Efficacy of a Broad Host Range Lytic Bacteriophage Against E. coli Adhered to Urothelium

Abstract. Lingga R, Budiarti S, Rusmana I, Wahyu AT. 2020. Isolation, characterization and efficacy of lytic bacteriophages against pathogenic Escherichia coli from hospital liquid waste. Biodiversitas 21: 3234-3241. Escherichia coli is known as a pathogenic contaminant bacteria in hospital wastewater hazardous to humans and the environment. Concerns about the emergence of chlorine- and antibiotic-resistant bacteria increase the urgency to find an alternative strategy to control pathogenic bacteria in hospital wastewater. One of the alternatives is using lytic bacteriophage. This study aimed to isolate, characterize, and examine the efficacy of lytic bacteriophage against pathogenic Escherichia coli from hospital wastewater. It isolated, characterized (plaque morphology, host range, virion electron micrograph, and sensitivity to temperature, pH, and chlorine treatments), and tested the efficacy of lytic bacteriophages in controlling pathogenic E. coli isolated from hospital wastewater. Five phages were successfully obtained, all of which had clear plaques (lytic phage character). Based on host range assay, most of the phages could lyse all tested E. coli strains but not for other species. Electron micrograph photography revealed that the phages belonged to Myoviridae. The phages showed stability in high temperature, broad-ranged pH, and high concentrations of chlorine treatments. Assay on phages efficacy suggested that the phages are capable of significantly reducing the E. coli population both in sterilized and non-sterilized wastewater. The combination of phage treatment and chlorine was more effective than single phage treatment. The efficacy test revealed that phage application in wastewater had the best result seen from cocktail treatment and a combination of phage treatment and chlorine. These results suggested that the phage can be a potential candidate for disinfection purposes.

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LamB, OmpC, and the Core Lipopolysaccharide of Escherichia coli K-12 Function as Receptors of Bacteriophage Bp7

Journal of Virology ◽

10.1128/jvi.00325-20 ◽

2020 ◽

Vol 94 (12) ◽

Cited By ~ 1

Author(s):

Peipei Chen ◽

Huzhi Sun ◽

Huiying Ren ◽

Wenhua Liu ◽

Guimei Li ◽

...

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Inner Core ◽

Phage Infection ◽

Broad Host Range ◽

Content Type ◽

E Coli ◽

Phage Adsorption ◽

Specific Receptors ◽

K 12

ABSTRACT Bp7 is a T-even phage with a broad host range specific to Escherichia coli, including E. coli K-12. The receptor binding protein (RBP) of bacteriophages plays an important role in the phage adsorption process and determines phage host range, but the molecular mechanism involved in host recognition of phage Bp7 remains unknown. In this study, the interaction between phage Bp7 and E. coli K-12 was investigated. Based on homology alignment, amino acid sequence analysis, and a competitive assay, gp38, located at the tip of the long tail fiber, was identified as the RBP of phage Bp7. Using a combination of in vivo and in vitro approaches, including affinity chromatography, gene knockout mutagenesis, a phage plaque assay, and phage adsorption kinetics analysis, we identified the LamB and OmpC proteins on the surface of E. coli K-12 as specific receptors involved in the first step of reversible phage adsorption. Genomic analysis of the phage-resistant mutant strain E. coli K-12-R and complementation tests indicated that HepI of the inner core of polysaccharide acts as the second receptor recognized by phage Bp7 and is essential for successful phage infection. This observation provides an explanation of the broad host range of phage Bp7 and provides insight into phage-host interactions. IMPORTANCE The RBPs of T4-like phages are gp37 and gp38. The interaction between phage T4 RBP gp37 and its receptors has been clarified by many reports. However, the interaction between gp38 and its receptors during phage adsorption is still not completely understood. Here, we identified phage Bp7, which uses gp38 as an RBP, and provided a good model to study the phage-host interaction mechanisms in an enterobacteriophage. Our study revealed that gp38 of phage Bp7 recognizes the outer membrane proteins (OMPs) LamB and OmpC of E. coli K-12 as specific receptors and binds with them reversibly. HepI of the inner-core oligosaccharide is the second receptor and binds with phage Bp7 irreversibly to begin the infection process. Determining the interaction between the phage and its receptors will help elucidate the mechanisms of phage with a broad host range and help increase understanding of the phage infection mechanism based on gp38.

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Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery

Journal of Bacteriology ◽

10.1128/jb.00621-10 ◽

2010 ◽

Vol 192 (24) ◽

pp. 6418-6427 ◽

Cited By ~ 132

Author(s):

Lionel Ferrières ◽

Gaëlle Hémery ◽

Toan Nham ◽

Anne-Marie Guérout ◽

Didier Mazel ◽

...

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Random Mutagenesis ◽

Broad Host Range ◽

Donor Strain ◽

Bacteriophage Mu ◽

Genetic Determinants ◽

Sensitive Species ◽

E Coli ◽

Original Construction

ABSTRACT Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.

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A broad-host-range event detector: expanding and quantifying performance between Escherichia coli and Pseudomonas species

Synthetic Biology ◽

10.1093/synbio/ysaa002 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Nymul Khan ◽

Enoch Yeung ◽

Yuliya Farris ◽

Sarah J Fansler ◽

Hans C Bernstein

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Memory Device ◽

Broad Host Range ◽

Complex Environments ◽

New Host ◽

E Coli ◽

Pseudomonas Fluorescens Sbw25 ◽

The Common ◽

New Applications

Abstract Modern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli. It is clear that new applications such as chemical sensing and event logging in complex environments will benefit from new host chassis. This study quantitatively compared how the same chemical event logger performed across four strains and three different microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads—Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two benchmark E. coli chassis including the probiotic Nissle 1917 and common cloning strain DH5α. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and downstream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5α in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.

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Broad-Host-Range Yersinia Phage PY100: Genome Sequence, Proteome Analysis of Virions, and DNA Packaging Strategy

Journal of Bacteriology ◽

10.1128/jb.01402-07 ◽

2007 ◽

Vol 190 (1) ◽

pp. 332-342 ◽

Cited By ~ 28

Author(s):

Dominik Schwudke ◽

Asgar Ergin ◽

Kathrin Michael ◽

Sven Volkmar ◽

Bernd Appel ◽

...

Keyword(s):

Host Range ◽

Proteome Analysis ◽

Open Reading Frames ◽

Broad Host Range ◽

Lytic Bacteriophage ◽

Coding Region ◽

Putative Gene ◽

Packaging Strategy ◽

Virion Particles ◽

Tail Proteins

ABSTRACT PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.

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Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria

mBio ◽

10.1128/mbio.01291-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 15

Author(s):

Jia Jia Zhang ◽

Xiaoyu Tang ◽

Michelle Zhang ◽

Darlene Nguyen ◽

Bradley S. Moore

Keyword(s):

Heterologous Expression ◽

Host Range ◽

Regulatory Elements ◽

Cloning And Expression ◽

Broad Host Range ◽

Implicit Assumption ◽

Gram Negative ◽

E Coli ◽

Expression Platform ◽

Violacein Production

ABSTRACTHeterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC fromPseudoalteromonas luteoviolacea2ta16 revealed robust production in two proteobacterial hosts,Pseudomonas putidaKT2440 andAgrobacterium tumefaciensLBA4404, but very little production of the antibiotic in various laboratory strains ofEscherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor fromP. luteoviolacea2ta16, PviR, that increases pathway transcription and violacein production inE. coliby ∼60-fold independently of acyl-homoserine lactone autoinducers. AlthoughE. coliharbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of variousE. colitranscription factors did not result in a statistically significant increase in violacein production, while overexpression of twoA. tumefaciensPviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.IMPORTANCEAlthough Gram-positive heterologous hosts such asStreptomyceshave been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs in Gram-negative proteobacterial expression hosts, and we leverage this platform to uncover regulatory elements that influence violacein expression fromPseudoalteromonas. Although it is often assumed that BGCs will be more successfully expressed in more closely related hosts, our work suggests that this may not be a general rule of thumb, as heterologous production of natural products can be influenced by specific host regulatory and/or biosynthetic elements, and the identity and effectiveness of those elements are difficult to predict. We argue for the use of a diverse set of heterologous hosts, which may also provide insights into the BGC biosynthetic mechanism and the biological function of BGCs.

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Expanding the toolbox of broad host-range transcriptional terminators for Proteobacteria through metagenomics

10.1101/485938 ◽

2018 ◽

Author(s):

Vanesa Amarelle ◽

Ananda Sanches-Medeiros ◽

Rafael Silva-Rocha ◽

María-Eugenia Guazzaroni

Keyword(s):

Escherichia Coli ◽

Synthetic Biology ◽

Pseudomonas Putida ◽

Host Range ◽

Broad Host Range ◽

Functional Metagenomics ◽

Alternative Hosts ◽

E Coli ◽

Broad Host Range Plasmid

AbstractAs the field of synthetic biology moves towards the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacteria Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in Pseudomonas putida. Further characterization allowed the identification of 4 unique sequences between 58 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

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Inhibition of Antimicrobial-Resistant Escherichia coli Using a Broad Host Range Phage Cocktail Targeting Various Bacterial Phylogenetic Groups

Frontiers in Microbiology ◽

10.3389/fmicb.2021.699630 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jinshil Kim ◽

Haejoon Park ◽

Sangryeol Ryu ◽

Byeonghwa Jeon

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Host Range ◽

Microscopic Analysis ◽

Broad Host Range ◽

Phylogenetic Groups ◽

Electron Microscopic ◽

E Coli ◽

Phage Cocktail ◽

Transmission Electron Microscopic Analysis

Antimicrobial-resistant (AMR) commensal Escherichia coli is a major reservoir that disseminates antimicrobial resistance to humans through the consumption of contaminated foods, such as retail poultry products. This study aimed to control AMR E. coli on retail chicken using a broad host range phage cocktail. Five phages (JEP1, 4, 6, 7, and 8) were isolated and used to construct a phage cocktail after testing infectivity on 67 AMR E. coli strains isolated from retail chicken. Transmission electron microscopic analysis revealed that the five phages belong to the Myoviridae family. The phage genomes had various sizes ranging from 39 to 170 kb and did not possess any genes associated with antimicrobial resistance and virulence. Interestingly, each phage exhibited different levels of infection against AMR E. coli strains depending on the bacterial phylogenetic group. A phage cocktail consisting of the five phages was able to infect AMR E. coli in various phylogenetic groups and inhibited 91.0% (61/67) of AMR E. coli strains used in this study. Furthermore, the phage cocktail was effective in inhibiting E. coli on chicken at refrigeration temperatures. The treatment of artificially contaminated raw chicken skin with the phage cocktail rapidly reduced the viable counts of AMR E. coli by approximately 3 log units within 3 h, and the reduction was maintained throughout the experiment without developing resistance to phage infection. These results suggest that phages can be used as a biocontrol agent to inhibit AMR commensal E. coli on raw chicken.

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Isolation and Analysis of Donor Chromosomal Genes Whose Deficiency Is Responsible for Accelerating Bacterial and Trans-Kingdom Conjugations by IncP1 T4SS Machinery

Frontiers in Microbiology ◽

10.3389/fmicb.2021.620535 ◽

2021 ◽

Vol 12 ◽

Author(s):

Fatin Iffah Rasyiqah Mohamad Zoolkefli ◽

Kazuki Moriguchi ◽

Yunjae Cho ◽

Kazuya Kiyokawa ◽

Shinji Yamamoto ◽

...

Keyword(s):

Host Range ◽

Gene Knockout ◽

Synergistic Effects ◽

Conjugal Transfer ◽

Homologous Gene ◽

Broad Host Range ◽

Double Knockout ◽

E Coli ◽

Knockout Mutants ◽

A Genome

Conjugal transfer is a major driving force of genetic exchange in eubacteria, and the system in IncP1-type broad-host-range plasmids transfers DNA even to eukaryotes and archaea in a process known as trans-kingdom conjugation (TKC). Although conjugation factors encoded on plasmids have been extensively analyzed, those on the donor chromosome have not. To identify the potential conjugation factor(s), a genome-wide survey on a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection) as donors to Saccharomyces cerevisiae recipients was performed using a conjugal transfer system mediated by the type IV secretion system (T4SS) of the IncP1α plasmid. Out of 3,884 mutants, three mutants (ΔfrmR, ΔsufA, and ΔiscA) were isolated, which showed an increase by one order of magnitude in both E. coli–E. coli and E. coli–yeast conjugations without an increase in the mRNA accumulation level for the conjugation related genes examined. The double-knockout mutants for these genes (ΔfrmRΔsufA and ΔiscAΔfrmR) did not show synergistic effects on the conjugation efficiency, suggesting that these factors affect a common step in the conjugation machinery. The three mutants demonstrated increased conjugation efficiency in IncP1β-type but not in IncN- and IncW-type broad-host-range plasmid transfers, and the homologous gene knockout mutants against the three genes in Agrobacterium tumefaciens also showed increased TKC efficiency. These results suggest the existence of a specific regulatory system in IncP1 plasmids that enables the control of conjugation efficiency in different hosts, which could be utilized for the development of donor strains as gene introduction tools into bacteria, eukaryotes, and archaea.

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Characterization of novel bacteriophage phiC119 capable of lysing multidrug-resistant Shiga toxin-producingEscherichia coliO157:H7

PeerJ ◽

10.7717/peerj.2423 ◽

2016 ◽

Vol 4 ◽

pp. e2423 ◽

Cited By ~ 7

Author(s):

Luis Amarillas ◽

Cristóbal Chaidez ◽

Arturo González-Robles ◽

Yadira Lugo-Melchor ◽

Josefina León-Félix

Keyword(s):

Host Range ◽

Shiga Toxin ◽

Pathogenic Bacteria ◽

Genetic Material ◽

Multidrug Resistant ◽

Broad Host Range ◽

Antibiotic Resistant ◽

E Coli ◽

Tract Infections

BackgroundShiga toxin-producingEscherichia 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-resistantE. colistrains 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.MethodsIn this study, we describe the characterization of a bacteriophage designated phiC119, which can infect and lyse several multidrug-resistant STEC strains and someSalmonellastrains. The phage genome was screened to detect thestx-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.ResultsAnalysis of the bacteriophage particles by transmission electron microscopy showed that it had an icosahedral head and a long tail, characteristic of the familySiphoviridae. The phage exhibits broad host range against multidrug-resistant and highly virulentE. coliisolates. 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.ConclusionThese 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.

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