Correlation analysis between Raman spectral signature and transcriptomic features of carbapenem-resistant Klebsiella pneumoniae.

The Raman microspectroscopy technology has been successfully applied to evaluate the molecular composition of living cells for identifying cell types and states, but the rationale behind it was not well investigated. In this study, we acquired single-cell Raman spectra (SCRS) of three Klebsiella pneumoniae (K. pneumoniae) strains with different Carbapenem resistant mechanisms and analyzed them with machine learning algorithm. Two carbapenem resistant Klebsiella pneumoniae (CRKP) strains can be successfully distinguished from susceptible strain and CRKP with KPC or IMP carbapenemases can be classified with an overall accuracy achieving 100 %. Furthermore, we performed a correlation analysis between transcriptome and Raman spectra, and found that Raman shifts such as 752 and 1039 cm-1 highly correlated with drug resistance genes expression and could be regarded as Raman biomarkers for CRKP with different mechanisms. The findings of the study provide a theoretical basis for identifying the relationship between Raman spectra and transcriptome of bacteria, as well as a novel method for rapid identification of CRKP and their carbapenemases types.

Heat shock potentiates aminoglycosides against gram-negative bacteria by enhancing antibiotic uptake, protein aggregation, and ROS.

The potentiation of antibiotics is a promising strategy for combatting antibiotic-resistant/tolerant bacteria. Herein, we report that a 5-min sublethal heat shock enhances the bactericidal actions of aminoglycoside antibiotics by six orders of magnitude against both exponential- and stationary-phase Escherichia coli. This combined treatment also effectively kills various E. coli persisters, E. coli clinical isolates, and numerous gram-negative but not gram-positive bacteria and enables aminoglycosides at 5% of minimum inhibitory concentrations to eradicate multidrug-resistant pathogens Acinetobacter baumannii and Klebsiella pneumoniae. Mechanistically, the potentiation is achieved comprehensively by heat shock-enhanced proton motive force that thus promotes the bacterial uptake of aminoglycosides, as well as by increasing irreversible protein aggregation and reactive oxygen species that further augment the downstream lethality of aminoglycosides. Consistently, protonophores, chemical chaperones, antioxidants, and anaerobic culturing abolish heat shock-enhanced aminoglycoside lethality. We also demonstrate as a proof of concept that infrared irradiation- or photothermal nanosphere-induced thermal treatments potentiate aminoglycoside killing of Pseudomonas aeruginosa in a mouse acute skin wound model. Our study advances the understanding of the mechanism of actions of aminoglycosides and demonstrates a high potential for thermal ablation in curing bacterial infections when combined with aminoglycosides.

Leveraging single-cell Raman spectroscopy and single-cell sorting for the detection and identification of yeast infections.

Invasive fungal infection serves as a great threat to human health. Discrimination between fungal and bacterial infections at the earliest stage is vital for effective clinic practice; however, traditional culture-dependent microscopic diagnosis of fungal infection usually requires several days, meanwhile, culture-independent immunological and molecular methods are limited by the detectable type of pathogens and the issues with high false-positive rates. In this study, we proposed a novel culture-independent phenotyping method based on single-cell Raman spectroscopy for the rapid discrimination between fungal and bacterial infections. Three Raman biomarkers, including cytochrome c, peptidoglycan, and nucleic acid, were identified through hierarchical clustering analysis of Raman spectra across 12 types of most common yeast and bacterial pathogens. Compared to those of bacterial pathogens, the single cells of yeast pathogens demonstrated significantly stronger Raman peaks for cytochrome c, but weaker signals for peptidoglycan and nucleic acid. A two-step protocol combining the three biomarkers was established and able to differentiate fungal infections from bacterial infections with an overall accuracy of 94.9%. Our approach was also used to detect ten raw urinary tract infection samples. Successful identification of fungi was achieved within half an hour after sample obtainment. We further demonstrated the accurate fungal species taxonomy achieved with Raman-assisted cell ejection. Our findings demonstrate that Raman-based fungal identification is a novel, facile, reliable, and with a breadth of coverage approach, that has a great potential to be adopted in routine clinical practice to reduce the turn-around time of invasive fungal disease (IFD) diagnostics.

Design, Synthesis, and Application of Carbon Dots With Synergistic Antibacterial Activity.

The diversity of bacteria and their ability to acquire drug resistance lead to many challenges in traditional antibacterial methods. Photothermal therapies that convert light energy into localized physical heat to kill target microorganisms do not induce resistance and provide an alternative for antibacterial treatment. However, many photothermal materials cannot specifically target bacteria, which can lead to thermal damage to normal tissues, thus seriously affecting their biological applications. Here, we designed and synthesized bacteria-affinitive photothermal carbon dots (BAPTCDs) targeting MurD ligase that catalyzes the synthesis of peptidoglycan (PG) in bacteria. BAPTCDs presented specific recognition ability and excellent photothermal properties. BAPTCDs can bind to bacteria very tightly due to their chiral structure and inhibit enzyme activity by competing with D-glutamic acid to bind to MurD ligases, thus inhibiting the synthesis of bacterial walls. It also improves the accuracy of bacteria treatment by laser irradiation. Through the synergy of biochemical and physical effects, the material offers outstanding antibacterial effects and potentially contributes to tackling the spread of antibiotic resistance and facilitation of antibiotic stewardship.

In Vitro Anticancer Drug Sensitivity Sensing through Single-Cell Raman Spectroscopy.

Traditional in vitro anticancer drug sensitivity testing at the population level suffers from lengthy procedures and high false positive rates. To overcome these defects, we built a confocal Raman microscopy sensing system and proposed a single-cell approach via Raman-deuterium isotope probing (Raman-DIP) as a rapid and reliable in vitro drug efficacy evaluation method. Raman-DIP detected the incorporation of deuterium into the cell, which correlated with the metabolic activity of the cell. The human non-small cell lung cancer cell line HCC827 and human breast cancer cell line MCF-7 were tested against eight different anticancer drugs. The metabolic activity of cancer cells could be detected as early as 12 h, independent of cell growth. Incubation of cells in 30% heavy water (D2O) did not show any negative effect on cell viability. Compared with traditional methods, Raman-DIP could accurately determine the drug effect, meanwhile, it could reduce the testing period from 72-144 h to 48 h. Moreover, the heterogeneity of cells responding to anticancer drugs was observed at the single-cell level. This proof-of-concept study demonstrated the potential of Raman-DIP to be a reliable tool for cancer drug discovery and drug susceptibility testing.

Development of a Fast Raman-Assisted Antibiotic Susceptibility Test (FRAST) for the Antibiotic Resistance Analysis of Clinical Urine and Blood Samples.

Human health is at great risk due to the spreading of antimicrobial resistance (AMR). The lengthy procedure of conventional antimicrobial susceptibility testing (AST) usually requires a few days. We developed a fast Raman-assisted antibiotic susceptibility test (FRAST), which detects single bacterial metabolic activity in the presence of antibiotics, using Raman single-cell spectroscopy. It was found that single-cell Raman spectra (SCRS) would show a clear and distinguishable Raman band at the "silent zone" (2000-2300 cm-1), due to the active incorporation of deuterium from heavy water (D2O) by antibiotic-resistant bacteria. This pilot study has compared the FRAST and the conventional AST for six clinical standard quality controls (four Gram-negative and two Gram-positive bacteria strains) in response to 38 antibiotics. In total, 3200 treatments have been carried out and approximately 64 000 SCRS have been acquired for FRAST analysis. The result showed an overall agreement of 88.0% between the FRAST and the conventional AST assay. The gram-staining classification based on the linear discriminant analysis (LDA) model of SCRS was developed, seamlessly coupling with the FRAST to further reduce the turnaround time. We applied the FRAST to real clinical analysis for nine urinary infectious samples and three sepsis samples. The results were consistent with MALDI-TOF identification and the conventional AST. Under the optimal conditions, the "sample to report" of the FRAST could be reduced to 3 h for urine samples and 21 h for sepsis samples. The FRAST provides fast and reliable susceptibility tests, which could speed up microbiological analysis for clinical practice and facilitate antibiotic stewardship.

High photoluminescence nitrogen, phosphorus co-doped carbon nanodots for assessment of microbial viability.

Assessment of microbial viability plays a key role in human health protection. Optical imaging based on fluorescent dyes is a simple and convenient way to assess microbial viability. However, it is still a challenge to obtain stable, nontoxic and low-cost dyes. Herein, we prepared a nitrogen and phosphorus co-doped carbon nanodots (N, P-CDs) via a one-step solvothermal method. The prepared CDs possess plenty of functional groups and exhibit high stability, good biocompatibility, excellent photoluminescent and low toxicity. Especially, the properties of high quantum yield (89.9%) and highly negative surface charge (-41.9 mV) make the prepared N, P-CDs ideal materials for microbial differentiation. Compared with commercial dyes, our CDs are more stable, cost less, which can rapidly distinguish dead microorganisms from living ones with higher specificity.

A novel "on-off-on" fluorescence assay for the discriminative detection of Cu(ii) and l-cysteine based on red-emissive Si-CDs and cellular imaging applications.

Copper ions (Cu2+) and l-cysteine (l-Cys) in the human body always play critical roles in various physiological processes, while abnormal Cu2+ and l-Cys concentrations in the biological system lead to many diseases. In this manuscript, Si-doped carbon dots (Si-CDs) with near-infrared fluorescence were designed for the detection of Cu2+ and l-Cys through the fluorescence "on-off-on" mode. The carbon dots exhibited not only excellent optical merits including good stability against photobleaching and high chemical stability, but also superior biological compatibility. Interestingly, due to the abundant amino groups distributed on the surface of Si-CDs, they could bind to copper ions to form cupric amine complexes and then quench the fluorescence of Si-CDs due to an electron transfer process. In addition, upon the addition of l-Cys, the FL intensity of Si-CDs could be effectively recovered accompanied with complexation between Cu2+ and the functional groups in l-Cys, due to which Cu2+ was removed from the surface of Si-CDs. Notably, as far as we know, these are the first red-emitting carbon dots for copper ion and l-Cys assays in water samples and human plasma samples. Furthermore, this strategy was successfully applied to the determination of Cu2+ and l-Cys in living systems, demonstrating great practicability in biomedical applications.

One-step synthesis of green emission carbon dots for selective and sensitive detection of nitrite ions and cellular imaging application.

In recent years, carbon dot (CD)-based fluorescent sensors for selective ions or small biomolecules have drawn great attention. In this work, highly fluorescent CDs (QY = 21%) were prepared from 2,3-diamino pyridine as the precursor through a facile solvothermal process. The CDs showed high stability and a green emission in aqueous, and the optimal emission wavelength of CDs is 508 nm under the excitation wavelength of 438 nm. Interestingly, a CDs-based nanoprobe was developed for a selective and sensitive fluorescence quenching response to NO2 - in water, and the quenching mechanism was investigated in the work. Besides, the recovery rates of NO2 - in the range of 98-103.5% were found to be acceptable, indicating that the proposed CDs could be act as potential candidates for determination of nitrite ions in real samples. Meanwhile, the nanoprobe was also successfully employed in a visualization biosensing platform for determination of NO2 - in living cells due to its eminent biocompatibility.

A highly selective fluorescent probe for cyanide ion and its detection mechanism from theoretical calculations.

A new cyanide probe has been prepared by one-step synthesis and evaluated by UV-vis and fluorescent method. This probe is combined by a fluorene part and a hemicyanine group through a conjugated linker, which is found to show rapid response, high selectivity and sensitivity for cyanide anions with significant dual colorimetric and fluorescent signal changes in aqueous solution. An intramolecular charge transfer (ICT) effect plays a key role in the CN- sensing properties, and the details of this mechanism are further supported by DFT and TD-DFT calculations. The theoretical study shows that the introduction of CN- twists the original plane structure and blocks the ICT process in the whole molecule, which brings about the absorption blue-shift and the fluorescence quenching.

One-step synthesis of nitrogen, sulfur co-doped carbon nanodots and application for Fe3+ detection.

Carbon nanodots (CDs) are novel forms of zero-dimensional carbonaceous nanomaterials, which have attracted the attention of researchers. Long wavelength emission decreases the interference of auto-fluorescence of tissue and can also penetrate more deeply. However, it is still a challenge to develop red emissive CDs. In this work, nitrogen, sulfur co-doped CDs are synthesized with red light emission through one-step solvothermal treatment. The as-synthesized CDs exhibit excellent properties such as high chemical stability, good biocompatibility, excellent photoluminescence properties, good water solubility, and low toxicity, and also show high sensitivity and selectivity towards Fe3+ sensing. The detection limit is as low as 17.3 nM. In addition, the CDs are successfully utilized for monitoring Fe3+ in living cells, which demonstrates their promising applicability in bioimaging. Furthermore, a test paper based on the CQDs has been prepared. This developed nanosensor can detect Fe3+ visually and sensitively without any further steps which will make a great contribution to the development of ion sensors.

Theoretical Study on the Photoinduced Electron Transfer Mechanisms of Different Peroxynitrite Probes.

The development of probes for rapid and selective detection of peroxynitrite in vivo is of great importance in biological science. We investigate different photoinduced electron transfer (PIET) processes of two generations of peroxynitrite probes. Each has fluorescein and phenol moieties; one is conjugated by an ether linkage while the other is conjugated via an amine linkage. Using theoretical calculations, we demonstrated that the PIET in the probe with an ether linkage occurs from the benzoic acid to the xanthene moiety. In contrast, the PIET in the probe with an amine linkage occurs from the phenol moiety to the fluorescein. This suggests that better sensitivity can be accomplished in probes with an amine linkage than with an ether linkage. Following this model, we designed two novel peroxynitrite probes and simulated their detection capabilities in the near-infrared region.