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.

Bone marrow haematopoiesis in patients with COVID-19.

AIMS: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection broadly affects organ homeostasis, including the haematopoietic system. Autopsy studies are a crucial tool for investigation of organ-specific pathologies. Here we perform an in-depth analysis of the impact of severe coronavirus disease 2019 (COVID-19) on bone marrow haematopoiesis in correlation with clinical and laboratory parameters. METHODS AND RESULTS: Twenty-eight autopsy cases and five controls from two academic centres were included in the study. We performed a comprehensive analysis of bone marrow pathology and microenvironment features with clinical and laboratory parameters and assessed SARS-CoV-2 infection of the bone marrow by quantitative polymerase chain reaction (qPCR) analysis. In COVID-19 patients, bone marrow specimens showed a left-shifted myelopoiesis (19 of 28, 64%), increased myeloid-erythroid ratio (eight of 28, 28%), increased megakaryopoiesis (six of 28, 21%) and lymphocytosis (four of 28, 14%). Strikingly, a high proportion of COVID-19 specimens showed erythrophagocytosis (15 of 28, 54%) and the presence of siderophages (11 of 15, 73%) compared to control cases (none of five, 0%). Clinically, erythrophagocytosis correlated with lower haemoglobin levels and was more frequently observed in patients from the second wave. Analysis of the immune environment showed a strong increase in CD68+ macrophages (16 of 28, 57%) and a borderline lymphocytosis (five of 28, 18%). The stromal microenvironment showed oedema (two of 28, 7%) and severe capillary congestion (one of 28, 4%) in isolated cases. No stromal fibrosis or microvascular thrombosis was found. While all cases had confirmed positive testing of SARS-CoV-2 in the respiratory system, SARS-CoV-2 was not detected in the bone marrow by high-sensitivity PCR, suggesting that SARS-CoV-2 does not commonly replicate in the haematopoietic microenvironment. CONCLUSIONS: SARS-CoV-2 infection indirectly impacts the haematological compartment and the bone marrow immune environment. Erythrophagocytosis is frequent and associated with lower haemoglobin levels in patients with severe COVID-19.

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.

Human Streptococcal Necrotizing Fasciitis Histopathology Mirrored in a Murine Model.

Streptococcal necrotizing fasciitis (NF) causes high morbidity and mortality despite state-of-the-art therapy. Low incidence and rapid disease progression, necessitating immediate initiation of therapy, have proven challenging aspects for setting up prospective randomized trials. This has resulted in little therapeutic progress over the past decade. The validation of reliable murine NF models to study both pathogenesis and optimized therapeutic regimens of streptococcal NF are thus essential. In this study, we characterized a murine NF model and compared the pathology with an in-depth tissue analysis of streptococcal NF in patients. We found that the streptococcal murine NF model closely reflected all histologic characteristics encountered in human streptococcal NF. This murine NF model helps understanding of human NF pathology better in a time-dependent manner and will allow studying novel therapeutic options in the future.

Immunoglobulin Attenuates Streptokinase-Mediated Virulence in Streptococcus dysgalactiae Subspecies equisimilis Necrotizing Fasciitis.

Background: Necrotizing fasciitis (NF) retains a very high mortality rate despite prompt and adequate antibiotic treatment and surgical debridement. Necrotizing fasciitis has recently been associated with Streptococcus dysgalactiae subspecies equisimilis (SDSE). Methods: We investigated the causes of a very severe clinical manifestation of SDSE-NF by assessing both host and pathogen factors. Results: We found a lack of streptokinase-function blocking antibodies in the patient resulting in increased streptokinase-mediated fibrinolysis and bacterial spread. At the same time, the clinical SDSE isolate produced very high levels of streptokinase. Exogenous immunoglobulin Gs (ex-IgGs) efficiently blocked streptokinase-mediated fibrinolysis in vitro, indicating a protective role against the action of streptokinase. In vivo, SDSE infection severity was also attenuated by ex-IgGs in a NF mouse model. Conclusions: These findings illustrate for the first time that the lack of specific antibodies against streptococcal virulence factors, such as streptokinase, may contribute to NF disease severity. This can be counteracted by ex-IgGs.

Treatment of non-alcoholic steatohepatitis patients with vitamin D: a double-blinded, randomized, placebo-controlled pilot study.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is defined by liver inflammation and consecutive fibrotic damage caused by a deposition of fat in the liver. No licensed medical treatments exist and lifestyle modification is difficult to incorporate into everyday life. We investigated the efficacy and safety of a 48-week treatment with vitamin D3 in NASH patients. METHODS: Histologically determined NASH patients with elevated alanine aminotransferase (ALT) and decreased 25-OH vitamin D level at baseline received vitamin D3 or placebo orally over a 48-week period. The primary endpoint of this study was the change in ALT from baseline to the end-of-treatment. Steatohepatitis was categorized according to the Steatosis, Activity and Fibrosis Score and disease activity was assessed using the NAFLD activity score. RESULTS: Serum 25-OH vitamin D levels significantly increased only in the vitamin D3 group over the 48-week treatment phase indicating compliance. In contrast to placebo, patients in the vitamin D group had markedly decreased ALT levels after the end-of-treatment phase. A significant decrease during treatment with vitamin D was also observed for cytokeratin-18 fragments compared with placebo. The study was not powered to detect changes in histological score, hence only descriptive results for histopathological characteristics are available. CONCLUSIONS: Treatment with 2100 IE vitamin D q.d. over 48 weeks was well tolerated and led to a significant improvement of serum ALT levels in patients with hypovitaminosis D and histology-proven NASH as the primary endpoint together with a trend toward reduction of hepatic steatosis, which was not significant due to a small number of available biopsy specimens.

Clindamycin Affects Group A Streptococcus Virulence Factors and Improves Clinical Outcome.

Group A Streptococcus (GAS) has acquired an arsenal of virulence factors, promoting life-threatening invasive infections such as necrotizing fasciitis. Current therapeutic regimens for necrotizing fasciitis include surgical debridement and treatment with cell wall-active antibiotics. Addition of clindamycin (CLI) is recommended, although clinical evidence is lacking. Reflecting the current clinical dilemma, an observational study showed that only 63% of the patients with severe invasive GAS infection received CLI. This work thus aimed to address whether CLI improves necrotizing fasciitis outcome by modulating virulence factors of CLI-susceptible and CLI-resistant GAS in vitro and in vivo. Treatment with CLI reduced extracellular DNase Sda1 and streptolysin O (SLO) activity in vivo, whereas subinhibitory CLI concentrations induced expression and activity of SLO, DNase, and Streptococcus pyogenes cell envelope protease in vitro. Our in vivo results suggest that CLI should be administered as soon as possible to patients with necrotizing fasciitis, while our in vitro studies emphasize that a high dosage of CLI is essential.

Molecular detection of hepatitis E virus (HEV) in liver biopsies after liver transplantation.

We aimed to determine the rate of hepatitis E virus (HEV) infection, a recently increasingly recognized disease in the Western world, in liver transplant patients by direct molecular testing of liver tissue. A RT-PCR assay was designed for detecting the HEV open reading frame (ORF) 2/3 gene region in formalin-fixed, paraffin-embedded tissues, and applied to all liver biopsies (n=683) taken 4 weeks or later from all patients (n=282) after liver transplantation of two large academic centers. HEV-RNA was detected in ten biopsies from four different patients (rate: 1%). Histology in early HEV infection was variable including cases with only few hepatocellular apoptoses, no or only minute inflammation. Hepatitis lasted for at least 6 months in 3/4 patients. Serologic testing for HEV-RNA in a subcohort (159 patients) was positive in five patients (rate: 3%), resulting in an overall HEV detection rate of 3% (8/282). In case both liver tissue and sera of a patient were available from the same time period, all cases tested positive in one material were also tested positive in the other material, respectively. All patients had de novo autochthonous infection with HEV genotype 3. Our data confirm that HEV infection is a relevant cause of liver injury after liver transplantation. Molecular testing for HEV in routinely processed transplant liver biopsies is powerful for evaluating patients with elevated transaminases of unknown origin. Histology of HEV infection under immunosuppression in the early phase is distinct from HEV infection in immunocompetent individuals.

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.

Apoptotic colonopathy under immunosuppression: mycophenolate-related effects and beyond.

BACKGROUND/AIMS: Diarrhea developing in patients under immunosuppression has a variety of underlying causes. A broad spectrum of histological findings can be found in intestinal biopsies taken for the diagnostic workup of diarrhea including a pathologically increased proportion of epithelial cell apoptosis in the colon, for which the descriptive term 'apoptotic colonopathy' was coined. In recent years, the immunosuppressive drug mycophenolate (mycophenolic acid, MPA) has been identified as a prototypical cause of apoptotic colonopathy, but other conditions may show similar or overlapping histological pictures. METHODS: Cases of likely or possible MPA colonopathy (n = 18) were retrospectively identified from the archive files. Clinical information on patient history, clinical presentation and endoscopic findings were recorded. All cases were routinely processed, i.e. stained by hematoxylin and eosin (HE) stain, optionally supplemented by special histochemical and immune stains. RESULTS AND CONCLUSION: Histopathological hallmarks of MPA treatment-related changes in the colon mucosa are reviewed with respect to the major histopathological differential diagnoses including normal and near-normal findings, infectious diseases, graft-versus-host disease and inflammatory bowel diseases. Furthermore, the challenge of multiple concomitant pathologies while on MPA treatment, in particular infectious diseases, is discussed, and some open questions concerning the effects of MPA on colon pathology are pointed out.

Oral, esophageal and cutaneous lichen ruber planus controlled with alitretinoin: case report and review of the literature.

BACKGROUND: Therapy-resistant lichen planus (LP) can be a challenging condition for dermatologists. There are some case reports about successful treatments with alitretinoin of cutaneous and oral, but not of esophageal LP. OBJECTIVE: We present the unique case of a patient with cutaneous, oral and esophageal LP which was refractory to classical treatment options (topical clobetasol propionate and pimecrolimus, intramuscular triamcinolone acetonide); because of systemic side effects the patient did not tolerate systemic acitretin dosed up to 25 mg daily. METHODS: Oral alitretinoin was used at a dose of 30 mg daily. RESULTS: Both oral and skin changes as well as dysphagia completely resolved within 4 weeks without any severe side effects and the drug was used for 6 months. No papules, intraoral striae or dysphagia recurred during the 6 months of treatment. After 4 months the patient relapsed with mucosal patches so that a second cycle was initiated for 6 months where oral LP lesions resolved after 4 weeks also (with sporadic mild headache). CONCLUSION: Further studies are needed to better understand the impact of alitretinoin in LP. Our observation suggests alitretinoin as a new, well-tolerated treatment option for esophageal LP after failed response to conventional treatments.

Determinants of Advanced Liver Fibrosis in Adult Patients After Fontan Palliation: Usefulness of Ultrasound Transient Elastography.

BACKGROUND: Fontan-associated liver disease is an increasing concern. Our aim was to assess prevalence and predictors of advanced liver fibrosis with a specific focus on utility of liver stiffness measurement by ultrasound transient elastography. METHODS: A total of 97 adult Fontan patients (55% male, median age: 23.1 years, interquartile range [IQR]: 18.7-30.6); 92 (95%) were evaluated with transient elastography, and 50 (52%) underwent transjugular liver biopsy. Advanced liver fibrosis was defined as congestive hepatic fibrosis score 3 or 4. RESULTS: Only 4 patients (4%) had liver stiffness values < 10 kilopascal (kPa). Liver-stiffness measurements correlated weakly with peak oxygen uptake on exercise testing and Fontan pressure but not with Model for End-Stage Liver Disease excluding INR (MELD-XI) score or spleen size. Serial follow-up liver stiffness measurements in 73 clinically stable patients showed large variability among individual patients. Advanced liver fibrosis was present in 35 of 50 (70%) patients on liver biopsy and was associated to MELD-XI-Score ≥ 11 and splenomegaly but not to liver-stiffness measurements. Advanced liver fibrosis was not associated with patient age or time since Fontan operation but with younger age at completion of Fontan (3.7 years, IQR: 2.3-6.3 vs 6.8 years; IQR: 3.5-12.1; P = 0.037). CONCLUSIONS: In our cohort, advanced liver fibrosis was present in the majority of adult Fontan patients. Liver stiffness as measured by transient elastography was not associated with the degree of liver fibrosis. Because of its high variability on serial measurements, it seems not to be useful for clinical decision making. The unexpected finding that younger age at completion of Fontan was associated with advanced liver fibrosis merits further evaluation.

A retrospective analysis of blood culture-negative endocarditis at a tertiary care centre in Switzerland.

AIMS OF THE STUDY: Numerous studies from different countries have contributed to an improved understanding of blood culture-negative infective endocarditis. However, little is known about its epidemiology and microbiology in Switzerland. We aimed to assess the epidemiology and microbiology of blood culture-negative endocarditis at the University Hospital Zurich, Switzerland. METHODS: We screened all patients hospitalised between 1997 and 2020 with possible or definite endocarditis at our institution. Thereof, we identified all cases with blood culture-negative endocarditis and retrospectively retrieved patient characteristics, microbiological, histopathological, radiographic and surgical data from medical records. RESULTS: Among 861 patients screened, 66 (7.7%) cases of blood culture-negative endocarditis were identified. Thereof, 31 cases could be microbiologically documented or not documented (n = 30), and in five cases a non-infectious aetiology was confirmed. Endocarditis predominantly affected men (77%) and the left heart (79%); predisposing factors were prosthetic valves (42%), congenital heart disease (35%) and prior endocarditis (14%). The most common reasons for negative blood cultures were antibiotic treatment prior to blood culture sampling (35%), fastidious and slow growing microorganisms (30%) and definite non-infective endocarditis (8%). Coxiella burnetii and Bartonella spp. were the most common fastidious bacteria identified. In addition to serology, identification of causative microorganisms was possible by microbiological and/or histopathological analysis of tissue samples, of which polymerase chain reaction testing (PCR) of the 16S ribosomal RNA proved to be most successful. CONCLUSIONS: The present study provides a detailed analysis of blood culture-negative endocarditis over a time span of more than 20 years in Zurich, Switzerland. Antibiotic treatment prior to blood collection, and fastidious and slow growing organisms were identified as main reasons for sterile blood cultures. Typical culture-negative bacteria were mainly found by PCR and/or culture of tissue samples.

Primary cutaneous nocardiosis of the head and neck in an immunocompetent patient.

Nocardiosis is known to be an opportunistic infection most commonly affecting immunocompromised patients that can lead to life-threatening conditions. Primary cutaneous disease remains a rare manifestation and unlike pulmonary or disseminated nocardiosis, it usually affects immunocompetent individuals. We present a case of a primary cutaneous nocardiosis of the head and neck after an insect bite in a healthy 50-year-old woman who had recently travelled from Greece. She presented with a painful right-sided swelling of her face and neck and an ulcerated plaque over the right temple. Biopsy of the plaque revealed inflammation with abscess formation indicating underlying infection. Culture from the biopsy showed growth of Nocardia spp and 16S rRNA gene sequence analysis identified Nocardia brasiliensis The patient was treated with trimethoprim/sulfamethoxazole and subsequently switched to amoxicillin/clavulanic acid due to a drug eruption. Antibiotic therapy was continued for a total of 3 months with complete resolution of the skin lesions.

Group A Streptococcal DNase Sda1 Impairs Plasmacytoid Dendritic Cells' Type 1 Interferon Response.

Group A Streptococcus causes severe invasive infections, including necrotizing fasciitis. The expression of an array of virulence factors targeting specific host immune functions impedes successful bacterial clearance. The virulence factor streptococcal DNase Sda1 was previously shown to interfere with the entrapment of bacteria through neutrophil extracellular traps and TLR9 signaling. In this study, we showed that plasmacytoid dendritic cells are recruited to the infected tissue during group A streptococcal necrotizing fasciitis. We found that the streptococcal DNase Sda1 impairs plasmacytoid dendritic cell recruitment by reducing IFN-1 levels at the site of infection. We found that streptococcal DNase Sda1 interferes with stabilization of the DNA by the host molecule HMGB1 protein, which may account for decreased IFN-1 levels at the site of infection.

Simultaneous endoscopic full-thickness resection of two synchronous colonic granular cell tumours.

Granular cell tumours (GCTs) are rare soft tissue tumours originating from Schwann cells. Due to potential malignant transformation, complete endoscopic resection should be aimed for. We report on a 49-year-old patient with two synchronous GCTs found in the caecum and the ascending colon, respectively. Synchronous endoscopic full-thickness resection (EFTR) using an all-in-one full-thickness resection device (FTRD) was performed under propofol sedation. Completeness of resection was proven histologically. No adverse events occurred. We report safe and complete simultaneous EFTR of two synchronous colonic GCTs.

Asymptomatic congenital tuberculosis: A case report.

RATIONALE: Congenital tuberculosis (TB) is described as a rare, but severe disease. In contrast to the cases with severe symptoms reported so far, we describe a child with asymptomatic congenital TB. PATIENT CONCERNS: An 8-week-old girl was investigated because of newly diagnosed TB in her mother, which complained about cough since 21 weeks gestation. Lung biopsy tissue specimens of the mother revealed necrotizing granuloma with a single acid-fast bacillus (AFB) and Mycobacterium tuberculosis (MTB) was detected by polymerase chain reaction. Bronchoalveolar lavage was negative for AFB smear and culture, arguing against postnatal transmission of MTB. TB contact investigations were negative. The child, at the age of 8 weeks at first assessment, was in an excellent general condition and diagnosed with congenital TB by culture-positive lung TB and exclusion of postnatal transmission. DIAGNOSES: The child fulfilled Cantwell criteria to diagnose congenital TB. INTERVENTIONS: Ambulatory anti-tuberculosis treatment was initiated for 6 months. OUTCOMES: The 18 months follow-up was uneventful. LESSONS: This case of asymptomatic congenital TB in a young child illustrates the diagnostic difficulties in congenital TB and raises the question whether congenital TB is underestimated.

Lymphadenopathy driven by TCR-Vγ8Vδ1 T-cell expansion in FAS-related autoimmune lymphoproliferative syndrome.

FAS-dependent apoptosis in Vδ1 T cells makes the latter possible culprits for the lymphadenopathy observed in patients with FAS mutations.Rapamycin and methylprednisolone resistance should prompt clinicians to look for Vδ1 T cell proliferation in ALPS-FAS patients.

Cutaneous Melanoma with Brain Metastasis: Report of 193 Patients with New Observations.

BACKGROUND: Brain metastasis is a common endpoint in patients suffering from malignant melanoma. However, little is known about factors that predispose to brain metastases. OBJECTIVE: We performed a retrospective clinical and pathological investigation of melanoma patients with brain metastases in order to better characterise this patient population. METHODS: 193 melanoma patients with brain metastasis histologically diagnosed between 1990 and 2015 at the University Hospital Zurich were retrospectively identified and further specified for sex, age at diagnosis and detection of brain metastasis, and localisation. In addition, data were extracted regarding the subtype of primary melanoma, Breslow tumour thickness, Clark Level, mutation status, extent of metastatic spread and history of a second melanoma. RESULTS: We found a significant male predominance (n = 126/193; 65%; p < 0.001). Breslow tumour thickness showed a wide range from 0.2 to 12.0 mm (n = 99; median 2.3 mm). 14 of 101 melanomas (14%) were classified as T1, thereof 11 (79%) were found in men. In 32 of 193 patients (17%), the primary melanoma was unknown. CONCLUSIONS: Of special interest in our series is the high incidence of male predominance (79%) in cases of thin metastasing melanoma (14%), implicating genetic or epigenetic (hormonal) gender differences underlying tumour progression. Additionally, the high percentage of unknown primary melanoma (17%), at least partly representing completely regressed melanomas, indicates the importance of immune surveillance in melanoma progression.