CHAPTER 1.................................................................................................................................. 1
2.3 Results
2.3.14 Sponge-associated Bacillus spp. extracts as inhibitors of specific P. aeruginosa QS
aeruginosa QS systems
The P. aeruginosa QS network can be broken down into three distinct QS systems (excluding the newly integrated IQS network). These three systems are the long chain las QS system, the short chain rhl QS system and the quinolone-based PQS system (Holm and Vikstrom, 2014;
Boopathi et al., 2017). As such, the virulence factors tested were themselves broken up into these three systems. Virulence factors primary under control of the las system are the production of elastase LasB and protease, while those under the control of the rhl system are rhamnolipid production and, due to the influence of rhamnolipids on this process, mature biofilm formation (Lee and Zhang, 2015). Pyocyanin and pyoverdine production receive their influence primarily through the PQS system (Welsh and Blackwell, 2016), although not exclusively, while the exact QS control mechanism in regard to swimming and swarming motility as well as initial biofilm formation remains to be fully elucidated and may consist of mixed influences. Due to the partially segregated nature of virulence factor production by P.
aeruginosa in terms of its QS network, the top three inhibitors of each of these assays were examined in order to see if a trend was revealed in regard to any specific sponge-associated Bacillus spp. extract or isolate.
In terms of the short chain rhl-AHL QS system, maximal inhibitions of over 75% were observed for rhamnolipid production while an inhibition of 86.28% was observed for mature
86
biofilm formation (Fig. 2.7). One bacterial isolate which stood out was B. weihenstephanensis SP5-AB7, which filled three of the top six spots as inhibitors of these two virulence factors.
This extract clearly displays then a proficiency for short chain QS inhibition.
Figure 2.7: Three of the best inhibitors of rhamnolipid and mature biofilm production which are primarily mediated through the short chain rhl system in P. aeruginosa. Extract SP5-AB7 (Mann) was identified as the lead inhibitor of rhamnolipid production (93.88%) as well as the third highest inhibitor of mature biofilm formation (15.71%). The medium 5294 extract of this isolate additionally resulted in a 79.96% rhamnolipid inhibition. Bacillus toyonensis extract SP2-AB7 (Mann) resulted in an 86.03% rhamnolipid inhibition while SP-AB2 (Mann) was the key inhibitor of mature biofilm formation (86.28%).
Similar to the observed inhibition in rhl-mediated virulence factor production, extract SP5-AB7 (Mann) proved once again to be a major inhibitor, this time of elastase production (71.32%) (Fig. 2.8). Also making a reappearance was B. toyonensis strain SP2-AB7 (Mann), which in addition to its rhamnolipid-inhibitory potential seen above, inhibited elastase production by 51.76% while the medium 5294 extract of the same isolate resulted in a 59.93%
decrease in protease production. Similarly, SP1-AB4 (5294) was one of the top inhibitors of mature biofilm formation, and the mannitol extract of the same organism inhibited both elastase and protease production by 50%, suggesting a broad spectrum QSI ability of this organism in addition to SP5-AB7 and SP2-AB7.
87
Figure 2.8: Three of the best inhibitors of elastase and protease production, which are primarily mediated through the long chain las system in P. aeruginosa. Extract SP5-AB7 (Mann) was identified as the lead inhibitor of elastase production (71.32%). This was followed by Bacillus toyonensis strain SP2-AB7 (Mann) and SP1-AB4 (Mann) with elastase inhibition of 51.76% and 50.32%, respectively. Medium 5294 extract of the B. toyonensis strain further inhibited protease production by 59.93% while SP1-AB4 (Mann) was additionally able to inhibit protease production by 50.07%, in conjunction with its elastase-inhibitory activity. The key inhibitor of protease production proved to be SP2-W6 with an inhibition of 63.81%.
For the quinolone-based PQS system in P. aeruginosa, similar trends in inhibition were observed. Extract SP5-AB7 (Mann) was the top inhibitor of both elastase and rhamnolipid production, and similarly it was the top inhibitor of pyocyanin production (39.36%). The medium 5294 extract was a top inhibitor of mature biofilm formation, as well as one of the top inhibitors of pyoverdine production (33.4%) (Fig. 2.9). Pyocyanin inhibition by SP5-AB7 (Mann) was followed by that of SP2-W6 (5294), which resulted in a decrease of 36.76% in conjunction with its previously observed protease-inhibitory capabilities. In conjunction to its pyoverdine-inhibitory activity (31.43%), SP1-AB4 (Mann) was a major inhibitor of protease and elastase production. Extract SP-AB2 (5294) inhibited pyocyanin production by 36.21% and SP-AB2 (Mann) inhibited pyoverdine production by 38.43%, the highest observed pyoverdine inhibition. This extract was not a top inhibitor of any of the previously discussed virulence factors, suggesting its QSI potential may be specific to the PQS system.
88
Figure 2.9: Three of the best inhibitors of pyocyanin and pyoverdine production, which are primarily mediated through the quinolone-based PQS system in P. aeruginosa. Extract SP5- AB7 (Mann) was identified as the lead inhibitor of pyocyanin production (39.36%) while the medium 5294 extract of the same was identified as one of the lead inhibitors of pyoverdine production (33.4%). Extract SP-AB2 (5294) resulted in a 36.21% decrease in pyocyanin production while the mannitol of extract of this organism resulted in the highest inhibition of pyoverdine production (38.43%). Extract SP2-W6 (5294) resulted in a pyocyanin inhibition of 36.76% while SP1-AB4 (Mann) resulted in a 31.43% reduction in pyoverdine production.
While the QS systems primarily mediating many of the P. aeruginosa virulence factors are well understood, in some instances there are too many systems with influence over the production of a virulence factor for the correlations to be so clear. Swimming and swarming motility as well as initial biofilm production are examples of such virulence factors (Fig. 2.10), although the PQS system has been putatively suggested to play a role in initial biofilm formation (Boopathi et al., 2017). Although unelucidated, these systems are themselves clearly linked, as similar extracts form the top inhibitors of each virulence factor. Extract SP1- AB4 (5294) swarming and initial biofilm formation by 56.96% and 69.39%, respectively. The mannitol extract of the same organism was the best inhibitor of initial biofilm formation, resulting in a reduction of 73.58%. Extract SP2-AB7 (5294) inhibited swimming and swarming (40.29% and 55.7%) while the mannitol extract of this organism was the major inhibitor of swarming motility, resulting in an inhibition of 59.49%. Both these organisms were proficient inhibitors of the rhl and las-mediated virulence factors, while SP1-AB4 additionally inhibited pyoverdine production, suggesting a broad spectrum QSI activity for these extracts. Extract SP5-AB7 (Mann) inhibited initial biofilm formation by 66.51%, in addition to being the top inhibitor of pyocyanin, LasB and rhamnolipid production. The medium 5294 extract of the
89
same organism resulted in the strongest inhibition in swimming motility (55.88%). This isolate clearly then possesses the ability to inhibit a broad range of QS systems.
Figure 2.10: The mediation of swimming and swarming motility as well as initial biofilm formation are closely intertwined, however the exact QS influence of these three virulence factors is not yet completely understood. They are either not known or highly variable. Here we see the best three inhibitors each of swimming, swarming and initial biofilm production.
Bacillus toyonensis extract SP2-AB7 (5294) resulted in >40% inhibition in both swimming and swarming motility, while the mannitol extract of this organism resulted in the highest swarming inhibition (59.49%). Extract SP1-AB4 (5294) resulted in a 56.96% reduction in swarming motility as well as a 69.39% reduction in initial biofilm formation. The mannitol extract of the same organism (SP1-AB4) resulted in the highest inhibition in initial biofilm formation, 73.58%. Extract SP2-W6 (Mann) resulted in a 39.55% reduction in swimming motility and SP5-AB7 (Mann) a 66.51% reduction in initial biofilm formation while SP5-AB7 (5294) resulted in a 55.88% reduction in swimming motility.