Horizontal Gene Transfer in Planktonic and Biofilm Modes - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 82

5 Monitoring of Horizontal Gene Transfer

A variety of monitoring techniques, either PCR-based or fluorescence microscopy
based, have been designed in the last two decades to quantitatively follow the
horizontal spread of metabolic traits or virulence factors under different conditions
and in distinct environments.
Jussila et al. ( 2007 ) designed a molecular profiling method for HGT of
aromatics-degrading plasmids. The method was successfully applied during
rhizomediation and conjugation in vivo. It is based on the PCR detection of the
TOL plasmid-specific xylE gene.
With increasing areas of transgenic crops during the last decades and the
necessity of biological control agents as alternative to chemical pesticides, the
establishment of techniques for environmental risk assessment has become neces-
sary for the evaluation of biological control microorganisms released into the
environment (Kim et al. 2012 ). Kim and coworkers investigated the possible
HGT between released recombinant agricultural microorganisms and indigenous
soil microorganisms. A recombinant B. subtilis strain and a recombinant plant
growth-promoting P. fluorescens strain were used as model microorganisms (Kim
et al. 2012 ). Soils of cucumber or tomato plants cultivated in the greenhouse were
inoculated with the recombinant bacteria. For a 6-month period the soils were
investigated for the presence of the recombinant bacteria by PCR, real-time PCR,
Southern hybridization, and terminal restriction fragment length polymorphism
fingerprinting. No positive signals for the recombinant B. subtilis and
P. fluorescens strains were detected in the soils suggesting that horizontal gene
flow from B. subtilis or P. fluorescens to soil bacteria in the greenhouse did not
occur during the 6-month period (Kim et al. 2012 ).
In complex microbial communities with a high background of antibiotic resis-
tance genes detection of HGT of resistance genes is challenging. One option to
overcome the problem is labeling the antibiotic resistance gene. This approach was
carried out by Haug et al. ( 2010 ). The conjugative multiresistance plasmid pRE25,
originating from E. faecalis , was tagged with a 34-bp random sequence marker
spliced by tet (M). The plasmid construct, denominated pRE25*, was introduced
into E. faecalis CG110/ gfp , a strain containing a gfp gene as chromosomal marker.
The plasmid pRE25* was shown to be fully functional compared with its parental
pRE25 and could be transferred to Listeria monocytogenes and Listeria innocua at
frequencies of 6 10 8 transconjugants per donor. Different markers

on the chromosome and the plasmid enabled independent quantification of donor
and plasmid via specific quantitative PCR, even if antibiotic resistance genes
occurred at high numbers in the background ecosystem. Haug and coworkers
concluded that E. faecalis CG110/ gfp /pRE25* is a potent tool for the study of
horizontal antibiotic resistance transfer in complex environments such as biofilms,
food matrices, or colonic models (Haug et al. 2010 ).
Over the last decade, advances in reporter gene technology have provided new
insights into the extent and spatial frequency of HGT in vitro and in natural

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