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Morphological, biological, and genetic characteristics of a virulent Siphoviridae phage, named vB-EcoS-95, is reported. This phage was isolated from urban sewage. It was found to infect some Escherichia coli strains giving clear plaques. The genome of this phage is composed of 50,910 bp and contains 89 ORFs. Importantly, none of the predicted ORFs...
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... Bacteriophage-based hydrogel-mediated delivery offers a precise strategy for modulating the expression of specific genes in individual microbes in the intestine, thereby promoting gut homeostasis and human health. The development of bacteriophage resistance is also a general consideration when delivering lytic bacteriophages (128). Therefore, the use of multiple bacteriophages (bacteriophage cocktails) may be necessary to ensure successful lytic activity and achieve the desired outcomes, broadening the host range of targeted microbes and suppressing the development of resistance (129). ...
In the advancement of Inflammatory Bowel Disease (IBD) treatment, existing therapeutic methods exhibit limitations; they do not offer a complete cure for IBD and can trigger adverse side effects. Consequently, the exploration of novel therapies and multifaceted treatment strategies provides patients with a broader range of options. Within the framework of IBD, gut microbiota plays a pivotal role in disease onset through diverse mechanisms. Bacteriophages, as natural microbial regulators, demonstrate remarkable specificity by accurately identifying and eliminating specific pathogens, thus holding therapeutic promise. Although clinical trials have affirmed the safety of phage therapy, its efficacy is prone to external influences during storage and transport, which may affect its infectivity and regulatory roles within the microbiota. Improving the stability and precise dosage control of bacteriophages—ensuring robustness in storage and transport, consistent dosing, and targeted delivery to infection sites—is crucial. This review thoroughly explores the latest developments in IBD treatment and its inherent challenges, focusing on the interaction between the microbiota and bacteriophages. It highlights bacteriophages’ potential as microbiome modulators in IBD treatment, offering detailed insights into research on bacteriophage encapsulation and targeted delivery mechanisms. Particular attention is paid to the functionality of various carrier systems, especially regarding their protective properties and ability for colon-specific delivery. This review aims to provide a theoretical foundation for using bacteriophages as microbiome modulators in IBD treatment, paving the way for enhanced regulation of the intestinal microbiota.
... Mid-exponential growing phase cultures of S. enterica (pKM101) were inoculated with phage Lu221 or Hi226 at MOI 0.001 (62). Aliquots of 500 µL were taken (0, 15,30,45,60,75,90,105, and 120 min) and centrifuged 2 min at 14,100 g. ...
Plasmid-dependent phages infect bacteria carrying conjugative plasmids by recognizing the plasmid-encoded pilus. Despite the high abundance of conjugative plasmids in diverse environments, plasmid-dependent phages have not been widely studied. Since conjugative plasmids often carry antimicrobial resistance genes (ARGs), interfering with conjugation could reduce the spread of ARGs and avoid the appearance of multiresistant superbugs. Our aim was to isolate and characterize plasmid-dependent phages able to infect bacteria carrying diverse conjugative plasmids belonging to the most common plasmid families among Gram-negative pathogens. We isolated two lytic phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncN plasmid pKM101. Both phages, named Lu221 and Hi226, are novel dsDNA viruses within the class Caudoviricetes with genomes of approximately 76 kb. They showed broad host range infecting Escherichia coli, S. enterica, Kluyvera sp., and Enterobacter sp. carrying conjugative plasmids. They recognize plasmid-encoded receptors from 12 out of 15 tested plasmids, all of them carrying resistance determinants. Phages Lu221 and Hi226 could have the potential to help combat the antimicrobial resistance crisis by reducing ARGs present in conjugative plasmids.
IMPORTANCE
This work was undertaken because plasmid-dependent phages can reduce the prevalence of conjugative plasmids and can be leveraged to prevent the acquisition and dissemination of ARGs by bacteria. The two novel phages described in this study, Lu221 and Hi226, can infect Escherichia coli, Salmonella enterica, Kluyvera sp. and Enterobacter sp. carrying conjugative plasmids. This was verified with plasmids carrying resistance determinants and belonging to the most common plasmid families among Gram-negative pathogens. Therefore, the newly isolated phages could have the potential to help control the spread of ARGs and thus help combat the antimicrobial resistance crisis.
... The overnight culture medium was centrifuged for 20 min, and the supernatant was extracted. The phage was isolated by the drop plate method [17], in which the lower plate was poured with an appropriate amount of solid LB medium, and the upper plate was poured with a mixture of 400 µL of host bacterial liquid and 3 mL of semi-solid LB medium at the appropriate temperature (around 45 • C). After centrifugation, 5 µL of the supernatant was dropped on the plate and placed in the incubator at 37 • C for 4 h. ...
Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are becoming increasingly common within clinical settings, requiring the development of alternative therapies. In this study, we isolated, characterized, and sequenced the genome of a CRKP phage, Phage168. The total genomic DNA of Phage168 was 40,222 bp in length, encoding 49 predicted proteins. Among these proteins, Dep40, the gene product of ORF40, is a putative tail fiber protein that exhibits depolymerase activity based on the result of bioinformatics analyses. In vitro, we confirmed that the molecular weight of the Phage168 depolymerase protein was about 110 kDa, the concentration of the produced phage 168 depolymerase protein was quantified as being 1.2 mg/mL, and the depolymerase activity was still detectable after the dilution of 1.2 µg/mL. This recombinant depolymerase exhibited enzyme activity during the depolymerization of the formed CRKP biofilms. We also found that depolymerase, when combined with polymyxin B, was able to enhance the bactericidal effect of polymyxin B on CRKP strains by disrupting their biofilm. When recombinant depolymerase was used in combination with human serum, it enhanced the sensitivity of the CRKP strain UA168 to human serum, and the synergistic bactericidal effect reached the strongest level when the ratio of depolymerase to human serum was 3:1. Our results indicated that depolymerase encoded by Phage168 may be a promising strategy for combating infections caused by drug-resistant CRKP formed within the biofilm.
... Detection of Bt phages was performed by the spot test technique; to 5mL of soft warm agar (60 °C, 0.7% agar) 100 µl of a log phase Bt culture was added, the agar was gently mixed and overlaid on to NA plate, and left for 20 min to solidify, The nutrient agar amended with the bacterial lawn inoculated with 10 µl of the Bt phage crude lysates, and incubated at 30 °C for 24 h, then the presence or absence of plaques was observed. This step was repeated three times before judging the sample as negative (Elmaghraby et al. 2015;Abo-senna 2017;Topka et al. 2019). According to the morphology of plaques, the samples were prepared for phage lysate by using a sensitive double agar layer, i.e., DAL (Phumkhachorn and Rattanachaikunsopon 2015). ...
Insecticidal crystal proteins (ICPs) produced by Bacillus thuringiensis (Bt) exhibit strong toxicity. Soil bacteriophages destroy the ICPs in nature. Also, environmental pH, temperature, and ultraviolet (UV) radiation shorten the ICPs’ validity and infectivity. To enhance the validity of ICPs of Bt, the soil Bt phages and the environmental parameters such as soil pH, temperature, and UV should be subjected to continuous evaluation.
In this study, five Bt bacteriophages were isolated, characterized, and named BtØ3, BtØ5, BtØ7, BtØ9, and BtØ11. Electron microscopy investigation showed that the five phages have an icosahedral head and a long contractile tail. In addition, the restriction endonuclease BamHI enzyme cleaves the phage genomic DNA suggesting that all five phages have double-stranded DNA (dsDNA) belonging to the order Caudovirales. The various inter simple sequence repeat restriction patterns suggested that the five phages genetically are not similar, and similarity metrics analysis placed the five phages into two clusters.
The reported lytic activity of phages against Bt was as follows: BtØ7 (100%), BtØ9 (100%), BtØ3(83%), BtØ5(83%), and BtØ11(75%). Moreover, the phages were 17% more effective in lysing Bt than the commercial antibiotics.
Bt phages isolated from this study highlighted the importance of regular assessment of soil conditions and the lytic potentials of naturally occurring Bt phages to protect Bt sp from being attacked or destroyed, and to calculate the exact Bt dose concentration of successful application in pest control, this will enhance the environmental health, food security, and crop safety.
... Sewage sites are recognized as an important reservoir for E. coli and also, for their specific phages, which can be isolated directly from the samples as previously reported. [40][41][42][43] Both the direct and enrichment procedures yielded phages, and as expected, the phage UP30 was biased toward the bacterial strain (PA5) used for its enrichment procedure. This concurs with previous work on Salmonella and Shigella and may have notable benefits for the targeted selection toward specific bacterial strains. ...
Background: The antimicrobial resistance catastrophe is a growing global health threat and predicted to be worse in developing countries. Phages for Global Health (PGH) is training scientists in these regions to isolate relevant therapeutic phages for pathogenic bacteria within their locality, and thus contributing to making phage technology universally available.
Materials and Methods: During the inaugural PGH workshop in East Africa, samples from Ugandan municipal sewage facilities were collected and two novel Escherichia coli lytic phages were isolated and characterized.
Results: The phages, UP19 (capsid diameter ∼100 nm, contractile tail ∼120/20 nm) and UP30 (capsid diameter ∼70 nm, noncontractile tail of ∼170/20 nm), lysed ∼82% and ∼36% of the 11 clinical isolates examined, respectively. The genomes of UP19 (171.402 kb, 282 CDS) and UP30 (49.834 kb, 75 CDS) closely match the genera Dhakavirus and Tunavirus, respectively.
Conclusion: The phages isolated have therapeutic potential for further development against E. coli infections.
... The optimum stability was observed under pH values of 7, 8, and 9 with phage titre of ∼9.5 Log 10 PFU/mL, meaning that phage ZCEC13 favours the neutral pH condition. It only lost its activity when it was incubated under pH 13 while other previously studied E. coli phages could not be viable under pH 2 and 13 after one hour of incubation [78][79][80]. ...
... These results reveal that ZCEC13 phage tolerates a wide range of temperatures as shown in (Figure 6(c)). Some of the previously isolated E. coli phages were stable at 65°C under thermal incubation for only 20 min, and when the duration increased to 30 min, the phage titres were dramatically decreased below the detection limit [79][80][81]. The lytic activity of the previously isolated AS1 phage was stable at 60°C, dramatically decreased at 70°C, and completely lost its viability at 80°C when it was thermally incubated for 30 min [80]. ...
The spread and development of Multi-Drug Resistant (MDR) bacteria in wastewater became beyond control and a global public health concern. The conventional disinfectants used in wastewater treatment methods have been becoming increasingly ineffective against a range of pathogenic and MDR bacteria. Bacteriophages are considered a novel approach to microbial control. Therefore, this study aims to explore the possibility of using phages against pathogenic and MDR Escherichia coli strains isolated from wastewater treatment plants. The wastewater samples were collected from two different treatment plants for E. coli isolation. The antibiotic sensitivity profile and occurrence of virulence and resistant genes were tested in twenty-eight (28) E. coli isolates. Phage ZCEC13 was selected based on its promising activity and host range to undergo identification and characterization. ZCEC13 was evaluated by transmission electron microscopy, genomic sequencing, in vitro lytic activity and tested for its stability under different conditions such as pH, Ultraviolet light exposure, and temperature. The results reported that ZCEC13 belongs to the Caudoviricetes class, with a high antibacterial dynamic. Phage ZCEC13 displayed high stability at different pH values ranging from 2 to 12, good tolerance to temperatures from -4 to 65 oC, and high stability at UV exposure for 120 mins. Respectively, the findings showed stability of the phage under several conditions and high efficiency in killing MDR bacteria isolated from the treatment plants. Further studies are encouraged to analyze the efficacy of phages as a microbial control agent in wastewater treatment plants.
... Detection of Bt phages was performed by the spot test technique; to 5ml of soft warm agar (60°C, 0.7% agar)100µl of a log phase Bt culture was added, the agar was gently mixed and overlaid on to NA plate, and left for 20 min to solidify, The nutrient agar amended with the bacterial lawn inoculated with 10µl of the Bt phage crude lysates, and incubated at 30°C for 24 hrs, then the presence or absence of plaques watched. This step was repeated three times before judging the sample as negative (Elmaghraby et al. 2015; Abo-senna 2017; Topka et al. 2019). According to the morphology of plaques the samples for preparation of phage lysate by using a sensitive double agar layer (DAL (Phumkhachorn and Rattanachaikunsopon 2015). ...
Background Insecticidal crystal proteins (ICPs) produced by Bacillus thuringiensis exhibit strong toxicity. Soil bacteriophages destroy the ICPs in nature. Also, environmental pH, temperature, and ultraviolet (UV) radiation shorten the ICP's validity and infectivity. To Enhance the validity of B. thuringiensis insecticidal (ICPs) the soil Bt phages & the environmental parameters such as soil pH, temperature, and UV should be subjected to continuous evaluation.
Result In this study, five B. thuringiensis bacteriophages were isolated, characterized, and named BtØ3, BtØ5, BtØ7, BtØ9, BtØ11. Electron microscopy investigation showed that the five phages have an icosahedral head and a long contractile tail. In addition, the restriction endonuclease BamHI enzyme cleaves the phage genomic DNA suggesting that all five phages have double-stranded DNA (dsDNA) belonging to the order Caudovirales. The various ISSR restriction patterns suggested that the five phages genetically are not similar, and similarity metrics analysis placed the five phages into two clusters.
The reported lytic activity of phages against B. thuringiensis was as follows BtØ7 (100%), BtØ9 (100%), BtØ3(83%), BtØ5(83%), and BtØ11(75%). Moreover, the phages were 17% more effective in lysing B. thuringiensis than the commercial antibiotics.
Conclusion B. thuringiensis phages isolated from this study highlighted the importance of regular assessment of soil conditions and the lytic potentials of naturally occurring Bt phages to protect B. thuringiensis sp, from being attacked or destroyed, and to calculate the exact Bt dose concentration of successful application in pest control, this will enhance the environmental health, food security, and crop safety.
... Phage activity at various ranges of pH (4, 5, 6 and 8) was tested after 1 h and 5 h at room temperature and then compared to the control lysate incubated at pH 7.1. The above-mentioned experiments were carried out according to the previously described methods with further modifications [44,45]. ...
... x FOR PEER REVIEW 11 of 33 All tested phages indicated a rapid increase in phage titer within 80 min after infection at initial MOI = 1 (Figure 2a-c). Interestingly, adsorption of the tested phages was much lower when compared to phages described in the literature [45,46] and its peak was noted later (10 to 30 min.) (Figure 1a). ...
... The differences could result from different All tested phages indicated a rapid increase in phage titer within 80 min after infection at initial MOI = 1 (Figure 2a-c). Interestingly, adsorption of the tested phages was much lower when compared to phages described in the literature [45,46] and its peak was noted later (10 to 30 min.) (Figure 1a). ...
In recent years, multidrug-resistant (MDR) strains of Klebsiella pneumoniae have spread globally, being responsible for the occurrence and severity of nosocomial infections. The NDM-1-kp, VIM-1 carbapenemase-producing isolates as well as extended-spectrum beta lactamase-producing (ESBL) isolates along with Klebsiella oxytoca strains have become emerging pathogens. Due to the growing problem of antibiotic resistance, bacteriophage therapy may be a potential alternative to combat such multidrug-resistant Klebsiella strains. Here, we present the results of a long-term study on the isolation and biology of bacteriophages active against K. pneumoniae, as well as K. oxytoca strains. We evaluated biological properties, morphology, host specificity, lytic spectrum and sensitivity of these phages to chemical agents along with their life cycle parameters such as adsorption, latent period, and burst size. Phages designated by us, vB_KpnM-52N (Kpn52N) and VB_KpnM-53N (Kpn53N), demonstrated relatively broad lytic spectra among tested Klebsiella strains, high burst size, adsorption rates and stability, which makes them promising candidates for therapeutic purposes. We also examined selected Klebsiella phages from our historical collection. Notably, one phage isolated nearly 60 years ago was successfully used in purulent cerebrospinal meningitis in a new-born and has maintained lytic activity to this day. Genomic sequences of selected phages were determined and analyzed. The phages of the sequenced genomes belong to the Slopekvirus and Jiaodavirus genus, a group of phages related to T4 at the family level. They share several features of T4 making them suitable for antibacterial therapies: the obligatorily lytic lifestyle, a lack of homologs of known virulence or antibiotic resistance genes, and a battery of enzymes degrading host DNA at infection.
... Characterization of plaque morphology refers to Topka et al. [83] with minor modifications. Bacteriophage stock was diluted using CaCl 2 /MgCl 2 (1 M CaCl 2 , 80 mM MgCl 2 ) buffer, and then 100 µL of the dilution was mixed with 100 µL of A. tumefaciens bacteria before adding 4.8 mL of top agar (0.2% (w/v)) yeast extract; 5% (v/v) glycerol; 1.1% (w/v) CaCl2, 0.4%(w/v) agar). ...
Overgrowth of Agrobacterium tumefaciens has frequently been found in Agrobacterium-mediated plant transformation. This overgrowth can reduce transformation efficiency and even lead to explant death. Therefore, this research investigates an alternative way to mitigate or eliminate Agrobacterium after transformation using a bacteriophage. To develop this alternative method, we conducted effectiveness studies of two lytic bacteriophages (ΦK2 and ΦK4) and performed an application test to control Agrobacterium growth after transformation. According to plaque morphological characterization and molecular analysis, the two bacteriophages used in this experiment were distinct. Moreover, some stability physicochemical and growth kinetics, such as adsorption time and susceptibility test, also showed that both bacteriophages differed. On the other hand, the optimum temperature and pH of both phages were the same at 28–30 °C and pH 7. Further investigation showed that both ΦK2 and ΦK4 were able to reduce the overgrowth of A. tumefaciens post transformation. Moreover, applying the cocktail (mixture of ΦK2 and ΦK4) with antibiotic application eradicated A. tumefaciens (0% overgrowth percentage). This result indicates that the application of bacteriophage could be used as an alternative way to eradicate the overgrowth of A. tumefaciens subsequent to transformation.
... The determination of phage adsorption was carried out as previously described [24] with some modifications. Briefly, purified phage lysates were added at multiplicity of infection (MOI) of 0.1 to bacteria host grown at mid-exponential phase (OD 600 = 0.3,~1 × 10 8 CFU/mL) in a 20 mL LB medium. ...
... One-step growth curve experiments were conducted as described previously [24] with slight modifications. Briefly, purified phage lysates Details for isolated phage taxonomic classification at family level and its population dynamic is also listed in this table. ...
Antibiotic resistance genes are often carried by plasmids, which spread intra- and inter genera bacterial populations, and also play a critical role in bacteria conferring phage resistance. However, it remains unknown about the influence of plasmids present in bacterial hosts on phage isolation and subsequent infectivity. In this study, using both Escherichia coli and Pseudomonas putida bacteria containing different plasmids, eight phages were isolated and characterized in terms of phage morphology and host range analysis, in conjunction with DNA and protein sequencing. We found that plasmids can influence both the phage isolation process and phage infectivity. In particular, the isolated phages exhibited different phage plaquing infectivity towards the same bacterial species containing different plasmids. Furthermore, the presence of plasmids was found to alter the expression of bacteria membrane protein, which correlates with bacterial cell surface receptors recognized by phages, thus affecting phage isolation and infectivity. Given the diverse and ubiquitous nature of plasmids, our findings highlight the need to consider plasmids as factors that can influence both phage isolation and infectivity.
