In this study, we found that independent of the antibiotic mode of action, certain sub-MICs increased the level of biofilm formation by the S. intermedius WT but not by the S. intermedius luxS mutant. At these sub-MICs, the S. intermedius WT biofilms displayed increased viable counts, while the luxS mutant biofilm counts decreased. In planktonic cell cultures, the luxS mutant exhibited increased susceptibility to the antibiotics at several sub-MICs compared to that of the WT planktonic cell cultures.
Several bacteria have been reported to display increased biofilm formation either as a specific response to certain antibiotics or as a result of a broader antibiotic stress reaction (11
). In our study, the level of biofilm formation by S. intermedius
increased upon exposure to antibiotics with diverse targets, indicating the involvement of a general response. This is in accordance with current findings indicating that stress conditions may promote biofilm formation (12
). Adoption of a protective biofilm lifestyle may thus provide a survival advantage to bacterial cells.
Notably, the increase in the S. intermedius
biofilm mass and viable counts in the presence of the antibiotics at certain sub-MICs was observed in the WT but not the luxS
mutant. This supports the assumption that the mechanisms leading to increased biofilm formation in the WT depends on the AI-2/LuxS system. Another possibility is that the increased susceptibility of the luxS
mutant to the antibiotics resulted in the availability of fewer viable cells to form biofilms. It is interesting, however, that in the WT, increased levels of biofilm formation were observed at concentrations that affected planktonic cell viability. The results indicated, therefore, that both increased and decreased biofilm formation may be observed concomitantly with an inhibitory effect on bacterial planktonic cell viability. In accordance with this finding, various antibiotics show either a restrictive or a provocative effect on biofilm viability, in addition to their inhibitory effects on growth (8
Since LuxS possesses an integral function in the methyl-activated cycle, increased antibiotic susceptibility and enhanced biofilm formation could also arise from metabolic defects following luxS
). We therefore investigated whether the AI-2 precursor DPD may complement the altered antibiotic response of the luxS
mutant. DPD restored the defective biofilm formation and the viability counts of the luxS
mutant to the WT levels at all sub-MICs tested. Our results indicate, therefore, that AI-2 molecules are implicated in the biofilm-forming and susceptibility responses observed.
In Streptococcus pneumoniae
, penicillin at the MIC50
significantly upregulates luxS
). We therefore explored the possibility that S. intermedius luxS
expression would be affected upon exposure to lower sub-MICs. Our results revealed a modest upregulation of luxS
expression in response to sub-MICs that increased the level of S. intermedius
WT biofilm formation. These subtle changes may be crucial to the synthesis of threshold AI-2 levels. It is difficult to ascertain whether such a modest effect would play a significant role in the biofilm outcomes observed. However, since LuxS is engaged in the global regulatory genetic networks of several bacteria (21
), complex mechanisms may be involved in antibiotic stress responses. The diverse role of AI-2/LuxS in bacteria suggests that numerous pathways may be involved in biofilm formation (9
). Nonetheless, the molecular basis for the role of AI-2/LuxS in increased biofilm formation by S. intermedius
in the presence of sub-MICs of various antibiotics calls for further investigation.
In conclusion, the AI-2/LuxS system in S. intermedius was involved in increased biofilm formation and susceptibility to various antibiotics at several sub-MICs. Our results support the significance of intercellular signaling in bacterial survival strategies and emerging views on interference with bacterial signaling as a novel means of fighting infections.