The regulatory network comprising ArcAB-RpoS-RssB influences motility in Vibrio cholerae.

The diarrheal disease cholera is caused by the versatile and responsive bacterium Vibrio cholerae, which is capable of adapting to environmental changes. Among others, the alternative sigma factor RpoS activates response pathways, including regulation of motility- and chemotaxis-related genes under nutrient-poor conditions in V. cholerae. Although RpoS has been well characterised, links between RpoS and other regulatory networks remain unclear. In this study, we identified the ArcAB two-component system to control rpoS transcription and RpoS protein stability in V. cholerae. In a manner similar to that seen in Escherichia coli, the ArcB kinase not only activates the response regulator ArcA but also RssB, the anti-sigma factor of RpoS. Our results demonstrated that, in V. cholerae, RssB is phosphorylated by ArcB, which subsequently activates RpoS proteolysis. Furthermore, ArcA acts as a repressor of rpoS transcription. Additionally, we determined that the cysteine residue at position 180 of ArcB is crucial for signal recognition and activity. Thus, our findings provide evidence linking RpoS response to the anoxic redox control system ArcAB in V. cholerae.

Deciphering the human antibody response against Burkholderia pseudomallei during melioidosis using a comprehensive immunoproteome approach.

Introduction: The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients. Methods and results: In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio. Conclusion: Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.

Vibrio cholerae's ToxRS bile sensing system.

The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae's rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies for example bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS's environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio's survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.

Cholera is a contagious diarrheal disease that leads to about 20,000 to 140,000 yearly deaths. It is caused by a bacterium called Vibrio cholerae, which can survive in harsh conditions and many environments. It often contaminates water, where it lives in an energy-conserving mode. But when humans consume Vibrio cholerae-contaminated water or food, the bacterium can sense its new environment and switch into a high-energy consuming state, causing fever, diarrhea, and vomiting. Vibrio cholerae recognizes bile acid in the human stomach, which signals that the bacterium has reached ideal conditions for causing disease. So far, it has been unclear, how exactly the bacterium detects bile acid. Understanding how these bacteria sense bile acid, could help scientists develop new ways to prevent cholera outbreaks or treat infections. Gubensäk et al. analysed two proteins from the Vibrio cholerae bacterium, called ToxR and ToxS, which are located below the bacteria's protective membrane. More detailed analyses showed that the two proteins bind together, forming a bile-binding pocket. When correctly assembled, this bile-sensing machine detects bile concentrations in the body, allowing the bacterium to adapt to the local conditions. Using crystal structures, a series of interaction studies, and modeling software, Gubensäk et al. detailed step-by-step how the two proteins sense bile acid and help the bacteria adapt and thrive in the human body. The results confirm the results of previous studies that implicated ToxR and ToxS in bile sensing and provide new details about the process. Scientists may use this information to develop new ways to interfere with the bacteria's bile-sensing and gut adaptation processes. They may also use the information to screen for existing drugs that block bile sensing and then test as cholera treatments or prevention strategies in clinical trials. New cholera treatment or prevention approaches that don't rely on antibiotics may help public health officials respond to growing numbers of cholera outbreaks and to prevent the spread of antibiotic-resistant bacteria.

Real-Time Nanopore Q20+ Sequencing Enables Extremely Fast and Accurate Core Genome MLST Typing and Democratizes Access to High-Resolution Bacterial Pathogen Surveillance.

Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches. In this study, we investigated whether novel nanopore Q20+ long-read chemistry enables standardized and easily accessible high-resolution typing combined with core genome multilocus sequence typing (cgMLST). We set high requirements for discriminatory power by using the slowly evolving bacterium Bordetella pertussis as a model pathogen. Our results show that the increased raw read accuracy enables the description of epidemiological scenarios and phylogenetic linkages at the level of gold-standard short reads. The same was true for our variant analysis of vaccine antigens, resistance genes, and virulence factors, demonstrating that nanopore sequencing is a legitimate competitor in the area of next-generation sequencing (NGS)-based high-resolution bacterial typing. Furthermore, we evaluated the parameters for the fastest possible analysis of the data. By combining the optimized processing pipeline with real-time basecalling, we established a workflow that allows for highly accurate and extremely fast high-resolution typing of bacterial pathogens while sequencing is still in progress. Along with advantages such as low costs and portability, the approach suggested here might democratize modern bacterial typing, enabling more efficient infection control globally.

Complete Genome Sequence of Klebsiella oxytoca Strain AHC-6, Isolated from a Patient during Acute Antibiotic-Associated Hemorrhagic Colitis.

Klebsiella oxytoca is a ubiquitous bacterium that is increasingly associated with inflammatory diseases. Here, we report the hybrid assembled genome for cytotoxic K. oxytoca strain AHC-6. The genome comprises a total of 5.7 Mbp, with a GC content of 55.2% and 5,258 coding sequences after assembly and annotation.

Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics.

Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.

Lower Levels of ABO Anti-A and Anti-B of IgM, IgG and IgA Isotypes in the Serum but Not the Saliva of COVID-19 Convalescents.

Individuals with ABO type O, naturally possessing anti-A and anti-B antibodies in their serum, are underrepresented among patients infected with SARS-CoV-2 compared with healthy controls. The ABO antibodies might play a role in the viral transmission. Therefore, we aimed to quantify anti-A/anti-B, including their subclasses IgM, IgG and IgA, in the serum and saliva of Caucasians (n = 187) after mild COVID-19 to compare them with individuals who had never been infected with SARS-CoV-2. Two samples were collected within two months after the diagnosis (median days: 44) and two months later. ABO antibodies were determined by flow cytometry. Additionally, total IgA in saliva and antibodies specific to SARS-CoV-2 were tested by ELISA. COVID-19 convalescents had significantly lower levels of anti-A/anti-B IgM, IgG and IgA in their serum than control subjects (p < 0.001). Interestingly, no significant differences were observed in saliva. ABO antibody levels remained stable over the period considered. No relation of ABO to the level of SARS-CoV-2-specific antibodies was observed. Total IgA was lower in convalescents than in controls (p = 0.038). Whereas ABO antibodies in the saliva may not contribute to the pathogenesis of COVID-19, individual pre-existing high serum concentrations of anti-A/anti-B may have a protective effect against SARS-CoV-2 infection.

Environmental Factors Associated With Soil Prevalence of the Melioidosis Pathogen Burkholderia pseudomallei: A Longitudinal Seasonal Study From South West India.

Melioidosis is a seasonal infectious disease in tropical and subtropical areas caused by the soil bacterium Burkholderia pseudomallei. In many parts of the world, including South West India, most cases of human infections are reported during times of heavy rainfall, but the underlying causes of this phenomenon are not fully understood. India is among the countries with the highest predicted melioidosis burden globally, but there is very little information on the environmental distribution of B. pseudomallei and its determining factors. The present study aimed (i) to investigate the prevalence of B. pseudomallei in soil in South West India, (ii) determine geochemical factors associated with B. pseudomallei presence and (iii) look for potential seasonal patterns of B. pseudomallei soil abundance. Environmental samplings were performed in two regions during the monsoon and post-monsoon season and summer from July 2016 to November 2018. We applied direct quantitative real time PCR (qPCR) together with culture protocols to overcome the insufficient sensitivity of solely culture-based B. pseudomallei detection from soil. A total of 1,704 soil samples from 20 different agricultural sites were screened for the presence of B. pseudomallei. Direct qPCR detected B. pseudomallei in all 20 sites and in 30.2% (517/1,704) of all soil samples, whereas only two samples from two sites were culture-positive. B. pseudomallei DNA-positive samples were negatively associated with the concentration of iron, manganese and nitrogen in a binomial logistic regression model. The highest number of B. pseudomallei-positive samples (42.6%, p < 0.0001) and the highest B. pseudomallei loads in positive samples [median 4.45 × 103 genome equivalents (GE)/g, p < 0.0001] were observed during the monsoon season and eventually declined to 18.9% and a median of 1.47 × 103 GE/g in summer. In conclusion, our study from South West India shows a wide environmental distribution of B. pseudomallei, but also considerable differences in the abundance between sites and within single sites. Our results support the hypothesis that nutrient-depleted habitats promote the presence of B. pseudomallei. Most importantly, the highest B. pseudomallei abundance in soil is seen during the rainy season, when melioidosis cases occur.

Development of a Rapid Live SARS-CoV-2 Neutralization Assay Based on a qPCR Readout.

Measuring SARS-CoV-2 neutralizing antibodies after vaccination or natural infection remains a priority in the ongoing COVID-19 pandemic to determine immunity, especially against newly emerging variants. The gold standard for assessing antibody-mediated immunity against SARS-CoV-2 are cell-based live virus neutralization assays. These assays usually take several days, thereby limiting test capacities and the availability of rapid results. In this study, therefore, we developed a faster live virus assay, which detects neutralizing antibodies through the early measurement of antibody-mediated intracellular virus reduction by SARS-CoV-2 qRT-PCR. In our assay, Vero E6 cells are infected with virus isolates preincubated with patient sera and controls. After 24 h, the intracellular viral load is determined by qRT-PCR using a standard curve to calculate percent neutralization. Utilizing COVID-19 convalescent-phase sera, we show that our novel assay generates results with high sensitivity and specificity as we detected antiviral activity for all tested convalescent-phase sera, but no antiviral activity in prepandemic sera. The assay showed a strong correlation with a conventional virus neutralization assay (rS = 0.8910), a receptor-binding domain ELISA (rS = 0.8485), and a surrogate neutralization assay (rS = 0.8373), proving that quantifying intracellular viral RNA can be used to measure seroneutralization. Our assay can be adapted easily to new variants, as demonstrated by our cross-neutralization experiments. This characteristic is key for rapidly determining immunity against newly emerging variants. Taken together, the novel assay presented here reduces turnaround time significantly while making use of a highly standardized and sensitive SARS-CoV-2 qRT-PCR method as a readout.

Open-Source, Adaptable, All-in-One Smartphone-Based System for Quantitative Analysis of Point-of-Care Diagnostics.

Point-of-care (POC) diagnostics, in particular lateral flow assays (LFA), represent a great opportunity for rapid, precise, low-cost and accessible diagnosis of disease. Especially with the ongoing coronavirus disease 2019 (COVID-19) pandemic, rapid point-of-care tests are becoming everyday tools for identification and prevention. Using smartphones as biosensors can enhance POC devices as portable, low-cost platforms for healthcare and medicine, food and environmental monitoring, improving diagnosis and documentation in remote, low-resource locations. We present an open-source, all-in-one smartphone-based system for quantitative analysis of LFAs. It consists of a 3D-printed photo box, a smartphone for image acquisition, and an R Shiny software package with modular, customizable analysis workflow for image editing, analysis, data extraction, calibration and quantification of the assays. This system is less expensive than commonly used hardware and software, so it could prove very beneficial for diagnostic testing in the context of pandemics, as well as in low-resource countries.

Toxin-Producing Klebsiella oxytoca in Healthy Infants: Commensal or Pathobiont?

OBJECTIVES: Klebsiella oxytoca is a gastrointestinal pathobiont with the potential to produce the toxins tilivalline and tilimycin, which cause antibiotic-associated hemorrhagic colitis. Overgrowth of toxigenic K oxytoca has recently been implicated in necrotizing enterocolitis. K oxytoca colonizes 2-9% of healthy adults, however, there is no systematic data on colonization in healthy children. We investigated K oxytoca colonization and its toxigenic properties in healthy infants. METHODS: We sampled stool of healthy infants and determined K oxytoca colonization using stool culture and PCR (pehX). Toxin in stool was measured with HPLC/high-resolution mass spectrometry. K oxytoca isolates were typed using multi-locus sequence typing (MLST) and K oxytoca toxin PCR (npsA/B). Cytotoxin production of isolates was analyzed by MTT assay. RESULTS: K oxytoca was detected in 30 of 61 infants (49%) using stool culture and in 45 of 61 (73%) using PCR (pehX). Toxin marker PCR (npsA/B) was positive in 66% of stool samples positive for K oxytoca PCR. Stool toxin levels were too low for quantitation but traces of tilivalline were detected. Contrarily, 49% of K oxytoca isolates demonstrated toxicity in the MTT assay. MLST revealed 36 distinct sequence types affiliated with all known K oxytoca sequence type clusters (A, B1 and B2). CONCLUSIONS: More than 70% of healthy infants were colonized with K oxytoca. Toxin quantities in stool of colonized healthy infants were below detection level, yet half of the isolates produced toxin in vitro demonstrating their pathobiont potential. The high occurrence of toxigenic K oxytoca in healthy infants has to be considered for future disease association studies.

A Novel High-Throughput Nanopore-Sequencing-Based Strategy for Rapid and Automated S-Protein Typing of SARS-CoV-2 Variants.

Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics capacities. In this study, we aimed to establish a rapid and user-friendly protocol for high-throughput S-gene sequencing and subsequent automated identification of variants. We designed two new primer pairs to amplify only the immunodominant part of the S-gene for nanopore sequencing. Furthermore, we developed an automated "S-Protein-Typer" tool that analyzes and reports S-protein mutations on the amino acid level including a variant of concern indicator. Validation of our primer panel using SARS-CoV-2-positive respiratory specimens covering a broad Ct range showed successful amplification for 29/30 samples. Restriction to the region of interest freed sequencing capacity by a factor of 12-13, compared with whole-genome sequencing. Using either the MinION or Flongle flow cell, our sequencing strategy reduced the time required to identify SARS-CoV-2 variants accordingly. The S-Protein-Typer tool identified all mutations correctly when challenged with our sequenced samples and 50 deposited sequences covering all VOCs (December 2021). Our proposed S-protein variant screening offers a simple, more rapid, and low-cost entry into NGS-based SARS-CoV-2 analysis, compared with current whole-genome approaches.

Structural and DNA-binding properties of the cytoplasmic domain of Vibrio cholerae transcription factor ToxR.

ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a "DNA-catcher" to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.

RegAB Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence.

Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain its physiology and virulence under hypoxia, but the crucial regulatory networks employed to switch from oxygen dependent respiration to alternative terminal electron acceptors (TEA) like nitrate, remains poorly understood. Here, we combined a Tn5 transposon mutagenesis screen and an anaerobic growth screen to identify a two-component signal transduction system with homology to RegAB. We show that RegAB is not only essential for anaerobic growth, but also for full virulence in cell lines and a mouse infection model. Further investigations of the RegAB regulon, using a global transcriptomic approach, identified 20 additional regulators under transcriptional control of RegAB, indicating a superordinate role of RegAB in the B. pseudomallei anaerobiosis regulatory network. Of the 20 identified regulators, NarX/L and a FNR homolog were selected for further analyses and a role in adaptation to anaerobic conditions was demonstrated. Growth experiments identified nitrate and intermediates of the denitrification process as the likely signal activateing RegAB, NarX/L, and probably of the downstream regulators Dnr or NsrR homologs. While deletions of individual genes involved in the denitrification process demonstrated their important role in anaerobic fitness, they showed no effect on virulence. This further highlights the central role of RegAB as the master regulator of anaerobic metabolism in B. pseudomallei and that the complete RegAB-mediated response is required to achieve full virulence. In summary, our analysis of the RegAB-dependent modulon and its interconnected regulons revealed a key role for RegAB of B. pseudomallei in the coordination of the response to hypoxic conditions and virulence, in the environment and the host.

Development and Validation of a Burkholderia pseudomallei Core Genome Multilocus Sequence Typing Scheme To Facilitate Molecular Surveillance.

Burkholderia pseudomallei causes the severe disease melioidosis. Whole-genome sequencing (WGS)-based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills, and variable bioinformatic approaches, with the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,351 accessory targets. The scheme was validated with 320 WGS data sets. We compared our novel typing scheme with single nucleotide polymorphism-based approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigating not only closely related strains but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugarcane field as the presumable source of a recent melioidosis case. In summary, we developed a robust cgMLST scheme that integrates high resolution, maximized standardization, and fast analysis for the nonbioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

The periplasmic domains of Vibriocholerae ToxR and ToxS are forming a strong heterodimeric complex independent on the redox state of ToxR cysteines.

The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases.

Burkholderia pseudomallei triggers canonical inflammasome activation in a human primary macrophage-based infection model.

Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1ß release, and caspase-1 and gasdermin D processing. IFN-γ, known to promote non-canonical inflammasome activation, did not influence pyroptosis induction or IL-1ß release from infected primary human macrophages. Nevertheless, it reduced intracellular B. pseudomallei loads, an effect which was partially antagonist by the inhibition of NADPH oxidase. Overall, our data implicate T3SS-3 dependent inflammasome activation and IFN-γ induced immune mechanisms as critical defense mechanisms of human macrophages against B. pseudomallei. In addition, our infection model provides a versatile tool to study human host-pathogen interactions and has the potential to elucidate the role of human individual genetic variations in B. pseudomallei infections.

An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020.

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites.

Melioidosis DS rapid test: A standardized serological dipstick assay with increased sensitivity and reliability due to multiplex detection.

BACKGROUND: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. METHODS AND PRINCIPAL FINDINGS: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97-100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. CONCLUSIONS: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts.

Monitoring fast chemical processes by reaction-interrupted excitation transfer (ExTra) NMR spectroscopy.

NMR spectroscopy is generally used to investigate molecules under equilibrium conditions. Despite recent technological and methodogical developments to study on-going reactions, tracing the fate of individual atoms during an irreversible chemical reaction is still a challenging and elaborate task. Reaction-interrupted excitation transfer (ExTra) NMR provides a selective tracking of resonances from atoms, which undergo chemical conversion. We show that reactions triggered either by rapid mixing or by photo-excitation can be conveniently followed at a sub-second time scale using standard NMR equipment. In ExTra NMR we use the selectively inverted magnetization of a selected atom to follow its conversion in the course of a fast chemical reaction. The chemical reaction has to be started within the relaxation period of an initial inverting 180° pulse. The presented protocol provides a generally applicable NMR method for reaction monitoring.