Raman spectroscopy (RS) is a widely used analytical technique based on the detection of molecular vibrations in a defined system, which generates Raman spectra that contain unique and highly resolved fingerprints of the system. However, the low intensity of normal Raman scattering effect greatly hinders its application. Recently, the newly emerged surface enhanced Raman spectroscopy (SERS) technique overcomes the problem by mixing metal nanoparticles such as gold and silver with samples, which greatly enhances signal intensity of Raman effects by orders of magnitudes when compared with regular RS. In clinical and research laboratories, SERS provides a great potential for fast, sensitive, label-free, and non-destructive microbial detection and identification with the assistance of appropriate machine learning (ML) algorithms. However, choosing an appropriate algorithm for a specific group of bacterial species remains challenging, because with the large volumes of data generated during SERS analysis not all algorithms could achieve a relatively high accuracy. In this study, we compared three unsupervised machine learning methods and 10 supervised machine learning methods, respectively, on 2,752 SERS spectra from 117 Staphylococcus strains belonging to nine clinically important Staphylococcus species in order to test the capacity of different machine learning methods for bacterial rapid differentiation and accurate prediction. According to the results, density-based spatial clustering of applications with noise (DBSCAN) showed the best clustering capacity (Rand index 0.9733) while convolutional neural network (CNN) topped all other supervised machine learning methods as the best model for predicting Staphylococcus species via SERS spectra (ACC 98.21%, AUC 99.93%). Taken together, this study shows that machine learning methods are capable of distinguishing closely related Staphylococcus species and therefore have great application potentials for bacterial pathogen diagnosis in clinical settings.

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Comparative Analysis of Machine Learning Algorithms on Surface Enhanced Raman Spectra of Clinical Staphylococcus Species.

With the low-cost, label-free, and nondestructive features, Raman spectroscopy is becoming an attractive technique with great potential to discriminate bacterial infections. In this pilot study, we analyzed surfaced-enhanced Raman spectroscopy (SERS) spectra via supervised machine learning algorithms, through which we confirmed the application potentials of the SERS technique in rapid and accurate discrimination of Klebsiella pneumoniae strains with different antibiotic resistance profiles. ABSTRACT In clinical settings, rapid and accurate diagnosis of antibiotic resistance is essential for the efficient treatment of bacterial infections. Conventional methods for antibiotic resistance testing are time consuming, while molecular methods such as PCR-based testing might not accurately reflect phenotypic resistance. Thus, fast and accurate methods for the analysis of bacterial antibiotic resistance are in high demand for clinical applications. In this pilot study, we isolated 7 carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains and 8 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from clinical samples. Surface-enhanced Raman spectroscopy (SERS) as a label-free and noninvasive method was employed for discriminating CSKP strains from CRKP strains through computational analysis. Eight supervised machine learning algorithms were applied for sample analysis. According to the results, all supervised machine learning methods could successfully predict carbapenem sensitivity and resistance in K. pneumoniae, with a convolutional neural network (CNN) algorithm on top of all other methods. Taken together, this pilot study confirmed the application potentials of surface-enhanced Raman spectroscopy in fast and accurate discrimination of Klebsiella pneumoniae strains with different antibiotic resistance profiles. IMPORTANCE With the low-cost, label-free, and nondestructive features, Raman spectroscopy is becoming an attractive technique with great potential to discriminate bacterial infections. In this pilot study, we analyzed surfaced-enhanced Raman spectroscopy (SERS) spectra via supervised machine learning algorithms, through which we confirmed the application potentials of the SERS technique in rapid and accurate discrimination of Klebsiella pneumoniae strains with different antibiotic resistance profiles.

https://doi.org/10.1128/spectrum.02409-21

Discrimination between Carbapenem-Resistant and Carbapenem-Sensitive Klebsiella pneumoniae Strains through Computational Analysis of Surface-Enhanced Raman Spectra: a Pilot Study

With its low-cost, label-free and non-destructive features, Raman spectroscopy is becoming an attractive technique with high potential to discriminate the causative agent of bacterial infections and bacterial infections per se. However, it is challenging to achieve consistency and accuracy of Raman spectra from numerous bacterial species and phenotypes, which significantly hinders the practical application of the technique. In this study, we analyzed surfaced enhanced Raman spectra (SERS) through machine learning algorithms in order to discriminate bacterial pathogens quickly and accurately. Two unsupervised machine learning methods, K-means Clustering (K-Means) and Agglomerative Nesting (AGNES) were performed for clustering analysis. In addition, eight supervised machine learning methods were compared in terms of bacterial predictions via Raman spectra, which showed that convolutional neural network (CNN) achieved the best prediction accuracy (99.86%) with the highest area (0.9996) under receiver operating characteristic curve (ROC). In sum, machine learning methods can be potentially applied to classify and predict bacterial pathogens via Raman spectra at general level. Graphical Abstract

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Rapid Discrimination of Clinically Important Pathogens Through Machine Learning Analysis of Surface Enhanced Raman Spectra

Abstract The worldwide increase of antimicrobial resistance (AMR) is a serious threat to human health. To avert the spread of AMR, fast reliable diagnostics tools that facilitate optimal antibiotic stewardship are an unmet need. In this regard, Raman spectroscopy promises rapid label- and culture-free identification and antimicrobial susceptibility testing (AST) in a single step. However, even though many Raman-based bacteria-identification and AST studies have demonstrated impressive results, some shortcomings must be addressed. To bridge the gap between proof-of-concept studies and clinical application, we have developed machine learning techniques in combination with a novel data-augmentation algorithm, for fast identification of minimally prepared bacteria phenotypes and the distinctions of methicillin-resistant (MR) from methicillin-susceptible (MS) bacteria. For this we have implemented a spectral transformer model for hyper-spectral Raman images of bacteria. We show that our model outperforms the standard convolutional neural network models on a multitude of classification problems, both in terms of accuracy and in terms of training time. We attain more than 96% classification accuracy on a dataset consisting of 15 different classes and 95.6% classification accuracy for six MR–MS bacteria species. More importantly, our results are obtained using only fast and easy-to-produce training and test data. 

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Accurate and fast identification of minimally prepared bacteria phenotypes using Raman spectroscopy assisted by machine learning

In this study, we investigated 30 bacterial species belonging to 9 different bacterial genera that were isolated from clinical samples via surfaced enhanced Raman spectroscopy (SERS). A total of 17,149 SERS spectra were harvested from a Raman spectrometer and were further analyzed via machine learning approaches, the results of which showed that the convolutional neural network (CNN) deep learning algorithm could achieve the highest prediction accuracy for recognizing pathogenic bacteria at both the genus and species levels. ABSTRACT The rapid and accurate identification of the causing agents during bacterial infections would greatly improve pathogen transmission, prevention, patient care, and medical treatments in clinical settings. Although many conventional and molecular methods have been proven to be efficient and reliable, some of them suffer technical biases and limitations that require the development and application of novel and advanced techniques. Recently, due to its cost affordability, noninvasiveness, and label-free feature, Raman spectroscopy (RS) is emerging as a potential technique for fast bacterial detection. However, the method is still hampered by many technical issues, such as low signal intensity, poor reproducibility, and standard data set insufficiency, among others. Thus, it should be cautiously claimed that Raman spectroscopy could provide practical applications in real-world settings. In order to evaluate the implementation potentials of Raman spectroscopy in the identification of bacterial pathogens, we investigated 30 bacterial species belonging to 9 different bacterial genera that were isolated from clinical samples via surfaced enhanced Raman spectroscopy (SERS). A total of 17,149 SERS spectra were harvested from a Raman spectrometer and were further analyzed via machine learning approaches, which showed that a convolutional neural network (CNN) deep learning algorithm achieved the highest prediction accuracy for recognizing pathogenic bacteria at both the genus and species levels. In summary, the SERS technique holds a promising potential for fast bacterial pathogen identification in clinical laboratories with the integration of machine learning algorithms, which might be further developed and sharpened for the direct identification and prediction of bacterial pathogens from clinical samples. IMPORTANCE In this study, we investigated 30 bacterial species belonging to 9 different bacterial genera that were isolated from clinical samples via surfaced enhanced Raman spectroscopy (SERS). A total of 17,149 SERS spectra were harvested from a Raman spectrometer and were further analyzed via machine learning approaches, the results of which showed that the convolutional neural network (CNN) deep learning algorithm could achieve the highest prediction accuracy for recognizing pathogenic bacteria at both the genus and species levels. Taken together, we concluded that the SERS technique held a promising potential for fast bacterial pathogen diagnosis in clinical laboratories with the integration of deep learning algorithms, which might be further developed and sharpened for the direct identification and prediction of bacterial pathogens from clinical samples.

Identification of Bacterial Pathogens at Genus and Species Levels through Combination of Raman Spectrometry and Deep-Learning Algorithms

Infectious diseases caused by bacterial pathogens are important public issues. In addition, due to the overuse of antibiotics, many multi-drug resistant bacterial pathogens have been widely encountered in clinical settings. Thus, the fast identification of bacteria pathogens and profiling of antibiotic resistance could greatly facilitate the precise treatment strategy of infectious diseases. So far, many conventional and molecular methods, both manual or automatized, have been developed for in vitro diagnostics, which have been proven to be accurate, reliable, and time efficient. Although Raman spectroscopy is an established technique in various fields such as geochemistry and material science, it is still considered as an emerging tool in research and diagnosis of infectious diseases. Based on current studies, it is too early to claim that Raman spectroscopy may provide practical guidelines for microbiologists and clinicians because there is still a gap between basic research and clinical implementation. However, due to the promising prospects of label-free detection and non-invasive identification of bacterial infections and antibiotic resistance in several single steps, it is necessary to have an overview of the technique in terms of its strong points and shortcomings. Thus, in this review, we went through recent studies of Raman spectroscopy in the field of infectious diseases, highlighting the application potentials of the technique and also current challenges that prevent its real-world applications.

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Wei Liu

Papers

Applications of Raman Spectroscopy in Bacterial Infections: Principles, Advantages, and Shortcomings.