eDNA Release Research Articles

Application of eDNA for monitoring freshwater bivalve Nodularia nipponensis and its glochidium larvae

AbstractHabitat loss, the introduction of invasive species, and climate change due to human activity have threatened many freshwater unionid bivalves worldwide. Unionid bivalves represent important members of freshwater ecosystems, providing bitterling fish with spawning grounds and contributing to water clarification via the filtration of suspended solids. This study examined an environmental DNA (eDNA) approach for monitoring the unionid bivalve Nodularia nipponensis in a lake environment. We developed oligonucleotide primers for the specific detection of mitochondrial DNA from N. nipponensis and evaluated the decay and release rates of eDNA in a laboratory tank experiment under two bivalve densities (49 and 164 individuals m−2) selected to ensure sufficient eDNA yields. The eDNA release rate was weakly and positively correlated with bivalve density but strongly and positively correlated with inorganic nitrogen and phosphorus excretion from the bivalve. In a eutrophic lake, Lake Hachiro, 67% of the samples (16 out of 24) collected from June to July yielded detectable amounts of eDNA and were not detected at all in May and August (0 out of 20). This difference was probably due to the release of N. nipponensis glochidium larvae. During these months, unionid glochidia adhered to bundles of polyvinylidene chloride fibers placed on the lake bottom for 8 h, confirmed by microscopic inspection. We successfully obtained N. nipponensis eDNA from all bundles examined over the months, demonstrating the efficacy of bundle capture in quantifying unionid bivalve eDNA. Our eDNA‐based technique with specific oligonucleotide primers and fiber bundles may be a promising tool for monitoring N. nipponensis in freshwater environments. Fiber bundles are useful for tracing eDNA derived from glochidium larvae, even at low unionid bivalve densities, which may be conducive to conservation efforts.

Challenges in eDNA detection of the invasive European green crab, Carcinus maenas

The early detection of invasive species is essential to cease the spread of the species before it can cause irreversible damage to the environment. The analysis of environmental DNA (eDNA) has emerged as a non-harmful method to detect the presence of a species before visual detection and is a promising approach to monitor invasive species. Few studies have investigated the use of eDNA for arthropods, as their exoskeleton is expected to limit the release of eDNA into the environment. We tested published primers for the invasive European green crab, Carcinus maenas, in the Gulf of Maine and found them not species-specific enough for reliable use outside of the area for which they were designed for. We then designed new primers, tested them against a broad range of local faunal species, and validated these primers in a field study. We demonstrate that eDNA analyses can be used for crustaceans with an exoskeleton and suggest that primers and probe sequences must be tested on local fauna at each location of use to ensure no positive amplification of these other species.

Clemastine Inhibits the Biofilm and Hemolytic of Staphylococcus aureus through the GdpP Protein.

ABSTRACTStaphylococcus aureus poses a significant threat to human health due to its virulence and multidrug resistance. In addition, recalcitrant biofilm formation of S. aureus often results in chronic infection and the treatment tolerance toward the traditional antibiotics. Thus, the development of novel antimicrobial agents capable to inhibit or eradicate S. aureus biofilm formation does matter. Here, we demonstrated that clemastine showed slight bacteriostatic activity and enhanced the antibacterial activity of oxacillin against S. aureus. Moreover, the dramatic inhibition of biofilm formation was found in clinical S. aureus strains by clemastine. Clemastine inhibited the release of eDNA during the biofilm formation and decreased the S. aureus hemolytic activity. Moreover, the S. aureus SA113 treated with clemastine displayed the decreased transcriptional level of the biofilm formation relevant genes (fnbB, icaA, and icaB), virulence genes (hlg, hld, lukde, lukpvl, beta-PSM, delta-PSM, and cap5A), and the regulatory genes agrA. The proteomics analysis of SA113 treated with clemastine demonstrated the significant changes in levels of biofilm-related proteins (stress response regulators ClpB and GroS, ATP-binding proteins, and urease metabolism), virulence-related proteins (SspA, superantigen, and VWbp), and methicillin resistance-related proteins (glutamine metabolism). The genetic mutations on gdpP (cyclic di-AMP phosphodiesterase) were found in the clemastine-induced tolerant derivative isolate by whole-genome sequencing. Furthermore, the interaction between clemastine and GdpP protein was demonstrated by the molecular docking, gdpP overexpression experiment, and thermal stability assay. Conclusively, clemastine might exert its inhibitory effects against the biofilm formation and hemolysis in S. aureus through targeting GdpP protein.IMPORTANCE The biofilm formation, which protects bacteria from stresses, including antibiotics and host immune responses, can be commonly found in clinical S. aureus isolates worldwide. Treatment failure of traditional antibiotics in biofilm-associated S. aureus infections remains a serious challenge. The novel anti-biofilm drug is urgently needed to address the looming crisis. In this study, clemastine, which is a histamine receptor H1 (HRH1) antagonist, was found to have a novel role of the significant inhibition against the biofilm formation and hemolytic activity of S. aureus and enhanced antibacterial activity against S. aureus when used in combination with oxacillin by targeting the GdpP protein. The discovery of this study identified novel use and mechanism of action of clemastine as a potential anti-biofilm drug for clinical application for S. aureus infectious.

Phenotypic and transcriptional study of the antimicrobial activity of silver and zinc oxide nanoparticles on a wastewater biofilm-forming Pseudomonas aeruginosa strain

The extensive use of nanoparticles (NPs) in industrial processes makes their potential release into the environment an issue of concern. Ag and ZnO NPs are among the most frequently used NPs, potentially reaching concentrations of 1–4 and 64 mg/kg, respectively, in Wastewater Treatment Plants (WWTPs), with unknown effects over microbial populations. Thus, we examined, in depth, the effect of such NPs on a P. aeruginosa strain isolated from a WWTP. We evaluated the growth, ROS production and biofilm formation, in addition to the transcriptomic response in presence of Ag and ZnO NPs at concentrations potentially found in sewage sludge. The transcriptomic and phenotypic patterns of P. aeruginosa in presence of Ag NPs were, in general, similar to the control treatment, with some specific transcriptional impacts affecting processes involved in biofilm formation and iron homeostasis. The biofilms formed under Ag NPs treatment were, on average, thinner and more homogeneous. ZnO NPs also alters the biofilm formation and iron homeostasis in P. aeruginosa, however, the higher and more toxic concentrations utilized caused an increase in cell death and eDNA release. Thus, the biofilm development was characterized by EPS production, via eDNA release. The number of differentially expressed genes in presence of ZnO NPs was higher compared to Ag NPs treatment. Even though the responses of P. aeruginosa to the presence of the studied metallic NPs was at some extent similar, the higher and more toxic concentrations of ZnO NPs produced greater changes concerning cell viability and ROS production, causing disruption in biofilm development.

EDNA Inactivation and Biofilm Inhibition by the PolymericBiocide Polyhexamethylene Guanidine Hydrochloride (PHMG-Cl).

The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA–PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

Aqueous extracts of spices inhibit biofilm in Listeria monocytogenes by downregulating release of eDNA

Listeria monocytogenes is a Gram-positive mesophilic foodborne pathogen whose pathogenicity is regulated by various environmental stress factors. The ability to form robust biofilm makes it difficult to eradicate this bacteria from food processing environments. Also, there is an increase in the antibiotic resistant food isolates of L. monocytogenes. Therefore, there is a constant need for the development of novel food preservation techniques and therapeutics. In this study, we sought to determine the effectiveness of clove and cinnamon water extracts on the listerial biofilm initiation, dispersion, and expression of the virulence genes. It was observed that the spice water extracts not only inhibited the biofilm formation of L. monocytogenes but also accelerated the dispersion. The spice extracts at higher concentrations were found to be bactericidal to L. monocytogenes. The extracts downregulated the expression of virulence genes and the release of extracellular DNA (eDNA) in the biofilm. The decreased ability to form biofilm could be attributed to the inhibition of eDNA secretion. Thus the water extracts of cinnamon and clove can be used as food preservatives against the biofilm formation of L. monocytogenes.

Andrographolide Sulfonate Is a Promising Treatment to Combat Methicillin-resistant Staphylococcus aureus and Its Biofilms

Methicillin-resistant Staphylococcus aureus (MRSA) is a drug-resistant pathogen threatening human health and safety. Biofilms are an important cause of its drug resistance and pathogenicity. Inhibition and elimination of biofilms is an important strategy for the treatment of MRSA infection. Andrographolide sulfonate (AS) is an active component of the traditional herbal medicine Andrographis paniculata. This study aims to explore the inhibitory effect and corresponding mechanisms of AS on MRSA and its biofilms. Three doses of AS (6.25, 12.5, and 25 mg/ml) were introduced to MRSA with biofilms. In vitro antibacterial testing and morphological observation were used to confirm the inhibitory effect of AS on MRSA with biofilms. Real-time PCR and metabonomics were used to explore the underlying mechanisms of the effect by studying the expression of biofilm-related genes and endogenous metabolites. AS displayed significant anti-MRSA activity, and its minimum inhibitory concentration was 50 μg/ml. Also, AS inhibited biofilms and improved biofilm permeability. The mechanisms are mediated by the inhibition of the expression of genes, such as quorum sensing system regulatory genes (agrD and sarA), microbial surface components–recognizing adhesion matrix genes (clfA and fnbB), intercellular adhesion genes (icaA, icaD, and PIA), and a gene related to cellular eDNA release (cidA), and the downregulation of five biofilm-related metabolites, including anthranilic acid, D-lactic acid, kynurenine, L-homocitrulline, and sebacic acid. This study provided valuable evidence for the activity of AS against MRSA and its biofilms and extended the methods to combat MRSA infection.

Intraspecific genetic variability and diurnal activity affect environmental DNA detection in Japanese eel.

Environmental DNA (eDNA) analysis with species-specific primer/probe sets is promising as a tool to quantify fish abundance and distribution. Nevertheless, several factors could reduce the accuracy of this method. Here, we aimed to analyze whether intraspecific variability and diel activity rhythm affect eDNA detection in Japanese eels (Anguilla japonica). For this purpose, we performed tank experiments focusing on two points. First, we assessed the effects of base pair sequences with probe region polymorphism on eDNA detection. Next, we evaluated the influences of diel rhythm, activity, and individual differences in eDNA release rate on eDNA concentration. We examined the base pair sequences of the probe regions of 20 individuals and found genetic mismatches in two of them. The eDNA concentration was estimated to be much lower in these variants than it was in the other individuals. We conducted a rearing experiment on four non-variant individuals to explore the influences of diel activity and inter-individual differences in eDNA detection. Nocturnal eel activity was reflected in the eDNA detection but the inter-individual differences remained large. The observed weak positive correlations between eDNA concentration and activity suggest that eDNA emission is highly dependent on basal metabolism. The present study suggests that consideration of polymorphic sites at the probe region and diel activity rhythms should improve the accuracy and precision of abundance estimation through eDNA. Such fine-tuning is applicable not only for eels but also for other fishes to be targeted by eDNA technology.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit.

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Extracellular DNA released by glycine-auxotrophic Staphylococcus epidermidis small colony variant facilitates catheter-related infections

Though a definitive link between small colony variants (SCVs) and implant-related staphylococcal infections has been well-established, the specific underlying mechanism remains an ill-explored field. The present study analyzes the role SCVs play in catheter infection by performing genomic and metabolic analyses, as well as analyzing biofilm formation and impacts of glycine on growth and peptidoglycan-linking rate, on a clinically typical Staphylococcus epidermidis case harboring stable SCV, normal counterpart (NC) and nonstable SCV. Our findings reveal that S. epidermidis stable SCV carries mutations involved in various metabolic processes. Metabolome analyses demonstrate that two biosynthetic pathways are apparently disturbed in SCV. One is glycine biosynthesis, which contributes to remarkable glycine shortage, and supplementation of glycine restores growth and peptidoglycan-linking rate of SCV. The other is overflow of pyruvic acid and acetyl-CoA, leading to excessive acetate. SCV demonstrates higher biofilm-forming ability due to rapid autolysis and subsequent eDNA release. Despite a remarkable decline in cell viability, SCV can facilitate in vitro biofilm formation and in vivo survival of NC when co-infected with its normal counterparts. This work illustrates an intriguing strategy utilized by a glycine-auxotrophic clinical S. epidermidis SCV isolate to facilitate biofilm-related infections, and casts a new light on the role of SCV in persistent infections.

A global transcriptomic analysis of Staphylococcus aureus biofilm formation across diverse clonal lineages.

A key characteristic of Staphylococcus aureus infections, and one that also varies phenotypically between clones, is that of biofilm formation, which aids in bacterial persistence through increased adherence and immune evasion. Though there is a general understanding of the process of biofilm formation – adhesion, proliferation, maturation and dispersal – the tightly orchestrated molecular events behind each stage, and what drives variation between S. aureus strains, has yet to be unravelled. Herein we measure biofilm progression and dispersal in real-time across the five major S. aureus CDC-types (USA100-USA500) revealing adherence patterns that differ markedly amongst strains. To gain insight into this, we performed transcriptomic profiling on these isolates at multiple timepoints, compared to planktonically growing counterparts. Our findings support a model in which eDNA release, followed by increased positive surface charge, perhaps drives initial abiotic attachment. This is seemingly followed by cooperative repression of autolysis and activation of poly-N-acetylglucosamine (PNAG) production, which may indicate a developmental shift in structuring the biofilm matrix. As biofilms mature, diminished translational capacity was apparent, with 53 % of all ribosomal proteins downregulated, followed by upregulation of anaerobic respiration enzymes. These findings are noteworthy because reduced cellular activity and an altered metabolic state have been previously shown to contribute to higher antibiotic tolerance and bacterial persistence. In sum, this work is, to our knowledge, the first study to investigate transcriptional regulation during the early, establishing phase of biofilm formation, and to compare global transcriptional regulation both temporally and across multiple clonal lineages.

Quantifying shedding and degradation rates of environmental DNA (eDNA) from Pacific crown-of-thorns seastar (Acanthaster cf. solaris)

Population outbreaks of the corallivorous crown-of-thorns seastar (CoTS; Acanthaster spp.) are significant threats to the Indo-Pacific reefs. Although recent research demonstrated that environmental DNA (eDNA) techniques could improve CoTS monitoring, the interpretation of surveillance results has been limited by uncertainties about eDNA dynamics. Here, we conducted aquarium experiments to identify biotic and abiotic factors affecting the shedding and degradation rates of CoTS eDNA. In the first experiment, we investigated the effect of two temperatures (24 and 28 °C) and three feeding treatments (no coral, coral accessible and coral inaccessible) on eDNA shedding rate of laboratory-raised 8-month-old juvenile CoTS. In the second experiment, we quantified CoTS eDNA degradation rate under three temperatures (24, 26 and 28 °C). We found that eDNA shedding rate was affected by feeding treatment (p < 0.0001) but not temperature. Specifically, the shedding rate under coral accessible treatment was about seven times higher than that of coral inaccessible treatment (p < 0.0001), whereas the presence of coral reduced the shedding rate by half (coral inaccessible vs no coral, p = 0.0249). Degradation of CoTS eDNA was rapid (half-life = 14 h) and not affected by temperature. Our results demonstrated that feeding activity increased eDNA release, but some of the released DNA was lost, potentially due to binding to coral surface mucus layer or skeleton. The rapid degradation rate indicated that results of eDNA surveillance likely reflects recent and local occurrence of CoTS. Although further testing is needed, this study provided support for using eDNA as a novel detection tool for early life stages of CoTS on coral reefs.

Understanding the Matrix: The Role of Extracellular DNA in Oral Biofilms.

Dental plaque is the key etiological agent in caries formation and the development of the prevalent chronic oral inflammatory disease, periodontitis. The dental plaque biofilm comprises a diverse range of microbial species encased within a rich extracellular matrix, of which extracellular DNA (eDNA) has been identified as an important component. The molecular mechanisms of eDNA release and the structure of eDNA have yet to be fully characterized. Nonetheless, key functions that have been proposed for eDNA include maintaining biofilm structural integrity, initiating adhesion to dental surfaces, acting as a nutrient source, and facilitating horizontal gene transfer. Thus, eDNA is a potential therapeutic target for the management of oral disease–associated biofilm. This review aims to summarize advances in the understanding of the mechanisms of eDNA release from oral microorganisms and in the methods of eDNA detection and quantification within oral biofilms.

EDNA metabarcoding survey of fish communities in the Danube and its tributaries

In complement to the JDS4 traditional fish survey (TFS, mainly by electrofishing), a fish eDNA metabarcoding-based survey has been implemented within the framework of the monitoring program organized by DNAqua-Net and in collaboration with the INTERREG "MEASURES" program. Water samples were collected at 29 sites from the source to the mouth of the Danube River, and at 18 tributaries. Two water samples (mean volume 29 L per sample) were collected at each site and the water filtered in situ. Twelve PCR replicates were performed per sample using teleo primers. In total, 80 taxa were detected, of which 19 corresponded mainly to farmed fish or food fish due to eDNA release in waste waters, most often downstream of large cities. Of the remaining 61 taxa, 50 taxa were identified at the species level, six taxa at the genus level, and five taxa at a higher taxonomic level. Concerning the Danube river itself, 69 and 57 species/markers were detected along the Danube River by TFS and eDNA surveys, respectively. 50 of these taxa were detected by both methods. Nine species were captured by TFS alone, mainly due to gaps in reference librairies and too colosely related barcodes. Eight species were only detected by eDNA. Except for the Salvelinus group, these were all benthic species, which are difficult to catch by electrofishing in large rivers (Acipenser ruthenus, Acipenser stellatus, Benthophilus sp., Romanogobio uranoscopus, Sabanejewia balcanica, Umbra krameri). The species richness tended to increase from upstream to downstream and the relative number of DNA sequences per taxa showed a clear succession of species along the river. Barbatula barbatula, Cottus gobio, Hucho hucho, Lampletra planeri, Phoxinus phoxinus and Thymallus thymallus were restricted to the Upper Danube whereas A. ruthenus, Neogobius fluviatilis, S. balcanica and Scardinius erythrophtalmus were detected from Vienna to the Danube river mouth. Abramis brama, Alburnus alburnus, Cyprinus carpio, Silurus glanis, Sander spp, Zingel streber were detected all along the river course; Alosa spp. and Syngnathus abaster downstream from the Iron Gate; A. stellatus and U. krameri only on the most downstream site (Danube delta). The calculation of a fish index, based on the common metrics used to intercalibrate national fish assessment methods on a European scale, classified most of the sites as being in moderate ecological status. Comparison of the indicative ecological status calculated using the same assessment method based on TSF and eDNA data at the common Danube sites showed a similar classification for six of the 13 sites and a difference of one class for the remaining seven sites.

The Effect of Activity, Energy Use, and Species Identity on Environmental DNA Shedding of Freshwater Fish

The quantitative measurement of environmental DNA (eDNA) from field-collected water samples is gaining importance for the monitoring of fish communities and populations. The interpretation of these signal strengths depends, among other factors, on the amount of target eDNA shed into the water. However, shedding rates are presumably associated with species-specific traits such as physiology and behavior. Although such differences between juvenile and adult fish have been previously detected, the general impact of movement and energy use in a resting state on eDNA release into the surrounding water remains hardly addressed. In an aquarium experiment, we compared eDNA shedding between seven fish species occurring in European freshwaters. The investigated salmonids, cyprinids, and sculpin exhibit distinct adaptions to microhabitats, diets, and either solitary or schooling behavior. The fish were housed in aquaria with constant water flow and their activity was measured by snapshots taken every 30 s. Water samples for eDNA analysis were taken every 3 h and energy use was determined in an intermittent flow respirometer. After controlling for the effect of fish mass, our results demonstrate a positive correlation between target eDNA quantities as measured with digital PCR, fish activity, and energy use, as well as species-specific differences. For cyprinids, the model based on data from individual fish was only partly transferable to groups, which showed lower activity and higher energy use. Our findings highlight the importance of fish physiology and behavior for the comparative interpretation of taxon-specific eDNA quantities. Species traits should therefore be incorporated into eDNA-based monitoring and conservation efforts.

PspC domain-containing protein (PCP) determines Streptococcus mutans biofilm formation through bacterial extracellular DNA release and platelet adhesion in experimental endocarditis.

Bacterial extracellular DNA (eDNA) and activated platelets have been found to contribute to biofilm formation by Streptococcus mutans on injured heart valves to induce infective endocarditis (IE), yet the bacterial component directly responsible for biofilm formation or platelet adhesion remains unclear. Using in vivo survival assays coupled with microarray analysis, the present study identified a LiaR-regulated PspC domain-containing protein (PCP) in S. mutans that mediates bacterial biofilm formation in vivo. Reverse transcriptase- and chromatin immunoprecipitation-polymerase chain reaction assays confirmed the regulation of pcp by LiaR, while PCP is well-preserved among streptococcal pathogens. Deficiency of pcp reduced in vitro and in vivo biofilm formation and released the eDNA inside bacteria floe along with reduced bacterial platelet adhesion capacity in a fibrinogen-dependent manner. Therefore, LiaR-regulated PCP alone could determine release of bacterial eDNA and binding to platelets, thus contributing to biofilm formation in S. mutans-induced IE.

Multiple holins contribute to extracellular DNA release in Pseudomonas aeruginosa biofilms

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell–cell and cell–substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

Targeting Bacterial Gyrase With Cystobactamid, Fluoroquinolone and Aminocoumarin Antibiotics Induces Distinct Molecular Signatures in &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt;

The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at sub-inhibitory concentrations could be clearly distinguished by RNA sequencing as well as metabolomics. We observed a strong (2.8-212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with eDNA release. Gyrase B-binding aminocoumarins induced the most pronounced changes, including a strong downregulation of phenazine and rhamnolipid virulence factors. Cystobactamids led to a downregulation of a glucose catabolism pathway. The study implies that clustering cellular mechanisms of action according to the primary target needs to take class-dependent variances into account.

SpoVG Modulates Cell Aggregation in Staphylococcus aureus by Regulating sasC Expression and Extracellular DNA Release.

Biofilm formation is involved in numerous Staphylococcus aureus infections such as endocarditis, septic arthritis, osteomyelitis, and infections of indwelling medical devices. In these diseases, S. aureus forms biofilms as cell aggregates interspersed in host matrix material. Here, we have observed that the level of cell aggregation was significantly higher in the isogenic spoVG-deletion strain than in the wild-type strain. Reverse transcription-quantitative PCR data indicated that SpoVG could repress the expression of sasC, which codes for S. aureus surface protein C and is involved in cell aggregation and biofilm accumulation. Electromagnetic mobility shift assay demonstrated that SpoVG could specifically bind to the promoter region of sasC, indicating that SpoVG is a negative regulator and directly represses the expression of sasC In addition, deletion of the SasC aggregation domain in the spoVG-deletion strain indicated that high-level expression of sasC could be the underlying cause of significantly increased cell aggregation formation. Our previous study showed that SpoVG is involved in oxacillin resistance of methicillin-resistant S. aureus by regulating the expression of genes involved in cell wall synthesis and degradation. In this study, we also found that SpoVG was able to negatively modulate the S. aureus drug tolerance under conditions of a high concentration of oxacillin treatment. These findings can broaden our understanding of the regulation of biofilm formation and drug tolerance in S. aureusIMPORTANCE This study revealed that SpoVG can modulate cell aggregation by repressing sasC expression and extracellular DNA (eDNA) release. Furthermore, we have demonstrated the potential linkage between cell aggregation and antibiotic resistance. Our findings provide novel insights into the regulatory mechanisms of SpoVG involved in cell aggregation and in biofilm development and formation in Staphylococcus aureus.

Advanced Glycation End Products Enhance Biofilm Formation by Promoting Extracellular DNA Release Through sigB Upregulation in Staphylococcus aureus.

Bacterial biofilms do serious harm to the diabetic foot ulcer (DFU) because they play a crucial role in infection invasion and spread. Staphylococcus aureus, the predominant Gram-positive bacteria in diabetic foot infection (DFI), is often associated with colonization and biofilm formation. Through biofilm formation tests in vitro, we observed that S. aureus bacteria isolated from DFU wounds were more prone to form biofilms than those from non-diabetic patients, while there was no difference in blood sugar between the biofilm (+) diabetics (DB+) and biofilm (-) diabetics (DB-). Furthermore, we found that advanced glycation end products (AGEs) promoted the biofilm formation of S. aureus in clinical isolates and laboratory strains in vitro, including a methicillin-resistant strain. Analysis of biofilm components demonstrated that the biofilms formed mainly by increasing extracellular DNA (eDNA) release; remarkably, the S. aureus global regulator sigB was upregulated, and its downstream factor lrgA was downregulated after AGE treatments. Mechanism studies using a sigB-deleted mutant (Newman-ΔsigB) confirmed that AGEs decreased expression of lrgA via induction of sigB, which is responsible for eDNA release and is a required component for S. aureus biofilm development. In conclusion, the present study suggests that AGEs promote S. aureus biofilm formation via an eDNA-dependent pathway by regulating sigB. The data generated by this study will provide experimental proof and theoretical support to improve DFU infection healing.