Project description:The genomes of many strains of Escherichia coli have been sequenced, as this organism is a classic model bacterium. Here, we report the genome sequence of Escherichia coli DH5?, which is resistant to a T4 bacteriophage (CCTCC AB 2015375), while its other homologous E. coli strains, such as E. coli BL21, DH10B, and MG1655, are not resistant to phage invasions. Thus, understanding of the genome of the DH5? strain, along with comparative analysis of its genome sequence along with other sequences of E. coli strains, may help to reveal the bacteriophage resistance mechanism of E. coli.
Project description:In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria.
Project description:Chicken colibacillosis is caused by some pathogenic Escherichia coli strains. Thirty-five pathogenic antibiotic-resistant E. coli strains were used in the host range detection of bacteriophage Bp7. The phage showed a wide range of E. coli hosts (46%). The complete genome of bacteriophage Bp7 was sequenced, assembled, and analyzed. The results revealed a linear double-stranded DNA sequence of 168,066 bp harboring 791 open reading frames. The major findings from its annotation are described.
Project description:A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.
Project description:Bacteriophages screened and isolated from sewage water samples exhibited antibacterial activities against multi-drug-resistant Escherichia coli strains. Five different water samples from Canadian habitats such as Kamloops Wastewater Treatment Center, Domtar, the Pacific Ocean, Bisaro Anima Cave, and alkali ponds, were used in this study. Four Enterobacteriaceae strains including one non-resistant and three clinical multi-drug Escherichia coli strains (E. coli 15-102, E. coli 15-124, and E. coli 15-318) were selected as target bacteria to screen for the bacteriophages from these collected water samples. Seeded agar assay technique was implemented for the screening. It was found that only sewage water sample exhibited a significant number of plaques count with the E. coli 15-318 (1.82 × 10² plaques/plate) cells in comparison to E. coli non-resistant strain K12 (8 plaques/plate). The phage did not produce plaques in the E. coli 15-124 and E. coli 15-102 strains. The bacteriophage, designated EMCL318, was isolated, purified, characterized, and identified to belong to the G4 species of the Family Microviridae, GenBank accession number MG563770. This is an explorative study conducted in order to reveal the viruses as alternative potentials to fight against emerging and existing multi-drug-resistant infectious diseases.
Project description:Bacteria and virulent bacteriophages are in a prey-predator relationship. Experimental models under simplified conditions with the presence of bacteria and bacteriophages have been used to elucidate the mechanisms that have enabled both prey and predator to coexist over long periods. In experimental coevolution conducted with Escherichia coli and the virulent RNA bacteriophage Q? in serial transfer, both coexisted for at least for 54 days, during which time they continued to change genetically and phenotypically. By day 16, an E. coli strain partially resistant to Q? appeared and caused an approximately 10(4)-fold decrease in Q? amplification. Whole-genome analysis of this strain suggested that a single mutation in TraQ was responsible for the partially resistant phenotype. TraQ interacts with propilin, encoded by the traA gene and a precursor of pilin, which is a component of the F pilus. The present study was performed to elucidate the mechanism underlying the coexistence of E. coli and Q? by investigating how a mutation in TraQ altered the physiological state of E. coli, and thus the amplification of Q?. Overexpression of wild-type TraQ in the partially resistant E. coli strain resulted in recovery of both TraA protein content, including propilin and pilin, and Q? amplification to levels comparable to those observed in the susceptible strain. Intriguingly, overexpression of the mutant TraQ in the partially resistant strains also increased the levels of TraA protein and Q? amplification, but these increases were smaller than those observed in the wild-type strain or the partially resistant strain expressing wild-type TraQ. The results of this study represent an example of how E. coli can become partially resistant to RNA bacteriophage infection via changes in a protein involved in maturation of a receptor rather than in the receptor itself and of how E. coli can stably coexist with virulent RNA bacteriophages.
Project description:The objective of this study was to determine the genomic changes that underlie coevolution between Escherichia coli B and bacteriophage T3 when grown together in a laboratory microcosm. We also sought to evaluate the repeatability of their evolution by studying replicate coevolution experiments inoculated with the same ancestral strains. We performed the coevolution experiments by growing Escherichia coli B and the lytic bacteriophage T3 in seven parallel continuous culture devices (chemostats) for 30 days. In each of the chemostats, we observed three rounds of coevolution. First, bacteria evolved resistance to infection by the ancestral phage. Then, a new phage type evolved that was capable of infecting the resistant bacteria as well as the sensitive bacterial ancestor. Finally, we observed second-order resistant bacteria evolve that were resistant to infection by both phage types. To identify the genetic changes underlying coevolution, we isolated first- and second-order resistant bacteria as well as a host-range mutant phage from each chemostat and sequenced their genomes. We found that first-order resistant bacteria consistently evolved resistance to phage via mutations in the gene, waaG, which codes for a glucosyltransferase required for assembly of the bacterial lipopolysaccharide (LPS). Phage also showed repeatable evolution, with each chemostat producing host-range mutant phage with mutations in the phage tail fiber gene T3p48 which binds to the bacterial LPS during adsorption. Two second-order resistant bacteria evolved via mutations in different genes involved in the phage interaction. Although a wide range of mutations occurred in the bacterial waaG gene, mutations in the phage tail fiber were restricted to a single codon, and several phage showed convergent evolution at the nucleotide level. These results are consistent with previous studies in other systems that have documented repeatable evolution in bacteria at the level of pathways or genes and repeatable evolution in viruses at the nucleotide level. Our data are also consistent with the expectation that adaptation via loss-of-function mutations is less constrained than adaptation via gain-of-function mutations.
Project description:The emergence of antibiotic-resistant foodborne bacteria is a global health problem that requires immediate attention. Bacteriophages are a promising biotechnological alternative approach against bacterial pathogens. However, a detailed analysis of phage genomes is essential to assess the safety of the phages prior to their use as biocontrol agents. Therefore, here we report the complete genome sequence of bacteriophage phiE142, which is able to lyse Salmonella and multidrug-resistant Escherichia coli O157:H7 strains. Bacteriophage phiE142 belongs to the Myoviridae family due to the presence of long non-flexible tail and icosahedral head. The genome is composed of 121,442 bp and contains 194 ORFs, and 2 tRNAs. Furthermore, the phiE142 genome does not contain any genes coding for food-borne allergens, antibiotics resistance, virulence factors, or associated with lysogenic conversion. The bacteriophage phiE142 is characterized by broad host range and compelling genetic attributes making them potential candidates as a biocontrol agent.
Project description:BACKGROUND:Shiga toxin-producing Escherichia coli (STEC) is one of the most common and widely distributed foodborne pathogens that has been frequently implicated in gastrointestinal and urinary tract infections. Moreover, high rates of multiple antibiotic-resistant E. coli strains have been reported worldwide. Due to the emergence of antibiotic-resistant strains, bacteriophages are considered an attractive alternative to biocontrol pathogenic bacteria. Characterization is a preliminary step towards designing a phage for biocontrol. METHODS:In this study, we describe the characterization of a bacteriophage designated phiC119, which can infect and lyse several multidrug-resistant STEC strains and some Salmonella strains. The phage genome was screened to detect the stx-genes using PCR, morphological analysis, host range was determined, and genome sequencing were carried out, as well as an analysis of the cohesive ends and identification of the type of genetic material through enzymatic digestion of the genome. RESULTS:Analysis of the bacteriophage particles by transmission electron microscopy showed that it had an icosahedral head and a long tail, characteristic of the family Siphoviridae. The phage exhibits broad host range against multidrug-resistant and highly virulent E. coli isolates. One-step growth experiments revealed that the phiC119 phage presented a large burst size (210 PFU/cell) and a latent period of 20 min. Based on genomic analysis, the phage contains a linear double-stranded DNA genome with a size of 47,319 bp. The phage encodes 75 putative proteins, but lysogeny and virulence genes were not found in the phiC119 genome. CONCLUSION:These results suggest that phage phiC119 may be a good biological control agent. However, further studies are required to ensure its control of STEC and to confirm the safety of phage use.