However, the theoretical projection often does not match the
practical reality. One such reason that this might occur is simply the
lack of enough differential characteristics, particularly when trying
to distinguish between very closely related species. A second reason
is the observational “end point” nature of phenotypic testing and
biochemical assays. Biochemical assays typically rely on the metabolism of specific chemical substrates, often resulting in chromogenic changes that are easily observable. Thus, the observed
phenotype is actually a surrogate used to infer the presence or
absence of a particular pathway or other feature rather than direct
evidence of the feature itself. Such tests therefore tend to provide
“consistent with” results rather than reliable species identification,
especially between closely related species, and are thus more useful
in ruling out incorrect organisms or contamination. For example,
observation of a culture’s phenotypic characteristics may not be
able to reliably identify whether the culture consists of M. gordonae
or M. paragordonae, but it would help to differentiate between
M. gordonae and Staphylococcus aureus.
However, relying on such phenotypic end points can be misleading, particularly in light of the complex nature of biochemical
pathways. A negative result for such an assay may be an indication
that the entire biochemical pathway is absent. On the other hand, a
complex, multifactor pathway that results in an observable phenotype (whether a biochemical reaction or a more global phenotype)
could be easily misinterpreted by an inactivation of an upstream
part of the pathway, such as an earlier step in an enzymatic cascade
or a critical regulatory protein. A single mutation could manifest
with the same visible results, implying the absence of the pathway
when in fact it is only functionally deactivated. One extreme example of such misleading results comes not from Mycobacterium, but
rather from one of the main organisms of concern for biodefense,
Bacillus anthracis, the etiological agent of the disease anthrax.
B. anthracis belongs to a group of very closely related species,
the Bacillus cereus group. At present, the B. cereus group contains
16 additional species, including B. cereus and B. thuringiensis.
These three species have been known for over a century, and the
proven pathogenic potential of B. anthracis dates back to Robert
Koch and his postulates. Thus, differential identification of
B. anthracis versus its much less pathogenic relatives has always
been important. Two of the primary differential characteristics
between these species are that B. cereus and B. thuringiensis typically
exhibit both motility and β-hemolysis, whereas B. anthracis is both
nonmotile and nonhemolytic. Differences in the expression of
major characteristics such as motility and hemolysis would seem
to imply significant differences between the species with respect to
the presence or absence of all the structural genes and proteins
involved. However, this is in fact an erroneous conclusion.
420
Marco A. Riojas et al.
practical reality. One such reason that this might occur is simply the
lack of enough differential characteristics, particularly when trying
to distinguish between very closely related species. A second reason
is the observational “end point” nature of phenotypic testing and
biochemical assays. Biochemical assays typically rely on the metabolism of specific chemical substrates, often resulting in chromogenic changes that are easily observable. Thus, the observed
phenotype is actually a surrogate used to infer the presence or
absence of a particular pathway or other feature rather than direct
evidence of the feature itself. Such tests therefore tend to provide
“consistent with” results rather than reliable species identification,
especially between closely related species, and are thus more useful
in ruling out incorrect organisms or contamination. For example,
observation of a culture’s phenotypic characteristics may not be
able to reliably identify whether the culture consists of M. gordonae
or M. paragordonae, but it would help to differentiate between
M. gordonae and Staphylococcus aureus.
However, relying on such phenotypic end points can be misleading, particularly in light of the complex nature of biochemical
pathways. A negative result for such an assay may be an indication
that the entire biochemical pathway is absent. On the other hand, a
complex, multifactor pathway that results in an observable phenotype (whether a biochemical reaction or a more global phenotype)
could be easily misinterpreted by an inactivation of an upstream
part of the pathway, such as an earlier step in an enzymatic cascade
or a critical regulatory protein. A single mutation could manifest
with the same visible results, implying the absence of the pathway
when in fact it is only functionally deactivated. One extreme example of such misleading results comes not from Mycobacterium, but
rather from one of the main organisms of concern for biodefense,
Bacillus anthracis, the etiological agent of the disease anthrax.
B. anthracis belongs to a group of very closely related species,
the Bacillus cereus group. At present, the B. cereus group contains
16 additional species, including B. cereus and B. thuringiensis.
These three species have been known for over a century, and the
proven pathogenic potential of B. anthracis dates back to Robert
Koch and his postulates. Thus, differential identification of
B. anthracis versus its much less pathogenic relatives has always
been important. Two of the primary differential characteristics
between these species are that B. cereus and B. thuringiensis typically
exhibit both motility and β-hemolysis, whereas B. anthracis is both
nonmotile and nonhemolytic. Differences in the expression of
major characteristics such as motility and hemolysis would seem
to imply significant differences between the species with respect to
the presence or absence of all the structural genes and proteins
involved. However, this is in fact an erroneous conclusion.
420
Marco A. Riojas et al.