The Use of DNA Methods to Characterize Biofilm Infection - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 21

methods do not provide any clear information on which species are producing the
infection and which species are merely contaminants.
New methods are rapidly developing where microRNA (Martens-Uzunova
et al. 2013 ) and messenger RNA (Mutz et al. 2013 ) can be sequenced and identified.
This will provide critical information as to the inner workings of microbial cells
which should provide insight as to strategies being used to cause infection. This
may shed light on which microorganisms within the community are behaving as
pathogens.
Before a bacterial species can be deemed a pathogen, or more importantly before
that species can be dismissed as a contaminant, the clinician must take into account
the synergies which arise within a polymicrobial infection. By including multiple
bacterial and/or fungal species into a single community, the biofilm achieves
numerous advantages such as passive resistance (Elias and Banin 2012 ), metabolic
cooperation (Fischbach and Sonnenburg 2011 ), by-product influence (Elias and
Banin 2012 ), quorum-sensing systems, an enlarged gene pool with more efficient
DNA sharing (Madsen et al. 2012 ), and many other synergies that give the
polymicrobial infection a competitive advantage. It is best to view a biofilm as a
single entity possessing multiple genetic resources to allow it to adapt and thrive
regardless of the stresses it encounters. In general, a more diverse population (i.e.,
greater the gene pool) will make the biofilm more robust in terms of its survivability
(Tuttle et al. 2011 ).
Metabolic cross feeding has been well established between genetically distinct
species. It has been shown that Streptococcus gordonii produces peroxide that can
cause Aggregatibacter actinomycetemcomitans (Aa) to produce a factor H binding
protein which limits the host's ability to kill Aa through a complement mediated
lysis (Ramsey et al. 2011 ). This metabolic cooperation has been identified in
numerous polymicrobial models (Dalton et al. 2011 ; Mikx and van der Hoeven
1975 ; Kuboniwa et al. 2006 ).
Waste products, molecules that bacteria produce that are end products and are of
no benefit to the metabolizing member, are released into the local biofilm environ-
ment. Many of these metabolites such as ammonia, lactic acid, and carbon dioxide
can have significant influence on the surrounding microorganisms (Elias and Banin
2012 ). Studies have demonstrated that Fusobacterium nucleatum and Prevotella
intermedia generate ammonia which raises the pH suitable for Porphyromonas
gingivalis (Takahashi 2003 ) and that F. nucleatum also provides an increased
carbon dioxide environment which increases the pathogenicity of P. gingivalis
(Diaz et al. 2002 ).
Passive resistance is when one of the members in the biofilm possesses a
resistance factor that can protect other members of the biofilm which do not have
the factor. There are numerous biofilm defenses which limit the effectiveness of
antibiotics. For example, a beta-lactamase producing strain of Haemophilus influ-
enza was cocultured with Streptococcus pneumoniae deficient in any resistance
factors. Haemophilus influenza increased the MIC/MBC of S. pneumoniae by
amoxicillin (Weimer et al. 2011 ).