eDNA Release Research Articles

Antibiofilm efficacy of emodin alone or combined with ampicillin against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as a significant global health concern. The development of resistance to a broad spectrum of antibiotics, particularly following biofilm formation, renders conventional therapeutic options for MRSA ineffective. Three MRSA clinical isolates were examined in vitro to assess their biofilm-forming capacity and the disruptive effects on pre-established biofilm (via crystal violet staining and scanning electron microscopy), and quantify extracellular DNA (eDNA) release after exposed to emodin alone or in combination with ampicillin. In addition, real-time PCR was employed to investigate the impact of emodin on the expression of biofilm-related genes in MRSA biofilms. The inhibitory effect of emodin on biofilm formation and disruption was observed in a dose dependent manner. The antagonistic activity of emodin in combination with ampicillin against MRSA biofilms was confirmed through adhesion assays. Real-time PCR analysis revealed that emodin, either alone or in combination with ampicillin, effectively downregulated the transcriptional levels of the biofilm-related genes fnbpB, clfA and atlA, but not icaA. In addition, drug treatment resulted in a significant reduction in eDNA release and protein contain in EPS (extracellular polymeric substances), which corresponded to the markedly decreased transcript level of atlA and fnbpB, respectively. These observations suggest that emodin, either alone or in combination with ampicillin, holds potential as a therapeutic approach for MRSA biofilm-related infections.

The potential impact of iron supply on the development of starved Enterococcus faecalis biofilm by modulating the liberation of extracellular DNA.

Enterococcus faecalis (E. faecalis) is commonly associated with persistent periapical infections. Even after multiple courses of root canal therapy, the infection is difficult to eradicate due to its drug resistance and adaptability. However, root canal treatment will remove nutrients from the root canal and make the remaining E. faecalis near starvation. Iron is an essential element for the growth and metabolism of E. faecalis, but previous studies were mostly based on bacterial nutrient sufficient conditions. Therefore, in this study, the starvation state was used as the breakthrough point to explore the mechanism of iron on the biofilm formation of E. faecalis, so as to be more suitable for clinical practice. In this study, we first constructed a starving E. faecalis model. Subsequently, we found that iron supply promoted biofilm formation in starved E. faecalis, with more eDNA in the biofilm. Iron starvation induced by the iron competitive inhibitor gallium nitrate reduced biofilm formation but increased the proportion of eDNA. In contrast, high iron levels in the environment counteracted this inhibition of biofilm formation. Following DNase I treatment, both the eDNA content and viable bacteria within the biofilm of the iron-supply group exhibited a statistically significant reduction. These results suggest that iron supply may regulate the proliferation of active bacteria by regulating eDNA release, thereby promoting biofilm formation of starved E. faecalis and providing a new perspective on its survival strategy under stress.

Capture—Incubate—Release: An Animal‐Friendly Approach to Assess Local Aquatic Macroinvertebrate Species Diversity Through Environmental DNA Metabarcoding

ABSTRACTMetabarcoding of environmental DNA (eDNA) from water samples has become an important tool for aquatic biodiversity assessment because it is minimally invasive, time‐efficient, and generates comprehensive taxa lists. Nevertheless, species lists differ noticeably from those obtained via bulk metabarcoding of the local benthic community because of eDNA traces transported in the water column. However, it is important for several assessments to capture local biodiversity signals. Our goal was to test whether we can combine the advantages of both methods, that is, obtaining a local signal and being minimally invasive. Therefore, our developed method includes capturing local benthic invertebrates, incubating them in a water container for eDNA enrichment and analysis, and releasing them back to their habitat. We first quantified eDNA release over time for 10 invertebrate species in a laboratory setting using qPCR. We found that a 5‐min incubation is sufficient to successfully detect 50% of the replicates for six of the 10 species. Three of the species showed a significant increase in eDNA molecules over time. However, the experiment showed a species‐specific eDNA release pattern that was not directly linked to body sclerotization nor biomass. As a second experiment, we sampled bulk samples at three field sites and incubated the bulk samples for 0, 20, 40, and 80 min in containers filled with stream water to compare taxa lists obtained via metabarcoding of bulk and the enriched eDNA samples. Our results showed a much higher overlap between bulk and enriched eDNA metabarcoding (55%–60%) in comparison to reported overlaps between bulk and stream eDNA metabarcoding from other studies (often < 20%). This overlap did not change with incubation time. Thus, our study demonstrates that it is possible to detect most locally occurring species via eDNA metabarcoding after shortly incubating them in water. Therefore, this approach has great potential for point‐sample eDNA analysis of macroinvertebrates without compromising animal welfare.

Release and Degradation of Environmental DNA and RNA From Eels in Aotearoa New Zealand

ABSTRACTEnvironmental DNA (eDNA) has become a crucial tool for detecting rare species and monitoring biodiversity. However, the prolonged persistence of eDNA in water complicates the precise determination of an organism's location based on an eDNA signal alone. In contrast, environmental RNA (eRNA) degrades faster, potentially offering a more accurate detection proxy. To test this, we analyzed eDNA and eRNA release concentrations and decay rates from six longfin (Anguilla dieffenbachii) and six shortfin (Anguilla australis) eels under controlled conditions. Eels were placed in aquaria for 30 h and, after their removal, temporal water sampling was conducted over 7 days to assess the eels' eDNA and eRNA dynamics. Concentrations of eDNA and eRNA were estimated using validated droplet digital PCR assays for each species (cytb and 16S mitochondrial genes). Temporal eDNA and eRNA dynamics followed an exponential decay function over time, demonstrating a predictable decline in their concentrations. Moreover, higher decay rates of eRNA could represent a slightly more accurate proxy than eDNA for the location determination of rare species. Variability in the release and decay could be linked to the type of nucleic acid, marker genes, or eel species. Understanding these dynamics will help fine‐tune detection models based on eDNA and eRNA.

Resveratrol inhibits the formation of Staphylococcus aureus biofilms by reducing PIA, eDNA release, and ROS production.

Staphylococcus aureus is a zoonotic pathogen that is difficult to control. Resveratrol (RES) has been shown to have significant antibacterial effects. The present study aimed to investigate the inhibitory effect of RES on the formation of Staphylococcus aureus biofilms and their molecular mechanism. First, the minimum inhibitory concentration and inhibitory action curve of RES against Staphylococcus aureus were obtained through testing. Second, we found that RES can inhibit biofilm formation by reducing the release of polysaccharide intercellular adhesion (PIA) and extracellular DNA (eDNA) from Staphylococcus aureus. RES treatment significantly reduced the production of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) in Staphylococcus aureus, indicating that ROS and NADPH are closely related to biofilm formation. This study demonstrates that RES inhibits the formation of Staphylococcus aureus biofilms by reducing PIA, eDNA release, and ROS production, and these results provide new ideas for the clinical application of RES in the treatment of Staphylococcus aureus infection.

Environmental DNA for Assessing Population and Spatial Distribution of Spinibarbus caldwelli in the Liuxi River

The wild resources of Spinibarbus caldwelli, once an important economic fish in southern China, have been drastically reduced in recent years due to environmental changes and human activities. The Liuxi River S. caldwelli National Aquatic Germplasm Reserve was established in Conghua District, Guangzhou city, and the release of S. caldwelli was carried out. However, traditional fishery resource survey methods yield less accurate results when the abundance of the surveyed species is low or when they are difficult to catch. As a non-destructive and non-invasive approach, environmental DNA (eDNA) is widely employed in aquatic species monitoring, though its detection efficiency may be affected by environmental conditions. Therefore, this study explored the eDNA monitoring methods of S. caldwelli in Liuxi River from the following four aspects: (1) the relationship between eDNA release and biomass/abundance; (2) the concentration and diffusion range of eDNA over time in a lentic ecosystem; (3) the diffusion range of eDNA in a lotic ecosystem; and (4) the effects of eDNA application in field monitoring. Our results showed a correlation between eDNA concentration and abundance/biomass of S. caldwelli. eDNA of S. caldwelli can diffuse up to 18 m in lentic ecosystems within 2 h and decreases with distance. eDNA of S. caldwelli released by 10 individuals in Liuxi River could be detected 900 m downstream. Field studies in Liuxi River showed that the eDNA method has high sensitivity in detecting the presence or absence of species and is highly consistent with the results of traditional methods. This study explored the application of environmental DNA technology in species monitoring in Liuxi River. Our aim was to evaluate the applicability and potential of eDNA in ecological monitoring of stream fishes.

Release of extracellular DNA by Pseudomonas sp. as a major determinant for biofilm switching and an early indicator for cell population control.

Release of extracellular DNA by Pseudomonas sp. as a major determinant for biofilm switching and an early indicator for cell population control.

Experimental assessment of Acanthopagrus schlegelii biomass based on environmental DNA technology

The Environmental DNA (eDNA) technology has attracted significant attention due to its convenience and high sensitivity. However, the variations of eDNA across diverse environments and biological species remain complex. Therefore, a detailed exploration of the release patterns of eDNA for specific species under different environments is crucial for the scientific utilization of eDNA detection techniques. This study conducted an experiment involving the aquaculture of Acanthopagrus schlegelii to explore the release and degradation mechanisms of eDNA. It also analyzed the influence of salinity and biomass on the concentration of eDNA in water. Through model simulations, the variation patterns of A. schlegelii eDNA were revealed. The study achieved three key findings: (1) The research on the release and degradation mechanisms of A. schlegelii eDNA indicated that the Generalized Additive Model (GAM) effectively fits the variation patterns of eDNA concentration. The peak concentration of eDNA released by A. schlegelii was observed at 42 h, and the degradation process exhibited two stages: rapid and slow degradation, with a negative correlation between eDNA concentration and time. (2) By investigating the relationship between the concentration of A. schlegelii eDNA and biomass, it was demonstrated that Linear Models (LM) effectively captured this relationship, indicating a correlation between eDNA concentration and biomass. (3) The detection of A. schlegelii eDNA concentration under different salinity conditions revealed that the GAM model better reflected the relationship between eDNA and salinity, exhibiting a negative correlation. As salinity increased, the concentration of eDNA decreased. This study lays a foundation for future assessments of the A. schlegelii biomass in natural waters using eDNA quantitative detection techniques, and provides relevant references for quantitative eDNA detection techniques in other marine fish species.

Biofilm formation by Staphylococcus aureus is triggered by a drop in the levels of a cyclic dinucleotide

The bacterial pathogen Staphylococcus aureus forms multicellular communities known as biofilms in which cells are held together by an extracellular matrix principally composed of repurposed cytoplasmic proteins and extracellular DNA. These biofilms assemble during infections or under laboratory conditions by growth on medium containing glucose, but the intracellular signal for biofilm formation and its downstream targets were unknown. Here, we present evidence that biofilm formation is triggered by a drop in the levels of the second messenger cyclic-di-AMP. Previous work identified genes needed for the release of extracellular DNA, including genes for the cyclic-di-AMP phosphodiesterase GdpP, the transcriptional regulator XdrA, and the purine salvage enzyme Apt. Using a cyclic-di-AMP riboswitch biosensor and mass spectrometry, we show that the second messenger drops in abundance during biofilm formation in a glucose-dependent manner. Mutation of these three genes elevates cyclic-di-AMP and prevents biofilm formation in a murine catheter model. Supporting the generality of this mechanism, we found that gdpP was required for biofilm formation by diverse strains of S. aureus. We additionally show that the downstream consequence of the drop in cyclic-di-AMP is inhibition of the "accessory gene regulator" operon agr, which is known to suppress biofilm formation through phosphorylation of the transcriptional regulator AgrA by the histidine kinase AgrC. Consistent with this, an agr mutation bypasses the block in biofilm formation and eDNA release caused by a gdpP mutation. Finally, we report the unexpected observation that GdpP inhibits phosphotransfer from AgrC to AgrA, revealing a direct connection between the phosphodiesterase and agr.

High release of Candida albicans eDNA as protection for the scaffolding of polymicrobial biofilm formed with Staphylococcus aureus and Streptococcus mutans against the enzymatic activity of DNase I.

This study aimed to determine the protective role of the high release of C. albicans extracellular DNA (eDNA) in a polymicrobial biofilm formed by S. aureus and S. mutans in the course of DNase I treatment. A tube-flow biofilm bioreactor was developed to mimic biofilm formation in the oral cavity. eDNA release was quantified by real-time PCR (qPCR) and confocal microscopy analysis were used to determine the concentration and distribution of eDNA and intracellular DNA (iDNA). The mean amount of eDNA released by each species in the polymicrobial was higher than that in monospecies biofilms (S. aureus: 3.1 × 10-2ng/μl polymicrobial versus 5.1 × 10-4ng/μl monospecies; S. mutans: 3 × 10-1ng/μl polymicrobial versus 2.97 × 10-2ng/μl monospecies; C. albicans: 8.35ng/μl polymicrobial versus 4.85ng/μl monospecies). The large amounts of eDNA released by C. albicans (96%) in polymicrobial biofilms protects the S. aureus and S. mutans cells against the degradation by DNase I and dampens the effect of clindamycin.

Mosquitoes on a chip-environmental DNA-based detection of invasive mosquito species using high-throughput real-time PCR.

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.

Functional Study of desKR: a Lineage-Specific Two-Component System Positively Regulating Staphylococcus aureus Biofilm Formation

PurposeBiofilms significantly contribute to the persistence and antibiotic resistance of Staphylococcus aureus infections. However, the regulatory mechanisms governing biofilm formation of S. aureus remain not fully elucidated. This study aimed to investigate the function of the S. aureus lineage-specific two-component system, desKR, in biofilm regulation and pathogenicity.MethodsBioinformatic analysis was conducted to assess the prevalence of desKR across various S. aureus lineages and to examine its structural features. The impact of desKR on S. aureus pathogenicity was evaluated using in vivo mouse models, including skin abscess, bloodstream infection, and nasal colonization models. Crystal violet staining and confocal laser scanning microscopy were utilized to examine the impact of desKR on S. aureus biofilm formation. Mechanistic insights into desKR-mediated biofilm regulation were investigated by quantifying polysaccharide intercellular adhesin (PIA) production, extracellular DNA (eDNA) release, autolysis assays, and RT-qPCR.ResultsThe prevalence of desKR varied among different S. aureus lineages, with notably low carriage rates in ST398 and ST59 lineages. Deletion of desKR in NCTC8325 strain resulted in decreased susceptibility to β-lactam and glycopeptide antibiotics. Although desKR did not significantly affect acute pathogenicity, the ΔdesKR mutant exhibited significantly reduced nasal colonization and biofilm-forming ability. Overexpression of desKR in naturally desKR-lacking strains (ST398 and ST59) enhanced biofilm formation, suggesting a lineage-independent effect. Phenotypic assays further revealed that the ΔdesKR mutant showed reduced PIA production, decreased eDNA release, and lower autolysis rates. RT-qPCR indicated significant downregulation of icaA, icaD, icaB, and icaC genes, along with upregulation of icaR, whereas autolysis-related genes remained unchanged.ConclusionThe desKR two-component system positively regulates S. aureus biofilm formation in a lineage-independent manner, primarily by modulating PIA synthesis via the ica operon. These findings provide new insights into the molecular mechanisms of biofilm formation in S. aureus and highlight desKR as a potential target for therapeutic strategies aimed at combating biofilm-associated infections.

Beyond the double helix: the multifaceted landscape of extracellular DNA in Staphylococcus aureus biofilms.

Staphylococcus aureus forms biofilms consisting of cells embedded in a matrix made of proteins, polysaccharides, lipids, and extracellular DNA (eDNA). Biofilm-associated infections are difficult to treat and can promote antibiotic resistance, resulting in negative healthcare outcomes. eDNA within the matrix contributes to the stability, growth, and immune-evasive properties of S. aureus biofilms. eDNA is released by autolysis, which is mediated by murein hydrolases that access the cell wall via membrane pores formed by holin-like proteins. The eDNA content of S. aureus biofilms varies among individual strains and is influenced by environmental conditions, including the presence of antibiotics. eDNA plays an important role in biofilm development and structure by acting as an electrostatic net that facilitates protein-cell and cell-cell interactions. Because of eDNA's structural importance in biofilms and its ubiquitous presence among S. aureus isolates, it is a potential target for therapeutics. Treatment of biofilms with DNase can eradicate or drastically reduce them in size. Additionally, antibodies that target DNABII proteins, which bind to and stabilize eDNA, can also disperse biofilms. This review discusses the recent literature on the release, structure, and function of eDNA in S. aureus biofilms, in addition to a discussion of potential avenues for targeting eDNA for biofilm eradication.

GlmS plays a key role in the virulence factor expression and biofilm formation ability of Staphylococcus aureus promoted by advanced glycation end products

ABSTRACT Staphylococcus aureus (S. aureus) is well known for its biofilm formation ability and is responsible for serious, chronic refractory infections worldwide. We previously demonstrated that advanced glycation end products (AGEs), a hallmark of chronic hyperglycaemia in diabetic tissues, enhanced biofilm formation by promoting eDNA release via sigB upregulation in S. aureus, contributing to the high morbidity and mortality of patients presenting a diabetic foot ulcer infection. However, the exact regulatory network has not been completely described. Here, we used pull-down assay and LC-MS/MS to identify the GlmS as a candidate regulator of sigB in S. aureus stimulated by AGEs. Dual-luciferase assays and electrophoretic mobility shift assays (EMSAs) revealed that GlmS directly upregulated the transcriptional activity of sigB. We constructed NCTC 8325 ∆glmS for further validation. qRT-PCR analysis revealed that AGEs promoted both glmS and sigB expression in the NCTC 8325 strain but had no effect on NCTC 8325 ∆glmS. NCTC 8325 ∆glmS showed a significant attenuation in biofilm formation and virulence factor expression, accompanied by a decrease in sigB expression, even under AGE stimulation. All of the changes, including pigment deficiency, decreased haemolysis ability, downregulation of hla and hld expression, and less and sparser biofilms, indicated that sigB and biofilm formation ability no longer responded to AGEs in NCTC 8325 ∆glmS. Our data extend the understanding of GlmS in the global regulatory network of S. aureus and demonstrate a new mechanism by which AGEs can upregulate GlmS, which directly regulates sigB and plays a significant role in mediating biofilm formation and virulence factor expression.

C A Review of Genes Related to Biofilm Formation in Enterococcus

Background: Enterococcus faecium is of great importance among Enterococcus species due to its antibiotic resistance and biofilm formation. The ability to form a biofilm on living surfaces is considered one of the most important pathogenic factors in this bacterium. E. faecium is involved in endocarditis, urinary tract infection, dental root infections, and eye infections by forming biofilms. Various factors, including the antibiotic resistance of E. faecium and the expression of genes involved in pathogenicity and biofilm formation, are involved in the stability of this bacterium in different conditions and the spread of infection. Objectives: Considering the special importance of E. faecium in biofilm production, the aim of this study was to review the presence and role of genes effective in biofilm formation in E. faecium bacteria. Materials and Methods: PubMed, Embase, and Web of Science databases were searched using the keywords "genes", "biofilm formation", "Enterococcus", and "quorum sensing". Studies that investigated the role of genes in the occurrence of biofilm in E. faecium were also included in the study. Results: Biofilm formation in enterococci involves a complex interaction of genes and virulence factors, including gelatinase, cytolysin, secreted antigen A, pili, and MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Polysaccharides also play a role in enterococcal biofilm formation. They are secreted in the form of capsules or exopolysaccharides. The genes involved in the production of polysaccharides include Sal, atn, epa, DltA, ebpABC, and bee. These genes increase the production of biofilm and attachment to the extracellular matrix. Quorum sensing, a process of intercellular communication, mediated by peptide pheromones such as Cob, Ccf, and Cpd, by targeting gene expression and regulation, plays an important role in biofilm development. The Fsr locus contains fsrA, fsrB, and fsrC genes. Furthermore, the regulation of extracellular DNA (eDNA) release has emerged as an essential component in biofilm formation. Conclusion: Biofilm formation in E. faecium is dependent on a large number of factors, including multiple genetic factors, a series of environmental conditions, and also a series of environmental signals, many of which need to be identified and researched. A complete understanding of the role of genetic and environmental factors involved in biofilm development may lead to the improvement and development of strategies for biofilm control in enterococci.

Antibacterial activity of camel colostrum against pathogenic strain of Escherichia coli F17-associated with calf diarrhea.

Many protective proteins, including lactoferrin and heavy chain antibodies, are present in camel colostrum, giving it a distinctive composition. Beyond a broad spectrum of pathogens, these proteins demonstrate antibacterial properties. The current research assessed the prophylactic properties of camel colostrum against Escherichia coli F17. A microbroth dilution method was employed to assess the efficacy of camel colostrum, whereas a crystal violet assay was utilized to determine its antibiofilm potential. Extracellular deoxyribonuclease acid (eDNA) release, swarming, and swimming motilities were also examined. Showed that camel colostrum significantly reduced E. coli-F17 growth by 70% and above at different incubation periods (6-24 hours). The rate of cell attachment gradually decreased from approximately 40% to 24% as the concentration increased from 12.5 to 50 mg/ml. E. coli-F17 developed a biofilm at a rate of 54.8% when exposed to 50 mg/ml of camel colostrum. In contrast, the greatest level of biofilm formation against the tested bacteria (94%) was observed at a concentration of 1.5 mg/ml. A halo zone of camel colostrum ranging from 10 to less than 30 mm at concentrations between 6 and 50 mg/ml also inhibited swimming and swarming capabilities. The treated cells yielded no eDNA. According to these results, camel colostrum inhibits the growth of E. coli-F17 by impeding the swarming and swimming motilities, and biofilm formation. Additionally, camel colostrum incubation with E. coli-F17 diminishes eDNA. To evaluate the potential protective effects of camel colostrum in an animal model, additional research is recommended.

Hidden in plain sight: The invasive macroalga Caulerpa prolifera evades detection by environmental DNA methods

Abstract Environmental managers need a rapid and cost‐effective monitoring tool for tracking the spread of invasive species, particularly at the onset of introduction. The macroalgae Caulerpa prolifera is considered an invasive species outside its native range, colonizing large patches of seafloor, reducing native species, and altering ecosystem functioning. Here, we developed a droplet digital PCR assay for detection of C. prolifera from environmental DNA seawater samples using the internal transcribed spacer (ITS) region. While the assay itself was confirmed to be highly efficient, we discovered concentrations of C. prolifera eDNA were present below detectable levels in the water column surrounding an outbreak. To understand why, we conducted tank‐based experiments for two California invasive algae species, Caulerpa prolifera and Sargassum horneri. The steady‐state eDNA concentration (eDNA copies/ gram of biomass detected) of C. prolifera was found to be two orders of magnitude lower than S. horneri. A meta‐analysis of steady‐state concentrations reported in the literature showed a remarkable range from ~104–1011 (copies/g), revealing C. prolifera to have the lowest recorded steady‐state concentrations of eDNA of any known species. We attribute C. prolifera's low steady‐state eDNA concentration to its unique biology as a unicellular macroscopic algae which reduces the possible modes of eDNA release compared to similarly sized multicellular organisms. Critically our results demonstrate the potential limits of eDNA approaches, the influence of shedding rates in the reliability of species detections, and the vital importance of benchmarking and validating eDNA assays in both field and laboratory settings.

Genotoxic stress stimulates eDNA release via explosive cell lysis and thereby promotes streamer formation of Burkholderia cenocepacia H111 cultured in a microfluidic device

DNA is a component of biofilms, but the triggers of DNA release during biofilm formation and how DNA contributes to biofilm development are poorly investigated. One key mechanism involved in DNA release is explosive cell lysis, which is a consequence of prophage induction. In this article, the role of explosive cell lysis in biofilm formation was investigated in the opportunistic human pathogen Burkholderia cenocepacia H111 (H111). Biofilm streamers, flow-suspended biofilm filaments, were used as a biofilm model in this study, as DNA is an essential component of their matrix. H111 contains three prophages on chromosome 1 of its genome, and the involvement of each prophage in causing explosive cell lysis of the host and subsequent DNA and membrane vesicle (MV) release, as well as their contribution to streamer formation, were studied in the presence and absence of genotoxic stress. The results show that two of the three prophages of H111 encode functional lytic prophages that can be induced by genotoxic stress and their activation causes DNA and MVs release by explosive cell lysis. Furthermore, it is shown that the released DNA enables the strain to develop biofilm streamers, and streamer formation can be enhanced by genotoxic stress. Overall, this study demonstrates the involvement of prophages in streamer formation and uncovers an often-overlooked problem with the use of antibiotics that trigger the bacterial SOS response for the treatment of bacterial infections.

Rhamnolipid Micelles Assist Azithromycin in Efficiently Disrupting Staphylococcus aureus Biofilms and Impeding Their Re-Formation

IntroductionBiofilm is highly resistant to antibiotics due to its heterogeneity and is implicated in over 80% of chronic infections; these refractory and relapse-prone infections pose a huge medical burden.MethodsIn this study, rhamnolipid (RHL), a biosurfactant with antibiofilm activity, was loaded with the antibiotic azithromycin (AZI) to construct a stable nanomicelle (AZI@RHL) that promotes Staphylococcus aureus (S. aureus) biofilm disruption.ResultsAZI@RHL micelles made a destruction in biofilms. The biofilm biomasses were reduced significantly by 48.2% (P<0.05), and the main components polysaccharides and proteins were reduced by 47.5% and 36.8%, respectively. These decreases were about 3.1 (15.9%), 7.3 (6.5%), and 1.9 (19.5%) times higher compared with those reported for free AZI. The disruption of biofilm structure was observed under a confocal microscope with fluorescent labeling, and 48.2% of the cells in the biofilm were killed. By contrast, the clearance rates of cells were only 20% and 17% when treated alone with blank micelles or free AZI. Biofilm formation was inhibited up to 92% in the AZI@RHL group due to effects on cell auto-aggregation and eDNA release. The rates for the other groups were significantly lower, with only 27.7% for the RHL group and 12% for the AZI group (P<0.05). The low cell survival and great formation inhibition could reduce biofilm recolonization and re-formation.ConclusionThe antibiofilm efficacy of rhamnolipid was improved through micellar nanoparticle effects when loading azithromycin. AZI@RHL provides a one-step solution that covers biofilm disruption, bacteria inactivation, recolonization avoidance, and biofilm re-formation inhibition.

Landscape of antibiotic resistance genes and bacterial communities in groundwater on the Tibetan Plateau, and distinguishing their difference with low-altitude counterparts

Landscape of antibiotic resistance genes and bacterial communities in groundwater on the Tibetan Plateau, and distinguishing their difference with low-altitude counterparts