Effects of Norspermidine and Spermidine on Biofilm Formation by Potentially Pathogenic Escherichia coli and Salmonella enterica Wild-Type Strains

Abstract

Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.T he ability to form complex multicellular communities such as biofilms has been linked to persistence and survival of pathogens in food and feed processing environments (1-3). Due to the increased biocide tolerance of bacteria in biofilms, finding molecules that can control biofilm attracts considerable interest (4). Recent publications have indicated that certain polyamines may possess such properties.Polyamines are small aliphatic hydrocarbon molecules with quaternary nitrogen groups that have a net positive charge at physiological pH. They are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including intercellular signaling, stress resistance, and RNA and protein synthesis, as well as motility and virulence (5-13). Some polyamines have also been shown to play an essential role in biofilm formation, i.e., putrescine in Yersinia pestis (14) and Escherichia coli (15), spermidine in Bacillus subtilis (16), and norspermidine in Vibrio cholerae (17). Burell et al. suggested that a role in biofilm formation may be an ancient physiological function of polyamines in bacteria (16).In addition to de novo polyamine synthesis systems, bacteria have transport systems that allow uptake of extracellular polyamines. Interestingly, exogenous spermidine has been reported to inhibit biofilm formation by Vibrio cholerae (18) and Neisseria gonorrhoeae (19). Kolkodin-Gal et al. reported that Bacillus subtilis produced biofilm disassembly factors, one of which was ...

Activity of Norspermidine on Bacterial Biofilms of Multidrug-Resistant Clinical Isolates Associated with Persistent Extremity Wound Infections

“…Our observations of a limited effect of norspermidine on bacterial cell growth for most species contrast with previous findings reported by Hobley et al, who noted significantly impaired growth of B. subtilis at concentrations ≥ 1 mM (Hobley et al 2014). Our findings showing minimal effect of norspermidine on planktonic growth of the Gram negative species evaluated herein are consistent previous studies evaluating the effect of norspermidine at concentrations up to 5 mM on the planktonic growth of E. coli and Salmonella enterica (Nesse et al 2015). Similarly, studies evaluating the activity of the related polyamine, spermine, on biofilms of Neisseria gonorrhoeae have reported that concentrations as high as 4 mM inhibited biofilm formation but had no notable effect on bacterial growth (Goytia et al 2013).…”

“…The activity of norspermidine was observed to be both species- and strain-dependent, with A. baumannii isolates being the most sensitive to these effects. Strain- and species-dependent differences in the response of isolates to norspermidine are consistent with previous studies and have been previously documented (Nesse et al 2015; Ramon-Perez et al 2014). This may be due differences between strains in their ability to develop biofilms on abiotic surfaces, differences in the composition of polymeric matrix, and undefined mechanisms impeding biofilm formation or dispersal initiation.…”

“…Importantly, for P. aeruginosa , exposure to norspermidine in some cases significantly enhanced biofilm formation (Figure 2A-B). As with the results in the preliminary screenings, the activity of norspermidine required much higher concentrations to achieve a significant effect compared to other previously characterized polyamines, putrescine and spermidine (Goytia et al 2013; Nesse et al 2015; Ramon-Perez et al 2014). As a qualitative confirmation of the above findings, visualization of bacterial biofilms by confocal laser microscopy (CLSM) showed similar effects of norspermidine on biofilm inhibition and dispersion, with A. baumannii and K. pneumoniae having the most visually significant changes in the biofilm phenotype (Figure 2C).…”

“…Notably, while the dispersive activity of norspermidine was observed primarily against A. baumannii and P. aeruginosa , significant inhibitory activity was observed against the majority of the representative clinical isolates tested (Fig 1B). Importantly, while norspermidine was observed to have dispersive and inhibitory activity herein, this activity was only observed at much higher concentrations compared to other classes of polyamines previously shown to have antibiofilm activity, including putrescine and spermidine (Goytia et al 2013; Nesse et al 2015; Ramon-Perez et al 2014) (Fig 1A-B). …”

“…This may be due differences between strains in their ability to develop biofilms on abiotic surfaces, differences in the composition of polymeric matrix, and undefined mechanisms impeding biofilm formation or dispersal initiation. Notably, while anti-biofilm activity was observed with norspermidine, this activity required much higher concentrations compared to putrescine and spermidine (Goytia et al 2013; Nesse et al 2015; Ramon-Perez et al 2014). Because other polyamines have similar chemical properties and are suggested to have similar mechanisms of biofilm disruption to norspermidine, given the specific activity on biofilm but not planktonic growth, it is possible that relative differences may arise from differences in solubility, and/or other undefined mechanisms of activity, for the species evaluated herein.…”