Identification of pathways required for Salmonella to colonize alfalfa using TraDIS-Xpress.

Enteropathogenic bacteria, such as Salmonella, have been linked to numerous fresh produce outbreaks, posing a significant public health threat. The ability of Salmonella to persist on fresh produce for extended periods is partly attributed to its capacity to form biofilms, which pose a challenge to food decontamination and can increase pathogenic bacterial load in the food chain. Preventing Salmonella colonization of food products and food processing environments is crucial for reducing the incidence of foodborne outbreaks. Understanding the mechanisms of establishment on fresh produce will inform the development of decontamination approaches. We used Transposon-Directed Insertion site Sequencing (TraDIS-Xpress) to investigate the mechanisms used by Salmonella enterica serovar Typhimurium to colonize and establish on fresh produce over time. We established an alfalfa colonization model and compared the findings to those obtained from glass surfaces. Our research identified distinct mechanisms required for Salmonella establishment on alfalfa compared with glass surfaces over time. These include the type III secretion system (sirC), Fe-S cluster assembly (iscA), curcumin degradation (curA), and copper tolerance (cueR). Shared pathways across surfaces included NADH hydrogenase synthesis (nuoA and nuoB), fimbrial regulation (fimA and fimZ), stress response (rpoS), LPS O-antigen synthesis (rfbJ), iron acquisition (ybaN), and ethanolamine utilization (eutT and eutQ). Notably, flagellum biosynthesis differentially impacted the colonization of biotic and abiotic environments over time. Understanding the genetic underpinnings of Salmonella establishment on both biotic and abiotic surfaces over time offers valuable insights that can inform the development of targeted antibacterial therapeutics, ultimately enhancing food safety throughout the food processing chain. IMPORTANCE: Salmonella is the second most costly foodborne illness in the United Kingdom, accounting for £0.2 billion annually, with numerous outbreaks linked to fresh produce, such as leafy greens, cucumbers, tomatoes, and alfalfa sprouts. The ability of Salmonella to colonize and establish itself in fresh produce poses a significant challenge, hindering decontamination efforts and increasing the risk of illness. Understanding the key mechanisms of Salmonella to colonize plants over time is key to finding new ways to prevent and control contamination of fresh produce. This study identified genes and pathways important for Salmonella colonization of alfalfa and compared those with colonization of glass using a genome-wide screen. Genes with roles in flagellum biosynthesis, lipopolysaccharide production, and stringent response regulation varied in their significance between plants and glass. This work deepens our understanding of the requirements for plant colonization by Salmonella, revealing how gene essentiality changes over time and in different environments. This knowledge is key to developing effective strategies to reduce the risk of foodborne disease.

Common food preservatives impose distinct selective pressures on Salmonella Typhimurium planktonic and biofilm populations.

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 Salmonella enterica serovar Typhimurium responded to subinhibitory concentrations of four 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. 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. All three preservatives 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. In conclusion, we showed that preservatives affect biofilm formation and bacterial growth in a compound specific manner. We showed trade-offs between biofilm formation and preservative tolerance, but no antibiotic cross-tolerance. This indicates that bacterial adaptation to continuous preservative exposure, is unlikely to affect food safety or contribute to antibiotic resistance.

An efficient method for high molecular weight bacterial DNA extraction suitable for shotgun metagenomics from skin swabs.

The human skin microbiome represents a variety of complex microbial ecosystems that play a key role in host health. Molecular methods to study these communities have been developed but have been largely limited to low-throughput quantification and short amplicon-based sequencing, providing limited functional information about the communities present. Shotgun metagenomic sequencing has emerged as a preferred method for microbiome studies as it provides more comprehensive information about the species/strains present in a niche and the genes they encode. However, the relatively low bacterial biomass of skin, in comparison to other areas such as the gut microbiome, makes obtaining sufficient DNA for shotgun metagenomic sequencing challenging. Here we describe an optimised high-throughput method for extraction of high molecular weight DNA suitable for shotgun metagenomic sequencing. We validated the performance of the extraction method, and analysis pipeline on skin swabs collected from both adults and babies. The pipeline effectively characterised the bacterial skin microbiota with a cost and throughput suitable for larger longitudinal sets of samples. Application of this method will allow greater insights into community compositions and functional capabilities of the skin microbiome.

Molecular mechanisms of antibiotic resistance revisited.

Antibiotic resistance is a global health emergency, with resistance detected to all antibiotics currently in clinical use and only a few novel drugs in the pipeline. Understanding the molecular mechanisms that bacteria use to resist the action of antimicrobials is critical to recognize global patterns of resistance and to improve the use of current drugs, as well as for the design of new drugs less susceptible to resistance development and novel strategies to combat resistance. In this Review, we explore recent advances in understanding how resistance genes contribute to the biology of the host, new structural details of relevant molecular events underpinning resistance, the identification of new resistance gene families and the interactions between different resistance mechanisms. Finally, we discuss how we can use this information to develop the next generation of antimicrobial therapies.

Functionally distinct mutations within AcrB underpin antibiotic resistance in different lifestyles.

Antibiotic resistance is a pressing healthcare challenge and is mediated by various mechanisms, including the active export of drugs via multidrug efflux systems, which prevent drug accumulation within the cell. Here, we studied how Salmonella evolved resistance to two key antibiotics, cefotaxime and azithromycin, when grown planktonically or as a biofilm. Resistance to both drugs emerged in both conditions and was associated with different substitutions within the efflux-associated transporter, AcrB. Azithromycin exposure selected for an R717L substitution, while cefotaxime for Q176K. Additional mutations in ramR or envZ accumulated concurrently with the R717L or Q176K substitutions respectively, resulting in clinical resistance to the selective antibiotics and cross-resistance to other drugs. Structural, genetic, and phenotypic analysis showed the two AcrB substitutions confer their benefits in profoundly different ways. R717L reduces steric barriers associated with transit through the substrate channel 2 of AcrB. Q176K increases binding energy for cefotaxime, improving recognition in the distal binding pocket, resulting in increased efflux efficiency. Finally, we show the R717 substitution is present in isolates recovered around the world.

Single-cell genomics reveals population structures from in vitro evolutionary studies of Salmonella.

Single-cell DNA sequencing has the potential to reveal detailed hierarchical structures in evolving populations of cells. Single cell approaches are increasingly used to study clonal evolution in human ageing and cancer but have not yet been deployed to study evolving clonal microbial populations. Here, we present an approach for single bacterial genomic analysis for in vitro evolution experiments using FACS isolation of individual bacteria followed by whole-genome amplification and sequencing. We apply this to the experimental evolution of a hypermutator strain of Salmonella in response to antibiotic stress (ciprofloxacin). By analysing sequence polymorphisms in individual cells from populations we identified the presence and prevalence of sub-populations which have acquired polymorphisms in genes previously demonstrated to be associated with ciprofloxacin susceptibility. We were also able to identify that the population exposed to antibiotic stress was able to develop resistance whilst maintaining diversity. This population structure could not be resolved from bulk sequence data, and our results show how high-throughput single-cell sequencing can enhance experimental studies of bacterial evolution.

ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa.

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.

Cefotaxime Exposure Selects Mutations within the CA-Domain of envZ Which Promote Antibiotic Resistance but Repress Biofilm Formation in Salmonella.

Cephalosporins are important beta lactam antibiotics, but resistance can be mediated by various mechanisms including production of beta lactamase enzymes, changes in membrane permeability or active efflux. We used an evolution model to study how Salmonella adapts to subinhibitory concentrations of cefotaxime in planktonic and biofilm conditions and characterized the mechanisms underpinning this adaptation. We found that Salmonella rapidly adapts to subinhibitory concentrations of cefotaxime via selection of multiple mutations within the CA-domain region of EnvZ. We showed that changes in this domain affect the ATPase activity of the enzyme and in turn impact OmpC, OmpF porin expression and hence membrane permeability leading to increased tolerance to cefotaxime and low-level resistance to different classes of antibiotics. Adaptation to cefotaxime through EnvZ also resulted in a significant cost to biofilm formation due to downregulation of curli. We assessed the role of the mutations identified on the activity of EnvZ by genetic characterization, biochemistry and in silico analysis and confirmed that they are responsible for the observed phenotypes. We observed that sublethal cefotaxime exposure selected for heterogeneity in populations with only a subpopulation carrying mutations within EnvZ and being resistant to cefotaxime. Population structure and composition dynamically changed depending on the presence of the selection pressure, once selected, resistant subpopulations were maintained even in extended passage without drug. IMPORTANCE Understanding mechanisms of antibiotic resistance is crucial to guide how best to use antibiotics to minimize emergence of resistance. We used a laboratory evolution system to study how Salmonella responds to cefotaxime in both planktonic and biofilm conditions. In both contexts, we observed rapid selection of mutants within a single hot spot within envZ. The mutations selected altered EnvZ which in turn triggers changes in porin production at the outer membrane. Emergence of mutations within this region was repeatedly observed in parallel lineages in different conditions. We used a combination of genetics, biochemistry, phenotyping and structural analysis to understand the mechanisms. This data show that the changes we observe provide resistance to cefotaxime but come at a cost to biofilm formation and the fitness of mutants changes greatly depending on the presence or absence of a selective drug. Studying how resistance emerges can inform selective outcomes in the real world.

Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells.

For antibiotics with intracellular targets, effective treatment of bacterial infections requires the drug to accumulate to a high concentration inside cells. Bacteria produce a complex cell envelope and possess drug export efflux pumps to limit drug accumulation inside cells. Decreasing cell envelope permeability and increasing efflux pump activity can reduce intracellular accumulation of antibiotics and are commonly seen in antibiotic-resistant strains. Here, we show that the balance between influx and efflux differs depending on bacterial growth phase in Gram-negative bacteria. Accumulation of the fluorescent compound ethidium bromide (EtBr) was measured in Salmonella enterica serovar Typhimurium SL1344 (wild type) and efflux deficient (ΔacrB) strains during growth. In SL1344, EtBr accumulation remained low, regardless of growth phase, and did not correlate with acrAB transcription. EtBr accumulation in the ΔacrB strains was high in exponential phase but dropped sharply later in growth, with no significant difference from that in SL1344 in stationary phase. Low EtBr accumulation in stationary phase was not due to the upregulation of other efflux pumps but instead was due to decreased permeability of the envelope in stationary phase. Transcriptome sequencing (RNA-seq) identified changes in expression of several pathways that remodel the envelope in stationary phase, leading to lower permeability. IMPORTANCE This study shows that efflux is important for maintaining low intracellular accumulation only in actively growing cells and that envelope permeability is the predominant factor in stationary-phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex infections with nongrowing or slow-growing bacteria, where intracellular accumulation may be low; (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing; and (iii) the remodeling of the cell envelope prior to stationary phase could provide novel drug targets.