Rapid microbial analysis is important in many fields such as industrial clean room maintenance, food processing, and medical diagnostics. If a pure specimen of a single microbial species is available, for instance, if cultured on an agar plate or if studied at the single-cell level, then an analysis method only needs to identify what microbe is present. More typically, however, a sample contains a mixture of different microbes. In such cases, relative concentrations of different species become relevant parameters. In medical cases, prompt and accurate acquisition of this information can be crucial to delivering proper treatment. In laboratory settings as well, it is desirable to specify the relative bacterial content of biological samples, which often contain similar species.
One important example of polymicrobial mixtures is dental plaque, which accumulates on the surfaces of teeth and is dominated by Streptococcal
bacteria in the human oral cavity.1,2
Among the various supragingival streptococcal species present on tooth surfaces, Streptococcus mutans
is particularly important because of its unique role associated with tooth decay. Elevated levels of S. mutans
have been strongly associated with dental caries.2–4
The level of S. mutans
is often compared to that of a competitive species, Streptococcus sanguis
, which is by far the most prevalent bacterial constituent in healthy plaque.1
The S. mutans
to S. sanguis
concentration ratio can be regarded as an indicator of oral health. Routine quantitative measurement of the relative concentration of S. mutans
in oral plaque would be valuable for studying animal models of caries as well as for human clinical purposes.
There are many methods of identifying a bacterial colony's species, including conventional biological or biochemical methods,5
more recently developed molecular biology assays such as polymerase chain reaction (PCR),6,7
and analytical spectroscopic methods, including pyrolysis mass spectrometry (PyMS),8,9
Fourier transform infrared spectroscopy (FTIR),10,11
surface-enhanced Raman scattering (SERS),12
ultraviolet (UV) resonance Raman,15,16
and near-infrared Raman spectroscopy.19–25
To date, the PCR methods offer the best discrimination ability while the spectroscopic methods usually take the least time and do not require additional chemicals.
In principle, all of these approaches could be developed into quantitative methods to analyze concentrations in polymicrobial specimens. Examples of existing approaches include “viable counting” (i.e., visually tabulating the number of colonies) after conventional agar culturing26
and quantitative PCR (q-PCR).27,28
Viable counting is by far the most widely used in laboratory and clinical applications; however, it usually takes 12 to 48 hours to obtain results, and uncertainties in transferal and growth efficiency limit the accuracy of the measured ratio to order-of-magnitude at best.6,29
Q-PCR is currently the gold standard for quantitative analysis of polymicrobial specimens. At present, however, the approach requires many pre- and post-processing steps, multiple chemical agents, and hours of sample incubation. This limits its routine application, particularly in clinical situations.
Recently, we reported an idea for a quantitative approach to polymicrobial analysis based on Raman spectroscopy.30
In an initial test, concentrations of S. mutans
and S. sanguis
in prepared mixtures were determined from their Raman spectra using chemometric calibration. The relative fraction of S. mutans
, on a scale from 0 to 1, was predicted with an error of ±0.04. In this study, however, there was complete correlation between the two species’ fractions, since an increase in one meant a decrease in the other. This artificial correlation presumably aided the calibration process and generated unreliably low prediction errors. Extrapolation to mixtures of more than two species, where such correlation would not exist, was not straightforward. The next logical step in developing the Raman method for analysis of complex human and animal specimens, therefore, was to study mixtures of more than two microbial species.
In the experiments we present in this paper, three bacterial species, S. mutans, S. sanguis, and S. gordonii were included in the mixture system. Previously acquired Raman spectra of pure cultures of these three species () had shown that they could be unambiguously classified using the area underneath selected peak regions, as shown in . Below, we present quantitative estimation of relative bacterial concentration in three-species mixture samples. The results demonstrate that the Raman technique, as a general method, is able to obtain quantitative bacterial information in multi-species bacterial mixtures without strong concentration correlations and holds promise specifically for analysis of oral plaque specimens.
Fig.1 (a) Typical Raman spectra of S. mutans, S. sanguis, and S. gordonii cultured in liquid Todd-Hewitt medium. See the Methods section for system details and culture/acquisition protocols. (b) Identification of pure S. mutans, S. sanguis, and S. gordonii (more ...)