Tuesday, October 7, 2008

Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains

Mette Burmølle, Martin Iain Bahl, Lars Bogø Jensen, Søren J. Sørensen and Lars Hestbjerg Hansen
Microbiology (2008), 154, 187–195

In this paper researchers from University of Copenhagen and The National Food Institute in Denmark bring our attention to a conjugative plasmid pOLA52 which features and genetic content occurred to be disturbing as they regard human and animal health.
pOLA52 plasmid was first isolated from swine manure and was shown to encode multidrug efflux pump which provides resistance to many antimicrobial agents such as olaquindox (which was or still is commonly used as a growth factor in pig farming), chloramphenicol, ethidium bromide, other antibiotics, detergents and disinfectants. It also carries
bla gene conferring resisitance to β-lactam antibiotics, such as ampicilin. Apart from multidrug resistance, pOLA52 plasmid carries 5.6 kb operon consisting of five genes, homologous to mrkABCDF genes contained in the mrk operon of Klebsiella pneumoniae, encoding type 3 fimbriae, which are known to be involved in attachment of bacteria to different kinds of biotic and abiotic surfaces, and thus increased biofilm formation.
Authors of the paper have previously observed that E. coli CSH26 strain harbouring pOLA52 plasmid formed higher amounts of biofilm and thus wanted to investigate if the operon conferring type 3 fimbriae could be responsible for observed feature.
In this study authors randomly introduced entranceposon pENTRANCEPOSON (KanR) into pOLA52 plasmid, electroporated it into E. coli Genehogs, selected tranformants on Kan and checked their ability to form biofilms on urinary catheters. Some clones occured to be biofilm negative and sequencing revealed that inserts were located inside type 3 fimbriae operon. Not surprisingly, biofilm positive clones had the inserts outside the operon.
Researchers have checked expression of mrk genes with RT-PCR and showed with immunoblotting that biofilm positive clones expressed type 3 fimbriae, whereas biofilm negative did not.
Later, they have conducted conjugation of pOLA52 plasmid into potentially pathogenic Enterobacteriaceae strains (such as Klebsiella pneumoniae, Salmonella typhimurium, Kluyvera sp., Enterobacter aerogenes) and tested the ability of transconjugants to form biofilms. It occured that transconjugants harbouring plasmid with transposon inside mrk operon showed significantly lower rate of plasmid transfer comparing to strains carrying wild type plasmid. Futhermore, transconjugants harbouring wild type pOLA52 plasmid formed biofilms, whereas strains with operon mrk mutated plasmid showed much lower biofilm formation.
This study proves how important and potentially dangerous pOLA52 plasmid is, as it can be transferred via conjugation to other bacteria, including pathogenic strains, providing them with new antibiotic resistances and type 3 fimbriae increasing their ability of spread plasmids and to form biofilms. Those newly accuaired features can lead to higher antibiotic persistence and increased spreading of pathogens on biological surfaces, such as tissues, as well as abiotic ones, for example catheters or artificial heart valves.
As pOLA52 plasmid is the first of probably many more plasmids with similar genetic content, there is a risk that one day they could be used as another dangerous weapon in hands of pathogenic bacteria. Let's hope we will be well prepared if this day would come...

Sylwia Deneka
Visiting Scholar
University of Idaho

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