Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus. It is characterized by inflammation and infection in the lung parenchyma, often presenting with symptoms such as cough, fever, and difficulty breathing. Empyema, on the other hand, is a severe complication of pneumonia marked by the accumulation of pus in the pleural cavity. Streptococcus pyogenes (S. pyogenes), also known as group A Streptococcus (GAS), is a bacterium that can cause various infections, including pharyngitis and skin infections. In rare cases, it can lead to community-acquired pneumonia. In our case report, we describe a 32-year-old female with a history of mild persistent asthma who contracted influenza B virus, eventually developing pneumonia caused by GAS, S. pyogenes.Pneumonia is a common respiratory infection typically caused by pathogens such as  Streptococcus pyogenes (S. pyogenes) is a major human pathogen that causes a wide range of infections, from minor to severe and invasive diseases with major morbidity and mortality. The M protein (emm type) plays an important role in pathogenicity [S. pyogenes is a relatively uncommon cause of community-acquired pneumonia [S. pyogenes pneumonia. Acute viral respiratory tract infection, including influenza, is followed by pneumonia caused by S. pyogenes [S. pyogenes.genicity . Typicalneumonia . Diabetepyogenes . Our cas9 cells/L , lactate of 4.2 mmol/L , blood urea nitrogen concentration of 43 mg/dL , and procalcitonin of >100.0 ng/mL . All of these variables were above the normal range. Arterial blood gas showed a pH of 7.16 , pCO2 of 61.9 mmHg , pO2 of 79.0 mmHg  and HCO3 of 21 mmol/L . Urinalysis was positive for protein (>500), esterase (trace), WBC (10-19), WBC clumps, and red blood cells (30-49). Urine Legionella and Streptococcus pneumoniae antigens were negative. A chest radiograph showed moderate bilateral pleural effusions  with complaints of myalgias, chills, nausea, and vomiting. She was diagnosed with influenza B virus infection by a respiratory pathogen panel in the ED and was discharged with advice for symptomatic therapy with acetaminophen and ibuprofen. After four days, she presented to the ED again with worsening shortness of breath and productive cough with blood-tinged sputum. She was hemodynamically unstable on presentation with a blood pressure of 70/48 mmHg, pulse rate of 103 beats/minute, respiratory rate of 25 breaths/minute, and temperature of 97.5°F. Her oxygen saturation was 91%. A physical examination revealed crackles in bilateral lungs and sternal retractions. Laboratory tests showed a high white blood cell (WBC) count of 18.47 x 10Computed tomography (CT) of the chest without contrast confirmed the moderate bilateral pleural effusions Figure . It alsoEchocardiography showed no abnormalities. Sputum culture and blood culture samples were collected at the time of admission.2); however, following the CT scan, she developed increased work of breathing and restlessness as well as worsening hypoxia requiring up-titration of FiO2. Given her restlessness, prior intolerance to BiPAP, hypercarbia on venous blood gas (pH -> 7.14 pCO2 -> 65.4 pO2 -> 43.0), and altered mentation, intubation was favored. She was admitted to the intensive care unit (ICU) with the diagnosis of acute hypercarbic respiratory failure secondary to acute exacerbation of asthma due to influenza infection. Intravenous administration of cefepime (1 g/day) and vancomycin was started along with oral administration of azithromycin 500 mg once a day for broad-spectrum coverage. She also received nebulization with bronchodilators. Following admission, a sputum smear detected no acid-fast bacteria and blood cultures showed no growth. Notably, beta-hemolytic group A streptococci were detected in sputum culture and were identified as S. pyogenes. Due to the methicillin-resistant Staphylococcus aureus test being negative, vancomycin was discontinued. As cultures did not show evidence of gram-negative rods, cefepime was discontinued and intravenous ceftriaxone 2 g/day was started, as directed by Infectious Disease specialists. Despite antibiotic therapy, the patient’s oxygenation worsened, indicated by a falling PaO2/FiO2 ratio. Ventilator settings were optimized in accordance with the acute respiratory distress protocol and the patient was paralyzed and proned. Reevaluation with a CT angiogram performed on day eight revealed enlarging bilateral pleural effusions  was done in the ED. She did not tolerate BiPAP and oxygen supplementation with a high-flow nasal cannula was attempted. She was initially saturating well on 50% fraction of inspired oxygen  and 0.67 on the left. The patient received tissue plasminogen activator (t-PA) and DNase in both chest tubes. The dose of DNase was 5 mg, and the dose of t-PA was 10 mg. They were given intrapleurally twice daily for three days, followed by clamping of the drain to permit the drugs to stay in the pleural space for one hour. Following this therapy, imaging showed reduced bilateral effusions , and other Streptococcus spp. [S. pyogenes. Without any significant trend throughout the course of the study period, there were between one and eight occurrences per year  [Pneumonia is commonly caused by cus spp. ,5. Our cto 2.29) .Streptococcus (GAS) can colonize humans asymptomatically or usually cause pharynx and skin infections. However, S. pyogenes, as a rare cause of community-acquired pneumonia, may exhibit a high mortality rate [S. pyogenes pneumonia and reported a high mortality rate (47%). S. pyogenes pneumonia usually occurs in winter and spring. Common comorbidities in patients with S. pyogenes pneumonia include diabetes, hypertension, and COPD. Pleural effusion, cavity formation in the lungs, septicemia, and septic shock are reported complications of S. pyogenes pneumonia. Sakai et al. documented a case of invasive GAS infection in an otherwise healthy adult male. This infection initially presented as community-acquired pneumonia and subsequently led to the development of pleural empyema. Ultimately, the patient experienced streptococcal toxic shock syndrome as a severe complication of this infection [S. pyogenes pneumonia cases, is the most common symptom suggesting this diagnosis, as seen in our patient. In rare cases, symptoms similar to scarlet fever, such as a desquamating rash, may be noted [S. pyogenes pneumonia to hospitalization can range from one to nine days, with a mean duration of 4.8 ± 2.4 days [Group A ity rate , respondity rate . Barnhamity rate  reviewedty rate 4%. S. pyoty rate 4%. S. pyoS. pyogenes pneumonia, patients had a history of respiratory illnesses  and showed co-infection of influenza B virus and S. pyogenes. Acute viral tract infections have been associated with S. pyogenes pneumonia due to virus-induced epithelial damage, altered or decreased ciliary activity of the respiratory epithelium, which favors bacterial infection, and transient immune suppression [EMM gene and resistant to phagocytosis, is a significant bacterial virulence factor. Among 82 invasive GAS isolates in Japan, the EMM1 gene was the most common (32.9%) and was substantially associated with poor outcomes [S. pyogenes illness, with M1 and M3 strains being the most commonly isolated and linked with pneumonia and deep soft tissue infections [S. pyogenes pneumonia [S. pyogenes pneumonia who have a history of influenza virus infection. GAS appears to cause higher morbidity and a longer hospital stay compared to S. pneumonia patients. It is more commonly isolated from pleural fluid in the absence of bacteremia, prompting a pleural fluid tap for appropriate diagnosis.In previous similar case reports on pression . Many baommon 32.% and wasfections . Accordifections . These cfections ,13. In oS. pyogenes infections in people who had received an influenza vaccination [Influenza vaccination can lower the risk of secondary bacterial infections. Studies have shown a decrease in cination ,15. ImmeIn June 2022, Mainali et al. reported a case of a 57-year-old female with a large right exudative effusion complicated with parapneumonic effusion treated with t-PA and DNAse therapy leading to the formation of hemothorax on the first treatment with t-PA . On the S. pyogenes vaccinations leaves our coverage of bacterial vaccines insufficient. Additionally, one of the limitations of our research is our inability to define the M protein serotype of the S. pyogenes strain discovered in our patient. Further study is needed to discover additional factors, such as previous viral infections other than influenza, and mechanisms for worsening S. pyogenes pneumonia.Nosocomial outbreaks and secondary cases in close contacts are common; therefore, infection control measures and prophylactic antibiotics for high-risk contacts are crucial aspects of disease control. However, the lack of S. pyogenes pneumonia in a patient with a history of asthma following influenza virus B infection. The rarity of S. pyogenes as a cause of community-acquired pneumonia underscores the importance of considering less common pathogens, especially in individuals with underlying respiratory conditions. The clinical course of this patient, marked by severe respiratory distress, pleural effusion, and prolonged mechanical ventilation, serves as a reminder of the potential severity of S. pyogenes pneumonia. Early recognition and initiation of appropriate antibiotic therapy are essential in managing such cases, and vaccination against influenza plays a critical role in reducing the risk of secondary bacterial infections. Healthcare providers should maintain a high index of suspicion for rare pathogens, even in cases of community-acquired pneumonia that do not fit the typical profile.This case report highlights the unique presentation of


Porphyromonas gingivalis contributes to the chronic oral disease periodontitis, triggering the activation of host inflammatory responses, inducing cellular stresses such as oxidation. During stress, host cells can activate the Integrated Stress Response (ISR), a pathway which determines cellular fate, by either downregulating protein synthesis and initiating a stress–response gene expression program, or by initiating programmed cell death. Recent studies have implicated the ISR within both host antimicrobial defenses and the pathomechanism of certain microbes. In this study, using a combination of immunofluorescence confocal microscopy and immunoblotting, the molecular mechanisms by which P. gingivalis infection alters translation attenuation during oxidative stress-induced activation of the ISR in oral epithelial cells were investigated. P. gingivalis infection alone did not result in ISR activation. In contrast, infection coupled with stress caused differential stress granule formation and composition. Infection heightened stress-induced translational repression independently of core ISR mediators. Heightened translational repression during stress was observed with both P. gingivalis–conditioned media and outer membrane vesicles, implicating a secretory factor in this exacerbated translational repression. The effects of gingipain inhibitors and gingipain-deficient P. gingivalis mutants confirmed these pathogen-specific proteases as the effector of exacerbated translational repression. Gingipains are known to degrade the mammalian target of rapamycin (mTOR) and the findings of this study implicate the gingipain-mTOR axis as the effector of host translational dysregulation during stress.  The oral cavity harbors a wide array of biofilm-forming bacteria, which form a symbiotic relationship with their host . HoweverPorphyromonas gingivalis, the keystone pathogen, as well as Tannerella forsythia and Treponema denticola ..P. gingiP. gingivalis infection and heightened oxidative stress-induced translational stalling independently of p-eIF2α (P. gingivalis gingipains, which are known to degrade and inhibit mTOR [P. gingivalis, where they exist both freely and packaged within OMVs [P. gingivalis-conditioned media and OMVs exhibiting a similar phenotype to internalized bacteria, the role of gingipains was investigated. Inhibition of secreted gingipains in conditioned media by gingipain-specific inhibitors inhibited the heightened translational attenuation observed with the conditioned media (P. gingivalis secreted lysine-specific gingipains for mTOR degradation [P. gingivalis-negative cells of the exposed population, as OMVs containing gingipains only enter around 8% of cells [P. gingivalis and oxidative stress (P. gingivalis invasion and internalization of host cells [As the heightened translational repression was eIF2α-independent and downstream mTORC1 targets were altered, rapamycin, a potent mTOR inhibitor, was used to further probe the pathway. During oxidative stress, rapamycin induced the same phenotype as  p-eIF2α A,B, furtbit mTOR ,39, are hin OMVs ,81. Withed media A. This ied media B. Since ed media D, which radation . This maof cells . In conte stress B. Furthest cells ,84, it cP. gingivalis dysregulates translational control and stress granule formation during oxidative stress, a condition phenotypic of the chronic inflammatory environment induced during periodontitis and caused by P. gingivalis [P. gingivalis to modulate host response, given that these pathways feed into cellular survival and the wider immune and inflammatory response [P. gingivalis. Furthermore, dysregulation of the ISR, translational control, and stress granule dynamics have been implicated in a range of diseases from cancer to neurodegeneration [This study, for the first time, demonstrates that the periodontopathogen ngivalis  (Illustrresponse , can conneration ,85,86.P. gingivalis virulence factors including capsule, fimbriae, and various effector proteins may also converge onto these pathways. This study utilized a single-cell model and mono-species P. gingivalis infection, and to build on and extend these findings, it will be useful to look at the effect of polymicrobial infection on translational control. This is particularly relevant as induction of NOD1 signaling has been reported in periodontic mouse models [P. gingivalis infection, and other diseases.While these findings implicate gingipains as defined effectors of translational dysregulation, studies into other e models  and NOD1e models . Further


Psychosomatic symptoms are an important problem that is frequently presented in medical consultations. These symptoms are often associated with psychiatric disorders, especially depressive and anxiety disorders.To study the association between anxiety disorders and psychosomatic symptoms in a sample of patients referred for pathology of functional origin.We made a descriptive retrospective study through the use of electronic medical records. The symptom onset and diagnosis were obtained for all patients referred to outpatients for psychosomatic symptoms during a 1-year period. We performed χ² Tests to assess the association of the diagnosis with the occurrence of psychosomatic symptoms.The only diagnosis that presented statistically significant association was anxiety disorder .Our study finds results that follow the line of other studies that show this association, such as Campo’s study which finds that functional somatic symptoms are consistently associated cross-sectionally with anxiety and depressive symptoms  or Imran’s study which finds that higher levels of somatization independently and significantly predicted higher anxiety  . However, our results show no association with depressive disorders whereas frequent associations are found in the literature; for example, a recent meta-analysis found that neuroticism and depression had the strongest influence on the association of medically unexplained physical symptoms and frequent healthcare use . This lack of association is probably due to greater ease in identifying depressive disorders as the main pathology versus anxiety disorders.Campo J. V. (2012). Annual research review: functional somatic symptoms and associated anxiety and depression--developmental psychopathology in pediatric practice. Journal of child psychology and psychiatry, and allied disciplines, 53(5), 575–592. den Boeft, M., Twisk, J. W., Terluin, B., Penninx, B. W., van Marwijk, H. W., Numans, M. E., van der Wouden, J. C., & van der Horst, H. E. (2016). The association between medically unexplained physical symptoms and health care use over two years and the influence of depressive and anxiety disorders and personality traits: a longitudinal study. BMC health services research, 16, 100Imran, N., Ani, C., Mahmood, Z., Hassan, K. A., & Bhatti, M. R. (2014). Anxiety and depression predicted by medically unexplained symptoms in Pakistani children: a case-control study. Journal of psychosomatic research, 76(2), 105–112.None Declared


Mycobacterium tuberculosis (M. tb) and increased infection risk. Investigating further clinical trials and their use of pathway inhibitors is necessary in order to ascertain their potential for TB treatment. This paper is particularly focused on the drug everolimus, an mTOR inhibitor. One of the first clinical trials sponsored by the Aurum Institute showed potential benefit in using everolimus as an adjunctive therapy for tuberculosis. Infection with tuberculosis is associated with a metabolic shift from oxidative phosphorylation towards glycolysis. The everolimus arm in the clinical trial showed further reduction than the control for both maximal and peak glycolytic activity. Compared with control, those receiving everolimus demonstrated increased lung function through forced expiratory volume in 1 s (FEV1) measurements, suggesting that everolimus may mitigate inflammation contributing to lung damage. The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (P13K/AKT/mTOR) pathway plays a key role in tuberculosis (TB) pathogenesis and infection. While the activity levels of this pathway during active infection are still debated, manipulating this pathway shows potential benefit for host-directed therapies. Some studies indicate that pathway inhibitors may have potential for TB treatment through upregulation of autophagy, while other studies do not encourage the use of these inhibitors due to possible host tissue destruction by  Mycobacterium tuberculosis (M. tb), is acquired via inhalation of aerosols containing the pathogen, which most commonly affects the lungs [M. tb infection manifests mostly in two forms: active disease and latent infection. In individuals with latent TB Infection (LTBI), the bacteria do not cause disease symptoms and the host is unable to spread the bacteria to others [M. tb develop active TB disease (primary TB) soon after infection, depending on both the host- and pathogen-derived factors [Despite significant progress in disease control measures, vaccination, and antibiotic regimen treatment options, tuberculosis (TB) remains one of the leading causes of death in humans worldwide. TB, a bacterial infectious disease caused by he lungs . Howeverhe lungs . The outo others . About 1Treatment for TB requires multiple antibiotics to be taken over the course of several months. The World Health Organization’s (WHO) policy of a directly observed therapy, short course (DOTS) strategy for TB has been shown to improve outcomes, especially in resource-poor countries . This stM. tb infection is not fully understood. In this systematic review, we analyze studies on everolimus-mediated immune regulation and discuss the effect of everolimus on autophagy pathways. in addition, we summarize the findings on preclinical and clinical trials as well as case studies in which everolimus has been tested.Host-directed therapy (HDT) is emerging as a novel concept to improve the antibiotics-mediated treatment outcome for TB . In thisM. tb pathogenesis, a literature search was conducted during May to July 2023 in PubMed, which is the National Institute of Health–National Library of Medicine database. The search keywords included the following terms: “everolimus”, “Mycobacterium tuberculosis”, “autophagy”, “mTOR”, “pathogenesis”, and “host-directed therapy”. All articles published in the last 25 years were screened for relevancy and reviewed, and their citations were examined. Particular attention was placed on the year of publication for each of the articles to create a chronological order of findings relevant to everolimus and M. tb.To identify articles reporting the effects of everolimus on host immune responses to TB is the second leading infectious cause of death globally, behind COVID-19 and above HIV/AIDS . In 2021While tuberculosis infection is distributed across the globe, certain regions experience higher rates of infection compared to others. The highest rates of infection are seen in India, sub-Saharan Africa, Micronesia, and Southeast Asian islands . The lowM. tb is one of the five mycobacterial species that cause TB [M. tb is waxy and has a high-lipid content, which allows it to resist antibiotics and survive in hostile conditions. Its capsule has a high mycolic acid content, which makes it a challenge for the body’s macrophages to destroy the bacteria [M. tb can resist oxidative stress via the action of catalase-peroxidase [M. tb-infected individuals do not develop active TB because their adaptive immune system limits the progression of infection and contains it as LTBI [cause TB . The bacbacteria . In addibacteria . M. tb croxidase . Despite as LTBI . The bod as LTBI .M. tb upon inhalation into the lungs [M. tb can successfully mount an infection through phagocytic entry and intracellular survival within the AM. The M. tb-infected AM secretes proinflammatory cytokines and chemokines, which facilitate the extravasation of other immune cells, including the neutrophils, dendritic cells, and NK cells. Although the non-specific effector functions of the innate immune cells contribute to the control of bacterial dissemination, in most cases effective control of M. tb occurs only after the onset of adaptive immunity, which is mediated by T and B lymphocytes. The organized cellular structure at the site of M. tb infection, composed of innate and adaptive immune cells, constitutes the granuloma, which is a pathological hallmark of TB [At the cellular level, the innate immune cells, including the alveolar macrophages (AM), are the primary cells encountered by he lungs . In a nark of TB . Within Autophagy, also known as type II programmed cell death, is a cellular catabolically-driven process in which a double membrane-bound crescent-shaped vesicle termed a phagophore matures into an autophagosome that envelops long-lived proteins and damaged organelles ,8,9. TheIn past years, everolimus  has generated excitement as a therapeutic agent for several ailments, including cancerous tumors and the prevention of organ transplant rejection ,12,13,14M. tb can exploit this autophagy inhibition and prevent normally programmed cell death of infected cells. In light of the ability of everolimus to inhibit these pathways and promote cellular autophagy, researchers have begun investigating the use of everolimus for the treatment of TB. Despite their similar mechanisms, large differences exist between rapamycin and everolimus. Everolimus was developed to provide an added bioavailability and a shorter half-life (15–35 h) compared to the half-life of rapamycin (58–63 h). Moreover, everolimus was developed in attempts to improve the low oral bioavailability seen in certain rapamycin treatment regimens [As an analog of rapamycin, an established TB treatment, everolimus has similar structure, targets, and actions. Both agents contain an identical mTOR binding site and FKBP binding site. At the variable carbon-40 position, everolimus replaces the R-OH group of rapamycin with R-OCCOH  Figure . The comregimens ,20.Even though rapamycin and everolimus play very similar roles in the inhibition of mTOR pathways and stimulation of cellular autophagy, researchers have suggested that rapamycin may have a somewhat limited impact on TB. This may be due in part to the host cell metabolism via the cytochrome p450 3A4 (CYP3A4) enzyme, which might decrease the bioavailability of rapamycin ,21. ThesThe P13K/AKT/mTOR pathway, which is commonly associated with autophagy, offers multiple potential targets for HDT to treat TB. In this section, we focus on autophagy in the context of the P13K/AKT/mTOR pathway, where everolimus has a role as an allosteric inhibitor of mTOR . In the M. tb, the pathogen remains able to negatively influence the regulatory pathway, allowing it to counteract this immune response and leading to active infection. These studies suggest that by targeting the P13K/AKT/mTOR pathway with, for example, an mTOR inhibitor, autophagy can be further activated in the host cells as a way to fight against the pathogen.Active TB influences the P13K/AKT/mTOR signaling pathway; whether this pathway is stimulated or inhibited is complex, and may depend on the particular type of infected cells, such as peripheral blood mononuclear cell (PMBC) versus cell lines or T cells ,28. PatiM. tb infection, there are significantly reduced levels of pathway components in cells, including CD3+AKT+ cells, CD3+p-AKT+ cells, CD3+mTOR+ cells, and CD3+p-mTOR+ cells in T lymphocytes and in CD4+CD25+FoxP3+Treg cells. This reduced activity leads to increased FoxP3+Treg cell activation, which aids in poor host control of infection and the potential for M. tb immune evasion [M. tb. This inhibition resulted in increased secretion and gene expression of matrix metalloproteinase-1 (MMP-1), a peptidase critical for tissue remodeling during TB pathogenesis. In addition, P13K inhibition in infected macrophages upregulated the expression of proinflammatory markers, including cytokines, chemokines, and growth factors [M. tb infection.Inhibition of the P13K/AKT/mTOR pathway in T-lymphocytes and regulatory T cells (Treg) cells has been reported in patients with active TB . During  evasion . Further factors . These sM. tb and increased risk for disease progression. An analysis of clinical trials detailing the roles of specific P13K/AKT/mTOR inhibitors in TB infection is needed in order to evaluate their potential use as HDT for TB treatment.Altogether, while the activity levels of the P13K/AKT/mTOR pathway during active TB remain debated, it is clear that these regulatory pathways play a crucial role in TB pathogenesis. While certain studies highlight the potential for host cell regulatory pathway inhibitors in TB treatment through upregulation of autophagy, others discourage these inhibitors in the context of host tissue destruction by M. tb for 8 days [M. tb, and combinatorial effects of everolimus with the antibiotics isoniazid and pyrazinamide on the level of intracellular oxidative stress, autophagy, mTOR expression, and TNF-α production. This study revealed a significant difference in bacterial viability between 1 and 2 nM concentrations of everolimus-treated cells compared to the untreated control at 8 days post-treatment, indicating the potential of everolimus to induce suppression of M. tb growth in vitro [Research on the effects of everolimus in modulating the host immune response has demonstrated its potential as an adjunctive therapy for TB. As previously mentioned, everolimus is an inhibitor of the mTOR pathway and promotes autophagy in host cells . In 2020r 8 days . The stuin vitro .M. tb after 8 days of exposure in in vitro granulomas with or without everolimus, isoniazid (INH), or pyrazinamide (PZA) and in granulomas treated with a combination of everolimus and INH or PZA. Compared to the control, a 60% reduction in bacterial growth was noted at 1 and 2 nm everolimus-treated human in vitro granulomas, supporting the effect of everolimus in reducing the survival of M. tb [M. tb-containing in vitro granulomas with 2 nM everolimus demonstrated a significant increase (about 50%) in the intensity of LC3B staining, which corresponds to increased autophagy in comparison to the sham-treated control group. Similarly, the INH plus everolimus treatment group showed a significant increase in levels of LC3B; interestingly, the INH-alone treatment group showed an increase in levels of LC3B compared to the untreated controls, showing that antibiotic treatment alone was effective in increasing autophagy marker expression in the M. tb-containing in vitro granulomas. However, although everolimus treatment significantly decreased the expression of mTOR compared to the control, as was expected, there was no clear trend in the expression of mTOR in granulomas treated with the antibiotics INH and PZA in conjunction with everolimus [The same study investigated the survival of of M. tb . Interesof M. tb . Furtherof M. tb . In addiof M. tb . To deteerolimus . TherefoM. tb-containing in vitro granulomas only treated with INH. However, everolimus did not have any additive effect with PZA, which further suggests that there is complexity in the mechanism of everolimus action depending on the type of front-line antibiotics used along with everolimus in antimycobacterial studies.TNF-α is a pro-inflammatory cytokine that plays an important role in the formation and maintenance of granulomas . EveroliM. tb-containing in vitro granulomas generated from immune cells isolated from eight individuals with type II diabetes (T2DM) [M. tb in the in vitro granulomas at both 8 and 15 days post-infection, further supporting the potential of everolimus against M. tb infection in vitro [M. tb infection [M. tb after 72 h of infection in THP-1 macrophages. in addition, treatment with everolimus significantly decreased levels of lipid bodies inside THP-1 macrophages [Another preclinical study by Cao et al. evaluated the effects of everolimus on s (T2DM) . The stuin vitro . A specinfection . In thisrophages .Mycobacterium bovis BCG strain [M. bovis BCG infection were investigated by sampling for the cytokines IFN-γ, TNF-α, IF-6, and IF-10 [M. tb. In conclusion, these trials highlight the immune-enhancing effects of everolimus, with potential to modulate M. tb burden. The high global prevalence of T2DM poses a serious threat to the health of those with M. tb infection. Therefore, this study indicates the possibility of everolimus as an adjunctive therapy to help in modulating the immune response in those affected by TB and T2DM.A double-blind clinical trial study was conducted in 2021 at Western University of Health Sciences to determine the effects of glutathione supplementation after 3 months of treatment in individuals with type 2 diabetes with the addition of in vitro everolimus on G strain . The stuG strain . the effnd IF-10 . The resnd IF-10 . As everM. tb strain from the patient was tested with everolimus to determine its viability in an axenic culture and in a PMBC infection model. Everolimus’ direct anti-mycobacterial activity was tested against M. tb GM2659 and M. tb H37Rv strains in an axenic culture at 3.13 nM and 8.35 nM [M. tb viability at either dose on either strain. Next, everolimus’ indirect antimycobacterial activity was tested in a PMBC infection model. PMBCs were infected with the M. tb GM2659 and M. tb H37Rv strains and treated with 3.13 nM and 8.35 nM everolimus, then were assessed for bacterial burden at 8 days post-infection. To validate the bacterial load assay, varying concentrations of M. tb H37Rv were plated on 7H11 solid growth medium containing everolimus at different concentrations . In addition, PMBCs treated with everolimus at different concentrations were challenged with M. tb H37Rv 4 days post-infection. There was no significant difference in M. tb growth on solid medium and no significant difference in CFU counting between the with and without everolimus conditions. These findings suggest that everolimus does not have a direct or indirect effect on M. tb viability at the administered concentrations [In a case study, a 62-year-old East-Asian male oncological patient treated with everolimus for a neuroendocrine pancreatic neoplasia developed active TB twice in a year and a half . The M. M. tb H37Rv, GM2659, MtbH3, and MtbEAI after treatment with everolimus at maximum therapeutic concentration. The results showed no increase in ROS production despite everolimus treatment [M. tb.The same study examined the effect of everolimus on the levels of intracellular reactive oxygen species (ROS). ROS levels were measured in THP-1 macrophages that were previously infected with M. tb. For 180 days, all study participants received oral rifabutin-substituted standard TB treatment. However, participants who were randomized into the everolimus arm received an additional 0.5 mg/day of everolimus from day 1–112 of treatment [A phase 2 randomized open-label controlled trial was conducted in South Africa to determine the safety and preliminary efficacy of four potential HDT candidates for TB treatment ,38. One Mtb-specific and general immune function, and pulmonary function [M. tb infection impacts glycolysis remains unclear. One study by Lachmandas et al. (2016) that was briefly discussed earlier suggested that there is a shift from oxidative phosphorylation towards glycolysis in macrophages infected with certain strains of M. tb [M. tb [The study looked at six measures, including safety, microbiologic effects in sputum and blood, PET/CT imaging, serum markers of inflammation, effects on function ,38. In tfunction . Microbifunction . The micfunction . Again, function . Glycolyof M. tb . Howeverb [M. tb . Everolib [M. tb . Nonetheb [M. tb . The clib [M. tb . No signb [M. tb .p = 0.044). Although a 6% increase in FEV1 might seem modest, this is equivalent to an increase of 200 mL, and is considerably higher than the difference deemed to be clinically relevant in trials of chronic obstructive pulmonary disease [Finally, this clinical trial assessed the lung function of subjects via spirometry. Despite favorable microbiological effects, long term sequelae from TB are common among cases treated with anti-TB drugs. Most of these cases develop bronchiectasis and lung fibrosis . These a disease .Overall, this study showed potential benefits of using everolimus as adjunctive therapy in treating TB. Because there was no dose-ranging component to the trial and because pharmacokinetic analysis was not performed, the selected dose may have not been optimal for the indication or severity of disease . AlthougM. tb poses a great threat to human populations worldwide. Currently, TB treatment revolves around antibiotics, including INH in combination with rifampin, pyrazinamide, and ethambutol. HDT is a new modality of M. tb treatment that has gained traction in recent years as a way to augment the host immune response. Everolimus, a potential HDT, has shown promising effects in its ability to modulate the host immune response against M. tb. Several preclinical trials have demonstrated the ability of everolimus to impact the immune response by inhibiting mTOR and modulating autophagy during M. tb infection. To date, one successful clinical trial that has shown host-beneficial effects of everolimus as an adjuvant therapy with standard antibiotics for TB. However, in another case study, everolimus treatment did not have any effect on M. tb viability, and there was no increase in ROS production intracellularly in infected macrophages. These findings highlight the need for more studies in order to determine whether everolimus can decrease M. tb burden, especially in TB patients with comorbidities, as a way to further address the threat of M. tb globally. While the available data are insufficient to recommend everolimus as an adjunctive therapy, they support calls for further studies to explore everolimus and its ability to modulate the host immune response against M. tb.


A 79-year-old woman with an elevated body mass index presented with a painful, pruritic, tender plaque on her inferior abdomen, extending to her superior mons pubis and progressing to her right lateral thigh, intermittently recurring with rigors and night sweats for over 6 years . She hadQuestion 1: What is the most likely diagnosis?A.Pyoderma gangrenosum (PG)B.Erythema nodosum (EN)C.Sweet syndrome (SS)D.Acute inflammatory edema (AIE)E.Neutrophilic eccrine hidradenitis (NEH)Answers:A.PG – Incorrect. Although PG is consistent with the histopathology and history of malignancy presented here, PG is more commonly characterized by the presence of nonhealing ulcers, which were not observed in this case.B.EN – Incorrect. EN typically manifests as tender, erythematous nodules or plaques on the bilateral shins, although it can rarely be observed on the thighs and buttocks, as observed here. Septal panniculitis is typically the predominant feature on EN histopathology.C.SS – Correct. SS is the most likely diagnosis. This case meets both major diagnostic criteria for SS, including the acute onset of painful erythematous plaques and histologic evidence of a dense neutrophilic infiltrate without vasculitis, as well as all 4 minor criteria, including fever, elevated inflammatory marker levels, appropriate response to systemic treatment and occurrence with an associated disease. More specifically, the widespread and relapsing giant lesions in this patient with an elevated body mass index are most consistent with the giant cellulitis-like variant of SS, as previously described.D.AIE – Incorrect. AIE is consistent with this case in that it typically involves areas of dependence while sparing those of pressure, with laboratory findings of leukocytosis and neutrophilic infiltrates on histopathology. However, AIE is more commonly bilateral, edematous, and associated with fluid overload.E.NEH – Incorrect. The association with malignancy is consistent with NEH; however, NEH is more likely to present on the extremities, trunk, or face in patients receiving chemotherapy, and histopathology characteristically reveals neutrophilic infiltration of eccrine glands.Question 2: Which of the following is not a characteristic histopathologic feature of this disease?A.Papillary dermal edemaB.Dense neutrophilic infiltrate in the upper and mid dermisC.Leukocytoclasis,D.Vasculitis,E.Endothelial swellingAnswers:A.Papillary dermal edema – Incorrect. Massive superficial dermal edema is common in SS.B.Dense neutrophilic infiltrate in the upper and mid-dermis – Incorrect. This is a characteristic histopathologic feature of SS.C.Leukocytoclasis – Incorrect. Interstitial leukocytoclastic nuclear debris is typically observed.D.Vasculitis – Correct. The absence of true vasculitic changes is characteristic of SS.E.Endothelial swelling – Incorrect. This is a characteristic histopathologic feature of SS.Question 3: Which of the following is the most appropriate first-line therapy for this patient?A.Oral corticosteroidsB.Topical corticosteroidsC.BiologicsD.Oral retinoidsE.IndomethacinAnswers:A.Oral corticosteroids – Correct. Systemic glucocorticoids are recommended as first-line therapy for adults with extensive cutaneous involvement and systemic symptoms. Dapsone, colchicine, and potassium iodide have also been successfully used as first-line therapies in patients where avoidance of systemic steroids is indicated due to comorbidities or other factors.B.Topical corticosteroids – Incorrect. There is evidence supporting high-potency topical or intralesional corticosteroids alone in patients with limited SS lesions (less than 5% body surface area) in the absence of systemic symptoms.C.Biologics – Incorrect. Although emerging reports have supported the efficacy of tumor necrosis factor inhibitors, anti-CD20 antibodies, interleukin 12/23 inhibitors, interleukin 1 inhibitors, interleukin 6 inhibitors, and JAK inhibitors in refractory cases of SS,D.Oral retinoids – Incorrect. Although oral retinoids have been used successfully in patients with SS recalcitrant to systemic steroids,E.Indomethacin – Incorrect. High-dose indomethacin showed benefit in an uncontrolled study of 18 patients with SSNone disclosed.


Cryptococcus is one of the most problematic fungal organisms causing substantial mortality and morbidity worldwide. Clinically, this high incidence of cryptococcosis is most commonly seen in immunocompromised patients, especially those who lack an adaptive T cell response, such as HIV/AIDS patients. However, patients with other underlying immunodeficiencies are also at an increased risk for cryptococcosis. The adaptive immune response, in particular the Th1/Th17 T-cell-mediated responses, to pulmonary Cryptococcus infections are required for host protection. Dendritic cells (DCs), encompassing multiple subsets identified to date, are recognized as the major professional antigen-presenting cell (APC) subset essential for the initiation and execution of T-cell immunity. Apart from their prominent role in orchestration of the adaptive arm of the immune defenses, DCs are fully armed cells from the innate immune system capable of the recognition, uptake, and killing of the fungal cells. Thus, DCs serve as a critical point for the endpoint outcomes of either fungal control or unrestrained fungal infection. Multiple studies have shown that DCs are required for anti-cryptococcal defense in the lungs. In addition, the role of DCs in Cryptococcus gattii infections is just starting to be elucidated. C. gattii has recently risen to prominence with multiple outbreaks in the US and Canada, demonstrating increased virulence in non-immunocompromised individuals. C. gattii infection fails to generate an inflammatory immune response or a protective Th1/Th17 T cell response, at least in part, through a lack of proper DC function. Here we summarize the multiple roles of DCs, including subsets of DCs in both mouse and human models, the roles of DCs during cryptococcal infection, and mechanisms by cryptococcal cells to attempt to undermine these host defenses.Fungal infections are an increasingly growing public health concern, and  Cryptococcus-species clusters that have been subsequently subdivided into several species including C. neoformans, C. deneoformans, and C. gattii, primarily affecting immunocompromised hosts with different levels of immunosuppression [Cryptococcosis is an infectious disease caused by pression . These fpression . Dendritpression . Comparepression ,5, servipression ,7,8, sug+ conventional pulmonary DCs (cDC2), CD103+ conventional pulmonary DCs (cDC1), plasmacytoid DCs (pDCs), Langerhans DCs (LCs), and inflammatory monocyte-derived DCs (MoDCs) [+ conventional DCs (cDC2) and CD103+ conventional DCs (cDC1) as well as a very small population of pDCs [+ CD14− DCs, CD1c+ CD14+ DCs, langerin+ (CD207+) DCs, and pDCs  ,15,16,17 of pDCs ,19. In hand pDCs  [14,20,2Cryptococcus, and, upon IFN-γ activation, upregulate the set of classical activation markers  and antifungal mechanisms [+ CD14− and CD1c+ CD14+ human conventional pulmonary DC subsets interact differently with C. neoformans in terms of cryptococcal uptake, but each is able to efficiently kill C. neoformans [Several studies in the mouse model have shown that cDC2 cells migrate to the lungs in response to cryptococcal infection ,30. Howechanisms . Whetherchanisms . In studchanisms . The datchanisms . Recent oformans .Cryptococcus recognition by DCs have been characterized best.The initial mobilization of host defenses against pathogens is triggered by the recognition of pathogen-associated molecular patterns (PAMPs) by specialized pattern recognition receptors (PRRs) that recognize these PAMPs. DCs were identified as the cell subset that abundantly expresses multiple classes of PRRs, of which many serve to recognize fungal components. Below, we will predominantly focus on the roles of toll-like receptors (TLRs) and c-type lectin receptors (CLRs), as their roles in −/− and TLR-4−/− mice infected with C. neoformans show normal pro-inflammatory cytokine responses and similar survival rates to their wild type counterparts [Cryptococcus. In TLR4 KO macrophages, phagocytosis is increased, which depends on the scavenger receptor MSR1 recognizing oxidized lipids [Cryptococcus. Unmethylated CpG DNA from C. neoformans stimulates BMDCs to become activated [−/− mice  have severely reduced DC activation in response to unmethylated CpG cryptococcal DNA [Cryptococcus via TLR9 to induce IL-12p40 production [−/− mice are more susceptible to pulmonary infection and have reduced fungal control in the lungs and spleen, and show impaired pulmonary lymphocyte recruitment during the adaptive phase of the immune response [C. gattii infections. TLR9−/− mice infected intratracheally with C. gattii are more susceptible to infection and have higher fungal burdens in their brains and spleens, organs associated with dissemination [−/− mice have lower levels of IFN-γ and IL-17A [TLRs are highly expressed by DCs, leading to the recognition of patterns expressed by a broad range of viral, bacterial, and fungal pathogens ,39,40,41terparts ,44, and d lipids . The intctivated . TLR9−/−ccal DNA . In addioduction . TLR9 sioduction . TLR9−/−response ,49. A simination . Interesd IL-17A .C. neoformans infection. Murine studies comparing Dectin-3-deficient and wild type mice did not show any significant differences in the anti-cryptococcal activity of Dectin-3−/− cDC2s or BMDCs [C. neoformans [C. gattii growth media conditions altering Dectin-2 signaling support this idea [C. neoformans was treated with liposomes delivering Amphotericin-B and soluble Dectin-2, the Dectin-2 targeting of the liposome packaged drug had an improved fungicidal effect [Another well-established family of receptors contributing to the detection of fungal pathogens by DCs are CLRs, including Dectin-1, Dectin-2, Dectin-3, Mincle, and the mannose receptor  . The CLRor BMDCs . Howeveror BMDCs . Dectin-or BMDCs . The ligoformans . Dectin-oformans . Multiploformans ,57. Howeoformans , when inoformans . Thus, this idea , and stul effect .Glycoproteins have been known to stimulate T cell responses against a wide variety of antigens. Mannose residues, which are the most common form of carbohydrate that is used by fungi for the synthesis of glycan, are usually present on the fungal glycoproteins . AnotherC. neoformans [Several other immune receptors are also important in controlling cryptococcal infections in the lungs. The complement receptor CR3 plays an important role in the in vitro phagocytosis of unencapsulated oformans . The scaoformans . In summC. neoformans opsonized with anti-capsular antibody or complement [C. neoformans-containing phagosomes [C. neoformans affects the lysosomal maturation pathway is not well understood in DCs, but early studies in macrophages show C. neoformans does not prevent phagolysosome fusion and can survive in an acidic environment [C. neoformans is capable of modulating the phagosome’s pH and development [DCs possess phagocytic and complement receptors that enable them to phagocytose and kill pathogens along with their antigen presentation capabilities ,74. In vmplement ,76  [+ T cells in the peripheral lymph nodes of WT infected mice, but not in MR KO mice [C. neoformans strain expressing murine TNF-α is able to improve fungal control by enhancing activation of cDC1 cells [C. neoformans, engineered to produce murine IFN-γ, also increases the infiltration of cDC2 cells to the lung during a protective immune response [Cryptococcus) [+ T cell response during infection [Following cryptococcal degradation, DCs play an essential role in inducing both protective and non-protective cell-mediated responses against cryptococcal species by presenting antigens to T cells and inducing T cell proliferation to cryptococcal antigens . DCs areof cDC1s ), and CCd MoDCs)  mice repd MoDCs) ,93. Ther KO mice . The ana KO mice . Proper  KO mice ,96. The  KO mice . This noC1 cells . This weC1 cells . Pulmonaresponse . DCs thaococcus) . This isococcus) . These Tnfection .+ and CD8+ T cells expressing an activated phenotype are detectable in the lymph nodes, spleen, or blood [−/− mice [+ T cell re-stimulation in the lungs. CCR2 KO mice fail to recruit enough cDC2s to the bronchovascular infiltrates, which represent tertiary lymphoid tissue [+ T cells in these bronchovascular infiltrates generate an increased expression of protective IL-12 and IFN-γ, while in CCR2 KO mice this is significantly impaired leading to increased IL-4 production and a non-protective Th2 response [In pulmonary cryptococcal infections, the full activation and polarization of T cells is not complete in the lymphatic tissue. Few CD4or blood . Insteador blood . Althougor blood . CCR2 is−/− mice ,99. Thisd tissue . In WT mresponse . It is lC. neoformans and complement or opsonizing antibodies [C. neoformans, including DC lysosomal proteins Coronin, HNE, NOSTRIN, MMP25, and MPO, although the mechanisms of their antifungal activity remains to be determined [C. neoformans is the induction of lysosomal damage, which favors its survival inside macrophages. Although lysosomal damage has not been reported in DCs, when macrophages are classically activated using IFN-γ, lysosomal damage is abrogated [Cryptococcus through the generation of ROS [Cryptococcus [C. gattii they are completely capable of recognizing and killing these cells, even though they do not mature for antigen presentation purposes [C. neoformans, the infection fails to trigger a coordinated immune response. Following ex vivo interaction with C. neoformans, RNA-sequencing data show that the cDC2s significantly downregulated the expression of MHC-I, suggesting these cells may have problems with antigen presentation. These DCs also significantly upregulate CYP1B1, a cytochrome P450 enzyme, suggesting that metabolism plays a role in the antifungal activity (or lack thereof) in specific DC subsets [While DCs play a critical role in generating a protective T cells response, they can also serve as terminal effector cells, and are capable of killing cryptococcal cells. This cryptococcal killing was observed when murine BMDCs and human monocyte-derived DCs were incubated with tibodies . Studiestibodies ,78. Sevetibodies . One suctermined . Althougbrogated . In addin of ROS . Inducibtococcus . Interespurposes . However subsets .Cryptococcus during later exposure [C. neoformans [DCs also have a major role in establishing a protective memory against cryptococcal infections via “trained immunity”. The mechanism of this DC memory response relies on epigenetic changes to the DCs, leading them to respond to exposure ,102,103.exposure ,105. DCsoformans . In addioformans .C. gattii infections. Immunization studies reveal that DC-based vaccines improve responses against pulmonary infection by increasing the levels of IL-17A, TNF-α, and IFN-γ and producing lymphocytes during C. gattii infection [C. gattii [C. gattii [In addition, DC-based vaccines have received growing interest as a way to protect against nfection . Studies. gattii . BMDCs, . gattii .Cryptococcus is no exception. Several virulence factors in Cryptococcus have been identified that alter their interactions with DCs. Several of the major factors that contribute to DC–Cryptococcus interactions involve the fungal cell wall and capsule composition. As much has been written on this topic [No discussion of the role of DCs in infections would be complete without considering the ways in which pathogens try to evade these cells, and is topic ,109,110,Cryptococcus has large glucuronoxylomannan (GXM) polysaccharide capsules, which are usually phagocytosed by macrophages and DCs when shed during infection. However, the GXM can persist in the serum and CSF of patients even after antifungal therapy, thereby acting as a major virulence factor. GXM from C. neoformans acts to non-specifically inhibit T cell proliferation following antigen stimulation by DCs, leading to reduced T cell responses [C. gattii, GXM blocks extracellular receptor signaling dependent on TNF-α and p38MAPK that is required for the maturation of human-monocyte-derived DCs along with reducing the phagocytic process, which is important for DC maturation and antigen processing, respectively [C. gattii is better at suppressing the inflammatory cytokines from DCs than the C. neoformans capsule due to altered deacetylation of glucuronoxylomannan (GXM) in the C. gattii capsule [The capsule which surrounds the cryptococcal cell wall has been identified as a major player in how the fungal cells interact with the host DCs. esponses . In C. gectively . In addi capsule .C. neoformans reveal that the presence of a capsule is an obstacle to the maturation and activation of DCs and their subsequent stimulation of pro-inflammatory cytokines and induction of chemokine genes [C. neoformans avoids efficient T cell responses [Cryptococcal mannoprotein (MP), another soluble component of capsules, stimulates murine BMDCs to upregulate IL-10 and IL-10R, which skews them to a nonprotective DC type 2 activation . Studiesne genes ,117,118.esponses . Howeveresponses . In totaRegarding immune-modulatory cell-wall components, chitin received a lot of well-deserved attention. Chitin is a major component of the fungal cell wall and has a variety of impacts on the interactions of the fungi with various immune cells. Chitin abundance impacted cryptococcal-antigen-specific Th2 cell accumulation in the lungs, coordinated by resident cDC2 cells. This was dependent on chitotriosidase, a mammalian chitinase, cleaving fungal chitin during infection . AnotherCryptococcus. In a pulmonary murine model of infection, a urease deficient (ure1) mutant had reduced fungal loads and is required for the fungi to alter the host immune response to a non-protective Th2 response [Other additional cryptococcal virulence factors that aid in their avoidance of a protective DC response are abundant. Urease, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide, is a well-known virulence factor in response . In addiresponse . This suresponse .C. neoformans is the F-box protein 1 (Fbp1) [+ monocytes to the lungs and their rapid differentiation into DCs [Cryptococcus can alter DC interactions. For example, it has been shown that cryptococcal culture supernatants can suppress DC activation when DCs are activated by cryptococcal DNA through TLR9 signaling, but not when DCs are stimulated with DNA from other organisms [Cryptococcus interferes with DC responses that we have yet to uncover.Another major virulence factor for 1 (Fbp1) ,123. FBP1 (Fbp1) . In a muinto DCs . While trganisms . The exaC. gattii is particularly capable of suppressing DC responses which is a major factor for its increased virulence. Several studies have begun to unravel the way in which C. gattii suppresses the immune system through its interactions with DCs. C. gattii infections have been shown to generate a reduced Th1 and Th17 response in mice pulmonary infections [C. gattii reduces the ability of DCs to differentiate T cells in Th1 or Th17 cells via the reduced expression of co-stimulatory molecules and a lower expression of IL-12 and IL-23 by DCs [C. gattii has shown that while cryptococcal cells can be efficiently phagocytosed and killed by human monocyte-derived DCs, these cells do not mature, leading to impaired T cell proliferation responses and immune dysregulation [C. gattii induces is linked to the generation of an F-actin cage which prevents the maturation of DCs that phagocytose C. gattii cells [C. gattii can subvert DC-mediated immunity.fections . C. gatt3 by DCs . Furthergulation . The DC ii cells . HoweverCryptococcus depends on the quality of the T cell response, DCs are essential in inducing the protective immune response by performing APC functions: (a) antigen sensing; (b) phagocytosis and degradation of C. neoformans antigens; (c) presenting antigen to the T cells; (d) inducing antigen-specific T-cell proliferation; and (e) orchestrating T cell polarization. This occurs by the delivery of classical signals of antigen specific T cell activation and the production of immune polarizing cytokines. In addition, DCs promote effector T cell responses by providing antigen-specific restimulation at the site of infection to trigger the effector cytokine production. Finally, DCs act as distal effector cells, actively killing the ingested fungal cells and serving as a mechanism of direct fungal control, with emerging evidence of memory-type responses. While the importance of DCs in cryptococcal infections is well-established, identifying and characterizing the various subsets of DCs that carry out these functions is still a rapidly developing area. It is clear that DCs exist in multiple distinct subsets, each possibly playing a differential role in antifungal defenses. In addition, each of these subsets interact uniquely with specific fungal factors to lead to differential responses that either promote immunoprotection or fungal exploitation of the immune system to its own advantage. Thus, we predict that DCs will remain a center of focus in antifungal defense research and will be exploited for their potential to boost antifungal host responses and for the generation of antifungal vaccines.DCs are key players in anti-cryptococcal host defenses, serving multiple functions in the control of this fungal pathogen. As effective control of


Background: Urinary tract infection (UTI) is one of the most commonly encountered bacterial infections. Due to the misuse or excessive use of antibiotics, the upsurge of multidrug-resistance cases in UTIs has now become a global threat to public health. Exploring a newer or safer treatment using green synthesized nanoparticles (NPs) is another substitute for eliminating multidrug-resistant pathogens.Syzygium cumini was used for green synthesis of gold NPs. Synthesis of Syzygium cumini gold nanoparticles (ScAu-NPs) was achieved by optimizing various reaction parameters. These ScAu-NPs were characterized through UV-visible spectroscopy, transmission electron microscope, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction. ScAu-NPs were then processed for antibacterial activity against clinically isolated multidrug-resistant pathogens like Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Acinetobacter baumannii, Staphylococcus aureus, and Enterococcus faecalis.Methodology: Leaf extract of Results: Characterization of NPs revealed that biosynthesized NPs were spherical in shape. FTIR spectroscopy showed the presence of phenolics and aromatic compounds. Biosynthesized NPs exhibit good antibacterial activity with a significant bacterial reduction seen against all bacterial isolates compared to the controls.Conclusion: From the results of the present study, the formulation of biosynthesized ScAu-NPs can be utilized in drug development for eliminating infections caused by multidrug-resistant pathogens. Enterobacteriaceae members of uropathogens, which produce enzymes like carbapenemase and extended-spectrum β-lactamases (ESBL) [Syzygium cumini. Essential oil of the leaves of S. cumini already showed substantial antioxidant and antibacterial activity [S. cumini gold nanoparticles (ScAu-NPs) and also investigate the minimum inhibitory concentration (MIC) of ScAu-NPs required to inhibit the growth of multidrug-resistant uropathogens.Urinary tract infections (UTIs) are one of the most common bacterial infections that contribute significantly to mortality and morbidity by affecting 150 million individuals annually . A highes (ESBL) . A larges (ESBL) . To tacks (ESBL) . Accordis (ESBL) ,8. Plants (ESBL) ,10. Theractivity . TherefoExperimental setupSynthesis of Gold NanoparticlesS. cumini were washed, shed dried at room temperature, and ground to obtain a fine powder. Ten grams of leaves were dissolved in distilled water, sonicated, and filtered followed by lyophilization. Synthesis of AuNPs was achieved by optimizing the reaction parameters such as concentration of salt and plant extract, temperature, reaction time, and pH. After optimizing, these NPs were then characterized through UV-visible spectroscopy (UV-Vis) . The shape and size of NPs were estimated by transmission electron microscopy (TEM) , and crystalline nature was detected through X-ray diffraction (XRD) patterns and selected area electron diffraction (SAED)  with a 2θ range of 5-90°. The functional group was detected through Fourier transform infrared spectroscopy (FTIR)  with 400-4000 cm-1.Leaves of Antimicrobial Susceptibility of Bacterial Isolates and Antibacterial Activity of AuNPsBacterial isolates were isolated from 200 mid-stream urine samples. These isolates were identified through biochemical tests, and antimicrobial susceptibility testing was performed. Antibacterial activity was accomplished through the Kirby-Bauer disk diffusion method. To test the antibacterial activity of ScAu-NPs, the bacterial inoculum was grown in Luria-Bertani (LB) broth and adjusted to 0.5 McFarland. ScAu-NPs were impregnated into the sterile disc in Mueller Hinton agar (MHA) plates. Quality control of the MHA medium was performed before performing the experiments. These plates were then incubated at 37°C, followed by a measurement of the zone of inhibition. MIC was calculated using the micro-broth dilution method (CLSI M07-A8). All bacterial inoculums were treated with different concentrations  of ScAu-NPs and incubated at 37°C. MIC was then calculated through visual observation and bacterial absorbance at an optical density (OD) of 600 nm.Characterization of gold nanoparticlesPreliminary identification of the biosynthesis of AuNPs was made through color change. A quick change in color from yellow to purple-red indicates the synthesis of AuNPs and it gets intensified over time due to surface plasmon resonance (SPR) phenomena Figure .Reaction parameters like plant extract, salt concentration, pH, time, and temperature were optimized for ScAu-NPs synthesis. Results of the optimization of reaction parameters showed that a higher yield of NPs was observed at a 1 mM concentration of salt with 200-500 ul of plant extract in an acidic pH at a temperature of 30-600°C. This optimized solution was subjected to UV-Vis spectroscopy that showed the formation of a bell-shaped curve at 520-535 nm wavelength Figure .TEM was carried out to determine the morphology and size of the biosynthesized ScAu-NPs. Briefly, a drop of AuNP solution is placed in a copper grid and the grid is allowed to dry completely. It was noticed that biosynthesized ScAu-NPs were predominantly spherical in shape with an approximate size of 50-60 nm in size measured through ImageJ software  Figure .S. cumini leaf extracts were used as a reducing agent for AuNP synthesis. FTIR spectrum showed absorption bands at 3251.75, 1718.41, 1618.84, 1275.51, 1260.64, 1207.79, 1042.85, 764.29, and 750.17 cm-1, which corresponds to 0-H, C-H, C=O, and C=C stretching bands that indicate the presence of carboxylic acid, aromatic compounds, phenolic, conjugated alkene, and aryl ether functional groups in the biosynthesized AuNPs . The average zones of inhibition formed by ScAu-NPs against S. aureus, A. baumannii, E. coli, P. aeruginosa, E. faecalis, K. pneumoniae, and P. vulgaris are 17.87 mm, 17.69 mm, 17.65 mm, 17.79 mm, 17.65 mm, 16.7 mm, and 20.05 mm, respectively, which display considerable antibacterial activity of AuNPs, as shown in Figure In this study, the antibacterial activity of biosynthesized ScAu-NPs was checked through the disk diffusion method against seven multidrug-resistant microorganisms, i.e., P. vulgaris, P. aeruginosa, S. aureus, and A. baumannii. MICs of E. coli and K. pneumoniae were achieved at 450 µg/mL and 250 µg/mL for E. faecalis with a sharp reduction in OD when compared to positive control. MIC of ScAu-NPs against all multidrug-resistant uropathogens can be seen in Figure From the above findings of the disk diffusion assay, it is evident that ScAu-NPs exhibit considerable antibacterial activity. To see at which concentration ScAu-NPs inhibit bacterial growth, MIC was also checked against these pathogens. MIC against all uropathogens was determined through visual observation and absorbance at OD 600 nm. It was observed that no growth was seen at a concentration of 350 µg/mL for S. cumini, also known as Jamun, is a tropical evergreen tree in India and previous reports have shown the antibacterial activity of the leaf extract of this plant against multidrug-resistant strains [S. cumini were used to synthesize AuNPs. From our results, it is evident that the leaf extract of S. cumini reduces the gold ions by changing the color of the solution into a reddish-purple that indicates the synthesis of AuNPs [UTI has now become a challenge in a hospital setting due to the exploitation or overuse of antibiotics in the treatment, which led to the emergence of multidrug resistance in urinary tract pathogens . To over strains . Therefoof AuNPs . The forof AuNPs ,18. The of AuNPs .S. cumini were used as a reducing agent for the synthesis of AuNPs to see their efficacy against multidrug-resistant urinary tract pathogens. A similar study synthesized silver NPs using the inflorescence of Tridax procumbens and concluded their medicinal properties in inhibiting multidrug-resistant urinary tract isolates [S. cumini but their NP formulation against multidrug-resistant urinary tract pathogens is yet to be explored [In context with the current scenario of multidrug resistance in uropathogens, researchers have synthesized NPs using the plant as a reducing agent. In this study, leaves of isolates . Few stuexplored ,22. The explored . Our finexplored ,25. MIC explored .S. cumini that aid in AuNP synthesis. Also, this is a complete in vitro study and provides a foundation for newer drug development by bringing out antimicrobial compounds from S. cumini to treat bacterial infections caused by these multidrug-resistant strains. Still, in vivo studies are further required to see its potency on animal models.There are a few limitations of the study. Though this study showed satisfactory and good antibacterial activity of AuNPs against multidrug resistance but it is also constrained in assessing the possible phytochemicals present in the leaf extract of S. cumini. Hence, further drug development by incorporating biosynthesized AuNPs could be a useful tool for eliminating the UTI infection caused by multidrug resistance.The existing situation of the emergence of multidrug resistance in urinary tract pathogens led researchers to seek an alternative therapeutic agent. Nanotechnology is itself an asset in the biomedical field and is widely used in drug delivery, drug development, and diagnostics. Therefore, AuNPs were used as an antibacterial agent against multidrug-resistant urinary tract pathogens. In this study, the greener approach of ScAu-NPs synthesis showed significant antibacterial activity against multidrug-resistant pathogens. These biosynthesized ScAu-NPs inhibit the growth of all multidrug-resisting uropathogens. To the best of the authors' knowledge, this is the first report that checked the antibacterial activity of AuNPs against multidrug resistance of UTI using the leaf extract of


Staphylococcus rostri, which is a newly identified NAS. Staphylococcus rostri is rarely reported or characterized in domestic animals, and, to our knowledge, never as a causative agent of dairy mastitis. With the present study, we report the finding of 81 Staphylococcus rostri isolates from nine dairy herds and mainly from subclinical mastitis. We characterized these S. rostri isolates with whole genome sequencing analysis. The results showed a limited distribution of known genes associated with virulence  and antibiotic resistance . However, we found that the S. rostri isolates consisted of herd-specific clones, indicating that each herd had separate introduction source(s). Overall, this study suggests that S. rostri acts as a mastitis pathogen, despite the exact pathogenetic mechanisms of S. rostri still requiring full characterization and remaining unknown.Mastitis is one of the most common diseases in dairy cows. To control mastitis in dairy cows, it is vital to understand the causative agents including their respective mode of action. Several bacterial species may cause bovine mastitis although non-aureus Staphylococci (NAS) are often reported as the most frequently observed cause of subclinical mastitis in dairy cows. As species of NAS may differ in their epidemiology and pathogenicity, performing diagnostics at the species level is crucial. This study is about S. rostri isolates from bovine mastitis . The isolates were first identified as S. microti by MALDI-TOF MS, but later whole genome sequencing analysis allowed reclassification as S. rostri. The isolates were derived from 52 cows and nine dairy herds in Denmark. To describe the pathogenicity of S. rostri, we used whole genome sequencing to infer the distribution of genes associated with virulence, antibiotic resistance, and mobile genetic elements. Also, we performed a core-genome phylogeny analysis to study the genetic relatedness among the isolates. All 81 isolates expressed the same virulence profile comprising two putative virulence genes, clpP and clpC. Three isolates carried a resistance gene encoding streptomycin (str) or lincomycin (lnuA) resistance. The distribution of plasmids suggested the detected antibiotic resistance genes to be plasmid-mediated. Phages were abundant among the isolates, and the single isolate from clinical mastitis acquired a phage disparate from the rest, which potentially could be involved with virulence in S. rostri. The core genome phylogeny revealed a strong genetic intra-herd conservation, which indicates the source of introduction being herd-specific and might further imply the ability of S. rostri to adapt to the bovine niche and spread from cow-to-cow in a contagious manner. With this study, we aim to acquaint clinicians and professionals with the existence of S. rostri which might have been overlooked so far.This study characterizes 81  Staphylococcus aureus is considered one of the major pathogens owing to its frequency and severity as a mastitis agent. All the non-aureus Staphylococci (NAS) are oppositely referred to as minor pathogens since they are primarily associated with subclinical (SCM) or mild clinical mastitis (CM). In terms of numbers, however, NAS are reported as the most prevalent mastitis-associated bacteria causing increased somatic cell counts (SCCs) despite the absence of severe infection. Thereby, NAS can negatively affect the milk quality and subsequently comprise a burden to milk production [Mastitis is one of the most common diseases in dairy cows, and Staphylococci are key agents in this regard . In relaoduction ,3. Traditionally, NAS has been considered a homogeneous group; hence, species differentiation has not been a routine procedure at diagnostic laboratories or in research. More recent research, however, demonstrates that species-specific differences among NAS exist, e.g., regarding their epidemiology, virulence factors, and antibiotic susceptibility. Therefore, it is now recommended to consider NAS as a heterogeneous group, which starts with studying each member individually and accordingly conducting diagnostics on species level ,5. ConveS. chromogenes, S. simulans, S. haemolyticus, S. xylosus, and S. epidermidis as the most frequently identified species [Staphylococcus microti and Staphylococcus rostri are two examples of novel NAS members. They were first discovered in 2010 from common voles  with Brucella infection and noses of healthy pigs, respectively [S. microti up till now has been reported in milk from bovine mastitis three times  [S. rostri is only once encountered in the literature in association with bovine mastitis (United States 2019) [S. rostri has been isolated related to dairy cows previously. First, in a single bulk-tank milk sample (Belgium 2017) [S. microti and S. rostri are rarely reported in the literature and characterization studies on these NAS species in relation to bovine mastitis remain few. This might suggest that these species are seldom found in the milk from bovine mastitis or alternatively that they have simply been overlooked hitherto . To date, more than 20 different NAS species have been isolated from bovine mastitis, including  species . On the ectively ,12. Whilny 2020) ,13,14, Sum 2017) , and secS. rostri isolates from bovine mastitis (mainly SCM) in Denmark. The isolates were initially identified as S. microti by MALDI-TOF MS followed by whole genome sequencing (WGS), which revealed that the isolates were misclassified as S. microti at first. The present characterization of S. rostri focuses on genetic relatedness among the isolates, and the presence of virulence genes, antibiotic resistance genes, and mobile genetic elements (MGEs). To our knowledge, this study comprises the first genotypic characterization of S. rostri in bovine mastitis.The present study provides a genotypic description of 81 n = 80/81) originated from another research study focusing on bacteria associated with bovine SCM in Denmark, which was conducted at Centre for Diagnostics, Technical University of Denmark  (unpublished). In that study, quarter milk samples were collected during 2019 to 2020 from dairy cows with SCM, i.e., cows with a SCC of ≥200.00 cells/mL and no visible signs of infection. The samples were collected just after the monthly milk recording, performed by the national Registration and Milk Recording organization (RYK) which monitors all DHI herds in Denmark. The milk samples were classified as either new or persistent infections. We defined persistent infection as cows having had an increased SCC of ≥200.000 cells/mL measured at least two times during the last three consecutive and monthly RYK recordings. New infection was defined as cows having had one SCC ≥200.00 cells/mL measured for the last three RYK recordings. The last isolate (n = 1/81) in the present study, came from a CM milk sample, submitted to CfD, DTU in 2019 by a veterinary clinic for diagnostic analysis.Most of the isolates in this study  and incubated overnight at 37 °C. Pure subcultures were identified by MALDI-TOF MS  as described by Astrup and associates, 2022 . A MALDIS. microti by MALDI-TOF MS at CfD, DTU from 2019 to June 2020 were whole genome sequenced to perform the genotypic characterization. DNA extraction and sequencing were outsourced to Novogene (Novogene (UK) Co., Ltd., Cambridge, UK). In brief, DNA extraction was performed using an AllPrep DNA/RNA kit  according to the manufacturer’s instructions. Extracted DNA was sequenced using the Illumina NovaSeq6000 platform in a 2 × 150 bp paired-end configuration. All isolates identified as S. microti DSM 22147 (GenBank Accession no. GCA_002902635), S. rostri DSM 21968 (GenBank Accession no. GCA_002902145), and S. muscae ATCC 49910 (GenBank Accession no. GCA_003019275) [The quality of the raw reads was examined using FastQC v.0.11.9). SPAdes (v.3.13.1) was used for genome assembly, applying the settings “-k 21,33,55,77 --careful” . The ass.9. SPAdeS. rostri DSM 21968 as reference [https://github.com/tseemann/snp-dists (accessed on 2 June 2023)). The GFF3 files were additionally used with the query_pan_genome function with roary to study potential gene differences between sets of isolates [p < 0.05) [Prokka (v. 1.14.6) was used for gene annotation using the eference . The GFFeference . Here, teference , which weference . The corisolates . Chi-squ < 0.05) .https://cytoscape.org/ (accessed on 8 May 2023)). The Abricate pipeline (https://github.com/tseemann/abricate (accessed on 21 April 2023)) was applied combined with VFDB (70% identity and coverage) [PhiSpy (v. 4.2.21) was used to predict prophage sequences  and vConoverage) , ResFindoverage) ,31,32, aoverage)  to invesS. microti at the diagnostic laboratory of CfD, DTU. As S. microti is a NAS rarely encountered nor described in the literature, we performed WGS on the isolates to characterize this novel species. The mean assembly length and GC-content were 2.30 Mb and 38.47%, respectively  under BioProject no. PRJNA988321.To enable future comparative studies on S. rostri SCM isolates, 35 isolates were classified as new SCM and 45 as persistent SCM. The last isolate (n = 1/81) came from CM  being located in Zealand, Denmark, all the other herds are distributed across Jutland, Denmark which represents the main geographical area of dairy herds in Denmark. The number of isolates among each herd varied from one isolate up to twenty-five isolates . For 30 quarters . p = 0.4741). Twenty-four isolates (29.6%) were found in pure cultures and 57 isolates (70.3%) in mixed cultures, i.e., cultures containing two different bacterial species . The CM S. rostri was identified in combination with 16 different bacterial species in total (n = 34/57), including S. simulans, S. epidermidis, and S. haemolyticus (S. rostri in mixed culture with another NAS was associated with the type of mastitis (new SCM/persistent SCM), but no statistical significance was found (p = 0.2025). For all herds, a mixed culture containing S. rostri with another NAS, was found in a minimum of one case, and none of the NAS species were exclusively related to specific herds, i.e., S. simulans, S. epidermidis, and S. haemolyticus, etc., were identified from several herds. At cow-level, 22 cows had S. rostri detected in >2 quarters. Out of these twenty-two cows, only four cows showed the same pair of S. rostri + another NAS in >2 quarters; one cow had S. rostri + S. chromogenes in two quarters, one cow had S. rostri + S. simulans in two quarters, while two other cows had S. rostri in three quarters where either S. epidermidis or S. simulans, respectively, were detected in two of the same quarters ). A pan-genome analysis was completed to deduce the genetic content and relatedness among the 81 isolates. For this, the porcine S. rostri DSM21968 strain with S. microti DSM 22147 as a reference, and it additionally dropped to 370 genes when using S. muscae ATCC 49910 as the reference. These observations suggest a somewhat high level of interspecies diversity among these three NAS species, although they are each other’s nearest relatives [S. rostri.Overall, a pan-genome of 2761 genes was identified in the isolates, of which 1509 genes were assigned a name and function whilst 1252 genes were annotated as hypothetical proteins (with unknown function). The core genome comprised 1941 genes and thereby formed 70% of the pan-genome, which is within the range of core genome sizes observed for other NAS from bovine mastitis . The corelatives ,13, and S. rostri, we evaluated the distribution of genes associated with virulence and antibiotic resistance. In this regard, we further looked for MGEs and discussed their potential association to virulence and antibiotic resistance. To explore the pathogenicity of S. rostri DSM 21968 strain carried two putative virulence genes: the ATP-dependent protease subunits clpP and clpC , which can regulate virulence in Gram-positive bacteria regarding, e.g., stress tolerance, biofilm formation, and motility [clpP and clpC genes were not detected in the same contig for any of the isolates (nor in S. rostri DSM 21968 strain), and thereby they were presumably not forming such a complex; thus, their exact virulence properties in S. rostri remain uncertain. All 81 isolates and the and clpC . Overallmotility ,37. Howestr (encoding streptomycin resistance) carried by SR51_herd3.cow13, and lnuA (encoding lincomycin resistance) carried by SR54_herd5.cow2 and SR77_herd8.cow6. The resistant isolates were further found to carry at least one plasmid replicon in the same contig as the resistance gene  replicon although no resistance genes were observed here.Three isolates were found to carry a resistance gene . These gnce gene , which m in 2007 , but botS. rostri isolates. We therefore speculated if other genes would be present with pathogenetic influence in S. rostri. To explore this, we evaluated the gene differences among the bovine isolates according to infection type, i.e., new SCM vs. persistent SCM and CM vs. SCM; additionally, differences in gene content between the bovine isolates and the porcine S. rostri DSM 21968 reference strain were evaluated. The latter, to explore the presence of genes potentially involved with host specificity. In this context, we did not predict any genes exclusively associated with SCM overall, or new SCM or persistent SCM  the pathogenicity of S. rostri and (b) the host-specificity of S. rostri. To explore this, we investigated the overall distribution of phages among the S. rostri population and evaluated their relation to each other via a viral clustering network  . While 28 strain . Of thes network . S. rostri isolates, with the exception of a single isolate from herd 3 (SR_36_herd3.cow6) where no phages were found. Overall, the phage prediction suggested nine different phage-species each distributed as a viral cluster (VC) , thereby suggesting some bovine-unique phages for S. rostri. Within these three VCs, more herds  were represented, although the VCs were all small . Five phages were observed in the CM isolate (SR81_herd9.cow1), where four out of five clustered together with other phages from SCM isolates and/or the reference strain. The remaining 1/5 predicted phage was a singleton, i.e., was not part of a VC as it did not share gene similarity with any other of the predicted phages. This could demonstrate a phage with impact on the pathogenicity of S. rostri, but this needs further investigation.One to five phages were predicted for each of all the ter (VC) . No phagS. rostri isolates, a phylogenetic tree was constructed based on the core genome alignment of the entire S. rostri population, including the porcine S. rostri DSM 21968 strain  . The C1 clade consisted solely of the two isolates from herd 7 and the C2 clade of the remaining seventy-nine bovine isolates and eight herds. The C2 clade further displayed two subclades (C2a and C2b), with C2a covering all isolates from herd 1 (n = 25 isolates) and C2b covering the isolates from the remaining herds  (n = 54 isolates). Of note, the isolates in C2 clustered together into smaller groups (subclades) according to their herd of origin, which indicates the presence of herd-specific S. rostri strains. To further investigate the genetic relatedness among the 8 strain . Two oven = 25), included a large subclade containing 22/25 isolates, which could demonstrate more S. rostri genotypes within herd 1. For this large subclade, the intra SNP value was 33 while it increased to 77 when including all isolates from herd 1 (the entire clade C2a). Clade C2b consisted of two subclades named clade C2b1  and C2b2  (n = 25), 11 for the subclade including the two isolates from herd 5, and 6 for the subclade covering all isolates from herd 6 (n = 14). The clearly observed clustering pattern of the isolates according to herd of origin, combined with the low intra-herd SNPs, revealed that the S. rostri strains consisted of herd-specific clones. Clade C2a, covering all isolates from herd 1 (solates) . C2b1 insolates) . The intS. rostri isolated from bovine mastitis. The isolates were initially identified as S. microti by MALDI-TOF MS, but reclassified as S. rostri following WGS and ANI analysis including the S. microti DSM 22147, S. rostri DSM 21968, and S. muscae ATCC 49910 as controls. These reference species were chosen due to previous studies demonstrating them to be the nearest relatives to each other [S. rostri was not present in the applied MALDI-TOF MS spectrum library, which probably led to the misidentification of the isolates at first. We therefore accentuate the importance of integrating spectra of novel bovine strains to MALDI-TOF MS libraries to enhance species identification and future diagnostic performance based on MALDI-TOF MS. This study presents the first characterization of ch other ,13. Of nS. rostri as a possible mastitis agent, we investigated the distribution of virulence and antibiotic resistance genes. Only two putative virulence genes were detected among the S. rostri isolates (clpP and clpC). Despite the fact that several virulence genes have been observed among NAS overall, some studies report virulence genes being less frequent among NAS compared to the major pathogen S. aureus [To explore the pathogenesis of . aureus ,35, and S. rostri have been studied previously, but for isolates with origins other than bovine mastitis and the observed resistance levels seem to vary. Stegmann and associates found S. rostri isolates from pigs resistant towards tetracycline, penicillin, streptomycin, clindamycin, erythromycin, and trimethoprim [S. rostri from pigs. These isolates were positive for the mecA gene, but they were beta-lactam-susceptible phenotypically [S. rostri from dairy water buffaloes susceptible to all antibiotics tested [S. rostri isolates and found all isolates expressing both phenotypic and genotypic resistance towards beta-lactams [S. rostri previously [S. rostri from bovine mastitis may become a relevant topic in future research. In Denmark, bovine mastitis is the main indication for antibiotic usage in adult dairy cows (cows > 1 year old), and beta-lactamase sensitive penicillins is the class counting for most of the antibiotic usage in this regard . Howeverethoprim . Vanderhypically . Locatels tested . Finally-lactams . We did eviously ,41, survS. rostri isolates. However, it is worth mentioning that a possible explanation for these observations could be due to the sequencing strategy applied, i.e., 2. generation (short-read) sequencing which, unlike 3. generation (long-read) sequencing, provides contigs instead of a closed genome; hence, some genes might not be detected due to fragment overlap or sequence gaps.To summarize the virulence and resistance gene distribution, a limited distribution of virulence and antibiotic resistance genes were found among the S. rostri alone, another pathogen than S. rostri, or by the combined presence of S. rostri and other pathogen(s) over time. Therefore, a possible reason for not finding any genetic differences explicitly among the group of new versus persistent SCM isolates in this study might be that the persistent SCM cases were not related to S. rostri alone. Likewise, our study cannot exclude that cow-factors are more predictive for the outcome of acute versus persistent SCM, nor for the outcome of SCM versus CM when S. rostri is present in the udder, as cow-factors have not been included in our study. Also, the low number of clinical isolates hampers the comparison between CM versus SCM.Regarding the classification of mastitis, NAS are often involved in persistent SCM, but the genetic factors behind this capability are still not clear. Importantly, in this study, persistent SCM isolates were classified as such, when isolates derived from cows with an increased SCC measured for at least 2/3 of the last RYK recordings (corresponding to approximately the last 2–3 months). By this, the historic SCC data served as an indicator of SCM persistency, but whether the previously measured increased SCCs were due to one continuous, possibly intermittent infection, or to several new infections remains unclear. Hence, it is unknown if the persistent SCM was due to S. rostri was infecting the cows separately from a single unknown source specific for each herd, or (b) that S. rostri can adapt to the bovine mammary gland following cow-to-cow transmission, and thereby that S. rostri can act as a contagious mastitis pathogen. Considering the possible transmission routes, the core genome phylogeny combined with the low intra SNPs measured suggested the presence of herd-specific clones. Such findings are of epidemiological importance and imply that each herd probably had its own introductory source(s). In addition, such findings may suggest two possible scenarios: (a) that S. rostri from domestic animals and in general are very sparse, although S. rostri has been described as a natural colonizer of the nasal cavities in healthy pigs [S. rostri were acquired from a porcine-related source. However, none of the herds in the present study were performing pig husbandry at the time this study was conducted, even though herds 1, 3, 5, and 6 were previously farming pigs according to the national Central Husbandry Register (CHR) database [S. rostri DSM 21968 reference  of the S. rostri isolates from SCM as pure cultures, and of note, the single S. rostri isolate from CM as a pure culture as well, which indicate the ability of S. rostri to act as a mastitis pathogen itself. Furthermore, we identified 12 additional S. rostri isolates, which were excluded from the overall study analysis, as they were found in samples with ≥2 other bacterial species, hence classified as contaminated. These isolates were from some of the present study herds  and a single isolate was from a herd not represented in the present study (herd 10). When performing a pan-genome analysis followed by a phylogenetic tree of the core genome alignment after adding these 12 additional S. rostri isolates, we did not observe any distinctive changes in the clustering pattern . Furthermore, no genes were found to be unique for the group of contamination-related S. rostri isolates  versus the lower number of contaminated samples (n = 12), that contain S. rostri indicates that S. rostri is more likely to be related to the milk rather than to contamination of it. If S. rostri should be considered a contaminant rather than a pathogen, we would expect more contaminated samples and fever mixed and pure cultures. Discussing the pathogenetic potential of  E. coli . We cannisolates . Accordi present . Hence, S. rostri was found with another pathogen (mixed samples) there was no pattern of what pathogens were found together with S. rostri, except that the other pathogen was most often another NAS. Hence, there was no indication that S. rostri was merely accompanying flora of more important pathogens.Last but not least, when S. rostri should be considered at least an opportunistic mastitis pathogen.Summing up on the pathogenetic potential, the relatively high proportions of pure- and mixed culture samples, the relatively low proportion of contaminated samples, and the lack of pattern in the mixed samples indicates that S. rostri isolates from bovine mastitis. The isolates were identified as S. microti by MALDI-TOF MS followed by WGS and later classified as S. rostri by ANI analysis. The isolates came from 52 cows from nine Danish dairy herds. Overall, the results showed limited presence of virulence and resistance genes. All isolates carried two putative genes involved with virulence, clpP and clpC, and three isolates carried a single resistance gene encoding streptomycin or lincomycin resistance, respectively. A pan-genome analysis revealed an overall high gene conservation in S. rostri, although a core genome phylogeny analysis showed that the S. rostri isolates consisted of herd-specific clones. Also, we observed that S. rostri was found only rarely in contaminated samples. Altogether, our study raises the hypothesis that S. rostri might act as a mastitis pathogen and that it persists on farms either via cow-to-cow transmission or via unknown environmental niche(s).The present study reports the finding and characterization of 81