BACKGROUND
bacteria have evolved a variety of strategies to resist antibiotics such as enzymatic inactivation, target alteration, efflux, and permeability changes.
antibiotic resistance can be intrinsic or acquired.
intrinsic resistance is intimately associated to the biology of the organism and usually involves the ability to resist a large number of different classes of antibiotics, while acquired resistance occurs when a bacterium that is sensitive to antibiotics develops resistance by mutation or by acquisition of new dna.
bacterial intrinsic drug resistance was thought to be a passive mechanism, based on the absence of the drug target or on the lack of permeability of the bacteria to a given drug.
however, it is becoming increasingly evident that the intrinsic resistance of many bacteria to antibiotics depends on the constitutive or inducible expression of active efflux systems  <cit> .
a typical example is pseudomonas aeruginosa, which was long thought to be poorly susceptible to a large range of antibiotics of different classes due to the low level of permeability of its outer membrane to drugs.
however, disruption of the gene encoding the mexb pump dramatically increases the susceptibility of p. aeruginosa to beta-lactams, tetracyclines, fluoroquinolones, and chloramphenicol  <cit> , indicating that the resistance was mediated by efflux.
in the prokaryotic kingdom there are five major families of efflux transporters: mf , mate , rnd , smr , and abc .
all these systems utilize the proton motive force as an energy source, apart from the abc family, which utilizes atp hydrolysis to drive the export of substrates  <cit> .
rnd transporters exist in all kingdoms of living organisms, but seem to be involved in drug resistance especially in gram-negative bacteria  <cit> .
the rnd transporters function as a protein complex spanning from the cytoplasmic membrane to the outer membrane.
the recent resolution of the three-dimensional structures of tolc and acrb from escherichia coli and mexa and oprm from p. aeruginosa gave rise to a better understanding of the efflux mechanism in gram-negative bacteria  <cit> .
the efflux pump systems of the rnd family are organized as tripartite efflux pumps.
the pump in e. coli and other gram-negative bacteria has three components: a transporter  protein in the inner membrane , a periplasmic accessory protein , and an outer membrane protein  channel   <cit> .
acrb captures its substrates within either the phospholipid bilayer of the inner membrane of the bacterial cell envelope or the cytoplasm  <cit>  and transports them into the external medium via tolc  <cit> .
the cooperation between acrb and tolc is mediated by the periplasmic protein acra.
the genetic organizations of the genes encoding these tripartite efflux systems are also similar among different species.
typically, the genes are organized as an operon: the regulator gene is located adjacent to the gene encoding the periplasmic accessory protein, which is located adjacent to the gene encoding the efflux pump protein, which is located next to that for the omp.
the membrane fusion protein and the pump protein are usually cotranscribed.
for some systems and/or species, the omp is not collocated with the other genes, e.g., e. coli acrab and tolc  <cit>  and p. aeruginosa mexxy and oprm  <cit> .
rnd efflux systems are also found in bacteria that exhibit cell wall properties to p. aeruginosa  <cit> .
the burkholderia cepacia complex  is noted for their ability to cause opportunistic infections in humans, particularly in patients with cystic fibrosis   <cit> .
bcc infections of cf lung can result in asymptomatic carriage, chronic infection or "cepacia syndrome", which is characterized by a rapid decline in lung function that can include invasive disease  <cit> .
the bcc contains at least nine closely related species  <cit> , of which burkholderia cenocepacia  is the most prevalent and has been most commonly associated with epidemic spread and increased clinical virulence  <cit> .
a hallmark of bcc infections is the inability to eradicate the organism because of high-level multiple intrinsic antibiotic resistance  <cit> .
the multiple antibiotic resistance of bcc has been attributed to reduced outer membrane permeability  <cit> , production of modifying enzymes such as β-lactamases  <cit> , and alteration of antibiotic targets  <cit> .
much less information is available on drug efflux systems, and only a few multi-drug efflux pumps of the mfs and mate families have been described in bcc species  <cit> .
screening for chloramphenicol resistance of a cosmid library constructed from a cf clinical isolate allowed the identification of the first rnd efflux operon in b. cenocepacia  <cit> .
within this operon, genes encoding a periplasmic lipase-like protein , a periplasmic linker , a cytoplasmic membrane component , and an outer membrane protein  were identified  <cit> .
ceoa, ceob, and opcm have homologs in p. aeruginosa and other gram-negative bacteria.
a significant difference from other prokaryotic multidrug efflux systems, including all rnd pumps described to date, is the inclusion of a gene encoding a lipase-like protein in the b. cenocepacia efflux operon  <cit> .
aim of this study was to identify rnd genes in the b. cenocepacia genome and start to investigate their involvement into antimicrobial resistance.
fourteen homologues of rnd efflux pump genes were identified by in silico homology searches.
by rt-pcr it was found that some of them were expressed at detectable levels in not inducing condition, i.e.
growth in lb medium, while others were detected in inducing conditions, i.e.
growth in lb medium in the presence of an antibiotic.
further, orf <dig> gene conferred a multidrug resistant phenotype, when over-expressed in escherichia coli kam <dig> strain.
moreover, an energy-dependent ethidium bromide efflux was observed in e. coli kam <dig> cells harbouring orf <dig> gene.
RESULTS
analysis of rnd drug transporters
the b. cenocepacia j <dig> strain was isolated in  <dig> in edimburgh  from the sputum of a cystic fibrosis patient .
the genome sequence of this strain is available within the sanger website  <cit> .
to identify the b. cenocepacia drug efflux proteins belonging to rnd, we first scanned the b. cenocepacia predicted proteins for the presence of the transporter family signature characterized by a number of strongly conserved amino acid residues.
the sequences of the motifs are: motif a , motif b , motif c , and motif d   <cit> , where x indicate any amino acid, capital letters show amino acids most frequently observed in a single position in more than 70% of the transport proteins, and lowercase letters display amino acid occurring in more than 40% of rnd.
by this method, fourteen possible rnd homologues have been identified .
in table  <dig>  the position of the residues encompassing the four typical motifs is indicated; it is noteworthy that the relative distances among the specific motifs appear well conserved in all the identified proteins.
members of the rnd family have  <dig> tms, with two large loops between tms <dig> and tms <dig>  and tms <dig> and tms <dig>  respectively  <cit> .
this organization appears well conserved in all the b. cenocepacia hypothetical rnd transporters, except for orfs  <dig> and  <dig>  which present  <dig> and  <dig> tms, respectively .
in order to assess a putative function of the identified transporters, an integrative analysis of protein motif databases was carried out, by using the interproscan program.
the proteins orf1–orf <dig> show the acriflavinrp, a 9-element fingerprint that provides a signature for members of the acriflavin resistance protein family as acrb of e. coli  <cit> , while the proteins orf11–orf <dig> show a 6– <dig> element fingerprint .
all hypothetical rnds contain two transmembrane functional domains located within tms <dig> and tms <dig> that coincide with the transporter family signature motifs c and d, respectively .
the crystal structure of multidrug efflux transporter acrb of e. coli  <cit>  reveals that this protein is characterized by the four subdomains pn <dig>  pn <dig>  pc <dig>  and pc <dig>  the structural motifs pn <dig> and pn <dig> comprise the polypeptide segment between tms <dig> and tms <dig>  while pc <dig> and pc <dig> comprise the segment between tms <dig> and tms <dig>  all the b. cenocepacia rnd transporters  show the four subdomains; the region with subdomains pn <dig> and pn <dig> coincides always with the transporter family signature motif a .
the tolc docking domain is composed of two subdomains, dn and dc, and these subdomains are found in all hypothetical rnd proteins except the orf <dig> that present only the dc subdomain .
in order to obtain a more exhaustive analysis about the structural and conformational properties of the orfs, the amino acid sequences were analysed by phyre program and the data were reported in table  <dig>  the orf1– <dig> were compared to the rnd proteins of e. coli and p. aeruginosa by using the blastp program.
particularly, orf <dig>  orf <dig>  and orf <dig> show strong identity with e. coli acrb  and with p. aeruginosa mexb .
orf <dig> and orf <dig> show identity with p. aeruginosa mexd , while orf <dig> and orf <dig> show identity with p. aeruginosa mexf .
orf <dig> and orf <dig> are homologues of metal efflux transporters belonging to the rnd family of e. coli and p. aeruginosa .
particularly, orf <dig> is highly homologous to czra protein of p. aeruginosa, involved in cadmium and zinc resistance  <cit> .
analysis of the upstream and downstream regions of the rnd coding genes confirmed the association with both an acra/mexa homolog and a tolc/oprm homolog in a variety of arrangements .
indeed, orf <dig>  orf <dig>  orf <dig>  orf <dig>  orf <dig>  orf <dig>  orf <dig> , orf <dig>  orf <dig>  orf <dig>  and orf <dig> seem to be part of an operon that also included a mexa and an oprm homologs, thus containing all three of the components of the tripartite pump complex.
differently, orf <dig> appears to have only the membrane component, while orf <dig> and orf <dig> are apparently co-transcribed and in operon with a periplasmic membrane fusion protein .
in p. aeruginosa, the genes encoding the rnd transporter  and periplasmic membrane fusion protein  are always present, while the gene encoding the om channel  is not always present  <cit> .
in e. coli, the genes for transporter and membrane fusion proteins  occur together as an operon and the gene for the tolc occurs elsewhere on the genome  <cit> .
in b. cenopacia the genetic organization of the rnd orfs seems to resemble in part that of p. aeruginosa and in part that of e. coli.
as acrd pump of e. coli, orf <dig> seem to be transcribed alone in the genome of b. cenocepacia  <cit> , while orf <dig> and orf <dig> are organized as the genes pa2526-2527- <dig> of p. aeruginosa that contains an additional rnd transporter  <cit> .
many pump component-encoding operons contain a physically linked regulatory gene coding for either a repressor or an activator  <cit> .
for example, expression of the mexab-oprm operon is regulated by the product of the upstream and divergently transcribed mexr gene.
not surprisingly, clinical isolates that overexpress the mexab-oprm system often carry mutations in the mexr gene  <cit> .
in the case of orf <dig>  orf <dig>  orf <dig>  orf6– <dig>  orf <dig>  orf <dig>  orf <dig>  and orf <dig> a hypothetical regulatory gene has been identified, divergently transcribed for all genes, except for the hypothetical operon containing the orf <dig>  orf <dig>  and orf6– <dig>  respectively .
the websidd program was used to analyse sequences encompassing the coding and intergenic regions of the hypothetical rnd operons for the presence of sidd  sites.
these sites have a specific and statistically highly significant pattern of association with transcriptional regions, specifically with promoters because strand separation is a necessary step in the initiation of transcription  <cit> .
all the orfs present sidd sites localized upstream of the first gene of the entire operon, except for the orf <dig>
rt-pcr analysis of the b. cenocepacia genes coding for the rnd transporters
we found  <dig> genes codifying hypothetical rnd drug transporters into b. cenocepacia genome.
by rt-pcr analysis, we studied the expression of all these genes at the exponential phase of cells grown in lb medium.
a clear expression of orf <dig>  orf <dig>  orf <dig>  and orf <dig> was observed , while orf <dig> appeared to be weakly expressed .
the expression of the other orfs  was not detectable, at least in our conditions .
it has been demonstrated that orf <dig> corresponds to the previously described ceob gene belonging to ceo operon, which has been shown to be strongly induced by salicylate and chloramphenicol  <cit> .
we decide to evaluated the expression of the  <dig> genes in b. cenocepacia cells grown in the presence of chloramphenicol .
in b. cenocepacia j <dig> strain, the basal level of expression of the orf <dig>  was strongly increased by chloramphenicol .
no induction could be detected in the case of orf <dig>  orf <dig>  orf <dig>  orf <dig>  orf6– <dig>  orf <dig>  orf <dig>  and orf <dig>  and no increased expression could be observed in the case of orf <dig>  orf <dig>  orf <dig>  and orf <dig> .
however, the b. cenocepacia rrna 16s expression was not affected by different growth conditions , thus demonstrating that the differences detected in the amount of orf <dig> mrna, under different growth conditions, are genuine.
as the ceo operon has been discovered as responsible for the mdr phenotype of a clinical strain  <cit> , we can hypothesize that this rnd efflux system is expressed as a result of a mutation.
in our conditions, no transcriptional induction of orf <dig> could be detected also in the presence of salicylate .
orf <dig> showed 71% identity with p. aeruginosa mexy, an inducible efflux system that contributes to the natural resistance of p. aeruginosa to antibiotics  <cit> .
experiments involving real-time pcr in p. aeruginosa pao <dig> showed induction of gene mexy by chloramphenicol, tetracycline, macrolides, and aminoglycosides  <cit> .
to date, no information is available on the regulation of mexxy expression, except that mutations occurring in the divergently transcribed repressor gene mexz are frequently isolated from the sputa of cystic fibrosis patients  <cit> .
in p. aeruginosa, with the exception of mexab-oprm, the expression of most of the rnd efflux systems is tightly regulated and overexpression of these rnd pumps is usually caused by mutations in genes encoding regulatory proteins  <cit> .
notably, overexpression of multidrug resistance pumps in clinical isolates of p. aeruginosa, resulting in increased bacterial resistance, is usually due to mutations in these regulatory genes  <cit> .
it would be interesting to further investigate if the expression of orf <dig>  orf <dig>  orf <dig>  and orf <dig> is due to a constitutive expression or to a transcriptional deregulation in the b. cenocepacia clinical isolate analysed in this study, which showed a multidrug resistance phenotype as it is resistant to ampicillin and aztreonam , ceftazidime , meropenem , piperacillin  amikacin , norfloxacin , rifampicin , streptomycin , tetracycline , chloramphenicol , and gentamicin .
cloning and phenotypic analysis of b. cenocepacia orf <dig> into e. coli kam3
one approach to test the role of the identified genes in drug efflux is to clone them into suitable hosts by selecting on different compounds.
in spite of several attempts, we met many problems in cloning experiments since a lot of rearranged clones were found in the host strains .
the difficulties that we met in cloning experiments could rely both on plasmid instability and/or toxic effect of the gene product .
in fact, the failure could be due to the toxicity of the gene product for the host bacterial cells and still b. multivorans as a host causes many troubles .
to overcome this problem we used the e. coli kam <dig> strain as host, which lacks the acrab genes and is sensitive to many drugs that are known as substrates of the acrab system  <cit> .
we decided to characterize one of the  <dig> hypothetical drug efflux pumps of b. cenocepacia by cloning the orf2-coding region from b. cenocepacia genomic dna into pbad <dig> expression vector.
the pbad <dig> vector and the recombinant plasmid  were transformed into e. coli kam <dig>  we chose orf <dig> because of its high homology to mexb, responsible for antibiotic resistance in many pseudomonas clinical isolates  <cit> .
particularly, studies with mutants that overproduce or lack mexab-oprm demonstrated that this efflux system extrudes quinolones, macrolides, tetracycline, chloramphenicol, novobiocin, and most β-lactams but not imipenem  <cit> .
the mics of e. coli kam <dig> strains harbouring pbad <dig> and pbad/orf <dig>  respectively, were determined by streaking the cultures onto lb containing different concentrations of the following compounds: streptomycin, ethidium bromide, nalidixic acid, several quinolones , chloramphenicol, erythromycin, tetraphenylphosphonium, and tetracycline.
plates were incubated at 37°c for  <dig> days and the growth was visually evaluated.
compared to the control strain, the overexpression of orf <dig> into e. coli kam <dig> cells conferred resistance to streptomycin , tetraphenylphosphonium , ethidium bromide , nalidixic acid , ciprofloxacin , ofloxacin , and norfloxacin  .
no difference in drug susceptibility was found with sparfloxacin, chloramphenicol, erythromycin, and tetracycline .
these data suggest that orf <dig> is expressed and functional in e. coli kam <dig>  interestingly, in p. aeruginosa, it has been found that fluoroquinolones like nalidixic acid and norfloxacin can commonly select mutants that constitutively overproduce the mexab-oprm efflux pump system  <cit> .
from these data it appeared that streptomycin and norfloxacin could be substrates of orf <dig>  we decided to perform rt-pcr of b. cenocepacia grown in the presence of sub-inhibitory concentration of streptomycin  and norfloxacin .
in our condition, orf <dig> transcript could not be detected by rt-pcr .
the expression of orf <dig> was also evaluated by southern blotting of the rt-pcr agarose gel and subsequent hybridization with a labelled 511-bp fragment internal to orf <dig> coding region.
no hybridisation signals were observed , indicating that orf <dig> is not expressed following treatment of b. cenocepacia cells with streptomycin or norfloxacin.
three different hypotheses could be given to explain the fact that orf <dig> is not expressed.
first, the experimental approaches utilized in this work  are not enough sensitive to detect very small amount of orf <dig> mrna.
real-time pcr is an attractive method for estimating gene expression of efflux pumps in bacteria because of its great sensitivity and wide effective range.
recently, the real-time pcr appeared to be a useful alternative method for assessing the expression of efflux pumps mexb and mexy in p. aeruginosa  <cit> .
second, not all orf <dig> substrates in e. coli necessarily must be inducers of the expression of this pump in b. cenocepacia.
a relevant example of such a behaviour is given by the mexcd-oprj efflux pump of p. aeruginosa, which extrudes antibiotics that do not induce its expression  <cit> .
instead, expression of this efflux system was induced by clinical important disinfectants such as benzalkonium chloride and chlorhexidine gluconate  <cit> .
finally, the orf <dig> expression may occur in a phase of cellular growth different from the exponential one we tested.
in conclusion, we could not determined neither the real inducing agent nor the culture conditions in which induction of orf <dig> expression is triggered; therefore, it could be interesting to test other organic compounds for the ability to induce expression of this efflux pump.
ethidium bromide efflux activity
ethidium bromide efflux experiment was performed to determine whether e. coli kam <dig> cells, carrying the recombinant plasmid pbad/orf <dig>  were more resistant to this dye due to an active efflux mechanism.
as shown in figure  <dig>  cells harboring the cloning vector pbad <dig> take up ethidium bromide rapidly and achieve a steady-state level of accumulation within about  <dig> min of incubation.
as observed in several experiments, accumulation of ethidium bromide was approximately 20% lower when cells contained the pbad/orf <dig> recombinant plasmid.
a reduced level of accumulation of the drug may be caused either by a decreased level of drug permeation or by active drug extrusion through the cytoplasmic membrane.
to study the effect of membrane deenergization on the uptake of tetracycline, the protonophore carbonyl cyanide m-chlophenylhydrazone  was added to cells containing ethidium bromide.
upon the addition of cccp, the level of ethidium bromide accumulation increased in the case of the pbad/orf2-harboring strain and reached a level almost equal to that observed in the case of the strain containing only the cloning vector pbad <dig> .
on the contrary, under our conditions, cccp had no significant effect on the level of ethidium bromide accumulation in the strain carrying the cloning vector .
these data indicate that orf <dig> pumped out ethidium bromide in an energy-dependent process, presumably by using proton motive force.
we could conclude that orf <dig>  belonging to rnd transporters, is an efflux pump of b. cenocepacia j <dig>
CONCLUSIONS
pulmonary infection with bcc in patients with cf is often associated with a more rapid decline in lung function.
because of the resistance of bcc to many antibiotics, treatment options are often limited.
the emergence of active efflux as a major causative factor in antibiotic resistance has been one of the most significant trend in anti-infective chemotherapy over the last decade and strategies to identify efflux pump inhibitors are in progress  <cit> .
for these reasons, the identification and the characterization of such efflux systems in b. cenocepacia are a very important topic in the field of antibiotic resistance.
taken together the results described in this paper fit with the prediction that b. cenocepacia contains homologs of the p. aeruginosa mex-opr efflux pump systems.
moreover, orf <dig>  orf <dig>  orf <dig>  and orf <dig> are expressed in b. cenocepacia j <dig>  even if, at this stage, it is not possible to state if this expression is constitutive or due to mutations in regulatory genes.
finally, the results described in this paper suggested that at least one of the rnd-type efflux pumps identified, extrude out of the cell some antibiotics as well as other compounds .
to demonstrate that this pump is really involved in the multidrug resistant phenotype of the b. cenocepacia j <dig>  further studies are necessary.
the generation of mutants with pump overproduction and/or genetic deletion of genes encoding pumps in a controlled laboratory environment provides invaluable information on the potential impact of multidrug efflux pumps on the intrinsic resistance of b. cenocepacia j <dig>  because of the difficulties in cloning and expression experiments, knock-out approach represents the only alternative method to physiologically characterize these transporters and to confirm if the encoded pumps play a significant role in the antimicrobial resistance of this microorganism.
a greater understanding of the genetic determinants that play a role in antibiotic resistance will lead to the development of new strategies for the treatment of b. cenocepacia infections in patients with cystic fibrosis.

 bacteria have evolved a variety of strategies to resist antibiotics such as enzymatic inactivation, target alteration, efflux, and permeability changes. antibiotic resistance can be intrinsic or acquired. intrinsic resistance is intimately associated to the biology of the organism and usually involves the ability to resist a large number of different classes of antibiotics, while acquired resistance occurs when a bacterium that is sensitive to antibiotics develops resistance by mutation or by acquisition of new dna. bacterial intrinsic drug resistance was thought to be a passive mechanism, based on the absence of the drug target or on the lack of permeability of the bacteria to a given drug. however, it is becoming increasingly evident that the intrinsic resistance of many bacteria to antibiotics depends on the constitutive or inducible expression of active efflux systems. a typical example is pseudomonas aeruginosa, which was long thought to be poorly susceptible to a large range of antibiotics of different classes due to the low level of permeability of its outer membrane to drugs. however, disruption of the gene encoding the mexb pump dramatically increases the susceptibility of p. aeruginosa to beta-lactams, tetracyclines, fluoroquinolones, and chloramphenicol, indicating that the resistance was mediated by efflux. in the prokaryotic kingdom there are five major families of efflux transporters: mf, mate, rnd, smr, and abc. all these systems utilize the proton motive force as an energy source, apart from the abc family, which utilizes atp hydrolysis to drive the export of substrates. rnd transporters exist in all kingdoms of living organisms, but seem to be involved in drug resistance especially in gram-negative bacteria. the rnd transporters function as a protein complex spanning from the cytoplasmic membrane to the outer membrane. the recent resolution of the three-dimensional structures of tolc and acrb from escherichia coli and mexa and oprm from p. aeruginosa gave rise to a better understanding of the efflux mechanism in gram-negative bacteria. the efflux pump systems of the rnd family are organized as tripartite efflux pumps. the pump in e. coli and other gram-negative bacteria has three components: a transporter protein in the inner membrane, a periplasmic accessory protein, and an outer membrane protein channel. acrb captures its substrates within either the phospholipid bilayer of the inner membrane of the bacterial cell envelope or the cytoplasm and transports them into the external medium via tolc. the cooperation between acrb and tolc is mediated by the periplasmic protein acra. the genetic organizations of the genes encoding these tripartite efflux systems are also similar among different species. typically, the genes are organized as an operon: the regulator gene is located adjacent to the gene encoding the periplasmic accessory protein, which is located adjacent to the gene encoding the efflux pump protein, which is located next to that for the omp. the membrane fusion protein and the pump protein are usually cotranscribed. for some systems and/or species, the omp is not collocated with the other genes, e.g., e. coli acrab and tolc and p. aeruginosa mexxy and oprm. rnd efflux systems are also found in bacteria that exhibit cell wall properties to p. aeruginosa. the burkholderia cepacia complex is noted for their ability to cause opportunistic infections in humans, particularly in patients with cystic fibrosis. bcc infections of cf lung can result in asymptomatic carriage, chronic infection or "cepacia syndrome", which is characterized by a rapid decline in lung function that can include invasive disease. the bcc contains at least nine closely related species, of which burkholderia cenocepacia is the most prevalent and has been most commonly associated with epidemic spread and increased clinical virulence. a hallmark of bcc infections is the inability to eradicate the organism because of high-level multiple intrinsic antibiotic resistance. the multiple antibiotic resistance of bcc has been attributed to reduced outer membrane permeability, production of modifying enzymes such as β-lactamases, and alteration of antibiotic targets. much less information is available on drug efflux systems, and only a few multi-drug efflux pumps of the mfs and mate families have been described in bcc species. screening for chloramphenicol resistance of a cosmid library constructed from a cf clinical isolate allowed the identification of the first rnd efflux operon in b. cenocepacia. within this operon, genes encoding a periplasmic lipase-like protein, a periplasmic linker, a cytoplasmic membrane component, and an outer membrane protein were identified. ceoa, ceob, and opcm have homologs in p. aeruginosa and other gram-negative bacteria. a significant difference from other prokaryotic multidrug efflux systems, including all rnd pumps described to date, is the inclusion of a gene encoding a lipase-like protein in the b. cenocepacia efflux operon. aim of this study was to identify rnd genes in the b. cenocepacia genome and start to investigate their involvement into antimicrobial resistance. fourteen homologues of rnd efflux pump genes were identified by in silico homology searches. by rt-pcr it was found that some of them were expressed at detectable levels in not inducing condition, i.e. growth in lb medium, while others were detected in inducing conditions, i.e. growth in lb medium in the presence of an antibiotic. further, orf gene conferred a multidrug resistant phenotype, when over-expressed in escherichia coli kam strain. moreover, an energy-dependent ethidium bromide efflux was observed in e. coli kam cells harbouring orf gene.  analysis of rnd drug transporters the b. cenocepacia j strain was isolated in in edimburgh from the sputum of a cystic fibrosis patient. the genome sequence of this strain is available within the sanger website. to identify the b. cenocepacia drug efflux proteins belonging to rnd, we first scanned the b. cenocepacia predicted proteins for the presence of the transporter family signature characterized by a number of strongly conserved amino acid residues. the sequences of the motifs are: motif a, motif b, motif c, and motif d, where x indicate any amino acid, capital letters show amino acids most frequently observed in a single position in more than 70% of the transport proteins, and lowercase letters display amino acid occurring in more than 40% of rnd. by this method, fourteen possible rnd homologues have been identified. in table the position of the residues encompassing the four typical motifs is indicated; it is noteworthy that the relative distances among the specific motifs appear well conserved in all the identified proteins. members of the rnd family have tms, with two large loops between tms and tms and tms and tms respectively. this organization appears well conserved in all the b. cenocepacia hypothetical rnd transporters, except for orfs and which present and tms, respectively. in order to assess a putative function of the identified transporters, an integrative analysis of protein motif databases was carried out, by using the interproscan program. the proteins orf1–orf show the acriflavinrp, a 9-element fingerprint that provides a signature for members of the acriflavin resistance protein family as acrb of e. coli, while the proteins orf11–orf show a 6– element fingerprint. all hypothetical rnds contain two transmembrane functional domains located within tms and tms that coincide with the transporter family signature motifs c and d, respectively. the crystal structure of multidrug efflux transporter acrb of e. coli reveals that this protein is characterized by the four subdomains pn pn pc and pc the structural motifs pn and pn comprise the polypeptide segment between tms and tms while pc and pc comprise the segment between tms and tms all the b. cenocepacia rnd transporters show the four subdomains; the region with subdomains pn and pn coincides always with the transporter family signature motif a. the tolc docking domain is composed of two subdomains, dn and dc, and these subdomains are found in all hypothetical rnd proteins except the orf that present only the dc subdomain. in order to obtain a more exhaustive analysis about the structural and conformational properties of the orfs, the amino acid sequences were analysed by phyre program and the data were reported in table the orf1– were compared to the rnd proteins of e. coli and p. aeruginosa by using the blastp program. particularly, orf orf and orf show strong identity with e. coli acrb and with p. aeruginosa mexb. orf and orf show identity with p. aeruginosa mexd, while orf and orf show identity with p. aeruginosa mexf. orf and orf are homologues of metal efflux transporters belonging to the rnd family of e. coli and p. aeruginosa. particularly, orf is highly homologous to czra protein of p. aeruginosa, involved in cadmium and zinc resistance. analysis of the upstream and downstream regions of the rnd coding genes confirmed the association with both an acra/mexa homolog and a tolc/oprm homolog in a variety of arrangements. indeed, orf orf orf orf orf orf orf, orf orf orf and orf seem to be part of an operon that also included a mexa and an oprm homologs, thus containing all three of the components of the tripartite pump complex. differently, orf appears to have only the membrane component, while orf and orf are apparently co-transcribed and in operon with a periplasmic membrane fusion protein. in p. aeruginosa, the genes encoding the rnd transporter and periplasmic membrane fusion protein are always present, while the gene encoding the om channel is not always present. in e. coli, the genes for transporter and membrane fusion proteins occur together as an operon and the gene for the tolc occurs elsewhere on the genome. in b. cenopacia the genetic organization of the rnd orfs seems to resemble in part that of p. aeruginosa and in part that of e. coli. as acrd pump of e. coli, orf seem to be transcribed alone in the genome of b. cenocepacia, while orf and orf are organized as the genes pa2526-2527- of p. aeruginosa that contains an additional rnd transporter. many pump component-encoding operons contain a physically linked regulatory gene coding for either a repressor or an activator. for example, expression of the mexab-oprm operon is regulated by the product of the upstream and divergently transcribed mexr gene. not surprisingly, clinical isolates that overexpress the mexab-oprm system often carry mutations in the mexr gene. in the case of orf orf orf orf6– orf orf orf and orf a hypothetical regulatory gene has been identified, divergently transcribed for all genes, except for the hypothetical operon containing the orf orf and orf6– respectively. the websidd program was used to analyse sequences encompassing the coding and intergenic regions of the hypothetical rnd operons for the presence of sidd sites. these sites have a specific and statistically highly significant pattern of association with transcriptional regions, specifically with promoters because strand separation is a necessary step in the initiation of transcription. all the orfs present sidd sites localized upstream of the first gene of the entire operon, except for the orf rt-pcr analysis of the b. cenocepacia genes coding for the rnd transporters we found genes codifying hypothetical rnd drug transporters into b. cenocepacia genome. by rt-pcr analysis, we studied the expression of all these genes at the exponential phase of cells grown in lb medium. a clear expression of orf orf orf and orf was observed, while orf appeared to be weakly expressed. the expression of the other orfs was not detectable, at least in our conditions. it has been demonstrated that orf corresponds to the previously described ceob gene belonging to ceo operon, which has been shown to be strongly induced by salicylate and chloramphenicol. we decide to evaluated the expression of the genes in b. cenocepacia cells grown in the presence of chloramphenicol. in b. cenocepacia j strain, the basal level of expression of the orf was strongly increased by chloramphenicol. no induction could be detected in the case of orf orf orf orf orf6– orf orf and orf and no increased expression could be observed in the case of orf orf orf and orf. however, the b. cenocepacia rrna 16s expression was not affected by different growth conditions, thus demonstrating that the differences detected in the amount of orf mrna, under different growth conditions, are genuine. as the ceo operon has been discovered as responsible for the mdr phenotype of a clinical strain, we can hypothesize that this rnd efflux system is expressed as a result of a mutation. in our conditions, no transcriptional induction of orf could be detected also in the presence of salicylate. orf showed 71% identity with p. aeruginosa mexy, an inducible efflux system that contributes to the natural resistance of p. aeruginosa to antibiotics. experiments involving real-time pcr in p. aeruginosa pao showed induction of gene mexy by chloramphenicol, tetracycline, macrolides, and aminoglycosides. to date, no information is available on the regulation of mexxy expression, except that mutations occurring in the divergently transcribed repressor gene mexz are frequently isolated from the sputa of cystic fibrosis patients. in p. aeruginosa, with the exception of mexab-oprm, the expression of most of the rnd efflux systems is tightly regulated and overexpression of these rnd pumps is usually caused by mutations in genes encoding regulatory proteins. notably, overexpression of multidrug resistance pumps in clinical isolates of p. aeruginosa, resulting in increased bacterial resistance, is usually due to mutations in these regulatory genes. it would be interesting to further investigate if the expression of orf orf orf and orf is due to a constitutive expression or to a transcriptional deregulation in the b. cenocepacia clinical isolate analysed in this study, which showed a multidrug resistance phenotype as it is resistant to ampicillin and aztreonam, ceftazidime, meropenem, piperacillin amikacin, norfloxacin, rifampicin, streptomycin, tetracycline, chloramphenicol, and gentamicin. cloning and phenotypic analysis of b. cenocepacia orf into e. coli kam3 one approach to test the role of the identified genes in drug efflux is to clone them into suitable hosts by selecting on different compounds. in spite of several attempts, we met many problems in cloning experiments since a lot of rearranged clones were found in the host strains. the difficulties that we met in cloning experiments could rely both on plasmid instability and/or toxic effect of the gene product. in fact, the failure could be due to the toxicity of the gene product for the host bacterial cells and still b. multivorans as a host causes many troubles. to overcome this problem we used the e. coli kam strain as host, which lacks the acrab genes and is sensitive to many drugs that are known as substrates of the acrab system. we decided to characterize one of the hypothetical drug efflux pumps of b. cenocepacia by cloning the orf2-coding region from b. cenocepacia genomic dna into pbad expression vector. the pbad vector and the recombinant plasmid were transformed into e. coli kam we chose orf because of its high homology to mexb, responsible for antibiotic resistance in many pseudomonas clinical isolates. particularly, studies with mutants that overproduce or lack mexab-oprm demonstrated that this efflux system extrudes quinolones, macrolides, tetracycline, chloramphenicol, novobiocin, and most β-lactams but not imipenem. the mics of e. coli kam strains harbouring pbad and pbad/orf respectively, were determined by streaking the cultures onto lb containing different concentrations of the following compounds: streptomycin, ethidium bromide, nalidixic acid, several quinolones, chloramphenicol, erythromycin, tetraphenylphosphonium, and tetracycline. plates were incubated at 37°c for days and the growth was visually evaluated. compared to the control strain, the overexpression of orf into e. coli kam cells conferred resistance to streptomycin, tetraphenylphosphonium, ethidium bromide, nalidixic acid, ciprofloxacin, ofloxacin, and norfloxacin. no difference in drug susceptibility was found with sparfloxacin, chloramphenicol, erythromycin, and tetracycline. these data suggest that orf is expressed and functional in e. coli kam interestingly, in p. aeruginosa, it has been found that fluoroquinolones like nalidixic acid and norfloxacin can commonly select mutants that constitutively overproduce the mexab-oprm efflux pump system. from these data it appeared that streptomycin and norfloxacin could be substrates of orf we decided to perform rt-pcr of b. cenocepacia grown in the presence of sub-inhibitory concentration of streptomycin and norfloxacin. in our condition, orf transcript could not be detected by rt-pcr. the expression of orf was also evaluated by southern blotting of the rt-pcr agarose gel and subsequent hybridization with a labelled 511-bp fragment internal to orf coding region. no hybridisation signals were observed, indicating that orf is not expressed following treatment of b. cenocepacia cells with streptomycin or norfloxacin. three different hypotheses could be given to explain the fact that orf is not expressed. first, the experimental approaches utilized in this work are not enough sensitive to detect very small amount of orf mrna. real-time pcr is an attractive method for estimating gene expression of efflux pumps in bacteria because of its great sensitivity and wide effective range. recently, the real-time pcr appeared to be a useful alternative method for assessing the expression of efflux pumps mexb and mexy in p. aeruginosa. second, not all orf substrates in e. coli necessarily must be inducers of the expression of this pump in b. cenocepacia. a relevant example of such a behaviour is given by the mexcd-oprj efflux pump of p. aeruginosa, which extrudes antibiotics that do not induce its expression. instead, expression of this efflux system was induced by clinical important disinfectants such as benzalkonium chloride and chlorhexidine gluconate. finally, the orf expression may occur in a phase of cellular growth different from the exponential one we tested. in conclusion, we could not determined neither the real inducing agent nor the culture conditions in which induction of orf expression is triggered; therefore, it could be interesting to test other organic compounds for the ability to induce expression of this efflux pump. ethidium bromide efflux activity ethidium bromide efflux experiment was performed to determine whether e. coli kam cells, carrying the recombinant plasmid pbad/orf were more resistant to this dye due to an active efflux mechanism. as shown in figure cells harboring the cloning vector pbad take up ethidium bromide rapidly and achieve a steady-state level of accumulation within about min of incubation. as observed in several experiments, accumulation of ethidium bromide was approximately 20% lower when cells contained the pbad/orf recombinant plasmid. a reduced level of accumulation of the drug may be caused either by a decreased level of drug permeation or by active drug extrusion through the cytoplasmic membrane. to study the effect of membrane deenergization on the uptake of tetracycline, the protonophore carbonyl cyanide m-chlophenylhydrazone was added to cells containing ethidium bromide. upon the addition of cccp, the level of ethidium bromide accumulation increased in the case of the pbad/orf2-harboring strain and reached a level almost equal to that observed in the case of the strain containing only the cloning vector pbad. on the contrary, under our conditions, cccp had no significant effect on the level of ethidium bromide accumulation in the strain carrying the cloning vector. these data indicate that orf pumped out ethidium bromide in an energy-dependent process, presumably by using proton motive force. we could conclude that orf belonging to rnd transporters, is an efflux pump of b. cenocepacia j  pulmonary infection with bcc in patients with cf is often associated with a more rapid decline in lung function. because of the resistance of bcc to many antibiotics, treatment options are often limited. the emergence of active efflux as a major causative factor in antibiotic resistance has been one of the most significant trend in anti-infective chemotherapy over the last decade and strategies to identify efflux pump inhibitors are in progress. for these reasons, the identification and the characterization of such efflux systems in b. cenocepacia are a very important topic in the field of antibiotic resistance. taken together the results described in this paper fit with the prediction that b. cenocepacia contains homologs of the p. aeruginosa mex-opr efflux pump systems. moreover, orf orf orf and orf are expressed in b. cenocepacia j even if, at this stage, it is not possible to state if this expression is constitutive or due to mutations in regulatory genes. finally, the results described in this paper suggested that at least one of the rnd-type efflux pumps identified, extrude out of the cell some antibiotics as well as other compounds. to demonstrate that this pump is really involved in the multidrug resistant phenotype of the b. cenocepacia j further studies are necessary. the generation of mutants with pump overproduction and/or genetic deletion of genes encoding pumps in a controlled laboratory environment provides invaluable information on the potential impact of multidrug efflux pumps on the intrinsic resistance of b. cenocepacia j because of the difficulties in cloning and expression experiments, knock-out approach represents the only alternative method to physiologically characterize these transporters and to confirm if the encoded pumps play a significant role in the antimicrobial resistance of this microorganism. a greater understanding of the genetic determinants that play a role in antibiotic resistance will lead to the development of new strategies for the treatment of b. cenocepacia infections in patients with cystic fibrosis. 
as resistance-nodulation-division 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. 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. a hallmark of b. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. genome analysis and homology searches revealed open reading frames encoding putative drug efflux pumps belonging to rnd family in b. cenocepacia j strain. burkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. by reverse transcription -pcr analysis, it was found that orf orf orf and orf were expressed at detectable levels, while orf appeared to be weakly expressed in b. cenocepacia. the 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. 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. the orf conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in escherichia coli kam 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.
 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 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.  genome analysis and homology searches revealed open reading frames encoding putative drug efflux pumps belonging to rnd family in b. cenocepacia j strain. by reverse transcription -pcr analysis, it was found that orf orf orf and orf were expressed at detectable levels, while orf appeared to be weakly expressed in b. cenocepacia. futhermore, orf was strongly induced by chloramphenicol. the orf conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in escherichia coli kam 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.  the 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 orf pump, one of these 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. 