Fatty Acids and Lipids in Microbial Forensics

35J.B. Cliff et al. (eds.), Chemical and Physical Signatures for Microbial Forensics, Infectious Disease,DOI 10.1007/978-1-60327-219-3_3, © Springer Science+Business Media, LLC 2012

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Abstract

Lipids are an integral component of the bacterial membrane and show great structural diversity within the cell. While lipid composition has been an important signature of bacterial phylogeny for many decades, it also has the potential to provide information on the resources and procedures used to culture an organism. Chemical factors like nutritional substrates, temperature, and physical dynamics during growth all can infl uence the types of lipids and their relative ratios inside the cell and potentially leave diagnostic biosignatures that are unique to a specifi c production process.

In this chapter, we examine the structural diversity of lipids in the cell and the factors that affect lipid composition during laboratory culturing with particular emphasis on Bacillus organisms. Methods used to extract, separate, and detect fatty acids are reviewed. In addition, the potential util-ity of lipid profi les in forensic investigations is discussed in the context of specifi c examples from the literature. Lastly, validation considerations and quality assurance are highlighted as important aspects in the implementa-tion of lipid analysis for microbial forensics.

J. M. Robertson , Ph.D. FBI Laboratory Division , Counterterrorism and Forensic Science Research Unit , 2501 Investigation Parkway , Quantico , VA 22135 , USA

C. J. Ehrhardt , Ph.D. (*) Virginia Commonwealth University, Department of Forensic Sciences, Box 843079, Grace E. Harris Hall South, Second Floor 1015 Floyd Avenue, Richmond, VA 23284-3079, USA e-mail: [email protected]

J. Bannan , Ph.D. FBI Laboratory Division , Chemical and Biological Sciences Unit , Quantico , VA , USA

Fatty Acids and Lipids

James M. Robertson , Christopher J. Ehrhardt , and Jason Bannan

3.1 Introduction

3.1.1 Lipids and Their Role in Microbes

Lipids refer to a class of biological molecules such as fats, fatty acids, and sterols that are more soluble in organic solvents such as chloroform and hexane than in aqueous solutions. Generally, lipids can be considered as chains of alkane units (phospholipids and glycolipids) or as structures of isoprene units (terpenoid lipids). The hydro-phobic character of the hydrocarbon unit of the lipid may be augmented by constituents that pro-vide a hydrophilic character to the molecule, such

36 J.M. Robertson et al.

as a hydroxyl or phosphate group. Thus, the number and type of functional groups can make the lipid either neutral (nonpolar) or charged (polar or amphiphilic). The hydrocarbon unit usually has a greater infl uence on the solubility of the lipid molecule in water than the polar units. Categories of lipids include fatty acids, hydro-carbons, fatty esters (e.g., glycerolipids), phos-pholipids, sphingolipids, glycolipids (e.g., lipopolysaccharides), ether lipids, sulpholipids, lipoproteins, and terpenoid lipids (steroids, ste-rols, prenols, carotenes, quinones). Some lipids are characteristic of a particular microorganism. A treatise of microbial lipids has been compiled, and the reader is encouraged to refer to this com-prehensive text for information on the types of lipids found in microbes [ 1 ] .

Lipids are the major constituents of the mem-branes of microorganisms. Depicted in Fig. 3.1 are simplifi ed drawings of the membranes of a Gram-negative bacterium (e.g., Vibrio spp.) and of a Gram-positive bacterium (e.g., Bacillus spp.), where the lipid bilayer is emphasized. Peptidoglycan, composed of sugars cross-linked to short peptides, forms a mesh-like layer (illus-trated in the fi gure by wavy, discontinuous lines connected by bridges) and provides mechanical strength and rigidity to the cell. The outer layer of peptidoglycan in Gram-positive organisms is relatively thick as compared to the peptidoglycan layer located between the cytoplasmic and outer membranes in Gram-negative organisms. The peptidoglycan layer in Gram-positive organisms is tethered to the cytoplasmic membrane by lipoteichoic acids. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS) that is composed of a phospholipid (lipid A) conjugated to a polysaccharide chain (O anti-gen) through a core oligosaccharide. This entire LPS complex, also called the endotoxin, is the target of medical research toward reducing infl ammation associated with bacterial infection [ 2 ] . The cytoplasmic membrane of eukaryotes contains sterols which give the membrane more rigidity due to their planar structures. Sterols have been reported to be useful for distinguishing eukaryotic microbes of fungi and protozoa [ 3 ] ,

and their detection in drinking water indicates contamination by microeukaryotes.

Bacteria contain a wide variety of fatty acids in their cytoplasmic membrane. The great diversity in the structure of fatty acids derives from differ-ences in chain length, the presence or absence of double bonds along with their position in the chain, and the lack or presence of residues such as methyl groups, cyclic structures, and hydroxyl groups. Examples of four different fatty acids representing saturated, unsaturated, and two branched chain structures are depicted in Fig. 3.2 . The presence of fatty acids of a wide structural scope helps the bac-teria adapt to environmental changes [ 1 ] . For the research biologist, the variety in structures of the fatty acids is benefi cial for studying bacterial clas-sifi cation because it provides a means to character-ize the organisms. Ecologists and environmental researchers utilize the phospholipid ester-linked fatty acids (PLFA) in membranes of viable bacte-ria to study bacterial communities [ 4– 6 ] .

3.2 Lipid Variation in Microbes

3.2.1 Fatty Acids

Whole-cell fatty acid analysis has been used for decades as a tool for characterizing a wide range of bacteria and other microbial species [ 7 ] . Extensive effort has been spent characterizing the fatty acids of organisms within the genus Bacillus for both historical reasons and as a tool to help resolve the taxonomy of the group [ 8 ] . Bacillus organisms are of particular interest to forensic studies due to their pathogenic potential and capability of certain species to be used as bioweapons (i.e., B. anthracis ). For these rea-sons, much of this review will focus on the fatty acid biomarkers and profi les within the genus Bacillus. The general concepts presented here should be applicable to lipid analysis of other threat agents or forensically relevant organisms and their data interpretation. For information on how fatty acids contribute to the overall study of bacterial taxonomy, the reader is referred to a comprehensive review on the subject [ 9 ] .

Fig. 3.1 Illustration of the envelopes of Gram-positive organisms (e.g., Bacillus spp.) and Gram-negative organ-isms (e.g., Vibrio spp.). The depiction concentrates on the lipid bilayer and is not meant to represent all the molecu-lar features of membranes. Accordingly, pores, most membrane protein complexes, and surface molecules are not depicted. Refer to Chap. 2 for more in-depth informa-tion on bacterial structure and physiology. Peptidoglycan ( Pg ), phospholipids ( PL ), and transmembrane proteins ( Pr ) are features common to both Gram-positive and

Gram-negative cells. Capsular polysaccharides are depicted as appendages on the outer layer of the Gram-positive membrane. Gram-negative membranes possess an outer membrane ( OM ) composed of O antigen ( OA ), lipid A ( LA ), and lipoprotein ( LP ). The fi gure is an adapta-tion of two previously published illustrations (Reprinted from Microbial lipids , Vol. 1, C. Ratledge and S.G. Wilkinson, Editors; Chap. 1: An overview of micro-bial lipids, p 12 and Chap. 7: Gram-negative bacteria, p 302; 1988 [ 1 ]


3.2.1.1 Factors Affecting Fatty Acid Composition: Taxonomy and Cell Type

The majority of fatty acids found within Bacillus species have a branched confi guration (iso and anteiso forms, Fig. 3.2 ) and are between 14 and 17 carbons in length [ 1, 10 ] . For some species,

the combined proportion of 15:0 iso, 15:0 anteiso, 17:0 iso, and 17:0 anteiso can comprise between 60% and 90% of the total cellular fatty acids [ 8, 10, 11 ] . The branched 15-carbon fatty acid structures (both iso and anteiso forms) are typi-cally the most abundant in Bacillus organisms [ 10, 11 ] , and their relative proportions can be

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Fig. 3.2 Structures of common fatty acids. The nomen-clature of the fatty acids involves listing the number of carbon atoms and following a colon, the number of double bonds. The position ( w ) of the double bond is denoted as the number of carbon atoms from the carbon terminus with the isomerization described as c for cis and t for

trans . The position of substituents (e.g., hydroxyl group) is denoted as the number of carbon atoms counting from the carboxyl terminus. A branching group (e.g., methyl) located on the carbon terminus is denoted as iso and as anteiso if located on the second carbon from the carbon terminus

393 Fatty Acids and Lipids

indicative of certain species. For instance, in vegetative cells of B. anthracis , B. cereus , B. thu-ringiensis , and B. mycoides , the 15:0 iso fatty acid is present in much higher abundance than 15:0 anteiso, whereas these two fatty acids are present in approximately equal amounts in B. subtilis [ 8 ] .

In addition to the differences between species, fatty acid composition can also vary between Bacillus cell types. Generally, spores show higher proportions of branched chain saturated fatty acids and vegetative cells show higher percent-ages of straight chain and unsaturated fatty acids [ 12, 13 ] . For B. anthracis and B. cereus organ-isms, elevated percentages of anteiso fatty acids (specifi cally, the 13:0, 15:0, 17:1, and 17:0 anteiso fatty acids) and lower cumulative propor-tions of iso fatty acids have been observed in spores compared to vegetative cells of the same species [ 12 ] . Spore fatty acids that are completely absent in vegetative cell profi les have also been reported [ 8, 12 ] , but these are likely not universal across different Bacillus species [ 8 ] .

3.2.1.2 Growth and Culturing Conditions Because biochemical precursors for fatty acid synthesis are supplied exogenously in laboratory cultures, the choice of medium recipe plays a large role in determining an organism’s fatty acid composition. Within the Bacillus group, the three main structure classes of branched fatty acids (iso-odd, iso-even, anteiso) are synthe-sized via separate biosynthetic pathways, each of which starts with a specifi c branched amino acid: leucine, valine, and isoleucine, respec-tively [ 10, 14 ] . Consequently, directly varying the concentration of the three branched amino acid in the growth medium has a profound infl u-ence on the proportions corresponding iso and anteiso fatty acids in vegetative cells [ 14– 18 ] and in spores [ 13, 19 ] .

Amino acid precursors are most commonly supplied to bacterial cultures in the form of com-plex nutrients such as yeast extract, tryptone, peptone, and soy. Bacillus spores grown on complex nutrients have shown systematic fatty acid differences when compared to chemically

defi ned media where all components (glucose, amino acids, minerals…) are added individually to the formulation [ 19, 20 ] . Also, because amino acid content will vary among different nutrient derivatives (i.e., milk protein, animal tissue, soy, etc.), each source can impart unique fatty acid content on cultured Bacillus organisms [ 20, 21 ] .

The composition of branched fatty acids can also be infl uenced by variations in carbon source. For example, inclusion of glucose to a complex nutrient formulation was found to increase the proportion of even-numbered, branched fatty acids of B. thuringiensis [ 22 ] . Substituting other carbon substrates such as acetate and galactose for glucose can increase the total percentages of branched odd-numbered fatty acids compared to branched even fatty acids [ 14, 23 ] .

Non-nutritional factors during laboratory growth have been found to change fatty acid composition. Comparisons of fatty acid profi les of an organism grown on an agar-supplemented medium with profi les from organisms grown in liquid broth have shown several differences. Kaneda [ 22 ] observed that Bacillus species grown on agar plates showed elevated levels of palmitic acid (16:0) and concomitant decreases in 16:1 unsaturated fatty acids when compared to profi les of identical cultures grown in liquid medium of the same composition. Other studies have shown that Bacillus cells grown on a solid medium exhibit varying ratios of anteiso, iso, and unsaturated fatty acids when compared to cells grown in the corresponding liquid medium [ 20, 21, 24 ] .

Abiotic factors including pH, dissolved gas content, and temperature all can infl uence fatty acid composition in bacteria [ 1, 25 ] . For Bacillus organisms, the latter parameter can have a key role in determining proportions of different branched fatty acid structures. Bacillus subtilis adapts to temperature changes by producing short chain–length fatty acids at cooler temperatures and long chain–length molecules at higher temperatures [ 26 ] . Other Bacillus organisms have been observed with increased proportions of 15:0 iso when grown at higher temperatures [ 10, 21 ] . This is sometimes accompanied by a decrease in specifi c unsaturated


fatty acids, mainly 16:1 w 7c–OH and 16:1 w 7c [ 21 ] . In line with these observations, it has been reported that lowering the temperature during growth and sporulation of Bacillus subtilis resulted in a decrease of iso relative to the anteiso branched chain fatty acids [ 27, 28 ] . However, caution must be invoked in making generalizations regarding the infl uence of environmental effects, since not all organisms behave similarly. For example, the ratio of anteiso to iso fatty acids did not change with temperature or carbon source for a thermo-philic Bacillus species [ 23 ] .

3.2.1.3 Methods for Extracting Fatty Acids from Whole Cells

Chemical Extraction of Fatty Acids The most common procedure to analyze fatty acids extracted from microbial samples is the fatty acid methyl ester (FAME) method, which is used in hundreds of analytical laboratories world-wide. Instrumentation, software, and databases have been commercialized for FAME analysis (MIDI Inc., Newark, Delaware, USA).

Following the standard MIDI method, the analyst cultures the microbe on agar and takes a large inoculating loop (~30–40 mg wet weight) of the vegetative form of the microorganism that is used directly in the extraction procedure (i.e., without a subculture). The cellular fatty acids are fi rst saponifi ed and released from the cell with high-temperature (100°C) incubation in a sodium hydroxide/methanol solution. This is followed by a second, high-temperature (80°C) incubation in an acidic solution. Extraction of the esterifi ed fatty acids is performed with an organic solvent (ether and hexane), but prior to chromatography, the organic extract is typically washed again in NaOH solution to prevent tailing of the peaks [ 29 ] . The entire procedure takes approximately 1 h [ 30 ] . A more gentle method for extraction of fatty acid using a lower temperature and concen-tration of alkali has been described and should yield ester-linked fatty acids without free fatty acids [ 31 ] . Comparisons of the MIDI method with several extraction methods have been described, and the results indicate that specifi c classes of FAMEs are favored by the type of extraction method [ 31, 32 ] .

Recently, another method has been developed by MIDI Inc., called “Instant FAME.” While conceptually similar to the previous protocol, the fatty acid extraction procedure is shortened to approximately 5 min per sample. In addition, several samples (up to 50) can be easily processed in tandem. In this simplifi ed procedure, samples are mixed with a potassium hydroxide/methanol solution that extracts, hydrolyzes, and methylates cellular fatty acids in a single step. Next, an organic extraction with hexane solution is per-formed. Finally, a solution containing a dye in dilute hydrochloric acid is added to help facilitate removal of the organic phase for chromatographic analysis. An added convenience of the Instant FAME procedure is that it requires no high-tem-perature incubations.

In Situ Fatty Acid Extraction Using supercritical fl uid extraction (SCF) on whole cells, fatty acids can be hydrolyzed and derivatized directly during the extraction step, thus saving time and labor [ 33 ] . The FAMEs pro-duced by this method could be analyzed without additional treatment. Other authors have described in situ treatments on whole cells using pyrolysis in a microreactor to reduce the FAME sample preparation time to a few minutes and the required sample mass to the microgram level [ 34 ] . FAME profi les obtained by the fast pyrolysis method, when compared with those obtained with the standard chemical hydrolysis and esterifi cation method, were compared by one group and were found to be congruent [ 35 ] . Hendricker et al. [ 36 ] examined 17 strains of pathogenic and related bacteria grown on (>10) various media, isolating them at different growth stages. Using the in situ thermal hydrolysis and methylation technique and analyzing the FAMEs, the authors observed greater than 98% correct classifi cation of the bac-teria in blinded measurements [ 36 ] .

Phospholipid Fatty Acid (PLFA) Extraction Lipids are classically extracted from cell suspen-sions using methanol and chloroform and then fractionated on a silica column into three frac-tions based on polarity. The most polar fraction contains the phospholipids, which is derivatized


for separation of the FAMEs on GC and analyzed on a coupled mass spectrometer [ 6 ] . An enhanced extraction method has been developed which reduces the extraction time several hours by employing pressurized accelerated hot solvent extraction and provides a greater yield of PLFA from Bacillus sp. spores [ 5 ] .

3.2.1.4 Methods for Separation and Detection of Fatty Acid Derivatives

Gas Chromatography Since fatty acid methyl esters are volatile, they can be injected into a gas chromatograph (GC) equipped with a fl ame ionization detector (GC-FID) to generate a profi le of the mixture. The gas chromatograph typically consists of four modules: the injector, vaporizing (fl ame or inductively coupled) device, chromatographic column, and detector. The system may include a carousel for automated sample processing. Sample separation is achieved by exploiting the variable affi nity of the vaporized FAME mole-cules with the liquid fi lm inside the fused silica column. This effect is caused by variations in chain length, conformation, substitutions, and branches of the vaporized FAME molecules. Identifi cation of individual fatty acids is facili-tated by software packages that convert retention times on capillary GC systems to equivalent chain lengths (ECLs) through calibration stan-dards [ 30 ] . MIDI offers various libraries, one of which targets biological threat agents. The soft-ware determines the correlation of the sample profi le with those of known samples in the library and reports the signifi cance of the comparisons. The reader is advised to consult one of the sev-eral texts on gas chromatography and review papers covering GC analysis of fatty acids [ 37 ] for in-depth knowledge.

Fatty acid extracts can also be analyzed with other detection systems such as HPLC coupled to MS/MS detectors [ 38 ] . However, these techniques are often more laborious and require substantial operator expertise. Therefore, they may not be practical for routine microbial identifi cation and should be reserved for characterization of unusual or unidentifi ed fatty acid compounds [ 21 ] .

PLFA and MIDI-FAME extraction procedures give comparable results for laboratory cultures [ 21 ] , and each has been used in a variety of envi-ronments [ 39– 41 ] . It is important to note that each extraction procedure targets different lipid sources within the cell and may give different fatty acid profi les depending on the nature of the sample. In the MIDI-FAME procedure, lipids are derivatized and extracted from a variety of sources within the sample including the cell membrane bilayer, lipopolysaccharides (LPS), and storage lipids as well as biologic detritus or nonmicrobial materials that may be present in the sample [ 4 ] . In contrast, the PLFA procedure [ 42 ] can be used to extract selectively phospholipids from the membranes of living cells at the exclu-sion of detritus or other exogenous fatty acids in the sample [ 4 ] .

Mass Spectrometry Microbial characterization by lipid analysis using mass spectrometry has been employed for two decades. Papers from a 1992 American Chemical Society Symposium reviewed practice and issues in the characterization and identifi cation of phos-pholipids, glycolipids, and lipooligosaccharides [ 3, 26, 43– 45 ] . Since the early endeavors, mass spectrometry has become more routine for lipid analysis [ 46 ] and is now described as a tool for studying lipidomics [ 47, 48 ] . An advantage that mass spectrometry offers is the capability to show ion peaks derived from various types of molecules, such as fatty acids and dipicolinic acid in the same spectrum [ 36 ] . Presence of the ion for dipicolinic acid indicates the spore form was present in the sample. Specifi c functional groups in the lipid structure can also be identifi ed with mass spectrometry techniques [ 49 ] . A thor-ough analysis of lipopeptide profi les from Bacillus strains has been described using MALDI-ToF mass spectrometry [ 50 ] . Today, mass spec trometry is often used to confi rm pre-sumptive, unusual fatty acids present in FAME GC profi les and for analysis of novel molecular structures. Researchers have also attempted to identify and quantify fatty acids using mass spec-trometry in a single assay [ 51, 52 ] . The results of the quantitation by mass spectrometry compared


favorably with those of GC fl ame ionization detection [ 51 ] . A few types of specialized mass spectrometry methods warrant mentioning because they have been used for identifi cation and characterization of bacterial strains and show promise for forensic applications (see below). However, due to the complexities and cost of the instrumentation and the requirement for substan-tial operator expertise, mass spectrometry will probably remain a technique only for the special-ized laboratory [ 21 ] .

High-Performance Liquid Chromatography (HPLC) HPLC has been used for separation and identifi -cation of very high molecular weight branched fatty acids of Mycobacterium species [ 38, 53 ] . As with GC, the retention times are determined by the fatty acid chain length, degree of saturation, and the presence of branch structures. A commer-cial database for Mycobacteria identifi cation using HPLC is available (SMIS, MIDI, Inc).

Infrared Spectroscopy Vibrational spectroscopy has been used for rapid analysis of bacteria that may contaminate foods [ 54 ] . Using 40 mg whole cells, FAMEs were pre-pared the usual way [ 30 ] , and 1.25 mL extract was concentrated to ~2 m L for analysis by attenu-ated total refl ection (ATR)-FTIR spectroscopy. The ATR-FTIR spectra were subjected to multi-variate analysis. Advantages of this approach include the elimination of the GC separation step, a short (2 min) analysis time period, and improved precision of the measurements. The potential of the technique was demonstrated with a small set of 14 pathogens that included B. anthracis , B. cereus , and four strains of Escherichia coli . The E. coli could be identifi ed to the strain level. Experiments with actual food samples spiked with bacteria were not reported.

Isotope Ratio Mass Spectrometry (IRMS) Bacterial community analysis of PLFAs using GC and IRMS to measure incorporation of 13 C-labeled substrates [ 6 ] has recently been reviewed [ 55 ] . The method obviates the need for culture and therefore is useful when the

organisms are refractory toward growth outside their natural environment. In another report, a forensic-like investigation was performed on conventional and organic-labeled milk products in order to establish mislabeled products. The authors used a combination of IRMS and GC analysis of FAMEs [ 56 ] . Although this work does not involve microbes, it demonstrates how IRMS can be used as an orthogonal measurement to support the conclusions from the analysis of the FAMEs. An in-depth review of IRMS use for microbial forensics is provided in Chap. 7 of this volume.

3.2.2 Lipopolysaccharides (LPS)

LPS Variation: Lipid A Lipid A is the hydrophobic phospholipid mono-layer that anchors the LPS to the outer membrane (as described in Sect. 3.1.1 ). The fatty acids asso-ciated with lipid A include dodecanoic (12:0), tetradecanoic (14:0), hexadecanoic (16:0), hexa-decenoic (16:1), and, in the greatest relative amount, 3-hydroxy acids (either 3-OH-10:0, 3-OH-12:0, or 3-OH-14:0) [ 57 ] . The structure and chemistry of lipid A can show variability between different species in the number of fatty acids present, the linkages between fatty acids and the polar head compounds, and the functional groups present along the hydrophobic chain [ 58, 59 ] . Growth conditions and environmental fac-tors can also infl uence the heterogeneity of the lipid A layer [ 60 ] . Recently, the structure of lipid A extracted from Yersinia pestis has been charac-terized using mass spectrometry [ 61 ] .

3.2.2.2 O Antigen The other components of the LPS include an oli-gosaccharide anchored to the lipid A (“non-repeating core”). This is connected distally to another oligosaccharide chain called the O anti-gen. While the former does not show much vari-ability within genera [ 2 ] , the O antigen component shows considerable structural diversity. Examples of this variation include the type of monosaccha-ride subunits present, the stereochemistry of gly-cosidic linkages, and the type of noncarbohydrate


constituents or other functional groups present [ 2 ] . Growth conditions can also infl uence the structural characteristics of the O antigen in some organisms [ 62 ] .

3.2.2.3 Methods for LPS Extraction The LPS can be extracted from whole cells using lysozyme-phenol/chloroform treatment and iso-lated by ultracentrifugation [ 63 ] . The fraction is purifi ed by treatment with a panel of enzymes to remove extraneous proteins and nucleic acids followed by centrifugation. Lipid A is obtained from the LPS by heating with sodium dodecyl sulfate (SDS) at 100°C and purifi ed by ethanol extraction, centrifugation, and extraction of the pellet with a mixture of chloroform/methanol/water [ 64 ] .

3.2.2.4 Methods for Separation/Detection LPS

Mass Spectrometry Lipid A, the immunogenic region portion of the LPS found on the outer membrane of Gram-negative bacteria, is very heterogeneous. The residues of lipid A are environmentally regulated, differing for example, when the bacteria are grown at different temperatures. Mass spectrom-etry has often been used to help elucidate the complex structures of the lipid A molecule [ 65– 67 ] . In pyrolysis-gas chromatography, ther-mal breakdown products of different LPS com-pounds are characterized via mass spectrometry. This technique has been used successfully in sev-eral bacterial discrimination studies [ 58, 68– 70 ] .

HPLC Another analytical technique that shows promise for forensic and fi eld applications is the “fl ash” microbial typing system [ 38 ] . The protocol uses a series of chemical extractions which sequentially target neutral lipid, polar lipid, spore-specifi c, and LPS biomarkers. Structural characterizations were performed with HPLC/MS/MS systems. In addition to the increased sensitivity (amol/ m l), portability, and automation potential of this tech-nique, the authors were able to successfully iden-tify different Gram-negative pathogens based on variations in the LPS chemistry.

3.3 Forensic Analysis Using Lipids

3.3.1 Characteristics of Forensically Informative Lipid Signatures

A perfect biomarker, in the context of this chap-ter, is one that is indicative of one type of organ-ism or of a defi ned culture or environmental condition. For bacterial lipids, biomarkers can be two different forms: (1) the presence/absence of a single, structurally distinct fatty acid and (2) the presence/absence of multiple fatty acids and their respective abundances within the cell. As dis-cussed in Sect. 3.2.1.1 and 3.2.1.2 , there are sin-gle fatty acid structures that are characteristic for some organisms and processes. However, unique signatures are rare. More often, the entire fatty acid profi le will be characteristic of a species, a cell type (e.g., spore and vegetative cell), or a par-ticular environment. Thus, in certain cases, the composite fatty acid profi le may be a more appro-priate forensic signature.

When choosing a unique biomarker within a lipid profi le, it is best to select one of medium to high signal intensity and avoid those that have intensities close to the threshold. Low signals will not be noticed when the amount of sample or effi ciency of extraction is low. In addition, it is more diffi cult to give signifi cance to subtle dif-ferences than large ones. If the distinguishing biomarker is naturally of low abundance in the cell, then it is very important to make sure the extraction effi ciency is optimal and that steps are taken to suppress lipolysis [ 1 ] . Although there will be stochastic effects in any type of measure-ment, unique signatures should have the same retention time in HPLC and GC measurements and charge/mass ratio for the mass spectrometric examinations, if the procedure is performed iden-tically from run-to-run and providing the instru-mentation is kept calibrated with appropriate reference standards. Likewise, the relative peak height of the selected biomarker should not change very much when different samples of the same material are examined. The cell wall lysis protocol may need to be enhanced in order to pro-vide suffi cient fatty acids for distinguishing some


bacteria [ 71 ] . In addition, since the evidence may be dirty or contaminated with materials other than bacteria, there may be background signals that will mask a low-level marker signal. Of course, these issues also pertain to signatures that are comprised of several signals (fatty acids), if they are of relative low intensity.

3.3.2 Example of Fatty Acid Analysis on Biological Sample

A typical fatty acid profi le and scoring table derived by the MIDI software are shown in Fig. 3.3 . The example illustrates the results for the fatty acids extracted from B. cereus T-strain grown on brain heart infusion (BHI) agar. The prepara-tion was judged to be almost pure spores by phase microscopy, and fatty acids were extracted accord-ing to previously described protocols [ 20 ] . The table presents information on the relative abun-dance of each entity. The software compares the profi le of the sample with those in the various libraries that are delivered with the MIDI system. A similarity index, ranging from 0.0 to 1.0, is cal-culated to indicate the degree of similarity between the sample profi le and those in the database. The names of the species that have similar profi les to the sample profi le are provided in the list. Regardless of the similarity index, if the fi rst name on the list is that of the sample, the result is considered correct. As one sees in Fig. 3.3 , the listing suggests that the sample is B. cereus , which is correct. To make the identifi cation reliable, the MIDI database has several profi les of all Bacillus species strains. When the similarity index for the fi rst choice is examined, it is apparent that there must be several differences between the two pro-fi les. This result is explained by the facts that the commercial repositories are populated only with profi les from vegetative cells, and, as discussed in Sect. 3.2.1.1 , the FAME profi les from spores dif-fer somewhat from those of the vegetative cell. Thus, a value of 0.646 for the similarity index is not surprising. To obtain high degrees of similar-ity, a separate repository for the spore fatty acid profi les should be constructed for the library com-parisons. As shown in the library list, this principle has been demonstrated at the proof-of-concept

level as indicated by the high similarity index (0.987) obtained for the B. cereus spores grown on BHI agar when the sample profi le was com-pared to those in a custom library composed of profi les of the same spores grown in various media.

3.3.3 Investigative Applications

3.3.3.1 Species and Subspecies Identifi cation

Species identifi cation is integral to recognizing when a biocrime has been committed and deter-mining the subsequent risk to public health [ 72 ] . While the phylogenetic specifi city of lipid profi l-ing makes it a viable technique for identifi cation of forensic samples, it is not likely to supplant genetic or traditional culture-based methods. However, lipid profi ling could be used as an orthogonal technique to confi rm biothreat identi-fi cations. Some pathogens like B. anthracis that are diffi cult to resolve genetically from closely related, but nonpathogenic, organisms (e.g., B. anthracis from B. cereus and B. thuringiensis [ 73 ] ) do show robust differences in lipid compo-sition [ 8, 74 ] . In the case of B. anthracis , vali-dated assays currently exist for identifi cation using fatty acid profi les [ 75, 76 ] .

For attribution purposes, subspecies identifi -cation can provide additional criteria with which to exclude facilities or link biocrime evidence to a source. Even though lipid signatures are gener-ally conserved within bacterial species, strain-specifi c fatty acid biomarkers have been reported for B. anthracis [ 8 ] , suggesting that lipid varia-tion may have potential for certain subspecies characterization and differentiation.

3.3.3.2 Identifi cation of Growth Environment and Laboratory Attribution

In addition to using FAME biomarkers for taxo-nomic characterizations, lipid profi ling may pro-vide information on the conditions used to grow a pathogen. As discussed in 2.1.2, choice of nutri-tional substrates infl uences the types and propor-tions of fatty acids found within the cell, particularly for Bacillus organisms. Examples of

RT Response AR/HT RFact ECL Peak Name Percent Comment 1 Comment2

Fig. 3.3 Report of FAME analysis by MIDI software. Shown are the data table, library list showing the similarity indices obtained by comparison of the sample profi le to those in the library, and a sample fatty acid profi le. B. cereus T-strain spores were grown on brain heart infusion (BHI) agar and compared against two different bacterial libraries of vegetative cells (NOOHC6 and FSTNOH; MIDI, Inc.). The spore profi le was most similar to profi les belonging to B. cereus – GC subgroup C and B. thuringiensis , respec-tively. When an internally developed library specifi c for B. cereus T-strain spores grown on various media was

selected, BHI was selected as the most likely medium (from a set of 17 different media profi les). The table depicts the relative fatty acid composition of the sample. “RT,” reten-tion time; “Response,” peak intensity; “ECL,” equivalent chain length (translates RT to carbon length); “Percent,” relative amount (that the fatty acid makes in the composite profi le of named features). “Sum In Feature 2”: sum of the contribution of the two 16:1 unsaturated fatty acids that can-not be resolved. “Sim Index”: similarity index. The higher the Sim Index value, the more similar is the library profi le to the sample profi le (Unpublished data of the authors)


the effect that medium recipe has on the fatty acid composition of B. cereus spores are shown in Figs. 3.4 and 3.5 (data from a previously pub-lished study [ 20 ] ).

In Fig. 3.4 , FAME profi les of B. cereus spores grown in a chemically defi ned sporulation broth and a medium containing complex nutrient addi-tives are compared. The top panel shows a FAME profi le from spores produced on Columbia agar, a complex medium containing meat peptone, tryp-tone, and yeast extract that was also supplemented with sheep blood. The lower panel shows the pro-fi le of spores produced on a “chemically defi ned sporulation medium” (CDSM [ 77 ] ) that was made by adding several compounds and salts of known concentrations and contained no blood products. In this example, it is readily apparent by casual inspection that the two profi les are dif-ferent. Close inspection reveals that for the spores grown in the presence of blood, there is an extra peak identifi ed by the MIDI software as oleic acid (18:1 w 9c, far right in the top panel).

This fatty acid is not observed in spores harvested from other, non-blood-containing sporulation media (bottom panel, [ 20 ] ). In addition, oleic acid has been observed in other bacterial cultures grown on sheep blood–supplemented media, specifi cally Helicobacter pylori [ 78 ] , suggesting that this fatty acid has the potential to be a unique biomarker for this particular growth medium additive.

It is important to note that changes in the growth environment, in most cases, will not cause changes in the presence and absence of certain fatty acids but rather in the relative proportion of different fatty acids, as is the case with Bacillus organisms [ 20– 22, 24 ] . This effect can be seen in the B. cereus spore profi les shown in Figs. 3.4 and 3.5 . For example, the relative intensity of the 14:0 iso and 15:0 iso peaks are signifi cantly dif-ferent in the CDSM profi le compared to spores grown on the blood-containing Columbia agar and on Schaeffer’s medium (Fig. 3.4 ). In addi-tion, the ratio of 17:0 iso to 17:0 anteiso is roughly

Fig. 3.4 Fatty acid profi les for B. cereus T-strain spores grown on media containing undefi ned blood additives and grown in chemically defi ned sporulation medium


equal, whereas the 17:0 iso fatty acid has a greater relative abundance in the other three media.

A similar effect is observed when other non-nutritional attributes of the medium are varied. In Fig. 3.5 , the spores were harvested from either broth (top panel) or solid preparations of the same medium (Schaeffer’s sporulation medium [ 79 ] ). In this example, a cursory glance at both profi les reveals few differences in the types of fatty acids present. However, variation does exist in the rela-tive peak heights of some fatty acids such as between 17:0 iso and 17:0 anteiso and between 16:0 iso and 16:0. This example serves to demon-strate that the relative ratio of specifi c spore fatty acids can act as the signature.

Given the vast combinations of growth condi-tions and medium substrates available for cultur-ing microorganisms, the limited number of fatty acids present in cells (typically <20), and that the biochemical response of each organism to envi-ronmental stress and culturing conditions is likely

to be different, identifying fatty acid biomarkers that are indicative of a particular growth medium or specifi c culturing conditions will continue to be a challenge [ 1 ] . To overcome these obstacles, phenotypic libraries of forensically relevant organisms prepared under various culturing con-ditions could be combined with novel types of quantitative analysis to extract informative bio-signatures from profi les. Multivariate statistical strategies like discriminant function analysis and artifi cial neural networks have been shown to minimize nonspecifi c variation in fatty acid pro-fi les and correctly classify organisms by species [ 74 ] or by the growth medium formulation used for culturing [ 20 ] .

3.3.3.3 Analysis of Mixed Biological Samples

Many protocols for fatty acid analysis utilize plate-grown, fresh cultures. Forensic evidence, how-ever, may contain mixed cultures and impurities.

Fig. 3.5 Fatty acid profi les for B. cereus spores grown on solid medium or in broth. Schaeffer’s medium was used to culture the bacteria for both examples, except the spores were isolated at stationary phase of growth by centrifugation

( top panel ) or scraped from agar plates ( bottom panel ). The bacteria were greater than 95% spores, as indicated by phase contrast microscopy


For example, a fl ask containing material that looks like an old culture may be found in a garage and have mold or debris from dead, lysed cells as well as intact vegetative cells. The evidence may be scrapings or vacuum-collected material that con-tains dust, soil, fungi, and plant material. Thus, lipids from environmental fl ora might be present in the profi le of such samples as well as spurious peaks from the dirt. However, these issues may not adversely affect the analysis. Ecologists have been successfully studying soil (sediment and mud) bacterial and marine communities using FAMEs for several years. In addition, insight regarding analysis and interpretation of environ-mental mixtures is available from published stud-ies of bacterial communities. For example, fatty acids specifi c for a microbe could be used to iden-tify an organism in a sample containing multiple species mixed in equal proportions [ 21 ] . The opti-mal discriminatory fatty acids were ones that dis-played a characteristic intensity. The authors reported no new or spurious peaks when FAMEs were mixed. However, other authors have sug-gested that method adaptation may be required to move from the standard protocols for pure cultures to one that is successful with mixtures [ 80 ] . A sys-tematic study with forensically relevant samples spiked with target organisms should be carried out before using the standard FAME protocol on evidence.

3.3.4 Validation Considerations

As discussed in the previous sections, there are several methods to analyze the lipids associated with bacteria. In particular, fatty acid analysis has been used for decades to characterize microbes in diverse fi elds such as agriculture [ 80 ] , medical diagnostics [ 7, 81– 83 ] , food-borne bacteria mon-itoring [ 12, 84 ] , and even law enforcement [ 85 ] . Methods for FAME analysis of bacteria are well accepted by the scientifi c community, and there are hundreds of peer-reviewed publications describing its use in multiple disciplines. Standards for the science of FAME analysis are high. Thus, it may be reasonable to submit a por-tion of the evidence collected in a microbial forensic event to a laboratory for characterization

by FAME analysis to develop investigative leads. However, since it is important to conserve evi-dence, it would be appropriate to investigate ways to reduce the amount of material consumed in the fatty acid analysis (typically ~10 mg powder with standard protocols) without affecting the quality of the data. Furthermore, the bacterial sample could be mixed with additives like stabilizers or dust so that 10 mg powder would contain less than the required amount of material for the stan-dard measurement. It may not be diffi cult to reduce the amount of evidence required for fatty acid analysis, since studies have reported fatty acid profi les obtained from <5 mg dry vegetative cells or spores with excellent reproducibility both within batch and among batches with the MIDI GC-FID procedure [ 8, 20 ] . Further proof that the biomass may be reduced without affecting the data quality was provided in a report from the US Department of Agriculture in which a single col-ony was used for the FAME analysis [ 86 ] .

3.3.5 Quality Assurance

Before submission of the evidence, it is very important to ensure that the laboratory has a pro-gram of quality assurance in place so the results can be regarded as reliable. Data security should be included in the quality assurance program to ensure that only authorized personnel have access to the data, in accordance with US Food and Drug Administration requirements [ 87 ] . In addition, a set of specifi c guidelines for microbial forensics has been formulated by scientists from various disciplines encompassing clinical and agricultural microbiology, medicine, and forensics [ 88 ] . The features of the guidelines include training, educa-tional requirements, validation, use of reference standards, routine quality control practices, and profi ciency testing. Referral to the microbial foren-sics guidelines will help the analyst select the best practice for the microbial forensic examinations. Overall, adherence to the individual laboratory quality assurance program and the general micro-bial forensics guidelines will help standardize lipid analyses. This, in turn, will make the results of such tests more trustworthy and acceptable to the demands of the judicial system.


3.4 Summary

Lipids exhibit a variety of different structures particularly within their long hydrocarbon chains. Lipid structures can also have polar and hydro-phobic functional groups attached. A large num-ber of potential lipid biomarkers exist in microbial cells that can be extracted, separated, and charac-terized by a variety of chromatographic and mass spectrometry techniques. The specifi c features of a lipid profi le are determined by the type of organism present and the growth conditions used during its cultivation. The presence of certain lipid biomarkers or variation in the proportion of multiple biomarkers may provide useful informa-tion during a forensic investigation about the spe-cifi c culturing conditions used to grow a microorganism. The fact that many validated protocols for lipid analyses currently exist sug-gests that a method could be developed to ana-lyze evidence of a suspected biothreat incident. However, a rigorous quality assurance program needs to be implemented to provide reliability to the data.

Disclaimer The views and conclusions contained in this document are those of the authors and should not be implied as necessarily representing the offi cial policies, either expressed or implied, by the US Government. This is publication 08–07 of the Federal Bureau of Investigation. Names of commercial manufacturers are provided for identifi cation only, and inclusion does not imply endorsement by the Federal Bureau of Investigation.

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Fatty Acids and Lipids in Microbial Forensics

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