Effect of a staphylococcin on Neisseria gonorrhoeae.
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Phage group 2 staphylococcal strain UT0002 contains a large 56S virulence plasmid with genes that code for both exfoliative toxin and a specific staphylococcin termed Bac R(1). Four penicillinase-producing strains and three penicillin-susceptible strains of Neisseria gonorrhoeae were killed by Bac R(1). After 30 min of growth of the penicillin-resistant TR1 strain in 62.5 arbitrary units of Bac R(1) per ml, loss of viability was approximately 90%, and, after 5 h, an approximately 99.99% loss of viability was observed. Lysis did not accompany cell death, and 84% of the Bac R(1) added to the growth medium was adsorbed to the gonococcal cells. The extracellular supernatant fluid from a substrain of staphylococcal strain UT0002 cured of the plasmid for Bac R(1) production had no lethal effect on the gonococcal strains. Bac R(1) was also shown to have bactericidal activity against an L-form of N. meningitidis, indicating that the outer envelope of a neisserial cell is not needed for bacteriocin activity. Ten different normal human sera were unable to neutralize Bac R(1) activity. The bacteriocin lacks adsorption specificity. It binds to but does not kill Escherichia coli cells, indicating that the cell envelope of gram-negative organisms can provide protection against the staphylococcin. (+info)
Prodigious substrate specificity of AAC(6')-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci.
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BACKGROUND: High-level gentamicin resistance in enterococci and staphylococci is conferred by AAC(6')-APH(2"), an enzyme with 6'-N-acetyltransferase and 2"-O-phosphotransferase activities. The presence of this enzyme in pathogenic gram-positive bacteria prevents the successful use of gentamicin C and most other aminoglycosides as therapeutic agents. RESULTS: In an effort to understand the mechanism of aminoglycoside modification, we expressed AAC(6')-APH(2") in Bacillus subtilis. The purified enzyme is monomeric with a molecular mass of 57 kDa and displays both the expected aminoglycoside N-acetyltransferase and O-phosphotransferase activities. Structure-function analysis with various aminoglycosides substrates reveals an enzyme with broad specificity in both enzymatic activities, accounting for AAC(6')-APH(2")'s dramatic negative impact on clinical aminoglycoside therapy. Both lividomycin A and paromomycin, aminoglycosides lacking a 6'-amino group, were acetylated by AAC(6')-APH(2"). The infrared spectrum of the product of paromomycin acetylation yielded a signal consistent with O-acetylation. Mass spectral and nuclear magnetic resonance analysis of the products of neomycin phosphorylation indicated that phosphoryl transfer occurred primarily at the 3'-OH of the 6-aminohexose ring A, and that some diphosphorylated material was also present with phosphates at the 3'-OH and the 3"'-OH of ring D, both unprecedented observations for this enzyme. Furthermore, the phosphorylation site of lividomycin A was determined to be the 5"-OH of the pentose ring C. CONCLUSIONS: The bifunctional AAC(6')-APH(2") has the capacity to inactivate virtually all clinically important aminoglycosides through N- and O-acetylation and phosphorylation of hydroxyl groups. The extremely broad substrate specificity of this enzyme will impact on future development of aminoglycosides and presents a significant challenge for antibiotic design. (+info)
The staphylococcal transferrin-binding protein is a cell wall glyceraldehyde-3-phosphate dehydrogenase.
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Staphylococcus aureus and Staphylococcus epidermidis possess a 42-kDa cell wall transferrin-binding protein (Tpn) which is involved in the acquisition of transferrin-bound iron. To characterize this protein further, cell wall fractions were subjected to two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis blotted, and the N-terminus of Tpn was sequenced. Comparison of the first 20 amino acid residues of Tpn with the protein databases revealed a high degree of homology to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Analysis of staphylococcal cell wall fractions for GAPDH activity confirmed the presence of a functional enzyme which, like Tpn, is regulated by the availability of iron in the growth medium. To determine whether Tpn is responsible for this GAPDH activity, it was affinity purified with NAD+ agarose. Both S. epidermidis and S. aureus Tpn catalyzed the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. In contrast, Staphylococcus saprophyticus, which lacks a Tpn, has no cell wall-associated GAPDH activity. Native polyacrylamide gel electrophoresis of the affinity-purified Tpn revealed that it was present in the cell wall as a tetramer, consistent with the structures of all known cytoplasmic GAPDHs. Furthermore, the affinity-purified Tpn retained its ability to bind human transferrin both in its native tetrameric and SDS-denatured monomeric forms. Apart from interacting with human transferrin, Tpn, in common with the group A streptococcal cell wall GAPDH, binds human plasmin. Tpn-bound plasmin is enzymatically active and therefore may contribute to the ability of staphylococci to penetrate tissues during infections. These studies demonstrate that the staphylococcal transferrin receptor protein, Tpn, is a multifunctional cell wall GAPDH. (+info)
Molecular characterization of the nitrite-reducing system of Staphylococcus carnosus.
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Characterization of a nitrite reductase-negative Staphylococcus carnosus Tn917 mutant led to the identification of the nir operon, which encodes NirBD, the dissimilatory NADH-dependent nitrite reductase; SirA, the putative oxidase and chelatase, and SirB, the uroporphyrinogen III methylase, both of which are necessary for biosynthesis of the siroheme prosthetic group; and NirR, which revealed no convincing similarity to proteins with known functions. We suggest that NirR is essential for nir promoter activity. In the absence of NirR, a weak promoter upstream of sirA seems to drive transcription of sirA, nirB, nirD, and sirB in the stationary-growth phase. In primer extension experiments one predominant and several weaker transcription start sites were identified in the nir promoter region. Northern blot analyses indicated that anaerobiosis and nitrite are induction factors of the nir operon: cells grown aerobically with nitrite revealed small amounts of full-length transcript whereas cells grown anaerobically with or without nitrite showed large amounts of full-length transcript. Although a transcript is detectable, no nitrite reduction occurs in cells grown aerobically with nitrite, indicating an additional oxygen-controlled step at the level of translation, enzyme folding, assembly, or insertion of prosthetic groups. The nitrite-reducing activity expressed during anaerobiosis is switched off reversibly when the oxygen tension increases, most likely due to competition for electrons with the aerobic respiratory chain. Another gene, nirC, is located upstream of the nir operon. nirC encodes a putative integral membrane-spanning protein of unknown function. A nirC mutant showed no distinct phenotype. (+info)
Changing susceptibilities of coagulase-negative staphylococci to teicoplanin in a teaching hospital.
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The susceptibility of two collections of coagulase-negative staphylococci (CNS) isolated from clinical specimens for teicoplanin and vancomycin were compared. They comprised 91 and 101 isolates, collected in 1985 and 1994 respectively, from different departments of a teaching hospital. MICs of vancomycin and teicoplanin were determined by a modified Etest method. Additionally, a disc diffusion test was performed for teicoplanin. All isolates were susceptible to vancomycin (MIC < or = 4 mg/L). Two of the 91 isolates collected in 1985 were intermediate to teicoplanin (MIC between 8 and 32 mg/L), whereas in 1994 the number of intermediate isolates was 20 out of 101 (P < 0.01). The correlation between MICs, as determined by the modified Etest assay, and disc diffusion zones was poor (r = -0.35). Results show that resistance to teicoplanin in CNS has increased in the study hospital over a period of 9 years. This increase is likely to be correlated with the introduction of teicoplanin. Furthermore, a disc diffusion method does not appear to be the first method of choice for detection of strains of CNS with diminished susceptibility to teicoplanin. (+info)
Evidence for nasal carriage of methicillin-resistant staphylococci colonizing intravascular devices.
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Nasal surveillance cultures were performed for 54 patients exhibiting >/=10(3) CFU of methicillin-resistant coagulase-negative staphylococci per ml in central venous catheter (CVC) rinse cultures over a 6-month period. Forty-two of the nasal cultures yielded growth of methicillin-resistant coagulase-negative staphylococci, and 33 of the 42 cultures contained organisms that belonged to the same species as the CVC isolates. Of the 33 same-species isolates, 20 appeared to be identical strains by pulsed-field gel electrophoresis analysis. These data suggest that measures should be taken to reduce cross-contamination between the respiratory tract and intravascular devices. However, the potential interest in detecting methicillin-resistant coagulase-negative staphylococcus carriage in high-risk patients is hampered by the lack of sensitivity of nasal surveillance cultures. (+info)
Penicillin-binding protein-mediated resistance in pneumococci and staphylococci.
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Target alteration underlies resistance to beta-lactam antibiotics in both Staphylococcus species and Streptococcus pneumoniae. The penicillin-binding protein (PBP) targets in penicillin-resistant strains of S. pneumoniae are modified, low-binding-affinity versions of the native PBPs. Multiple PBP targets may be modified by transformation and homologous recombination with DNA from PBP genes of viridans streptococci. The level of resistance is determined by how many and to what extent targets are modified. In contrast, methicillin resistance in staphylococci is due to expression of PBP 2a, a novel, low-affinity PBP for which there is no homologue in methicillin-susceptible strains. PBP 2a is encoded by mecA, a highly conserved gene most likely acquired by a rare transposition from Staphylococcus sciuri or a closely related ancestor. Expression of resistance can be highly variable, but this seems not to be determined by PBP modifications. Several non-PBP factors are required for high-level resistance. (+info)
Preparation of labeled staphylococcal enterotoxin A with high specific activity.
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Staphylococcal enterotoxin A (SEA) was labeled by the chloramine-T method with 125I to a specific activity of 68 to 300 muCi per mug of SEA and with 131I to specific activity of 8 to 218 muCi per mug of SEA. SEA was partially damaged and aggregated during the labeling and storage. The damage seemed not to be greatly dependent on the specific activity of labeled entertoxin. Crossed immunoelectrophoresis showed two antigenically active and three inactive components in the ascending part of the labeled enterotoxin peak during fractionation by gel chromatography. During storage at 4 degrees C, the antigenic activity of label decreased faster when labeling had been with 131I than when with 125I. The antigenic activity of labeled SEA was lowered remarkably in the ascending part of the protein peak. Greatest release of radioiodine during storage was in the same part of protein peak. According to these results, the most suitable label for radioimmunoassay is obtained from the descending part of protein peak. (+info)