Switching from membrane disrupting to membrane crossing, an effective strategy in designing antibacterial polypeptide.

Drug-resistant bacterial infections have caused serious threats to human health and call for effective antibacterial agents that have low propensity to induce antimicrobial resistance. Host defense peptide-mimicking peptides are actively explored, among which poly-ß-l-lysine displays potent antibacterial activity but high cytotoxicity due to the helical structure and strong membrane disruption effect. Here, we report an effective strategy to optimize antimicrobial peptides by switching membrane disrupting to membrane penetrating and intracellular targeting by breaking the helical structure using racemic residues. Introducing ß-homo-glycine into poly-ß-lysine effectively reduces the toxicity of resulting poly-ß-peptides and affords the optimal poly-ß-peptide, ßLys50HG50, which shows potent antibacterial activity against clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) and MRSA persister cells, excellent biosafety, no antimicrobial resistance, and strong therapeutic potential in both local and systemic MRSA infections. The optimal poly-ß-peptide demonstrates strong therapeutic potential and implies the success of our approach as a generalizable strategy in designing promising antibacterial polypeptides.

Weight-based cefuroxime dosing provides comparable orthopedic target tissue concentrations between weight groups - a microdialysis porcine study.

Antibiotic prophylaxis is a key element in prevention of surgical site infections. For the majority of orthopedic procedures, antibiotic administration follows fixed dosing regimens irrespective of weight. However, this may result in insufficient antibiotic target tissue concentrations and higher risk of surgical site infections in obese individuals. The aim of this study was to investigate the effect of weight-based cefuroxime dosing on plasma and target tissue concentrations. Eighteen female pigs were allocated into three groups differentiated by weight: 53-57 kg, 73-77 kg, and 93-97 kg. Microdialysis catheters were placed for continuous sampling in bone, muscle, and subcutaneous tissue during an 8h sampling interval. Blood samples were collected as reference. Cefuroxime was administered intravenously as a bolus according to weight (20 mg/kg). The primary endpoint was the time above the cefuroxime minimal inhibitory concentration for Staphylococcus aureus (T > MIC (4 µg/mL)). Comparable target tissue T > MICs (4 µg/mL) were found between weight groups. Mean T > MIC ranged between 116-137 min for plasma, 118-154 min for bone, 109-146 min for the skeletal muscle, and 117-165 min for subcutaneous tissue across the groups. Weight-based cefuroxime (20 mg/kg) dosing approach provides comparable perioperative plasma and target tissue T > MIC (4 µg/mL) in animals between 50-100 kg body weight, and thus a comparable prophylaxis of surgical site infections.

Basophil-derived IL-4 promotes cutaneous Staphylococcus aureus infection.

Superficial cutaneous Staphylococcus aureus (S. aureus) infection in humans can lead to soft tissue infection, an important cause of morbidity and mortality. IL-17A production by skin TCRγδ+ cells in response to IL-1 and IL-23 produced by epithelial and immune cells is important for restraining S. aureus skin infection. How S. aureus evades this cutaneous innate immune response to establish infection is not clear. Here we show that mechanical injury of mouse skin by tape stripping predisposed mice to superficial skin infection with S. aureus. Topical application of S. aureus to tape-stripped skin caused cutaneous influx of basophils and increased Il4 expression. This basophil-derived IL-4 inhibited cutaneous IL-17A production by TCRγδ+ cells and promoted S. aureus infection of tape-stripped skin. We demonstrate that IL-4 acted on multiple checkpoints that suppress the cutaneous IL-17A response. It reduced Il1 and Il23 expression by keratinocytes, inhibited IL-1+IL-23-driven IL-17A production by TCRγδ+ cells, and impaired IL-17A-driven induction of neutrophil-attracting chemokines by keratinocytes. IL-4 receptor blockade is shown to promote Il17a expression and enhance bacterial clearance in tape-stripped mouse skin exposed to S. aureus, suggesting that it could serve as a therapeutic approach to prevent skin and soft tissue infection.

Method development and characterisation of the low-molecular-weight peptidome of human wound fluids.

The normal wound healing process is characterised by proteolytic events, whereas infection results in dysfunctional activations by endogenous and bacterial proteases. Peptides, downstream reporters of these proteolytic actions, could therefore serve as a promising tool for diagnosis of wounds. Using mass-spectrometry analyses, we here for the first time characterise the peptidome of human wound fluids. Sterile post-surgical wound fluids were found to contain a high degree of peptides in comparison to human plasma. Analyses of the peptidome from uninfected healing wounds and Staphylococcus aureus -infected wounds identify unique peptide patterns of various proteins, including coagulation and complement factors, proteases, and antiproteinases. Together, the work defines a workflow for analysis of peptides derived from wound fluids and demonstrates a proof-of-concept that such fluids can be used for analysis of qualitative differences of peptide patterns from larger patient cohorts, providing potential biomarkers for wound healing and infection.

Infected wounds and burns represent a serious risk to patients: they can delay healing and, if left untreated, can lead to generalised infection or sepsis, organ failure and death. Wounds and burns get infected when harmful micro-organisms, such as bacteria, enter the wound. Predicting the risk of infections, and detecting them early, could reduce their impact and make treating them easier. A way to distinguish between healing and infected wounds is to study how proteins are broken down in each situation. Proteases are the enzymes that break down proteins, and they are different in healing wounds and infected wounds that are failing to heal. This is because, while the body produces proteases, the bacteria that cause infection do so too. Each protease breaks down proteins in a specific way, resulting in a different set of protein fragments, known as peptides. Together, all the peptides in a wound are referred to as the wound's 'peptidome'. Studying the peptidome of a wound could show whether it is infected, and even what type of bacteria might be responsible, which could help identify suitable treatments. Van der Plas et al. used a technique called mass spectrometry to study the peptidome of wounds after surgery. Sterile post-surgical wounds showed high levels of peptides compared to plasma, the liquid component of blood, with up to 4,300 different peptides. Comparing healing wounds to ones infected with the bacterium Staphylococcus aureus revealed that infected wounds contained peptides from about 150 proteins not found in uninfected wounds, while peptides from 90 proteins were unique to uninfected wounds. The peptides exclusive to uninfected wounds included some linked to antimicrobial activity and immune system activity. Van der Plas et al.'s results suggest that analysing the peptidome may be an approach to tracking the healing status of wounds, making it easier to detect infection before symptoms are apparent. The next step will be to study more wounds and identify the reliable peptide markers to use them for diagnostic tests.

Host-pathogen interaction between macrophage co-cultures with Staphylococcus aureus biofilms.

The ability of Staphylococcus aureus to form biofilms is an important virulence factor. During the infectious process, the interaction between biofilms and immune cells is determinant; however, the properties that make biofilms resistant to the immune system are not well characterized. In order to better understand this, we evaluated the in vitro interaction of macrophages during the early stages of S. aureus biofilm formation. Biofilm formation was evaluated by crystal violet staining, light microscopy, and confocal scanning laser microscopy. Furthermore, different activation on L-arginine pathways such as nitric oxide (NO•) release and the arginase, the production of reactive oxygen species (ROS), the total oxidative stress response (OSR), and levels of cytokine liberation, were determined. Our findings show that the interaction between biofilms and macrophages results in stimuli for catabolism of L-arginine via arginase, but not for NO•, an increase of ROS production, and activation of the non-enzymatic OSR. We also observed the production of IL-6, but not of TNFα o IL-10 in these co-cultures. These results contribute to a better understanding of host-pathogen interactions and suggest that biofilms increase resistance against immune cell mechanisms, a phenomenon that could contribute to the ability of S. aureus biofilms to establish mature biofilms.

Bilateral Endogenous Methicillin-Resistant Staphylococcus aureus Endophthalmitis in a Young Athlete: A Story of Full Recovery

Endogenous endophthalmitis (EE) is an ophthalmological emergency. We report the long-term outcome of bilateral methicillin-resistant Staphylococcus aureus EE in a 23-year-old healthy immunocompetent athlete who presented with EE secondary to pelvic abscess and remained with excellent vision.

Bacterial Skin Infections in Hospitalized Patients with Bullous Pemphigoid.

OBJECTIVE: To explore the features and risk factors of bacterial skin infections (BSIs) in hospitalized patients with bullous pemphigoid (BP). METHODS: Records were retrospectively reviewed for 110 hospitalized patients with BP admitted to Peking University First Hospital between 2013 and 2019. Bacterial species and drug resistance were assessed, and then the underlying risk factors for BSIs were evaluated. RESULTS: Infections were present in 40% (44/110) of the patients. Staphylococcus aureus (72.7%, 32/44) was the most common bacterium, and it was highly resistant to penicillin (81.3%, 26/32), erythromycin (62.5%, 20/32), and clindamycin (56.3%, 18/32), but 100.0% sensitive to vancomycin and tigecycline. Coronary heart disease (P = .02; odds ratio [OR], 12.68), multisystem comorbidities (P = .02; OR, 3.67), hypoalbuminemia (P = .04; OR, 3.70), high levels of anti-BP180 antibodies (>112.4 U/mL; P = .003; OR, 6.43), and season (spring: reference; summer: P = .002; OR, 23.58; autumn: P = .02; OR, 12.19; winter: P = .02; OR, 13.19) were significantly associated with BSIs. CONCLUSIONS: Hospitalized patients with BP had a high incidence of BSIs, and those patients with underlying risk factors require careful management to prevent and control BSIs.

Liposomes with Water as a pH-Responsive Functionality for Targeting of Acidic Tumor and Infection Sites.

A lipid named DCPA was synthesized under microwave-assisted heating. DCPA possesses a pyridine betaine, hydrophilic group that can be complexed with water through hydrogen bonding (DCPA-H2 O). DCPA-H2 O liposomes became protonated relatively fast already at pH<6.8, due to the high HOMO binding energy of DCPA-H2 O. In murine models, DCPA-H2 O liposomes had longer blood circulation times than natural DPPC or cationic DCPM liposomes, while after tail-vein injection DCPA-H2 O liposomes targeted faster to solid tumors and intra-abdominal infectious biofilms. Therapeutic efficacy in a murine, infected wound-healing model of tail-vein injected ciprofloxacin-loaded DCPA-H2 O liposomes exceeded the ones of clinically applied ciprofloxacin as well as of ciprofloxacin-loaded DPPC or DCPM liposomes.

Staphylococcus aureus lipoproteins promote abscess formation in mice, shielding bacteria from immune killing.

Despite being a major bacterial factor in alerting the human immune system, the role of Staphylococcus aureus (S. aureus) lipoproteins (Lpp) in skin infections remains largely unknown. Here, we demonstrated that subcutaneous injection of S. aureus Lpp led to infiltration of neutrophils and monocytes/macrophages and induced skin lesions in mice. Lipid-moiety of S. aureus Lpp and host TLR2 was responsible for such effect. Lpp-deficient S. aureus strains exhibited smaller lesion size and reduced bacterial loads than their parental strains; the altered phenotype in bacterial loads was TLR2-independent. Lpp expression in skin infections contributed to imbalanced local hemostasis toward hypercoagulable state. Depletion of leukocytes or fibrinogen abrogated the effects induced by Lpp in terms of skin lesions and bacterial burden. Our data suggest that S. aureus Lpp induce skin inflammation and promote abscess formation that protects bacteria from innate immune killing. This suggests an intriguing bacterial immune evasion mechanism.

Cathelicidin ΔPb-CATH4 derived from Python bivittatus accelerates the healing of Staphylococcus aureus-infected wounds in mice.

The effects of ΔPb-CATH4, a cathelicidin derived from Python bivittatus, were evaluated against Staphylococcus aureus-infected wounds in mice. These effects were comparable to those of classical antibiotics. ΔPb-CATH4 was resistant to bacterial protease but not to porcine trypsin. A reduction in the level of inflammatory cytokines and an increase in the migration of immune cells was observed in vitro. Thus, ΔPb-CATH4 can promote wound healing by controlling infections including those caused by multidrug-resistant bacteria via its immunomodulatory effects.

Ultrastructure of a late-stage bacterial endocarditis valve vegetation.

Infective endocarditis (IE) remains a severe illness with high mortality rate, despite advances in antibiotic therapy and cardiac surgery. If infectious bacteria and platelets are two key players of human IE vegetation developmental process, their interactions and respective roles in fully developed late-stage IE vegetations remain obscure. The objective of this study was to better understand the organization of the different components of the IE vegetation and to provide a detailed description of this vegetation ultrastructure. A late stage Staphylococcal endocarditic vegetation was provided from a 13 years teenager patient. After reception of the surgical piece, we carried out a histological study using routine methods, notably the hematoxylin-eosin-saffron staining. Labeling with the anti-CD 61 antibody was also carried out. In a second step, we used transmission electron microscopy to describe the different regions making up the vegetation. Our ultrastructural study revealed vegetation was clearly composed by three different regions and identified the specific location of the bacteria and platelets in the vegetation tissues. Histological analysis showed that platelets and Staphylococcus aureus were not co-localized. Electron microscopy study confirmed that S. aureus were found at distance from platelets, as well from immune cells, embedded in a biofilm and/or a necrotic area. These results reveal a development of a deep bacteria-only niche in vegetation, raising questions about medication access to these microorganisms. Vegetation composed of three regions: a region rich in bacteria incorporated into the necrotic tissue, the second region composed of fibrin filaments and the third region rich in platelets and free of bacteria.

Longitudinal study on the effects of intramammary infection with non-aureus staphylococci on udder health and milk production in dairy heifers.

We conducted a longitudinal study to evaluate the effect of non-aureus staphylococci (NAS) causing subclinical intramammary infections (IMI) on quarter milk somatic cell count (qSCC) and quarter milk yield (qMY). In total, 324 quarters of 82 Holstein Friesian heifers were followed from calving to 130 d in milk (DIM) and were sampled 10 times each at 14-d intervals. The IMI status of each quarter was determined based on bacterial culture results at the current and previous or next sampling day, or both. The qSCC was determined on each sampling day and the average qMY on sampling day was available through stored daily milk weight data in the management program of the automatic milking system. A transient IMI (tIMI) was defined as a case where a specific pathogen was isolated from a quarter on only one sampling day and not on the previous or next sampling day. When the same bacterial strain, as defined by random amplification of polymorphic DNA-PCR, was isolated from the same quarter on multiple sampling days, it was defined as a persistent IMI (pIMI) status on those sampling days; a pIMI episode was defined as the combination of multiple consecutive pIMI statuses with the same bacterial strain on different sampling days. During this study, 142 subclinical IMI with NAS occurred in 116 different quarters from 64 animals, yielding in total 304 NAS isolates belonging to 17 different species. The prevalence of NAS was highest in the first 4 DIM. Overall, the predominant species was Staphylococcus chromogenes (52% of the isolates), followed by S. epidermidis (9.2%), S. xylosus (8.2%), and S. equorum (5.9%). Staphylococcus chromogenes was the only species for which an effect on qSCC and qMY could be analyzed separately; the other NAS species were considered as a group because of their low prevalence. Eighteen out of 40 IMI (45%) caused by S. chromogenes persisted over at least 2 sampling days, whereas only 10 of 102 (9.8%) IMI caused by other NAS species persisted for at least 2 sampling days. The average duration of pIMI episodes was 110.4 d for S. chromogenes and 70 d for the other NAS species. Remarkably, 17 of the 18 pIMI episodes with S. chromogenes started within the first 18 DIM. The qSCC was highest in quarters having a pIMI with a major pathogen, followed by quarters having a pIMI with S. chromogenes, and a pIMI with other NAS. Transient IMI with other NAS or with a major pathogen caused a small but significantly higher qSCC, whereas the qSCC in quarters having a tIMI with S. chromogenes was not statistically different compared with noninfected quarters. No significant differences in qMY were observed between quarters having a pIMI or tIMI with S. chromogenes or with the other NAS species compared with noninfected quarters, despite the higher qSCC. Quarters having a pIMI with major pathogens showed significantly lower daily milk production. Surprisingly, quarters that cured from an IMI with S. chromogenes had a significantly lower qMY than noninfected quarters.

Cystic fibrosis with non-G551D gating mutations in Italy: Epidemiology and clinical characteristics.

BACKGROUND: Cystic fibrosis transmembrane conductance regulator (CFTR) gating mutations (GMs) result in CFTR that is present at the cell surface but nonfunctional. Patients with the G551D mutation, the most prevalent worldwide, have been well studied. Italian GM patients have mainly non-G551D mutations. We studied their epidemiology and clinical characteristics in the period spanning the pre/post ivacaftor introduction to the Italian market. METHODS: Data from the Italian CF Registry were used to describe patients with GMs and compare them with F508del homozygous (F/F) patients. RESULTS: In total, 186 patients with GMs (median [range] age, 21.96 [0.13-63.38] years) were identified among the 5552 patients included in the study (3.3%). They had lower sweat chloride values at diagnosis than the F/F and a lower ratio of males. In the GM group, examining the data of the years 2012 and 2017 and comparing with F/F, lung infection by Staphylococcus aureus and diabetes became less prevalent, and better FEV1 and nutritional status were observed in 2017. The cross-sectional evaluation year-by-year from 2012 to 2017 of the GM group showed improving trends in lung function and body mass index, and the decreasing prevalence of diabetes compared with F/F. Longitudinal evaluation of GM patients showed improvement in percent predicted (pp)FEV1 and nutrition in the 2012-2017 period. These variations correspond to the introduction of treatment with the CFTR potentiator ivacaftor (2014/2015). CONCLUSIONS: Italian patients with GMs are few and are characterized by milder phenotypes than F/F patients. Improved outcomes are likely influenced by treatment with ivacaftor.

Intracellular Staphylococcus aureus Perturbs the Host Cell Ca2+ Homeostasis To Promote Cell Death.

The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca2+ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca2+ rise led to an increase in mitochondrial Ca2+ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+ homeostasis and induces cytoplasmic Ca2+ overload, which results in both apoptotic and necrotic cell death in parallel or succession.IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca2+ homeostasis.

A Case of Lymphangiomatosis With Infected Lymphangiomas Effectively Treated by Thoracoscopic Debridement and Drainage.

A 40-year-old woman with lymphangiomatosis also had an intrathoracic lymphangioma infection. Since the age of 8 years, the patient had undergone repeated abdominal and mediastinal cyst surgeries and had received a diagnosis of lymphangiomatosis. At this time, she showed a high fever of 38.5°C. Cultures of both blood and fluid aspirated from the cyst were positive for Staphylococcus aureus. Chest CT imaging revealed an enlarged right-sided mediastinal cystic lymphangioma with new septa in it. A chest tube was inserted into the cyst to remove effusion. The patient was then started with the antibacterial drug cefazolin at 3 g/d. But effective drainage was difficult because of the high viscosity of the effusion and septa working as barriers. We removed these components with endoscopic surgical instruments and via a pulsed-lavage system under general anesthesia. Postoperative CT images showed reexpanded lung structure and reduced cyst size. The patient has taken a favorable course for 2 years.

Temperature Trajectory Subphenotypes Correlate With Immune Responses in Patients With Sepsis.

OBJECTIVES: We recently found that distinct body temperature trajectories of infected patients correlated with survival. Understanding the relationship between the temperature trajectories and the host immune response to infection could allow us to immunophenotype patients at the bedside using temperature. The objective was to identify whether temperature trajectories have consistent associations with specific cytokine responses in two distinct cohorts of infected patients. DESIGN: Prospective observational study. SETTING: Large academic medical center between 2013 and 2019. SUBJECTS: Two cohorts of infected patients: 1) patients in the ICU with septic shock and 2) hospitalized patients with Staphylococcus aureus bacteremia. INTERVENTIONS: Clinical data (including body temperature) and plasma cytokine concentrations were measured. Patients were classified into four temperature trajectory subphenotypes using their temperature measurements in the first 72 hours from the onset of infection. Log-transformed cytokine levels were standardized to the mean and compared with the subphenotypes in both cohorts. MEASUREMENTS AND MAIN RESULTS: The cohorts consisted of 120 patients with septic shock (cohort 1) and 88 patients with S. aureus bacteremia (cohort 2). Patients from both cohorts were classified into one of four previously validated temperature subphenotypes: "hyperthermic, slow resolvers" (n = 19 cohort 1; n = 13 cohort 2), "hyperthermic, fast resolvers" (n = 18 C1; n = 24 C2), "normothermic" (n = 54 C1; n = 31 C2), and "hypothermic" (n = 29 C1; n = 20 C2). Both "hyperthermic, slow resolvers" and "hyperthermic, fast resolvers" had high levels of G-CSF, CCL2, and interleukin-10 compared with the "hypothermic" group when controlling for cohort and timing of cytokine measurement (p < 0.05). In contrast to the "hyperthermic, slow resolvers," the "hyperthermic, fast resolvers" showed significant decreases in the levels of several cytokines over a 24-hour period, including interleukin-1RA, interleukin-6, interleukin-8, G-CSF, and M-CSF (p < 0.001). CONCLUSIONS: Temperature trajectory subphenotypes are associated with consistent cytokine profiles in two distinct cohorts of infected patients. These subphenotypes could play a role in the bedside identification of cytokine profiles in patients with sepsis.

Staphylococcus aureus Bacteremia: Contemporary Management.

Staphylococcus aureus bacteremia (SAB) is a serious cause of bloodstream infection associated with significant morbidity and mortality. Complications include deep-seated foci of infection including infective endocarditis, device-associated infection, osteoarticular metastases, pleuropulmonary involvement, and recurrent infection. With the 30-day all-cause mortality being around 20%, a collaborative effort of early Infectious Diseases (ID) consultation and Antimicrobial Stewardship Program (ASP) involvement will show improved SAB outcomes and therapy optimization.1.

Pathogen effects on milk yield and composition in chronic subclinical mastitis in dairy cows.

This study aimed to evaluate the effects of chronic subclinical mastitis (CSM) on milk production and component yields in dairy cows. A total of six herds located in the Midwest area of São Paulo State, Brazil were selected. Herds were visited once every 2 weeks to measure milk yield and to collect milk samples from lactating Holstein cows. Milk samples were collected at two stages (1 and 2), and each stage comprised three milk samplings. In stage 1, a total of 117 of 647 cows were diagnosed with CSM based on at least two of three repeated somatic cell counts (SCC) > 2000,000 cells/mL and positive bacterial milk culture results (BC). Cows with CSM were selected for the second stage. In stage 2, selected cows had quarter sampling aseptically collected for BC analyses prior to milking, and quarter milk yield was measured. Milk components (total protein, fat, lactose, and total solids) were measured using mid-infrared spectroscopy. Mammary quarters were considered healthy if all three repeated SCC results were ≤ 200,000 cells/mL and no bacterial growth was detected on BC. All quarters with positive bacterial growth were classified as having (non-chronic) subclinical mastitis when only one of three SCC results were > 200,000 cells/mL, and CSM when at least two of three SCC results were > 200,000 cells/mL. The effects of CSM by type of pathogen on milk and components yield were assessed using a linear mixed model. Mammary quarters with CSM caused by major pathogens had milk loss of 1.1 kg/quarter milking in comparison to healthy quarters. Milk losses were 0.8 and 1.3 kg/quarter milking when CSM was caused by Staphylococcus aureus or environmental streptococci, respectively. In addition, healthy quarters produced more milk components than quarters with CSM caused by major pathogens. Minor pathogens causing CSM (non-aureus staphylococci and Corynebacterium spp.) had no effect on milk yield. Quarters with CSM had lower milk and component yields when compared with healthy quarters. Milk losses varied according to the type of pathogen and were higher when associated with major pathogens such as S. aureus and environmental streptococci compared with healthy quarters.

α-Hemolysin suppresses osteogenesis by inducing lipid rafts accumulation in bone marrow stromal cells.

α-hemolysin (Hla) is considered an essential virulent factor for Staphylococcus aureus (S. aureus) toxicity, the mechanism by which Hla affect bone metabolism is poorly understood. In this study, 2-month-old C57BL/6 mice were treated with Hla (40 µg/kg, i.p.) or S. aureus (1 × 106 CFU/ml, 100 µl, i.v.) with the presence or absence of methyl-ß-cyclodextrin (MßCD) (300 mg/kg, i.p.). MicroCT analysis showed progressive bone loss from week 2 to week 4 after Hla treatment, accompanied by a decreased osteoblasts and increased osteoclasts in femoral metaphysis in mice. Further, Hla stimulated the expression of Caveolin-1 in vivo and in vitro, activated lipid rafts accumulation in cell membrane of bone marrow stromal cells (BMSCs), and suppressed osteogenesis of BMSCs. Destruction of lipid rafts with MßCD or inhibition of Caveolin-1 with Daidzein blocked the detrimental effect of Hla on osteogenesis of BMSCs. Importantly, treating mice with MßCD rescued the loss of osteoblasts and increased osteoclastogenesis induced by Hla as well as the bone loss induced by S. aureus infection. Together, we demonstrate that Hla induces bone destruction directly by suppressing osteogenesis and indirectly by stimulating osteoclastogenesis, and that lipid rafts may mediate the detrimental effect of Hla and S. aureus on osteogenesis and bone formation.