Differential infection properties of three inducible prophages from an epidemic strain of Pseudomonas aeruginosa
- Chloe E James1Email author,
- Joanne L Fothergill1,
- Hannah Kalwij1,
- Amanda J Hall1,
- Jennifer Cottell1,
- Michael A Brockhurst†2 and
- Craig Winstanley†1
© James et al.; licensee BioMed Central Ltd. 2012
Received: 15 May 2012
Accepted: 19 September 2012
Published: 21 September 2012
Pseudomonas aeruginosa is the most common bacterial pathogen infecting the lungs of patients with cystic fibrosis (CF). The Liverpool Epidemic Strain (LES) is transmissible, capable of superseding other P. aeruginosa populations and is associated with increased morbidity. Previously, multiple inducible prophages have been found to coexist in the LES chromosome and to constitute a major component of the accessory genome not found in other sequenced P. aerugionosa strains. LES phages confer a competitive advantage in a rat model of chronic lung infection and may, therefore underpin LES prevalence. Here the infective properties of three LES phages were characterised.
This study focuses on three of the five active prophages (LESφ2, LESφ3 and LESφ4) that are members of the Siphoviridae. All were induced from LESB58 by norfloxacin. Lytic production of LESφ2 was considerably higher than that of LESφ3 and LESφ4. Each phage was capable of both lytic and lysogenic infection of the susceptible P. aeruginosa host, PAO1, producing phage-specific plaque morphologies. In the PAO1 host background, the LESφ2 prophage conferred immunity against LESφ3 infection and reduced susceptibility to LESφ4 infection. Each prophage was less stable in the PAO1 chromosome with substantially higher rates of spontaneous phage production than when residing in the native LESB58 host. We show that LES phages are capable of horizontal gene transfer by infecting P. aeruginosa strains from different sources and that type IV pili are required for infection by all three phages.
Multiple inducible prophages with diverse infection properties have been maintained in the LES genome. Our data suggest that LESφ2 is more sensitive to induction into the lytic cycle or has a more efficient replicative cycle than the other LES phages.
KeywordsPseudomonas aeruginosa Prophage induction Bacteriophage infection Cystic fibrosis Host range Type IV pili
Pseudomonas aeruginosa is a versatile Gram-negative bacterium, able to metabolise multiple carbon sources and exploit diverse ecological niches, e.g. soil, water, plants and animal hosts [1, 2]. This opportunistic pathogen causes a range of human infections, including acute infections of severe wounds  and burns [4, 5] and chronic lung infections in cystic fibrosis (CF) patients . P. aeruginosa forms biofilms in the CF lung that are highly resistant to antibiotics and clearance by the immune system . Once established, such biofilms cannot be eradicated and are associated with greatly increased morbidity and mortality .
Several CF-associated transmissible strains of P. aeruginosa, capable of between patient transmission, have been identified in the UK, Europe, Australia and North America . The Liverpool Epidemic Strain (LES), a UK transmissible strain, was first isolated in 1996 at Alder Hey Children’s Hospital (AHCH), Liverpool . This strain is capable of super-infection, supplanting pre-existing P. aeruginosa populations in the CF lung . Chronic infection with LES is associated with increased morbidity and mortality compared to other P. aeruginosa strains . The LES is highly prevalent within individual hospital CF units  and is the most abundant P. aeruginosa strain amongst CF patients in the UK . It was also recently isolated from the sputa of CF patients in North America .
Sequencing of the earliest LES isolate, LESB58, demonstrated that the genome shares 95% similarity with the lab strain PAO1. However, its core genome is punctuated by multiple norfloxacin-inducible prophages . Specifically, there are five inducible prophage genomes (LESφ2; LESφ3 LESφ4 LESφ5 and LESφ6) that are mosaic in nature. The gene organisation of LESφ2 and LESφ3 resembles that of lambdoid phages. These two phage genomes share 82.2% identity across a 13.6-kb region at their 3’ ends that makes up 32% of the phage genomes. The closest known relative to both these phages is the Pseudomonas phage F10 . LESφ3 also contains a 7.5 kb region that shares 99.8% homology with LESφ5, which exhibits a considerable sequence similarity to the O-antigen converting phage D3 . LESφ4 is a transposable Mu-like phage that closely resembles phage D3112 . The LESφ6 sequence resembles a pf1-like filamentous phage .
Temperate phages have been shown to confer selective, beneficial traits to a range of P. aeruginosa hosts . For example, phage D3 orchestrates O antigen conversion from O5 to O16 in PAO1, which may aid evasion of the immune system and resistance to phage superinfection [18, 21]. Phage φCTX infection of PAS10 results in conversion to a toxigenic strain  and the filamentous phage, Pf1, has been associated with biofilm disruption and dispersal . LES prophages have been suggested to contribute to the competitiveness of their bacterial host in vivo. LESB58 mutants, with disrupted prophage genes, exhibited 10 to 1000-fold decreased competitiveness in a rat model of chronic lung infection compared to wild type LESB58 . The LES phages are induced by exposure to clinically relevant antibiotics, e.g. ciprofloxacin , and free LES phages and other tailed-phage virions have been detected in CF patient sputa [25, 26].
Temperate phages are key vectors of horizontal gene transfer (HGT) . Therefore, it is important to assess the ability of the LES phages to infect other bacterial hosts to which they may confer traits beneficial to life in the CF lung environment. Here we describe the infection characteristics of three of the five LES prophages LESφ2, LESφ3 and LESφ4, induced from the sequenced CF lung isolate LESB58.
LES phage morphology
Three different Siphoviridae phages were induced from LESB58 cultures and visualised using electron microscopy. The phages possessed icosahedral heads (50–60 nm diameter) and long flexible tails (approximately 200 nm). Plaque assay of each phage on PAO1 resulted in the formation of small turbid plaques with different phage-specific morphologies. LESφ3 plaques were the largest (2–3 mm), with well-defined lysogen islands, whereas LESφ2 plaques were considerably smaller (0.5-1.5 mm). LESφ4 produced plaques with small, clear centres surrounded by a turbid halo. The identity of each LES phage responsible for the different plaque morphologies was confirmed using a multiplex PCR assay.
Differential induction of LES phages from LESB58
Lysogenic infection of a model PAO1 host
Differential Immunity profiles of each LES phage in PAO1
Efficiency of plating values
PAO1 naive host
Single φ2 lysogen
< 1x10 -9
< 1x10 -9
Single φ3 lysogen
< 1x10 -9
Single φ4 lysogen
LES phages integrate at the same sites in different bacterial host strains
The LESφ2 int probe hybridised to an additional DNA fragment in all lysogens containing LESφ2, including LESB58. The size of the additional hybridised fragment corresponds to one of two possibilities: 1) the integration of a second LESφ2 copy in to the chromosome directly downstream of the first; 2) an extra copy of LESφ2 in circular form (Figure 4). The published LESB58 genome sequence clearly shows a single LESφ2 copy in the chromosome. Since the hybridisation pattern of the PAO1 LESφ2 lysogen matches that of LESB58, a second chromosomal copy can be ruled out. This suggests that the extra copy is circular, which may represent phage replication resulting from spontaneous activation of the lytic life cycle. Alternatively, the extra copy may indicate pseudolysogeny, in which stable circular copies are maintained.
The LESφ4 cI probe indicated that LESφ4 is able to integrate in several chromosomal locations in PAO1.
LES phages infect a narrow host range in a type IV pilus-dependant manner
Susceptibility of a panel of Pseudomonas isolates to LES phages 2, 3 and 4
Isolate source (#)
Reference strains (2)
Keratitis patient (12)
Non-LES child (8)
Non-LES adult (6)
Anomalous LES (6)
A non-piliated PAO1 mutant (pilA - ) was resistant to infection by all 3 phages, suggesting that LESφ2, 3 and 4 all require type IV pili for infection. The hyper-piliated mutant (pilT - ) was also resistant to the LES phages, whilst an alternative hyper-piliated mutant (pilU-) remained fully susceptible.
Differential induction among co-infecting prophages
Induction experiments demonstrated that LESφ2 virions were produced from LESB58 in greater numbers than the other phages. These data suggest that LESφ2 replication is more efficient than the other phages and could out number and therefore out compete the other, co-infecting LES phages during the lytic cycle. Potentially supporting this hypothesis, we detected an extra copy of this phage in the LESφ2 lysogen genome. Southern analysis suggests the presence of either a pseudo-lysogenic plasmid form , or a highly active replicative form of LESφ2 during spontaneous phage production.
The implications of within-host competition between co-infecting prophages has been little studied, however Refardt et al. observed hierarchical competition between multiple prophages in E. coli, which suggested that the sensitivity of the lytic switch can determine dominance of one prophage over another in a polylysogen. Carriage of phages that are very prone to activation of the lytic lifecycle may represent a significant cost to their host cells, and thus could be selected against in natural populations. However, while natural isolates of LES sampled from CF patient sputa often lack one or more of the LES prophages , there is no evidence that LESφ2 is more or less stably maintained than LESφ3 or LESφ4.
LES phages exhibit different immunity profiles
Each phage conferred inhibition of superinfection by the same phage, although the Mu-like phage, LESφ4 was observed to infect LESφ4 lysogens at a very low frequency. This may represent the development of rare mutations that affect immunity functions. There are several examples of such mutations in phage Mu . Repressor/operator coevolution has been suggested to be the driving force for the evolution of superinfection immunity groups of lambdoid phages . The same may hold true for Mu-like phages. For example, mutation of the operator region has been shown to affect binding of the repressor in Mu vir mutants .
Sequential infection of PAO1 with different LES phages revealed an interesting superinfection hierarchy. LESφ3 lysogens remained susceptible to LESφ2 and LESφ4; and LESφ4 lysogens were susceptible to LESφ2 and LESφ3. However, LESφ2 prevented infection by LESφ3 and greatly reduced susceptibility to LESφ4. Such uni-directional infection exclusion has been reported between other phages, and is commonly associated with super-infection exclusion genes such as the lambda rex genes  and sieA, sieB and a1 in the Salmonella phage, P22 [35–38].
It is likely that LESφ3 and LESφ4 prophages would have been acquired before LESφ2, because the infection hierarchy suggests that prior acquisition of LESφ2 would have prevented subsequent LESφ3 and LESφ4 infection.
LES prophages in PAO1 undergo spontaneous activation to the lytic cycle at a far higher rate than in LESB58
High levels of spontaneous induction were observed in PLPLs, suggesting that lysogeny is relatively unstable in the PAOl genetic background. We show that phage production remained high between PLPLs containing one, two or three LES prophages, suggesting that polylysogens were no more or less stable than any single lysogens. Southern analysis confirmed that LESφ2 and LESφ3 integrated into the same position in PLPLs as they did in LESB58. Therefore, the instability of PLPLs was not due to prophage integration into unstable sites. LESφ4 integrated in several alternative sites in PLPLs. The sequence of this phage shares a high level of genome synteny and homology with the transposable Mu-like phage D3112 , whose random integration has been demonstrated to create mutations within the host chromosome. LESφ4 may play a similar role in LES genome evolution.
The LES phages exhibit a narrow host-range
Our investigation of the LES phage host range revealed narrow, overlapping host specificity. No association between bacterial clone-type and phage susceptibility was observed, although testing more strains may have identified a pattern. Despite the high proportion of resistant clinical isolates, our data show that LES phages are capable of infecting some P. aeruginosa strains isolated from keratitis patients and non-LES infected CF patients. LES phages have been detected in CF patients’ sputa, and may therefore allow mobilisation of genes between P. aeruginosa strains [25, 26]. By contrast, LES phages may allow LES to displace other P. aeruginosa strains during superinfection in the CF lung  by lysing susceptible resident strains .
LES phage infection is Type IV pilus-dependent
We demonstrate that LES phage infection is dependent on the type IV pilus, which is required by P. aeruginosa for adhesion, biofilm formation and twitching motility [40–42]. This important surface structure is commonly used as a receptor by diverse Pseudomonas phages . Both non-piliated (pilA - ) and hyper-piliated (pilT - ) PAO1 mutants were resistant to infection by all three LES phages. However, a different hyper-piliated mutant (pilU - ) remained susceptible. These findings mirror other pilin-dependent P. aeruginosa phage studies [43–45]. Hyper-piliated mutants are incapable of twitching motility due to abrogated pili retraction. These data suggest that retraction is involved in the infection process by LESφ2 LESφ3 and LESφ4.
Despite infecting via an important and common surface structure, all three LES phages exhibited narrow host ranges and each showed strain specificities. For example, LESφ4 was able to infect PA14 and several keratitis isolates that were resistant to infection by the other LES phages. It is likely that many clinical strains of P. aeruginosa harbour prophages that may belong to the same immunity group and therefore exclude super-infection by one or more of the LES phages . Alternatively, resistance could be achieved by loss or modification of the type IV pili receptor [44, 45].
In summary, we demonstrate that the LES phages exhibit differential sensitivities to induction, narrow host ranges and divergent infection behaviour in the model host PAO1 compared with the native LESB58 host background. Extensive genotypic and phenotypic variation has been observed in clinical LES populations , including changes in the number of resident LES prophages . These phages may, therefore, be important contributors to diversity of the LES populations.
Bacterial strains and growth conditions
Bacterial strains and sources
Strain (1Clone type)
Laboratory P. aeruginosa strains:
PAO1 pilA-; PAO1 pilU-; PAO1 pilT- 2
Clinical LES isolates:
LESB 58 (T) - Sequenced isolate
LES 431 (T) - Lacks LES prophage 2
Anomalous LES isolates3:
O69574 (T); 0521 (T); 43513 (T); 079444 (T); 0342 (T).
P. aeruginosa isolates from keratitis patients4:
39015 (B); 39115 (A); 39103 (A2); 39145 (A3); 39053 (A5); 39135 (C); 39016 (D); 39421 (F); 39061 (I); 39284 (L); 39376 (U); 39129 (V).
P. aeruginosa isolates from non-LES infected CF patients:
CHILDREN: AH23 (B); AH4 (A); AH19 (A3); AH14 (C); AH1 (D); AH6 (L); AH9 (U); AH7 (A4);
ADULTS: NL28 (A); NL20 (C); NL25 (F); NL16 (U); NL21 (A4); NL14 (A7).
Environmental Pseudomonas spp:
WC5365; F113; ATCC 17400; pf5; pf01.
‘tomato’ DC300; B728a
P. syringae pv. Coriandricola
P. syringae pv. maculocola
P. syringae pv. antirrhini
F-A1-1; LMG 13010.
P. aeruginosa LESB58 was grown to mid-exponential phase (OD600 0.5) and LES phages were induced into the lytic cycle by exposure to the minimum inhibitory concentration of norfloxacin (50 μg ml-1) for 1 h . Induced cultures were sub-cultured (1:10) into fresh LB to enable recovery for 2 h before filtration (0.2 μm Millipore). Active phage particles in the induced supernatants were enumerated by standard plaque assay using PAO1 host cells.
LES phages were isolated from induced LESB58 cultures using plaque assays with PAO1 host cultures as described previously . Phages were purified by picking individual plaques that were suspended in LB (1 ml), filter sterilized (0.2 μm Millipore) and used in a second plaque assay with PAO1. This process was repeated twice to ensure purity. Phage purity was confirmed using PCR assays. Amplification of phage stocks was achieved by modifying previous methods . Briefly, mid-exponential phase PAO1 cultures (100 ml) were infected with purified LES phage (MOI = 0.1), at 37°C for 2 h. Lysed cultures were filter-sterilized.
Phage suspensions (1x109 – 1x1010 p.f.u. ml-1) were concentrated by centrifugation, negatively stained with 2% (w/v) uranyl acetate , and examined by transmission electron microscopy (magnification x 200,000).
Multiplex PCR to confirm pure phage stocks and lysogens
95°C, 4 min then 30 cycles: 95°C, 30 s; 58°C, 30 s; 72°C, 30 s; final extension step, 72°C, 7 min;
95°C, 10 min; then 40 cycles: 95°C, 10 s; 60°C, 15 s; 72°C s.
Preparation of DIG-labeled probes:
95°C, 4 min. 30 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 1 min s; final extension step, 72°C, 7 min
Quantifying production of each LES phage from LESB58
Replication of each LES phage in response to induction of the lytic cycle was compared using Q-PCR to distinguish and enumerate each specific phage type. LESB58 induction experiments were performed on three separate occasions in the presence and absence of norfloxacin for 30 and 60 min exposure times before the 2 h recovery step. DNA was prepared from each replicate using the Bacterial and Virus DNA extraction kit (QIAGEN) and the automated QIAsymphony machine (QIAGEN; pathogen complex 200 protocol). Q-PCR was performed using six specific primer sets to differentiate between prophage and total copies of each phage.
Q-PCR was used to quantify LES phages by comparing the number of specific amplicon copies in extracted DNA from induction experiments to a concentration gradient of known standards. Primer sets with the prefixes, “tot” (total) and “pro” (prophage) were designed to amplify unique regions within, and flanking, each LES phage genome (Figure 1D). All primer sequences and amplification details are listed in Table 4. Amplicon copy number μl-1 was calculated using the formula [(6.023 x 1023 x [DNA] g/ml)/(molecular weight of product)]/1,000 . Molecular weight was calculated as number of base pairs x 6.58 x 102 g. A 10-fold dilution series of each DNA standard was prepared for quantification of phage numbers in each sample.
Q-PCR reactions (25 ul) contained 1 uM each primer pair and 1X Rotorgene-SYBR green supermix (QIAGEN). Phage numbers were quantified from DNA samples (1 μl) in triplicate using a Rotorgene cycler (QIAGEN). Q-PCR data were analyzed using Rotorgene Q series software 1.7 (QIAGEN). Total phage and prophage numbers from each sample were quantified in separate reactions using “tot” and “pro” primer sets for each phage and comparing fluorescent signals to those from standard concentration gradients. The level of free phage in a given sample was calculated by subtracting prophage numbers from total phage numbers.
Specific phage sequences were quantified in triplicate from each of 3 experimental replicates using Q-PCR, and technical replicates were averaged prior to analyses. Differences in phage numbers, with and without norfloxacin and between time-points were analysed using separate ANOVAs for each phage, fitting induction (2 levels), time (2 levels) and their interaction as fixed factors.
Isolation of PAO1 lysogens
PAO1 LES phage lysogens (PLPLs) were isolated from turbid islands in the centre of well-separated plaques using a sterile toothpick and streaked on to Columbia agar (Oxoid) to obtain single colonies. Individual lysogen colonies were analysed by multiplex PCR assays to confirm the presence of LES prophages.
Lawns of PAO1 and each PLPL were created by adding mid-exponential phase (OD600 0.5) cultures (100 ul) to molten 0.4% (v/v) agar and pouring onto Columbia agar plates to set. A 10-fold dilution series of each purified phage suspension (1010 – 103 p.f.u ml-1) was spotted (20 ul) onto lawns of each host. Countable plaques were observed at varying dilutions depending on the phage-host combination. The efficiency of plating (eop) value was calculated as the ratio of assay titre/most permissive titre. The most permissive titre was obtained on non-lysogenic PAO1.
Southern blot analysis
Southern analysis was performed as previously described . Specific probes were prepared using the digoxigenin (DIG) PCR labelling kit (Roche). DIG-labelled probes were designed to hybridize targets within either the LESφ2 int gene, the LESφ3 int gene, or the LESφ4 cI gene using primers: φ2intDIG F/R; φ3intDIG F/R and φ4cIDIG F/R (Table 4). Bacterial genomic DNA was extracted using a Wizard Genomic DNA extraction kit (Promega) and digested using PstI, AcuI or DraIII (NEB) according to the manufacturer’s instructions. Probes were hybridised to digested genomic DNA as described previously . Hybridized probe was detected using alkaline phosphatase-conjugated anti-DIG antibody (1:10,000) and CPDstar substrate (1:100) (Roche) according to the manufacturer’s instructions.
This work was supported by the Wellcome Trust (089215/Z/09/Z).
Thanks to Brian Getty (Institute of Infection and Global Health, University of Liverpool) for performing the electron microscopy; Dr Heather Allison for helpful discussions and to Professor Angus Buckling and Dr Rob Jackson for kindly supplying pil mutants and environmental Pseudomonas strains respectively.
- Hardalo C, Edberg SC: Pseudomonas aeruginosa: assessment of risk from drinking water. Crit Rev Microbiol. 1997, 23: 47-75. 10.3109/10408419709115130.PubMedView ArticleGoogle Scholar
- Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, et al: Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature. 2000, 406: 959-964. 10.1038/35023079.PubMedView ArticleGoogle Scholar
- Gjodsbol K, Christensen JJ, Karlsmark T, Jorgensen B, Klein BM, Krogfelt KA: Multiple bacterial species reside in chronic wounds: a longitudinal study. Int Wound J. 2006, 3: 225-231. 10.1111/j.1742-481X.2006.00159.x.PubMedView ArticleGoogle Scholar
- Nasser S, Mabrouk A, Maher A: Colonization of burn wounds in Ain Shams University Burn Unit. Burns. 2003, 29: 229-233. 10.1016/S0305-4179(02)00285-1.PubMedView ArticleGoogle Scholar
- Chitkara YK, Feierabend TC: Endogenous and exogenous infection with Pseudomonas aeruginosa in a burns unit. Int Surg. 1981, 66: 237-240.PubMedGoogle Scholar
- Hutchison ML, Govan JR: Pathogenicity of microbes associated with cystic fibrosis. Microbes Infect. 1999, 1: 1005-1014. 10.1016/S1286-4579(99)80518-8.PubMedView ArticleGoogle Scholar
- Hoiby N, Ciofu O, Bjarnsholt T: Pseudomonas aeruginosa biofilms in cystic fibrosis. Future Microbiol. 2010, 5: 1663-1674. 10.2217/fmb.10.125.PubMedView ArticleGoogle Scholar
- Hassett DJ, Korfhagen TR, Irvin RT, Schurr MJ, Sauer K, Lau GW, Sutton MD, Yu H, Hoiby N: Pseudomonas aeruginosa biofilm infections in cystic fibrosis: insights into pathogenic processes and treatment strategies. Expert Opin Ther Targets. 2010, 14: 117-130. 10.1517/14728220903454988.PubMedView ArticleGoogle Scholar
- Fothergill JL, Walshaw MJ, Winstanley C: Transmissible strains of Pseudomonas aeruginosa in Cystic Fibrosis lung infections. Eur Respir J. 2012, 40: 227-238. 10.1183/09031936.00204411.PubMedView ArticleGoogle Scholar
- Cheng K, Smyth RL, Govan JR, Doherty C, Winstanley C, Denning N, Heaf DP, van Saene H, Hart CA: Spread of beta-lactam-resistant Pseudomonas aeruginosa in a cystic fibrosis clinic. Lancet. 1996, 348: 639-642. 10.1016/S0140-6736(96)05169-0.PubMedView ArticleGoogle Scholar
- McCallum SJ, Corkill J, Gallagher M, Ledson MJ, Hart CA, Walshaw MJ: Superinfection with a transmissible strain of Pseudomonas aeruginosa in adults with cystic fibrosis chronically colonised by P aeruginosa. Lancet. 2001, 358: 558-560. 10.1016/S0140-6736(01)05715-4.PubMedView ArticleGoogle Scholar
- Al-Aloul M, Crawley J, Winstanley C, Hart CA, Ledson MJ, Walshaw MJ: Increased morbidity associated with chronic infection by an epidemic Pseudomonas aeruginosa strain in CF patients. Thorax. 2004, 59: 334-336. 10.1136/thx.2003.014258.PubMedPubMed CentralView ArticleGoogle Scholar
- Panagea S, Winstanley C, Parsons YN, Walshaw MJ, Ledson MJ, Hart CA: PCR-based detection of a cystic fibrosis epidemic strain of Pseudomonas aeruginosa. Mol Diagn. 2003, 7: 195-200. 10.2165/00066982-200307030-00009.PubMedView ArticleGoogle Scholar
- Scott FW, Pitt TL: Identification and characterization of transmissible Pseudomonas aeruginosa strains in cystic fibrosis patients in England and Wales. J Med Microbiol. 2004, 53: 609-615. 10.1099/jmm.0.45620-0.PubMedView ArticleGoogle Scholar
- Aaron SD, Vandemheen KL, Ramotar K, Giesbrecht-Lewis T, Tullis E, Freitag A, Paterson N, Jackson M, Lougheed MD, Dowson C, et al: Infection with transmissible strains of Pseudomonas aeruginosa and clinical outcomes in adults with cystic fibrosis. JAMA. 2010, 304: 2145-2153. 10.1001/jama.2010.1665.PubMedView ArticleGoogle Scholar
- Winstanley C, Langille MG, Fothergill JL, Kukavica-Ibrulj I, Paradis-Bleau C, Sanschagrin F, Thomson NR, Winsor GL, Quail MA, Lennard N, et al: Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa. Genome Res. 2009, 19: 12-23.PubMedPubMed CentralView ArticleGoogle Scholar
- Kwan T, Liu J, Dubow M, Gros P, Pelletier J: Comparative genomic analysis of 18 Pseudomonas aeruginosa bacteriophages. J Bacteriol. 2006, 188: 1184-1187. 10.1128/JB.188.3.1184-1187.2006.PubMedPubMed CentralView ArticleGoogle Scholar
- Kuzio J, Kropinski AM: O-antigen conversion in Pseudomonas aeruginosa PAO1 by bacteriophage D3. J Bacteriol. 1983, 155: 203-212.PubMedPubMed CentralGoogle Scholar
- Rehmat S, Shapiro JA: Insertion and replication of the Pseudomonas aeruginosa mutator phage D3112. Mol Gen Genet. 1983, 192: 416-423. 10.1007/BF00392184.PubMedView ArticleGoogle Scholar
- Ceyssens PJ, Lavigne R: Bacteriophages of Pseudomonas. Future Microbiol. 2010, 5: 1041-1055. 10.2217/fmb.10.66.PubMedView ArticleGoogle Scholar
- Holloway BW, Cooper GN: Lysogenic conversion in Pseudomonas aeruginosa. J Bacteriol. 1962, 84: 1321-1324.PubMedPubMed CentralGoogle Scholar
- Hayashi T, Baba T, Matsumoto H, Terawaki Y: Phage-conversion of cytotoxin production in Pseudomonas aeruginosa. Mol Microbiol. 1990, 4: 1703-1709. 10.1111/j.1365-2958.1990.tb00547.x.PubMedView ArticleGoogle Scholar
- Rice SA, Tan CH, Mikkelsen PJ, Kung V, Woo J, Tay M, Hauser A, McDougald D, Webb JS, Kjelleberg S: The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage. ISME J. 2009, 3: 271-282. 10.1038/ismej.2008.109.PubMedPubMed CentralView ArticleGoogle Scholar
- Fothergill JL, Mowat E, Walshaw MJ, Ledson MJ, James CE, Winstanley C: Effect of antibiotic treatment on bacteriophage production by a cystic fibrosis epidemic strain of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2011, 55: 426-428. 10.1128/AAC.01257-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Fothergill JL, Mowat E, Ledson MJ, Walshaw MJ, Winstanley C: Fluctuations in phenotypes and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during pulmonary exacerbations. J Med Microbiol. 2010, 59: 472-481. 10.1099/jmm.0.015875-0.PubMedView ArticleGoogle Scholar
- Ojeniyi B, Birch-Andersen A, Mansa B, Rosdahl VT, Hoiby N: Morphology of Pseudomonas aeruginosa phages from the sputum of cystic fibrosis patients and from the phage typing set. An electron microscopy study. APMIS. 1991, 99: 925-930. 10.1111/j.1699-0463.1991.tb01280.x.PubMedView ArticleGoogle Scholar
- Brussow H, Canchaya C, Hardt WD: Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev. 2004, 68: 560-602. 10.1128/MMBR.68.3.560-602.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Wiehlmann L, Wagner G, Cramer N, Siebert B, Gudowius P, Morales G, Kohler T, van Delden C, Weinel C, Slickers P, Tummler B: Population structure of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2007, 104: 8101-8106. 10.1073/pnas.0609213104.PubMedPubMed CentralView ArticleGoogle Scholar
- Miller RV, Pemberton JM, Clark AJ: Prophage F116: evidence for extrachromosomal location in Pseudomonas aeruginosa strain PAO. J Virol. 1977, 22: 844-847.PubMedPubMed CentralGoogle Scholar
- Refardt D: Within-host competition determines reproductive success of temperate bacteriophages. ISME J. 2011, 5: 1451-1460. 10.1038/ismej.2011.30.PubMedPubMed CentralView ArticleGoogle Scholar
- Priess H, Kamp D, Kahmann R, Brauer B, Delius H: Nucleotide sequence of the immunity region of bacteriophage Mu. Mol Gen Genet. 1982, 186: 315-321. 10.1007/BF00729448.PubMedView ArticleGoogle Scholar
- Berngruber TW, Weissing FJ, Gandon S: Inhibition of superinfection and the evolution of viral latency. J Virol. 2010, 84: 10200-10208. 10.1128/JVI.00865-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Vanvliet F, Couturier M, Desmet L, Faelen M, Toussaint A: Virulent Mutants of Temperate Phage-Mu-1. Mol Gen Genet. 1978, 160: 195-202. 10.1007/BF00267481.View ArticleGoogle Scholar
- Benzer S: Fine Structure of a Genetic Region in Bacteriophage. Proc Natl Acad Sci U S A. 1955, 41: 344-354. 10.1073/pnas.41.6.344.PubMedPubMed CentralView ArticleGoogle Scholar
- Susskind MM, Botstein D, Wright A: Superinfection exclusion by P22 prophage in lysogens of Salmonella typhimurium. III. Failure of superinfecting phage DNA to enter sieA+ lysogens. Virology. 1974, 62: 350-366. 10.1016/0042-6822(74)90398-5.PubMedView ArticleGoogle Scholar
- Susskind MM, Botstein D: Superinfection exclusion by lambda prophage in lysogens of Salmonella typhimurium. Virology. 1980, 100: 212-216. 10.1016/0042-6822(80)90571-1.PubMedView ArticleGoogle Scholar
- Susskind MM, Botstein D: Molecular genetics of bacteriophage P22. Microbiol Rev. 1978, 42: 385-413.PubMedPubMed CentralGoogle Scholar
- Heo YJ, Chung IY, Choi KB, Lau GW, Cho YH: Genome sequence comparison and superinfection between two related Pseudomonas aeruginosa phages, D3112 and MP22. Microbiology. 2007, 153: 2885-2895. 10.1099/mic.0.2007/007260-0.PubMedView ArticleGoogle Scholar
- Brown SP, Le Chat L, De Paepe M, Taddei F: Ecology of microbial invasions: amplification allows virus carriers to invade more rapidly when rare. Curr Biol. 2006, 16: 2048-2052. 10.1016/j.cub.2006.08.089.PubMedView ArticleGoogle Scholar
- Irvin RT, Doig P, Lee KK, Sastry PA, Paranchych W, Todd T, Hodges RS: Characterization of the Pseudomonas aeruginosa pilus adhesin: confirmation that the pilin structural protein subunit contains a human epithelial cell-binding domain. Infect Immun. 1989, 57: 3720-3726.PubMedPubMed CentralGoogle Scholar
- O’Toole GA, Kolter R: Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol. 1998, 30: 295-304. 10.1046/j.1365-2958.1998.01062.x.PubMedView ArticleGoogle Scholar
- Mattick JS: Type IV pili and twitching motility. Annu Rev Microbiol. 2002, 56: 289-314. 10.1146/annurev.micro.56.012302.160938.PubMedView ArticleGoogle Scholar
- Whitchurch CB, Mattick JS: Characterization of a gene, pilU, required for twitching motility but not phage sensitivity in Pseudomonas aeruginosa. Mol Microbiol. 1994, 13: 1079-1091. 10.1111/j.1365-2958.1994.tb00499.x.PubMedView ArticleGoogle Scholar
- Brockhurst MA, Buckling A, Rainey PB: The effect of a bacteriophage on diversification of the opportunistic bacterial pathogen, Pseudomonas aeruginosa. Proc Biol Sci. 2005, 272: 1385-1391. 10.1098/rspb.2005.3086.PubMedPubMed CentralView ArticleGoogle Scholar
- Chibeu A, Ceyssens PJ, Hertveldt K, Volckaert G, Cornelis P, Matthijs S, Lavigne R: The adsorption of Pseudomonas aeruginosa bacteriophage phiKMV is dependent on expression regulation of type IV pili genes. FEMS Microbiol Lett. 2009, 296: 210-218. 10.1111/j.1574-6968.2009.01640.x.PubMedView ArticleGoogle Scholar
- Mowat E, Paterson S, Fothergill JL, Wright EA, Ledson MJ, Walshaw MJ, Brockhurst MA, Winstanley C: Pseudomonas aeruginosa population diversity and turnover in cystic fibrosis chronic infections. Am J Respir Crit Care Med. 2011, 183: 1674-1679. 10.1164/rccm.201009-1430OC.PubMedView ArticleGoogle Scholar
- Taylor TB, Buckling A: Competition and dispersal in Pseudomonas aeruginosa. Am Nat. 2010, 176: 83-89. 10.1086/652995.PubMedView ArticleGoogle Scholar
- Rahme LG, Stevens EJ, Wolfort SF, Shao J, Tompkins RG, Ausubel FM: Common virulence factors for bacterial pathogenicity in plants and animals. Science. 1995, 268: 1899-1902. 10.1126/science.7604262.PubMedView ArticleGoogle Scholar
- Salunkhe P, Smart CH, Morgan JA, Panagea S, Walshaw MJ, Hart CA, Geffers R, Tummler B, Winstanley C: A cystic fibrosis epidemic strain of Pseudomonas aeruginosa displays enhanced virulence and antimicrobial resistance. J Bacteriol. 2005, 187: 4908-4920. 10.1128/JB.187.14.4908-4920.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Fothergill JL, White J, Foweraker JE, Walshaw MJ, Ledson MJ, Mahenthiralingam E, Winstanley C: Impact of Pseudomonas aeruginosa genomic instability on the application of typing methods for chronic cystic fibrosis infections. J Clin Microbiol. 2010, 48: 2053-2059. 10.1128/JCM.00019-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Stewart RM, Wiehlmann L, Ashelford KE, Preston SJ, Frimmersdorf E, Campbell BJ, Neal TJ, Hall N, Tuft S, Kaye SB, Winstanley C: Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections. J Clin Microbiol. 2011, 49: 993-1003. 10.1128/JCM.02036-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Mahenthiralingam E, Coenye T, Chung JW, Speert DP, Govan JR, Taylor P, Vandamme P: Diagnostically and experimentally useful panel of strains from the Burkholderia cepacia complex. J Clin Microbiol. 2000, 38: 910-913.PubMedPubMed CentralGoogle Scholar
- Allison HE, Sergeant MJ, James CE, Saunders JR, Smith DL, Sharp RJ, Marks TS, McCarthy AJ: Immunity profiles of wild-type and recombinant shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens. Infect Immun. 2003, 71: 3409-3418. 10.1128/IAI.71.6.3409-3418.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Ackermann H-W, Heldal M: Basic electron microscopy of aquatic viruses. Manual of Aquatic Viral Ecology. Edited by: Wilhelm SW, Weinbauer MG, Suttle CA, Waco TX. 2010, American Society of Limnology and Oceanography, Inc, 182-192.View ArticleGoogle Scholar
- Yin JL, Shackel NA, Zekry A, McGuinness PH, Richards C, Putten KV, McCaughan GW, Eris JM, Bishop GA: Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I. Immunol Cell Biol. 2001, 79: 213-221. 10.1046/j.1440-1711.2001.01002.x.PubMedView ArticleGoogle Scholar
- Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975, 98: 503-517. 10.1016/S0022-2836(75)80083-0.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.