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Detection of Biological Agents: Looking for Bugs in All the Wrong Places

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focal point BY LAURA A. VANDERBERG LOS ALAMOS NATIONAL LABORATORY LOS ALAMOS, NEW MEXICO Detection of Biological Agents: Looking for Bugs in All the Wrong Places INTRODUCTION The threat of new and
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focal point BY LAURA A. VANDERBERG LOS ALAMOS NATIONAL LABORATORY LOS ALAMOS, NEW MEXICO Detection of Biological Agents: Looking for Bugs in All the Wrong Places INTRODUCTION The threat of new and potent pathogens has become a great concern over the last several years. Recent advances in biotechnology, the low cost and ease of producing potent pathogens, and their relative invisibility have increased the likelihood for biowarfare. Almost any pathogenic organism can be used as a biological warfare (BW) agent. Table I shows some of the more common pathogenic microorganisms that may be employed as agents of biowarfare. Currently, at least seven countries are thought to have active biothreat agent (BA) production and research programs. 1 On a weight-for-weight basis, BAs are more toxic than chemical warfare (CW) agents and can potentially provid e broader coverage than C W agents per pound of payload. Small quantities of biological material (for example, 1 kg of anthrax) may harm hundreds of thousands of people, depending on the delivery method and weather conditions. 2 The cost to produce BAs is minimal and does not require any special equipment. In fact, the same equipment that is used to produce biotechnological products (e.g., fermentation systems, centrifuges, chromatography column puri cation equipment, autoclaves, cell concentrators) can be used to produce BAs! This dual-use issue is daunting in terms of being able to detect proliferation of biological weapons research. Once disseminated, BAs can reproduce in the host to cause infection and be further disseminated. History. One of the rst recorded uses of biological warfare was in 1346, when bodies of Tartar soldiers who had died of the plague were catapulted over the walls of Kaffa, a besieged city. During the French and Indian War ( ) smallpox was used against Native Americans. A captain in the British forces gave blankets and a handkerchief from the smallpox hospital to the Native Americans and recorded an entry in his diary that read, I hope it will have the desired effect. 3 Both of these attempts to wage biological warfare happened at about the same time as outbreaks (epidemics) of the same diseases, respectively. Thus, the plague outbreak in Kaffa and the sm allpox epidemic among Native Am ericans could conceivably have been the result of natural outbreaks and not due solely to these incidents. 4 World War I enemies took advantage of m icrob iological ad vances and employed biological warfare at will. The Germans supposedly infected livestock that were to be exported to Allied forces with B. anthracis and B. mallei, and the U.S. attempted to contaminate livestock feed. 5,6 Despite the international diplomatic effort to limit proliferation of w eapo ns of m ass d estruction (both chemical and biological) following the war, a number of countries began research efforts to develop biological weapons. These included Poland, the Netherlands, Belgium, France, the Soviet Union, and Italy. 7 The Japanese used biolo gical weapons against the Manchurians during Examples of their use included contaminating wa- 376A Volume 54, Number 11, 2000 FIG. 1. Diagram of a (A) prokaryotic and (B) eukaryotic cell with major components labeled. Note that the prokaryotic (bacterial) cell is essentially a baggie of molecules, whereas the eukaryotic (mammalian, plant, etc.) cell is organized into discreet organelles. ter and food supplies in at least 11 Chinese cities with a number of different pathogenic bacteria and releasing plague-infested eas over China. 4 The United States began an offensive biological w eapons research program in 1942 and by the late 1960s had amassed a formidable arsenal of weapons including agents such as B. anthracis, botulinum toxin, F. tularensis, and several anticrop agents. Despite the allegations of deployment against several countries, th ese w eapon s w ere nev er used. The U.S. program was terminated in 1969, and stocks of the biological w eap ons w ere destroyed during The rati cation of the Biological Weapons Convention (BW C) in 1972 has not brought an end to the proliferation of biological warfare. In fact, several countries that signed the BW C have apparently continued their programs. In one notable incident, the Soviets allegedly used ricin, a potent toxin produced in castor beans, to execute a Russian defector. Ricin was placed into a drilled-out pellet, sealed with dissolvable wax, and red from a weapon disguised as an umbrella. 8 Detection. Detection of a BA attack is extremely dif cult. Moreover, effective detection for warning of a biological attack must be both extremely fast and very sensitive as the presence of as few as 10 organisms might be an infectious dose. 9 Speci- city is critical since attacks are likely to occur in complex environmental backgrounds, some of which contain naturally occurring pathogens or close relatives to the pathogen of interest. The ease with which these agents can be produced by using traditional biotechnology industry equipment or simple home-brew ing equipment makes detection quite dif cult. In addition, the human senses have no means to recognize when exposure has occurred, and the delay in onset of symptoms makes identi cation of the place and time of attack dif cult. BW attacks may resemble and be attributed to a natural outbreak of a disease, particularly if a country is not at war. In addition, the environmental background against which biothreat agents must be detected is biolo gically com p lex, and m an y nonbiological particles may interfere with various detection schemes. The background might also contain naturally occurring populations of the BA that one is attempting to detect. The aim of this Focal Point article is to explain the biological basis for BA detection and provide some examples of spectroscopic methods for such. In particular, detection of the entire bacterial cell and excreted biomolecules is presented. BACKGRO UND A Little M icrobiology. Bacteria are fairly simple single-celled organisms. Bacterial cells are generally categorized into three shapes rods, cocci, or spirella and they are APPLIED SPECTROSCOPY 377A focal point FIG. 2. IR spectral contours from a pathogen, Morganella morganii. Spectral range cm 21. (A) Original spectrum normalized to equal absorbance; (B) rst derivative of A; (C) second derivative of A. Reprinted with permission from Naumann et al., TABLE I. BA bacteria, rickettsia, and fungi. Agent Bacteria Bacillus anthracis Clostridium botulinum Yersinia pestis Brucella melitensis Fracisella tularensis Vibrio cholera Corynebacterium diphtheriae Burkholderia mallei Salmonella typhi Rickettsia Coxiella burnetii Rickettsia prowazeki Rickettsia mooseri Rickettsia rickettsi Fungi Coccidioides immitis Tilletia Puccinia graminis a Crop pathogens. Disease Anthrax Botulism Bubonic plague Brucellosis Tularemia Cholera Diphtheria Glanders Typhoid fever Q fever Epidemic typhus Endemic typhus Rocky mountain spotted fever Coccidiodomycosis Wheat smut Wheat smut Lethality if untreated (from Ref. 1) Fatal Fatal Fatal Low Intermediate High Low Fatal Low Low Intermediate Low High Low 67 Low a Low a structurally different from mammalian cells (Fig. 1). Shape is maintained by a cell wall, and the selectively permeable boundary between the cell and the environment is its cell membrane. Other structural co m po nents (i.e., enzym es, rib o- somes, and nuclear material) are found in the cytoplasm, the aqueous uid of the cell where metabolism takes place. The cell wall is a rigid structure predominantly made of peptidoglycan, a polymer composed of N-acetylglucosamine and N-acetylmuramine cross linked by short peptides. Bacillus an thracis, the causative agent of anthrax, is a Gram positive microbe and contains a thick layer of peptidoglycan. Yersinia pestis, the causative agent of the plague, is a Gram negative microbe and has a thin layer of peptidoglycan. Coxiella burnetti, the causative agent of Q fever, is a rickettsial organism, something like a cross between bacteria and viruses. The organisms have a cell membrane, but are completely dependent upon their host for survival. Some bacteria can form dormant structures, spores, that are formed under adverse environmental conditions. These are highly dessicated structures, akin to a nut, with several layers of protection. The core contains the cell proper, dipicolinic acid (DPA), a chemical unique to bacterial spores, and calcium ions. The dipicolinic acid and calcium ions are thought to provide heat resistance. The next layer is the cortex, and surrounding it is the spore coat, composed of densely packed, less crosslinked peptidoglycan. 10 Spores are metabolically inactive and have tremendous heat, chemical, and radiation resistance. W hen conditions become favorable (i.e., when there is fo od availab le), spo re s germ inate and become vegetative cells once again. The cell membrane, found in all microbes, is a uid structure composed of phospholipids and proteins. As many as seven different phospholipids and 200 proteins have been found in the membrane of common 378A Volume 54, Number 11, 2000 bacteria such as Escherichia coli. 11 The interactions of these different proteins and phospholipids may provide a diagnostic ngerprint for a particular microbe. 12 M any intracellular molecules in biological systems (not just bacteria) are associated with energy-yielding reactions. Some have speci c electronic excitation and emission spectra, providing a spectroscopic signature. For example, the amino acids tryptophan, phenylalanine, tyrosine, and histidine, which are components of proteins, can be excited by radiation at nm. Nicotinamide adenine dinucleo tid e (pho sp hate) (NAD(P)H) is excited at 325 nm and has an emission band from 420 to 580 nm. These features can be used to distinguish biological from nonbiological particles, but not necessarily to distinguish between bacteria and other biologically derived material. Pathogenic bacteria have speci c mechanisms by which they affect host cells. Generally, these organisms must be equipped with a means of invading the host and obtaining nutrients to perm it their survival and replication in vivo. 13 Most pathogens produce proteinaceous toxins that can be quite lethal. For example, just mg/kg of botulinum toxin (produced by the pathogen Clostridium botulinum) constitutes an infective dose. 14 Toxins are typically considered as CW agents, but since spectroscopic detection may be quite useful in detecting these small molecules, they have been included in this discussion. DISCUSSIO N C onvention al D etection M ethods. The culturing of unknown microbes on solid surfaces has been accepted as standard practice for over 100 years. 15 This procedure involves placement of a sample on a growth surface, allowing it to reproduce to form visible colonies with further examination of the morphological, biochemical, and physiological characteristics of one particular isolate. At this time, no single test can provide a de nitive identi cation of any particular organism. In some cases, direct microscopic examination of a sample may provide top-level information (morphology, staining characteristics) about a potential pathogen, but additional time-consuming con rmatory testing must be done. These types of techniques can require days to complete testing and identi cation. R apid bio chem ical testing an d identi cation may be accomplished by using any one of a variety of com m ercially available k its (e.g., API, Analytab Products, Plainview, NY; Biolog, Biolog, Inc., Hayward, CA). These kits provide a suite of key tests that can distinguish between similar microbes, but they require that an unknown microbe be isolated and grown in a pure culture again, a time-consuming front-end process. Interpretation of the results from rapid biochemical testing can also be a challenge as culture density and other parameters have a signi cant effect on testing outcomes. Immunological methods have long been employed for the identi cation of pathogenic microbes. Almost all microbial species have at least one unique antigen. Identi cation and puri cation of that antigen enable one to generate antibodies to that antigen. These antibodies can then be used to detect the presence of the original antigen through a number of methods. ELISA, or enzyme-linked immunosorbent assay, is perhaps the most well-known and frequently used immunological technique. In this technique, speci c antibodies are adsorbed onto the walls of a well that is part of a microtiter plate. A suspension that contains, or is suspected to contain, the antigen of interest is added to the well. If it is present, the antigen will react with the bound antibody. The well is rinsed to remove unbound materials. A second antibody (one that also binds to the antigen) with a conjugated reporter enzyme is added. This antibody binds to the antigen, excess material is rinsed away, and a substrate for the reporter enzyme is added. The sandwiched antigen is detected by assaying for reporter enzyme activity. This activity is typically a colorimetric reaction. 11 M olecular biological techniques such as in situ hybridization and polymerase chain reaction (PCR) have been developed and are extremely sensitive. These DNA/RNAbased techniques allow for the rapid identi cation of an organism when extremely small amounts of genetic material are present. 16 In situ hybridization uses speci c sequences of DNA that contain a uorescent tag. W hen these sequences bind to the DNA of interest, they can be readily seen under the microscope or by ow cytometry. 17 Extensive sample preparation time is needed for in situ hybridization, making it only a small improvement over culture-based methods. PCR is used to amplify minute quantities of speci c DNA sequences and can be used to unequivocally identify microorganisms without prior culturing; however, automation may be dif cult, and the presence of contamination is detrimental. 18 As with in situ hybridization, extensive and time-consuming sample clean-up and preparation is required. Toxin production can be detected through a number of methods. A common method involves exposure of mammalian or plant cell culture to a putative toxin and observation of cell necrosis or death. 19,20 Immunoassays such as ELISA and assays using PCR are also employed Spectroscopic Methods for Detection and Identi cation. To address the need for rapid and sensitive pathogen detection, particularly the detection of BA, there is a need for rapid, compact, eld-portable, userfriendly equipment. S pectroscopic methods have the potential to meet these challenging criteria; indeed, a number of biosensors have been developed that address one or more of these requirements. The rem ainder of this Focal Point article will examine some of these technologies in detail and indicate shortfalls and further developmental needs, improvements, and other requirements. Measurements of the intrinsic uo- APPLIED SPECTROSCOPY 379A focal point FIG. 3. Schematic diagram of surface plasmon resonance detection for molecular interactions. Modi ed from Jordan and Corn, rescence associated with BA simulants have been examined in detail. Examples include work by Bronk and Reinisch, Faris et al., Christesen and Ong, and Stephens Despite voluminous published research in this area, uorescence spectroscopy has not been accepted as a standard method for the identi cation of biothreat agents due to the lack of speci city of uorescing biomolecules. To illustrate, Bronk and Reinisch examined the uorescence emission spectra of four different bacterial species (Bacillus cereus, Bacillus subtilis, Staphylococcus epidermidis, and E. coli) following excitation at 290 nm. Spores of different bacterial species were also interrogated. It was concluded that well-de ned invariant features are not found in the UV-induced bacterial auto uorescence and that the use of emission spectra could provide only preliminary information for identi cation of pathogens. 24 However, this methodology has been combined with particle sizing for the initial detection of biological materials in aerosols Vibrational Spectroscopy. Infrared spectroscopy was investigated as a rapid detection method back in the fties, but was deemed unacceptable. 31 Modern Fourier transform infrared (FT-IR) spectroscopy has been examined more recently, with better prospects. 32 Naumann and his coworkers have pioneered the development of FT-IR methods for differentiation of bacteria at the species and even strain level (Fig. 2) Bacterial FT-IR spectra provide a ngerprint for both species and in some cases strains. 33 These spectra are derived from the molecular structure of an organism that is, the intracellular, membrane, and surface material of the cells. This approach results in a complex spectrum, some features of which are common to many different organisms. The common absorption bands occur at 2930 cm 21 (CH 2 CH 3 ), 1653 cm 21 (amide I), 1541 cm 21 (amide II), 1236 cm 21 (phosphate), and 1082 cm 21 (phosphate and sugars). 37 Naumann and co-workers divided the entire infrared spectrum into ve main regions based on their discriminative power and speci c information. These regions are as follows: the fatty acid region ( cm 21 ), which is dominated by the CH 3, the.ch 2, and the 5CH stretching vibrations of functional groups typically present in the cell membrane; the amide region ( cm 21 ), which is dominated by amide I and amide II stretchings of peptides and proteins; the mixed region ( cm 21 ), where proteins and fatty acids are seen; the phosphate and polysaccharide region ( cm 21 ), which is dominated by carbohydrates in the cell wall and compounds containing phosphates; and the true ngerprint region ( cm 21 ), which shows very speci c patterns that have not yet been assigned to speci c functional groups Species and strain identi cation can be accomplished by performing a cluster analysis on the rst derivatives of a selected wavelength region or the entire spectrum Closely related strains of Staphylococcus, Streptococcus, and Clostridium were identi ed by using this analysis. The wavelength regions employed w ere , , and cm 21. This technique was also effective at identifying the different species of Streptococcus and Staphylococcus in unknown clinical specimens. 35 Raman spectroscopy has the distinct advantage over infrared spectroscopy in that water, a main component of biological matrices, is only a minimal interferent. Raman spectra, ho w ever, can b e com p letely masked by the uorescence of many biological materials. With the advent of near-infrared Fourier transform Raman (N IR-FT-Raman) spectroscopy, this problem is circumvented. 38 Keller and colleagues showed that NIR-FT-R aman spectroscopy can be used to characterize some medically important biomaterials and food products with minimal sample preparation. 39,40 More recently, Naumann et al. extended their earlier work with FT-IR to combine FT-IR with NIR-FT-Ram an spectrosco py to ch ara cterize bacteria. 41 It is suggested that this method provides enhanced sensitivity due to the complementarity of 380A Volume 54, Number 11, 2000 FIG. 4. DPA release from different Bacillus species spores. Spores were suspended in sterile distilled water, heat shocked at 65 8 C for 15 min, and mixed with the germinant combination (20 mm L-alanine, 20 mm L-asparagine, 20 mm D-glucose). DPA release was detected by terbium complexation and photoluminescence. B. globigii (black); B. cereus (royal blue); B. thuringiensis (red); B. megaterium (green); and B. subtilis (turquoise). Raman and FT-IR measurements. In particular, the NIR-FT-Raman spectra are dominated by characteristic amino acid side-chain vibrations, by some nucleic acid base-ring vibrations, and by the C H stretching and bending modes of fatty acids. A signi cant drawback to current FT-IR methods is the need to have a pure culture of the unknown organism.
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