The strains of Pseudomonas aeruginosa used in this study and their origin are listed in Table 2.1. All strains were stored in Lysogeny Broth (LB) (Appendix A), with the addition of 25% (v/v) glycerol, and frozen at -80˚C. When required, a small aliquot of frozen cells was plated onto Columbia agar (Oxoid) and incubated overnight at 37˚C. When liquid cultures were required, a single colony was transferred into a glass universal containing a 5 ml aliquot of LB and incubated at 37˚C, with shaking at 180 r.p.m. until desired growth was achieved.
Table 2.1 Bacterial strains used in this study and sources Name Pseudomonas aeruginosa strain LES prophages present in genome Description Origin PAO1 (aka PAO1φ-)
PAO1 None Wild-type PAO1 Winstanley strain
collection: well studied laboratory reference strain (Stover et al., 2000) PAO1 GFP a.k.a. PAO1 GmR
PAO1 None Mini-Tn7 transposon marker
inserted at a neutral and site- specific location on
chromosome. Expresses green fluorescent protein (GFP) and gentamicin resistance. Winstanley strain collection. Constructed by Dr. Chloe James (unpublished) PAO1 DsRed- Express
PAO1 None Mini-Tn7 transposon inserted
at a neutral and site-specific location on chromosome. Expresses DsRed-Express protein and gentamicin resistance. Winstanley strain collection. Constructed by Dr. Chloe James (unpublished)
PAO1ΔmutS PAO1 None Displays hypermutable
phenotype
Winstanley strain collection
PAO1pilA-
::TetR
PAO1 None Mini-Tn5lux transposon
mutant (tetracycline resistance). Insertion in pilA gene results in absence of typeIV pili.
(Taylor & Buckling, 2010)
PA14 PA14 None Wild-type PA14 Winstanley strain
collection: well- characterised laboratory reference strain (He et al., 2004)
PLPLφ2 PAO1 LESφ2 LESφ2 lysogen Winstanley strain
collection.
Constructed by Chloe James (James et al.,
38
2012).
PLPLφ2 GFP PAO1 LESφ2 Marker as for PAO1 GFP.
LESφ2 lysogen Winstanley strain collection. Constructed by Chloe James (unpublished) PLPLφ2 DsRed- Express
PAO1 LESφ2 Marker as for PAO1 DsRed-
Express. LES φ2 lysogen
PLPLφ3 PAO1 LESφ3 LESφ3 lysogen Winstanley strain
collection.
Constructed by Chloe James (James et al., 2012).
PLPLφ3 GFP PAO1 LESφ3 Marker as for PAO1 GFP.
LESφ3 lysogen Winstanley strain collection. Constructed by Chloe James (unpublished) PLPLφ3 DsRed- Express
PAO1 LESφ3 Marker as for PAO1 DsRed-
Express. LESφ3 lysogen
PLPLφ4 PAO1 LESφ4 LESφ4 lysogen Winstanley strain
collection.
Constructed by Chloe James (James et al., 2012).
PLPLφ4 GFP PAO1 LESφ4 Marker as for PAO1 GFP.
LESφ4 lysogen Winstanley strain collection. Constructed by Chloe James (unpublished) PLPLφ4 DsRed- Express
PAO1 LESφ4 Marker as for PAO1 DsRed-
Express. LESφ4 lysogen
PLPLφtriple PAO1 LESφ2, 3 and 4 Triple LES phage lysogen Winstanley strain
collection.
Constructed by Chloe James (James et al., 2012).
PLPLφtriple GFP
PAO1 LESφ2, 3 and 4 Marker as for PAO1 GFP.
Triple LES phage lysogen
Winstanley strain collection. Constructed by Chloe James (unpublished) PLPLφtriple DsRed- Express
PAO1 LESφ2, 3 and 4 Marker as for PAO1 DsRed-
Express. Triple LES phage lysogen
LESB58 LES LESφ1:6 (full
complement)
Clinical strain; CF isolate Winstanley strain
collection
(Winstanley et al., 2009)
LESB65 LES LESφ1:4, 6 Clinical strain; CF isolate Winstanley strain
collection (Fothergill et al., 2007; Jeukens et al., 2014)
LES400 LES LESφ1:6 (full
complement)
Clinical strain; CF isolate Winstanley strain
collection (Jeukens et al., 2014; Parsons et al., 2002)
LES431 LES LESφ1,3:6 Clinical strain; Isolate from
non-CF parent of child with CF
Winstanley strain collection (Jeukens et al., 2014; McCallum et al., 2001)
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2.1.1 Quantification of viable bacteria in a sample
Bacterial cultures were serially diluted 10-fold and 6 x 20 µl spots pipetted onto Columbia agar for each dilution and allowed to dry. Plates were incubated overnight, the resulting colonies counted, and the colony forming units (c.f.u.) ml-1 calculated. If bacterial colonies were required for further processing, then a spread-plate method was used, to ensure numerous, well-separated colonies. 75 µl of a dilution was spread evenly across the surface of the plate with a sterile L-shaped spreader and allowed to dry. All platings were conducted in triplicate.
2.1.2 Determination of the minimal inhibitory concentration (MIC) of
antibiotics on bacterial strains
Antibiotic powders were purchased from Sigma-Aldrich (unless otherwise stated) and dissolved in 1 ml sterile distilled water (SDW), with the exceptions of
norfloxacin, which was dissolved in 1% (v/v) glacial acetic acid and water, and nitrocefin and rifampicin, which were dissolved in 100 mg ml-1 dimethyl sulfoxide (DMSO). SDW was added and the antibiotic solution sterile filtered through a 0.2 µm syringe filter (Millipore). The concentrated stocks (100, 10 or 1 mg ml-1) were frozen at -20˚C until required, or made fresh, depending on the manufacturer’s recommendations. Stocks were diluted in Mueller Hinton broth (MHB (Oxoid)) to obtain required working concentrations.
2.1.2.1 Broth microdilution method
Two-fold serial dilutions of MHB/antibiotic were dispensed into rows of a sterile 96- well clear, flat-bottomed microtitre plate (50 µl per well). Overnight liquid bacterial cultures were diluted to the turbidity equivalent of a McFarland standard 0.5
(Beckton Dickinson), followed by a further 10-2 dilution, and 50 µl of this was added
to each well and pipetted up and down to mix thoroughly. A growth control (MHB and bacteria only) and a sterility control (MHB only) were included on each plate. Plates were incubated for 18 hours at 37 ˚C and the MIC (i.e. lowest antibiotic concentration at which growth is inhibited) was determined by manual inspection of wells. Four wells per strain were tested on each plate, and each strain was tested on three individual plates.
40
2.1.2.2 Determination of the MIC for two antibiotics used in conjunction
The MICs of two antibiotics when used in combination may differ due to synergism or antagonism. To determine the MICs of two antibiotics used together, the
checkerboard broth microdilution method was used. As in (2.1.2.1), all dilutions were performed in MHB. Two-fold serial dilutions of antibiotic 1 were dispensed into the rows of a 96 well plate, and two fold serial dilutions of antibiotic 2 were dispensed into the columns. Each well contained 25 µl of each antibiotic. Bacteria were added to the wells and controls included on each plate (2.1.2.1). The
combination MIC was determined as the lowest concentration for each antibiotic at which no bacterial growth was observed. One strain was tested per plate, and nine replicate plates were tested for each strain, necessary because of the inherent unreliability of this test (White et al., 1996).
2.1.2.3 Disk diffusion method
For high-throughput testing of multiple isolates, the standardised disc diffusion method was used, according to the British Society of Antimicrobial Chemotherapy (BSAC) guidelines (Andrews & Howe, 2011). Antibiotic susceptibility was
determined by referring to the published breakpoints for P. aeruginosa (EUCAST, 2013). All isolates were tested in duplicate.