Staphylococcus capitis: insights into epidemiology, virulence, and antimicrobial resistance of a clinically relevant bacterial species.

SUMMARYStaphylococcus capitis is divided into two subspecies, S. capitis subsp. ureolyticus (renamed urealyticus in 1992; ATCC 49326) and S. capitis subsp. capitis (ATCC 27840), and fits with the archetype of clinically relevant coagulase-negative staphylococci (CoNS). S. capitis is a commensal bacterium of the skin in humans, which must be considered an opportunistic pathogen of interest particularly as soon as it is identified in a clinically relevant specimen from an immunocompromised patient. Several studies have highlighted the potential determinants underlying S. capitis pathogenicity, resistance profiles, and virulence factors. In addition, mobile genetic element acquisitions and mutations contribute to S. capitis genome adaptation to its environment. Over the past decades, antibiotic resistance has been identified for S. capitis in almost all the families of the currently available antibiotics and is related to the emergence of multidrug-resistant clones of high clinical significance. The present review summarizes the current knowledge concerning the taxonomic position of S. capitis among staphylococci, the involvement of this species in human colonization and diseases, the virulence factors supporting its pathogenicity, and the phenotypic and genomic antimicrobial resistance profiles of this species.

Analysis of in-patient evolution of Escherichia coli reveals potential links to relapse of bone and joint infections.

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of the case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill understood adaptation of the pathogen to the host. Here, we investigated three pairs of Escherichia coli strains from BJI cases and their relapses to unravel in-patient adaptation. Whole genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of two virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

Activity of Exebacase (CF-301) against Biofilms Formed by Staphylococcus epidermidis Strains Isolated from Prosthetic Joint Infections.

Staphylococcus epidermidis is one of the main pathogens responsible for bone and joint infections, especially those involving prosthetic materials, due to its ability to form biofilms. In these cases, biofilm formation, combined with increased antimicrobial resistance, often results in therapeutic failures. In this context, the development of innovative therapies active against S. epidermidis is a priority. The aim of this study was to evaluate the in vitro activity of the lysin exebacase (CF-301) against biofilms formed by 19 S. epidermidis clinical strains isolated from prosthetic joint infections (PJI). We determined the biomass and the remaining viable bacteria inside biofilms after 24 h of exposure to exebacase. Exebacase activity was compared to that of rifampicin, vancomycin, and daptomycin. The use of exebacase in addition to antibiotics was also assessed. Exebacase displayed (i) a significant anti-biomass activity on S. epidermidis biofilms at concentrations ≥5 mg/L (mean decrease up to 66% at 150 mg/L), (ii) significant bactericidal activity on biofilms at concentrations ≥50 mg/L (mean decrease up to 1.7 log CFU at 150 mg/L), (iii) synergistic effects when used in addition to rifampicin, vancomycin, or daptomycin. The extent of these activities varied by isolate. Exebacase can be considered a promising therapy in addition to rifampicin, vancomycin, or daptomycin in the context of PJI. Further in vitro studies are needed to understand its mechanism of action on S. epidermidis biofilms and in vivo investigations are required to confirm these data.

In vitro antibiotic activity against intraosteoblastic Staphylococcus aureus: a narrative review of the literature.

Staphylococcus aureus - a major aetiological agent of bone and joint infection (BJI) - is associated with a high risk of relapse and chronicity, in part due to its ability to invade and persist in non-professional phagocytic bone cells such as osteoblasts. This intracellular reservoir protects S. aureus from the action of the immune system and most antibiotics. To date, the choice of antimicrobial strategies for BJI treatment mostly relies on standard susceptibility testing, bone penetration of antibiotics and their 'antibiofilm' activity. Despite the role of intracellular persistent S. aureus in the development of chronic infection, the ability of antibiotics to target the S. aureus intraosteoblastic reservoir is not considered in therapeutic choices but might represent a key determinant of treatment outcome. This review provides an overview of the intracellular pharmacokinetics of antistaphylococcal drugs used in the treatment of BJI and of their ability to target intraosteoblastic S. aureus. Thirteen studies focusing on the intraosteoblastic activity of antibiotics against S. aureus were reviewed, all relying on in vitro models of osteoblast infection. Despite varying incubation times, multiplicities of infection, bacterial strains, and the types of infected cell lines, rifamycins and fluoroquinolones remain the two most potent antimicrobial classes for intraosteoblastic S. aureus eradication, consistent with clinical data showing a superiority of this combination therapy in S. aureus orthopaedic device-related infections.

Evaluation of intraosteoblastic activity of dalbavancin against Staphylococcus aureus in an ex vivo model of bone cell infection.

OBJECTIVES: Long-acting lipoglycopeptides are promising therapeutic options in Staphylococcus aureus bone and joint infections (BJIs). This study evaluated the ability of dalbavancin to eradicate the intraosteoblastic reservoir of S. aureus, associated with BJI chronicity. METHODS: Osteoblastic cells were infected with a standardized inoculum of the S. aureus reference strain HG001 and incubated for 24 h with dalbavancin, vancomycin or rifampicin using the MIC, 10×MIC, 100×MIC and/or the intraosseous concentrations reached using standard therapeutic doses (i.e. vancomycin, 10 mg/L; rifampicin, 2 mg/L; and dalbavancin, 6 mg/L). The remaining intracellular bacteria were quantified by plating cell lysates. RESULTS: MICs of dalbavancin, vancomycin and rifampicin were 0.125, 1 and 0.004 mg/L, respectively. Dalbavancin significantly reduced the intracellular inoculum of S. aureus starting at a concentration equal to the MIC, with a significant dose effect, ranging from a reduction of 31.4% (95% CI = 17.6%-45.2%) at MIC to 51.6% (95% CI = 39.8%-63.4%) at 100×MIC compared with untreated cells. Of note, dalbavancin was the only molecule to significantly reduce the intraosteoblastic inoculum at low concentration (MIC). At intraosseous concentrations, dalbavancin reduced the intracellular inoculum by 49.6% (95% CI = 45.1%-54.1%) compared with untreated cells (P < 0.001), with no significant difference compared with vancomycin (38.1%; 95% CI = 19.2%-57.0%; P = 0.646), and was less efficient than rifampicin (69.0%; 95% CI = 63.2-74.8; P < 0.001). CONCLUSIONS: Dalbavancin was able to decrease the intraosteoblastic S. aureus inoculum by 50% at intraosseous concentrations reached during standard human therapeutic dosing, with no difference compared with vancomycin, and remained less efficient than rifampicin. However, it was the only molecule significantly active at low concentration.

Evaluation of the Activity of a Combination of Three Bacteriophages Alone or in Association with Antibiotics on Staphylococcus aureus Embedded in Biofilm or Internalized in Osteoblasts.

Staphylococcus aureus is responsible for difficult-to-treat bone and joint infections (BJIs). This is related to its ability to form biofilm and to be internalized and persist inside osteoblasts. Recently, bacteriophage therapy has emerged as a promising option to improve treatment of such infections, but data on its activity against the specific bacterial lifestyles presented above remain scarce. We evaluated the activity of a combination of three bacteriophages, recently used for compassionate treatment in France, against S. aureus HG001 in a model of staphylococcal biofilm and a model of osteoblasts infection, alone or in association with vancomycin or rifampin. The activity of bacteriophages against biofilm-embedded S. aureus was dose dependent. In addition, synergistic effects were observed when bacteriophages were combined with antibiotics used at the lowest concentrations. Phage penetration into osteoblasts was observed only when the cells were infected, suggesting a S. aureus-dependent Trojan horse mechanism for internalization. The intracellular bacterial count of bacteria in infected osteoblasts treated with bacteriophages as well as with vancomycin was significantly higher than in cells treated with lysostaphin, used as a control condition, owing to the absence of intracellular activity and the rapid killing of bacteria released after the death of infected cells. These results suggest that bacteriophages are both inactive in the intracellular compartment after being internalized in infected osteoblasts and present a delayed killing effect on bacteria released after cell lysis into the extracellular compartment, which avoids preventing them from infecting other osteoblasts. The combination of bacteriophages tested was highly active against S. aureus embedded in biofilm but showed no activity against intracellular bacteria in the cell model used.

Antibiofilm and intraosteoblastic activities of rifamycins against Staphylococcus aureus: promising in vitro profile of rifabutin.

BACKGROUND: Targeting biofilm-embedded and intraosteoblastic Staphylococcus aureus, rifampicin gained a pivotal role in bone and joint infection (BJI) treatment. Two other rifamycins, rifabutin and rifapentine, may represent better-tolerated alternatives, but their activity against bacterial reservoirs associated with BJI chronicity has never been evaluated. OBJECTIVES: To evaluate the activities of rifampicin, rifabutin and rifapentine in osteoblast infection models. METHODS: Using three S. aureus isolates, rifamycins were compared regarding: (i) their intracellular activity in 'acute' (24 h) and 'chronic' (7 days) osteoblast infection models at 0.1× MIC, 1× MIC, 10× MIC and 100× MIC, while impacting infection-induced cytotoxicity (MTT assay), intracellular phenol-soluble modulin (PSM) secretion (RT-PCR), resistance selection and small colony variant (SCV) emergence; and (ii) their minimal biofilm eradication concentration (MBEC) and their MIC to prevent biofilm formation (bMIC). RESULTS: At 0.1× MIC, only rifabutin significantly reduced intracellular inoculum and PSM secretion. All rifamycins allowed a 50% reduction of intraosteoblastic inoculum at higher concentrations, with no difference between acute and chronic infection models, while reducing infection-induced cytotoxicity and PSM secretion. Dose-dependent emergence of intracellular SCVs was observed for all molecules. No intracellular emergence of resistance was detected. bMICs were equivalent for all molecules, but MBEC90s of rifapentine and rifabutin were 10- to 100-fold lower than those of rifampicin, respectively. CONCLUSIONS: All rifamycins are efficient in reducing the S. aureus intraosteoblastic reservoir while limiting infection-induced cytotoxicity, with a higher activity of rifabutin at low concentrations. All molecules prevent biofilm formation, but only rifapentine and rifabutin consistently reduce formed biofilm-embedded bacteria for all isolates. The activity of rifabutin at lower doses highlights its therapeutic potential.

Investigation of a Staphylococcus argenteus Strain Involved in a Chronic Prosthetic-Joint Infection.

Staphylococcus argenteus is an emerging species responsible for infections comparable to those induced by Staphylococcus aureus. It has been involved in few chronic or persistent infections so far. In this study, we described a case of a persistent prosthetic-joint infection (PJI) affecting a young woman. We investigated in vitro the virulence traits of the incriminated S. argenteus strain (bone cell invasion, biofilm formation and induction of inflammation) and analyzed its genome, in comparison with two other strains of S. argenteus and two S. aureus isolates. It appeared that this S. argenteus PJI strain combined biofilm formation, osteoblast invasion and intracellular persistence abilities together with genes potentially involved in the escape of the host immune defenses, which might explain the chronicization of the infection.

Evaluation of the ability of linezolid and tedizolid to eradicate intraosteoblastic and biofilm-embedded Staphylococcus aureus in the bone and joint infection setting.

OBJECTIVES: Prolonged use of linezolid for bone and joint infection (BJI) is limited by its long-term toxicity. The better safety profile of tedizolid, a recently developed oxazolidinone, could offer an alternative. However, its efficacy against biofilm-embedded and intracellular Staphylococcus aureus, the two main bacterial reservoirs associated with BJI chronicity, is unknown. METHODS: Using three S. aureus strains (6850 and two clinical BJI isolates), linezolid and tedizolid were compared regarding their ability: (i) to target the S. aureus intracellular reservoir in an in vitro model of osteoblast infection, using three concentrations increasing from the bone concentration reached with standard therapeutic doses (Cbone = 2.5 × MIC; Cplasm = 10 × MIC; Cmax = 40 × MIC); (ii) to eradicate mature biofilm [minimal biofilm eradication concentration (MBEC)]; and (iii) to prevent biofilm formation [biofilm MIC (bMIC) and confocal microscopy]. RESULTS: Linezolid and tedizolid weakly reduced the intracellular inoculum of S. aureus in a strain-dependent manner despite the similar MICs for the tested strains, but improved cell viability even in the absence of an intracellular bactericidal effect. Conversely, linezolid and tedizolid were ineffective in eradicating mature biofilm formed in vitro, with MBEC >2000 and >675 mg/L, respectively. bMICs of tedizolid were 4-fold lower than those of linezolid for all strains. CONCLUSIONS: Linezolid and tedizolid alone are not optimal candidates to target bacterial phenotypes associated with chronic forms of BJI. Despite weak intracellular activity, they both reduce infection-related cytotoxicity, suggesting a role in modulating intracellular expression of staphylococcal virulence factors. Although inactive against biofilm-embedded S. aureus, both-but particularly tedizolid-are able to prevent biofilm formation.

LipoParticles: a lipid membrane coating onto polymer particles to enhance the internalization in osteoblast cells.

LipoParticles, core-shell assemblies consisting of a polymer core coated by a lipid membrane, are promising carriers for drug delivery applications with intracellular targets. This is of great interest since it is actually challenging to treat infections involving intracellular bacteria such as bone and joint infections where the bacteria are hidden in osteoblast cells. The present work reports for the first time to the best of our knowledge the proof of enhanced internalization of particles in osteoblast cells thanks to a lipid coating of particles (= LipoParticles). The ca. 300 nm-sized assemblies were elaborated by reorganization of liposomes (composed of DPPC/DPTAP 10/90 mol/mol) onto the surface of poly(lactic-co-glycolic acid) (PLGA) particles, and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and zetametry. Optimization of these assemblies was also performed by adding poly(ethylene glycol) (PEG) chains on their surface (corresponding to a final formulation of DPPC/DPTAP/DPPE-PEG5000 8/90/2 mol/mol/mol). Interestingly, this provided them colloidal stability after their 20-fold dilution in PBS or cell culture medium, and made possible their freeze-drying without forming aggregates after their re-hydration. Their non-cytotoxicity towards a human osteoblast cell line (MG63) was also demonstrated. The enhanced internalization of LipoParticles in this MG63 cell line, in comparison with PLGA particles, was proven by observations with a confocal laser scanning microscope, as well as by flow cytometry assays. Finally, this efficient internalization of LipoParticles in MG63 cells was confirmed by TEM on ultrathin sections, which also revealed localization close to intracellular Staphylococcus aureus.

Prophylactic Antibiofilm Activity of Antibiotic-Loaded Bone Cements against Gram-Negative Bacteria.

Gram-negative bacilli can be responsible for prosthetic joint infection (PJI) even if staphylococci are the main involved pathogens. Gram-negative PJIs (GN-PJI) are considered difficult-to-treat infections due to the increase in antimicrobial resistance and biofilm formation. To minimize the risk of infection in cases of arthroplasties with cemented prosthesis, bone cement can be loaded with antibiotics, especially gentamicin. In this study, we aimed to compare the prophylactic antibiofilm activity of ready-to-use antibiotic-loaded bone cements (ALBC), already commercialized or new prototypes. We compared ALBCs containing gentamicin alone, gentamicin plus vancomycin, gentamicin plus clindamycin, gentamicin plus Fosfomycin, and fosfomycin alone, to plain cement (no antibiotic); these comparisons were conducted to investigate the biofilm formation of three strains of Escherichia coli, three strains of Pseudomonas aeruginosa and two strains of Klebsiella pneumoniae, with or without specific resistance to gentamicin or fosfomycin. We reported that ALBC containing gentamicin and clindamycin (COPAL G+C) seems to be the most interesting ALBC of our tested panel for the prevention of biofilm formation by gentamicin-susceptible strains, even if clindamycin is not effective against Gram-negative bacteria. However, gentamicin-resistant strains are still a problem, and further studies are needed to identify an antibiotic to associate with gentamicin for an efficient dual ALBC against Gram-negative bacteria.

Patients at a high risk of PJI: Can we reduce the incidence of infection using dual antibiotic-loaded bone cement?

Prosthetic joint infection (PJI) is one of the most devastating complications of orthopedic surgery. However, not all patients are equally at the risk of severe infection. The incidences of PJI vary with the host and surgery-related risk factors. It is now generally accepted that some important medical comorbidities may predispose the patients to a high risk of PJI. Time-consuming and invasive surgical procedures, such as revision arthroplasties, are also associated with a high incidence of PJI, presumably due to the increased risk of surgical site contamination. Effective infection-preventing strategies should begin with identifying and optimizing the patients at a high risk of infection prior to surgery. Optimizing the operating room environment and antibiotic prophylaxis are also essential strategies that help minimize the overall incidence of infection in orthopedic surgery. The ideal antibiotic prophylaxis is still under debate, and discussions have emerged about whether variations or adjustments to the standard protocol are justified in patients at a high risk of infection. This also includes evaluating the possible benefits and risks of using high-dose dual antibiotic-loaded bone cement instead of low-dose single antibiotic-loaded bone cement in arthroplasty. This review summarizes the evidence showing that the combination of two local antibiotics in bone cement exerts a strong and longer-lasting antimicrobial effect against PJI-associated pathogens. This conclusion is consistent with the preliminary clinical studies showing a low incidence of PJI in high-risk patients undergoing cemented hemiarthroplasty, cemented revision, and primary arthroplasty if dual ALBC is used. These results may encourage clinicians to consolidate this hypothesis in a wider clinical range.

Methods to monitor bacterial growth and replicative rates at the single-cell level.

The heterogeneity of bacterial growth and replicative rates within a population was proposed a century ago notably to explain the presence of bacterial persisters. The term "growth rate" at the single-cell level corresponds to the increase in size or mass of an individual bacterium while the "replicative rate" refers to its division capacity within a defined temporality. After a decades long hiatus, recent technical innovative approaches allow population growth and replicative rates heterogeneity monitoring at the single-cell level resuming in earnest. Among these techniques, the oldest and widely used is time-lapse microscopy, most recently combined with microfluidics. We also discuss recent fluorescence dilution methods informing only on replicative rates and best suited. Some new elegant single cell methods so far only sporadically used such as buoyant mass measurement and stable isotope probing have emerged. Overall, such tools are widely used to investigate and compare the growth and replicative rates of bacteria displaying drug-persistent behaviors to that of bacteria growing in specific ecological niches or collected from patients. In this review, we describe the current methods available, discussing both the type of queries these have been used to answer and the specific strengths and limitations of each method.

Environmental Persistence of Staphylococcus capitis NRCS-A in Neonatal Intensive Care Units: Role of Biofilm Formation, Desiccation, and Disinfectant Tolerance.

The clone Staphylococcus capitis NRCS-A is responsible for late-onset sepsis in neonatal intensive care units (NICUs) worldwide. Over time, this clone has evolved into three subgroups that are increasingly adapted to the NICU environment. This study aimed to decipher the mechanisms involved in NRCS-A persistence in NICUs. Twenty-six S. capitis strains belonging to each of the three NRCS-A clone subgroups and two other non-NRCS-A groups from neonates (alpha clone) or from adult patients ("other strains") were compared based on growth kinetics and ability to form biofilm as well as tolerance to desiccation and to different disinfectants. S. capitis biofilm formation was enhanced in rich medium and decreased under conditions of nutrient stress for all strains. However, under conditions of nutrient stress, NRCS-A strains presented an enhanced ability to adhere and form a thin biofilm containing more viable and culturable bacteria (mean 5.7 log10 CFU) than the strains from alpha clone (mean, 1.1 log10 CFU) and the "other strains" (mean, 4.2 log10 CFU) (P < 0.0001). The biofilm is composed of bacterial aggregates with a matrix mainly composed of polysaccharides. The NRCS-A clone also showed better persistence after a 48-h desiccation. However, disinfectant tolerance was not enhanced in the NRCS-A clone in comparison with that of strains from adult patients. In conclusion, the ability to form biofilm under nutrient stress and to survive desiccation are two major advantages for clone NRCS-A that could explain its ability to persist and settle in the specific environment of NICU settings. IMPORTANCE Neonatal intensive care units (NICUs) host extremely fragile newborns, including preterm neonates. These patients are very susceptible to nosocomial infections, with coagulase-negative staphylococci being the species most frequently involved. In particular, a Staphylococcus capitis clone named NRCS-A has emerged worldwide specifically in NICUs and is responsible for severe nosocomial sepsis in preterm neonates. This clone is specifically adapted to the NICU environment and is able to colonize and maintain on NICU surfaces. The present work explored the mechanisms involved in the persistence of the NRCS-A clone in the NICU environment despite strict hygiene measures. The ability to produce biofilm under nutritional stress and to resist desiccation appear to be the two main advantages of NRCS-A in comparison with other strains. These findings are pivotal to provide clues for subsequent development of targeted methods to combat NRCS-A and to stop its dissemination.

Microscopy-based phenotypic profiling of infection by Staphylococcus aureus clinical isolates reveals intracellular lifestyle as a prevalent feature.

Staphylococcus aureus is increasingly recognized as a facultative intracellular pathogen, although the significance and pervasiveness of its intracellular lifestyle remain controversial. Here, we applied fluorescence microscopy-based infection assays and automated image analysis to profile the interaction of 191 S. aureus isolates from patients with bone/joint infections, bacteremia, and infective endocarditis, with four host cell types, at five times post-infection. This multiparametric analysis revealed that almost all isolates are internalized and that a large fraction replicate and persist within host cells, presenting distinct infection profiles in non-professional vs. professional phagocytes. Phenotypic clustering highlighted interesting sub-groups, including one comprising isolates exhibiting high intracellular replication and inducing delayed host death in vitro and in vivo. These isolates are deficient for the cysteine protease staphopain A. This study establishes S. aureus intracellular lifestyle as a prevalent feature of infection, with potential implications for the effective treatment of staphylococcal infections.

Lysosomal alkalization to potentiate eradication of intra-osteoblastic Staphylococcus aureus in the bone and joint infection setting.

OBJECTIVES: Beyond intracellular penetration, acidic lysosomal pH might affect the intracellular activity of some antimicrobials. This study evaluated the ability of lysosomotropic alkalizing agents to potentiate the antimicrobial eradication of an intra-osteoblastic Staphylococcus aureus reservoir in the setting of bone and joint infection (BJI). METHODS: MICs of 16 anti-staphylococcal molecules active against methicillin-sensitive S. aureus (MSSA) were evaluated at pH 5 and pH 7. Additionally, the lysosomal alkalizing potential (spectrofluorometry) and cytotoxicity (MTT assay) of hydroxychloroquine, amantadine and ammonium chloride were assessed. The results led to further investigation of clindamycin, cotrimoxazole, daptomycin and levofloxacin-alone or in combination with hydroxychloroquine-in an in vitro model of osteoblast infection. The impact of hydroxychloroquine on autophagy was finally investigated using Western blot detection of two autophagic flux indicators, the LC3 membrane protein and the SQSTM1 cargo protein. RESULTS: Daptomycin, cotrimoxazole, clindamycin and levofloxacin alone significantly decreased the intracellular staphylococcal reservoir (5.12 log10 CFU/100 000 cells) by 0.14 (95%CI 0.01-0.34), 0.25 (95%CI 0.12-0.43), 0.16 (95%CI 0.004-0.39) and 1.18 (95%CI 1.04-1.38) log10 CFU/100 000 cells, respectively (p < 10-3). Adding hydroxychloroquine (20 mg/L) increased intralysosomal pH from 4.8 to 7, and concomitantly the inoculum of each antimicrobial was reduced by 0.50 (95%CI 0.30-0.84), 0.73 (95%CI 0.59-0.96), 0.59 (95%CI 0.46-0.78) and 1.8 (95%CI 1.66-2.1) log10 CFU/100 000 cells, respectively (p < 10-4). Cellular levels of LC3II and SQSTM1 showed that hydroxychloroquine has direct activity on the autophagic flux, fostering the eradication of intracellular S. aureus by antimicrobials. CONCLUSION: At high concentrations, hydroxychloroquine used as an adjuvant to antimicrobials improves eradication of an S. aureus intra-osteoblastic reservoir in our in vitro cell infection model. These findings advocate further in vivo evaluation of alkalization efficacy and tolerance in S. aureus BJI.

YAP promotes cell-autonomous immune responses to tackle intracellular Staphylococcus aureus in vitro.

Transcriptional cofactors YAP/TAZ have recently been found to support autophagy and inflammation, which are part of cell-autonomous immunity and are critical in antibacterial defense. Here, we studied the role of YAP against Staphylococcus aureus using CRISPR/Cas9-mutated HEK293 cells and a primary cell-based organoid model. We found that S. aureus infection increases YAP transcriptional activity, which is required to reduce intracellular S. aureus replication. A 770-gene targeted transcriptomic analysis revealed that YAP upregulates genes involved in autophagy/lysosome and inflammation pathways in both infected and uninfected conditions. The YAP-TEAD transcriptional activity promotes autophagic flux and lysosomal acidification, which are then important for defense against intracellular S. aureus. Furthermore, the staphylococcal toxin C3 exoenzyme EDIN-B was found effective in preventing YAP-mediated cell-autonomous immune response. This study provides key insights on the anti-S. aureus activity of YAP, which could be conserved for defense against other intracellular bacteria.