Expansion of the Plaquing Host Range and Improvement of the Absorption Rate of a T5-like Salmonella Phage by Altering the Long Tail Fibers.

The high host specificity of phages is a real challenge in the therapy applications of the individual phages. This study aimed to edit the long tail fiber proteins (pb1) of a T5-like phage to obtain the engineered phages with expanded plaquing host range. Two T5-like Salmonella phages with high genome sequence homology but different plaquing host ranges, narrow-host range phage vB STyj5-1 (STyj5-1) and wide-host range phage vB BD13 (BD13), were isolated and characterized. The pb1 parts of STyj5-1 were replaced by the corresponding part of BD13 using homologous recombination method to obtain the engineered phages. The alterations of the whole pb1 part or the N-terminal amino acids 1-400 of pb1 of STyj5-1 could expand their plaquing host ranges (from 20 strains to 30 strains) and improve their absorption rates (from 0.28-28.84% to 28.10-99.49%). Besides, the one-step growth curves of these engineered phages with modified pb1 parts were more similar to that of STyj5-1. The burst sizes of phages BD13, STyj5-1 and the engineered phages were 250, 236, 166, and 223 PFU per cell, respectively. The expanded plaquing host range and improved absorption rates of these engineered phages revealed that the pb1 part might be the primary determinant of the host specificities of some T5-like phages. IMPORTANCE Genetic editing can be used to change or expand the host range of phages and have been successfully applied in T2, T4 and other phages to obtain engineered phages. However, there are hardly any similar reports on T5-like phages due to that the determinant regions related to their host ranges have not been completely clarified and the editing of T5-like phages is more difficult compared to other phages. This study attempted and successfully expanded the host range of a narrow-host range T5-like phage (STyj5-1) by exchanging its whole pb1 part or the N-terminal 1-400aa of that part by a broad-host range phage (BD13). These demonstrated the pb1 part might be the primary determinant of the host specificities for some T5-like phages and provided an effective method of extension plaquing host range of these phages.

Complete genome analysis of the novel Alcaligenes faecalis phage vB_AfaP_QDWS595.

A novel lytic phage named vB_AfaP_QDWS595 infecting Alcaligenes faecalis was isolated and characterized in this study. The genome of phage vB_AfaP_QDWS595 was sequenced and analyzed, and the result revealed that the phage contained 70,466 bp of double-stranded DNA with 41.12% GC content. There were 74 putative genes encoding proteins as well as 11 tRNAs predicted in the phage genome. Phenotype and phylogeny analysis indicated that this phage might be a new member of the family Schitoviridae.

The cell structure damage and embden-meyerhof-parnas pathway inhibition of Listeria monocytogenes induced by glycinin basic peptide.

Glycinin basic peptide (GBP) is a natural antibacterial peptide. This study aimed to explore the antibacterial characteristics of GBP against Listeria monocytogenes (L. monocytogenes) by measuring the membrane potential, membrane permeability, cell damage, morphological changes, respiration metabolism inhibition and DNA content. GBP increased the surface zeta potential and decreased the trans-membrane potential of L. monocytogenes in a dose-dependent manner. Compared with the control, the electrical conductivities of GBP-treated bacterial suspensions were significantly increased. The percentages of bacteria with damaged membrane increased from 6.40% to 70.90% with GBP from 0 to 0.8 mg/mL. Obvious rupture and deform of bacterial cells with GBP were observed by transmission electron microscope (TEM), showing the destructive effect of GBP on L. monocytogenes. GBP also inhibited the embden-meyerhof-parnas pathway of the bacterial respiration metabolism and reduced the activities of its key regulator enzymes. Besides, the content of DNA in GBP-treated L. monocytogenes was lower than that in control.

The potential of glycinin basic peptide derived from soybean as a promising candidate for the natural food additive and preservative: A review.

Glycinin basic peptide (GBP) is the basic polypeptide of soybean glycinin that is isolated using cheap and readily available raw materials (soybean meals). GBP can bear high-temperature processing and has good functional properties, such as emulsification and adhesion properties et al. GBP exhibits broad-spectrum antimicrobial activities against Gram-positive and Gram-negative bacteria as well as fungi. Beyond that, GBP shows enormous application potential to improve the quality and extend the shelf life of food products. This review will systematically provide information on the purification, physicochemical and functional properties of GBP. Moreover, the antimicrobial activities and multi-target antimicrobial mechanism of GBP as well as the applications of GBP in different food products are also reviewed and discussed in detail. This review aims to offer valuable insights for the applications of GBP in the food industry as a promising natural food additive and preservative.

Development of the phage lysin-loaded liposomes as preservatives for live clams.

Exogenous applications of phage lysins against Vibrio parahaemolyticus (V. parahaemolyticus) are a challenge due to the gram-negative bacteria outer membrane barrier. This study aimed to improve the antibacterial effect of V. parahaemolyticus phage lysin Lysqdvp001 (Lys), the best-characterized lysin with lytic activity against multiple species of Vibrios, by using liposome delivery. Various kinds of Lys-loaded liposome (Lys-lip) systems were designed and tested. The antibacterial activities of cationic guar gum (CGG) containing liposomes were much higher than the other liposomes, causing >5 log10CFU/mL of reductions of V. parahaemolyticus in buffer and severely damaging the bacterial cell structure. Moreover, some CGG liposome formulations retained high antibacterial effect after both 60-80 °C heat treatments and freeze-drying. Besides, the most stable liposome formulation killed 99 % of V. parahaemolyticus in the seawater with live clams, and its depuration rate against the bacterial contaminated clams also reached 99 %.

Tn5 Transposon-based Mutagenesis for Engineering Phage-resistant Strains of Escherichia coli BL21 (DE3).

Escherichia coli is a preferred strain for recombinant protein production, however, it is often plagued by phage infection during experimental studies and industrial fermentation. While the existing methods of obtaining phage-resistant strains by natural mutation are not efficient enough and time-consuming. Herein, a high-throughput method by combining Tn5 transposon mutation and phage screening was used to produce Escherichia coli BL21 (DE3) phage-resistant strains. Mutant strains PR281-7, PR338-8, PR339-3, PR340-8, and PR347-9 were obtained, and they could effectively resist phage infection. Meanwhile, they had good growth ability, did not contain pseudolysogenic strains, and were controllable. The resultant phage-resistant strains maintained the capabilities of producing recombinant proteins since no difference in mCherry red fluorescent protein expression was found in phage-resistant strains. Comparative genomics showed that PR281-7, PR338-8, PR339-3, and PR340-8 mutated in ecpE, nohD, nrdR, and livM genes, respectively. In this work, a strategy was successfully developed to obtain phage-resistant strains with excellent protein expression characteristics by Tn5 transposon mutation. This study provides a new reference to solve the phage contamination problem.

Biological characterization and complete genome analysis of a novel Stenotrophomonas maltophilia phage vB_SM_ytsc_ply2008005c.

Multidrug-resistant bacteria have become a major threat to global public health. Bacteriophages are regarded as a promising substitute. Here, we present a novel lytic Stenotrophomonas maltophilia phage, vB_SM_ytsc_ply2008005c, which was isolated from sewage water samples in Qingdao, east China. Virion morphology of phage particles indicated that ply2008005c has an icosahedral head (56 ± 5 nm in diameter) and a noncontractile sheathed tail (129 ± 6 nm in length), which are the typical characteristics of phages belonging to the family Siphoviridae. Phage ply2008005c could be used for phage therapy for its stability in a wide pH (4 to 12) range and high temperature (up to 70°C) environment. Genome analysis revealed that ply2008005c has a circular double-strand DNA of 42,318 bp with a G+C content of 63.02%. It shared the closest relationship with phage vB_PaeS_PAO1_Ab18, but the homology coverage is just 20%. There were 54 open reading frames predicted in its genome, including three unique proteins and 34 functional genes in different modules. The phylogenetic analysis revealed that ply2008005c forms a distinct branch of the family Siphoviridae. These results demonstrated that ply2008005c was supposed to be a representative new member within the family Siphoviridae, which could be considered a potential bioagent against multidrug-resistant S. maltophilia.

Development of cationic peptide chimeric lysins based on phage lysin Lysqdvp001 and their antibacterial effects against Vibrio parahaemolyticus: A preliminary study.

Cationic peptide chimeric lysins, Lysqdvp001-5aa, Lysqdvp001-10aa and Lysqdvp001-15aa, were designed based on lysin Lysqdvp001 from Vibrio parahaemolyticus (V. parahaemolyticus) phage qdvp001. These chimeric lysins showed equivalent peptidoglycan hydrolysis activities with Lysqdvp001 and could lyse the bacteria from the outside. The antibacterial activity as well as outer and inner membrane permeabilization of Lysqdvp001 and chimeric lysins against V. parahaemolyticus were Lysqdvp001-15aa>Lysqdvp001-10aa>Lysqdvp001-5aa>Lysqdvp001. Lysqdvp001-15aa exhibited an excellent antibacterial activity with minimum inhibition and bactericidal concentrations (MIC and MBC) of 0.2 and 0.4 mg/mL, respectively, and its antibacterial spectrum was much broader than phage qdvp001. Membrane hyperpolarization and membrane phospholipid exposure of V. parahaemolyticus were observed after Lysqdvp001-15aa treatments. Transmission electron microscope (TEM) showed Lysqdvp001-15aa destroyed structure integrity of V. parahaemolyticus. Besides, MIC and MBC of Lysqdvp001-15aa decreased V. parahaemolyticus counts in oyster by 3.20 and 4.03 log10CFU/g. Lysqdvp001-15aa at MBC eradicated about 50% of V. parahaemolyticus biofilms and inhibited over 90% of the formation of the bacterial biofilms.

Synergistic effects of endolysin Lysqdvp001 and ε-poly-lysine in controlling Vibrio parahaemolyticus and its biofilms.

The synergistic antibacterial effects between endolysin Lysqdvp001 and ε-poly-lysine (ε-PL) against Vibrio parahaemolyticus (V. parahaemolyticus) were investigated in this study. Lysqdvp001 combined with ε-PL exhibited a strong antibacterial synergism against V. parahaemolyticus. The combinations of Lysqdvp001 (≥60 U/mL) and ε-PL (≥0.2 mg/mL) dramatically decreased cell density of the bacterial suspensions at both 25 °C and 37 °C. Surface zeta potential increment and membrane hyperpolarization of V. parahaemolyticus were observed after treatment by ε-PL and its combination with Lysqdvp001. More ß-lactamase and ß-galactosidase were leaked from V. parahaemolyticus with combined treatment of Lysqdvp001 and ε-PL than from the bacteria treated with single Lysqdvp001 or ε-PL. Fluorescence and transmission electron microscope revealed that Lysqdvp001 and ε-PL synergistically induced the damage and morphological destruction of V. parahaemolyticus cells. When applying in Gadus macrocephalus, Penaeus orientalis and oyster, the two antimicrobials' cocktail allowed for 3.75, 4.16 and 2.50 log10CFU/g reductions of V. parahaemolyticus, respectively. Besides, Lysqdvp001 in combination with ε-PL removed approximately 44%-68% of V. parahaemolyticus biofilms on polystyrene, glass and stainless steel surfaces. These results demonstrated that Lysqdvp001 and ε-PL might be used together for controlling V. parahaemolyticus and the bacterial biofilms in food industry.

Characterizations of the endolysin Lys84 and its domains from phage qdsa002 with high activities against Staphylococcus aureus and its biofilms.

Staphylococcus aureus (S. aureus), particularly methicillin-resistant S. aureus (MRSA), and its biofilms are great threats in the food industry. Bacteriophage-encoded endolysins are promising tools to inhibit pathogens and to eliminate their biofilms. In this work, a virulent phage qdsa002 against S. aureus ATCC43300 (MRSA) was isolated, and the phage's endolysin (Lys84) and its domains were expressed and purified. Morphological and genome analyses demonstrated that qdsa002 is a Twort-like phage from Myoviridae. Lys84 contains two catalytic domains (CHAP and Amidase_2) and one cell binding domain (SH3b). This endolysin exhibits a strong lytic activity against S. aureus and has a wider bactericidal spectrum than qdsa002. Moreover, Lys84 exceed 10 µM effectively removed around 90 % of the biofilms of S. aureus. Besides, CHAP and Amidase_2 domains remained 61.20 % and 59.46 % of lytic activity as well as 84.31 % and 70.11 % of anti-biofilm activity of Lys84, respectively. The lytic and anti-biofilm activities of the combination of CHAP and Amidase_2 were close to 90 % of those of Lys84. These results indicated that Lys84 and its domains might be alternative antimicrobials for controlling S. aureus and its biofilms.