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Bacteriophages offer an opportunity for chemical-free, precise control of problematic bacteria, but this approach can be limited when lytic phages are difficult to obtain for the target host. In such cases, phage-based targeting of cooperating or cross-feeding bacteria (e.g., Streptococcus gordonii) can be an effective approach to control the problematic bacteria (e.g., Porphyromonas gingivalis). Using a dual-species biofilm system, phage predation of S. gordonii (108 PFU·mL-1) decreased the abundance of pathogenic P. gingivalis by >99% compared with no-treatment controls, while also inhibiting the production of cytotoxic metabolic end products (butyric and propionic acids). Phage treatment upregulated genes associated with interspecies co-adhesion (5- to 8-fold) and quorum sensing (10-fold) in residual P. gingivalis, which is conducive to increased potential to bind to S. gordonii. Counterintuitively, lower-titer phage applications (104 PFU·mL-1) increased the production of extracellular polymeric substance (EPS) by 22% and biofilm biomass by 50%. This overproduction of EPS may contribute to the phenomenon where the biofilm separated into two distinct species layers, as observed by confocal laser scanning microscopy. Although more complex mixed-culture systems should be considered to delineate the merits and limitations of this novel biocontrol approach (which would likely require the use of phage cocktails), our results offer proof of concept that indirect phage-based targeting can expand the applicability of phage-based control of pathogenic bacteria for public health protection. IMPORTANCE: Lytic phages are valuable agents for targeted elimination of bacteria in diverse applications. Nevertheless, lytic phages are difficult to isolate for some target pathogens. We offer proof of concept that this limitation may be overcome via indirect phage targeting, which involves knocking out species that interact closely with and benefit the primary problematic target bacteria. Our target (P. gingivalis) only forms a periodontal pathogenic biofilm if the pioneer colonizer (S. gordonii) offers its surface for P. gingivalis to attach. Phage predation of the co-adhesive S. gordonii significantly reduced abundance of the target pathogen by >99%, decreased the total biofilm biomass by >44%, and suppressed its production of cytotoxic metabolic byproducts. Thus, this research extends the scope of phage-based biocontrol for public health protection.

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Introduction: Fusobacterium necrophorum, a human and animal pathogen, is the primary etiologic agent of bovine liver abscesses and a driving factor for prophylactic antibiotic use in the fed cattle industry. Considering calls to reduce agricultural antibiotic use, we isolated phages capable of killing F. necrophorum as an alternative or complementary biocontrol strategy. Methods: Six novel phages (φFN37, φRTG5, φKSUM, φHugo, φPaco, and φBB) were isolated from rumen fluid or ruminal F. necrophorum isolates and subjected to host range testing on both F. necrophorum subspecies. Four F. necrophorum subspecies, necrophorum phages, were tested for cross-resistance and host growth inhibition individually and in pairs. Additionally, genomic sequencing, annotation, and analysis were performed.s. Results: Four of six isolated phages were able to form lysogens, although all six contained lysogeny-related genes. φKSUM and φBB, did not form lysogens and were able to infect both subspecies. Four phages could infect F. necrophorum 8L1 (a liver abscess model challenge strain) in vitro. Genomic analysis showed that these phages belong to class Caudoviricetes with genome sizes ranging from 35 kbp to 111 kbp and GC values ranging from 26% to 36% and have extremely limited similarity to other deposited phage genomes infecting Fusobacterium or other genera. Conclusions: Although all phages isolated contained sequences bearing similarities to genes implicated in lysogeny, the four selected for use in cocktails showed potential in inhibiting host growth, with several demonstrating promising attributes for biocontrol and therapeutic applications. Phage cocktails that may offer enhanced antibacterial activity were also identified, indicating the potential of some lysogenic phages to be adapted for biocontrol or therapeutic purposes when lytic phages are difficult to obtain.

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Fusobacterium varium has been generally overlooked in cattle rumen microbiome studies relative to the presumably more abundant liver abscess-causing Fusobacterium necrophorum. However, F. varium was found to be more abundant in the rumen fluid of cattle and under culture conditions tailored to enrich F. necrophorum. Using near-full length 16S ribosomal ribonucleic acid sequencing, we demonstrate that F. varium grows under restrictive conditions commonly used to enumerate F. necrophorum, suggesting that previous F. necrophorum abundance assessment may have been inaccurate and that F. varium may be an underestimated member of the ruminal bacterial community. Fusobacterium varium were not as susceptible as F. necrophorum to in-feed antibiotics conventionally used in feedlots. Exposure to tylosin, the current gold standard for liver abscess reduction strategies in cattle, consistently hindered growth of the F. necrophorum strains tested by over 67% (P < 0.05) relative to the unexposed control. In contrast, F. varium strains were totally or highly resistant (0%-13% reduction in maximum yield, P < 0.05). Monensin, an ionophore antibiotic, had greater inhibitory activity against F. necrophorum than F. varium. Finally, preliminary genomic analysis of two F. varium isolates from the rumen revealed the presence of virulence genes related to those of pathogenic human F. varium isolates associated with active invasion of mammalian cells. The data presented here encourage further investigation into the ecological role of F. varium within the bovine rumen and potential role in liver abscess development, and proactive interventions.

The conventional method of liver abscess prevention in feedlot cattle is in-feed use of tylosin to target Fusobacterium necrophorum, which has been presumed to be the most common Fusobacterium species within the ruminal compartment. Our investigation into ruminal Fusobacterium, however, revealed a different species, Fusobacterium varium, to be abundant and ubiquitous in ruminal content samples. Furthermore, growth conditions tailored to enrich F. necrophorum consistently promoted growth of F. varium, and the bovine isolates tested had much lower susceptibilities to the commonly fed antibiotics tylosin and monensin compared to F. necrophorum. Fusobacterium varium is an emerging pathogen in humans and preliminary genome sequencing of two ruminal F. varium isolates revealed genes linked to pathogenicity. While the ecological role of F. varium in the rumen is still not fully understood, our findings draw attention to this pathogen and its potential implication in liver abscesses.

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The adverse health effects of phthalate esters (PAEs) and perfluoroalkyl substances (PFAS) in drinking water have attracted considerable attention. Our study investigated the effects of PAEs and PFAS on the bacterial community and the growth of potential human pathogenic bacteria in rural drinking water distribution systems. Our results showed that the total concentration of PAEs and PFAS ranged from 1.02 × 102 to 1.65 × 104 ng/L, from 4.40 to 1.84 × 102 ng/L in rural drinking water of China, respectively. PAEs concentration gradually increased and PFAS slowly decreased along the pipeline distribution, compared to concentrations in the effluents of rural drinking water treatment plants. The co-occurrence of higher concentrations of PAEs and PFAS changed the structure and function of the bacterial communities found within these environments. The bacterial community enhanced their ability to respond to fluctuating environmental conditions through up-regulation of functional genes related to extracellular signaling and interaction, as well as genes related to replication and repair. Under these conditions, co-occurrence of PAEs and PFAS promoted the growth of potential human pathogenic bacteria (HPB), therefore increasing the risk of the development of associated diseases among exposed persons. The main HPB observed in this study included Burkholderia mallei, Mycobacterium tuberculosis, Klebsiella pneumoniae, Acinetobacter calcoaceticus, Escherichia coli, and Pseudomonas aeruginosa. Contaminants including particles, microorganisms, PAEs and PFAS were found to be released from corrosion scales and deposits of pipes and taps, resulting in the increase of the cytotoxicity and microbial risk of rural tap water. These results are important to efforts to improve the safety of rural drinking water.

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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.

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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.

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Microorganisms in activated sludge are widely recognized for their roles in wastewater treatment. However, previous studies were mainly concerned with the diversity and driving factors of microbial communities within domestic wastewater treatment, and those of domestic wastewater treatment systems mixed with industrial wastewater are poorly understood. In this research, three different full-scale aerobic activated sludge (AS) wastewater treatment systems fed with municipal, textile-dyeing, and mixed wastewater, respectively, were monitored over the operation course of three months. 16S rRNA amplicon sequencing analysis revealed that the microbial communities in textile-dyeing wastewater activated sludge (AS) exhibited significantly lower richness and diversity (p < 0.01, Adonis) compared to those fed with municipal wastewater. In contrast, textile-dyeing derived AS selectively enriched microbial taxa with aromatic degradation and denitrification potentials. Further, FARPROTAX and metabolomics indicated the inhibition of 72.5% metabolic functions (p < 0.01) in AS from the system fed with textile-dyeing wastewater, including the pathways of pentose phosphate metabolism, purine metabolism, and glycerophospholipid metabolism. Overall, this study corroborates textile-dyeing wastewater is a novel microbial niche and could suppress sludge performance by inhibiting microbial activity and metabolism, raising concerns on AS-based systems for industrial wastewater treatment.

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BACKGROUND: Microbe-virus interactions have broad implications on the composition, function, and evolution of microbiomes. Elucidating the effects of environmental stresses on these interactions is critical to identify the ecological function of viral communities and understand microbiome environmental adaptation. Heavy metal-contaminated soils represent a relevant ecosystem to study the interplay between microbes, viruses, and environmental stressors. RESULTS: Metagenomic analysis revealed that Cr pollution adversely altered the abundance, diversity, and composition of viral and bacterial communities. Host-phage linkage based on CRISPR indicated that, in soils with high Cr contamination, the abundance of phages associated with heavy metal-tolerant hosts increased, as did the relative abundance of phages with broad host ranges (identified as host-phage linkages across genera), which would facilitate transfection and broader distribution of heavy metal resistance genes in the bacterial community. Examining variations along the pollutant gradient, enhanced mutualistic phage-bacterium interactions were observed in the face of greater environmental stresses. Specifically, the fractions of lysogens in bacterial communities (identified by integrase genes within bacterial genomes and prophage induction assay by mitomycin-C) were positively correlated with Cr contamination levels. Furthermore, viral genomic analysis demonstrated that lysogenic phages under higher Cr-induced stresses carried more auxiliary metabolic genes regulating microbial heavy metal detoxification. CONCLUSION: With the intensification of Cr-induced environmental stresses, the composition, replication strategy, and ecological function of the phage community all evolve alongside the bacterial community to adapt to extreme habitats. These result in a transformation of the phage-bacterium interaction from parasitism to mutualism in extreme environments and underscore the influential role of phages in bacterial adaptation to pollution-related stress and in related biogeochemical processes. Video Abstract.

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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.

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This study explores the prevalence, emission, and reduction of five ARGs (sulI, tetA, mphB, qnrD, and mcr-1) and integron (intI) through a distributed swine wastewater purification facility and the effluent-receiving environment. Typical metal resistance genes (MRGs), pathogenic bacterial indicators, the bacterial community, and wastewater properties were also explored to determine their effects on the fates of ARGs. Results indicated that the purification process could hardly effectively remove ARGs' prevalence. 3.1 × 104 -7.1 × 108  copies/L were present after purification, and 4%-57% of them persisted in the subsequent creek and adjacent soil. 16S rRNA sequencing suggested that the discharge of wastewater significantly changed the bacterial community in receiving creek and soil. Molecular ecological networks analysis detected the wide co-occurrence among ARGs, MRGs, and PBGs, which could further facilitate the propagation of antibiotic resistance. ARG incidence and specific bacterial genera were closely correlated, suggesting an extensive hosting relationship. Redundancy analyses showed wastewater organics and nutrients showed positive correlation to most ARGs' abundance, but negatively correlated to their relative abundance. PRACTITIONER POINTS: Fate of five ARGs and intI was studied in a swine wastewater treatment system. The treatment process could not effectively reduce ARGs' abundance. ARGs and pathogens in wastewater were transferred to the receiving creek and soil. The network analysis found wide co-occurrence among ARGs, metal resistance genes, and pathogens. Wastewater nutrients positively correlated to ARG's abundance but negatively correlated to their relative abundance.

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