The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan networ

Isolation and in-vitro and in-vivo characterisation of a mutant of Pseudomonas aeruginosa PAO1 that exhibited a reduced postantibiotic effect in response to imipenem

The postantibiotic effect (PAE) is the persistent inhibition of bacterial growth after a brief exposure to an antibiotic. Most β-lactams do not induce a PAE for Gram-negative bacteria, but PAEs have been reported for carbapenems and penems. This study investigated the effect of sequential doses of imipenem on the PAE for Pseudomonas aeruginosa and Escherichia coli cultures in a chemostat. The PAE for the bacterial population did not change even after six successive exposures to imipenem. Nevertheless, screening of colonies isolated after repeated drug exposure identified a single P. aeruginosa mutant whose imipenem PAE was shortened, although the MIC was unchanged. The PAEs for the parent and mutant were studied in vitro in batch culture by monitoring: (i) viable counts; (ii) electrical impedance of the culture medium; (iii) incorporation of radiolabelled N-acetyl-D-glucosamine and (iv) cell volume changes. PAEs for the parent and mutant were found to be significantly different by all in-vitro methods used. Moreover, the median cell volume in antibiotic-exposed cultures remained much smaller and less heterogeneous than in the control cultures, even though both cultures were growing at the same rate. The mutant was found to have a reduced expression of a 52 kDa outer membrane protein. These observations suggest that factors in addition to suppression of bacterial growth should be considered when studying the PAE. The PAEs of imipenem for the parent and mutant were studied in a thigh infection model in leucopenic mice. Similar PAEs were observed in vivo for both parent and mutant in one experiment and no PAEs for either organism were found in a second experiment. This study showed that although the PAE is a stable in-vitro phenomenon, the lack of correlation between the in-vitro and in-vivo results warrants caution in attributing clinical significance to the PAE of imipene

MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus

MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureu

Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response

The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S. aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell division. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S. aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S. aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S. aureus cell envelope biogenesi

An acid-stable laccase from sclerotium rolfsii with potential for wool dye decolourization

The plant pathogen basidiomycete S. rolfsii secretes two laccases (SRL1 and SRL2) with molecular weights of 55 and 86 kDa, respectively. Laccase production was shown to be inducible by the addition of 2,5-xylidine to the cultural media. After treatment with a combination of chitinase and -1,3-glucanase, two different laccases were isolated from the sclerotia depending on the stage of sclerotia development. The more prominent laccase, SRL1, was purified and found to decolourize the industrially important wool azo dye Diamond Black PV 200 without the addition of redox mediators. The enzyme (pI 5.2) was active in the acidic pH range, showing an optimal activity at pH 2.4, with ABTS as substrate. The optimum temperature for activity was determined to be 62 ◦C. Enzyme stability studies revealed that SRL1 was notably stable at 18 ◦C and pH 4.5, retaining almost full activity after a week. Oxidation of tyrosine was not detectable under the reaction conditions but the enzyme did oxidize a variety of the usual laccase substrates. SRL1 was strongly inhibited by sodium azide and fluoride. Dye solutions decolourized with the immobilized laccase were successfully used for redyeing.(undefined

Cell wall-associated redox enzymes in white rot fungi

Comunicación presentada al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.Many enzymes of white rot fungi involved in wood degradation belong to the class of redox enzymes. The most important are laccase (copper-containing polyphenol oxidase), lignin peroxidase, manganese- dependent peroxidase and manganese-independent peroxidase. However, the role of these enzymes in wood degradation remains unclear and complex redox processes or unknown redox enzymes also may contribute to this process. Several oxidative enzymes secreted by white rot fungi into the environment have been studied in the past, but little attention has been paid to the cell wall-associated redox enzymes. Cell wall-associated laccase activity in the purified cell walls of copper induced cultures of Trametes versicolor has been found. Laccases have been extracted by establishing new methods for cell wall purification and for protein release from the cell walls of basidiomycetes.the author's laboratory is supported by the Deutsche Bundesstiftung Umwelt (DBU)

Copper in fruiting body development of Coprinus cinereus

Resumen del poster presentado al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.The model homobasidiomycete Coprinopsis cinerea grows best at 37°C, but, normally, it produces fruiting bodies only at moderate temperatures around 25-28°C. Light is needed to induce fruiting and also for fruiting body maturation. Cultures kept after fruiting induction predominantly in the dark form structures with an extended stipe and an underdeveloped cap (so-called “etiolated stipes”). In a day/night rhythm, caps develop further, basidia are formed, in which karyogamy and meiosis occurs and of which the basidiospores bud off. Besides light, fruiting body development in basidiomycetes has been repeatedly linked to enzymes belonging to the group of phenoloxidases, in particular the multi-copper containing laccases. However, their roles in fruiting remain unclear. In attempts to induce laccase production in liquid standing cultures at 37°C, to our surprise we found unusual inititation of fruiting body development. However, the abundantly formed primordia did never develop into mature fruiting bodies but into large-sized etiolated stipes, both in dark and in light. Laccase under these conditions was not detected in the medium but bound to the fruiting initiating mycelium. Moreover, enzyme production and etiolated stipe formation correlated with an increase from pH 5.5 to a slightly alkaline pH. Ammonium was found to be produced and nitrate reductase activity has enzymatically been shown. Under normal fruiting conditions, addition of copper to cultures enhances fruiting initiation in time and number. To further unravel the potential involvement of laccases in fruiting as well as of proteins influencing ammonia secretion, we are studying expression of corresponding genes during vegetative growth and fruiting body development. Work in our laboratory is supported by DBU (Deutsche Bundesstiftung Umwelt). MNG holds a CONACYT (Mexico) PhD studentship.Work in the author's laboratory is supported by DBU (Deutsche Bundesstiftung Umwelt)

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus

Background: Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore β-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. Results: In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. Conclusion: Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors

Prospective monitoring of cefepime in intensive care unit adult patients

INTRODUCTION: Cefepime has been associated with a greater risk of mortality than other beta-lactams in patients treated for severe sepsis. Hypotheses for this failure include possible hidden side-effects (for example, neurological) or inappropriate pharmacokinetic/pharmacodynamic (PK/PD) parameters for bacteria with cefepime minimal inhibitory concentrations (MIC) at the highest limits of susceptibility (8 mg/l) or intermediate-resistance (16 mg/l) for pathogens such as Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. We examined these issues in a prospective non-interventional study of 21 consecutive intensive care unit (ICU) adult patients treated with cefepime for nosocomial pneumonia. METHODS: Patients (median age 55.1 years, range 21.8 to 81.2) received intravenous cefepime at 2 g every 12 hours for creatinine clearance (CLCr) >or= 50 ml/min, and 2 g every 24 hours or 36 hours for CLCr < 50 ml/minute. Cefepime plasma concentrations were determined at several time-points before and after drug administration by high-pressure liquid chromatography. PK/PD parameters were computed by standard non-compartmental analysis. RESULTS: Seventeen first-doses and 11 steady states (that is, four to six days after the first dose) were measured. Plasma levels varied greatly between individuals, from two- to three-fold at peak-concentrations to up to 40-fold at trough-concentrations. Nineteen out of 21 (90%) patients had PK/PD parameters comparable to literature values. Twenty-one of 21 (100%) patients had appropriate duration of cefepime concentrations above the MIC (T>MIC >or= 50%) for the pathogens recovered in this study (MIC <or= 4 mg/l), but only 45 to 65% of them had appropriate coverage for potential pathogens with cefepime MIC >or= 8 mg/l. Moreover, 2/21 (10%) patients with renal impairment (CLCr < 30 ml/minute) demonstrated accumulation of cefepime in the plasma (trough concentrations of 20 to 30 mg/l) in spite of dosage adjustment. Both had symptoms compatible with non-convulsive epilepsy (confusion and muscle jerks) that were not attributed to cefepime-toxicity until plasma levels were disclosed to the caretakers and symptoms resolved promptly after drug arrest. CONCLUSIONS: These empirical results confirm the suspected risks of hidden side-effects and inappropriate PK/PD parameters (for pathogens with upper-limit MICs) in a population of ICU adult patients. Moreover, it identifies a safety and efficacy window for cefepime doses of 2 g every 12 hours in patients with a CLCr >or= 50 ml/minute infected by pathogens with cefepime MICs <or= 4 mg/l. On the other hand, prompt monitoring of cefepime plasma levels should be considered in case of lower CLCr or greater MICs

Multiple hydrophobin genes in mushrooms

Comunicación presentada al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.Hydrophobins are small secreted fungal proteins that form amphipathic films on the hyphal surfaces. In the wood-rotting fungus Schizophyllum commune, four different hydrophobins are known with well established functions during vegetative growth and fruiting body development. Our study aims at elucidating the role of these proteins in wood penetration and lignocellulose degradation. Blast searches of the genome of the dung fungus Coprinopsis cinerea revealed a surprising number of 34 different hydrophobin genes in this species. Functional analysis of these genes is in progress.Part of this work was conducted within the NHN (Niedersächsisches Kompetenznetz für Nachhaltige Holznutzung). Financial support by the Ministry for Science and Culture of Lower Saxony and by EFRE (European Fund for Regional Development, grant 2001.085). The section Molecular Wood Biotechnology at the Institute for Forest Botany is funded by the DBU (Deutsche Bundesstiftung Umwelt)