The Use of DNA Methods to Characterize Biofilm Infection - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 23

highlight the main inadequacy of molecular methods, which is the lack of antibiotic
sensitivity data similar to that provided by culture methods.
There are several strategies for managing chronic biofilm infections with the
lack of antibiotic sensitivity information. First, if the infection is accessible to
topical treatment, high concentrations of antibiotics far in excess of resistance
factors can overwhelm most mobile genetic element-induced antibiotic resistance.
Second, if systemic antibiotics will be necessary then certain mobile genetic
elements with limited diversity, such as mecA cassettes, van genes, and others
can be identified by real-time PCR. Third, if sensitivity data is still critical, then
molecular diagnosis is still very often the quickest and most cost-effective way to
proceed because many microbes are not initially grown in routine clinical culture.
By first identifying the microbes of interest by molecular methods, custom nutrients
and methods can be used to cultivate microbes for sensitivity work or genomic
study (Sibley et al. 2011 ). With the emerging massive increase in capacity per run,
advances in bioinformatics and computing, along with steady decreases in costs, it
is becoming feasible to evaluate all the genes in a sample which may allow
molecular methods to eventually assess resistance directly in the near future.
Dealing with diversity is made easier by the data provided by DNA-based
diagnostics, but caveats remain. Sequencing provides a relative abundance for
each species identified in the sample; however, it yields no “absolute” quantifica-
tion for how much microbial material is present. Real-time PCR has the ability to
give reproducible estimates of the number of microbes per gram of tissue (such as
10 5 /g) which is termed the “microbial load” or “bacterial load.” Several factors can
fictitiously lower the value for “microbial load,” such as inefficient extraction,
decreased primer efficiency, and small variations throughout the analysis. As a
result, a low “microbial load” should never be discounted as “not a significant
infection.” The diagnosis of infection is a clinical decision; therefore, chronic
infection itself should always dictate treatment. To evaluate the progression or
improvement of an infection it may be necessary to have the lab run the initial
sample with subsequent samples in the same run to mitigate these variations, which
allows for better comparison.
Quantification of microbes in the polymicrobial infections often encountered in
biofilm infection is indispensable. For example, if a sample contains just 1 %
MRSA but the bacterial load is 10 8 /g then there are still 10 6 MRSA even though
it is a minor component of the biofilm. So MRSA coverage would be reasonable.
But 1 % MRSA with a bacterial load of 10 5 /g (10 3 MRSA) requires only observa-
tion which can greatly reduce the use of first-line MRSA antibiotics.
The diversity can be daunting at first, but it is amazing how the many disparate
microbes resolve down to treatment groups that require only one or two treating
agents. For example, a group of microbes in chronic wounds consisting of MRSA,
Streptococcus , Peptoniphilus , Anaerococcus , Bacteroides , Pseudomonas , and
Serratia can effectively be treated with the use of clindamycin and amikacin. By
collapsing the gram positives and anaerobes into one treatment group covered by
clindamycin and then covering the gram negatives with amikacin, only two