Introduction

P. aeruginosa is able to survive in and colonise a wide variety of environments including fresh and salt water, soil, plants, insects, nematodes and mammals [1-3]. The adaptability of P. aeruginosa is clearly demonstrated by survival in these environments, and this allows interaction with a plethora of other microbial species. These species, at a basic level, can be seen as genomic libraries of potential benefit to P. aeruginosa. P. aeruginosa may utilize horizontal gene transfer mechanisms to accumulate and express random, or perhaps even targeted, regions of foreign DNA. Any beneficial genomic additions are likely to pass through successive generations, and potentially create novel isolated epidemic strains. Indeed, epidemic strains of P. aeruginosa are known to occur in isolates from cystic fibrosis [4-6] and these can be differentiated on their genetic make-up.

Inflammation and infection of the cornea caused by P. aeruginosa can rapidly lead to scarring and loss of sight unless treated quickly and effectively [7]. In the eye, a combination of several P. aeruginosa virulence factors are associated with cellular damage and induction of the host immune response [8]. These include exoenzymes S (exoS), and U (exoU), [9] elastase (lasB), [10] alkaline protease (aprA) [11] and protease IV (prpL) [12]. P. aeruginosa also possess many transcriptional regulators such as Vfr, [13,14] AlgR [15] and quorum sensing systems [16] which control the expression of many keratitis-related virulence genes. Vfr controls the production of elastase, exotoxin A and twitching motility via type IV pili partly by controlling the las quorum sensing system in P. aeruginosa [13,14,17]. AlgR also regulates twitching motility, the production of hydrogen cyanide, pyocyanin and pyoverdin and quorum sensing via the rhl system [15,18,19].

However, there are both virulent and avirulent phenotypes of P. aeruginosa, [20,21] with the avirulent types causing the non-infectious inflammatory condition termed contact lens-induced acute red eye [22]. Even within virulent types there are those that cause acute cytotoxicity of corneal epithelial cells (cytotoxic strains) and those that can invade corneal epithelial cells (Invasive Strains). These two phenotypes are distinguished genotypically by possession of two type III secretion genes, exoS or exoU [23]. Indeed, the possession of exoU, along with a small group of other genes, has been shown to identify a specific sub-population or clone that is associated predominantly with infectious keratitis [23,24]. ExoU possessing strains are also associated with increased resistance to fluoroquinolones and beta-lactam antibiotics [25]. Using multilocus sequence typing, two other clonal types have been associated with keratitis, ST308 in Indian strains [26] and ST235 in UK strains [27].

The technique known as suppression subtractive hybridization (SSH) can be used to identify genetic differences between related strains. This technique essentially hybridizes the cDNA of two strains, with pieces of cDNA that differ between the strains remaining unhybridized. Polymerase chain reaction is then used to amplify this remaining cDNA which represents differentially expressed sequences or different genomic sequences. SSH has been used to study differences in many bacteria [28]. This technique was used to identify a unique genetic locus that can be used to detect the presence of clonal cystic fibrosis isolates from Australia [6] and the UK, [4] to identify the accessory genome of cystic fibrosis isolates, [5] or virulence genes (using the similar technique of representational difference analysis) in a highly pathogenic strain of P. aeruginosa [29]. SSH has not been used with ocular isolates of P. aeruginosa. Furthermore, most previous studies have used isolates from infections to study genetic relatedness. However, as strains can be isolated from asymptomatic people and from non-infectious events that can occur during contact lens wear, we were interested to study the relationship between these types of strains and those isolated from microbial keratitis.

Methods

Bacterial Strains, Growth Conditions

The P. aeruginosa strains used in this study are given in (Table 1). Strains were isolated from contact lens wearing subjects following a corneal inflammatory event (Paer1), routine microbiology analyses of asymptomatic patients (Paer2 and -3) or after corneal infection (Paer17, -26, 6294 and 6206) [28,30]. Previously, these strains had been characterized in terms of their serotype, [31,32] production of proteases (elastase, alkaline protease and protease IV), [30,32,33] pyoverdine and rhamnolipids production, [33,34] production of quorum sensing acylated homoserine lactones, [30] possession of genes for protease IV, [33] type IV secretion, [32,33] and quorum sensing, [34] as well as resistance to antibiotics, [32] phenotype of invasion or cytotoxicity to epithelial cells, [30] and virulence in a mouse model of corneal infection [20,21,34]. All bacteria were stored at -80°C in Trypticase Soya Broth (TSB; Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia) containing 30% glycerol, and were cultured at 37°C on Trypticase Soya agar (Thermo Fisher Scientific Australia). For general laboratory work, colonies of P. aeruginosa were inoculated into 5 ml TSB and incubated at 37°C with vigorous shaking for 18 hours to reach stationary phase.

Polymerase Chain Reactions for Detection of Virulence Genes

Oligonucleotide primers used in this study and their appropriate annealing temperatures are shown in (Table 2). All primers were synthesized by Invitrogen (Mulgrave, Vic, Australia). Vfr primers were designed using Prime (GCG) on the Biomanager suite (Australian National Genomic Information Services website: www.angis.org.au), using the PAO1 sequence from the Pseudomonas database. For template DNA in each polymerase chain reaction (PCR), bacterial DNA was released by incubating 1 ml of fresh overnight bacterial broth culture with 19 ml of microLYSIS^TM buffer (Microzone Ltd., Sussex, UK) which was heated in a thermal cycler using the following conditions: 65°C, 5 minutes; 96°C, 2 minutes; 65°C, 4 minutes; 96°C, 1 minute; 65°C, 1 minute; 96°C, for 30 seconds and then cooled to 25°C. DNA prepared by this method was used immediately or stored in small aliquoted amounts below -70°C. Template DNA was not exposed to repeat freeze thaw cycles. PCR was carried out in a reaction mixture containing 12.5 ml of BioMix Red (32 mM (NH₄)₂SO₄, 125 mM Tris-HCl, pH 8.8, 0.02% Tween 20, 2 mM dNTP’s, 2.5 mM MgCl₂, DNA polymerase 0.05 units/µl; BioLine GmbH, Luckenwalde, Germany), 100 pmol of each primer, 1 µl of Microlysis/bacterial DNA template, and brought to a reaction volume of 25 ml with nuclease free water. Parameters for the amplification cycles were: denaturation for 5 minutes at 94°C, then 30 cycles of 94°C for 30 seconds, annealing at 52-62°C for 30 seconds, and extension at 72°C for 90 seconds, followed by a final extension period of 72°C for 7 minutes. The expected amplification product size for each reaction is listed in (Table 2). PCR reactions were analyzed by electrophoresis though 1.5% agarose (in 45 mM Tris, pH 8.0, 45 mM boric acid and 1mM ethylenediaminetetraacetic acid buffer) gel, stained with SybrSafe (In vitro gen) and viewed under ultra-violet illumination. PCRs were performed at least twice for each primer pair and used freshly prepared template DNA on each separate occasion.

Suppression Subtractive Hybridization (SSH)

The oligonucleotides used for subtractive hybridizations are shown in (Table 2) [35]. SSH was used to compare the genomes of P. aeruginosa Paer3 and 6294. The SSH protocol used in this study was adapted from the methods originally described for Helicobacter pylori DNA, [35] initially using 6294 DNA as the tester and Paer3 as the driver DNA. The following minor changes to the original protocol [35] were made: RsaI restriction enzyme was used to digest the DNA; in the first PCR reaction, the final 72°C extension was increased to 7 minutes; the products of the second PCR were inserted into the pCR®4-TOPO® TA cloning kit (ThermoFisher Scientific, North Ryde, Australia) according to the manufacturer’s instructions. During sequencing, any vector DNA was removed in silico from each sequence using VecScreen at http://www.ncbi.nlm.nih.gov/VecScreen. Sequences were then submitted to the Genbank Database (National Centre for Biotechnology Information, Bethesda, MD) under accession numbers DQ436444-DQ436454 and screened for similarity to known protein sequences (BLASTx; http://www. ncbi.nlm.nih.gov/BLAST). The SSH protocol was repeated twice using identical techniques but reversing the 6294 and Paer3 DNA pools to determine whether Paer3 contained any additional DNA sequences to 6294.

Dot Blot Hybridizations

To determine the possession of the potential unique sequences in clones and other P. aeruginosa strains, 5mL of PCR product from each subtracted library clone or 10ml genomic DNA from other P. aeruginosa strains to be probed was heat denatured (95°C for 10 minutes, then 0°C for 10 minutes) and applied to Hybond N+ membrane and fixed by baking at 80°C for 2 hours. Labelled 6294 and Paer3 DNA was prepared by incubating 1 mg of heat denatured RsaI digested genomic DNA with 0.25 mM random primer (Promega Australia, Sydney, Australia), 1 mM ATP, CTP, GTP (each), 0.65 mM UTP, 0.35 mM DIG-11-UTP (Roche Applied Science, Mannheim, Germany), reaction buffer (50 mM Tris-HCl, pH 7.2, 10 mM MgSO4, 0.1 mM DTT) and 10 units of DNA polymerase I (Promega Australia) in a total volume of 50 μl at 37°C for 18 hours. A positive control used prpL primers (forward 5’-AGAGCCACTCCAGACCAAAC-3’; reverse 5’-GGATAAACGGCGGATAACAC-3’) and PAO1 DNA extract as template for the PCR reaction. The probes were purified using the Wizard SV Gel and PCR Cleanup Kit (Promega Australia), and the concentration estimated by spectrophometric absorbance readings at 260/280 nm. Pre-hybridization of the membrane occurred in nuclease free glass tubes (Hybaid) containing 0.125 μl/ cm2 DIG Easy Hyb solution (Roche Applied Science) for 2 hours at 42°C in a rotisserie-style hybridizing oven. The appropriate amount of probe was denatured by incubation for 5 minutes in a boiling water bath and then held on ice for 5 minutes. A small portion of the pre-hybridization solution was removed and mixed with the appropriate volume of denatured DIG labelled probe (final concentration 5 ng/ml). The probe solution was added back to the tube and allowed to hybridize at 42°C overnight. Unbound probe was removed by performing two successive low stringency washes for 5 minutes each in 5 X SSC buffer (0.75 M NaCl, 0.075 M Na citrate, pH 7.0), 0.1% (w/v) SDS (sodium dodecyl sulfate) at 25°C. This was followed by a high stringency wash in 1 X SSC, 0.1% (w/v) SDS at 42°C; and finally in 0.1 X SSC, 0.1% (w/v) SDS at 42°C. Membranes were then incubated for 5 minutes in maleic acid buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5) containing 3% (v/v) Tween 20 and blocked for 2 hours in maleic acid buffer containing 1% (w/v) blocking reagent (Roche Applied Science). The membranes were then incubated with a 1:5000 dilution of alkaline phosphatase-conjugated anti-DIG antibody (Roche Applied Science) in the blocking solution for 30 minutes. Membranes were washed twice for 15 minutes each in maleic acid buffer containing 3% (v/v) Tween 20, and DNA spots that bound the labelled probe were detected with Nitroblue Tetrazolium-5- Bromo-4-Chloro-3-Indolylphosphate (NBT-BCIP) in alkaline phosphatase detection buffer (100 mM Tris HCl, 100 mM NaCl, pH 9.5) until suitable development had occurred. Image analysis was performed by using Quantity One software and GS-800 calibrated densitometer (Bio-Rad Australia, Gladesville, NSW, and Australia) to determine the percentage of probe bound to the membrane, in comparison to the wildtype strain PAO1.

Gel Analysis and Sequencing

A 5 μl aliquot of each 6294-specific amplified fragment was analyzed by electrophoresis though a 1.5% (w/v) agarose gel in TBE buffer (45 mM Tris, pH 8.0, 45 mM boric acid and 1 mM EDTA), stained with SybrSafe (Invitrogen) and viewed under ultra-violet illumination. A DNA size comparison was included to allow size estimation of each clone fragment (BenchTop 1 kb Ladder, Promega Australia). Clones that produced a PCR product with a clear single band of a unique size to all other fragments were selected for subsequent analysis. Fragments were purified from the original PCR reaction using a Wizard SV Gel and PCR Cleanup Kit (Promega Australia), following the manufacturer’s instructions. The DNA sequence of each fragment was analyzed using an ABI Prism 3700 Automated DNA platform. Matches to each sequence were then found using the BLASTx search tool on the NCBI database website.

Results

All isolates were positive for vfr and algR by PCR. After SSH, a total of 144 clones were picked at random over both experiments. After dot blot screening (Figure 1), 31 of the DNA fragment inserts were identified to be present in strain 6294 and not in Paer3. Clones were then screened using PCR amplification and gel electrophoresis. Eleven clones were deemed suitable for further analysis and sent to be DNA sequenced (Lanes 1, 4, 5, 11, 13, 16, 18, 22, 23, 28, 30) (Figure 1).

Seven of the 11 sequences had significant similarity (>96%) to published P. aeruginosa genes. One sequence was too short to produce significant similarity to the database and was therefore excluded from further analysis. The analysis of each sequenced library fragment is shown in (Table 3). Two fragments (E5, B6) were of a higher G+C content, two (C18, D18) of lower G+C content and three (E12, E13 and F15) had approximately the average G+C content of PAO1. All fragments were submitted to Genbank and their accession numbers are given in (Table 3).

Each clone identified from the library screening was used to probe each of the strains used in this study (Table 4). The majority of the subtracted library clones (D1, E5, B6, E12, E13, F15, C18) were found to be present in all of the screened P. aeruginosa strains tested except Paer1 and Paer3. The sequences of clones B14, C15 and H17 were absent in all strains other than 6294. There was no significant hybridization between any of the probes and the negative control strains of P. putida and E. coli. All strains hybridized with DNA to prpL, except the negative controls.

After reversing the 6294 and Paer3 DNA pools, no DNA fragments were identified that were unique to the Paer3 genome once homologous 6294 DNA sequences were removed. This suggests that there were no additional genes or significant differences within the Paer3 genome.

Discussion

The P. aeruginosa strains used in the current study were chosen based upon the diversity of the ocular conditions they were isolated from and genotypic and phenotypic traits. All of these strains possessed the vrf and algR genes that encode for their transcription factors. Vrf is a global regulator of virulence factor expression, controlling the expression of exotoxin A, protease, type IV pili, a type III secretion system as well as another transcription regulatory system, the las quorum-sensing, which controls the expression of hundreds of additional genes, including multiple virulence factors [13,14,17,36,37]. The type III secretion effectors ExoS, ExoT, and ExoU have been shown to have important roles in the pathogenesis of P. aeruginosa keratitis, [38,39] as has exotoxin [40] Type IV pili that mediate twitching motility, [41] AlgR activates alginate production and twitching motility but represses the P. aeruginosa quorum-sensing system Rhl which is responsible for rhamnolipid production [19] and hydrogen cyanide, [15] pyocyanin and pyoverdine production [18]. The Rhl system appears to be less important than the Las system during keratitis, [34] whereas loss of the ability to produce pyoverdine reduces the virulence of P. aeruginosa during keratitis [42]. These data suggest that vfr would be important for strains to induce keratitis, but algR may be less important. However, the fact that strains, isolated as far apart in years as 1954 (PAO1) to 1998 (Paer26), have retained these genes suggests that they are important in survival of the organism.

In this study we identified genetic elements of pathogenic P. aeruginosa strain 6294 that were not present or not transcribed or significantly mutated in the non-pathogenic strain Paer3. By selecting strain 6294 for this study, instead of using the type strain PAO1, we were not only able to identify genes absent from the Paer3 genome, but also novel genes added to the 6294 strain that could potentially contribute to virulence during keratitis.

Six of the nine genes identified from the 6294 genome were not present in the cDNA library of Paer3. These six gene fragments were also absent from the cDNA library of strain Paer1, another avirulent isolate. The identification that the gene for the quorum-sensing signal generating enzyme LasI (clone F15) was missing from the cDNA library of Paer1 and Paer3 was a significant finding from this study and confirms previous findings [34] thus validating the SSH technique used in the current study. This enzyme produces an acylated homoserine molecule involved in the regulation of the Las quorum-sensing system [43-46]. A previous study demonstrated that the lasI gene controls virulence of P. aeruginosa in the eye [34]. This confirms previous evidence that shows strains Paer1 and -3 were deficient in this gene and that this was at least in part responsible for the attenuated virulence seen in the scratched mouse cornea model [34].

The probable tyrosine-type recombinase/integrase (Clone E13) of P. aeruginosa is of interest. Whilst this particular gene has not been implicated in P. aeruginosa virulence, these types of integrases catalyze recombination between DNA sequences that share limited identity and so may facilitate genomic rearrangements and integrations of mobile genetic elements within the chromosome of P. aeruginosa. The absence of this integrase in Paer3 and Paer1 may be an indication that, at some point in the evolution of these strains, the loss of this gene, and possibly surrounding genetic information, was favorable to survival. Conversely, the integrase may have been damaged or lost, preventing horizontal transfer of important virulence factors, and perhaps contributing to the more limited genetic difference of Paer3 compared to 6294. The role of these genes and their proteins in ocular virulence of P. aeruginosa requires further investigation with site-specific mutants.

Three of the nine genes identified by this study were unique to the cDNA of 6294 when compared to all the other strains used in this study, but two had homologues with genes from other P. aeruginosa strains. These two DNA fragments contained an AAA family ATPase or a hypothetical protein. AAA family ATPases couple energy generation from ATP hydrolysis to mechanical force and have been associated with flagella expression and rapid translation from a motile state to the sessile biofilm state [47], and also several other cellular functions. Perhaps possession or transcription of these genes gives certain strains of P. aeruginosa a competitive advantage. The ATPase is identical to that of several Pseudomonas aeruginosa strains (192S190811BSL_PA1, 192S190811BSL_PA2 192S190811BSL_PA3) isolated from sputa of a cystic fibrosis patients in Canada, and the hypothetical protein was identical to a gene from strains from China (such as WCHPA075045, WCHPA075022) and from throat swabs of cystic fibrosis patients in Germany (strains OY3, BJ2) and a microbial keratitis isolate (PA40) from India. The hypothetical protein may be involved in pathogenesis or other important functions of the virulent strains.

The unique DNA fragment B14 might be used to create a specific probe to identify 6294 and very closely related strains. Alternatively, lack of DNA fragments D1, E5, B6, E12, E13, F15 or C18 has the possibility of identifying avirulent strains such as Paer1 or Paer3. This might be important when people present with Contact Lens-Induced Acute Red Eye (CLARE). Having the ability to clearly identify avirulent strains that might be cultured from contact lenses at the time of a CLARE event may reduce the use of antibiotics, which are not needed to treat CLARE (Simply removing the contact lens is an appropriate treatment), [48] and so reduce the possibility of creating antibiotic resistance in strains. There is clear evidence that topical use of fluoroquinolones is associated with increased isolation of resistant strains from eyes [49,50].

Conclusion

In conclusion, this study identified several genes in the pathogenic microbial keratitis isolate that were absent in two strains that had been isolated from either a non-infectious inflammatory condition associated with bacterial colonisation of contact lenses, CLARE, or a strain isolated from the contact lens disinfecting storage case of an asymptomatic contact lens wearer, both of which had previously been shown to be avirulent in a mouse keratitis model. These genes might confer virulence phenotypes to P. aeruginosa and this should be followed up using strains deleted in those genes in the mouse model of keratitis.

Acknowledgement

This study was partly funded by a grant from the National Health and Medical Research Council, Australia, APP350900.

References

1. Rahme LG, Tan MW, Wong SM, Tompkins RG, Calderwood B, et al. (1997) Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc Natl Acad Sci USA 94: 13245-13250.
2. Jander G, Rahme LG, Ausubel FM (2000) Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects. J Bacteriol 182: 3843-3845.
3. Tan MW, Mahajan-Miklos S, Ausubel FM (1999) Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc Natl Acad Sci USA 96: 715-720.
4. Smart CH, Scott FW, Wring EA, Walshaw MJ, Hart CA, et al. (2006) Development of a diagnostic test for the Midlands 1 cystic fibrosis epidemic strain of Pseudomonas aeruginosa. J Med Microbiol 55: 1085-1091.
5. Smart CH, Walshaw MJ, Hart CA, Winstanley C (2006) Use of suppression subtractive hybridization to examine the accessory genome of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa. J Med Microbiol 55: 677-688.
6. Williams HL, Turnbull L, Thomas SJ, Murphy A, Stinear T, et al. (2010) A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa. Ann Clin Microbiol Antimicrob 9: 18.
7. Sankaridurg PR, Sharma S, Willcox M, Naduvilath TJ, Sweeney DF, et al. (2000) Bacterial colonization of disposable soft contact lenses is greater during corneal infiltrative events than during asymptomatic extended lens wear. J Clin Microbiol 38: 4420-4424.
8. Hazlett LD (2002) Pathogenic mechanisms of aeruginosa keratitis: a review of the role of T cells, Langerhans cells, PMN, and cytokines. DNA Cell Biol 21: 383-390.
9. Fleiszig SM, Wiener-Kronish JP, Miyazaki H, Vallas V, Mostov KE, et al. (1997) Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun 65: 579-586.
10. Kessler E, Blumberg S (1987) Specific inhibitors of Pseudomonas aeruginosa elastase as potential drugs for the treatment of Pseudomonas keratitis. Antibiot Chemother 39: 102-112.
11. Twining SS, Kirschner SE, Mahnke LA, Frank DW (1993) Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins. Invest Ophthalmol Vis Sci 34: 2699-2712.
12. O'Callaghan RJ, Engel LS, Hobden JA, Callegan MC, Green LC, et al. (1996) Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence. Invest Ophthalmol Vis Sci 37: 534-543.
13. Albus AM, Pesci EC, Runyen-Janecky LJ, West SE, Iglewski BH (1997) Vfr controls quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179: 3928-3935.
14. Beatson SA, Whitchurch CB, Sargent JL, Levesque RC, Mattick JS (2002) Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 184: 3605-3613.
15. Lizewski SE, Schurr JR, Jackson DW, Frisk A, Carterson AJ, et al. (2004) Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis. J Bacteriol 186: 5672-5684.
16. Lee J, Zhang L (2015) The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 6: 26-41.
17. West SE, Sample AK, Runyen-Janecky JL (1994) The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. J Bacteriol 176: 7532-7542.
18. Little AS, Okkotsu Y, Reinhart AA, Damron FH, Barbier M, et al. (2018) Pseudomonas aeruginosa AlgR phosphorylation status differentially regulates pyocyanin and pyoverdine production. mBio 9.
19. Okkotsu Y, Tieku P, Fitzsimmons LF, Churchill ME, Schurr MJ (2013) Pseudomonas aeruginosa AlgR phosphorylation modulates rhamnolipid production and motility. J Bacteriol 195: 5499-5515.
20. Cole N, Willcox MD, Fleiszig SM, Stapleton F, Bao B, et al. (1998) Different strains of Pseudomonas aeruginosa isolated from ocular infections or inflammation display distinct corneal pathologies in an animal model. Curr Eye Res 17: 730-735.
21. Cowell BA, Willcox MD, Hobden JA, Schneider RP, Hazlett LD (1998) An ocular strain of Pseudomonas aeruginosa is inflammatory but not virulent in the scarified mouse model. Exp Eye Res 67: 347-56.
22. Holden BA, Hood DL, Newton-Howes GJ, Baleriola-Lucas C, Willcox MD, et al. (1996) Gram-negative bacteria can induce contact lens related acute red eye (CLARE) responses. CLAO J 22: 47-52.
23. Lomholt JA, Poulsen K Kilian M (2001) Epidemic population structure of Pseudomonas aeruginosa: evidence for a clone that is pathogenic to the eye and that has a distinct combination of virulence factors. Infect Immun 69: 6284-6295.
24. Stewart RM, Wiehlmann L, Ashelford KE, Preston SJ, Frimmersdorf E, et al. (2011) Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections. J Clin Microbiol 49: 993-1003.
25. Subedi D, Vijay AK, Kohli GS, Rice SA, Willcox M, et al. (2018) Association between possession of ExoU and antibiotic resistance in Pseudomonas aeruginosa. PLoS One 13: e0204936.
26. Subedi D, Vijay Ak. Kohli GS, Rice SA (2018) Comparative genomics of clinical strains of Pseudomonas aeruginosa strains isolated from different geographic sites. Sci Rep 8: 15668.
27. Hall AJ, Fothergill JL, Kaye TJ, Neal SB, McNamara PS, et al. (2013) Intraclonal genetic diversity amongst cystic fibrosis and keratitis isolates of Pseudomonas aeruginosa. J Med Microbiol, 2013. 62(Pt 2): p. 208-16.
28. Winstanley C (2002) Spot the difference: applications of subtractive hybridisation to the study of bacterial pathogens. J Med Microbiol 51: 459-467.
29. Choi JY, SIfri CD, Goumnerov BC, Rahme LG, Ausubel FM, et al. (2002) Identification of virulence genes in a pathogenic strain of Pseudomonas aeruginosa by representational difference analysis. J Bacteriol 184: 952-961.
30. Zhu H, Thuruthyil SJ, Willcox MD (2002) Determination of quorum-sensing signal molecules and virulence factors of Pseudomonas aeruginosa isolates from contact lens-induced microbial keratitis. J Med Microbiol 51: 1063-1070.
31. Thuruthyil SJ, Zhu H, Willcox MD (2001) Serotype and adhesion of Pseudomonas aeruginosa isolated from contact lens wearers. Clin Exp Ophthalmol 29: 147-149.
32. Zhu H, Conibear TCR, Bandara R, Aliwarga Y, Stapleton F, et al. (2006) Type III secretion system-associated toxins, proteases, serotypes, and antibiotic resistance of Pseudomonas aeruginosa isolates associated with keratitis. Curr Eye Res 31: 297-306.
33. Conibear TC, Willcox MDP, Flanagan JL, Zhu H (2012) Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates. J Med Microbiol 61: 180-190.
34. Zhu H, Bandara R, Conibear TCR, Thuruthyil SJ, Rice SA, et al. (2004) Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection. Invest Ophthalmol Vis Sci 45: 1897-1903.
35. Akopyants NS, Fradkov A, Diatchenko L, Hill JE, Siebert PD, et al. (1998) PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc Natl Acad Sci USA 95: 108-113.
36. Schuster M, Lostroh P, Ogi T, Greenberg EP (2003) Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185: 2066-2079.
37. Wagner VE, Bushnell D, Passador L, Brooks AI, Iglewski BH (2003) Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J Bacteriol 185: 2080-2095.
38. Sun Y, Karmakar M, Taylon PR, Rietsch A, Pearlman E (2012) ExoS and ExoT ADP ribosyltransferase activities mediate Pseudomonas aeruginosa keratitis by promoting neutrophil apoptosis and bacterial survival. J Immunol 188: 1884-1895.
39. Tam C, Lewis SE, Lee E, Evans DJ, Fleiszig SMJ (2007) Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity. Exp Eye Res 85: 799-805.
40. Pillar CM, Hobden JA (2002) Pseudomonas aeruginosa exotoxin A and keratitis in mice. Invest Ophthalmol Vis Sci 43: 1437-1444.
41. Zolfaghar I, Evans DJ, Fleiszig SM (2003) Twitching motility contributes to the role of pili in corneal infection caused by Pseudomonas aeruginosa. Infect Immun 71: 5389-5393.
42. Suzuki T, Okamoto S, Oka N, Hayashi N, Gotoh N, et al. (2018) Role of pvdE pyoverdine synthesis in Pseudomonas aeruginosa Cornea 37: S99-S105.
43. Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A, et al. (1994) Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci USA 91: 197-201.
44. Latifi A, Foglino M, Tanaka K, Wiliams P, Lazduski A (1996) A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol 21: 1137-1146.
45. Pearson JP, Pesci EC, Iglewski BH (1997) Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179: 5756-5767.
46. Passador L, Cook JM, Gambello MJ, Rust L (1993) Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260: 1127-1130.
47. Banerjee P, Chanchal, Jain D (2019) Sensor I regulated ATPase activity of FleQ is essential for motility to biofilm transition in Pseudomonas aeruginosa. ACS Chem Biol 14: 1515-1527.
48. Sweeney DF, Jalbert I, Covey M, Sankaridurg PR, Vajdic C, et al. (2003) Clinical characterization of corneal infiltrative events observed with soft contact lens wear. Cornea 22: 435-442.
49. Fintelmann RE, Hoskins EN, Lietman TM, Keenan JD, Gaynor BD, et al. (2011) Topical fluoroquinolone use as a risk factor for in vitro fluoroquinolone resistance in ocular cultures. Arch Ophthalmol 129: 399-402.
50. Ray KJ, Prajna L, Srinivasan M, Geetha M, Karpagam R, et al. (2013) Fluoroquinolone treatment and susceptibility of isolates from bacterial keratitis. JAMA Ophthalmol 131: 310-313.