Extracellular Resistance Research Articles

ECM Remodeling and an Abrupt, Stochastic Transition to Arrest Determine Tissue Growth Kinetics

During development, multicellular organisms undergo stereotypical patterns of tissue growth in space and time, but how this is orchestrated remains unclear. Drosophila histoblast nests are small clusters of progenitor epithelial cells that undergo extensive growth to give rise to the abdominal epidermis and are amenable to live-imaging. Our quantitative analysis of the temporal dynamics of abdomen growth reveals that histoblasts display oscillatory cell division rates and that growth termination occurs through the rapid emergence of arrested cells rather than a gradual increase in cell cycle time. By examining tissue mechanics throughout histoblast expansion, we uncover an increase in tissue stress together with a decrease in extracellular matrix resistance as the histoblasts expand. The decrease in resistance is driven by timely extracellular matrix remodeling, which is necessary to trigger tissue expansion. Thus, changes in the tissue microenvironment, and a rapid cell cycle exit, control the formation of the adult Drosophila abdomen.

Effects of extracellular DNA on dual-species biofilm formed by Streptococcus mutans and Candida albicans

Streptococcus mutans is the most important acid-producing pathogen that causes dental caries, while Candida albicans is an opportunistic fungal pathogen that is frequently detected in conjunction with heavy infection by S. mutans. Their interactions in dental plaque biofilms remain unclear. Extracellular DNA (eDNA) is found in oral biofilms, but its effects have not been thoroughly defined. In this study, the role of eDNA in dual-species biofilms formed by S. mutans and C. albicans was investigated. With eDNA removal, the growth of both strains was not affected, but the formation of dual-species biofilms obviously decreased. In addition, the removal of eDNA spatially disrupted the structure of the dual-species biofilm. It was also shown that eDNA mainly affected the initial attachment and development stages of the dual-species biofilms but not the well-developed biofilms. A similar phenomenon was also observed in the cell viability of dual-species biofilms after DNase I treatment. To further exploration, we analyzed the expression of genes associated with biofilm formation in both S. mutans and C. albicans. We determined that the co-cultivation of S. mutans and C. albicans promotes the expression of genes related to extracellular polysaccharide production (e.g., gtfC), adhesion (e.g., spaP, epa1), mycelial transformation (e.g., hwp1), and drug resistance (e.g., cdr2). However, these genes were significantly downregulated when the eDNA of the dual-species biofilm was removed by adding DNase I compared to those untreated groups. Altogether, eDNA removal, such as that by DNase I treatment, could be considered a promising strategy to control oral biofilms and biofilm-associated oral diseases.

Comparative Transcriptome Analysis Providing Resistance Mechanism of Aspergillus oryzae Under Arsenate Stress

The molecular resistance mechanism of Aspergillus oryzae (A. oryzae) under arsenate (As(V)) stress was investigated by comparative transcriptome analysis. In total, 4911 differentially expressed genes (DEGs) of A. oryzae were detected under As(V) stress, including 2103 up-regulated genes and 2808 down-regulated genes. Genes induced by As(V) were involved in metal ion binding and transportation, redox homeostasis, efflux carrier, DNA repair, volatilization, etc. Moreover, eight genes were selected for qRT-PCR verification, and their trends were consistent with RNA-Seq analysis, which proved that RNA-Seq analysis was efficient and reliable. The results indicated that the extracellular and intracellular synergistic resistance mechanisms contributed to the improvement of A. oryzae resistance to As (V).

Intracellular and Extracellular Antibiotic Resistance Genes in Airborne PM2.5 for Respiratory Exposure in Urban Areas

The current research paradigm for the environmental dissemination of antibiotic resistance genes (ARGs) focuses on those harbored within bacteria (i.e., intracellular fraction). Understanding both intracellular and extracellular ARGs has particular implications for the flux and fate of ARGs that are airborne via fine particulate matter (PM₂.₅) from the ambient atmosphere to the human airway. In this study, we developed an operationally defined protocol for urban PM₂.₅ to quantify the abundance of ARGs occurring in free and phage-associated DNA, in addition to their counterparts commonly analyzed within bacterial cells. The results demonstrated that the extracellular fraction comprises a significant proportion of total ARGs in PM₂.₅, with subtype-specific dominance in either free or phage-associated DNA. A comparison between temperate and subtropical cities revealed geographical disparities of PM₂.₅-associated intracellular and extracellular ARGs due to the influences of regional meteorological factors and oxidative gases. Considering the potential for horizontal gene transfers and the efficiency of respiratory deposition, the extracellular fraction could represent >60% of the modeled inhalational intake of most of the analyzed ARGs in some cities. This study highlights the importance of the dynamics of ARGs in airborne PM₂.₅ and their health implications across climate zones and pollution gradients.

Removal of extracellular antibiotic resistance genes using magnetic biochar/quaternary phosphonium salt in aquatic environments: A mechanistic study

The proliferation and spread of antibiotic resistance genes (ARGs) is becoming a worldwide crisis. Extracellular DNA encoding ARGs (eARGs) in aquatic environment plays a critical role in the dispersion of antimicrobial resistance genes. Strategies to control the dissemination of eARGs are urgently required for ecological safety and human health. Towards this goal, magnetic biochar/quaternary phosphonium salt (MBQ), was used to investigate the efficiency and removal mechanism for eARGs. Magnetic biochar modified by quaternary phosphonium salt enhanced the adsorption capacity of extracellular DNA to approximately 9 folds, compared to that of the unmodified. DNA adsorption by MBQ was mainly dominated by chemisorption in heterogeneous systems and was promoted in acidic and low-salt environment. The generation of •OH and MBQ colloid jointly cleaved DNA into fragments, facilitating the adsorption of the phosphate backbone of DNA onto MBQ through electrostatic force as well as the conformational transition of DNA. Furthermore, quantification of extracellular DNA after MBQ was applied in water demonstrated that over 92.7% of resistance genes were removed, indicating a significantly reduced risk of propagation of antimicrobial resistance in aquatic environments. These findings have a practical significance in the application of MBQ in mitigating the spread of ARGs in aquatic environment.

Fate and persistence of antibiotic-resistant bacteria and genes through a multi-barrier treatment facility for direct potable reuse

Abstract Given the availability of technological solutions and guidelines for safe drinking water, direct potable reuse of reclaimed water has become a promising option to overcome severe lack of potable water in arid regions. However, the growing awareness of the presence of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) in corresponding raw wastes has led to new safety concerns. This study investigated the fate of ARB and intracellular and extracellular ARG after each treatment step of an advanced water treatment facility in Windhoek, Namibia. The New Goreangab Water Reclamation Plant (NGWRP) produces drinking water from domestic secondary wastewater treatment plant effluent and directly provides for roughly a quarter of Windhoek's potable water demand. Procedures to study resistance determinants were based on both molecular biology and culture-based microbiological methods. TaqMan real-time PCR was employed to detect and quantify intracellular resistance genes sul1, ermB, vanA, nptII and nptIII as well as extracellular resistance gene sul1. The NGWRP reduced the amount of both culturable bacterial indicators as well as the resistance genes to levels below the limit of detection in the final product. The main ozonation and the ultrafiltration had the highest removal efficiencies on both resistance determinants.

Microplastics affect the ammonia oxidation performance of aerobic granular sludge and enrich the intracellular and extracellular antibiotic resistance genes

Microplastics (MPs) and antibiotic resistance genes (ARGs), as emerging pollutants, are frequently detected in wastewater treatment plants, and their threats to the environment have received extensive attentions. However, the effects of MPs on the nitrification of aerobic granular sludge (AGS) and the spread patterns of intracellular and extracellular ARGs (iARGs and eARGs) in AGS were still unknown. In this study, the responses of AGS to the exposure of 1, 10 and 100 mg/L of typical MPs (polyvinyl chloride (PVC), polyamide (PA), polystyrene (PS) and polyethylene (PE)) and tetracycline were focused on in 3 L nitrifying sequencing batch reactors. 10 mg/L MPs decreased the nitrification function, but nitrification could recover. Furthermore, MPs inhibited ammonia-oxidizing bacteria and enriched nitrite-oxidizing bacteria, leading partial nitrification to losing stability. PVC, PA and PS stimulated the secretion of extracellular polymeric substances and reactive oxygen species. PE had less negative effect on AGS than PVC, PA and PS. The abundances of iARGs and eARGs (tetW, tetE and intI1) increased significantly and the intracellular and extracellular microbial communities obviously shifted in AGS system under MPs stress. Potential pathogenic bacteria might be the common hosts of iARGs and eARGs in AGS system and were enriched in AGS and MPs biofilms.

Theranostic Radiopharmaceuticals Targeting Cancer-Associated Fibroblasts.

The tumor microenvironment is a dynamic ecosystem where malignant cells interact with the stromal cells sustaining and promoting tumor growth and metastasis. Cancer-associated fibroblasts (CAFs) are the major component of tumor stroma. CAFs control key tumorigenic activities by participating in immune evasion and suppression, extracellular matrix remodeling, neo-angiogenesis, and drug resistance. Therefore, targeting CAFs emerges as an attractive anti-cancer strategy. This review summarized recent advancements in targeting CAFs with diagnostic and therapeutic radiopharmaceuticals using clinically-promising biomarkers. The efforts to improve clinical outcomes via the application of new radiotheranostic compounds are discussed in the context of radionuclide, the pharmacophore, and, more generally, in terms of biomarker specificity and expression across different cancers and CAF phenotypes.

Significant increase in the secretion of extracellular vesicles and antibiotics resistance from methicillin-resistant Staphylococcus aureus induced by ampicillin stress

Extracellular vesicles (EVs) containing specific cargo molecules from the cell of origin are naturally secreted from bacteria. EVs play significant roles in protecting the bacterium, which can contribute to their survival in the presence of antibiotics. Herein, we isolated EVs from methicillin-resistant Staphylococcus aureus (MRSA) in an environment with or without stressor by adding ampicillin at a lower concentration than the minimum inhibitory concentration (MIC). We investigated whether EVs from MRSA under stress condition or normal condition could defend susceptible bacteria in the presence of several β-lactam antibiotics, and directly degrade the antibiotics. A comparative proteomic approach was carried out in both types of EVs to investigate β-lactam resistant determinants. The secretion of EVs from MRSA under antibiotic stressed conditions was increased by 22.4-fold compared with that of EVs without stress. Proteins related to the degradation of β-lactam antibiotics were abundant in EVs released from the stressed condition. Taken together, the present data reveal that EVs from MRSA play a crucial role in the survival of β-lactam susceptible bacteria by acting as the first line of defense against β-lactam antibiotics, and antibiotic stress leads to release EVs with high defense activity.

Fates of intracellular and extracellular antibiotic resistance genes during a pilot-scale aerobic granular sludge cultivation process

• Both i-ARGs and e-ARGs were enriched and had different shift patterns in this study. • I-ARGs had higher relative abundance but lower growth rate than e-ARGs. • Different oligotypes led to distinct co-occurrence patterns among taxa and i/e-ARGs. • E-ARGs might be carried by e-integrons, allowing them to persist outside the cell. • Both microbes and environmental factors played key roles in shaping e-ARGs pattern. Antibiotic resistance genes (ARGs) were considered as emerging genetic contaminants, which were threatening public health worldwide. ARGs had two forms of existence: intracellular ARGs (i-ARGs) and extracellular ARGs (e-ARGs), and their emergence and spread patterns were rarely revealed in aerobic granular sludge (AGS) system. The results showed that both i-ARGs and e-ARGs were enriched but had different shift patterns during AGS cultivation process, and e-ARGs had higher growth rate. Most functional bacteria enriched in AGS might play important roles in accumulating of i/e-ARGs, and their oligotypes might be the key reason for the different correlations between taxa and i/e-ARGs. E-ARGs might be transformed from i-ARGs and mainly carried by e-integrons, and three major modules of co-occurrence among e-ARGs were observed. The combined action of environmental factors and bacterial community contributed most (35.43%) to the shift pattern of e-ARGs. This study deciphered the migrations and accumulations of i/e-ARGs during aerobic granulation process, and provided several meaningful comprehensions on i/e-ARGs dissemination during practical AGS application.

Nanoparticles as vectors for antibiotic resistance: The association of silica nanoparticles with environmentally relevant extracellular antibiotic resistance genes

A relevant but yet unconsidered subset of particles that may alter the fate of extracellular antibiotic resistance genes (eARGs) are nano-scale particles (NPs), which are ubiquitous in natural environments and have unique properties. In this study, sorption isotherms were developed describing the association of linear DNA fragments isolated from widespread eARGs (blaI and nptII) with either micon-sized kaolinite or silica nanoparticles (SNPs), to determine if sorption capacity was enhanced at the nanoscale. For each isotherm, eARG fragments were added at five starting concentrations (5–40 μg/mL) to mixed batch systems with 0.25 g of particles and nuclease-free water. Sorption was quantified by the removal of DNA from solution, as detected by a Qubit fluorimeter. Isotherms were developed for eARGs of various fragment lengths (508, 680 and 861 bp), guanine-cytosine (GC) contents (34%, 47% and 54%) and both double and single stranded eARGs, to assess the impact of DNA properties on particle association. Sorption isotherms were also developed in systems with added humic acid and/or CaCl2, to assess the impact of these environmental parameters on sorption. FTIR analysis was performed to analyze the conformation of sorbed eARGs. Desorption of eARGs was studied by quantifying the removal of eDNA from washed and vortexed post-sorption particles. Statistically significant irreversible sorption of eARGs to environmentally relevant NPs (humic acid functionalized silica nanoparticles) was demonstrated for the first time. Nano-emergent properties did not increase sorption capacity of eARGs, but led to a unique compressed conformation of sorbed eARGs. The addition of humic acid, increased CaCl2 concentration and small DNA fragment size favored sorption. NPs showed a slight preference for the sorption of single-stranded DNA over double-stranded DNA. These findings suggest that NP association with eARGs may be a significant and unique environmental phenomenon that could influence the spread of antibiotic resistance.

Distribution of extracellular and intracellular antibiotic resistance genes in sludge fractionated in terms of settleability

The widespread proliferation of antibiotic resistance genes (ARGs) is a serious environmental and human health issue. Wastewater treatment facilities (WWTFs) are potential sources to spread ARGs to natural environment, for which, the presence state of ARGs in the sludge, as extracellular ones (eARGs) or intracellular ones (iARGs), along with the sludge settleability, are very important factors. The sludge settleability is closely associated with its floc size and density, bacterial activity, and the proportion of intact/damaged bacterial cells that aggregate together to form flocs for separation in the sedimentation process. It is reasonable to hypothesize that the distribution of eARGs and iARGs may differ with the sludge fractions of different settleability, a topic of great academic and practical significance requiring clarification. In this study, sludge samples from the aerobic contact tank of six household WWTFs were fractionated into fractions with different settling velocities: sludge of low settleability (LS), medium settleability (MS) and high settleability (HS); and the distribution of eARGs and iARGs in the obtained fractions for the widely detected tet G, tet M and sul 1 in water environment was evaluated based on the PMA-qPCR method, together with the evaluation for the well reported mobile genomic element intl 1 and total bacterial 16S rDNA. For the LS fractions, which contained more damaged bacterial cells, the distribution percentages of eARGs were generally higher than those of iARGs. For the HS fractions, which contained flocs with larger sizes formed by both intact and damaged bacterial cells, the relative abundances of ARGs and intl 1 were found apparently lower even if the presence percentages of eARGs were comparatively higher. It is thus inferable that sludge fractions of LS may possess higher transfer potential for ARGs and enhancing their settleability through optimization of the operation conditions is important for mitigating the proliferation of ARGs.

Microbe-EDTA mediated approach in the phytoremediation of lead-contaminated soils using maize (Zea mays L.) plants

In the current study, we investigated the potential of Cronobacter sakazakii- ethylenediaminetetraacetic acid (EDTA) assisted phytoremediation potential of Zea mays L. to remediate lead (Pb)-contaminated soils. The C. sakazakii exhibited various stress tolerance mechanisms via plant growth promoting (PGP) traits, intrinsic extracellular enzyme production and antibiotic resistance. A greenhouse experiment was conducted to examine the dual effects of plant growth promoting endophytic bacteria (PGPEB)-chelator synergy in maize plants under different Pb contaminated soil regimes. C. sakazaii-EDTA (5 mM EDTA kg−1) complex significantly (p < 0.05) enhanced plant growth and biomass (48.91%); chlorophyll a, b and carotenoid contents (27.26%, 25.02% and 42.09%); relative water content (61.33%); proline content (63.60%); root and shoot Pb accumulation capacity (52.31% and 44.71%) in Pb contaminated soils. This may suggest the efficacy of current approach in enhancing plant tolerance capability toward Pb-uptake and phytoremediation capacity. Moreover, maize plants showed differential response to Pb availability in soil-1 (S1; Pb spiked soil, 500 mg kg−1) and soil-2 (S2; aged-contaminated soil) under various treatments. We describe the intriguing role of C. sakazakii-EDTA-maize system for Pb decontamination which can be used as a base line to explore the proposed combinatorial approach for long-term trails under field conditions for reclamation of Pb-contaminated soils. Highlights The PGPEB-EDTA mediated potential of Z. mays against Pb spiked and industrial contaminated soils is noticed. Increased tolerance of Z. mays against Pb in association with C. sakazakii, and EDTA is reported first time. Enhanced accumulation of metals by Z. mays is reported under combined treatment of C. sakazakii, and EDTA. Inoculation of plants with C. sakazakii, and EDTA has positive effects on growth and accumulation of Pb by Z. mays.

Enhanced Membrane Anti-fouling Ability of Anaerobic Membrane Bioreactor Equipped with Bio-electrochemical System Using Nano-zero-valent Iron and Its Regulation Mechanism

Membrane fouling is the biggest challenge of membrane bioreactor industrialization. In this study, a bio-electrochemical system (BES)-anaerobic membrane bioreactor (AnMBR) system was constructed, and the effect of nano-zero-valent iron (nZVI) on membrane anti-fouling ability and methane production was investigated. The results showed that the BES-AnMBR system was stable and the chemical oxygen demand (COD) removal rate was maintained at approximately 95%. The optimum condition was observed to be nZVI 0.1 g·g-1(VS). Under this condition, transmembrane pressure (TMP) was reduced by 28.1%, the membrane flux had a slight improvement, and methane production was up to 81.3 mL·g-1(CODremoved). This was 12.1% higher than that of the control. In addition, a further analysis of extracellular polymeric substances (EPS) fraction and membrane resistance showed that nZVI enhanced EPS decomposition, promoted the formation of an iron-rich layer of inorganic and organic matters on the membrane surface, and changed the distribution of organics and inorganics, thereby significantly alleviating membrane fouling. This study will enrich basic theory of conventional AnMBR and provide a new solution for efficient sludge treatment and resource utilization.

The impedance analysis of small intestine fusion by pulse source.

The radiofrequency-induced intestine fusion has been widely studied as an alternative for traditional suture in surgery, but fusion quality cannot be evaluated directly. Impedance measurement can evaluate fusion quality, but the relation between impedance and the fusion quality needs optimization for best results. The present study reports the optimum resistance of small intestine fusion. As the feedback signal, resistance was considered the indicator of the fusion completion for the device design of intestine fusion and an in-depth study of microstructure change. A self-design pulse source was used for the small intestine fusion with adjustable voltage, duty ratio, frequency and output time. A frequency of 440 kHz was set, whereas voltage, output time and compression pressure (CP) of the small intestine were independent variables. Different conditions of voltage, CP and time were investigated for achieving the highest burst pressure (BP) measured with a pressure gauge and a peristaltic pump. Each parameter of the equivalent circuit model was calculated by an experimental waveform. Hematoxylin–eosin staining of fusion samples was used for assessing the quality of fusion. The real-time current was measured and recorded during the fusion for the calculation of capacitance and resistance. The highest BP of 38.9 mmHg was achieved with a CP of 900 kPa, a voltage of 50 V and a time of 5 s. Finally, an optimum extracellular resistance range of 61.0–86.2 Ω was found as the optimum resistance for the end of fusion, thus indicating automatic fusion with the best fusion quality.

Impact factors of the accumulation, migration and spread of antibiotic resistance in the environment.

Antibiotic resistance is a great concern, which leads to global public health risks and ecological and environmental risks. The presence of antibiotic-resistant genes and antibiotic-resistant bacteria in the environment exacerbates the risk of spreading antibiotic resistance. Among them, horizontal gene transfer is an important mode in the spread of antibiotic resistance genes, and it is one of the reasons that the antibiotic resistance pollution has become increasingly serious. At the same time, free antibiotic resistance genes and resistance gene host bacterial also exist in the natural environment. They can not only affect horizontal gene transfer, but can also migrate and aggregate among environmental media in many ways and then continue to affect the proliferate and transfer of antibiotic resistance genes. All this shows the seriousness of antibiotic resistance pollution. Therefore, in this review, we reveal the sensitive factors affecting the distribution and spread of antibiotic resistance through three aspects: the influencing factors of horizontal gene transfer, the host bacteria of resistance genes and the migration of antibiotic resistance between environmental media. This review reveals the huge role of environmental migration in the spread of antibiotic resistance, and the environmental behavior of antibiotic resistance deserves wider attention. Meanwhile, extracellular antibiotic resistance genes and intracellular antibiotic resistance genes play different roles, so they should be studied separately.

Predictive assembling model reveals the self-adaptive elastic properties of lamellipodial actin networks for cell migration

Branched actin network supports cell migration through extracellular microenvironments. However, it is unknown how intracellular proteins adapt the elastic properties of the network to the highly varying extracellular resistance. Here we develop a three-dimensional assembling model to simulate the realistic self-assembling process of the network by encompassing intracellular proteins and their dynamic interactions. Combining this multiscale model with finite element method, we reveal that the network can not only sense the variation of extracellular resistance but also self-adapt its elastic properties through remodeling with intracellular proteins. Such resistance-adaptive elastic behaviours are versatile and essential in supporting cell migration through varying extracellular microenvironments. The bending deformation mechanism and anisotropic Poisson’s ratios determine why lamellipodia persistently evolve into sheet-like structures. Our predictions are confirmed by published experiments. The revealed self-adaptive elastic properties of the networks are also applicable to the endocytosis, phagocytosis, vesicle trafficking, intracellular pathogen transport and dendritic spine formation.

The importance of water and hydraulic pressure in cell dynamics.

All mammalian cells live in the aqueous medium, yet for many cell biologists, water is a passive arena in which proteins are the leading players that carry out essential biological functions. Recent studies, as well as decades of previous work, have accumulated evidence to show that this is not the complete picture. Active fluxes of water and solutes of water can play essential roles during cell shape changes, cell motility and tissue function, and can generate significant mechanical forces. Moreover, the extracellular resistance to water flow, known as the hydraulic resistance, and external hydraulic pressures are important mechanical modulators of cell polarization and motility. For the cell to maintain a consistent chemical environment in the cytoplasm, there must exist an intricate molecular system that actively controls the cell water content as well as the cytoplasmic ionic content. This system is difficult to study and poorly understood, but ramifications of which may impact all aspects of cell biology from growth to metabolism to development. In this Review, we describe how mammalian cells maintain the cytoplasmic water content and how water flows across the cell surface to drive cell movement. The roles of mechanical forces and hydraulic pressure during water movement are explored.

Removal of Intracellular and Extracellular Antibiotic Resistance Genes from Swine Wastewater by Sequential Electrocoagulation and Electro-Fenton Processes

Antibiotics are often used excessively in the livestock industry, and the effluent from livestock wastewater treatment is one of the main sources of antibiotic resistance genes (ARGs) in the enviro...

Membrane Fouling Inversely Impacts Intracellular and Extracellular Antibiotic Resistance Gene Abundances in the Effluent of an Anaerobic Membrane Bioreactor.

Anaerobic membrane bioreactors (AnMBRs) can significantly reduce the release of antibiotic resistance elements to the environment. The purpose of this study was to elucidate the role of membrane fouling layers (biofilms) in mitigating the release of intracellular and extracellular antibiotic resistance genes (iARGs and eARGs) from an AnMBR. The AnMBR was equipped with three membrane modules, each exhibiting a different level of fouling. Results showed that the absolute abundance of ARGs decreased gradually in the suspended biomass during operation of the AnMBR. Normalized abundances of targeted ARGs and intI1 were found to be significantly higher in the fouling layers compared to the suspended biomass, implying adsorption or an increased potential for horizontal gene transfer of ARGs in the biofilm. Effluent ARG data revealed that the highly fouled (HF) membrane significantly reduced the absolute abundance of eARGs. However, the HF membrane effluent concomitantly had the highest absolute abundance of iARGs. Nevertheless, total ARG abundance (sum of iARG and eARG) in the effluent of the AnMBR was not impacted by the extent of fouling. These results suggest a need for a combination of different treatment technologies to effectively prevent antibiotic resistance proliferation associated with these two ARG fractions.