Comparison of the spectral emission of lux recombinant and bioluminescent marine bacteria

The purpose of the present paper was to study the influence of bacteria harbouring the luciferase‐encoding Vibrio harveyi luxAB genes upon the spectral emission during growth in batch‐culture conditions. In vivo bioluminescence spectra were compared from several bioluminescent strains, either naturally luminescent (Vibrio fischeri and Vibrio harveyi) or in recombinant strains (two Gram‐negative Escherichia coli::luxAB strains and a Gram‐positive Bacillus subtilis::luxAB strain). Spectral emission was recorded from 400 nm to 750 nm using a highly sensitive spectrometer initially devoted to Raman scattering. Two peaks were clearly identified, one at 491–500 nm (± 5 nm) and a second peak at 585–595 (± 5 nm) with the Raman CCD. The former peak was the only one detected with traditional spectrometers with a photomultiplier detector commonly used for spectral emission measurement, due to their lack of sensitivity and low resolution in the 550–650 nm window. When spectra were compared between all the studied bacteria, no difference was observed between natural or recombinant cells, between Gram‐positive and Gram‐negative strains, and growth conditions and growth medium were not found to modify the spectrum of light emission. Copyright © 2003 John Wiley & Sons, Ltd.     

Understanding the effects of culture conditions in bacterial growth: A biochemical perspective using Raman microscopy

Rapid, sensitive and label‐free methods to probe bacterial growth irrespective of the culture conditions can shed light on the mechanisms by which bacteria adapt to different environmental stimuli. Raman spectroscopy can rapidly and continuously monitor the growth of bacteria under varied conditions. In this study, the growth of Escherichia coli in Luria broth (nutrient rich conditions) and minimal media with either glucose or glycerol as carbon source (nutrient limiting conditions) is profiled using Raman spectroscopy. Moreover, the study also gives insights into the altered bacterial biochemistry upon exposure to low‐ (25°C) and high‐temperature (45°C) stress. Raman spectral measurement was performed on bulk bacteria cultured under laboratory conditions. A detailed analysis of the spectra as a function of bacterial growth reveals changes in Raman band intensities/area of biomolecules such as DNA, proteins and lipids. We also report five novel ratiometric markers (I₈₃₀/I₈₁₀, I₁₁₂₆/I₁₁₀₀, I₁₃₄₀/I₁₄₄₀, I₁₂₀₇/I₁₂₄₀ and I₁₅₈₀/I₁₄₄₀) that can identify the phase of growth, independent of the culture condition. Unsupervised multivariate methods like Principal Component Analysis also corroborate the aforementioned markers of growth. Altogether, our findings highlight the potential of Raman spectroscopy in yielding universal biochemical signatures that may be indicative of stress and aging in a growth milieu.     

Exploration research on the fusion of multimodal spectrum technology to improve performance of rapid diagnosis scheme for thyroid dysfunction

The spectral fusion by Raman spectroscopy and Fourier infrared spectroscopy combined with pattern recognition algorithms is utilized to diagnose thyroid dysfunction serum, and finds the spectral segment with the highest sensitivity to further advance diagnosis speed. Compared with the single infrared spectroscopy or Raman spectroscopy, the proposal can improve the detection accuracy, and can obtain more spectral features, indicating greater differences between thyroid dysfunction and normal serum samples. For discriminating different samples, principal component analysis (PCA) was first used for feature extraction to reduce the dimension of high‐dimension spectral data and spectral fusion. Then, support vector machine (SVM), back propagation neural network, extreme learning machine and learning vector quantization algorithms were employed to establish the discriminant diagnostic models. The accuracy of spectral fusion of the best analytical model PCA‐SVM, single Raman spectral accuracy and single infrared spectral accuracy is 83.48%, 78.26% and 80%, respectively. The accuracy of spectral fusion is higher than the accuracy of single spectrum in five classifiers. And the diagnostic accuracy of spectral fusion in the range of 2000 to 2500 cm^?1 is 81.74%, which greatly improves the sample measure speed and data analysis speed than analysis of full spectra. The results from our study demonstrate that the serum spectral fusion technique combined with multivariate statistical methods have great potential for the screening of thyroid dysfunction.     

Automated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins,cephalosporins, and fluoroquinolones using specific Raman marker bands

A Raman‐based, strain‐independent, semi‐automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch‐wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third‐generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug‐resistant Gram‐negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long‐term comparability of Raman measurements.     

Inhibitory effects of soluble MD‐2 and soluble CD14 on bacterial growth

The effects of the soluble forms of the endotoxin receptor molecules sMD‐2 and sCD14 on bacterial growth were studied. When Escherichia coli and Bacillus subtilis were incubated at 37°C for 18 hr with either sMD‐2 or sCD14, growth of these bacteria was significantly inhibited as evaluated by viable cell counts and NADPH/NADH activity. A mutant of sCD14 (sCD14d57‐64) lacking a region essential for LPS binding did not inhibit the growth of E. coli, whereas this mutant did inhibit the growth of B. subtilis. Addition of excess PG to the bacterial culture reversed the inhibitory effect of sMD‐2 on the growth of B. subtilis, but not on the growth of E. coli. Furthermore, when evaluated by ELISA, both sMD‐2 and sCD14 bound specifically to PG. Taken together, these results indicate that sMD‐2 and sCD14 inhibit the growth of both Gram‐positive and Gram‐negative bacteria and further suggest that binding to PG and LPS is involved in the inhibitory effect of sMD‐2 on Gram‐positive bacteria and of sCD14 on Gram‐negative bacteria, respectively.     

Bacterial cyclic β‐(1,2)‐glucans sequester iron to protect against iron‐induced toxicity

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron‐restricted conditions while also limiting the potential for the production of iron‐induced reactive oxygen species (ROS). Here, we report that membrane‐derived oligosaccharide (mdo) glucan, an intrinsic component of Gram‐negative bacteria, sequesters the ferrous form of iron. Iron‐binding, uptake, and localization experiments indicated that both secreted and periplasmic β‐(1,2) ‐ glucans bind iron specifically and promote growth under iron‐restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β‐(1,2) ‐ glucan accumulate low amounts of intracellular iron under iron‐restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron‐replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram‐negative bacteria.     

Review of established and innovative detection methods for carbapenemase‐producing Gram‐negative bacteria

The minimal antibiotic options for carbapenemase‐producing Gram‐negative bacteria necessitate their rapid detection. A literature review of a variety of phenotypic and genotypic methods is presented. Advances in culture methods and screening media are still subject to long incubation hours. Biochemical methods have shorter turnaround times and higher sensitivities and specificities, but cannot differentiate between various types and variants. Spectrophotometric methods are cheap and efficient, but are uncommon in many clinical settings, while the MALDI‐TOF MS is promising for species identification, typing and resistance gene determination. Although next generation sequencing (NGS) technologies provide a better platform to detect, type and characterize carbapenem‐resistant bacteria, the different NGS platforms, the large computer memories and space needed to process and store genomic data and the nonuniformity in data analysis platforms are still a challenge. The sensitivities, specificities and turnaround times recorded in the various studies reviewed favours the use of the biochemical tests (Carba NP or Rapid Carb screen tests) for the detection of putative carbapenemase‐producing isolates. MALDI‐TOF MS and/or molecular methods like microarray, loop‐mediated isothermal amplification and real‐time multiplex PCR assays could be used for further characterization in a reference laboratory. NGS may be used for advanced epidemiological and molecular studies.     

Development and first in‐human use of a Raman spectroscopy guidance system integrated with a brain biopsy needle

Navigation‐guided brain biopsies are the standard of care for diagnosis of several brain pathologies. However, imprecise targeting and tissue heterogeneity often hinder obtaining high‐quality tissue samples, resulting in poor diagnostic yield. We report the development and first clinical testing of a navigation‐guided fiberoptic Raman probe that allows surgeons to interrogate brain tissue in situ at the tip of the biopsy needle prior to tissue removal. The 900 μm diameter probe can detect high spectral quality Raman signals in both the fingerprint and high wavenumber spectral regions with minimal disruption to the neurosurgical workflow. The probe was tested in three brain tumor patients, and the acquired spectra in both normal brain and tumor tissue demonstrated the expected spectral features, indicating the quality of the data. As a proof‐of‐concept, we also demonstrate the consistency of the acquired Raman signal with different systems and experimental settings. Additional clinical development is planned to further evaluate the performance of the system and develop a statistical model for real‐time tissue classification during the biopsy procedure.      

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.     

Evaluation of sample preparation methods for MALDI-TOF MS identification of highly dangerous bacteria

Aims: To propose a universal workflow of sample preparation method for the identification of highly pathogenic bacteria by MALDI‐TOF MS. Methods and Results: Fifteen bacterial species, including highly virulent Gram‐positive (Bacillus anthracis and Clostridium botulinum) and Gram‐negative bacteria (Brucella melitensis, Burkholderia mallei, Francisella tularensis, Shigella dysenteriae, Vibrio cholerae, Yersinia pestis and Legionella pneumophila), were employed in the comparative study of four sample preparation methods compatible with MALDI‐TOF MS. The yield of bacterial proteins was determined by spectrophotometry, and the quality of the mass spectra, recorded in linear mode in the range of 2000–20 000 Da, was evaluated with respect to the information content (number of signals) and quality (S/N ratio). Conclusions: Based on the values of protein concentration and spectral quality, the method using combination of ethanol treatment followed by extraction with formic acid and acetonitrile was the most efficient sample preparation method for the identification of highly pathogenic bacteria using MALDI‐TOF MS. Significance and Impact of the Study: The method using ethanol/formic acid generally shows the highest extraction efficacy and the spectral quality with no detrimental effect caused by storage. Thus, this can be considered as a universal sample preparation method for the identification of highly virulent micro‐organisms by MALDI‐TOF mass spectrometry.     

A novel pathway for outer membrane protein biogenesis in Gram‐negative bacteria

The understanding of the biogenesis of the outer membrane of Gram‐negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram‐negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram‐negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β‐barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram‐negative outer membrane that is independent of the BAM complex and TAM.     

Cell classification with low‐resolution Raman spectroscopy (LRRS)

The identification of individual eukaryotic and prokaryotic cells is the backbone of clinical pathology and provides crucial information about the genesis and progression of a disease. While most commonly fluorescent‐label based methods are applied, label‐free methods, such as Raman spectroscopy, are elegant alternatives. A major disadvantage of Raman spectroscopy is the low signal yield resulting in long acquisition times, making it impractical for high‐throughput clinical analysis. As a rule, Raman‐based cell identification relies on high‐resolution Raman spectra. This comes at a cost of detected Raman photons. In this letter we show that while the proper biochemical characterization of cells requires high‐resolution Raman spectra, the proper classification of cells does not. By varying the slit‐width between 50 µm and 500 µm it is possible to show that detected Raman signal from eukaryotic cells increased up to seven‐fold. Raman‐based cell classification was performed on three cancer cell lines: Jurkat, MiaPaca2, and Capan1, at three different resolutions 8 cm^–1, 24 cm^–1, and 48 cm^–1. Moreover, we have simulated the resolution decrease due to low‐diffraction gratings by binning neighboring pixels together. In both cases the cells were well classifiable using support vectors machine (SVM).

For anyone working in the field of Raman spectroscopy this picture of Sir C.V. Raman is recognizable, even with reduced spatial resolution. Raman spectra of eukaryotic cells can also be recognized even with six fold reduced spectral resolution.     

Bioengineering and functional characterization of arenicin shortened analogs with enhanced antibacterial activity and cell selectivity

New bioengineering approaches are required for development of more active and less toxic antimicrobial peptides. In this study we used β‐hairpin antimicrobial peptide arenicin‐1 as a template for design of more potent antimicrobials. In particular, six shortened 17‐residue analogs were obtained by recombinant expression in Escherichia coli. Besides, we have introduced the second disulfide bridge by analogy with the structure of tachyplesins. As a result, a number of analogs with enhanced activity and cell selectivity were developed. In comparison with arenicin‐1, which acts on cell membranes with low selectivity, the most potent and promising its analog termed ALP1 possessed two‐fold higher antibacterial activity and did not affect viability of mammalian cells at concentration up to 50 μM. The therapeutic index of ALP1 against both Gram‐positive and Gram‐negative bacteria was significantly increased compared with that of arenicin‐1 while the mechanism of action remained the same. Like arenicin‐1, the analog rapidly disrupt membranes of both stationary and exponential phase bacterial cells and effectively kills multidrug‐resistant Gram‐negative bacteria. Furthermore, ALP1 was shown to bind DNA in vitro at a ratio of 1:1 (w/w). The circular dichroism spectra demonstrated that secondary structures of the shortened analogs were similar to that of arenicin‐1 in water solution, but significantly differed in membrane‐mimicking environments. This work shows that a strand length is one of the key parameters affecting cell selectivity of β‐hairpin antimicrobial peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)‐FTIR and HT‐Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.     

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

The human immune system can directly lyse invading micro‐organisms and aberrant host cells by generating pores in the cell envelope, called membrane attack complexes (MACs). Recent studies using single‐particle cryoelectron microscopy have revealed that the MAC is an asymmetric, flexible pore and have provided a structural basis on how the MAC ruptures single lipid membranes. Despite these insights, it remains unclear how the MAC ruptures the composite cell envelope of Gram‐negative bacteria. Recent functional studies on Gram‐negative bacteria elucidate that local assembly of MAC pores by surface‐bound C5 convertase enzymes is essential to stably insert these pores into the bacterial outer membrane (OM). These convertase‐generated MAC pores can subsequently efficiently damage the bacterial inner membrane (IM), which is essential for bacterial killing. This review summarizes these recent insights of MAC assembly and discusses how MAC pores kill Gram‐negative bacteria. Furthermore, this review elaborates on how MAC‐dependent OM damage could lead to IM destabilization, which is currently not well understood. A better understanding on how MAC pores kill bacteria could facilitate the future development of novel strategies to treat infections with Gram‐negative bacteria.     

Antimicrobial activity of Coniothyrium minitans and its macrolide antibiotic macrosphelide A

Aims: Assessment of antimicrobial activity of the mycoparasite Coniothyrium minitans and its macrolide antibiotic macrosphelide A. Methods and Results: Thirteen isolates of C. minitans were tested for ability to inhibit a number of filamentous fungi, yeasts, oomycetes and bacteria in agar based tests. Activity was found against some ascomycetes, basidiomycetes, oomycetes and Gram‐positive bacteria, but not against zygomycetes, yeasts or Gram‐negative bacteria tested. Six C. minitans isolates (Conio, Contans, IVT1, CM/AP/3118, B279/1, A1/327/1) were found to produce macrosphelide A in liquid culture and no other antibiotics were detected. On agar, macrosphelide A inhibited growth of some ascomycetes, basidiomycetes, oomycetes and all four Gram‐positive bacteria tested, including the medically important Staphylococcus aureus with a minimum inhibitory concentration of ≤500 μg ml^?1. There was no inhibition observed against the yeasts and Gram‐negative bacteria when macrosphelide A was tested at 700 μg ml^?1. Conclusions: The spectrum and level of activity of macrosphelide A produced by C. minitans against micro‐organisms are extended markedly compared to previous reports. Significance and Impact of the Study: Macrosphelide A was effective against Staph. aureus. Further study on the control of this bacterium is merited in view of the development of antibiotic resistance.     

Bacterial membrane vesicles: Biogenesis,immune regulation and pathogenesis

Outer membrane vesicles were first described approximately 50 years ago and for many years were considered to be an artifact of bacterial growth. Since that initial discovery, it has become evident that outer membrane vesicles are produced by almost all Gram‐negative bacteria as part of their normal growth in addition to driving pathogenesis within the host. More recently, the identification of membrane vesicle (MV) production by some Gram‐positive bacteria, parasites, fungi, mycobacteria and infected host cells has significantly broadened the field of MV research and emphasized their importance to pathogenesis. In this review, we will focus on discussing recent advances in the field of bacterial MV biogenesis and the mechanisms whereby they modulate immunity and contribute to pathogenesis. We will highlight findings identifying the contribution of extracellular vesicles produced by Gram‐positive bacteria, fungi, parasites, and infected host cells in mediating pathogenesis in addition to the functions of MVs produced by commensal bacteria. Finally, we will discuss recent progress in the development of bacterial MVs as novel vaccines capable of mediating cellular and humoral immune responses.     

Antioxidant,Cytotoxic and Antimicrobial Activity of Chitosan Preparations Extracted from Ganoderma Lucidum Mushroom

Two chitosan extracts were prepared by chemical and enzymatic treatment of Ganoderma lucidum mushroom, as an alternative source to crustacean shells. The molecular weight of the enzymatic extract was lower than that of the chemical one and of shrimp chitosan, as determined by viscosity measurements. Characteristic signals were identified in the ¹H‐NMR spectra and high deacetylation degree indicated good physico‐chemical properties for both mushroom chitosan extracts. The scavenging capacity of mushroom chitosan extracts was moderate against the synthetic radicals of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), but higher values were observed for the enzymatic extract, compared to the chemical extract and shrimp chitosan. In vitro cytotoxicity was evaluated in L929 mouse fibroblast cell lines and the results of MTT assay showed good cytocompatibility in the tested range of concentrations. The growth of Gram‐positive bacteria was inhibited more than Gram‐negative bacteria in the presence of mushroom chitosan extracts, in particular by the chemical one, indicating their efficiency as antimicrobial agents. All these results strengthen the evidence of mushroom polysaccharide preparations availability for biomedical applications.     

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid‐phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram‐positive and Gram‐negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain‐specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species‐specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples.     

The proline‐rich domain of TonB possesses an extended polyproline II‐like conformation of sufficient length to span the periplasm of Gram‐negative bacteria

TonB from Escherichia coli and its homologues are critical for the uptake of siderophores through the outer membrane of Gram‐negative bacteria using chemiosmotic energy. When different models for the mechanism of TonB mediated energy transfer from the inner to the outer membrane are discussed, one of the key questions is whether TonB spans the periplasm. In this article, we use long range distance measurements by spin‐label pulsed EPR (Double Electron–Electron Resonance, DEER) and CD spectroscopy to show that the proline‐rich segment of TonB exists in a PPII‐like conformation. The result implies that the proline‐rich segment of TonB possesses a length of more than 15 nm, sufficient to span the periplasm of Gram‐negative bacteria.