Optical fingerprinting in bacterial epidemiology: Raman spectroscopy as a real-time typing method

Abstract

Hospital-acquired infections (HAI) increase morbidity and mortality and constitute a high financial burden on health care systems. An effective weapon against HAI is early detection of potential outbreaks and sources of contamination. Such monitoring requires microbial typing with sufficient reproducibility and discriminatory power. Here, a microbial-typing method is presented, based on Raman spectroscopy. This technique provides strain-specific optical fingerprints in a few minutes instead of several hours to days, as is the case with genotyping methods. Although the method is generally applicable, we used 118 Staphylococcus aureus isolates to illustrate that the discriminatory power matches that of established genotyping techniques (numerical index of diversity [D]=0.989) and that concordance with the gold standard (pulsed-field gel electrophoresis) is high (95%). The Raman clustering of isolates was reproducible to the strain level for five independent cultures, despite the various culture times from 18 h to 24 h. Furthermore, this technique was able to classify stored (-80 degrees C) and recent isolates of a methicillin-resistant Staphylococcus aureus-colonized individual during surveillance studies and did so days earlier than established genotyping techniques did. Its high throughput and ease of use make it suitable for use in routine diagnostic laboratory settings. This will set the stage for continuous, automated, real-time epidemiological monitoring of bacterial infections in a hospital, which can then be followed by timely corrective action by infection prevention teams.

FIG. 1.

Overview of Raman procedure and spectrometer. Biomass from a bacterial culture (a) on TSA medium is collected using a 1-μl inoculation loop and suspended in 5 μl of demineralized water (b). After a brief centrifugation step to remove air bubbles, the wet pellet is transferred onto a fused silica slide (c), where it is allowed to dry (a typical slide holds 24 samples). The slide with the dried biomass is placed in the measurement stage (d), where the samples are illuminated with laser light (e). The Raman signal generated is collected along the same optical path and separated from the laser light using an optical filter (f) that only reflects light of a higher wavelength than the laser. The laser light is passed through. The wavelength of the Raman signal is dispersed on an optical grating (g) and collected using a near-infrared-optimized charge-coupled device detector (h). The Raman spectra are gathered, stored, and analyzed on a personal computer (i).

FIG. 2.

Reproducibility of Raman spectroscopy. Shown are the results of hierarchical cluster analysis of the repeated measurements of five MRSA isolates measured after different culturing times. Each isolate is indicated with a colored label. a.u., arbitrary units.

FIG. 3.

Hierarchical cluster analysis of the MRSA isolates of collection I. Clusters are indicated in red and are based on the correlation coefficient of the five isolates with identical PFGE patterns (pattern A). For each node with a correlation coefficient lower than the cutoff, the corresponding value is indicated. a.u., arbitrary units.

FIG. 4.

Contact screening of January 2007. Directly after the first MRSA isolate was isolated, the contact screening was started. Two days later, a second MRSA isolate was found and sent to the RIVM for analysis. At the same time, all isolates were analyzed using Raman spectroscopy. Within 24 h, the Raman results showed that the two MRSA isolates were not identical. This was confirmed by PFGE 4 days later.