Evaluation of infrared spectroscopy as a bacterial identification method

Summary A study motivated by the recent revival of interest in the use of IR spectroscopy to identify bacteria is reported. A library of FT-IR spectra of dried bacterial films was compled using 16 different strains. A test set was complied from spectra of the same strains grown several months later. The test set was quantitatively compared with the library on the basis of spectral similarity in the region 980–1190 cm^–1. Six of the strains in the test set were not matched with the correct strain in the library despite efforts to reproduce the conditions under which cells were grown and prepared. The results suggest that reproducibility of the bacterial spectra is a potential difficulty that must be addressed by any attempts to develop FT-IR spectroscopy as a bacterial identification method.     

Interaction of monolayers of concanavalin A with mono- and polysaccharides A study by means of infrared-attenuated total reflectance spectroscopy

The effects of pH, Mn²⁺ and Ca²⁺ and urea denaturation on the interaction of monolayers of concanavalin A on saline with the polysaccharide dextran B-1355 and the monosaccharides methyl α-d-mannopyranoside and d-galactose have been investigated. Infrared absorption spectra of compressed monolayers of the protein and the protein-dextran complex coated on a germanium plate have been obtained by means of attenuated total reflectance spectroscopy. Except in one case of denaturation, the amide I absorption of concanavalin A peaked around 1631 cm^?1, indicating a predominance of the β-pleated sheet conformation, in agreement with its secondary structure in the solution and crystalline phases. The contribution to the absorbance of the concanavalin A-dextran films at 3300 cm^?1 due to absorption by the O-H stretching modes of the polysaccharide is a measure of its binding. Increasing the pH from 6.1 to 7.5 appreciably reduced the dextran binding, at pH 9.3 the binding was zero. Adding 1 mM Mn²⁺ and Ca²⁺ to the subphase at pH 7.5 restored both the dextran binding and the affinity of concanavalin A for methyl α-d-mannopyranoside to that of the native protein at pH 6.1. At this latter pH, the weak binding of dextran to monolayers of demetallized concanavalin A (apo-concanavalin A) was also restored to that for the native molecule by the addition of these divalents. This indicates the requirement of concanavalin A for these ions to maintain the integrity of the saccharide-binding site. The loss of dextran binding with urea denaturation was also observed. These results parallel those for solutions of the protein, indicating the validity of the monolayer system for the study of these interactions.     

Meningococcal group W-135 and Y capsular polysaccharides paradoxically enhance activation of the alternative pathway of complement

Although capsular polysaccharide (CPS) is critical for meningococcal virulence, the molecular basis of alternative complement pathway (AP) regulation by meningococcal CPSs remains unclear. Using serum with only the AP active, the ability of strains to generate C3a (a measure of C3 activation) and subsequently deposit C3 fragments on bacteria was studied in encapsulated group A, B, C, W-135, and Y strains and their isogenic unencapsulated mutants. To eliminate confounding AP regulation by membrane-bound factor H (fH; AP inhibitor) and lipooligosaccharide sialic acid, the meningococcal fH ligands (fHbp and NspA) and lipooligosaccharide sialylation were deleted in all strains. Group A CPS expression did not affect C3a generation or C3 deposition. C3a generated by encapsulated and unencapsulated group B and C strains was similar, but CPS expression was associated with reduced C3 deposition, suggesting that these CPSs blocked C3 deposition on membrane targets. Paradoxically, encapsulated W-135 and Y strains (including the wild-type parent strains) enhanced C3 activation and showed marked C3 deposition as early as 10 min; at this time point C3 was barely activated by the unencapsulated mutants. W-135 and Y CPSs themselves served as a site for C3 deposition; this observation was confirmed using immobilized purified CPSs. Purified CPSs bound to unencapsulated meningococci, simulated findings with naturally encapsulated strains. These data highlight the heterogeneity of AP activation on the various meningococcal serogroups that may contribute to differences in their pathogenic mechanisms.     

An innovative spectroelectrochemical reflection cell for rapid protein electrochemistry and ultraviolet/visible/infrared spectroscopy

A novel electrochemical reflection cell combining electrochemical techniques and spectroscopy which uses a solid gold working electrode as an optical mirror is described. This cell can be used at path lengths as low as a few micrometers and thus is suitable for ultraviolet/visible (UV/Vis) and infrared spectroscopy even for aqueous solutions and suspensions. The cell was designed for small sample volumes of only a few microliters, thus reducing the effort for sample preparation. Due to the short path length of some micrometers, the entire volume is within the Nernst diffusion layer, hence resulting in fast equilibration. Evaluation of the technique is described with direct electrochemistry of horse heart cytochrome c at the gold electrode modified with 4,4'-dithiodipyridine. Cyclic voltammograms indicate rapid and reversible electrochemistry with the correct midpoint potential (52 mV vs Ag/AgCl/3 M KCl). Chronoamperometry and coulometry confirm rapid and complete oxidation and reduction; the cell volume can be entirely fully reduced within less than 10-20 s. Spectroscopy in the UV/Vis region, with potentials at the working electrode stepped between -390 and 390 mV, show perfect titration of the cytochrome c heme bands. A Nernst fit of the alpha band absorption, with redox potential Em and number of electrons n left as parameters, yields a midpoint potential of 49 mV and n=0.9. The potential of this cell in the investigation of biological electron transfer reactions and in the study of bioenergetic systems is discussed.     

An automated approach for fringe frequency estimation and removal in infrared spectroscopy and hyperspectral imaging of biological samples

In infrared spectroscopy of thin film samples, interference introduces distortions in spectra, commonly referred to as fringes. Fringes may alter absorbance peak ratios, which hampers the spectral analysis. We have previously introduced extended multiplicative signal correction (EMSC) for fringes correction. In the current article, we provide a robust open-source algorithm for fringe correction in infrared spectroscopy and propose several improvements to the Fringe EMSC model. The suggested algorithm achieves a more precise fringe frequency estimation by mean centering of the measured spectrum and applying a window function prior to the Fourier transform. It selects two frequencies from a user defined number of maxima in the Fourier domain. The improved Fringe EMSC algorithm is validated on two experimental datasets, one of them being a hyperspectral image. Techniques for separating sample spectra from background spectra in hyperspectral images, and techniques to identify spectra affected by fringes are also provided.     

Comparative study of non-invasive monitoring via infrared spectroscopy for mammalian cell cultivations

Process analytical technology (PAT) is a guide to improve process development in biotech industry. Optical sensors such as near and mid infrared spectrometers fulfill an essential part for PAT. NIRS and MIRS were investigated as non-invasive on line monitoring tools for animal cell cultivations in order to predict critical process parameters, like cell parameters as well as substrate and metabolite concentrations. Eight cultivations were performed with frequent sampling. Variances between cultivations were induced by spiking experiments with intent to break correlations between analytes; to keep causality of the models; and to increase model robustness.     

Water near lipid membranes as seen by infrared spectroscopy

The ordering and H-bonding characteristics of the hydration water of the lipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were studied using polarized infrared spectroscopy by varying either the temperature or the relative humidity of the ambient atmosphere of multibilayer samples. The OH-stretching band of lipid-bound water was interpreted by a simplified two-state model of well-structured, low density “network” water and of less-structured dense “multimer” water. The IR-spectroscopic data reflect a rather continuous change of the water properties with increasing distance from the membrane and with changing temperature. Network and multimer water distribute across the whole polar interphase with changing composition and orientation. Upon dehydration the fraction of network water increases from about 30 to 60%, a value which is similar to that in supercooled water at −25°C. The highly ordered gel phase gives rise to an increased fraction of structured network water compared with the liquid crystalline phase. The IR order parameter shows that the water dipoles rearrange from a more parallel towards a more perpendicular orientation with respect to the membrane normal with progressive hydration. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Probing conformational changes of proteins by quantitative second-derivative infrared spectroscopy

Probing protein conformational changes plays a crucial role in protein structure and function studies. However, the lack of efficient biophysical techniques makes it difficult to obtain the distinct behaviors of different secondary structure elements in a protein upon perturbation. This paper presents a discussion of the two major problems, the effect of sidelobes and different half-width at half-height (HWHH) values, encountered in quantitative second-derivative infrared (QSD-IR) spectroscopy and introduces the development of two criteria for checking the validity of the results obtained using the QSD-IR method. It was found that neither the sidelobes nor the HWHH significantly affected the quantitative result of protein conformational changes by using poly-l-lysine and hemoglobin as model proteins. A case study of bovine serum albumin (BSA) thermal aggregation suggested that the thermal transition of BSA was a process involving sequential events, and the two helical components were found to have a distinct response to heat perturbation. These results were confirmed by two-dimensional infrared correlation spectroscopy and by results in literature, suggesting that the QSD-IR method might be a potentially powerful tool to probe the distinct response of different secondary structures to perturbation.     

惰性材料表面细菌生物膜构建的研究

目的构建惰性材料塑料输液管内壁细菌生物膜体外模型,观察细菌生物膜的结构,探讨输液管内壁细菌生物膜形成影响因素。方法建立铜绿假单胞菌生物膜和铜绿假单胞菌、肺炎克雷伯菌混合生物膜,分别于培养1、3和7d用扫描电镜动态观察生物膜形成过程。结果混合菌生物膜的生长速度高于铜绿假单胞菌单独成膜。结论输液管是形成细菌生物膜的良好支持材料,混合细菌培养可以加速细菌形成生物膜。     

An inexpensive infrared growth sensor array for detection of bacterial antibiotic susceptibility

Abstract An inexpensive infrared sensor was constructed and used for the rapid testing of bacterial antibiotic susceptibility by detection of changes in absorbance at 950 nm. By comparing cultures of clinical isolates together with control strains ( Escherichia coli NCTC 10418, Staphylococcus aureus NCTC 6571 or Pseudomonas aeruginosa NCTC 10662) after addition of an antibiotic, results on susceptibility were obtained within 3–5 h from the original plate culture. Representative strains of E. coli, P. aeruginosa , and S. aureus were tested successfully against ampicillin, penicillin, gentamicin or ciprofloxacin.     

NO detection in biological samples: Differentiation of NO and NO using infrared laser spectroscopy

Accurate characterization of the biochemical pathways of nitric oxide (NO) is essential for investigations in the field of NO research. To analyze the different reaction pathways of enzymatic and non-enzymatic NO formation, determination of the source of NO is crucial. Measuring NO-related products in biological samples distinguishing between ¹⁴NO and ¹⁵NO offers the opportunity to specifically analyze NO signaling in blood and tissue. The aim of this study was to establish a highly sensitive technique for the specific measurement of NO in an isotopologue-selective manner in biological samples.With the cavity leak-out spectroscopy setup (CALOS) a differentiation between ¹⁴NO and ¹⁵NO is feasible. We describe here the employment of this method for measurements in biological samples. Certified gas mixtures of ¹⁴NO/N₂ and ¹⁵NO/N₂ were used to calibrate the system. and of aqueous and biological samples were reduced in a triiodide solution, and the NO released was detected via CALOS. Gas-phase chemiluminescence detection (CLD) was used for evaluation.The correlation received for both methods for the detection of NO in the gas phase was r = 0.999, p < 0.0001. Results obtained using aqueous and biological samples verified that CALOS enables NO measurements with high accuracy (detection limit for 0.3 pmol and 0.5 pmol; correlation ¹⁴NO: p < 0.0001, r = 0.975, ¹⁵NO: p < 0.0001, r = 0.969).The CALOS assay represents an extension of NO measurements in biological samples, allowing specific investigations of enzymatic and non-enzymatic NO formation and metabolism in a variety of samples.     

Membrane-induced peptide structural changes monitored by infrared and circular dichroism spectroscopy

As more peptide secondary structures deduced by infrared spectroscopy (IR) have been reported in the literature, there have been overlaps in assignments of elements of secondary structure to carbonyl vibrational frequencies. We have investigated this phenomenon with regards to the use of IR for monitoring membrane-induced structural changes using conformationally diverse peptides. These IR studies, complemented by circular dichroism (CD) experiments, revealed that peptide–solvent interactions can mask membrane-induced conformational changes monitored by IR. A structural transition from random coil to α-helix upon the binding of mastoparan X to a membrane was clearly observed by CD but obscured in the amide I region of the IR spectrum. In addition, unlike the buried helical peptides gramicidin D and P16 in micelles, the amide II peak for mastoparan X was absent, likely due to H–D exchange. This suggests information on the peptide's membrane-bound solvent accessibility could be obtained from this region of the spectrum.     

Quantum dot-based assay for Cu quantification in bacterial cell culture

A simple and sensitive method for quantification of nanomolar copper with a detection limit of 1.2 × 10⁻¹⁰ M and a linear range from 10⁻⁹ to 10⁻⁸ M is reported. For the most useful analytical concentration of quantum dots, 1160 μg/ml, a 1/K_sv value of 11 μM Cu²⁺ was determined. The method is based on the interaction of Cu²⁺ with glutathione-capped CdTe quantum dots (CdTe–GSH QDs) synthesized by a simple and economic biomimetic method. Green CdTe–GSH QDs displayed the best performance in copper quantification when QDs of different sizes/colors were tested. Cu²⁺ quantification is highly selective given that no significant interference of QDs with 19 ions was observed. No significant effects on Cu²⁺ quantification were determined when different reaction matrices such as distilled water, tap water, and different bacterial growth media were tested. The method was used to determine copper uptake kinetics on Escherichia coli cultures. QD-based quantification of copper on bacterial supernatants was compared with atomic absorption spectroscopy as a means of confirming the accuracy of the reported method. The mechanism of Cu²⁺-mediated QD fluorescence quenching was associated with nanoparticle decomposition.     

Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa

Mapping the distribution of phylogenetically distinct bacteria in natural environments is of primary importance to an understanding of ecological dynamics. Here we present a quantitative PCR (qPCR) assay for the analysis of higher taxa composition in natural communities that advances previously available methods by allowing quantification of several taxa during the same qPCR run. Existing primers targeting the 16S rRNA gene specific for Firmicutes, Actinobacteria, Bacteroidetes and for the α and γ subdivisions of the Proteobacteria were improved by largely increasing the coverage of the taxon they target without diminishing their specificity. The qPCR assay was validated in vitro testing artificial mixtures of 16S rRNA sequences and used to characterise the composition of natural communities developing in young marine biofilms. The possible contribution of the proposed technique in revealing ecological dynamics affecting higher bacterial taxa is discussed.     

A radioisotope assay for the quantification of hydrocarbon biodegradation potential in environmental samples

An enrichment culture method is described for quantifying the activity of hydrocarbon oxidizing bacteria in water and sediments. Application of the procedure indicated that the hydrocarbon oxidizing potential of environmental samples reflects the hydrocarbon burden of the area, the ability of the microflora to utilize hydrocarbons, and that lakes with large aquatic plant communities contain populations of hydrocarbon bacteria comparable to those found in oil-polluted harbors.     

Fourier transform infrared spectroscopy as a tool to characterize molecular composition and stress response in foodborne pathogenic bacteria

Vibrational spectroscopy techniques have shown capacity to provide non-destructive, rapid, relevant information on microbial systematics, useful for classification and identification. Infrared spectroscopy enables the biochemical signatures from microbiological structures to be extracted and analyzed, in conjunction with advanced chemometrics. In addition, a number of recent studies have shown that Fourier Transform Infrared (FT-IR) spectroscopy can help understand the molecular basis of events such as the adaptive tolerance responses expressed by bacteria when exposed to stress conditions in the environment (e.g. those that cells confront in food and during food processing). The current review gives an overview of the published experimental techniques, data-processing algorithms and approaches used in FT-IR spectroscopy to assess the mechanisms of bacterial inactivation by food processing technologies and antimicrobial compounds, to monitor the spore and membrane properties of foodborne pathogens in changing environments, to detect stress-injured microorganisms in food-related environments, to assess dynamic changes in bacterial populations, and to study bacterial tolerance responses.     

Advances of infrared spectroscopy in natural product research

Natural product's properties are related to certain classes of compounds such as alkaloids, flavonoids, essential oils and others. Traditionally, separation techniques including thin layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE) even hyphenated to mass spectrometry (MS) were used for the elucidation, qualitative and quantitative analysis of individual compounds.In food industry, spectroscopic investigations using infrared radiation have been used to monitor and evaluate the composition and quality already since the early sixties. During the last four decades near-infrared spectroscopy (NIR; 800–2500 nm; 12,500–4000 cm⁻¹) has become one of the most attractive and used methods for analysis for the following reasons: it represents a non-invasive analytical tool allowing a fast and simultaneous qualitative and quantitative characterization of natural products and their constituents. Additionally, the development of custom-made hand-held instruments enables in-field measurement for determining the optimum harvest time.Attenuated total reflection (ATR) and Fourier transform infrared (FTIR) spectroscopic imaging are suitable not only for the differentiation of different plant species, but also to distinct various ingredients within a plant. FTIR spectroscopic microscopy enables molecular imaging of complex botanical samples and therefore the detection and characterization of the molecular components of biological tissue.In the present contribution, the principle, technique and methodology of the different infrared spectroscopic methods are described followed by a discussion of quantitative and qualitative application possibilities in the field of natural product analysis.