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1.  Genome Sequences of Sequence Type 45 (ST45) Persistent Methicillin-Resistant Staphylococcus aureus (MRSA) Bacteremia Strain 300-169 and ST45 Resolving MRSA Bacteremia Strain 301-188 
Genome Announcements  2014;2(2):e00174-14.
Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (positive blood cultures after ≥7 days) represents a challenging subset of invasive MRSA infections. The comparison of genome sequences of persistent (300-169) and resolving (301-188) MRSA bacteremia isolates with similar genetic background (sequence type 45 [ST45]) will help us to better understand underlying mechanisms of persistent MRSA bacteremia.
doi:10.1128/genomeA.00174-14
PMCID: PMC3953194  PMID: 24625873
2.  Reduced Vancomycin Susceptibility in an In Vitro Catheter-Related Biofilm Model Correlates with Poor Therapeutic Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus 
Staphylococcus aureus is the most common cause of endovascular infections, including catheter sepsis and infective endocarditis (IE). Vancomycin (VAN) is the primary choice for treatment of methicillin-resistant S. aureus (MRSA) infections. However, high rates of VAN treatment failure in MRSA infections caused by VAN-susceptible strains have been increasingly reported. Biofilm-associated MRSA infections are especially prone to clinical antibiotic failure. The present studies examined potential relationships between MRSA susceptibility to VAN in biofilms in vitro and nonsusceptibility to VAN in endovascular infection in vivo. Using 10 “VAN-susceptible” MRSA bloodstream isolates previously investigated for VAN responsiveness in experimental IE, we studied the mechanism(s) of such in vivo VAN resistance, including: (i) VAN binding to MRSA organisms; (ii) the impact of VAN on biofilm formation and biofilm composition; (iii) VAN efficacy in an in vitro catheter-related biofilm model; (iv) effects on cell wall thickness. As a group, the five strains previously categorized as VAN nonresponders (non-Rsp) in the experimental IE model differed from the five responders (Rsp) in terms of lower VAN binding, increased biofilm formation, higher survival in the presence of VAN within biofilms in the presence or absence of catheters, and greater biofilm reduction upon proteinase K treatment. Interestingly, sub-MICs of VAN significantly promoted biofilm formation only in the non-Rsp isolates. Cell wall thickness was similar among all MRSA strains. These results suggest that sublethal VAN levels that induce biofilm formation and reduce efficacy of VAN in the in vitro catheter-associated biofilms may contribute to suboptimal treatment outcomes for endovascular infections caused by “VAN-susceptible” MRSA strains.
doi:10.1128/AAC.02073-12
PMCID: PMC3591927  PMID: 23295925
3.  In vitro Endothelial Cell Damage is Positively Correlated with Enhanced Virulence and Poor Vancomycin Responsiveness in Experimental Endocarditis due to Methicillin Resistant Staphylococcus aureus 
Cellular microbiology  2011;13(10):1530-1541.
Summary
The pathogenesis of Staphylococcus aureus infective endocarditis (IE) is postulated to involve invasion and damage of endothelial cells (ECs). However, the precise relationships between S. aureus – EC interactions in vitro and IE virulence and treatment outcomes in vivo are poorly defined. Ten methicillin-resistant S. aureus (MRSA) clinical isolates previously tested for their virulence and vancomycin responsiveness in an experimental IE model were assessed in vitro for their hemolytic activity, protease production, and capacity to invade and damage ECs. There was a significant positive correlation between the in vitro EC damage caused by these MRSA strains and their virulence during experimental IE (in terms of bacterial densities in target tissues; P < 0.02). Importantly, higher EC damage was also significantly correlated with poor microbiologic response to vancomycin in the IE model (P < 0.001). Interestingly, the extent of EC damage was unrelated to a strain's ability to invade ECs, hemolytic activity and protease production, or β-toxin gene transcription. Inactivation of the agr locus in two MRSA strains caused ∼20% less damage as compared to the corresponding parental strains, indicating that a functional agr is required for maximal EC damage induction. Thus, MRSA-induced EC damage in vitro is a unique virulence phenotype that is independent of many other prototypical MRSA virulence factors, and may be a key biomarker for predicting MRSA virulence potential and antibiotic outcomes during endovascular infections.
doi:10.1111/j.1462-5822.2011.01639.x
PMCID: PMC3173605  PMID: 21777408
4.  Divergent Responses of Different Endothelial Cell Types to Infection with Candida albicans and Staphylococcus aureus 
PLoS ONE  2012;7(6):e39633.
Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs) to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.
doi:10.1371/journal.pone.0039633
PMCID: PMC3382135  PMID: 22745797
5.  Relationship of agr Expression and Function with Virulence and Vancomycin Treatment Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus ▿ 
Antimicrobial Agents and Chemotherapy  2011;55(12):5631-5639.
The accessory gene regulator (agr) locus has been shown to be important for virulence in several animal models of Staphylococcus aureus infection. However, the role of agr in human infections, and specifically in antibiotic treatment, is controversial. Interestingly, agr dysfunction has been associated with reduced vancomycin responses. To systematically investigate the role of agr in virulence and treatment outcome in the context of endovascular infection, 10 well-characterized vancomycin-susceptible methicillin-resistant S. aureus (MRSA) bloodstream isolates (5 agr-I [clonal complex 45, or CC45] and 5 agr-II [CC5]) were studied for (i) agr function, (ii) RNAIII transcriptional profiles, (iii) agr locus sequences, (iv) intrinsic virulence and responses to vancomycin therapy in an experimental infective endocarditis (IE) model, and (v) in vivo RNAIII expression. Significant differences in agr function (determined by delta-hemolysin activity) correlated with the time point of RNAIII transcription (earlier RNAIII onset equals increased agr function). Unexpectedly, four MRSA strains with strong delta-hemolysin activities exhibited significant resistance to vancomycin treatment in experimental IE. In contrast, five of six MRSA strains with weak or no delta-hemolysin activity were highly susceptible to vancomycin therapy in the IE model. agr sequence analyses showed no common single-nucleotide polymorphism predictive of agr functionality. In vivo RNAIII expression in cardiac vegetations did not correlate with virulence or vancomycin treatment outcomes in the IE model. Inactivation of agr in two strains with strong delta-hemolysin activity did not affect virulence or the in vivo efficacy of vancomycin. Our findings suggest that agr dysfunction does not correlate with vancomycin treatment failures in this experimental IE model in two distinct MRSA genetic backgrounds.
doi:10.1128/AAC.05251-11
PMCID: PMC3232782  PMID: 21968365
6.  Combinatorial Phenotypic Signatures Distinguish Persistent from Resolving Methicillin-Resistant Staphylococcus aureus Bacteremia Isolates ▿  
Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) (positive blood cultures after ≥7 days of therapy) represents a clinically challenging subset of invasive MRSA infections. In this investigation, we examined the potential correlation of specific virulence signatures with PB versus resolving MRSA bacteremia (RB) (negative blood cultures within 2 to 4 days of therapy) strains. Thirty-six MRSA isolates from patients enrolled in a recent multinational clinical trial were studied for (i) susceptibility to host defense cationic peptides (HDPs) (i.e., thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide 1 [hNP-1]); (ii) adherence to host endovascular ligands (fibronectin) and cells (endothelial cells); and (iii) biofilm formation. We found that PB isolates exhibited significantly reduced susceptibilities to tPMPs and hNP-1 (P < 0.001 and P = 0.023, respectively). There was no significant association between the PB outcome and fibronectin binding, endothelial cell binding, or biofilm formation (P = 0.25, 0.97, and 0.064 versus RB strains, respectively). However, multiple logistic regression analysis revealed that the PB outcome was significantly associated with the combination of reduced susceptibilities to HDPs and extent of biofilm formation (P < 0.0001). Similar results were obtained in a second analysis using days of bacteremia as a continuous outcome, showing that reduced HDP susceptibilities and increased biofilm formation cocontributed to predict the duration of bacteremia. Our data indicate that PB isolates have specific pathogenic signatures independent of conventional antimicrobial susceptibility. These combinatorial mosaics can be defined and used to prospectively distinguish PB from RB strains in advance and potentially to predict ultimate clinical outcomes.
doi:10.1128/AAC.01028-10
PMCID: PMC3028773  PMID: 21098242
7.  Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus 
BMC Microbiology  2009;9:95.
Background
The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions.
Results
The addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA) cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.
Conclusion
This study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.
doi:10.1186/1471-2180-9-95
PMCID: PMC2697999  PMID: 19450265
8.  CcpA Mediates the Catabolite Repression of tst in Staphylococcus aureus▿  
Infection and Immunity  2008;76(11):5093-5099.
Some clinical isolates of Staphylococcus aureus produce the superantigenic toxic shock syndrome toxin 1 (TSST-1), encoded by tst, located on pathogenicity islands. The expression of tst is complex and is influenced by environmental conditions such as pH, CO2, and glucose. We identified a putative catabolite-responsive element (cre) in the promoter regions of all known tst genes, indicating that tst transcription may be regulated by the catabolite control protein CcpA. By introducing tst genes under the control of their native promoters or tst promoter-reporter gene fusions in wild-type strain Newman, we showed that glucose was able to repress tst transcription and TSST-1 production, whereas glucose repression was abolished in the corresponding ΔccpA mutant. Stabilizing the pH ruled out a pH effect due to acid production during glucose catabolism. CcpA thus directly regulates tst transcription, linking carbohydrate utilization to virulence gene expression in S. aureus.
doi:10.1128/IAI.00724-08
PMCID: PMC2573359  PMID: 18710862
9.  Staphylococcus aureus CcpA Affects Biofilm Formation▿  
Infection and Immunity  2008;76(5):2044-2050.
Biofilm formation in Staphylococcus aureus under in vitro growth conditions is generally promoted by high concentrations of sugar and/or salts. The addition of glucose to routinely used complex growth media triggered biofilm formation in S. aureus strain SA113. Deletion of ccpA, coding for the catabolite control protein A (CcpA), which regulates gene expression in response to the carbon source, abolished the capacity of SA113 to form a biofilm under static and flow conditions, while still allowing primary attachment to polystyrene surfaces. This suggested that CcpA mainly affects biofilm accumulation and intercellular aggregation. trans-Complementation of the mutant with the wild-type ccpA allele fully restored the biofilm formation. The biofilm produced by SA113 was susceptible to sodium metaperiodate, DNase I, and proteinase K treatment, indicating the presence of polysaccharide intercellular adhesin (PIA), protein factors, and extracellular DNA (eDNA). The investigation of several factors which were reported to influence biofilm formation in S. aureus (arlRS, mgrA, rbf, sarA, atl, ica, citZ, citB, and cidABC) showed that CcpA up-regulated the transcription of cidA, which was recently shown to contribute to eDNA production. Moreover, we showed that CcpA increased icaA expression and PIA production, presumably over the down-regulation of the tricarboxylic acid cycle genes citB and citZ.
doi:10.1128/IAI.00035-08
PMCID: PMC2346702  PMID: 18347047
10.  σB and the σB-Dependent arlRS and yabJ-spoVG Loci Affect Capsule Formation in Staphylococcus aureus▿  
Infection and Immunity  2007;75(9):4562-4571.
The alternative transcription factor σB of Staphylococcus aureus affects the transcription of the cap gene cluster, required for the synthesis of capsular polysaccharide (CP), although this operon is lacking an apparent σB-dependent promoter. Regulation of cap expression and CP production in S. aureus strain Newman was shown here to be influenced by σB, the two-component signal transduction regulatory system ArlRS, and the yabJ-spoVG locus to different extents. Inactivation of arlR or deletion of the sigB operon strongly suppressed capA (CP synthesis enzyme A) transcription. Deletion of spoVG had a polar effect on yabJ-spoVG transcription and resulted in a two- to threefold decrease in capA transcription. Interestingly, immunofluorescence showed that CP production was strongly impaired in all three mutants, signaling that the yabJ-spoVG inactivation, despite its only partial effect on capA transcription, abolished capsule formation. trans-Complementation of the ΔspoVG mutant with yabJ-spoVG under the control of its native promoter restored CP-5 production and capA expression to levels seen in the wild type. Northern analyses revealed a strong impact of σB on arlRS and yabJ-spoVG transcription. We hypothesize that ArlR and products of the yabJ-spoVG locus may serve as effectors that modulate σB control over σB-dependent genes lacking an apparent σB promoter.
doi:10.1128/IAI.00392-07
PMCID: PMC1951174  PMID: 17635871
11.  Staphylococcus aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance 
Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the ΔccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding α-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.
doi:10.1128/AAC.50.4.1183-1194.2006
PMCID: PMC1426959  PMID: 16569828

Results 1-11 (11)