Fraction Of Phage Research Articles

Bacteriophages from faecal contamination are an important reservoir for AMR in aquatic environments

Bacteriophages have been shown to play an important role in harbouring and propagating antibiotic resistance genes (ARGs). Faecal matter contains high levels of phages, suggesting that faecal contamination of water bodies may lead to increased antimicrobial resistance (AMR) levels due to increased phage loading in aquatic environments. In this study, we assessed whether faecal pollution of three rivers (Rivers Liffey, Tolka, and Dodder) was responsible for increased levels of ARGs in phage particles using established phage-faecal markers, focusing on four ARGs (blaTEM, tet(O), qnrS, and sul1). We observed all four ARGs in phage fractions in all three rivers, with ARGs more frequently observed in agricultural and urban sampling sites compared to their source. These findings highlight the role of faecal pollution in environmental AMR and the impact of agricultural and urban activities on water quality. Furthermore, our results suggest the importance of including phages as indicators when assessing environmental AMR, as they serve as significant reservoirs of resistance genes in aquatic environments. This study provides important insights into the role of faecal pollution and phages in the prevalence of AMR in the environment and the need for their inclusion in future studies to provide a comprehensive understanding of environmental AMR.

Antibiotic resistance genes in bacteriophages from wastewater treatment plant and hospital wastewaters

Antibiotic resistant bacteria (ARB) are a major health risk caused particularly by anthropogenic activities. Acquisition of antibiotic resistances by bacteria is known to have happened before the discovery of antibiotics and can occur through different routes. Bacteriophages are thought to have an important contribution to the dissemination of antibiotic resistance genes (ARGs) in the environment. In this study, seven ARGs (blaTEM, blaSHV, blaCTX-M, blaCMY, mecA, vanA, and mcr-1) were investigated, in the bacteriophage fraction, in raw urban and hospital wastewaters. The genes were quantified in 58 raw wastewater samples collected at five WWTPs (n = 38) and hospitals (n = 20). All genes were detected in the phage DNA fraction, with the bla genes found in higher frequency. On the other hand, mecA and mcr-1 were the least frequently detected genes. Concentrations varied between 102 copies/L and 106 copies/L. The gene coding for the resistance to colistin (mcr-1), a last-resort antibiotic for the treatment of multidrug-resistant Gram-negative infections, was identified in raw urban and hospital wastewaters with positivity rates of 19 % and 10 %, respectively. ARGs patterns varied between hospital and raw urban wastewaters, and within hospitals and WWTP. This study suggests that phages are reservoirs of ARGs, and that ARGs (with particularly emphasis on resistance to colistin and vancomycin) in the phage fraction are already widely widespread in the environment with potential large implications for public health.

Prevalence of bacterial genes in the phage fraction of food viromes

Antibiotic resistance genes (ARGs) have been identified in viral DNA isolated from different kinds of food, but little is known about their origin. In this study, twenty-one viromes were analyzed from samples of food previously reported to carry ARGs, including meat (poultry, veal, and pork), fish (Mediterranean, Atlantic, frozen, farmed and shellfish) and vegetables (lettuce, cucumber, and spinach). Classification of the contigs by Kraken revealed a large percentage of unclassified contigs (43.7–98.2%) in all the viromes. Only 0.05–7.1% of the contigs were identified as viral and of these, more than 91% belonged to different bacteriophage families, Podophages and Siphophages being the most prevalent. According to VirSorter, the largest number of viral contigs were derived from viromes of shellfish, followed by spinach. Spinach viromes also included the largest number of phage sequences identified by PHASTER. The abundant presence of bacterial genes in the viromes, including 16S rRNA genes, was attributed to the phage packaging of the bacterial genome fragments, as no bacterial DNA was found outside the viral capsids. The detection of 16S rRNA genes in the different viromes allowed diverse phage bacterial hosts to be identified. The three major functional groups of genes determined were related to metabolism, detoxification/resistance, and above all, biosynthesis. Various ARGs were quantified in the viromes by qPCR, the most prevalent being β-lactamases, particularly blaTEM. Analysis of ARG diversity in the viromes by Prokka and CARD revealed various resistance-related genes, whereas a more restrictive search by ResFinder identified blaTEM in all the food viromes, blaOXA in Atlantic fish-1 and spinach-2, oqxB in lettuce-1, and dfr in spinach-2. The presence of ARGs in the food viromes points to bacterial DNA mobilization by transduction mechanisms. Transduction of resistances by phage particles may therefore contribute to the emergence of resistant strains along the food chain and should be monitored.

Using the forces of hydrodynamic countercurrent chromatography for the study of bacteriophages.

Bacteriophages (phages) are viruses that target bacteria, with the ability to lyse and kill host bacterial cells. Due to this, they have been of some interest as a therapeutic since their discovery in the early 1900s, but with the recent increase in antibiotic resistance, phages have seen a resurgence in attention. Current methods of isolation and purification of phages can be long and tedious, with caesium chloride concentration gradients the gold standard for purifying a phage fraction. Isolation of novel phages requires centrifugation and ultrafiltration of mixed samples, such as water sources, effluent or faecal samples etc, to prepare phage filtrates for further testing. We propose countercurrent chromatography as a novel and alternative approach to use when studying phages, as a scalable and high-yield method for obtaining phage fractions. However, the full extent of the usefulness and resolution of separation with this technique has not been researched; it requires optimization and ample testing before this can be revealed. Here we present an initial study to determine survivability of two phages, T4 and ϕX174, using only water as a mobile phase in a Spectrum Series 20 HPCCC. Both phages were found to remain active once eluted from the column. Phages do not fully elute from the column and sodium hydroxide is necessary to flush the column between runs to deactivate remaining phages.

Nano-etched fused-silica capillary used for on-line preconcentration and electrophoretic separation of bacteriophages from large blood sample volumes with off-line MALDI-TOF mass spectrometry identification.

The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10μL of the prepared sample at the optimized flow rate of 6.5μLmin-1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 =0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (±) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract.

β-lactam resistance genes in bacteriophage and bacterial DNA from wastewater, river water, and irrigation water in Washington State

Our objective was to determine whether β-lactamase genes are carried within bacteriophage capsids, as a first step towards exploring the possible role of bacteriophages as vehicles for dispersal of antimicrobial resistance genes through an agricultural region of Washington State. Water samples (n = 178) from municipal wastewater treatment plants, river and irrigation canals were collected over a period of eight months. The occurrence of four β-lactam resistance gene groups (blaTEM, blaCTX-M, blaPSE and blaCMY-2) and three carbapenem resistance genes (blaKPC, blaOXA-48-like, and blaNDM) in bacterial and phage fractions of water samples was evaluated by PCR. All of the seven targeted resistance genes were detected both in wastewater and river water samples. Relatively high proportions of samples (7.3%–64.9%) positive for resistance genes were found in bacteriophage fractions of water samples compared to the bacterial fractions (5.4%–36.8%). blaOXA-48-like (57.3%) and blaTEM (64.0%) were the most prevalent antimicrobial resistance genes detected at all the sampling points. Resistance genes are commonly present in treated wastewater flowing through municipal and agricultural environments, indicating a plausible role for this water in the dissemination of antimicrobial resistance traits, including blaCTX-M.

Presence & mobility of antimicrobial resistance in Gram-negative bacteria from environmental samples in coastal Karnataka, India.

To understand antimicrobial resistance (AMR) patterns and mechanisms of horizontal gene transfer in human-associated environments is essential to AMR surveillance. Gram-negative bacteria (1122 isolates) from food-animal environments were characterized for antimicrobial susceptibility and AMR genes. Seventy five per cent of the isolates (837 of 1122) were resistant to at least one of the antibiotics tested. Resistance to more than three groups of antimicrobials (multidrug resistance) was observed in 43 isolates with most often encountered (12 of 43) resistance to β-lactams, tetracycline, quinolones and nitrofurantoin. The profile of frequently reported plasmid-mediated resistance gene in these isolates was determined. The mobility of these elements as plasmids or phages was examined. The blaCTX-M gene was present in the plasmid of 61 per cent and packed in induced phage fractions in 72 per cent of the isolates and blaTEM in 69 per cent phage fractions compared to 15 per cent presence in the plasmid.

Antibiotic resistance genes in phage particles isolated from human faeces and induced from clinical bacterial isolates

Phage particles have emerged as elements with the potential to mobilise antibiotic resistance genes (ARGs) in different environments, including the intestinal habitat. This study aimed to determine the occurrence of ARGs in phage particles present in faecal matter and induced from strains isolated from faeces. Nine ARGs (blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, blaOXA-48, qnrA, qnrS, mecA, sul1 and armA) were quantified by qPCR in the phage DNA fractions of 150 faecal samples obtained from healthy individuals who had not received antibiotic treatment or travelled abroad in the 3 months prior to sample collection. On the suspicion that the detected particles originated from bacterial flora, 82 Escherichia coli and Klebsiella pneumoniae isolates possessing at least one identified ARG (blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, armA, qnrA, qnrS and sul1) were isolated and their capacity to produce phage particles carrying these ARGs following induction was evaluated. Of 150 samples, 72.7% were positive for at least one ARG, with blaTEM and blaCTX-M-9 group being the most prevalent and abundant. Of the 82 isolates, 51 (62%) showed an increase in the number of copies of the respective ARG in the phage fraction following induction, with blaTEM, blaCTX-M-1 group, blaCTX-M-9 group and sul1 being the most abundant. Phages induced from the isolates were further purified and visualised using microscopy and their DNA showed ARG levels of up to 1010 gene copies/mL. This study highlights the abundance of phage particles harbouring ARGs and indicates that bacterial strains in the intestinal habitat could be source of these particles.

Contribution of bacteriophage and plasmid DNA to the mobilization of antibiotic resistance genes in a river receiving treated wastewater discharges

In this study, we quantified eleven antibiotic compounds and nine antibiotic resistance genes (ARGs) in water samples collected upstream and downstream of the discharge point from a municipal wastewater treatment plant (WWTP) into the Ter River. Antibiotics were analyzed by liquid chromatography coupled to mass spectrometry, whereas the concentration of ARGs in bacterial, phage and plasmid DNA fractions was determined by real-time PCR to explore their contribution to environmental antibiotic resistance. WWTP discharges resulted in higher concentrations of antibiotic residues as well as ARGs in water samples collected downstream the impact point. Specifically, genes conferring resistance to macrolides (ermB), fluoroquinolones (qnrS) and tetracyclines (tetW) showed significant differences (p<0.05) between upstream and downstream sites in the three DNA fractions (i.e. bacteria, plasmids and phages). Interestingly, genes conferring resistance to β-lactams (blaTEM, blaNDM and blaKPC) and glycopeptides (vanA) only showed significant differences (p<0.05) between upstream and downstream sites in phage and plasmid DNA but not in the bacterial DNA fraction. Our results show for the first time the extent to which phages and plasmids contribute to the mobilization of ARGs in an aquatic environment exposed to chronic antibiotic pollution via WWTP discharges. Accordingly, these mobile genetic elements should be included in further studies to get a global view of the spread of antibiotic resistance.

The occurrence of antibiotic resistance genes in a Mediterranean river and their persistence in the riverbed sediment

The spread of antibiotic resistance genes (ARGs) in the environment is a serious concern. Bacterial ARGs can spread via different mobile genetic elements as phage particles, which thereby emerge as novel vectors for environmental dissemination. To assess how climate events, such as heavy rains or water scarcity, could affect the spread of ARGs, it is necessary to know their prevalence and abundance in aquatic environments as well as the potential reservoirs from which they could become mobile. This study evaluates the occurrence of ARGs in the water and sediment of a Mediterranean river. Six clinically relevant ARGs (blaTEM, blaCTX-M, qnrA, qnrS, mecA and sul1) were quantified by qPCR in the bacterial and phage fractions of 69 water and 70 sediment samples from the River Llobregat (NE Spain), collected during both dry and rainy periods. blaTEM and sul1 were the most prevalent and abundant ARGs; the others were more variable. Significant seasonal differences in ARG prevalences and abundances were observed. Since ARGs were detected in the sediment, the persistence of the most abundant ARGs naturally occurring in that sediment (blaTEM and sul1) was evaluated under three conditions. No ARG inactivation occurred in fresh sediment over 14 days; while the ARGs declined by less than 2 log10 units over 35 days in semi-dry and dry sediment. The occurrence of ARGs in water and sediment is influenced by seasonal conditions and they can be mobilized by bacteria and phage particles. In sediment, ARGs persist for long periods and hence sediment can be a natural reservoir of ARGs, from where they can spread and cause the emergence of new resistant strains.

The use of bacteriophages to biocontrol oral biofilms

Infections induced by oral biofilms include caries, as well as periodontal, and peri-implant disease, and may influence quality of life, systemic health, and expenditure. As bacterial biofilms are highly resistant and resilient to conventional antibacterial therapy, it has been difficult to combat these infections. An innovative alternative to the biocontrol of oral biofilms could be to use bacteriophages or phages, the viruses of bacteria, which are specific, non-toxic, self-proliferating, and can penetrate into biofilms. Phages for Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis, Fusobacterium nucleatum, Lactobacillus spp., Neisseria spp., Streptococcus spp., and Veillonella spp. have been isolated and characterised. Recombinant phage enzymes (lysins) have been shown to lyse A. naeslundii and Streptococcus spp. However, only a tiny fraction of available phages and their lysins have been explored so far. The unique properties of phages and their lysins make them promising but challenging antimicrobials. The genetics and biology of phages have to be further explored in order to determine the most effective way of applying them. Studying the effect of phages and lysins on multispecies biofilms should pave the way for microbiota engineering and microbiota-based therapy.

Persistence of naturally occurring antibiotic resistance genes in the bacteria and bacteriophage fractions of wastewater

The emergence and prevalence of antibiotic resistance genes (ARGs) in the environment is a serious global health concern. ARGs from bacteria can be mobilized by mobile genetic elements, and recent studies indicate that phages and phage-derived particles, among others, could play a role in the spread of ARGs through the environment. ARGs are abundant in the bacterial and bacteriophage fractions of water bodies and for successful transfer of the ARGs, their persistence in these environments is crucial. In this study, three ARGs (blaTEM, blaCTX-M and sul1) that naturally occur in the bacterial and phage fractions of raw wastewater were used to evaluate the persistence of ARGs at different temperatures (4 °C, 22 °C and 37 °C) and pH values (3, 7 and 9), as well as after various disinfection treatments (thermal treatment, chlorination and UV) and natural inactivation in a mesocosm. Gene copies (GC) were quantified by qPCR; then the logarithmic reduction and significance of the differences between their numbers were evaluated. The ARGs persisted for a long time with minimal reductions after all the treatments. In general, they showed greater persistence in the bacteriophage fraction than in the bacterial fraction. Comparisons showed that the ARGs persisted under conditions that reduced culturable Escherichia coli and infectious coliphages below the limit of detection. The prevalence of ARGs, particularly in the bacteriophage fraction, poses the threat of the spread of ARGs and their incorporation into a new bacterial background that could lead to the emergence of new resistant clones.

Risperidone-induced weight gain is mediated through shifts in the gut microbiome and suppression of energy expenditure

Risperidone is a second-generation antipsychotic that causes weight gain. We hypothesized that risperidone-induced shifts in the gut microbiome are mechanistically involved in its metabolic consequences. Wild-type female C57BL/6J mice treated with risperidone (80μg/day) exhibited significant excess weight gain, due to reduced energy expenditure, which correlated with an altered gut microbiome. Fecal transplant from risperidone-treated mice caused a 16% reduction in total resting metabolic rate in naïve recipients, attributable to suppression of non-aerobic metabolism. Risperidone inhibited growth of cultured fecal bacteria grown anaerobically more than those grown aerobically. Finally, transplant of the fecal phage fraction from risperidone-treated mice was sufficient to cause excess weight gain in naïve recipients, again through reduced energy expenditure. Collectively, these data highlight a major role for the gut microbiome in weight gain following chronic use of risperidone, and specifically implicates the modulation of non-aerobic resting metabolism in this mechanism.

Abundance of Antibiotic Resistance Genes in Bacteriophage following Soil Fertilization with Dairy Manure or Municipal Biosolids, and Evidence for Potential Transduction

Animal manures and municipal biosolids recycled onto crop production land carry antibiotic-resistant bacteria that can influence the antibiotic resistome of agricultural soils, but little is known about the contribution of bacteriophage to the dissemination of antibiotic resistance genes (ARGs) in this context. In this work, we quantified a set of ARGs in the bacterial and bacteriophage fractions of agricultural soil by quantitative PCR. All tested ARGs were present in both the bacterial and phage fractions. We demonstrate that fertilization of soil with dairy manure or human biosolids increases ARG abundance in the bacterial fraction but not the bacteriophage fraction and further show that pretreatment of dairy manure can impact ARG abundance in the bacterial fraction. Finally, we show that purified bacteriophage can confer increased antibiotic resistance to soil bacteria when combined with selective pressure. The results indicate that soilborne bacteriophage represents a substantial reservoir of antibiotic resistance and that bacteriophage could play a significant role in the horizontal transfer of resistance genes in the context of an agricultural soil microbiome. Overall, our work reinforces the advisability of composting or digesting fecal material prior to field application and suggests that application of some antibiotics at subclinical concentrations can promote bacteriophage-mediated horizontal transfer of ARGs in agricultural soil microbiomes.

Bacteriophages as a reservoir of extended-spectrum β -lactamase and fluoroquinolone resistance genes in the environment

Six antibiotic resistance genes (blaCTX-M, blaSHV, blaTEM, qnrA, qnrB and qnrS) were quantified by qPCR in both phage and bacterial DNA fractions of environmental water samples in order to determine the contribution of phages to the dissemination of antibiotic resistance genes (ARGs) in the environment. Although the highest copy numbers (p <0.05) of ARGs were detected in the bacterial DNA fraction, qnrS and blaSHV genes were found in the phage DNA from all samples analysed, reaching up to 4 log10 copy numbers/mL in hospital samples. These results indicate that bacteriophages are a potential reservoir of resistance genes and may act as efficient vehicles for horizontal gene transfer.

Sludge as a potential important source of antibiotic resistance genes in both the bacterial and bacteriophage fractions.

The emergence and prevalence of antibiotic resistance genes (ARGs) in the environment is a serious global health concern. ARGs found in bacteria can become mobilized in bacteriophage particles in the environment. Sludge derived from secondary treatment in wastewater treatment plants (WWTPs) constitutes a concentrated pool of bacteria and phages that are removed during the treatment process. This study evaluates the prevalence of ARGs in the bacterial and phage fractions of anaerobic digested sludge; five ARGs (blaTEM, blaCTX-M, qnrA, qnrS, and sul1) are quantified by qPCR. Comparison between the wastewater and sludge revealed a shift in the prevalence of ARGs (blaTEM and sul1 became more prevalent in sludge), suggesting there is a change in the bacterial and phage populations from wastewater to those selected during the secondary treatment and the later anaerobic mesophilic digestion of the sludge. ARGs densities were higher in the bacterial than in the phage fraction, with high densities in both fractions; particularly for blaTEM and sul1 (5 and 8 log10 gene copies (GC)/g, respectively, in bacterial DNA; 5.5 and 4.4 log10 GC/g, respectively, in phage DNA). These results question the potential agricultural uses of treated sludge, as it could contribute to the spread of ARGs in the environment and have an impact on the bacterial communities of the receiving ecosystem.

Inactivation of F-specific bacteriophages during flocculation with polyaluminum chloride – A mechanistic study

Bacteriophages are often used as surrogates for enteric viruses in spiking experiments to determine the efficiencies of virus removal of certain water treatment measures, like e.g. flocculation or filtration steps. Such spiking experiments with bacteriophages are indispensable if the natural virus concentrations in the raw water of water treatment plants are too low to allow the determination of elimination levels over several orders of magnitude. In order to obtain reliable results from such spiking tests, it is essential that bacteriophages behave comparable to viruses and remain stable during the experiments. To test this, the influence of flocculation parameters on the bacteriophages MS2, Qβ and phiX174 was examined. Notably, the F-specific phages MS2 and Qβ were found to be inactivated in flocculation processes with polyaluminum chloride (PACl). In contrast, other aluminum coagulants like AlCl3 or Al2(SO4)3 did not show a comparable effect on MS2 in this study. In experiments testing the influence of different PACl species on MS2 and Qβ inactivation during flocculation, it could be shown that cationic dissolved PACl species (Al13) interacted with the MS2 surface and hereby reduced the surviving phage fraction to c/c0 values below 1*10−4 even at very low PACl concentrations of 7 μmol Al/L. Other inactivation mechanisms like the irreversible adsorption of phages to the floc structure or the damage of phage surfaces due to entrapment into the floc during coagulation and floc formation do not seem to contribute to the low surviving fraction found for both F-specific bacteriophages. Furthermore, no influence of phage agglomeration or pH drops during the flocculation process on phage inactivation could be observed. The somatic coliphage phiX174 in contrast did not show sensitivity to chemical stress and in accordance only slight interaction between Al13 and the phage surface was observed. Consequently, F-specific phages like MS2 should not be used as surrogate for viruses in flocculation experiments with PACl to determine the removal rates of viruses, as the results are influenced by a strong inactivation of the bacteriophages due to the experimental conditions.

CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome

The bacterial community in the human gut has crucial health roles both in metabolic functions and in protection against pathogens. Phages, which are known to significantly affect microbial community composition in many ecological niches, have the potential to impact the gut microbiota, yet thorough characterization of this relationship remains elusive. We have reconstructed the content of the CRISPR bacterial immune system in the human gut microbiomes of 124 European individuals and used it to identify a catalog of 991 phages targeted by CRISPR across all individuals. Our results show that 78% of these phages are shared among two or more individuals. Moreover, a significant fraction of phages found in our study are shown to exist in fecal samples previously derived from American and Japanese individuals, identifying a common reservoir of phages frequently associated with the human gut microbiome. We further inferred the bacterial hosts for more than 130 such phages, enabling a detailed analysis of phage–bacteria interactions across the 124 individuals by correlating patterns of phage abundance with bacterial abundance and resistance. A subset of phages demonstrated preferred association with host genomes as lysogenized prophages, with highly increased abundance in specific individuals. Overall, our results imply that phage–bacterial attack–resistance interactions occur within the human gut microbiome, possibly affecting microbiota composition and human health. Our finding of global sharing of gut phages is surprising in light of the extreme genetic diversity of phages found in other ecological niches.

Apparent heterogeneity in the pIII-peptide fusion protein in single-phage clones isolated from peptide libraries

Ligand homogeneity is an important issue in affinity chromatography. Using phages expressing peptides on the pIII protein, a heterogeneity in the binding of monoclonal phages was observed during affinity chromatography on supermacroporous cryogels. Fractions with different apparent binding affinities could be separated by stepwise elution. When these different fractions were re-applied, the respective differences in affinity were retained. However, when phage fractions with different apparent affinities were first amplified, an offspring was generated with again variable affinities. As the sequence of the peptide insert was the same, the heterogeneity must be ascribed to differences in avidity and although no direct evidence could be generated, we hypothesize that this is possibly due to phages displaying different numbers of the same peptide as a consequence of either proteolytic or packaging events during the amplification step in Escherichia coli.

Optimization of lytic phage manufacturing in bioreactor using monolithic supports

A process for manufacturing large quantities of lytic bacteriophages was developed. Determination of cultivation termination was found to be essential to achieve high phage quantity and purity. When optimal cultivation termination is missed, phage fraction was found to be highly contaminated with deoxyribonucleic acid released from Escherichia coli cells. Besides, an already established method for monitoring of phage cultivation based on optical density, where its peak indicates point when maximal phage titer is achieved, a new indirect chromatographic method using methacrylate monoliths is proposed for on-line estimation of phage titer. It is based on the measurement of released E. coli deoxyribonucleic acid and shows high correlation with phage titer obtained from plaque assay. Its main advantage is that the information is obtained within few minutes. In addition, the same method can also be used to determine purity of a final phage fraction. Two strategies to obtain highly pure phage fractions are proposed: an immediate purification of phage lysate using monolithic columns or an addition of EDTA before chromatographic purification. The developed protocol was shown to give phage purity above 90% and it is completed within one working day including cultivation and phage titer in the final formulation using developed chromatographic method.