Search Results (74)

Wastewater is a hotspot for the spread of antimicrobial resistance (AR); therefore, bacteriophages offer a promising biocontrol alternative to overcome the limitations of conventional disinfection. This study evaluated the efficacy of bacteriophages and cocktails for the biocontrol of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) (CG258 and ST307) and Escherichia coli producers of extended-spectrum β-lactamases (ESBL-Ec) (ST131) in simulated wastewater. A synthetic wastewater matrix was prepared in which bacterial viability and bacteriophage stability were assessed for 72 h. CR-Kp or ESBL-Ec strain were treated with individual bacteriophages or phage-cocktails (dosed in different ways) and bacterial loads were monitored for 54 h. The Klebsiella phages FKP3 and FKP14 eliminated 99% (−2.9 Log) of CR-Kp-CG258 at 54 h, and FKP10 reduced 99% (−2.15 Log) of the CR-Kp-ST307 strains. The Klebsiella phage-cocktail in a single dose reduced to 99.99% (−4.12 Log) of the CR-Kp-CG258 at 36 h. Coliphage FEC1 reduced to 2.12 Log (99%) of ESBL-Ec-bla_CTX-M-G9, and FEC2 and FEC4 reduced approximately 1 Log (90%) of ESBL-Ec-bla_CTX-M-G9 and bla_CTX-M-G1. The coliphage cocktail increased the reduction up to 2.2 Logarithms. This study provides evidence supporting the use of bacteriophage cocktails for the control of resistant bacteria in wastewater, a sustainable intervention to mitigate the spread of AR and support water reuse safety. Full article

The problem of the multidrug resistance of pathogenic bacteria is a serious concern, one which only becomes more pressing with every year that passes, motivating scientists to look for new therapeutic agents. In this situation, phage therapy, i.e., the use of phages to combat bacterial infections, is back in the spotlight of research interest. Bacterial viruses are highly strain-specific towards their hosts, which makes them particularly valuable for targeting pathogenic variants amidst non-pathogenic microflora, represented by such commensals of animals and humans as E. coli, S. aureus, etc. However, selecting phages for the treatment of bacterial infections is a complex task. The prospective candidates should meet a number of criteria; in particular, the selected phage must not contain potentially dangerous genes (e.g., antibiotic resistance genes, genes of toxins and virulence factors etc.)—or be capable of transferring them from their hosts. This work introduces a new approach to selecting T4-related coliphages; it allows one to identify strains which may be safer in terms of involvement in the horizontal gene transfer. The approach is based on the search for genes that reduce the frequency of genetic transduction. Full article

Flow virometry (FVM) offers a promising approach for monitoring viruses and virus-like particles (VLPs) in environmental samples. This study compares levels of non-specific VLPs across a wastewater treatment plant (WWTP) with levels of somatic coliphage, (F+) specific coliphage, Pepper Mild Mottle Virus (PMMoV), CrAssphage (CrAss), and Tomato Brown Rugose Fruit Virus (ToBRFV). All targets were quantified in influent, secondary-treated effluent, and tertiary-treated effluent at the University of California, Davis Wastewater Treatment Plant (UCDWWTP) over 11 weeks. We established an FVM-gating boundary for VLPs using bacteriophages T4 and ϕ6 as well as four phages isolated from wastewater. We then utilize T4 alongside three submicron beads as quality controls in the FVM assay. Coliphage was measured by standard plaque assays, and genome copies of PMMoV, CrAss, and ToBRFV were measured by digital droplet (dd)PCR. FVM results for wastewater revealed distinct microbial profiles at each treatment stage. However, correlations between VLPs and targeted viruses were poor. Trends for virus inactivation and removal, observed for targeted viruses during wastewater treatment, were consistent with expectations. Conversely, VLP counts were elevated in the WWTP effluent relative to the influent. Additional sampling revealed a decrease in VLP counts during the filtration treatment step following secondary treatment but a substantial increase in VLPs following ultraviolet disinfection. Defining application boundaries remain crucial to ensuring meaningful data interpretation as flow cytometry and virometry take on greater significance in water quality monitoring. Full article

Wastewater-based epidemiology (WBE) is a valuable tool for monitoring pathogen spread in communities; however, current protocols mainly target non-enveloped viruses. This study addresses the need for standardized methods to detect both enveloped and non-enveloped viruses by testing four aluminum hydroxide adsorption–precipitation techniques. Wastewater samples were spiked with an enveloped virus surrogate (Φ6 bacteriophage) and a non-enveloped virus surrogate (MS2 coliphage), and viral recovery was assessed using reverse-transcription quantitative PCR (RT-qPCR). The highest recovery for the enveloped virus was achieved with AlCl₃ at pH 3.5, a 15 min flocculation time, and a 3% elution solution concentration. For the non-enveloped virus, optimal recovery was found with AlCl₃ at pH 6.0, no flocculation time, and a 10% elution solution. The best method for recovering both virus types used AlCl₃ at pH 6.0, 15 min flocculation, and a 3% elution solution concentration. This study shows that while optimal conditions vary between virus types, a standardized AlCl₃ flocculation protocol can efficiently recover both, providing a cost-effective approach for outbreak monitoring in Grenada. Full article

Coliphage N4 is a representative species of the Schitoviridae family of bacteriophages. Originally structurally studied in 2008, the capsid structure was solved to 14 Å to reveal an interesting arrangement of Ig-like decoration proteins across the surface of the capsid. Herein, we present a high-resolution N4 structure, reporting a 2.45 Å map of the capsid obtained via single particle cryogenic-electron microscopy. Structural analysis of the major capsid proteins (MCPs) and decoration proteins (gp56 and gp17) of phage N4 reveals a pattern of interactions across the capsid that are mediated by structurally homologous domains of gp17. In this study, an analysis of the complex interface contacts allows us to confirm that the gp17 Ig-like decoration proteins of N4 are likely employed by the virus to increase the capsid’s structural integrity. Full article

Using Escherichia coli BW25113 as a host, we isolated a novel lytic phage from the commercial poly-specific therapeutic phage cocktail Sextaphage^® (Microgen, Russia). We provide genetic and phenotypic characterization of the phage and describe its host range on the ECOR collection of reference E. coli strains. The phage, hereafter named Sxt1, is a close relative of classical coliphage T3 and belongs to the Teetrevirus genus, yet its internal virion proteins, forming an ejectosome, differ from those of T3. In addition, the Sxt1 lateral tail fiber (LTF) protein clusters with those of the phages from the Berlinvirus genus. A comparison of T7, T3, and Sxt1 LTFs reveals the presence of insertions leading to the elongation of Sxt1 tail fibers, which, together with the difference in the HRDRs (host range-determining regions), might explain the expanded host specificity for the Sxt1. Full article

Escherichia coli and its bacteriophages are among the most studied model microorganisms. Bacteriophages for various E. coli strains can typically be easily isolated from environmental sources, and many of these viruses can be harnessed to combat E. coli infections in humans and animals. However, some relatively rare E. coli strains pose significant challenges in finding suitable phages. The uropathogenic strain E. coli UPEC124, isolated from a patient suffering from neurogenic bladder dysfunction, was found to be resistant to all coliphages in our collections, and initial attempts to isolate new phages failed. Using an improved procedure for phage enrichment, we isolated the N4-related phage Mimir124, belonging to the Gamaleyavirus genus, which was able to lyse this “difficult” E. coli strain. Although Mimir124 is a narrow-spectrum phage, it was effective in the individualized treatment of the patient, leading to pathogen eradication. The primary receptor of Mimir124 was the O antigen of the O101 type; consequently, Mimir124-resistant clones were rough (having lost the O antigen). These clones, however, gained sensitivity to some phages that recognize outer membrane proteins as receptors. Despite the presence of nine potential antiviral systems in the genome of the UPEC124 strain, the difficulty in finding effective phages was largely due to the efficient, non-specific cell surface protection provided by the O antigen. These results highlight the importance of an individualized approach to phage therapy, where narrow host-range phages—typically avoided in pre-fabricated phage cocktails—may be instrumental. Furthermore, this study illustrates how integrating genomic, structural, and functional insights can guide the development of innovative therapeutic strategies, paving the way for broader applications of phage therapy in combating multidrug-resistant bacterial pathogens. Full article

A conventional, group-specific PCR method was developed to identify each of the four previously defined major taxa (Myoviridae, Siphoviridae, Podoviridae and Microviridae) of somatic coliphages and used to classify isolates from sewage. Somatic coliphage infectivity detection, occurrence and survival in primary human sewage effluent was observed over time to further understand the presence and behavior of the groups of somatic coliphages at two environmental temperatures (4 and 25 °C). Over time, the taxonomic composition of the somatic coliphage population in sewage changed, with the Microviridae family becoming the most prevalent family in the sewage population after several weeks. Based on their persistence and prevalence in environmental waters, phages belonging to the Microviridae family provide supporting information on sewage contamination and possibly of human enteric viruses in sewage-contaminated water. Full article

With the aim of developing an innovative water treatment approach for developing countries in the Global South, we have applied the method of treating a cavitating water stream with a plasma discharge under real conditions. To this end, we have optimised the approach after investigating the effects that occur in the treated medium during such a treatment. Based on the obtained light absorption curves of treated model solutions of titanium oxysulphate and potassium bichromate, it was found that inside the reactor the main role in the destruction of chemical contaminants is played by hydroxide ions, while outside the reactor the main chemical interaction takes place with hydrogen peroxide. The plasma treatment unit was tested in the biological wastewater treatment plant of a health resort in the territory of the Russian Federation (Almetyevsk, Republic of Tatarstan). Water samples taken directly from the tertiary decantation tank were used as real wastewater samples instead of adding chemical reagents for disinfection. It was found that with different modes of operation of the plasma treatment plant, the concentration levels of coliform bacteria, coliphages and Escherichia coli decreased significantly and fell below the limit of permissible concentrations for wastewater discharge. At the same time, the possible effect of the plasma on persistent inorganic compounds was investigated. It was shown that the plasma discharge in the flow of the incoming liquid can almost completely destroy compounds that are difficult to remove, such as hydrogen sulphide and chlorides. In the course of the study, the optimum frequency of electrical pulses of 68 kHz was selected, which ensures the lowest consumption of electrical energy while maintaining the required efficiency. Full article

Background/Objectives. Bacteriophages have gained significant interest as a potential solution to combat harmful bacteria, especially in the fight against antimicrobial resistance. With the rise in drug-resistant microorganisms, the medical community is increasingly exploring new alternatives to traditional antibiotics, and bacteriophages offer several advantages in this regard. However, phage applications still face some challenges, such as host specificity. Methods. In this study, a somatic Siphovirus-like coliphage (SOM7) was tested for inhibiting the biofilm-forming capacity of the non-target strain Pseudomonas aeruginosa (ATTC 10145). The phage-sensitive strain E. coli WG5 was used as a control. The selected microorganisms were first tested for growth in the presence of SOM7 at three different concentrations (10⁵, 10⁷, and 10⁹ PFU/mL). Results. As expected, the phage-sensitive E. coli WG5 was fully inhibited by the coliphage, and no phage-related affection on the growth rate was observed for the SOM7-resistant P. aeruginosa. More notably, increasing concentrations of SOM7 significantly reduced both the biofilm-forming capacity and the amount of pre-established bacterial biofilm of the phage-insensitive P. aeruginosa (24.9% and 38.8% reduction in the biofilm-forming ability, and 18.8% and 28.0% biofilm degradation for 10⁷ PFU/mL and 10⁹ PFU/mL SOM7, respectively; p < 0.05). These results were supported by transmission electron microscopy (TEM) imaging, providing unprecedent evidence for the interaction of the somatic coliphage with the non-host strain. Conclusions. Although more studies in other biofilm models are necessary, our results show for the very first time that bacteriophages could potentially be used as an alternative to achieve desired anti-biofilm and biofilm-degrading activity in non-host bacterial strains. Full article

Climate change, unpredictable weather patterns, and droughts are depleting water resources in some parts of the globe, where recycling and reusing wastewater is a strategy for different purposes. To counteract this, the EU regulation for water reuse sets minimum requirements for the use of reclaimed water for agricultural irrigation, including a reduction in human enteric viruses. In the present study, the occurrence of several human enteric viruses, including the human norovirus genogroup I (HuNoV GI), HuNoV GII, and rotavirus (RV), along with viral fecal contamination indicator crAssphage was monitored by using (RT)-qPCR methods on influent wastewater and reclaimed water samples. Moreover, the level of somatic coliphages was also determined as a culturable viral indicator. To assess the potential viral infectivity, an optimization of a capsid integrity PMAxx-RT-qPCR method was performed on sewage samples. Somatic coliphages were present in 60% of the reclaimed water samples, indicating inefficient virus inactivation. Following PMAxx-RT-qPCR optimization, 66% of the samples tested positive for at least one of the analyzed enteric viruses, with concentrations ranging from 2.79 to 7.30 Log₁₀ genome copies (gc)/L. Overall, most of the analyzed reclaimed water samples did not comply with current EU legislation and contained potential infectious viral particles. Full article

This study investigates the influence of urban pollution and climate dynamics on water quality and the bacterial communities in an Argelian watershed. Twenty-one sampling campaigns were conducted over two years at six sites along the Oued Boussellam, a river impacted by the effluent of a sewage treatment plant, from a low-polluted site to a water reservoir within a 50 km distance. Fecal indicators and the human fecal marker crAssphage were monitored. Illumina 16S rRNA amplicon sequencing was used to assess water microbial populations’ changes. Urban sewage discharge had an impact on the river quality and microbial ecosystem, which was attenuated along the river course. Significant reductions (>4 log₁₀ for E. coli and somatic coliphages, >3 log₁₀ for crAssphage) occurred, particularly during high-temperature periods. crAssphage correlated strongly with somatic coliphages downstream the river. Seasonal differences were observed in the diversity of the bacterial communities, with higher values during the high-temperature period. The genus-level community structure was similar at highly polluted river sites, also displaying seasonal differences. Despite high pollution levels, natural processes reduced fecal indicators to acceptable levels in the reservoir as well as shaped the bacterial communities along the river, highlighting the importance of understanding indicator persistence and microbial community resilience for effective water quality management within the context of the global warming scenario. Full article

We conducted laboratory experiments under varied solar radiation and salinity levels to investigate their influences on the natural attenuation of multiple promising microbial indicators including fecal bacteria and two types of bacteriophages. Inactivation coefficients were estimated and compared following first-order kinetics. Somatic coliphage was found to be the most resistant, while fecal bacteria exhibited higher susceptibility to both factors. The estimated inactivation coefficients of E. coli were applied to a 3D water quality model and validated with a daily basis monitoring dataset. The validation revealed high consistency among modelled and monitored concentrations, with a less than 1-log concentration difference. Further, the effect of actual solar radiation and salinity on E. coli inactivation after a rainfall event was calculated and compared. The results exhibited that solar radiation is a stronger influential factor. Simulation illustrated that lower-strength radiation exposure can limit E. coli inactivation, enabling them to survive up to one week after combined sewer overflow (CSO) discharge. The model revealed a promising capacity as a tool for the timely prediction of the CSO-induced severity of microbial contamination and associated risk, as well as associated natural attenuation; thus, this model can enhance the competency of public water managers for decision making. Full article

In most Gram-negative bacteria, outer membrane (OM) lipopolysaccharide (LPS) molecules carry long polysaccharide chains known as the O antigens or O polysaccharides (OPS). The OPS structure varies highly from strain to strain, with more than 188 O serotypes described in E. coli. Although many bacteriophages recognize OPS as their primary receptors, these molecules can also screen OM proteins and other OM surface receptors from direct interaction with phage receptor-binding proteins (RBP). In this review, I analyze the body of evidence indicating that most of the E. coli OPS types robustly shield cells completely, preventing phage access to the OM surface. This shield not only blocks virulent phages but also restricts the acquisition of prophages. The available data suggest that OPS-mediated OM shielding is not merely one of many mechanisms of bacterial resistance to phages. Rather, it is an omnipresent factor significantly affecting the ecology, phage–host co-evolution and other related processes in E. coli and probably in many other species of Gram-negative bacteria. The phages, in turn, evolved multiple mechanisms to break through the OPS layer. These mechanisms rely on the phage RBPs recognizing the OPS or on using alternative receptors exposed above the OPS layer. The data allow one to forward the interpretation that, regardless of the type of receptors used, primary receptor recognition is always followed by the generation of a mechanical force driving the phage tail through the OPS layer. This force may be created by molecular motors of enzymatically active tail spikes or by virion structural re-arrangements at the moment of infection. Full article

Phages influence microbial communities, can be applied in phage therapy, or may serve as bioindicators, e.g., in (waste)water management. We here characterized the Escherichia phage vB_EcoS-EE09 isolated from an urban wastewater treatment plant effluent. Phage vB_EcoS-EE09 belongs to the genus Dhillonvirus, class Caudoviricetes. It has an icosahedral capsid with a long non-contractile tail and a dsDNA genome with an approximate size of 44 kb and a 54.6% GC content. Phage vB_EcoS-EE09 infected 12 out of the 17 E. coli strains tested. We identified 16 structural phage proteins, including the major capsid protein, in cell-free lysates by protein mass spectrometry. Comparative proteomics of protein extracts of infected E. coli cells revealed that proteins involved in amino acid and protein metabolism were more abundant in infected compared to non-infected cells. Among the proteins involved in the stress response, 74% were less abundant in the infected cultures compared to the non-infected controls, with six proteins showing significant less abundance. Repressing the expression of these proteins may be a phage strategy to evade host defense mechanisms. Our results contribute to diversifying phage collections, identifying structural proteins to enable better reliability in annotating taxonomically related phage genomes, and understanding phage–host interactions at the protein level. Full article