Broad Lytic Spectrum Research Articles

Isolation and characteristic of Bacillus cereus phage Z3 and its application in rice and milk

Bacillus cereus is a foodborne pathogen that widely exists in various food products. In this study, a new B. cereus phage, named Z3, was isolated from the soil and characterized. Transmission electron microscopy (TEM) observations revealed that Z3 had an icosahedral head and a retractable tail, belonging to myxovirus. The double-stranded linear DNA of Z3 was 159,116 bp in length with a CG% content of 39.96%. Genomic analysis indicated that Z3 represented a new species belonging to the Caeruleovirus genus. Z3 showed broad lytic spectrum, as it could lyse 10 out of 13 tested strains of B. cereus. The in vitro phage killing assay and the one-step growth curve showed that Z3 could effectively inhibit the bacteria growth within 10 h and it had a short latent period and fast multiplication speed. Besides, stability tests showed that Z3 was tolerant to a wide range of temperature (4 °C–55 °C) and pH levels (4–10), and was UV-resistant for 100 min. Furthermore, the applications of Z3 in rice and milk demonstrated its effective antimicrobial properties against B. cereus in food matrix. These findings suggested that Z3 may be a promising biocontrol agent against B. cereus in food industry.

Characterization and genomic analysis of a broad-spectrum lytic phage HZ2201 and its antibiofilm efficacy against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a clinically common conditionally pathogenic bacterium, and the abuse of antibiotics has exacerbated its drug resistance in recent years. This has resulted in extensive reports about the usage of Pseudomonas aeruginosa phage as a novel antibacterial drug. In this study, we isolated a novel phage HZ2201 with a broad lytic spectrum. The lytic rate of this phage against Pseudomonas aeruginosa reached 78.38% (29/37), including 25 multi-drug- and carbapenem-resistant Pseudomonas aeruginosa strains. Transmission electron microscopy revealed that phage HZ2201 belongs to the class Caudoviricetes. Biological characterization showed that phage HZ2201 had an latent period of 40 min, a lytic period of 20 min, and a burst size of 440 PFU/cell, with improved tolerance to temperature and pH. Considering genomic analysis, the HZ2201 genome was a circular double-stranded DNA with a size of 45,431 bp and a guanine-cytosine (G + C) content of 52.16%, and contained 3 tRNAs. 27 of the 74 open reading frames (ORFs) annotated by the Rapid Annotation using Subsystem Technology (RAST) tool could be matched to the genomes of known functions, and no genes related to virulence and antibiotic resistance were found. The phylogenetic tree suggests that phage HZ2201 is highly related to the phage ZCPS1 and PaP3, and ORF57 and ORF17 are predicted to encode a holin and an endolysin, respectively. Cell lysis by HZ2201 proceeds through the holin-endolysin system, suggesting that it is a novel phage. Additionally, we demonstrated that phage HZ2201 has a high inhibitory capacity against Pseudomonas aeruginosa biofilms. The results of our study suggest that phage HZ2201 is a novel potential antimicrobial agent for treating drug-resistant Pseudomonas aeruginosa infection.

Phenotypic Characterization and Comparative Genomic Analysis of Novel Salmonella Bacteriophages Isolated from a Tropical Rainforest.

Salmonella infections across the globe are becoming more challenging to control due to the emergence of multidrug-resistant (MDR) strains. Lytic phages may be suitable alternatives for treating these multidrug-resistant Salmonella infections. Most Salmonella phages to date were collected from human-impacted environments. To further explore the Salmonella phage space, and to potentially identify phages with novel characteristics, we characterized Salmonella-specific phages isolated from the Penang National Park, a conserved rainforest. Four phages with a broad lytic spectrum (kills >5 Salmonella serovars) were further characterized; they have isometric heads and cone-shaped tails, and genomes of ~39,900 bp, encoding 49 CDSs. As the genomes share a <95% sequence similarity to known genomes, the phages were classified as a new species within the genus Kayfunavirus. Interestingly, the phages displayed obvious differences in their lytic spectrum and pH stability, despite having a high sequence similarity (~99% ANI). Subsequent analysis revealed that the phages differed in the nucleotide sequence in the tail spike proteins, tail tubular proteins, and portal proteins, suggesting that the SNPs were responsible for their differing phenotypes. Our findings highlight the diversity of novel Salmonella bacteriophages from rainforest regions, which can be explored as an antimicrobial agent against MDR-Salmonella strains.

Bacteriophages as an alternative for biological control of biofilm-forming Salmonella enterica.

Salmonellosis is one of the most common foodborne diseases worldwide. Surface adherence and biofilm formation are among the main strategies evolved by Salmonella to survive under harsh conditions and are risk factors for its spread through the food chain. Owing to the increase in antimicrobial resistance, there is a growing need to develop other methods to control foodborne pathogens, and bacteriophages have been suggested as a potential alternative for this purpose. The aim of this study was to evaluate bacteriophages as a biological control of Salmonella enterica serotypes to inhibit and remove bacterial biofilms. A total of 12 S. enterica isolates were selected for this study, all of which were biofilm producers. Seven bacteriophages were tested, individually and in a cocktail, for their host range and efficiency of plating (EOP). The phage cocktail was evaluated for its antibiofilm effect against the Salmonella biofilms. Phages UPF_BP1, UPF_BP2, UPF_BP3, UPF_BP6, and 10:2 possessed a broad lytic spectrum and could infect all S. enterica strains. Phages 10:2, UPF_BP6, and UPF_BP3 had high EOP in 10, 9, and 9 out of the 12 S. enterica strains, respectively. The cocktail was able to infect all S. enterica strains and had a high EOP in 10 out of 12 S. enterica isolates, presenting a broader host range than any of the tested single phages. A wide variation of inhibition among strains was observed, ranging from 14.72% to 88.53%. Multidrug-resistant and strong biofilm producer strains showed high biofilm inhibition levels by phage cocktail. Our findings demonstrate the ability of the cocktail to prevent biofilm formation and remove formed biofilms of Salmonella. These results indicate that the phage cocktail is a promising candidate to be used as an alternative for the control of Salmonella biofilms through surface conditioning.

Broad lytic spectrum of novel Salmonella phages on ciprofloxacin-resistant Salmonella contaminated in the broiler production chain.

Background and Aim:Ciprofloxacin (CIP) is recommended for salmonellosis treatment as the drug of choice; however, overuse of this drug can cause drug resistance issues and failure to treat diseases. Phage therapy is an alternative approach for combatting CIP-resistant infection. This study aimed to estimate the prevalence of CIP-resistant Salmonella isolated from the broiler production chain and evaluated the lytic ability of novel Salmonella phages isolated from water samples.Materials and Methods:Samples were obtained from the broiler production chain and used for Salmonella isolation. serovar and CIP resistance of each isolate were characterized through latex agglutination and agar disk diffusion test, respectively. Water samples from different sources were acquired for phage isolation. The lytic activity of novel-isolated phages was also examined.Results:In this study, 51 Salmonella isolates were recovered from the broiler production chain (two commercial farms, one free-range farm, two slaughterhouses, and three stalls from the wet market). Kentucky was the major serovar characterized (16), followed by Typhimurium (9), Agona (5), Corvalis (5), Schwarzengrund (5), Singapore (3), Weltevreden (3), Mbandaka (2), Give (2), and Albany (1). The serovars that exhibited CIP resistance were 14/16 isolates of serovar Kentucky (87.5%) and one isolate of serovar Give (50%), whereas eight other serovars were susceptible to this drug. Overall, the prevalence of CIP-resistant Salmonella recovered from the sources included in this study was 29.4%. This study identified 11 Salmonella phages isolated from wastewater samples derived from broiler farms, wastewater treatment stations, and natural reservoirs. Our phages showed the total percentage of lysis ability ranging from 33.3% to 93.3% against CIP-resistant isolates. However, only one bacterial isolate, namely 210SL, recovered from the food contact surface of a wet market stall and was resistant to all phages.Conclusion:Diverse serovars of Salmonella were recovered in the broiler production chain in this study, while the isolates presenting CIP-resistant Salmonella were as high as 29.4%. Overall, Salmonella phages showed high lysis ability against these CIP-resistant Salmonella isolates, suggesting the potential application of phage-based treatments or biocontrol in the broiler production chain.

Isolation and Characterization of Novel Lytic Bacteriophage vB_RsoP_BMB50 infecting Ralstonia solanacearum.

Ralstonia solanacearum is a soil-borne phytopathogen, and it can cause bacterial wilt disease in a variety of key crops around the world, thus resulting in enormous financial losses. However, there is a lack of effective, green, and safe prevention and control measures against increasingly devastating bacterial wilt disease. Bacteriophages (phages) are considered as potential biocontrol agents against bacterial wilt disease. Although many phages infecting R. solanacearum have been isolated, so far, these Ralstonia phages are still insufficient to deal with the diversity of the bacteria of R. solanacearum. In this study, a novel lytic bacteriophage vB_RsoP_BMB50 infecting multiple R. solanacearum was isolated from tomato fields in Dalian, China. Transmission electron microscopy and genomics analysis indicated that vB_RsoP_BMB50 belonged to the subfamily Okabevirinae, Autographiviridae family, and order Caudovirales, and it comprised a double-stranded DNA with a full length of 43,665bp and a mean G+C content of 61.79%, containing 53 open reading frames (ORFs). This novel phage exhibited a large burst size, high temperature stability (4-50 °C), and strong pH tolerance (pH 5-10). Comparative analyses and phylogenetic analyses revealed that vB_RsoP_BMB50 represented a novel Ralstonia phage genus since it exhibited a low sequence similarity to other phages in the GenBank database. Due to its broad lytic spectrum, high thermal stability, and strong pH tolerance, vB_RsoP_BMB50 is considered as an effective candidate biocontrol agent against bacterial wilt disease caused by R. solanacearum.

Phenotypic and Genetic Characterization of Aeromonas hydrophila Phage AhMtk13a and Evaluation of Its Therapeutic Potential on Simulated Aeromonas Infection in Danio rerio.

Phage therapy can be an effective alternative to standard antimicrobial chemotherapy for control of Aeromonas hydrophila infections in aquaculture. Aeromonas hydrophila-specific phages AhMtk13a and AhMtk13b were studied for basic biological properties and genome characteristics. Phage AhMtk13a (Myovirus, 163,879 bp genome, 41.21% CG content) was selected based on broad lytic spectrum and physiologic parameters indicating its lytic nature. The therapeutic potential of phage AhMtk13a was evaluated in experimental studies in zebrafish challenged with A. hydrophila GW3-10 via intraperitoneal injection and passive immersion in aquaria water. In experimental series 1 with single introduction of AhMtk13a phage to aquaria water at phage–bacteria ratio 10:1, cumulative mortality 44% and 62% was registered in fish exposed to phage immediately and in 4 h after bacterial challenge, correspondingly, compared to 78% mortality in the group with no added phage. In experimental series 2 with triple application of AhMtk13a phage at ratio 100:1, the mortality comprised 15% in phage-treated group compared to the 55% in the control group. Aeromonas hydrophila GW3-10 was not detectable in aquaria water from day 9 but still present in fish at low concentration. AhMtk13a phage was maintained in fish and water throughout the experiment at the higher concentration in infected fish.

Characterization and genome analysis of a broad lytic spectrum bacteriophage P479 against multidrug-resistant Escherichia coli

The increase of multi-drug resistant and multi-serotypes of pathogenic Escherichia coli has brought more severe challenge to control infection. Nowadays, bacteriophage is a promising tool to treat colibacillosis as an alternative of antibiotics. A coliphage P479, isolated from sewage of poultry farm, could lyse multiple serotypes, including not only O1, O2, O8, O9, O21, O78, O83, O145 of Avian pathogenic E. coli, but O157:H7 of Enterohaemorrhagic E. coli and O18:K1:H7 Neonatal meningitis E. coli. Additionally, P479 could also lyse multi-drug resistant E. coli. These indicated that P479 had good lytic ability. One-step growth curve revealed that the latent time period of P479 was 10 min and the burst size was about 318 PFU/cell. Stability tests demonstrated that P479 had good stability under various temperature (4 to 50 °C) and pH (3 to11) conditions. P479 contained of a linear, double-stranded DNA molecule of 172,033 bp with 40.3% GC content. P479 contained 296 putative coding sequences (CDSs) and two tRNA genes. Based on genomic comparison, P479 was classified as a member of genus Gaprivervirus, subfamily Tevenvirinae, family Myoviridae, order Caudovirales. No known virulent or lysogenic genes were detected in the genome of P479, manifesting P479 was safe to adhibit. Antibacterial activity in vitro manifested that P479 has varying degrees bacteriostatic activity against different bacteria. According to the above properties, P479 has the potential to be applied in phage therapy in the future.

Молекулярно-генетическая характеристика трех новых бактериофагов Klebsiella pneumoniae, перспективных для применения в фаговой терапии

The Klebsiella pneumoniae bacterium is capable of causing the broad range of human nosocomial infections associated with antibiotic resistance and high mortality. Virulent bacteriophage therapy is one of the promising alternatives to antibiotic treatment of such infections. The study was aimed to isolate virulent bacteriophages effective against the relevant clinical K. pneumoniae strains, and to perform the molecular genetic characterization of these phages. Bacteriophages were isolated from the river water samples using the enrichment method. The whole-genome sequencing was performed on the MiSeq platform (Illumina). Three novel K. pneumoniae bacteriophages belonging to families Autographiviridae (vB_KpnP_NER40, GenBank MZ602146) and Myoviridae (vB_KpnM_VIK251, GenBank MZ602147; vB_KpnM_FRZ284, GenBank MZ602148) have been isolated and characterized. On the collection of 105 K. pneumoniae clinical strains, it has been found that bacteriophages vB_KpnP_NER40 and vB_KpnM_VIK251 have a narrow lytic spectrum (22% and 11%), which is limited to strains of the capsular types К2 and К20 respectively. In contrast, bacteriophage vB_KpnM_FRZ284 has a broad lytic spectrum (37%), causing the lysis of strains with different types of capsular polysaccharide. The phages are strictly virulent and have no genes encoding integrases, toxins or pathogenicity factors in their genomes. Genes of depolymerases, encoding the potential receptor binding proteins, have been found in the genomes of the capsular-specific bacteriophages vB_KpnP_NER40 and vB_KpnM_VIK251. The cocktail of three bacteriophages has lysed about 65% of the studied collection of K. рneumoniae strain and is potentially applicable for therapeutic purposes.

Bacteriophage-Based Biosensing of Pseudomonas aeruginosa: An Integrated Approach for the Putative Real-Time Detection of Multi-Drug-Resistant Strains.

During the last decennium, it has become widely accepted that ubiquitous bacterial viruses, or bacteriophages, exert enormous influences on our planet’s biosphere, killing between 4–50% of the daily produced bacteria and constituting the largest genetic diversity pool on our planet. Currently, bacterial infections linked to healthcare services are widespread, which, when associated with the increasing surge of antibiotic-resistant microorganisms, play a major role in patient morbidity and mortality. In this scenario, Pseudomonas aeruginosa alone is responsible for ca. 13–15% of all hospital-acquired infections. The pathogen P. aeruginosa is an opportunistic one, being endowed with metabolic versatility and high (both intrinsic and acquired) resistance to antibiotics. Bacteriophages (or phages) have been recognized as a tool with high potential for the detection of bacterial infections since these metabolically inert entities specifically attach to, and lyse, bacterial host cells, thus, allowing confirmation of the presence of viable cells. In the research effort described herein, three different phages with broad lytic spectrum capable of infecting P. aeruginosa were isolated from environmental sources. The isolated phages were elected on the basis of their ability to form clear and distinctive plaques, which is a hallmark characteristic of virulent phages. Next, their structural and functional stabilization was achieved via entrapment within the matrix of porous alginate, biopolymeric, and bio-reactive, chromogenic hydrogels aiming at their use as sensitive matrices producing both color changes and/or light emissions evolving from a reaction with (released) cytoplasmic moieties, as a bio-detection kit for P. aeruginosa cells. Full physicochemical and biological characterization of the isolated bacteriophages was the subject of a previous research paper.

The strategy of biopreservation of meat product against MRSA using lytic domain of lysin from Staphylococcus aureus bacteriophage

Staphylococcus aureus is a notorious foodborne pathogen capable of producing various toxins including heat-stable enterotoxin, forming biofilm, evolving to multidrug-resistant strains including methicillin-resistant S. aureus (MRSA) against antibiotics, and threatening public health, of which the biocontrol garners increasing interest from both researchers and food industry. Recently, bacteriophage lysins, degrading cell wall that is indispensable for bacteria, are deemed promising antimicrobial agents. In this work, the lysin LysGH15 from MRSA phage GH15 demonstrated a broad lytic spectrum to S. aureus, cleaving six test S. aureus strains including MRSA, as well as its catalytic domain CHAPLysGH15 alone. LysGH15 and CHAPLysGH15 exhibited different characteristics to one MRSA strain (MRSA 2701), reaching the highest activity at different conditions (35 °C and pH of 6.0 for LysGH15, 40 °C and pH of 9.0 for CHAPLysGH15). Especially, the different sensitivities of two enzymes to NaCl concentration in foods were found, where 500 mM of NaCl was indispensable for LysGH15's efficient lytic action while high concentration of NaCl impaired the activity of CHAPLysGH15. The susceptibility to NaCl and high optimal antimicrobial pH (9.0) of CHAPLysGH15 enabled its application in low-salt and non-acid foods such as fresh meat, of which the preservation is always an issue in food industry. LysGH15 and CHAPLysGH15 presented a promising biocontrol effect in high-salt food and low-salt food, respectively. The 0.4 nmol/cm2 of LysGH15 and CHAPLysGH15 were applied in Chinese bacon and fresh pork, where they could efficiently decrease MRSA from 104 to 0 CFU/cm2 after 2 h (LysGH15 to bacon, and CHAPLysGH15 to fresh pork).

Identification and Characterization of New Bacteriophages to Control Multidrug-Resistant Pseudomonas aeruginosa Biofilm on Endotracheal Tubes.

Studies involving antimicrobial-coated endotracheal tubes are scarce, and new approaches to control multidrug-resistant Pseudomonas aeruginosa biofilm on these devices should be investigated. In this study, five new P. aeruginosa bacteriophages from domestic sewage were isolated. All of them belong to the order Caudovirales, Myoviridae family. They are pH and heat stable and produce 27 to 46 particles after a latent period of 30 min at 37°C. Their dsDNA genome (ranging from ∼62 to ∼65 kb) encodes 65 to 89 different putative proteins. They exhibit a broad lytic spectrum and infect 69.7% of the P. aeruginosa strains tested. All the bacteriophages were able to reduce the growth of P. aeruginosa strains in planktonic form. The bacteriophages were also able to reduce the biofilm viability rates and the metabolic activity of P. aeruginosa strains in a model of biofilms associated with endotracheal tubes. In addition, scanning electron microscopy micrographs showed disrupted biofilms and cell debris after treatment of bacteriophages, revealing remarkable biofilm reduction. The lytic activity on multidrug-resistant P. aeruginosa biofilm indicates that the isolated bacteriophages might be considered as good candidates for therapeutic studies and for the application of bacteriophage-encoded products.

Characterization of Endolysin LysECP26 Derived from rV5-like Phage vB_EcoM-ECP26 for Inactivation of Escherichia coli O157:H7.

With an increase in the consumption of non-heated fresh food, foodborne shiga toxin-producing Escherichia coli (STEC) has emerged as one of the most problematic pathogens worldwide. Endolysin, a bacteriophage-derived lysis protein, is able to lyse the target bacteria without any special resistance, and thus has been garnering interest as a powerful antimicrobial agent. In this study, rV5-like phage endolysin targeting E. coli O157:H7, named as LysECP26, was identified and purified. This endolysin had a lysozyme-like catalytic domain, but differed markedly from the sequence of lambda phage endolysin. LysECP26 exhibited strong activity with a broad lytic spectrum against various gram-negative strains (29/29) and was relatively stable at a broad temperature range (4°C- 55°C). The optimum temperature and pH ranges of LysECP26 were identified at 37°C-42°C and pH 7- 8, respectively. NaCl supplementation did not affect the lytic activity. Although LysECP26 was limited in that it could not pass the outer membrane, E. coli O157: H7 could be effectively controlled by adding ethylenediaminetetraacetic acid (EDTA) and citric acid (1.44 and 1.14 log CFU/ml) within 30 min. Therefore, LysECP26 may serv as an effective biocontrol agent for gram-negative pathogens, including E. coli O157:H7.

Isolation and characterization of a novel phage Xoo-sp2 that infects Xanthomonas oryzae pv. oryzae.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a serious bacterial disease in rice-growing regions worldwide. Phage therapy has been proposed as a potential measure to treat bacterial infections. In this study, a novel phage, Xoo-sp2, which infects Xoo was isolated from soil. The characteristics of Xoo-sp2, including the morphology, one-step growth curve and host range, were analysed. The genome of phage Xoo-sp2 was sequenced and annotated. The results demonstrated that Xoo-sp2 is a siphovirus and has a broad lytic spectrum, infecting 9 out of 10 representative Xoo strains. Genome analysis showed that the Xoo-sp2 genome consists of a linear double-stranded DNA molecule of length 60 370 bp. Annotation of the whole genome indicated that Xoo-sp2 encodes 79 putative open reading frames (ORFs). Comparative genomics analysis of Xoo-sp2 showed that it shares significant similarity only with Pseudomonas and Stenotrophomonas phages (with maximum identity reaching 80 % along 69 % of the genome), and thus represents a novel Xanthomonas phage. Xoo-sp2 significantly inhibited Xoo growth in liquid culture. An experiment with potted plants indicated that Xoo-sp2 could efficiently control BLB in living rice. In summary, our work characterized a novel Xanthomonas phage and demonstrated its potential as a prophylactic agent in the control of BLB in rice.

Characterization of a newly isolated phage infecting pathogenic Escherichia coli and analysis of its mosaic structural genes

The outbreak of multidrug-resistant pathogenic bacteria made the discovery of novel control strategies necessary. Phages have regained attention for their specific lytic activity against pathogenic bacterium. A newly isolated phage infecting the clinical Escherichia coli isolates, including several multidrug-resistant strains, was isolated, and this phage showed high control effects against the tested pathogenic E. coli strains. Host range analysis revealed that although the phage exhibited broad lytic spectrum against the tested E. coli strains, it could not lyse strains from the other species. Comparative genomic analysis showed that phages had undergone at least three genome recombination events during the evolutionary process at the position of the three phage tail genes, which was reported to be associated with the host range determination of the phage. The recombinant tail proteins contained functional domains that were highly similar with genes of the Salmonella phage and genes of Pseudomonas and Neisseria. The findings of this study not only provide resources for developing phage therapy against E. coli, but also showed the highly variable genome structure of the phage.

A lytic bacteriophage of the newly emerging rainbow trout pathogen Weissella ceti

This study was conducted to isolate and characterize a bacteriophage of a newly emerging pathogen, Weissella ceti, which causes weissellosis outbreaks of intensively farmed rainbow trout worldwide. The phage appeared together with the cultured Weissella ceti during isolation of pathogen from kidney of diseased rainbow trout. The morphological, physiological, proteomic and lytic spectrum were characterized. This phage, named PWc, belonged to the family Siphoviridae and possessed an isometric head (approximately 65 nm in diameter) and a flexible, non-contractile tail of 170–180 nm in length. The latent time and burst size of PWc were approximately 25 min and 16 PFU/infected cells, respectively. The PWc was relatively stable over a wide range of temperatures and pH values and possessed a broad lytic spectrum, lysing all 36 tested W. ceti strains isolated from diseased rainbow trout in Japan. The protein profile of the phage was obtained using SDS-PAGE analysis, and the potential packaging strategy was determined based on terminase large subunit sequence analysis. This is the first study to investigate a lytic bacteriophage of a newly emerging pathogen W. ceti that causes infectious disease in rainbow trout.

Morphological characterization and biocontrol effects of Vibrio vulnificus phages against Vibriosis in the shrimp aquaculture environment

The re-emerging field of phage therapy is the potential biocontrol agents for the transfer of virulence factor and to kill their bacterial hosts. In this study, the lytic Vibrio vulnificus phages were studied to provide a better understanding of phage-host interactions and development of phage therapy. Four new V. vulnificus phages were detected from shrimp aquaculture system, named VV1, VV2, VV3 and VV4. All lytic V. vulnificus phages are the Tectiviruses of the family Tectiviridae with typical double layered elongated icosahedral head and tailless morphology. Lytic V. vulnificus phages which infect other Vibrio isolates were further characterized by long term storage, enzymes treatment, organic solvents treatment, detergents treatment, pH stability, temperature stability, agar bioassay method and one-step growth experiment. The effects of chloroform, acetone, ethyl alcohol, methyl alcohol, ribonuclease (RNase), trypsin, protease, Triton-X100 treatments were not affected the growth of VV1, VV2, VV3 and VV4 phages. These phages (VV1-VV4) were inactivated completely with temperature (over 60 °C), pH (<3 or >12), lysozyme and sodium dodecyl sulfate (SDS) treatment. One-step growth experiments indicated that the latent period was at 3 h and burst size was at 37 °C. Agar bioassay method indicated that the percentage of inhibition was 75% (VV1) and 70% (VV2, VV3 & VV4), respectively. SDS-PAGE analysis of all V. vulnificus phages had similar protein patterns with molecular weight masses of 260, 249, 204, 148, 63, 59, 22 and 15 kDa. Hence, it could be concluded that V. vulnificus phage had a broad lytic spectrum and potential biocontrol of luminous Vibriosis in the shrimp aquaculture system.

Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity.

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

The lytic activity of recombinant phage lysin LysKΔamidase against staphylococcal strains associated with bovine and human infections in the Jiangsu province of China

We investigated the lytic activity of the bacteriophage endolysin (lysin) LysKΔamidase against live methicillin-resistant and-susceptible staphylococcal strains clinically isolated from bovine milk and humans from different origins of China. Antibiotic resistance patterns, multilocus sequence typing and SCCmec type of 137 staphylococcal strains isolated from bovine milk associated with bovine mastitis and human diseases were studied. A lytic enzyme, LysKΔamidase, was constructed by fusing the N-terminal 220 amino acids with the C-terminal 105 amino acids of staphylococcal phage lysin LysK. Herein, the antimicrobial activity of LysKΔamidase against 66 methicillin-resistant staphylococcal strains and 71 methicillin-susceptible staphylococcal strains isolated from bovine milk and from humans in China were studied. Our results show that the lysin displayed a broad lytic spectrum; in vitro treatment killed all 137 of the milk and clinical isolates of staphylococci strains tested, including MRSA, methicillin-susceptible S. aureus (MSSA), MR-Staphylococcus hominis ssp. homins, MR-Staphylococcus epidermidis and MR-Staphylococcus haemolyticus as evidenced by scanning electron microscopy, transmission electron microscopy, turbidity reduction assay and disruption of biofilms. The present results suggest that LysKΔamidase has the potential to be an alternative therapeutic agent against pathogenic methicillin-resistant and-susceptible staphylococcal strains isolated from China.

Isolation and characterization of a T4-like phage with a relatively wide host range within Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, resulting in severe economic losses worldwide. Coliphages represent alternative antibacterial substitutes based on high lytic efficiency and few side-effects. However, the complete genome sequences information of APEC phages are limited, and knowledge of undesired genes and the narrow host range restrict their applications. In this study, we isolated a virulent phage QL01, with a relatively broad lytic spectrum (41 of 78 APEC strains). Transmission electron micrography showed it belonged to the family Myoviridae with an elongated head and a contractile tail. Whole genome sequencing revealed a linear double-stranded DNA (170,527 kb; GC content, 39.6%) with 275 possible ORFs. Comparative genome analysis revealed high homology between QL01 and other T4-like phages. However, it also showed some unique features, for example, ORF142 and ORF143, which encode IP9 and IP8, respectively, and may counteract host resistance only exist in a few T4-like phages such as IME08 and vB_EcoM_VR5. Furthermore, phage therapy in artificially infected ducks showed a 26.67% decrease in mortality compared with the untreated group. Our study indicates the potential antibacterial function of phage QL01 against APEC infections and highlights unique molecular features underlying the relatively broad host range.