New pyrazinoquinazoline alkaloids Isolated from a culture of Stenotrophomonas maltophilia QB-77.

Two new pyrazinoquinazoline alkaloids, epi-fiscalin D (1) and epi-fiscalin E (2), as well as three known analogues, norquinadoline A (3), quinadoline A (4), and fiscalin C (5), were isolated from ethyl acetate extract of the fermentation broth of Stentrophomonas maltophilia QB-77. The structures of new compounds were elucidated on the basis of extensive spectroscopic data analysis including UV, HRESIMS, and 1D and 2D NMR experiments. All the isolated compounds were tested for their in vitro cytotoxicity against five human cancer cell lines (SMMC-7721, MCF-7, HL-60, SW480, and A-549) and antibacterial activities against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus.

Utility of MALDI-TOF mass spectrometry in an outbreak investigation of acute endophthalmitis following intravitreal injection.

Bevacizumab, a recombinant humanized monoclonal antibody to inhibit tumour angiogenesis, is available for off-label use for treating choroidal neovascularization. Outbreaks have been reported after use of this drug, either because of contamination during manufacturing or the practice of multiple doses being administered from a single vial to different patients. In our institute we investigated one endophthalmitis outbreak following intravitreal injection of bevacizumab, rapidly identified the Stenotrophomonas maltophilia as the causative agent, and established the contaminated bevacizumab vial as the source using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) typing. The study emphasizes the need for early diagnosis of the source and pathogen for appropriate control measures.

Delineation of Stenotrophomonas maltophilia isolates from cystic fibrosis patients by fatty acid methyl ester profiles and matrix-assisted laser desorption/ionization time-of-flight mass spectra using hierarchical cluster analysis and principal component analysis.

Stenotrophomonas maltophilia is an opportunist multidrug-resistant pathogen that causes a wide range of nosocomial infections. Various cystic fibrosis (CF) centres have reported an increasing prevalence of S. maltophilia colonization/infection among patients with this disease. The purpose of this study was to assess specific fingerprints of S. maltophilia isolates from CF patients (n = 71) by investigating fatty acid methyl esters (FAMEs) through gas chromatography (GC) and highly abundant proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and to compare them with isolates obtained from intensive care unit (ICU) patients (n = 20) and the environment (n = 11). Principal component analysis (PCA) of GC-FAME patterns did not reveal a clustering corresponding to distinct CF, ICU or environmental types. Based on the peak area index, it was observed that S. maltophilia isolates from CF patients produced significantly higher amounts of fatty acids in comparison with ICU patients and the environmental isolates. Hierarchical cluster analysis (HCA) based on the MALDI-TOF MS peak profiles of S. maltophilia revealed the presence of five large clusters, suggesting a high phenotypic diversity. Although HCA of MALDI-TOF mass spectra did not result in distinct clusters predominantly composed of CF isolates, PCA revealed the presence of a distinct cluster composed of S. maltophilia isolates from CF patients. Our data suggest that S. maltophilia colonizing CF patients tend to modify not only their fatty acid patterns but also their protein patterns as a response to adaptation in the unfavourable environment of the CF lung.

A novel highly charged exopolysaccharide produced by two strains of Stenotrophomonas maltophilia recovered from patients with cystic fibrosis.

Stenotrophomonas maltophilia is a non-fermenting Gram-negative microorganism capable of causing chronic pulmonary infection in cystic fibrosis patients and its ability to form biofilms on polystyrene and glass surfaces, as well as on cystic fibrosis-derived bronchial epithelial IB3-I cells was recently demonstrated. The latter evidence might explain the power of S. maltophilia to produce persistent lung infections, despite intensive antibiotic treatment. In addition to being important components of the extracellular biofilm matrix, polysaccharides are involved in virulence, as they contribute to bacterial survival in a hostile environment. With the aim of contributing to the elucidation of S. maltophilia virulence factors, the exopolysaccharides produced by two mucoid clinical isolates of S. maltophilia obtained from two cystic fibrosis patients were completely characterised, mainly by means of ESI-MS and NMR spectroscopy. The results showed that, although the two isolates were recovered from two different patients living in different countries (Italy and France), the exopolysaccharides produced have an identical primary structure, with the following repeating unit: The exopolysaccharide is highly negatively charged for the presence of three uronic acids on four residues in the repeating unit. Moreover, an ether-linked d-lactate substituent is located on C-3 and one O-acetyl group on C-4 of the galacturonic acid side chain. Another O-acetyl group substitutes C-2 of the galacturonic acid in the backbone, making this primary structure unique.

Pathogen and autoantigen homologous regions within the cystic fibrosis transmembrane conductance regulator (CFTR) protein suggest an autoimmune treatable component of cystic fibrosis.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel provides the glutathione and hypochlorous acid necessary for bactericidal/viricidal actions. CFTR mutations block these effects, diminishing pathogen defence and allowing extracellular pathogen accumulation, where antibody encounter is likely. KEGG pathway analysis of the CFTR interactome shows that CFTR is involved in pathogen entry pathways and immune defence as well as in pathways relevant to comorbid conditions (diabetes, cardiomyopathies and sexual organ development). Pseudomonas aeruginosa and Staphylococcus aureus infections decrease the lifespan of cystic fibrosis patients and Stenotrophomonas maltophilia colonization is increased. Autoantibodies, targeting myeloperoxidase, the bactericidal/permeability-increasing protein and calgranulin may further compromise pathogen defence. Short consensus sequences, within immunogenic extracellular regions of the CFTR protein, are homologous to proteins expressed by P. aeruginosa, S. aureus and S. maltophilia, and to several autoantigens, with a universal overlap between autoantigen/pathogen/CFTR consensi. Antibodies to pathogens are thus likely responsible for the creation of these autoantibodies, which, with pathogen antibodies, may target the CFTR protein acting as antagonists, further compromising its function. This creates a feedforward cycle, diminishing the function of the CFTR protein and increasing the probability of pathogen accumulation and antibody production at every turn. Interruption of this cycle by antibody adsorption or immunosuppressant therapy may be beneficial in cystic fibrosis.

Structure of Stenotrophomonas maltophilia FeoA complexed with zinc: a unique prokaryotic SH3-domain protein that possibly acts as a bacterial ferrous iron-transport activating factor.

Iron is vital to the majority of prokaryotes, with ferrous iron believed to be the preferred form for iron uptake owing to its much better solubility. The major route for bacterial ferrous iron uptake is found to be via an Feo (ferrous iron-transport) system comprising the three proteins FeoA, FeoB and FeoC. Although the structure and function of FeoB have received much attention recently, the roles played by FeoA and FeoC have been little investigated to date. Here, the tertiary structure of FeoA from Stenotrophomonas maltophilia (Sm), a vital opportunistic pathogen in immunodepressed hosts, is reported. The crystal structure of SmFeoA has been determined to a resolution of 1.7 A using an Se single-wavelength anomalous dispersion (Se-SAD) approach. Although SmFeoA bears low sequence identity to eukaryotic proteins, its structure is found to adopt a eukaryotic SH3-domain-like fold. It also bears weak similarity to the C-terminal SH3 domain of bacterial DtxR (diphtheria toxin regulator), with some unique characteristics. Intriguingly, SmFeoA is found to adopt a unique dimer cross-linked by two zinc ions and six anions (chloride ions). Since FeoB has been found to contain a G-protein-like domain with low GTPase activity, FeoA may interact with FeoB through the SH3-G-protein domain interaction to act as a ferrous iron-transport activating factor.

Analysis of bacterial fatty acids by flow modulated comprehensive two-dimensional gas chromatography with parallel flame ionization detector/mass spectrometry.

Comprehensive two-dimensional gas chromatography (GCxGC) offers an interesting tool for profiling bacterial fatty acids. Flow modulated GCxGC using a commercially available system was evaluated, different parameters such as column flows and modulation time were optimized. The method was tested on bacterial fatty acid methyl esters (BAMEs) from Stenotrophomonas maltophilia LMG 958T by using parallel flame ionization detector (FID)/mass spectrometry (MS). The results are compared to data obtained using a thermal modulated GCxGC system. The data show that flow modulated GCxGC-FID/MS method can be applied in a routine environment and offers interesting perspectives for chemotaxonomy of bacteria.