Inactivation of the Response Regulator AgrA Has a Pleiotropic Effect on Biofilm Formation, Pathogenesis and Stress Response in Staphylococcus lugdunensis.

Staphylococcus lugdunensis is a coagulase-negative Staphylococcus that emerges as an important opportunistic pathogen. However, little is known about the regulation underlying the transition from commensal to virulent state. Based on knowledge of S. aureus virulence, we suspected that the agr quorum sensing system may be an important determinant for the pathogenicity of S. lugdunensis. We investigated the functions of the transcriptional regulator AgrA using the agrA deletion mutant. AgrA played a role in cell pigmentation: ΔargA mutant colonies were white while the parental strains were slightly yellow. Compared with the wild-type strain, the ΔargA mutant was affected in its ability to form biofilm and was less able to survive in mice macrophages. Moreover, the growth of ΔagrA was significantly reduced by the addition of 10% NaCl or 0.4 mM H2O2 and its survival after 2 h in the presence of 1 mM H2O2 was more than 10-fold reduced. To explore the mechanisms involved beyond these phenotypes, the ΔagrA proteome and transcriptome were characterized by mass spectrometry and RNA-Seq. We found that AgrA controlled several virulence factors as well as stress-response factors, which are well correlated with the reduced resistance of the ΔagrA mutant to osmotic and oxidative stresses. These results were not the consequence of the deregulation of RNAIII of the agr system, since no phenotype or alteration of the proteomic profile has been observed for the ΔRNAIII mutant. Altogether, our results highlighted that the AgrA regulator of S. lugdunensis played a key role in its ability to become pathogenic. IMPORTANCE Although belonging to the natural human skin flora, Staphylococcus lugdunensis is recognized as a particularly aggressive and destructive pathogen. This study aimed to characterize the role of the response regulator AgrA, which is a component of the quorum-sensing agr system and known to be a major element in the regulation of pathogenicity and biofilm formation in Staphylococcus aureus. In the present study, we showed that, contrary to S. aureus, the agrA deletion mutant produced less biofilm. Inactivation of agrA conferred a white colony phenotype and impacted S. lugdunensis in its ability to survive in mice macrophages and to cope with osmotic and oxidative stresses. By global proteomic and transcriptomic approaches, we identified the AgrA regulon, bringing molecular bases underlying the observed phenotypes. Together, our data showed the importance of AgrA in the opportunistic pathogenic behavior of S. lugdunensis allowing it to be considered as an interesting therapeutic target.

The surreptitious survival of the emerging pathogen Staphylococcus lugdunensis within macrophages as an immune evasion strategy.

Staphylococcus lugdunensis is a commensal bacterium that can cause serious infection suggesting an ability to circumvent aspects of host immunity. We demonstrate here that macrophages fail to kill ingested S. lugdunensis and the bacteria persist for extended periods, without replicating, within mature LAMP-1-positive phagolysosomes. Phagocytosed S. lugdunensis also do not intoxicate host cells in contrast to Staphylococcus aureus. Optimal survival of S. lugdunensis requires O-acetylated peptidoglycan because an oatA mutant, which is more sensitive to killing by lysozyme than wild type, survived to a lesser extent in macrophages. In vitro models of macrophage infection reveal that viable intracellular S. lugdunensis bacteria can be made to grow by pharmacologic perturbation of phagosome function or by phagocyte intoxication by S. aureus toxins. Remarkably, replicating S. lugdunensis is not constrained by LAMP-1 and phosphatidylserine-positive endomembranes, which is distinct from S. aureus that replicates within phagolysosomes. In vivo, S. lugdunensis can also reside in the murine Kupffer cell where the bacteria persist without replicating and require oatA to resist killing in vivo. The intracellular environment of the macrophage represents a niche where S. lugdunensis can exist while protected from extracellular immune factors and may serve as a reservoir from which these bacteria could disseminate.

Clinical and microbiological properties of Staphylococcus lugdunensis skin infections.

Staphylococcus lugdunensis is an emerging pathogen in skin and soft tissue infections that was previously considered a commensal. The aim of this study was to elucidate the characteristics of skin infections by S. lugdunensis and its appropriate management, in a tertiary referral medical center. The clinical files, bacterial cultures and histopathology reports of all S. lugdunensis isolates from skin infections over a period of 8 years (September 2009-September 2017) were reviewed. S. lugdunensis was isolated from 29 patients with skin infections, aged 7-89 years (mean 33.3 years). A state of immune suppression (drug-induced, malignancy or diabetes) was present in five patients (17%). Folliculitis and cutaneous pustulosis were the most common presentations (16 cases, 55%), followed by secondary infection of hidradenitis suppurativa (five cases, 17%). Other sources of isolation were infected molluscum contagiosum (two cases), folliculitis decalvans (one case), dissecting cellulitis (one case), abscess (one case), cyst (one case), impetigo (one case) and granuloma after trauma (one case). The in vitro antibiotic sensitivity tests showed susceptibility to most tested antibiotics, although a few isolates were resistant to gentamycin, penicillin and oxacillin. In 19 of 20 patients for whom follow ups were available, cutaneous manifestations improved or resolved with proper local and/or oral antibiotic therapy. S. lugdunensis may play a role as a primary or secondary pathogen in various skin infections, most commonly folliculitis and pustulosis. Proper antibiotic therapy may lead to improvement or resolution.

Truncated Autoinducing Peptides as Antagonists of Staphylococcus lugdunensis Quorum Sensing.

Competitive quorum sensing (QS) antagonism offers a novel strategy for attenuating current multidrug resistant staphylococcal infections. To this end, a series of 10 truncated analogues based on the parent autoinducing peptides (AIPs) of Staphylococcus lugdunensis (groups I and II) and Staphylococcus epidermidis (groups I-III) were sequentially assessed against a newly developed Staphylococcus lugdunensis group I QS reporter strain. The truncated analogues based upon Staphylococcus lugdunensis AIP-1 (1) and AIP-2 (2) displayed respective IC50 values of 0.2 ± 0.01 µM and 0.3 ± 0.01 µM, while the truncated analogue of the Staphylococcus epidermidis AIP-1 (3) elicited an IC50 value of 2.7 ± 0.1 µM. These findings demonstrate the potential of cognate and "crosstalk" competitive quorum sensing inhibition using truncated AIPs as a means of attenuating staphylococcal infections in species beyond Staphylococcus aureus.

Shear-Resistant Binding to von Willebrand Factor Allows Staphylococcus lugdunensis to Adhere to the Cardiac Valves and Initiate Endocarditis.

BACKGROUND: Staphylococcus lugdunensis is an emerging cause of endocarditis. To cause endovascular infections, S. lugdunensis requires mechanisms to overcome shear stress. We investigated whether platelets and von Willebrand factor (VWF) mediate bacterial adhesion to the vessel wall and the cardiac valves under flow. METHODS: S. lugdunensis binding to VWF, collagen, and endothelial cells was studied in a parallel flow chamber in the absence and presence of platelets. In vivo adhesion of S. lugdunensis was evaluated in a mouse microvasculature perfusion model and a new mouse model of endocarditis. RESULTS: Contrary to other coagulase-negative staphylococci, S. lugdunensis bound to VWF under flow, thus enabling its adhesion to endothelial cells and to the subendothelial matrix. In inflamed vessels of the mesenteric circulation, VWF recruited S. lugdunensis to the vessel wall. In a novel endocarditis mouse model, local inflammation and the resulting release of VWF enabled S. lugdunensis to bind and colonize the heart valves. CONCLUSIONS: S. lugdunensis binds directly to VWF, which proved to be vital for withstanding shear forces and for its adhesion to the vessel wall and cardiac valves. This mechanism explains why S. lugdunensis causes more-aggressive infections, including endocarditis, compared with other coagulase-negative staphylococci.

Orthopedic implant infections: Incompetence of Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis to invade osteoblasts.

Septic failure is still the major complication of prosthetic implants. Entering host cells, bacteria hide from host immune defenses, shelter from extracellular antibiotics, and cause chronic infection. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter bone cells and induce osteoblast apoptosis, osteoclast recruitment, and highly destructive osteomyelitis. Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis are opportunistic pathogens causative of implant-related infections. This study investigated the ability to internalize into osteoblastic MG63 cells of 22 S. epidermidis, 9 S. lugdunensis, and 21 E. faecalis clinical isolates from orthopedic implant infections. Isolates were categorized in clusters by ribotyping. Internalization assay was carried out by means of a microtiter plate-based method. S. epidermidis, S. lugdunensis, and E. faecalis strains turned out incompetent to enter osteoblasts, exhibiting negligible internalization into MG63 cells, nearly three orders of magnitude lower than that of S. aureus. Osteoblast invasion does not appear as a pathogenetic mechanism utilized by S. epidermidis, S. lugdunensis, or E. faecalis for infecting orthopedic implants. Moreover, it can be inferred that intracellularly active antimicrobials should not be necessary against implant infections caused by the three bacterial species. Finally, implications with the uptake of biomaterial microparticles by nonphagocytic cells are enlightened. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 788-801, 2016.

Coagulase negative staphylococci - a fast emerging threat.

OBJECTIVE: To determine the frequency of isolation of coagulase-negative staphylococci and their resistance to methicillin over a period of time. METHODS: The descriptive cross-sectional study was carried out at Army Medical College, Rawalpindi, from June 2009 to May 2012, and comprised clinical samples mostly from patients admitted to the intensive care unit. They were inoculated onto appropriate culture media depending upon the specimen. After 24-hour incubation at 35°C, coagulase-negative staphylococci were identified on the basis of colony morphology, gram staining, a positive catalase and a negative tube coagulase test.Methicillin resistance among the isolated staphylococci was determined using a 30µg Cefoxitin disc as per the Clinical and Laboratory Standards Institute protocol. Number of coagulase-negative staphylococci for each year and their methicillin resistance rates were calculated. A comparison was made with methicillin resistant staphylococcus aureus) isolated during the same period. RESULTS: Of the total 1331 specimens studies over three years, 581(43.65%) were coagulase-negative staphylococci. The rate of coagulase-negative staphylococci and methicillin resistance was higher each year; 110(26.6%) in May 2009-Jun 2010, 134(36.5%) in 2011, and 337(61%) in 2012. Methicillin resistance rates also increased from 25(22.7%) to 46(34.3%) and then to 201(59.6%) in 2012.Maximum isolated specimens came from blood 311(53.5%), followed by pus/swabs 204(35.1%). CONCLUSIONS: The frequency of isolation of coagulase-negative staphylococci and its methicillin resistance among hospitalised patients is on the rise.

The adhesive properties of the Staphylococcus lugdunensis multifunctional autolysin AtlL and its role in biofilm formation and internalization.

Although it belongs to the group of coagulase-negative staphylococci, Staphylococcus lugdunensis has been known to cause aggressive courses of native and prosthetic valve infective endocarditis with high mortality similar to Staphylococcus aureus. In contrast to S. aureus, only little is known about the equipment of S. lugdunensis with virulence factors including adhesins and their role in mediating attachment to extracellular matrix and plasma proteins and host cells. In this study, we show that the multifunctional autolysin/adhesin AtlL of S. lugdunensis binds to the extracellular matrix and plasma proteins fibronectin, fibrinogen, and vitronectin as well as to human EA.hy926 endothelial cells. Furthermore, we demonstrate that AtlL also plays an important role in the internalization of S. lugdunensis by eukaryotic cells: The atlL-deficient mutant Mut17 adheres to and becomes internalized by eukaryotic cells to a lesser extent than the isogenic wild-type strain Sl253 and the complemented mutant Mut17 (pCUatlL) shows an increased internalization level in comparison to Mut17. Thus, surface localized AtlL that exhibits a broad binding spectrum also mediates the internalization of S. lugdunensis by eukaryotic cells. We therefore propose an internalization pathway for S. lugdunensis, in which AtlL plays a major role. Investigating the role of AtlL in biofilm formation of S. lugdunensis, Mut17 shows a significantly reduced ability for biofilm formation, which is restored in the complemented mutant. Thus, our data provide evidence for a significant role for AtlL in adherence and internalization processes as well as in biofilm formation of S. lugdunensis.

Genotypes and phenotypes of Staphylococcus lugdunensis isolates recovered from bacteremia.

BACKGROUND: Staphylococcus lugdunensis is a member of coagulase-negative staphylococci, which has the potential to cause serious infections, such as endocarditis, bone and joint infections, and septicemia. Differences in phenotypic/genotypic characterization may be linked to different diseases. METHODS: Genotypes of 11 S. lugdunensis isolates from bacteremia were determined by pulsed field gel electrophoresis and accessory gene regulator (agr) typing. The SCCmec elements in two oxacillin-resistant isolates were sequenced. Phenotypes were tested by antimicrobial susceptibility testing, biofilm formation assessments, and virulence factor analysis (hemolytic and protease activities). RESULTS: Among the 11 isolates, six pulsotypes were found, and seven isolates belonged to two major pulsotypes. Two agr types (agr-1sl or agr-2sl) were found. The 11 isolates were susceptible to most antimicrobial agents tested. The SCCmec elements in two oxacillin-resistant isolates belonged to the SCCmec type V, but with additional ccrAB2 genes. The agr-2sl isolates (n = 7) displayed higher hemolytic and protease activities than the agr-1sl isolates. All isolates contained the icaA gene but with variable biofilm activities. The results suggest that protein might play an important part in S. lugdunensis biofilms, possibly through an ica-independent pathway. Of the 11 patients with S. lugdunensis bacteremia, one patient had a community-onset infection, and others had a hospital-acquired infection, which were mostly central venous catheter-related infections. CONCLUSION: The 11 S. lugdunensis bacteremia isolates displayed various genotypes and phenotypes. Two oxacillin-resistant isolates contained SCCmec type V and carried additional ccrAB2 genes. Correlation of genotypes and phenotypes with infections needs further studies.

IsdC from Staphylococcus lugdunensis induces biofilm formation under low-iron growth conditions.

Staphylococcus lugdunensis is a coagulase-negative staphylococcus that is a commensal of humans and an opportunistic pathogen. It can cause a spectrum of infections, including those that are associated with the ability to form biofilm, such as occurs with endocarditis or indwelling medical devices. The genome sequences of two strains revealed the presence of orthologues of the ica genes that are responsible for synthesis of poly-N-acetylglucosamine (PNAG) that is commonly associated with biofilm in other staphylococci. However, we discovered that biofilm formed by a panel of S. lugdunensis isolates growing in iron-restricted medium was susceptible to degradation by proteases and not by metaperiodate, suggesting that the biofilm matrix comprised proteins and not PNAG. When the iron concentration was raised to 1 mM biofilm formation by all strains tested was greatly reduced. A mutant of strain N920143 lacking the entire locus that encodes iron-regulated surface determinant (Isd) proteins was defective in biofilm formation under iron-limited conditions. An IsdC-null mutant was defective, whereas IsdK, IsdJ, and IsdB mutants formed biofilm to the same level as the parental strain. Expression of IsdC was required both for the primary attachment to unconditioned polystyrene and for the accumulation phase of biofilm involving cell-cell interactions. Purified recombinant IsdC protein formed dimers in solution and Lactococcus lactis cells expressing only IsdC adhered to immobilized recombinant IsdC but not to IsdJ, IsdK, or IsdB. This is consistent with a specific homophilic interaction between IsdC molecules on neighboring cells contributing to accumulation of S. lugdunensis biofilm in vivo.

Small changes in environmental parameters lead to alterations in antibiotic resistance, cell morphology and membrane fatty acid composition in Staphylococcus lugdunensis.

Staphylococcus lugdunensis has emerged as a major cause of community-acquired and nosocomial infections. This bacterium can rapidly adapt to changing environmental conditions to survive and capitalize on opportunities to colonize and infect through wound surfaces. It was proposed that S. lugdunensis would have underlying alterations in metabolic homeostasis to provide the necessary levels of adaptive protection. The aims of this project were to examine the impacts of subtle variations in environmental conditions on growth characteristics, cell size and membrane fatty acid composition in S. lugdunensis. Liquid broth cultures of S. lugdunensis were grown under varying combinations of pH (6-8), temperature (35-39°C) and osmotic pressure (0-5% sodium chloride w/w) to reflect potential ranges of conditions encountered during transition from skin surfaces to invasion of wound sites. The cells were harvested at the mid-exponential phase of growth and assessed for antibiotic minimal inhibitory concentration (MIC), generation time, formation of small colony variants, cell size (by scanning electron microscopy) and membrane fatty acid composition. Stress regimes with elevated NaCl concentrations resulted in significantly higher antibiotic resistance (MIC) and three of the combinations with 5% NaCl had increased generation times (P<0.05). It was found that all ten experimental growth regimes, including the control and centroid cultures, yielded significantly different profiles of plasma membrane fatty acid composition (P<0.0001). Alterations in cell size (P<0.01) were also observed under the range of conditions with the most substantial reduction occurring when cells were grown at 39°C, pH 8 (514±52 nm, mean ± Standard Deviation) compared with cells grown under control conditions at 37°C with pH 7 (702±76 nm, P<0.01). It was concluded that S. lugdunensis responded to slight changes in environmental conditions by altering plasma membrane fatty acid composition, growth rates and morphology to achieve optimal adaptations for survival in changing environments.

The major autolysin of Staphylococcus lugdunensis, AtlL, is involved in cell separation, stress-induced autolysis and contributes to bacterial pathogenesis.

Staphylococcus lugdunensis is a human skin commensal organism, but it is considered as a virulent Staphylococcus species. In a previous study, we described the first S. lugdunensis autolysin, AtlL. This enzyme displays two enzymatic domains and generates two peptidoglycan hydrolases, an N-acetylmuramoyl-l-alanine amidase and an N-acetylglucosaminidase. In this study, to further investigate the functions of this autolysin, a ΔatlL mutant was constructed. The microscopic examination of the mutant showed cell aggregates and revealed a rough outer cell surface demonstrating, respectively, the roles of AtlL in cell separation and peptidoglycan turnover. This ΔatlL mutant exhibited a lower susceptibility to Triton X-100-induced autolysis assays and appears to be more resistant to cell wall antibiotic-induced lysis and death compared with its parental strain. The atlL mutation affected the biofilm formation capacity of S. lugdunensis. Furthermore, the ΔatlL mutant showed trends toward reduced virulence using the Caenorhabditis elegans model. Overall, AtlL appears as a major cell wall autolysin of S. lugdunensis implicated in cell separation, in stress-induced autolysis and in bacterial pathogenesis.

A Brazilian lineage of Staphylococcus lugdunensis presenting rough colony morphology may adhere to and invade lung epithelial cells.

Staphylococcus lugdunensis is an unusually virulent coagulase-negative species, which causes serious infection similar to S. aureus. We evaluated the expression of virulence factors such as S. lugdunensis synergistic haemolysin (SLUSH), fibrinogen-binding protein (Fbl), biofilm production and biofilm-production-related genes in 23 S. lugdunensis clinical isolates and one type strain that had been previously characterized for their genotypes. In addition, the biofilm composition and the ability of isolates to adhere to and invade human epithelial lung cells were also investigated. The PCR method used detected the presence of slush and intercellular adhesin (ica) virulence genes in all isolates. All isolates produced the Fbl protein and, with the exception of the type strain, all isolates produced the SLUSH haemolysin. Fourteen (60.9 %) isolates produced biofilms. The detachment assay, using sodium metaperiodate or proteolytic enzymes to analyse the biofilm composition, showed protein-mediated biofilms in two representative isolates, one for each colony type (rough and smooth). All strongly biofilm-producing isolates, including three with rough colony morphology, had the same prevalent PFGE pattern. However, among the representative strains tested, only the S. lugdunensis isolate that formed rough colonies was able to adhere to and invade A549 cell monolayers in the same quantities as those observed with S. aureus isolates (P = 1.000). No significant adhesion or invasion was observed for the other isolates in comparison with the S. aureus isolate, independent of biofilm production or clonality. Our results could explain the incredible ability of this pathogen to cause infections that are as aggressive as S. aureus. In addition, the ability of S. lugdunensis to adhere to and invade eukaryotic cells was also noticed for isolates with rough colony morphology, reinforcing the increased virulence in this species.