Occurrence of Vibrio parahaemolyticus and Its Specific Phages from Shrimp Ponds in East Coast of India

Vibrio parahaemolyticus is a natural microflora of marine and coastal water bodies and associated with mortality of larval shrimp in penaeid shrimp in ponds. Bacteriophages occur virtually in all places where their hosts exist. In this study, total distribution of V. parahaemolyticus and its phages were examined in shrimp ponds, seawater, estuary, animal surface, and tissues. Total vibrio count in sediments of two ponds was found to be 2.6 × 10³ and 5.6 × 10³ cfu/g. Incidence of V. parahaemolyticus in the ponds was close, while it was markedly higher in the animal surface and tissue samples. Biochemically identified eight strains of V. parahaemolyticus (V1, V3–V6, V9, V11, and V12) were taken for further infection studies with bacteriophage. Totally five bacteriophages capable of infecting V. parahaemolyticus MTCC-451 strain were isolated from all the samples. One of the isolated bacteriophage Vp1 from estuary was able to lyse all the isolated V. parahaemolyticus strains we used. Therefore, the morphology of Vp1 was estimated in TEM. Vp1 phage head measuring approximately about 50–60 nm diameter with icosahedral outline and a contractile tails of diameter 7 nm and length 100 nm and it was identified as Myoviridae. Therefore, the phages have the potential application in destroying bacterial pathogens.     

Restriction and Modification in Group N Streptococci: Effect of Heat on Development of Modified Lytic Bacteriophage

The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 × 10⁻⁷ for tr plaqued on 799 and 2.1 × 10⁻⁷ for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50°C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.     

Isolation of Psychrophilic Bacteriophage-Host Systems from Refrigerated Food Products

Thirty-eight bacteriophage-host systems were isolated from 22 of 45 refrigerated food products examined under psychrophilic conditions. Isolates were obtained from ground beef, pork sausage, chicken, raw skim milk, and oysters, whereas no isolations were made from liquid egg whites and processed meat products. Thirty of the 38 psychrophilic bacterial hosts were gram-negative rods, and 27 of these were classified within the genus Pseudomonas; three were members of the family Enterobacteriaceae. The remaining eight were gram-positive cocci, which were tentatively classified as Leuconostoc. Plate counts of psychrophilic bacteria were greater than 2.2 × 10⁵/ml (g) in all but one sample which contained phage, whereas phage titers ranged from less than 100 to 6.3 × 10⁶ plaque-forming units/ml (g). Phage isolates showed limited host ranges usually attacking only those hosts upon which they were isolated. Of eight phages tested against 13 cultures of known identity, one showed lytic action, and this was against strains of P. fragi.     

Vibriophages Differentially Influence Biofilm Formation by Vibrio anguillarum Strains

Vibrio anguillarum is an important pathogen in marine aquaculture, responsible for vibriosis. Bacteriophages can potentially be used to control bacterial pathogens; however, successful application of phages requires a detailed understanding of phage-host interactions under both free-living and surface-associated growth conditions. In this study, we explored in vitro phage-host interactions in two different strains of V. anguillarum (BA35 and PF430-3) during growth in microcolonies, biofilms, and free-living cells. Two vibriophages, ΦH20 (Siphoviridae) and KVP40 (Myoviridae), had completely different effects on the biofilm development. Addition of phage ΦH20 to strain BA35 showed efficient control of biofilm formation and density of free-living cells. The interactions between BA35 and ΦH20 were thus characterized by a strong phage control of the phage-sensitive population and subsequent selection for phage-resistant mutants. Addition of phage KVP40 to strain PF430-3 resulted in increased biofilm development, especially during the early stage. Subsequent experiments in liquid cultures showed that addition of phage KVP40 stimulated the aggregation of host cells, which protected the cells against phage infection. By the formation of biofilms, strain PF430-3 created spatial refuges that protected the host from phage infection and allowed coexistence between phage-sensitive cells and lytic phage KVP40. Together, the results demonstrate highly variable phage protection mechanisms in two closely related V. anguillarum strains, thus emphasizing the challenges of using phages to control vibriosis in aquaculture and adding to the complex roles of phages as drivers of prokaryotic diversity and population dynamics.     

Complete Genome Sequence of Vibrio parahaemolyticus Bacteriophage vB_VpaM_MAR

Vibrio parahaemolyticus is a major pathogen that is mainly associated with seafood and is a global food safety issue. Our objective was to isolate and completely sequence a specific phage against this bacterium. Phage vB_VpaM_MAR is able to lyse 76% of the V. parahaemolyticus strains tested. MAR belongs to the Myoviridae family and has a genome comprised of double-stranded DNA with a size of 41,351 bp, a G+C content of 51.3%, and 62 open reading frames (ORFs). Bioinformatic analysis showed that phage MAR is closely related to Vibrio phages VHML, VP58.5, and VP882 and Halomonas aquamarina phage ΦHAP-1.     

Isolation and Characterization of a Filamentous Phage,Vf33, Specific for Vibrio parahaemolyticus

Phage Vf33, a filamentous phage about 1,400 nm long and 7 nm wide, specific for Vibrio parahaemolyticus, was isolated and characterized. The buoyant density of Vf33 in CsCl was 1.292 g/cm³. As with other filamentous phages, the lytic activity of Vf33 was resistant to heating below 80 C and to treatment with diethylether, acetone or methanol but sensitive to chloroform. The nucleic acid of this phage is single-stranded circular DNA 8.4 kb in size. The viral genome was converted to a double-stranded replicative form in the host cell. Among the strains tested, only V. parahaemolyticus strains possessing K38 antigen was sensitive to the phage.     

Genome-driven elucidation of phage-host interplay and impact of phage resistance evolution on bacterial fitness

When considering the interactions between bacteriophages and their host, the issue of phage-resistance emergence is a key element in understanding the ecological impact of phages on the bacterial population. It is also an essential parameter for the implementation of phage therapy to combat antibiotic-resistant pathogens. This study investigates the phenotypic and genetic responses of five Pseudomonas aeruginosa strains (PAO1, A5803, AA43, CHA, and PAK) to the infection by seven phages with distinct evolutionary backgrounds and recognised receptors (LPS/T4P). Emerging phage-insensitivity was generally accompanied by self and cross-resistance mechanisms. Significant differences were observed between the reference PAO1 responses compared to other clinical representatives. LPS-dependent phage infections in clinical strains selected for mutations in the “global regulatory” and “other” genes, rather than in the LPS-synthesis clusters detected in PAO1 clones. Reduced fitness, as proxied by the growth rate, was correlated with large deletion (20–500 kbp) and phage carrier state. Multi-phage resistance was significantly correlated with a reduced growth rate but only in the PAO1 population. In addition, we observed that the presence of prophages decreased the lytic phage maintenance seemingly protecting the host against carrier state and occasional lytic phage propagation, thus preventing a significant reduction in bacterial growth rate.Subject terms: Bacteriophages, Biodiversity     

Phages Infecting Vibrio vulnificus Are Abundant and Diverse in Oysters (Crassostrea virginica) Collected from the Gulf of Mexico

Phages infecting Vibrio vulnificus were abundant (>10⁴ phages g of oyster tissue⁻¹) throughout the year in oysters (Crassostrea virginica) collected from estuaries adjacent to the Gulf of Mexico (Apalachicola Bay, Fla.; Mobile Bay, Ala.; and Black Bay, La.). Estimates of abundance ranged from 10¹ to 10⁵ phages g of oyster tissue⁻¹ and were dependent on the bacterial strain used to assay the sample. V. vulnificus was near or below detection limits (<0.3 cell g⁻¹) from January through March and was most abundant (10³ to 10⁴ cells g⁻¹) during the summer and fall, when phage abundances also tended to be greatest. The phages isolated were specific to strains of V. vulnificus, except for one isolate that caused lysis in a few strains of V. parahaemolyticus. Based on morphological evidence obtained by transmission electron microscopy, the isolates belonged to the Podoviridae, Styloviridae, and Myoviridae, three families of double-stranded DNA phages. One newly described morphotype belonging to the Podoviridae appears to be ubiquitous in Gulf Coast oysters. Isolates of this morphotype have an elongated capsid (mean, 258 nm; standard deviation, 4 nm; n = 35), with some isolates having a relatively broad host range among strains of V. vulnificus. Results from this study indicate that a morphologically diverse group of phages which infect V. vulnificus is abundant and widely distributed in oysters from estuaries bordering the northeastern Gulf of Mexico.     

Two Phages,phiIPLA-RODI and phiIPLA-C1C,Lyse Mono- and Dual-Species Staphylococcal Biofilms

Phage therapy is a promising option for fighting against staphylococcal infections. Two lytic phages, vB_SauM_phiIPLA-RODI (phiIPLA-RODI) and vB_SepM_phiIPLA-C1C (phiIPLA-C1C), belonging to the Myoviridae family and exhibiting wide host ranges, were characterized in this study. The complete genome sequences comprised 142,348 bp and 140,961 bp and contained 213 and 203 open reading frames, respectively. The gene organization was typical of Spounavirinae members, with long direct terminal repeats (LTRs), genes grouped into modules not clearly separated from each other, and several group I introns. In addition, four genes encoding tRNAs were identified in phiIPLA-RODI. Comparative DNA sequence analysis showed high similarities with two phages, GH15 and 676Z, belonging to the Twort-like virus genus (nucleotide identities of >84%); for phiIPLA-C1C, a high similarity with phage phiIBB-SEP1 was observed (identity of 80%). Challenge assays of phages phiIPLA-RODI and phiIPLA-C1C against planktonic staphylococcal cells confirmed their lytic ability, as they were able to remove 5 log units in 8 h. Exposure of biofilms to phages phiIPLA-RODI and phiIPLA-C1C reduced the amount of adhered bacteria to about 2 log units in both monospecies and dual-species biofilms, but phiIPLA-RODI turned out to be as effective as the mixture of both phages. Moreover, the frequencies of bacteriophage-insensitive mutants (BIMs) of Staphylococcus aureus and S. epidermidis with resistance to phiIPLA-RODI and phiIPLA-C1C were low, at 4.05 × 10⁻⁷ ± 2.34 × 10⁻⁹ and 1.1 × 10⁻⁷ ± 2.08 × 10⁻⁹, respectively. Overall, a generally reduced fitness in the absence of phages was observed for BIMs, which showed a restored phage-sensitive phenotype in a few generations. These results confirm that lytic bacteriophages can be efficient biofilm-disrupting agents, supporting their potential as antimicrobials against staphylococcal infections.     

Phage Application for the Protection from Acute Hepatopancreatic Necrosis Disease (AHPND) in <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus has been one of the most problematic diseases in marine shrimp aquaculture throughout Southeast Asia and Latin America. To evaluate the effectiveness of a bacteriophage (phage) treatment for AHPND, a series of bioassays were carried out in a marine shrimp (Penaeus vannamei) model using an AHPND-V. parahaemolyticus strain that is highly pathogenic to shrimp. We monitored the mortality and histopathological changes during phage treatment. Shrimps treated with phage prophylaxis and phage therapy displayed significant protection from AHPND and survived a lethal bacterial challenge.     

A new group of cosmopolitan bacteriophages induce a carrier state in the pandemic strain of Vibrio parahaemolyticus

A clonal population of pathogenic Vibrio parahaemolyticus O3 : K6 serovar has spread in coastal waters, causing outbreaks worldwide since 1996. Bacteriophage infection is one of the main factors affecting bacterial strain concentration in the ocean. We studied the occurrence and properties of phages infecting this V. parahaemolyticus pandemic strain in coastal waters. Analysing 143 samples, phages were found in 13. All isolates clustered in a closely related group of podophages with at least 90% nucleotide sequence identity in three essential genes, despite distant geographical origins. These bacteriophages were able to multiply on the V. parahaemolyticus pandemic strain, but the impact on host concentration and subsequent growth was negligible. Infected bacteria continued producing the phage but were not lysogenized. The phage genome of prototype strain VP93 is 43 931 nucleotides and contains 337 bp direct terminal repeats at both ends. VP93 is the first non‐Pseudomonas phage related to the ΦKMV‐like subgroup of the T7 supergroup. The lack of a major effect on host growth suggests that these phages exert little control on the propagation of the pandemic strain in the environment. This form of phage growth can be modelled if phage‐sensitive and ‐resistant cells that convert to each other with a high frequency are present in clonal cultures of pandemic V. parahaemolyticus.     

Isolation and Characterisation of Lytic Bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca

Klebsiella bacteria have emerged as an increasingly important cause of community-acquired nosocomial infections. Extensive use of broad-spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains that frequently produce extended-spectrum β-lactamases and/or other defences against antibiotics. Many of these strains are highly virulent and exhibit a strong propensity to spread. In this study, six lytic Klebsiella bacteriophages were isolated from sewage-contaminated river water in Georgia and characterised as phage therapy candidates. Two of the phages were investigated in greater detail. Biological properties, including phage morphology, nucleic acid composition, host range, growth phenotype, and thermal and pH stability were studied for all six phages. Limited sample sequencing was performed to define the phylogeny of the K. pneumoniae- and K. oxytoca-specific bacteriophages vB_Klp_5 and vB_Klox_2, respectively. Both of the latter phages had large burst sizes, efficient rates of adsorption and were stable under different adverse conditions. Phages reported in this study are double-stranded DNA bacterial viruses belonging to the families Podoviridae and Siphoviridae. One or more of the six phages was capable of efficiently lysing ~63 % of Klebsiella strains comprising a collection of 123 clinical isolates from Georgia and the United Kingdom. These phages exhibit a number of properties indicative of potential utility in phage therapy cocktails.     

Isolation of lytic bacteriophage against Vibrio harveyi

Aims: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Methods and Results: Water samples from discharge channels and grow‐out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage‐enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Conclusions: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Significance and Impact of the Study: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.     

Bacteriophage ?MAM1, a Viunalikevirus,Is a Broad-Host-Range,High-Efficiency Generalized Transducer That Infects Environmental and Clinical Isolates of the Enterobacterial Genera Serratia and Kluyvera

Members of the enterobacterial genus Serratia are ecologically widespread, and some strains are opportunistic human pathogens. Bacteriophage ϕMAM1 was isolated on Serratia plymuthica A153, a biocontrol rhizosphere strain that produces the potently bioactive antifungal and anticancer haterumalide oocydin A. The ϕMAM1 phage is a generalized transducing phage that infects multiple environmental and clinical isolates of Serratia spp. and a rhizosphere strain of Kluyvera cryocrescens. Electron microscopy allowed classification of ϕMAM1 in the family Myoviridae. Bacteriophage ϕMAM1 is virulent, uses capsular polysaccharides as a receptor, and can transduce chromosomal markers at frequencies of up to 7 × 10⁻⁶ transductants per PFU. We also demonstrated transduction of the complete 77-kb oocydin A gene cluster and heterogeneric transduction of a plasmid carrying a type III toxin-antitoxin system. These results support the notion of the potential ecological importance of transducing phages in the acquisition of genes by horizontal gene transfer. Phylogenetic analyses grouped ϕMAM1 within the ViI-like bacteriophages, and genomic analyses revealed that the major differences between ϕMAM1 and other ViI-like phages arise in a region encoding the host recognition determinants. Our results predict that the wider genus of ViI-like phages could be efficient transducing phages, and this possibility has obvious implications for the ecology of horizontal gene transfer, bacterial functional genomics, and synthetic biology.     

Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36–42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13–15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10–33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.     

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.     

Vibrio parahaemolyticus and Its Specific Bacteriophages as an Indicator in Cockles (Anadara granosa) for the Risk of V. parahaemolyticus Infection in Southern Thailand

Correlation between the numbers of Vibrio parahaemolyticus and its specific bacteriophages in cockles was investigated from June 2009 to May 2010 in Hat Yai, Songkhla, Thailand. Cockles obtained monthly from a local market were sampled to determine the numbers of V. parahaemolyticus and bacteriophages that could form plaques on ten strains of pandemic and nonpandemic V. parahaemolyticus. In addition, V. parahaemolyticus isolates from clinical samples from Hat Yai hospital over the same period were investigated. All 139 cockles sampled were positive for V. parahaemolyticus. However, only 76 of them were positive for bacteriophages. During the testing period, the number of bacteriophages was not significantly correlated with the incidence of V. parahaemolyticus-infected patients, but the numbers of V. parahaemolyticus isolates from the cockle samples were closely related to the number of infected patients. The bacteriophages isolated from V. parahaemolyticus also infected Vibrio alginolyticus and Vibrio mimicus, suggesting that the broad host range of phages may be a factor of providing the possibility of their participation in the processes of genetic exchange between V. parahaemolyticus and closely related Vibrio spp. In conclusion, this study indicated that the number of V. parahaemolyticus in cockles may be a useful tool for predicting the relative risk of infection by V. parahaemolyticus in this area of Thailand.     

Isolation and Characterization of Campylobacter Bacteriophages from Retail Poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.     

PhREEPred: Phage Resistance Emergence Prediction Web Tool to Foresee Encapsulated Bacterial Escape from Phage Cocktail Treatment

Phages, as well as phage-derived proteins, especially lysins and depolymerases, are intensively studied to become prospective alternatives or supportive antibacterials used alone or in combination. In the common phage therapy approach, the unwanted emergence of phage-resistant variants from the treated bacterial population can be postponed or reduced by the utilization of an effective phage cocktail. In this work, we present a publicly available web tool PhREEPred (Phage Resistance Emergence Prediction) (https://phartner.shinyapps.io/PhREEPred/), which will allow an informed choice of the composition of phage cocktails by predicting the outcome of phage cocktail or phage/depolymerase combination treatments against encapsulated bacterial pathogens given a mutating population that escapes single phage treatment. PhREEPred simulates solutions of our mathematical model calibrated and tested on the experimental Klebsiella pneumoniae setup and Klebsiella-specific lytic phages: K63 type-specific phage KP34 equipped with a capsule-degrading enzyme (KP34p57), capsule-independent myoviruses KP15 and KP27, and recombinant capsule depolymerase KP34p57. The model can calculate the phage-resistance emergence depending on the bacterial growth rate and initial density, the multiplicity of infection, phage latent period, its infectiveness and the cocktail composition, as well as initial depolymerase concentration and activity rate. This model reproduced the experimental results and showed that (i) the phage cocktail of parallelly infecting phages is less effective than the one composed of sequentially infecting phages; (ii) depolymerase can delay or prevent bacterial resistance by unveiling an alternative receptor for initially inactive phages. In our opinion, this customer-friendly web tool will allow for the primary design of the phage cocktail and phage-depolymerase combination effectiveness against encapsulated pathogens.     

A novel termini analysis theory using HTS data alone for the identification of Enterococcus phage EF4-like genome termini

Background

Enterococcus faecalis and Enterococcus faecium are typical enterococcal bacterial pathogens. Antibiotic resistance means that the identification of novel E. faecalis and E. faecium phages against antibiotic-resistant Enterococcus have an important impact on public health. In this study, the E. faecalis phage IME-EF4, E. faecium phage IME-EFm1, and both their hosts were antibiotic resistant. To characterize the genome termini of these two phages, a termini analysis theory was developed to provide a wealth of terminal sequence information directly, using only high-throughput sequencing (HTS) read frequency statistics.

Results

The complete genome sequences of phages IME-EF4 and IME-EFm1 were determined, and our termini analysis theory was used to determine the genome termini of these two phages. Results showed 9 bp 3′ protruding cohesive ends in both IME-EF4 and IME-EFm1 genomes by analyzing frequencies of HTS reads. For the positive strands of their genomes, the 9 nt 3′ protruding cohesive ends are 5′-TCATCACCG-3′ (IME-EF4) and 5′-GGGTCAGCG-3′ (IME-EFm1). Further experiments confirmed these results. These experiments included mega-primer polymerase chain reaction sequencing, terminal run-off sequencing, and adaptor ligation followed by run-off sequencing.

Conclusion

Using this termini analysis theory, the termini of two newly isolated antibiotic-resistant Enterococcus phages, IME-EF4 and IME-EFm1, were identified as the byproduct of HTS. Molecular biology experiments confirmed the identification. Because it does not require time-consuming wet lab termini analysis experiments, the termini analysis theory is a fast and easy means of identifying phage DNA genome termini using HTS read frequency statistics alone. It may aid understanding of phage DNA packaging.