<italic>In vivo</italic> studies
The earliest study on the role of iron acquisition mechanisms in the virulence of members of the Bcc was carried out prior to the discovery of the primary siderophore, ornibactin, in this group of bacteria (Sokol, 1986). Here, it was observed that from a collection of 43 “P. cepacia” CF isolates, ~50% produced detectable levels of pyochelin during iron limited growth in vitro (Pch+ phenotype). However, 86% of the Pch+ strains were associated with infections which had led to the death of the patient or were responsible for severe infections, whereas only 41% of Pch− strains were associated with such outcomes. Thus, while the ability to colonize a CF patient could not be linked to the ability to produce this siderophore, there was a link between pyochelin production and the morbidity/mortality of disease in CF patients. Similarly, while addition of exogenous pyochelin to two pyochelin-negative Bcc strains did not increase bacterial numbers or bacterial persistence in infected rat lungs, it did increase the severity of infection as assessed by lung pathology (Sokol and Woods, 1988). It was proposed that the observed enhancement of lung damage brought about by exogenous addition of pyochelin to these strains was most likely due to increased dissemination of the bacteria throughout the lungs. However, it is not clear from these studies whether it was the role of pyochelin in iron acquisition that was responsible for the more severe outcome or some other effect of the siderophore. In this regard, it is known that apart from binding iron and other metals (Cuppels et al., 1987; Visca et al., 1992; Baysse et al., 2000; Braud et al., 2009) pyochelin possesses an inherent chemical reactivity that may contribute to disease severity. For example, it can promote the degradation of organotin derivatives (Sun et al., 2006), and perhaps pertinently, it can catalyse the generation of ROS such as hydroxyl radicals that result in tissue damage (Coffman et al., 1990; Britigan et al., 1994, 1997; Adler et al., 2012). The latter property is proposed to contribute to the known antibiotic activity of this siderophore (Adler et al., 2012; Ong et al., 2016). It should be noted that one important Bcc pathogen of CF patients, B. multivorans, does not produce pyochelin (Table 1) and the clonally related B. cenocepacia CF epidemic strains K56-2 and J2315 produce very little of this siderophore (see below).
A direct genotypic-phenotypic link between iron acquisition and the virulence of B. cenocepacia was observed during an investigation of the virulence potential of ornibactin deficient mutants in rodent models of both chronic and acute respiratory infection (Sokol et al., 1999). In this study, mutants derived from the highly transmissible epidemic B. cenocepacia strain, K56-2, which contained an insertionally inactivated pvdA gene that is required for ornibactin synthesis (Figure 3), were generated by transposon mutagenesis (pvdA::Tn5-OT182) and allelic replacement (pvdA::tp). Both mutants were significantly attenuated in these models. Thus, in a rat lung chronic infection model, the number of pvdA::Tn5-OT182 bacterial cells recovered from the lung was 4 logs lower than that of the wild type K56-2 strain at 28 days post infection. Furthermore, the pvdA::tp strain could not be recovered from the lungs after the same length of time, suggesting the infection had been cleared. The degree of pathology, as determined by the amount of inflammatory cell infiltration and exudate in the lungs, was also significantly reduced in the K56pvdA::tp strain compared to that of K56-2. Aerosol administration of K56-2 and the pvdA::Tn5-OT182 mutant into neutropenic mice as an acute respiratory infection model revealed that whereas the wild type strain was able to persist in the lung 7 days post infection, the pvdA mutant was cleared from most of the mice after 3 days. These experiments suggested the importance of ornibactin-mediated iron acquisition by the bacteria for initial colonization, persistence and resulting pathological changes within the host. However, given the difference in the iron content of the lungs of a healthy individual compared to those of a CF patient, it is not clear to what extent the conclusions from this study, which did not involve CF mice or rats, can be extrapolated to the situation in the CF lung.
While the data indicated an important role for ornibactin in lung colonization in these models, the K56-2 strain (and other members of the highly transmissible ET12 epidemic lineage) produces very low amounts of the siderophore pyochelin compared to other B. cenocepacia strains due to a frameshift mutation in pchF (Darling et al., 1998; Holden et al., 2009) (Based on perusal of the genome sequence, we presume pyochelin biosynthesis in these strains occurs through independent initiation of translation from an internal in-frame GUG codon located upstream of the frameshift site that results in production of PchF as two separate components). Thus, the role of pyochelin in the lung infection model could not be established using ET12 strains. However, given that ET12 strains cause life-threatening infections in CF patients, this would again appear to rule out an important role for pyochelin in colonization of the CF lung by B. cenocepacia. Later work using a B. cenocepacia strain (Pc715jorbA::tp) that produced normal amounts of pyochelin but was unable to utilize ferric-ornibactin due to disruption of the gene encoding the ferric-ornibactin TBDR, OrbA, revealed that it was cleared from rat lungs much more quickly than the WT strain (Visser et al., 2004). A ferric-pyochelin receptor mutant (Pc715jfptA::tp) persisted with the same efficiency as that of the WT. These data suggested that while pyochelin may have a role in the severity of infection, it is unable to compensate for the loss of a functional ornibactin utilization system. Therefore, it is ornibactin which appears to be important in order to establish an infection in this system. This may be explained by the presumed lower affinity of pyochelin than ornibactin for iron (Cox and Graham, 1979; Visca et al., 1993).
The importance of the ornibactin system for the virulence of B. cenocepacia has also been assessed in other infection models, including invertebrates and plants. Both a K56-2 orbA mutant and a K56-2 pvdA mutant were attenuated in the Caenorhabditis elegans and Galleria mellonella invertebrate models. The pvdA mutant was also slightly attenuated in the plant alfalfa model (Uehlinger et al., 2009). An orbJ mutant of the B. cenocepacia CF strain, H111, that is also deficient in the production of ornibactin, was also attenuated in the G. mellonella system (Mathew et al., 2014). Consistent with the virulence of the K56-2 strain in the rat lung chronic infection model, an H111 ΔpchAB pyochelin deficient mutant was still virulent in G. mellonella.
Using a modified signature-tagged mutagenesis (STM) procedure to identify genes required for survival in the rat chronic lung infection model, one of the attenuated B. cenocepacia K56-2 mutants which could not survive for 10 days in this model contained a transposon inserted just upstream of an ORF that encoded a haem TBDR-like protein (Hunt et al., 2004). The authors of this study indicated that the gene was the first in a cluster of genes associated with haem uptake that were located on chromosome 2, and the encoded protein was very similar to the 79 kDa RS03722 gene product of Ralstonia solanacearum strain GMI1000 (now reannotated as RSp0244). As RSp0244 is highly similar to BhuR we conclude that the plasposon insertion exerted polar effects on expression of the bhuRSTUV operon that impaired or abolished haem uptake. In contrast, genes involved in the biosynthesis and transport of ornibactin and pyochelin were not implicated in this study (Hunt et al., 2004). These observations would suggest that haem acquisition, and not siderophore-mediated iron acquisition, is an essential trait for persistence in the rat lung. Notwithstanding the different time courses of the chronic lung infection models, it is not clear why the ornibactin system should be implicated in some studies (Sokol et al., 1999; Visser et al., 2004) but not in the STM study, particularly as the ornibactin gene cluster presents a large target for plasposon-mediated disruption. One possible explanation is the selection procedure employed to construct the B. cenocepacia transposon mutant library. Here, mutants were selected on a mineral salts based medium in order to exclude auxotrophs. A K56-2 mutant in which the plasposon has disrupted a gene required for ornibactin synthesis or utilization would effectively be unable to obtain iron in a siderophore-dependent manner. We have observed that B. cenocepacia siderophore deficient mutants grow more slowly that the parental wild type strain on mineral salts medium (see for example Asghar et al., 2011), and it is possible that such mutants were omitted from the library of Hunt et al. This is not a complete explanation, however, as the haem uptake deficient mutants in the STM study can still produce ornibactin and so they might be expected to retain virulence based on other studies. This may suggest that a combination of both iron acquisition systems (haem- and ornibactin-mediated) is required for efficient colonization and persistence in the rat lung model.
Finally, the B. cenocepacia FtrABCD system was also investigated for a potential role in virulence in the Galleria wax moth model. Whereas deletion of the ftr system in isolation did not result in reduced virulence, when deleted in a strain that was unable to biosynthesise ornibactin and pyochelin, the mutant was more attenuated in comparison to an ornibactin-negative strain. This observation suggests that while ornibactin is the more important iron acquisition system for virulence in this model, the FtrABCD system can play a role in iron acquisition during infection in the absence of siderophores (Mathew et al., 2014).
Gene expression and omics studies
Transcriptomic and proteomic analyses can be used to identify sets of genes or proteins that may be required for survival under particular conditions by virtue of their differential expression. A few such studies have been carried out with B. cenocepacia that have suggested an important role for iron uptake mechanisms in bacterial persistence within CF patients. However, as we discuss below, the experimental set up may not necessarily be appropriate for addressing this particular question. The first notable study looked at global gene expression during growth of B. cenocepacia J2315 in a basal salts medium supplemented with CF sputum in comparison to growth in unsupplemented medium (Drevinek et al., 2008). The microarray revealed upregulation of 287 genes and downregulation of 437 other genes during growth in CF sputum medium. However, only two of the upregulated genes were associated with characterized iron acquisition systems in this bacterium. These two genes (pchR and pchD) encode the transcription activator of the pyochelin gene cluster and an enzyme required for biosynthesis of the pyochelin precursor, salicylic acid, respectively, but their expression increased only 2-fold. Transcription of the ornibactin genes was not upregulated, although the gene encoding the global iron repressor, Fur, that represses the ornibactin gene cluster, was found to be downregulated 2- to 3-fold (Agnoli et al., 2006; Drevinek et al., 2008).
The authors of this work measured the iron content of their sputum medium and found it to be ~35 μM, which is more than adequate to sustain growth of B. cenocepacia in standard laboratory medium without upregulating siderophore biosynthesis (Drevinek et al., 2008; Madeira et al., 2013; our unpublished results). Despite the presence of CF sputum in the medium, which has been argued by some investigators to sequester iron due to the presence of various iron binding components (Wang et al., 1996; Palmer et al., 2007), their results imply that sufficient iron is available to effect repression of the ornibactin system. Accordingly, the observed induction of the pch genes may not be related to iron depletion but is rather a response to the presence of another component in CF sputum. It is noteworthy that genes encoding other components of the pyochelin biosynthesis machinery (particularly those enzymes required to assemble pyochelin from salicylate and cysteine) and alternative iron uptake systems (FtrABCD and BhuRSTUV) were also not upregulated. The authors contend that the CF medium they employed restricted the available iron based on the observed gross upregulation of the BCAL0270 gene, which they considered to be involved in iron acquisition (described in the study as a “ferric reductase-like transmembrane component”). However, in a basal salts medium without iron supplementation, this gene was upregulated only 2-fold compared to medium containing the standard amount of ferrous sulfate (43 μM). Moreover, this gene is currently annotated in the NCBI database as encoding a “sulfoxide reductase heme-binding subunit YedZ” and it is transcriptionally linked to BCAL0269, a gene that encodes a YedY-homologous protein. Our own bioinformatics analysis supports this annotation (results not shown). The E. coli YedYZ complex is a membrane anchored haem-molybdoenzyme that serves to reduce an as yet unknown S- or N-oxide (Iobbi-Nivol and Leimkuhler, 2013). Therefore, there is little evidence to support the suggestion that BCAL0270 is involved in iron acquisition. The possibility that the CF sputum medium of Drevinek et al. (2008) is iron sufficient accords with the high iron concentration included in the basal salts medium used to generate the medium. Notwithstanding the fact that the sputum is present at only 10% (w/v) in the medium, the inability of the added CF sputum to induce iron acquisition systems may suggest that its ability to sequester iron is somewhat limited and/or it is iron replete.
The results of Drevinek et al. (2008) contrast with those of Palmer et al. (2005), who monitored gene expression in the CF pathogen P. aeruginosa growing in a mineral salts based medium containing only CF sputum as the source of carbon and energy (“MOPS-sputum medium”), and noted that a large number of genes specifying the biosynthesis of the major siderophore, pyoverdine, as well as the entire cluster of genes specifying the biosynthesis and transport of the secondary siderophore, pyochelin, were considerably upregulated relative to their transcription in cells growing in sputum-free MOPS-glucose medium. However, although MOPS-sputum medium contained a similar amount of CF sputum to that used by Drevinek et al. (2008), the iron content (3.5 μM) was approximately one tenth of that present in the medium used in the B. cenocepacia experiment (in both cases iron was added as ferrous sulfate). Thus, although it should be borne in mind that two different CF pathogens are being compared, each possessing a different primary siderophore system, the amount of iron added ranged from iron replete (in the B. cenocepacia experiment) to a concentration that will support bacterial growth but may require a degree of upregulation of the siderophore-mediated iron acquisition system (in the P. aeruginosa experiment), (pyoverdine synthesis is fully repressed at ~4 μM iron and is upregulated to a progressively greater degree as concentrations of iron are decreased below 4 μM; Meyer and Abdallah, 1978). The reader may wish to consider which version of CF sputum medium more closely represents the true environment of the CF lung with respect to iron availability. The other variable at play that may influence the iron content is the source of the sputum. As discussed above, the iron content of sputum shows marked variation among CF patients, particularly in relation to the severity of the disease (Stites et al., 1998; Reid et al., 2002; Hunter et al., 2013). To illustrate the potential for a different outcome that may reflect variation in the iron content of CF sputum, in an IVET study conducted on P. aeruginosa growing in a mineral salts medium containing 10% CF mucus, and otherwise with no iron supplementation, only a single iron-regulated gene, fptA (encoding the ferric-pyochelin outer membrane receptor), was identified as being upregulated (Wang et al., 1996).
Global changes in B. cenocepacia gene expression have also been analyzed in a synthetic CF sputum medium (SCFM). SCFM is a defined (i.e., sputum-free) medium containing the average concentrations of ions, free amino acids, glucose and lactate as those found in the sputum of CF patients and has been shown to support similar growth rates and elicit similar changes in expression of some subsets of genes in P. aeruginosa to those observed during growth in MOPS-sputum medium (Palmer et al., 2007). It also contains 3.6 μM ferrous iron (i.e., similar to that of MOPS-sputum medium). Although the iron concentration of SCFM may be low enough to cause upregulation of P. aeruginosa siderophore gene expression relative to iron replete conditions, these genes were not (unsurprisingly) upregulated relative to cells growing in a MOPS-glucose based mineral salts medium containing an almost identical concentration of iron (Palmer et al., 2007). The contrasting high level of siderophore gene expression observed in P. aeruginosa growing in MOPS-sputum medium relative to cells growing in MOPS-glucose medium (Palmer et al., 2005) was rationalized on the basis that CF sputum also contains iron sequestering components which have been proposed to restrict the availability of iron (Palmer et al., 2007). Other analogous attempts to mimic CF sputum conditions using semi-synthetic media, such as ASMDM or Modified ASMDM, and comparing gene expression in P. aeruginosa to that in cells growing in standard laboratory media have likewise not suggested a requirement for the main siderophore-mediated iron acquisition systems in synthetic sputum-like media [apart from one case where a small (2-fold) increase in some pyochelin biosynthesis and transport genes was observed; Fung et al., 2010; Hare et al., 2012].
In apparent contrast to the observations with P. aeruginosa, the entire ornibactin gene cluster of B. cenocepacia, as well as genes encoding a number of TBDRs and a cluster of genes that encode a putative bacterioferritin-associated ferredoxin and a TonB system (BCAL2290-BCAL2293) were found to be strongly upregulated during growth of B. cenocepacia J2315 in SCFM in comparison to growth in soil extract medium, although the pyochelin biosynthesis genes were not upregulated in SCFM (Yoder-Himes et al., 2010). These results also contrast with those observed for B. cenocepacia growing in a basal salts medium supplemented with glucose, casamino acids and CF sputum (see above; Drevinek et al., 2008). The most likely reason for the latter difference is that there was a 10-fold higher concentration of iron in the CF sputum medium in comparison to SCFM. It might seem intriguing that SCFM stimulates ornibactin gene expression in B. cenocepacia but not expression of pyoverdine genes in P. aeruginosa. However, again one must proceed with caution in interpreting these data, as the fold induction of gene expression in P. aeruginosa cells growing in SCFM was expressed relative to medium containing the same iron concentration, whereas the comparator for the B. cenocepacia experiment were cells growing in soil extract medium, which has an indeterminate iron concentration. In fact, it is likely that relative to cells growing in an iron replete, nutrient rich laboratory medium, both the pyoverdine and ornibactin gene clusters may actually be upregulated in cells growing in SCFM.
To summarize the above, it is difficult to make informed judgements regarding the requirement or otherwise for various iron acquisition systems based on gene expression analysis in cells growing in defined or semi-defined media that seek to mimic CF conditions when the concentration of iron and its relative availability may not accurately reflect the situation in the patient. As an example, we note that in some more recent attempts to mimic CF conditions using synthetic media, a higher iron concentration has been used by including ferritin to better reflect the prevailing view that the iron content of the CF lung is relatively high (Hare et al., 2012). Moreover, experiments involving media which incorporate CF-derived sputa may be prone to a high degree of experimental variation according to the disease severity and consequential iron status of the sputa. Finally, obvious though this must appear, consideration of the comparator is fundamentally important in assessing whether or not iron acquisition genes are upregulated in such media.
The difficulty in reproducing CF conditions in vitro can be bypassed by measuring gene expression in bacterial pathogens that are present in sputum following collection of samples from CF patients. As an example, in one such study, a microarray experiment was performed using P. aeruginosa mRNA isolated from sputum obtained from a single patient (Son et al., 2007). In this investigation, genes specifying the biosynthesis of pyochelin were upregulated but not those encoding the biosynthesis and transport of the major siderophore pyoverdine. In a later study, an RT-qPCR analysis of gene expression in P. aeruginosa strains that were present in the lungs of a cohort of CF patients suggested that the siderophore pyoverdine was likely to contribute to iron acquisition in this context (Konings et al., 2013), and indeed the presence of the siderophore could be detected in CF sputa (Martin et al., 2011). In contrast, to our knowledge, studies involving direct sampling of RNA from Bcc bacteria colonizing the CF lung have not been carried out. However, sampling of B. cenocepacia mRNA directly from an animal model of a chronic lung infection has been carried out (O'Grady and Sokol, 2011). As discussed earlier, the ability to biosynthesise ornibactin plays an important role in chronic infections of the rat lung by B. cenocepacia (7 and 14 days post-infection; Visser et al., 2004). However, microarray data in which gene expression in B. cenocepacia K56-2 cells that were recovered from the rat lung model 3 days post-infection was compared to cells that were grown to stationary phase in a nutrient-rich broth (iron replete medium), showed no difference in ornibactin gene expression levels (O'Grady and Sokol, 2011). Therefore, the simplest interpretation of these data is that ornibactin is not required to establish an infection in this particular model system but it is required for persistence. Moreover, in contrast to the STM analysis of Hunt et al. (2004), the microarray analysis did not reveal a difference in expression of the bhuR and bhuS genes (referred to as huvA and hmuS by the authors) in the rat lung model compared to growth in iron replete medium (O'Grady and Sokol, 2011). As the STM study was conducted with animals that were infected for 10 days, one possible explanation is that haem utilization becomes important for longer term infections as has been observed in P. aeruginosa (see below).
Transcriptomics has been used to monitor B. cenocepacia adaptation to the host over time, although in this case RNA was isolated following in vitro culture of the bacteria. In one such study, gene expression was compared in two clonal variants that were isolated 3 years apart from a CF patient who died of cepacia syndrome. This study revealed that in the later clone, seven genes located within the ornibactin gene cluster were upregulated 1.9- to 5.9-fold compared to those in the earlier isolate when both strains were cultured on a nutrient-rich agar. Other genes potentially involved in iron uptake were also found to be more transcriptionally active in this isolate, including three genes that encode TBDRs that are not involved in ornibactin or pyochelin uptake, and two genes, bhuR and bhuS (referred to as huvA and hmuS by the authors), from the bhuRSTUV gene cluster that is proposed to be required for the uptake of haem (Figure 4C; Mira et al., 2011). A subsequent proteomic study employing the same pair of isolates, along with a third isolate collected just before the death of the patient, showed that the two later isolates exhibited an increased abundance of four proteins involved in siderophore-mediated iron uptake compared to the earliest clone (Madeira et al., 2013). These proteins included two TBDRs (one of which was FptA) that had increased in abundance by <2-fold, and one component of the ferric-ornibactin cytoplasmic membrane transporter (OrbC) which showed a relatively small increase in abundance (~50%) in the third isolate compared to the first. However, in contrast to the transcriptomic study, a general increase in abundance of iron acquisition proteins was not observed. Moreover, based on a CAS assay, the latter two isolates were considered to be more tolerant to low iron concentrations (siderophore production was induced at 4 or 5 μM iron, whereas in the primary isolate siderophore production was upregulated at 6 μM iron).
In a later study carried out on the same sequential clonal isolates, the upregulation of ornibactin gene expression observed in response to the iron chelating activity of exogenously added pyoverdine was significantly less pronounced in the last isolate compared to the earlier isolates (Tyrrell et al., 2015). These observations are consistent with a scenario in which selection for genetic alterations has occurred upon long term colonization of the CF lung that lead to a decreased reliance on siderophore-dependent iron acquisition by the bacterium. This may indicate a switchover to another means of iron acquisition or it may reflect a general downregulation of iron acquisition mechanisms due to increased inflammatory damage that occurs in the CF lung as the disease progresses and the consequent increased availability of iron (Cohen and Prince, 2012).
The results are analogous to those obtained from studies carried out on P. aeruginosa, in which it was observed that during infection of the CF lung mutations accrue in the bacterial population that result in a reduction or abolition of production of the major siderophore, pyoverdine (Marvig et al., 2014; Nguyen et al., 2014 and refs within). Evidence was also provided for increased haem usage as an iron source concomitant with a reduction in pyoverdine synthesis during the later stages of infection (Marvig et al., 2014; Nguyen et al., 2014). Consistent with this, in a separate microarray study carried out on mRNA isolated directly from a P. aeruginosa-infected CF patient, two genes from the pyochelin gene cluster (pchA and pchC) were observed to be upregulated 2- to 3-fold, but no pyoverdine genes were identified as being upregulated (Son et al., 2007). Although these results were obtained from work on a different CF pathogen, they are consistent with the idea that siderophore-mediated iron acquisition in the CF lung may not be of major importance to some pathogenic bacteria, particularly later on in an infection.
While these types of analyses may provide useful pointers as to the iron acquisition mechanisms that may be employed by pathogenic bacteria colonizing the CF lung, particularly if the isolate has accrued mutations that have inactivated a particular uptake system, the fact that these systems are subject to genetic regulation means that a true understanding of the iron uptake mechanisms at play during an infection will also require analysis of bacterial gene expression in vivo, i.e., in the CF lung.
Studies on the effects of other environmental parameters on global gene expression in B. cenocepacia have also been conducted that have potential implications for iron acquisition in this organism. In one investigation, the effect of oxygen depletion on gene expression was assessed, as there is evidence to suggest that within the CF lung a steep oxygen gradient is generated due to increased activity of airway epithelial Na+-K+-ATPase pumps and excessive mucin secretion. This results in the deepest layers of mucus providing a hypoxic environment that can support high densities of micro-oxic and anaerobic microbes in the CF lung (Tunney et al., 2008). In this study it was shown that B. cenocepacia can grow in an atmosphere with oxygen concentrations as low as 0.1% (Pessi et al., 2013). The possibility that B. cenocepacia may therefore occupy a low oxygen niche when colonizing the lungs of CF patients prompted an RNA-seq and shotgun proteome analysis of B. cenocepacia H111 growing under micro-oxic conditions (0.5% oxygen) in comparison to aerobic growth (21% oxygen). RNA-seq showed strong down regulation of the pyochelin biosynthesis genes pchD, pchE, and pchF in addition to the gene encoding the ferric-pyochelin outer membrane receptor, FptA, in micro-oxic conditions. Overall production of siderophores was also reduced in micro-oxic conditions as assessed by the CAS agar assay (Pessi et al., 2013). This kind of environment promotes the increased stabilization of Fe(II) and may favor the use of ferrous iron transport systems, such as the FtrABCD system in B. multivorans, over siderophore based systems.
Another environmental parameter that appears to affect the expression of iron acquisition genes that may be pertinent in the context of CF lung infections is oxidative stress. Thus, it has been observed that exposure of B. cenocepacia biofilms to hydrogen peroxide causes increased expression of the first few genes of the ornibactin gene cluster (orbS, orbH, and orbG; Peeters et al., 2010). While the oxygen and oxidative stress status of the niche occupied by B. cenocepacia in the CF lung has not yet been established, these observations bring into focus the requirement to mimic, as close as possible, the conditions of the CF lung when assessing the potential role of iron acquisition mechanisms in the virulence of respiratory pathogens.
To summarize, there appears to be a role for ornibactin-mediated iron acquisition in the virulence of B. cenocepacia in both vertebrate and invertebrate models of infection, including infections of the respiratory tract. However, in the context of a CF infection the role of ornibactin is less certain and there is a possibility that haem acquisition may come into play. Moreover, the severity of the disease and the particular niche that is occupied by Bcc bacteria in the lung—for example, whether it is oxygen rich or hypoxic—are also likely to dictate which iron acquisition mechanism(s) are primarily deployed. Currently, there is little evidence to support a role for the secondary siderophores in colonization and persistence by Bcc bacteria, although this is partly due to the fact that no studies on the possible roles of cepaciachelin and cepabactin have been reported. Further studies are required to establish the relative importance of the various iron acquisition mechanisms available to Bcc bacteria for colonization of the CF lung.