ELÍAS: HOMBRE Y PROFETA
LA VENIDA DE ELÍAS EL PROFETA
2.3.1 Bacterial strains and culture conditions
A list of strains and plasmids used in this study can be found in Table 2.2. Bacteria were routinely cultured in glass universal bottles in a volume of 5 ml LB broth inoculated from single colonies on LB plates for 16 hours at 37°C with shaking at 220 rpm (this procedure is referred to in this thesis as ‘overnight culture’). Unless otherwise stated, for experiments, overnight cultures were used to sub-inoculate 1:1000 in 25 ml LB broth in 250 ml sterilize Erlenmeyer flasks and grown at 37°C with 220 rpm agitation. For growth in minimal media, 1 ml of overnight culture was harvested by centrifugation at 12,000 x g at room temperature for 1 minute. Cell pellets were washed three times in minimal media and sub-inoculated 1:500 in 25 ml of minimal media in 250 ml sterilized Erlenmeyer flasks and grown at 37°C with 220 rpm agitation. Optical density (OD) measurements were taken using a Jenway 67- series spectrophotometer at a wavelength of 600 nm. When colony forming unit (CFU) counts were required, 10-fold serial dilutions of cultures (up to a factor of 10-8) were carried out in sterile LB broth and triplicate 10 µl drops plated on LB agar plates (Miles and Misra method). CFU counts are given in units per ml (CFU/ml). Frozen stocks of all bacterial strains were kept at -80°C for the duration of the work. To prepare frozen stocks, overnight cultures were mixed with sterile (autoclaved) 50% glycerol to a final concentration of 20% in a 1.5 ml volume in a sterile 2 ml cryovial tube (i.e. 900 µl overnight and 600 µl 50% glycerol). Bacterial colonies were maintained on agar plates at 4°C for a maximum of 1 week.
2.3.2 Motility assays
0.3% LB agar was made-up and whilst molten, exactly 25 ml of agar were aliquoted into petri dishes to ensure minimal variation in agar depth between plates. The plates were left to dry at room temperature overnight (16 hours). 3 µl of overnight cultures were spotted on to the 0.3% LB agar plates. Care was taken not to pierce the agar surface with the pipette tip. Plates were left at room temperature for 30 minutes before incubation at 37°C for 5 hours. Motility was assessed by measurement of the diameter of migration. All strains were tested independently in triplicate.
2.3.3 RDAR morphotype assays
The methodology used to assay RDAR morphology was based a recent publication (Singletary et al., 2016). 2 µl of overnight bacterial cultures was spotted onto two sets of LB plates without NaCl and supplemented with 40 µg/ml Congo red (Table 2.1). Plates were incubated in parallel at 25°C and 37°C for 7 days without inversion. To avoid drying of the agar over the prolonged incubation time, plates were incubated
37 within a plastic bag. All experiments were conducted in triplicate. After 7 days colonial morphology was photographed using the ImageQuant LAS 4000 imager (GE Healthcare). The presence of a filamentous, patterned texture of the colony at 25°C indicated RDAR morphology, whereas smooth colony morphology was judged to be RDAR negative. RDAR morphology does not form at 37°C therefore this condition acted as a negative control.
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Table 2.2 All bacterial strains used in this thesis.
Bacterial strains Description a References/origin S. Typhimurium
D23580 derivatives
JH3621 D23580 WT (Kingsley et al., 2009)
JH3810 ∆BTP1::FRT This study
JH3798 ∆BTP5:FRT This study
JH3874 ∆Gifsy-1 (Owen et al., 2017)
JH3875 ∆Gifsy-2 (Owen et al., 2017)
JH3877 ∆BTP1 (Owen et al., 2017)
JH3878 ∆BTP5 (Owen et al., 2017)
JH3881 ∆BTP1 ∆BTP5 (Owen et al., 2017)
JH3940 ∆BTP1 ∆BTP5 ∆Gifsy-1 (Owen et al., 2017)
JH3942 ∆BTP1 ∆BTP5 ∆Gifsy-1 ∆Gifsy-2 (Owen et al., 2017)
JH3949 ∆Φ (∆BTP1 ∆BTP5 ∆Gifsy-1 ∆Gifsy-2 ∆ST64B) (Owen et al., 2017)
JH3983 ∆Gifsy-1, aph-galE496; KmR (Owen et al., 2017)
JH3984 ∆Gifsy-2, aph-galE496; KmR (Owen et al., 2017)
JH3986 ∆ST64B (Owen et al., 2017)
JH3987 PdinI-gfoAfrom 14028s (C→T substitution,
position 2,790,162 in D23580 chromosome) (Owen et al., 2017) D23580 BTP5::KmR BTP5::KmR (insertion of aph in BTP5); KmR This study
D23580∆BTP1 BTP5::KmR
∆BTP1 BTP5::KmR (insertion of aph in BTP5);
KmR This study
D23580 ΔrecA D23580 with recA gene replaced with nptII from pKD13 and subsequently flipped out using the Flp recombinase-encoding pCP20 plasmid
(Owen et al., 2017)
D23580 ST313-tdSTOP
D23580 with two stop codons introduced into the beginning of the ST313-td gene sequence (total change of 4 nucleotides)
This study 14028s derivatives
MA5958 14028s, WT L. Bossi
MA6684 ∆Gifsy-1 ∆Gifsy-2 bio-106::Tn10 galE496; TcR L. Bossi (Campoy et al.,
2006) LT2 derivatives
LT2 LT2, WT (Zinder and Lederberg,
1952) MA8508 Gifsy-1[-] Gifsy-2[-] Fels-2[-] Fels-1[Δ(int-
attR)104::cat]
L. Bossi (Lemire et al., 2011)
LT2 [BTP1] LT2 derivative MA8508 lysogenised with phage
BTP1 (Owen et al., 2017)
LT2 [P22] LT2 derivative MA8508 lysogenised with phage
P22 (wildtype) (Owen et al., 2017)
LT2 Δrec
LT2 derivative MA8508 with recA gene replaced with nptII from pKD13 and subsequently flipped out using Flp recombinase encoding pCP20 plasmid
(Owen et al., 2017) 4/74 derivative
4/74, WT (Rankin and Taylor, 1966)
JH3864 ∆Gifsy-2 N. Wenner
JH3865 ∆Gifsy-1 N. Wenner
JH3866 ∆Gifsy-1 ∆Gifsy-2 N. Wenner
JH3880 ∆ΦSopE N. Wenner
E. coli
S17-1 λpir pro thi hsdR recA chromosome::RP4-2 Tc::Mu Km::Tn7/λpir; TpR, SmR (Simon et al., 1983) a Relevant antibiotic resistances are indicated by R: Ap, ampicillin; Gm, gentamicin; Km, kanamycin, OxyTc, oxytetracyline; Sm,
streptomycin; Tc, tetracycline; Tp, trimethoprim. For clarity purposes, the natural resistances of D23580 and its derivative are not indicated.
39 2.3.4 Stationary phase catalase activity assay
Catalase activity was assayed based on previously described methods (Singletary et al., 2016). 20 µl of 20% aqueous H2O2 was added to 1 ml of bacterial overnight culture in 1 cm diameter glass test tubes and briefly vortexed to homogenise the suspension. Tubes were incubated at room temperature for 5 minutes and subsequently photographed and the height of the bubble column measured. Assays were conducted in triplicate.
2.3.5 Antibiotic resistance typing
Phenotypic antimicrobial susceptibility testing was carried out at Public Health England, Collindale for all UK-isolated ST313 strains. The antimicrobial susceptibility testing was done using breakpoint concentrations. Briefly, an agar dilution method involving Iso-sensitest agar or Muller-Hinton agar was used to determine if isolates were sensitive or resistant to a set concentration of individual antimicrobials (Table 2.3).
Antibiotic Concentrations tested
Ampicillin 8 mg/L Chloramphenicol 8 and 16 mg/L Colistin 2 mg/L Sulphonamide 256 mg/L Gentamicin 2 mg/L Tobramycin 8 mg/L Amikacin 8 mg/L Streptomycin 16 mg/L Tetracycline 8 mg/L Trimethoprim 2 mg/l Nalidixic Acid 16 mg/L Ciprofloxacin 0.064 and 0.5 mg/L Ceftazidime 1 and 2 mg/L Cefotaxime 0.5 and 1 mg/L Cefoxitin 8 mg/L Cefpirome 8 mg/L Ertapenem 0.064 and 0.5 mg/L Temocillin 128 mg/L.
Table 2.3 Antibiotic concentrations used for antimicrobial resistance typing of UK-isolated ST313 strains carried out by PHE (Chapter 4).
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2.3.6 Microscopy and preparation of samples
An EVOS FL cell imaging system (Thermo Fisher) fitted with a GFP light cube (470/22 nm excitation, 525/50 nm emission) was used to visualise cells under transmitted light and fluorescent light with an EVOS 40X flurorite coverslip-corrected objective (Thermo Fisher, AMEP4699) and an Olympus 100X super-apochromat, coverslip- corrected oil objective (Thermo Fisher, AMEP4733). 2 µl of cell samples were pipetted onto glass microscopy slides and covered with a glass coverslip. Where cell fixing was required, cell samples were pelleted by centrifugation at 11,000 Xg for 2 minutes, and washed in PBS buffer (Melford, P3203). Cells were further pelleted and resuspended in 4% paraformaldehyde PBS for 20 minutes at room temperature. Cells were again pelleted and resuspended in PBS ready for microscopy. Where the 100X objective was used, a drop of immersion oil (Sigma-Aldrich, 56822) was applied to the objective before imaging.