Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome.
BACKGROUND
Burkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. A hallmark of B. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. As Resistance-Nodulation-Division (RND) efflux systems are responsible for much of the intrinsic multidrug resistance in Gram-negative bacteria, this study aims to identify RND genes in the B. cenocepacia genome and start to investigate their involvement into antimicrobial resistance.


RESULTS
Genome analysis and homology searches revealed 14 open reading frames encoding putative drug efflux pumps belonging to RND family in B. cenocepacia J2315 strain. By reverse transcription (RT)-PCR analysis, it was found that orf3, orf9, orf11, and orf13 were expressed at detectable levels, while orf10 appeared to be weakly expressed in B. cenocepacia. Futhermore, orf3 was strongly induced by chloramphenicol. The orf2 conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in Escherichia coli KAM3, a strain lacking the multidrug efflux pump AcrAB. The orf2-overexpressing E. coli also accumulate low concentrations of ethidium bromide, which was restored to wild type level in the presence of CCCP, an energy uncoupler altering the energy of the drug efflux pump.


CONCLUSIONS
The 14 RND pumps gene we have identified in the genome of B. cenocepacia suggest that active efflux could be a major mechanism underlying antimicrobial resistance in this microorganism. We have characterized the ORF2 pump, one of these 14 potential RND efflux systems. Its overexpression in E. coli conferred resistance to several antibiotics and to ethidium bromide but it remains to be determined if this pump play a significant role in the antimicrobial intrinsic resistance of B. cenocepacia. The characterization of antibiotic efflux pumps in B. cenocepacia is an obligatory step prior to the design of specific, potent bacterial inhibitors for the improved control of infectious diseases. Consequently, the topic deserves to be further investigated and future studies will involve systematic investigation on the function and expression of each of the RND efflux pump homologs.
Guglierame
Paola
P
Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, Via Ferrata 1, 27100 Pavia, Italy. paola.g@ipvgen.unipv.it
Pasca
Maria Rosalia
MR
De Rossi
Edda
E
Buroni
Silvia
S
Arrigo
Patrizio
P
Manina
Giulia
G
Riccardi
Giovanna
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042602:Burkholderia cepacia complex / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D003470:Culture Media; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism; D016680:Genome, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003470:Culture Media; D004996:Ethidium