From Bulk to Single-Cell Classification of the Filamentous Growing Streptomyces Bacteria by Means of Raman Spectroscopy

Abstract

Classification of Raman spectra recorded from single cells is commonly applied to bacteria that exhibit small sizes of approximately 1 to 2 μm. Here, we study the possibility to adopt this classification approach to filamentous bacteria of the genus Streptomyces. The hyphae can reach extensive lengths of up to 35 μm, which can correspond to a single cell identified in light microscopy. The classification of Raman bulk spectra will be demonstrated. Here, ultraviolet resonance Raman (UV RR) spectroscopy is chosen to classify six Streptomyces species by the application of a tree-like classifier. For each knot of the hierarchical classifier, estimated classification accuracies of over 94% are accomplished. In contrast to the classification of bulk spectra, the classification of single-cell spectra requires a homogenous substance distribution within the cell. Consequently, the bacterial cell chemistry can be represented by one individual spectrum. This requirement is not fulfilled when different spectra are processed from different locations within the cell. Bacteria of the investigated genus Streptomyces exhibit, besides the normal bacterial spectra, lipid-rich spectra. The occurrence of lipid enrichment depends on culture age and nutrition availability. With this study, we investigate the cell substance distribution, especially of lipid-rich fractions. The classification utilizing a tree-like classifier is also applied to the Streptomyces single-cell spectra, resulting in classification accuracies between 80 and 93% for the investigated Streptomyces species.

Raman spectroscopy: techniques and applications in the life sciences

“…Using data analysis techniques that will be discussed in Section 5, Raman spectroscopy has been used to classify bacteria [14,15], nano-bio-materials [138][139][140], cells [141,142], and animal and human tissues [143,144]. Because Raman spectroscopy is non-labeling and non-destructive, repeated measurements can be made of the same sample.…”

“…This continues for a set number of layers until the final layer makes a classification decision. Jürgen Popp and his group have used ANN to classify Raman spectra from various bacterial species [15]. One criticism of this model is its "black box" nature; it can be difficult to extract which features were responsible for the classification.…”

“…The high accuracy of Raman spectroscopy has received a great deal of attention as a potential diagnostic tool. Raman spectroscopy has been used to classify bacteria [14][15][16] and diagnose a broad range of diseases [17][18][19].…”

Spectral signatures for the classification of microbial species using Raman spectra

“…The spectra of di181 isolate display intense, sharp peaks at 1,106, 1,154, and 1,508 cm −1 . These three peaks are spectral signatures of carotenoid-family molecular species [21,22]. The strong carotenoid spectral signature in isolate di181 clearly distinguishes it from isolates di202 and di307.…”

IR and Raman Spectroscopy for Pathogen Detection

“…This results in different spectra at different measurement positions and hence one spectrum may not be representative of the whole organism anymore. For example, Walter et al investigated filamentously growing (hyphal structured) Streptomyces bacteria at the single-cell level [190]. They cultured two Streptomyces species for up to 7 days and performed age-dependent measurements.…”