NHP BurkPx: A multiplex serodiagnostic bead assay to monitor Burkholderia pseudomallei exposures in non-human primates.

BACKGROUND: Melioidosis is a disease caused by the bacterium Burkholderia pseudomallei, infecting humans and non-human primates (NHP) through contaminated soil or water. World-wide there are an estimated 165,000 human melioidosis cases each year, but recordings of NHP cases are sporadic. Clinical detection of melioidosis in humans is primarily by culturing B. pseudomallei, and there are no standardized detection protocols for NHP. NHP are an important animal model for melioidosis research including clinical trials and development of biodefense countermeasures. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the diagnostic potential of the multiple antigen serological assay, BurkPx, in NHP using two sera sets: (i) 115 B. pseudomallei-challenged serum samples from 80 NHP collected each week post-exposure (n = 52) and at euthanasia (n = 47), and (ii) 126 B. pseudomallei-naïve/negative serum samples. We observed early IgM antibody responses to carbohydrate antigens followed by IgG antibody recognition to multiple B. pseudomallei protein antigens during the second week of infection. B. pseudomallei negative serum samples had low to intermediate antibody cross reactivity to the antigens in this assay. Infection time was predicted as the determining factor in the variation of antibody responses, with 77.67% of variation explained by the first component of the principal component analysis. A multiple antigen model generated a binary prediction metric ([Formula: see text]), which when applied to all data resulted in 100% specificity and 63.48% sensitivity. Removal of week 1 B. pseudomallei challenged serum samples increased the sensitivity of the model to 95%. CONCLUSION/SIGNIFICANCE: We employed a previously standardized assay for humans, the BurkPx assay, and assessed its diagnostic potential for detection of B. pseudomallei exposure in NHP. The assay is expected to be useful for surveillance in NHP colonies, in investigations of suspected accidental releases or exposures, and for identifying vaccine correlates of protection.

Acute Respiratory Distress in Aged, SARS-CoV-2-Infected African Green Monkeys but Not Rhesus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a wide range of disease severity, ranging from asymptomatic infection to a life-threating illness, particularly in the elderly population and individuals with comorbid conditions. Among individuals with serious coronavirus 2019 (COVID-19) disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. Animal models of SARS-CoV-2 infection that manifest severe disease are needed to investigate the pathogenesis of COVID-19-induced ARDS and evaluate therapeutic strategies. We report two cases of ARDS in two aged African green monkeys (AGMs) infected with SARS-CoV-2 that had pathological lesions and disease similar to severe COVID-19 in humans. We also report a comparatively mild COVID-19 phenotype characterized by minor clinical, radiographic, and histopathologic changes in the two surviving, aged AGMs and four rhesus macaques (RMs) infected with SARS-CoV-2. Notable increases in circulating cytokines were observed in three of four infected, aged AGMs but not in infected RMs. All the AGMs had increased levels of plasma IL-6 compared with baseline, a predictive marker and presumptive therapeutic target in humans infected with SARS-CoV-2. Together, our results indicate that both RMs and AGMs are capable of modeling SARS-CoV-2 infection and suggest that aged AGMs may be useful for modeling severe disease manifestations, including ARDS.

Predictive Capacity of Oxygen Delivery During Cardiopulmonary Bypass on Acute Kidney Injury.

BACKGROUND: The randomized goal-directed perfusion trial confirmed retrospective findings that a goal-directed perfusion strategy to maintain oxygen delivery index (DO2i) during cardiopulmonary bypass greater than 280 mL/min/m2 reduces the incidence of acute kidney injury (AKI). We developed a predictive model for AKI using data from the Australian and New Zealand Collaborative Perfusion Registry to determine whether these findings could be validated in a real-world clinical setting and to identify an optimal DO2i threshold for predictive diagnostic accuracy. METHODS: Data in 19,410 cardiopulmonary bypass procedures were randomly divided into training (n = 9705) and validation (n = 9705) datasets. Multivariate logistic regression was used to determine the best predictive models for AKI (RIFLE [renal Risk, Injury, Failure, Loss of renal function and End-stage renal disease] classification), incremental predictive value of minimum cardiopulmonary bypass DO2i, and optimal threshold. RESULTS: Minimum DO2i was significantly associated with any AKI, AKI risk, and AKI injury or greater class in both datasets (validation dataset; any AKI odds ratio [OR], 0.993; 95% confidence interval [CI], 0.991-0.995; P < .001; AKI risk OR, 0.994; 95% CI, 0.992-0.996; P < .001, AKI injury or greater 0.993; 95% CI, 0.991-0.996; P < .001), representing on average a 7% increase in the likelihood of AKI for every 10-mL/min/m2 decrease in DO2i. Diagnostic accuracy was similar for both datasets, with an optimal DO2i threshold of 270 mL/min/m2. The odds of any AKI were increased by 52% in those below the threshold (OR, 1.52; 95% CI, 1.29-1.77; P < .001). CONCLUSIONS: This study confirms previous findings that minimum DO2i during cardiopulmonary bypass is independently associated with AKI, supporting previous findings in a broader-risk, multicenter cohort.

Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease.

The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples from Borrelia burgdorferi-infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection of B. burgdorferi antigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease.

Bioassays of quorum sensing compounds using Agrobacterium tumefaciens and Chromobacterium violaceum.

In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

A previously uncharacterized gene, yjfO (bsmA), influences Escherichia coli biofilm formation and stress response.

Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical stresses than their unattached, planktonic counterparts. Using transcriptional profiling and quantitative PCR, we observed a previously uncharacterized gene, yjfO to be upregulated during Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfO mutant, developed through targeted-insertion mutagenesis, and a yjfO-complemented strain, were obtained for further characterization. While bacterial surface colonization levels (c.f.u. cm(-2)) were similar in all three strains, the mutant strain exhibited reduced microcolony formation when observed in flow cells, and greatly enhanced flagellar motility on soft (0.3 %) agar. Complementation of yjfO restored microcolony formation and flagellar motility to wild-type levels. Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat stress. Planktonic cultures from all three strains showed similar responses to these stresses. Regardless of the presence of yjfO, planktonic E. coli withstood alkali stress better than biofilm populations. Complementation of yjfO restored viability following exposure to peroxide stress, but did not restore acid resistance. Based on its influence on biofilm maturation and stress response, and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm stress and motility).

Soil factors controlling the toxicity of copper and zinc to microbial processes in Australian soils.

Abstract-Two soil microbial processes, substrate-induced nitrification (SIN) and substrate-induced respiration (SIR), were measured in the topsoils of 12 Australian field trials that were amended separately with increasing concentrations of ZnSO4 or CuSO4. The median effect concentration (EC50) values for Zn and Cu based on total metal concentrations varied between 107 and 8,298 mg kg(-1) for Zn and 108 and 2,155 mg kg(-1) Cu among soils. The differences in both Zn and Cu toxicity across the 12 soils were not explained by either the soil solution metal concentrations or CaCl2-extractable metal concentrations, because the variation in the EC50 values was larger than those using total concentrations. Toxicity of Zn and Cu decreased with increasing soil pH for SIN. For Cu, also increasing cation exchange capacity (CEC) and percent clay decreased the toxicity towards SIN. In contrast to SIN, soil pH had no significant effect on toxicity values of SIR. Significant relationships were found between the EC50 values for SIR and background Zn and CEC for Zn, and percent clay and log CEC for Cu. Relationships such as those developed in this study will permit Australian environmental regulation to move from single-value national soil quality guidelines to soil-specific quality guidelines and permit soil-specific risk assessments to be undertaken.

Cell-cell influences on bacterial community development in aquatic biofilms.

Dialysis tubing containing spent culture media, when placed in a lake, was colonized by a low diversity of bacteria, whereas abiotic controls had considerable diversity. Changes were seen in the presence and absence of acylated homoserine lactones, suggesting that these molecules and other factors may influence adherent-population composition.