Bacterial biofilms have great significance for public health, since biofilm-associated microorganisms exhibit dramatically decreased susceptibility to antimicrobial agents and treatments. To date most attention has focused on biofilms that arise from the colonization of solid-liquid or solid-air interfaces. It is of interest that colonization of the interface between air and liquid, which can be selectively advantageous for aerobic or facultative aerobic bacteria, has been rarely studied, although it may present a major problem in industrial aquatic systems. In this work we investigated the role of a biofilm at the interface between air and liquid (pellicle) in the susceptibility of Salmonella enterica serovar Typhimurium to stress conditions. For a control we used a mutant that had lost its ability to synthesize cellulose and thin aggregative fimbriae and thus did not produce the pellicle. Resistance of bacteria from the pellicle to heat, acidification, and chlorination was compared to resistance of planktonic cells from the logarithmic and stationary phases of growth. Pellicle cells were significantly more resistant to chlorination, and thus the surrounding matrix conferred protection against the reactive sodium hypochlorite. However, the stress management of pellicle cells in response to heat and low pH was not enhanced compared to that of stationary-phase cells. A long-period of incubation resulted in endogenous hydrolysis of the pellicle matrix. This phenomenon provides a potential new approach to combat microbial cells in biofilms.

Triclosan is a potent biocide that is included in a diverse range of products. This research was aimed to investigate the susceptibility of planktonic and biofilm-associated Salmonella enterica serovar Typhimurium to triclosan, and to identify potential mechanisms of adaptation. The effect of triclosan was studied on planktonic Salmonella (log and stationary phases), on biofilm-associated cells, on bacteria derived from disrupted biofilms and on a biofilm-deficient mutant. An eight-log reduction of exponentially growing cells was observed with 1000 micro g mL(-1) triclosan within 10 min, a 3.6-log reduction in stationary cells and a 6.3-log reduction in stationary cells of a biofilm-deficient mutant (P<0.05). Biofilm-associated cells were tolerant (1-log reduction). However, biofilm-derived cells showed sensitivity to triclosan similar to stationary-phase cells. Triclosan induced the transcription of fabI and micF. Within biofilms, triclosan also up-regulated the transcription of acrAB, encoding for an efflux pump, marA, and the cellulose-synthesis-coding genes bcsA and bcsE. Thus, Salmonella within biofilms could experience reduced influx, increased efflux and enhanced exopolysaccharides production. Our results demonstrated that the tolerance of Salmonella towards triclosan in the biofilm was attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance to triclosan and to other antimicrobials.

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The gene SRE1, encoding the GATA transcription factor siderophore biosynthesis repressor (Sre1), was identified in the genome of the maize pathogen Cochliobolus heterostrophus and deleted. Mutants were altered in sensitivity to iron, oxidative stress, and virulence to the host. To gain insight into mechanisms of this combined regulation, genetic interactions among SRE1 (the nonribosomal peptide synthetase encoding gene NPS6, which is responsible for extracellular siderophore biosynthesis) and ChAP1 (encoding a transcription factor regulating redox homeostasis) were studied. To identify members of the Sre1 regulon, expression of candidate iron and oxidative stress-related genes was assessed in wild-type (WT) and sre1 mutants using quantitative reverse-transcription polymerase chain reaction. In sre1 mutants, NPS6 and NPS2 genes, responsible for siderophore biosynthesis, were derepressed under iron replete conditions, whereas the high-affinity reductive iron uptake pathway associated gene, FTR1, was not, in contrast to outcomes with other well-studied fungal models. C. heterostrophus L-ornithine-N(5)- monooxygenase (SIDA2), ATP-binding cassette (ABC6), catalase (CAT1), and superoxide dismutase (SOD1) genes were also derepressed under iron-replete conditions in sre1 mutants. Chap1nps6 double mutants were more sensitive to oxidative stress than either Chap1 or nps6 single mutants, while Chap1sre1 double mutants showed a modest increase in resistance compared with single Chap1 mutants but were much more sensitive than sre1 mutants. These findings suggest that the NPS6 siderophore indirectly contributes to redox homeostasis via iron sequestration, while Sre1 misregulation may render cells more sensitive to oxidative stress. The double-mutant phenotypes are consistent with a model in which iron sequestration by NPS6 defends the pathogen against oxidative stress. C. heterostrophus sre1, nps6, Chap1, Chap1nps6, and Chap1sre1 mutants are all reduced in virulence toward the host, compared with the WT.

Triclosan is a biocide whose wide use has raised a debate about the potential benefits vs. hazards of the incorporation of antimicrobials in consumer products. The purpose of the present study was to determine whether exposure of biofilms of Salmonella enterica serovar Typhimurium to triclosan influences the tolerance of the bacteria towards antibiotics such as ciprofloxacin and vice versa. A synergistic antibiofilm activity was observed when the biofilms were treated with triclosan before or together with ciprofloxacin, and an additive activity was observed with planktonic cells. For example 500 microg mL(-1) triclosan and 500 microg mL(-1) ciprofloxacin reduced the number of viable cells in the biofilm by 1.6 and 0.5 log, respectively. However, the sequential treatment of 500 microg mL(-1) triclosan followed by ciprofloxacin resulted in 4.8 log reduction. Combination indexes (CI) for biofilms treated with triclosan followed by ciprofloxacin were 0.7, 0.32 and 0.25 for reduction of 90%, 99% and 99.9%, respectively, indicating a synergism. For planktonic cells, CIs were 1+/-0.1, indicating an additive effect. Therefore, it was suggested that triclosan weakens the ability of biofilm-associated cells to survive exposure to ciprofloxacin in the biofilm, probably by improving the permeability or the activity of ciprofloxacin.