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.

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.

Biofilm-responsive encapsulated-phage coating for autonomous biofouling mitigation in water storage systems.

Biofilms in water storage systems may harbor pathogens that threaten public health. Chemical disinfectants are marginally effective in eradicating biofilms due to limited penetration, and often generate harmful disinfection byproducts. To enhance biofouling mitigation in household water storage tanks, we encapsulated bacteriophages (phages) in chitosan crosslinked with tri-polyphosphate and 3-glycidoxypropyltrimethoxysilane. Phages served as self-propagating green biocides that exclusively infect bacteria. This pH-responsive encapsulation (244 ± 11 nm) enabled autonomous release of phages in response to acidic pH associated with biofilms (corroborated by confocal microscopy with pH-indicator dye SNARF-4F), but otherwise remained stable in pH-neutral tap water for one month. Encapsulated phages instantly bind to plasma-treated plastic and fiberglass surfaces, providing a facile coating method that protects surfaces highly vulnerable to biofouling. Biofilm formation assays were conducted in tap water amended with 200 mg/L glucose to accelerate growth and attachment of Pseudomonas aeruginosa, an opportunistic pathogen commonly associated with biofilms in drinking water distribution and storage systems. Biofilms formation on plastic surfaces coated with encapsulated phages decreased to only 6.7 ± 0.2% (on a biomass basis) relative to the uncoated controls. Likewise, biofilm surface area coverage (4.8 ± 0.2 log CFU/mm2) and live/dead fluorescence ratio (1.80) were also lower than the controls (6.6 ± 0.2 log CFU/mm2 and live/dead ratio of 11.05). Overall, this study offers proof-of-concept of a chemical-free, easily implementable approach to control problematic biofilm-dwelling bacteria and highlights benefits of this bottom-up biofouling control approach that obviates the challenge of poor biofilm penetration by biocides.

Uncover the secret of the stability and interfacial Gibbs free energy of aerobic granular sludge.

Interfacial Gibbs free energy (IGFE) as a thermodynamic indicator characterize the stability of the natural system. For aerobic granular sludge (AGS), how IGFE determines the stability of sludge remains to be determined. The Gibbs free energy change at the AGS-water interface (ΔGswa) and AGS interfaces (ΔGsc) were selected as the main interfacial thermodynamic factors. Results indicated that the stable AGS was guaranteed with ΔGsc at the range of -31 to - 46 J m-2. Pearson correlation coefficients between ΔGswa/ ΔGsc and relative hydrophobicity, water content, SVI30, integrity coefficient were -0.9, 0.8, 0.85, and 0.84, which illustrated that the IGFE could be a more comprehensive thermodynamic indicator. Microbial community and EPS analysis verified the importance of denitrifiers, Amide III, protein-like substances for AGS stability. This work offers a new insight into the development of AGS stability based on IGFE.

Bacterial Concentrations and Water Turbulence Influence the Importance of Conjugation Versus Phage-Mediated Antibiotic Resistance Gene Transfer in Suspended Growth Systems.

Despite the abundance of phage-borne antibiotic resistance genes (ARGs) in the environment, the frequency of ARG propagation via phage-mediated transduction (relative to via conjugation) is poorly understood. We investigated the influence of bacterial concentration and water turbulence level [quantified as Reynold's number (Re)] in suspended growth systems on the frequency of ARG transfer by two mechanisms: delivery by a lysogenic phage (phage λ carrying gentamycin-resistance gene, genR) and conjugation mediated by the self-transmissible plasmid RP4. Using Escherichia coli (E. coli) as the recipient, phage delivery had a comparable frequency (1.2 ± 0.9 × 10-6) to that of conjugation (1.1 ± 0.9 × 10-6) in suspensions with low cell concentration (104 CFU/mL) and moderate turbulence (Re = 5 × 104). Turbulence affected cell (or phage)-to-cell contact rates and detachment (due to shear force), and thus, it affected the relative importance of conjugation versus phage delivery. At 107 CFU/mL, no significant difference was observed between the frequencies of ARG transfer by the two mechanisms under quiescent water conditions (2.8 ± 0.3 × 10-5 for conjugation vs 2.2 ± 0.5 × 10-5 for phage delivery, p = 0.19) or when Re reached 5 × 105 (3.4 ± 1.5 × 10-5 for conjugation vs 2.9 ± 1.0 × 10-5 for phage delivery, p = 0.52). Transcriptomic analysis of genes related to conjugation and phage delivery and simulation of cell (or phage)-to-cell collisions at different Re values corroborate that the importance of phage delivery relative to conjugation increases under either quiescent or turbulent conditions. This finding challenges the prevailing view that conjugation is the dominant ARG transfer mechanism and underscores the need to consider and mitigate potential ARG dissemination via transduction.

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.

Redistribution of intracellular and extracellular free & adsorbed antibiotic resistance genes through a wastewater treatment plant by an enhanced extracellular DNA extraction method with magnetic beads.

Due to the limitations of current extraction methods, extracellular DNA (eDNA) is rarely discerned from intracellular DNA (iDNA) despite having unique contributions to antibiotic resistance genes (ARGs) propagation. Furthermore, eDNA may be free (f-eDNA) or adsorbed to or suspended solids, including cells (a-eDNA), which affects ARG persistence and transmissivity. We developed a novel method using magnetic beads to separate iDNA, a-eDNA, and f-eDNA to assess how these physical states of ARGs change across a wastewater treatment plant. This method efficiently extracted eDNA (>85.3%) with higher recovery than current methods such as alcohol precipitation, CTAB-based extraction, and DNA extraction kits (<10%). Biological treatment and UV disinfection decreased the concentration of intracellular ARGs (iARGs) and adsorbed extracellular ARGs (a-eARGs), causing an increase of released free extracellular ARGs (f-eARGs). More ARGs were discharged through the wasted biosolids than in the effluent; iARGs and a-eARGs are prevalent in wasted biosolids ((73.9 ±â€¯22.5) % and (23.4 ±â€¯15.3) % of total ARGs respectively), while f-eARGs were prevalent in the effluent ((90.3 ±â€¯16.5) %). Bacterial community analysis showed significant correlations between specific genera and ARGs (e.g., Aeromonas, Pseudomonas and Acinetobacter were strongly correlated with multidrug-resistance gene blaTEM). This treatment system decreased the discharge of iARGs to receiving environments, however, increased eARG concentrations were present in the effluent, which may contribute to the environmental resistome.

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.

A rapid hydrophilic interaction liquid chromatographic determination of glimepiride in pharmaceutical formulations.

Glimepiride is one of the most widely prescribed antidiabetic drugs and contains both hydrophobic and hydrophilic functional groups in its molecules, and thus could be analyzed by either reversed-phase high performance liquid chromatography (HPLC) or hydrophilic interaction liquid chromatography (HILIC). In the literature, however, only reversed-phase HPLC has been reported. In this study, a simple, rapid and accurate hydrophilic interaction liquid chromatographic method was developed for the determination of glimepiride in pharmaceutical formulations. The analytical method comprised a fast ultrasound-assisted extraction with acetonitrile as a solvent followed by HILIC separation and quantification using a Waters Spherisorb S5NH2 hydrophilic column with a mobile phase consisting of acetonitrile and aqueous acetate buffer (5.0 mM). The retention time of glimepiride increased slightly with decrease of mobile phase pH value from 6.8 to 5.8 and of acetonitrile content from 60% to 40%, indicating that both hydrophilic, ionic, and hydrophobic interactions were involved in the HILIC retention and elution mechanisms. Quantitation was carried out with a mobile phase of 40% acetonitrile and 60% aqueous acetate buffer (5.0 mM) at pH 6.3, by relating the peak area of glimepiride to that of the internal standard, with a detection limit of 15.0 µg/L. UV light absorption responses at 228 nm were linear over a wide concentration range from 50.0 µg/L to 6.00 mg/L. The recoveries of the standard added to pharmaceutical tablet samples were 99.4-103.0% for glimepiride, and the relative standard deviation for the analyte was less than 1.0%. This method has been successfully applied to determine the glimepiride contents in pharmaceutical formulations.