Antimicrobial Resistance Studies Using Raman Spectroscopy on Clinically Relevant Bacterial Strains.

There has been a steep rise in the emergence of antibiotic-resistant bacteria in the past few years. A timely diagnosis can help in initiating appropriate antibiotic therapy. However, conventional techniques for diagnosing antibiotic resistance are time-consuming and labor-intensive. Therefore, we investigated the potential of Raman spectroscopy as a rapid surveillance technology for tracking the emergence of antibiotic resistance. In this study, we used Raman spectroscopy to differentiate clinical isolates of antibiotic-resistant and -sensitive bacteria of Escherichia coli, Acinetobacter baumannii, and Enterobacter species. The spectra were collected with or without exposure to various antibiotics (ciprofloxacin, gentamicin, meropenem, and nitrofurantoin), each having a distinct mechanism of action. Ciprofloxacin- and meropenem-treated sensitive strains showed a decrease in the intensity of Raman bands associated with DNA (667, 724, 785, 1378, 1480, and 1575 cm-1) and proteins (640 and 1662 cm-1), coupled with an increase in the intensity of lipid bands (891, 960, and 1445 cm-1). Gentamicin- and nitrofurantoin-treated sensitive strains showed an increase in the intensity of nucleic acid bands (668, 724, 780, 810, 1378, 1480, and 1575 cm-1) while a decrease in the intensity of protein bands (640, 1003, 1606, and 1662 cm-1) and the lipid band (1445 cm-1). The Raman spectral changes observed in the antibiotic-resistant strains were opposite to that of antibiotic-sensitive strains. The Raman spectral data correlated well with the antimicrobial susceptibility test results. The Raman spectral dataset was used for partial least-squares (PLS) analysis to validate the biomarkers obtained from the univariate analysis. Overall, this study showcases the potential of Raman spectroscopy for detecting antibiotic-resistant and -sensitive bacteria.

Raman spectroscopy study of CdS nanorods and strain induced by the adsorption of 4-mercaptobenzoic acid.

In this study, near- and off-resonance Raman spectra of cadmium sulfide (CdS) quantum rods (NRs) and 4-mercaptobenzoic acid (4-MBA) adsorbed CdS NRs are reported. The envelopes of characteristic optical phonon modes in the near-resonance Raman spectrum of CdS NRs are deconvoluted by following the phonon confinement model. As compared with off-resonant Raman spectra, optical phonon modes scattering cross section is amplified significantly in near-resonance Raman spectra through the Fröhlich interaction. The Huang-Rhys factor defining the strength of the Fröhlich interaction is estimated (∼0.468). Moreover, the adsorption of different concentrations of 4-mercaptobenzoic acid (4-MBA) onto CdS NRs produces surface strain in CdS NRs originating due to surface reconstruction and consequently blue and red shifts in off-resonance (514.5 nm) Raman spectra depending on the concentration of 4-MBA. These consequences are attributed to compressive and tensile strains, respectively. Relative to bulk CdS powder as the reference, strain in CdS NRs increases with decreasing 4-MBA concentrations. In off-resonance Raman spectra of 4-MBA adsorbed CdS NRs, the full width at half maxima of phonon modes (1-LO and 2-LO) and intensity ratio I2-LO/I1-LO increase with decreasing 4-MBA concentration.

Herbicides 2,4-Dichlorophenoxy Acetic Acid and Glyphosate Induce Distinct Biochemical Changes in E. coli during Phenotypic Antibiotic Resistance: A Raman Spectroscopic Study.

Antibiotic resistance is a major global health concern. The increased use of herbicides may lead to multiple antibiotic resistance in bacteria. Conventional techniques for diagnosing antibiotic resistance are laborious, time-intensive, expensive, and lack information about antibiotic susceptibility. On the other hand, Raman spectroscopy is a rapid, label-free, noninvasive alternative to traditional techniques to detect antibiotic resistance. In this study, two popular herbicides 2,4-dichlorophenoxy acetic acid (2,4-D) and N-(phosphonomethyl)glycine (glyphosate) were used to study their effects on the emergence of antibiotic resistance. The Escherichia coli wild-type (WT) MG1655 strain and two isogenic mutants, Δlon and ΔacrB, were used together with Raman spectroscopy. The WT E. coli is sensitive to antibiotics, but exposure to both herbicides induces antibiotic resistance. Using an excitation wavelength of 785 nm, the intensity ratios (e.g., I740/I785, I740/I1003, I1480/I1445, I2934/I2868, and I2934/I2845) were identified as biomarkers to study the induction of antibiotic resistance in bacteria but not NaCl-mediated stress. Using an excitation wavelength of 633 nm, the peak intensity at 740 cm-1 assigned to cytochrome bd decreases under antibiotic stress but increases upon exposure to both herbicides and antibiotics, indicating the development of resistance. Thus, this study can be applied to monitor antibiotic resistance using Raman spectroscopy.

Culture-Independent Raman Spectroscopic Identification of Bacterial Pathogens from Clinical Samples Using Deep Transfer Learning.

The rapid identification of bacterial pathogens in clinical samples like blood, urine, pus, and sputum is the need of the hour. Conventional bacterial identification methods like culturing and nucleic acid-based amplification have limitations like poor sensitivity, high cost, slow turnaround time, etc. Raman spectroscopy, a label-free and noninvasive technique, has overcome these drawbacks by providing rapid biochemical signatures from a single bacterium. Raman spectroscopy combined with chemometric methods has been used effectively to identify pathogens. However, a robust approach is needed to utilize Raman features for accurate classification while dealing with complex data sets such as spectra obtained from clinical isolates, showing high sample-to-sample heterogeneity. In this study, we have used Raman spectroscopy-based identification of pathogens from clinical isolates using a deep transfer learning approach at the single-cell level resolution. We have used the data-augmentation method to increase the volume of spectra needed for deep-learning analysis. Our ResNet model could specifically extract the spectral features of eight different pathogenic bacterial species with a 99.99% classification accuracy. The robustness of our model was validated on a set of blinded data sets, a mix of cultured and noncultured bacterial isolates of various origins and types. Our proposed ResNet model efficiently identified the pathogens from the blinded data set with high accuracy, providing a robust and rapid bacterial identification platform for clinical microbiology.

Infrared Microspectroscopy With Multivariate Analysis to Differentiate Oral Hyperplasia From Squamous Cell Carcinoma: A Proof of Concept for Early Diagnosis.

BACKGROUND AND OBJECTIVES: Despite having numerous advances in therapeutics, mortality and morbidity due to oral cancer incidence are still very high. Early detection can improve the chances of survival in most patients. However, diagnosis at early stages can be challenging as premalignant conditions are usually asymptomatic. Currently, histological assessment remains the gold standard for diagnosis. Early diagnosis poses challenges to pathologists due to less severe morphological changes associated with early stages. Therefore, a fast and robust method of detection based on molecular changes is needed for early diagnosis. © 2021 Wiley Periodicals LLC. STUDY DESIGN/MATERIAL AND METHODS: In the present study, Fourier transform infrared (FTIR) spectroscopic imaging has been used to differentiate early-stage oral hyperplasia from adjacent normal (AN) and oral squamous cell carcinoma (OSCC). Hyperplasia is often considered as an initial event in the pathogenesis of oral cancer and OSCC is the most common advanced stage of malignancy. Differentiating normal versus hyperplasia and hyperplasia versus OSCC can remain quite challenging on occasion using conventional staining as the histological assessment is based on morphological changes. RESULTS: Unsupervised hierarchical cluster analysis (UHCA) has been performed on FTIR images of multiple tissues together that provided some degree of classification among tissue groups. The AN epithelium clustered distinctively using UHCA from both hyperplasia and grades 1 and 2 of OSCC. An increase in the content of DNA, denaturation of protein, and altered lipid structures were more clearly elucidated with spectral analysis. CONCLUSION: This study demonstrates a simple strategy to differentiate early-stage oral hyperplasia from AN and OSCC using UHCA. This study also proposes a future alternative method where FTIR imaging can be used as a diagnostic tool for cancer at early stages.

Cell-free hemoglobin is a marker of systemic inflammation in mouse models of sepsis: a Raman spectroscopic study.

Sepsis is a life-threatening condition caused by heightened host immune responses post infection. Despite intensive research, most of the existing diagnostic methods remain non-specific, labour-intensive, time-consuming or are not sensitive enough for rapid and timely diagnosis of the onset and progression of sepsis. The present work was undertaken to explore the potential of Raman spectroscopy to identify the biomarkers of sepsis in a label-free and minimally invasive manner using different mouse models of inflammation. The sera of BALB/c mice infected with Salmonella Typhimurium reveal extensive hemolysis, as indicated by the Raman bands that are characteristic of the porphyrin ring of hemoglobin (668, 743, 1050, 1253 and 1397 cm-1) which increase in a kinetic manner. These markers are also observed in a lipopolysaccharide-induced endotoxic shock model, but not in a thioglycollate-induced sterile peritonitis model. These data demonstrate that hemolysis is a signature of systemic, but not localised, inflammation. To further validate our observations, sepsis was induced in the nitric oxide synthase 2 (Nos2-/-) deficient strain which is more sensitive to infection. Interestingly, Nos2-/- mice exhibit a higher degree of hemolysis than C57BL/6 mice. Sepsis-induced hemolysis was also confirmed using resonance Raman spectroscopy with 442 nm excitation which demonstrated a pronounced increase in the resonant Raman bands at 670 and 1350 cm-1 in sera of the infected mice. This is the first study to identify inflammation-induced hemolysis in mouse models of sepsis using Raman spectral signatures for hemoglobin. The possible implications of this method in detecting hemolysis in different inflammatory pathologies, such as the ongoing COVID-19 pandemic, are discussed.

Potential of Raman spectroscopic techniques to study proteins.

Proteins are large, complex molecules responsible for various biological processes. However, protein misfolding may lead to various life-threatening diseases. Therefore, it is vital to understand the shape and structure of proteins. Despite numerous techniques, a mechanistic understanding of the protein folding process is still unclear. Therefore, new techniques are continually being explored. In the present article, we have discussed the importance of Raman spectroscopy, Raman Optical Activity (ROA) and various other advancements in Raman spectroscopy to understand protein structure and conformational changes based on the review of our earlier work and recent literature. A Raman spectrum of a protein provides unique signatures for various secondary structures like helices, beta-sheets, turns, random structures, etc., and various amino acid residues such as tyrosine, tryptophan, and phenylalanine. We have shown how Raman spectra can differentiate between bovine serum albumin (BSA) and lysozyme protein based on their difference in sequence and structure (primary, secondary and tertiary). Although it is challenging to elucidate the structure of a protein using a Raman spectrum alone, Raman spectra can be used to differentiate small changes in conformations of proteins such as BSA during melting. Various new advancements in technique and data analyses in Raman spectroscopic studies of proteins have been discussed. The last part of the review focuses on the importance of the ROA spectrum to understand additional features about proteins. The ROA spectrum is rich in information about the protein backbone due to its rigidity compared to its side chains. Furthermore, the ROA spectra of lysozyme and BSA have been presented to show how ROA provides extra information about the solvent properties of proteins.

Understanding phase transition and vibrational mode coupling in ammonium nitrate using 2D correlation Raman spectroscopy.

Ammonium nitrate (AN) is an important component of the chemical industry such as an active ingredient in fertilizers, as an oxidizer in explosive compositions and propellants, and as a blasting agent in civil explosives. Numerous accidents have been reported in the past which concerns its thermal instability and poses a big threat to its processing, transportation, and storage. Despite much literature being reported to understand its thermal instability, a mechanistic view remains unclear. In the present work, we have studied the behavior of AN to temperature change using a mathematical approach called 2D correlation (2D Cos) Raman spectroscopy to provide complete insight into the detailed dynamical nature of the interactions between the species (ionic or molecular) occurring with an increase in temperature. We have analyzed various libration and translational modes of nitrate in the low-frequency region using this mathematical tool. It is observed from 2D maps that the phase transition of AN starts with changes in libration modes followed by various nitrate modes and ammonium modes which further precedes low-frequency translational modes. Further, the 2D correlation could differentiate between modes splitting and shifting based on specific 2D Cos pattern. The changes occurring in the N-O deformation modes, symmetric stretching modes as well as anti-symmetric stretching modes which have been attributed to the weakening of the hetero-ionic coupling between the NH4+ and the NO3- ions could be clearly distinguished in the 2D synchronous and asynchronous plots. Besides, moving window analysis was performed to visualize the transition temperature at which phase change of AN takes place.

Profiling antibiotic resistance in Escherichia coli strains displaying differential antibiotic susceptibilities using Raman spectroscopy.

The rapid identification of antibiotic resistant bacteria is important for public health. In the environment, bacteria are exposed to sub-inhibitory antibiotic concentrations which has implications in the generation of multi-drug resistant strains. To better understand these issues, Raman spectroscopy was employed coupled with partial least squares-discriminant analysis to profile Escherichia coli strains treated with sub-inhibitory concentrations of antibiotics. Clear differences were observed between cells treated with bacteriostatic (tetracycline and rifampicin) and bactericidal (ampicillin, ciprofloxacin, and ceftriaxone) antibiotics for 6 hr: First, atomic force microscopy revealed that bactericidal antibiotics cause extensive cell elongation whereas short filaments are observed with bacteriostatic antibiotics. Second, Raman spectral analysis revealed that bactericidal antibiotics lower nucleic acid to protein (I812 /I830 ) and nucleic acid to lipid ratios (I1483 /I1452 ) whereas the opposite is seen with bacteriostatic antibiotics. Third, the protein to lipid ratio (I2936 /I2885 and I2936 /I2850 ) is a Raman stress signature common to both the classes. These signatures were validated using two mutants, Δlon and ΔacrB, that exhibit relatively high and low resistance towards antibiotics, respectively. In addition, these spectral markers correlated with the emergence of phenotypic antibiotic resistance. Overall, this study demonstrates the efficacy of Raman spectroscopy to identify resistance in bacteria to sub-lethal concentrations of antibiotics.

Probing the Stepwise Unfolding of Bovine Serum Albumin Using 2D Correlation Raman Spectroscopic Analysis.

Protein denaturation involves a change in the protein structure with the loss of activity, which proceeds via various intermediates. The possible intermediate structures account largely for understanding the process of unfolding. Hence, considerable attention is required to characterize partially unfolded protein states and to gain more insight into the information about the sequence and steps involved in protein folding mechanisms. In this report, a stepwise unfolding of bovine serum albumin (BSA) with guanidine hydrochloride (GuHCl) has been investigated using Raman spectroscopy in the amide I and III regions. Two-dimensional (2D) correlation analysis has been applied to reveal information on the sequential order and the dynamic properties of interaction during the unfolding process. Raman spectral signatures in the amide I region revealed that there is no significant change in secondary structures up to 2 M concentration of GuHCl. However, 2D correlation analysis further supports the observation by inferring the strengthening of secondary structure at the expense of tertiary structure. At a higher concentration of GuHCl (2-4 M), there is an accumulation of random and ß-sheet structures that is mediated by small connecting segments of helices. It further accelerates the unfolding of helices and a complete collapse of structure. These analyses establish the ability of Raman spectroscopy to estimate the ensemble of secondary structures present in proteins. The results reveal the mechanistic details of unfolding, characterizing structure of intermediates even at high concentrations, and understanding the evolution of various secondary structures with respect to each other during unfolding. Such observations can be helpful in understanding the factors affecting the shape and size of proteins during folding/unfolding.

Biofilms of the non-tuberculous Mycobacterium chelonae form an extracellular matrix and display distinct expression patterns.

Mycobacterium chelonae is an environmental, non-tuberculous mycobacterial species, capable of causing infections in humans. Biofilm formation is a key strategy used by M. chelonae in colonising niches in the environment and in the host. We studied a water-air interface (pellicle) biofilm of M. chelonae using a wide array of approaches to outline the molecular structure and composition of the biofilm. Scanning electron micrographs showed that M. chelonae biofilms produced an extracellular matrix. Using a combination of biochemical analysis, Raman spectroscopy, and fluorescence microscopy, we showed the matrix to consist of proteins, carbohydrates, lipids and eDNA. Glucose was the predominant sugar present in the biofilm matrix, and its relative abundance decreased in late (established) biofilms. RNA-seq analysis of the biofilms showed upregulation of genes involved in redox metabolism. Additionally, genes involved in mycolic acid, other lipid and glyoxylate metabolism were also upregulated in the early biofilms.

Identification of a resonance Raman marker for cytochrome to monitor stress responses in Escherichia coli.

Raman spectroscopy and resonance Raman spectroscopy are widely used to study bacteria and their responses to different environmental conditions. In the present study, the identification of a novel resonance Raman peak for Escherichia coli, recorded with 633 nm laser excitation is discussed. A peak at 740 cm-1 is observed exclusively with 633 nm excitation but not with 514 nm or 785 nm excitation. This peak is absent in the lag phase but appears in the log phase of bacterial growth. The intensity of the peak increases at high temperature (45 °C) compared with growth at low temperature (25 °C) or the physiological temperature (37 °C). Although osmotic stress lowered bacterial growth, the intensity of this peak was unaffected. However, treatment with chemical uncouplers of oxidative phosphorylation resulted in significantly lower intensity of this Raman band, indicating its possible involvement in respiration. Cytochromes, a component of bacterial respiration' can show resonance enhancement at 633 nm due to the presence of a shoulder in that region depending on the type and conformation of cytochrome. Therefore, the peak intensity was monitored in different genetic mutants of E. coli lacking cytochromes. This peak is absent in the Escherichia coli mutant lacking cydB, but not ccmE, demonstrating the contribution of cytochrome bd subunit II in the peak's origin. In future, this newly found cytochrome marker can be used for biochemical assessment of bacteria exposed to various conditions. Overall, this finding opens the scope for use of red laser excitation in resonance Raman in monitoring stress and respiration in bacteria. Graphical abstract.

Design of P-Doped Mesoporous Carbon Nitrides as High-Performance Anode Materials for Li-Ion Battery.

Herein, we demonstrate a simple and unique strategy for the preparation of P-doped into the substructure of mesoporous carbon nitride materials (P-MCN-1) with ordered porous structures as a high-energy and high-power Li-ion battery (LIB) anode. The P-MCN-1 as an anode in LIB delivers a high reversible discharge capacity of 963 mAh g-1 even after 1000 cycles at a current density of 1 A g-1, which is much higher than that of other counterparts comprising s-triazine (C3H3N3, g-C3N4), pristine MCN-1, and B-containing MCN-1 (B-MCN-1) subunits or carbon allotropes like CNT and graphene (rGO) materials. The P-MCN-1 electrode also exhibits exceptional rate capability even at high current densities of 5, 10, and 20 A g-1 delivering 685, 539, and 274 mAh g-1, respectively, after 2500 cycles. The high electrical conductivity and Li-ion diffusivity (D), estimated from electrochemical impedance spectra (EIS), very well support the extraordinary electrochemical performance of the P-MCN-1. Higher formation energy, lower bandgap value, and high Li-ion adsorption ability predicted by first principle calculations of P-MCN-1 are in good agreement with experimentally observed high lithium storage, stable cycle life, high power capability, and minimal irreversible capacity (IRC) loss. To the best of our knowledge, it is an entirely new material with the combination of ordered mesostructures with P codoping in carbon nitride substructure which offers superior performance for LIB, and hence we believe that this work will create new momentum for the design and development of clean energy storage devices.

Rapid detection of bacterial infection and viability assessment with high specificity and sensitivity using Raman microspectroscopy.

Infectious diseases caused by bacteria still pose major diagnostic challenges in spite of the availability of various molecular approaches. Irrespective of the type of infection, rapid identification of the causative pathogen with a high degree of sensitivity and specificity is essential for initiating appropriate treatment. While existing methods like PCR possess high sensitivity, they are incapable of identifying the viability status of the pathogen and those which can, like culturing, are inherently slow. To overcome these limitations, we developed a diagnostic platform based on Raman microspectroscopy, capable of detecting biochemical signatures from a single bacterium for identification as well as viability assessment. The study also establishes a decontamination protocol for handling live pathogenic bacteria which does not affect identification and viability testing, showing applicability in the analysis of sputum samples containing pathogenic mycobacterial strains. The minimal sample processing along with multivariate analysis of spectroscopic signatures provides an interface for automatic classification, allowing the prediction of unknown samples by mapping signatures onto available datasets. Also, the novelty of the current work is the demonstration of simultaneous identification and viability assessment at a single bacterial level for pathogenic bacteria. Graphical abstract.

Pathogenic Escherichia coli (E. coli) detection through tuned nanoparticles enhancement study.

OBJECTIVE: This study aims to detect pathogenic Escherichia coli (E. coli) bacteria using non-destructive fluorescence microscopy and micro-Raman spectroscopy. RESULTS: Raman vibrational spectroscopy provides additional information regarding biochemical changes at the cellular level. We have used two nanomaterials zinc oxide nanoparticles (ZnO-NPs) and gold nanoparticles (Au-NPs) to detect pathogenic E. coli. The scanning electron microscope (SEM) with energy dispersive X-ray (EDAX) spectroscopy exhibit surface morphology and the elemental composition of the synthesized NPs. The metal NPs are useful contrast agents due to the surface plasmon resonance (SPR) to detect the signal intensity and hence the bacterial cells. The changes due to the interaction between cells and NPs are further correlated to the change in the surface charge and stiffness of the cell surface with the help of the fluorescence microscopic assay. CONCLUSIONS: We conclude that when two E. coli strains (MTCC723 and MTCC443) and NPs are respectively mixed and kept overnight, the growth of bacteria are inhibited by ZnO-NPs due to changes in cell membrane permeability and intracellular metabolic system under fluorescence microscopy. However, SPR possessed Au-NPs result in enhanced fluorescence of both pathogens. In addition, with the help of Raman microscopy and element analysis, significant changes are observed when Au-NPs are added with the two strains as compared to ZnO-NPs due to protein, lipid and DNA/RNA induced conformational changes.

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.

Thermodynamically Stable Mesoporous C3 N7 and C3 N6 with Ordered Structure and Their Excellent Performance for Oxygen Reduction Reaction.

Carbon nitrides with a high N/C atomic ratio (>2) are expected to offer superior basicity and unique electronic properties. However, the synthesis of these nanostructures is highly challenging since many parts of the CN frameworks in the carbon nitride should be replaced with thermodynamically less stable NN frameworks as the nitrogen content increases. Thermodynamically stable C3 N7 and C3 N6 with an ordered mesoporous structure are synthesized at 250 and 300 °C respectively via a pyrolysis process of 5-amino-1H-tetrazole (5-ATTZ). Polymerization of the precursor to the ordered mesoporous C3 N7 and C3 N6 is clearly proved by X-ray and electron diffraction analyses. A combined analysis including diverse spectroscopy and FDMNES and density functional theory (DFT) calculations demonstrates that the NN bonds are stabilized in the form of tetrazine and/or triazole moieties in the C3 N7 and C3 N6 . The ordered mesoporous C3 N7 represents the better oxygen reduction reaction (ORR) performances (onset potential: 0.81 V vs reversible hydrogen electrode (RHE), electron transfer number: 3.9 at 0.5 V vs RHE) than graphitic carbon nitride (g-C3 N4 ) and the ordered mesoporous C3 N6 . The study on the mechanism of ORR suggests that nitrogen atoms in the tetrazine moiety of the ordered mesoporous C3 N7 act as active sites for its improved ORR activity.

Raman spectroscopy reveals distinct differences between two closely related bacterial strains, Mycobacterium indicus pranii and Mycobacterium intracellulare.

A common technique used to differentiate bacterial species and to determine evolutionary relationships is sequencing their 16S ribosomal RNA genes. However, this method fails when organisms exhibit high similarity in these sequences. Two such strains that have identical 16S rRNA sequences are Mycobacterium indicus pranii (MIP) and Mycobacterium intracellulare. MIP is of significance as it is used as an adjuvant for protection against tuberculosis and leprosy; in addition, it shows potent anti-cancer activity. On the other hand, M. intracellulare is an opportunistic pathogen and causes severe respiratory infections in AIDS patients. It is important to differentiate these two bacterial species as they co-exist in immuno-compromised individuals. To unambiguously distinguish these two closely related bacterial strains, we employed Raman and resonance Raman spectroscopy in conjunction with multivariate statistical tools. Phenotypic profiling for these bacterial species was performed in a kinetic manner. Differences were observed in the mycolic acid profile and carotenoid pigments to show that MIP is biochemically distinct from M. intracellulare. Resonance Raman studies confirmed that carotenoids were produced by both MIP as well as M. intracellulare, though the latter produced higher amounts. Overall, this study demonstrates the potential of Raman spectroscopy in differentiating two closely related mycobacterial strains. Graphical abstract.

Spectroscopic and computational insights into the ion-solvent interactions in hydrated aprotic and protic ionic liquids.

Ionic liquids (ILs) and their aqueous solutions are emerging media for solving and manipulating biochemical molecules such as proteins. Unleashing the full potential however requires a detailed mechanistic understanding of how suitable protic and aprotic ILs behave in the presence of water in the first place. The present work aims at making an important step by performing a combined experimental and computational study of two selected ILs and their mixtures with water: the aprotic cholinium propionate ([Chl][Pro]) and the protic N-methyl-2-pyrrolidonium propionate ([NMP][Pro]). IR and Raman spectroscopy reveal stronger ion-solvent interactions in [Chl][Pro]-H2O systems compared to [NMP][Pro]-H2O mixtures. This can be explained by the tightly packed ion-pair associations in [NMP][Pro] comprising the protic -N+-H counterpart, which allows the establishment of highly directional and strong interionic hydrogen bonds. The spectral decomposition of the O-D stretching band into three sub-peaks showed that the protic [NMP][Pro] favors the self-association of water molecules. On the other hand, the predominant fraction of water-anion/cation aggregates exists in aprotic [Chl][Pro]. These hydrated systems can be envisaged using quantum-chemical calculations in the following way: H2O[Chl]+H2O[Pro]-H2O and H2O[NMP]+[Pro]-H2O, which implied preferable solvent-shared ion-pair (SIP) configurations for [Chl][Pro]-H2O systems, whereas the contact ion-pair (CIP) state prevails for the [NMP][Pro]-H2O systems. The latter holds even in the water-rich regime. In future work, these findings will be the basis for an understanding of the underlying principles that govern the interactions of ions with bio-molecules in aqueous solutions.

Sulfur-Doped Mesoporous Carbon Nitride with an Ordered Porous Structure for Sodium-Ion Batteries.

Mesoporous carbon nitride (MCN) with well-ordered porous structures is a promising anode material for secondary ion batteries owing to their unique physico- and electrochemical properties. However, the practical application of these MCNs in sodium-ion batteries (SIBs) is still limited because of their confined interlayer distance, which results in restricted accommodation of Na ions inside the lattice. Here, we report on the synthesis of highly ordered sulfur-doped MCN (S-MCN) through a hard template approach by employing dithiooxamide (DTO) as a single molecular precursor containing carbon, nitrogen, and sulfur elements. The interlayer distance of carbon nitride is significantly expanded upon the introduction of larger S ions on the MCN lattice, which enables high capability of Na ion accommodation. We also demonstrate through the first-principles density functional theory calculation that the present S-MCN is highly optimized not only for the chemical structure but also for uptaking abundant Na ions with high adsorption energy. The specific discharge capacity of SIBs appears to be remarkably enhanced for S-MCN (304.2 mA h g-1) compared to the nonporous S-CN (167.9 mA h g-1) and g-C3N4 (5.4 mA h g-1), highlighting the pivotal roles of the highly ordered mesoporous structure and S-doping in enhancing the electrochemical functionality of carbon nitride as an anode material for SIBs.