The Use of DNA Methods to Characterize Biofilm Infection - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 14

The human microbiome project (HMP) has forever changed how microorganisms
will be identified (Chain et al. 2009 ). The HMP was established to identify and to
quantitate bacteria living in normal human environments such as the gut, oral
cavity, skin, urogenital, etc. Several challenges for the project were that the
microbes in these host environments are polymicrobial, they are not quantifiable
by cultivation methods, and they generally exist in a biofilm phenotype. In fact, the
vast majority of the species known to inhabit normal host environments are not
routinely culturable (Petrosino et al. 2009 ), which is characteristic of the biofilm
phenotype (Fux et al. 2005 ). These facts led investigators to employ molecular
methods.
Molecular methods are based on the idea of direct examination of the bacterial
DNA existing in the sample to allow for identification of the bacteria that are
present. There has been a very rapid and fluid progression of molecular technolo-
gies that can analyze microbial DNA. However, to get any of these molecular
technologies to give a meaningful analysis, high-quality DNA first must be
obtained. Therefore, one of the most important obstacles to using molecular
methods for identifying and quantitating microorganisms in human infections is
obtaining good microbial DNA from the sample (i.e., the process of DNA extrac-
tion). There are a number of excellent kits and laboratory methods for obtaining
microbial DNA from mixed samples (samples that contain both microbial and
human DNA). However, each method has different extraction efficiencies, and
these efficiencies may vary for the different species within the sample. Yet even
with these challenges, many extraction methods can approach 96 % efficiency
(Fitzpatrick et al. 2010 ).
The process of DNA extraction, especially from samples that contain some of
the host products, also can extract substances that inhibit later analysis of the
microbial DNA. For example, polymerase chain reaction (PCR) is a common
method used to amplify microbial DNA, yet the process can be inhibited by sub-
stances found in the sample. These PCR inhibitors include complex polysaccha-
rides, bile salts, hemoglobin degradation products, polyphenolic compounds, heavy
metals, and, most frequently, large amounts of human DNA (Stauffer et al. 2008 ).
Many of the more common PCR inhibitors can be effectively mitigated, but if the
inhibitors cannot be identified and controlled, resampling may be necessary. Once
good DNA is obtained from the sample, most current molecular instrumentation
can obtain reliable clinical results.
PCR is a widely used method of processing DNA that has a relatively long
history of use in the clinic (Krishna and Cunnion 2012 ; Reddington et al. 2013 ).
PCR utilizes primers that attach to complementary regions of bases in the microbial
DNA and, through a polymerase reaction, create copies of this area. This copying
process doubles the amount of target sequence with every cycle of the PCR. Real-
time PCR has the ability to quantitate, in an absolute sense, how much microbial
DNA is in the original sample. The number of cycles required before the real-time
signal reaches a detection threshold (cycle threshold number or ct number) can be
correlated to an absolute number of microbes present in the original sample. This is
an extremely powerful feature of real-time PCR that can used to quantify “bacterial