Antimicrobial effect and mode of action of terpeneless cold-pressed Valencia orange essential oil on methicillin-resistant Staphylococcus aureus.

AIMS: The objectives of this study were to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic-resistant Staphylococcus aureus strains. METHODS AND RESULTS: The inhibitory effect of commercial orange essential oil (EO) against six Staph. aureus strains was tested using disc diffusion and agar dilution methods. The mechanism of EO action on MRSA was analysed by transcriptional profiling. Morphological changes of EO-treated Staph. aureus were examined using transmission electron microscopy. Results showed that 0·1% of terpeneless cold-pressed Valencia orange oil (CPV) induced the cell wall stress stimulon consistent with the inhibition of cell wall synthesis. Transmission electron microscopic observation revealed cell lysis and suggested a cell wall lysis-related mechanism of CPV. CONCLUSIONS: CPV inhibits the growth of Staph. aureus, causes gene expression changes consistent with the inhibition of cell wall synthesis, and triggers cell lysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple antibiotics resistance is becoming a serious problem in the management of Staph. aureus infections. In this study, the altered expression of cell wall-associated genes and subsequent cell lysis in MRSA caused by CPV suggest that it may be a potential antimicrobial agent to control antibiotic-resistant Staph. aureus.

Genetic changes that correlate with the pine-oil disinfectant-reduced susceptibility mechanism of Staphylococcus aureus.

AIMS: To identify factors associated with the Staphylococcus aureus pine-oil disinfectant-reduced-susceptibility (PD(RS)) mechanism and to describe one possible PD(RS) model. METHODS AND RESULTS: Comparative genomic sequencing (CGS) and microarray analysis were utilized to detect mutations and transcriptome alterations that occur in a S. aureus PD(RS) mutant. Mutant analysis, antimicrobial gradient plates, growth studies and 3-hydroxy-3-methylglutaryl coenzyme A synthase assays were then performed to confirm the biological consequences of the 'omics' alterations detected in a PD(RS) mutant. CGS uncovered three mutations in a PD(RS) mutant in a(n): alcohol dehydrogenase (adh), catabolite control protein A (ccpA) and an NADPH-flavin oxidoreductase (frp). These mutations lead to increased growth rates; increased transcription of an NAD-dependent D-lactate dehydrogenase gene (ddh); and increased flux through the mevalonate pathway. PD(RS) mutants demonstrated reduced susceptibility to bacitracin and farnesol, and one PD(RS) mutant displayed upregulation of bacA, a bacitracin-resistance gene. Collectively, this evidence demonstrates altered undecaprenol metabolism in PD(RS) mutants. CONCLUSIONS: The PD(RS) mechanism proposed results from increased catabolic capabilities and increased flux through the mevalonate pathway as well as altered bactoprenol physiology. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel mechanism that bacteria utilize to overcome the killing effects of PD formulations is proposed that is unique from the PD(RS) mechanism of the enterobacteraciae.

The key role of peptidoglycan in the opsonization of Staphylococcus aureus.

In an effort to determine the staphylococcal cell surface component(s) of importance in opsonization, cell walls (peptidoglycan and teichoic acid) and peptidoglycan were isolated from Staphylococcus aureus strain H grown in [3H]glycine-containing broth. After incubation of the cell walls and peptidoglycan with various opsonic sources, uptake by human polymorphonuclear leukocytes was measured. The opsonic requirements for phagocytosis of cell walls and peptidoglycan were found to be similar to those of intact bacteria. Removal of teichoic acid from the cell wall did not affect opsonization. Likewise, a teichoic acid-deficient mutant strain of S. aureus H was opsonized in a manner similar to that of the parent strain. Immunoglobulin G functioned as the major heat-stable opsonic factor and both the classical and alternative pathways participated in opsonization. Kinetic studies revealed that opsonization of peptidoglycan, as well as C3-C9 consumption by peptidoglycan, proceeded at a slower rate via the alternative pathway (C2-deficient serum) than when the classical pathway was present (normal serum). The ability of peptidoglycan to activate C3-C9 was significantly reduced when normal and C2-deficient sera were preabsorbed with peptidoglycan at 2 degrees C suggesting that antibodies to peptidoglycan may be involved in activation of both the classical and alternative complement pathways. Thus, peptidoglycan appears to be the key cell wall component involved in staphylococcal opsonization, and it is suggested that host response to peptidoglycan, a major cell wall component of most gram-positive bacteria, may be related to the development of "natural immunity" to this group of microorganisms.

Sodium-dependent uptake of taurine in encapsulated Staphylococcus aureus strain M.

A novel uptake system for the unusual sulfonated amino acid taurine was discovered in the prokaryote, encapsulated Staphylococcus aureus strain M. This strain has been shown previously to contain taurine in its capsular polysaccharide. Taurine uptake by whole cells incubated in buffer showed a saturable dependency upon Na+ and taurine uptake was itself a saturable process, stimulated by glucose, and markedly affected by temperature. No evidence was found for the inducibility of taurine uptake. In the presence of 10 mM NaCl Lineweaver-Burk plots revealed a Km of 42 microM and Vmax of 4.6 nmol/min per mg dry weight for taurine uptake at 37 degrees C. Increasing concentrations of Na+ decreased the Km of the system and appeared to increase the Vmax. Of various other cations tested only Li+ supported marked taurine uptake. Excess unlabelled taurine did not cause efflux of radioactivity taken up. Taurine was taken up into cold trichloroacetic acid-soluble material and did not chromatograph as taurine, indicating rapid metabolism during or closely following uptake. Taurine uptake appeared to occur via a highly specific system because amino acids representing the major known groups of amino acid transport systems in S. aureus did not inhibit taurine uptake, and uptake was only slightly diminished by the structurally closely related compounds hypotaurine and 3-amino-1-propane sulfonic acid. Sulfhydryl group reagents, electron transport inhibitors, an uncoupler and inhibitors of Na+-linked transport processes inhibited taurine uptake. A variety of other metabolic inhibitors had little effect on taurine uptake.

Cyanide-resistant electron transport in sporulating Bacillus megaterium KM.

The NADH oxidase activity of stage V mother-cell membranes, isolated from sporulating Bacillus megaterium KM, shows a greater inhibition by cyanide and displays this response at lower concentrations of cyanide than the stage V forespore inner membrane. Comparison of the effects of various respiratory inhibitors reveals that the difference in cyanide sensitivity between these membranes is located on the oxidase side of the 2-heptyl-4-hydroxyquinoline N-oxide-sensitive step. Both membranes contain cytochromes a+a3, b-562, b-555, c and d, with three potential oxidases: cytochromes a+a3, o and d. Cyanide difference spectra suggest that cytochromes b-562 and d may be the components involved in the cyanide-resistant electron transport pathway. Membrane ascorbate-N,N,N',N'-tetramethylphenylenediamine and ascorbate 2,6-dichlorophenolindophenol oxidase activities are highly sensitive to cyanide. Evidence is presented for terminal branching of the respiratory chain with branches differing in cyanide sensitivity. The cyanide sensitivity of the NADH oxidase of membranes prepared from various stages of sporulation is compared. Morphogenesis of the mother-cell plasma membrane to a cyanide-sensitive form during stages II and III of sporulation is postulated.

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes.

Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures. Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L. monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L. monocytogenes, presumably by maintaining membrane fluidity. The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L. monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region. tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C. However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1. No evidence was seen for a clear lipid phase transition in any sample. We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L. monocytogenes membrane that permits growth at refrigeration temperatures.

Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.

The escalating challenge of vancomycin resistance in Staphylococcus aureus.

The glycopeptide antibiotic vancomycin is considered indispensable for the treatment of multidrug-resistant Staphylococcus aureus infections, and so the acquisition by these organisms of transmissible glycopeptide resistance elements from enterococci had been anticipated with apprehension. It was therefore a considerable surprise when vancomycin-intermediate S. aureus (VISA) clinical isolates were reported in 1997, with a novel, borderline-resistance phenotype acquired without genetic exchange. Clinical vancomycin-resistant S. aureus (VRSA) were not reported until 2002, expressing high level, transmissible resistance by virtue of vanA resistance determinants within enterococcal transposable elements residing on staphylococcal plasmids. This review will provide an update on the frustratingly variable characteristics of the VISA phenotype, focus on the progress made in understanding the molecular basis of the VISA resistance mechanism from the viewpoint of genetic regulation and cell wall stress response, and summarize the information currently available on VRSA. Finally, alternatives to vancomycin that are already available or nearing approval will be briefly reviewed, with attention to their limitations and potential for resistance development.

Transdermal nicotine and haloperidol in Tourette's disorder: a double-blind placebo-controlled study.

BACKGROUND: Preclinical animal and open-trial clinical trials using nicotine gum and the transdermal nicotine patch found that treatment with nicotine potentiates the effects of neuroleptics in reducing the dyskinetic symptoms of Tourette's disorder. We sought to verify and expand these findings in a prospective double-blind placebo-controlled trial. METHOD: Seventy patients with DSM-IV Tourette's disorder were treated with either transdermal nicotine (7 mg/24 hours) or placebo patches in a 33-day, randomized, double-blind study. Each patient received an individually based optimal dose of haloperidol for at least 2 weeks prior to random assignment to nicotine or placebo treatment. A new patch was worn each day for the first 5 days. On the sixth day, the dose of haloperidol was reduced by 50%. Daily patch applications were then continued for an additional 2 weeks (through day 19), at which time the patch was discontinued, but the 50% dose of haloperidol was continued for an additional 2 weeks (through day 33). Clinical and safety assessments were made at each visit. RESULTS: Patients who completed all 19 days of nicotine (N = 27) or placebo (N = 29) patch treatment were used in efficacy analyses. As documented by the Clinician- and Parent-rated Global Improvement scales, transdermal nicotine was superior to placebo in reducing the symptoms of Tourette's disorder. The Yale Global Tic Severity Scale was less sensitive in detecting a placebo/drug difference than were the global improvement scores, suggesting that some of the improvement may not have been related to treatment-related changes in tic severity, but to the emotional and behavioral symptoms. The side effects of nausea and vomiting were significantly more common in the nicotine group (71% [N = 25] and 40% [N = 14]) than in the placebo group (17% [N = 6] and 9% [N = 3]) (nausea, p = .0001; vomiting, p = .004). CONCLUSION: Transdermal nicotine was superior to placebo in reducing behavioral symptoms when patients were receiving an optimal dose of haloperidol, when the dose of haloperidol was reduced by 50%, and when the patch had been discontinued for 2 weeks. These findings confirm earlier open-label findings and suggest that combining nicotinic receptor modulation and neuroleptics could be a therapeutic option for the treatment of Tourette's disorder. While side effects limit chronic use of nicotine, it may be useful on a p.r.n. basis. Further clinical research is warranted to investigate the use of novel nicotinic receptor modulating agents with improved safety profiles over nicotine.

Neutrophil chemotactic activity of peptidoglycan. A comparison between Staphylococcus aureus and Staphylococcus epidermidis.

The ability of peptidoglycan from Staphylococcus epidermidis and Staphylococcus aureus to generate in human serum chemoattractant for peripheral blood neutrophils was studied. It was shown that PG from the two bacteria was able to induce chemotactic activity in normal human serum. Sonication of PG was required to generate this activity. Very little or no activity was generated in heat-treated or C5-deficient human serum by PG, indicating that PG treatment of serum resulted in generation of chemoattractants by activation of complement. Kinetics studies employing C2-deficient or MgEGTA-chelated serum revealed that S. epidermidis induced chemotactic activity by activating the alternative complement pathway. The alternative complement activation induced by S. epidermidis occurred rapidly and was completed after 15 min, whereas S. aureus activated the alternative pathway much more slowly, with activation reaching a maximum at 60 min. The rapid activation of the alternative complement pathway by S. epidermidis PG may partly explain why this bacterium does not normally cause infections in healthy individuals.

Attachment of staphylococci to silicone catheters in vitro.

The adherence of radiolabeled staphylococci to silicone catheters was investigated in vitro. Staphylococcus aureus and Staphylococcus epidermidis strains bound to the same extent to the catheters. Also, S. epidermidis strains isolated from patients with plastic-related infections showed binding similar to that of other S. epidermidis strains. By preincubation of catheters the influence of purified staphylococcal cell surface components on the binding was evaluated. The most potent inhibitors of the binding of S. aureus were the two surface proteins, clumping factor and protein A, and the cytoplasmic membrane. Surface proteins and the cell membrane of S. epidermidis also blocked the binding. Only protein-containing surface proteins inhibited the binding. The production of slime correlated with the degree of S. epidermidis binding. Human plasma and serum, as well as purified albumin and IgG, inhibited the binding of both staphylococcal species. Fibrinogen, and to a certain extent fibronectin, inhibited the binding of S. epidermidis, while both these purified plasma proteins enhanced the binding of S. aureus.

A microdilution plating method for population analysis of antibiotic-resistant staphylococci.

The microdilution plating method, using colony-forming units (CFU)/ml determinations from 10-microl droplets, was compared with the standard plate count in population analyses of methicillin-resistant and glycopeptide-intermediate Staphylococcus aureus (MRSA and GISA) strains. Efficiency of plating plots yielded similar population resistance profiles for both methods with MRSA class 1-4 strains, laboratory-selected GISA strains of varying susceptibilities, two clinical GISA strains, as well as susceptible strains. A single heterogeneous MRSA, plated by both methods in 41 trials with and without 50 microg/ml oxacillin present, demonstrated no significant difference between the results of the two methods of colony counting (p > 0.05, and r = 0.67). Standard plating and microdilution plating produced mean resistant subpopulation determinations of one cell in 1.19 x 10(4) and 1.36 x 10(4), respectively. Population analyses carried out by microdilution plating require one-fourth or fewer of the plates used for standard plating, and both plating and colony counting required less time to perform.

Lower autolytic activity in a homogeneous methicillin-resistant Staphylococcus aureus strain compared to derived heterogeneous-resistant and susceptible strains.

It has been proposed that in addition to production of a penicillin-binding protein with low affinity for beta-lactam antibiotics, control of autolysin activity is involved in the mechanism of staphylococcal methicillin resistance. A homogeneous methicillin-resistant Staphylococcus aureus strain (DU4916) had lower rates of unstimulated, NaCl- and Triton X-100-stimulated autolysis, and daptomycin (LY146032)-induced lysis than a heterogeneous methicillin-resistant strain (DU4916-K7) and a methicillin-susceptible strain (DU4916S) derived from DU4916.

Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach.

Proteins produced in elevated amounts in response to oxacillin challenge of Staphylococcus aureus strain RN450, were studied by comparing Coomassie blue stained two-dimensional gels of cellular proteins. At least nine proteins were produced in elevated amounts following exposure to growth inhibitory concentrations of oxacillin. N-terminal sequences were obtained for five of the proteins and the databases were searched to tentatively identify them. The proteins were identified as homologs of (i) methionine sulfoxide reductase (MsrA); (ii) a signal transduction protein (TRAP) involved in regulating RNAIII production encoded by the agr locus; (iii) transcription elongation factor GreA; (iv) the heat shock protein GroES; and (v) the enzyme IIA component of the phosphoenolpyruvate:sugar phosphotransferase system. A similar induction response was observed with the other cell wall-active antibiotics, but not with antibiotics that affect other cellular targets. Increased transcription of the msrA and groEL genes in response to cell wall-active antibiotics was also demonstrated. Although net protein synthesis is inhibited subsequent to inhibition of peptidoglycan biosynthesis by cell wall-active antibiotics, some proteins are induced in S. aureus, presumably in an attempt by the cell to counter the inhibitory effects of these agents.

Conditions that induce Staphylococcus aureus heat shock proteins also inhibit autolysis.

When Staphylococcus aureus strain 8325 was grown at 30 degrees C and heat shocked at 40 degrees C the rate of cell autolysis in buffer with or without Triton X-100 was reduced. Treatment of growing cells with other agents (CdCl2, ethanol, NaCl) known to induce heat shock proteins also resulted in cells that showed a decreased rate of autolysis. Heat shocked cells showed lower rates of freeze-thaw autolysin activity on purified cell walls, and isolated crude cell walls from heat shocked cells had lower rates of autolytic activity compared to controls. No differences in the peptidoglycan hydrolase activity profiles of control and heat shocked cells were detected by renaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is proposed that autolysins are damaged by heat shock and their targeting to the cell wall is impaired, possibly by complexing with heat shock proteins, which may also inhibit autolysin activity. Heat shock also inhibited the autolytic activity of methicillin-resistant and related-susceptible strains, and the possible relationship of this to the expression of methicillin resistance is discussed.

Hydrophobicity-hydrophilicity of staphylococci.

The hydrophobicity-hydrophilicity of various strains of Staphylococcus has been studied by a technique involving partitioning of the cells between aqueous and hydrocarbon phases. S. aureus was typically hydrophobic, and to a greater degree in stationary- than in exponential-phase cultures. Mutants that lacked teichoic acid, protein A or coagulase production were hydrophobic, indicating that none of these factors was responsible for hydrophobicity. The presence of a capsule rendered strains hydrophilic. Thus, determination of hydrophobicity may be a useful additional test for capsulation. However, a non-capsulate S. aureus strain was hydrophilic. Trypsin treatment converted strains from hydrophobic to hydrophilic. Isolated bacterial cell wall preparation, either crude or purified, and peptidoglycan were hydrophilic. These results indicate that the determinant of hydrophobicity is a protein or protein-associated molecule localised at the cell surface of the organism, i.e., a component of either the cell wall, cell membrane, or both. Twenty-five strains of twelve coagulase-negative species were examined and most (18) were hydrophobic, again indicating that protein A is not a major determinant of hydrophobicity in these staphylococci. Four of seven hydrophilic strains were capsulate; three strains of S. sciuri were hydrophilic but non-capsulate.

Autolytic properties of glycopeptide-intermediate Staphylococcus aureus Mu50.

Whole-cell autolytic activity of prototypical glycopeptide-intermediate Staphylococcus aureus (GISA) Mu50 was reduced versus that of hetero-GISA Mu3 and glycopeptide-susceptible S. aureus, consistent with other GISA strains. In contrast, autolytic activity was relatively high in Mu50 crude cell walls and autolysin extracts against purified cell walls, reflecting the complexities of autolytic activity regulation.

Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners.

AIMS: To characterize mutants of Staphylococcus aureus expressing reduced susceptibility to house cleaners (HC), assess the impact of the alternative sigma factor SigB on HC susceptibility, and determine the MIC of clinical methicillin-resistant S. aureus (MRSA) to a HC. METHODS AND RESULTS: Susceptibility to HC, HC components, H2O2, vancomycin and oxacillin and physiological parameters were determined for HC-reduced susceptibility (HCRS) mutants, parent strain COL and COLsigB::kan. HCRS mutants selected with three HC expressed reduced susceptibility to multiple HC, HC components, H2O2 and vancomycin. Two unique HCRS mutants also lost the methicillin resistance determinant. In addition, all HCRS mutants exhibited better growth at two temperatures, and one HCRS mutant expressed reduced carotenoid production. COLsigB::kan demonstrated increased susceptibility to all HC and many HC components. sigB operon mutations were not detected in one HCRS mutant background. Of 76 clinical MRSA, 20 exhibited reduced susceptibility to a HC. CONCLUSIONS: HCRS mutants demonstrate altered susceptibility to multiple antimicrobials. While sigB is required for full HC resistance, one HCRS mechanism does not involve sigB operon mutations. Clinical MRSA expressing reduced susceptibility to a common HC were detected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that HCRS mutants are not protected against, nor selected by, practical HC concentrations.

Staphylococcus aureus cell surface: capsule as a barrier to bacteriophage adsorption.

Encapsulated Staphylococcus aureus strains M and Smith diffuse bound phage 84 and 52A much less efficiently than their unencapsulated counterparts, M variant and Smith compact. It is proposed that the capsule acts as a barrier excluding phage from interaction with their receptor (cell wall peptidoglycan and teichoic acid). Inefficient phage adsorption by encapsulated staphylococci may explain, in part, the poor phage typing of such strains.

Increased outer membrane ornithine-containing lipid and lysozyme penetrability of Paracoccus denitrificans grown in a complex medium deficient in divalent cations.

Paracoccus denitrificans grown in a complex medium was highly susceptible to lysozyme, in contrast to cells grown in a complex medium supplemented with Mg2+ and Ca2+ or in a succinate-salts medium. The complex medium was deficient in divalent cations needed for optimum outer membrane stability. The major change in molecular compositions of outer membranes isolated from cells grown under the different conditions was a higher ratio of ornithine-containing lipid to phospholipid in complex-medium-grown cells (0.63) than in cells grown in complex medium with Mg2+ and Ca2+ (0.22) or in succinate-salts medium (0.14). We suggest that the dipolarionic ornithine-containing lipid is less dependent than acidic phospholipids on divalent cations for its incorporation into the outer membrane.