Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions
© Garbe et al; licensee BioMed Central Ltd. 2010
Received: 21 May 2010
Accepted: 26 November 2010
Published: 26 November 2010
Pseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF). Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages.
We isolated and characterized a phage, named JG024, which infects a broad range of clinical and environmental P. aeruginosa strains. Sequencing of the phage genome revealed that the phage JG024 is highly related to the ubiquitous and conserved PB1-like phages. The receptor of phage JG024 was determined as lipopolysaccharide. We used an artificial sputum medium to study phage infection under conditions similar to a chronic lung infection. Alginate production was identified as a factor reducing phage infectivity.
Phage JG024 is a suitable broad host range phage which could be used in phage therapy. Phage infection experiments under simulated chronic lung infection conditions showed that alginate production reduces phage infection efficiency.
Pseudomonas aeruginosa is well known as an opportunistic human pathogen characterized by a high intrinsic antibiotic tolerance [1, 2]. In humans, P. aeruginosa can cause urinary tract, respiratory tract, and burn wound infections [3–5]. Respiratory tract infections caused by P. aeruginosa are dreaded in patients suffering from the genetic disorder Cystic Fibrosis (CF) [2, 6, 7]. CF patients exhibit an increased mucus production in the lung . Bacteria like P. aeruginosa are able to colonize this mucus and cause chronic infections, which cannot be eradicated by antibiotic treatment . Several hypothesis exist explaining the observed high antibiotic tolerance of P. aeruginosa in the CF-lung, which is caused by special growth conditions. These include growth as biofilm-like microcolonies, which have been shown to increase antibiotic tolerance up to 1000-fold [9, 10]. A couple of in vitro model systems have been described to simulate a CF lung infection caused by P. aeruginosa[11–13]. The artificial sputum medium is a complex medium based on components measured in the CF sputum . It mimics the CF-lung environment during infection and causes typical P. aeruginosa phenotypes as mucoidy and microcolony formation . Since eradication of chronic P. aeruginosa infections by antibiotics fails, phage therapy is a possibility to treat bacterial infections. Advantages over antibiotics are the specificity of phages and that phages can be isolated and investigated rapidly . For this reason, several suitable P. aeruginosa broad host range phages have been characterized. The Pseudomonas infecting PB1-like phages are widespread in nature and possess highly conserved genomes. Comparative genome analysis of five PB1-like (PB1, SN, 14-1, LMA2 and LBL3) phages was recently published and is the first genome report for these phages . PB1-like phages belong to the Myoviridae phage family and the genome sizes vary between 64,427 and 66,530 bp. The genomes encode for 88 (LBL3) to 95 proteins (LMA2) . More than 42 phages have been reported to be PB1-like. These results are mainly based on DNA hybridization and morphological studies [15, 16]. More recently, PB1-like phages as phage 14-1 have been reported as part of a well defined phage cocktail to treat P. aeruginosa burn wound infections . The application of phages as a therapeutical agent requires an in depth understanding of the phage biology . Moreover, phages which multiply well under in vitro conditions can fail to replicate during treatment in vivo. Therefore, phages and especially the ability of the phage to infect the host in vivo should be investigated carefully prior to use. Here we describe the in depth characterization of a broad host range PB1-like phage with a slight prevalence to clinical isolates. We used an artificial sputum medium to simulate the conditions in the CF lung and investigated the ability of phage JG024 to infect P. aeruginosa and multiply under these conditions.
Results and Discussion
Isolation and host range of phage JG024
Strains and phages used in this study.
Bacterial strain or phage
Phenotype or genotype
mucoid CF isolate
PAO1 mucA::aacC1-gfp GmR
Sabrina Thoma, this laboratory, unpublished
pilA inactivated by allelic displacement; tagged with eGFP, TcR, GmR
fliM inactivated by allelic displacement; tagged with eGFP, TcR, GmR
PAO1 algC ::aacC1-gfp GmR
Julia Garbe, this laboratory, unpublished
BT2, BT72, BT73, RN3, RN43, RN45, NN84
clinical CF isolates
Medical Highschool Hannover, Germany
PACF15, PACF21, PAKL1, PAKL4, PACF60, PACF61, PACF62, PACF63
clinical CF isolate
Gerd Döring, Tübingen, Germany
Nr. 18, 19, 26, 29
urinary tract infection isolate
Michael Hogardt, München, Germany
Katherina Selezska, HZI Braunschweig, Germany
wild type PAO1 LPS specific lytic bacteriophage
Family affiliation of JG024
To determine family affiliation of phage JG024, we determined the nature of the nucleic acids and the morphology of the phage to assign the family by comparison . Nucleic acids were isolated as described in Methods and identified as dsDNA due to its sensitivity to restriction endonucleases like Sac II, which cut only dsDNA. Sac II produced distinct fragments of approximately 30 kb, 25 kb and 8 kb (data not shown). Computational analysis of the Sac II restriction sites in the sequenced genome (see below) revealed slightly different fragment sizes of 28,348 kb and 21,719 kb, respectively as well as two fragments with a size of 8,49 kb and 7.718 kb, which we observed as one 8 kb fragment.
Receptor of phage JG024
We used different P. aeruginosa mutants to identify the receptor of phage JG024 as outlined by others . Aflagella mutant (ΔfliM), a pili mutant (ΔpilA) and an LPS mutant (ΔalgC) were infected with the phage JG024. After incubation, lysis was investigated on bacterial lawns (data not shown). JG024 lyses the pili- and the flagella mutant but not the P. aeruginosa ΔalgC mutant. The algC gene encodes an enzyme with phosphoglucomutase and phosphomannomutase activity. A P. aeruginosa ΔalgC mutant produces a truncated LPS core and lacks common antigen suggesting that these structures might constitute the host receptor for JG024 attachment [24, 25].
JG024 is a PB1-like phage
Comparison of the JG024 genome to the genomes of PB1-like phages 15.
Genome size (bp)
GC content (%)
DNA identity (%) to JG024
Features of the JG024 genome
Since these phages share a high sequence similarity a comparative ORF prediction was possible. First, the heuristic approach of GeneMark was used to identify genes in small genomes under 100 kb . With this approach a total of 84 putative ORFs were identified. In a second approach we used the NCBI ORF Finder program coupled with the program blastp and compared the translated proteins with the proteins of the PB1-like phages [26, 32]. Combination of the results of both approaches revealed a total of 94 predicted ORFs as well as one unique ORF in phage JG024.
No RNA polymerase was detected suggesting that this phage uses the host transcriptional machinery, as it was also suggested for the PB1-like family of phages.
We detected a putative structural gene cluster which contains genes encoding for putative head structure proteins (ORF 18 and 19) as well as for tail and baseplate proteins (ORF 22-47). Moreover, ORF 40 was designated as a lytic tail protein. It was shown for the phages 14-1 and LBL3 that this protein has a transglycosylase domain with a N-acetyl-D-glucosamine binding site, which shows a specific degradation of peptidoglycan . ORF 48 encodes a putative endolysin with a high similarity to the endolysin of phage LMA2 (98.6%) and belongs to a lysozyme-like superfamily. A putative holin may be encoded by ORF 52, which shares a 100% identity to ORF 50 of phage F8 and to ORF 51 of phage 14-1. It was suggested that these ORFs encode probable holins since they are located near the endolysin gene and they encode a small protein (201 aa) containing three transmembrane domains .
Potential regulatory elements and intergenic motifs of the JG024 genome.
dG (kcal mol-1)
putative σ 70-dependent promoter elements:
ATGTTTGAATCTCTTTTGAA CGT TTGATGTTTCCCCTATAA TAAGC GCACA
TCATCTATAAGTAACGTTAT AAC ATAACGTCAATTTATATGCT CTA GACGT
putative rho-independent terminator elements:
AAGCCCGGA GCGATCCGGGCTT T TCTGTGTT
GGCCGG GTTTCCGGCCTT TGTT
AAAAGGCCGCT TATTCAGCGGCC TTTT TGCTTT
AAAAGGCCGCT GAATAAGCGGCC TTTT CTTTT
AGGCCGCC TTCGGGCGGTCT TTT CTTT
AAAGCCCCGG ACTCT AGTTCAGA A TCCGGGGCTTT CTTTT
AAAGCCCCGG ATTCT GAACTAGA G TCCGGGGCTTT GTCGCTTCT
ASM infection assay
In contrast to the P. aeruginosa PAO1 strain the CF-isolate BT73 is mucoid and secretes the exopolysaccharide alginate. We wondered if alginate overproduction could explain the observed results. It was recently published that even non-mucoid strains like the wild type PAO1 express the exopolysaccharide alginate in response to oxygen-limiting conditions . We also observed that cultures of PAO1 in ASM, which mimics the CF lung, were highly viscous compared to the cultures in LB medium, suggesting a high production of alginate by the wild type PAO1 in this medium. If alginate is the factor in our experimental setup which decreases phage infection efficiency, a mucoid variant of strain PAO1 should show a similar result as the clinical isolate BT73. Therefore, we repeated the phage infection experiments in LB and ASM with a P. aeruginosa mucA mutant strain. We observed again only a 1.6-fold decrease in ASM and an overall approximately tenfold reduction in phage particles when compared with P. aeruginosa PAO1 (Figure 4). These results are in agreement with our hypothesis that alginate overproduction reduces phage infection efficiency. Moreover, they point to alginate as the dominant factor for the decrease in phage infection efficiency in ASM. To verify this result, we performed the same experiment with P. aeruginosa PAO1 in LB medium and increasing alginate concentrations. We chose alginate concentrations of 50, 100, 200, 500 μ g/ml up to 1000 μ g/ml, since non-mucoid P. aeruginosa strains have been reported to produce 50-200 μ g/ml alginate, while mucoid isolates produce up to 1000 μ g/ml alginate [34–36]. In accordance with our hypothesis, the presence of alginate reduced phage multiplication in our test assay. A concentration of 50 to 200 μ g/ml alginate resulted in an almost 20-fold reduction of phage particles compared to LB medium alone in accordance with the 50-fold reduction of phage particles observed in ASM compared to LB. This effect is even more obvious with alginate concentations of 500 and 1000 μ g/ml, where we observed a reduction of phage particles by a factor of 130 and almost 2800, respectively.
We could show that the phage JG024 belongs to the PB1-like phages and shares several characteristic features of this group. These phages are widespread in nature and very successful. A new member of this group, phage JG024, was isolated and characterized. General growth characteristics as well as the genome were investigated, showing that JG024 is able to pass one infection cycle in approximately 50 min. Genome analysis revealed the strong relatedness to the PB1-like phages. Moreover, we could show that JG024 has broad spectrum activity with a prevalence to clinical isolates. Also, infection of the host P. aeruginosa was even possible under challenging conditions like the ASM medium which mimics the CF lung. High viscosity and microcolony growth of the host were only small obstacles for JG024 to infect and multiply under these conditions. These results show that this group of bacteria could be an important contribution to phage therapy. Moreover, we established a method to investigate the possibility of a phage to lyse bacteria under infection conditions prior to use for phage therapy in vivo.
Bacterial strains and growth conditions
Table 1 shows the genotype and phenotypes of the bacteria and phage JG024 used in this study. The 100 environmental Pseudomonas aeruginosa strains used in this study origin from a comprehensive screen of approx. 400 environmental river strains. These were genetically characterized using the ArrayTube hybridization chip . The 100 strains used here are all different in their core genomic SNP pattern and were chosen such to represent the entire population genetic diversity currently known for P. aeruginosa. Details of the comprehensive screen will be published elsewhere. P. aeruginosa strains were routinely propagated in Luria Bertani (LB) broth medium aerobically at 37°C. The composition of the artificial sputum medium (ASM) is described elsewhere .
Phages were isolated from sewage following a simple enrichment procedure. Samples from a sewage plant Steinhof in Braunschweig, Germany were centrifuged for 5 min at 4100 × g (Biofuge fresco). Ten ml of the supernatant were mixed with 5 ml of a P. aeruginosa overnight culture and incubated in 50 ml LB broth at room temperature. After an incubation of 48 h, the cells were sedimented by centrifugation at 4100 × g (Biofuge fresco) for 10 min and the supernatant was transferred to a clean tube. To kill remaining bacteria, several drops of chloroform were added to the supernatant and the emulsion was mixed for 30 s. To separate the phages, appropriate dilutions of the phage lysate were spotted onto bacterial lawns of top-agar plates. Top-agar plates were produced by adding approximately 5*108 cells/ml of P. aeruginosa from an overnight LB broth to 3.5 ml of LB top-agar (0.75%). The inoculated top-agar was overlaid on an LB agar plate and allowed to solidify. After incubation at 37°C for 10 to 16 h zones of lysis were monitored. Single plaques, derived from a single phage, were separated by stinging with a pipette tip into the plaque followed by resuspending the phages in SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl, pH 7.5). The resulting phage lysate was stored at 4°C.
The morphology of the phages was detected by negative staining with uranyl acetate and transmission electron microscopy. Phages were allowed to absorbe onto a thin carbon film, prepared on mica, from a liquid sample for different time points, washed in TE buffer (10 mM TRIS, 2 mM EDTA, pH 6.9) and distilled water. Phages were negatively stained by floating the carbon film for approx. 15 sec on a drop of 2% aqueous uranyl acetate. Then, the carbon film was picked up with copper grids (300 mesh), blotted semi-dry with filter paper and was subsequently air dried. Samples were examined in a Zeiss EM910 transmission electron microsope at an acceleration voltage of 80 kV and at calibrated magnifications. Images were recorded digitally with a Slow-Scan CCD-Camera (ProScan, 1024 × 1024, Scheuring, Germany) with ITEM-Software (Olympus Soft Imaging Solutions, Münster, Germany). Brightness and contrast were adjusted with Adobe Photoshop CS3.
Phage host range spectrum and detection of host receptor
To determine the phage host range, top-agar plates with the potential host lawn were prepared. Top-agar plates were produced by adding approximately 5*108 cells/ml of P. aeruginosa from an overnight LB broth to 3.5 ml of LB top agar (0.75%). Ten μ l of a phage stock solution were spotted on the top-agar plate and incubated at 37°C for 12 to 16 h. After incubation, the appearance of the lysis zones at the site where the phage suspension was added, was examined. Each phage was tested against each bacterial strain in triplicate in independent experiments. The lysis was categorized as clear (+), turbid (0) and no reaction (-) as described . For detection of the phage receptor molecule, we used a P. aeruginosa flagella mutant (ΔfliM), a pili mutant (ΔpilA) and an LPS mutant (ΔalgC), which were infected with the phage JG024 as described above. The strains for the receptor identification are derived from a PAO1 wildtype and therefore belong to the same serotype as PAO1, namely serotype O5 . An effect on the efficiency of plating was not observed for the strains with intact LPS.
Phage growth characteristics
To determine phage growth characteristics like burst size and duration of the infection cycle, single step growth experiments were performed as previously described with some modifications [40, 41]. P. aeruginosa was grown aerobically in 10 ml LB medium until exponential growth phase. After the bacteria reached an OD578 of 0.3, an aliquot containing 5*108 phages was added to the culture which corresponds to a multiplicity of infection (MOI) of 0.16. Therefore, it is likely that a cell is infected by only one phage and that the amount of infected bacteria is equal to the amount of the initial phage concentration. After addition of the phages, one aliquot was immediately used for determination of the phage titer. Then, phages were allowed to adsorb for 15 min. Afterwards, cultures were diluted in LB 104-, 105-, 106- and 107 -fold and incubated at 37°C for 60 min. Samples for phage enumeration were taken aseptically at different time points after infection. The burst size was determined as: (phage titer at the end of the single step growth curve at time point 55 min minus phage titer at time point 20 min) divided by phage titer at time point 20 min. The latent phase was estimated at the midpoint of the exponential phase of a one step growth experiment [40, 41].
Sequencing, analysis and annotation of phage genomes
To isolate phage DNA, phages were propagated in top-agar plates as described above. After growth at 37°C the plates were overlayed with 10 ml SM buffer and incubated with shaking at 4°C for 4 h. The supernatant was sterile filtrated (0.22 μ m) and stored at 4°C. Phage DNA was isolated using the Qiagen Lambda Kit according to manufacturer's instructions. Ten ml phage lysate with a titer of at least 1*1010 phages/ml were used to isolate up to 1 μ g/μ l pure phage DNA. Digestion with restriction endonucleases was done following the protocols of the manufacturer. Whole genome sequencing of the phage JG024 was done at the McGill University and Génome Québec Innovation Centre (Montréal, QC, Canada) using the Genome Sequencer FLX and 454 Technology. A total of 66,684 reads with an average length of 344 bases was assembled to one single contig with a 300-fold coverage. The annotation of the unknown phage genes was done by using the software GeneMark.HMM . The Heuristic approach of GeneMark was used to identify genes in small genomes under 100 kb. The identified genes were compared with the NCBI ORF Finder . Nucleotide sequences were scanned for homologues using the Basic Alignment Search Tool (blastx) . To search for tRNA genes in the phage sequences the internet tool tRNAscan-SE 1.21 was used . Sequence comparison was conducted using ClustalW2 online analysis tool . Investigation of the codon usage was performed using a software tool based on JCat . The genome sequence as well as the annotation is deposited with the GenBank (National Center for Biotechnology Information) using the following accession number: GU815091.
Identification of promoter regions, terminator structures and other motifs
The genome of phage JG024 was scanned for the presence of sigma 70-dependent promoter regions using the web service SAK . Putative promoter regions with a score above 1 were scanned for the presence of conserved -10 and -35 regions using the Virtual Footprint software . Two promoter regions were identified in this way. Rho-independent terminator structures were identified using the TransTerm  and FindTerm (Softberry, Inc.) software tools. The program MEME was used for identification of conserved intergenic motifs in phage JG024 .
ASM infection assay
Phage susceptibility of P. aeruginosa in ASM medium was tested in 24 well plates. 1 ml ASM medium and as control LB medium were inoculated with indicated strains aerobically for 24 h at 37°C. An OD 578 of 0.5 was used for the inoculation. Afterwards, 1*105 phages were added which describes the initial phage concentration. After incubation for additional 24 h at 37°C the colony forming units (CFU) as well as the plaque forming units (PFU) were determined. To determine the change in phage concentration we divided the final phage concentration after 24 h by the initial phage concentration. To determine the effect of alginate the same experiment was performed in LB with purified alginate using increasing concentrations in a range of 50 μ g/ml to 1 mg/ml. Alginate was purified from mucoid P. aeruginosa strain FRD1  as described previously .
The authors thank Gerd Döring, Burkhard Tümmler and Michael Hogardt for providing the clinical P. aeruginosa strains. We thank Petra Tielen for the gift of isolated alginate. JG was supported by the DFG-European Graduate College 653.
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