Since the 1970s, microbiologists have realized that bacteria grow predominantly as biofilms in a large diversity of environments, rather than as free-living planktonic cells (22
). Within these biofilms, the bacteria are better protected from external stress factors like antibacterial agents and the immune system of the host (9
In this study, we have synthesized and screened a small focused library of brominated furanones for their activities against Salmonella biofilm formation. Since we envisaged the main application of compounds that inhibit biofilm formation in the environment outside the host, in order to limit the spread and the survival of this pathogen, we studied Salmonella biofilm formation under nutrient-poor conditions at 16°C. We focused on differences in the alkyl chain lengths of the furanones to investigate whether this feature is important for their activity in Salmonella. The following structure-activity relationship can be derived from the results depicted in Table . Furanones without an alkyl chain (Fur-1 to Fur-3) were the strongest biofilm inhibitors but were also more toxic for Salmonella than the alkylated furanones, which might be correlated with the higher water solubility of the nonalkylated compounds. Of the 3-alkylated furanones, only molecules with one bromine atom on the ring structure and one on the methylidene side chain (Fur-5, Fur-6, and Fur-8) showed Salmonella biofilm-inhibiting activities. No activity could be detected when a dibromomethylidene substituent was present (Fur-4 and Fur-7). Since the observed activities were limited to molecules with alkyl chains up to six carbon atoms long, Fur-9 to Fur-11 probably are too little water soluble to be biologically active in our experimental setup. To the best of our knowledge, this is the first report on brominated furanones inhibiting Salmonella biofilm formation. Given the higher toxicity of the nonalkylated furanones (Fur-1 to Fur-3), we conclude that the furanones Fur-5, Fur-6, and Fur-8 are the most interesting compounds among those tested. Interestingly, the nonbrominated furanone Fur-12 did not show any activity under the conditions used, stressing the importance of bromination of the furanones to be active as Salmonella biofilm inhibitors.
In addition, we explored the activities of the brominated furanones in combination with antibiotics. Table shows that interesting effects were observed for the three antibiotics tested, since the combined treatment with Fur-8 and antibiotic resulted in a stronger decrease in the number of viable cells than would be expected. However, it should be remarked that the observed effects were different for the three antibiotics used. The effect of the addition of Fur-8 was most pronounced in combination with tetracycline and least pronounced in combination with cefotaxime. As expected, ciprofloxacin was the most potent antibiotic tested both with and without the furanone. It is, however, of interest to note that the tested antibiotics were unable to kill all biofilm cells at concentrations that were 100 to 1,000 times higher than the MICs for planktonic cells. Similar observations have been made previously by others using different experimental setups (49
) and are presumably caused by a combination of several different factors, one of which is the observation that a small subpopulation of the biofilm consists of dormant, nongrowing “persister” cells that are tolerant to antibiotics (9
Next, we aimed at gaining insight into the mode of action of the brominated furanones on S. enterica
serovar Typhimurium by studying the Salmonella
genes that are differentially expressed in the presence of Fur-5 via microarray analysis. Since we hypothesize that the furanones prevent planktonic cells from forming a biofilm, this study focused on the gene expression of planktonic cells rather than biofilm cells. Similar gene expression analyses have previously been performed to study the effects of Fur-3 on P. aeruginosa
) and the effects of Fur-6 on E. coli
) and the gram-positive pathogen Bacillus subtilis
). When comparing the differential gene expression profiles of the four species after treatment with furanones, it is clear that the contact with furanones is experienced as a stress factor, since some genes involved in drug sensitivity and stress response are upregulated in all species (e.g., marA
in both Salmonella
and E. coli
). This suggests that the furanones affect the global stress response of the bacteria, but this results in growth retardation only in B. subtilis
(brominated furanones are generally known as inhibitors of the growth of gram-positive bacteria) (33
). Interestingly, Fur-5 affects the expression of many Salmonella
genes that are involved in metabolism. Such genes can also be found among the genes that are differently regulated by furanones in the three other species. Most interestingly, Fur-5 inhibited the expression of several Salmonella
genes that are involved in different stages of the flagellar biosynthesis (6
). Similarly, it has been shown that furanones inhibit the expression of flagellar biosynthesis genes in E. coli
), although no phenotypic analysis was performed. We focused on the flagellar biosynthesis to validate our microarray data. Therefore, we studied the number of flagella formed in the presence of the furanone and showed that almost no flagella were present after an incubation time of 4 h in the presence of Fur-5, while several flagella were formed per cell in the absence of the furanone (Fig. ). These results were confirmed by swimming experiments which showed that Fur-5 retards swimming of Salmonella
cells. Since it has been shown that the presence of functional flagella is of importance for the formation of a normal biofilm by Salmonella
), it is possible that interference with the flagellar assembly causes the observed biofilm defect. This would imply that furanones have less influence on already established Salmonella
biofilms, which has been confirmed in preliminary experiments (data not shown). However, it still remains to be determined whether the furanones have a specific target in Salmonella
. The interference with the flagellar biosynthesis might be caused by an interaction of the furanones with such a specific target or by the more global metabolic effect that was observed. Further experiments to unravel the mode of action of the furanones are currently ongoing in our laboratories.
Since brominated furanones are generally considered to interfere with QS systems in gram-negative bacteria (15
), it was surprising that none of the known target genes of the SdiA- and the AI-2-mediated QS systems of Salmonella
were differentially regulated by Fur-5. Several experiments with gene fusion reporter systems to measure the activity of these QS systems corroborated this finding. All experiments were performed at both 16°C and 37°C, as it has been shown for SdiA that this system is selectively activated by AHLs at 37°C but not at lower temperatures (62
). However, similar results were obtained under both conditions. Whereas in a number of bacterial species brominated furanones have been reported to exert their effects by interfering with QS systems, we have found no evidence of a link between the effects of the furanones on Salmonella
biofilm formation and the QS systems that are so far identified in Salmonella
. There are several possible explanations for our observations: (i) the furanones target another yet unknown Salmonella
QS system, (ii) the target of the furanones is not part of a Salmonella
QS system, or (iii) the observed inhibition of biofilm formation results from a combination of effects on several different targets.
In conclusion, we have shown that several brominated furanones have inhibitory effects on Salmonella biofilm formation. Additionally, pretreatment with furanones results in fewer biofilm cells surviving the treatment with several different antibiotics. In an effort to unravel the working mechanism of the furanones, we have determined the differential gene expression of Salmonella in the presence of a furanone. This analysis led to the finding that the furanones interfere with flagellar biosynthesis. Since our data suggest that the brominated furanones do not inhibit Salmonella biofilm formation by interference with the two putative QS systems of S. enterica serovar Typhimurium, we are currently investigating the specific targets of the furanones.