Laser desorption mass spectrometry for microbial DNA analysis.

Recently, we demonstrated that a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) can be used to determine the molecular weight of polymerase chain reaction (PCR) products of intact 16S rRNA regions and to profile their restriction digests. This is the first time that MALDI-TOF MS with ultraviolet (UV) photoionization has been used to analyze a PCR product of approximately 1600 nucleotides in length.     

Viscoelastic properties of Staphylococcus aureus and Staphylococcus epidermidis mono‐microbial biofilms

The viscoelastic properties of mono‐microbial biofilms produced by ocular and reference staphylococcal strains were investigated. The microorganisms were characterized for their haemolytic activity and agr typing and the biofilms, grown on stainless steel surface under static conditions, were analysed by Confocal Laser Scanning Microscopy. Static and dynamic rheometric tests were carried out to determine the steady‐flow viscosity and the elastic and viscous moduli. The analysed biofilms showed the typical time‐dependent behaviour of viscoelastic materials with considerable elasticity and mechanical stability except for Staphylococcus aureus ATCC 29213 biofilm which showed a very fragile structure. In particular, S. aureus 6ME biofilm was more compact than other staphylococcal biofilms studied with a yield stress ranging between 2 and 3 Pa. The data obtained in this work could represent a starting point for developing new therapeutic strategies against biofilm‐associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.     

Electric current-induced detachment of Staphylococcus epidermidis biofilms from surgical stainless steel

Biomaterial-centered infections of orthopedic percutaneous implants are serious complications which can ultimately lead to osteomyelitis, with devastating effects on bone and surrounding tissues, especially since the biofilm mode of growth offers protection against antibiotics and since removal frequently is the only ultimate solution. Recently, it was demonstrated that as a possible pathway to prevent infections of percutaneous stainless steel implants, electric currents of 60 to 100 microA were effective at stimulating the detachment of initially adhering staphylococci from surgical stainless steel. However, initially adhering bacteria are known to adhere more reversibly than bacteria growing in the later stages of biofilm formation. Hence, the aim of this study was to examine whether a growing Staphylococcus epidermidis biofilm can be stimulated to detach from surgical stainless steel by the use of electric currents. In separate experiments, four currents, i.e., 60 and 100 microA of direct current (DC) and 60 and 100 microA of block current (50% duty cycle, 1 Hz), were applied for 360 min to stimulate the detachment of an S. epidermidis biofilm that had grown for 200 min. A 100-microA DC yielded 78% detachment, whereas a 100-microA block current under the same experimental conditions yielded only 31% detachment. The same trend was found for 60 microA, with 37% detachment for a DC and 24% for a block current. Bacteria remaining on the surface after the current application were less viable than they were prior to the current application, as demonstrated by confocal laser scanning microscopy. In conclusion, these results suggest that DCs are preferred for curing infections.     

Antimicrobial activities of YycG histidine kinase inhibitors against Staphylococcus epidermidis biofilms

Staphylococcus epidermidis has become a significant pathogen causing infections due to biofilm formation on surfaces of indwelling medical devices. Biofilm-associated bacteria exhibit enhanced resistance to many conventional antibiotics. It is therefore, important to design novel antimicrobial reagents targeting S. epidermidis biofilms. In a static chamber system, the bactericidal effect of two leading compounds active as YycG inhibitors was assessed on biofilm cells by confocal laser scanning microscopy combined with viability staining. In young biofilms (6-h-old), the two compounds killed the majority of the embedded cells at concentrations of 100 microM and 25 microM, respectively. In mature biofilms (24-h-old), one compound was still effectively killing biofilm cells, whereas the other compound mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC (8 microg mL(-1)). Even at a high concentration (128 microg mL(-1)), vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure (6 h). The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.     

Modulation of poly-N-acetylglucosamine accumulation within mature Staphylococcus epidermidis biofilms grown in excess glucose

PNAG is a major component of Staphylococcus epidermidis biofilms involved in intercellular adhesion as well as in the interaction of the biofilm with components of the host immune response. Synthesis of PNAG has been found to be regulated by several environmental factors. In the present study, the effect of glucose metabolism-dependent culture medium acidification in PNAG accumulation was evaluated. Established S. epidermidis biofilms were allowed to grow in excess glucose with or without maintained pH conditions. PNAG accumulation in these biofilms was determined by flow cytometry and fluorescence microscopy using wheat germ agglutinin as a fluorescent probe. Biofilms grown in maintained pH conditions presented significantly higher amounts of this polymer as well as higher icaA expression than biofilms grown in acidic pH conditions. Moreover, PNAG accumulation in biofilms grown in non-maintained pH conditions occurred in association with cell death. Overall, we show that glucose metabolism by decreasing the culture pH affects biofilm physiology in respect to PNAG production and cell death. The reported in vitro modulation of PNAG accumulation within S. epidermidis biofilms further highlights the role of environment on determining the biofilm physiological state.     

Laser desorption ionization mass spectrometry of protein tryptic digests on nanostructured silicon plates

We report on the simple application of a new nanostructured silicon (NanoSi) substrate as laser desorption/ionization (LDI)-promoting surface for high-throughput identification of protein tryptic digests by a rapid MS profiling and subsequent MS/MS analysis. The NanoSi substrate is easily prepared by chemical etching of crystalline silicon in NH(4)F/HNO(3)/AgNO(3) aqueous solution. To assess the LDI performances in terms of sensitivity, repeatability and robustness, the detection of small synthetic peptides (380-1700Da) was investigated. Moreover, peptide sequencing was tackled. Various tryptic synthetic peptide mixtures were first characterized in MS and MS/MS experiments carried out on a single deposit. Having illustrated the capability to achieve peptide detection and sequencing on these ionizing surfaces in the same run, protein tryptic digests from Cytochrome C, β-Casein, BSA and Fibrinogen were then analyzed in the femtomolar range (from 50 fmol for Cytochrome C down to 2 fmol for Fibrinogen). Comparison of the NanoSi MS and MS/MS data with those obtained with sample conditioned in organic matrix demonstrated a great behavior for low mass responses. We demonstrated the capability of LDI on NanoSi to be a complementary method to MALDI peptide mass fingerprinting ensuring determination of peptide molecular weights and sequences for more efficient protein database searches.     

Cross-reactions between Staphylococcus epidermidis and 23 other bacterial species

By quantitative immunoelectrophoretic methods, 43 antigens were found in a mixture of sonicated preparations of four Staphylococcus epidermidis strains, using corresponding rabbit antiserum. Two of the antigens were identified as cell wall teichoic acid and a peptidoglycan antigen, respectively. Using this antigen/antibody reference system, cross-reactions between S. epidermidis antigens and antigens from other bacterial species were investigated. Fourteen of the S. epidermidis antigens cross-reacted with antigens from all S. aureus strains investigated. Only few cross-reactions were found between S. epidermidis and bacteria not belonging to the Micrococcaceae. The antigenic relatedness, expressed as a matching coefficient, seems promising for taxonomic work.     

Effects of growth in the presence of subinhibitory concentrations of dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus biofilms

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions of biofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.     

Anomalous growth of Staphylococcus epidermidis in the presence of Silastic and glycopeptide antibiotics

Abstract In the presence of supra-inhibitory concentrations of the glycopeptide antibiotics vancomycin and teicoplanin, biofilms of Staphylococcus epidermidis on a Silastic surface produce anomalous growwth. This takes the form of macroscopic, cohesive aggregates of cocci bound together with slime. This phenomenon was intermittent, independent of antibiotic concentrations between 20 and 50 μg ml⁻¹, occured more often with teicoplanin, and was found both with slime-negative strains.     

In vitro activity of alpha-mangostin in killing and eradicating Staphylococcus epidermidis RP62A biofilms

Applied Microbiology and Biotechnology - Alpha-mangostin (α-MG) has been reported to be an effective antibacterial agent against planktonic cells of many Gram-positive bacteria. However, the...     

Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometry.

We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 microL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Liquid chromatography-electrospray mass spectrometry of tunicamycin-type antibiotics

Several Streptomyces and Clavibacter species produce a family of tunicamycin-like antibiotics (tunicamycins, streptovirudins, corynetoxins, etc.) that inhibit the polyprenol-P:N-acetylhexosamine-1-P translocase family, thus blocking both bacterial cell wall biosynthesis and eukaryotic protein N-glycosylation. The mechanisms of biosynthesis and resistance to these toxins by the producing bacteria are largely unknown. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometric techniques are described that structurally assign tunicamycin N-acylated variants in the picomolar range. A voltage gradient across the ESI inlet port was used to generate fragmentation ions that were structurally diagnostic for the tunicamycins. The application of in-line reversed-phase high-performance liquid chromatography-electrospray MS (LC-ESI-MS) resulted in the identification of eight new tunicamycins. Based on these structural assignments a revised nomenclature for tunicamycins is proposed. Application of the LC-ESI-MS methodology to culture supernatants and cellular extracts of the tunicamycin-producing bacterium, Streptomyces lysosuperificus, confirmed tunicamycin production and showed it to be growth-temperature dependent, but did not detect corynetoxins production in culture by phage-infected Clavibacter toxicus.