Rapid Simulation of Decade-Scale Charcoal Aging in Soil: Changes in Physicochemical Properties and Their Environmental Implications.

In situ aging can change biochar properties, influencing their ecosystem benefits or risks over time. However, there is a lack of field verification of laboratory methods that attempt simulation of long-term natural aging of biochar. We exploited a decade-scale natural charcoal (a proxy for biochar) aging event to determine which lab-aging methods best mimicked field aging. We oxidized charcoal by ultraviolet A radiation (UVA), H2O2, or monochloramine (NH2Cl), and compared it to 10-year field-aged charcoal. We considered seven selected charcoal properties related to surface chemistry and organic matter release, and found that oxidation with 30% H2O2 most representatively simulated 10-year field aging for six out of seven properties. UVA aging failed to approximate oxidation levels while showing a distinctive dissolved organic carbon (DOC) release pattern. NH2Cl-aged charcoal was the most different, showing an increased persistent free radical (PFR) concentration and lower hydrophilicity. All lab oxidation techniques overpredicted polycyclic aromatic hydrocarbon release. The O/C ratio was well-correlated with DOC release, PFR concentration, surface charge, and charcoal pH, indicating the possibility to accurately predict biochar aging with a reduced suite of physicochemical properties. Overall, our rapid and verified lab-aging methods facilitate research toward derisking and enhancing long-term benefits of biochar application.

Enhanced Bacterium-Phage Symbiosis in Attached Microbial Aggregates on a Membrane Surface Facing Elevated Hydraulic Stress.

Phages are increasingly recognized for their importance in microbial aggregates, including their influence on microbial ecosystem services and biotechnology applications. However, the adaptive strategies and ecological functions of phages in different aggregates remain largely unexplored. Herein, we used membrane bioreactors to investigate bacterium-phage interactions and related microbial functions within suspended and attached microbial aggregates (SMA vs AMA). SMA and AMA represent distinct microbial habitats where bacterial communities display distinct patterns in terms of dominant species, keystone species, and bacterial networks. However, bacteria and phages in both aggregates exhibited high lysogenicity, with 60% lysogenic phages in the virome and 70% lysogenic metagenome-assembled genomes of bacteria. Moreover, substantial phages exhibited broad host ranges (34% in SMA and 42% in AMA) and closely interacted with habitat generalist species (43% in SMA and 49% in AMA) as adaptive strategies in stressful operation environments. Following a mutualistic pattern, phage-carried auxiliary metabolic genes (pAMGs; 238 types in total) presumably contributed to the bacterial survival and aggregate stability. The SMA-pAMGs were mainly associated with energy metabolism, while the AMA-pAMGs were mainly associated with antioxidant biosynthesis and the synthesis of extracellular polymeric substances, representing habitat-dependent patterns. Overall, this study advanced our understanding of phage adaptive strategies in microbial aggregate habitats and emphasized the importance of bacterium-phage symbiosis in the stability of microbial aggregates.

Characterization of a novel Vibrio parahaemolyticus host-phage pair and antibacterial effect against the host.

Vibrio parahaemolyticus is a widely recognized pathogen that has caused numerous outbreaks and is prevalent in the marine environment. In this study, we investigated the characteristics of the novel V. parahaemolyticus strain BTXS2 and its associated phage, VB_VpP_BT-1011, isolated from the Bohai Coast (Tianjin, China). Strain BTXS2 is a short coryneform bacterium with a terminal flagellum and is able to utilize and metabolize a wide variety of organic matter because of its unique carbon source utilization and enzyme activity. It grows well in medium between pH 5.0 and 9.0 and salinities of simulated freshwater, estuary water, and seawater (NaCl 0.5%-3%). Multiple antibiotic resistance genes and virulence genes that endanger human health were found in the BTXS2 genome. Phage VB_VpP_BT-1011, which infects BTXS2, is a 40,065-bp double-stranded DNA virus of the family Myoviridae with a latent time of 30 min and burst size of 24 PFU/cell. Like its host, the phage tolerates a broad range of environmental conditions (salinity, 0-3% NaCl; pH 5.0-9.0; temperature, 4-37°C). A host range test showed that the phage only infected and inhibited isolate BTXS2. In summary, we investigated a novel V. parahaemolyticus host-phage pair and the antibacterial effect of the phage on V. parahaemolyticus, providing insights into marine microbial ecology and risks.

Phage Predation Promotes Filamentous Bacterium Piscinibacter Colonization and Improves Structural and Hydraulic Stability of Microbial Aggregates.

Although bacteria-phage interactions have broad environmental applications and ecological implications, the influence of phage predation on bacterial aggregation and structural stability remains largely unexplored. Herein, we demonstrate that inefficient lytic phage predation can promote host filamentous bacterium Piscinibacter colonization onto non-host Thauera aggregates, improving the structural and hydraulic stability of the dual-species aggregates. Specifically, phage predation at 103-104 PFU/mL (i.e., multiplication of infection at 0.01-0.1) promoted initial Piscinibacter colonization by 10-15 folds and resulted in 29-31% higher abundance of Piscinibacter in the stabilized aggregates than that in the control aggregates without phage predation. Transcriptomic analysis revealed upregulated genes related to quorum sensing (by 15-92 folds) and polysaccharide secretion (by 10-90 folds) within the treated aggregates, which was consistent with 120-172% higher content of polysaccharides for the treated dual-species aggregates. Confocal laser scanning microscopic images further confirmed the increase of filamentous bacteria and polysaccharides (both with wider distribution) within the dual-species aggregates. Accordlingly, the aggregates' structural strength (via atomic force microscopes) and shear resistance (via hydraulic stress tests) increased by 77 and 42%, respectively, relative to the control group. In the long-term experiments, the enhanced hydraulic stability of the treated aggregates could facilitate dwelling bacteria propagation in flow-through conditions. Overall, our study demonstrates that phage predation can promote bacterial aggregation and enhance aggregate structural stability, revealing the beneficial role of lytic phage predation on bacterial symbiosis and environmental adaptivity.

Bacterial Inactivation and Biofilm Disruption through Indigenous Prophage Activation Using Low-Intensity Cold Atmospheric Plasma.

Biofilms can be pervasive and problematic in water treatment and distribution systems but are difficult to eradicate due to hindered penetration of antimicrobial chemicals. Here, we demonstrate that indigenous prophages activated by low-intensity plasma have the potential for efficient bacterial inactivation and biofilm disruption. Specifically, low-intensity plasma treatment (i.e., 35.20 W) elevated the intracellular oxidative reactive species (ROS) levels by 184%, resulting in the activation of prophage lambda (λ) within antibiotic-resistant Escherichia coli K-12 (lambda+) [E. coli (λ+)]. The phage activation efficiency was 6.50-fold higher than the conventional mitomycin C induction. Following a cascading effect, the activated phages were released upon the lysis of E. coli (λ+), which propagated further and lysed phage-susceptible E. coli K-12 (lambda-) [E. coli (λ-)] within the biofilm. Bacterial intracellular ROS analysis and ROS scavenger tests revealed the importance of plasma-generated ROS (e.g., •OH, 1O2, and •O2-) and associated intracellular oxidative stress on prophage activation. In a mixed-species biofilm on a permeable membrane surface, our "inside-out" strategy could inactivate total bacteria by 49% and increase the membrane flux by 4.33-fold. Furthermore, the metagenomic analysis revealed that the decrease in bacterial abundance was closely associated with the increase in phage levels. As a proof-of-concept, this is the first demonstration of indigenous prophage activations by low-intensity plasma for antibiotic-resistant bacterial inactivation and biofilm eradication, which opens up a new avenue for managing associated microbial problems.

Phthalate Esters Released from Plastics Promote Biofilm Formation and Chlorine Resistance.

Phthalate esters (PAEs) are commonly released from plastic pipes in some water distribution systems. Here, we show that exposure to a low concentration (1-10 µg/L) of three PAEs (dimethyl phthalate (DMP), di-n-hexyl phthalate (DnHP), and di-(2-ethylhexyl) phthalate (DEHP)) promotes Pseudomonas biofilm formation and resistance to free chlorine. At PAE concentrations ranging from 1 to 5 µg/L, genes coding for quorum sensing, extracellular polymeric substances excretion, and oxidative stress resistance were upregulated by 2.7- to 16.8-fold, 2.1- to 18.9-fold, and 1.6- to 9.9-fold, respectively. Accordingly, more biofilm matrix was produced and the polysaccharide and eDNA contents increased by 30.3-82.3 and 10.3-39.3%, respectively, relative to the unexposed controls. Confocal laser scanning microscopy showed that PAE exposure stimulated biofilm densification (volumetric fraction increased from 27.1 to 38.0-50.6%), which would hinder disinfectant diffusion. Biofilm densification was verified by atomic force microscopy, which measured an increase of elastic modulus by 2.0- to 3.2-fold. PAE exposure also stimulated the antioxidative system, with cell-normalized superoxide dismutase, catalase, and glutathione activities increasing by 1.8- to 3.0-fold, 1.0- to 2.0-fold, and 1.2- to 1.6-fold, respectively. This likely protected cells against oxidative damage by chlorine. Overall, we demonstrate that biofilm exposure to environmentally relevant levels of PAEs can upregulate molecular processes and physiologic changes that promote biofilm densification and antioxidative system expression, which enhance biofilm resistance to disinfectants.

Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity.

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control.

Renaissance for Phage-Based Bacterial Control.

Bacteriophages (phages) are an underutilized biological resource with vast potential for pathogen control and microbiome editing. Phage research and commercialization have increased rapidly in biomedical and agricultural industries, but adoption has been limited elsewhere. Nevertheless, converging advances in DNA sequencing, bioinformatics, microbial ecology, and synthetic biology are now poised to broaden phage applications beyond pathogen control toward the manipulation of microbial communities for defined functional improvements. Enhancements in sequencing combined with network analysis make it now feasible to identify and disrupt microbial associations to elicit desirable shifts in community structure or function, indirectly modulate species abundance, and target hub or keystone species to achieve broad functional shifts. Sequencing and bioinformatic advancements are also facilitating the use of temperate phages for safe gene delivery applications. Finally, integration of synthetic biology stands to create novel phage chassis and modular genetic components. While some fundamental, regulatory, and commercialization barriers to widespread phage use remain, many major challenges that have impeded the field now have workable solutions. Thus, a new dawn for phage-based (chemical-free) precise biocontrol and microbiome editing is on the horizon to enhance, suppress, or modulate microbial activities important for public health, food security, and more sustainable energy production and water reuse.

Chlorination (but Not UV Disinfection) Generates Cell Debris that Increases Extracellular Antibiotic Resistance Gene Transfer via Proximal Adsorption to Recipients and Upregulated Transformation Genes.

To advance the understanding of antibiotic resistance propagation from wastewater treatment plants, it is important to elucidate how different effluent disinfection processes affect the dissemination of predominantly extracellular antibiotic resistance genes (eARGs). Here, we show that, by facilitating proximal adsorption to recipient cells, bacterial debris generated by chlorination (but not by UV irradiation) increases the natural transformation frequency of their adsorbed eARG by 2.9 to 7.2-fold relative to free eARGs. This is because chlorination increases the bacterial surface roughness by 1.1 to 6.7-fold and the affinity toward eARGs by 1.6 to 5.8-fold, and 98% of the total eARGs released after chlorination were adsorbed to cell debris. In contrast, UV irradiation released predominantly free eARGs with 18% to 56% lower transformation frequency. The collision theory indicates that the ARG donor-recipient collision frequency increased by 35.1-fold for eARGs adsorbed onto chlorination-generated bacterial debris, and the xDLVO model infers a 29% lower donor-recipient contact energy barrier for these ARGs. Exposure to chlorination-generated bacterial debris also upregulated genes associated with natural transformation in Vibrio vulnificus (e.g., tfoX encoding the major activator of natural transformation) by 2.6 to 5.2-fold, likely due to the generation of chlorinated molecules (5.1-fold higher Cl content after chlorination) and persistent reactive species (e.g., carbon-centered radicals) on bacterial debris. Increased proximal eARG adsorption to bacterial debris was also observed in the secondary effluent after chlorination; this decreased eARG decay by 64% and increased the relative abundance of ARGs by 7.2-fold. Overall, this study highlights that different disinfection approaches can result in different physical states of eARGs that affect their resulting dissemination potential via transformation.

Aminoglycosides Antagonize Bacteriophage Proliferation, Attenuating Phage Suppression of Bacterial Growth, Biofilm Formation, and Antibiotic Resistance.

The common cooccurrence of antibiotics and phages in both natural and engineered environments underscores the need to understand their interactions and implications for bacterial control and antibiotic resistance propagation. Here, aminoglycoside antibiotics that inhibit protein synthesis (e.g., kanamycin and neomycin) impeded the replication of coliphage T3 and Bacillus phage BSP, reducing their infection efficiency and mitigating their hindrance of bacterial growth, biofilm formation, and tolerance to antibiotics. For example, treatment with phage T3 reduced subsequent biofilm formation by Escherichia coli liquid cultures to 53% ± 5% of that of the no-phage control, but a smaller reduction of biofilm formation (89% ± 10%) was observed for combined exposure to phage T3 and kanamycin. Despite sharing a similar mode of action with aminoglycosides (i.e., inhibiting protein synthesis) and antagonizing phage replication, albeit to a lesser degree, tetracyclines did not inhibit bacterial control by phages. Phage T3 combined with tetracycline showed higher suppression of biofilm formation than when combined with aminoglycosides (25% ± 6% of the no-phage control). The addition of phage T3 to E. coli suspensions with tetracycline also suppressed the development of tolerance to tetracycline. However, this suppression of antibiotic tolerance development disappeared when tetracycline was replaced with 3 mg/liter kanamycin, corroborating the greater antagonism with aminoglycosides. Overall, this study highlights this overlooked antagonistic effect on phage proliferation, which may attenuate phage suppression of bacterial growth, biofilm formation, antibiotic tolerance, and maintenance of antibiotic resistance genes. IMPORTANCE The coexistence of residual antibiotics and phages is common in many environments, which underscores the need to understand their interactive effects on bacteria and the implications for antibiotic resistance propagation. Here, aminoglycosides acting as bacterial protein synthesis inhibitors impeded the replication of various phages. This alleviated the suppressive effects of phages against bacterial growth and biofilm formation and diminished bacterial fitness costs that suppress the emergence of tolerance to antibiotics. We show that changes in bacteria caused by environmentally relevant concentrations of sublethal antibiotics can affect phage-host dynamics that are commonly overlooked in vitro but can result in unexpected environmental consequences.

Hitchhiking Behavior in Bacteriophages Facilitates Phage Infection and Enhances Carrier Bacteria Colonization.

Interactions between bacteriophages (phages) and biofilms remain poorly understood despite the broad implications for microbial ecology, water quality, and microbiome engineering. Here, we demonstrate that lytic coliphage PHH01 can hitchhike on carrier bacteria Bacillus cereus to facilitate its infection of host bacteria, Escherichia coli, in biofilms. Specifically, PHH01 could adsorb onto the flagella of B. cereus, and thus phage motility was increased, resulting in 4.36-fold more effective infection of E. coli in biofilm relative to free PHH01 alone. Moreover, phage infection mitigated interspecies competition and enhanced B. cereus colonization; the fraction of B. cereus in the final biofilm increased from 9% without phages to 43% with phages. The mutualistic relationship between the coliphage and carrier bacteria was substantiated by migration tests on an E. coli lawn: the conjugation of PHH01 and B. cereus enhanced B. cereus colonization by 6.54-fold compared to B. cereus alone (6.15 vs 0.94 cm2 in 24 h) and PHH01 migration by 5.15-fold compared to PHH01 alone (10.3 vs 2.0 mm in 24 h). Metagenomic and electron microscopic analysis revealed that the phages of diverse taxonomies and different morphologies could be adsorbed by the flagella of B. cereus, suggesting hitchhiking on flagellated bacteria might be a widespread strategy in aquatic phage populations. Overall, our study highlights that hitchhiking behavior in phages can facilitate phage infection of biofilm bacteria, promote carrier bacteria colonization, and thus significantly influence biofilm composition, which holds promise for mediating biofilm functions and moderating associated risks.

Going Viral: Emerging Opportunities for Phage-Based Bacterial Control in Water Treatment and Reuse.

Water security to protect human lives and support sustainable development is one of the greatest global challenges of this century. While a myriad of water pollutants can impact public health, the greatest threat arises from pathogenic bacteria that can be harbored in different components of water treatment, distribution, and reuse systems. Bacterial biofilms can also promote water infrastructure corrosion and biofouling, which substantially increase the cost and complexity of many critical operations. Conventional disinfection and microbial control approaches are often insufficient to keep up with the increasing complexity and renewed relevance of this pressing challenge. For example, common disinfectants cannot easily penetrate and eradicate biofilms, and are also relatively ineffective against resistant microorganisms. The use of chemical disinfectants is also curtailed by regulations aimed at minimizing the formation of harmful disinfection byproducts. Furthermore, disinfectants cannot be used to kill problematic bacteria in biological treatment processes without upsetting system performance. This underscores the need for novel, more precise, and more sustainable microbial control technologies. Bacteriophages (phages), which are viruses that exclusively infect bacteria, are the most abundant (and perhaps the most underutilized) biological resource on Earth, and hold great promise for targeting problematic bacteria. Although phages should not replace broad-spectrum disinfectants in drinking water treatment, they offer great potential for applications where selective targeting of problematic bacteria is warranted and antimicrobial chemicals are either relatively ineffective or their use would result in unintended detrimental consequences. Promising applications for phage-based biocontrol include selectively suppressing bulking and foaming bacteria that hinder activated sludge clarification, mitigating proliferation of antibiotic resistant strains in biological wastewater treatment systems where broad-spectrum antimicrobials would impair pollutant biodegradation, and complementing biofilm eradication efforts to delay corrosion and biofouling. Phages could also mitigate harmful cyanobacteria blooms that produce toxins in source waters, and could also serve as substitutes for the prophylactic use of antibiotics and biocides in animal agriculture to reduce their discharge to source waters and the associated selective pressure for resistant bacteria. Here, we consider the phage life cycle and its implications for bacterial control, and elaborate on the biochemical basis of such potential application niches in the water supply and reuse cycle. We also discuss potential technological barriers for phage-based bacterial control and suggest strategies and research needs to overcome them.

Hormetic Promotion of Biofilm Growth by Polyvalent Bacteriophages at Low Concentrations.

Interactions between bacteriophages (phages) and biofilms are poorly understood despite their broad ecological and water quality implications. Here, we report that biofilm exposure to lytic polyvalent phages at low concentrations (i.e., 102-104 phages/mL) can counterintuitively promote biofilm growth and densification (corroborated by confocal laser scanning microscopy (CLSM)). Such exposure hormetically upregulated quorum sensing genes (by 4.1- to 24.9-fold), polysaccharide production genes (by 3.7- to 9.3-fold), and curli synthesis genes (by 4.5- to 6.5-fold) in the biofilm-dwelling bacterial hosts (i.e., Escherichia coli and Pseudomonas aeruginosa) relative to unexposed controls. Accordingly, the biofilm matrix increased its polysaccharide and extracellular DNA content relative to unexposed controls (by 41.8 ± 2.3 and 81.4 ± 2.2%, respectively), which decreased biofilm permeability and increased structural integrity. This contributed to enhanced resistance to disinfection with chlorine (bacteria half-lives were 6.08 ± 0.05 vs 3.91 ± 0.03 min for unexposed controls) and to subsequent phage infection (biomass removal was 18.2 ± 1.2 vs 32.3 ± 1.2% for unexposed controls), apparently by mitigating diffusion of these antibacterial agents through the biofilm. Overall, low concentrations of phages reaching a biofilm may result in unintended biofilm stimulation, which might accelerate biofouling, biocorrosion, or other biofilm-related water quality problems.

Selective Adsorption and Photocatalytic Degradation of Extracellular Antibiotic Resistance Genes by Molecularly-Imprinted Graphitic Carbon Nitride.

There is a growing need to mitigate the discharge of extracellular antibiotic resistance genes (ARGs) from municipal wastewater treatment systems. Here, molecularly-imprinted graphitic carbon nitride (MIP-C3N4) nanosheets were synthesized for selective photocatalytic degradation of a plasmid-encoded ARG (blaNDM-1, coding for multidrug resistance New Delhi metallo-ß-lactamase-1) in secondary effluent. Molecular imprinting with guanine enhanced ARG adsorption, which improved the utilization of photogenerated oxidizing species to degrade blaNDM-1 rather than being scavenged by background nontarget constituents. Consequently, photocatalytic removal of blaNDM-1 in secondary effluent with MIP-C3N4 (k = 0.111 ± 0.028 min-1) was 37 times faster than with bare graphitic carbon nitride (k = 0.003 ± 0.001 min-1) under UVA irradiation (365 nm, 3.64 × 10-6 Einstein/L·s). MIP-C3N4 can efficiently catalyze the fragmentation of blaNDM-1, which decreased the potential for ARG repair by transformed bacteria. Molecular imprinting also changed the primary degradation pathway; electron holes (h+) were the predominant oxidizing species responsible for blaNDM-1 removal with MIP-C3N4 versus free radicals (i.e., ·OH and O2-) for coated but nonimprinted C3N4. Overall, MIP-C3N4 efficiently removed blaNDM-1 from secondary effluent, demonstrating the potential for molecular imprinting to enhance the selectivity and efficacy of photocatalytic processes to mitigate dissemination of antibiotic resistance from sewage treatment systems.

Pilot-Scale Pyrolytic Remediation of Crude-Oil-Contaminated Soil in a Continuously-Fed Reactor: Treatment Intensity Trade-Offs.

Pyrolytic treatment offers the potential for the rapid remediation of contaminated soils. However, soil fertility restoration can be highly variable, underscoring the need to understand how treatment conditions affect soil detoxification and the ability to support plant growth. We report here the first pilot-scale study of pyrolytic remediation of crude-oil-contaminated soil using a continuously fed rotary kiln reactor. Treatment at 420 °C with only 15 min of residence time resulted in high removal efficiencies for both total petroleum hydrocarbons (TPH) (99.9%) and polycyclic aromatic hydrocarbons (PAHs) (94.5%) and restored fertility to clean soil levels (i.e., Lactuca sativa biomass dry weight yield after 21 days increased from 3.0 ± 0.3 mg for contaminated soil to 8.8 ± 1.1 mg for treated soil, which is similar to 9.0 ± 0.7 mg for uncontaminated soil). Viability assays with a human bronchial epithelial cell line showed that pyrolytic treatment effectively achieved detoxification of contaminated soil extracts. As expected, TPH and PAH removal efficiencies increased with increasing treatment intensity (i.e., higher temperatures and longer residence times). However, higher treatment intensities decreased soil fertility, suggesting that there is an optimal system-specific intensity for fertility restoration. Overall, this study highlights trade-offs between pyrolytic treatment intensity, hydrocarbon removal efficiency, and fertility restoration while informing the design, optimization, and operation of large-scale pyrolytic systems to efficiently remediate crude-oil-contaminated soils.

Bacterial Endospores as Phage Genome Carriers and Protective Shells.

Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.

Easily Recoverable, Micrometer-Sized TiO2 Hierarchical Spheres Decorated with Cyclodextrin for Enhanced Photocatalytic Degradation of Organic Micropollutants.

Micrometer-sized titanium dioxide hierarchical spheres (TiO2-HS) were assembled from nanosheets to address two common limitations of photocatalytic water treatment: (1) inefficiency associated with scavenging of oxidation capacity by nontarget water constituents and (2) energy-intensive separation and recovery of the photocatalyst slurry. These micrometer-sized spheres are amenable to low-energy separation, and over 99% were recaptured from both batch and continuous flow reactors using microfiltration. Using nanosheets as building blocks resulted in a large specific surface area-3 times larger than that of commercially available TiO2 powder (Evonik P25). Anchoring food-grade cyclodextrin onto TiO2-HS (i.e., CD-TiO2-HS) provided hydrophobic cavities to entrap organic contaminants for more effective utilization of photocatalytically generated reactive oxygen species. CD-TiO2-HS removed over 99% of various contaminants with dissimilar hydrophobicity (i.e., bisphenol A, bisphenol S, 2-naphthol, and 2,4-dichlorophenol) within 2 h under a low-intensity UVA input (3.64 × 10-6 einstein/L/s). As with other catalyst (including TiO2 slurry), periodic replacement or replenishment would be needed to maintain high treatment efficiency (e.g., we demonstrate full reactivation through simple reanchoring of CD). Nevertheless, this task would be offset by significant savings in photocatalyst separation. Thus, CD-TiO2-HS is an attractive candidate for photocatalytic water and wastewater treatment of recalcitrant organic pollutants.

Can single disease payment system based on clinical pathway reduce hospitalization costs in rural area? A case study of uterine leiomyoma in Anhui, China.

BACKGROUND: Single disease payment program based on clinical pathway (CP-based SDP) plays an increasingly important role in reducing health expenditure in china and there is a clear need to explore the scheme from different perspectives. This study aimed at evaluating the effect of the scheme in rural county public hospitals within Anhui, a typical province of China,using uterine leiomyoma as an example. METHODS: The study data were extracted from the data platform of the New Rural Cooperative Medical Office of Anhui Province using stratified-random sampling. Means, constituent ratios and coefficients of variations were calculated and/or compared between control versus experiment groups and between different years. RESULTS: The total hospitalization expenditure (per-time) dropped from 919.08 ± 274.92 USD to 834.91 ± 225.29 USD and length of hospital stay reduced from 9.96 ± 2.39 days to 8.83 ± 1.95 days(P < 0.01), after CP-based SDP had implemented. The yearly total hospitalization expenditure manifested an atypical U-shaped trend. Medicine expense, nursing expense, assay cost and treatment cost reduced; while the fee of operation and examination increased (P < 0.05). The expense constituent ratios of medicine, assay and treatment decreased with the medicine expense dropped the most (by 4.4%). The expense constituent ratios of materials, ward, operation, examination and anesthetic increased,with the examination fee elevated the most (by 3.9%).The coefficient of variation(CVs) of treatment cost declined the most (- 0.360); while the CV of materials expense increased the most (0.186). CONCLUSION: There existed huge discrepancies in inpatient care for uterine leiomyoma patients. Implementation of CP-based SDP can help not only in controlling hospitalization costs of uterine leiomyoma in county-level hospitals but also in standardizing the diagnosis and treatment procedures.

Suppression of Enteric Bacteria by Bacteriophages: Importance of Phage Polyvalence in the Presence of Soil Bacteria.

Bacteriophages are widely recognized for their importance in microbial ecology and bacterial control. However, little is known about how phage polyvalence (i.e., broad host range) affects bacterial suppression and interspecies competition in environments harboring enteric pathogens and soil bacteria. Here we compare the efficacy of polyvalent phage PEf1 versus coliphage T4 in suppressing a model enteric bacterium (E. coli K-12) in mixtures with soil bacteria (Pseudomonas putida F1 and Bacillus subtilis 168). Although T4 was more effective than PEf1 in infecting E. coli K-12 in pure cultures, PEf1 was 20-fold more effective in suppressing E. coli under simulated multispecies biofilm conditions because polyvalence enhanced PEf1 propagation in P. putida. In contrast, soil bacteria do not propagate coliphages and hindered T4 diffusion through the biofilm. Similar tests were also conducted under planktonic conditions to discern how interspecies competition contributes to E. coli suppression without the confounding effects of restricted phage diffusion. Significant synergistic suppression was observed by the combined effects of phages plus competing bacteria. T4 was slightly more effective in suppressing E. coli in these planktonic mixed cultures, even though PEf1 reached higher concentrations by reproducing also in P. putida (7.2 ± 0.4 vs 6.0 ± 1.0 log10PFU/mL). Apparently, enhanced suppression by higher PEf1 propagation was offset by P. putida lysis, which decreased stress from interspecies competition relative to incubations with T4. In similar planktonic tests with more competing soil bacteria species, P. putida lysis was less critical in mitigating interspecies competition and PEf1 eliminated E. coli faster than T4 (36 vs 42 h). Overall, this study shows that polyvalent phages can propagate in soil bacteria and significantly enhance suppression of co-occurring enteric species.