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Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis Thomas J Evans*

ABSTRACT Pseudomonas aeruginosa is the most common pathogen that colonizes the lungs of patients with cystic fibrosis. Isolates from sputum are typically all derived from the same strain of bacterium but show extensive phenotypic heterogeneity. One of these variants is the so-called small colony variant, which also shows increased ability to form a biofilm and is frequently resistant to multiple antibiotics. The presence of small colony variants in the sputum of patients with cystic fibrosis is associated with a worse clinical condition. The underlying mechanism responsible for generation of the small colony phenotype remains unclear, but a final common pathway would appear to be elevation of intracellular levels of cyclic di-GMP. This phenotypic variant is thus not just a laboratory curiosity, but a significant bacterial adaptation that favors survival within the lung of patients with cystic fibrosis and contributes to the pulmonary damage caused by P. aeruginosa. “She was a vixen when she went to school. And though she be but little, she is fierce.” – Shakespeare. A Midsummer Night’s Dream. Act III, Scene 2 All organisms of a single species generally show some degree of phenotypic variation, reflecting small genotypic differences that arise from usually random mutagenic events. These differences are acted upon by natural selection, resulting in elimination of the less favorable and spread of those conferring selective advantage [1] . Bacteria demonstrate this phenomenon very clearly: antibiotic resistance being an example with which we are sadly all too familiar. However, there are other important phenotypic differences in bacteria that also can confer selective advantage under certain circumstances. One of these is the ‘small colony’ or ‘dwarf colony’ variant (Figure 1) , a phenotype named for its most obvious feature – small colony size – but, as we shall discuss, possessing a number of other important differences from the parent bacterial strain. This phenotypic variant is found in a number of important human pathogens that can cause persistent infection [2] . It clearly confers a selective advantage to the bacterium in allowing it to survive under adverse environmental conditions, such as within the respiratory tract, and is commonly associated with bacteria that can establish chronic colonization. Despite having been described for some decades, the molecular basis for this phenotypic change and the clinical correlates of infection with small-colony variants (SCVs) remain poorly defined. The autosomal recessive condition cystic fibrosis is characterized by chronic colonization of the respiratory tract by bacteria [4] . In early childhood, infections with Staphylococcus aureus predominate [5] , but as the patients get older, infections with Pseudomonas aeruginosa are most prevalent [6] . Initially these are intermittent and can be eradicated by antibiotic therapy [7] , but by late teens or early twenties this temporary colonization gives way to a permanent chronic colonization with

KEYWORDS 

• antibiotic resistance • biofilm • bronchiectasis • cystic fibrosis • exopolysaccharide • lung infection • Pseudomonas aeruginosa • small colony

variant

*Institute of Infection, Immunology & Inflammation, University of Glasgow, Glasgow, UK; Tel.: +44 141 330 8418; Fax: +44 141 330 4297; [email protected]

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Figure 1. Colonies of Pseudomonas aeruginosa showing the parent strain (left), a small-colony variant that arose under antibiotic selection (middle) and a large-colony revertant arising from the small-colony variant (right). Figure taken from reference [3], published under the Creative Commons Attribution license.

P. aeruginosa. This transition is associated with a progressive decline in lung function that for about 80–90% of patients will ultimately result in respiratory failure [8] . It is during the period of chronic colonization that the SCVs of P. aeruginosa have been isolated. In this review, we will consider what phenotypic characteristics these variants possess, their clinical correlates, the molecular basis of the phenotype, and the relationship of the SCVs with other phenotypic variants of P. aeruginosa that are also selected during this period. Phenotypic characteristics of small colony P. aeruginosa variants Early publications on the properties of P. aeruginosa noted phenotypic variation in colonial morphology, a phenomenon that was termed ‘colonial dissociation’ [9] . This described visual differences between mucoid and nonmucoid, smooth versus rough, iridescent and noniridescent and pigmented versus nonpigmented. In addition, small or ‘dwarf’ colonies were reported in cultures of sputum from patients with CF, but were usually present at much lower levels than the more readily recognized mucoid colonies of P. aeruginosa that seemed to be specifically linked with CF. They have also been found in the paranasal sinuses of CF patients [10] . Analysis of the genetic relatedness between different colonies of P. aeruginosa isolated from a given patient shows that most individuals are infected with one clonal variety [11,12] . This was

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initially carried out using relatively insensitive techniques such as restriction fragment length differences as detected by pulsed field gel electrophoresis or phage typing. More modern analysis using next generation DNA sequencing has by and large corroborated these earlier findings [13] , but also has shown that there are significant polymorphisms between isolates from a given individual at any one time [14] . No doubt these differences can account for the observed phenotypic heterogeneity of P. aeruginosa in the CF lung, although the relationship between genotype and phenotype is not well understood, as discussed in more detail below. The first study to examine in depth the occurrence and properties of SCVs of P. aeruginosa in patients with CF appeared in 1999 [15] . Over a 2-year period, 3.0% of P. aeruginosa positive specimens from chronically colonized patients with CF were SCVs. These had the typical small colonial morphology, with diameters ranging between 1 and 3 mm. The authors studied the stability of the SCV phenotype by serial passage in BHI medium. Revertants to the large colonial morphology were recovered with varying frequencies: 6.8% reverted with frequencies greater than 1 in 100; 81.8% reverted at a rate of less than 1 in 104 and only after serial passage; 11.4% never showed reversion, even after more than 50 passages. Ultrastructure, as judged by electron microscopy, was identical for SCVs and revertants, and metabolic profile and pulsed field gel electrophoresis also showed no differences.

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Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis  MICs of SCVs to 12 different antipseudomonal antibiotics were two- to eight-fold higher than those of the revertants. These findings have been consolidated and extended by other studies. The smaller colony size of the SCV – the defining attribute – is not generally reflected in differences in growth rate. Several studies have found very similar growth rates for the SCVs compared with parent clonal large colony isolates or revertants [16] . However, other studies have found a delayed growth pattern in liquid media [3] , although the growth rate finally attained and the resulting final cell density were very similar between SCVs and wild type. Certain SCV strains can outcompete their wildtype counterparts [17] . SCVs of P. aeruginosa are reported to be hyperpiliated, auto-aggretative and to have enhanced capacity to form biofilms [17] , although this latter observation has only been made in vitro. Loss of flagellar motility has also been found in SCVs of P. aeruginosa. Biofilm formation proceeds with a distinct developmental pathway. Initial attachment requires flagellar driven motility, but thereafter loss of flagella and increase in piliation allows the bacteria to form a tightly adherent mass [18,19] . Increased secretion of exopolysaccharides then stabilizes this adherent bacterial mass to form a robust biofilm. The loss of flagellar driven motility and increase in piliation noted above in SCVs thus all favor biofilm formation. SCVs also increase their rate of production of exopolysaccharides. P. aeruginosa produces three exopolysaccharides that are important in biofilm production: alginate, composed of mannuronic and guluronic acids; glucose-rich exopolysaccharide synthesized by genes in the pel gene cluster; and products of the psl gene cluster, which are rich in mannose, glucose and rhamnose [20] . Alginate is a very well characterized exopolysaccharide responsible for the mucoid phenotype commonly observed in strains of P. aeruginosa recovered from patients with CF. However, the psl and pel directed exopolysaccharides are essential for biofilm formation, since deletion of these gene clusters completely prevents biofilm formation [21] . SCVs of P. aeruginosa have been found to have upregulation of the psl and pel gene clusters, which will thus also contribute to the enhanced biofilm production characteristic of these variants [22,23] . A transcriptional study of a SCV compared with its clonal wild type and revertant showed that there was significant upregulation of the type III protein secretion system (T3SS) [24] .

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This system mediates secretion of exotoxins directly into the host cell and is important in the virulence of the organism in acute models of infection. A similar pattern of upregulation of T3SS components and secreted exotoxins was noted in a proteomic analysis of the secretome of SCVs [25] . Strains of P. aeruginosa isolated from CF patients tend to have downregulated the T3SS [26] . This study, therefore, suggests that T3SS-mediated cytotoxicity may be important in CF patients chronically colonized with SCVs of P. aeruginosa. Other phenotypic differences have been seen in SCVs. Production of the siderophores pyoverdin and pyochelin was found to be downregulated in SCVs compared with wild-type organisms grown in iron-rich media [16] . However, under iron limitation, siderophore production was the same as wild type. Since iron concentrations in bronchoalveolar lavage specimens of CF patients are considerably higher than healthy controls [27] , this downregulation of siderophore production by SCVs under iron-replete conditions may reflect no requirement for these proteins in the iron-rich conditions pertaining in the CF lung. Quorum sensing has also been investigated in SCV strains. One study found a dramatic downregulation of the quorum sensing molecules of the 4-quinolone family (4Qs) [3] . 4Qs have been shown to promote biofilm formation in P. aeruginosa [28] , so this lack of 4Qs in an SCV seems opposite to the other phenotypic changes in SCVs, which enhance biofilm formation. However, although 4Qs are produced at high level in P. aeruginosa strains isolated from CF patients in the first three years of life, their production thereafter significantly drops off [28] . Thus, it may be that the 4Qs are important in early adaptation in the CF lung and are no longer required under the conditions when SCVs are colonizing. The study showing downregulation of the 4Qs in the SCVs included a global transcriptional analysis of differentially regulated genes in the SCV that extends the findings of the microarray study described above [3] . The authors report increased expression of the efflux pumps MexXY and MexAB, porins OprH and F, and an LPS modification system. All of these changes are linked to the observed increase in resistance of the SCV strain to a broad range of antibiotics. They also found downregulation of the cytochrome c ccoN gene, which has also been associated with the SCV phenotype and aminoglycoside resistance. This study highlights the complex and

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Review Evans extensive genotypic and phenotypic changes that occur in the SCVs. The broad conclusion from all these investigations is that the phenotypic changes observed in the SCV would be expected to confer survival advantage in the environment of the CF lung, particularly in the face of repeated antibiotic usage in these patients. Selection & maintenance of diverse phenotypic strains of P. aeruginosa in the CF lung SCVs are only one of a number of phenotypic varieties of P. aeruginosa found in CF – the ‘colonial dissociation’ referred to in the early literature. If SCVs are so well adapted to the environment within the CF lung, why are they not the only phenotype of P. aeruginosa that is found? Many studies have shown that there is extraordinary phenotypic diversity of this organism in chronically colonized patients. For example, a study by Mowat et al. analyzed sputum samples from 10 chronically colonized CF patients over the course of a year [29] . Forty isolates were grown from each sample and tested for 15 different phenotypic traits – a total of 1720 isolates were examined. Although each patient was colonized by the same strain – the Liverpool Epidemic Strain – the phenotypic variation was very large. About half of the total variation was found within a single sample of sputum. Moreover, the turnover of a particular variant in any one patient was high. Another study by Workentine et al. of P. aeruginosa phenotypic differences in one chronically colonized patient over time analyzed 169 clonal isolates [14] . Again, the phenotypic variation was very high. Moreover, even isolates with the same morphotype, for example, an SCV showed a variety of different phenotypic traits in other ­characteristics such as antibiotic sensitivity. A number of possible explanations could account for this extraordinary phenotypic diversity. The CF lung is no doubt a complex environment comprising a variety of different ecological niches, with varying oxygen concentrations, pH, host immune cells and cytokines and so on. Such a diverse landscape provides specialist niches which will encourage adaptive radiation, and the appearance of ‘niche specialists,’ specific strains of P. aeruginosa, that are adapted to a particular environment within the respiratory tract. Such adaptive changes were beautifully demonstrated by Rainey and Travisano in their classic study of experimental adaptive radiation in P. fluorescens [30] . Even in a simple static broth culture,

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different phenotypes of an original homogeneous strain appeared within 3 days. These were of thee main morphs: smooth, wrinkly spreader and fuzzy spreader. For example, the wrinkly spreader occupied the air-broth interface and flourished in this location, forming a self-supporting mat. However, its fitness declined with time as the weight of the mat caused it to sink, allowing the other phenotypes greater opportunity to propagate. Thus, even such a simple environment can produce phenotypic diversity that fluctuates over time. Antibiotic exposure is also another important initiator of the SCV. In the laboratory, exposure to a variety of antibiotics can also produce SCV in cultures of P. aeruginosa, with many of the features of the naturally occurring SCV found in CF [31,32] However, the range of phenotypes recovered from the CF lung seems out of proportion to the possible different niches it might contain. Additionally, many of the phenotypes, including the SCVs, are unstable and show phase variation. Another explanation to account for this diversity and instability follows from the Red Queen hypothesis [33] . This hypothesis was originally put forward to explain the origins of sex and recombination as a means for a host to escape the pressure of parasites by generating rare offspring genotypes that are resistant to parasite removal and hence will be at a selective advantage. The Red Queen is one of the characters that Alice met in Lewis Carroll’s ‘Alice Through the Looking Glass’, who had to run fast just to stay in the same place. By analogy, the Red Queen hypothesis proposed that variation by sexual recombination is advantageous as it provides the organism with the ability to generate individuals resistant to future evolution of the parasite i.e., constant change to remain viable. The CF lung is thus a possible ‘reverse’ Red Queen kingdom. In this case, the host is presenting a changing environment as lung damage evolves, and different antibiotics are given over time. How do the colonizing P. aeruginosa respond? Bacteria are haploid, so sexual variation does not occur, but the large population size of colonizing P. aeruginosa with a very short generation time allows for a reasonably rapid appearance of mutants, of which some will have a selective advantage, Under these conditions, there may indeed be selection for more rapidly mutating strains –the hypermutator phenotype, which has been seen in CF [34] . We as yet have only a basic understanding of the genotypic changes that can give rise

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Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis  to the observed phenotypic variation (discussed below), but the generation of such diversity would allow the colonizing P. aeruginosa to overcome the changing environment within the CF lung and the effects of different antibiotics used in treatment. Clinical correlations of the SCV phenotype in CF Correlation between clinical status of a patient and the growth of a SCV from sputum presents some difficulties in interpretation. Causality is difficult to infer, since if SCVs are recovered more frequently from those with a worse clinical condition it is not possible to conclude that the SCV caused the worse condition or that the SCV is able to survive better in the lung environment found in those patients who have more serious disease, due to some other cause. Moreover, given that one of the major phenotypic properties of the SCVs is increased adherence and biofilm formation, it may well be that estimation of SCV numbers in sputum give an erroneous picture of the degree to which the airway of a particular patient is colonized with SCVs of P. aeruginosa. With that proviso in mind, it is clear, however, that a number of different studies have shown a clear relationship between the isolation of SCVs and a worse clinical condition. The study of Hauβler et al. was one of the first to correlate the recovery of SCVs from sputum in CF and clinical features of the patient [15] . They studied 86 patients who had chronic colonization with P. aeruginosa. Carriers of SCVs (n = 33; 38%) had worse lung function, with a FEV1 of 56% predicted compared with the SCV negative patients, who had a predicted FEV1 of 80%, a highly significant difference. The SCV carrying patients also were often those receiving daily inhaled antibiotics. Further studies have by and large confirmed these initial findings. For example, the study of Schneider et al. prospectively examined CF sputum samples for SCV over a three month period [35] . They studied 98 patients with CF; 86 were colonized with P. aeruginosa or S. aureus or both. Of these colonized patients, 9.2% grew a P. aeruginosa SCV. This is somewhat lower than the percentages found by Hauβler et al., which reflects the shorter study period and that only one sputum sample per patient was analyzed. Comparisons were made of a number of clinical characteristics of those colonized with SCVs compared with patients colonized without SCV. FEV1 of the SCV group was significantly

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lower at 39% predicted compared with 65.5% of the non-SCV patients. pO2 was also lower in the SCV group at 64 mmHg compared with 71.5 mmHg in those without SCVs. The SCV patients also had lower body mass (mean 14.0% underweight compared with 2.7% of non-SCV patients). No significant difference was found in antibiotic exposure between SCV and non-SCV groups. As outlined above, SCVs usually have an extended range of resistance to antibiotics when compared with their normal colony morphology parents. The clinical impact of this change is not clear. Antipseudomonal antibiotic therapy does improve symptoms and lung function in CF exacerbations [36,37] . However, this improvement does not correlate with the changes in bacterial load consequent to treatment. For example, McLaughlin et al. in a study of CF patients chronically colonized with P. aeruginosa, found that a variety of antipseudomonal antibiotic regimens had similar effects on lung function, and gave about a 2-log reduction in sputum bacterial load [38] . However, there was no correlation between the improvement in lung function and the change in sputum bacterial concentration or the sensitivities of the bacteria to the antibiotic regimen used. Moreover, at follow up three months after treatment, the levels of bacteria in sputum were back to virtually the same levels that had been present at the time of the pulmonary exacerbation, despite a continued stable clinical state. Other studies have also shown antipseudomonal therapy improving clinical status during pulmonary exacerbations in CF, but with no apparent correlation with the antibiotic sensitivities of the bacteria within sputum [39] . Using both conventional culture and PCR based quantitation, Deschaght et al. found that during antibiotic treatment of exacerbations 62% of P. aeruginosa detected in sputum by PCR were not cultivatable [40] . Whether these bacteria were dormant or dead but intact is not clear. Thus, it is not certain what contribution SCV antibiotic resistance makes to outcome in treatment of pulmonary exacerbations of CF. A study that quantified the levels of P. aeruginosa before, during and after an exacerbation would be very informative as to the effect antibiotics have on SCVs. The other important issue is whether the organisms recovered in sputum truly reflect the total population of P. aeruginosa in the lung. The highly adherent SCVs might be very abundant in the lower airways but not shed readily into sputum. Further studies to define the phenotype

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Review Evans of bacteria recovered from bronchial biopsies low down in the respiratory tract would help to resolve this issue. Molecular basis of phenotype The molecular basis of the dramatic phenotypic change that results in the development of SCVs remains elusive. However, a number of studies have shown the importance of the bacterial signaling molecular cyclic di-GMP in the generation of SCVs. A seminal study by Drenkard and Ausubel analyzed the molecular basis behind a SCV of P. aeruginosa they isolated through growth in inhibitory concentrations of kanamycin [31] . These SCVs had all the typical phenotypic features considered above. They identified a gene, phenotype variant regulator, pvrR, that when overexpressed caused the SCV to revert at high frequency to the parent large-colony phenotype. Although inactivation of this gene did not produce the SCV phenotype, it increased the frequency with which SCVs appeared on exposure to kanamycin. This suggested that the product of the pvrR gene might act upstream of a proximal switch to inhibit its action. The pvrR gene encodes a protein containing an EAL domain that is found within proteins that have phosphodiesterase activity and are thought to degrade cyclic di-GMP [41] . D’Argenio et al. also studied genes that control the SCV phenotype and identified a gene WspR which when upregulated could confer the SCV morphology [42] . WspR encodes a protein Planktonic

that contains a GGDEF domain, found in proteins that have diguanylate cyclase activity and hence raise cyclic di-GMP levels [41] . A further study using a genetic screen for SCV revertants to large-colony morphology [43] also identified genes that are involved in controlling cyclic diGMP levels as important in generation of the SCV phenotype. A number of proteins with a GGDEF domain when mutated were found to cause the SCV to revert to the large-colony phenotype. Levels of cyclic di-GMP were elevated in the SCV and reduction of the levels by the phosphodiesterase pvrR resulted in reversion to the wild-type colony morphology. Overexpression of the GGDEF domain proteins induced formation of fimbrial expression that mediates the increased adherence of the SCV but did not reproduce the SCV phenotype morphology. Thus, elevation of cyclic di-GMP levels alone was not sufficient to induce a switch to the SCV morphology. Another study analyzing the origin of SCVs of P. aeruginosa [23] also found elevated levels of cyclic di-GMP in both clinical and laboratory SCVs. These strains had all the typical features of the SCVs outlined above. The authors engineered a strain that over produced cyclic di-GMP and determined which genes were differentially regulated in response to a rise in intracellular cyclic di-GMP. Over 50 such genes were identified, including those within the pel and psl gene clusters, while downregulation of flagellum and pilus genes were noted.

Biofilm initiation

Mature biofilm

• • •

• Hyperpiliated • Adherent • Loss of flagella

Sessile Highly adherent Secretion of exopolysaccharide

• Flagellated • Free swimming

• • •

SCV and other variants prevalent Increased antibiotic resistance ? Decreased virulence factors

Figure 2. Evolution of Pseudomonas aeruginosa in colonizing the respiratory epithelium.

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Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis  Recent studies have identified the yfiBNR operon as important in SCV generation [44,45] . The YfiN protein encoded within this operon acts as a diguanylate cyclase, elevating cyclic di-GMP levels, inducing a SCV phenotype and upregulating production of the Psl and Pel exopolysaccharides that are important in biofilm production in the SCVs. What controls the activity of the YfiN cyclase or indeed the nature of the genotypic change underlying the SCV phenotype is unclear. Genome sequencing has revealed mutations in a number of candidate genes that could contribute to the development of the SCV phenotype [46,47] . The filamentous phage pf4 has been implicated in inducing the SCV phenotype in the P. aeruginosa strain PAO1 [48] , although this has not been found to be a general mechanism in other pseudomonal strains [49] . Phage can produce transacting factors that mediate DNA inversions [50] . Large-scale DNA inversion has been found to be responsible for the generation of a SCV in S. aureus [51] . Fifty percent of strains of P. aeruginosa from CF lung are found to have such large chromosomal inversions that may be related to the production of phenotypic variation [52] . The mobile element IS6100 has been found to be involved in the generation of these inversions [53] . It is naïve to assume that there will be one common mechanism underlying the generation of SCVs; more likely, a number of different genetic mechanisms exist. Epigenetic modifications are also possible [54,55] . Further work is required to determine if the environment of the CF lung specifically induces the formation of the SCVs, or if these variants arise spontaneously at low frequency and are then

Review

selected for their enhanced survival in the CF airway. Conclusion SCVs of P. aeruginosa are commonly found colonizing patients with CF. They have a very distinctive phenotype, many of whose features give them a selective advantage in the harsh environment of the CF airways. The colonization of the airway by this bacterium progresses through a number of sequential steps, summarized in Figure 2. SCVs are one of many phenotypic variants found in CF sputum culture, but have received far less attention than the mucoid variants. However, as hopefully this review has made clear, they do play an important part in the survival of P. aeruginosa in the lung and are associated with clinical deterioration. Rather as Helena comments on her childhood friend, Hermia, in Shakespeare’s ‘A Midsummer Night’s Dream’ in the quote at the beginning of this review, although the SCVs are little they are certainly fierce. Future perspective The next 5–10 years will see considerable advances in our understanding of SCVs of P. aeruginosa. The region of the CF lung where the SCVs are most abundant will be defined, and the relationship of this abundance to the characteristics of the particular niche they inhabit will be defined. Genomic and transcriptome sequencing will allow the molecular nature of the switch(s) to the SCV phenotype to be understood. Prospective studies will define better the relationship between acquisition of SCVs and deterioration in clinical condition in patients

EXECUTIVE SUMMARY Phenotypic characteristics of small-colony variants ●●

Distinctive small colonial phenotype that arises in cystic fibrosis patients or can be induced by growth in inhibitory antibiotic concentrations.

●●

Associated features include increased biofilm formation and resistance to multiple antibiotics.

Clinical correlations of the small-colony variants in cystic fibrosis ●●

Small colony variants are more associated with worse clinical condition in cystic fibrosis.

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Whether this is a cause or an effect is difficult to determine.

Molecular basis of phenotype ●●

Raised levels of cyclic di-GMP are associated with the generation of the small-colony phenotype.

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The genetic changes underlying this biochemical change are not yet clear.

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Review Evans with CF. Finally, the effects of new therapies, such as ivacaftor, on the frequency of SCVs in CF sputum will be delineated. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial

fibrosis children have implications for chronic lung infection. ISME J. 6(1), 31–45 (2012).

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