disease severity are conserved between zebrafish and mammals. By using transgenic zebrafish with fluorescent phagocytes, we showed that a mutation of an established A. baumannii global virulence regulator led to marked changes in neutrophil behavior involving rapid neutrophil influx to a localized site of infection, followed by prolonged neutrophil dwelling. This neutrophilic response augmented bacterial clearance and was secondary to an impaired A. baumannii phenylacetic acid catabolism pathway, which led to accumulation of phenylacetate. Purified phenylacetate was confirmed to be a neutrophil chemoattractant. These data identify a previously unknown mechanism of bacterial-guided neutrophil chemotaxis in vivo, providing insight into the role of bacterial metabolism in host innate immune evasion. Furthermore, the work provides a potentially new therapeutic paradigm of targeting a bacterial metabolic pathway to augment host innate immune responses and attenuate diseas