T he anthrax attacks that occurredsoon after September 11, 2001,paralyzed the U.S. Postal Service,

sickened 22 people, killing five of them,and brought home the need to rapidlydetect a biological attack and deal with itquickly before national panic ensues.There is also a real need for such method-ology in the hospital setting and in doc-tors’ offices, where patients frequentlyshow up with life-threatening diseases of natural origin, such as pneumonia ormeningitis, and require rapid diagnosisfor optimal therapy.

Generally, health care workers stillisolate bacteria by growing them on agarplates (Fig. 1), and then use physiologicalor DNA-based molecular biology meth-ods to identify the bacteria. Although thepolymerase chain reaction (PCR) workswell for non-culture-based detection ofinfection in clinical samples, it remainstechnically cumbersome and requireshighly trained personnel.

Unfortunately, a PCR-based biodetec-tion technology that can identify bacteriain environmental samples in real timerequires further development. In recentyears, renewed interest has also been

26

A 21st CenturyChallenge toSpectroscopists

Alvin Fox

The real threat of bioterrorism has driven demand for techniques

that can detect organisms used in an attack quickly and without the

need for prior culture. However, no current technology can rapidly

distinguish one or a few characteristic microbial components

released during a terrorist incident from those in other organisms

already in the environment or another chemical background.

Innovations in spectroscopy could provide the necessary tools.

27

dictable fashion—as the result of draftsor the settling of particles in indoor oroutdoor air, for example.

Modern discrimination of bacterialspecies relies on DNA or proteinsequences. PCR—one of the most widelyused techniques—enzymatically ampli-fies a genetic region with a characteristicDNA sequence; a flanking set of primers(short pieces of complementary DNA)focuses the enzyme on the DNA region of interest. Classical PCR detects a PCRproduct by electrophoretic mobility on a gel, an effective but time-consumingapproach. Real-time PCR differs fromclassical PCR in that it avoids elec-trophoresis (Fig. 2). The PCR product is detected simply by an increase in fluo-rescence, which can be performed with or without prior culture.

The analysis of proteins by peptidesequences relies now on mass spectrom-etry and tandem mass spectrometry, pri-marily matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). ESI generates chargedions of characteristic mass from a liquidstream and MALDI from a surface whenhit with a laser beam. However, the sensi-

tivity of both techniques is currently limited to the analysis of bacteria in pure cultures.

In classical trace analysis, the analyti-cal chemist uses chromatography in a gas (GC) or liquid (LC) prior to massspectrometry to concentrate an analytepresent in low concentration and to separate it from high abundance back-ground peaks that would otherwise con-found the analysis. When using PCR,however, such separations are oftenunnecessary, even when performingtrace analysis on complex clinical orenvironmental samples.

Advances in mass spectrometryWhen I was a Ph.D. student in the mid-1970s, I envisioned using a mass spec-trometer for the trace detection ofchemical markers in bacteria present incomplex matrices—an idea to whichsome in the scientific community dis-played considerable skepticism. Later,we defined a chemical marker as a com-pound present in all bacteria or somebacterial species but absent in human orother higher cell types. Muramic acidprovided one such marker because it is

Figure 1. Bacteria are generally grown (cultured) on agar plates; each individual cellbecomes a colony, which is visible to the naked eye. Different colony types can be distin-guished by morphology.

Bacillus cereus Bacillus anthracis

CDC/Dr. James Feeley

focused on immunoassay, which detectsantigens by their interaction with specificantibodies. However, the sensitivity ofimmunoassays is often poor because thetechnique does not involve biologicalamplification of the protein target priorto analysis.

My purpose here is to draw on myexperience working at the interface ofanalytical chemistry and microbiology todiscuss what has been achieved with clas-sical microbiology approaches and mod-ern instrumental methods—includingoptical approaches—and to suggest spec-troscopic methodologies that I would liketo see developed in the future. I envisionthe development of optical biodetectionapproaches that are similar to the Tri-corder on Star Trek, which was used toscan for aliens and other life forms.(When the editors of OPN invited me to write this article, they did not requireme to stay within the bounds of reality,so I make no apologies!)

Challenges to detection of organisms in complex matricesMolecular structure can be characterizedby direct or indirect chemical properties,and mass spectrometric and spectro-scopic techniques rank high among themost powerful approaches. However,detecting or identifying the molecularstructure of a bacterial cell or other com-plex organism is more complicated whenthe compound of interest is just onecomponent among many. The ultimatedifficulty occurs when bacteria are pre-sent at trace levels in a complex clinicalor, worse, environmental matrix.

In a clinical sample, only one or a fewbacterial species are present and the sam-ple matrix does not vary appreciablyfrom one individual to another. At theother extreme, an environmental samplemay include thousands of bacterialspecies, many of which are close relativesof the organism of interest and, thus,have very similar chemical and physicalproperties. The presence of numerousinorganic and organic contaminants in a sample makes deciphering it even morecomplex. Organics may include materialof bacterial, fungal, plant and animal origin. Furthermore, the matrix of anenvironmental sample may vary bothtemporally and spatially in an unpre-

present in almost all bacteria cell wallsbut absent elsewhere in nature.

Between 1977 and 1980, from ourwork and others, a few articles demon-strated that it was possible, using thetechnique of gas chromatography-massspectrometry (GC-MS), to detect bacte-ria in complex matrices without culture.

Almost two decades later, in the mid-1990s, the potential for detecting organ-isms in complex mixtures was fullyrecognized with the availability of themore advanced gas chromatograph-tan-dem mass spectrometer (GC-MS-MS).

Using GC-MS-MS methodology, wehave searched for bacterial contamination

without culturing a sample in a variety of matrices, ranging from human bodyfluids and dust samples collected indiverse environments to fines broughtback from the moon. However, suchanalysis remains extremely time-consum-ing. It was not until the development ofMALDI and ESI mass spectrometry tech-niques—and the ability to detect natu-rally present peptides that discriminatebacterial species—that the potential forreal-time biodetection in complex matri-ces became possible.

One potential advantage of spec-troscopy is that it does not require theinstrument to come directly into contactwith a sample. Although one could placea pure bacterial specimen—a bloodor dust sample, for example—into anultraviolet Raman or Fourier transforminfrared spectroscope or microscope, it isalso possible to observe a signal from asample of airborne bacteria in a distantcloud after a biological attack.

Unfortunately, such a spectroscopicsignal represents a mixture derived fromall components present in a bacterial cell.Only a few compounds—such as polyhy-droxybutyrate, a storage compound,and dipicolinic acid, which is found inspores—have features unique enough todetect spectroscopically. But they are notspecies-specific. Dipicolinic acid is presentin bacilli, including Bacillus anthracis(the causative agent of anthrax), andclostrodria, which include Clostridiumbotulinum (the organism that makesbotulinum toxin). Individual componentsof the spectra do not reflect species-spe-cific components, such as peptide or DNAsequences, but rather represent the sum ofcomponents, such as much less discrimi-natory peptide bonds.

Differentiating the spectra of organismsSpectra are visually quite similar amongrelated and even some distantly relatedorganisms, and small spectral shifts must be discriminated. Accordingly,researchers have devised complicatedalgorithms, including the use of neuralnets, in their efforts to define discrimi-nating features of an organism’s spec-trum. Thus, we have seen only verylimited use of spectroscopic methods ofthis type in mainstream microbiology.

28

A CHALLENGE TO SPECTROSCOPISTS

Figure 2. Amplification of genes or genetic regions using real-time PCR employs a non-fluorescent dye present in the amplification media. The dye only becomes fluorescent onbinding to double stranded (ds) DNA. Thus, the amount of DNA-amplified fluorescenceincreases is measured in real-time.

Figure 3. Individual colonies are examined by light microscopy after Gram stain for sizeand shape and Gram staining characteristics. Whether they are Gram positive or negativereflects underlying differences in the chemistry of the cell envelope. Generally, commonpathogens are identified by simple physiological tests that detect the presence of prod-ucts generated by enzymatic action, such as bubbling, an effect that results from the production of oxygen from hydrogen peroxide by the action of the enzyme catalase.

Gram stain

Dry on slide

Crystal violet stain

Iodine fix

Alcohol de-stain

Safranin stain

Cycle one

Cycle two

Cycle 30

Dye

ds DNA

Primer

Primer

230

Gram negative Gram positive

29

One should not confuse this approachwith the simple and widely used spectro-scopic methods for measuring broadclasses of compounds, such as proteindetection by tryptophan absorption at 280 nm.

Biosensors that detect natural fluores-cence and particle concentration are avail-able. However, because of their lack ofspecificity, they have little practical utilityin the detection of a bioterrorist event,although they could prove very useful indetermining the total bacterial bioload asa measure of indoor air quality.

Anyone looking at a cloud would findall the spectral features of the numerousbacterial and nonbacterial particlesmixed together. Thus, the situation posesfar greater challenges than the analysis ofpure cultures. However, having the abilityto analyze single particles one at a timefor their chemical or physical featureswould help to simplify the mixture.Achieving this feat requires a second levelof algorithm—one that lumps like spec-tra together to define the levels of indi-vidual particles present at low levels in a mixture of other particles present atmuch higher concentrations.

Note that Gram staining, one of themore widely used techniques after cultur-

ing, is followed by light microscopy,which provides a great deal of informa-tion on the shape and size of individualbacteria and whether they occur singly,in clusters or in chains (Fig. 3). For exam-ple, this technique can readily discrimi-nate the B. cereus group from the relatedB. subtilis group. Although it is not possi-ble to speciate by Gram stain alone, rarelywould anyone attempt to identify anorganism without first performing aGram stain.

Making spectroscopy match current techniquesIt is illuminating to compare spectro-scopic results with what one can achievein a more time-consuming fashion withan electron microscope. A single cell ofB. anthracis looks very different from itscousin B. subtilis on an electron micro-graph; the two species cannot be con-fused (Fig. 4). The spore of B. anthracis ischaracterized by the presence of an exter-nal exosporium that consists of a basallayer surrounded by a glycoprotein nap.The exosporium surrounds a coat layer.The glycoprotein nap of B. anthracis dis-plays finger-like projections, while the B. subtilis spore is distinguished by a less defined amorphous appearance.

The coat of B. subtilis is also quite dif-ferent from B. anthracis. It consists of twolayers: a lightly staining inner coat and a darker outer coat, which are tightlybound to one another.

Thus, physical information on themorphology of characteristic layers pre-sent within the cell envelope also pro-vides important information in additionto gross cellular morphology, such asshape and size.

Surely, with the huge amount ofchemical and physical informationpotentially available in a spectral signal,it should be possible to develop opticalmethods at least as powerful as currentmicrobiology and molecular biologyapproaches but without the need forsample processing prior to instrumentalobservation. I will leave as a challengefor the spectroscopists of the 21st cen-tury the decision as to what is actuallypractical versus what might be sciencefiction.

Alvin Fox (afox@med.sc.edu) is a professor ofpathology and microbiology at the University ofSouth Carolina School of Medicine in Columbia. Heis editor-in-chief of the Journal of MicrobiologicalMethods and an editor of the book “AnalyticalMicrobiology: Chromatography and MassSpectrometry.” He also helps organize internationalsymposia on the interface between analytical chemistry and microbiology.

A CHALLENGE TO SPECTROSCOPISTS

Figure 4. Bacterial spores, (a) B. anthracis and (b) B. subtilis, as viewed by electron microscopy.

ExosporiumOuter coat

Coat

(a) (b)

Inner coat

Exosporium