Isolation and characterization of bacteriophages against E.coli urinary tract infection and evaluating their anti-biofilm activity and antibiotic synergy.

Urinary tract infections (UTIs) by Uropathogenic Escherichia coli (UPEC) are a significant health concern, especially due to the increasing prevalence of antibiotic resistance. This study focuses on isolating and characterizing bacteriophages specific to UPEC strains isolated from UTI samples. The isolated phages were assessed for their ability to target and lyse UPEC in vitro, focusing on their efficacy in disrupting biofilms, a key virulence factor contributing to UTI recurrence and antibiotic resistance. The morphological structure observed by TEM belongs to Myoviridae, the phage exhibited icosahedral symmetry with a long non-constricting tail, the approximate measurement of the phage head was 39 nm in diameter, and the phage tail was 105.317 nm in length. One-step growth experiments showed that the latent period was approximately 20 min, followed by a rise period of 40 min, and a growth plateau was reached within 20 min and the burst size observed was 26 phages/infected bacterial cells. These phages were capable of killing cells within the biofilms, leading to a reduction in living cell counts after a single treatment. This study highlights the potential of phages to play a significant role in disrupting, inactivating, and destroying Uropathogenic Escherichia coli (UPEC) biofilms. Such findings could be instrumental in developing treatment strategies that complement antibiotics and disinfectants. The phage-antibiotic synergistic activity was compared to have the possibility to facilitate the advancement of focused and enduring alternatives to traditional antibiotic therapies for UTIs.

Discovery and characterisation of new phage targeting uropathogenic Escherichia coli.

Antimicrobial resistance is an escalating threat with few new therapeutic options in the pipeline. Urinary tract infections (UTIs) are one of the most prevalent bacterial infections globally and are prone to becoming recurrent and antibiotic resistant. We discovered and characterized six novel Autographiviridae and Guernseyvirinae bacterial viruses (phage) against uropathogenic Escherichia coli (UPEC), a leading cause of UTIs. The phage genomes were between 39,471 bp - 45,233 bp, with 45.0%-51.0% GC%, and 57-84 predicted coding sequences per genome. We show that tail fiber domain structure, predicted host capsule type, and host antiphage repertoire correlate with phage host range. In vitro characterisation of phage cocktails showed synergistic improvement against a mixed UPEC strain population and when sequentially dosed. Together, these phage are a new set extending available treatments for UTI from UPEC, and phage vM_EcoM_SHAK9454 represents a promising candidate for further improvement through engineering.

Characterization of bacteriophages infecting multidrug-resistant uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections, and strains that are resistant to antibiotics are a major problem in treating these infections. Phage therapy is a promising alternative approach that can be used to treat infections caused by polyresistant bacterial strains. In the present study, 16 bacteriophages isolated from sewage and surface water were investigated. Phage host specificity was tested on a collection of 77 UPEC strains. The phages infected 2-44 strains, and 80% of the strains were infected by at least one phage. The susceptible E. coli strains belonged predominantly to the B2 phylogenetic group, including strains of two clones, CC131 and CC73, that have a worldwide distribution. All of the phages belonged to class Caudoviricetes and were identified as members of the families Straboviridae, Autographiviridae, and Drexlerviridae and the genera Kagunavirus, Justusliebigvirus, and Murrayvirus. A phage cocktail composed of six phages - four members of the family Straboviridae and two members of the family Autographiviridae - was prepared, and its antibacterial activity was tested in liquid medium. Complete suppression of bacterial growth was observed after 5-22 hours of cultivation, followed by partial regrowth. At 24 hours postinfection, the cocktail suppressed bacterial growth to 43-92% of control values. Similar results were obtained when testing the activity of the phage cocktail in LB and in artificial urine medium. The results indicate that our phage cocktail has potential to inhibit bacterial growth during infection, and they will therefore be preserved in the national phage bank, serving as valuable resources for therapeutic applications.

Characterization and in vitro activity of a lytic phage RDN37 isolated from community sewage water active against MDR Uropathogenic E. coli.

PURPOSE: Due to the emergence of multidrug-resistant Uropathogenic E. coli (MDR-UPEC) strains, alternatives to antibiotics like phage therapy have been sought. The present study was planned to characterize and test the activity of a phage (RDN37) which was isolated from community sewage water of Chandigarh and was found to be active against MDR-UPEC. MATERIALS AND METHODS: We studied the morphology of the phage by transmission electron microscopy and determined one-step growth curve analysis and stability of the phage at various temperature and pH ranges. PCR amplification and Sanger sequencing were performed to confirm the phage family. Genome sequences from 12 related phages (BLASTn identity >95%) were obtained from the NCBI database in GenBank format. A phylogenetic analysis was conducted using the neighbour-joining method in ClustalX2 and MEGAX. Host range and lytic activity were tested by spot assay and time-kill experiment, respectively. RESULTS: Phage RDN37 had a large burst size and belonged to the Myoviridae family as per transmission electron microscopy and Sanger sequencing results. It was stable over wide range of temperature (-20°,4°, 25°, 37 °C) and pH (6,7,8). The phylogenetic analysis of amplified PCR product (major coat protein gp23) grouped the phage RDN37 with Escherichia phage vB_EcoM_IME537 (MT179807) isolated from community sewage water in China. RDN37 phage was active against MDR-UPEC strains resistant to third generation cephalosporins, aminoglycosides, carbapenems, fluoroquinolones and cotrimoxazole. The multiplicity of infection (MOI) of 0.01 was found to be optimum to reduce the bacterial cell density in the time-kill assay. CONCLUSIONS: RDN37 is a stable lytic phage with large burst size, specific to E. coli, has a therapeutic potential to treat UTI caused by highly drug resistant UPEC. A cocktail of multiple phages will be required to overcome its narrow host range.

Bacteriophage therapy for inhibition of multi drug-resistant uropathogenic bacteria: a narrative review.

Multi-Drug Resistant (MDR) uropathogenic bacteria have increased in number in recent years and the development of new treatment options for the corresponding infections has become a major challenge in the field of medicine. In this respect, recent studies have proposed bacteriophage (phage) therapy as a potential alternative against MDR Urinary Tract Infections (UTI) because the resistance mechanism of phages differs from that of antibiotics and few side effects have been reported for them. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are the most common uropathogenic bacteria against which phage therapy has been used. Phages, in addition to lysing bacterial pathogens, can prevent the formation of biofilms. Besides, by inducing or producing polysaccharide depolymerase, phages can easily penetrate into deeper layers of the biofilm and degrade it. Notably, phage therapy has shown good results in inhibiting multiple-species biofilm and this may be an efficient weapon against catheter-associated UTI. However, the narrow range of hosts limits the use of phage therapy. Therefore, the use of phage cocktail and combination therapy can form a highly attractive strategy. However, despite the positive use of these treatments, various studies have reported phage-resistant strains, indicating that phage-host interactions are more complicated and need further research. Furthermore, these investigations are limited and further clinical trials are required to make this treatment widely available for human use. This review highlights phage therapy in the context of treating UTIs and the specific considerations for this application.

Managing urinary tract infections through phage therapy: a novel approach.

Upsurge in the instances of antibiotic-resistant uropathogenic Escherichia .coli (UPECs) strains has repositioned the attention of researchers towards a century old antimicrobial approach popularly known as phage therapy. Rise of extended spectrum beta lactamase (ESBL) and biofilm producing strains has added another step of hurdle in treatment of uropathogens with conventional antibiotics, thus providing a further impetus for search for exploring new therapeutic measures. In this direction, bacteriophages, commonly called phages, are recently being considered as potential alternatives for treatment of UPECs. Phages are the tiniest form of viruses which are ubiquitous in nature and highly specific for their host. This review discusses the possible ways of using natural phages, genetically engineered phages, and phage lytic enzymes (PLEs) as an alternative antimicrobial treatment for urinary tract infections. The review also sheds light on the synergistic use of conventional antibiotics with phages or PLEs for treatment of uropathogens. These methods of using phages and their derivatives, alone or in combination with antibiotics, have proved fruitful so far in in vitro studies. However, in vivo studies are required to make them accessible for human use. The present review is a concerted effort towards putting together all the information available on the subject.

Alternative treatment approaches of urinary tract infections caused by uropathogenic Escherichia coli strains.

Urinary tract infections (UTIs) are the most widespread and annoying infections affecting millions of people every year annually. The biggest problems of urinary diseases are recurrences, increasing resistance of uropathogens to commonly used antibiotics, as well as the high health care costs of afflicted persons. Uropathogenic Escherichia coli strains (UPECs) are the most dominant etiologic agents of community-acquired infections of this type. During UTI pathogenesis, UPECs utilize various virulence factors, especially mono- and polyadhesive appendages of the chaperone-usher secretion pathway (CUP) required for adhesion, invasion and biofilm formation. Commonly used antibiotics for UTI treatment are usually effective, but their long-term utility may affect gut microbiota of the treated individuals and cause selection of drug resistant uropathogenic variants. Due to increasing resistance of UPEC strains to antibiotics via the evolution of specific defense mechanisms, there is a need to develop alternative methods and therapeutic strategies to fight UTIs (vaccines, receptor analogues, pilicides and curlicides, bacterial interference or phagotherapy). Such therapeutic approaches usually target processes enabling uropathogens to survive within the urinary tract and cause recurrent infections.

Identification of novel bacteriophage vB_EcoP-EG1 with lytic activity against planktonic and biofilm forms of uropathogenic Escherichia coli.

Urinary tract infections are one of the most common infectious diseases worldwide. Uropathogenic Escherichia coli (UPEC) is a major cause of unary tract infection. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the UPECs are urgently needed. In this respect, phage therapy has been demonstrated to be a good candidate. Here, we described a novel bacteriophage named vB_EcoP-EG1, which can infect several strains of UPEC. Phage morphology and genome sequencing analysis show that vB_EcoP-EG1 belongs to the T7-like Podoviridae. vB_EcoP-EG1 possesses a genome (39,919 bp) containing 51 predicted genes and 149 bp terminal repeats. vB_EcoP-EG1 genome does not encode toxic proteins or proteins related to lysogeny. And no known virulent proteins were found in purified phage particles by mass spectrometry. vB_EcoP-EG1 appeared to be relatively specific and sensitive to clinical UPEC strains, which could infect 10 out of 21 clinical multidrug-resistant UPEC strains. In addition, vB_EcoP-EG1 suspension can eliminate biofilm formed by E. coli MG1655 and multidrug-resistant UPEC strain 390G7. Therefore, we concluded that vB_EcoP-EG1 has desirable characteristics for potential therapy, which may serve as an alternative to antibiotic therapy against urinary tract infections caused by multidrug-resistant UPEC.

Bacteriophages with the ability to degrade uropathogenic Escherichia coli biofilms.

Escherichia coli-associated urinary tract infections (UTIs) are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC) have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages) as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12) which were able to lyse 80.5% of a subset (42) of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages' genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2-12 h of incubation.

Phylogenetic and pathotype analysis of Escherichia coli swine isolates from Southern Brazil / Análise filogenética e de patotipos de Escherichia coli isoladas de suínos no Sul do Brazil

The current study evaluated the presence of virulence factors by a multiplex PCR technique and then phylogenetically classified the studied strains into groups A, B1, B2 and D, according to Clermont et al. (2000), in 152 intestinal and extraintestinal swine isolates of Escherichia coli. Seventy seven isolates tested were positive for virulence factors. Phylogenetic characterization placed 21 samples into group A, 65 into B1, 19 into B2 and 47 into D. Fourteen urine samples were classified as uropathogenic E. coli (UPEC), nine were both UPEC and enterotoxigenic E. coli (ETEC) and four were ETEC only. The most common phylogenetic classifications were B1 and D groups. Of the analyzed fecal samples, 25 were classified as ETEC. Phylogenetically, the group of higher occurrence was B1, followed by B2, A and D. For the small intestine samples, 20 were classified as ETEC. Phylogenetic analysis found groups B1 and A to be the most commons in these samples. Six isolated tissue samples were classified as ETEC and most of them were designated as group D by phylogenetic classification. The phylogenetic analysis could be employed in veterinary laboratories in the E. coli isolates screening, including the possibility of vaccine strain selection and epidemiological searches.

O presente estudo teve por objetivo avaliar a presença de diferentes fatores de virulência em 152 isolados de Escherichia coli intestinais e extra-intestinais provenientes de suínos pela técnica de PCR multiplex e classificá-los nos grupos filogenéticos A, B1, B2 e D, de acordo com Clermont et al. (2000). Setenta e sete isolados foram positivos para pelo menos um fator de virulência. Através da caracterização filogenética, 21 isolados foram caracterizados como pertencentes ao grupo A, 65 ao grupo B1, 19 ao grupo B2 e 47 isolados ao grupo D. Quatorze isolados de urina foram caracterizados como E. coli uropatogênica (UPEC); nove apresentaram fatores de UPEC e E. coli enterotoxigênica (ETEC) simultaneamente e quatro foram classificados como ETEC. Na classificação filogenética, os isolados provenientes de amostras de urina classificaram-se principalmente nos grupos D e B1. Das amostras de fezes analisadas, 25 demonstraram fatores de virulência característicos do patotipo ETEC. Filogeneticamente, o grupo de maior ocorrência foi o B1 seguido de B2, A e D. Em relação às cepas isoladas de intestino delgado, 20 foram caracterizadas como ETEC. Pela filogenia, 23 isolados classificaram-se nos grupos A ou B1. Seis isolados de tecidos foram qualificados como ETEC e a maioria deles foram designados como pertencentes ao grupo D, pela classificação filogenética. A análise filogenética pode ser empregada em laboratórios de diagnóstico veterinário como um screening para isolados de E. coli, incluindo a possibilidade de seleção de cepas vacinais e levantamentos epidemiológicos.

Isolation of generalized transducing bacteriophages for uropathogenic strains of Escherichia coli.

The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.