Hyperinflammatory environment drives dysfunctional myeloid cell effector response to bacterial challenge in COVID-19.

COVID-19 displays diverse disease severities and symptoms including acute systemic inflammation and hypercytokinemia, with subsequent dysregulation of immune cells. Bacterial superinfections in COVID-19 can further complicate the disease course and are associated with increased mortality. However, there is limited understanding of how SARS-CoV-2 pathogenesis and hypercytokinemia impede the innate immune function against bacterial superinfections. We assessed the influence of COVID-19 plasma hypercytokinemia on the functional responses of myeloid immune cells upon bacterial challenges from acute-phase COVID-19 patients and their corresponding recovery-phase. We show that a severe hypercytokinemia status in COVID-19 patients correlates with the development of bacterial superinfections. Neutrophils and monocytes derived from COVID-19 patients in their acute-phase showed an impaired intracellular microbicidal capacity upon bacterial challenges. The impaired microbicidal capacity was reflected by abrogated MPO and reduced NETs production in neutrophils along with reduced ROS production in both neutrophils and monocytes. Moreover, we observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes, in line with suppressed autocrine and paracrine cytokine signaling. This phenotype was characterized by a high expression of CD66b, CXCR4 and low expression of CXCR1, CXCR2 and CD15 in neutrophils and low expression of HLA-DR, CD86 and high expression of CD163 and CD11b in monocytes. Furthermore, the impaired antibacterial effector function was mediated by synergistic effect of the cytokines TNF-α, IFN-γ and IL-4. COVID-19 patients receiving dexamethasone showed a significant reduction of overall inflammatory markers in the plasma as well as exhibited an enhanced immune response towards bacterial challenge ex vivo. Finally, broad anti-inflammatory treatment was associated with a reduction in CRP, IL-6 levels as well as length of ICU stay and ventilation-days in critically ill COVID-19 patients. Our data provides insights into the transient functional dysregulation of myeloid immune cells against subsequent bacterial infections in COVID-19 patients and describe a beneficial role for the use of dexamethasone in these patients.

Mucosa-Associated Invariant T Cell Hypersensitivity to Staphylococcus aureus Leukocidin ED and Its Modulation by Activation.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite Ags presented by MHC class Ib-related protein (MR1) and play important roles in immune control of microbes that synthesize riboflavin. This includes the pathobiont Staphylococcus aureus, which can also express a range of virulence factors, including the secreted toxin leukocidin ED (LukED). In this study, we found that human MAIT cells are hypersensitive to LukED-mediated lysis and lost on exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid and occurred at toxin concentrations lower than those required for toxicity against conventional T cells. Furthermore, this coincided with high MAIT cell expression of CCR5, and loss of these cells was efficiently inhibited by the CCR5 inhibitor maraviroc. Interestingly, exposure and preactivation of MAIT cells with IL-12 and IL-18, or activation via TCR triggering, partially protected from LukED toxicity. Furthermore, analysis of NK cells indicated that LukED targeted the mature cytotoxic CD57+ NK cell subset in a CCR5-independent manner. Overall, these results indicate that LukED efficiently eliminates immune cells that can respond rapidly to S. aureus in an innate fashion without the need for clonal expansion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED secretion may allow S. aureus to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

Pulmonary Surfactant Proteins Are Inhibited by Immunoglobulin A Autoantibodies in Severe COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Molecular reprogramming and phenotype switching in Staphylococcus aureus lead to high antibiotic persistence and affect therapy success.

Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat.

Antiphospholipid antibodies are enriched post-acute COVID-19 but do not modulate the thrombotic risk.

BACKGROUND AND OBJECTIVES: COVID-19-associated coagulopathy, shown to increase the risk for the occurrence of thromboses and microthromboses, displays phenotypic features of the antiphospholipid syndrome (APS), a prototype antibody-mediated autoimmune disease. Several groups have reported elevated levels of criteria and non-criteria antiphospholipid antibodies (aPL), assumed to cause APS, during acute or post-acute COVID-19. However, disease heterogeneity of COVID-19 is accompanied by heterogeneity in molecular signatures, including aberrant cytokine profiles and an increased occurrence of autoantibodies. Moreover, little is known about the association between autoantibodies and the clinical events. Here, we first aim to characterise the antiphospholipid antibody, anti-SARS-CoV-2 antibody, and the cytokine profiles in a diverse collective of COVID-19 patients (disease severity: asymptomatic to intensive care), using vaccinated individuals and influenza patients as comparisons. We then aim to assess whether the presence of aPL in COVID-19 is associated with an increased incidence of thrombotic events in COVID-19. METHODS AND RESULTS: We conducted anti-SARS-CoV-2 IgG and IgA microELISA and IgG, IgA, and IgM antiphospholipid line immunoassay (LIA) against 10 criteria and non-criteria antigens in 155 plasma samples of 124 individuals, and we measured 16 cytokines and chemokines in 112 plasma samples. We additionally employed clinical and demographic parameters to conduct multivariable regression analyses within multiple paradigms. In line with recent results, we find that IgM autoantibodies against annexin V (AnV), ß2-glycoprotein I (ß2GPI), and prothrombin (PT) are enriched upon infection with SARS-CoV-2. There was no evidence for seroconversion from IgM to IgG or IgA. PT, ß2GPI, and AnV IgM as well as cardiolipin (CL) IgG antiphospholipid levels were significantly elevated in the COVID-19 but not in the influenza or control groups. They were associated predominantly with the strength of the anti-SARS-CoV-2 antibody titres and the major correlate for thromboses was SARS-CoV-2 disease severity. CONCLUSION: While we have recapitulated previous findings, we conclude that the presence of the aPL, most notably PT, ß2GPI, AnV IgM, and CL IgG in COVID-19 are not associated with a higher incidence of thrombotic events.

Assessing Antibiotic Tolerance of Staphylococcus aureus Derived Directly from Patients by the Replica Plating Tolerance Isolation System (REPTIS).

Antibiotic-tolerant Staphylococcus aureus poses a great challenge to clinicians as well as to microbiological laboratories and is 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 Staphylococcus 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 h 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 rifampin. 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. Further studies should evaluate the impact of rapid detection of antibiotic tolerance as a clinical decision-making tool for tailored antibiotic treatments.

Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing.

Chronic wounds are a major disease burden worldwide. The breach of the epithelial barrier facilitates transition of skin commensals to invasive facultative pathogens. Therefore, we investigated the potential effects of Staphylococcus aureus (SA) on dermal fibroblasts as key cells for tissue repair. In co-culture systems combining live or heat-killed SA with dermal fibroblasts derived from the BJ-5ta cell line, healthy individuals, and patients with systemic sclerosis, we assessed tissue repair including pro-inflammatory cytokines, matrix metalloproteases (MMPs), myofibroblast functions, and host defense responses. Only live SA induced the upregulation of IL-1ß/-6/-8 and MMP1/3 as co-factors of tissue degradation. Additionally, the increased cell death reduced collagen production, proliferation, migration, and contractility, prerequisite mechanisms for wound closure. Intracellular SA triggered inflammatory and type I IFN responses via intracellular dsDNA sensor molecules and MyD88 and STING signaling pathways. In conclusion, live SA affected various key tissue repair functions of dermal fibroblasts from different sources to a similar extent. Thus, SA infection of dermal fibroblasts should be taken into account for future wound management strategies.

Antibiotic resistance and persistence-Implications for human health and treatment perspectives.

Antimicrobial resistance (AMR) and persistence are associated with an elevated risk of treatment failure and relapsing infections. They are thus important drivers of increased morbidity and mortality rates resulting in growing healthcare costs. Antibiotic resistance is readily identifiable with standard microbiological assays, and the threat imposed by antibiotic resistance has been well recognized. Measures aiming to reduce resistance development and spreading of resistant bacteria are being enforced. However, the phenomenon of bacteria surviving antibiotic exposure despite being fully susceptible, so-called antibiotic persistence, is still largely underestimated. In contrast to antibiotic resistance, antibiotic persistence is difficult to measure and therefore often missed, potentially leading to treatment failures. In this review, we focus on bacterial mechanisms allowing evasion of antibiotic killing and discuss their implications on human health. We describe the relationship between antibiotic persistence and bacterial heterogeneity and discuss recent studies that link bacterial persistence and tolerance with the evolution of antibiotic resistance. Finally, we review persister detection methods, novel strategies aiming at eradicating bacterial persisters and the latest advances in the development of new antibiotics.

Polyester Vascular Graft Material and Risk for Intracavitary Thoracic Vascular Graft Infection1.

Prosthetic vascular graft infections of the thoracic aorta are rare but can be fatal. Our comparison of collagen- and gelatin-coated grafts showed that collagen-coated grafts were associated with increased biofilm formation and bacterial adherence in vitro and with higher rates of perioperative vascular graft infections in vivo.

Antibiotics Stimulate Formation of Vesicles in Staphylococcus aureus in both Phage-Dependent and -Independent Fashions and via Different Routes.

Bacterial membrane vesicle research has so far focused mainly on Gram-negative bacteria. Only recently have Gram-positive bacteria been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MV formation, the underlying mechanisms are poorly understood. In this study, we show that antibiotics can induce MVs through different routes in the important human pathogen Staphylococcus aureus DNA-damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains of S. aureus but not in their phage-devoid counterparts. The ß-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is greater than that for MVs formed by ß-lactam antibiotic-induced blebbing. The purified MVs derived from S. aureus protected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, both in vitro and ex vivo in whole blood. In addition, the MVs protected S. aureus from killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.

Investigating group A Streptococcus antibiotic tolerance in necrotizing fasciitis.

Group 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. Difficult-to-treat infections are often characterized by the presence of bacteria surviving prolonged antibiotic exposure without displaying genetic resistance, referred to as persisters. In the present study, we investigated the presence of GAS persisters in tissue freshly debrided from patients as well as in an in vivo mouse model of NF and examined the phenomenon of antibiotic tolerance. Time-lapse imaging of GAS plated directly upon isolation from NF debrided tissue and an antibiotic challenge-based persisters assay were used to assess the presence of persisters. We show for the first time that GAS recovered directly from freshly debrided NF tissue is characterized by heterogeneous and overall delayed colony appearance time, suggesting the presence of persisters. Acidic pH or nutrient stress exposure, mimicking the NF-like environment in vitro, led to a similar phenotypic heterogeneity and resulted in enhanced survival upon antibiotic challenge, confirming the presence of GAS persisters. GAS persisters might contribute to NF treatment failure, despite extensive surgery and adequate antibiotic treatment.IMPORTANCEDifficult-to-treat and recurrent infections are a global problem burdening society and the health care system alike. Unraveling the mechanisms by which bacteria can survive antibiotic treatment without developing genetic resistance is of utmost importance to lay the foundation for new, effective therapeutic approaches. For the first time, we describe the phenomenon of antibiotic tolerance in group A Streptococcus (GAS) isolated from necrotizing fasciitis (NF) patients. Dormant, non-replicating cells (persisters) are tolerant to antibiotics and their occurrence in vivo is reported in an increasing number of bacterial species. Tailored treatment options, including the use of persisters-targeting drugs, need to be developed to specifically target dormant bacteria causing difficult-to-treat and recurrent infections.

Single-cell patterning and characterisation of antibiotic persistent bacteria using bio-sCAPA.

In microbiology, accessing single-cell information within large populations is pivotal. Here we introduce bio-sCAPA, a technique for patterning bacterial cells in defined geometric arrangements and monitoring their growth in various nutrient environments. We demonstrate bio-sCAPA with a study of subpopulations of antibiotic-tolerant bacteria, known as persister cells, which can survive exposure to high doses of antibiotics despite lacking any genetic resistance to the drug. Persister cells are associated with chronic and relapsing infections, yet are difficult to study due in part to a lack of scalable, single-cell characterisation methods. As >105 cells can be patterned on each template, and multiple templates can be patterned in parallel, bio-sCAPA allows for very rare population phenotypes to be monitored with single-cell precision across various environmental conditions. Using bio-sCAPA, we analysed the phenotypic characteristics of single Staphylococcus aureus cells tolerant to flucloxacillin and rifampicin killing. We find that antibiotic-tolerant S. aureus cells do not display significant heterogeneity in growth rate and are instead characterised by prolonged lag-time phenotypes alone.

Antibacterial Neutrophil Effector Response: Ex Vivo Quantification of Regulated Cell Death Associated with Extracellular Trap Release.

Regulated cell death (RCD) and the concomitant release of extracellular traps by neutrophils (NETs) constitute an important antibacterial effector response. Usually, the dynamic processes of RCD and NETs release are assessed independently of each other by either unspecific or time-consuming methods. Here, we describe a flow cytometry-based high-throughput analysis method incorporating neutrophil RCD and NETs release with visual live-imaging conformation upon ex vivo bacterial challenge. This combined approach allows to quantify and closely follow the kinetics of the dynamic neutrophil effector response towards bacterial infection.

Limited Adaptation 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 adaptation traits are understudied. We therefore assessed phenotypic and genotypic profiles of 11 S. aureus isolate pairs sampled from colonized patients simultaneously suffering from invasive S. aureus infections. Ten out of 11 isolate pairs displayed the same spa and multilocus sequence type, suggesting colonization as an origin for the invasive infection. Systematic analysis of colonizing and invasive isolate pairs showed similar adherence, hemolysis, reproductive fitness properties, antibiotic tolerance, and virulence in a Galleria mellonella infection model, as well as minimal genetic differences. Our results provide insights into the similar phenotypes associated with limited adaptation between colonizing and invasive isolates. Disruption of the physical barriers of mucosa or skin was identified in the majority of patients, further emphasizing colonization as a major risk factor for invasive disease. IMPORTANCE S. aureus is a major pathogen of humans, causing a wide range of diseases. The difficulty to develop a vaccine and antibiotic treatment failure 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 been effective 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 both host and 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 among colonizing and invasive isolates, our work suggests that barrier breaches are a key event in the disease continuum of S. aureus.

Systemic application of bone-targeting peptidoglycan hydrolases as a novel treatment approach for staphylococcal bone infection.

IMPORTANCE: The rising prevalence of antimicrobial resistance in S. aureus has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria.

Serine-threonine phosphoregulation by PknB and Stp contributes to quiescence and antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus can cause infections that are often chronic and difficult to treat, even when the bacteria are not antibiotic resistant because most antibiotics act only on metabolically active cells. Subpopulations of persister cells are metabolically quiescent, a state associated with delayed growth, reduced protein synthesis, and increased tolerance to antibiotics. Serine-threonine kinases and phosphatases similar to those found in eukaryotes can fine-tune essential bacterial cellular processes, such as metabolism and stress signaling. We found that acid stress-mimicking conditions that S. aureus experiences in host tissues delayed growth, globally altered the serine and threonine phosphoproteome, and increased threonine phosphorylation of the activation loop of the serine-threonine protein kinase B (PknB). The deletion of stp, which encodes the only annotated functional serine-threonine phosphatase in S. aureus, increased the growth delay and phenotypic heterogeneity under different stress challenges, including growth in acidic conditions, the intracellular milieu of human cells, and abscesses in mice. This growth delay was associated with reduced protein translation and intracellular ATP concentrations and increased antibiotic tolerance. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified targets of serine-threonine phosphorylation that may regulate bacterial growth and metabolism. Together, our findings highlight the importance of phosphoregulation in mediating bacterial quiescence and antibiotic tolerance and suggest that targeting PknB or Stp might offer a future therapeutic strategy to prevent persister formation during S. aureus infections.

Clonal complexes and virulence factors of Staphylococcus aureus from several cities in India.

BACKGROUND: Diseases from Staphylococcus aureus are a major problem in Indian hospitals and recent studies point to infiltration of community associated methicillin resistant S. aureus (CA-MRSA) into hospitals. Although CA-MRSA are genetically different from nosocomial MRSA, the distinction between the two groups is blurring as CA-MRSA are showing multidrug resistance and are endemic in many hospitals. Our survey of samples collected from Indian hospitals between 2004 and 2006 had shown mainly hospital associated methicillin resistant Staphylococcus aureus (HA-MRSA) carrying staphylococcal cassette chromosome mec (SCCmec) type III and IIIA. But S. aureus isolates collected from 2007 onwards from community and hospital settings in India have shown SCCmec type IV and V cassettes while several variations of type IV SCCmec cassettes from IVa to IVj have been found in other parts of the world. In the present study, we have collected nasal swabs from rural and urban healthy carriers and pus, blood etc from in patients from hospitals to study the distribution of SCCmec elements and sequence types (STs) in the community and hospital environment. We performed molecular characterization of all the isolates to determine their lineage and microarray of select isolates from each sequence type to analyze their toxins, virulence and immune-evasion factors. RESULTS: Molecular analyses of 68 S. aureus isolates from in and around Bengaluru and three other Indian cities have been carried out. The chosen isolates fall into fifteen STs with all major clonal complexes (CC) present along with some minor ones. The dominant MRSA clones are ST22 and ST772 among healthy carriers and patients. We are reporting three novel clones, two methicillin sensitive S. aureus (MSSA) isolates belonging to ST291 (related to ST398 which is live stock associated), and two MRSA clones, ST1208 (CC8), and ST672 as emerging clones in this study for the first time. Sixty nine percent of isolates carry Panton- Valentine Leucocidin genes (PVL) along with many other toxins. There is more diversity of STs among methicillin sensitive S. aureus than resistant ones. Microarray analysis of isolates belonging to different STs gives an insight into major toxins, virulence factors, adhesion and immune evasion factors present among the isolates in various parts of India. CONCLUSIONS: S. aureus isolates reported in this study belong to a highly diverse group of STs and CC and we are reporting several new STs which have not been reported earlier along with factors influencing virulence and host pathogen interactions.

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

OBJECTIVES: Difficult-to-treat infections caused by antibiotic-susceptible strains have been linked to the occurrence of persisters, a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. These persisters can be identified phenotypically by 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 remains unknown. METHODS: We 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. We retained 36 Staphylococcus aureus monocultures for further analysis. We investigated clinical factors associated with increased colony growth-delay with regression analyses. We corroborated the clinical findings using in vitro grown static biofilms exposed to distinct antibiotics. RESULTS: The extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients (from no delay to a maximum of 57.6 hours). Increased heterogeneity coincided with increased median colony growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay (13.3 hours; 95% CI 7.13-19.6 hours; p < 0.001). S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with clindamycin or levofloxacin exhibited prolonged growth-delay (p < 0.05 for 11 of 12 comparisons), correlating with a strain-dependent increase in antibiotic tolerance. DISCUSSION: Colony-size heterogeneity upon direct sampling of difficult-to-treat S. aureus infections was frequently observed. Hence, future studies are needed to assess the potential benefit of phenotypic heterogeneity quantification for staphylococcal infection prognosis and treatment guidelines.

SARS-CoV-2 leads to a small vessel endotheliitis in the heart.

BACKGROUND: SARS-CoV-2 infection (COVID-19 disease) can induce systemic vascular involvement contributing to morbidity and mortality. SARS-CoV-2 targets epithelial and endothelial cells through the ACE2 receptor. The anatomical involvement of the coronary tree is not explored yet. METHODS: Cardiac autopsy tissue of the entire coronary tree (main coronary arteries, epicardial arterioles/venules, epicardial capillaries) and epicardial nerves were analyzed in COVID-19 patients (n = 6). All anatomical regions were immunohistochemically tested for ACE2, TMPRSS2, CD147, CD45, CD3, CD4, CD8, CD68 and IL-6. COVID-19 negative patients with cardiovascular disease (n = 3) and influenza A (n = 6) served as controls. FINDINGS: COVID-19 positive patients showed strong ACE2 / TMPRSS2 expression in capillaries and less in arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor and had only mild intimal inflammation. Epicardial capillaries had a prominent lympho-monocytic endotheliitis, which was less pronounced in arterioles/venules. The lymphocytic-monocytic infiltrate strongly expressed CD4, CD45, CD68. Peri/epicardial nerves had strong ACE2 expression and lympho-monocytic inflammation. COVID-19 negative patients showed minimal vascular ACE2 expression and lacked endotheliitis or inflammatory reaction. INTERPRETATION: ACE2 / TMPRSS2 expression and lymphomonocytic inflammation in COVID-19 disease increases crescentically towards the small vessels suggesting that COVID-19-induced endotheliitis is a small vessel vasculitis not involving the main coronaries. The inflammatory neuropathy of epicardial nerves in COVID-19 disease provides further evidence of an angio- and neurotrophic affinity of SARS-COV2 and might potentially contribute to the understanding of the high prevalence of cardiac complications such as myocardial injury and arrhythmias in COVID-19. FUNDING: No external funding was necessary for this study.

Bacterial pulmonary superinfections are associated with longer duration of ventilation in critically ill COVID-19 patients.

The impact of secondary bacterial infections (superinfections) in coronavirus disease 2019 (COVID-19) is not well understood. In this prospective, monocentric cohort study, we aim to investigate the impact of superinfections in COVID-19 patients with acute respiratory distress syndrome. Patients are assessed for concomitant microbial infections by longitudinal analysis of tracheobronchial secretions, bronchoalveolar lavages, and blood cultures. In 45 critically ill patients, we identify 19 patients with superinfections (42.2%). Superinfections are detected on day 10 after intensive care admission. The proportion of participants alive and off invasive mechanical ventilation at study day 28 (ventilator-free days [VFDs] at 28 days) is substantially lower in patients with superinfection (subhazard ratio 0.37; 95% confidence interval [CI] 0.15-0.90; p = 0.028). Patients with pulmonary superinfections have a higher incidence of bacteremia, virus reactivations, yeast colonization, and required intensive care treatment for a longer time. Superinfections are frequent and associated with reduced VFDs at 28 days despite a high rate of empirical antibiotic therapy.