A high speed detection platform based on surface-enhanced Raman scattering for monitoring antibiotic-induced chemical changes in bacteria cell wall

Abstract

Rapid and accurate diagnosis for pathogens and their antibiotic susceptibility is critical for controlling bacterial infections. Conventional methods for determining bacterium's sensitivity to antibiotic depend mostly on measuring the change of microbial proliferation in response to the drug. Such "biological assay" inevitably takes time, ranging from days for fast-growing bacteria to weeks for slow-growers. Here, a novel tool has been developed to detect the "chemical features" of bacterial cell wall that enables rapid identification of drug resistant bacteria within hours. The surface-enhanced Raman scattering (SERS) technique based on our newly developed SERS-active substrate was applied to assess the fine structures of the bacterial cell wall. The SERS profiles recorded by such a platform are sensitive and stable, that could readily reflect different bacterial cell walls found in Gram-positive, Gram-negative, or mycobacteria groups. Moreover, characteristic changes in SERS profile were noticed in the drug-sensitive bacteria at the early period (i.e., approximately 1 hr) of antibiotic exposure, which could be used to differentiate them from the drug-resistant ones. The SERS-based diagnosis could be applied to a single bacterium. The high-speed SERS detection represents a novel approach for microbial diagnostics. The single-bacterium detection capability of SERS makes possible analyses directly on clinical specimen instead of pure cultured bacteria.

Figure 1. The SERS detection platform and dataset normalization.

(A) Five SERS spectra were taken from the same S. aureus bacteria (black traces), different clusters of bacteria present on the same substrate (blue traces), or bacteria on different substrates (green traces). The raw dataset (I) were subjected to three steps (II, III, IV) of processing to normalize the spectra by the constant value of 732 cm⁻¹. (B–C) Normalized SERS spectra from five data points of bacteria on the same substrate (B), or bacteria on different substrates (C) are superimposed with each other. (D) Normalized SERS spectra of the same S. aureus bacteria that were recorded over time as indicated. Standard deviations along the spectrum are shown in red.

Figure 2. SERS spectra of bacteria with different cell wall compositions.

Spectra of (A) various Gram-positive bacteria are presented as well as that of cell wall-less protoplasts of S. aureus (blue trace), (B) various Gram-negative bacteria are presented as indicated as well as that of cell wall-less spheroplasts of E. coli (green trace), and (C) two species of Mycobacterium. Each SERS profile stands for the mean spectrum averaged from more than 10 samples.

Figure 3. SERS spectra of S. aureus and E. coli obtained at different growth phases.

(A–C) Gram-positive S. aureus was grown to OD₆₀₀ 0.4, 1.5 and 2.5 and then harvested (open circles, A); the spectra (B) and SEM images (C) were then recorded. No significant changes were present. (D–G) Gram-negative E. coli was grown to OD₆₀₀ 0.4, 1.5 and 2.0 and then harvested (open circles, D); the spectra (E) and SEM images (F) were then recorded. Six SERS peaks (#1∼#6) showed the progressive changes as the OD₆₀₀ value of the culture increased. (G) Quantification (mean±S.D.) of the altered SERS peaks is shown in (E).

Figure 4. Antibiotic-induced SERS spectral changes are indicative of the bacteria's antibiotic sensitivity.

(A) Sequential SERS recordings of S. aureus with or without the exposure to oxacillin. Time periods of drug exposure are indicated. (B) Sequential SERS recordings of E. coli with or without the exposure to ampicillin. Noted that the decrease in the peaks at 725 and 1095 cm⁻¹ indicates an inhibition of bacterial proliferation. (C) Spectra of S. aureus after treated with various different antibiotics as indicated. All of the antibiotics target the bacterial cell wall. The time period shown for each antibiotic treatment corresponds to the beginning of observing significant spectral changes. (D) Spectra of S. aureus after treated with antibiotics that inhibited bacterial protein synthesis for time periods as indicated. A characteristic SERS response was not noted until after 9∼13 hr of treatment.

Figure 5. SERS-based microbial diagnostics of a single bacterium.

(A) A single S. aureus resolved under light microscopy (arrow, inset) was subjected to SERS detection every 10 min for 90 min (various colored traces); the standard deviation among all recordings along the spectrum are shown in red. (B) Sequential SERS spectral evolution of a single live bacterium of S. aureus on exposure to vancomycin, which is known to actively disrupted bacterial cell wall.