Edited by: Giovanna Suzzi, University of Teramo, Italy
Reviewed by: David Damian Tomat, Faculty of Biochemical and Pharmaceutical Science, Argentina; Douwe Van Sinderen, University College Cork, Ireland
*Correspondence: Josefina León-Félix
This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology
†These authors have contributed equally to this work.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Foodborne diseases are a serious and growing problem, and the incidence and prevalence of antimicrobial resistance among foodborne pathogens is reported to have increased. The emergence of antibiotic-resistant bacterial strains demands novel strategies to counteract this epidemic. In this regard, lytic bacteriophages have reemerged as an alternative for the control of pathogenic bacteria. However, the effective use of phages relies on appropriate biological and genomic characterization. In this study, we present the isolation and characterization of a novel bacteriophage named phiLLS, which has shown strong lytic activity against generic and multidrug-resistant
Foodborne diseases are an important cause of morbidity and mortality worldwide, and their incidence has increased globally (Torgerson et al.,
Recently, multidrug-resistant
The worldwide emergence of antibiotic-resistant bacterial strains creates the need for implementing means to control these threats. The viral–lytic organisms termed bacteriophages (phages) have reemerged as a promising alternative for the control of pathogenic bacteria (Hagens and Loessner,
The T-even type of bacteriophages are known by a strictly lytic (virulent) life style, degradation of the host chromosome, and broad host ranges against pathogenic bacteria (Onodera,
Additionally, although not strongly correlated, the virion morphology characteristic is another factor that may also be an important criterion for selecting phages for biocontrol applications. Usually,
Phages may encode virulence factor genes. Therefore, the complete genome needs to be sequenced to determine whether bacteriophages are suitable to control pathogenic bacteria and whether it is useful to expand our understanding of phage characteristics (Clark and March,
The aim of this study was to isolate and characterize a polyvalent phage with a wide spectrum of activity as a potential biocontrol agent of multidrug-resistant strains of
The bacterial strains used in this study are listed in Table
Wild-type bacterial strains used for the host range spectrum of the bacteriophage phiLLS.
RM8745 | O73:H4 | − |
RM8746 | O73:H4 | − |
RM8747 | O15: NT | + |
RM8748 | O73: NT | + |
RM8749 | O20:H4 | + |
RM8750 | O20:H4 | + |
RM8751 | O20:H4 | + |
RM8752 | O75:H8 | + |
RM8755 | O111:H8 | − |
RM8756 | O146:H21 | − |
RM8757 | O146:H21 | − |
RM8758 | O146:H21 | − |
RM8760 | O75:H8 | + |
RM8761 | O146:H21 | − |
RM8762 | O146:H8 | − |
RM8763 | O75:H8 | + |
RM8764 | O75:H8 | − |
RM8765 | O75:H8 | + |
RM8772 | O8:H19 | − |
RM8773 | O8:H19 | − |
RM8774 | O8:H19 | − |
RM8775 | O8:H19 | − |
RM8776 | O8:H19 | − |
RM8778 | O75:H8 | + |
RM8779 | O75:H8 | + |
RM8780 | O75:H8 | + |
RM8916 | O111:H8 | − |
RM8917 | O168: NT | + |
RM8929 | O75:H8 | + |
RM8930 | O75:H8 | + |
RM8744 | O157:H7 | + |
RM8753 | O157:H7 | + |
RM8754 | O157:H7 | + |
RM8759 | O157:H7 | + |
RM8767 | O157:H7 | + |
RM8768 | O157:H7 | + |
RM8769 | O157:H7 | + |
RM8771 | O157:H7 | + |
RM8781 | O157:H7 | + |
RM8921 | O157:H7 | + |
RM8922 | O157:H7 | + |
RM8927 | O157:H7 | + |
RM8928 | O157:H4 | + |
RM9450 | O157:H7 | + |
RM9451 | O157:H7 | + |
RM9452 | O157:H7 | + |
RM9453 | O157:H7 | + |
RM9454 | O157:H7 | + |
RM9455 | O157:H7 | + |
RM9456 | O157:H7 | + |
RM9457 | O157:H7 | + |
RM9458 | O157:H7 | − |
RM9459 | O157:H7 | − |
RM9460 | O157:H7 | + |
RM9461 | O157:H7 | + |
RM9462 | O157:H7 | − |
RM9463 | O157:H7 | + |
Pond water and wastewater samples were collected between November and December 2015 in different regions in Sinaloa, Mexico. The samples were assayed for the presence of phages capable of forming plaques on
Electron micrographs of purified phage particles were obtained according to standard method. Suspension phage sample was dropped (approximately 30 μL) onto 400-mesh carbon-coated Formvar covered grids placed in a vacuum evaporator (JEE400, JEOL Ltd. Tokyo, Japan), stained with 2% (wt/vol) phosphotungstic acid (pH 7.2), and air dried. Samples were examined in a transmission electron microscope (JEM-1011, JEOL Ltd. Tokyo, Japan) at an acceleration voltage of 80 kV, and phage particles were examined at 15,000–25,000 times magnification.
The host range of the phage was tested against 57 strains environmental isolated by the spot method (Kutter,
The experiment to determine the latent period and phage burst size was carried as described previously (Goodridge et al.,
Phage bacteriolytic activity was determined
Phage phiLLS was propagated using the double agar overlay technique as described previously by Jamalludeen et al. (
The identification of phage packaging strategies, and the type of physical ends of bacteriophage genome can often be deduced based on phylogenetic analysis of amino acid sequences of terminase large subunit of phage compared to other phages with known DNA packaging strategies (Wittmann et al.,
The predicted amino acid sequences of the large terminase subunits genes of dsDNA coliphages were retrieved from National Center for Biotechnology Information (NCBI) and were used for phylogenetic analysis. The bacteriophages included in this study has been molecularly analyzed independently from investigators throughout the world and contains the well-characterized dsDNA bacteriophages with different types of packaging strategies depend on terminase actions (headful, 5′-extended cos ends, 3′-extended cos ends and direct terminal repeats) experimentally determined. The phage large terminase proteins included are listed below with their respective accession numbers. All sequences were aligned using ClustalW in Geneious with default parameters. Phylogenetic trees were inferred using neighbor-joining algorithm and statistical support for the internal nodes was determined by 1,000 bootstrap replicates in Geneious version R9.
Additionally, the genome ends were determined as described by Casjens and Gilcrease (
Detection of the genes encoding Shiga toxin 1 (
The phage genome was sequenced using a TruSeq protocol on an Illumina HiSeq platform, with pair-end read sizes of 100 bp. The raw reads were quality checked through FastQC and trimmed with FASTQ Quality Trimmer (minimum Q30 score) available on the public Galaxy server (
Several water samples, which included ponds, creeks, streams, and canal ways, were tested for the presence of bacteriophages against
phiLLS formed clear plaques, with sizes ranging from 1.5 to 2.0 mm in diameter, and well-defined boundaries against the
Transmission electron microscopy analysis revealed that phiLLS had an isometric and icosahedral head with an estimated diameter of 56 ± 2 nm. The phage presented a non-contractile, long flexible and extremely thin tail, measuring 135 ± 5 nm in length and 15 ± 1 nm in width. The presence of a neck, a base plate, spikes, or fiber, is not seen in the mature phage. To date, bacteriophages are classified based on differences in the morphology of their virion characteristics. According to their morphological characteristics and based on guidelines of the International Committee on Taxonomy of Viruses (Fauquet et al.,
Transmission electron microscopy images of phiLLS negatively stained using 2% uranyl acetate. Negatively stained electron micrographs of phiLLS virions showing the typical morphology of phages within the family
Shiga toxin (Stx) is one of the most potent bacterial toxins, and genes encoding these toxins are located on different bacteriophages, which are integrated into the bacterial chromosome (Mauro and Koudelka,
The ability of new isolated phage to lyse pathogenic
Based on the host range studies, the newly isolated phage phiLLS possesses a broad lytic spectrum. The broad host range infectivity against a diverse collection of
The one-step growth studies were conducted to investigate the different phases of the phage infection process such as the latent period and the burst size of phage phiLLS. According to the one-step growth experiment, the latent period of phiLLS propagated on
One-step growth curve of phage phiLLS. Shown are the pfu per infected cell in the cultures at different time points. Each data point represent mean from three independent experiments, and the error bars indicate standard deviations.
To investigate the ability of phage phiLLS to lyse
Bacterial challenge test of phage phiLLS with
In general, the results of host-cell lysis caused by phage phiLLS demonstrated that the bactericidal activity was related to the MOI. In this study, the MOI 100 ratio showed the highest reduction rate of viable bacteria count. Previous studies also found that the higher MOI resulted in lower numbers of viable bacteria. However, it is also important consider that high MOI effects may attenuate bacteriophage proliferation in natural systems, which that as the result of adsorption of large numbers of phage causing destabilization of the outer membrane and subsequent bacterial lysis, preventing phage replication and release “lysis from without” (Brown and Bidle,
The ability of bacteriophages to facilitate horizontal gene transfer through transduction is an important consideration for using them for the control of pathogenic bacteria (Meaden and Koskella,
The phylogenetic analysis of the large terminase subunit suggests that phiLLS is a headful packaging phage containing a circularly permuted genome (Figure
Neighbor-joining phylogenetic tree of terminase large subunit of phiLLS and their comparison to other coliphages with known packaging mechanisms. Bootstrap analysis was performed with 1,000 repetitions. The terminase large subunits were compared using the ClustalW in Geneious program version R9. Colored boxes indicate the phages grouped into similar cluster that share same packaging strategy.
According to the results obtained, phiLLS was clustered with the terminases of phages RB49 and T4, both with DTR in their chromosome ends, this cluster share high identity indicates strong phylogenetic relationship between theses phages. Based on the close association with the large terminases of phages that have an experimentally confirmed packaging strategy, it is predicted that the genome of the phiLLS has possibly circularly permuted direct terminal repeats. To support this finding, the phage genome was treated with different restriction enzymes.
To determine whether phiLLS has cohesive ends, restriction enzyme digestion was performed and then analyzed by agarose gel electrophoresis (Figure
Enzymatic analysis of phiLLS genomic DNA. A restriction map of the genomic DNA of phage phiLLS was constructed using the restriction endonucleases
Usually, virulent
To further our understanding of phage biology, the phage phiLLS genome was sequenced. A
Features of the open reading frames of bacteriophage phiLLS and homology to proteins databases.
1 | 116 | 1,843 | + | Putative tail tip protein (PHAGE_Entero_SSL2009a_NC_012223) | 36 | 2.43E−40 | 90 |
2 | 1,899 | 2,141 | − | Hypothetical protein [Shigella phage SHSML-45] | 98 | 2.00E−43 | 85 |
3 | 2,119 | 2,565 | − | Putative deoxyUTP pyrophosphatase [Escherichia phage T5] | 91 | 2.00E−98 | 100 |
4 | 2,562 | 3,437 | − | Flap endonuclease [Escherichia phage APCEc03] | 99 | 0 | 99 |
5 | 3,437 | 3,919 | − | D14 protein [Escherichia phage T5] | 99 | 4.00E−116 | 100 |
6 | 3,923 | 5,761 | − | Putative recombination endonuclease subunit D13 [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
7 | 5,742 | 6,719 | − | Calcineurin-like phosphoesterase superfamily domain protein [Escherichia phage slur09] | 99 | 0 | 99 |
8 | 6,756 | 7,529 | − | D11 protein [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 100 |
9 | 7,522 | 7,806 | − | Hypothetical protein T5.125 [Escherichia phage T5] | 98 | 8.00E−62 | 100 |
10 | 8,027 | 9,379 | − | Putative ATP-dependent helicase [Salmonella phage Spc35] | 99 | 0 | 99 |
11 | 9,376 | 9,873 | − | Hypothetical protein CPT_Shivani113 [Salmonella phage Shivani] | 99 | 0 | 99 |
12 | 9,866 | 12,433 | − | DNA polymerase [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
13 | 12,527 | 12,856 | − | Hypothetical protein SLUR09_00081 [Escherichia phage slur09] | 99 | 9.00E−62 | 85 |
14 | 12,885 | 13,775 | − | Putative DNA replication primase [Salmonella phage Spc35] | 99 | 0 | 100 |
15 | 13,772 | 15,244 | − | Putative replicative DNA helicase [Escherichia phage Akfv33] | 99 | 0 | 100 |
16 | 15,327 | 16,094 | − | Portal vertex protein [SHSML-45] | 93 | 8.00E−163 | 100 |
17 | 16,087 | 16,866 | − | NAD-dependent DNA ligase, subunit B [Escherichia phage vB_EcoS_FFH1] | 100 | 0 | 98 |
18 | 17,069 | 18,040 | − | NAD-dependent DNA ligase, subunit A [Escherichia phage T5] | 99 | 0 | 99 |
19 | 18,041 | 18,313 | − | Hypothetical protein | |||
20 | 18,400 | 18,708 | − | Hypothetical protein T5.115 [Escherichia phage T5] | 99 | 8.00E−70 | 100 |
21 | 18,759 | 19,055 | − | Hypothetical protein T5.114 [Escherichia phage T5] | 98 | 6.00E−52 | 100 |
22 | 19,092 | 19,502 | − | D3 protein [Escherichia phage T5] | 99 | 3.00E−67 | 100 |
23 | 19,606 | 19,857 | − | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 2.00E−51 | 96 |
24 | 19,850 | 20,554 | − | D2 protein [Escherichia phage T5] | 99 | 8.00E−171 | 99 |
25 | 20,625 | 20,867 | − | Hypothetical protein SPC35_0100 [Salmonella phage Spc35] | 95 | 1.00E−48 | 99 |
26 | 20,842 | 23,631 | − | Putative replication origin binding protein [Salmonella phage Spc35] | 99 | 0 | 99 |
27 | 24,251 | 24,643 | − | Hypothetical protein APCEc03_120 [Escherichia phage APCEc03] | 99 | 5.00E−88 | 95 |
28 | 24,653 | 25,081 | − | Hypothetical protein SLUR09_00096 [Escherichia phage slur09] | 99 | 2.00E−98 | 98 |
29 | 25,084 | 25,590 | − | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 2.00E−109 | 99 |
30 | 25,577 | 25,759 | − | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 3.00E−36 | 98 |
31 | 25,750 | 26,574 | − | Putative Sir2-like protein [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 96 |
32 | 26,574 | 26,768 | − | Hypothetical protein [Escherichia phage Akfv33] | 98 | 6.00E−36 | 92 |
33 | 26,737 | 26,940 | − | Hypothetical protein SPC35_0092 [Salmonella phage Spc35] | 98 | 6.00E−39 | 100 |
34 | 26,937 | 27,296 | − | Hypothetical protein APCEc03_129 [Escherichia phage APCEc03] | 98 | 2.00E−77 | 97 |
35 | 27,395 | 29,269 | − | Anaerobic ribonucleoside triphosphate reductase [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
36 | 29,463 | 29,954 | + | Putative HNH endonuclease family protein (PHAGE_Ralsto_RSK1_NC_022915) | |||
37 | 30,140 | 30,892 | + | Phosphate starvation-inducible protein [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
38 | 30,894 | 31,115 | + | Tail length tape-measure protein 1 (PHAGE_Salmon_NR01_NC_031042) | 98 | 2.00E−45 | 100 |
39 | 31,258 | 33,588 | + | Aerobic ribonucleoside diphosphate reductase large subunit [Salmonella phage NR01] | 97 | 0 | 99 |
40 | 33,690 | 34,190 | + | Putative H-N-H-endonuclease P-TflVIII [Salmonella phage Spc35] | 98 | 8.00E−71 | 66 |
41 | 34,190 | 35,335 | + | Aerobic ribonucleoside diphosphate reductase, small subunit [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
42 | 35,332 | 35,865 | + | Putative dihydrofolate reductase [Escherichia phage APCEc03] | 99 | 2.00E−123 | 97 |
43 | 35,865 | 36,704 | + | Putative thymidylate synthase [Escherichia phage T5] | 99 | 0 | 99 |
44 | 36,805 | 37,188 | + | Hypothetical protein NR01_0022 [Salmonella phage NR01] | 99 | 1.00E−78 | 95 |
45 | 37,181 | 37,729 | + | Putative HNH endonuclease [Salmonella phage NR01] | 97 | 2.00E−71 | 61 |
46 | 37,726 | 38,202 | + | Ribonuclease H [Escherichia phage T5] | 99 | 2.00E−115 | 99 |
47 | 38,279 | 38,557 | + | Tail fibers protein | 98 | 6.00E−60 | 100 |
48 | 38,641 | 39,156 | + | Virion structural protein | 80 | 5.00E−98 | 99 |
49 | 39,220 | 39,435 | + | Baseplate wedge subunit | 98 | 9.00E−41 | 99 |
50 | 39,567 | 39,713 | + | Hypothetical protein NR01_0017 [Salmonella phage NR01] | 97 | 8.00E−27 | 98 |
51 | 39,742 | 40,443 | + | Putative metallopeptidase [Salmonella phage Spc35] | 99 | 3.00E−174 | 99 |
52 | 40,514 | 40,696 | + | Hypothetical protein SLUR09_00119 [Escherichia phage slur09] | 98 | 2.00E−34 | 100 |
53 | 40,750 | 41,388 | + | Hypothetical protein [Escherichia phage Akfv33] | 99 | 4.00E−149 | 99 |
54 | 41,831 | 42,148 | + | Hypothetical protein APCEc03_147 [Escherichia phage APCEc03] | 99 | 9.00E−72 | 99 |
55 | 42,154 | 42,603 | + | Cell wall hydrolase SleB [Escherichia phage Akfv33] | 99 | 1.00E−80 | 100 |
56 | 42,672 | 42,842 | + | Hypothetical protein SPC35_0072 [Salmonella phage Spc35] | 98 | 2.00E−30 | 98 |
57 | 42,842 | 43,285 | + | Hypothetical protein SPC35_0071 [Salmonella phage Spc35] | 99 | 1.00E−103 | 100 |
58 | 44,245 | 45,192 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 1.00E−179 | 99 |
59 | 45,517 | 45,933 | − | Hypothetical protein NR01_0007 [Salmonella phage NR01] | 99 | 5.00E−36 | 98 |
60 | 45,955 | 46,938 | − | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 2.00E−177 | 99 |
61 | 47,205 | 47,723 | + | Hypothetical protein SPC35_0067 [Salmonella phage Spc35] | 99 | 1.00E−112 | 100 |
62 | 47,937 | 48,125 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 2.00E−36 | 100 |
63 | 48,225 | 48,392 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 1.00E−32 | 100 |
64 | 48,385 | 48,591 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 5.00E−40 | 100 |
65 | 48,682 | 48,957 | + | Hypothetical protein CPT_Shivani65 [Salmonella phage Shivani] | 98 | 3.00E−59 | 98 |
66 | 49,505 | 49,777 | + | Hypothetical protein SLUR09_00141 [Escherichia phage slur09] | 98 | 5.00E−48 | 98 |
67 | 49,944 | 50,462 | + | Hypothetical protein [Escherichia phage Bf23] | 99 | 2.00E−111 | 98 |
68 | 50,619 | 50,804 | + | Hypothetical protein T5.068 [Escherichia phage T5] | 98 | 3.00E−33 | 98 |
69 | 50,916 | 51,263 | + | Hypothetical protein NR01_0002 [Salmonella phage NR01] | 99 | 2.00E−77 | 99 |
70 | 51,525 | 51,692 | + | Hypothetical protein [Salmonella phage 5] | 96 | 6.00E−19 | 70 |
71 | 51,899 | 52,057 | + | Hypothetical protein [Salmonella phage 118970_sal2] | 98 | 2.00E−17 | 69 |
72 | 52,244 | 52,411 | + | Hypothetical protein [Salmonella phage 5] | 98 | 4.00E−31 | 98 |
73 | 53,664 | 53,795 | + | Hypothetical protein NR01_0148 [Salmonella phage NR01] | 97 | 6.00E−18 | 81 |
74 | 53,810 | 54,004 | + | Hypothetical protein [Escherichia phage Akfv33] | 98 | 1.00E−36 | 91 |
75 | 54,133 | 54,753 | + | No significant similarity found | − | – | − |
76 | 54,834 | 55,085 | + | Hypothetical protein APCEc03_006 [Escherichia phage APCEc03] | 98 | 2.00E−45 | 100 |
77 | 55,078 | 55,242 | + | Hypothetical protein APCEc03_007 [Escherichia phage APCEc03] | 98 | 2.00E−28 | 96 |
78 | 55,402 | 55,692 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 1.00E−62 | 98 |
79 | 55,803 | 55,886 | + | Hypothetical protein [Escherichia phage Bf23] | 96 | 1.00E−10 | 100 |
80 | 55,994 | 56,188 | + | Hypothetical protein [Escherichia phage Akfv33] | 98 | 2.00E−39 | 100 |
81 | 56,231 | 56,599 | + | Putative acetyltransferase-like protein [Salmonella phage NR01] | 99 | 1.00E−73 | 98 |
82 | 56,681 | 56,995 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 1.00E−60 | 99 |
83 | 57,117 | 57,464 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 5.00E−79 | 100 |
84 | 57,541 | 57,822 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 6.00E−61 | 100 |
85 | 57,815 | 58,114 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 2.00E−64 | 98 |
86 | 58,107 | 58,502 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 3.00E−82 | 100 |
87 | 58,480 | 58,776 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 1.00E−62 | 94 |
88 | 58,773 | 59,057 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 3.00E−61 | 99 |
89 | 59,168 | 59,512 | + | Hypothetical protein [Escherichia phage Akfv33] | 99 | 3.00E−63 | 95 |
90 | 59,667 | 60,365 | + | Hypothetical protein [Escherichia phage Akfv33] | 99 | 2.00E−171 | 99 |
91 | 60,322 | 60,771 | + | Putative terminase | 99 | 4.00E−90 | 99 |
92 | 60,702 | 61,055 | + | Hypothetical protein SLUR09_00180 [Escherichia phage slur09] | 99 | 7.00E−81 | 97 |
93 | 61,055 | 61,807 | + | Deoxynucleoside-5-monophosphate kinase [Escherichia phage APCEc03] | 99 | 0 | 100 |
94 | 61,820 | 62,416 | + | Putative ATP-dependent Clp protease [Escherichia phage vB_EcoS_FFH1] | 99 | 4.00E−145 | 98 |
95 | 62,573 | 63,229 | + | Holin [Salmonella phage Shivani] | 99 | 8.00E−159 | 99 |
96 | 63,226 | 63,639 | + | Lysozyme [Escherichia phage Akfv33] | 99 | 8.00E−97 | 100 |
97 | 63,717 | 64,133 | + | Hypothetical protein SLUR09_00005 [Escherichia phage slur09] | 99 | 2.00E−94 | 100 |
98 | 64,209 | 64,640 | + | Hypothetical protein T5.038 [Escherichia phage T5] | 99 | 9.00E−86 | 100 |
99 | 64,633 | 64,923 | + | Putative thioredoxin [Escherichia phage Akfv33] | 98 | 4.00E−65 | 100 |
100 | 65,051 | 65,428 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 8.00E−52 | 100 |
101 | 65,433 | 65,678 | + | Major capsid protein | 98 | 6.00E−48 | 95 |
102 | 65,681 | 66,544 | + | Serine/threonine-protein phosphatase 2 [Escherichia phage slur09] | 99 | 0 | 99 |
103 | 66,544 | 66,843 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 1.00E−66 | 100 |
104 | 66,833 | 67,423 | + | Putative serine/threonine protein phosphatase [Escherichia phage vB_EcoS_FFH1] | 99 | 3.00E−143 | 100 |
105 | 67,416 | 67,538 | + | No significant similarity found | − | – | – |
106 | 67,591 | 68,022 | + | Hypothetical protein T5.033 [Escherichia phage T5] | 99 | 2.00E−100 | 99 |
107 | 68,101 | 68,352 | + | Hypothetical protein T5.032 [Escherichia phage T5] | 98 | 3.00E−52 | 100 |
108 | 68,352 | 68,513 | + | Hypothetical protein SPC35_0029 [Salmonella phage Spc35] | 98 | 5.00E−26 | 92 |
109 | 68,513 | 68,794 | + | Tail sheath monomer | 98 | 3.00E−54 | 95 |
110 | 68,791 | 69,036 | + | Hypothetical protein SPC35_0027 [Salmonella phage Spc35] | 98 | 2.00E−49 | 98 |
111 | 69,026 | 69,352 | + | Hypothetical protein APCEc03_041 [Escherichia phage APCEc03] | 99 | 1.00E−70 | 97 |
112 | 69,452 | 69,652 | + | Hypothetical protein SPC35_0025 [Salmonella phage Spc35] | 98 | 2.00E−38 | 98 |
113 | 69,649 | 70,110 | + | Hypothetical protein SLUR09_00019 [Escherichia phage slur09] | 99 | 1.00E−109 | 99 |
114 | 70,058 | 70,429 | + | Hypothetical protein T5.025 [Escherichia phage T5] | 99 | 1.00E−80 | 98 |
115 | 70,410 | 70,688 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 72 | 6.00E−42 | 100 |
116 | 70,690 | 71,136 | + | Putative terminase | 99 | 1.00E−108 | 99 |
117 | 71,129 | 71,419 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 6.00E−63 | 98 |
118 | 71,407 | 71,640 | + | Minor tail protein | 98 | 1.00E−45 | 99 |
119 | 71,640 | 71,825 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 45 | 3.00E−09 | 96 |
120 | 71,825 | 72,385 | + | Hypothetical protein APCEc03_050 [Escherichia phage APCEc03] | 99 | 2.00E−132 | 94 |
121 | 72,522 | 73,406 | + | Hypothetical protein T5.018 [Escherichia phage T5] | 99 | 0 | 99 |
122 | 75,140 | 75,343 | − | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 1.00E−41 | 100 |
123 | 75,362 | 75,520 | − | Hypothetical protein | |||
124 | 75,517 | 75,861 | − | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 2.00E−78 | 99 |
125 | 75,863 | 76,075 | − | Hypothetical protein T5.014 [Escherichia phage T5] | 98 | 1.00E−42 | 100 |
126 | 76,078 | 76,227 | − | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 98 | 5% | 92 |
127 | 76,274 | 76,504 | − | Hypothetical protein [Escherichia phage Akfv33] | 98 | 7.00E−49 | 97 |
128 | 76,622 | 77,626 | − | DNA N-6-adenine methyltransferase | 96 | 4.00E−176 | 87 |
129 | 78,765 | 78,968 | + | Hypothetical protein NR01_0098 [Salmonella phage NR01] | 98 | 2.00E−40 | 100 |
130 | 79,197 | 79,448 | + | Baseplate wedge protein | 98 | 2.00E−41 | 96 |
131 | 79,547 | 79,954 | + | Virion structural protein | 99 | 6.00E−92 | 99 |
132 | 80,013 | 80,210 | + | Putative membrane protein [Enterobacteria phage DT57C] | 98 | 4.00E−36 | 94 |
133 | 80,307 | 81,977 | + | Baseplate wedge subunit | 99 | 0 | 97 |
134 | 82,051 | 82,443 | + | Baseplate wedge subunit | 99 | 4.00E−82 | 90 |
135 | 82,518 | 83,237 | + | Minor tail protein | 98 | 4.00E−174 | 97 |
136 | 83,402 | 83,647 | − | Hypothetical protein SPC35_0145 [Salmonella phage Spc35] | 98 | 5.00E−40 | 96 |
137 | 83,640 | 83,831 | − | Tail assembly protein | 98 | 2.00E−15 | 88 |
138 | 83,828 | 83,944 | − | Tail fiber protein | 97 | 2.00E−06 | 97 |
139 | 83,935 | 84,063 | − | Hypothetical protein [Escherichia phage Akfv33] | 97 | 2.00E−10 | 100 |
140 | 84,240 | 84,506 | − | Receptor-blocking protein [Escherichia phage Akfv33] | 98 | 2.00E−58 | 100 |
141 | 84,592 | 86,349 | + | Super-infection exclusion protein | 99 | 0 | 98 |
142 | 86,360 | 86,842 | + | Hypothetical protein SLUR09_00049 [Escherichia phage slur09] | 99 | 1.00E−79 | 99 |
143 | 86,842 | 88,158 | + | Terminase, large subunit [Escherichia phage T5] | 99 | 0 | 99 |
144 | 88,273 | 88,710 | + | Hypothetical protein APCEc03_075 [Escherichia phage APCEc03] | 99 | 2.00E−91 | 100 |
145 | 88,710 | 89,927 | + | Portal protein [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
146 | 89,924 | 90,418 | + | Tail fibers protein [Shigella phage SHSML-45] | 99 | 1.00E−93 | 99 |
147 | 90,422 | 91,054 | + | Putative prohead protease [Escherichia phage vB_EcoS_FFH1] | 99 | 9.00E−155 | 100 |
148 | 91,072 | 92,448 | + | Major head protein precursor [Escherichia phage T5] | 99 | 0 | 98 |
149 | 92,508 | 93,020 | + | Hypothetical protein APCEc03_080 [Escherichia phage APCEc03] | 99 | 4.00E−124 | 100 |
150 | 93,020 | 93,787 | + | Hypothetical protein CPT_Shivani137 [Salmonella phage Shivani] | 99 | 0 | 99 |
151 | 93,791 | 94,276 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 5.00E−117 | 100 |
152 | 94,303 | 95,709 | + | Putative major tail protein [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
153 | 95,714 | 96,616 | + | Minor tail protein [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
154 | 96,609 | 97,013 | + | Hypothetical protein [Escherichia phage vB_EcoS_FFH1] | 99 | 5.00E−94 | 99 |
155 | 97,111 | 97,443 | + | Hypothetical protein [Escherichia phage Akfv33] | 99 | 2.00E−63 | 100 |
156 | 97,527 | 101,207 | + | Pore-forming tail tip protein [Escherichia phage Akfv33] | 99 | 0 | 99 |
157 | 101,317 | 101,931 | + | DNA polymerase I | 99 | 1.00E−144 | 99 |
158 | 101,928 | 104,777 | + | Tail protein Pb3 [Escherichia phage vB_EcoS_FFH1] | 99 | 0 | 99 |
159 | 104,777 | 106,834 | + | Tail protein [Escherichia phage APCEc03] | 99 | 0 | 98 |
160 | 106,840 | 107,262 | + | Putative phage tail protein [Escherichia phage Akfv33] | 99 | 4.00E−95 | 97 |
The genome of phiLLS is double-stranded DNA genome consisting of 107,263 bp with a GC content of 39.0%. In total, 160 putative ORFs were predicted in phage genome, with 112 ORFs on the positive strand and 48 ORFs on the negative strand (Figure
Map of the genome organization of bacteriophage phiLLS
Based on the result of BLAST analyses, the predicted amino acid sequences from 42 ORFs of phiLLS display significant similarity to the T5-like phages, especially to the coliphages vB_EcoS_FFH1 (GenBank accession number:
Furthermore, the BLAST analysis indicates that the phages phiLLS, vB_EcoS_FFH1, and bV_EcoS_AKFV33, are related phylogenetically with a minimum 87% of query cover and, 70% shared orthologous proteins. The genetic similarities among these phages may correlate with their biological properties because the conserved core genes include the replication and morphogenesis modules of each genome, interestingly these bacteriophages are effective in limiting contamination with
In an attempt to define the origin and terminus of replication of the phage genome, a cumulative GC skew analysis was performed. The results of GC skew analysis in the genome of the phiLLS phage (Figure
Cumulative GC skew analysis of the phage genome sequence. The global minimum and maximum are displayed in the cumulative graph were calculated by using a window size of 1,000 bp and a step size of 100 bp. The GC-skew and the cumulative GC-skew are represented by blue and red lines, respectively. The minimum and maximum of a GC-skew can be used to predict the origin of replication (27179 nt) and the terminus location (103791 nt).
Based on the information obtained from an exhaustive search of the NCBI GenBank database, it was possible to determine that the coliphages have very different genome sizes (Supplementary Material
The phages within the family
The bacteriophage phiLLS genome has a high gene density—1.64 genes per kilobase. The genome analysis suggests that the phage phiLLS is strictly lytic and does not carry genes associated with virulence factors and/or potential immunoreactive allergens in their genomes. Therefore, this phage has desirable genetic features as a biocontrol agent. However, further oral toxicity testing is needed to ensure the safety of phage use.
The molecular GC content was calculated at 39.0%, which is significantly lower than of
The phiLLS genome is organized in a modular gene structure that is common of tailed bacteriophage genomes (Krupovic et al.,
In addition, the phiLLS genome was found to contain 16 tRNA genes with anticodons for Arg, Ser, Met, Leu, Glu, Cys, Asn, Pro, Lys, Gln, Gly, and Ile, located around a region at position 44,136–55,956 bp of the genome (Figure
Comparison of codon usage and tRNAs between phiLLS and host.
In summary, phage phiLLS genome sequence analysis revealed valuable information concerning its biology. Detailed genomic analysis showed a modular organization, which is different from other identified enterobacteriophages. Nevertheless, it demonstrated a high degree of identity with ORFs from some other phages, especially with T5-like bacteriophages. Moreover, the phiLLS phage does not encode lysogenic genes. The phiLLS genome encodes several putative proteins with lytic activity, which may be exploited for other biotechnological applications. This study identified the groups of enzymes responsible for producing bacterial lysis. The practical use of the phiLLS genome will be derived from the understanding of its organization. Based on the genetic information of this phage, future work may be performed to obtain enzymes with antimicrobial activity for the biocontrol of pathogenic bacteria.
In conclusion, we have isolated and characterized a new lytic phage, phiLLS, with lytic activity against multidrug-resistant
6 Preliminary challenge trials were performed to evaluate the potential of the isolated phages as 7 antimicrobials against
LA, LR, and JL conceived, designed and coordinated the study. LA, LR, and AG carried out the experimentation. LA, LR, and JL analyzed the results. Contributed reagents/materials/analysis tools: JL, CC, and AG. CC edited the English grammar of the manuscript. All authors wrote, read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank the LANIIA, CIAD for experimental support. We appreciate the support provided by Dra Bianca Amézquita-López of the Universidad Autonóma de Sinaloa. This investigation was partially supported by Fundación Produce Sinaloa. The authors are thankful to QFB Sergio Juan Manuel González de León and QFB Jesús Héctor Carrillo Yáñez for critical technical assistance.
The Supplementary Material for this article can be found online at: