More Is Not Always Better-the Double-Headed Role of Fibronectin in Staphylococcus aureus Host Cell Invasion.

While Staphylococcus aureus has classically been considered an extracellular pathogen, these bacteria are also capable of being taken up by host cells, including nonprofessional phagocytes such as endothelial cells, epithelial cells, or osteoblasts. The intracellular S. aureus lifestyle contributes to infection development. The predominant recognition and internalization pathway appears to be the binding of the bacteria via a fibronectin bridge to the α5ß1-integrin on the host cell membrane, followed by phagocytosis. Although osteoblasts showed high expression of α5ß1-integrin and fibronectin, and bacteria adhered to osteoblasts to a high proportion, here we demonstrate by internalization assays and immunofluorescence microscopy that S. aureus was less engulfed in osteoblasts than in epithelial cells. The addition of exogenous fibronectin during the infection of cells with S. aureus resulted in an increased uptake by epithelial cells but not by osteoblasts. This contrasts with the previous conception of the uptake mechanism, where high expression of integrin and fibronectin would promote the bacterial uptake into host cells. Extracellular fibronectin surrounding osteoblasts, but not epithelial cells, is organized in a fibrillary network. The inhibition of fibril formation, the short interfering RNA-mediated reduction of fibronectin expression, and the disruption of the fibronectin-fibril meshwork all resulted in a significant increase in S. aureus uptake by osteoblasts. Thus, the network of fibronectin fibrils appears to strongly reduce the uptake of S. aureus into a given host cell, indicating that the supramolecular structure of fibronectin determines the capacity of particular host cells to internalize the pathogen. IMPORTANCE Traditionally, Staphylococcus aureus has been considered an extracellular pathogen. However, among other factors, the frequent failure of antimicrobial therapy and the ability of the pathogen to cause recurrent disease have established the concept of eukaryotic invasion of the pathogen, thereby evading the host's immune system. In the current model of host cell invasion, bacteria initially bind to α5ß1 integrin on the host cell side via a fibronectin bridge, which eventually leads to phagocytosis of S. aureus by host cells. However, in this study, we demonstrate that not the crude amount but the supramolecular structure of fibronectin molecules deposited on the eukaryotic cell surface plays an essential role in bacterial uptake by host cells. Our findings explain the large differences of S. aureus uptake efficacy in different host cell types as well as in vivo differences between courses of bacterial infections and the localization of bacteria in different clinical settings.

Antibiotic Treatment and Age Are Associated With Staphylococcus aureus Carriage Profiles During Persistence in the Airways of Cystic Fibrosis Patients.

BACKGROUND: Staphylococcus aureus is one of the most isolated pathogens from the airways of cystic fibrosis (CF) patients. There is a lack of information about the clonal nature of S. aureus cultured from CF patients and their impact on disease. We hypothesized that patients would differ in their clinical status depending on S. aureus clonal carriage profiles during persistence. METHODS: During a 21-months prospective observational multicenter study (Junge et al., 2016), 3893 S. aureus isolates (nose, oropharynx, and sputa) were cultured from 183 CF patients (16 German centers, 1 Austrian center) and subjected to spa-sequence typing to assess clonality. Data were associated to lung function, age, gender, and antibiotic treatment by multivariate regression analysis. RESULTS: Two hundred and sixty-five different spa-types were determined with eight prevalent spa-types (isolated from more than 10 patients): t084, t091, t008, t015, t002 t012, t364, and t056. We observed different carriage profiles of spa-types during the study period: patients being positive with a prevalent spa-type, only one, a dominant or related spa-type/s. Patients with more antibiotic cycles were more likely to be positive for only one spa-type (p = 0.005), while older patients were more likely to have related (p = 0.006), or dominant spa-types (p = 0.026). Two percent of isolates were identified as methicillin-resistant S. aureus (MRSA) and evidence of transmission of clones within centers was low. CONCLUSION: There was a significant association of antibiotic therapy and age on S. aureus carriage profiles in CF patients indicating that antibiotic therapy prevents acquisition of new clones, while during aging of patients with persisting S. aureus, dominant clones were selected and mutations in the spa-repeat region accumulated.

Comparative in vitro activity of bacteriophage endolysin HY-133 against Staphylococcus aureus attached to vascular graft surface.

Novel strategies are needed for combating Staphylococcus aureus biofilm in vascular graft infections. We investigated the in vitro activity of bacteriophage endolysin HY-133, daptomycin and rifampin against S. aureus attached to vascular graft surface. Daptomycin showed rapid bactericidal effect on surface-associated S. aureus, while the activity of HY-133 on graft surface-adherent cells was moderate and rifampin did not achieve bactericidal effect. Even in the highest concentrations, all antimicrobials used failed in a complete eradication of the surface-adherent bacteria.

A retrospective analysis of the pathogens in the airways of patients with primary ciliary dyskinesia.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare genetically heterogeneous disorder of motile cilia, which leads to recurrent and chronic airway infections. Detailed information about infection causing pathogens is scarce. With this study, we aimed to determine the prevalence and susceptibility of the most common respiratory pathogens in PCD patients retrospectively in a cross-sectional and the dynamics of the microbiological diversity in a longitudinal study. METHODS: Microbiological and clinical data of 106 patients between 2010 and 2016 were analysed cross-sectionally and of 28 patients longitudinally. Dynamics in microbiological diversity were assessed by calculating the mean rate of alteration (MRA). RESULTS: Haemophilus influenzae was the most common pathogen (n = 41; 38.7%) followed by Staphylococcus aureus (n = 36; 34%), Moraxella catarrhalis (n = 18; 17%) and Pseudomonas aeruginosa (n = 16; 15.1%). Nontuberculous mycobacteria were cultured from two patients (1.9%). H. influenzae was the most prevalent pathogen in children (n = 31; 45.6%), S. aureus in adults (n = 15; 39%). Two patients were infected by methicillin-resistant S. aureus. P. aeruginosa was mostly susceptible to standard antibiotics with highest rates of resistance against fosfomycin (63.6%; 7/11). The culture of P. aeruginosa correlated negatively with age adjusted FEV1% predicted (p = 0.04), while the MRA was positively associated with age (rho 0.411, p = 0.032). DISCUSSION: In PCD patients, the prevalence of pathogens differed in children and adults with H. influenzae and S. aureus being the most common pathogens in children, S. aureus and P. aeruginosa in adults, respectively. Unexpectedly, the MRA increased by age.

The prevalence of Staphylococcus aureus with mucoid phenotype in the airways of patients with cystic fibrosis-A prospective study.

BACKGROUND: Staphylococcus aureus is one of the most frequently isolated pathogens in the respiratory tract of CF patients. Recently, we characterized peculiar mucoid S. aureus isolates, which are excessive biofilm formers and which carried a 5bp-deletion within the intergenic region of the ica operon. In this prospective study, we determined the prevalence of mucoid S. aureus-isolates in the airways of CF-patients during a 3-months period. METHODS: We analyzed specimens (sputa, throat swabs) from 81 CF patients who attended two CF centers in Münster, Germany. Ten S. aureus isolates were randomly picked from every S. aureus-positive airway specimen and evaluated for mucoidy using Congo Red agar and phenotypic tests. Mucoid isolates were characterized by spa sequence typing, biofilm production and sequencing of the intergenic region of the ica operon to screen for the 5bp-deletion. RESULTS: In 7 of 81 examined patients (8.6%), we detected mucoid S. aureus phenotypes (37 out of 1050 isolates; 3.5%). Twenty-five mucoid isolates carried the 5bp-deletion. Mucoid isolates produced excessive biofilm and were significantly more resistant to certain antibiotics. CONCLUSIONS: In our prospective study, mucoid S. aureus was present in 8.6% of S. aureus-positive CF-patients. In 6 of 7 patients, mucoid isolates carried the 5bp-deletion, indicating that also other so far not identified mechanisms cause excessive biofilm formation. Further studies are necessary to ascertain the clinical impact of mucoid S. aureus phenotypes on the severity of the CF disease.

Complement 5a Receptor Polymorphisms Are Associated With Panton-Valentine Leukocidin-positive Staphylococcus aureus Colonization in African Pygmies.

Panton-Valentine leukocidin (PVL) is common in African Staphylococcus aureus and can be associated with skin and soft tissue infection. PVL-positive S. aureus colonization is associated with a variant of complement receptor 5a, the cellular target of the lukS PVL subunit.

Bactericidal activity of bacteriophage endolysin HY-133 against Staphylococcus aureus in comparison to other antibiotics as determined by minimum bactericidal concentrations and time-kill analysis.

Methicillin-resistant Staphylococcus aureus (MRSA) decolonization is expensive and time consuming, and new agents are necessary due to increasing resistance rates. The administration of bacteriophages or particularly their endolysins may offer an alternative treatment strategy and could provide a solution to overcome the selection pressure due to classical antibiotics. Here, the bactericidal activity was characterized for the recombinant chimeric bacteriophage endolysin HY-133 in comparison to other antimicrobials. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for 2 reference strains, 24 clinical MRSA and methicillin-susceptible S. aureus (MSSA) isolates, as well as 6 isolates with high-level mupirocin resistance. Additionally, HY-133 activity against bacteria in stationary or exponential growth phase was compared in 12 isolates. Time-kill curves were performed with 2 representative isolates to investigate the pharmacodynamics until 48-h incubation time. All experiments were performed in comparison to daptomycin and mupirocin. The MIC50/90 and MBC50/90 values were in the range 0.12-0.5 mg/L for all 3 growth conditions comparable to daptomycin with 0.5/0.5 mg/L, respectively. The MBC was almost always equal the MIC and without considerable differences between MSSA and MRSA. Time-kill curves revealed a rapid bactericidal effect of HY-133 within the first 2 h, similar to daptomycin. Even with low concentrations, the recombinant endolysin HY-133 was highly active against all tested MSSA and MRSA isolates including mupirocin-resistant isolates. The application of this alternative agent may offer a future strategy for MRSA/MSSA decolonization and, potentially, for treatment purposes.

The Energy-Coupling Factor Transporter Module EcfAA'T, a Novel Candidate for the Genetic Basis of Fatty Acid-Auxotrophic Small-Colony Variants of Staphylococcus aureus.

Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA'T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA'T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid-auxotrophic staphylococcal SCVs.

Metabolic conversion of CI-1040 turns a cellular MEK-inhibitor into an antibacterial compound.

Influenza virus (IV) infections cause severe respiratory illnesses that can be complicated by bacterial super-infections. Previously, we identified the cellular Raf-MEK-ERK cascade as a promising antiviral target. Inhibitors of MEK, such as CI-1040, showed potent antiviral activity. However, it remained unclear if this inhibitor and its active form, ATR-002, might sensitize host cells to either IV or secondary bacterial infections. To address these questions, we studied the anti-pathogen activity of ATR-002 in comparison to CI-1040, particularly, its impact on Staphylococcus aureus (S. aureus), which is a major cause of IV super-infections. We analysed IV and S. aureus titres in vitro during super-infection in the presence and absence of the drugs and characterized the direct impact of ATR-002 on bacterial growth and phenotypic changes. Importantly, neither CI-1040 nor ATR-002 treatment led to increased bacterial titres during super-infection, indicating that the drug does not sensitize cells for bacterial infection. In contrast, we rather observed reduced bacterial titres in presence of ATR-002. Surprisingly, ATR-002 also led to reduced bacterial growth in suspension cultures, reduced stress- and antibiotic tolerance without resistance induction. Our data identified for the first time that a particular MEK-inhibitor metabolite exhibits direct antibacterial activity, which is likely due to interference with the bacterial PknB kinase/Stp phosphatase signalling system.

Staphylococcus aureus requires less virulence to establish an infection in diabetic hosts.

Staphylococcus aureus is the most frequent pathogen causing diabetic foot infections. Here, we investigated the degree of bacterial virulence required to establish invasive tissue infections in diabetic organisms. Staphylococcal isolates from diabetic and non-diabetic foot ulcers were tested for their virulence in in vitro functional assays of host cell invasion and cytotoxicity. Isolates from diabetes mellitus type I/II patients exhibited less virulence than isolates from non-diabetic patients, but were nevertheless able to establish severe infections. In some cases, non-invasive isolates were detected deep within diabetic wounds, even though the strains were non-pathogenic in cell culture models. Testing of defined isolates in murine footpad injection models revealed that both low- and high-virulent bacterial strains persisted in higher numbers in diabetic compared to non-diabetic hosts, suggesting that hyperglycemia favors bacterial survival. Additionally, the bacterial load was higher in NOD mice, which have a compromised immune system, compared to C57Bl/6 mice. Our results reveal that high as well as low-virulent staphylococcal strains are able to cause soft tissue infections and to persist in diabetic humans and mice, suggesting a reason for the frequent and endangering infections in patients with diabetes.

Author Correction: The Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The Novel Phage-Derived Antimicrobial Agent HY-133 Is Active against Livestock-Associated Methicillin-Resistant Staphylococcus aureus.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates are increasingly migrating from livestock into human and animal health care settings. Alternative substances are needed to overcome the drawbacks of currently available drugs used for MRSA eradication. The recombinant bacteriophage endolysin HY-133 has proved to be an active agent against S. aureus Here, the in vitro activity of HY-133 was studied against a large collection of genetically diverse LA-MRSA isolates revealing its high activity against mecA-, mecB-, and mecC-positive LA-MRSA.

Management of superficial and deep-seated Staphylococcus aureus skin and soft tissue infections in sub-Saharan Africa: a post hoc analysis of the StaphNet cohort.

PURPOSE: The incidence of Staphylococcus aureus skin and soft tissue infection (SSTI) is high in sub-Saharan Africa. This is fueled by a high prevalence of Panton-Valentine leukocidin (PVL), which can be associated with necrotizing disease. The aim was to describe the clinical presentation and the treatment of SSTI in the African setting and to identify challenges in the management. METHODS: Patients (n = 319) were recruited in DR Congo (n = 56, 17.6%), Gabon (n = 89, 27.9%), Mozambique (n = 79, 24.8%) and Tanzania (n = 95, 29.8%) during the prospective observational StaphNet cohort study (2010-2015). A physician recorded the clinical management in standardized questionnaires and stratified the entity of SSTI into superficial (sSSTI) or deep-seated (dSSTI). Selected virulence factors (PVL, ß hemolysin) and multilocus sequence types (MLST) were extracted from whole genome sequencing data. RESULTS: There were 220/319 (69%) sSSTI and 99/319 (31%) dSSTI. Compared to sSSTI, patients with dSSTI were more often hospitalized (13.2 vs. 23.5%, p = 0.03), HIV-positive (7.6 vs. 15.9%, p = 0.11), and required more often incision and drainage (I&D, 45.5 vs. 76.5%, p = 0.04). The proportion of an adequate antimicrobial therapy increased marginally from day 1 (empirical therapy) to day 3 (definite therapy), for sSSTI (70.7 to 72.4%) and dSSTI (55.4 to 58.9%). PVL was a risk factor for I&D (OR = 1.7, p = 0.02) and associated with MLST clonal complex CC121 (OR = 2.7, p < 0.001). CONCLUSION: Appropriate antimicrobial agents and surgical services to perform I&D were available for the majority of patients. Results from susceptibility testing should be considered more efficiently in the selection of antimicrobial therapy.

Staphylococcus aureus in the airways of cystic fibrosis patients - A retrospective long-term study.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive disease associated with chronic airway infections by Staphylococcus aureus as one of the earliest and most prevalent pathogens. We conducted a retrospective study to determine the S. aureus infection status of CF patients treated since 1994 at two certified CF-centres in Münster, Germany, to get insights into the dynamics of S. aureus airway infection and the clinical impact on lung function on a long-term perspective. MATERIALS AND METHODS: We used data from our microbiological database collected between 1994 and 2016 for patients treated at two centres in Münster, Germany, respectively, to determine the infection status for S. aureus. Furthermore, the resistance to selected antibiotics was determined for all patients' isolates and for 15 patients on a longitudinal basis. In addition, the prevalence of adaptive phenotypes such as small colony variants (SCVs) and mucoid S. aureus was assessed. RESULTS: For this study, 2867 patient years with respiratory specimens (mean of 9.3 years for every patient, range 1-22 years) were evaluated for 283 CF patients (median age of 7 years at the beginning of the observation period, range 0-57 years, 51% male). 18% of patients were rarely infected by S. aureus (≤24% of observation years), 20% of patients intermittently (25-49%) and 61% persistently (≥50% of observation period). Susceptibility testing for 12969 S. aureus isolates resulted in resistance to methicillin in 9%, trimethoprim/sulfamethoxazole in 10%, levofloxacin in 14%, gentamicin in 20%, erythromycin and/or clindamycin in 30% and penicillin in 80% of all isolates. S. aureus isolates of 15 patients revealed dynamics of resistance with increase, decrease and loss of resistant isolates to the analysed antibiotics during the study period. SCVs were isolated at least once from 42% (n = 118) of patients and mucoid isolates from 2% (n = 7) of patients. In the last study year, 89 patients were infected by S. aureus only, 44 patients by S. aureus and Pseudomonas aeruginosa and 18 by P. aeruginosa only. Patients infected by S. aureus only were younger and had better lung function compared to the other two groups. CONCLUSIONS: We determined a high percentage of patients with persistent S. aureus infection. During persistence, mostly fluctuation of resistance against various antibiotics was observed in the isolates indicating acquisition and loss of resistance genes by S. aureus. The prevalence of adaptive phenotypes during long-term persistence was high for SCVs (42% of patients), but low for mucoid isolates (2% of patients), which might be underestimated for mucoid phenotypes due to the retrospective study design and the difficulty to detect mucoid isolates in primary cultures. While patients with S. aureus only had better lung function and were younger, no difference was found between the group of P. aeruginosa and S. aureus co-infection and P. aeruginosa only with previous S. aureus infection.

Panton-Valentine Leukocidin associated with S. aureus osteomyelitis activates platelets via neutrophil secretion products.

Globalization and migration promote the spread of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus strains. The toxin PVL is linked to the development of thrombosis in association with osteomyelitis. The mechanisms by which PVL drives thrombosis development are however still unknown. We demonstrate that PVL-damaged neutrophils activate platelets via neutrophil secretion products, such as α-defensins and the myeloperoxidase product HOCl, as well as the formation of HOCl-modified proteins. Neutrophil damage by PVL is blocked by anti-PVL-antibodies, explaining why especially young osteomyelitis patients with a low antibody titre against PVL suffer from thrombotic complications. Platelet activation in the presence of PVL-damaged neutrophils is prevented by α-defensin inhibitors and by glutathione and resveratrol, which are both inhibitors of HOCl-modified protein-induced platelet activation. Remarkably, intravenously infused glutathione also prevents activation of human platelets in an ex vivo assay. We here describe a new mechanism of PVL-neutrophil-platelet interactions, which might be extrapolated to other toxins that act on neutrophils. Our observations may make us think about new approaches to treat and/or prevent thrombotic complications in the course of infections with PVL-producing S. aureus strains.

Staphylococcus aureus triggers a shift from influenza virus-induced apoptosis to necrotic cell death.

Superinfections with Staphylococcus aureus are a major complication of influenza disease, causing excessive inflammation and tissue damage. This enhanced cell-damaging effect is also observed in superinfected tissue cultures, leading to a strong decrease in overall cell viability. In our analysis of the underlying molecular mechanisms, we observed that, despite enhanced cell damage in superinfection, S. aureus did not increase but rather inhibited influenza virus (IV)-induced apoptosis in cells on the level of procaspase-8 activation. This apparent contradiction was solved when we observed that S. aureus mediated a switch from apoptosis to necrotic cell death of IV-infected cells, a mechanism that was dependent on the bacterial accessory gene regulator ( agr) locus that promotes bacterial survival and spread. This so far unknown action may be a bacterial strategy to enhance dissemination of intracellular S. aureus and may thereby contribute to increased tissue damage and severity of disease.-Van Krüchten, A., Wilden, J. J., Niemann, S., Peters, G., Löffler, B., Ludwig, S., Ehrhardt, C. Staphylococcus aureus triggers a shift from influenza virus-induced apoptosis to necrotic cell death.

Adaptation of Staphylococcus aureus to Airway Environments in Patients With Cystic Fibrosis by Upregulation of Superoxide Dismutase M and Iron-Scavenging Proteins.

Adaptation of S. aureus to the hostile environment of CF airways resulted in changed abundance of proteins involved in energy metabolism, cellular processes, transport and binding, but most importantly in an iron-scavenging phenotype and increased activity of superoxide dismutase M.

In Vitro Susceptibility of Clinical Staphylococcus aureus Small-Colony Variants to ß-Lactam and Non-ß-Lactam Antibiotics.

The Staphylococcus aureus small-colony variant (SCV) phenotype has been associated with relapsing and antibiotic-refractory infections. However, little is known about the activities of antibiotics on clinical SCVs. Here, we demonstrated that SCVs without detectable auxotrophies were at least as susceptible to most ß-lactam and non-ß-lactam antibiotics in vitro as their corresponding clonally identical strains with a normal phenotype. After prolonged incubation, a regrowth phenomenon has been observed in gradient diffusion inhibition zones irrespective of the strains' phenotype.

Plasmid-Encoded Transferable mecB-Mediated Methicillin Resistance in Staphylococcus aureus.

During cefoxitin-based nasal screening, phenotypically categorized methicillin-resistant Staphylococcus aureus (MRSA) was isolated and tested negative for the presence of the mecA and mecC genes as well as for the SCCmec-orfX junction region. The isolate was found to carry a mecB gene previously described for Macrococcus caseolyticus but not for staphylococcal species. The gene is flanked by ß-lactam regulatory genes similar to mecR, mecI, and blaZ and is part of an 84.6-kb multidrug-resistance plasmid that harbors genes encoding additional resistances to aminoglycosides (aacA-aphD, aphA, and aadK) as well as macrolides (ermB) and tetracyclines (tetS). This further plasmidborne ß-lactam resistance mechanism harbors the putative risk of acceleration or reacceleration of MRSA spread, resulting in broad ineffectiveness of ß-lactams as a main therapeutic application against staphylococcal infections.