Biology subjects

Brugger, S. D.

Publications and source records attributed to Brugger, S. D..

18 recordsLinked to original sources

bioRxiv2026

Inhibition of multidrug-resistant Staphylococcus aureus by commensal bacterial species from the human nose

Staphylococcus aureus is an important human pathogen. With the growing threat of antimicrobial resistance, exemplified by methicillin-resistant S. aureus (MRSA) infections requiring last-line antibiotics, alternative strategies to combat S. aureus and MRSA colonisation are needed. One promising direction is to harness interactions between S. aureus and commensal bacteria resident in the nasal passage. However, it remains unclear which bacterial species or combinations are most effective at inhibiting S. aureus, and whether this inhibition is affected by resistance to last-line antibiotics. Here, we address this gap using systematic in vitro experiments to identify commensal species or combinations that inhibit MRSA, including strains resistant to key last-resort therapies. In a combinatorial screen, we detected widespread inhibition of MRSA by commensal species, with inhibitory strength dependent on species identity and community composition. Staphylococcal commensal species produced the strongest MRSA inhibition, but we achieved similarly strong inhibition by combining Corynebacterium species with Dolosigranulum pigrum. This was linked to positive growth interactions between commensals. Importantly, we observed similar levels of MRSA inhibition by commensal species when we tested MRSA strains resistant to key anti-MRSA antibiotics, including clinical isolates. Inhibition was sustained during serial passaging experiments, with minimal MRSA adaptation upon prolonged exposure. Our results show defined combinations of nasal commensals can robustly inhibit MRSA, including strains resistant to last-resort antibiotics. This supports the potential of microbiota-based therapies to prevent or eliminate S. aureus colonisation from the human nose, including in the context of antibiotic resistance.

microbiology↗

bioRxiv2026

Nanopore metagenomic sequencing links clinically relevant resistance determinants to pathogens

Culture-independent metagenomics enables the detection of plasmid-encoded antimicrobial resistance (AMR) genes directly from clinical samples; however, the clinical significance of these genes depends on their bacterial host and genomic context, which metagenomics cannot fully infer. Nanopore sequencing technology intrinsically encodes epigenetic modifications such as methylation, which can be leveraged for plasmid-host associations from metagenomic data. Existing methods rely on the recovery of metagenome-assembled genomes (MAGs), which can introduce bias toward abundant taxa and leave clinically relevant, low-abundance pathogens unassociated. To address this limitation, we extended methylation-based plasmid-host association from the MAG level to individual assembly contigs and sequencing reads. The CUPID pipeline implements the calculation of contig and read similarity scores, which compare weighted mean methylation rates across motifs genetically shared between any contig or read pair. We validated this approach on a mock metagenomic community composed of ten carbapenem-resistant Enterobacterales isolates, where we achieved 93.8% accuracy at the contig level and 100% at the read level for carbapenemase plasmid-host associations. When applied to metagenomic and quasimetagenomic data of sixteen patient rectal swabs collected during routine hospital surveillance, our approach assigned every detected plasmid-encoded carbapenemase to its correct bacterial host at the contig level, using matched culture-based diagnostics and whole-genome sequencing as a ground truth. Read-level analysis identified additional associations that were missed at the contig level, including a multi-host plasmid confirmed by established diagnostics. These findings demonstrate a pathway from rapid AMR gene detection using metagenomics to actionable surveillance for infection prevention, transmission tracing, and outbreak investigation. Impact statementCulture-independent metagenomics can detect antimicrobial resistance genes, but their clinical significance depends on the bacterial host and genomic context. Here, we show that nanopore-derived bacterial DNA methylation patterns can link carbapenemase genes to pathogenic hosts and plasmid context directly from patient samples. This provides a route from rapid antimicrobial resistance gene detection to actionable public health surveillance. Data summaryAll sequencing data after human content filtering have been deposited at the European Nucleotide Archive (ENA, BioProject accession PRJEB108076, with all isolate sequencing data for mock community generation available under the sample accession numbers SAMEA121375149-58, all isolate sequencing data from the rectal swabs available at SAMEA121334008-24, all metagenomic data from the rectal swabs available at SAMEA121325220-27, and all quasimetagenomic data available at SAMEA122914816-23, SAMEA122920068-74). All code is available at GitHub: https://github.com/harikaurel/cupid. All other supporting data are provided in the article and supplementary tables.

genomics↗

bioRxiv2026

Individual bacterial taxa drive colonisation resistance to methicillin-resistant Staphylococcus aureus in human nasal microbiome samples

Identifying bacterial interactions that determine susceptibility of human microbiomes to colonisation by pathogenic bacteria has important implications for understanding health and disease and, consequently, improving treatment and prevention. Here, we show how microbiome composition of healthy-human nasal passage samples determines susceptibility to colonisation by methicillin-resistant Staphylococcus aureus (MRSA) using a replicated microcosm approach. We find that variable MRSA population growth among samples from different individuals is associated with differences in microbial community composition. To identify individual taxa contributing to in vitro colonisation resistance, we isolate bacteria from inhibitory samples and measure their effects on MRSA in co-culture. This reveals strong MRSA inhibition by multiple Enterobacteriaceae isolates, including some which fully suppress MRSA growth in our system. Finally, by assembling nasal model communities reflecting the composition of natural nasal microbiome samples and combining drop-in and drop-out designs, we show that individual taxa can drive community-level resistance to MRSA growth. Together, our results demonstrate causal links between taxonomic composition of nasal microbiome samples and resistance to S. aureus, informing potential new microbiome-targeted interventions to manage S. aureus colonisation.

microbiology↗

bioRxiv2025

Cross-scale effect of microbiota dynamics on resistance

Antimicrobial resistance (AMR) is often carried asymptomatically in the human gut microbiota, yet the role of this carriage in AMR persistence and transmission remains poorly understood. To address this gap, we developed a cross-scale model that links within-host microbiota dynamics of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing bacteria with transmission across hospital and community settings. The model incorporates plasmid exchange, antibiotic effects, and host heterogeneity. It integrates diverse data sources, including microbiota dynamics, community antibiotic consumption, and detailed surveillance data from a large university hospital, complemented by literature estimates. This model identifies mechanisms that enable coexistence of resistant and sensitive strains at both individual and population levels, revealing host heterogeneity as a critical driver of resistance persistence. Unlike classic epidemiological models, our analysis finds a continuum of colonization states within individuals rather than discrete resistant or sensitive categories, and highlights that realistic detection thresholds may substantially underestimate resistance prevalence. Finally, we use this framework to evaluate interventions such as hospital screening and isolation, showing that their impact strongly depends on local transmission dynamics and resistance prevalence. By bridging within-host and population processes, our model provides a novel tool for investigating AMR spread and optimizing prevention strategies in healthcare and community environments.

ecology↗

bioRxiv2025

Fsr quorum sensing system restricts biofilm growth and activates inflammation in enterococcal infective endocarditis

Infective endocarditis (IE) is a life-threatening biofilm-associated infection, yet the factors driving biofilm formation remain poorly understood. Here, we identified the Fsr quorum sensing (QS) system of Enterococcus faecalis as a potent negative regulator of IE pathogenesis. Using microfluidic and in vivo models, we show that Fsr is induced in late IE when bacteria become shielded from blood flow. Deleting Fsr altered biofilm metabolism and promoted robust biofilm growth and gentamicin tolerance in vivo. Furthermore, Fsr inactivation attenuated inflammation by disrupting IL-1{beta} cleavage and activation via the Fsr-regulated gelatinase (gelE), allowing biofilm to grow unchecked by the immune system. Consistent with our pre-clinical findings, analysis of two IE patient cohorts linked naturally occurring Fsr-deficient E. faecalis to prolonged bacteremia. Overall, our findings provide insights into the role of QS in biofilm growth, persistence, and immune evasion in enterococcal IE.

microbiology↗

bioRxiv2023

Group A Streptococcus strains causing meningitis without distinct invasive phenotype

Group A streptococcal (GAS, aka Streptococcus pyogenes) meningitis is a fulminant disease associated with high morbidity and mortality. To elucidate the mechanisms underlying the invasiveness of GAS in meningitis, we compared GAS isolates derived from five cases of meningitis, to otitis and colonizing isolates. We did not observe differences in adherence to and invasion of human brain microvascular endothelial cells, virulence factors activity or barrier disruption. Whole genome sequencing did not reveal particular invasiveness traits. Most patients previously suffered from otitis media suggesting that meningitis likely resulted from a continuous spread of the infection rather than being attributable to changes in pathogens virulence.

microbiology↗

bioRxiv2022

Developing and optimizing a personalized probiotic treatment regime for nasal endogenous Staphylococcus aureus decolonization

AbstractDespite the increasing burden of antibiotic resistance and persistence, current approaches to eradicate nasal pathobionts such as Staphylococcus aureus and Streptococcus pneumoniae are based on the use of antibacterial agents. An alternative approach is the artificial inoculation of commensal bacteria, i.e., probiotic treatment, which is supported by the increasing evidence for commensal-mediated inhibition of pathogens. To systematically investigate the potential and the limitations of this approach, we developed a quantitative framework simulating the dynamics of the nasal bacterial microbiome by combining mathematical modeling with longitudinal microbiota data. By inferring the microbial interaction parameters using 16S rRNA amplicon sequencing data and simulating the nasal microbial dynamics of patients colonized with S. aureus, we compared the decolonization performance of probiotic and antibiotic treatments under different assumptions on patients bacterial community composition and susceptibility profile. To further compare the robustness of these treatments, we simulated a S. aureus challenge following each treatment and quantified the recolonization probability. Eventually, using nasal swabs of adults colonized with S. aureus, we confirmed that after antibiotic treatment, recolonization of S. aureus was inhibited in samples treated with a probiotic mixture compared to the non-treated control. Our results suggest that probiotic treatment clearly outperforms antibiotics in terms of decolonization performance, recolonization robustness, and leads to less collateral reduction of the microbiome diversity. Moreover, we find that recolonization robustness is highest in those patients that were not initially colonized by Dolosigranulum pigrum. Thus, probiotic treatment may provide a promising alternative to combat antibiotic resistance, with the additional advantage of personalized treatment options via using the patients own metagenomic data to tailor the intervention. The combination of an in silico framework with in vitro confirmatory experiments using clinical samples reported in this work is an important step forward to further investigate this alternative in clinical trials. ImportanceThe development of new antimicrobial agents is declining while antibiotic resistance is rising, which is particularly concerning for upper respiratory tract pathogens S. pneumoniae and S. aureus. Combating such resistant infections will only become more challenging unless alternative treatment strategies are explored. Despite the accumulating evidence on using commensal bacteria for pathobiont decolonization, it is still not commonly practiced. To investigate the potential of commensal-mediated inhibition of pathogens systematically, we developed a quantitative framework describing the dynamics of the nasal microbiome by merging mathematical modeling and metagenomic data. We show that probiotic treatment outperforms antibiotics regarding decolonization performance and recolonization robustness while preserving the microbiome diversity with the additional advantage of personalized treatment options via using the patients own microbiota data. Moreover, we validated the approach by using nasal swabs from adults with nasal S. aureus colonization, demonstrating that probiotic treatment prevents recolonization with S. aureus in vitro. The framework developed in this work is an important step forward for the translation of experimental and clinical data into mainstream clinical practice in a systematic and controlled manner.

microbiology↗

bioRxiv2022

Group A Streptococcus antibiotic tolerance in necrotizing fasciitis

ObjectivesGroup A Streptococcus (GAS) necrotizing fasciitis (NF) is a difficult-to-treat bacterial infection associated with high morbidity and mortality despite extensive surgery and targeted antibiotic treatment. Bacteria surviving prolonged antibiotic exposure without displaying genetic resistance, so-called persisters, are associated with difficult-to-treat infections, such as GAS-NF. In the present study, we investigated the presence of persistent GAS in tissue freshly debrided from three NF patients and examined more in depth the persisters phenomenon in GAS-NF clinical isolates. MethodsTime-lapse imaging of freshly isolated GAS-NF clinical isolates, the image analysis software ColTapp and antibiotic challenge-based persisters assays were used to assess the presence of persisters. ResultsWe show for the first time that GAS recovered directly from freshly debrided NF patients tissue are characterized by increased colony appearance time heterogeneity, indicating the presence of persisters. Acidic pH or nutrient stress exposure, mimicking the NF-like environment in vitro, similarly leads to phenotypic heterogeneity resulting in enhanced antibiotic survival and confirming the presence of GAS persisters. ConclusionsGAS persisters are present in the tissue freshly debrided from GAS-NF patients and might be one explanation for antibiotic treatment failure and surgery requirement in GAS-NF. Tailored treatment options, including the use of persisters-targeting drugs, need to be developed to increase GAS-NF therapy success.

microbiology↗

bioRxiv2022

Rapid detection of Staphylococcus aureus and Streptococcus pneumoniae by real-time analysis of volatile metabolites

Rapid detection of pathogenic bacteria is needed for rapid diagnostics allowing adequate and timely treatment. In this study, we aimed to evaluate the technical feasibility of Secondary Electro-Spray Ionization-High Resolution Mass Spectrometry (SESI-HRMS) as a diagnostic tool for rapid detection of bacterial infections and compare its performance with the current standard of diagnostics. We compared the time required to confirm growth of the pathogenic bacteria Staphylococcus aureus and Streptococcus pneumoniae by conventional detection by culture and MAL-DI-TOF vs. detection of specific volatile organic compounds (VOCs) produced by these human pathobionts. SESI-HRMS could consistently detect VOCs produced by S. aureus or S. pneumoniae on blood agar plates within minutes, allowing to positively identify bacteria within hours. Unique S. aureus and S. pneumoniae features were detected already at bacterial densities as low as [~]103 colony forming units. Rich mass spectral fingerprints allowed for the distinction of these two bacteria on a species and even strain level. To give an incentive towards clinical application of this technology, further analyzed 17 clinical samples previously diagnosed by conventional methods. We predominantly obtained a separation of samples which showed growth (i.e. presence of living bacteria) compared to samples with no bacterial growth (i.e. presence of dead bacteria). We conclude that SESI-HRMS allows rapid identification of unique bacterial features. Further development of real-time analysis of clinical samples by SESI-HRMS will shorten the time required for microbiological diagnosis with a high level of confidence and sensitivity and should help to improve patients tailored treatment. IMPORTANCEA timely identification of a pathogenic bacteria causing the infection is of pivotal importance for the initiation of an adequate antimicrobial therapy. In this regard, different technologies have been developed with the aim to achieve a highly reliable, specific, and overall fast identification of pathogenic bacteria. However, conventional diagnostic techniques still require long preprocessing times (hours to days) to acquire enough biological material for an accurate identification of the pathogen. Therefore, in this work, we aimed to further shorten the detection time of current gold standards for microbiological diagnostics by providing a system capable of a fast, sensitive and specific discrimination of different pathogenic bacteria. This system relies on the real-time mass spectrometric detection of volatile organic compounds (VOCs) produced by a given organism during its growth, potentially leading to a significant shortening of the time required to obtain a positive reliable diagnostic.

microbiology↗

bioRxiv2022

Intervertebral disc cell chondroptosis elicits neutrophil response in Staphylococcus aureus spondylodiscitis

ObjectiveTo understand the pathophysiology of spondylodiscitis due to Staphylococcus aureus, an emerging infectious disease of the intervertebral disc (IVD) and vertebral body with a high complication rate, by combining clinical insights and experimental approaches. DesignClinical data and histological material of nine patients suffering from S. aureus spondylodiscitis were retrospectively collected at a single center. To mirror the clinical findings experimentally, we developed a novel porcine ex vivo model mimicking acute S. aureus spondylodiscitis and assessed the interaction between S. aureus and IVD cells within their native environment. In addition, the inflammatory features underlying this interaction were assessed in primary human IVD cells. Finally, mirroring the clinical findings, we assessed primary human neutrophils for their ability to respond to secreted inflammatory modulators of IVD cells upon S. aureus challenge. ResultsAcute S. aureus spondylodiscitis in patients was characterized by tissue necrosis and neutrophil infiltration. Additionally, the presence of empty IVD cells lacunae was observed. This was mirrored in the ex vivo porcine model, where S. aureus induced extensive IVD cell death, leading to empty lacunae. Concomitant engagement of the apoptotic and pyroptotic cell death pathways was observed in primary human IVD cells, resulting in cytokine release. Among the released cytokines, functionally intact neutrophil-priming as well as broad pro- and anti-inflammatory cytokines known for their involvement in IVD degeneration were found. ConclusionsIn patients as well as ex vivo in a novel porcine model, S. aureus spondylodiscitis infection caused IVD cell death, resulting in empty lacunae, which was accompanied by release of inflammation markers and recruitment of neutrophils. These findings offer valuable insights into the important role of inflammatory IVD cell death during the onset of spondylodiscitis and potential future therapeutic approaches.

immunology↗

bioRxiv2021

Limited adaptive evolution of Staphylococcus aureus during transition from colonization to invasive infection

Staphylococcus aureus carriage is a risk factor for invasive infections. Unique genetic elements favoring the transition from colonizing to invasive phenotype have not yet been identified and phenotypic traits are understudied. We therefore assessed pheno- and genotypic profiles of eleven S. aureus isolate pairs sampled from colonized patients simultaneously suffering from invasive S. aureus infections. Ten out of 11 isolate pairs presented the same spa and MLST suggesting colonization as origin for the invasive infection. Systematic analysis of colonizing and invasive isolate pairs showed similar adherence, hemolysis and reproductive fitness properties, minimal genetic differences, identical antibiotic tolerance and bacterial virulence in Galleria mellonella. Our results provide insights into the similar phenotype associated with limited adaptive genetic evolution between the colonizing and invasive isolates. Disruption of the physical barriers mucosa or skin were identified in the majority of patients further emphasizing colonization as a major risk factor for invasive disease. ImportanceS. aureus is a major pathogen of humans causing a wide range of disease. The failure to develop a vaccine and antibiotic warrant the exploration of novel treatment strategies. Asymptomatic colonization of the human nasal passages is a major risk factor for invasive disease and decolonization procedures have shown efficacy in preventing invasive infections. However, the transition of S. aureus from a benign colonizer of the nasal passages to a major pathogen is not well understood and host as well as bacterial properties have been discussed as being relevant for this behavioral change. We conducted a thorough investigation of patient-derived strain pairs reflecting colonizing and invasive isolates in a given patient. Although we identified limited genetic adaptation in certain strains as well as slight differences in adherence capacity of colonizing and invasive isolates, our work suggests that barrier breaches are a key event in the disease continuum of S. aureus.

microbiology↗

bioRxiv2021

Quantification of within patient Staphylococcus aureus phenotypic heterogeneity as a proxy for presence of persisters across clinical presentations

BackgroundDifficult-to-treat infections caused by antibiotic susceptible strains have been linked with the occurrence of persisters. Persisters are a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. They can be identified phenotypically upon plating on nutrient agar because of their altered growth dynamics, resulting in colony size heterogeneity. The occurrence of within-patient bacterial phenotypic heterogeneity in various infections and clinical determinants of persister formation remain unknown. MethodsWe plated bacteria derived from 132 patient-samples of difficult-to-treat infections directly on nutrient-rich agar and monitored colony growth by time-lapse imaging. Of these, we retained 36 Staphylococcus aureus mono-cultures for further analysis. We investigated clinical factors potentially associated with increased colony growth-delay with regression analyses. Additionally, we corroborated the clinical findings using in vitro grown static biofilms, exposed to distinct antibiotics. ResultsThe extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients. Increased heterogeneity coincided with increased median growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay. S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with either clindamycin or levofloxacin exhibited prolonged growth-delay, correlating with a strain-dependent increase in antibiotic tolerance. ConclusionsUpon direct cultivation on nutrient-rich agar, S. aureus from difficult-to-treat infections commonly exhibited colony size heterogeneity. This was due to heterogeneous delays in growth resumption, with delays larger than two days in the most extreme cases. Since bacteria in a dormant state are tolerant to antibiotics, the observation of large growth-delays might have direct clinical implications. Future studies are needed to assess the potential of bacterial phenotypic heterogeneity quantification for staphylococcal infections prognosis.

microbiology↗

bioRxiv2021

Assessing antibiotic tolerance of Staphylococcus aureus derived directly from patients by the Replica Plating Tolerance Isolation System- REPTIS

Antibiotic tolerant Staphylococcus aureus pose a great challenge to clinicians as well as to microbiological laboratories and are one reason for treatment failure. Antibiotic tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro. Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required. We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in S. aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics. One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 hours of incubation and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation. Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA-replication, transcription and cell wall synthesis, with the exception of rifampicin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates. In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Rapid detection of antibiotic tolerance offers a new diagnostic readout that might allow more tailored treatments in the future.

microbiology↗

bioRxiv2021

Genomic Stability and Genetic Defense Systems in Dolosigranulum pigrum a Candidate Beneficial Bacterium from the Human Microbiome

Dolosigranulum pigrum is positively associated with indicators of health in multiple epidemiological studies of human nasal microbiota. Knowledge of the basic biology of D. pigrum is a prerequisite for evaluating its potential for future therapeutic use; however, such data are very limited. To gain insight into D. pigrums chromosomal structure, pangenome and genomic stability, we compared the genomes of 28 D. pigrum strains that were collected across 20 years. Phylogenomic analysis showed closely related strains circulating over this period and closure of 19 genomes revealed highly conserved chromosomal synteny. Gene clusters involved in the mobilome and in defense against mobile genetic elements (MGEs) were enriched in the accessory genome versus the core genome. A systematic analysis for MGEs identified the first candidate D. pigrum prophage and insertion sequence. A systematic analysis for genetic elements that limit the spread of MGEs, including restriction modification (RM), CRISPR-Cas, and deity-named defense systems, revealed strain-level diversity in host defense systems that localized to specific genomic sites including one RM system hotspot. Analysis of CRISPR spacers pointed to a wealth of MGEs against which D. pigrum defends itself. These results reveal a role for horizontal gene transfer and mobile genetic elements in strain diversification while highlighting that in D. pigrum this occurs within the context of a highly stable chromosomal organization protected by a variety of defense mechanisms. IMPORTANCEDolosigranulum pigrum is a candidate beneficial bacterium with potential for future therapeutic use. This is based on its positive associations with characteristics of health in multiple studies of human nasal microbiota across the span of human life. For example, high levels of D. pigrum nasal colonization in adults predicts the absence of Staphylococcus aureus nasal colonization. Also, D. pigrum nasal colonization in young children is associated with healthy control groups in studies of middle ear infections. Our analysis of 28 genomes revealed a remarkable stability of D. pigrum strains colonizing people in the U.S. across a 20-year span. We subsequently identified factors that can influence this stability, including genomic stability, phage predators, the role of MGEs in strain-level variation and defenses against MGEs. Finally, these D. pigrum strains also lacked predicted virulence factors. Overall, these findings add additional support to the potential for D. pigrum as a therapeutic bacterium.

microbiology↗

bioRxiv2021

Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients

Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

immunology↗

bioRxiv2020

Neutrophil and monocyte dysfunctional effector response towards bacterial challenge in critically-ill COVID-19 patients

COVID-19 displays diverse disease severities and symptoms. Elevated inflammation mediated by hypercytokinemia induces a detrimental dysregulation of immune cells. However, there is limited understanding of how SARS-CoV-2 pathogenesis impedes innate immune signaling and function against secondary bacterial infections. We assessed the influence of COVID-19 hypercytokinemia on the functional responses of neutrophils and monocytes upon bacterial challenges from acute and corresponding recovery COVID-19 ICU patients. We show that severe hypercytokinemia in COVID-19 patients correlated with bacterial superinfections. Neutrophils and monocytes from acute COVID-19 patients showed severely impaired microbicidal capacity, reflected by abrogated ROS and MPO production as well as reduced NETs upon bacterial challenges. We observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes leading to a suppressive autocrine and paracrine signaling during bacterial challenges. Our data provide insights into the innate immune status of COVID-19 patients mediated by their hypercytokinemia and its transient effect on immune dysregulation upon subsequent bacterial infections

immunology↗

bioRxiv2020

Quantifying variation in bacterial reproductive fitness with and without antimicrobial resistance: a high-throughput method

BackgroundTo evaluate changes in reproductive fitness of bacteria, e.g., after acquisition of antimicrobial resistance, a low-cost high-throughput method to analyse bacterial growth on agar is desirable for broad usability, including in low-resource settings. MethodIn our bacterial quantitative fitness analysis (baQFA), cultures are spotted in a predefined array on agar plates and photographed sequentially while growing. These time-lapse images are analysed using a purpose-built open source software to derive normalised image intensity values for each culture spot. Subsequently, a Gompertz growth model is fitted to these optical intensity values of each culture spot and fitness is calculated from parameters of the model. For image segmentation validation, we investigated the association between normalised intensity values and colony-forming unit (CFU) counts. To represent a range of clinically important pathogenic bacteria, we used different strains of Enterococcus faecium, Escherichia coli and Staphylococcus aureus, with and without antimicrobial resistance. Relative competitive fitness (RCF) was defined as the mean fitness ratio of two strains growing competitively on one plate. ResultsbaQFA permitted the accurate construction of growth curves from bacteria grown on semisolid agar plates and fitting of Gompertz models: Normalised image intensity values showed a strong association with the total CFU/ml count per spotted culture (p<0.001) for all strains of the three species. Bacterial QFA showed relevant reproductive fitness differences between individual strains, suggesting substantial higher fitness of methicillin-resistant S. aureus JE2 than Cowan (RCF 1.60, p<0.001). Similarly, the vancomycin-resistant E. faecium ST172b showed higher competitive fitness than susceptible E. faecium ST172 (RCF 1.72, p<0.001). ConclusionOur baQFA adaptation allows detection of fitness differences between our bacterial strains, and is likely to be applicable to other bacteria. In the future, baQFA may help to estimate epidemiological antimicrobial persistence or contribute to the prediction of clinical outcomes in severe infections at a low cost.

microbiology↗

bioRxiv2019

Dolosigranulum pigrum cooperation and competition in human nasal microbiota

BackgroundMultiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae. ResultsUsing a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro. Whereas, robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together, and not either alone. D. pigrum L-lactic-acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum encoded a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions. ConclusionsThese new insights into D. pigrums functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments.

microbiology↗