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Abstract Ageing is a major risk factor that contributes to increased mortality and morbidity rates during influenza A virus (IAV) infections. Macrophages are crucial players in the defense against viral infections and display impaired function during ageing. However, the impact of ageing on macrophage function in response to an IAV infection remains unclear and offers potential insight for underlying mechanisms. In this study, we investigated the immune response of young and aged human monocyte-derived macrophages to two different H1N1 IAV strains. Interestingly, macrophages of aged individuals showed a lower interferon response to IAV infection, resulting in increased viral load. Transcriptomic data revealed a reduced expression of stimulator of interferon genes (STING) in aged macrophages albeit the cGAS-STING pathway was upregulated. Our data clearly indicate the importance of STING signaling for interferon production by applying a THP-1 STING knockout model. Evaluation of mitochondrial function during IAV infection revealed the release of mitochondrial DNA to be the activator of cGAS-STING pathway. The subsequent induction of apoptosis was attenuated in aged macrophages due to decreased STING signaling. Our study provides new insights into molecular mechanisms underlying age-related immune impairment. To our best knowledge, we are the first to discover an age-dependent difference in gene expression of STING on a transcriptional level in human monocyte-derived macrophages possibly leading to a diminished interferon production.

The telomerase RNA component (Terc) constitutes a non-coding RNA critical for telomerase function, commonly associated with aging and pivotal in immunomodulation during inflammation. Our study unveils heightened susceptibility to pneumonia caused by Staphylococcus aureus (S. aureus) in Terc knockout (Tercko/ko) mice compared to both young and old infected counterparts. The exacerbated infection in Tercko/ko mice correlates with heightened inflammation, manifested by elevated interleukin-1β (IL-1β) levels and activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome within the lung. Employing mRNA sequencing methods alongside in vitro analysis of alveolar macrophages (AMs) and T cells, our study elucidates a compelling correlation between Tercko/ko, inflammation, and impaired T cell functionality. Terc deletion results in compromised T cell function, characterized by dysregulation of the T cell receptor and absence of CD247, potentially compromising the host’s capacity to mount an effective immune response against S. aureus. This investigation provides insights into the intricate mechanisms governing increased vulnerability to severe pneumonia in the context of Terc deficiency, which might also contribute to aging-related pathologies, while also highlighting the influence of Terc on T cell function.

IntroductionInfluenza-associated pulmonary aspergillosis is associated with high mortality rates and limited treatment options. The current standard practice involves treating each pathogen separately. However, the use of antifungal drugs can lead to serious side effects, and the presence of triazole-resistant Aspergillus strains can complicate antifungal therapy. In addition, drug-resistant influenza viruses are becoming an increasing concern in clinics. A drug that affects fungal and viral propagation could overcome these disadvantages. Thus, we conducted a study to examine the antifungal and antiviral properties of ProcCluster® and procaine hydrochloride (HCl), which are prodrugs derived from the local anesthetic procaine.MethodsConidia of different A. fumigatus strains, A. flavus and A. terreus were treated with the test substances in a human cell-free system and antifungal properties were analyzed either by fluorescence microscopy or absorption measurements. Changes in metabolic activity and intracellular Ca2+ distribution during treatment of A. fumigatus with ProcCluster® were observed using fluorescence microscopy. In addition, antifungal and antiviral properties of ProcCluster® and procaine HCl were investigated during in vitro coinfection of lung epithelial cells with A. fumigatus and influenza A viruses (IAV). Analysis was performed by fluorescence microscopy, standard plaque assay and Western blot assay.ResultsBoth substances inhibited the growth of the fungus, even when applied after germination or in the presence of purified IAV particles. ProcCluster® remained effective against triazole-resistant A. fumigatus strains. However, the addition of CaCl2 reversed the antifungal effect, indicating that ProcCluster® inhibited fungal growth by disrupting fungal Ca2+ homeostasis. Furthermore, in vitro studies showed that ProcCluster® and procaine HCl reduced the pathogen load of IAV and A. fumigatus during coinfection. Finally, the combination of ProcCluster® with the antiviral drug favipiravir exhibited increased antipathogenic activity, particularly against IAV replication.DiscussionThis research highlights ProcCluster® and procaine HCl as substances with anti-infective properties against various pathogens.

IntroductionTreatment of influenza A virus infections is currently limited to few direct acting antiviral substances. Repurposing other established pharmaceuticals as antivirals could aid in improving treatment options.MethodsThis study investigates the antiviral properties of ProcCluster® and procaine hydrochloride, two derivatives of the local anesthetic procaine, in influenza A virus infection of A549, Calu-3 and MDCK cells.ResultsBoth substances inhibit replication in all three of these cell lines in multi-cycle experiments. However, cell line-dependent differences in the effects of the substances on viral RNA replication and subsequent protein synthesis, as well as release of progeny viruses in single-cycle experiments can be observed. Both ProcCluster® and procaine hydrochloride delay endosome fusion of the virus early in the replication cycle, possibly due to the alkaline nature of the active component procaine. In A549 and Calu-3 cells an additional effect of the substances can be observed at late stages in the first replication cycle. Interestingly, this effect is absent in MDCK cells. We demonstrate that ProcCluster® and procaine hydrochloride inhibit phospholipase A2 (PLA2) enzymes from A549 but not MDCK cells and confirm that specific inhibition of calcium independent PLA2 but not cytosolic PLA2 has antiviral effects.DiscussionWe show that ProcCluster® and procaine hydrochloride inhibit influenza A virus infection at several stages of the replication cycle and have potential as antiviral substances.

Rapid molecular assays can be used to identify Candida pathogens directly from positive blood cultures (BCs) in a timely manner compared to standard methods using subcultures. In this study, the eazyplex® Candida ID assay, which is based on loop-mediated amplification (LAMP) and is currently for research use only, was evaluated for the identification of the most common fungal species. A total of 190 BCs were analysed. Sensitivity and specificity were 93.88% and 99.26% for C. albicans, 89.13% and 100% for Nakaseomyces glabratus (N. glabratus), 100% and 100% for Pichia kudravzevii (P. kudriavzevii), 100% and 100% for C. tropicalis, and 100% and 99.44% for C. parapsilosis. Sample preparation took approximately 11 min and positive amplification results were obtained between 8.5 and 19 min. The eazyplex® Candida ID LAMP assay is an easy-to-use diagnostic tool that can optimise the management of patients with candidemia.

Staphylococcus aureus (S. aureus) is a facultative pathogenic bacterium that can cause infections in various tissue types in humans. Fluorescence imaging techniques have been employed to visualize the bacteria in ex-vivo samples mostly in research, aiding in the understanding of the etiology of the pathogen. However, the multispectral images generated from fluorescence microscopes are complex, making it difficult to locate bacteria across image files, especially in consecutive planes with different imaging depths. To address this issue, we present Findaureus, an open-source application specifically designed to locate and extract critical information about bacteria, especially S. aureus. Due to the limited availability of datasets, we tested the application on a dataset comprising fluorescence-labelled infected mouse bone tissue images, achieving an accuracy of 95%. We compared Findaureus with other state-of-the-art image analysis tools and found that it performed better, given its specificity toward bacteria localization. The proposed approach has the potential to aid in medical research of bone infections and can be extended to other tissue and bacteria types in the future.

Early identification of acute gastroenteritis (AGE) pathogens via PCR may improve the management of patients presenting to the emergency department (ED). In this study, we evaluated the implementation of a testing algorithm for ED patients with AGE using the BD MAX automated PCR system. Data from 133 patients were analyzed. A total of 56 patients (42%) tested positive via PCR for at least one bacterial or viral pathogen. The median time to report PCR results was 6.17 h compared to 57.28 h for culture results for bacterial pathogens. The most common pathogen was Clostridioides difficile (n = 20, 15%). In total, 14 of the 20 C. difficile-positive patients were aged >65 years and 17 of the 20 patients (85%) were diagnosed with a clinically relevant infection based on typical symptoms and laboratory values. They received antibiotics, mostly oral vancomycin, starting a median of 11.37 h after ED admission. The introduction of PCR for the diagnosis of AGE infection in patients presenting to the ED may have the greatest impact on the rapid identification of C. difficile and the timely administration of antibiotics if necessary.

Abstract Background The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory bowel disease with significant morbidity and mortality. Although the precise cause remains unknown, disturbances in the intestinal microbial community have been linked to its pathogenesis. Randomized controlled trials in UC and relapsing Clostridioides difficile infection (CDI) have established fecal microbiota (FM) transfer (FMT) as an effective therapy. In this context, preliminary results indicated that the transfer of sterile fecal microbiota filtrates (

Understanding persistent cellular and humoral immune responses to SARS-CoV-2 will be of major importance to terminate the ongoing pandemic. Here, we assessed long-term immunity in individuals with mild COVID-19 up to 1 year after a localized SARS-CoV-2 outbreak. CoNAN was a longitudinal population-based cohort study performed 1.5 months, 6 months, and 12 months after a SARS-CoV-2 outbreak in a rural German community. We performed a time series of five different IgG immunoassays assessing SARS-CoV-2 antibody responses on serum samples from individuals that had been tested positive after a SARS-CoV-2 outbreak and in control individuals who had a negative PCR result. These analyses were complemented with the determination of spike-antigen specific TH cell responses in the same individuals. All infected participants were presented as asymptomatic or mild cases. Participants initially tested positive for SARS-CoV-2 infection either with PCR, antibody testing, or both had a rapid initial decline in the serum antibody levels in all serological tests but showed a persisting TH cell immunity as assessed by the detection of SARS-CoV-2 specificity of TH cells for up to 1 year after infection. Our data support the notion of a persistent T-cell immunity in mild and asymptomatic cases of SARS-CoV-2 up to 1 year after infection. We show that antibody titers decline over 1 year, but considering several test results, complete seroreversion is rare.Trial registrationGerman Clinical Trials Register DRKS00022416.

A large proportion of clinical S. aureus isolates that carry an inactive Agr system are associated with persistent infection that is difficult to treat. Once S. aureus is inside the bloodstream, it can cross the endothelial barrier and invade almost every organ in the human body. Endothelial cells can either be lysed by this pathogen or they serve as a niche for its intracellular long-term survival. Following phagocytosis, several vesicles such as phagosomes and autophagosomes, target intracellular S. aureus for elimination. S. aureus can escape from these vesicles into the host cytoplasm through the activation of phenol-soluble modulins (PSMs) αβ. Thereafter, it replicates and lyses the host cell to disseminate to adjacent tissues. Herein we demonstrate that staphylococcal strains which lack the expression of PSMs employ an alternative pathway to better persist within endothelial cells. The intracellular survival of S. aureus is associated with the co-localization of the autophagy marker LC3. In cell culture infection models, we found that the absence of psmαβ decreased the host cell lysis and increased staphylococcal long-term survival. This study explains the positive selection of agr-negative strains that lack the expression of psmαβ in chronic infection due to their advantage in surviving and evading the clearance system of the host.

To confirm discrepant SARS-CoV-2-IgG results in four standard assays we applied for the first time a prototype of a coronavirus IgG-line-blot which employs antigens from seasonal coronaviruses, SARS-1 and SARS-CoV-2 combined with avidity testing as a confirmatory tool in a follow-up of five cases including pre-pandemic samples.

(1) Background: In the oral environment, sound enamel and dental restorative materials are immediately covered by a pellicle layer, which enables bacteria to attach. For the development of new materials with repellent surface functions, information on the formation and maturation of salivary pellicles is crucial. Therefore, the present in situ study aimed to investigate the proteomic profile of salivary pellicles formed on different dental composites. (2) Methods: Light-cured composite and bovine enamel samples (controls) were exposed to the oral cavity for 30, 90, and 120 min. All samples were subjected to optical and mechanical profilometry, as well as SEM surface evaluation. Acquired pellicles and unstimulated whole saliva samples were analyzed by SELDI–TOF–MS. The significance was determined by the generalized estimation equation and the post-hoc bonferroni adjustment. (3) Results: SEM revealed the formation of homogeneous pellicles on all test and control surfaces. Profilometry showed that composite surfaces tend to be of higher roughness compared to enamel. SELDI–TOF–MS detected up to 102 different proteins in the saliva samples and up to 46 proteins in the pellicle. Significant differences among 14 pellicle proteins were found between the composite materials and the controls. (4) Conclusions: Pellicle formation was material- and time-dependent. Proteins differed among the composites and to the control.

Rapid testing for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) of patients presenting to emergency departments (EDs) facilitates the decision for isolation on admission to hospital wards. Differences in the sensitivity of molecular assays have implications for diagnostic workflows. This study evaluated the performance of the cobas® Liat® RT-PCR, which is routinely used as the initial test for ED patients in our hospitals, compared with the eazyplex® RT-LAMP. A total of 378 oropharyngeal and nasal swabs with positive Liat® results were analysed. Residual sample aliquots were tested using NeuMoDx™, cobas® RT-PCR, and the eazyplex® assay. Patients were divided into asymptomatic (n = 157) and symptomatic (n = 221) groups according to the WHO case definition. Overall, 14% of positive Liat® results were not confirmed by RT-PCR. These samples were mainly attributed to 26.8% of asymptomatic patients, compared to 3.8% of the symptomatic group. Therefore, positive Liat® results were used to provisionally isolate patients in the ED until RT-PCR results were available. The eazyplex® assay identified 62% and 90.6% of RT-PCR-confirmed cases in asymptomatic and symptomatic patients, respectively. False-negative eazyplex® results were associated with RT-PCR Ct values > 30, and were more frequent in the asymptomatic group than in the symptomatic group (38.1% vs. 5.1%, respectively). Both the Liat® and eazyplex® assays are suitable for testing symptomatic patients. Their use in screening asymptomatic patients depends on the need to exclude any infection or identify those at high risk of transmission.

Abstract Background Monitoring pathogens of bloodstream infections (BSI) and their antibiotic susceptibility is important to guide empiric antibiotic treatment strategies and prevention programs. This study assessed the epidemiology of BSI and antibiotic resistance patterns at the German Federal State of Thuringia longitudinally. Methods A surveillance network consisting of 26 hospitals was established to monitor BSIs from 01/2015 to 12/2019. All blood culture results, without restriction of age of patients, of the participating hospitals were reported by the respective microbiological laboratory. A single detection of obligate pathogens and a repeated detection of coagulase-negative staphylococci, Bacillus spp., Corynebacterium spp., Micrococcus spp. and Propionibacterium spp., within 96 h were regarded as a relevant positive blood culture. If one of the aforementioned non-obligate pathogens has been detected only once within 96 h, contamination has been assumed. Logistic regression models were applied to analyse the relationship between resistance, year of BSI and hospital size. Generalized estimating equations were used to address potential clustering. Results A total of 343,284 blood cultures (BC) of 82,527 patients were recorded. Overall, 2.8% (n = 9571) of all BCs were classified as contaminated. At least one relevant pathogen was identified in 13.2% (n = 45,346) of BCs. Escherichia coli (25.4%) was the most commonly detected pathogen, followed by Staphylococcus aureus (15.2%), Staphylococcus epidermidis (8.1%) and Klebsiella pneumoniae (4.6%). In S. aureus, we observed a decline of methicillin resistance (MRSA) from 10.4% in 2015 to 2.5% in 2019 (p

Chronic staphylococcal osteomyelitis can persist for long time periods causing bone destruction. The ability of Staphylococcus aureus to develop chronic infections is linked to its capacity to invade and replicate within osteoblasts and osteocytes and to switch to a dormant phenotype called small colony variants. Recently, osteocytes were described as a main reservoir for this pathogen in bone tissue. However, the mechanisms involved in the persistence of S. aureus within these cells are still unknown. Here, we investigated the interaction between S. aureus and osteoblasts or osteocytes during infection. While osteoblasts are able to induce a strong antimicrobial response and eliminate intracellular S. aureus, osteocytes trigger signals to recruit immune cells and enhance inflammation but fail an efficient antimicrobial activity to clear the bacterial infection. Moreover, we found that extracellular signals from osteocytes enhance intracellular bacterial clearance by osteoblasts. Even though both cell types express Toll-like receptor (TLR) 2, the main TLR responsible for S. aureus detection, only osteoblasts were able to increase TLR2 expression after infection. Additionally, proteomic analysis indicates that reduced intracellular bacterial killing activity in osteocytes is related to low antimicrobial peptide expression. Nevertheless, high levels of lipid mediators and cytokines were secreted by osteocytes, suggesting that they can contribute to inflammation. Taken together, our results demonstrate that osteocytes contribute to severe inflammation observed in osteomyelitis and represent the main niche for S. aureus persistence due to their poor capacity for intracellular antimicrobial response.

We introduce a magnetic bead-based sample preparation scheme for enabling the Raman spectroscopic differentiation of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-positive and -negative samples. The beads were functionalized with the angiotensin-converting enzyme 2 (ACE2) receptor protein, which is used as a recognition element to selectively enrich SARS-CoV-2 on the surface of the magnetic beads. The subsequent Raman measurements directly enable discriminating SARS-CoV-2-positive and -negative samples. The proposed approach is also applicable for other virus species when the specific recognition element is exchanged. A series of Raman spectra were measured on three types of samples, namely SARS-CoV-2, Influenza A H1N1 virus and a negative control. For each sample type, eight independent replicates were considered. All of the spectra are dominated by the magnetic bead substrate and no obvious differences between the sample types are apparent. In order to address the subtle differences in the spectra, we calculated different correlation coefficients, namely the Pearson coefficient and the Normalized cross correlation coefficient. By comparing the correlation with the negative control, differentiating between SARS-CoV-2 and Influenza A virus is possible. This study provides a first step towards the detection and potential classification of different viruses with the use of conventional Raman spectroscopy.

The acquisition of hypervirulence-associated genes by carbapenemase-producing Klebsiella pneumoniae is being increasingly observed, and easy-to-use diagnostic tests are needed for the surveillance of the hypervirulent K. pneumoniae (hvKp). In this pilot study, 87 K. pneumoniae isolates from invasive infections collected in 2022 and 2023 were analysed using the LAMP-based eazyplex® Superbug CRE and hvKp assays for the simultaneous identification of carbapenemases and virulence genes (rmpA/A2, iuC, iroC, ybt, clb). Nine isolates showed a Kleborate virulence score of 4 or 5 (10.3%). The time for the results of the eazyplex® assays ranged from 6.5 to 13 min, and the total turnaround time, including sample preparation, was less than 30 min. Five isolates, three of which produced New Delhi metallo-beta lactamase (NDM), were subjected to whole-genome sequencing (WGS) analysis for further characterisation. The eazyplex® test results for beta-lactamase and virulence genes were confirmed. The eazyplex® hvKp, currently only available as a Research Use Only assay, may be a useful tool for the rapid identification of hvKp without significant additional workload when combined with the eazyplex® Superbug CRE assay for the detection of carbapenemases.

Staphylococcus aureus evades antibiotic therapy and antimicrobial defenses by entering human host cells. Bacterial transcriptomic analysis represents an invaluable tool to unravel the complex interplay between host and pathogen. Therefore, the extraction of high-quality RNA from intracellular S. aureus lays the foundation to acquire meaningful gene expression data. In this study, we present a novel and straightforward strategy to isolate RNA from internalized S. aureus after 90 min, 24 h, and 48 h postinfection. Real-time PCR data were obtained for the target genes agrA and fnba, which play major roles during infection. The commonly used reference genes gyrB, aroE, tmRNA, gmk, and hu were analyzed under different conditions: bacteria from culture (condition I), intracellular bacteria (condition II), and across both conditions I and II. The most stable reference genes were used for the normalization of agrA and fnbA. Delta Cq (quantification cycle) values had a relatively low variability and thus demonstrated the high quality of the extracted RNA from intracellular S. aureus during the early phase of infection. The established protocol allows the extraction and purification of intracellular staphylococcal RNA while minimizing the amount of host RNA in the sample. This approach can leverage reproducible gene expression data to study host–pathogen interactions.

Abstract Background It is essential to avoid admission of patients with undetected corona virus disease 2019 (COVID-19) to hospitals’ general wards. Even repeated negative reverse transcription polymerase chain reaction (RT-PCR) results do not rule-out COVID-19 with certainty. The study aimed to evaluate a rule-out strategy for COVID-19 using chest computed tomography (CT) in adults being admitted to the emergency department and suspected of COVID-19. Methods In this prospective, single centre, diagnostic accuracy cohort study, consecutive adults (≥ 18 years) presenting with symptoms consistent with COVID-19 or previous contact to infected individuals, admitted to the emergency department and supposed to be referred to general ward were included in March and April 2020. All participants underwent low-dose chest CT. RT-PCR- and specific antibody tests were used as reference standard. Main outcome measures were sensitivity and specificity of chest CT. Predictive values were calculated based on the theorem of Bayes using Fagan’s nomogram. Results Of 165 participants (56.4% male, 71 ± 16 years) included in the study, the diagnosis of COVID-19 was confirmed with RT-PCR and AB tests in 13 participants (prevalence 7.9%). Sensitivity and specificity of chest CT were 84.6% (95% confidence interval [CI], 54.6–98.1) and 94.7% (95% CI, 89.9–97.7), respectively. Positive and negative likelihood ratio of chest CT were 16.1 (95% CI, 7.9–32.8) and 0.16 (95% CI, 0.05–0.58) and positive and negative predictive value were 57.9% (95% CI, 40.3–73.7) and 98.6% (95% CI, 95.3–99.6), respectively. Conclusion At a low prevalence of COVID-19, chest CT could be used as a complement to repeated RT-PCR testing for early COVID-19 exclusion in adults with suspected infection before referral to hospital’s general wards. Trial registration ClinicalTrials.gov: NCT04357938 April 22, 2020.

Staphylococcus aureus bloodstream infections are associated with a high morbidity and mortality. Nevertheless, significance of a positive blood culture with this pathogen is often underestimated or findings are misinterpreted as contamination, which can result in inadequate diagnostic and therapeutic consequences. We here review and discuss current diagnostic and therapeutic key elements and open questions for the management of Staphylococcus aureus bloodstream infections.

Clinical observations indicate that COVID-19 is a systemic disease. An investigation of the viral distribution within the human body and its correlation with tissue damage can aid in understanding the pathophysiology of SARS-CoV-2 infection. We present a detailed mapping of the viral RNA in 61 tissues and organs of 11 deceased patients with COVID-19. The autopsies were performed within the early postmortem interval (between 1.5 and 15 hr, mean: 5.6 hr) to minimize the bias due to viral RNA and tissue degradation. Very high viral loads (>104copies/ml) were detected in most patients' lungs, and the presence of intact viral particles in the lung tissue could be verified by transmission electron microscopy. Interestingly, viral RNA was detected throughout various extrapulmonary tissues and organs without visible tissue damage. The dissemination of SARS-CoV-2-RNA throughout the body supports the hypothesis that there is a maladaptive host response with viremia and multiorgan dysfunction.

Staphylococcus aureus is able to survive within host cells by switching its phenotype to the small-colony variant (SCV) phenotype. The emergence of SCVs is associated with the development of persistent infections, which may be both chronic and recurrent. This slow-growing subpopulation of S. aureus forms small colonies on solid-medium agar, is induced within host cells, presents a non-homogenous genetic background, has reduced expression of virulence factors and presents a variable phenotype (stable or unstable). While virtually all SCVs isolated from clinical specimens can revert to the parental state with rapid growth, the stable SCVs recovered in clinical specimens have been found to contain specific mutations in metabolic pathways. In contrast, other non-stable SCVs are originated from regulatory mechanisms involving global regulators (e.g., sigB, sarA, and agr) or other non-defined mutations. One major characteristic of SCVs was the observation that SCVs were recovered from five patients with infections that could persist for decades. In these five cases, the SCVs had defects in electron transport. This linked persistent infections with SCVs. The term “persistent infection” is a clinical term wherein bacteria remain in the host for prolonged periods of time, sometimes with recurrent infection, despite apparently active antibiotics. These terms were described in vitro where bacteria remain viable in liquid culture medium in the presence of antibiotics. These bacteria are called “persisters”. While SCVs can be persisters in liquid culture, not all persisters are SCVs. One mechanism associated with the metabolically variant SCVs is the reduced production of virulence factors. SCVs have consistently shown reduced levels of RNAIII, a product of the accessory gene regulatory (agrBDCA) locus that controls a quorum-sensing system and regulates the expression of a large number of virulence genes. Reduced Agr acitivity is associated with enhanced survival of SCVs within host cells. In this review, we examine the impact of the SCVs with altered metabolic pathways on agr, and we draw distinctions with other types of SCVs that emerge within mammalian cells with prolonged infection.

Humoral immunity after infection or after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been attributed a key part in mitigating the further transmission of the virus. In this study, we used a commercial anti-Spike immunoglobulin G (S-IgG) assay and developed a cell culture-based neutralization assay to understand the longitudinal course of neutralizing antibodies in both SARS-CoV2 infected or vaccinated individuals. We show that even more than one year after infection, about 78% of observed study participants remained seropositive concerning S-IgG antibodies. In addition, the serum of the individuals had stable neutralization capacity in a neutralization assay against a SARS-CoV-2 patient isolate from March 2020. We also examined volunteers after either homologous BNT162b2 prime-boost vaccination or heterologous AZD1222 prime/mRNA-based booster vaccination. Both the heterologous and the homologous vaccination regimens induced higher levels of neutralizing antibodies in healthy subjects when compared to subjects after a mild infection, showing the high effectiveness of available vaccines. In addition, we could demonstrate the reliability of S-IgG levels in predicting neutralization capacity, with 94.8% of seropositive samples showing a neutralization titer of ≥10, making it a viable yet cheap and easy-to-determine surrogate parameter for neutralization capacity.

Staphylococcus aureus has acquired resistance to antibiotics since their first use. The S. aureus protein NorA, an efflux pump belonging to the major facilitator superfamily (MFS), contributes to resistance to fluoroquinolones (e.g., ciprofloxacin), biocides, dyes, quaternary ammonium compounds, and antiseptics. Different compounds have been identified as potential efflux pump inhibitors (EPIs) of NorA that result in increased intracellular concentration of antibiotics, restoring their antibacterial activity and cell susceptibility. However, none of the currently known EPIs have been approved for clinical use, probably due to their toxicity profiles. In the present study, we screened approved drugs for possible efflux pump inhibition. By screening a compound library of approximately 1200 different drugs, we identified nilotinib, a tyrosine kinase inhibitor, as showing the best efflux pump inhibitory activity, with a fractional inhibitory concentration index of 0.1875, indicating synergism with ciprofloxacin, and a minimum effective concentration as low as 0.195 μM. Moreover, at 0.39 μM, nilotinib, in combination with 8 μg/mL of ciprofloxacin, led to a significant reduction in biofilm formation and preformed mature biofilms. This is the first description of an approved drug that can be used as an efflux pump inhibitor and to reduce biofilms formation at clinically achievable concentrations.

Influenza virus (IV) infections are considered to cause severe diseases of the respiratory tract. Beyond mild symptoms, the infection can lead to respiratory distress syndrome and multiple organ failure. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti-influenza drugs. Interestingly, the virus-supportive function of the cellular phosphatidylinositol 3-kinase (PI3K) suggests that this signaling module may be a potential target for antiviral intervention. In the sense of repurposing existing drugs for new indications, we used Pictilisib, a known PI3K inhibitor to investigate its effect on IV infection, in mono-cell-culture studies as well as in a human chip model. Our results indicate that Pictilisib is a potent inhibitor of IV propagation already at early stages of infection. In a murine model of IV pneumonia, the in vitro key findings were verified, showing reduced viral titers as well as inflammatory response in the lung after delivery of Pictilisib. Our data identified Pictilisib as a promising drug candidate for anti-IV therapies that warrant further studying. These results further led to the conclusion that the repurposing of previously approved substances represents a cost-effective and efficient way for development of novel antiviral strategies.

Staphylococcus aureus is an opportunistic and versatile pathogen that can cause several diseases, which range from acute and destructive, to chronic and difficult-to-treat infections [...]

Human beings are exposed to microorganisms every day. Among those, diverse commensals and potential pathogens including Staphylococcus aureus (S. aureus) compose a significant part of the respiratory tract microbiota. Remarkably, bacterial colonization is supposed to affect the outcome of viral respiratory tract infections, including those caused by influenza viruses (IV). Since 30% of the world’s population is already colonized with S. aureus that can develop metabolically inactive dormant phenotypes and seasonal IV circulate every year, super-infections are likely to occur. Although IV and S. aureus super-infections are widely described in the literature, the interactions of these pathogens with each other and the host cell are only scarcely understood. Especially, the effect of quasi-dormant bacterial subpopulations on IV infections is barely investigated. In the present study, we aimed to investigate the impact of S. aureus small colony variants on the cell intrinsic immune response during a subsequent IV infection in vitro. In fact, we observed a significant impact on the regulation of pro-inflammatory factors, contributing to a synergistic effect on cell intrinsic innate immune response and induction of harmful cell death. Interestingly, the cytopathic effect, which was observed in presence of both pathogens, was not due to an increased pathogen load.

Pneumonia is the leading cause of hospitalization worldwide. Besides viruses, bacterial co-infections dramatically exacerbate infection. In general, surfactant protein-A (SP-A) represents a first line of immune defense. In this study, we analyzed whether influenza A virus (IAV) and/or Staphylococcus aureus (S. aureus) infections affect SP-A expression. To closely reflect the situation in the lung, we used a human alveolus-on-a-chip model and a murine pneumonia model. Our results show that S. aureus can reduce extracellular levels of SP-A, most likely attributed to bacterial proteases. Mono-epithelial cell culture experiments reveal that the expression of SP-A is not directly affected by IAV or S. aureus. Yet, the mRNA expression of SP-A is strongly down-regulated by TNF-α, which is highly produced by professional phagocytes in response to bacterial infection. By using the human alveolus-on-a-chip model, we show that the down-regulation of SP-A is strongly dependent on macrophages. In a murine model of pneumonia, we can confirm that S. aureus decreases SP-A levels in vivo. These findings indicate that (I) complex interactions of epithelial and immune cells induce down-regulation of SP-A expression and (II) bacterial mono- and super-infections reduce SP-A expression in the lung, which might contribute to a severe outcome of bacterial pneumonia.

Development of organ dysfunction discriminates sepsis from uncomplicated infection. The paradigm shift implicated by the new sepsis-3 definition holds that initial impairment of any organ can pave the way for multiple organ dysfunction and death. Moreover, the role of the systemic inflammatory response, central element in previous sepsis definitions, has been questioned. Most strikingly, a so far largely underestimated defense mechanism of the host, i.e., “disease tolerance,” which aims at maintaining host vitality without reducing pathogen load, has gained increasing attention. Here, we summarize evidence that a dysregulation of critical cellular signaling events, also in non-immune cells, might provide a conceptual framework for sepsis-induced dysfunction of parenchymal organs in the absence of significant cell death. We suggest that key signaling mediators, such as phosphoinositide 3-kinase, mechanistic target of rapamycin, and AMP-activated protein kinase, control the balance of damage and repair processes and thus determine the fate of affected organs and ultimately the host. Therapeutic targeting of these multifunctional signaling mediators requires cell-, tissue-, or organ-specific approaches. These novel strategies might allow stopping the domino-like damage to further organ systems and offer alternatives beyond the currently available strictly supportive therapeutic options.

We describe the first isolation of Mycobacterium hassiacum , a rapid-growing, partial acid-resistant mycobacterium, in a respiratory specimen from a patient with exacerbated chronic obstructive pulmonary disease. To provide therapeutic recommendation for future cases, antibiotic susceptibility testing of 3 clinical isolates was performed by broth microdilution. All strains tested showed susceptibility to clarithromycin, imipenem, ciprofloxacin, and doxycycline. The role of M hassiacum as a respiratory pathogen remains unclear and needs to be evaluated by future reports.

Acute cholangitis (AC) requires the immediate initiation of antibiotic therapy in addition to treatment for biliary obstruction. Against a background of an increasing prevalence of multi-drug resistant (MDR) bacteria, the risk factors for the failure of empiric therapy must be defined.Using a pathogen-based approach, 1764 isolates from positive bile duct cultures were retrospectively analyzed to characterize the respective pathogen spectra in two German tertiary centers. Using a patient-based approach, the clinical and laboratory data for 83 patients with AC were assessed to identify risk factors for AC with pathogens resistant to the applied empiric therapy.Bile cultures were predominantly polymicrobial, and empiric antibiotic therapies did not cover the full biliary pathogen spectrum in 78% of cases. MDR bacteria were isolated from the bile of 24/83 (29%) patients. The univariate risk factors for biliary MDR bacteria were male sex, nosocomial AC, prior antibiotic exposure and prior biliary stenting, of which biliary stenting was the only independent risk factor according to multivariate analysis (OR = 3.8; 95% CI 1.3-11.0, P = 0.013). Although there were no significant differences in survival or hospital stay in AC patients with and without detected biliary MDR pathogens, the former more often had a concomitant bloodstream infection (58% vs. 24%; P = 0.019), including those involving MDR pathogens or fungi (21% vs. 2%; P = 0.007).Patients with biliary stents who develop AC should receive empiric therapy covering enterococci and extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. These patients are at an increased risk for bloodstream infections by MDR pathogens or fungi.

[This corrects the article DOI: 10.1371/journal.pone.0169900.].

Staphylococcus aureus colonizes epithelial surfaces, but it can also cause severe infections. The aim of this work was to investigate whether bacterial virulence correlates with defined types of tissue infections. For this, we collected 10–12 clinical S. aureus strains each from nasal colonization, and from patients with endoprosthesis infection, hematogenous osteomyelitis, and sepsis. All strains were characterized by genotypic analysis, and by the expression of virulence factors. The host–pathogen interaction was studied through several functional assays in osteoblast cultures. Additionally, selected strains were tested in a murine sepsis/osteomyelitis model. We did not find characteristic bacterial features for the defined infection types; rather, a wide range in all strain collections regarding cytotoxicity and invasiveness was observed. Interestingly, all strains were able to persist and to form small colony variants (SCVs). However, the low-cytotoxicity strains survived in higher numbers, and were less efficiently cleared by the host than the highly cytotoxic strains. In summary, our results indicate that not only destructive, but also low-cytotoxicity strains are able to induce infections. The low-cytotoxicity strains can successfully survive, and are less efficiently cleared from the host than the highly cytotoxic strains, which represent a source for chronic infections. The understanding of this interplay/evolution between the host and the pathogen during infection, with specific attention towards low-cytotoxicity isolates, will help to optimize treatment strategies for invasive and therapy-refractory infection courses.

Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.

Staphylococcus aureus is a major pathogen causing bone infections that can become chronic and difficult to treat. Recently, we described the mechanism employed by S. aureus to switch to small colony variants (SCVs) and trigger intracellular bacterial persistence through the global stress regulator SigB. Here, we studied the role of SigB in the formation of chronic osteomyelitis. We used a murine hematogenous osteomyelitis model, where the mice were infected via the tail vein and subsequently developed chronic osteomyelitis. Mice were infected with S. aureus LS1, LS1ΔsigB and LS1ΔsigB complemented and kidney and bone tissues were analyzed six weeks after infection. S. aureus LS1ΔsigB formed a high rate of abscesses in kidneys, but the bacterial loads and the weight loss of the animals were lower in comparison with animals infected with the wild type and the complemented strain, indicating a more rapid and efficient bacterial clearing by the host immune system. Moreover, the sigB-mutant was not able to form SCV phenotypes either in kidney or in bone tissue. Our results demonstrate that staphylococcal SigB is important to avoid bacterial elimination by the host immune response, establish a bone infection and mediate bacterial adaptation (SCV-formation) for persistent infections

ABSTRACT Osteomyelitis is a difficult-to-eradicate bone infection typically caused by Staphylococcus aureus. In this study, we investigated the in vivo transcriptional adaptation of S. aureus during bone infection. To this end, we determined the transcriptome of S. aureus during the acute (day 7) and chronic (day 28) phases of experimental murine osteomyelitis using RNA sequencing (RNA-Seq). We identified a total of 180 genes significantly more highly expressed by S. aureus during acute or chronic in vivo infection than under in vitro growth conditions. These genes encoded proteins involved in gluconeogenesis, proteolysis of host proteins, iron acquisition, evasion of host immune defenses, and stress responses. At the regulatory level, sarA and -R and saeR and -S as well as the small RNA RsaC were predominantly expressed by S. aureus during in vivo infection. Only nine genes, including the genes encoding the arginine deiminase (ADI) pathway and those involved in the stringent response, were significantly more highly expressed by S. aureus during the chronic than the acute stage of infection. Analysis by quantitative reverse transcription-PCR (qRT-PCR) of a subset of these in vivo-expressed genes in clinical specimens yielded the same results as those observed in the murine system. Collectively, our results show that during acute osteomyelitis, S. aureus induced the transcription of genes that mediate metabolic adaptation, immune evasion, and replication. During the chronic phase, however, S. aureus switched its transcriptional response from a proliferative to a persistence mode, probably driven by the severe deficiency in nutrient supplies. Interfering with the survival strategies of S. aureus during chronic infection could lead to more effective treatments. IMPORTANCE The key to the survival success of pathogens during an infection is their capacity to rapidly adjust to the host environment and to evade the host defenses. Understanding how a pathogen redirects and fine-tunes its gene expression in response to the challenges of infection is central to the development of more efficient anti-infective therapies. Osteomyelitis is a debilitating infection of the bone predominantly caused by S. aureus. In this study, we evaluated the transcriptional response of S. aureus during bone infection. Our results indicate that S. aureus reprograms its genetic repertoire during the acute phase of infection to adapt to nutrient availability and to replicate within the host. During the chronic phase, S. aureus upregulates a survival genetic program activated in response to nutrient starvation. Thus, we have uncovered key survival pathways of S. aureus during acute and chronic osteomyelitis that can be used as therapeutic targets.

Infective endocarditis (IE) is a severe and often fatal disease, lacking a fast and reliable diagnostic procedure. The purpose of this study was to establish a mouse model of Staphylococcus aureus-induced IE and to develop a MRI technology to characterize and diagnose IE. To establish the mouse model of hematogenous IE, aortic valve damage was induced by placing a permanent catheter into right carotid artery. 24 h after surgery, mice were injected intravenously with either iron particle-labeled or unlabeled S. aureus (strain 6850). To distinguish the effect of IE from mere tissue injury or recruited macrophages, subgroups of mice received sham surgery prior to infection (n = 17), received surgery without infection (n = 8), or obtained additionally injection of free iron particles to label macrophages (n = 17). Cardiac MRI was performed 48 h after surgery using a self-gated ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration, 12∶08 min) to obtain high-resolution, artifact-free cinematographic images of the valves. After MRI, valves were either homogenized and plated on blood agar plates for determination of bacterial titers, or sectioned and stained for histology. In the animal model, both severity of the disease and mortality increased with bacterial numbers. Infection with 105 S. aureus bacteria reliably caused endocarditis with vegetations on the valves. Cinematographic UTE MRI visualised the aortic valve over the cardiac cycle and allowed for detection of bacterial vegetations, while mere tissue trauma or labeled macrophages were not detected. Iron labeling of S. aureus was not required for detection. MRI results were consistent with histology and microbial assessment. These data showed that S. aureus-induced IE in mice can be detected by MRI. The established mouse model allows for investigation of the pathophysiology of IE, testing of novel drugs and may serve for the development of a clinical diagnostic strategy.

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.