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Objectives This study aimed to isolate lytic bacteriophage to control EHEC and ETEC, characterize, and apply their ability to control these two bacteria in food. Results One of the bacteria that contaminate the food is Diarrheagenic Escherichia coli (DEC) which was consist of Enterohaemorrhagic E. coli and Enteropathogenic E. coli . Preservation methods were used to improve shelf-life to reduce microbial growth but it might have side effect to the nutrition and human health. Therefore, it is required to explore an alternative method that can be used to be a biocontrol agent for food, including the bacteriophage approach. Our study showed that these two phages are promising to be used as biocontrol agent in food. Genomic DNA sequencing performed that this phage being exclusively lytic.

Background The formation of biofilms and subsequent encasement of bacterial cells in a complex matrix can enhance resistance to antimicrobials and sterilizing agents making these organisms difficult to eradicate and control. The aim of this study was to evaluate and compare the capacity of 40 E. coli O26 isolates of enterohemorrhagic E. coli (EHEC, n  = 27), potential EHEC (pEHEC, n  = 3), atypical enteropathogenic E. coli (aEPEC, n  = 8) and non-toxigenic E. coli (NTEC, n  = 2) from human and cattle sources to form biofilms on different surfaces, and determine whether extracellular matrix (ECM) components (cellulose, curli), motility, prophage insertion in mlrA and cell surface hydrophobicity could influence biofilm formation. Finally, the influence of biofilm formation on the sensitivity of isolates to quaternary ammonium compounds (QACs; Profoam, Kwiksan 22) and peracetic acid-based sanitizer (Topactive Des.) for 2 min on polystyrene plate were also evaluated. Results Biofilm production on one surface may not indicate biofilm formation on a different surface. Biofilm was formed by different pathotypes on polystyrene (70%), stainless steel (87.5%) and glass slides (95%), however only 50% demonstrated pellicle formation. EHEC isolates were significantly more likely to form a pellicle at the air-liquid interface and biofilms on polystyrene surface at 48 h than aEPEC. Strains that don’t produce ECM (curli or cellulose), harbor a prophage insertion in mlrA, and are non-motile have lower biofilm forming capacities than those isolates possessing combinations of these attributes. Hydrophobicity had no impact on biofilm formation. After 2 min exposure, none of the disinfectants tested were able to completely inactivate all cells within a biofilm regardless of pathotypes and the amount of biofilm formed. Conclusion Pathotypes of E. coli O26 showed varying capacities to form biofilms, however, most EHEC strains had the capacity to form biofilm on all surfaces and at the air-liquid interface under the conditions used in this study. Biofilms provided a protective effect to E. coli O26 strains against the three sanitizers, previously shown to successfully control the growth of their planktonic counterparts. Whether the characteristics of biofilm forming and non-biofilm forming strains observed in this study reflect their attributes within the food and meat-processing environments is unknown. Further studies that represent the food and meat-processing environments are required.

Background Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases. Results We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains. Conclusions Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a Bsu BI/ Pst I methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.

Background Enterohemorrhagic Escherichia coli (EHEC) O145 are among the top non-O157 serogroups associated with severe human disease worldwide. Two serotypes, O145:H25 and O145:H28 have been isolated from human patients but little information is available regarding the virulence repertoire, origin and evolutionary relatedness of O145:H25. Hence, we sequenced the complete genome of two O145:H25 strains associated with hemolytic uremic syndrome (HUS) and compared the genomes with those of previously sequenced O145:H28 and other EHEC strains. Results The genomes of the two O145:H25 strains were 5.3 Mbp in size; slightly smaller than those of O145:H28 and other EHEC strains. Both strains contained three nearly identical plasmids and several prophages and integrative elements, many of which differed significantly in size, gene content and organization as compared to those present in O145:H28 and other EHECs. Furthermore, notable variations were observed in several fimbrial gene cluster and intimin types possessed by O145:H25 and O145:H28 indicating potential adaptation to distinct areas of host colonization. Comparative genomics further revealed that O145:H25 are genetically more similar to other non-O157 EHEC strains than to O145:H28. Conclusion Phylogenetic analysis accompanied by comparative genomics revealed that O145:H25 and O145:H28 evolved from two separate clonal lineages and that horizontal gene transfer and gene loss played a major role in the divergence of these EHEC serotypes. The data provide further evidence that ruminants might be a possible reservoir for O145:H25 but that they might be impaired in their ability to establish a persistent colonization as compared to other EHEC strains.

Bacteriophages, simply phages, have long been used as a potential alternative to antibiotics for livestock due to their ability to specifically kill enterotoxigenic Escherichia coli (ETEC), which is a major cause of diarrhea in piglets. However, the control of ETEC infection by phages within intestinal epithelial cells, and their relationship with host immune responses, remain poorly understood. In this study, we evaluated the effect of phage EK99P-1 against ETEC K99-infected porcine intestinal epithelial cell line (IPEC-J2). Phage EK99P-1 prevented ETEC K99-induced barrier disruption by attenuating the increased permeability mediated by the loss of tight junction proteins such as zonula occludens-1 (ZO-1), occludin, and claudin-3. ETEC K99-induced inflammatory responses, such as interleukin (IL)-8 secretion, were decreased by treatment with phage EK99P-1. We used a IPEC-J2/peripheral blood mononuclear cell (PBMC) transwell co-culture system to investigate whether the modulation of barrier disruption and chemokine secretion by phage EK99P-1 in ETEC K99-infected IPEC-J2 would influence immune cells at the site of basolateral. The results showed that phage EK99P-1 reduced the mRNA expression of ETEC K99-induced pro-inflammatory cytokines, IL-1β and IL-8 , from PBMC collected on the basolateral side. Together, these results suggest that phage EK99P-1 prevented ETEC K99-induced barrier dysfunction in IPEC-J2 and alleviated inflammation caused by ETEC K99 infection. Reinforcement of the intestinal barrier, such as regulation of permeability and cytokines, by phage EK99P-1 also modulates the immune cell inflammatory response.

A novel polyvalent broad-spectrum phage, vB_EcoM_XAM237 (XAM237), was isolated from pig farm sewage. It can simultaneously lyse multiple strains of pathogenic Escherichia coli ( E. coli ), demonstrating a broad host range. When the enteropathogenic E. coli (EPEC) strain E711 was used as the host bacterium, the phage XAM237 exhibited a short latent period, high stability at different temperatures and pH values and good tolerance to chloroform. Moreover, phage XAM237 can efficiently adsorb and lyse host bacteria in vitro. Whole-genome sequencing revealed that XAM237 is a double-stranded DNA (dsDNA) phage consisting of 170 541 bp with a G + C content of 35%. Phylogenetic analysis confirmed that XAM237 belongs to the family Straboviridae , genus Tequatrovirus . In addition, the genome of XAM237 did not contain genes related to lysogenicity, virulence or antimicrobial resistance. The effects of phage XAM237 in treating EPEC infections in vivo were evaluated in a mouse model. Phage XAM237 was able to reduce the number of colonized aEPEC strain E711 in the small intestine, liver, spleen, and kidney. This study suggested that phage XAM237 may be a promising candidate biologic agent for controlling pathogenic E. coli infections.

E. coli mastitis is a major production disease in dairy cattle and requires alternative treatments to antibiotics. In this context, phages are of growing interest, but their host specificity remains a challenge in therapy. This study aimed to characterize eight newly isolated phages for the control of E. coli mastitis, to assess their efficacy in milk and to investigate their specificity. Physicochemical characterization of the phages was performed, followed by in vitro stability and lytic activity assays in raw and heat-treated milks. Genome sequencing of phages and bacteria was performed to investigate phage attachment. Phage stability was maintained across physiological pH and temperature ranges, as well as in raw milk. Phage lytic activity demonstrated bacterial decreases below detection level, but regrowth occurred in raw milk after 5 h of incubation with 3/8 phages. A narrow host range was linked to the diversity of the bacterial collection and to the presence of two receptor-binding proteins among Tevenvirinae . Indeed, structural analysis of the proteins revealed a variable region in the long tail fiber and a conserved short tail fiber. In conclusion, phage specificity was mainly associated with the long tail fiber and milk components didn’t hinder the efficacy of phages to control bovine mastitis, although resistance should be investigated.

Prophages are prevalent features of bacterial genomes that can reduce susceptibility to infection by competing phages, yet the mechanisms involved are often elusive. Here, we identify a small RNA (svsR) encoded by the lambdoid prophage NC-SV in adherent-invasive Escherichia coli strain NC101 that limits infection by virulent coliphages. Comparative genomics revealed that NC-SV–like prophages and svsR homologs are broadly conserved across Enterobacteriaceae. Transcriptomic analyses show that svsR represses maltodextrin transport genes, including lamB , which encodes the outer membrane maltoporin LamB, a known receptor for numerous coliphages. Deletion of the lamB gene reveals that while LamB is not required for replication of the virulent phages tested, it contributes to plaque expansion, indicating a role in phage spread but not as an essential receptor. Nutrient supplementation experiments further linked maltodextrin and glucose availability to changes in plaque expansion and phage adsorption. In vivo, we compared wild-type NC101 and a prophage-deletion strain (NC101 ∆NC-SV ) in mice to assess the impact of NC-SV on lytic phage susceptibility. Although intestinal E. coli densities remained stable across groups, animals colonized with NC101 exhibited markedly reduced phage burdens in both the intestinal lumen and mucosa compared to mice colonized with NC101 ∆NC-SV . This reduced phage pressure was associated with increased dissemination of E. coli to extraintestinal tissues, including the spleen and liver. Together, these findings highlight a nutrient-responsive, prophage-encoded mechanism that protects E. coli from phage predation and may promote bacterial persistence in and dissemination from the mammalian gut.

The global rise of multidrug-resistant Escherichia coli ST410 presents a growing clinical threat. We isolated a highly lytic phage targeting ST410, but rapid resistance emerged both in vitro and in vivo . Beyond receptor mutations, we identified a hypermucoid mutant that evades phage infection primarily through colanic acid overproduction, providing broad phage resistance. This colanic acid overproduction also led to diminished macrophage phagocytosis in vitro and accelerated mortality in mice. Transcriptomic and genetic analyses linked this phenotype to activation of the Rcs pathway. Notably, naturally occurring ST410 isolates with colanic acid-overproducing phenotypes were also observed, suggesting that capsule-mediated immune evasion may arise in clinical populations. To overcome resistance, we designed a rational four-phage cocktail with diverse receptors, achieving robust therapeutic efficacy in infection models. Our findings highlight that phage resistance can be accompanied by enhanced virulence potential through capsule-mediated immune evasion, with colanic acid playing a central role, emphasizing important considerations for phage therapy design.

Avian Pathogenic Escherichia coli (APEC) cause colibacillosis leading to significant economic losses in the poultry industry. This laboratory-based study aimed at establishing stocks of avian pathogenic Escherichia coli lytic bacteriophages, for future development of cocktail products for colibacillosis management. The study determined the antibiotic susceptibility; phylogenetic categories, occurrence of selected serotypes and virulence genes among Escherichia coli stock isolates from chicken colibacillosis cases; and evaluated bacteriophage activity against the bacteria. Escherichia coli characterization was done through phenotypic and multiplex PCR methods. Bacteriophage isolation and preliminary characterization was achieved using the spot assay and overlay plating techniques. Fifty-six (56) isolates were phenotypically confirmed as E . coli and all exhibited resistance to at least one antimicrobial agent; while multi-drug resistance (at least three drugs) was encountered in 50 (89.3%) isolates. The APEC isolates mainly belonged to phylogroups A and D, representing 44.6% and 39.3%, respectively; whereas serotypes O1, O2 and O78 were not detected. Of the 56 isolates, 69.6% harbored at least one virulence gene, while 50% had at least four virulence genes; hence confirmed as APEC. Virulence genes, ompT and iutA were the most frequent in 33 (58.9%) and 32 (57.1%) isolates respectively; while iroN least occurred in 23 (41.1%) isolates. Seven lytic bacteriophages were isolated and their host range, at 1×10 8 PFU/ml, varied from 1.8% to 17.9% of the 56 APEC isolates, while the combined lytic spectrum was 25%. Phage stability was negatively affected by increasing temperatures with both UPEC04 and UPEC10 phages being undetectable at 70°C; whereas activity was detected between pH 2 and 12. The high occurrence of APEC isolates resistant against the commonly used antibiotics supports the need for alternative strategies of bacterial infections control in poultry. The low host range exhibited by the phages necessitates search for more candidates before in-depth phage characterization and application.