trans-complementation of a Staphylococcus aureus agr mutant by Staphylococcus lugdunensis agr RNAIII

RNAIII from Staphylococcus lugdunensis (RNAIII-sl) in a Staphylococcus aureus agr mutant partially restored the Agr phenotype. A chimeric construct consisting of the 5' end of RNAIII-sl and the 3' end of RNAIII from S. aureus restored the Agr phenotype to a greater extent, suggesting the presence of independent regulatory domains.     

SarA level is a determinant of agr activation in Staphylococcus aureus

Shortage of reliable plasma assays has hampered studies of cholecystokinin (CCK). The assay problems are low plasma concentrations, extensive molecular heterogeneity, and close homology of CCK to gastrin, which circulates in higher concentrations. To develop an accurate CCK RIA, antibodies were raised in rabbits, guinea pigs, and mice in titers from 200 to 4000000. The specificity of the antisera was tested with homologous peptides, and tissue and plasma extracts. Rabbit 92128 produced antibodies in high titer (> or =500000) with sufficient avidity (K(o)eff > or = 10(12) mol(-1)) and the desired specificity. The antiserum binds the bioactive forms of CCK with equimolar potency and displays no reactivity with gastrin. CCK concentrations in plasma from healthy humans rose from 1.13 +/- 0.10 pmol/L (mean +/- SE, n = 26) to 4.92 +/- 0.34 pmol/L after a mixed meal. Chromatography of human plasma revealed traces of CCK-58, a predominance of CCK-33 and CCK-22, and moderate amounts of CCK-8. The results show that it is possible to produce specific CCK-antisera using a sulfated CCK-12 analog.     

Genetic instability of the global regulator agr explains the phenotype of the xpr mutation in Staphylococcus aureus KSI9051

Staphylococcus aureus KSI9051 has a complex mutation that was associated with the aberrant expression of cell surface and extracellular proteins (M. S. Smeltzer, M. E. Hart, and J. J. Iandolo, J. Bacteriol. 61:919-925, 1993). This mutation was named xpr, although no specific gene was identified. Here this mutation is referred to as Delta1058::Tn551. In this study, we show that in strain KSI9051, the Delta1058::Tn551 mutation occurred coincidentally with a frameshift in agrC that is expected to truncate the sensor component of the known staphylococcal global regulatory locus agr. Remarkably, pleiotropic mutations affecting cell surface and extracellular proteins are generated at frequencies approaching 50% upon the transduction of erythromycin resistance (Emr) encoded by Delta1058::Tn551 from S. aureus KSI905 back to its parental strain, S6C. Three independent isolates created in the manner of KSI9051 contained mutations within agrC. Each isolate had different mutations, suggesting that the transduction of Emr encoded by Delta1058::Tn551 affects the stability of agrC in S6C. In similar experiments with strains from an S. aureus 8325 genetic background, a mutant AgrC phenotype could not be isolated, implying that strain S6 has aberrant genetic behavior. A comparison of the nucleotide sequences of AgrC from several strains revealed seven errors in the GenBank entry for agr (X52543); these data were confirmed with plasmid pRN6650, the original wild-type clone of agr.     

Impact of the high-affinity proline permease gene (putP) on the virulence of Staphylococcus aureus in experimental endocarditis

Staphylococcus aureus causes a wide variety of invasive human infections. However, delineation of the genes which are essential for the in vivo survival of this pathogen has not been accomplished to date. Using signature tag mutagenesis techniques and large mutant pool screens, previous investigators identified several major gene classes as candidate essential gene loci for in vivo survival; these include genes for amino acid transporters, oligopeptide transporters, and lantibiotic synthesis (W. R. Schwan, S. N. Coulter, E. Y. W. Ng, M. H. Langhorne, H. D. Ritchie, L. L. Brody, S. Westbrock-Wadman, A. S. Bayer, K. R. Folger, and C. K. Stover, Infect. Immun. 66:567-572, 1998). In this study, we directly compared the virulence of four such isogenic signature tag mutants with that of the parental strain (RN6390) by using a prototypical model of invasive S. aureus infection, experimental endocarditis (IE). The oligonucleotide signature tag (OST) mutant with insertional inactivation of the gene (putP) which encodes the high-affinity transporter for proline uptake exhibited significantly reduced virulence in the IE model across three challenge inocula (10(4) to 10(6) CFU) in terms of achievable intravegetation densities (P, <0.05). The negative impact of putP inactivation on in vivo survival in the IE model was confirmed by simultaneous challenge with the original putP mutant and the parental strain as well as by challenge with a putP mutant in which this genetic inactivation was transduced into a distinct parental strain (S6C). In contrast, inactivation of loci encoding an oligopeptide transporter, a purine repressor, and lantibiotic biosynthesis had no substantial impact on the capacity of OST mutants to survive within IE vegetations. Thus, genes encoding the uptake of essential amino acids may well represent novel targets for new drug development. These data also confirm the utility of the OST technique as an important screening methodology for identifying candidate genes as requisite loci for the in vivo survival of S. aureus.     

Deletion of the alternative sigma factor sigmaB in Staphylococcus aureus reveals its function as a global regulator of virulence genes

A deletion of the sigB operon was constructed in three genetically distinct Staphylococcus aureus strains, and the phenotypes of the resulting mutants were analyzed. Compared to the corresponding wild-type strains, the DeltasigB mutants showed reduced pigmentation, accelerated sedimentation, and increased sensitivity to hydrogen peroxide during the stationary growth phase. A cytoplasmic protein missing in the DeltasigB mutants was identified as alkaline shock protein 23, and an extracellular protein excreted at higher levels in one of the DeltasigB mutants was identified as staphylococcal thermonuclease. Interestingly, most sigB deletion phenotypes were only seen in S. aureus COL and Newman and not in 8325, which was found to contain an 11-bp deletion in the regulator gene rsbU. Taken together, our results show that sigmaB is a global regulator which modulates the expression of several virulence factors in S. aureus and that laboratory strain 8325 is a sigmaB-defective mutant.     

The virulence of Staphylococcus aureus isolates differing by siderophore production

Pentraxins are a family of pentameric serum proteins that have been conserved in evolution and share sequence homology, similar subunit assembly and the capacity for calcium-dependent ligand binding. The classical pentraxins are human C-reactive protein (CRP) and serum amyloid P component (SAP). The sequence homology and gene organization indicate that they arose from a gene duplication of an ancestral pentraxin gene. They are usually isolated based on their affinity for phosphorylcholine and agarose, respectively. We have used this method for isolation of pentraxin-like proteins from normal serum of Atlantic salmon (Salmo salar), common wolffish (Anarhichas lupus), cod (Gadus morhua) and halibut (Hippoglossus hippoglossus). Although pentraxin structures have not been verified, the isolated proteins all appear to be pentraxin-like based on their binding specificity, molecular weight of subunits, cross-reactivity with antibodies to human pentraxins and N-terminal amino acid sequences. However, with the described method only one pentraxin-like protein was detected in each of the fish species.     

Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus

Staphylococcus aureus causes pathologies ranging from minor skin infections to life-threatening diseases. Pathogenic effects are largely due to production of bacterial toxin, which is regulated by an RNA molecule, RNAIII. The S. aureus protein called RAP (RNAIII activating protein) activates RNAIII, and a peptide called RIP (RNAIII inhibiting peptide), produced by a nonpathogenic bacteria, inhibits RNAIII. Mice vaccinated with RAP or treated with purified or synthetic RIP were protected from S. aureus pathology. Thus, these two molecules may provide useful approaches for the prevention and treatment of diseases caused by S. aureus.     

Cytokine expression in a rat model of Staphylococcus aureus endophthalmitis

PURPOSE: To examine the ability of viable Staphylococcus aureus to induce the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, cytokine-induced neutrophil chemoattractant (CINC), and interferon (IFN)-gamma after intravitreal injection. METHODS: Experimental rat eyes were injected with a 25-microl volume of approximately 80 colony-forming units of viable S. aureus; control eyes received sterile saline. Eyes were graded daily for signs of clinical inflammation and were removed 6, 24, 48, and 72 hours after injection. One group was prepared for histologic analysis, and vitreous was removed from the other group for cytokine analysis, using standard enzyme-linked immunosorbent assay procedures. RESULTS: TNF-alpha, IL-1beta, CINC, and IFN-gamma were detected in experimental vitreous samples at increased levels that peaked at 24 hours. TNF-alpha, IL-1beta, and CINC declined at 48 hours, but IFN-gamma remained elevated. At 72 hours, levels returned to baseline. Statistically significant elevations of TNF-alpha, IL-1beta, and CINC were detected in experimental samples at 24, but not at 6 and 48 hours compared with levels in saline control samples (P < 0.03). A statistically significant increase in IFN-gamma was detected at 24 and 48 hours compared with control levels (P < 0.03). In experimental animals, clinical inflammation and inflammatory cells peaked at 24 hours, persisted at 48 hours, and began to decline thereafter. Neutrophils were the predominant inflammatory cell detected at 24 (72.3% of cells) and 48 (60.1%) hours. By 72 hours, the total number of inflammatory cells had decreased by 75.0%, and the cellular infiltrate had changed so that neutrophils equaled monocytes-macrophages. CONCLUSIONS: S. aureus induced the expression of TNF-alpha, IL-1beta, CINC, and IFN-gamma. The time course of these cytokine levels could account for the clinical inflammatory responses and the entry and decline of vitreous cells in this model of bacterial endophthalmitis.     

The Staphylococcus aureus alternative sigma factor sigmaB controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model

The role of sigmaB, an alternative sigma factor of Staphylococcus aureus, has been characterized in response to environmental stress, starvation-survival and recovery, and pathogenicity. sigmaB was mainly expressed during the stationary phase of growth and was repressed by 1 M sodium chloride. A sigB insertionally inactivated mutant was created. In stress resistance studies, sigmaB was shown to be involved in recovery from heat shock at 54 degreesC and in acid and hydrogen peroxide resistance but not in resistance to ethanol or osmotic shock. Interestingly, S. aureus acquired increased acid resistance when preincubated at a sublethal pH 4 prior to exposure to a lethal pH 2. This acid-adaptive response resulting in tolerance was mediated via sigB. However, sigmaB was not vital for the starvation-survival or recovery mechanisms. sigmaB does not have a major role in the expression of the global regulator of virulence determinant biosynthesis, staphylococcal accessory regulator (sarA), the production of a number of representative virulence factors, and pathogenicity in a mouse subcutaneous abscess model. However, SarA upregulates sigB expression in a growth-phase-dependent manner. Thus, sigmaB expression is linked to the processes controlling virulence determinant production. The role of sigmaB as a major regulator of the stress response, but not of starvation-survival, is discussed.     

Various possible pathogenicity factors in Staphylococcus aureus (proceedings)

Parametric and symptomatologic studies of the EEG in large populations have served to define stages of maturation and criteria of "normality". The younger the subjects, however, the wider are the variations. In the first stage of life only massive cerebral lesions afford unquestionable EEG evidence: in evaluating dysrhythmias it is necessary to bear in mind their high incidence in normal subjects. Hence their meaning is to be understood only by prolonged longitudinal studies. The reference here is not only to diffuse or focal slow anomalies, but also to specific pathologic rhythms. The difficulty of interpreting such anomalies is particularly evident when epileptogenic potentials are found, since they may be signs of a lesion but their clinical correlations are uncertain. The concept of "masked epilepsy" must be rejected. Only the diagnoses of latent or proven epilepsy are admissible, and these epilepsies may be without direct relationship to the clinical, psychiatric or central nervous findings in the affected subject. Numerous genetic, afferential, emotional or biologic factors are involved in the development of non-lesional disorders. Accordingly, the EEG has only a minor contribution to make in the definition of mild focal or diffuse pathologic anatomy of the brain in the very young subject.     

Translation of RNAIII, the Staphylococcus aureus agr regulatory RNA molecule, can be activated by a 3''-end deletion

A case of enteropathy associated T-cell lymphoma (EATCL) in a 62-year-old female with a previous history of coeliac disease, complicated during the clinical course by massive blood and tissue eosinophilia is described. The patient's serum contained a factor capable of stimulating the in vitro growth of eosinophilic colonies (CFU-Eo), that was absent in the serum of normal donors. We suggest that such factor was Interleukin-5 (IL-5), as indicated by the presence in the monoclonal tumor T cells of IL-5 encoding mRNA, usually absent in the normal enterocytes of the jejunum.     

Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model

OBJECTIVE: The purpose of this study was to determine whether increased cytosolic phospholipase A2 activity mediated arachidonic acid mobilization for prostaglandin synthesis in amnion at parturition. STUDY DESIGN: Amnion was collected immediately after delivery from four groups of patients: preterm (<37 weeks) with no labor or labor and term (>37 weeks) with no labor or labor and stored at -70 degrees C. Tissues were homogenized and centrifuged for 1 hour at 100,000 g, and cytosol was assayed for cytosolic phospholipase A2 activity with use of carbon 14-labeled 1-stearoyl-2 arachidonyl phosphatidylcholine plus 10 micromol/L unlabeled substrate and 5 mmol/L calcium in 10 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.4. Incubations were performed in duplicate +/- 10 micromol/L arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic phospholipase A2 activity, at 30 degrees C for 45 minutes. RESULTS: Total cytosolic phospholipase A2 activity (in picomoles of arachidonic acid per minute per milligram of protein) calculated as the difference between the activity in the presence and absence of arachidonyl trifluoromethyl ketone was (mean +/- SE) as follows: preterm no labor (n = 7) 8.94 +/- 3.08, preterm with labor (n = 6) 6.79 +/- 2.31, term no labor (n = 7) 14.85 +/- 1.66, and term with labor (n = 5) 5.51 +/- 1.52. Enzyme activity increased with gestational age and was highest in the term no labor group. A significant decrease in cytosolic phospholipase A2 activity occurred with labor (p < 0.05). The greatest decrease in activity was in the term group (p < 0.05). CONCLUSION: Total cellular cytosolic phospholipase A2 activity in amnion is highest in anticipation of labor but during labor total activity is depleted, resulting in the low activity measured after delivery of the placenta. The substrate specificity and changes in amnion total cytosolic phospholipase A2 activity with labor strongly suggests a role in mediation of arachidonic acid mobilization and prostaglandin synthesis at labor.     

The genome of Staphylococcus aureus: a review

The genome of Staphylococcus aureus consists of a single circular chromosome (2.7-2.8 mbp) plus an assortment of extrachromosomal accessory genetic elements: conjugative and nonconjugative plasmids, mobile elements (IS, Tn, Hi), prophages and other variable elements. Plasmids (1-60 kbp) are classified into 4 classes and there are 15 known incompatibility groups. Mobile elements of the genome (0.8-18 kbp) appear in the chromosome or in plasmids of classes II and III. Prophages (45-60 kbp) are integrated in the bacterial chromosome, and they are UV- or mitomycin-inducible. Temperate bacteriophages of S. aureus are members of the Siphoviridae and the serological groups A, B and F occur most frequently. In the paper presented, the characteristics of chromosome, plasmids, transposons and other genetic elements of S. aureus genome are given and an alphabetical list of known genes of this species is included.     

Molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy

The molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy was studied in a five-month period in 1996, during which all S. aureus isolated were collected. All MRSA isolates (95) and a sample of methicillin-susceptible S. aureus (20) were typed with a variety of phenotypic and genotypic methods. Clonal identities were determined by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests and, for MRSA isolates, by probing ClaI digests with a mecA probe and a Tn554 probe. Overall, MRSA represented 32.3% of all isolates, with very high percentages from the intensive care units (adult and neonatal). PFGE after restriction with SmaI resolved genomic DNA of 95 MRSA strains into 26 major PFGE patterns. The use of southern blot hybridization of ClaI genomic digests with mecA and Tn554 allowed us a significant increase in discrimination, differentiating at least 32 different clones. Two major clones, however, each sharing common ClaI-mecA and Tn554 type and PFGE pattern as well as a common resistance phenotype, represented more than 50% of all MRSA isolates. The recovery of these two clones in the majority of the isolates of adult and neonatal intensive care units, respectively, is indicative of typical nosocomial outbreaks and clonal spread. It is concluded that intensive care units are major areas requiring preventative interventions.     

Staphylococcus aureus and Candida albicans infection (animal experiments)

During a certain period of the course of infection in white mice inoculated intraperitoneally with the pure culture of a proteolysing strain of Candida albicans, Staphylococcus aureus and C. albicans both, were isolated simultaneously from the peritoneal abscesses, especially those adhering to the stomach, duodenum, pancreas and the upper part of small intestine. This concommitant occurrence of the two pathogens was further corroborated by histopathological examination which revealed large number of staphylococci present in the close neighbourhood of Candida cells, usually in the center of the granulomata caused by the fungus. In view of the facts that the proteolytic end-products of C. albicans can offer a good substratum for the growth of S. aureus and the latter may be isolated from the intestinal tract of apparently healthy mice, possibly as a constituent of the transient microflora, the co-existence of these two important aetiologic agents of endogenous infections as encountered during this study appears to be of great clinical interest. Furthermore, these observations also demonstrate the importance of controlling the bacterial flora of mice for pure mycological studies.     

The pH of the host niche controls gene expression in and virulence of Candida albicans

Little is known of the biological attributes conferring pathogenicity on the opportunistic fungal pathogen Candida albicans. Infection by this pathogen, as for bacterial pathogens, may rely upon environmental signals within the host niche to regulate the expression of virulence determinants. To determine if C. albicans responds to the pH of the host niche, we tested the virulence of strains with mutations in either of two pH-regulated genes, PHR1 and PHR2. In vitro, PHR1 is expressed when the ambient pH is at 5.5 or higher and deletion of the gene results in growth and morphological defects at neutral to alkaline pHs. Conversely, PHR2 is expressed at an ambient pH below 5.5, and the growth and morphology of the null mutant is compromised below this pH. A PHR1 null mutant was avirulent in a mouse model of systemic infection but uncompromised in its ability to cause vaginal infection in rats. Since systemic pH is near neutrality and vaginal pH is around 4.5, the virulence phenotype paralleled the pH dependence of the in vitro phenotypes. The virulence phenotype of a PHR2 null mutant was the inverse. The mutant was virulent in a systemic-infection model but avirulent in a vaginal-infection model. Heterozygous mutants exhibited partial reductions in their pathogenic potential, suggesting a gene dosage effect. Unexpectedly, deletion of PHR2 did not prevent hyphal development in vaginal tissue, suggesting that it is not essential for hyphal development in this host niche. The results suggest that the pH of the infection site regulates the expression of genes essential to survival within that niche. This implies that the study of environmentally regulated genes may provide a rationale for understanding the pathobiology of C. albicans.     

MRSA (methicillin-resistant Staphylococcus aureus) infections in patients with pulmonary diseases

Methicillin-resistant Staphylococcus aureus (MRSA) has become a major nosocomial pathogen. We investigated MRSA-infections in patients with pulmonary diseases referring to epidemiological aspects. Between 9/92 and 2/92 we found MRSA-infections in our hospital in 24 patients (11 female, 13 male, average age 54.6 years). Clinical presentation, main and accompanying disorders and previous antibiotic therapy regimens were registered. Strains were typed using DNA-RFLP and lysotyping. MRSA detection were done in specimen from sputum (12/24) and from the bronchial secret (9/24). In 18/24 cases the MRSA-colonisation was associated with infection. In 15/24 cases the first acquisition of MRSA happened in our hospital, 6/24 times the germ was carried off other institutions and in 3/24 cases it was possibly community acquired. Most frequently patients suffered from bronchial cancer (6/24), from chronical bronchitis (5/24), from pneumonia (4/24) or Cystic fibrosis (4/24). Usually the patients showed other severe comorbidity. 13/24 patients had an antibiotic course before detecting MRSA. Typing revealed a strain already known in different hospitals of Berlin, another known strain of northern Germany and two so far unknown strains. Of interest was a different behaviour of resistance and the lost of resistance of strains in the course. MRSA-infection in pulmonary medicine emerged as a problem mostly in patients with multimorbidity and severe underlying diseases. Change of resistance in strains and new strains were observed.     

Septic arthritis following Staphylococcus aureus infection in mice lacking inducible nitric oxide synthase

Nitric oxide (NO), produced in large amounts by inducible NO synthase (iNOS), has emerged recently as an important microbicidal and immunomodulatory mediator. We have investigated its role in bacterial septic arthritis caused by Staphylococcus aureus infection using iNOS-deficient mice. The incidence, rate of development, and severity of arthritis were greater in iNOS-deficient than in heterozygous or wild-type control mice. Similarly, the incidence and severity of septicemia and mortality were significantly higher in iNOS-deficient mice compared with controls. Increased TNF-alpha synthesis in vivo and in vitro and enhanced IFN-gamma compared with IL-4 production in vitro in iNOS-mutant mice demonstrated exaggerated Th1 polarization of the host response. These data indicate that high output NO production is not a prerequisite for severe articular destruction and imply that NO is of importance in synovial defense against staphylococcal infection.