Staphylococcal Esx Proteins Modulate Apoptosis and Release of Intracellular Staphylococcus aureus during Infection in Epithelial Cells

The opportunistic pathogen Staphylococcus aureus is one of the major causes of health care-associated infections. S. aureus is primarily an extracellular pathogen, but it was recently reported to invade and replicate in several host cell types. The ability of S. aureus to persist within cells has been implicated in resistance to antimicrobials and recurrent infections. However, few staphylococcal proteins that mediate intracellular survival have been identified. Here we examine if EsxA and EsxB, substrates of the ESAT-6-like secretion system (Ess), are important during intracellular S. aureus infection. The Esx proteins are required for staphylococcal virulence, but their functions during infection are unclear. While isogenic S. aureus esxA and esxB mutants were not defective for epithelial cell invasion in vitro, a significant increase in early/late apoptosis was observed in esxA mutant-infected cells compared to wild-type-infected cells. Impeding secretion of EsxA by deleting C-terminal residues of the protein also resulted in a significant increase of epithelial cell apoptosis. Furthermore, cells transfected with esxA showed an increased protection from apoptotic cell death. A double mutant lacking both EsxA and EsxB also induced increased apoptosis but, remarkably, was unable to escape from cells as efficiently as the single mutants or the wild type. Thus, using in vitro models of intracellular staphylococcal infection, we demonstrate that EsxA interferes with host cell apoptotic pathways and, together with EsxB, mediates the release of S. aureus from the host cell.     

Internalization of Staphylococcus aureus by Endothelial Cells Induces Apoptosis

The ability of Staphylococcus aureus to invade and survive within endothelial cells is believed to contribute to its propensity to cause persistent endovascular infection with endothelial destruction. In the present study, we show that following invasion of human umbilical vein endothelial cells, intracellular S. aureus organisms remain viable over a 72-h period and, as determined by transmission electron microscopic examination, that the bacteria exist within vacuoles and free within the cytoplasm. We also demonstrate that endothelial cell death following S. aureus invasion occurs at least in part by apoptosis as shown by DNA fragmentation and changes in nuclear morphology. Apoptotic changes were evident as early as 1 h after infection of endothelial cells. Internalization of S. aureus rather than adherence appears to be necessary, since use of the phagocytosis inhibitor cytochalasin D prevented apoptosis. UV-killed staphylococci, although retaining the capacity to be internalized, were not capable of inducing apoptosis, suggesting that apoptosis is dependent upon a factor associated with viable organisms. The studies demonstrate that viable intracellular S. aureus induces apoptosis of endothelial cells and that internalized staphylococci can exist free within the cytoplasm.     

Analysis of Staphylococcus aureus proteins secreted inside infected human epithelial cells

Staphylococcus aureus, an opportunistic pathogen is able to invade into and persist inside non-professional phagocytic cells. To do so, this bacterium possesses a wide range of secreted virulence factors which enable attachment to the host as well as intracellular survival. Hence, a monitoring of virulence factors specifically produced upon internalization might reveal targets for prevention or therapy of S. aureus infections. However, previous proteome approaches enriching S. aureus from lysed host cells after infection did not cover secreted virulence factors.Therefore, we used density gradient centrifugation and mass spectrometry to identify S. aureus HG001 proteins which were secreted into compartments of infected human bronchial epithelial S9 cells. Because shotgun mass spectrometry revealed only few bacterial proteins amongst 1905 host proteins, we used highly sensitive and selective single reaction monitoring mass spectrometry as an alternative approach and quantified 37 bacterial proteins within the S. aureus containing host cell compartment 2.5 h and 6.5 h post infection. Among them were secreted bacterial virulence factors like lipases, pore forming toxins, and secreted adhesins which are usually hard to detect from infected sample material by proteomics approaches due to their low abundance. S. aureus adapted its proteome to improve its response to oxidative and cell wall stress occurring inside the host, but also, increased the amounts of some adhesins and pore-forming toxins, required for attachment and host cell lysis.     

Staphylococcus aureus host cell invasion and post-invasion events

Staphylococcus aureus is now recognized as a facultative intracellular pathogen. The aim of this review is to discuss novel data regarding the invasion mechanism and post-invasion events with a focus on the fate of the infected phagosome in non-professional phagocytes and the role of S. aureus α-toxin.     

Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells

We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes.

Staphylococcus aureus is a pathogen with a broad host range and is a leading cause of infections in humans and domesticated animals worldwide. The rapidly increasing frequency of methicillin-resistant isolates and the looming threat of resistance to vancomycin by this organism have recently caused considerable alarm within the medical community. Infections associated with this organism are extremely common and often life threatening; therefore, there is serious potential for S. aureus to cause increased morbidity and mortality. S. aureus is also the leading cause of intramammary infection in ruminants, which is the most economically important disease to the dairy industry in the United States, with approximately one-half of dairy cows afflicted with some form of mastitis. This disease accounts for approximately 70% of the total expenses for dairy farmers (14) and results in the loss of billions of dollars each year (3). Thus, a thorough understanding of the pathogenesis of staphylococcal disease could have a profound impact on public health and agriculture in the United States and worldwide.The mechanism of persistence of staphylococci in its hosts, despite the induction of seemingly sufficient levels of humoral and mucosal antibodies, remains unexplained. This is an important issue for both humans and animals, which can have repeated occurrences of staphylococcal infections and toxigenic diseases such as toxic shock syndrome (7). While there is some evidence that immunosuppression induced by superantigens is partially responsible for both the persistence of infection and reduced antibody levels against some staphylococcal products, not all human and animal cases are attributed to superantigen-producing organisms. For example, less than half of bovine mastitis staphylococcal isolates produce known superantigen exotoxins (26).One confirmed mechanism employed by some bacteria to evade humoral immunity is to become internalized in host cells. For example, organisms such as Listeria monocytogenes and Mycobacterium tuberculosis are facultative intracellular pathogens that require cell-mediated immunity to be eliminated most efficiently; the presence of antibodies alone is ineffective during infection by these pathogens. S. aureus is generally not considered to be an intracellular pathogen of the magnitude associated with classical facultative intracellular pathogens (i.e., Listeria, Mycobacterium, Salmonella, and Shigella spp.). However, it is well documented that S. aureus can be internalized in epithelial cells (1) and endothelial cells (18, 37). Little is known regarding the mechanisms involved in internalization, the potential role of internalization of S. aureus by the host cell, or host cell responses.The objective of this study was to investigate the invasion of mammary epithelial cells by a strain of S. aureus known to cause bovine mastitis. We now report that S. aureus cells successfully invade epithelial cells, exist free in the cytoplasm after effecting release from the endosome, and induce the host cells to undergo apoptosis.     

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection

The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.     

Mechanisms of folate metabolism-related substances affecting Staphylococcus aureus infection

Staphylococcus aureus (S. aureus) is one of the critical clinical pathogens which can cause multiple diseases ranging from skin infections to fatal sepsis. S. aureus is generally considered to be an extracellular pathogen. However, more and more evidence has shown that S. aureus can survive inside various cells. Folate plays an essential role in multiple life activities, including the conversion of serine and glycine, the remethylation of homocysteine to methionine, and the de novo synthesis of purine /dTMP, et al. More and more studies reported that S. aureus intracellular infection requires the involvement of folate metabolism. This review focused on the mechanisms of folate metabolism and related substances affecting S. aureus infection. Loss of tetrahydrofolic acid (THF)-dependent dTMP directly inhibits the nucleotide synthesis pathway of the S. aureus due to pabA deficiency. Besides, trimethoprim-sulfamethoxazole (TMP/SMX), a potent antibiotic that treats S. aureus infections, interferes in the process of the folate mechanism and leads to the production of thymidine-dependent small-colony variants (TD-SCVs). In addition, S. aureus is resistant to lysostaphin in the presence of serine hydroxymethyltransferase (SHMT). We provide new insights for understanding the molecular pathogenesis of S. aureus infection.     

Host–pathogen interactions in epidermolysis bullosa patients colonized with Staphylococcus aureus

Patients with the genetic blistering disease epidermolysis bullosa (EB) often have chronic wounds that can become colonized by different bacteria, especially the opportunistic pathogen Staphylococcus aureus. We therefore determined the S. aureus colonization rates in EB patients from the Netherlands by collecting swabs from their anterior nares, throats and wounds. Within a period of ∼2 years, more than 90% of the sampled chronic wounds of EB patients were found to be colonized by S. aureus. Molecular typing revealed that EB patients were not colonized by a single S. aureus type. Rather the S. aureus population structure in the sampled EB patients mirrored the local S. aureus population structure within the Netherlands. Furthermore, multiple types of S. aureus were found in close proximity to each other within individual chronic wounds, indicating that these S. aureus types are not mutually exclusive. Over time, strong fluctuations in the S. aureus types sampled from individual EB patients were observed. This high exposure to different S. aureus types is apparently reflected by high plasma levels of antistaphylococcal IgG's, especially in patients carrying multiple S. aureus types. It remains to be determined to what extent this strong immune response protects EB patients against serious staphylococcal infections. Lastly, further research is needed to define the impact of staphylococcal colonization of chronic wounds on the development, exacerbation and healing of such wounds in patients with EB.     

An in vitro investigation of the protective role of Staphylococcus Epidermidis extracts on Staphylococcus Aureus induced toxicity in human keratinocytes

PurposeStaphylococcus aureus infections are a major cause of concern in nosocomial infections and especially so, in the hospitalized immunocompromised patients. Staphylococcus epidermidis is a skin commensal that could have a role in preventing colonization on human skin by potential pathogen.MethodsThe probable protective role of S. epidermidis, its lysate (S.epi lysate) and spent culture fluid (SCF) has been explored against S. aureus using human epidermal keratinocytes as a model system. The viability of keratinocytes and bacterial adhesion was investigated pre- and post-exposure to S. epi lysate and SCF.ResultsThe viability of keratinocytes was significantly reduced when incubated with S. aureus for 24 ?h while S. epidermidis and its extracts exhibited no significant effect. S. aureus infected keratinocytes showed increased viability when incubated with viable S. epidermidis which was even greater with its lysate and SCF. The timing of the application of lysate and SCF affected the degree of protection conferred to the keratinocytes against S. aureus induced toxicity. Co-exposed and post-exposed keratinocytes were afforded equal protection. However, a pre-exposure of 2 ?h was not efficient enough to provide significant protection. S. epi lysate and SCF reduced the number of adherent cells considerably even after 8 ?h of pathogen exposure.ConclusionsS. epidermidis and its extracts protect human epidermal keratinocytes from the toxic effects of S. aureus by competitive displacement of pathogen and reduction in adhesion. S. epi lysate and SCF are safer options for the treatment of pathogen induced skin damage.     

Staphylococcus aureus strategies to evade the host acquired immune response

Staphylococcus aureus poses a significant public-health problem. Infection caused by S. aureus can manifest as acute or long-lasting persistent diseases that are often refractory to antibiotic and are associated with significant morbidity and mortality. To develop more effective strategies for preventing or treating these infections, it is crucial to understand why the immune response is incapable to eradicate the bacterium. When S. aureus first infect the host, there is a robust activation of the host innate immune responses. Generally, S. aureus can survive this initial interaction due to the expression of a wide array of virulence factors that interfere with the host innate immune defenses. After this initial interaction the acquired immune response is the arm of the host defenses that will try to clear the pathogen. However, S. aureus is capable of maintaining infection in the host even in the presence of a robust antigen-specific immune response. Thus, understanding the mechanisms underlying the ability of S. aureus to escape immune surveillance by the acquired immune response will help uncover potentially important targets for the development of immune-based adjunctive therapies and more efficient vaccines. There are several lines of evidence that lead us to believe that S. aureus can directly or indirectly disable the acquired immune response. This review will discuss the different immune evasion strategies used by S. aureus to modulate the different components of the acquired immune defenses.     

Activation of the alternative sigma factor SigB of Staphylococcus aureus following internalization by epithelial cells – An in vivo proteomics perspective

Staphylococcus aureus is a versatile pathogen that can be a commensal but also cause a wide range of different infections. This broad disease spectrum is a reflection of the complex regulation of a large collection of virulence factors that together with metabolic fitness allow adaptation to different niches. The alternative sigma factor SigB is one of the global regulators mediating this adaptation. However, even if SigB contributes to expression of many virulence factors its importance for successful infection greatly varies with the strain and the infection setting analyzed. We have recently established a proteomics workflow that combines high efficiency cell sorting with sensitive mass spectrometry and allows monitoring of global proteome adaptations with roughly one million bacterial cells. Thus, we can now approach the adaptation of pathogens to the intracellular milieu. In the current study this proteomics workflow was used in conjunction with qRT-PCR and confocal fluorescence microscopy to comparatively analyze the adaptation of the S. aureus wild type strain HG001 and its isogenic sigB mutant to the intracellular milieu of human S9 bronchial epithelial cells. The study revealed fast and transient activation of SigB following internalization by human host cells and the requirement of SigB for intracellular growth. Loss of SigB triggered proteome changes reflecting the different residual growth rates of wild type and sigB mutant, respectively, the resistance to methicillin, adaptation to oxidative stress and protein quality control mechanisms.     

Wall teichoic acid is an essential component of Staphylococcus aureus for the induction of human dendritic cell maturation

Staphylococcus aureus is a Gram-positive pathogen that can cause chronic skin inflammation, pneumonia, and septic shock. The immunomodulatory functions of wall teichoic acid (WTA), a glycopolymer abundantly expressed on the Gram-positive bacterial cell wall, are poorly understood. Here, we investigated the role of WTA in the phenotypic and functional activation of human monocyte-derived dendritic cells (DCs) treated with ethanol-killed S. aureus. WTA-deficient S. aureus mutant (ΔtagO) exhibited attenuated binding and internalization to DCs compared to the wild-type. ΔtagO induced lower expression of maturation markers on and cytokines in DCs than the wild-type S. aureus. Furthermore, autologous human peripheral blood mononuclear cells cocultured with ΔtagO-treated DCs exhibited a marked reduction in T cell proliferative activity, the expression of activation markers, and the production of cytokines compared to the wild-type S. aureus-stimulated DCs. Collectively, these results suggest that WTA is an important cell wall component of S. aureus for the induction of DC maturation and activation.     

A proteomic view of cell physiology and virulence of Staphylococcus aureus

Staphylococcus aureus is a human pathogen that has advanced to a main problem in hospital settings since effective treatment options for infections caused by this pathogen are limited. Thus, new strategies to prevent and treat S. aureus infections and rapid diagnostic tools are urgently needed. The course of an S. aureus infection largely depends on successful adaptation to the host environment and a very complex and poorly understood interplay of bacterial virulence factors with each other and with host components. Over the last years, genome sequences of different S. aureus strains have been published permitting a high-throughput proteomic analysis of this pathogen. This review summarizes the impact of 2D gel- and mass spectrometry-based proteomic approaches on a more comprehensive understanding of S. aureus pathophysiology and virulence. We show that only a combination of both techniques allows a proteomic view which adequately considers all subproteomic fractions of a bacterium, i.e. cytosolic, membrane, cell surface-associated, and extracellular proteins. By this means, the majority of proteins expressed in S. aureus can be identified and even quantified. In addition, posttranslational processes such as protein secretion, modification, processing, damages, and degradation can be adequately studied.     

Advanced glycation end products reduce macrophage-mediated killing of Staphylococcus aureus by ARL8 upregulation and inhibition of autolysosome formation

Staphylococcus aureus, a pathogen most frequently found in diabetic foot ulcer infection, was recently suggested as an intracellular pathogen. Autophagy in professional phagocytes like macrophages allows selective destruction of intracellular pathogens, and its dysfunction can increase the survival of internalized pathogens, causing infections to worsen and spread. Previous works have shown that S. aureus infections in diabetes appeared more severe and invasive, and coincided with the suppressed autophagy in dermal tissues of diabetic rat, but the exact mechanisms are unclear. Here, we demonstrated that accumulation of advanced glycation end products (AGEs) contributed to the diminished autophagy-mediated clearance of S. aureus in the macrophages differentiated from PMA-treated human monocytic cell line THP-1. Importantly, infected macrophages showed increased S. aureus containing autophagosome, but the subsequent fusion of S. aureus containing autophagosome and lysosome was suppressed in AGEs-pretreated cells, suggesting AGEs blocked the autophagic flux and enabled S. aureus survival and escape. At the molecular level, elevated lysosomal ARL8 expression in AGEs-treated macrophages was required for AGEs-mediated inhibition of autophagosome-lysosome fusion. Silencing ARL8 in AGEs-treated macrophages restored autophagic flux and increased S. aureus clearance. Our results therefore demonstrate a new mechanism, in which AGEs accelerate S. aureus immune evasion in macrophages by ARL8-dependent suppression of autophagosome-lysosome fusion and bactericidal capability.     

Human mast cell activation by Staphylococcus aureus: interleukin-8 and tumor necrosis factor alpha release and the role of Toll-like receptor 2 and CD48 molecules

The ability of Staphylococcus aureus to invade and survive within host cells is believed to contribute to its propensity to cause persistent and metastatic infections. In addition, S. aureus infections often are associated with atopic diseases such as dermatitis, rhinitis, and asthma. Mast cells, the key cells of allergic diseases, have a pivotal role in innate immunity and have the capacity of phagocytosis, and they can destroy some pathogenic bacteria. However, little is known about the ability of some other bacteria to survive and overcome mast cell phagocytosis. Therefore, we were interested in evaluating the interplay between mast cells and S. aureus. In this study, we show that human cord blood-derived mast cells (CBMC) can be infected by pathogenic S. aureus. S. aureus displayed a high adherence to mast cells as well as invasive and survival abilities within them. However, when infections were performed in the presence of cytochalasin D or when CBMC were preincubated with anti-Toll-like receptor 2 (TLR2) or anti-CD48 antibodies, the invasiveness and the inflammatory response were abrogated, respectively. Furthermore, we observed an increase of TLR2 and CD48 molecules on CBMC after S. aureus infection. The infection of CBMC with S. aureus also caused the release of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8). Both live and killed S. aureus organisms were found to trigger TNF-α and IL-8 release by CBMC in a time-dependent manner. Cumulatively, these findings suggest that S. aureus internalizes and survives in mast cells. This may play an important role in infections and in atopic diseases associated with S. aureus.     

Staphylococcus chromogenes,a Coagulase-Negative Staphylococcus Species That Can Clot Plasma

Staphylococcus chromogenes is one of the main coagulase-negative staphylococci isolated from mastitis of dairy cows. We describe S. chromogenes isolates that can clot plasma. Since the main pathogen causing mastitis is coagulase-positive Staphylococcus aureus, the coagulase-positive phenotype of S. chromogenes described here can easily lead to misidentification.     

Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages

Methicillin-resistant Staphylococcus aureus (MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports that S. aureus bacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of the S. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains of S. aureus survived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and β-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or other S. aureus strains, suggesting that S. aureus perturbs acquisition of lysosomal enzymes. We examined the impact of acidification on S. aureus intramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulator agr but had little effect on expression of sarA, saeR, or sigB. Bacterial exposure to acidic pH in vitro increased agr expression. Together, these results suggest that S. aureus survives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival.     

Discovery of potential anti-infectives against Staphylococcus aureus using a Caenorhabditis elegans infection model

Background

The limited antibiotic options for effective control of methicillin-resistant Staphylococcus aureus infections has led to calls for new therapeutic approaches to combat this human pathogen. An alternative approach to control MRSA is through the use of anti-infective agents that selectively disrupt virulence-mediated pathways without affecting microbial cell viability or by modulating the host natural immune defenses to combat the pathogen.

Methods

We established a C. elegans – S. aureus liquid-based assay to screen for potential anti-infectives against S. aureus. The assay was utilized to screen 37 natural extracts and 29 synthetic compounds for the ability to extend the lifespan of infected nematodes. Disc diffusion and MIC microdilution tests were used to evaluate the anti-microbial properties of these natural extracts and synthetic compounds whilst in vivo bacterial CFU within the C. elegans gut were also enumerated.

Results

We screened a total of 37 natural extracts and 29 synthetic compounds for anti-infective properties. The screen successfully revealed 14 natural extracts from six plants (Nypa fruticans, Swietenia macrophylla, Curcuma longa, Eurycoma longifolia, Orthosiphon stamineus and Silybum eburneum) and one marine sample (Faunus ater) that improved the survival of S. aureus-infected worms by at least 2.8-fold as well as 14 synthetic compounds that prolonged the survival of S. aureus-infected nematodes by 4-fold or greater. An anti-microbial screen of all positive hits demonstrated that 8/28 hits had no effect on S. aureus growth. Of these 8 candidates, 5 of them also protected the worms from MRSA infection. We also noted that worms exposed to N. fruticans root and O. stamineus leaf extracts showed reduced intestinal colonization by live S. aureus. This suggests that these extracts could possibly activate host immunity to eliminate the bacteria or interfere with factor/s that prevents pathogen accumulation.

Conclusion

We have successfully demonstrated the utility of this liquid-based screen to identify anti-infective substances that prolong S. aureus-infected host survival without affecting bacterial cell viability.