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A novel method for rapid production and purification of exfoliative toxin A of Staphylococcus aureus.

The exfoliative toxins of Staphylococcus aureus are the causative agents of the scalded-skin syndrome. Previously described methods of toxin production and purification require large quantities of culture medium, take a long time and often produce low yields of toxin. A novel method of toxin production and purification using a dialysis sac to separate the culture medium from the staphylococci is described. This method produces up to 12 mg of crude toxin per ml of bacterial cell culture bathing the surface of the dialysis sac within 36 h and almost 10 mg of purified toxin per ml of cell culture within 3 days, in contrast to previous procedures that took over a week to produce 0.1-1.0 mg ml(-1) crude toxin and less than 0.01 mg ml(-1) purified toxin. This rapid method of toxin production should speed up future research into the pathogenesis of the staphylococcal scalded-skin syndrome.

Bacteriological Techniques↗

Cloning, characterization, and inactivation of the gene pbpC, encoding penicillin-binding protein 3 of Staphylococcus aureus.

The gene pbpC from Staphylococcus aureus was sequenced: it encodes a 691-amino-acid protein with all of the conserved motifs of a class B high-molecular-weight penicillin-binding protein (PBP), including the transpeptidase conserved motifs SXXK, SXN, and KTG. Insertional inactivation of pbpC and introduction of the intact gene in a laboratory mutant missing PBP 3 showed that the pbpC gene encodes the staphylococcal PBP 3. Inactivation of pbpC caused no detectable change in the muropeptide composition of cell wall peptidoglycan and had only minimum, if any, effect on growth rates, but caused a small but significant decrease in rates of autolysis. Cells of abnormal size and shape and disoriented septa were produced when bacteria with inactivated pbpC were grown in the presence of a sub-MIC of methicillin.

Amino Acid Sequence↗

[Reduced susceptibility to triclosan in methicillin-resistant Staphylococcus aureus].

Susceptibility to triclosan in Staphylococcus aureus was determined. The study was carried out on 200 strains, including 100 resistant (MRSA) and 100 susceptibile (MSSA) to methicillin. The examined strains were isolated from varied clinical samples and patients in 18 medical centers, in majority from hospitals in the region of Gdansk. The susceptibility was estimated by the MIC (minimal inhibitory concentration) using dilution test in Mueller-Hinton agar. The antimicrobial resistance patterns were determined, including resistance to methicillin and mupirocin. The most of MRSA strains (62%) demonstrated reduced susceptibility to triclosan (MIC 2mg/L), while 93% of MSSA strains were highly sensitive to this antibacterial agent (MIC 0,031mg/L). The majority (66,1%) of MRSA strains with reduced susceptibility to triclosan demonstrated the same antimicrobial resistance pattern. Reduced susceptibility to triclosan was observed in 8 from 9 high - level mupirocin resistant strains, but the most of MRSA strains with reduced triclosan susceptibility (91,5%) were found among fusidic acid resistant strains.

Anti-Infective Agents, Local↗

Purification and characterization of a lipase from Staphylococcus aureus.

An extracellular lipase from Staphylococcus aureus (strain FN 37) was purified to homogeneity. A cell-free culture broth was subjected to ammonium sulphate precipitation, and the lipase was isolated from the resuspended pellet by adsorption chromatography on octyl-Sepharose. The purification was 957-fold, and the recovery of the octyl-Sepharose chromatography was about 100%. The specific activity of the purified lipase was 546 mU of lipase activity per micrograms protein. The purity of the final product was documented by SDS-polyacrylamide gel electrophoresis in which a homogeneous protein band of 43 kDa was found. In gel chromatography on Sephadex G-200 the lipase eluted as a homogeneous peak with an apparent molecular mass of 110 kDa, suggesting that the lipase may exist as an oligomer in physiological media. Analysis of the amino-acid composition revealed a predominance of polar, non-charged amino acids, with serine accounting for 24 mol% of the amino-acid residues.

Amino Acids↗

Methicillin-resistant Staphylococcus aureus bacteriuria.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteriuria was detected in 11 of 41 patients colonized or infected with MRSA. The patients with bacteriuria generally were older than 40 years of age, five were diabetic, seven had prior indwelling uretheral catheters, two had undergone other urologic manipulations, and only one was clinically symptomatic. Eight patients received variable combinations of antibiotic therapy prior to the diagnosis of MRSA bacteriuria, and seven were still on antibiotic therapy at the time the bacteriuria was detected. Bacteriuria lasted four days to 14 weeks, and was eradicated promptly with cephalosporin therapy in five six patients. Bacteriuria in the untreated patients cleared spontaneously in one month. A single MRSA serotype (83A) predominated. The MRSA isolates were resistant in vitro to most antibiotics except vancomycin. Resistant colonies were observed within cephalothin disc zones of inhibition at 30C (resistance was confirmed by microtube-dilution sensitivity testing). MRSA disc sensitivity testing for cephalothin may be unreliable when performed at 35C.

Adult↗

Measurement of 'blocking antibodies' to pollen antigens by radiolabelled protein A from Staphylococcus aureus.

Radiolabelled protein A from Staphylococcus aureus (125I-SpA) was utilized to measure the relative concentrations of the so-called 'blocking antibodies' (BA) in sera from patients subjected to two different forms of desensitization therapy. In addition, sera from rabbits immunized actively with pollen antigens were assayed. Antigens coupled to CNBr-activated papear discs were used as solid phase, and BA bound to the solid-phase antigens were detected by 125I-SpA. The results obtained with this technique correlated excellently with those of a double-antibody technique for IgG, indicating the 125I-SpA assay to be suitable for the measurement of the antigen-specific IgG. The BA concentration in sera from patients subjected to conventional injection therapy increased significantly during the treatment period, whereas no increase could be detected in sera from patients subjected to sublingual treatment. The increases in BA concentrations of rabbit antisera to pollen antigens could be readily traced by this technique.

Adolescent↗

An in vitro investigation of the intracellular bioactivity of amoxicillin, clindamycin, and erythromycin for Staphylococcus aureus.

The intraphagocytic bioactivities for Staphylococcus aureus of amoxicillin, clindamycin, and erythromycin (0.0075-20 micrograms/ml) were measured in human polymorphonuclear leukocytes (PMNLs) with the combination of a fluorochrome microassay and a radioassay. PMNLs with normal or depleted membrane-associated oxidative metabolism were used to investigate the interactions that may occur between the intrinsic O2-dependent antimicrobial systems of human phagocytes and antimicrobial agents in the elimination of intracellular microbial pathogens. Neutralization of O2-dependent antimicrobial systems with retention of phagocytic capacity was achieved with use of PMNLs from four children with chronic granulomatous disease (CGD) or NaF-pulsed normal PMNLs. None of the test antibiotics possessed intracellular bactericidal activity. Clindamycin and erythromycin possessed significant intracellular bacteriostatic activity relative to the modest activity of amoxicillin. Optimal intracellular bioactivity of all three antibiotics was obtained with normal PMNLs relative to NaF-pulsed or CGD PMNLs, a result indicating the existence of beneficial interactions between the antimicrobial agents and the O2-dependent antimicrobial systems of PMNLs.

Amoxicillin↗

Post-functional endoscopic sinus surgery methicillin-resistant Staphylococcus aureus sinusitis.

Methicillin-resistant Staphylococcus aureus (MRSA) is a highly virulent bacterium that is difficult to eradicate. It has become a common nosocomial pathogen, but it also causes sporadic infections in some outpatients. Among 358 chronic sinusitis patients who received functional endoscopic sinus surgery (FESS) for treatment between July 1995 and June 1997 in our department, 18 were infected postoperatively by MRSA by the end of August 1997. One patient was excluded because she received another nasal surgery, partial turbinectomy, and submucous resection of the nasal septum, after FESS. Most of 17 MRSA infected patients presented themselves with mucopurulent nasal discharge and/or nasal crust. The treatment was generally difficult because MRSAs were resistant to multiple antibiotics. When quinolone antibiotics were used to treat most patients, the improvement rate was 76.5%. We conclude that MRSA infections in post-FESS patients might affect the outcome of FESS.

Adolescent↗

[Risk factors and treatment of methicillin-resistant Staphylococcus aureus infections].

METICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS INFECTIONS (MRSA): The maintenance of a catheter in febrile neutropenic patients with MRSA bacteremia is justified, except when the latter persists 2 to 3 days after the initiation of treatment. Prior administration of fluoroquinolones (levofloxacine or ciprofloxacine) is a risk factor significantly associated with the isolation of MRSA. Meticillin resistance is not a factor of mortality in HIV-infected patients exhibiting S. aureus bacteremia. Surveillance blood cultures represent a simple method for identifying patients at high risk of secondary foci. INFECTIONS WITH GLYCOPEPTIDE-INTERMEDIATE MRSA (HETERO-GISA): In liver transplant recipients, the risk of acquiring a hetero-GISA is not associated with prior MRSA infection nor prior glycopeptide treatment. It is significantly associated with the occurrence of an infectious episode in the weeks preceding the transplant and with the administration of beta-lactams during the previous two months. Mortality is not increased in patients with hetero-GISA. NEW ANTIBIOTICS: Linezolide and quinupristine-dalfopristine are new agents available for the treatment of MRSA infection. Other antibiotics under development have demonstrated their activity: lipopeptides (daptomycin), semi-synthetic glycopeptides (oritavancin, dalbavancin), glycylglycines (tigecyclin), carbapenems (CP5609), and cephalosphorins (LB 11058). ANTIBIOTIC COMBINATIONS: Various studies have shown that antibiotic combinations must be used with care in the treatment of MRSA and GISA infections (glycopeptide intermediate strains) because of the antagonism between molecules belonging to different families.

Anti-Bacterial Agents↗

Community-acquired methicillin-resistant Staphylococcus aureus infections.

Methicillin-resistant Staphylococcus aureus (MRSA) should no longer be regarded as a strictly nosocomial pathogen. During the past decade, community-acquired MRSA (CA-MRSA) infections among young persons without healthcare-associated (HCA) risk factors have emerged in several areas worldwide. These infections are caused by strains that almost exclusively carry the staphylococcal cassette chromosome mec type IV element and the Panton-Valentine leukocidin genes and, unlike HCA-MRSA strains, are not multiresistant. Although the majority of CA-MRSA infections are mild skin and soft tissue infections, severe life-threatening cases of necrotizing pneumonia, necrotizing fasciitis, myonecrosis and sepsis have been reported. Clindamycin is an effective agent for skin and soft tissue infections, however attention should be paid to the possibility of the emergence of resistance during treatment in strains with the macrolide, lincosamide and group B streptogramin (MLS(B))-inducible resistance phenotype. For patients with invasive infections that may be caused be CA-MRSA, vancomycin, teicoplanin and linezolid represent appropriate empirical therapeutic options.

Anti-Bacterial Agents↗

Personal hygiene and methicillin-resistant Staphylococcus aureus infection.

Methicillin-resistant Staphylococcus aureus (MRSA) infections outside the healthcare setting are an increasing concern. We conducted a case-control study to investigate an MRSA outbreak during 2002-2003 in a Missouri prison and focused on hygiene factors. Information on sociodemographic characteristics, medical history, and hygiene practices of study participants was collected by interview and medical record review. Logistic regression was used to evaluate MRSA infection in relation to hygiene factors individually and as a composite hygiene score; potential confounding factors were controlled. Selected MRSA isolates were analyzed by pulsed-field gel electrophoresis (PFGE). MRSA infection was significantly associated with a low composite hygiene score. Transmission among prison inmates appeared to be responsible for this outbreak. PFGE analysis showed that isolates were indistinguishable and associated with community-onset MRSA infections in other US prisons. Improving hygiene practices and environmental conditions may help prevent and interrupt future MRSA outbreaks in prison settings.

Adult↗

Attenuation and persistence of and ability to induce protective immunity to a Staphylococcus aureus aroA mutant in mice.

Staphylococcus aureus is the most important etiological agent of bovine mastitis, a disease that causes significant economic losses to the dairy industry. Several vaccines to prevent the disease have been tested, with limited success. The aim of this study was to obtain a suitable attenuated aro mutant of S. aureus by transposon mutagenesis and to demonstrate its efficacy as a live vaccine to induce protective immunity in a murine model of intramammary infection. To do this, we transformed S. aureus RN6390 with plasmid pTV1ts carrying Tn917. After screening of 3,493 erythromycin-resistant colonies, one mutant incapable of growing on plates lacking phenylalanine, tryptophan, and tyrosine was isolated and characterized. Molecular characterization of the mutant showed that the affected gene was aroA and that the insertion occurred 756 bp downstream of the aroA start codon. Complementation of the aroA mutant with a plasmid carrying aroA recovered the wild-type phenotype. The mutant exhibited a 50% lethal dose (1 x 10(6) CFU/mouse) higher than that of the parental strain (4.3 x 10(4) CFU/mouse). The aroA mutant showed decreased ability to persist in the lungs, spleens, and mammary glands of mice. Intramammary immunization with the aroA mutant stimulated both Th1 and Th2 responses in the mammary gland, as ascertained by reverse transcription-PCR, and induced significant protection from challenge with either the parental wild-type or a heterologous strain isolated from a cow with mastitis.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus.

Most cases of severe Staphylococcus aureus disease cannot be explained by the action of a single virulence determinant, and it is likely that a number of factors act in combination during the infective process. This study examined the relationship between disease in humans and a large number of putative virulence determinants, both individually and in combination. S. aureus isolates (n = 334) from healthy blood donors and from patients with invasive disease were compared for variation in the presence of 33 putative virulence determinants. After adjusting for the effect of clonality, seven determinants (fnbA, cna, sdrE, sej, eta, hlg, and ica) were significantly more common in invasive isolates. All seven factors contributed independently to virulence. No single factor predominated as the major predictor of virulence, their effects appearing to be cumulative. No combinations of the seven genes were either more or less likely to cause disease than others with the same number of virulence-associated genes. There was evidence of considerable horizontal transfer of genes on a background of clonality. Our findings also suggested that allelic variants of a polymorphic locus can make different contributions to the disease process, further study of which is likely to expand our understanding of staphylococcal disease pathogenesis.

Adhesins, Bacterial↗

A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G.

Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.

Bacterial Proteins↗

Novel mechanism of antibiotic resistance originating in vancomycin-intermediate Staphylococcus aureus.

As an aggressive pathogen, Staphylococcus aureus poses a significant public health threat and is becoming increasingly resistant to currently available antibiotics, including vancomycin, the drug of last resort for gram-positive bacterial infections. S. aureus with intermediate levels of resistance to vancomycin (vancomycin-intermediate S. aureus [VISA]) was first identified in 1996. The resistance mechanism of VISA, however, has not yet been clarified. We have previously shown that cell wall thickening is a common feature of VISA, and we have proposed that a thickened cell wall is a phenotypic determinant for vancomycin resistance in VISA (L. Cui, X. Ma, K. Sato, et al., J. Clin. Microbiol. 41:5-14, 2003). Here we show the occurrence of an anomalous diffusion of vancomycin through the VISA cell wall, which is caused by clogging of the cell wall with vancomycin itself. A series of experiments demonstrates that the thickened cell wall of VISA could protect ongoing peptidoglycan biosynthesis in the cytoplasmic membrane from vancomycin inhibition, allowing the cells to continue producing nascent cell wall peptidoglycan and thus making the cells resistant to vancomycin. We conclude that the cooperative effect of the clogging and cell wall thickening enables VISA to prevent vancomycin from reaching its true target in the cytoplasmic membrane, exhibiting a new class of antibiotic resistance in gram-positive pathogens.

Cell Wall↗

Effect of storage and changes in bacterial growth phase and antibiotic concentrations on antimicrobial tolerance in Staphylococcus aureus.

Forty clinical isolates of Staphylococcus aureus were tested for tolerance to oxacillin and cephalothin by broth dilution susceptibility tests and killing curves. Most experiments were carried out with stationary-phase inocula, but nine tolerant isolates were retested with log-phase inocula. All 40 isolates were retested in killing curves at double the antibiotic concentrations used in initial tests. Isolates were retested for tolerance to oxacillin after storage at --70 degrees C for 1 year. In broth dilution tests, 23 of 40 (57.5%) and 20 of 40 (50%) isolates were tolerant to oxacillin and cephalothin, respectively. By killing curves, 25 of 40 (62.5%) and 22 of 40 (55%) isolates were tolerant to oxacillin and cephalothin, respectively. When nine tolerant isolates were retested with log-phase inocula, none manifested tolerance. Only 25 to 30% of the isolates were tolerant in killing curves performed with oxacillin and cephalothin at concentrations double those used in initial tests. After storage at --70 degrees C for 1 year, only two-thirds of the isolates remained tolerant. In isolates that remained tolerant, the degree of tolerance diminished to about 25% of that observed in initial tests.

Anti-Bacterial Agents↗

Granulation tissue formation in experimental wounds inoculated with Staphylococcus aureus.

The effects of inoculated Staphylococcus aureus microorganisms on developing granulation tissue were studied in rats. Hollow cylindrical viscose cellulose sponges were implanted subcutaneously as an inductive matrix for the growth of granulation tissue. The control implants were injected immediately after implantation with 1 ml of physiological saline while the experimental implants were injected with a corresponding volume of saline solution containing live staphylococci in the following concentrations: 10(2), 10(3), 10(4) or 10(5) microorganisms per milliliter. Analyses of wound fluid and granulation tissue were carried out two weeks after implantation. Implants inoculated with 10(3) or more organisms reproducibly developed infection which delayed healing while implants inoculated with 10(2) bacteria showed no infection and were usually able to clear themselves from the organisms. In the latter implants the number of wound fluid monocytes and macrophages was markedly elevated and the mean amount of collagen hydroxyproline exceeded the control level by 55%. This demonstrates that wound healing can be accelerated to a certain extent by inoculation of live staphylococci. The acceleration concerns only such bacterial concentrations which are able to promote a local inflammatory response but which can be governed by wound defence mechanisms.

Animals↗

Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus.

The sarA locus in Staphylococcus aureus controls the expression of many virulence genes. The sarA regulatory molecule, SarA, is a 14.7-kDa protein (124 residues) that binds to the promoter region of target genes. Here we report the 2.6 A-resolution x-ray crystal structure of the dimeric winged helix SarA protein, which differs from the published SarA structure dramatically. In the crystal packing, multiple dimers of SarA form a scaffold, possibly via divalent cations. Mutations of individual residues within the DNA-binding helix-turn-helix and the winged region as well as within the metal-binding pocket implicate basic residues R84 and R90 within the winged region to be critical in DNA binding, whereas acidic residues D88 and E89 (wing), D8 and E11 (metal-binding pocket), and cysteine 9 are essential for SarA function. These data suggest that the winged region of the winged helix protein participates in DNA binding and activation, whereas the putative divalent cation binding pocket is only involved in gene function.

Amino Acid Sequence↗