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Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates ( n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains ( n = 5) versus CC45 strains ( n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin ( P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA , which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB , particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections.

Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates ( P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.

Staphylococcus aureus clonal complex 75 (herein referred to as S. argenteus) lacks the carotenoid pigment operon, crtOPQMN, responsible for production of the putative virulence factor, staphyloxanthin. Although a common cause of community-onset skin infections among Indigenous populations in northern Australia, this clone is infrequently isolated from hospital-based patients with either bacteremic or nonbacteremic infections. We hypothesized that S. argenteus would have attenuated virulence compared to other S. aureus strains due to its staphyloxanthin “deficiency.” Compared to prototypical S. aureus strains, S. argenteus was more susceptible to oxidative stress and neutrophil killing in vitro and had reduced virulence in murine sepsis and skin infection models. Transformation with pTX-crtOPQMN resulted in staphyloxanthin expression and increased resistance to oxidative stress in vitro. However, neither resistance to neutrophil killing nor in vivo virulence was increased. Thus, reduced virulence of S. argenteus in these models is due to mechanisms unrelated to lack of staphyloxanthin production.

The impact of Panton-Valentine leukocidin (PVL) on the outcome in Staphylococcus aureus pneumonia is controversial. We genotyped S. aureus isolates from patients with hospital-acquired pneumonia (HAP) enrolled in two registrational multinational clinical trials for the genetic elements carrying pvl and 30 other virulence genes. A total of 287 isolates (173 methicillin-resistant S. aureus [MRSA] and 114 methicillin-susceptible S. aureus [MSSA] isolates) from patients from 127 centers in 34 countries for whom clinical outcomes of cure or failure were available underwent genotyping. Of these, pvl was detected by PCR and its product confirmed in 23 isolates (8.0%) (MRSA, 18/173 isolates [10.4%]; MSSA, 5/114 isolates [4.4%]). The presence of pvl was not associated with a higher risk for clinical failure (4/23 [17.4%] versus 48/264 [18.2%]; P = 1.00) or mortality. These findings persisted after adjustment for multiple potential confounding variables. No significant associations between clinical outcome and (i) presence of any of the 30 other virulence genes tested, (ii) presence of specific bacterial clone, (iii) levels of alpha-hemolysin, or (iv) delta-hemolysin production were identified. This study suggests that neither pvl presence nor in vitro level of alpha-hemolysin production is the primary determinant of outcome among patients with HAP caused by S. aureus .

Staphylococcus aureus is the most common cause of both infective endocarditis (IE) [1] and soft tissue infection (STI) [2] in the industrialized world. The frequency of S. aureus as a human pathogen is thought to be due in part to its diverse armamentarium of virulence-associated genes. Although substantial evidence suggests that clinical manifestations of S. aureus are influenced by the genetic characteristics of the infecting strain [3–7], the association between S. aureus genes and severity of illness is incompletely understood. Previously, we demonstrated a significant association between specific S. aureus isolates genotypes and infection severity [4]. We used multilocus sequence typing (MLST) to show that clonal complex (CC) 5 and CC30 were significantly associated with the presence of IE and bone and joint infection among 371 clinically well-characterized S. aureus isolates from a single geographical region. However, these findings must be confirmed prior to being considered broadly generalizable. The current investigation seeks to externally validate these previously observed associations between bacterial genotype and infection severity in S. aureus. To do this, we used bacterial isolates from 2 large multinational cohorts of patients with distinct forms of staphylococcal disease: IE and STI.

The role of Panton-Valentine leukocidin (PVL) in determining the severity and outcome of complicated skin and skin structure infections (cSSSI) caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is controversial. We evaluated potential associations between clinical outcome and PVL status by using MRSA isolates from patients enrolled in two large, multinational phase three clinical trials assessing telavancin for the treatment of cSSSI (the ATLAS program). MRSA isolates from microbiologically evaluable patients were genotyped by pulsed-field gel electrophoresis (PFGE) and PCR for pvl and 31 other putative virulence determinants. A single baseline pathogen of MRSA was isolated from 522 microbiologically evaluable patients (25.1%) among 2,079 randomized patients. Of these MRSA isolates, 83.2% (432/519) exhibited the USA300 PFGE genotype and 89.1% (465/522) were pvl positive. Patients with pvl -positive MRSA were more likely than those with pvl -negative MRSA to be young, to be North American, and to present with major abscesses ( P < 0.001 for each). Patients were significantly more likely to be cured if they were infected with pvl -positive MRSA than if they were infected with pvl -negative MRSA (91.6% versus 80.7%; P = 0.015). This observation remained statistically significant after adjustment for presence of abscess, fever, or leukocytosis; infection size; diabetes; patient age; and study medication received. The fnbA , cna , sdrC , map-eap , sed , seg , sei , sej , SCC mec type IV, and agr group II genes were also associated with clinical response ( P < 0.05). This contemporary, international study demonstrates that pvl was not the primary determinant of outcome in patients with MRSA cSSSI.

In the study of differential gene expression of Cryptococcus neoformans, a transcript of COX1 (cytochrome oxidase c subunit 1) was identified in a serotype A strain. The transcript was upregulated at 37 degrees C compared to 30 degrees C and expressed by yeasts infecting the central nervous system. Northern analysis of COX1 from the serotype A strain revealed two polycistronic transcripts, a temperature-upregulated 2.3 kb transcript and a 1.9 kb transcript that was not affected by temperature. In contrast, COX1 in a serotype D strain showed only a 1.9 kb polycistronic transcript plus a 1.6 kb monocistronic message, and temperature had no effect on the transcripts. The sequence of COX1 revealed similar coding regions between the two strains, but the serotype D strain had five introns whereas no introns were found in the serotype A strain. The serotype D strain had reduced growth rates compared to the serotype A strain at 37 degrees C, but in an AD hybrid strain the serotype D COX1 gene could support efficient high temperature growth. These studies have revealed mitochondrial molecular differences between serotype A and D strains which show evolutionary divergence. It will be important to determine whether differences in mitochondrial structure and function can influence cryptococcosis.

Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments.

Staphylococcus aureus CC30 is distinguished by its ability initiate complicated infections. This ability is due to acquisition of unique genes, SNPs and metabolic changes that attenuate virulence until the conditions become favorable for bacteremia and subsequent hematogenous seeding., Background. The contemporary Staphylococcus aureus clonal complex (CC) 30 lineage is associated with complicated infections, including endocarditis and osteomyelitis. This lineage diverged from the phage-type 80/81 S aureus clone responsible for a major bacterial epidemic of the 20th century. The genome and transcriptome features that contribute to complicated infections of the CC30 lineage are unknown. Methods. Twenty-nine clinical methicillin-resistant S aureus (MRSA) strains (8 from CC30 and 21 from other major CCs were evaluated for virulence using murine and Galleria mellonella sepsis models. Genomic features of CC30 were identified by comparative genome sequencing and RNA-Seq transcriptome analysis of the 29 strains and 31 previously sequenced S aureus genomes. Results. The CC30 isolates displayed lower virulence in the sepsis models compared with other CCs [P < .0001]. Comparisons of orthologous proteins and transcriptome analysis identified genes (eg, nitric oxide reductase) and changes in metabolic pathways (eg, pyrimidine metabolism) that contribute to the distinct CC30 phenotype. Previously reported nonsynonymous single-nucleotide polymorphisms (SNPs) were found in accessory gene regulator C (agrC) and α-hemolysin (hla), molecules important for virulence. Additional nonsynonymous SNPs conserved across clinical CC30 isolates when compared with the first sequenced contemporary CC30 clone, MRSA-16, were identified in multiple genes, suggesting continuing evolutionary divergence in this lineage. Conclusions. Genomic and transcriptional analyses suggest that the CC30 lineage has acquired metabolic features that contribute to persistent and complicated infections. Absence of sepsis-induced mortality in animal models may be due in part to its unique genomic profile and suggests that specific genotypes of S aureus elicit distinct types of infection types.

Functional genomics has become a major focus in the study of microbial pathogenesis. This study used a functional genomic tool, differential display reverse transcription-PCR, to identify a transcriptional profile of Cryptococcus neoformans cells as they produced meningitis in an immunosuppressed host. This serial global gene expression during infection allowed for the identification of up- and down-regulated genes during infection. During this profiling, a single gene for the enzyme isocitrate lyase (ICL1) was found to be up regulated at 1 week of infection in a rabbit meningitis model and during a time of maximum host cellular response. The finding suggested that this enzyme and the glyoxylate shunt pathway are important to this yeast's energy production during infection. However, site-directed icl1 mutants had no apparent virulence defect in two animal models and no growth defect within macrophages. These observations suggest that although the yeast responded to a certain environmental cue(s) by an increase in ICL1 expression during infection, this gene was not necessary for progression of a C. neoformans infection. Compounds that specifically target only ICL1 are unlikely to cripple C. neoformans growth in vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) first appeared clinically in the early 1960s [1]. MRSA isolates became endemic in most US hospitals by the late 1980s [2–4]. In the 1990s, a new wave of MRSA infections occurred in community settings [3–8]. In many centers, MRSA isolates predominate as a cause of S. aureus community-onset (CO) infections, including bacteremia [3–5, 9]. Rather than being “escaped” hospital-based MRSA clones, the community MRSA strains were derived from methicillin-susceptible S. aureus (MSSA) strains that acquired a novel resistance element, SCCmec type IV [2, 4, 10–12]. These strain types, especially the USA300 genetic background, were highly virulent, often susceptible to multiple non–β-lactam antibiotics, and carried signature toxin genes (most commonly Panton-Valentine leukocidin [PVL]) rarely found in the older, hospital-acquired strain types [13]. USA300 also had a constitutive upregulation of several key virulence genes [14]. Driven by emergence of USA300, the incidence of MRSA infections rose dramatically in the early 2000s. In 2000, the Centers for Disease Control and Prevention (CDC) estimated that there were >30 000 hospitalizations for MRSA bacteremia [15]; however, more recent published reports have described declines in invasive MRSA infection rates, particularly in healthcare settings [16–20]. In the United States and the United Kingdom, the decline in MRSA bacteremia rates appears to have preceded enhanced infection prevention efforts in hospitals [21]. In a study of US military personnel and their dependents, rates of MRSA bacteremia declined between 2008 and 2011 [22]. However, to date, few reports have documented the incidence of MSSA bacteremia in this time period, or assessed potential geographic variations in CO infection rates, as opposed to hospital-onset (HO) rates. The current study was conducted to define trends in the annual incidence of MRSA and MSSA bacteremia at 5 large, geographically dispersed US academic medical centers to assess the incidence of HO and CO MSSA and MRSA bacteremia during a 4-year period.

Two American football players on the same team were diagnosed with methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections on the same day. Our investigation, including whole genome sequencing, confirmed that players did not transmit MRSA to one another nor did they acquire the MRSA from a single source within the training facility.

Using A/J mice, which are susceptible to Staphylococcus aureus, we sought to identify genetic determinants of susceptibility to S. aureus, and evaluate their function with regard to S. aureus infection. One QTL region on chromosome 11 containing 422 genes was found to be significantly associated with susceptibility to S. aureus infection. Of these 422 genes, whole genome transcription profiling identified five genes (Dcaf7, Dusp3, Fam134c, Psme3, and Slc4a1) that were significantly differentially expressed in a) S. aureus –infected susceptible (A/J) vs. resistant (C57BL/6J) mice and b) humans with S. aureus blood stream infection vs. healthy subjects. Three of these genes (Dcaf7, Dusp3, and Psme3) were down-regulated in susceptible vs. resistant mice at both pre- and post-infection time points by qPCR. siRNA-mediated knockdown of Dusp3 and Psme3 induced significant increases of cytokine production in S. aureus-challenged RAW264.7 macrophages and bone marrow derived macrophages (BMDMs) through enhancing NF-κB signaling activity. Similar increases in cytokine production and NF-κB activity were also seen in BMDMs from CSS11 (C57BL/6J background with chromosome 11 from A/J), but not C57BL/6J. These findings suggest that Dusp3 and Psme3 contribute to S. aureus infection susceptibility in A/J mice and play a role in human S. aureus infection., Author Summary Staphylococcus aureus causes life-threatening infections in humans. Host genetic determinants influence the outcome of S. aureus infection, yet are poorly understood. Susceptible A/J and resistant C57BL/6J mice provide a unique platform to study the genetic difference responsible for variable host response to S. aureus infection. We showed that chromosome 11 in A/J was responsible for susceptibility to S. aureus. We further identified a QTL locus on Chromosome 11 significantly associated with S. aureus susceptibility. Five genes in the QTL (Dcaf7, Dusp3, Fam134c, Psme3, and Slc4a1) were significantly differently expressed in a) susceptible vs. resistant mice, and b) humans with S. aureus blood stream infection vs. healthy human subjects. Three genes (Dusp3, Psme3, and Dcaf7) were down-regulated in susceptible A/J mice. siRNA-mediated knockdown of Dusp3 and Psme3 in bone marrow derived macrophage (BMDMs) significantly enhanced cytokine responses through NF-κB activity upon S. aureus challenge in a pattern that was also present in S. aureus-challenged BMDMs from susceptible CSS11 (chr. 11 from A/J but otherwise C57BL/6J) mice, but not resistant C57BL/6J mice. These findings suggest that Dusp3 and Psme3 contribute to S. aureus infection susceptibility in A/J mice and play a role in human S. aureus infection.

Using heterologous probing of a genomic library, we have cloned and sequenced the actin gene from the pathogenic yeast Cryptococcus neoformans. The actin gene is 1371 bp in length, and exists as a single copy, as is the case for all fungi studied to date. The locations of the introns in the C. neoformans actin gene are unique among all other known actin genes, and the deduced coding sequence results in a 375 amino acid chain with very high homology to other actins. A phylogenetic tree comprising 31 actin-coding sequences from a wide variety of organisms shows that the C. neoformans actin gene is grouped on a distinct branch together with all other known fungal actin sequences. The availability of the C. neoformans actin gene will aid future phylogenetic and molecular studies of this important human pathogen.

A cryptococcal meningitis model in corticosteroid-treated rabbits was used to assess the requirement for the phosphoribosylaminoimidazole gene (ADE2) for virulence of Cryptococcus neoformans. A wild-type strain (H99), an ade2 auxotroph of H99 (M001), and a randomly selected prototrophic transformant of M001 (M001.1c) which had received the cloned ADE2 cDNA copy were inoculated intrathecally into immunosuppressed rabbits. While M001 was avirulent in the central nervous system model, virulence was completely restored to wild-type pathogenicity in the prototrophic transformant. This study identifies the pathogenic importance of an endogenous adenine pathway in this yeast and confirms that purine biosynthesis is a potential target for antifungal therapy. It also demonstrates that the virulence of C. neoformans can be molecularly changed and detected within a clinically relevant animal model.

International audience; BACKGROUND: The significance of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is unknown. Using a multinational collection of isolates from methicillin-resistant S. aureus (MRSA) infective endocarditis (IE), we characterized patients with IE with and without hVISA, and we genotyped the infecting strains. METHODS: MRSA bloodstream isolates from 65 patients with definite IE from 8 countries underwent polymerase chain reaction (PCR) for 31 virulence genes, pulsed-field gel electrophoresis, and multilocus sequence typing. hVISA was defined using population analysis profiling. RESULTS: Nineteen (29.2%) of 65 MRSA IE isolates exhibited the hVISA phenotype by population analysis profiling. Isolates from Oceania and Europe were more likely to exhibit the hVISA phenotype than isolates from the United States (77.8% and 35.0% vs 13.9%; P < .001). The prevalence of hVISA was higher among isolates with a vancomycin minimum inhibitory concentration of 2 mg/L (P = .026). hVISA-infected patients were more likely to have persistent bacteremia (68.4% vs 37.0%; P = .029) and heart failure (47.4% vs 19.6%; P = .033). Mortality did not differ between hVISA- and non-hVISA-infected patients (42.1% vs 34.8%, P = .586). hVISA and non-hVISA isolates were genotypically similar. CONCLUSIONS: In these analyses, the hVISA phenotype occurred in more than one-quarter of MRSA IE isolates, was associated with certain IE complications, and varied in frequency by geographic region.

The role of Panton-Valentine leukocidin (PVL) in determining the severity and outcome of complicated skin and skin structure infections (cSSSI) caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is controversial. We evaluated potential associations between clinical outcome and PVL status by using MRSA isolates from patients enrolled in two large, multinational phase three clinical trials assessing telavancin for the treatment of cSSSI (the ATLAS program). MRSA isolates from microbiologically evaluable patients were genotyped by pulsed-field gel electrophoresis (PFGE) and PCR for pvl and 31 other putative virulence determinants. A single baseline pathogen of MRSA was isolated from 522 microbiologically evaluable patients (25.1%) among 2,079 randomized patients. Of these MRSA isolates, 83.2% (432/519) exhibited the USA300 PFGE genotype and 89.1% (465/522) were pvl positive. Patients with pvl-positive MRSA were more likely than those with pvl-negative MRSA to be young, to be North American, and to present with major abscesses (P < 0.001 for each). Patients were significantly more likely to be cured if they were infected with pvl-positive MRSA than if they were infected with pvl-negative MRSA (91.6% versus 80.7%; P = 0.015). This observation remained statistically significant after adjustment for presence of abscess, fever, or leukocytosis; infection size; diabetes; patient age; and study medication received. The fnbA, cna, sdrC, map-eap, sed, seg, sei, sej, SCCmec type IV, and agr group II genes were also associated with clinical response (P < 0.05). This contemporary, international study demonstrates that pvl was not the primary determinant of outcome in patients with MRSA cSSSI.

We investigated associations between the genotypic and phenotypic features of Staphylococcus aureus bloodstream isolates and the clinical characteristics of bacteremic patients enrolled in a phase III trial of S. aureus bacteremia and endocarditis. Isolates underwent pulsed-field gel electrophoresis, PCR for 33 putative virulence genes, and screening for heteroresistant glycopeptide intermediate S. aureus (hGISA). A total of 230 isolates (141 methicillin-susceptible S. aureus and 89 methicillin-resistant S. aureus [MRSA]) were analyzed. North American and European S. aureus isolates differed in their genotypic characteristics. Overall, 26% of the MRSA bloodstream isolates were USA 300 strains. Patients with USA 300 MRSA bacteremia were more likely to be injection drug users (61% versus 15%; P < 0.001), to have right-sided endocarditis (39% versus 9%; P = 0.002), and to be cured of right-sided endocarditis (100% versus 33%; P = 0.01) than patients with non-USA 300 MRSA bacteremia. Patients with persistent bacteremia were less likely to be infected with Panton-Valentine leukocidin gene ( pvl )-constitutive MRSA (19% versus 56%; P = 0.005). Although 7 of 89 MRSA isolates (8%) exhibited the hGISA phenotype, no association with persistent bacteremia, daptomycin resistance, or bacterial genotype was observed. This study suggests that the virulence gene profiles of S. aureus bloodstream isolates from North America and Europe differ significantly. In this study of bloodstream isolates collected as part of a multinational randomized clinical trial, USA 300 and pvl -constitutive MRSA strains were associated with better clinical outcomes.

The impact of bacterial genetic characteristics on the outcome of patients with Staphylococcus aureus infections is uncertain. This investigation evaluated potential associations between bacterial genotype and clinical outcome using isolates collected as part of an international phase 2 clinical trial (FAST II) evaluating telavancin for the treatment of complicated skin and skin structure infections (cSSSI). Ninety S. aureus isolates from microbiologically evaluable patients with cSSSI enrolled in the FAST II trial from 11 sites in the United States (56 isolates, or 62%) and 7 sites in South Africa (34 isolates, or 38%) were examined for staphylococcal cassette chromosome mec , agr , and the presence of 31 virulence genes and subjected to pulsed-field gel electrophoresis (PFGE). South African methicillin-susceptible S. aureus (MSSA) isolates were more likely to carry certain virulence genes, including sdrD ( P = 0.01), sea ( P < 0.01), and pvl ( P = 0.01). All 44 (49%) methicillin-resistant S. aureus (MRSA) isolates were from the United States; 37 (84%) were strain USA 300 by PFGE. In the United States, MRSA isolates were more likely than MSSA isolates to carry genes for sdrC ( P = 0.03), map/eap ( P = 0.05), fnbB ( P = 0.11), tst ( P = 0.02), sea ( P = 0.04), sed ( P = 0.04), seg ( P = 0.11), sej ( P = 0.11), agr ( P = 0.09), V8 ( P = 0.06), sdrD , sdrE , eta , etb , and see ( P < 0.01 for all). MRSA isolates were more often clonal than MSSA isolates by PFGE. Isolates from patients who were cured were significantly more likely to contain the pvl gene than isolates from patients that failed or had indeterminate outcomes (79/84 [94%] versus 3/6 [50%]; P = 0.01). S. aureus strains from different geographic regions have different distributions of virulence genes.

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase ( TPS1 ), trehalose-6-phophate phosphatase ( TPS2 ), and neutral trehalase ( NTH1 ) genes. We found that both TPS1 and TPS2 are required for high-temperature (37°C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37°C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans . The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans , in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.

Cryptococcal meningitis is a fungal infection, caused by Cryptococcus neoformans , which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-β-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of Saccharomyces cerevisiae and Candida albicans mutants identify the membrane-spanning subunit of glucan synthase, encoded by the FKS genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against Candida and Aspergillus species but are much less potent against C. neoformans . In order to examine why C. neoformans cells are less susceptible to echinocandin treatment, we have cloned a homolog of S. cerevisiae FKS1 from C. neoformans . We have developed a generalized method to evaluate the essentiality of genes in Cryptococcus and applied it to the FKS1 gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the C. neoformans FKS1 gene is essential for viability. The C. neoformans FKS1 sequence is closely related to the FKS1 sequences from other fungal species and appears to be single copy in C. neoformans . Furthermore, amino acid residues known to be critical for echinocandin susceptibility in Saccharomyces are conserved in the C. neoformans FKS1 sequence.

The impact of Panton-Valentine leukocidin (PVL) on the severity of complicated skin and skin structure infections (cSSSI) caused by Staphylococcus aureus is controversial. We evaluated potential associations between clinical outcome and PVL presence in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates from patients enrolled in two large, multinational phase three clinical trials assessing ceftaroline fosamil for the treatment of cSSSI (the CANVAS 1 and 2 programs). Isolates from all microbiologically evaluable patients with monomicrobial MRSA or MSSA infections (n = 473) were genotyped by PCR for pvl and underwent pulsed-field gel electrophoresis (PFGE). Genes encoding pvl were present in 266/473 (56.2%) isolates. Infections caused by pvl-positive S. aureus were associated with younger patient age, North American acquisition, and presence of major abscesses (P

A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system. Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar frequencies. Examination of randomly selected transformants by biolistics showed that 15 to 40% were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.

We have cloned the phosphoribosyl anthranilate isomerase (PRAI)-encoding gene (TRP1) of Cryptococcus neoformans by genetic complementation in Saccharomyces cerevisiae. Sequence analysis of this gene revealed it to be 939 bp in length, and without known promoter or termination sequences. Unlike some of the filamentous fungi, where PRAI enzymatic activity is controlled by a trifunctional gene product, the C. neoformans PRAI appears to be unifunctional. PRAI of C. neoformans exhibits 39% amino acid (aa) sequence identity compared to the S. cerevisiae counterpart. The TRP1 gene of C. neoformans maps to different size chromosomes in strains with different serotypes. The cloning of this gene for vector constructions, and the demonstration that S. cerevisiae can be used as a surrogate for C. neoformans gene expression, should help with the molecular studies of this significant fungal pathogen in our increasing immunocompromised population.

Exposure to whole cigarette smoke from reference cigarettes results in the prompt (peak activity is 6 hrs), but fairly weak (similar to 2 fold), induction of murine pulmonary microsomal monooxygenase activity. This activity can be detected by using as substrates either benzo(a)pyrene or ethoxyresorufin, and can be inhibited by treatment with cycloheximide or actinomycin D. Unlike the induction of pulmonary monooxygenases following intratracheal administration of 3-methylcholanthrene, these cigarette smoke-induced increases were not unequivocally linked to the Ah locus. Whole smoke condensate and fractions derived from these condensates can; a) induce pulmonary monooxygenase activity, b) inhibit benzo(a)pyrene metabolism in vitro, c) be metabolized to forms mutagenic to Salmonella typhimurium tester strains TA153, or TA98, d) transform C3H 10T1/2 cells in vitro, and e) enhance the carcinogenicity of benzo(a)pyrene in murine pulmonary tissue. A potentially important observation is that whereas hepatic tissue is capable of activating whole cigarette smoke condensate to mutagenic forms in vitro, murine pulmonary tissue does not seem capable of such activation. Although these pulmonary-derived tissue homogenates have significant AHH activity and can metabolize Aflatoxin B1, 2-aminofluorene and 7, 8-dihydro-7,8-dihydroxybenzo(a)pyrene to mutagenic forms, these homogenates fail to activate both cigarette smoke condensate and the pro-mutagen, 6-aminochrysene. These results are discussed with reference to the concept that whole cigarette smoke may be both a potential "initiator" and "promotor" of lung cancer in mice, and that this latter property may be the most important in determining cancer risk.

Regenerating rat liver nuclei when sonicated and centrifuged in a Cs2SO4 gradient were fractionated into three distinct bands. These bands were designated as light band (LB), middle band (MB), and heavy band (HB) according to their density. LB and MB were shown to consist of large granular particles with varying electron densities, but LB also contained remnants of nuclear membrane. When analysed by gel electrophoresis, LB and MB displayed more than 35 bands of proteins. The third fraction, HB, consisted largely of small chromatin fibers and its proteins were predominantly the four histones of the nucleosomal core particle. Following short pulses with [3H]thymidine in vivo, the specific activity of DNA in LB and MB was significantly higher than that of bulk DNA contained in HB. DNA in all three fractions became equally labelled as the duration of the labelling interval increased beyond 30 min. Newly synthesized DNA was characterized by electrophoresis on analytical 1.7% acrylamide -0.5% agarose composite gels. After a 1-min labelling interval in vivo, 17% of the rapidly labelled DNA from LB and MB was stationary at the gel origin like replication forks from E. coli, but only 3% of HB DNA had zero mobility. Electron microscopy confirmed the presence of DNA replication forks in LB, MB, and HB. With increasing time of synthesis the proportion of labelled DNA exhibiting zero mobility decreased in all three fractions. Denaturation of DNA or digestion of single-stranded DNA with S1 nuclease released newly synthesized DNA from the gel origin. Ribonuclease was without effect. DNA recovered from LB and MB also had a higher molecular weight than the HB DNA. Together these results indicate (1) that LB and MB are enriched in newly replicated DNA; (2) that an increased proportion of newly replicated DNA in LB and MB is associated with DNA replication forks; and (3) that the replicating DNA recovered in LB and MB may be associated with other nuclear constituents in situ because this DNA appears to be protected from the more frequent chain breaks introduced into the bulk of chromatin (HB) by sonication.

C57BL/6Cum, DBA/2Cum, first filia (F1), and backcross progeny from these 2 parental strains of mice were evaluated for their susceptibility to 3-methylcholanthrene-induced lung cancers. In the crosses among these mice, aryl hydrocarbon hydroxylase (AHH) responsiveness segregated as a single autosomal dominant gene (the Ah locus). AHH responsive mice (Ahb allele) expressed 40-60 units AHH activity/g wet wt liver following intraperitoneal treatment with 3-methylcholanthrene (MCA) compared to AHH non-responsive mice (Ahd allele) which expressed 7-11 units AHH activity/g wet wt liver after MCA treatment. Intratracheal administration of 500 microgram MCA for a total of 4 times at weekly intervals yielded a variety of pulmonary cancers, including squamous cell carcinomas, alveolar adenocarcinomas, and adeno-squamous cell carcinomas among mice that survived 1 year after the carcinogen treatment. The AHH responsive C57BL/6Cum, F1, and C57BL/6Cum X F1 animals were much more susceptible to MCA-induced lung cancers than the AHH non-responsive DBA/2Cum mice. The lung cancers were also not randomly distributed in DBA/2Cum X F1 backcross progeny since significantly more lung cancers were found in AHH-responsive progeny than in AHH non-responsive mice. Data support genetic linkage between susceptibility to MCA-induced lung carcinomas and the Ahb allele.

Pulmonary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene (MCA). I.t. instillation of 188 mug MCA in sterile 0.2% gelatin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57BL/6Cum, C57BL/6J, BALB/cMai, C3H/fMai, and C57L/J were observed to be induced within 24 h after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/2J and RF/J expressed no such increase. At a dose of 500 mug MCA, the pulmonary tissue of DBA/2 mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by: (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7,8-benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.

Background Humans vary in their susceptibility to acquiring Staphylococcus aureus infection, and research suggests that there is a genetic basis for this variability. Several recent genome-wide association studies (GWAS) have identified variants that may affect susceptibility to infectious diseases, demonstrating the potential value of GWAS in this arena. Methods We conducted a GWAS to identify common variants associated with acquisition of S. aureus bacteremia (SAB) resulting from healthcare contact. We performed a logistic regression analysis to compare patients with healthcare contact who developed SAB (361 cases) to patients with healthcare contact in the same hospital who did not develop SAB (699 controls), testing 542,410 SNPs and adjusting for age (by decade), sex, and 6 significant principal components from our EIGENSTRAT analysis. Additionally, we evaluated the joint effect of the host and pathogen genomes in association with severity of SAB infection via logistic regression, including an interaction of host SNP with bacterial genotype, and adjusting for age (by decade), sex, the 6 significant principal components, and dialysis status. Bonferroni corrections were applied in both analyses to control for multiple comparisons. Results Ours is the first study that has attempted to evaluate the entire human genome for variants potentially involved in the acquisition or severity of SAB. Although this study identified no common variant of large effect size to have genome-wide significance for association with either the risk of acquiring SAB or severity of SAB, the variant (rs2043436) most significantly associated with severity of infection is located in a biologically plausible candidate gene (CDON, a member of the immunoglobulin family) and may warrant further study. Conclusions The genetic architecture underlying SAB is likely to be complex. Future investigations using larger samples, narrowed phenotypes, and advances in both genotyping and analytical methodologies will be important tools for identifying causative variants for this common and serious cause of healthcare-associated infection.