FEMS Microbiology Letters

The use of cyanobacteria as bio-factories for production of numerous compounds of interest (biofuels, bioplastics ...) has attracted lots of attention mainly due to their simple nutritional requirements, coupled with the decrease in atmospheric CO2 levels. However, although cyanobacteria are easily genetically manipulated, there are few genetic tools developed and, in some cases, the modifications necessary for metabolic engineering are limited for this reason. We have developed a new positive selection marker based on the arsenic resistance system of the cyanobacterium Synechocystis sp. PCC 6803. In this cyanobacterium, resistance to arsenic is mediated by the arsBHC operon in which arsB encodes an arsenite transporter whose mutation confers hypersensitivity to the presence of both arsenite and arsenate. Using arsB mutant strain (SARSB) as recipient we introduced plasmids containing both arsB and an antibiotic resistance gene and transformants were selected using either arsenic or the antibiotic with similar efficiency. The plasmids and conditions to use the arsB gene as a selectable marker have been optimized. Furthermore, we have generated an integrative vector to delete the whole arsBHC operon that allows easy introduction of regulated genes in this locus. Analysis of this strain have shown that the{Delta} arsBHC mutant has a higher sensitivity to arsenite than the SARSB strain, even when they are complemented with an arsB copy. These suggest that arsH, arsC or both could have an additional role in arsenite resistance.

Copper is frequently found in drinking water due to its presence in the natural environment and the widespread usage of copper pipes. This toxic metal has a well-known antimicrobial activity, an activity harnessed in copper-silver ionization (CSI) to eliminate the opportunistic pathogen Legionella pneumophila from engineered water systems. Despite utilizing the antimicrobial properties of copper in Legionella control, little is known about how copper containing environments affect L. pneumophila populations. The goal of this study is to understand how L. pneumophila responds to copper within a hot water distribution system (HWDS) environment. To answer this question, different sequence types and regulatory mutants were exposed to copper to compare their survival. L. pneumophila isolates of 4 sequence types from 3 different HWDSs exhibited a wide diversity of phenotypes after copper stress. The {Delta}letA and {Delta}letS mutants were sensitive to copper, indicating that the LetAS two component system is important for copper resistance. Additionaly, transmissive phase cultures were more resistant to copper than replicative phase cultures. Therefore, the regulation of entry into transmissive phase by the LetAS system is essential for L. pneumophilas ability to survive copper stress. In a water system, L. pneumophila replicates within eukaryotic hosts. When cocultured with the host ciliate Tetrahymena pyriformis, L. pneumophila was more resistant to copper than when the bacteria were in a monoculture. No difference in L. pneumophila replication inside of hosts in cocultures with or without copper was observed. This result confirms that the presence of host cells protects L. pneumophila from copper stress. Therefore, presence of host cells in water system may limit the efficacy of copper-based control strategies.

Membrane cardiolipin (CL) phospholipids play a fundamental role in the adaptation of bacteria to various environmental conditions, including saline stress. Here, we constructed deletion mutants of two CL synthetase genes, clsA and clsB, in the rhizobacterium Pseudomonas fluorescens UM270, and evaluated their role in plant growth promotion under salt stress. P. fluorescens UM270 {Delta}clsA and {Delta}clsB mutants showed a significant reduction in CL synthesis compared to the UM270 wild-type strain (58% {Delta}clsA and 53% {Delta}clsB), and their growth rate was not affected, except when grown at 100 and 200 mM NaCl. Additionally, the root colonization capacity of both mutant strains was impaired compared with that of the wild type. Concomitant with the deletion of clsA and clsB, some physiological changes were observed in the UM270 {Delta}clsA and {Delta}clsB mutants, such as a reduction in indole acetic acid and biofilm production. By contrast, an increase in siderophore biosynthesis was observed. Further, inoculation of the UM270 wild-type strain in tomato plants (Lycopersicon esculentum Saladette) grown under salt stress conditions (100 and 200 mM NaCl) resulted in an increase in root and shoot length, chlorophyll content, and dry weight. On the contrary, when each of the mutants ({Delta}clsA and {Delta}clsB) were inoculated in tomato plants, a reduction in root length was observed when grown at 200 mM NaCl, but the shoot length, chlorophyll content, and total plant dry weight parameters were significantly reduced under normal or saline conditions (100 and 200 mM NaCl), compared to UM270 wild-type-inoculated plants. In conclusion, these results suggest that CL synthesis in P. fluorescens UM270 plays an important role in the promotion of tomato plant growth under normal conditions, but to a greater extent, under salt-stress conditions.

Candida albicans is a human commensal, which causes opportunistic infections with high morbidity and mortality and is characterised by the production of resistant biofilms. Polyunsaturated fatty acids, such as arachidonic acid, can increase azole susceptibility of C. albicans biofilms significantly. However, the underlining mechanism is not known. We investigated the effect of arachidonic acid on known fluconazole resistance mechanisms namely, overexpression of ERG11, increased ergosterol content, oxidative stress resistance, as well as expression and activity of the efflux pump, Cdr1p. Upregulation of ERG11 was observed in biofilms exposed to fluconazole. However, this was reversed in the presence of arachidonic acid, even in the presence of fluconazole. Furthermore, arachidonic acid downregulated the fluconazole-induced oxidative stress response of C. albicans. Previous transcriptome data indicated a significant increase in CDR1 expression during early biofilm formation in the presence of arachidonic acid. However, we found that efflux activity was reduced in the presence of arachidonic acid, which indicates the loss of function of the membrane-associated protein. This contradictory phenomenon was further investigated by determining the effect of arachidonic acid on the localisation and phosphorylation of Cdr1p (ProteomeXchange identifier PXD070958). Our results show that the presence of arachidonic acid may cause mislocalisation and changes in the phosphorylation of Cdr1p; all of which could impact its activity. These results demonstrate that multiple mechanisms are potentially involved in the arachidonic acid-induced increased fluconazole susceptibility and allows for further exploration of lipid-mediated modulation of antifungal responses.

The ability of chaperonins to buffer mutations that affect protein folding pathways suggests that their abundance should be evolutionarily advantageous. Here, we investigate the effect of chaperonin overproduction on cellular fitness in Escherichia coli. We demonstrate that chaperonin abundance confers (a) an ability to tolerate higher temperatures, (b) improved cellular fitness and (c) enhanced folding of metabolic enzymes, which is expected to lead to enhanced energy harvesting potential.

Natural isolates of the soil-dwelling bacterium Bacillus subtilis form robust biofilms under laboratory conditions and colonize plant roots. B. subtilis biofilm gene expression displays phenotypic heterogeneity that is influenced by a family of Rap-Phr regulatory systems. Most Rap-Phr systems in B. subtilis have been studied independently, in different genetic backgrounds and under distinct conditions, hampering true comparison of the Rap-Phr systems impact on bacterial differentiation. Here, we investigated each of the 12 Rap-Phr systems of B. subtilis NCIB 3610 for their effect on biofilm formation. By studying single {Delta}rap-phr mutants, we show that despite redundancy between the cell-cell communication systems, deletion of each of the 12 Rap-Phr systems influences matrix gene expression, which could possibly enable fine-tuning of the timing and level of matrix production in response to specific conditions. Furthermore, some of the {Delta}rap-phr mutants demonstrated altered biofilm formation in vitro and colonization of Arabidopsis thaliana roots, but not necessarily similarly in both processes, indicating that the pathways regulating matrix gene expression and other factors important for biofilm formation may be differently regulated under these distinct conditions. Significance StatementNatural isolates of Bacillus subtilis form robust biofilms in vitro and on plant roots. The formation of these heterogeneous populations is regulated by diverse Rap-Phr systems. However, most Rap-Phr systems in B. subtilis have been studied independently and in different genetic backgrounds. Here, we report that all 12 Rap-Phr systems affect matrix gene expression, while some of them affect development of in vitro biofilms and plant root colonization. Our study highlights the importance of the Rap-Phr systems in environmental adaptation of B. subtilis, specifically during biofilm formation in the rhizosphere.

Nearly all microbes, including fungal pathogens, form biofilms, which are structured communities of microbial aggregates enclosed in self-produced extracellular polymeric substances (EPS) and attached to a surface. Studying plant-associated fungal biofilms can enhance understanding of fungal biology and knowledge of the links between fungal diseases and plants. However, only a few plant-associated fungi are reported to form biofilms. This study aimed to examine the ability of a mycotoxigenic fungus of maize, Fusarium verticillioides, to form biofilms under laboratory conditions. During our investigation, F. verticillioides stationary phase cultures incubated in liquid media developed a biofilm-like pellicle with a hyphal assemblage that appears in the form of a cloudy and thin slime material. Under the microscope, the biofilms exhibited a highly heterogeneous architecture made of dense, entangled, and compact hyphae, which were accompanied by a quantifiable EPS and extracellular DNA (eDNA). The biofilm was also found to respond to different abiotic conditions including pH and temperature, suggesting their relevance in a field setting. We further demonstrate the biofilm structural maintenance role of eDNA through treatment with DNase, which was only marginally effective during late biofilm stages, suggesting that it forms complex interactions with the EPS during biofilm maturation. Based on these results, we propose that F. verticillioides forms a true biofilm that may act as a potential virulence factor.

Candida auris is a multi-drug resistant human fungal pathogen that has become a global threat to human health due to its drug resistant phenotype, persistence in the hospital environment and propensity for patient to patient spread. Isolates display variable aggregation that may affect the relative virulence of strains. Therefore, dissection of this phenotype has gained substantial interest in recent years. We studied eight clinical isolates from four different clades (I-IV); four of which had a strongly aggregating phenotype and four of which did not. Genome analysis identified polymorphisms associated with loss of cell surface proteins were enriched in weakly-aggregating strains. Additionally, we identified down-regulation of chitin synthase and chitinase genes involved in the synthesis and dissolution of the chitinous septum. Characterisation of the cells revealed no ultrastructural defects in cytokinesis or cell separation in aggregating isolates. Strongly and weakly aggregating strains did not differ in net surface charge or in cell surface hydrophobicity. The capacity for aggregation and for adhesion to polystyrene microspheres were also not correlated. However, aggregation and extracellular matrix formation were all increased at higher growth temperatures, and treatment with the amyloid protein inhibitor Thioflavin-T markedly attenuated aggregation. Genome analysis further indicated strain specific differences in the genome content of GPI-anchored proteins including those encoding genes with the potential to form amyloid proteins. Collectively our data suggests that aggregation is a complex strain and temperature dependent phenomenon that may be linked in part to the ability to form extracellular matrix and cell surface amyloids. HIGHLIGHTSThe amyloid inhibitor Thioflavin-T inhibited C. auris aggregation. Aggregating isolates do not exhibit any defects in cell separation. Genomic differences were identified between strongly aggregating and weakly-aggregating strains of C. auris. Aggregation did not correlate with surface charge or hydrophobicity of yeast cells.

Acanthamoeba castellanii is an amphizoic free-living amoeba as it can be found in humans and in the environment. This amoeba represents an important reservoir of pathogenic microorganisms. Persistence of A. castellanii in the environment or in humans is allowed by the ability of the vegetative form to differentiate under cysts when surrounding conditions are unfavorable. In this study, we investigate the role of the ACA1_383450 gene during encystment of A. castellanii. This gene encodes a putative G-protein coupled receptor, which shares homology with human GPR107 and murine GPR108. Expression of the ACA1_383450 gene is transiently repressed at the early phase of encystment and its overexpression affects encystment of A. castellanii. This study reveals a new Acanthamoeba gene which could affect the encystment process. HighlightsO_LIThe ACA1_383450 gene encodes for a putative G-protein coupled receptor (GPCR). C_LIO_LIThe ACA1_383450 mRNA levels are down-regulated during the early phase of encystment. C_LIO_LIOverexpression of the ACA1_383450 gene affects formation of cysts. C_LI

KatG and Msrs are important enzymes associated with ROS homeostasis and bacterial survival under oxidative stress. Consistent to this notion, mutant strains in these enzymes showed hypersensitivity to oxidants and accumulates elevated levels of ROS. In current study we observed that a pan msr deletion ({Delta}5msr mutant) strain of S. Typhimurium accumulates significantly higher levels of ROS. However, unexpectedly, as compared to S. Typhimurium, the {Delta}5msr mutant strain exhibits more than 2000 folds resistance to H2O2. Transcriptional and mass spectrometry analyses reveal the upregulation of KatG in {Delta}5msr mutant strain. Further, {Delta}5msr mutant strain exhibits [~]6 folds higher KatG activity. Supplementation of {Delta}5msr mutant culture with reduced glutathione resulted in ROS neutralization, decreased KatG activity and abrogation of H2O2 resistance. However, {Delta}5msr mutant strain showed negligible KatE and KatN activities. The findings of current study suggest that the Salmonella have evolved the mechanism to upregulate one antioxidant gene in absence of others to mitigate oxidative stress.

The competence of bacteria to colonize different environmental niches is often determined by their ability to form biofilms. This depends on both cellular and extracellular factors, such as individual characteristics of a strain, type of colonized surface (abiotic or biotic) or availability and source of nutrients. Pseudomonas donghuensis P482 efficiently colonizes rhizosphere of various plant hosts, but a connection between plant tissue colonization and biofilm formation has not been verified for P482 up to date. Here we demonstrate that the ability of P482 to form biofilm on abiotic surfaces and the structural characteristics of the biofilm are connected to the type of carbon source available to the bacteria, with glycerol promoting formation of developed biofilm at early stages. Also, the type of substratum, polystyrene or glass, significantly influences the ability of P482 to attach to the surface, possibly due to hydrophobic effects. Moreover, mutants in genes associated with motility or chemotaxis, synthesis of polysaccharides, and encoding proteases or regulatory factors, affected in biofilm formation on glass were fully capable of colonizing root tissue of both tomato and maize hosts. This indicates that the ability to form biofilm on distinct abiotic surfaces does not simply correlate with the efficient colonization of rhizosphere and formation of biofilm on plant tissue by P482.

Biofilm formation likely plays an important role in the pathogenesis of implant-related infections caused by Cutibacterium acnes. Biofilms protect bacteria from antimicrobials and host defenses which makes biofilm-related infections difficult to treat. Here we demonstrate that the exopolysaccharide poly-N-acetylglucosamine (PNAG) contributes to C. acnes biofilm formation in vitro. By treating C. acnes cells and biofilms with the PNAG-degrading enzyme dispersin B, we found that PNAG mediates the attachment of C. acnes cells to polystyrene rods and the formation C. acnes biofilms in glass and polypropylene tubes. We further show that PNAG protects C. acnes biofilm cells from killing by tetracycline and benzoyl peroxide. PNAG may play an important role in biofilm formation, antibiotic tolerance, and virulence in this opportunistic pathogen.

Phelipanche aegyptiaca is an obligate holo-parasitic weed lacking a functional photosynthetic system, which subsists on roots of a wide range of host crops, causing severe losses in yield quality and quantity. The parasite and its host are connected through their vascular system, forming a unique ecological system that enables the exchange of various substances. In a previous study, it was suggested that endophytic bacteria, which naturally inhabit the internal tissues of plants, can also be transmitted from the parasitic weed to its host and vice versa. In the current study, we investigate the characteristics of a previously isolated Pseudomonas sp. PhelS10 strain, using both biochemical and molecular methods. Our results revealed that production of Pseudomonas aeruginosa quinolone signal (PQS) was 2.1 times higher than that of the standard Pseudomonas aeruginosa strain (PAO1), which contributed to a 22% higher biofilm formation capability. PhelS10 strain was detected in the xylem of tomato plants using FISH analysis. In addition, PhelS10 strain was found in the parasitic weeds inner tissues, confirming the hypothesis that endophytic bacteria traffic between the plant host and its parasitic weed.

Teredinibacter turnerae is an intracellular bacterial symbiont that resides in the gills of shipworms, wood-eating bivalve mollusks. This bacterium produces a catechol siderophore, turnerbactin, required for the survival of this bacterium under iron limiting conditions. The turnerbactin biosynthetic genes are contained in one of the secondary metabolite clusters conserved among T. turnerae strains. However, Fe(III)-turnerbactin uptake mechanisms are largely unknown. Here, we show that the first gene of the cluster, fttA a homologue of Fe(III)-siderophore TonB-dependent outer membrane receptor (TBDR) genes is indispensable for iron uptake via the endogenous siderophore, turnerbactin, as well as by an exogenous siderophore, amphi-enterobactin, ubiquitously produced by marine vibrios. Furthermore, three TonB clusters containing four tonB genes were identified, and two of these genes, tonB1b and tonB2, functioned not only for iron transport but also for carbohydrate utilization when cellulose was a sole carbon source. Gene expression analysis revealed that none of the tonB genes and other genes in those clusters were clearly regulated by iron concentration while turnerbactin biosynthesis and uptake genes were up-regulated under iron limiting conditions, highlighting the importance of tonB genes even in iron rich conditions, possibly for utilization of carbohydrates derived from cellulose.

All bacterial epibiotic predators are rich in secondary metabolites and most genera rich in secondary metabolites have demonstrable predatory abilities. Therefore it is likely that an antibiotic resistant and thereby predation resistant species may benefit not only by escaping predation but also by utilizing nutrients released by lysis of prey cells by predatory bacteria. The resistant organisms may enjoy greater fitness benefits than the predator since they get the benefit without investing in the predation machinery. In our experiment, a marine isolate of Streptomyces atrovirens showed good predatory activity on a range of species including Staphylococcus aureus and Proteus vulgaris. Escherichia coli was resistant to predation by this species. On slide culture with water agar when the predator, S. aureus and E. coli were grown together S. aureus population declined whereas the predation resistant E. coli increased their population as compared to controls. However the growth of E. coli did not affect growth of the predator unfavorably. This strengthens the possibility that evolution of antibiotic resistance not only gave a selective advantage of escaping predation, it also would have increased the fitness of the resistant organism by promoting growth on nutrients released from the prey cells lysed by the predator. When the predator was grown with S. aureus and P. vulgaris as prey, S. aureus declined rapidly whereas P. vulgaris was spared. This suggests that the predator appears to show preference towards prey and in that case even a partial or relative resistance may give substantial advantage to a population.

The nematode Caenorhabditis elegans has emerged as a popular model system to investigate cell biology and host-pathogen interactions. Presently, C. elegans is studied as a natural host of intracellular pathogens such as microsporidia and Orsay virus along with extracellular bacterial and fungal pathogens. The use of C. elegans as a model in host-pathogen research is limited by the number of naturally occurring pathogens to the organism. Through a sampling project to identify new pathogens of C. elegans, we identified the fungus Mucor hiemalis as a pathogen of Caenorhabditis species. We observed the fungus in the intestinal lumen of wild-caught Caenorhabditis briggsae, and co-culturing the wild-caught species with infection reporter C. elegans confirmed infection by M. hiemalis. This study characterizes the fungal infection by M. hiemalis in Caenorhabditis nematodes. Fluorescence microscopy with fungal staining revealed the life cycle of M. hiemalis within multiple Caenorhabditis species at varying growth stages. We observed the killing of nematodes by M. hiemalis via intestinal perforation and assessed its host range through a series of lifespan assays. We investigated the food preference of C. elegans and determined that nematodes show a preference towards food that contains M. hiemalis spores. Lastly, we evaluated common C. elegans transcriptional immune responses and found that M. hiemalis does not induce genes associated with the intracellular pathogen response or other responses seen with previously studied bacterial and fungal pathogens. Characterization of this fungal infection in Caenorhabditis nematodes will provide new insights into the biology of pathogenic fungi and host immune responses.

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

The opportunistic human pathogen Candida albicans is an important cause of nosocomial infections, in large part because of its propensity to form biofilms on indwelling medical devices such as catheters. The formation of these biofilms is controlled by a complex transcriptional network and involves over a thousand genes, many of which are uncharacterized. We have investigated three genes (ORF19.4654, ORF19.7608, and PBR1), found only in C. albicans and closely related species, that are highly induced under biofilm conditions and encode small proteins with N-terminal signal sequences. Through the construction of fluorescent protein fusions, we have examined the location of the encoded proteins in both planktonic and biofilm cells. Orf19.4654-Scarlet and Pbr1-Scarlet were localized to the vacuole under both conditions. In contrast, the Orf19.7608-GFP fusion generated a punctate pattern only under biofilm conditions and was designated Ppp1 (Punctate Pattern Protein 1). The Ppp1-GFP puncta were similar in location, stability, and size to those formed by the eisosome subunit Sur7, but co-localization studies suggest that Ppp1 and Sur7 define separate elements. The PPP1 mutation does not cause a distinct phenotype under various stress conditions or in the presence of antifungals and does not impact biofilm formation and biomass. These data suggest that while the expression and cellular localization of Ppp1 appear controlled by conditions generating biofilms, and define a unique subcellular localization pattern, Ppp1 protein function is not essential for biofilm formation. IMPORTANCEBiofilm formation is a virulence factor of medical importance in C. albicans. Identifying the biological function and cellular localization of biofilm-related proteins can help in their characterization and understanding of the biofilm network. Microscopy was employed to identify the subcellular localization of putative biofilm proteins, aiming to provide insight into their possible functions. Prb1 and Orf19.4654 localized to the vacuole, while Orf19.7608 (Ppp1) formed puncta. Phenotypic assays to investigate the uniqueness of PPP1 revealed that it does not play a major role in the stress response pathways or antifungal activity. Overall, our study provides insight into the localization of the products of Candida-specific biofilm genes.

The symbiotic bacterium Sinorhizobium meliloti contains a large number of adenylate cyclases (AC) for the control of different life styles. ACs produce cyclic AMP (cAMP) as a secondary messenger. Earlier, the redox responsive membrane-intrinsic AC CyaC has been shown to produce a trimeric signalling complex CyaCxCycRxcAMP-Clr. Here, use of the bacterial two-hybrid system BACTH showed that the LysR-type transcriptional regulator Smc01817 interacts in vivo with CyaC and the general cAMP-regulator Clr, suggesting the formation of a sensor-regulator complex CyaCxSmc01817xcAMP-Clr. Therefore, formation of sensor-regulator complexes from ACs and transcriptional regulators seems to be a means in S. meliloti to setup specific signalling routes in a background with a large number of signalling routes applying the same signalling molecule (cAMP). Use of caged cAMP allows to address a specific signalling route and set of regulators, which is at variance with the classical role of cAMP as a diffusing secondary messenger.

Food preservatives are crucial in controlling microbial growth in processed foods to maintain food safety. Bacterial biofilms pose a threat in the food chain by facilitating persistence on a range of surfaces and food products. Cells in a biofilm are often highly tolerant of antimicrobials and can evolve in response to antimicrobial exposure. Little is known about the efficacy of preservatives against biofilms and their potential impact on the evolution of antimicrobial resistance. In this study we investigated how the common food pathogen Salmonella enterica serovar Typhimurium responded to subinhibitory concentrations of four common food preservatives (sodium chloride, potassium chloride, sodium nitrite or sodium lactate) when grown planktonically and in biofilms. We found that each preservative exerted a unique selective pressure on S. Typhimurium populations grown planktonically and in a biofilm. Biofilm formation itself seemed to confer protection when exposed to each of the four preservatives, more so than previous exposure to sub-inhibitory concentrations of preservatives. There was a trade-off between biofilm formation and growth in the presence of three of the four preservatives, where prolonged preservative exposure resulted in reduced biofilm biomass and matrix production over time. Despite the differences in biofilm formation and preservative tolerance seen following three preservative stresses, they selected for mutations in global stress response regulators rpoS and crp. There was no evidence for any selection of cross-resistance to antibiotics after preservative exposure, and some evidence that antagonism between preservatives can be exploited in compound cocktails to reduce contamination in the food chain. HighlightsO_LIPreservative-specific evolutionary adaptation of Salmonella was shown over time. C_LIO_LIA trade-off between adaptation and biofilm formation was observed. C_LIO_LINo cross-resistance to antibiotics was seen after preservative exposure. C_LIO_LIMutations were found to be preservative-specific, with some common ones like rpoS and crp. C_LI