Effects of microbial metabolites on catalase activity and growth ofStaphylococcus aureus 6538 P

The effects of cell extracts and supernatants ofLactobacillus spp. andCorynebacterium spp. on catalase activity and growth ofStaphylococcus aureus 6538 P were studied. Intra- and extracellular metabolites of lactobacilli and corynebacteria inhibited catalase activity ofS. aureus 6538 P. The growth ofS. aureus 6538 P decreased after incubation with lactobacillus metabolites. The inhibitory effect of intra- and extracellular metabolites of lactobacilli and corynebacteria on catalase activity ofS. aureus is a possible pathway of microbial interrelations responsible for the formation and/or development of microbial biocenoses. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 7, pp. 80–82, July, 2000     

Effect of helium-neon laser on activity and optical properties of catalase

The effects of laser (632.8 nm) on functional and spectral properties of catalase at pH 6.0–7.4 were studied. Laser irradiation led to photoactivation of the enzyme at pH 7.1–7.4. Changes in the spectral properties of photomodified hemoprotein were found in the absorption spectrum of the protein component: apoenzyme displayed protective effects in relation to ferroporphyrin. Structural modifications of catalase induced by helium-neon laser irradiation correlated with its functional properties. These results can be used in clinical practice to design the individual management program. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 6, pp. 633–636, June, 2000     

Treatment of Staphylococcus aureus endocarditis using moxifloxacin

The conventional treatment of staphylococcal endocarditis requires in-patient administration, is inconvenient, and is potentially toxic. Increasing experience with well-absorbed, well-tolerated and highly active agents such as the new quinolones has prompted interest in their use as therapeutic alternatives for the treatment of such infections. We describe a case of staphylococcal endocarditis which failed to respond to conventional therapy, but where the addition of moxifloxacin, an 8-methoxyquinolone, was curative.     

Intracellular survival of Staphylococcus aureus: correlating production of catalase and superoxide dismutase with levels of inflammatory cytokines

BACKGROUND: Superoxide dismutase (SOD) and catalase are anti-oxidant enzymes potentially used by the bacteria to neutralize macrophage microbicidal molecules such as hydrogen peroxide (H(2)O(2)). OBJECTIVE: To investigate contribution of bacterial anti-oxidant enzymes in intracellular survival of Staphylococcus aureus (S. aureus) within macrophages. MATERIALS: Murine peritoneal macrophages and S. aureus (CMC-524, ICH-629 and ICH-757). TREATMENT: 10(6) colony forming units (CFU) of the 90 minutes (min) intracellularly viable S. aureus were administered (i.v.) per mouse through 0.1 ml saline. METHODS: Anti-oxidant enzyme assay, phagocytic activity, H(2)O(2) release, Zymography for catalase, serum tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) level were estimated. One-way Model I ANOVA and one tail Student's t-test were performed. RESULTS: Survival of S. aureus was least after 90 min of reincubation within macrophages. Maximum amount of bacterial anti-oxidant enzymes were released after 90 min of re-incubation. H(2)O(2) released after 90 min of re-incubation with S. aureus was maximum. Higher activity of catalase and SOD by S. aureus occurred in response to the gradual production of H(2)O(2). Serum IL-6 and TNF-alpha was also elevated 1h post infection. CONCLUSIONS: Bacterial catalase and SOD combat reactive oxygen species enabling S. aureus to persist within macrophages, inducing local inflammation, causing greater induction of serum TNF-alpha and IL-6.     

Heparan sulfate proteoglycans mediate Staphylococcus aureus interactions with intestinal epithelium

Staphylococcus aureus can be internalized by non-professional phagocytes, and may colonize the intestine in normal and antibiotic-treated individuals. Intestinal colonization may depend on the interactions of S. aureus with the intestinal epithelium. The best described mechanism of S. aureus binding to eukaryotic cells involves S. aureus fibronectin binding proteins (FnBPs), using fibronectin as a bridging molecule to β1 integrins on the eukaryotic cell surface. Because S. aureus can be internalized by enterocytes, and because S. aureus is known to bind heparan sulfate (HS), we hypothesized that heparan sulfate proteoglycans (HSPGs) widely expressed on epithelia may mediate S. aureus interactions with intestinal epithelial cells. Internalization of S. aureus RN6390 by cultured intestinal epithelial cells was inhibited in a dose-dependent fashion by the HS mimic heparin, and by HS itself. Internalization of S. aureus DU5883, which lacks expression of staphylococcal FnBPs, was also inhibited by heparin. S. aureus adherence to ARH-77 cells, transfected to express the HSPG syndecan-1, was greatly increased when compared to adherence to plasmid control ARH-77 cells which have little detergent extractable HS. In addition, compared to wild-type HS-expressing Chinese hamster ovary (CHO) cells, internalization of S. aureus was decreased using mutant CHO cells with decreased HS expression. These findings are consistent with a model wherein S. aureus internalization by intestinal epithelial cells (and perhaps other epithelia) is mediated by S. aureus binding to the HS moiety of cell-surface HSPGs, and this interaction appears independent of fibronectin binding.     

Staphylococcus aureus strain designation by agr and cap polymorphism typing and delineation of agr diversification by sequence analysis

The allelic variations of the regulatory operon agr (groups I-IV) and the cap polymorphism (capsular types 5 and 8) were used as a typing scheme for rapid strain designation in Staphylococcus aureus. In combining 10 agr subgroups resolved by restriction fragment length polymorphism (RFLP) analysis with the two cap polymorphisms 12 types could be defined. To assess whether this type designation is informative for the population structure of the species S. aureus, agr and cap types were determined in clonal lineages defined by pulsed-field gel electrophoresis (PFGE) of a collection of 219 isolates. agr groups and cap types were both linked to certain clone complexes. However, little correlation was found between the two polymorphic loci. By PFGE cluster analysis 11 prevalent and 52 sporadic clones were defined. Most of the prevalent clones (9/11) could be discriminated by agr/cap typing. Thus, this technique allows a first subdivision of isolates and an inter-center comparable designation of S. aureus clones preceding a more detailed clonal analysis by PFGE or multi-locus sequence typing (MLST). To get insight into agr diversification, sequence analysis of the variable and conserved part of agr from selected S. aureus clones was performed. Strains of agr-I displayed the highest sequence divergence on the nucleotide and amino acid level, suggesting an early diversification of this group. When analyzing the relationship between the four agr interference groups we could show: (i) one intermediate between agr-I and agr-IV alleles; (ii) agr-IV sequences seem to bridge the agr-I and -III groups and (iii) two cases of horizontal transfer of the variable gene cassette from an agr-I strain to an agr-II strain. Thus, stepwise evolutionary progression and rare events of recombination were evident in the diversification of the agr locus.     

The epidermolytic (exfoliative) toxins of Staphylococcus aureus

Two epidermolytic toxins, produced by different strains of Staphylococcus aureus, split human skin at a site in the upper epidermis. Clinical effects are most common in infants, but adults are susceptible. Epidermolysis may also be observed in the mouse, in vivo and in vitro, and in a few other mammals. Recent in vitro experiments have demonstrated an inhibition by chelators and point to metal-ion, possibly Ca²⁺, involvement. The epidermolysis effect is insensitive to a wide range of other metabolic inhibitors. The toxin amino acid sequences are similar to that of staphylococcal proteinase, and new experiments by chemical modification and site-directed mutagenesis have shown that toxicity depends on active serine residues of a catalytic triad similar to that found in serine proteases. Furthermore the toxins possess esterolytic activity, also dependent on the active serine sites. However, the toxins have low or undetectable activity towards a range of peptide or protein substrates. In histological and related studies, the toxins bound selectively to an intracellular skin protein, profilaggrin, but there was no evidence that the toxin can enter intact epidermal cells. Therefore, although the circumstantial evidence that the toxins act by proteolysis is convincing, a specific skin proteolytic substrate for the toxin has not been identified.     

Role of insertion elements and yycFG in the development of decreased susceptibility to vancomycin in Staphylococcus aureus

The glycopeptide antibiotic vancomycin acts by binding to the D-alanyl-D-alanine terminus of the cell wall precursor lipid II in the cytoplasmic membrane. The purpose of this study was the identification of genes that might be involved in the vancomycin resistance mechanism. To this end, the expression profiles of two vancomycin intermediately resistant Staphylococcus aureus (VISA) strains, the clinical isolate S. aureus SA137/93A (Etest: 8 microg/ml) and its laboratory mutant S. aureus SA137/93G (Etest: 12 microg/ml) were analyzed using an S. aureus full-genome chip. The results indicated that an essential two-component regulatory system, yycF (vicR) and yycG (vicK) was drastically up-regulated in strain SA137/93A. Sequencing of the yycFG promoter region of strain SA137/93A revealed an insertion of IS256 in the predicted promoter region creating a potentially stronger hybrid promoter. In strain SA137/93G, IS256 was not integrated in the yycFG promoter region but, in previous studies, a copy of IS256 had been found to inactivate the tcaA gene (Maki et al. Antimicrob. Agents and Chemother. 48, 1953-1959 (2004)). Detailed population analyses showed that, in addition to the loss of SCCmec, the inactivation of tcaA seems to cause at least part of the increase in teicoplanin and vancomycin resistance in strain SA137/93G.     

Ribonucleotide reductase class III, an essential enzyme for the anaerobic growth of Staphylococcus aureus, is a virulence determinant in septic arthritis

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.     

Bacterial evasion of innate host defenses--the Staphylococcus aureus lesson

Bacterial pathogens such as Staphylococcus aureus use highly efficient mechanisms to evade recognition and elimination by the innate immune system. S. aureus produces sophisticated anti-inflammatory molecules and it employs several mechanisms protecting the bacteria against host cationic antimicrobial molecules such as defensin-like peptides and bacteriolytic enzymes such as lysozyme. Cell wall teichoic acids and lipoteichoic acids, complex Gram-positive surface polymers, and modified membrane lipids such as lysylphosphatidylglycerol are crucial in defensin resistance and other important aspects of staphylococcal virulence such as nasal colonization and biofilm formation on biomaterials. Certain S. aureus genes conferring escape from innate host defenses are conserved in many human pathogens suggesting that the underlying mechanisms are of general significance in bacterial virulence.     

Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus

Thymidine-auxotrophic small colony variants (SCVs) of Staphylococcus aureus are frequently isolated from the chronically infected airways of patients suffering from cystic fibrosis. To date, little is known regarding the molecular mechanisms leading to the formation of this special phenotype, but the auxotrophism for thymidine suggests that impaired thymidine metabolism might play a major role. Sequence analysis of the thymidylate synthase-encoding thyA gene of six clinical thymidine-auxotrophic S. aureus SCVs revealed that all isolates had mutations within thyA. In five isolates the function of the thymidylate synthase was definitely impaired: three of them showed a truncation of the thyA coding sequence by nonsense or frame-shift mutations, in one further isolate the active site of the enzyme was affected by an internal 12-bp deletion, and another isolate had a 173-bp deletion spanning the 5'-terminal region of thyA and the preceding DNA sequence. The sixth isolate showed two amino acid substitutions within the thyA gene product. To confirm the importance of impaired thymidylate synthase synthesis or activity for the formation of the thymidine-auxotrophic SCV phenotype, we constructed a thyA knock-out mutant of a wild-type S. aureus strain. This mutant showed all characteristics of clinical SCVs, such as slow growth, decreased pigment production, reduced hemolytic activity, auxotrophism for thymidine, resistance to trimethoprim/sulfamethoxazol, and reduced plasma coagulase activity. Complementation of the thyA knock-out mutant with intact thyA in trans nearly restored the normal phenotype. In conclusion, these data confirm at the molecular level that impaired thymidylate synthase function is causative for the formation of the thymidine-auxotrophic SCV phenotype in S. aureus.     

Co-infection of the cotton rat (Sigmodon hispidus) with Staphylococcus aureus and influenza A virus results in synergistic disease

Bacterial super-infection of influenza patients is the primary cause of excess mortality during influenza pandemics, with Staphylococcus aureus (S. aureus) having the highest fatality rate. The cotton rat (Sigmodon hispidus) is an excellent model for both influenza and S. aureus pathogenesis, and therefore a potential tool to model co-infection. We compared physiologic and pathologic changes in cotton rats infected with both S. aureus and influenza A/Wuhan/359/95 (H3N2), with animals infected with each pathogen alone. Co-infected cotton rats demonstrated significantly higher mortality, lower temperatures on 2 and 3 days post-inoculation (p.i.), higher levels of bacteremia and pulmonary bacterial load 4 days p.i., and worse pathology 7 days p.i. Early indicators of exacerbated disease coincided with higher pulmonary mRNA levels for IL-1beta, IL-6, IL-10 and IFNy, supporting the idea that these may contribute to disease severity. Our results demonstrate that the cotton rat is a good model of influenza and S. aureus co-infection, with increased mortality and hypothermia as well as prolonged bacterial duration indicative of synergistic disease that may be the result of increased induction of both pro- and anti-inflammatory cytokines.     

Effect of the catalase inhibitor 3-amino-1,2,4-triazole on oxidation-phosphorylation coupling and the state of the mitochondrial adenosine system in the liver of albino rats

Incubation of the specific catalase inhibitor 3-amino-1,2,4-triazole with liver mitochondria and administration of this drug to intact rats are shown to uncouple oxidation and phosphorylation and to inhibit adenosine nucleotide synthesis in the animal liver. These disturbances apparently results from catalase inhibition. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 6, pp. 638–640, June, 1996     

Improved and rapid detection of methicillin-resistant Staphylococcus aureus nasal carriage using selective broth and multiplex PCR

To improve efficiency in detecting nasal methicillin-resistant Staphylococcus aureus (MRSA), we evaluated a multiplex PCR using pre-enrichment of the specimen in selective broths, and compared it with detection performed by routine tests in hospital laboratories. Nasal swab specimens from 311 patients were inoculated onto mannitol-salt agar (MSA) at the hospital laboratories and in two Mueller-Hinton broths with 7% NaCl containing oxacillin at concentrations of 2 and 4 micro g/ml. Isolates on MSA were identified as MRSA by classical laboratory tests (coagulase and oxacillin disk diffusion tests). Oxacillin broth cultures were subcultured on blood agar and MRSA isolates were identified by coagulase and susceptibility tests, including agar dilution and the oxacillin-screening method (gold standard method). Simultaneously, multiplex-PCR was performed from the selective broths to detect S. aureus species-specific and mecA gene segments (OxMPCR method). Thirty-two S. aureus isolates were recovered: 29 (90.6%) were MRSA strains and 3 (9.4%) were oxacillin-susceptible isolates. Twenty-eight (96.5%) MRSA isolates were detected by OxMPCR, while 17 (58.6%) were identified by routine tests (P=0.002). This new method for detection of MRSA nasal carriers showed higher sensitivity and led to faster reporting--i.e., within 24 h--of results.     

Antibacterial effect of silver nanoparticles on Staphylococcus aureus

Antibacterial activity of silver nanoparticles (AgNPs) was investigated using Staphylococcus aureus PTCC1431 as a model of Gram-positive bacteria. The mechanism of antibacterial activity of AgNPs was then studied by analyzing the growth, morphology, and molecular variations in the cell wall. Experimental data showed that AgNPs at a concentration of 4 μg/ml completely inhibited bacterial growth. Transmission electron microscopy results confirmed cell wall damage produced by AgNPs as well as accumulation of AgNPs in the bacterial membrane. Meanwhile, the AgNP-treated bacteria were monitored by circular dichroism to reveal peptidoglycan variations. Some degree of variation in the α-helix position of the peptide chain was observed. Moreover, increasing the AgNP concentration to 8 μg/ml resulted in release of muramic acid (MA) into the medium, which could be attributed to cell wall distraction. A gas chromatography-tandem mass spectrometry analysis and release of MA, as a bacterial indicator, showed that glycan strands may also be decomposed as a result of AgNP treatment.     

Purified Staphylococcus aureus leukotoxin LukM/F' does not trigger inflammation in the bovine mammary gland

An early recruitment of neutrophils in mammary tissue and milk is considered an important component of the defense of the mammary gland against Staphylococcus aureus. We investigated whether the leukotoxin LukM/F′, which is produced by a proportion of mastitis-causing strains of S. aureus, would be able to trigger inflammation in the udder. Infusion of purified LukM/F′ toxin in lactating mammary glands did not cause neutrophil influx in milk, showing that the toxin was not able to cause mastitis on its own. Purified LukM/F′ did not kill or stimulate mammary epithelial cells in culture. As expected, LukM bound to mammary macrophages and the complete LukM/F′ toxin killed these cells, but subcytotoxic LukM/F′ concentrations did not induce secretion of IL-8, TNF-α, IL-1β or IL-6 by macrophages. On the contrary, the production of these pro-inflammatory mediators by adhesion-stimulated macrophages was reduced. Overall, these results indicate that purified leukotoxin LukM/F′ is not likely to contribute to the initiation of the inflammatory response and could even play an anti-inflammatory role in the mammary gland by inactivating macrophages.