The Effect of Plasmids and Other Biomolecules on the Effectiveness of Antibiofilm Agents - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 156

capacity and bacterial inactivation. Some biocides, such as the QACs, are used
externally on the skin to prevent or limit microbial infection (antiseptics and topical
antimicrobials) and for preoperative skin disinfection and are incorporated into
pharmaceutical products (preservatives) to avoid microbial contamination (Russell
2003 ). The cationic biocide BDMDAC has already demonstrated strong
antibacterial activity, causing damage of the cytoplasmic membrane and the con-
sequent release of essential intracellular components (Ferreira et al. 2011 ). In the
second case-study presented on this chapter, the influence of potential interfering
substances (bovine serum albumin—BSA, alginate, yeast extract and humic acids)
was studied on the antimicrobial activity of other two QACs—benzalkonium
chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB)—against
B. cereus and P. fluorescens , as it was previously shown that the presence of
organic matter can affect the efficiency of biocides (Russell 2003 ). The biomole-
cules used throughout this experiment are recognised as potentially interfering
agents in the European Standard EN-1276 ( 1997 ) and biofilm matrix components
that have an important role in biofilm resistance against antimicrobial agents
(Cloete 2003 ).

1.1 The Influence of Plasmids on Biofilm Formation
and Resistance

A plasmid is an extrachromosomal, circular and double-stranded DNA molecule
that carries its own origin of replication. Under natural conditions, many plasmids
are transmitted to new hosts by conjugation, a procedure by which donor cells can
transfer genes to recipient cells. However, the plasmids used in this work—pET28
and pUC8—are incapable of directing their own conjugation because they lack the
tra gene and therefore they are non-conjugative plasmids (Ehlers 2000 ).
The pET28 vector (Fig. 1 ) is a 5.4 kb plasmid harbouring a kanamycin resistance
gene and a pMB1 origin of replication, and is usually present in about 20-60 copies
per cell (Prather et al. 2003 ). This vector is used for recombinant protein expression
using the transcriptional promoter and termination sequences from phage T7.
Recombinant proteins are expressed as fusions with histidine residues to enable
purification with a nickel affinity column. Expression of the T7 polymerase that will
transcribe the cloned gene is achieved through the inducible lacUV5 promoter that
is present in the host cell chromosome via a lysogenic insertion. To prevent “leaky”
expression from this promoter, a copy of the lacI repressor is also present on the
plasmid to reduce toxicity effects related with the expression of the cloned gene
prior to induction.
The pUC8 vector (Fig. 2 ) is a 2.7 kb plasmid containing the bla TEM-1 ampicillin-
resistance gene (Paterson and Bonomo 2005 ) and a mutated pMB1 origin of
replication, which is responsible for its high copy number (500-700 copies per
cell) (Prather et al. 2003 ). It also contains the β -lactamase promoter to transcribe the