BACKGROUND
burkholderia cepacia complex  bacteria are antibiotic resistant opportunistic pathogens known for their ability to infect individuals with cystic fibrosis .
the complex currently consists of  <dig> formally named species, of which b. multivorans and b. cenocepacia most often cause infection in cf  <cit> .
the b. cenocepacia et <dig> strain is one of the most problematic bcc strains infecting patients with cf.
it was most likely recognised as early as  <dig> in the pioneering studies of isles et al.
<cit>  that showed that "p. cepacia" could cause an invasive, frequently fatal infection that became known "cepacia syndrome."
subsequent studies demonstrated that et <dig> strains could transmit between cf patients by social contact  <cit> , had spread intercontinentally across north america and europe, and could replace infection with other bcc species .
isolates of the et <dig> strain can be identified using a number of characteristics including presence of the cable pilus gene and the burkholderia cepacia epidemic strain marker, a rapd  <dig> genotype fingerprint, a reca subgroup iii-a phylotype , and most recently as possessing the multilocus sequence type  st- <dig>  <cit> .
as a result of the devastating cf infections caused by the b. cenocepacia et <dig> strain, it has become one of the most studied bcc bacteria in terms of virulence  <cit>  and antimicrobial resistance  <cit> .
the b. cenocepacia et <dig> isolate, j <dig>  was recovered from an infected cf patient in edinburgh, uk, in  <dig>  <cit>  and has been subject to complete genome sequence analysis  <cit> .
this genomic resource enabled a b. cenocepacia microarray to be designed that has greatly enhanced our molecular understanding of this cf pathogen  <cit> .
b. cenocepacia encodes multiple pathways that it uses to resist killing by antimicrobial agents, including efflux pumps  <cit> , lipopolysaccharide, beta-lactamases and a trimethoprim resistant dihydrofolate reductase enzyme  <cit> .
a problematic feature of chronic bcc infection in cf is the ability of the infecting strain to adapt to very high levels of antibiotic resistance.
the emergence of pan resistant bcc strains, which are either untreatable or require combinations of multiple antibiotics to suppress exacerbations of infection, is of great concern  <cit> .
the molecular mechanisms behind the evolution of spontaneous antimicrobial resistance in b. cenocepacia are not known and how the multiple resistance pathways function on a global scale to allow b. cenocepacia to survive antibiotic therapy is poorly understood.
cationic drugs such as chlorpromazine and theophylline have been shown to reduce the minimal inhibition concentration  of certain antibiotics that are otherwise ineffective against bcc bacteria  <cit>  and a more complete understanding of how these non-antibiotic drugs act may also provide novel therapies.
in this study, we employed a transcriptomic, microarray-based strategy to investigate the following :  how exposure to sub-inhibitory concentrations of three antibiotics  that are widely used to treat cf infections  <cit>  and sub-inhibitory exposure to the cationic drug chlorpromazine affect gene expression in b. cenocepacia,  how b. cenocepacia alters gene expression after the selection of spontaneous resistant mutants by the three latter antibiotics, and finally,  how the antimicrobial resistance characteristics and global gene expression of the strain have naturally evolved during the  <dig> years it has circulated within the cf community since the isolation of the genome sequenced isolate, b. cenocepacia j <dig> in  <dig>
RESULTS
antibiotic susceptibility of b. cenocepacia j <dig> and derived mutants with spontaneous resistance
using the clinically defined mic breakpoints, wild type b. cenocepacia j <dig> was resistant to  <dig> of the  <dig> antibiotics tested; susceptibility to meropenem and ceftazidime was classified as intermediate .
synergy testing indicated no synergy in any of the combination of two antibiotics tested against j <dig>  the antibiotic potentiator chlorpromazine reduced the mic of amikacin , tobramycin  and azithromycin  at a concentration of  <dig>  mm; the same concentration of prochlorperazine produced slightly greater reductions in antibiotic mic .
these reductions were consistent with previous data obtained for "b. cepacia" strain atcc  <dig>  <cit>  which is a b. cenocepacia iiia strain, but not from the et <dig> lineage  of b. cenocepacia j <dig>  resistant mutants and outbreak isolates
spontaneous mutants with elevated resistance to amikacin , meropenem  and trimethoprim-sulfamethoxazole  were generated by plating on selective agar containing each respective antibiotic .
the mutants had mics that were increased over the wild type,  <dig> -,  <dig> - and 5-fold, for each respective antibiotic .
the growth rate of each mutant was unaltered in comparison to j <dig> and the resistant phenotype of each mutant was stable after  <dig> transfers on agar without antibiotics.
the spontaneous mutants appeared at a frequency of  <dig>   × 10- <dig> for amikacin,  <dig>   × 10- <dig> for meropenem, and  <dig>   × 10- <dig> for trimethoprim-sulfamethoxazole; no difference in mutation frequency was observed between j <dig> cultures that were plated in log phase or at the beginning of stationary phase.
mutant j2315-a showed an increased resistance to all other aminoglycosides tested, and interestingly also to the beta-lactams meropenem, ceftazidime, cefotaxime and aztreonam .
the meropenem-resistant mutant, j2315-m, demonstrated elevated resistance to all beta-lactams tested, as well as increases in tobramycin, chloramphenicol and tetracycline resistance.
mutant j2315-t was more resistant to fluoroquinolones, chloramphenicol, rifampicin and tetracycline .
antibiotic susceptibility and features of clonal outbreak strains circulating  <dig> years after isolation of b. cenocepacia j2315
two isolates from two cf patients involved in a recent et <dig> outbreak  <cit> , bcc <dig> and bcc <dig>  were compared to j <dig> in terms of phenotype and global gene expression genotype.
they represent the natural evolution of this epidemic strain as an infectious agent circulating in cf community since the recovery of j <dig> in  <dig>  bcc <dig> and bcc <dig> were both found to be clonally identical with j <dig> .
each was more resistant to aminoglycosides, macrolides, fluoroquinolones and tetracycline, than j <dig> ; bcc <dig> was less susceptible to all beta-lactams tested.
this is in concordance with previous observations of rapid evolution of elevated drug resistance within sequential et <dig> cf isolates, where isolates from episodes of clinical exacerbation were more resistant than those recovered during stable clinical conditions  <cit> .
the growth rate of bcc <dig> and bcc <dig> on iso-sensitest broth  was also slower than j <dig> .
p. aeruginosa isolates recovered from cf infection have reduced growth rates compared to those from other sources  <cit>  and a slower growth rate is also linked to increased antibiotic resistance in bacterial pathogens  <cit> .
both of the recent et <dig> clones were not motile in contrast to j <dig> .
loss of motility has been observed among p. aeruginosa isolates recovered from chronic cf infection  <cit> .
both j <dig> and bcc <dig> were auxotrophic for tyrosine and phenylalanine.
auxotrophy for amino acids has also been observed in cf isolates of p. aeruginosa and b. cepacia  <cit>  and is attributed to the cf lung environment being rich in amino acids and other nutrients, selecting for auxotrophic variants.
j <dig> and bcc <dig> both formed a brown pigment at the beginning of stationary phase on iso-sensitest and on lb medium.
p. aeruginosa cf isolates often produce a brown, melanin-like pigment, attributed to accumulation of homogentisate in the growth medium  <cit> .
the same phenomenon has been observed for b. cenocepacia  <cit>  with melanin production being correlated to increased resistance to oxidative stress.
in contrast to j <dig> and bcc <dig>  strain bcc <dig> was not auxotrophic and did not produce a brown pigment, illustrating the known phenotypic variability of these bacteria  <cit> .
b. cenocepacia global gene expression in the presence of antibiotics
transcriptomic analysis of strain j <dig> was performed on mid-log phase cultures exposed to amikacin, meropenem, trimethoprim-sulfamethoxazole, chlorpromazine and a combination of amikacin and chlorpromazine .
expression analysis of the spontaneous mutants was performed with and without each respective antibiotic on which the mutant had been selected; antibiotic exposure was performed at the same antibiotic concentration as used for j <dig> and levels above this to induce further responses in the spontaneous mutants .
validation of the microarray results was carried out by qpcr analysis of  <dig> protein coding genes of interest .
in all cases the up- or downregulation observed by qpcr correlated to the microarray results corroborating previous studies using the b. cenocepacia j <dig> microarray  <cit> .
a the number of genes differentially expressed were significantly different  in the resistant mutants compared to the corresponding condition of j <dig> exposed to each respective antibiotic.
very few significant alterations in the expression of features on the microarray were observed after exposure of j <dig> to amikacin , while exposure to meropenem and trimethoprim-sulfamethoxazole resulted in approximately three times this number of features altering in expression .
the presence of  <dig>  mm chlorpromazine also altered a limited number of features , and this doubled with the combination of chlorpromazine and amikacin .
overall, a greater number of genes were upregulated than downregulated in each antimicrobial exposure condition .
the number of differentially expressed genes was significantly higher in all experiments where the antibiotic resistant mutants were compared to j <dig> .
the number of differentially expressed genes was highest in the outbreak isolates bcc <dig> and bcc <dig>  with  <dig> and  <dig> differentially expressed genes compared to j <dig> .
gene expression in j <dig> and j2315-a in response to amikacin
the upregulation of trnas dominated the transcriptomic response of j <dig> to amikacin ; since this aminoglycoside targets protein synthesis, the alteration of trna expression may be correlated to the mode of action of amikacin but, further research will be need to validate this assumption.
in terms of protein encoding genes only two were significantly upregulated in the presence of  <dig> μg/ml amikacin: bcal <dig>  a protein with unknown function and the adjacent gene, bcal <dig>  a cds with homology to metal-dependent phosphohydrolases .
both were also upregulated in j2315-a .
the induction of bcal <dig> by the presence of amikacin was confirmed by qpcr .
bcal <dig> contains nucleotidyltransferase and phosphotransferase motifs that could possibly confer similar enzyme activities as those present in aminoglycoside-modifying enzymes, and which have been implicated in aminoglycoside resistance  <cit> .
introduction of the bcal1755/bcal <dig> region into b. vietnamiensis g <dig>  which lacks homologous genes in its genome did not result in transformants with an elevated resistance to amikacin.
mobilization of the same construct into b. cenocepacia k56- <dig> , a genetically amenable b. cenocepacia strain that is frequently used as a surrogate model for genetic manipulation due to difficulties associated with j <dig>  <cit> , did not increase the amikacin resistance of this strain by insertion of additional copies this locus.
the function of these genes was also tested by creating an isogenic knockout mutant in b. cenocepacia strain, k56- <dig> .
in correlation with the gene transfer experiment deletion of genes bcal1755- <dig> in mutant k56-2Δl1755- <dig> did not result in increased susceptibility to aminoglycosides or any other antimicrobial tested .
bcam0296
multidrug efflux system protein
increased susceptibility for azithromycin
bcam2187
abc-type efflux carrier protein
a the following antibiotics and antimicrobials were tested on all mutants, except k56-2Δs0167: amikacin, tobramycin, meropenem, ceftazidime, trimethoprim, azithromycin, erythromycin, levofloxacin, tetracycline, chloramphenicol, chlorhexidine
the amikacin-resistant mutant j2315-a demonstrated upregulation of different genes/gene clusters compared to the wild type, regardless of the present or absence of the antibiotic .
most conspicuous was the upregulation of a gene cluster on the large chromosome, bcal3148- <dig>  one of these genes, bcal <dig> encoded a sigma factor  and an adjacent anti-sigma factor, bcal <dig>  the bcal <dig> sigma factor is highly conserved among burkholderia species  <cit> .
ecf sigma factors are involved in sensing and regulating the response to changes in the environment, including maintaining cell envelope integrity under stress  <cit> .
the bcal3148- <dig> cluster was not induced in the wild type by sub-inhibitory concentrations of amikacin, but its constitutive upregulation in j2315-a suggested it may play a major role in the general stress response linked to the spontaneous adaptation to an elevated state of antimicrobial resistance.
another gene cluster found to be constitutively upregulated in j2315-a was a putative acetyltransferase, bcam0829a, and a nearby abc transporter gene, bcam <dig> .
limited upregulation of bcam0829a was observed when j <dig> was exposed to amikacin  but this was confirmed by qpcr .
acetyltransferases are known mediators of aminoglycoside resistance  <cit>  and abc transporters often contribute towards multi-drug resistance in e. coli  <cit> , hence, both genes, bcam0829a and bcam <dig> have plausible roles in functionally mediating resistance to amikacin.
however, transfer of the acetyltransferase bcam0829a into b. vietnamiensis g <dig> or b. cenocepacia k56- <dig> failed to alter their susceptibility to amikacin, gentamicin and tobramycin , and deletion of the associated abc transporter gene bcam <dig> in mutant k56-2Δm <dig> also did not reveal an antimicrobial phenotype .
gene expression in j <dig> and j2315-m in response to meropenem
the global gene expression response of b. cenocepacia to meropenem was characterised by upregulation of predictable factors such as beta-lactamases and of novel enzymes like those linked to the phenylacetic acid  degradation pathway .
bcas <dig>  a class c beta-lactamase encoded on the small chromosome was the most upregulated gene seen when the wild type was exposed to meropenem; a class a beta-lactamase, bcam <dig>  encoded on the medium chromosome was also highly expressed .
interestingly, neither beta-lactamase was significantly altered in expression in the meropenem resistant mutant in the absence of the antibiotic , indicating that these enzymes were not primarily involved in the elevated resistance phenotype of j2315-m .
located  <dig> bp upsteam of the class a beta-lactamase bcam <dig> was a lysr family regulator bcam <dig>  with 90% homology to and in a location corresponding to that of penr previously described in "p. cepacia"  <cit>  .
this region is conserved in all completed burkholderia genomes and trépanier et al.
<cit>  showed that penr controlled the transcription of the class a beta-lactamase pena with a putative binding site mapped to a region  <dig> bp upstream of the beta-lactamase, directly adjacent to the regulator sequence.
a motif search revealed the same binding site in j <dig>  upstream of both bcam <dig> and bcas <dig>  suggesting that they may be co-regulated and possibly under the control of the bcam <dig> regulator.
although both beta-lactamases were upregulated in the presence of meropenem, it was not known which, if any, is active against the antibiotic.
carbapenem hydrolyzing class a beta-lactamases have been reported for klebsiella pneumoniae  <cit> .
a class a penb beta-lactamase has been characterised in b. cenocepacia  <cit>  that is active against penicillin and cephalosporins, but has only a marginal ability to degrade meropenem and imipenem.
a plasmid-encoded class c beta-lactamase with weak imipenemase activity has also been reported  <cit> .
to clarify the involvement of bcam <dig> in meropenem inactivation by b. cenocepacia, clavulanic acid was tested as a class a-specific beta-lactamase-inhibitor, but it failed to reduce the mic for meropenem even at concentrations as high as  <dig> mg/l .
the role of the class c beta-lactamase, bcas <dig> was tested by complementation into b. vietnamiensis g <dig> which lacks this gene .
the resulting transformant showed an increase in mic for cefotaxime , ceftazidime , imipenem  and piperacillin , but the mic for meropenem  and aztreonam  remained unchanged from that of wild type b. vietnamiensis g <dig>  when the same construct was mobilized into b. cenocepacia k56- <dig>  the mic for cefotaxime  and ceftzidime  increased, but the remaining antibiotic mics were unaltered.
addition of chlorpromazine to the complemented strains did not alter their antibiotic susceptibility further.
a functional role for bcas <dig> in mediating high level beta-lactam resistance was therefore supported, but interestingly not for meropenem as a specific substrate for this class c beta-lactamase.
three gene clusters annotated as phenylacetic acid degradation enzymes  <cit>  were upregulated in mutant j2315-m regardless of the presence or absence of the meropenem .
in b. cenocepacia, these genes are organised as follows: two gene clusters on the large chromosome  and one gene cluster on the medium chromosome .
all three clusters appeared largely co-regulated from the microarray data and a meme search revealed a common regulatory motif  <cit>  for all three locations, upstream of bcal <dig>  bcal <dig> and bcam <dig> respectively.
a mast analysis revealed the same regulatory motif to be present upstream of the pa degradation genes in all genome sequenced burkholderia species.
upregulation of bcal <dig>  bcal <dig>  and bcal1711/ <dig> was confirmed by qpcr .
two sets of genes involved in glucose transport and metabolism via the pentose phosphate pathway were substantially down regulated in the resistant mutant j2315-m: bcal1804- <dig> and bcam1356- <dig> .
growth curve analysis showed that j2315-m grew at less than half the growth rate on glucose than j <dig>  the pa catabolic enzymes have been shown to be under glucose catabolite repression in b. cenocepacia  <cit> .
our data therefore suggested that for j2315-m to achieve the upregulation of pa genes and increase its meropenem resistance, it had to down regulate the glucose transport and metabolism genes to lower the intracellular glucose concentration.
the resulting slower growth rate of j2315-m in presence of glucose may also be an advantageous phenotype for surviving antimicrobial exposure, analogous to the alterations seen in small colony variants  <cit> .
to elucidate if the pa catabolic enzymes aid resistance to antibiotics, mic values in the presence and absence of glucose  in lb medium were determined.
the presence of glucose resulted in reductions of ≥ 2-fold in j2315's mic for meropenem , ceftazidime , chloramphenicol  and tetracycline ; no change was observed for amikacin, tobramycin, erythromycin, levofloxacin and trimethoprim-sulfamethoxazole.
a panel of  <dig> closely related b. cenocepacia were also tested and  <dig> out of  <dig> also demonstrated a reduction in meropenem mic in the presence of  <dig> mm glucose .
analysis of pa gene expression directly correlated to these phenotypic observations, being considerably reduced in j <dig>  in the presence of glucose.
expression of this gene was unaltered by addition of glucose for b. cenocepacia k56- <dig>  which did not demonstrate the same mic reduction for meropenem.
the strain to strain variability of this putative catabolite repression response suggest that the regulation of central metabolism in b. cenocepacia is highly complex; given the number of paralogous metabolic pathways present in burkholderia genomes  <cit>  such plasticity is not surprising.
qpcr showed that the expression of the beta-lactamases  was not affected by the presence of glucose in j <dig> indicating that these degradative antibiotic resistance enzymes were not catabolite repressed.
the role of the pa degradation enzymes in burkholderia is not yet fully understood, but recently it was shown that pa pathway is necessary for full pathogenicity during caenorhabditis elegans infection  <cit> .
the linkage of pa degradation to the phenomenon of spontaneous antibiotic resistance in b. cenocepacia adds further complexity to the central role aromatic metabolism plays in the biology of these microorganisms.
gene expression in j <dig> wt and j2315-t in response to trimethoprim-sulfamethoxazole
the gene encoding a homologue of aida , a quorum sensing regulated gene required for full virulence of b. cenocepacia h <dig> during c. elegans infection  <cit>  was the most upregulated gene when wild type j <dig> was exposed to sub-inhibitory levels of tmp-smx .
other quorum sensing regulated genes such as the zinc metalloprotease  <cit> , zmpa , and the lectin encoding bcam <dig>  <cit>  were also upregulated.
few other significant alterations were made by j <dig> in response of sub-lethal amounts of tmp-smx .
deletion of bcas <dig> together with the adjacent bcas <dig> in mutant k56-2Δs0293- <dig> did not alter its susceptibility to trimethoprim, sulfamethoxazole, or any other antimicrobials .
the tmp-smx resistant mutant j2315-t also altered the expression of a limited number of genes, however, the most conspicuous change it made was a strong and constitutive upregulation of an efflux pump of the rnd family, bcam2549- <dig> .
this is the j <dig> homologue of the ceo multidrug efflux pump operon described by nair et al.
<cit>  consisting of the three parts of the efflux pump  and a hydrolase/lipase .
the mic data for j2315-t showed increased resistance to trimethoprim, fluorquinolones, chloramphenicol and tetracyclines , a phenotype consistent with the substrate spectrum of the ceo efflux pump described by nair et al.
<cit>  and thus corroborating the microarray expression data.
upstream and divergently transcribed from the ceo efflux pump is a lysr family regulatory protein, bcam <dig>  that is a homologue of the ceor transcriptional activator  <cit> .
this regulator was constitutively upregulated in mutant j2315-t .
nair et al.
<cit>  had also shown that the pump was upregulated in the presence of salicylate.
in contrast to their findings made on b. cenocepacia strain pc <dig>  <cit> , where salicylate and chloramphenicol were found to induce expression of the ceo efflux pump, we found that both the efflux pump and ceor in b. cenocepacia j <dig> and k56- <dig> were not inducible by salicylate  or sub-inhibitory levels of tmp-smx .
gene expression in j <dig> wt in response to chlorpromazine
exposure of b. cenocepacia to this potentiator of aminoglycoside activity resulted in significant upregulation of two efflux gene clusters, bcam0923- <dig> and to a lesser extent, bcam2186- <dig> .
no specific link to amikacin-related gene expression, the antibiotic most potentiated by chlorpromazine, was observed .
the bcam2186- <dig> pump had homology to a macrolide-specific abc-type efflux pump system in e. coli  <cit> .
transfer of the efflux pump genes to b. vietnamiensis g <dig> or b. cenocepacia k56- <dig>  did not alter the antimicrobial susceptibility of these strains .
likewise creation of a b. cenocepacia deletion mutant k56-2Δm2186- <dig> did not produce an observable phenotype in relation to the antibiotics  examined in this study .
in relation to the other chlorpromazine activated efflux system, bcam <dig> and bcam <dig>  were closely related to mexd and mexc, respectively, components of the mexcd-oprj efflux pump in p. aeruginosa  <cit> .
bcam <dig>  an outer membrane protein, is most similar to oprm, which is part of the mexab-oprm efflux pump in p. aeruginosa  <cit> .
bcam <dig> is the response regulator part of a two component regulatory system that may control expression of the efflux pump; its role was investigated by the construction of a deletion mutant k56-2Δm <dig> .
the mutation did not alter its chlorpromazine or antibiotic mic .
the expression of the structural pump genes, bcam0926- <dig>  was also still induced by chlorpromazine in the deletion mutant suggesting that bcam <dig> was not critical for regulation of the adjacent efflux pump .
to further investigate the role of the efflux pump structural genes bcam0925- <dig>  a k56- <dig> deletion mutant spanning all  <dig> genes was constructed .
the k56-2Δm0925- <dig> mutant demonstrated increased susceptibility to the disinfectant chlorhexidine and the antibiotic azithromycin, but no alteration in susceptibility to other antibiotics.
growth curve analysis of the k56-2Δm0925- <dig> mutant revealed a 10% slower growth rate in the presence of chlorpromazine compared to the parent strain.
qpcr analysis of the k56- <dig> parent strain showed that chlorhexidine, as well as chlorpromazine induced the efflux pump gene bcam <dig>  345-fold in the presence of  <dig> μg/ml chlorhexidine and 125-fold in the presence of  <dig>  mm chlorpromazine.
chlorhexidine, like chlorpromazine is a cationic compound known to interact with and damage cellular membranes  <cit> , hence it is logical that both compounds induce the efflux pump and that the efflux mutant demonstrated sensitivity to this disinfectant.
chlorhexidine is also known to induce the pseudomonas aeruginosa mexcd-oprj efflux pump  <cit> .
since the bcam0925- <dig> efflux operon shares considerable homology with latter p. aeruginosa system, the mutant phenotype also suggests that the pump may play a role in chlorhexidine efflux in b. cenocepacia.
why deletion of the pump led to an alteration in susceptibility to azithromycin is not known.
another gene cluster upregulated in the presence of chlorpromazine was a squalene-hopene cyclase, bcas <dig>  and an adjacent, divergently transcribed response regulator, bcas <dig> .
squalene-hopene cyclase is involved in the synthesis of hopanoids, the prokaryotic equivalent of steroids  <cit>  which may function to counter the effect of membrane disrupting agents on bacterial cells.
a deletion mutant of bcas <dig>  k56-2Δs <dig>  demonstrated reduced growth rate in the presence of chlorpromazine and chlorhexidine, as well as an increased susceptibility to chlorhexidine .
since the squalene-hopene cyclase gene and surrounding region is highly conserved in several bcc genomes  these data suggest it plays an integral role in the intrinsic resistance of burkholderia to membrane disrupting antimicrobial agents.
gene expression in the pan-resistant cf outbreak isolates bcc <dig> and bcc1617
the most pronounced change in expression profiles of both et <dig> outbreak isolates was the downregulation of genes involved in flagella production and in chemotaxis  compared to j <dig>  bcc <dig> and bcc <dig> also downregulated expression of their cable pilus genes .
the lack of flagella and chemotaxis gene expression correlated directly to the non-motile phenotype of the outbreak isolates, in contrast to j <dig> which was motile .
during rapid growth in a sputum-based medium j <dig> upregulates genes involved in production of the flagellum  <cit> .
this may reflect a short term response of b. cenocepacia to early growth during infection, where flagella and motility serve as important virulence factors  <cit> .
lack of motility has been observed in p. aeruginosa cf isolates from chronic infection  <cit>  and as a global gene expression adaptation to growth in sputum  <cit> .
the stable non-motile phenotype and genotype of the recent et <dig> clinical strains suggest that b. cenocepacia may in time also adapt to the same phenotype associated with p. aeruginosa from chronic cf infection.
both the outbreak isolates had upregulated genes involved in transport and efflux, restriction modification, and transposition as follows .
an abc membrane transport protein  and adjacent hypothetical protein with possible hydrolytic activity  were highly expressed.
the transport protein bcas <dig> contains both an atp binding cassette and transmembrane components, and has homology to the mdlb gene of e. coli which is implicated in multidrug resistance  <cit> .
deletion of bcas <dig> in mutant k56-2Δs <dig> resulted in increased susceptibility to tetracycline and chlorhexidine, but did not alter the mic for the other antimicrobial agents tested .
these data validate the microarray observed upregulation of bcas <dig> and demonstrate it does play a role in the antimicrobial resistance of b. cenocepacia.
also highly upregulated in both strains were genes of a type i restriction modification system .
in j <dig>  the first gene in this operon is a pseudogene mutated with an insertion sequence  that may prevent expression of the entire downstream gene cluster.
pcr amplification and sequence analysis of this region revealed that bcc <dig> and bcc <dig> do not possess the same is mutation, hence allowing overexpression of the genes as detected by the microarray analysis.
mutation of the putative dna methylase, bcal <dig>  in this cluster results in the attenuation of b. cenocepacia virulence in a rat lung model of chronic lung infection .
the dna modification conferred by the gene cluster may also play a role in surviving the damage caused by hydroxyl radicals which are released when bacteria are exposed to bactericidal antibiotics  <cit> .
since the type i restriction modification cluster is clearly transcriptionally activated in the outbreak strains, it will be interesting to follow up if their expression has provided them with a resistance and virulence advantage compared to j <dig> as an ancestral et <dig> isolate.
another response conserved between the two outbreak isolates was the upregulation of transposase encoding genes within isbcen <dig> .
the is profiles of b. cenocepacia isolates of the same mlst strain type have been recently shown to vary markedly and oxidative stress was linked to this is movement  <cit> .
the upregulation of the isbcen <dig> transposase in these recent outbreak isolates may indicate that these strains are hypermutable with regard to this is.
this may allow very rapid phenotypic switches to occur within individual bacteria in the infecting population and alter their pathogenicity.
when examined individually, the two outbreak isolates demonstrated several global gene expression characteristics that had been observed in the antibiotic resistant b. cenocepacia j <dig> derivatives.
bcc <dig> upregulated the pa degradation enzyme genes , that were also constitutively upregulated in j2315-m .
these data re-enforce the hypothesis that the ability of b. cenocepacia to switch metabolism to alternative pathways in terms of aromatic hydrocarbon degradation plays a critical role in its spontaneous antibiotic resistance.
it had also upregulated the class a beta-lactamase  that was induced in j <dig> and j2315-m upon meropenem exposure .
phenotypically, bcc <dig> possessed the same elevated resistance to beta-lactam antibiotics that j2315-m had developed after meropenem selection , and its meropenem mic could be reduced by growth in the presence glucose .
genes found upregulated in the amikacin-resistant j2315-a derivative  were also constitutively over-expressed in bcc <dig>  again correlated to high aminoglycoside resistance of this isolate .
several differentially expressed genes were specific to isolate bcc <dig>  genes involved in the p-hydroxybenzoate degradation pathway were highly upregulated .
since the pa pathway genes were not significantly altered in bcc <dig>  it would be interesting to follow up if the p-hydroxybenzoate degradation pathway may provide an alternative metabolic shift for b. cenocepacia to evolve its antibiotic resistance.
a specific resistance determinant upregulated in bcc <dig> was an efflux pump  that is a homologue of the amrab  <cit>  and of bpeab  <cit>  systems encoded by b. pseudomallei.
both the latter systems mediate resistance to aminoglycosides and macrolides in b. pseudomallei and it was notable that bcc <dig> possessed very high levels of resistance to tobramycin, azithromycin and erythromycin .
an adjacent gene bcal <dig>  encoding a transcriptional regulator and inactive in j <dig> due to a frameshift mutation, does not have the frameshift in bcc <dig> as revealed by sequence analysis.
hence, this could in turn allow the upregulation of the efflux pump in the outbreak strain compared to j <dig>
bcc <dig> also over expressed the phosphohydrolase bcal <dig> which was stably upregulated in the j2315-a amikacin exposed mutant.
other factors possibly contributing to the overall antibiotic resistance in bcc <dig> compared to j <dig> is the absence of a frameshift mutation in gene bcal <dig>  a transport protein annotated as tetracycline resistance protein, and absence of an insertion sequence in bcam <dig>  annotated as multidrug resistance transporter.
both latter genes are pseudogenes in j <dig>  <cit>  and also in bcc <dig> .
although their expression did not change in bcc <dig>  by just being functionally expressed they may mediate increased resistance to antibiotics.
CONCLUSIONS
our novel transcriptomic analysis of the response of b. cenocepacia to antibiotic exposure has demonstrated that it expresses a multitude of genes and different pathways to achieve high levels of resistance.
b. cenocepacia j <dig> and the clonally related strains we studied are phenotypically and transcriptomically very versatile and can adapt quickly to environmental conditions.
a key finding was that once certain antibiotic resistance phenotypes in b. cenocepacia had been selected by a single antibiotic, the changes in the transcriptome were not transient, and were stably maintained in the spontaneous antibiotic resistant mutants.
this finding suggests that mutations selected in the spontaneously resistant b. cenocepacia j <dig> mutants are stably inherited and do not revert at a high frequency.
a change in antibiotic therapy may therefore not reverse a previously acquired b. cenocepacia spontaneous antibiotic resistance trait.
our molecular analysis corroborates the results of antibiotic susceptibility testing that demonstrate focussing on a single antibiotic or antibiotic class for therapy has limited efficacy and that multiple antibiotic combinations  <cit>  offer the best means to combat the spontaneous resistance of this pathogen.
we have identified multiple novel molecular pathways b. cenocepacia utilises to resist sub-mic levels of antibiotic.
however, for many of the genes altering expression in relation to antibiotic exposure we could not prove a direct involvement resistance, with only  <dig> of the  <dig> b. cenocepacia antibiotic resistance gene deletion mutants demonstrating increased antimicrobial susceptibility.
for spontaneous resistance to single antibiotics, b. cenocepacia expresses set of genes which are distinct from those altered by transient sub-mic exposure, and which are stably altered in expression irrespective of the presence of the priming antibiotic.
these findings suggest that spontaneous antibiotic resistance in b. cenocepacia selects multiple individual as well as pleiotropic mutations, stably altering the expression of many genetic pathways; uncovering the location of these mutations by genome re-sequencing and polymorphism analysis in future studies would shed light on why these changes are stably inherited traits in b. cenocepacia.
of the b. cenocepacia resistance mechanisms identified, those such as the beta-lactamases, phosphohydrolases, novel efflux pumps and phenylacetic acid pathways genes may be targeted to improve the efficacy of current antibiotics.
we may also be able to harness the phenomenon of bacterial catabolite repression by glucose as a novel means to improve the efficacy of aerosolised antibiotics against certain b. cenocepacia during cf infection.

 burkholderia cepacia complex bacteria are antibiotic resistant opportunistic pathogens known for their ability to infect individuals with cystic fibrosis. the complex currently consists of formally named species, of which b. multivorans and b. cenocepacia most often cause infection in cf. the b. cenocepacia et strain is one of the most problematic bcc strains infecting patients with cf. it was most likely recognised as early as in the pioneering studies of isles et al. that showed that "p. cepacia" could cause an invasive, frequently fatal infection that became known "cepacia syndrome." subsequent studies demonstrated that et strains could transmit between cf patients by social contact, had spread intercontinentally across north america and europe, and could replace infection with other bcc species. isolates of the et strain can be identified using a number of characteristics including presence of the cable pilus gene and the burkholderia cepacia epidemic strain marker, a rapd genotype fingerprint, a reca subgroup iii-a phylotype, and most recently as possessing the multilocus sequence type st-. as a result of the devastating cf infections caused by the b. cenocepacia et strain, it has become one of the most studied bcc bacteria in terms of virulence and antimicrobial resistance. the b. cenocepacia et isolate, j was recovered from an infected cf patient in edinburgh, uk, in and has been subject to complete genome sequence analysis. this genomic resource enabled a b. cenocepacia microarray to be designed that has greatly enhanced our molecular understanding of this cf pathogen. b. cenocepacia encodes multiple pathways that it uses to resist killing by antimicrobial agents, including efflux pumps, lipopolysaccharide, beta-lactamases and a trimethoprim resistant dihydrofolate reductase enzyme. a problematic feature of chronic bcc infection in cf is the ability of the infecting strain to adapt to very high levels of antibiotic resistance. the emergence of pan resistant bcc strains, which are either untreatable or require combinations of multiple antibiotics to suppress exacerbations of infection, is of great concern. the molecular mechanisms behind the evolution of spontaneous antimicrobial resistance in b. cenocepacia are not known and how the multiple resistance pathways function on a global scale to allow b. cenocepacia to survive antibiotic therapy is poorly understood. cationic drugs such as chlorpromazine and theophylline have been shown to reduce the minimal inhibition concentration of certain antibiotics that are otherwise ineffective against bcc bacteria and a more complete understanding of how these non-antibiotic drugs act may also provide novel therapies. in this study, we employed a transcriptomic, microarray-based strategy to investigate the following: how exposure to sub-inhibitory concentrations of three antibiotics that are widely used to treat cf infections and sub-inhibitory exposure to the cationic drug chlorpromazine affect gene expression in b. cenocepacia, how b. cenocepacia alters gene expression after the selection of spontaneous resistant mutants by the three latter antibiotics, and finally, how the antimicrobial resistance characteristics and global gene expression of the strain have naturally evolved during the years it has circulated within the cf community since the isolation of the genome sequenced isolate, b. cenocepacia j in  antibiotic susceptibility of b. cenocepacia j and derived mutants with spontaneous resistance using the clinically defined mic breakpoints, wild type b. cenocepacia j was resistant to of the antibiotics tested; susceptibility to meropenem and ceftazidime was classified as intermediate. synergy testing indicated no synergy in any of the combination of two antibiotics tested against j the antibiotic potentiator chlorpromazine reduced the mic of amikacin, tobramycin and azithromycin at a concentration of mm; the same concentration of prochlorperazine produced slightly greater reductions in antibiotic mic. these reductions were consistent with previous data obtained for "b. cepacia" strain atcc which is a b. cenocepacia iiia strain, but not from the et lineage of b. cenocepacia j resistant mutants and outbreak isolates spontaneous mutants with elevated resistance to amikacin, meropenem and trimethoprim-sulfamethoxazole were generated by plating on selective agar containing each respective antibiotic. the mutants had mics that were increased over the wild type, -, - and 5-fold, for each respective antibiotic. the growth rate of each mutant was unaltered in comparison to j and the resistant phenotype of each mutant was stable after transfers on agar without antibiotics. the spontaneous mutants appeared at a frequency of × 10- for amikacin, × 10- for meropenem, and × 10- for trimethoprim-sulfamethoxazole; no difference in mutation frequency was observed between j cultures that were plated in log phase or at the beginning of stationary phase. mutant j2315-a showed an increased resistance to all other aminoglycosides tested, and interestingly also to the beta-lactams meropenem, ceftazidime, cefotaxime and aztreonam. the meropenem-resistant mutant, j2315-m, demonstrated elevated resistance to all beta-lactams tested, as well as increases in tobramycin, chloramphenicol and tetracycline resistance. mutant j2315-t was more resistant to fluoroquinolones, chloramphenicol, rifampicin and tetracycline. antibiotic susceptibility and features of clonal outbreak strains circulating years after isolation of b. cenocepacia j2315 two isolates from two cf patients involved in a recent et outbreak, bcc and bcc were compared to j in terms of phenotype and global gene expression genotype. they represent the natural evolution of this epidemic strain as an infectious agent circulating in cf community since the recovery of j in bcc and bcc were both found to be clonally identical with j. each was more resistant to aminoglycosides, macrolides, fluoroquinolones and tetracycline, than j; bcc was less susceptible to all beta-lactams tested. this is in concordance with previous observations of rapid evolution of elevated drug resistance within sequential et cf isolates, where isolates from episodes of clinical exacerbation were more resistant than those recovered during stable clinical conditions. the growth rate of bcc and bcc on iso-sensitest broth was also slower than j. p. aeruginosa isolates recovered from cf infection have reduced growth rates compared to those from other sources and a slower growth rate is also linked to increased antibiotic resistance in bacterial pathogens. both of the recent et clones were not motile in contrast to j. loss of motility has been observed among p. aeruginosa isolates recovered from chronic cf infection. both j and bcc were auxotrophic for tyrosine and phenylalanine. auxotrophy for amino acids has also been observed in cf isolates of p. aeruginosa and b. cepacia and is attributed to the cf lung environment being rich in amino acids and other nutrients, selecting for auxotrophic variants. j and bcc both formed a brown pigment at the beginning of stationary phase on iso-sensitest and on lb medium. p. aeruginosa cf isolates often produce a brown, melanin-like pigment, attributed to accumulation of homogentisate in the growth medium. the same phenomenon has been observed for b. cenocepacia with melanin production being correlated to increased resistance to oxidative stress. in contrast to j and bcc strain bcc was not auxotrophic and did not produce a brown pigment, illustrating the known phenotypic variability of these bacteria. b. cenocepacia global gene expression in the presence of antibiotics transcriptomic analysis of strain j was performed on mid-log phase cultures exposed to amikacin, meropenem, trimethoprim-sulfamethoxazole, chlorpromazine and a combination of amikacin and chlorpromazine. expression analysis of the spontaneous mutants was performed with and without each respective antibiotic on which the mutant had been selected; antibiotic exposure was performed at the same antibiotic concentration as used for j and levels above this to induce further responses in the spontaneous mutants. validation of the microarray results was carried out by qpcr analysis of protein coding genes of interest. in all cases the up- or downregulation observed by qpcr correlated to the microarray results corroborating previous studies using the b. cenocepacia j microarray. a the number of genes differentially expressed were significantly different in the resistant mutants compared to the corresponding condition of j exposed to each respective antibiotic. very few significant alterations in the expression of features on the microarray were observed after exposure of j to amikacin, while exposure to meropenem and trimethoprim-sulfamethoxazole resulted in approximately three times this number of features altering in expression. the presence of mm chlorpromazine also altered a limited number of features, and this doubled with the combination of chlorpromazine and amikacin. overall, a greater number of genes were upregulated than downregulated in each antimicrobial exposure condition. the number of differentially expressed genes was significantly higher in all experiments where the antibiotic resistant mutants were compared to j. the number of differentially expressed genes was highest in the outbreak isolates bcc and bcc with and differentially expressed genes compared to j. gene expression in j and j2315-a in response to amikacin the upregulation of trnas dominated the transcriptomic response of j to amikacin; since this aminoglycoside targets protein synthesis, the alteration of trna expression may be correlated to the mode of action of amikacin but, further research will be need to validate this assumption. in terms of protein encoding genes only two were significantly upregulated in the presence of μg/ml amikacin: bcal a protein with unknown function and the adjacent gene, bcal a cds with homology to metal-dependent phosphohydrolases. both were also upregulated in j2315-a. the induction of bcal by the presence of amikacin was confirmed by qpcr. bcal contains nucleotidyltransferase and phosphotransferase motifs that could possibly confer similar enzyme activities as those present in aminoglycoside-modifying enzymes, and which have been implicated in aminoglycoside resistance. introduction of the bcal1755/bcal region into b. vietnamiensis g which lacks homologous genes in its genome did not result in transformants with an elevated resistance to amikacin. mobilization of the same construct into b. cenocepacia k56-, a genetically amenable b. cenocepacia strain that is frequently used as a surrogate model for genetic manipulation due to difficulties associated with j, did not increase the amikacin resistance of this strain by insertion of additional copies this locus. the function of these genes was also tested by creating an isogenic knockout mutant in b. cenocepacia strain, k56-. in correlation with the gene transfer experiment deletion of genes bcal1755- in mutant k56-2Δl1755- did not result in increased susceptibility to aminoglycosides or any other antimicrobial tested. bcam0296 multidrug efflux system protein increased susceptibility for azithromycin bcam2187 abc-type efflux carrier protein a the following antibiotics and antimicrobials were tested on all mutants, except k56-2Δs0167: amikacin, tobramycin, meropenem, ceftazidime, trimethoprim, azithromycin, erythromycin, levofloxacin, tetracycline, chloramphenicol, chlorhexidine the amikacin-resistant mutant j2315-a demonstrated upregulation of different genes/gene clusters compared to the wild type, regardless of the present or absence of the antibiotic. most conspicuous was the upregulation of a gene cluster on the large chromosome, bcal3148- one of these genes, bcal encoded a sigma factor and an adjacent anti-sigma factor, bcal the bcal sigma factor is highly conserved among burkholderia species. ecf sigma factors are involved in sensing and regulating the response to changes in the environment, including maintaining cell envelope integrity under stress. the bcal3148- cluster was not induced in the wild type by sub-inhibitory concentrations of amikacin, but its constitutive upregulation in j2315-a suggested it may play a major role in the general stress response linked to the spontaneous adaptation to an elevated state of antimicrobial resistance. another gene cluster found to be constitutively upregulated in j2315-a was a putative acetyltransferase, bcam0829a, and a nearby abc transporter gene, bcam. limited upregulation of bcam0829a was observed when j was exposed to amikacin but this was confirmed by qpcr. acetyltransferases are known mediators of aminoglycoside resistance and abc transporters often contribute towards multi-drug resistance in e. coli, hence, both genes, bcam0829a and bcam have plausible roles in functionally mediating resistance to amikacin. however, transfer of the acetyltransferase bcam0829a into b. vietnamiensis g or b. cenocepacia k56- failed to alter their susceptibility to amikacin, gentamicin and tobramycin, and deletion of the associated abc transporter gene bcam in mutant k56-2Δm also did not reveal an antimicrobial phenotype. gene expression in j and j2315-m in response to meropenem the global gene expression response of b. cenocepacia to meropenem was characterised by upregulation of predictable factors such as beta-lactamases and of novel enzymes like those linked to the phenylacetic acid degradation pathway. bcas a class c beta-lactamase encoded on the small chromosome was the most upregulated gene seen when the wild type was exposed to meropenem; a class a beta-lactamase, bcam encoded on the medium chromosome was also highly expressed. interestingly, neither beta-lactamase was significantly altered in expression in the meropenem resistant mutant in the absence of the antibiotic, indicating that these enzymes were not primarily involved in the elevated resistance phenotype of j2315-m. located bp upsteam of the class a beta-lactamase bcam was a lysr family regulator bcam with 90% homology to and in a location corresponding to that of penr previously described in "p. cepacia". this region is conserved in all completed burkholderia genomes and trépanier et al. showed that penr controlled the transcription of the class a beta-lactamase pena with a putative binding site mapped to a region bp upstream of the beta-lactamase, directly adjacent to the regulator sequence. a motif search revealed the same binding site in j upstream of both bcam and bcas suggesting that they may be co-regulated and possibly under the control of the bcam regulator. although both beta-lactamases were upregulated in the presence of meropenem, it was not known which, if any, is active against the antibiotic. carbapenem hydrolyzing class a beta-lactamases have been reported for klebsiella pneumoniae. a class a penb beta-lactamase has been characterised in b. cenocepacia that is active against penicillin and cephalosporins, but has only a marginal ability to degrade meropenem and imipenem. a plasmid-encoded class c beta-lactamase with weak imipenemase activity has also been reported. to clarify the involvement of bcam in meropenem inactivation by b. cenocepacia, clavulanic acid was tested as a class a-specific beta-lactamase-inhibitor, but it failed to reduce the mic for meropenem even at concentrations as high as mg/l. the role of the class c beta-lactamase, bcas was tested by complementation into b. vietnamiensis g which lacks this gene. the resulting transformant showed an increase in mic for cefotaxime, ceftazidime, imipenem and piperacillin, but the mic for meropenem and aztreonam remained unchanged from that of wild type b. vietnamiensis g when the same construct was mobilized into b. cenocepacia k56- the mic for cefotaxime and ceftzidime increased, but the remaining antibiotic mics were unaltered. addition of chlorpromazine to the complemented strains did not alter their antibiotic susceptibility further. a functional role for bcas in mediating high level beta-lactam resistance was therefore supported, but interestingly not for meropenem as a specific substrate for this class c beta-lactamase. three gene clusters annotated as phenylacetic acid degradation enzymes were upregulated in mutant j2315-m regardless of the presence or absence of the meropenem. in b. cenocepacia, these genes are organised as follows: two gene clusters on the large chromosome and one gene cluster on the medium chromosome. all three clusters appeared largely co-regulated from the microarray data and a meme search revealed a common regulatory motif for all three locations, upstream of bcal bcal and bcam respectively. a mast analysis revealed the same regulatory motif to be present upstream of the pa degradation genes in all genome sequenced burkholderia species. upregulation of bcal bcal and bcal1711/ was confirmed by qpcr. two sets of genes involved in glucose transport and metabolism via the pentose phosphate pathway were substantially down regulated in the resistant mutant j2315-m: bcal1804- and bcam1356-. growth curve analysis showed that j2315-m grew at less than half the growth rate on glucose than j the pa catabolic enzymes have been shown to be under glucose catabolite repression in b. cenocepacia. our data therefore suggested that for j2315-m to achieve the upregulation of pa genes and increase its meropenem resistance, it had to down regulate the glucose transport and metabolism genes to lower the intracellular glucose concentration. the resulting slower growth rate of j2315-m in presence of glucose may also be an advantageous phenotype for surviving antimicrobial exposure, analogous to the alterations seen in small colony variants. to elucidate if the pa catabolic enzymes aid resistance to antibiotics, mic values in the presence and absence of glucose in lb medium were determined. the presence of glucose resulted in reductions of ≥ 2-fold in j2315's mic for meropenem, ceftazidime, chloramphenicol and tetracycline; no change was observed for amikacin, tobramycin, erythromycin, levofloxacin and trimethoprim-sulfamethoxazole. a panel of closely related b. cenocepacia were also tested and out of also demonstrated a reduction in meropenem mic in the presence of mm glucose. analysis of pa gene expression directly correlated to these phenotypic observations, being considerably reduced in j in the presence of glucose. expression of this gene was unaltered by addition of glucose for b. cenocepacia k56- which did not demonstrate the same mic reduction for meropenem. the strain to strain variability of this putative catabolite repression response suggest that the regulation of central metabolism in b. cenocepacia is highly complex; given the number of paralogous metabolic pathways present in burkholderia genomes such plasticity is not surprising. qpcr showed that the expression of the beta-lactamases was not affected by the presence of glucose in j indicating that these degradative antibiotic resistance enzymes were not catabolite repressed. the role of the pa degradation enzymes in burkholderia is not yet fully understood, but recently it was shown that pa pathway is necessary for full pathogenicity during caenorhabditis elegans infection. the linkage of pa degradation to the phenomenon of spontaneous antibiotic resistance in b. cenocepacia adds further complexity to the central role aromatic metabolism plays in the biology of these microorganisms. gene expression in j wt and j2315-t in response to trimethoprim-sulfamethoxazole the gene encoding a homologue of aida, a quorum sensing regulated gene required for full virulence of b. cenocepacia h during c. elegans infection was the most upregulated gene when wild type j was exposed to sub-inhibitory levels of tmp-smx. other quorum sensing regulated genes such as the zinc metalloprotease, zmpa, and the lectin encoding bcam were also upregulated. few other significant alterations were made by j in response of sub-lethal amounts of tmp-smx. deletion of bcas together with the adjacent bcas in mutant k56-2Δs0293- did not alter its susceptibility to trimethoprim, sulfamethoxazole, or any other antimicrobials. the tmp-smx resistant mutant j2315-t also altered the expression of a limited number of genes, however, the most conspicuous change it made was a strong and constitutive upregulation of an efflux pump of the rnd family, bcam2549-. this is the j homologue of the ceo multidrug efflux pump operon described by nair et al. consisting of the three parts of the efflux pump and a hydrolase/lipase. the mic data for j2315-t showed increased resistance to trimethoprim, fluorquinolones, chloramphenicol and tetracyclines, a phenotype consistent with the substrate spectrum of the ceo efflux pump described by nair et al. and thus corroborating the microarray expression data. upstream and divergently transcribed from the ceo efflux pump is a lysr family regulatory protein, bcam that is a homologue of the ceor transcriptional activator. this regulator was constitutively upregulated in mutant j2315-t. nair et al. had also shown that the pump was upregulated in the presence of salicylate. in contrast to their findings made on b. cenocepacia strain pc, where salicylate and chloramphenicol were found to induce expression of the ceo efflux pump, we found that both the efflux pump and ceor in b. cenocepacia j and k56- were not inducible by salicylate or sub-inhibitory levels of tmp-smx. gene expression in j wt in response to chlorpromazine exposure of b. cenocepacia to this potentiator of aminoglycoside activity resulted in significant upregulation of two efflux gene clusters, bcam0923- and to a lesser extent, bcam2186-. no specific link to amikacin-related gene expression, the antibiotic most potentiated by chlorpromazine, was observed. the bcam2186- pump had homology to a macrolide-specific abc-type efflux pump system in e. coli. transfer of the efflux pump genes to b. vietnamiensis g or b. cenocepacia k56- did not alter the antimicrobial susceptibility of these strains. likewise creation of a b. cenocepacia deletion mutant k56-2Δm2186- did not produce an observable phenotype in relation to the antibiotics examined in this study. in relation to the other chlorpromazine activated efflux system, bcam and bcam were closely related to mexd and mexc, respectively, components of the mexcd-oprj efflux pump in p. aeruginosa. bcam an outer membrane protein, is most similar to oprm, which is part of the mexab-oprm efflux pump in p. aeruginosa. bcam is the response regulator part of a two component regulatory system that may control expression of the efflux pump; its role was investigated by the construction of a deletion mutant k56-2Δm. the mutation did not alter its chlorpromazine or antibiotic mic. the expression of the structural pump genes, bcam0926- was also still induced by chlorpromazine in the deletion mutant suggesting that bcam was not critical for regulation of the adjacent efflux pump. to further investigate the role of the efflux pump structural genes bcam0925- a k56- deletion mutant spanning all genes was constructed. the k56-2Δm0925- mutant demonstrated increased susceptibility to the disinfectant chlorhexidine and the antibiotic azithromycin, but no alteration in susceptibility to other antibiotics. growth curve analysis of the k56-2Δm0925- mutant revealed a 10% slower growth rate in the presence of chlorpromazine compared to the parent strain. qpcr analysis of the k56- parent strain showed that chlorhexidine, as well as chlorpromazine induced the efflux pump gene bcam 345-fold in the presence of μg/ml chlorhexidine and 125-fold in the presence of mm chlorpromazine. chlorhexidine, like chlorpromazine is a cationic compound known to interact with and damage cellular membranes, hence it is logical that both compounds induce the efflux pump and that the efflux mutant demonstrated sensitivity to this disinfectant. chlorhexidine is also known to induce the pseudomonas aeruginosa mexcd-oprj efflux pump. since the bcam0925- efflux operon shares considerable homology with latter p. aeruginosa system, the mutant phenotype also suggests that the pump may play a role in chlorhexidine efflux in b. cenocepacia. why deletion of the pump led to an alteration in susceptibility to azithromycin is not known. another gene cluster upregulated in the presence of chlorpromazine was a squalene-hopene cyclase, bcas and an adjacent, divergently transcribed response regulator, bcas. squalene-hopene cyclase is involved in the synthesis of hopanoids, the prokaryotic equivalent of steroids which may function to counter the effect of membrane disrupting agents on bacterial cells. a deletion mutant of bcas k56-2Δs demonstrated reduced growth rate in the presence of chlorpromazine and chlorhexidine, as well as an increased susceptibility to chlorhexidine. since the squalene-hopene cyclase gene and surrounding region is highly conserved in several bcc genomes these data suggest it plays an integral role in the intrinsic resistance of burkholderia to membrane disrupting antimicrobial agents. gene expression in the pan-resistant cf outbreak isolates bcc and bcc1617 the most pronounced change in expression profiles of both et outbreak isolates was the downregulation of genes involved in flagella production and in chemotaxis compared to j bcc and bcc also downregulated expression of their cable pilus genes. the lack of flagella and chemotaxis gene expression correlated directly to the non-motile phenotype of the outbreak isolates, in contrast to j which was motile. during rapid growth in a sputum-based medium j upregulates genes involved in production of the flagellum. this may reflect a short term response of b. cenocepacia to early growth during infection, where flagella and motility serve as important virulence factors. lack of motility has been observed in p. aeruginosa cf isolates from chronic infection and as a global gene expression adaptation to growth in sputum. the stable non-motile phenotype and genotype of the recent et clinical strains suggest that b. cenocepacia may in time also adapt to the same phenotype associated with p. aeruginosa from chronic cf infection. both the outbreak isolates had upregulated genes involved in transport and efflux, restriction modification, and transposition as follows. an abc membrane transport protein and adjacent hypothetical protein with possible hydrolytic activity were highly expressed. the transport protein bcas contains both an atp binding cassette and transmembrane components, and has homology to the mdlb gene of e. coli which is implicated in multidrug resistance. deletion of bcas in mutant k56-2Δs resulted in increased susceptibility to tetracycline and chlorhexidine, but did not alter the mic for the other antimicrobial agents tested. these data validate the microarray observed upregulation of bcas and demonstrate it does play a role in the antimicrobial resistance of b. cenocepacia. also highly upregulated in both strains were genes of a type i restriction modification system. in j the first gene in this operon is a pseudogene mutated with an insertion sequence that may prevent expression of the entire downstream gene cluster. pcr amplification and sequence analysis of this region revealed that bcc and bcc do not possess the same is mutation, hence allowing overexpression of the genes as detected by the microarray analysis. mutation of the putative dna methylase, bcal in this cluster results in the attenuation of b. cenocepacia virulence in a rat lung model of chronic lung infection. the dna modification conferred by the gene cluster may also play a role in surviving the damage caused by hydroxyl radicals which are released when bacteria are exposed to bactericidal antibiotics. since the type i restriction modification cluster is clearly transcriptionally activated in the outbreak strains, it will be interesting to follow up if their expression has provided them with a resistance and virulence advantage compared to j as an ancestral et isolate. another response conserved between the two outbreak isolates was the upregulation of transposase encoding genes within isbcen. the is profiles of b. cenocepacia isolates of the same mlst strain type have been recently shown to vary markedly and oxidative stress was linked to this is movement. the upregulation of the isbcen transposase in these recent outbreak isolates may indicate that these strains are hypermutable with regard to this is. this may allow very rapid phenotypic switches to occur within individual bacteria in the infecting population and alter their pathogenicity. when examined individually, the two outbreak isolates demonstrated several global gene expression characteristics that had been observed in the antibiotic resistant b. cenocepacia j derivatives. bcc upregulated the pa degradation enzyme genes, that were also constitutively upregulated in j2315-m. these data re-enforce the hypothesis that the ability of b. cenocepacia to switch metabolism to alternative pathways in terms of aromatic hydrocarbon degradation plays a critical role in its spontaneous antibiotic resistance. it had also upregulated the class a beta-lactamase that was induced in j and j2315-m upon meropenem exposure. phenotypically, bcc possessed the same elevated resistance to beta-lactam antibiotics that j2315-m had developed after meropenem selection, and its meropenem mic could be reduced by growth in the presence glucose. genes found upregulated in the amikacin-resistant j2315-a derivative were also constitutively over-expressed in bcc again correlated to high aminoglycoside resistance of this isolate. several differentially expressed genes were specific to isolate bcc genes involved in the p-hydroxybenzoate degradation pathway were highly upregulated. since the pa pathway genes were not significantly altered in bcc it would be interesting to follow up if the p-hydroxybenzoate degradation pathway may provide an alternative metabolic shift for b. cenocepacia to evolve its antibiotic resistance. a specific resistance determinant upregulated in bcc was an efflux pump that is a homologue of the amrab and of bpeab systems encoded by b. pseudomallei. both the latter systems mediate resistance to aminoglycosides and macrolides in b. pseudomallei and it was notable that bcc possessed very high levels of resistance to tobramycin, azithromycin and erythromycin. an adjacent gene bcal encoding a transcriptional regulator and inactive in j due to a frameshift mutation, does not have the frameshift in bcc as revealed by sequence analysis. hence, this could in turn allow the upregulation of the efflux pump in the outbreak strain compared to j bcc also over expressed the phosphohydrolase bcal which was stably upregulated in the j2315-a amikacin exposed mutant. other factors possibly contributing to the overall antibiotic resistance in bcc compared to j is the absence of a frameshift mutation in gene bcal a transport protein annotated as tetracycline resistance protein, and absence of an insertion sequence in bcam annotated as multidrug resistance transporter. both latter genes are pseudogenes in j and also in bcc. although their expression did not change in bcc by just being functionally expressed they may mediate increased resistance to antibiotics.  our novel transcriptomic analysis of the response of b. cenocepacia to antibiotic exposure has demonstrated that it expresses a multitude of genes and different pathways to achieve high levels of resistance. b. cenocepacia j and the clonally related strains we studied are phenotypically and transcriptomically very versatile and can adapt quickly to environmental conditions. a key finding was that once certain antibiotic resistance phenotypes in b. cenocepacia had been selected by a single antibiotic, the changes in the transcriptome were not transient, and were stably maintained in the spontaneous antibiotic resistant mutants. this finding suggests that mutations selected in the spontaneously resistant b. cenocepacia j mutants are stably inherited and do not revert at a high frequency. a change in antibiotic therapy may therefore not reverse a previously acquired b. cenocepacia spontaneous antibiotic resistance trait. our molecular analysis corroborates the results of antibiotic susceptibility testing that demonstrate focussing on a single antibiotic or antibiotic class for therapy has limited efficacy and that multiple antibiotic combinations offer the best means to combat the spontaneous resistance of this pathogen. we have identified multiple novel molecular pathways b. cenocepacia utilises to resist sub-mic levels of antibiotic. however, for many of the genes altering expression in relation to antibiotic exposure we could not prove a direct involvement resistance, with only of the b. cenocepacia antibiotic resistance gene deletion mutants demonstrating increased antimicrobial susceptibility. for spontaneous resistance to single antibiotics, b. cenocepacia expresses set of genes which are distinct from those altered by transient sub-mic exposure, and which are stably altered in expression irrespective of the presence of the priming antibiotic. these findings suggest that spontaneous antibiotic resistance in b. cenocepacia selects multiple individual as well as pleiotropic mutations, stably altering the expression of many genetic pathways; uncovering the location of these mutations by genome re-sequencing and polymorphism analysis in future studies would shed light on why these changes are stably inherited traits in b. cenocepacia. of the b. cenocepacia resistance mechanisms identified, those such as the beta-lactamases, phosphohydrolases, novel efflux pumps and phenylacetic acid pathways genes may be targeted to improve the efficacy of current antibiotics. we may also be able to harness the phenomenon of bacterial catabolite repression by glucose as a novel means to improve the efficacy of aerosolised antibiotics against certain b. cenocepacia during cf infection. 
subsequently, mm glucose was found to produce greater that 2-fold reductions in the mic of multiple antibiotics against b. cenocepacia j mutation of an rnd multidrug efflux pump locus and squalene-hopene cyclase gene, both upregulated after chlorpromazine exposure, confirmed their role in resistance. two pan-resistant et outbreak isolates recovered two decades after j were also compared to identify naturally evolved gene expression changes. spontaneous resistance in b. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. the phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. to map these pathways, transcriptomic analysis was performed on: strain j exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and on spontaneous mutants derived from j and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. global mapping of the genetic pathways which mediate antibiotic resistance in b. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy. mutation of an abc transporter gene upregulated in both outbreak strains, confirmed its role in b. cenocepacia resistance. burkholderia cenocepacia is a member of the burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. a novel role for the phenylacetic acid degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. b. cenocepacia isolate j has been genome sequenced and is representative of a virulent, epidemic cf strain. the recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance.
 burkholderia cenocepacia is a member of the burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. b. cenocepacia isolate j has been genome sequenced and is representative of a virulent, epidemic cf strain. its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. to map these pathways, transcriptomic analysis was performed on: strain j exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and on spontaneous mutants derived from j and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. two pan-resistant et outbreak isolates recovered two decades after j were also compared to identify naturally evolved gene expression changes.  spontaneous resistance in b. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. the phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. a novel role for the phenylacetic acid degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. subsequently, mm glucose was found to produce greater that 2-fold reductions in the mic of multiple antibiotics against b. cenocepacia j mutation of an rnd multidrug efflux pump locus and squalene-hopene cyclase gene, both upregulated after chlorpromazine exposure, confirmed their role in resistance. the recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. mutation of an abc transporter gene upregulated in both outbreak strains, confirmed its role in b. cenocepacia resistance.  global mapping of the genetic pathways which mediate antibiotic resistance in b. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy. 