Neu3 neuraminidase induction triggers intestinal inflammation and colitis in a model of recurrent human food-poisoning.

Intestinal inflammation is the underlying basis of colitis and the inflammatory bowel diseases. These syndromes originate from genetic and environmental factors that remain to be fully identified. Infections are possible disease triggers, including recurrent human food-poisoning by the common foodborne pathogen Salmonella enterica Typhimurium (ST), which in laboratory mice causes progressive intestinal inflammation leading to an enduring colitis. In this colitis model, disease onset has been linked to Toll-like receptor-4-dependent induction of intestinal neuraminidase activity, leading to the desialylation, reduced half-life, and acquired deficiency of anti-inflammatory intestinal alkaline phosphatase (IAP). Neuraminidase (Neu) inhibition protected against disease onset; however, the source and identity of the Neu enzyme(s) responsible remained unknown. Herein, we report that the mammalian Neu3 neuraminidase is responsible for intestinal IAP desialylation and deficiency. Absence of Neu3 thereby prevented the accumulation of lipopolysaccharide-phosphate and inflammatory cytokine expression in providing protection against the development of severe colitis.

Plasma Proteome Signature of Sepsis: a Functionally Connected Protein Network.

Sepsis is an extreme host response to infection that leads to loss of organ function and cardiovascular integrity. Mortality from sepsis is on the rise. Despite more than three decades of research and clinical trials, specific diagnostic and therapeutic strategies for sepsis are still absent. The use of LFQ- and TMT-based quantitative proteomics is reported here to study the plasma proteome in five mouse models of sepsis. A knowledge-based interpretation of the data reveals a protein network with extensive connectivity through documented functional or physical interactions. The individual proteins in the network all have a documented role in sepsis and are known to be extracellular. The changes in protein abundance observed in the mouse models of sepsis have for the most part the same directionality (increased or decreased abundance) as reported in the literature for human sepsis. This network has been named the Plasma Proteome Signature of Sepsis (PPSS). The PPSS is a quantifiable molecular readout that can supplant the current symptom-based approach used to diagnose sepsis. This type of molecular interpretation of sepsis, its progression, and its response to therapeutic intervention are an important step in advancing our understanding of sepsis, and for discovering and evaluating new therapeutic strategies.

An intrinsic mechanism of secreted protein aging and turnover.

The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell-Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease.

Intraspecies variation in the emergence of hyperinfectious bacterial strains in nature.

Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection. However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in virulence during the infective process- a prospect that has significant implications for human and animal health. Our findings indicate that the capacity to become hypervirulent (100-fold decreased LD(50)) was much more evident in certain S. enterica strains than others. Hyperinfectious salmonellae were among the most virulent of this species; restricted to certain serotypes; and more capable of killing vaccinated animals. Such strains exhibited rapid (and rapidly reversible) switching to a less-virulent state accompanied by more competitive growth ex vivo that may contribute to maintenance in nature. The hypervirulent phenotype was associated with increased microbial pathogenicity (colonization; cytotoxin production; cytocidal activity), coupled with an altered innate immune cytokine response within infected cells (IFN-ß; IL-1ß; IL-6; IL-10). Gene expression analysis revealed that hyperinfectious strains display altered transcription of genes within the PhoP/PhoQ, PhoR/PhoB and ArgR regulons, conferring changes in the expression of classical virulence functions (e.g., SPI-1; SPI-2 effectors) and those involved in cellular physiology/metabolism (nutrient/acid stress). As hyperinfectious strains pose a potential risk to human and animal health, efforts toward mitigation of these potential food-borne contaminants may avert negative public health impacts and industry-associated losses.

Microfluidic chip-based detection and intraspecies strain discrimination of Salmonella serovars derived from whole blood of septic mice.

Salmonella is a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar of Salmonella enterica subsp. enterica, which comprises over 99% of human-pathogenic S. enterica isolates. Point-of-care methods for detection and strain discrimination of Salmonella serovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination of Salmonella serovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination of S. enterica subsp. enterica serovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens.

Antimicrobial susceptibility testing to evaluate minimum inhibitory concentration values of clinically relevant antibiotics.

Antimicrobial susceptibility testing is used to determine the minimum inhibitory concentration (MIC), the standard measurement of antibiotic activity. Here, we present a protocol for evaluating MIC values of clinically relevant antibiotics against bacterial isolates cultured in standard bacteriologic medium and in mammalian cell culture medium. We describe steps for pathogen identification, culturing bacteria, preparing MIC plates, MIC assay incubation, and determining MIC. This protocol can potentially optimize the use of existing antibiotics while enhancing efforts to discover new ones. For complete details on the use and execution of this protocol, please refer to Heithoff et al.1.

Re-evaluation of FDA-approved antibiotics with increased diagnostic accuracy for assessment of antimicrobial resistance.

Accurate assessment of antibiotic susceptibility is critical for treatment of antimicrobial resistant (AMR) infections. Here, we examine whether antimicrobial susceptibility testing in media more physiologically representative of in vivo conditions improves prediction of clinical outcome relative to standard bacteriologic medium. This analysis reveals that ∼15% of minimum inhibitory concentration (MIC) values obtained in physiologic media predicted a change in susceptibility that crossed a clinical breakpoint used to categorize patient isolates as susceptible or resistant. The activities of antibiotics having discrepant results in different media were evaluated in murine sepsis models. Testing in cell culture medium improves the accuracy by which MIC assays predict in vivo efficacy. This analysis identifies several antibiotics for treatment of AMR infections that standard testing failed to identify and those that are ineffective despite indicated use by standard testing. Methods with increased diagnostic accuracy mitigate the AMR crisis via utilizing existing agents and optimizing drug discovery.

Innate mechanism of mucosal barrier erosion in the pathogenesis of acquired colitis.

The colonic mucosal barrier protects against infection, inflammation, and tissue ulceration. Composed primarily of Mucin-2, proteolytic erosion of this barrier is an invariant feature of colitis; however, the molecular mechanisms are not well understood. We have applied a recurrent food poisoning model of acquired inflammatory bowel disease using Salmonella enterica Typhimurium to investigate mucosal barrier erosion. Our findings reveal an innate Toll-like receptor 4-dependent mechanism activated by previous infection that induces Neu3 neuraminidase among colonic epithelial cells concurrent with increased Cathepsin-G protease secretion by Paneth cells. These anatomically separated host responses merge with the desialylation of nascent colonic Mucin-2 by Neu3 rendering the mucosal barrier susceptible to increased proteolytic breakdown by Cathepsin-G. Depletion of Cathepsin-G or Neu3 function using pharmacological inhibitors or genetic-null alleles protected against Mucin-2 proteolysis and barrier erosion and reduced the frequency and severity of colitis, revealing approaches to preserve and potentially restore the mucosal barrier.

A broad-spectrum synthetic antibiotic that does not evoke bacterial resistance.

BACKGROUND: Antimicrobial resistance (AMR) poses a critical threat to public health and disproportionately affects the health and well-being of persons in low-income and middle-income countries. Our aim was to identify synthetic antimicrobials termed conjugated oligoelectrolytes (COEs) that effectively treated AMR infections and whose structures could be readily modified to address current and anticipated patient needs. METHODS: Fifteen chemical variants were synthesized that contain specific alterations to the COE modular structure, and each variant was evaluated for broad-spectrum antibacterial activity and for in vitro cytotoxicity in cultured mammalian cells. Antibiotic efficacy was analyzed in murine models of sepsis; in vivo toxicity was evaluated via a blinded study of mouse clinical signs as an outcome of drug treatment. FINDINGS: We identified a compound, COE2-2hexyl, that displayed broad-spectrum antibacterial activity. This compound cured mice infected with clinical bacterial isolates derived from patients with refractory bacteremia and did not evoke bacterial resistance. COE2-2hexyl has specific effects on multiple membrane-associated functions (e.g., septation, motility, ATP synthesis, respiration, membrane permeability to small molecules) that may act together to negate bacterial cell viability and the evolution of drug-resistance. Disruption of these bacterial properties may occur through alteration of critical protein-protein or protein-lipid membrane interfaces-a mechanism of action distinct from many membrane disrupting antimicrobials or detergents that destabilize membranes to induce bacterial cell lysis. INTERPRETATION: The ease of molecular design, synthesis and modular nature of COEs offer many advantages over conventional antimicrobials, making synthesis simple, scalable and affordable. These COE features enable the construction of a spectrum of compounds with the potential for development as a new versatile therapy for an imminent global health crisis. FUNDING: U.S. Army Research Office, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute.

Coagulation factor protein abundance in the pre-septic state predicts coagulopathic activities that arise during late-stage murine sepsis.

BACKGROUND: Although sepsis accounts for 1 in 5 deaths globally, few molecular therapies exist for this condition. The development of effective biomarkers and treatments for sepsis requires a more complete understanding of host responses and pathogenic mechanisms at early stages of disease to minimize host-driven pathology. METHODS: An alternative to the current symptom-based approach used to diagnose sepsis is a precise assessment of blood proteomic changes during the onset and progression of Salmonella Typhimurium (ST) murine sepsis. FINDINGS: A distinct pattern of coagulation factor protein abundance was identified in the pre-septic state- prior to overt disease symptoms or bacteremia- that was predictive of the dysregulation of fibrinolytic and anti-coagulant activities and resultant consumptive coagulopathy during ST murine sepsis. Moreover, the changes in protein abundance observed generally have the same directionality (increased or decreased abundance) reported for human sepsis. Significant overlap of ST coagulopathic activities was observed in Gram-negative Escherichia coli- but not in Gram-positive staphylococcal or pneumococcal murine sepsis models. Treatment with matrix metalloprotease inhibitors prevented aberrant inflammatory and coagulopathic activities post-ST infection and increased survival. Antibiotic treatment regimens initiated after specific changes arise in the plasma proteome post-ST infection were predictive of an increase in disease relapse and death after cessation of antibiotic treatment. INTERPRETATION: Altered blood proteomics provides a platform to develop rapid and easy-to-perform tests to predict sepsis for early intervention via biomarker incorporation into existing blood tests prompted by patient presentation with general malaise, and to stratify Gram-negative and Gram-positive infections for appropriate treatment. Antibiotics are less effective in microbial clearance when initiated after the onset of altered blood proteomics as evidenced by increased disease relapse and death after termination of antibiotic therapy. Treatment failure is potentially due to altered bacterial / host-responses and associated increased host-driven pathology, providing insight into why delays in antibiotic administration in human sepsis are associated with increased risk for death. Delayed treatment may thus require prolonged therapy for microbial clearance despite the prevailing notion of antibiotic de-escalation and shortened courses of antibiotics to improve drug stewardship. FUNDING: National Institutes of Health, U.S. Army.

Establishment of blood glycosidase activities and their excursions in sepsis.

Glycosidases are hydrolytic enzymes studied principally in the context of intracellular catabolism within the lysosome. Therefore, glycosidase activities are classically measured in experimentally acidified assay conditions reflecting their low pH optima. However, glycosidases are also present in the bloodstream where they may retain sufficient activity to participate in the regulation of glycoprotein half-lives, proteostasis, and disease pathogenesis. We have, herein, established at physiological pH 7.4 in blood plasma and sera the normal ranges of four major glycosidase activities essential for blood glycoprotein remodeling in healthy mice and humans. These activities included ß-galactosidase, ß-N-acetylglucosaminidase, α-mannosidase, and α-fucosidase. We have identified their origins to include the mammalian genes Glb1, HexB, Man2a1, and Fuca1. In experimental sepsis, excursions of glycosidase activities occurred with differences in host responses to discrete bacterial pathogens. Among similar excursions in human sepsis, the elevation of ß-galactosidase activity was a prognostic indicator of increased likelihood of patient death.

Assessment of a Smartphone-Based Loop-Mediated Isothermal Amplification Assay for Detection of SARS-CoV-2 and Influenza Viruses.

Importance: A critical need exists in low-income and middle-income countries for low-cost, low-tech, yet highly reliable and scalable testing for SARS-CoV-2 virus that is robust against circulating variants. Objective: To assess whether a smartphone-based assay is suitable for SARS-CoV-2 and influenza virus testing without requiring specialized equipment, accessory devices, or custom reagents. Design, Setting, and Participants: This cohort study enrolled 2 subgroups of participants (symptomatic and asymptomatic) at Santa Barbara Cottage Hospital. The symptomatic group consisted of 20 recruited patients who tested positive for SARS-CoV-2 with symptoms; 30 asymptomatic patients were recruited from the same community, through negative admission screening tests for SARS-CoV-2. The smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) was first optimized for analysis of human saliva samples spiked with either SARS-CoV-2 or influenza A or B virus; these results then were compared with those obtained by side-by-side analysis of spiked samples using the Centers for Disease Control and Prevention (CDC) criterion-standard reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay. Next, both assays were used to test for SARS-CoV-2 and influenza viruses present in blinded clinical saliva samples obtained from 50 hospitalized patients. Statistical analysis was performed from May to June 2021. Exposures: Testing for SARS-CoV-2 and influenza A and B viruses. Main Outcomes and Measures: SARS-CoV-2 and influenza infection status and quantitative viral load were determined. Results: Among the 50 eligible participants with no prior SARS-CoV-2 infection included in the study, 29 were men. The mean age was 57 years (range, 21 to 93 years). SmaRT-LAMP exhibited 100% concordance (50 of 50 patient samples) with the CDC criterion-standard diagnostic for SARS-CoV-2 sensitivity (20 of 20 positive and 30 of 30 negative) and for quantitative detection of viral load. This platform also met the CDC criterion standard for detection of clinically similar influenza A and B viruses in spiked saliva samples (n = 20), and in saliva samples from hospitalized patients (50 of 50 negative). The smartphone-based LAMP assay was rapid (25 minutes), sensitive (1000 copies/mL), low-cost (<$7/test), and scalable (96 samples/phone). Conclusions and Relevance: In this cohort study of saliva samples from patients, the smartphone-based LAMP assay detected SARS-CoV-2 infection and exhibited concordance with RT-qPCR tests. These findings suggest that this tool could be adapted in response to novel CoV-2 variants and other pathogens with pandemic potential including influenza and may be useful in settings with limited resources.

Conditions that diminish myeloid-derived suppressor cell activities stimulate cross-protective immunity.

Immunity conferred by conventional vaccines is restricted to a narrow range of closely related strains, highlighting the unmet medical need for the development of vaccines that elicit protection against multiple pathogenic serotypes. Here we show that a Salmonella bivalent vaccine comprised of strains that lack and overproduce DNA adenine methylase (Dam) conferred cross-protective immunity to salmonella clinical isolates of human and animal origin. Protective immunity directly correlated with increased levels of cross-reactive opsonizing antibodies and memory T cells and a diminished expansion of myeloid-derived suppressor cells (MDSCs) that are responsible for the immune suppression linked to several conditions of host stress, including chronic microbial infections, traumatic insults, and many forms of cancer. Further, aged mice contained increased numbers of MDSCs and were more susceptible to Salmonella infection than young mice, suggesting a role for these cells in the immune declines associated with the natural aging process. These data suggest that interventions capable of reducing MDSC presence and activities may allow corresponding increases in B- and T-cell stimulation and benefit the ability of immunologically diverse populations to be effectively vaccinated as well as reducing the risk of susceptible individuals to infectious disease.

Human Salmonella clinical isolates distinct from those of animal origin.

The global trend toward intensive livestock production has led to significant public health risks and industry-associated losses due to an increased incidence of disease and contamination of livestock-derived food products. A potential factor contributing to these health concerns is the prospect that selective pressure within a particular host may give rise to bacterial strain variants that exhibit enhanced fitness in the present host relative to that in the parental host from which the strain was derived. Here, we assessed 184 Salmonella enterica human and animal clinical isolates for their virulence capacities in mice and for the presence of the Salmonella virulence plasmid encoding the SpvB actin cytotoxin required for systemic survival and Pef fimbriae, implicated in adherence to the murine intestinal epithelium. All (21 of 21) serovar Typhimurium clinical isolates derived from animals were virulent in mice, whereas many (16 of 41) serovar Typhimurium isolates derived from human salmonellosis patients lacked this capacity. Additionally, many (10 of 29) serovar Typhimurium isolates derived from gastroenteritis patients did not possess the Salmonella virulence plasmid, in contrast to all animal and human bacteremia isolates tested. Lastly, among serovar Typhimurium isolates that harbored the Salmonella virulence plasmid, 6 of 31 derived from human salmonellosis patients were avirulent in mice, which is in contrast to the virulent phenotype exhibited by all the animal isolates examined. These studies suggest that Salmonella isolates derived from human salmonellosis patients are distinct from those of animal origin. The characterization of these bacterial strain variants may provide insight into their relative pathogenicities as well as into the development of treatment and prophylactic strategies for salmonellosis.

Smartphone-based pathogen diagnosis in urinary sepsis patients.

BACKGROUND: There is an urgent need for rapid, sensitive, and affordable diagnostics for microbial infections at the point-of-care. Although a number of innovative systems have been reported that transform mobile phones into potential diagnostic tools, the translational challenge to clinical diagnostics remains a significant hurdle to overcome. METHODS: A smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) system was developed for pathogen ID in urinary sepsis patients. The free, custom-built mobile phone app allows the phone to serve as a stand-alone device for quantitative diagnostics, allowing the determination of genome copy-number of bacterial pathogens in real time. FINDINGS: A head-to-head comparative bacterial analysis of urine from sepsis patients revealed that the performance of smaRT-LAMP matched that of clinical diagnostics at the admitting hospital in a fraction of the time (~1 h vs. 18-28 h). Among patients with bacteremic complications of their urinary sepsis, pathogen ID from the urine matched that from the blood - potentially allowing pathogen diagnosis shortly after hospital admission. Additionally, smaRT-LAMP did not exhibit false positives in sepsis patients with clinically negative urine cultures. INTERPRETATION: The smaRT-LAMP system is effective against diverse Gram-negative and -positive pathogens and biological specimens, costs less than $100 US to fabricate (in addition to the smartphone), and is configurable for the simultaneous detection of multiple pathogens. SmaRT-LAMP thus offers the potential to deliver rapid diagnosis and treatment of urinary tract infections and urinary sepsis with a simple test that can be performed at low cost at the point-of-care. FUND: National Institutes of Health, Chan-Zuckerberg Biohub, Bill and Melinda Gates Foundation.

Accelerated Aging and Clearance of Host Anti-inflammatory Enzymes by Discrete Pathogens Fuels Sepsis.

Sepsis is a life-threatening inflammatory syndrome accompanying a bloodstream infection. Frequently secondary to pathogenic bacterial infections, sepsis remains difficult to treat as a singular disease mechanism. We compared the pathogenesis of murine sepsis experimentally elicited by five bacterial pathogens and report similarities among host responses to Gram-negative Salmonella and E. coli. We observed that a host protective mechanism involving de-toxification of lipopolysaccharide by circulating alkaline phosphatase (AP) isozymes was incapacitated during sepsis caused by Salmonella or E. coli through activation of host Toll-like receptor 4, which triggered Neu1 and Neu3 neuraminidase induction. Elevated neuraminidase activity accelerated the molecular aging and clearance of AP isozymes, thereby intensifying disease. Mice deficient in the sialyltransferase ST3Gal6 displayed increased disease severity, while deficiency of the endocytic lectin hepatic Ashwell-Morell receptor was protective. AP augmentation or neuraminidase inhibition diminished inflammation and promoted host survival. This study illuminates distinct routes of sepsis pathogenesis, which may inform therapeutic development.

Comparison of tissue-selective proinflammatory gene induction in mice infected with wild-type, DNA adenine methylase-deficient, and flagellin-deficient Salmonella enterica.

Mutants of Salmonella enterica serovar Typhimurium deficient in DNA adenine methylase (Dam) are attenuated for virulence in mice and confer heightened immunity in vaccinated animals. In contrast, infection of mice with wild-type (WT) strains or flagellin-deficient mutants of Salmonella causes typhoid fever. Here we examined the bacterial load and spatiotemporal kinetics of expression of several classes of host genes in Peyer's patches, the liver, and the spleen following oral infection of mice with WT, dam mutant, or flagellin-deficient (flhC) Salmonella. The genes evaluated included inflammatory (interleukin-1beta [IL-1beta], tumor necrosis factor alpha), chemokine (macrophage inflammatory protein 2), Th1/Th2 indicator (IL-12p40, IL-4), and interferon system (beta interferon [IFN-beta], IFN-gamma, protein Mx1 GTPase, RNA-dependent protein kinase, inducible nitric oxide synthase, suppressor of cytokine signaling 1) beacons. We showed that maximal interferon system and proinflammatory gene induction occurred by 5 days after infection and that the levels were comparable for the WT and flhC strains but were significantly lower for the dam mutant. Additionally, host gene expression in systemic tissues of individual animals was dependent on the bacterial load in the Peyer's patches for mice infected with WT, dam mutant, or flhC mutant Salmonella as early as 8 h after infection. Moreover, a bacterial load threshold in the Peyer's patches was necessary to stimulate the host gene induction in the liver and spleen. Taken together, these results suggest that bacterial load and the accompanying strain-specific cytokine signature are important determinants of the host innate immune response and associated disease manifestations observed in dam mutant Salmonella-infected animals compared to the immune response and disease manifestations observed in WT and flhC mutant Salmonella-infected animals.

Recurrent infection progressively disables host protection against intestinal inflammation.

Intestinal inflammation is the central pathological feature of colitis and the inflammatory bowel diseases. These syndromes arise from unidentified environmental factors. We found that recurrent nonlethal gastric infections of Gram-negative Salmonella enterica Typhimurium (ST), a major source of human food poisoning, caused inflammation of murine intestinal tissue, predominantly the colon, which persisted after pathogen clearance and irreversibly escalated in severity with repeated infections. ST progressively disabled a host mechanism of protection by inducing endogenous neuraminidase activity, which accelerated the molecular aging and clearance of intestinal alkaline phosphatase (IAP). Disease was linked to a Toll-like receptor 4 (TLR4)-dependent mechanism of IAP desialylation with accumulation of the IAP substrate and TLR4 ligand, lipopolysaccharide-phosphate. The administration of IAP or the antiviral neuraminidase inhibitor zanamivir was therapeutic by maintaining IAP abundance and function.

Correcting a Fundamental Flaw in the Paradigm for Antimicrobial Susceptibility Testing.

The emergence and prevalence of antibiotic-resistant bacteria are an increasing cause of death worldwide, resulting in a global 'call to action' to avoid receding into an era lacking effective antibiotics. Despite the urgency, the healthcare industry still relies on a single in vitro bioassay to determine antibiotic efficacy. This assay fails to incorporate environmental factors normally present during host-pathogen interactions in vivo that significantly impact antibiotic efficacy. Here we report that standard antimicrobial susceptibility testing (AST) failed to detect antibiotics that are in fact effective in vivo; and frequently identified antibiotics that were instead ineffective as further confirmed in mouse models of infection and sepsis. Notably, AST performed in media mimicking host environments succeeded in identifying specific antibiotics that were effective in bacterial clearance and host survival, even though these same antibiotics failed in results using standard test media. Similarly, our revised media further identified antibiotics that were ineffective in vivo despite passing the AST standard for clinical use. Supplementation of AST medium with sodium bicarbonate, an abundant in vivo molecule that stimulates global changes in bacterial structure and gene expression, was found to be an important factor improving the predictive value of AST in the assignment of appropriate therapy. These findings have the potential to improve the means by which antibiotics are developed, tested, and prescribed.

Development of a Salmonella cross-protective vaccine for food animal production systems.

Intensive livestock production is associated with increased Salmonella exposure, transmission, animal disease, and contamination of food and water supplies. Modified live Salmonella enterica vaccines that lack a functional DNA adenine methylase (Dam) confer cross-protection to a diversity of salmonellae in experimental models of murine, avian, ovine, and bovine models of salmonellosis. However, the commercial success of any vaccine is dependent upon the therapeutic index, the ratio of safety/efficacy. Herein, secondary virulence-attenuating mutations targeted to genes involved in intracellular and/or systemic survival were introduced into Salmonella dam vaccines to screen for vaccine candidates that were safe in the animal and the environment, while maintaining the capacity to confer cross-protective immunity to pathogenic salmonellae serotypes. Salmonella dam mgtC, dam sifA, and dam spvB vaccine strains exhibited significantly improved vaccine safety as evidenced by the failure to give rise to virulent revertants during the infective process, contrary to the parental Salmonella dam vaccine. Further, these vaccines exhibited a low grade persistence in host tissues that was associated with reduced vaccine shedding, reduced environmental persistence, and induction of cross-protective immunity to pathogenic serotypes derived from infected livestock. These data indicate that Salmonella dam double mutant vaccines are suitable for commercial applications against salmonellosis in livestock production systems. Reducing pre-harvest salmonellae load through vaccination will promote the health and productivity of livestock and reduce contamination of livestock-derived food products, while enhancing overall food safety.