To quantitatively investigate the effects of rhPON2 on PAO1 growth, QS, virulence and tobramycin susceptibility, PAO1 cultures were co-treated with rhPON2 and tobramycin. PAO1 cultures either in pre-exponential growth-phase (Figure 5.16) or mid-exponential growth (Figure 5.17) were treated with 0, 5 or 25 U/mL rhPON2 and either with 0 or 10 µg/mL (2 MIC) tobramycin. We hypothesised that treatment of pre-exponential cultures would prevent bacterial QS and that treatment of mid-exponential phase cultures would dampen QS-mediated virulence. Samples were collected from three independent
experiments at 0, 2, 6, 12, 14 and 24 hours and analysed to determine the CFU/ml and C12HSL concentration (both open and closed ring forms).
Treatment of pre-exponential phase cultures of PAO1 with 5 U/mL rhPON2-alone did not affect bacterial growth (as seen in Figure 5.16A) but prevented accumulation of C12HSL (closed-ring form) presumably by promoting C12 HSL lactonolysis (open-ring form, Figure 5.16B). The maximum amount of C12HSL (closed-ring form) occurred during exponential growth and was significantly reduced in the presence of rhPON2. There was a
corresponding increase in concentration of the C12HSL-acid (open-ring form) that reflected the extensive activity of rhPON2 over the entire 24 hour period. The increased C12HSL-acid detected in rhPON2-treated culture supernatants compared to those collected from not- infected controls suggested that the bacteria might be compensating for the lack of C12HSL in their growth environment by increasing their overall production of C12HSL, which in would have been hydrolysed to the acid form being detected (Fig. 5.16B)
When pre-exponential growth phase cultures were treated with 2 MIC tobramycin, no viable bacterial cells were able to be recovered 24 hours post treatment. There also appeared to be little, if any, effect on the rate of antibiotic-mediated killing when cultures were co-treated with 5 U/mL rhPON2 and 2 MIC tobramycin and no detectable C12HSL (open- or closed-ring form, data not shown), most likely as a consequence of the rapid bacterial cell death. In summary, treatment of pre-exponential phase cultures had no detectable effect on bacterial growth, prevented accumulation of C12HSL (by rapid
hydrolysis to form C12HSL-acid) and did not alter bacterial susceptibility to tobramycin (2 MIC) most likely because of rapid killing of the cells by tobramycin.
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A B
Figure 5.16 Time-dependent effect of rhPON2 in combination with tobramycin on pre-exponential phase PAO1. LB media was inoculated with 5 105 colony forming
units per mL (CFU/mL) of PAO1. Recombinant hPON2 (5 U/ml) with or without tobramycin (2 MIC) was added at the time of inoculation and the cultures were grown at 37C for 24 hours. Bacteria were enumerated and the C12HSL (lactone and acid) concentrations in the culture supernatants were determined at 0, 2, 6, 10, 14 and 24 hours. Figure shows A) Bacterial growth rate and B) C12HSL-lactone (solid line) and C12HSL-acid (dotted line) concentration. Error bars represent S.D. from three independent experiments. H o u r s p o s t t r e a t m e n t C F U /m L P a O 1 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 1 00 1 01 1 02 1 03 1 04 1 05 1 06 1 07 1 08 1 09 1 01 0 B u ffe r O n ly 5 U /m L r h P O N 2 1 0 u g /m L T o b r a m y c in 5 U /m L P O N 2 + 1 0 u g /m L T o b r a m y c in T im e ( H o u r s ) n M C 1 2 H S L ( L a c t o n e /A c id ) 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 B u ffe r O n ly ( L a c to n e ) 5 U /m L r h P O N 2 ( L a c to n e ) 5 U /m L r h P O N 2 ( A c id ) B u ffe r O n ly ( A c id )
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A B C
Figure 5.17 Time-dependent effect of rhPON2 in combination with tobramycin on mid-exponential phase PAO1. LB media was inoculated with 5 105 colony forming units per mL (CFU/mL) of PAO1. Recombinant hPON2 (5 U/ml) with or without tobramycin (2 MIC) was added six hours post-inoculation (mid-exponential growth) and the cultures were grown at 37C for 24 hours. Bacteria were enumerated and the C12HSL (lactone and acid) concentrations in the culture supernatants were determined at 0, 1, 6 and 24 hours. Figure shows A) Bacterial growth rate, B) C12HSL-lactone concentration and C) C12HSL-acid concentration. Error bars represent S.D. from three independent experiments.
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When mid-exponential phase cultures of PAO1 that had already accumulated significant levels of C12HSL (~800 nm) were treated with either 5 U/mL or 25 U/mL rhPON2, bacterial growth was unaffected (Figure 5.17A). There was, however, rapid (over 10-fold decrease within the first hour of treatment) and almost complete hydrolysis of the accumulated C12HSL (less than 5 nM C12HSL remaining, Figure 5.17B) to the open-ring C12HSL acid form (Figure 5.17C).
These mid-exponential cultures were also resistant to complete killing by 2 MIC
tobramycin (Figure 5.17A). Tobramycin treatment alone decreases C12HSL concentration because of the decrease in cell numbers, as seen in Figure 5.17B, without substantial increases in the open-ring C12HSL acid form (Figure 5.17C). This is most likely the result of rapid bacterial killing causing a decrease in C12HSL production. However, when these cultures were co-treated with rhPON2 and 2 MIC tobramycin there was a significant (t-
test, p < 0.05) dose-dependent increase in bacterial killing in the presence of rhPON2, which coincided with hydrolysis of C12HSL (Figure 5.17C).
5.3.5.4 Effect of rhPON2 with or without antibiotics on strain PAO1 QS and