Prediction of key amino acids of Salmonella phage endolysin LysST-3 and detection of its mutants' activity.

Salmonella Typhimurium, a zoonotic pathogen, causes systemic and localized infection. The emergence of drug-resistant S. Typhimurium has increased; treating bacterial infections remains challenging. Phage endolysins derived from phages have a broader spectrum of bacteriolysis and better bacteriolytic activity than phages, and are less likely to induce drug resistance than antibiotics. LysST-3, the endolysin of Salmonella phage ST-3, was chosen in our study for its high lytic activity, broad cleavage spectrum, excellent bioactivity, and moderate safety profile. LysST-3 is a promising antimicrobial agent for inhibiting the development of drug resistance in Salmonella. The aim of this study is to investigate the molecular characteristics of LysST-3 through the prediction of key amino acid sites of LysST-3 and detection of its mutants' activity. We investigated its lytic effect on Salmonella and identified its key amino acid sites of interaction with substrate. LysST-3 may be a Ca2+, Mg2+ - dependent metalloenzyme. Its concave structure of the bottom "gripper" was found to be an important part of its amino acid active site. We identified its key sites (29P, 30T, 86D, 88 L, and 89 V) for substrate binding and activity using amino acid-targeted mutagenesis. Alterations in these sites did not affect protein secondary structure, but led to a significant reduction in the cleavage activity of the mutant proteins. Our study provides a basis for phage endolysin modification to target drug-resistant bacteria. Identifying the key amino acid site of the endolysin LysST-3 provides theoretical support for the functional modification of the endolysin and the development of subsequent effective therapeutic solutions.

New insight into soluble extracellular metabolites during sludge bulking process based on excitation-emission matrix spectroscopy and ultrahigh-performance liquid chromatography-mass spectrometry.

Soluble extracellular metabolites (SEM) produced by microorganisms might significantly change during sludge bulking, which is a major operational problem caused by the excessive growth of filamentous bacteria. However, knowledge remains limited about the dynamics and potential role of SEM in the bulking of sludge. In this study, filamentous bulking was simulated in a laboratory-scale reactor and changes to SEM characteristics during the bulking process were investigated using excitation-emission matrix spectroscopy and ultrahigh-performance liquid chromatography-mass spectrometry. SEM components changed significantly at different phases of sludge bulking. Changes in SEM were closely correlated with the structure of the bacterial community. Based on the EEM profiles, significant increases in fulvic acid-like and humic acid-like substances in SEM were observed with the development of filamentous bulking. The degree of humification in SEM showed a clear increasing trend. Untargeted extracellular metabolomic analysis showed that the intensity of berberine and isorhamnetin in SEM increased significantly during the bulking phase, which might synergistically facilitate the development of filamentous bulking.

Isolation and characterization of SGF3, a novel Microviridae phage infecting Shigella flexneri.

In the context of widespread bacterial contamination and the endless emergence of antibiotic-resistant bacteria, more effective ways to control pathogen infection are urgently needed. Phages become potential bactericidal agents due to their bactericidal specificity and not easy resistance to bacteria. But an important factor limiting its development is the lack of phage species. Therefore, the isolation of more new phages and studying their biological and genomic characteristics is of great significance for subsequent applications. So, in this study, SGF3, a Microviridae phage, which has shown lytic activity against Shigella flexneri, was isolated, purified, and characterized. Morphological and phylogenetic analyses identified it as a phiX174 species belonging to the Microviridae family. The latent period of phage SGF3 was 20 min, with an average burst size of approximately 7.1. Host spectrum experiments indicated its strong host specificity. Furthermore, the biofilm removal efficiency was increased by 20%-25% when SGF3 was coupled with other phages. In conclusion, the phage SGF3 found in this study was a lytic phage belonging to the Microviral family, and could be added as an auxiliary material in the phage cocktail. Studies of its characteristics and bactericidal properties had enriched the germplasm resources of microphages, provided more potential material in fighting against emerging and existing multidrug-resistant bacteria.

Expression and biological activity of lytic proteins HolST-3 and LysST-3 of Salmonella phage ST-3.

Salmonella infection is a major public health concern. Several strategies for Salmonella infection prevention and control are currently available including vaccines and antibiotics. However, vaccines are expensive and inefficient, and the use of antibiotics can lead to antibiotic resistance. Thus, alternative strategies for the treatment of Salmonella remain warrant. In this study, recombinant holin HolST-3 and lysin LysST-3 from Salmonella phage ST-3 were expressed and purified, and their bactericidal properties were analyzed. HolST-3 and LysST-3 possessed a wider lysis spectrum and more efficient bactericidal effect than phage ST-3, and a synergistic bactericidal effect was observed when combined in vitro. In addition, we explored the bactericidal properties of HolST-3 and LysST-3 in vivo using zebrafish as a model organism, and found that the bactericidal effects of both HolST-3 and LysST-3 in vivo were comparable to those of cefotaxime, an antibiotic. This study provides a basis for the development of HolST-3 and LysST-3 as novel bactericidal agents for the prevention and treatment of infectious diseases caused by Salmonella spp.

Activity of the lyases LysSSE1 and HolSSE1 against common pathogenic bacteria and their antimicrobial efficacy in biofilms.

Bacillary dysentery is a common foodborne disease with an exaggerated mortality rate because of Shigella infection. With the increasing severity of Shigella infection, lyase has been considered as the most promising alternative to antimicrobial agents, owing to the emergence of resistant bacteria and the difficulty in disrupting and eliminating bacterial biofilms. In this study, we cloned and characterised HolSSE1 and LysSSE1, holin, and lysozyme from the S. dysenteriae phage SSE1 with extended bacterial host range against common gram-negative and gram-positive bacteria. In addition, the efficacy of HolSSE1 and LysSSE1 in removing bacterial biofilms was observed on polystyrene surfaces. Moreover, synergistic bacteriostasis was observed when they were used together. Alignment and structural model analysis showed that both HolSSE1 and LysSSE1 are T4 phage proteins that have not yet been identified. Therefore, HolSSE1 and LysSSE1 can be promising biocontrol agents for the prevention and treatment of various pathogenic infections.

Bacteriophage ecology in biological wastewater treatment systems.

Biological wastewater treatment (BWT) is currently the most widely applied approach for treating wastewater. The performance of BWT systems depends on the complex microbial communities they support. Although bacteriophages (phages), which are the viruses that infect prokaryotes, are recognized as the most abundant life entities, understanding of their ecological roles in BWT systems remains limited. Here, we review recent progress in phage-associated researches in BWT systems, including the interactions between phage and host, polyvalent phages, the influence of phage activity on BWT performance, and the potential applications of phage-based control for sludge bulking/foaming and pathogens. The challenges and perspectives of phage ecology are also outlined, which are expected to provide implications for future research and applications.Key points• Phage-host interactions in BWT systems are summarized• Impacts of phage activities on BWT performance• Potential applications of phages in BWT systems.

Differences in nitrification and ammonium-oxidising prokaryotes in the process of wetland restoration.

Ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) are ammonium oxidising prokaryotes that can drive soil nitrification in wetlands. During the restoration of wetlands, different types of land use soils (agricultural soil [AS], restored wetland soil [RS], and natural wetland soil [NWS]) are present. However, studies on the effects of changes in the types of land use in wetlands during restoration on nitrification and the community composition of AOA and AOB are still not well understood. In this study, the differences in the potential nitrification rate (PNR) and community composition of AOA and AOB in AS, RS, and NWS were compared and discussed. The results indicated that the PNRs in the AS, RS, and NWS were on the same order of magnitude. Nitrification was mainly driven by AOB. High-throughput sequencing results showed that the genus Nitrososphaera of AOA and unclassified_o_Nitrosomonadales of AOB were only detected in the AS. Redundancy analysis (RDA) results indicated that the community composition of AOA was mostly influenced by pH, while TC was the most influential variable on the community composition of AOB. Our study provides a basis for distinguishing the roles of ammonium-oxidising prokaryotes in nitrification and further understanding the changes in nitrifying activity in wetlands during restoration.

Characterization and Genome Analysis of a Novel Salmonella Phage vB_SenS_SE1.

Salmonella is a significant food-borne pathogen that infects a large number of people worldwide. In this study, a lytic bacteriophage vB_SenS_SE1 capable of infecting Salmonella is isolated from municipal wastewater in Beijing, and its biological and genomic features are analyzed. Transmission electron micrograph shows that vB_SenS_SE1 is likely a Siphoviridae virus, with an icosahedral head and a long non-contracted tail. The stability test in vitro reveals that it is stable at 4-50 °C and pH 4-12. Based on the one-step growth curve, vB_SenS_SE1 has a 60-min exponential phase and a low burst size (19 PFU per cell). Bioinformatics analysis reveals that vB_SenS_SE1 consists of a circular, double-stranded DNA molecule of 40,987 bp with a GC content of 51.2%. Its genome carries 63 predicted open reading frames (orfs), with 22 orfs encoding known proteins. Phylogenetic analysis of the large terminase subunit shows that vB_SenS_SE1 exhibits strong homology to Salmonella phage St161, St162, VSiP, and FSL SP-031. The CoreGenes analysis shows that it is a member of the virus genus Cornellvirus. The features of phage vB_SenS_SE1 suggest that it has the potential to be an agent to control Salmonella.

Removal of Fluoride and Arsenic by a Hybrid Constructed Wetland System.

A pilot-scale hybrid wetland system was constructed for the removal of fluoride and arsenic from synthetic wastewater. After five months of operation, the fluoride and arsenic removal rate were at the value of 65 % and 90 %, respectively. Through calculation, the accumulation of fluoride in plants only accounted for 1.63 % of the accumulation in substrates, and the accumulation of arsenic in plants accounted for 3.3 % of that in substrates. Both the accumulation of fluoride and arsenic in plants were much higher in roots than that in leaves. And for substrates, the accumulation in the first layer was higher than the second layer. The changes of microbial community in the substrate of the wetland during the operation were also analyzed to investigate the effects of operating condition on the microbial community and to study the role of microorganism on the removal of fluoride and arsenic. The results showed that the relative abundance of Firmicutes reduced, while the relative abundance of Proteobacteria increased, indicating that the fluoride and arsenic in solution had a great influence on the microbial community. Findings of this study suggest that the hybrid constructed wetland system may be a promising process for the removal of fluoride and arsenic from synthetic wastewater.

Shifts of the nirS and nirK denitrifiers in different land use types and seasons in the Sanjiang Plain, China.

Denitrification is a key nitrogen removal process that involves many denitrifying bacteria. In this study, the denitrification performance was estimated for soil samples from different land use types including farmland soil, restored wetland soil, and wetland soil. The quantitative real-time polymerase chain reaction results showed that the average abundance of nirS and nirK genes was notably affected by seasonal changes, increasing from 2.34 × 10 6 and 2.81 × 10 6 to 1.97 × 10 6 and 4.55 × 10 6 gene copies/g of dry soil, respectively, from autumn to spring. This suggests that the abundance of nirS and nirK denitrifiers in spring is higher than those in autumn. Furthermore, the abundance of nirS and nirK genes was higher in the farmland soil than in restored wetland soil and wetland soil in both seasons. According to the analyses of MiSeq sequencing of nirS and nirK genes, Halobacteriaceae could be used as a special strain to distinguish wetland soil from farmland soil and restored wetland soil. Furthermore, redundancy analysis indicated that the soil environmental variables of total carbon, total nitrogen, moisture content, and organic matter were the main factors affecting the community structures of nirS and nirK denitrifiers existing in wetland soil. These findings could contribute to understanding the differences in nirS and nirK denitrifiers between different land use types during seasonal changes.

Phage-host associations in a full-scale activated sludge plant during sludge bulking.

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73 ml/g). Bulking sludges (SVI > 125 ml/g) taken about 10 months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of "Candidatus Microthrix parvicella" led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with "Candidatus Accumulibacter phosphatis," suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.

The complete genome sequence of PE3-1, a novel E. coli O153 phage.

A novel virulent phage PE3-1 against E. coli O153 was isolated from an aeration tank in a wastewater treatment plant. Transmission electron microscopy images showed that phage PE3-1 had an icosahedral head and a short tail, which revealed that it was a member of the family Podoviridae of the order Caudovirales. The complete PE3-1 genome consisted of 39,093 bp and was a linear double-stranded DNA with an average GC content of 49.93 %. Phage PE3-1 showed homology to the T7-like phages in 48 open reading frames (ORFs), but it differed from previously reported E .coli phages in morphology and bioinformatics analysis. This indicated that phage PE3-1 is a new member of the genus T7 virus.

Characteristics of hydrocarbon hydroxylase genes in a thermophilic aerobic biological system treating oily produced wastewater.

Alkane and aromatic hydroxylase genes in a full-scale aerobic system treating oily produced wastewater under thermophilic condition (45-50 °C) in the Jidong oilfield, China, were investigated using clone library and quantitative polymerase chain reaction methods. Rather than the normally encountered integral-membrane non-haem iron monooxygenase (alkB) genes, only CYP153-type P450 hydroxylase genes were detected for the alkane activation, indicating that the terminal oxidation of alkanes might be mainly mediated by the CYP153-type alkane hydroxylases in the thermophilic aerobic process. Most of the obtained CYP153 gene clones showed distant homology with the reference sequences, which might represent novel alkane hydroxylases. For the aromatic activation, the polycyclic aromatic hydrocarbon-ring hydroxylating dioxygenase (PAH-RHD) gene was derived from Gram-negative PAH-degraders belonging to the Burkholderiales order, with a 0.72% relative abundance of PAH-RHD gene to 16S rRNA gene. This was consistent with the result of 16S rRNA gene analysis, indicating that Burkholderiales bacteria might play a key role in the full-scale process of thermophilic hydrocarbon degradation.

Removal of fluoride and arsenic by pilot vertical-flow constructed wetlands using soil and coal cinder as substrate.

This study evaluated the performance of soil and coal cinder used as substrate in vertical-flow constructed wetlands for removal of fluoride and arsenic. Two duplicate pilot-scale artificial wetlands were set up, planted respectively with cannas, calamus and no plant as blank, fed with a synthetic sewage solution. Laboratory (batch) incubation experiments were also carried out separately to ascertain the fluoride and arsenic adsorption capacity of the two materials (i.e. soil and coal cinder). The results showed that both soil and coal cinder had quite high fluoride and arsenic adsorption capacity. The wetlands were operated for two months. The concentrations of fluoride and arsenic in the effluent of the blank wetlands were obviously higher than in the other wetlands planted with cannas and calamus. Fluoride and arsenic accumulation in the wetlands body at the end of the operation period was in range of 14.07-37.24% and 32.43-90.04%, respectively, as compared with the unused media.

Molecular analysis of long-term biofilm formation on PVC and cast iron surfaces in drinking water distribution system.

To understand the impacts of different plumbing materials on long-term biofilm formation in water supply system, we analyzed microbial community compositions in the bulk water and biofilms on faucets with two different materials-polyvinyl chloride (PVC) and cast iron, which have been frequently used for more than10 years. Pyrosequencing was employed to describe both bacterial and eukaryotic microbial compositions. Bacterial communities in the bulk water and biofilm samples were significantly different from each other. Specific bacterial populations colonized on the surface of different materials. Hyphomicrobia and corrosion associated bacteria, such as Acidithiobacillus spp., Aquabacterium spp., Limnobacter thiooxidans, and Thiocapsa spp., were the most dominant bacteria identified in the PVC and cast iron biofilms, respectively, suggesting that bacterial colonization on the material surfaces was selective. Mycobacteria and Legionella spp. were common potential pathogenic bacteria occurred in the biofilm samples, but their abundance was different in the two biofilm bacterial communities. In contrast, the biofilm samples showed more similar eukaryotic communities than the bulk water. Notably, potential pathogenic fungi, i.e., Aspergillus spp. and Candida parapsilosis, occurred in similar abundance in both biofilms. These results indicated that microbial community, especially bacterial composition was remarkably affected by the different pipe materials (PVC and cast iron).

Bacteriophage EPP-1, a potential antibiotic alternative for controlling edwardsiellosis caused by Edwardsiella piscicida while mitigating drug-resistant gene dissemination.

Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo (P = 0.0035), achieving an efficacy comparable to that of florfenicol (P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.

Integration of Lysin into Chitosan Nanoparticles for Improving Bacterial Biofilm Inhibition.

Bacterial biofilms (BBFs) exhibit high drug resistance, antiphagocytosis, and extremely strong adhesion, and therefore can cause various diseases. They are also one of the important causes of bacterial infections. Thus, the effective removal of BBFs has attracted considerable research interest. Endolysins, which are efficient antibacterial bioactive macromolecules, have recently been receiving increasing attention. In this study, we overcame the deficiencies of endolysins via immobilization on chitosan nanoparticles (CS-NPs) by preparing LysST-3-CS-NPs using the ionic cross-linking reaction between CS-NPs and LysST-3, an endolysin purified using phage ST-3 expression. The obtained LysST-3-CS-NPs were verified and thoroughly characterized, their antimicrobial activity was investigated using microscopy, and their antibacterial efficacy on polystyrene surfaces was studied. The results obtained suggested that LysST-3-CS-NPs exhibit enhanced bactericidal properties and increased stability and can serve as reliable biocontrol agents for the prevention and treatment of Salmonella biofilm infections.

Metagenome sequencing reveals shifts in phage-associated antibiotic resistance genes from influent to effluent in wastewater treatment plants.

Antibiotic resistance poses a significant threat to global health, and the microbe-rich activated sludge environment may contribute to the dissemination of antibiotic resistance genes (ARGs). ARGs spread across various bacterial populations via multiple dissemination routes, including horizontal gene transfer mediated by bacteriophages (phages). However, the potential role of phages in spreading ARGs in wastewater treatment systems remains unclear. This study characterized the core resistome, mobile genetic elements (MGEs), and virus-associated ARGs (vir_ARGs) in influents (Inf) and effluents (Eff) samples from nine WWTPs in eastern China. The abundance of ARGs in the Inf samples was higher than that in the Eff samples. A total of 21 core ARGs were identified, accounting for 38.70 %-83.70 % of the different samples. There was an increase in MGEs associated with phage-related processes from influents to effluents (from 12.68 % to 21.10 %). These MGEs showed strong correlations in relative abundance and composition with the core ARGs in the Eff samples. Across the Inf and Eff samples, 58 unique vir_ARGs were detected, with the Eff samples exhibiting higher diversity of vir_ARGs than the Inf samples. Statistical analyses indicated a robust relationship between core ARG profile, MGEs associated with phage-related processes, and vir_ARG composition in the Eff samples. Additionally, the co-occurrence of MGEs and ARGs in viral genomes was observed, ranging from 22.73 % to 68.75 %. This co-occurrence may exacerbate the persistence and spread of ARGs within WWTPs. The findings present new information on the changes in core ARGs, MGEs, and phage-associated ARGs from influents to effluents in WWTPs and provide new insights into the role of phage-associated ARGs in these systems.

Changes of putative pathogenic species within the water bacterial community in large-scale drinking water treatment and distribution systems.

Although the management of microbes in drinking water is of paramount importance for public health, there remain challenges in comprehensively examining pathogenic bacteria in the water supply system at the species level. In this study, high-throughput sequencing of nearly full-length 16S rRNA genes was performed to investigate the changes of the water bacterial community in three large-scale drinking water treatment plants (DWTPs) and their corresponding distribution systems during winter and summer. Our findings revealed significant differences in the bacterial community structure between winter and summer water samples for each DWTP and its distribution management area (DMA). In the groundwater-fed DWTP, selective enrichment of mycobacterial species was observed in both seasons, and the subsequent DMA also exhibited strong selection for specific mycobacterial species. In one of the surface water-fed DWTPs, certain Legionella species present in the source water in winter were selectively enriched in the bacterial community after pre-oxidation, although they were susceptible to the subsequent purification steps. A variety of putative pathogenic species (n = 83) were identified based on our pathogen identification pipeline, with the dominant species representing opportunistic pathogens commonly found in water supply systems. While pathogen removal primarily occurred during the purification processes of DWTPs, especially for surface water-fed plants, the relative abundance of pathogenic bacteria in the DMA water flora was lower than that in the DWTP effluent flora, indicating a diminished competitiveness of pathogens within the DMA ecosystem.

Sophoricoside ameliorates methicillin-resistant Staphylococcus aureus-induced acute lung injury by inhibiting Bach1/Akt pathway.

BACKGROUND: The lack of effective treatments for methicillin-resistant Staphylococcus aureus (MRSA) infection, which often leads to severe acute lung injury (ALI), poses a grave threat to human life. Sophoricoside (SOP), an isoflavone glycoside abundant in the fruit of traditional Chinese herbal Sophora japonica l., showed anti-inflammatory effects against atopic dermatitis, allergic inflammation, and lipopolysaccharide-induced ALI. However, its effect and underlying mechanism on MRSA-induced ALI remain unclear. PURPOSE: The aim of this study is to assess the protective effect of SOP in MRSA-induced ALI and elucidate its underlying molecular mechanisms. METHODS: In vivo experiments were conducted using wild-type mice to establish MRSA-induced ALI mouse model, and the effects of SOP on ALI were evaluated by hematoxylin-eosin staining, flow cytometry, quantitative real-time polymerase chain reaction, and several biochemical indicators. Adoptive transfer experiments and BTB and CNC homology 1 knockout (Bach1-/-) mice were also utilized in this study. In vitro studies employed murine macrophages RAW264.7 cells, primary bone marrow-derived macrophages (BMDMs), and primary lung macrophages to explore the underlying molecular mechanisms. RESULTS: The administration of SOP ameliorated MRSA-induced ALI by improving pulmonary histological damages, reducing neutrophil infiltration, suppressing oxidative stress levels, and decreasing the expression of inflammatory cytokines. In isolation experiments with ALI mouse lung macrophages and macrophage adoptive transfer experiments, SOP prevented macrophage activation, thereby reducing the production of proinflammatory cytokines. In vitro experiments demonstrated that SOP decreased the expression of inflammatory mediators in lipoteichoic acid (LTA)-stimulated RAW264.7 cells, BMDMs, and primary lung macrophages. Additionally, SOP inhibited protein kinase B (Akt) phosphorylation and treatment with MK2206-a specific inhibitor of Akt-eliminated SOP's ability to suppress LTA-stimulated macrophage inflammation. Furthermore, stimulation with LTA or MRSA up-regulated Bach1 expression; however, deletion of Bach1 abolished the inhibitory effect of SOP on p-Akt activation as well as inflammation and ALI development. CONCLUSION: This study provides the first evidence that SOP effectively mitigates MRSA-induced ALI via suppressing macrophage activation through the inhibition of Bach1/Akt pathway. These findings highlight the potential of SOP as a novel therapeutic agent for treating MRSA-induced ALI.