Stimulation of astaxanthin formation in the yeast Xanthophyllomyces dendrorhous by the fungus Epicoccum nigrum

A fungal contaminant on an agar plate containing colonies of Xanthophyllomyces dendrorhous markedly increased carotenoid production by yeast colonies near to the fungal growth. Spent-culture filtrate from growth of the fungus in yeast-malt medium also stimulated carotenoid production by X. dendrorhous. Four X. dendrorhous strains including the wild-type UCD 67-385 (ATCC 24230), AF-1 (albino mutant, ATCC 96816), Yan-1 (beta-carotene mutant, ATCC 96815) and CAX (astaxanthin overproducer mutant) exposed to fungal concentrate extract enhanced astaxanthin up to approximately 40% per unit dry cell weight in the wild-type strain and in CAX. Interestingly, the fungal extract restored astaxanthin biosynthesis in non-astaxanthin-producing mutants previously isolated in our laboratory, including the albino and the beta-carotene mutant. The fungus was identified as Epicoccum nigrum by morphology of sporulating cultures, and the identity confirmed by genetic characterization including rDNA sequencing analysis of the large-subunit (LSU), the internal transcribed spacer, and the D1/D2 region of the LSU. These E. nigrum rDNA sequences were deposited in GenBank under accesssion numbers AF338443, AY093413 and AY093414. Systematic rDNA homology alignments were performed to identify fungi related to E. nigrum. Stimulation of carotenogenesis by E. nigrum and potentially other fungi could provide a novel method to enhance astaxanthin formation in industrial fermentations of X. dendrorhous and Phaffia rhodozyma.     

Antibody used to identify penicillin-binding protein 2' in methicillin-resistant strains of Staphylococcus aureus (MRSA)

We found that tau, one of the major microtubule-associated proteins, is a good substrate for protein kinase C. The phosphorylation occurred mainly on serine residues and the sites phosphorylated by protein kinase C were largely different from those phosphorylated by cAMP-dependent protein kinase as analyzed by phosphopeptide mapping. The protein kinase C-mediated phosphorylation of tau reduced its abilities to promote tubulin polymerization and to cross-link actin filaments. The reduction in its abilities was in proportion to the number of phosphates incorporated into tau.     

Immunological detection of penicillin-binding protein 2'' in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.     

Interactions of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with moenomycin.

Kinetics studies in homogeneous aqueous solution showed that solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus (a bacterial DD-peptidase) was inhibited by the amphiphilic glycolipid antibiotic moenomycin. Inhibition at the peptidase site was determined by competition experiments between moenomycin and the chromophoric beta-lactam nitrocefin. Under conditions of high salt concentration (1 M NaCl), pseudo-first-order rate constants for the reaction of moenomycin with sPBP2a leading to inhibition of acylation by nitrocefin varied with moenomycin concentration in a biphasic fashion. At low moenomycin concentration (<20 microM) little inhibition occurred, but at higher concentrations a linear increase in rate constant with moenomycin concentration was observed, yielding a second-order rate constant of inhibition of 120 s(-)(1) M(-)(1). Since the cmc of moenomycin under these conditions was shown to be ca. 20 microM, the inhibition was concluded to arise from reaction of sPBP2a with a moenomycin micelle. Protein fluorescence studies showed a pseudo-first-order decrease in fluorescence on reaction of the protein with moenomycin. The variation of this rate constant with moenomycin concentration was consistent with reaction of a moenomycin monomer with the protein with a second-order rate constant of 650 s(-)(1) M(-)(1). This monomer reaction did not occur at the DD-peptidase site since its rate was unaffected by prior acylation of the enzyme by benzylpenicillin; nor did it inhibit reaction at that site by beta-lactams. Under low salt conditions (0.175 M NaCl) where reaction could be studied over a greater range of monomer concentrations since the cmc was ca. 120 microM, similar reactions were involved. Under these circumstances, inhibition was concerted with the reaction of moenomycin monomers, although fast premicellar aggregation of moenomycin with the protein also occurred. All moenomycin interactions with sPBP2a were reversible, as revealed by detergent-extraction chromatography. Lower limits to moenomycin off-rates and equilibrium dissociation constants were 7.7 x 10(-)(4) s(-)(1) and 1.2 microM, respectively. Other amphiphiles did not react in exactly the same manner as moenomycin, indicating some degree of specificity in reactions of the latter. sPBP2a did not have detectable affinity for lipid surfaces (Triton X-114 and phosphatidylglycerol vesicles). A general scheme for reaction of moenomycin with sPBP2a is proposed.     

Shared functional attributes between the mecA gene product of Staphylococcus sciuri and penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus

The genome of Staphylococcus aureus is constantly in a state of flux, acquiring genes that enable the bacterium to maintain resistance in the face of antibiotic pressure. The acquisition of the mecA gene from an unknown origin imparted S. aureus with broad resistance to beta-lactam antibiotics, with the resultant strain designated as methicillin-resistant S. aureus (MRSA). Epidemiological and genetic evidence suggests that the gene encoding PBP 2a of MRSA might have originated from Staphylococcus sciuri, an animal pathogen, where it exists as a silent gene of unknown function. We synthesized, cloned, and expressed the mecA gene of S. sciuri in Escherichia coli, and the protein product was purified to homogeneity. Biochemical characterization and comparison of the protein to PBP 2a of S. aureus revealed them to be highly similar. These characteristics start with sequence similarity but extend to biochemical behavior in inhibition by beta-lactam antibiotics, to the existence of an allosteric site for binding of bacterial peptidoglycan, to the issues of the sheltered active site, and to the need for conformational change in making the active site accessible to the substrate and the inhibitors. Altogether, the evidence strongly argues that the kinship between the two proteins is deep-rooted on the basis of many biochemical attributes quantified in this study.     

The basis for resistance to beta-lactam antibiotics by penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus

Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available beta-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several beta-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by beta-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k2) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillin-sensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (Kd) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.     

Methicillin-resistant strains of Staphylococcus aureus; presence of identical additional penicillin-binding protein in all strains examined

Abstract The methicillin-resistant strain of Staphylococcus aureus MR-1 previously reported to possess a penicillin-binding protein 3 (PBP 3) with a decreased affinity for β-lactam antibiotics was re-examined and, in common with other resistant strains, found to contain an additional PBP (PBP 2'). Expression of the additional protein, which has a very low affinity for β-lactams, was not influenced by temperature or osmolarity of the medium in contrast with strains examined previously. It was the only PBP still available to bind radioactive β-lactams and therefore still active enzymically when strain MR-1 was grown in the presence of concentrations of β-lactam antibiotics sufficient to kill sensitive strains of S. aureus . Penicillin-peptides derived by partial proteolysis of PBP 2'-penicillin complexes of MR-1 and 3 other methicillin-resistant strains appeared to be identical and different from the penicillin-peptides derived from PBP 1, PBP 2 and PBP 3, each of which gave rise to a unique series of peptides containing covalently-bound penicillin.     

Characterisation of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2'' of methicillin-resistant Staphylococcus aureus

The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains.     

Construction of a modified penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus and purification by immobilized metal affinity chromatography.

The mecA-27r gene, which encodes PBP2a-27r, was modified by site-specific mutagenesis, resulting in replacement of the N-terminal membrane anchor with a short chelating peptide (CP-PBP2a-27r). CP-PBP2a-27r retained the same binding affinity for beta-lactam antibiotics as the wild-type enzyme. Approximately 95% pure CP-PBP2a-27r was recovered in a single step by use of chelating-peptide-immobilized metal ion affinity chromatography.     

Restoration of susceptibility of methicillin-resistant Staphylococcus aureus to beta-lactam antibiotics by acidic pH: role of penicillin-binding protein PBP 2a

Methicillin-resistant Staphylococcus aureus (MRSA) is a global scourge, and treatment options are becoming limited. The MRSA phenotype reverts to that of beta-lactam-sensitive S. aureus when bacteria are grown at pH 5.0 in broth and, more importantly from a medical perspective (protracted, relapsing infections), after phagocytosis by macrophages, where the bacteria thrive in the acidic environment of phagolysosomes. The central factor for the MRSA phenotype is the function of the penicillin-binding protein (PBP) 2a, which maintains transpeptidase activity while being poorly inhibited by beta-lactams because of a closed conformation of its active site. We document herein by binding, acylation/deacylation kinetics, and circular dichroism spectroscopy with purified PBP 2a that at acidic pH (i) beta-lactams interact with PBP 2a more avidly; (ii) the non-covalent pre-acylation complex exhibits a lower dissociation constant and an increased rate of acyl-enzyme formation (first-order rate constant) without change in hydrolytic deacylation rate; and (iii) PBP 2a undergoes a conformational change in the presence of the antibiotic consistent with the opening of the active site from the closed conformation. These observations argue that PBP 2a most likely evolved for its physiological function at pH 7 or higher by adopting a closed conformation, which is not maintained at acidic pH. Although at the organism level the effect of acidic pH on other biological processes in MRSA could not be discounted, our report should provide the impetus for closer examination of the properties of PBP 2a at low pH and thereby identifying novel points of intervention in combating this problematic organism.     

Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein.

A beta-lactam-sensitive strain of Staphylococcus aureus could be converted to methicillin resistance by the introduction of a plasmid carrying the 4.3-kilobase HindIII chromosomal DNA fragment which encoded the mecA gene from a methicillin-resistant S. aureus. Transformant cells produced methicillin-resistant S. aureus-specific penicillin-binding protein constitutively, and additional insertion of an inducible penicillinase plasmid caused production of the pencillin-binding protein to become inducible.     

Biofilm density and detection of biofilm-producing genes in methicillin-resistant Staphylococcus aureus strains

Many serious diseases caused by Staphylococcus aureus appear to be associated with biofilms. Therefore, we investigated the biofilm-forming ability of the methicillin-resistant S. aureus (MRSA) isolates collected from hospitalized patients. As many as 96?% strains had the ability to form biofilm in vitro. The majority of S. aureus strains formed biofilm in ica-dependent mechanism. However, 23?% of MRSA isolates formed biofilm in ica-independent mechanism. Half of these strains carried fnbB genes encoding surface proteins fibronectin-binding protein B involved in intercellular accumulation and biofilm development in S. aureus strains. The biofilm structures were examined via confocal laser scanning microscopy (CLSM) and three-dimensional structures were reconstructed. The images obtained in CLSM revealed that the biofilm created by ica-positive strains was different from biofilm formed by ica-negative strains. The MRSA population showed a large genetic diversity and we did not find a single clone that occurred preferentially in hospital environment. Our results demonstrated the variation in genes encoding adhesins for the host matrix proteins (elastin, laminin, collagen, fibronectin, and fibrinogen) and in the gene involved in biofilm formation (icaA) within the majority of S. aureus clones.     

Cyclooxygenase-2 enhances antimicrobial peptide expression and killing of Staphylococcus aureus

Antimicrobial peptides such as human β-defensins (hBDs) and cathelicidins are critical for protection against infection and can be induced by activation of TLRs, a pathway that also activates cyclooxygenase(Cox)-2 expression. We hypothesized that Cox-2 is induced by TLR activation and is necessary for optimal AMP production, and that inhibitors of Cox-2 may therefore inhibit antimicrobial action. Normal human keratinocytes (NHEKs) stimulated with a TLR2/6 ligand, macrophage-activating lipopeptide-2, or a TLR3 ligand, polyinosinic-polycytidylic acid, increased Cox-2 mRNA and protein and increased PGE(2), a product of Cox-2. Treatment with a Cox-2 selective inhibitor (SC-58125) or Cox-2 small interfering RNA attenuated hBD2 and hBD3 production in NHEKs when stimulated with macrophage-activating lipopeptide-2, polyinosinic-polycytidylic acid, or UVB (15 mJ/cm(2)), but it did not attenuate vitamin D3-induced cathelicidin. SC-58125 also inhibited TLR-dependent NF-κB activation. Conversely, treatment with Cox-derived prostanoids PGD(2) or 15-deoxy-Δ(12,14)-PGJ(2) induced hBD3 or hBD2 and hBD3, respectively. The functional significance of these observations was seen in NHEKs that showed reduced anti-staphylococcal activity when treated with a Cox-2 inhibitor. These findings demonstrate a critical role for Cox-2 in hBD production and suggest that the use of Cox-2 inhibitors may adversely influence the risk for bacterial infection.     

Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000

AIMS: To analyse population minimum inhibitory concentrations (MICs) data from clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa for changes over a 10-year period and to look for correlations between the antimicrobials tested. METHODS AND RESULTS: Data from the MIC study of 256 clinical isolates of Staph. aureus [169 methicillin-sensitive Staph. aureus (MSSA), 87 methicillin-resistant Staph. aureus (MRSA)] and 111 clinical isolates of Ps. aeruginosa against eight antimicrobial biocides and several clinically relevant antibiotics was analysed using anova, Spearman-Rho correlation and principal component analysis. Comparisons suggest that alterations in the mean susceptibility of Staph. aureus to antimicrobial biocides have occurred between 1989 and 2000, but that these changes were mirrored in MSSA and MRSA suggests that methicillin resistance has little to do with these changes. Between 1989 and 2000 a sub-population of MRSA has acquired a higher resistance to biocides, but this has not altered the antibiotic susceptibility of that group. In both Staph. aureus and Ps. aeruginosa several correlations (both positive and negative) between antibiotics and antimicrobial biocides were found. CONCLUSIONS: From the analyses of these clinical isolates it is very difficult to support a hypothesis that increased biocide resistance is a cause of increased antibiotic resistance either in Staph. aureus or in Ps. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation of negative correlations between antibiotics and biocides may be a useful reason for the continued use of biocides promoting hygiene in the hospital environment.     

Quantification of methicillin-resistant Staphylococcus aureus strains in marine and freshwater samples by the most-probable-number method

Recreational beach environments have been recently identified as a potential reservoir for methicillin-resistant Staphylococcus aureus (MRSA); however, accurate quantification methods are needed for the development of risk assessments. This novel most-probable-number approach for MRSA quantification offers improved sensitivity and specificity by combining broth enrichment with MRSA-specific chromogenic agar.     

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000-2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.     

Role of bacterial components in macrophage activation by the LAC and MW2 strains of community-associated, methicillin-resistant Staphylococcus aureus

We tested the contribution of four staphylococcal components – PSM-α, PSM-β, δ-toxin, and PVL – in triggering macrophage secretion of tumor necrosis factor (TNF) and interleukins 6 (IL-6) and 12 (IL-12) by two prominent, circulating strains of community-associated, methicillin-resistant Staphylococcus aureus (CA-MRSA): LAC, USA300; MW2, USA400. RAW 264.7 murine macrophages were stimulated with live, antibiotic-exposed bacteria, and cytokine secretion was quantitated in supernatants. Deletion of PSM-α expression in LAC led to >50% reduction in macrophage TNF and IL-6 secretion and a 20% reduction in IL-12 secretion, while PSM-α deletion in MW2 did not significantly reduce macrophage TNF secretion but resulted in a 15–20% reduction in IL-6 and IL-12 secretion. Deletion of δ-toxin in either strain led to more than 50% reduction in macrophage IL-6 secretion and smaller reductions in macrophage TNF and IL-12 secretion (8–25%). Our data implicate both PSM-α and δ-toxin in stimulating macrophage cytokine responses to CA-MRSA bacteria.     

Presence of cna, emp and pls genes and pathogenicity of methicillin-resistant Staphylococcus aureus strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are important etiological factors responsible for hospital-acquired infections. The aim of this study was to analyze the influence of the presence of emp, pls and cna genes on the pathogenicity of MRSA strains. The presence of these genes was tested by PCR in 302 MRSA strains isolated from hospitalized patients and from carriers. For each tested gene, proportions of positive and negative strains were similar among the infected patients and carriers. We did not find any obvious correlation between the presence of the three tested genes and the infectivity of strains. Our results may also suggest that a lack of emp and presence of pls may correlate with reduced virulence of these strains.