Transcriptional responses of Burkholderia cenocepacia to polymyxin B in isogenic strains with diverse polymyxin B resistance phenotypes
© Loutet et al; licensee BioMed Central Ltd. 2011
Received: 12 July 2011
Accepted: 29 September 2011
Published: 29 September 2011
Burkholderia cenocepacia is a Gram-negative opportunistic pathogen displaying high resistance to antimicrobial peptides and polymyxins. We identified mechanisms of resistance by analyzing transcriptional changes to polymyxin B treatment in three isogenic B. cenocepacia strains with diverse polymyxin B resistance phenotypes: the polymyxin B-resistant parental strain K56-2, a polymyxin B-sensitive K56-2 mutant strain with heptoseless lipopolysaccharide (LPS) (RSF34), and a derivative of RSF34 (RSF34 4000B) isolated through multiple rounds of selection in polymyxin B that despite having a heptoseless LPS is highly polymyxin B-resistant.
A heptoseless LPS mutant of B. cenocepacia was passaged through multiple rounds of selection to regain high levels of polymyxin B-resistance. This process resulted in various phenotypic changes in the isolate that could contribute to polymyxin B resistance and are consistent with LPS-independent changes in the outer membrane. The transcriptional response of three B. cenocepacia strains to subinhibitory concentrations of polymyxin B was analyzed using microarray analysis and validated by quantitative Real Time-PCR. There were numerous baseline changes in expression between the three strains in the absence of polymyxin B. In both K56-2 and RSF34, similar transcriptional changes upon treatment with polymyxin B were found and included upregulation of various genes that may be involved in polymyxin B resistance and downregulation of genes required for the synthesis and operation of flagella. This last result was validated phenotypically as both swimming and swarming motility were impaired in the presence of polymyxin B. RSF34 4000B had altered the expression in a larger number of genes upon treatment with polymyxin B than either K56-2 or RSF34, but the relative fold-changes in expression were lower.
It is possible to generate polymyxin B-resistant isolates from polymyxin B-sensitive mutant strains of B. cenocepacia, likely due to the multifactorial nature of polymyxin B resistance of this bacterium. Microarray analysis showed that B. cenocepacia mounts multiple transcriptional responses following exposure to polymyxin B. Polymyxin B-regulated genes identified in this study may be required for polymyxin B resistance, which must be tested experimentally. Exposure to polymyxin B also decreases expression of flagellar genes resulting in reduced swimming and swarming motility.
Burkholderia cenocepacia belongs to the B. cepacia complex (Bcc), a group of Gram-negative opportunistic pathogens infecting patients with cystic fibrosis (CF) and chronic granulomatous disease [1–3]. These infections are detrimental in CF patients because the bacteria can spread between patients via social contact , and in some cases patients develop an acute and fatal infection known as "cepacia syndrome" . Treatment of Bcc infections is difficult because the bacteria are resistant to many antibiotics [5–7], including antimicrobial peptides and polymyxins [8–11], a group of compounds that have been proposed as potential new therapeutics for treatment of Pseudomonas aeruginosa lung infections in CF patients [12, 13].
We have recently proposed a two-tier model of antimicrobial peptide resistance in B. cenocepacia  with the first and most significant tier consisting of the complete lipopolysaccharide (LPS) core oligosaccharide (OS) [9, 15] and the lipid A and core OS aminoarabinose residues that are essential for the viability of B. cenocepacia [16, 17]. This tier accounts for the low binding of polymyxin B to B. cenocepacia cells and poor permeabilization of the B. cenocepacia outer membrane . The second tier consists of other mechanisms that each contribute a small amount of antimicrobial peptide resistance but that as whole contribute significantly to the high resistance of this organism .
Based on the observation that about 1% of polymyxin B-sensitive B. cenocepacia heptoseless LPS mutant cells survive treatment with 500 μg/ml of the antimicrobial peptide polymyxin B for 24 hours (Loutet and Valvano, unpublished), we hypothesized that B. cenocepacia heptoseless LPS isolates with increased resistance to polymyxin B could be obtained. We cultured a polymyxin B-sensitive B. cenocepacia heptoseless LPS mutant, RSF34 , in a way that allowed for the isolation of clones with increased resistance to polymyxin B to identify other mechanisms of antimicrobial peptide resistance in this highly resistant organism. RSF34 has a polymyxin B minimum inhibitory concentration-50 (MIC50) of 32 μg/ml which is much lower than the full-length LPS strain from which it was derived, K56-2, which has a polymyxin B MIC50 of > 1024 μg/ml [14, 18]. B. cenocepacia strains with heptoseless LPS make an LPS molecule that consists of lipid A and the innermost core oligosaccharide sugars: a trisaccharide of 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), D-glycero-D-talo-oct-2-ulopyranosonic acid (Ko) and 4-amino-4-deoxy-L-arabinose (L-Ara4N). Our isolation procedure led to the generation of heptoseless LPS strains with an increasing range of polymyxin B resistance levels, some with at least 40-fold greater resistance than RSF34. Next, we determined how B. cenocepacia responds at the transcriptional level after treatment with polymyxin B, as a strategy for identifying additional mechanisms of antimicrobial peptide resistance in B. cenocepacia. We used three strains for this study: K56-2, the parental clinical isolate that is highly resistant to polymyxin B , RSF34, and RSF34 4000B, the isolate of RSF34 selected for the highest level of polymyxin B resistance. We established the baseline differences in transcription between the strains in the absence of polymyxin B challenge and identified genes transcriptionally regulated by the presence of polymyxin B in the three strains.
Isolation and characterization of polymyxin B-resistant B. cenocepacia heptoseless LPS clones
Strains and plasmids used in this study
Strain or Plasmid
Source or Reference
F-, ϕ80 lacZ ΔM15 Δ(lacZYA-argF) U169 deoR endA1 recA1 hsdR17 (rK- mK+) thi-1 glnV44
CF clinical isolate (polymxyin B-resistant)
K56-2, ΔhldA (polymyxin B-sensitive)
RSF34 colony isolated on 25 μg/ml polymyxin B
RSF34 25A colony isolated on 200 μg/ml polymyxin B
RSF34 200E colony isolated on 1 mg/ml polymyxin B
RSF34, selected through multiple rounds for polymyxin B resistance
K56-2, ΔfliCD, flagella-negative
Non-CF clinical isolate
RK2 derivative, KmR mob+ tra+ oricolE1
B. cenocepacia expression plasmid, rhamnose-inducible promoter, TpR CmR
pSL6, B. cenocepacia hldAD
Next, we tested whether or not the changes are stable or lost in the absence of selective pressure. Cells passaged for five days in the absence of polymyxin B grew as well on plates containing polymyxin B as cells grown only overnight in the absence of polymyxin B (Additional file 3 Figure S3). From this we concluded that the changes that have occurred in our polymyxin B-resistant RSF34 isolates are likely constitutive.
To measure cell envelope permeability, bacteria were treated with 1-N-phenylnaphthylamine (NPN). In a hydrophobic environment and excited at 350 nm, NPN will emit at 420 nm. When treated with NPN, RSF34 emitted at 420 nm at about twice the level as K56-2 (Figure 2B). The polymyxin B-resistant RSF34 isolates had decreased permeability to NPN compared to RSF34, with the permeability of RSF34 strains 200E, 1000D, and 4000B roughly 30% less than RSF34 (Figure 2B, p-values between 0.006 and 0.05). None of the polymyxin B-resistant isolates had permeability reduced to the level of the wild-type LPS strain K56-2 (Figure 2B). The decreased permeability of the polymyxin B-resistant RSF34 strains, suggest LPS-independent changes in the outer membrane of these mutants leading at least in part to increased polymyxin B resistance.
Consistent with this conclusion, changes in colony morphology were also noted: K56-2 colonies are round with smooth margins (Additional file 4 Figure S4), while those of RSF34 are irregularly shaped, have irregular margins and appear to have the dry and brittle morphology described as "crunchy" by Parker et al . The colony morphology of RSF34 25A is similar to RSF34, but RSF34 200E, RSF34 1000D, and RSF34 4000B all form colonies that are more similar to those of K56-2 (Additional file 4 Figure S4). Although the causes of these changes are unknown, they are likely independent of the presence of heptoseless LPS, which is common to all mutant strains.
Together, these results indicate that the selection process provided a series of clonal isolates with: (i) increasing polymyxin B resistance, (ii) a stable polymyxin B resistance phenotype, (iii) no detectable changes in the original heptoseless LPS structure of RSF34, (iv) no significant changes to bacterial fitness, (v) no decreased polymyxin B binding to whole cells, (vi) some decrease in membrane permeability, and (vii) a return to the colony morphology of the parental K56-2 strain. The absence of changes in LPS structure in the RSF34 4000B mutant, suggests additional LPS-independent mechanisms of resistance most likely targeting the bacterial outer membrane.
RSF34 1000D and RSF34 4000B have increased adherent growth in the presence of high concentrations of polymyxin B
Increased resistance is neither specific to polymyxin B nor a general phenomenon
The polymyxin B-resistant isolates were tested for increased resistance to honey bee melittin, an antimicrobial peptide that is structurally unrelated to polymyxin B. There were modest increases in melittin resistance in RSF34 200E, RSF34 1000D, and RSF34 4000B (all of which grew similarly in melittin), but the growth of all of the heptoseless LPS isolates was inhibited by at least 75% in 200 μg/ml melittin (Figure 3B).
Disk diffusion assay results
Zone of inhibition (mm)*
11.3 ± 0.4
22.0 ± 0.4
17.3 ± 1.1
18.6 ± 0.6
17.4 ± 0.3
15.6 ± 0.1
21.1 ± 0.3
27.2 ± 0.5
16.8 ± 1.4
20.6 ± 0.1
18.3 ± 0.5
36.7 ± 0.7
17.9 ± 0.6
29.3 ± 0.2
17.0 ± 0.7
23.8 ± 0.6
18.2 ± 0.8
35.9 ± 0.5
17.8 ± 0.4
30.0 ± 0.4
17.9 ± 0.9
22.7 ± 0.8
19.4 ± 0.1
25.9 ± 1.3
18.3 ± 0.8
30.0 ± 0.4
16.1 ± 1.6
21.2 ± 0.5
19.3 ± 0.5
18.2 ± 0.8
19.9 ± 0.5
29.8 ± 0.3
17.2 ± 1.6
14.2 ± 1.1
20.0 ± 0.5
17.1 ± 0.8
Experimental approach for microarray analysis
To obtain a comprehensive view of the transcriptional response of B. cenocepacia to polymyxin B, and to gain greater insight into the changes that have occurred through our selection process, microarray analysis was conducted on the transcriptional response to treatment with sub-inhibitory concentrations of polymyxin B in three strains (K56-2, RSF34, and RSF34 4000B). For each of the strains we compared transcription in the presence of polymyxin B to transcription in the presence of a vehicle control. Additionally, to obtain baseline differences between the strains we also compared transcription in the presence of the vehicle control between K56-2 and RSF34, and between RSF34 and RSF34 4000B. Concentrations of polymxyin B used in these studies were 25 μg/ml for RSF34 and 500 μg/ml for K56-2 and RSF34 4000B. These were the highest concentrations tested that did not inhibit growth of the strains under the conditions described in the methods (data not shown).
Establishment of baseline differences between K56-2, RSF34, and RSF34 4000B
Real-time PCR validation of selected genes identified by microarray analysis as differentially regulated
Gene (predicted function of encoded protein)
Genes differentially regulated in RSF34 compared to K56-2
BCAL3490 (Exported protein)
BCAM0083 (Hypothetical protein)
BCAM0537 (Serine peptidase)
BCAM0855 (UDP-glucose dehydrogenase)
BCAM1010 (putative UTP-glucose-1-phosphate)
Genes differentially regulated in RSF34 4000B compared to RSF34
BCAL1083 (Exported alkaline phosphatase)
BCAL1213 (2-oxoisovalerate dehydrogenase β subunit)
BCAL1270 (Phosphate transport, periplasmic)
BCAM0537 (Serine peptidase)
BCAM0855 (UDP-glucose dehydrogenase)
BCAM2195 (AMP-binding enzyme)
Genes differentially regulated in K56-2 upon polymyxin B treatment
BCAL0114 (Flagellin, FliC)
BCAL0140 (Flagellar biosynthesis)
BCAL0520 (Flagellar hook-length control, FlhB)
BCAL0566 (Flagellar basal body rod modification, FlgD)
BCAL1351 (Exported Protein)
BCAM0083 (Hypothetical protein)
BCAM2187 (Macrolide-specific ABC-type efflux)
Other genes significantly overexpressed in RSF34 compared to K56-2 that are not then altered between RSF34 4000B and RSF34 include many genes predicted to encode exported proteins, lipoproteins, as well as proteins involved in cell envelope biogenesis, carbohydrate transport and metabolism, an efflux system, and a lectin (Additional file 5 Table S1). BCAL3490, BCAM0083, BCAM0186, and BCAM1010 were chosen as representative up-regulated genes and this was confirmed by qRT-PCR (Table 3).
There were few genes downregulated to a large extent in RSF34 compared to K56-2 (Additional file 5 Table S1). The gene BCAL2945 shown in Additional file 5 Table S1 to be down-regulated 87.4-fold in RSF34 compared to K56-2 is hldA, the gene deleted in RSF34. Otherwise the only other gene down-regulated by 5-fold or more in RSF34 is BCAL0114 that encodes flagellin. This is consistent with the observation that RSF34 is less motile than K56-2 (Additional file 6 Figure S5); however, qRT-PCR found no differences in BCAL0114 expression between RSF34 and K56-2 (Table 3). All other genes are downregulated by 4-fold or less and more than 50% of these genes were predicted prophage-related genes in the genomic island BcenGI12, which spans from BCAM1024 to BCAM1096 [26, 27]. Again, there is reciprocal regulation of the genes in BcenGI12 and they are upregulated in RSF34 4000B compared to RSF34 (Additional file 5 Table S1).
Finally there were genes differentially expressed in RSF34 4000B compared to RSF34 that were not significantly altered between RSF34 and K56-2 (Additional file 5 Table S1). These genes include two clusters (BCAL1212 to BCAL1215 and BCAM2191 to BCAM2196) that are overexpressed by 10-fold or more and are predicted to encode proteins involved in energy production and lipid metabolism. Genes down-regulated by 10-fold or more include one cluster (BCAL1270 to BCAL1276) predicted to encode a phosphate ABC transport system and BCAL1083, which encodes a predicted exported alkaline phosphatase. qRT-PCR analysis of representative genes from these clusters (BCAL1213, BCAL2195, BCAL1270, and BCAL1083) confirmed these patterns of expression (Table 3). These genes are of interest because their changes in expression could contribute to the increased resistance of RSF34 4000B to polymyxin B compared to RSF34. With the baseline differences between K56-2, RSF34, and RSF34 4000B established, we next sought to investigate the polymyxin B transcriptional responses made by each of these three strains.
Cell motility associated genes are downregulated by B. cenocepacia K56-2 upon polymyxin B treatment
Swimming and swarming motility are impaired by polymyxin B
Assays were also conducted to determine if swarming motility is impaired by polymyxin B. K56-2 was able to swarm across plates containing the vehicle control (Figure 7D and 7F). The flagella-negative mutant RSF44 also grew across the surface of the plates with the vehicle control, although to a much less extent than K56-2 (Figure 7D and 7F). Motility of both of these strains was highly impaired in the presence of 500 μg/ml polymyxin B (Figure 7E and 7F). Swarming motility results in the absence of polymyxin B for RSF44 are similar to those reported for a Pseudomonas aeruginosa mutant lacking both flagella and the type IV pili, which began to undergo sliding motility when grown under conditions required for swarming motility . The authors of this study also found that sliding and swarming motility resulted from similar environmental cues, which could explain why both K56-2 and RSF44 appear non-motile in the presence of polymyxin B. These types of analyses were not completed for RSF34 and RSF34 4000B because both strains have motility similar to RSF44 in the absence of polymyxin B (Additional file 6 Figure S5).
Polymyxin B treatment upregulates genes with diverse functions
Treatment of K56-2 with polymyxin B led to the upregulation of thirty genes (Sheet 1 of Additional file 7 Table S2), predicted to encode proteins involved in a variety of pathways, particularly lipid transport and metabolism and cell envelope biogenesis (Figure 6). Genes that were amongst the most highly overexpressed include: a cluster spanning BCAM0082 to BCAM0084 that contains genes encoding proteins with predicted sugar modifying and transferase activities, BCAM1364, encoding a predicted NAD dependent epimerase/dehydratase, and BCAM2186 to BCAM2188, encoding a predicted macrolide efflux system. qRT-PCR conducted for BCAM0083 and BCAM2187 demonstrated that both genes were overexpressed (Table 3). qRT-PCR experiments were also attempted for BCAM1364. However, these experiments failed, as we could not obtain a primer pair that efficiently amplified the transcript or the gene from genomic DNA (data not shown). The genes upregulated upon treatment with polymyxin B are of interest as they may represent novel genes involved in the resistance of B. cenocepacia to polymyxin B.
Regulation by polymyxin B in RSF34 and RSF34 4000B
Since we have previously identified genes involved in polymyxin B resistance in RSF34 , we thought that it might be possible to identify additional polymyxin B-responsive genes in this strain that were not identified in K56-2. In total, 59 genes were upregulated in RSF34 upon polymyxin B treatment (Sheet 2 of Additional file 7 Table S2) and of the 30 genes upregulated by K56-2 in the presence of polymyxin B, more than a third appeared in the RSF34 data set as well. Of the remaining 48 genes upregulated in RSF34 in the presence of polymyxin B only two are upregulated by five-fold or more. The only cluster that stood out in this data set was a group of genes, BCAL1349 to BCAL1351, predicted to encode a two-component regulatory system and an outer membrane protein, transcribed in opposite orientations. This cluster is also upregulated in K56-2 in the presence of polymyxin B but not to the same extent (less than four-fold). qRT-PCR experiments for BCAL1351 using RNA extracted from K56-2 grown in the presence or absence of polymyxin B indicated that this cluster of genes is upregulated in the presence of polymyxin B (Table 3). Almost all genes downregulated by RSF34 in the presence of polymyxin B are involved in the assembly or function of the flagellum (Sheet 2 of Additional file 7 Table S2).
The picture is quite different in RSF34 4000B; 204 genes were found to be upregulated in the presence of polymyxin B, all of which were upregulated by less than five-fold (Sheet 3 of Additional file 7 Table S2). This list of genes did not include some of the largest changes described above, including the cluster spanning BCAM0082 to BCAM0084, or the fold-changes were less than those seen in K56-2 and/or RSF34. Only 32 genes were downregulated by RSF34 4000B in the presence of polymyxin B, a third of which were tRNA-encoding genes, and none of which were flagellar-related genes (Sheet 3 of Additional file 7 Table S2).
Two genomic approaches were utilized to study polymyxin B resistance in B. cenocepacia. A lineage of increasingly polymyxin B-resistant heptoseless LPS mutants was obtained through selection of resistant isolates on media that prohibited the replication of the vast majority of the cells plated. At each round of selection approximately one polymyxin B-resistant isolate per 105 CFU plated was obtained. Initial characterization of these isolates shows that they exhibited: (i) increased polymyxin B resistance that is relatively stable, since it is maintained after the cells are grown in the absence of polymyxin B for five days; (ii) no defects in growth rate, suggesting that the mutation or mutations do not affect the general fitness of the bacteria, and (iii) increased polymyxin B resistance in liquid media despite the fact that selection of the isolates occurred on solid media. These isolates show significant increases in resistance to imipenem and melittin, and in the case of the final isolate (RSF34 4000B), to gentamicin.
Although it must be noted that the pleiotropic changes seen in the polymyxin B-resistant isolates may not all contribute to the polymxyin B resistance of the isolates, many of the phenotypic changes seen in the isolates could be associated with increased antimicrobial peptide resistance. Subpopulations of P. aeruginosa and E. coli within biofilms [34, 35] have been shown to develop increased antimicrobial peptide resistance and the results presented here suggest that high concentrations of polymyxin B induce increased adherent growth of RSF34 1000D and RSF34 4000B, possibly resulting in protection of bacterial cells within the adherent growth from polymyxin B. The heptoseless LPS phenotype induces outer membrane instability that has pleiotropic effects on bacteria [36, 37] including changes in colony morphology changes [21, 38, 39]. The return to the wild-type colony morphology in RSF34 200E and later isolates could indicate that the polymyxin B-resistant isolates may have altered outer membrane properties that in some way stabilize the outer membrane. This interpretation is consistent with our observation of decreased NPN access to the outer membrane in the polymyxin B-resistant isolates. Experiments are currently underway to precisely determine the genes and/or proteins whose expression and/or function have been altered through the selection process to increase the polymyxin B resistance of RSF34.
Microarray analysis conducted to compare baseline changes in the gene expression between K56-2, RSF34 and RSF34 4000B show that there are many genes whose expression are substantially upregulated in RSF34 compared to K56-2 (Additional file 5 Table S1, and Table 3). The types of genes differentially expressed in RSF34 compared to K56-2 are consistent with observations in the literature that heptoseless LPS mutants tend to alter the synthesis of other polysaccharides [21, 38, 39], The data are also similar to a microarray study of a Salmonella enterica serovar Typhimurium heptoseless LPS mutant which showed changes in sugar metabolism and expression of genes predicted to encode outer membrane proteins and lipoproteins, as well as decreased expression of genes required for the flagellum . These types of changes are likely the consequence of the pleiotropic effects seen in heptoseless LPS mutants [36, 37]. Interestingly, RSF34 4000B reverses some but not all of these changes (Additional file 5 Table S1, and Table 3). This is similar to some of the phenotypic changes seen in this strain such as return to wild-type colony morphology (Additional file 4 Figure S4) and intermediate permeability to NPN (Figure 2B). Since some of the genes overexpressed in RSF34 are downregulated in RSF34 4000B, this isolate could have an altered cell envelope that decreases these pleiotropic effects, and also makes it less susceptible to polymyxin B challenge. There are also genes (BCAL1212 to BCAL1215, BCAM2191 to BCAM2196, BCAL1270 to BCAL1276, and BCAL1083) whose expression is significantly altered in RSF34 4000B compared to RSF34 but are not altered between K56-2 and RSF34. These changes in gene expression may contribute to the increased polymyxin B resistance in RSF34 4000B, which we are currently studying using mutagenesis strategies.
Downregulation of motility-associated gene expression is a major transcriptional response in B. cenocepacia upon treatment with polymyxin B under the conditions tested in this study which results in impairment of swimming and swarming motility in the presence of polymyxin B. None of the genes that have previously been implicated in the resistance of B. cenocepacia to antimicrobial peptides [9, 14–16, 41] were differentially regulated by polymyxin B under the conditions tested. It is possible that other conditions (such as higher concentrations of polymyxin B or treatment with other antimicrobial peptides) are required to see differential regulation of these genes. It is also just as possible that expression of these antimicrobial peptide resistance genes are not regulated by the presence of antimicrobial peptides in the environment and that major resistance mechanisms are constitutively active in B. cenocepacia, which could help to explain in part the high resistance of B. cenocepacia to these compounds. There are various genes upregulated by both K56-2 and RSF34 grown in the presence of polymyxin B, including genes encoding proteins involved in lipid transport and metabolism, cell envelope biogenesis, signal transduction, and transcription, as well as genes of unknown function. Characterization of potential roles in polymyxin B resistance for these genes is currently underway.
At least two microarray studies have been published on P. aeruginosa and its response to antimicrobial peptides that have identified differential regulation of genes associated with motility. Cummins et al showed that exposure of planktonic cells to subinhibitory concentrations of colistin (polymyxin E) lead to small decreases in expression of genes associated with motility . The authors of this study also found that the response of P. aeruginosa to colistin included upregulation of both known colistin resistance genes as well as genes that had not previously been shown to be involved in resistance (such as genes involved in quorum sensing and biofilm formation). Meanwhile, Overhage et al showed that exposure of P. aeruginosa grown as biofilms in the presence of subinhibitory concentrations of LL-37 led to increased expression of type IV pili genes and decreased expression of flagella genes . Phenotypically, the presence of subinhibitory concentrations of LL-37 led to increased twitching motility (which requires pili ), but had no effect on swarming or swimming motility.
Swarming motility is a multicellular bacterial lifestyle  and there are conflicting reports in the literature as to whether or not this protects bacteria from antimicrobial peptides. Lai and colleagues  showed that swarming cells of Escherichia coli, P. aeruginosa, and Bacillus subtilis were more resistant than planktonic cells to numerous antibiotics, except for the antimicrobial peptides polymyxin B and colistin; while Kim et al  showed that swarming Salmonella enterica serovar Typhimurium cells were more resistant than planktonic cells to polymxyin B and colistin. Our data indicates that a subinhibitory concentration of polymyxin B greatly impairs the ability of B. cenocepacia to both swim and swarm.
We demonstrate that it is possible to obtain heptoseless LPS strains of B. cenocepacia with high resistance to polymyxin B, and suggest that this may occur through LPS-independent changes that stabilize the outer membrane in some way. Furthermore, our data demonstrate that major transcriptional changes made by B. cenocepacia upon treatment with polymxyin B include downregulation of genes required for the synthesis and operation of the flagella and upregulation of a set of genes encoding proteins with diverse predicted functions. The contribution made by genes that are upregulated by B. cenocepacia upon treatment with polymyxin B to polymyxin B resistance must now be determined and is underway in the Valvano Laboratory. Decreased flagellar gene expression upon treatment with polymyxin B impairs both swimming and swarming motility, two processes that require the flagella. B. cenocepacia mutants lacking flagella have been shown to be less virulent in mice  and less able to invade A549 human respiratory epithelial cells . Additionally, upregulation of flagellar genes has been reported in B. cenocepacia when it is grown in CF sputum . Therefore, even if a therapeutically available antimicrobial peptide was incapable of killing B. cenocepacia it might still be useful for treating B. cenocepacia infections because the inhibition of motility may be detrimental to the pathogenicity of B. cenocepacia.
Bacterial strains, culture conditions, and reagents
All strains and plasmids used in this study are listed in Table 1. A clinical isolate of B. cenocepacia, strain K56-2 , and strains derived from K56-2, were used for microarray analysis. K56-2 is clonally related to the sequenced B. cenocepacia strain J2315  whose sequence was used to design the B. cenocepacia microarrays . Unless otherwise noted, all bacterial cell culturing was done at 37°C in either Luria Broth (LB) or LB solidified with 1.6% Bacto Agar. When required, antibiotics were used at the following concentrations: trimethoprim, 100 μg/ml for B. cenocepacia and 50 μg/ml for E. coli; kanamycin, 40 μg/ml for E. coli. All antibiotics and chemicals were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). All media was purchased from Becton, Dickinson, and Company (Franklin Lakes, New Jersey, USA). Polymyxin B was dissolved in 0.2% bovine serum albumin + 0.01% acetic acid, which was also used as a vehicle control in all experiments.
Isolation of colonies with increased resistance to polymyxin B
Four sequential rounds of selection were used to obtain RSF34 isolates able to grow on up to 4 mg/ml of polymyxin B. For each round of selection, cells were grown overnight to stationary phase and then diluted to approximately 5 × 106 CFU/mL in LB and 100 μl of cells (approximately 5 × 105 CFU) was plated on an LB agar plate supplemented with polymyxin B. The plate was incubated for 40 to 48 h and the colonies (typically 4-6 colonies) were selected for further study. In the first round of selection, RSF34 cells were plated on 25 μg/ml polymyxin B. In the second round of selection, one of the RSF34 isolates from the first round of selection, RSF34 25A, was used and bacteria were plated on 200 μg/ml polymyxin B. Next, an isolate from the second round of selection (RSF34 200E) was plated on 1000 μg/ml polymyxin B. Finally, an isolate from the third round of selection (RSF34 1000D) was plated on 4000 μg/ml polymyxin B and isolate RSF34 4000B was selected for study.
For complementation studies, plasmids pSL6 and pSL7 were transferred to RSF34 and the RSF34 resistant isolates by triparental mating with the pRK2013 helper plasmid . Gentamicin (50 μg/ml) was used to select against the E. coli donor and helper strains. Rhamnose (0.2% wt/vol) was used to induce gene expression from the plasmids.
For analysis by gel electrophoresis and silver staining, LPS was prepared and visualized as previously described [9, 50]. For more detailed chemical analyses, monosaccharides and fatty acids were identified using gas-liquid chromatography-mass spectrometry (GLC-MS) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) as previously described [15, 20].
PCR analysis of RSF34 mutation
Primers used in this study
Assays for resistance to antimicrobial agents
To assess the resistance of strains to polymyxin B on solid media, cells were grown overnight in LB, diluted to an optical density at 600 nm (OD600) of 1.0 and serially diluted in ten-fold increments to 10-4. Ten-μl drops were spotted on to LB-agar plates supplemented with the vehicle control, 25 μg/ml, 200 μg/ml, or 1000 μg/ml polymyxin B. Plates were incubated for 24 h and then scanned. To assess the stability of the increased resistance to polymyxin B, experiments were carried out as described above using cells that had been grown in liquid culture for 120 h in the absence of polymyxin B (with passaging of cells to fresh media every 24 h).
To assess the resistance of the strains to polymyxin B in liquid media, bacteria were grown overnight and diluted to an OD600 of 0.01. Three hundred-μl volumes were aliquoted in 1.5 mL Eppendorf tubes, polymyxin B was added at final concentrations of 0 (vehicle control), 25, 50, 100, 200, 400, 600, 800, and 1000 μg/ml, cells were grown for 24 h while rotating in a LabQuake (Barnstead Thermolyne, Dubuque, Iowa) and the final OD600 was measured. Similar experiments were performed with the antimicrobial peptide honey bee melittin, except Mueller-Hinton Broth (MHB) was used instead of LB and final melittin concentrations used were 0 (vehicle control), 5, 25, 50, 100, and 200 μg/ml.
Resistance to SDS, novobiocin, gentamicin, tetracycline, chloramphenicol, and imipenem was assessed using disk diffusion assays. Briefly cells were grown overnight, diluted to an OD600 of 0.2 and spread onto LB-agar plates. Blank paper disks were added to plates and inoculated with 8 μl of 10% (wt/vol) SDS, 0.5% (wt/vol) novobiocin, 10% (wt/vol) gentamicin, 0.5% (wt/vol) tetracycline, 0.5% (wt/vol) chloramphenicol, or 0.5% (wt/vol) imipenem. Plates were incubated for 24 h and zones of inhibition were measured.
Growth curves were completed as previously described for B. cenocepacia heptoseless mutants .
Dansyl-polymyxin B binding and 1-N-phenylnaphthylamine (NPN) permeability assays
Dansyl-polymyxin B was synthesized and quantified using the dinitrophenylation assay as previously described [51, 52]. Binding of dansyl-polymyxin B to whole B. cenocepacia cells was conducted as described by Ortega et al , cells were excited at 340 nm and emission at 485 nm was measured. Assays for NPN permeability were conducted similarly to the dansyl-polymyxin B assay except cells were treated with 20 μl of 50 μM of NPN in acetone. Cells were then excited at 350 nm and emission at 420 nm was measured.
Measurement of adherent growth in polymxyin B
Experiments were conducted as described above for the polymyxin B liquid challenge. The OD600 was recorded from cultured cells prior to vortexing tubes and after vigorous vortexing of tubes. A ratio of OD600 after vortexing to OD600 before vortexing was calculated.
Microscopic assessment of colony morphology
Cells were plated for isolated colonies on LB-agar plates and incubated for 48 h. Changes in colony morphology were recorded at 100× magnification using an Olympus IX71 inverted microscope and Image-Pro Plus Version 5.0 software.
Growth of bacteria and RNA extraction for microarray analysis
Bacteria were grown overnight and then diluted to an optical density at 600 nm (OD600) of 0.05 in 30 ml of LB. Cells were grown for 3 h to an OD600 between 0.3 and 0.4 and then treated with either polymyxin B (500 μg/ml for K56-2 and RSF34 4000B, 25 μg/ml for RSF34) or the vehicle control for 30 min (final OD600 between 0.4 and 0.5). RNA was prepared from B. cenocepacia using the RiboPure-Bacteria kit from Ambion, Inc. (Austin, TX, USA) and treated with DNAse 1 (also from Ambion), following the manufacturer's protocol; each treatment condition required RNA prepared with five columns of the kit. RNA from the five individual preparations were combined and concentrated with LiCl. Integrity of the RNA was assessed by agarose gel electrophoresis and by measuring the ratio of absorbance at 260 nm to 280 nm (values obtained between 2.0 and 2.2). RNA was subjected to a PCR reaction for the gene hisD using Taq Polymerase (Qiagen Inc., Mississauga, ON, Canada), primers hisD for and hisD rev (Table 4), and the following reaction conditions: 94°C for 2 min, 29 cycles of 94°C for 40 sec, 60°C for 40 sec, and 72°C for 40 sec, followed by a final extension at 72°C for 7 min. If a reaction product was PCR amplified the RNA sample was treated with DNAse 1 again and re-tested. RNA was prepared in three independent experiments for microarray analysis.
Labeled cDNA was synthesized from RNA and hybridized as previously described [48, 53] to a custom 2 × 11 K B. cenocepacia microarray developed with Agilent's two-color 60-mer inkjet synthesis platform (Agilent Technologies, Santa Clara, CA, USA). For each of the five comparisons (Additional file 6 Figure S5), the two sample cDNA pools were fluorescently labeled with either Cy3 or Cy5 dyes and directly compared to each other. For each of these comparisons, one of the biological replicate experiments was re-analyzed with the dyes swapped.
Data was imported into GeneSpring (version 7.3.1) and normalized using the "Agilent FE" procedure. Genes listed in the additional files were found to be significantly different between the two test conditions (t-test p-value < 0.05), passed the Benjamini-Hochberg false discovery rate test, and were differentially regulated by 2-fold or more (as an arbitrarily chosen cut-off).
The microarray dataset has been deposited in the ArrayExpress database http://www.ebi.ac.uk/arrayexpress/ under accession number E-MTAB-720.
Primers used for real-time PCR are listed in Table 4. All primer pairs had PCR efficiencies greater than 88%. RNA was extracted from 5 mL cultures as described above for bacterial growth and RNA extraction for microarray analysis. RNA was converted to cDNA using Transcriptor Reverse Transcriptase (Roche Diagnostics, Laval, Quebec, Canada) according to the manufacturer's instructions with modifications previously described . Real-time PCR reactions using FastStart SYBR Green Master (Roche Diagnostics) and a Rotor-Gene 6000 thermal cycler (Corbett Life Sciences, Sydney, Australia) were conducted as previously described . Data was analyzed using the manufacturer's software (Rotor-Gene 6000 Series Software Version 1.7). Fidelity of PCR amplifications was assessed using melt curve analysis and agarose gel electrophoresis. Fold changes in gene expression were calculated using the Pfaffl Method . All changes are relative to an internal control, hisD, a gene which previously used as an internal control for semi-quantitative and real-time PCR analysis [16, 55] and that was not found to be differentially expressed in any of the microarray analysis generated in this study.
For swimming motility, bacteria were grown overnight in liquid culture and diluted to an OD600 of 1.0 in LB. Two-μl of culture was added to semi-solid LB plates (solidified with 0.3% Bacto Agar) by puncturing the top of the agar. Plates were incubated lid side up for 24 h and growth was measured as the diameter across which the bacteria grew. Swarming motility assays were completed as above except that media consisted of Nutrient Broth + 0.2% Glucose solidified with 0.5% Bacto Agar and bacteria were spotted on to the surface of the plates.
All other statistical analyses (unpaired student's t-tests) were conducted with GraphPad Prism 4.0.
The authors thank Andrea Sass and Eshwar Mahenthiralingam from Cardiff University, who conducted the microarray hybridization experiments and assisted with microarray data analysis, as well as Hemant Kelkar and JP Jin from the Center for Bioinformatics at the University of North Carolina, Chapel Hill, who also assisted with microarray data analysis.
S.A.L. was supported by a scholarship from Cystic Fibrosis Canada. This work was supported by grants from Cystic Fibrosis Canada, the Canadian Institutes of Health Research Special Initiative on Novel Antimicrobials, and the B. cepacia microarray initiative from the U.S. Cystic Fibrosis Foundation. M.A.V. is a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis and a Cystic Fibrosis Canada Zeller's Senior Researcher Scientist. A. M. acknowledges COST Action BM1003. This work has been partially supported to A.M. by PRIN 2008 and by Italian Cystic Research Foundation, grant FFC 11#2010 with the contribution of Pastificio Giovanni Rana s.p.a.
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