Endpoint isolates were tested for resistance to each of the LES phages. Phage resistance was widespread in both treatments (Figure 4.7). The majority of isolates were resistant to all three phages in all but one population (#5), in which 93% of isolates remained susceptible to all three phages.
Control Phage 0 .0 0 0 .0 5 0 .1 0 0 .1 5 0 .2 0 0 .2 5 Treatment D iv e rs ity in d e x
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Figure 4.7 Phage resistance of endpoint isolates to LESφ2-4. (A.) Control and (B.) phage-treated populations. Bars are coloured according to the particular phage combination to which resistance was observed. High titre pure phage stocks were spotted onto a soft-agar lawn of each isolate and scored as resistant if no lysis was observed after overnight incubation at 37˚C.
4.3.2.5 Biofilm formation
In order to assess whether isolates evolved in ASM had altered in their capacity to form biofilms, a simple in vitro assay was used to measure surface-attached biofilm formation. Due to the inherent variability of this assay, a large number of replicates are required, so it was not feasible to test all the isolates; instead, the first 10 from each population were tested. After staining of the biofilm with crystal violet and solubilisation, the A590 was measured as a proxy of biofilm formation, and
normalised to the ancestor. Overall, there was no trend towards increased or decreased biofilm formation (Figure 4.8). There was large variability within many
121 populations, most notably population #3, which has a median biofilm formation 1.5 times that of the control, but included poor and hyperbiofilm producers in the population, resulting in a large range.
Figure 4.8 Biofilm formation of isolates obtained from endpoint populations. Biofilm formation was assessed by crystal violet staining of a surface-attached biofilm, followed by solubilisation and measurement at A590. Technical replicates were normalised the PAO1 control on the same plate. Two biological replicates were performed for each isolates, and 10 isolates were assayed per population. Boxes represent the interquartile range of the population, the middle black line the median, the whiskers the 5th and 95th percentiles, and the black circles outliers.
4.3.2.6 Antibiotic resistance
Disc diffusion assays revealed all 40 isolates from every population to be as susceptible to the selected panel of antipseudomonals as the ancestor. The method was then adapted to look for clinically resistant isolates that may be present at a low frequency in the entire population, by plating entire populations on agar containing antibiotics at clinical resistance breakpoint levels.
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The frequency of antibiotic resistant isolates varied according to population and to antibiotic, with certain populations containing a relatively high frequency of isolates displaying clinical resistance (Figure 4.9); tazobactam-piperacillin resistance was displayed in 0.08% of the population in population 11. Resistance occurred in a higher frequency of cells in this population than in the mutator strain, PAO1ΔmutS. Resistance to ceftazidime and tazobactam-piperacillin arose more frequently than resistance to other antibiotics. No meropenem resistant isolates were identified.
In order to assess the extent to which the mutator phenotype of the isolates is responsible for the high frequencies of antibiotic resistant cells, the proportion of hypermutators in a population was plotted against the (arcsine transformed) frequency of resistant isolates (Figure 4.10). This was done for ceftazidime and tazobactam-piperacillin, as these were the antibiotics to which the highest level of resistance was detected. A correlation was detected between the frequency of hypermutators and tazobactam-piperacillin resistance (Spearman’s rank correlation coefficient: ρ = 0.69, p < 0.05), and between the frequency of hypermutators and ceftazidime resistance (Spearman’s rank correlation coefficient: ρ = 0.72, p < 0.01).
123 Figure 4.9 Frequency of bacterial cells in whole populations displaying clinical resistance to a panel of anti- pseudomonals (arcsine transformed). Frequencies were calculated after plating endpoint populations onto antibiotic free media, and media containing antibiotics at the clinical resistance breakpoint level.
Planktonic PAO1 and PAO1 grown in ASM were included as negative controls. PAO1 ΔmutS was included as a reference. Error bars ±1 S.E.M.
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Figure 4.10 The correlation between the frequency of hypermutators in a population and the frequency of isolates (arcsine transformed) demonstrating clinical resistance to the antibiotics ceftazidime and
piperacillin/tazobactam (n=12).
In order to further characterise the nature of the spontaneously occurring antibiotic resistance, resistant colonies growing on ceftazidime or tazobactam-piperacillin from population #11 were isolated and MICs to both antibiotics determined (n = 20). The MICs were at least two-fold higher than the clinical resistance breakpoints, and there was a high degree of cross-resistance; all isolates found to be resistant to one
antibiotic were also resistant to the other (Table 4.1). This suggests a resistance mechanism common to both antibiotics. Ceftazidime and piperacillin are both beta- lactam antibiotics, and certain beta-lactamase enzymes are capable of degrading both, even in the presence of tazobactam (an inhibitor). In order to clarify if beta- lactmases were being produced by these isolates, a beta-lactamase assay was conducted on five randomly selected isolates from each antibiotic medium, using nitrocefin, a chromogenic substrate for beta-lactamases.
125 Beta-lactamase production was observed in all the isolates tested, to varying degrees, with no clear difference between isolates originally isolated from media containing ceftazidime or tazobatam-piperacillin (Figure 4.11).
Table 4.1 Minimum inhibitory concentrations (as determined by broth microdilution) of PAO1 isolates displaying resistance to ceftazidime or tazobactam-piperacillin, to each antibiotic.
Isolated from MICs CAZ (mg L-1) MICs TZP (mg L-1)
CAZ 32-128 128-256
TZP 32-64 128
Figure 4.11 Beta-lactamase production in ceftazidime and tazobactam-piperacillin resistant isolates. Activity was measured using nitrocefin, in a chromogenic assay by calculating the change in A486/390 per minute. The dashed line indicates the value of the negative control (PAO1). The MIC of CAZ required for all isolates tested that were originally isolated from CAZ was 64 µg ml-1.
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