Subtyping Method Characteristics
Typeability
An ideal typing method should be able to produce data that can lead to the establishment of subtypes for the majority, if not all, of the strains of the pathogen being studied. The degree with which a method achieves this can be expressed, in mathematical terms, as the percentage of typeable isolates among the total number of isolates being subtyped. All molecular subtyping methods (except plasmid profiling with some organisms) show very high typeability with the organ-isms that they target.
TABLE 1 Definitions commonly used in molecular epidemiologya
Term Definition
Isolate A population of microbial cells from a pure culture derived from a single colony on an isolation plate.
Strain An isolate or group of isolates exhibiting phenotypic and/or genotypic traits which are distinctive from those of other isolates of the same species.
Clone A group of isolates descending from a common ancestor as part of a direct chain of replication and transmission from host to host or from the environment to host. The term “outbreak strain” is often used with this meaning in the context of epidemiologic subtyping.
Subtype A specific pattern or set of markers displayed by a strain when a particular typing system is used.
Typeability The proportion of strains for which a subtype may be generated by a given subtyping method.
Reproducibility Same as repeatability. The ability of a subtyping method to produce the same results upon repeated testing. Usually stated as the proportion of strains in a given population that displays the same subtype upon repeated testing.
Stability The ability of a subtyping method to assign the same subtype to epidemiologically related strains, e.g., as part of the same single-strain outbreak or originating from the same patient or from serial passage in vitro or in vivo. Usually stated as the proportion of epidemiologically linked strains showing the same subtype.
Discriminatory The ability of a subtyping method to differentiate between power epidemiologically unrelated strains.
Epidemiological The ability of a subtyping method to link epidemiologically related strains.
concordance
Comparative Subtyping results generated in the same or in a very few experiments in subtyping the same laboratory may be compared due to poor interexperiment
reproducibility.
Definitive subtyping Subtyping results generated in different laboratories and/or at different times may be compared and stored in a reference library.
Library subtyping Subtyping results generated by definitive subtyping are stored in a database (library). Sometimes used synonymously with definitive subtyping.
Cluster The occurrence of clinical isolations of microbes with a particular subtype greater than would otherwise be expected in a particular time and place with no further supporting epidemiological information.
Outbreak Same as an epidemic. The occurrence of disease greater than would otherwise be expected in a particular time and place.
Sporadic Antonym to outbreak. Occurring with no clear relation to an outbreak.
Endemicity Constant presence of a disease at a significant frequency; typically restricted to, or peculiar to, a locality or region.
a
In part adopted from Van Belkum et al. (1) and Struelens (242).
Reproducibility and Stability
Reproducibility refers to the ability of a method to assign the same type to an isolate tested on independent occasions separated in time and/or place. It may be calculated as the percentage of strains that, upon repeated testing using the same parameters, yield the same result. This term is some-times also referred to as the repeatability. Typically, there is a direct correlation between the reproducibility and ro-bustness of a method and the quality of the data being generated. Reproducibility may be influenced by many steps in the procedure, such as preparation of materials (growth conditions, DNA extraction), different batches of reagents, different types of instruments, and, finally, bias in observing, analyzing, and interpreting results. Some methods have such a poor reproducibility that it is not possible to compare results generated indifferent experiments. Such methods are said to be comparative. Other methods are so
reproduci-ble that it is possireproduci-ble to recognize the same subtypes even though they have been generated at separate times and/or in separate places. Such methods are considered definitive subtyping methods.
Reproducibility has both intralaboratory and interlabora-tory dimensions. As will be discussed later, some subtyping me th od s, e. g. , a mp li fi ed fr ag me nt le ng th po ly mo rp hi sm (AFLP) analysis, show excellent reproducibility when per-formed on the same instrument in one laboratory (i.e., they have good intralaboratory reproducibility) but poor or subop-timal reproducibility when testing is performed in different laboratories (i.e., they may have a poor interlaboratory repro-ducibility).
Reproducibility is also indirectly affected by the stability of the genetic markers being targeted by the method. The assessed markers should remain stable during outbreaks and among multiple individual patient isolates not varying to a
TABLE 2 Characteristics and application of a number of subtyping methods
Quality of or applicability to:
SNP
Whole-
Whole-Characteristic
Plasmid PCR- Gene detection Mass
PFGE genome RAPD rep-PCR AFLP MLST MLVA genome
profiling ribotyping sequencing with DNA spectrometry
mapping sequencing
microarrays
Reproducibility Good Good Good Poor Poor Good Good Good Good Good Good Good Good
Stability Variable Good Good Poor Moderate Good Good Moderate to Moderate to Moderate to Good Moderate to Good
good good good good
Discriminatory Variable Excellent Excellent Good Good Good Excellent Low to Excellent Excellent Good to Excellent Poor
power moderate excellent
Universal Yes Yes Yes Yes Yes Yes Yes No No No No Yes No
applicability
Applicable for Yes Yes Yes No No/yes (in Yes Yes (in local Yes Yes Yes Yes Yes Yes
library local libraries)
subtypinga libraries)
Complexity of Simple Complex Complex Complex Complex Simple Complex Simple Simple Simple Simple Very complex Simple
data
Ease of usea Simple Moderately Moderately Simple Simple/simple Simple Moderate Simple to Simple Simple to Simple to Labor-intensive Simple
labor- labor- moderately moderately moderately
intensive intensive labor- labor-
labor-intensive intensive intensive
Costa Low Moderate High Low Low to Low Moderate Moderate Moderate Moderate Moderate High Low
moderate
Suggested use of SupplementOutbreak To aid in To answer As RAPD First-line Local Phylogenetic Outbreak Outbreak Outbreak Outbreak Subspecies the method to other surveillance, the specific subtyping outbreak studies, surveillance, surveillance, surveillance, investigation, level
methods large-scale assembly limited of C. surveillance, attribution large-scale large-scale large-scale (large-scale) (serotype,
libraries of next- epidemiological difficile suitable for of library library library library pathotype)
generation questions/ local Campylobacter, subtyping if subtyping, subtyping, subtyping, typing
short small-scale library potential standardized, phylogenetic phylogenetic phylogenetic
sequence outbreak subtyping forensic use potentially studies, studies, studies,
reads investigations good for forensic forensic forensic
forensic and microbiology, microbiology, microbiology, attribution attribution attribution attribution purposes
Comments Gold standard Semiautomated
for highly method may
discriminatory possibly be
bacterial used for
subtyping local
surveillance
a
Manual/automated version.
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degree that confuses the epidemiological picture. The ge-netic fingerprints generated by a method shouldalso not be affected by in vitro manipulations, such asfreeze-thaw cycles and serial passages.
Discriminatory Power
Discriminatory power is defined as the ability of a method to distinguish between unrelated strains. An objective meas-ure of discriminatory power can be obtained by calculating Simpson’s index of diversity (DI) (2), which is an estimate of the probability that two epidemiologically unrelated strains will display different subtypes. The formula reads
where N is the number of unrelated strains tested and aj is the number of strains with a subtype that is indistinguishable from the jth strain. The DI value of 1.0 indicates that all strains can be differentiated from each other. A value above 0.95 is a desirable cutoff for subtyping methods to be used for outbreak investigations.
The DI is a function of both the number of subtypes in the strain population (richness) and the proportion of strains representing each subtype, i.e., the evenness of the distribu-tion of the isolates among the different subtypes. It is not possible to determine the contribution of the richness or the evenness of a subtyping method to the DI from the number per se. This should also be taken into consideration when comparing different subtyping methods. Several other diversity indices have been devised for this purpose, among which Shannon’s index (H) (3) is the most commonly cited.
However, the maximal size of this index is a direct function
FIGURE 1 Hypothetical example of the subtype distribution of 100 epidemiologically unrelated microbial strains generated by two subtyping methods. doi:10.1128/9781555817381.ch10.f1
of the number of subtypes generated within a given strain population, and it is therefore difficult to interpret differ-ences in the sizes of the index unless the populations studied generate the same number of subtypes. An easier and more comprehensible way to judge the richnessof a subtyping method is to create a histogram showing the distribution of different subtypes. The results of a hypothetical experi-ment of subtyping the same population of 100 strains by two different methods are shown inFig. 1AandB. Method A has a slightly higher DI than method B. However, the difference between the methods becomes obvious when one observes the histograms: method A has a higher richness than method B. The 100 strains are differentiated into 74 subtypes, with all containing 5 or fewer strains by method A, whereas the 6 most common subtypes by method B contain half of the strains, and overall, only 43 subtypes are generated by this method.
An ideal method should have such a high discriminatory power that it is capable of discriminating all epidemiologi-cally unrelated isolates from each other (i.e., it has a high specificity). However,the method should also be ableto group together isolates that are associated with the same source (i.e., it has a high sensitivity). In other words, a method with high sensitivity and specificity generates epide-miologically relevant data. The ability of a method to group together epidemiologically related isolates is sometimes also referred to as the epidemiological concordance.
Convenience Parameters
There are many convenience parameters to be considered, i n c l u d i n g s p e e d o f a n a l y s i s , c o s t , t e c h n i c a l d e m a n d s , accessibility of the method, and ease of data analysis. The
ability of a method to generate data rapidly is affected by the throughput of the method, the number of steps involved, the amount of hands-on technical time required to perform the method, and whether the method is amenable for auto-mation. Ideally, typing results should be available within a single working day. The cost of performing a method de-pends on numerous factors, such as the initial investment in equipment and infrastructure, the price of reagents and consumables, and the number and skill level of staff needed.
Cost is usually also the most critical factor affecting the accessibility of the method to general microbiology laborato-ries. Ease of use encompasses technical simplicity, high throughput, and ease of scoring the results. For easy analysis, data should be objective, amenable for computerized analy-sis, and easily disseminated between laboratories.
Another convenience parameter rarely considered is the universal applicability of the method; can the same method be used to subtype a broad range of organisms while main-taining a universally high typeability, reproducibility, and discriminatory power? The broader the range of microbial species that can be studied, the more central the position of the method in the general typing laboratory will be.
Non-Target-Specific Methods
A short description of the principle of some non-target-specific methods is shown inFig. 2.
Plasmid Profiling
Plasmids are circular extrachromosomal autonomous self-replicating genetic elements that are found in bacteria and some eukaryotes. Their sizes range from approximately 1 kb to >1 Mb. Besi de s c ar ry in g g en es re gu la ti ng th ei r o wn
FIGURE 2 Procedural principles of some commonly used non-target-specific subtyping methods.
doi:10.1128/9781555817381.ch10.f2
replication and transmission, plasmids may carry genes that confer specific properties in the host organism, e.g., antimi-crobial resistance or toxins. Plasmid profiling was the first widely used nucleic acid-based bacterial subtyping method dating back to the 1970s. The rationale for using plasmid profiling for subtyping is that bacterial strains typically differ in the numbers and sizes of plasmids that they carry. The isolates are lysed using a method that disrupts chromosomal DNA while retaining the integrity of the plasmid, e.g., alkaline lysis (4), followed by separation of the plasmids by agarose gel electrophoresis (5), staining using a fluorescent dye, e.g., ethidium bromide, and visualization of the plasmids under UV light.
The advantages of this method are that it is universal (i.e., it may be used to characterize any organism that con-tains plasmids by using the same basic procedure) and that it is inexpensive, rapid, and simple, with no requirements for special equipment. The typeability of the method is variable, the discriminatory power varies between organ-isms, and the reproducibility is in general good, but since plasmids may be lost during strain propagation in vivo and in vitro, the stability is suboptimal. Additionally, plasmids can be unintentionally nicked, causing changes in their conformational structure (from supercoiled to a relaxed or linear form), impacting their migrating properties and result-ing in an incorrect profile. These changes may happen if the purification process is performed too harshly. Two or all three conformations may be present at the same time in significant amounts and be visible as two or three bands in the gel, and since they have different migration properties in agarose, interpretation of the profiles may be difficult.
In addition, large plasmids, those >100 kb in size, are poorly
separated by ordinary agarose gel electrophoresis. Unrelated plasmids with similar sizes may not be differentiated in an agarose gel. However, if they are digested with a restriction enzyme before electrophoresis, it will often be possible to differentiate between them.
Plasmid profiling has been used in numerous epidemiol-ogical investigations of a wide range of community-acquired or nosocomially acquired infections (6,7) and is still used as an adjunct in investigations of outbreaks of foodborne infections in some countries (8). Plasmids may spread be-tween different organisms and cause outbreaks of, for exam-ple, antimicrobial resistance. Plasmid profiling is the first step in the investigation of such outbreaks (9,10).
Restriction Fragment Length Polymorphisms
A restriction endonuclease is an enzyme that recognizes a short DNA sequence and cuts it at a specific location within the restriction site. Genomic DNA from a single microor-ganism will always be cut into fragments of the same size and number if it is digested by the same restriction enzyme.
The restriction fragments may be separated according to their size by electrophoresis using agarose or a similar me-dium as a matrix, followed by staining and visualization under UV light to reveal the DNA fingerprint or restriction profile of that organism. Since different strains have differ-ent genomic contdiffer-ents, even strains of the same species that are epidemiologically unrelated to each other will usually show different restriction profiles. Subtyping methods that explore the polymorphisms of restriction profiles are called restriction fragment length polymorphism (RFLP) methods, and the process is called restriction endonuclease analysis (REA). The method was introduced in the late 1970s and the 1980s for subtyping of viruses, parasites, fungi, and bacte-ria (11–14) using high-frequencycutting enzymes, which resulted in DNA fingerprints containing up to 500 restric-tion fragments ranging in size from <1 kb to 30 kb. Although the method had universal applicability, was simple, and required few resources, the resulting fragments were often difficult to resolve and the DNA fingerprints too complex to analyze accurately, especially for organisms with large genomes, e.g., bacteria, fungi, and parasites. For this reason, ways to simplify the RFLP DNA fingerprints while main-taining the discriminatory power of the method were sought.
This wasachieved by reducing the number of restriction fragments in the fingerprints by (i) reducing the number of fragments generated during the restriction reaction or (ii) reducing the number of fragments being visualized. Pulsed-field gel electrophoresis (PFGE) falls within the former cate-gory. There are numerous examples of the latter technique, among which IS6110 fingerprinting will be men ti on ed herein. Finally, optical mapping or whole-genome mapping (WGM), which does not involve electrophoresis but visual-izes high-frequency RFLP fingerprints on a glass surface with the restriction fragments inthe order that they occurin the chromosome, will be described.
Pulsed-Field Gel Electrophoresis
In pulsed-field gel electrophoresis (PFGE), the DNA finger-print is simplified by using rarely cutting restriction enzymes with the goal of reducing the number of restriction fragments to be analyzed (macrorestriction). The resulting fragments usually range between∼20 kb and >1 Mb in size. Because organisms differ in the guanine and cytosine (GC) contents of their DNA, the optimal restriction enzymes for PFGE vary between organisms (15). The optimal restriction enzyme(s) generates between approximately 8 and 25 DNA fragments
that are well separated and evenly distributed throughout the gel from each strain tested.
Large DNA fragments cannot efficiently be purified in a liquid suspension because this will cause random shearing.
To avoid that, the genomic DNA is released and purified from cells that have been embedded in a solid agarose plug.
The plug stabilizes the DNA against breaking or shearing as the cells are lysed chemically. The intact genomic DNA is then digested with an infrequently cutting restriction enzyme. Large restriction fragments cannot be resolved with conventional agarose gel electrophoresis, which works best for separation of fragments smaller than approximately 30 kb. This limitation is overcome by subjecting the plug with the macrorestricted DNA to electrophoresis in an alternat-ing or pulsing electric field using an agarose gel as the separation matrix. Since its introduction in 1984 (16), sev-eral different PFGE platforms havebeen developed (17–
19). In all formats, the electric field alternates in direction in a predefined manner throughout the course of the electro-phoresis. During PFGE, smaller DNA fragments reorient (i.e., make a directional change) in the electrical field faster than larger fragments and therefore move more rapidly through the gel, resulting in separation of the fragments in a size-dependent manner. In some platforms, the interval of time that the electrical current is applied in one direction before it is switched to another direction may be changed.
Usually the switch times are set to be short in the beginning of the electrophoresis and are then increased or ramped up during the course of the run.
PFGE has been performed since the 1980s and still re-mains the gold standard for bacterial molecular subtyping due to its universal applicability, virtual 100% typeability, high discriminatory power, good reproducibility, and stabil-ity. By nature, all electrophoresis-based methods are com-parative; i.e., only isolates investigated in the same experi-ment may be compared. However, this problem may be overcome by rigorous standardization of the procedure, with the choice of restriction enzyme, type and brand of agarose, and electrophoresis running conditions being critical. Since 1996, PulseNet USA, the national molecular subtyping net-work for foodborne disease surveillance, has used PFGE as the preferred method for molecular surveillance, thereby
PFGE has been performed since the 1980s and still re-mains the gold standard for bacterial molecular subtyping due to its universal applicability, virtual 100% typeability, high discriminatory power, good reproducibility, and stabil-ity. By nature, all electrophoresis-based methods are com-parative; i.e., only isolates investigated in the same experi-ment may be compared. However, this problem may be overcome by rigorous standardization of the procedure, with the choice of restriction enzyme, type and brand of agarose, and electrophoresis running conditions being critical. Since 1996, PulseNet USA, the national molecular subtyping net-work for foodborne disease surveillance, has used PFGE as the preferred method for molecular surveillance, thereby