Characterization and genomic analysis of a Demerecviridae phage SP76 with lytic multiple-serotypes of Salmonella.

With the increase in antimicrobial resistance of Salmonella, phages have been paid more attention to as an alternative to antibiotics. In this study, a phage designated as SP76 was isolated from sewage. It can lyse several serotypes of Salmonella, including S. typhimurium (21/33), S. enteritidis (7/7), S. dublin (4/4), S. pullorum (2/2) and S. choleraesuis (1/2). SP76 showed a latent time of about 10 min, and maintained good lytic activity at a pH range of 3-10 and temperatures between 4 and 37 °C. Moreover, its optimal multiplicity of infection (MOI) was 0.0001. Based on the results of genomic sequence and analysis, SP76 was found to have a genome of 111,639 bp that encoded 166 predicted ORFs and belong to the Demerecviridae family, order Caudovirales. No virulence or lysogen formation gene clusters were identified in the SP76 genome. A pan-genome analysis based on 100 phages within the subfamily Markadamsvirinae indicated that SP76 had 23 core genes and 1199 accessory genes. We grouped the subfamily Markadamsvirinae and found that the main difference was in group III. In vitro bacteriostasis, experiments showed that the phage SP76 reduced planktonic bacteria by 1.52 log10 CFU/mL, and biofilms (24 h old) by 0.372 log10 CFU/mL, respectively. Thus, we isolated a safe and efficient phage that might be a good antibacterial agent.

The effectiveness of extended binding affinity of prophage lysin PlyARI against Streptococcus suis infection.

Streptococcus suis is an important zoonotic pathogen. An increase in multi-drug-resistant strains has led to poor performance of traditional antibiotic therapies. Thus, alternative antibacterial agents are urgently needed. In this study, we identified a recombined and expressed lysin PlyARI derived from the novel serotype S. suis (Chz) prophage PhiARI0460-1. The recombinant PlyARI at a concentration of 10 µg/mL showed high bacteriolytic activity against 30 S. suis isolates. The minimum inhibitory concentration (MIC) of PlyARI against S. suis was found to be as low as 2 µg/mL, and the lytic efficiency could be maintained between the range of pH 4 and 12. Additionally, in a mouse infection model, a dose of 0.5 mg of PlyARI protected 10 out of 10 mice that were challenged with highly virulent S. suis strain HA9801. Furthermore, the binding specificity of PlyARI was evaluated by constructing a green fluorescent protein (GFP-ARIb), where GFP was fused with the PlyARI-SH3b (cell wall-binding domain, CBD), revealing a high affinity to S. suis, Staphylococcus aureus, and Streptococcus equi along with exhibiting a medium affinity to Streptococcus pneumoniae as well as Streptococcus agalactiae. Overall, our findings indicated that PlyARI may be an alternative antibacterial agent that was useful in treating and possibly the prevention of Streptococcal infections.

Identification of a phage-derived depolymerase specific for KL64 capsule of Klebsiella pneumoniae and its anti-biofilm effect.

The increasing prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to global health. Phages and phage-derived enzymes gained increasing attention for controling CRKP infections. In this study, a lytic phage P510 infecting KL64 type K. pneumoniae was isolated and characterized. Whole genome analysis and electron microscopy analysis showed that phage P510 belonged to genus Przondovirus, family Autographiviridae, the order Caudovirales. The tail fiber protein of the phage was predicted to encode capsule depolymerase. Further analysis demonstrated that recombinant depolymerase P510dep had polysaccharide-degrading activity against KL64-types capsule of K. pneumoniae, and its lysis spectrum matched to host range of phage P510. We also demonstrated that the recombinant depolymerase was able to significantly inhibit biofilm formation. The discovery of the phage-derived depolymerase lays the foundation for controlling the spread of CRKPs.

Characterization and genome analysis of Klebsiella phage P509, with lytic activity against clinical carbapenem-resistant Klebsiella pneumoniae of the KL64 capsular type.

The increasing population infected by carbapenem-resistant Klebsiella pneumoniae necessitates the development of alternative therapies. In this study, we isolated, characterized, and sequenced a bacteriophage, P509, which was able to specifically infect and lyse carbapenem-resistant K. pneumoniae of K locus type KL64. A one-step growth curve experiment showed that the latent time period of phage P509 was 5 min, and the burst size was about 85 phage particles/cell. Stability tests confirmed that P509 was stable over a wide range of temperatures (4 to 50 °C) and pH (3 to 11) conditions. Phage P509 was identified as a linear double-stranded DNA phage with a genome of 40,954 bp with 53.2% G + C content, encoding 50 predicted proteins. Genomic and morphological analysis suggested that P509 belonged to the genus Przondovirus, family Autographiviridae, order Caudovirales. Further analysis showed that no virulence-related genes or lysogen-formation gene clusters were detected in the genome, suggesting that P509 is a lytic phage, making it potentially suitable for clinical applications. In vitro, the number of viable cells in three phage-treated groups (MOI = 0.1, 0.01, 0.001) decreased by 3.75 log10 CFU/ml, 3.32 log10 CFU/ml and 3.21 log10 CFU/ml, respectively, after 80 min of incubation, in comparison to that in the untreated group. Based on these characteristics, phage P509 may be a promising candidate for future phage therapy applications.

Isolation and Characterization of Novel Lytic Bacteriophages Infecting Epidemic Carbapenem-Resistant Klebsiella pneumoniae Strains.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant clinical problem given the lack of therapeutic options available. Alternative antibacterial agents, such as bacteriophages, can be used as a valuable tool to treat the infections caused by these highly resistant bacteria. In this study, we isolated 54 phages from medical and domestic sewage wastewater between July and September 2019 and determined their host ranges against 54 clinical CRKP isolates, collected from a tertiary hospital in eastern China. The 54 CRKP isolates were from 7 sequence types (STs) and belonged to 9 capsular K locus types, harboring bla KPC- 2 (n = 49), bla NDM- 1 (n = 5), and bla IMP- 4 (n = 3). Among them, the epidemic KPC-2-producing ST11 strains were most predominant (88.9%). The 54 phages showed different host ranges from 7 to 52 CRKP isolates. The total host ranges of three phages can potentially cover all 54 CRKP isolates. Among the 54 phages, phage P545, classified as a member of Myoviridaes, order Caudovirales, had a relatively wide host range (96.3%), a short latent period of 20 min, and a medium burst size of 82 PFU/cell and was stably maintained at different pH values (4-10) and temperatures (up to 60°C). P545 showed the ability to inhibit biofilm formation and to degrade the mature biofilms. Taken together, the results of our study showed that the newly isolated phage P545 had a relatively wide host range, excellent properties, and antibacterial activity as well as antibiofilm activity against a clinical CRKP ST11 isolate, providing a promising candidate for future phage therapy applications.

Recombination of T4-like Phages and Its Activity against Pathogenic Escherichia coli in Planktonic and Biofilm Forms.

The increasing emergence of multi-drug resistant Escherichia coli (E. coli) has become a global concern, primarily due to the limitation of antimicrobial treatment options. Phage therapy has been considered as a promising alternative for treating infections caused by multi-drug resistant E. coli. However, the application of phages as a promising antimicrobial agent is limited by their narrow host range and specificity. In this research, a recombinant T4-like phage, named WGqlae, has been obtained by changing the receptor specificity determinant region of gene 37, using a homologous recombination platform of T4-like phages established by our laboratory previously. The engineered phage WGqlae can lyse four additional hosts, comparing to its parental phages WG01 and QL01. WGqlae showed similar characteristics, including thermo and pH stability, optimal multiplicity of infection and one-step growth curve, to the donor phage QL01. In addition, sequencing results showed that gene 37 of recombinant phage WGqlae had genetically stable even after 20 generations. In planktonic test, phage WGqlae had significant antimicrobial effects on E. coli DE192 and DE205B. The optical density at 600 nm (OD600) of E. coli in phage WGqlae treating group was significantly lower than that of the control group (P < 0.01). Besides, phage WGqlae demonstrated an obvious inhibitory effect on the biofilm formation and the clearance of mature biofilms. Our study suggested that engineered phages may be promising candidates for future phage therapy applications against pathogenic E. coli in planktonic and biofilm forms.