Label-Free Surface-Enhanced Raman Scattering Detection of Fire Blight Pathogen Using a Pathogen-Specific Bacteriophage.

Fire blight is one of the most devastating plant diseases, causing severe social and economic problems. Herein, we report a novel method based on label-free surface-enhanced Raman scattering (SERS) combined with an Erwinia amylovora-specific bacteriophage that allows detecting efficiently fire blight bacteria E. amylovora for the first time. To achieve the highest SERS signals for E. amylovora, we synthesized and compared plasmonic nanoparticles (PNPs) with different sizes, i.e., bimetallic gold core-silver shell nanoparticles (Au@AgNPs) and monometallic gold nanoparticles (AuNPs) and utilized the coffee-ring effect for the self-assembly of PNPs and enrichment of fire blight bacteria. Furthermore, we investigated the changes in the SERS spectra of E. amylovora after incubation with an E. amylovora-specific bacteriophage, and we found considerable differences in the SERS signals as a function of the bacteriophage incubation time. The results indicate that our bacteriophage-based label-free SERS analysis can specifically detect E. amylovora without the need for peak assignment on the SERS spectra but simply by monitoring the changes in the SERS signals over time. Therefore, our facile method holds great potential for the label-free detection of pathogenic bacteria and the investigation of viral-bacterial interactions.

Bacteriophage cocktail for biocontrol of soft rot disease caused by Pectobacterium species in Chinese cabbage.

Pectobacterium spp. are necrotrophic plant pathogens that cause the soft rot disease in Chinese cabbage, resulting in severe yield loss. The use of conventional antimicrobial agents, copper-based bactericides, and antibiotics has encountered several limitations, such as bioaccumulation on plants and microbial resistance. Bacteriophages (phages) are considered promising alternative antimicrobial agents against diverse phytopathogens. In this study, we isolated and characterized two virulent phages (phiPccP-2 and phiPccP-3) to develop a phage cocktail. Morphological and genomic analyses revealed that two phages belonged to the Tevenvirinae and Mccorquodalevirinae subfamilies, respectively. The phiPccP-2 and phiPccP-3 phages, which have a broad host range, were stable at various environmental conditions, such as various pHs and temperatures and exposure to ultraviolet light. The phage cocktail developed using these two lytic phages inhibited the emergence of phage-resistant bacteria compared to single-phage treatments in in vitro challenge assays. The phage cocktail treatment effectively prevented the development of soft rot symptom in matured Chinese cabbage leaves. Additionally, the phage cocktail comprising three phages (phiPccP-1, phiPccP-2, and phiPccP-3) showed superior biocontrol efficacy against the mixture of Pectobacterium strains in Chinese cabbage seedlings. These results suggest that developing phage cocktails is an effective approach for biocontrol of soft rot disease caused by Pectobacterium strains in crops compared to single-phage treatments. KEY POINTS: •Two newly isolated Pectobacterium phages, phiPccP-2 and phiPccP-3, infected diverse Pectobacterium species and effectively inhibited the emergence of phage-resistant bacteria. •Genomic and physiological analyses suggested that both phiPccP-2 and phiPccP-3 are lytic phages and that their lytic activities are stable in the environmental conditions under which Chinese cabbage grows. •Treatment using a phage cocktail comprising phiPccP-2 and phiPccP-3 efficiently suppressed soft rot disease in detached mature leaves and seedlings of Chinese cabbage, indicating the applicability of the phage cocktail as an alternative antimicrobial agent.

Characterization and genomic study of EJP2, a novel jumbo phage targeting antimicrobial resistant Escherichia coli.

The emergence of antimicrobial resistance (AMR) Escherichia coli has noticeably increased in recent years worldwide and causes serious public health concerns. As alternatives to antibiotics, bacteriophages are regarded as promising antimicrobial agents. In this study, we isolated and characterized a novel jumbo phage EJP2 that specifically targets AMR E. coli strains. EJP2 belonged to the Myoviridae family with an icosahedral head (120.9 ± 2.9 nm) and a non-contractile tail (111.1 ± 0.6 nm), and contained 349,185 bp double-stranded DNA genome with 540 putative ORFs, suggesting that EJP2 could be classified as jumbo phage. The functions of genes identified in EJP2 genome were mainly related to nucleotide metabolism, DNA replication, and recombination. Comparative genomic analysis revealed that EJP2 was categorized in the group of Rak2-related virus and presented low sequence similarity at the nucleotide and amino acid level compared to other E. coli jumbo phages. EJP2 had a broad host spectrum against AMR E. coli as well as pathogenic E. coli and recognized LPS as a receptor for infection. Moreover, EJP2 treatment could remove over 80% of AMR E. coli biofilms on 96-well polystyrene, and exhibit synergistic antimicrobial activity with cefotaxime against AMR E. coli. These results suggest that jumbo phage EJP2 could be used as a potential biocontrol agent to combat the AMR issue in food processing and clinical environments.

Characterization of the lytic phage MSP1 for the inhibition of multidrug-resistant Salmonella enterica serovars Thompson and its biofilm.

The increasing prevalence of multidrug-resistant (MDR) Salmonella is a serious public health threat. Intervention strategies available to control Salmonella mostly target Salmonella enterica serovars Typhimurium and Enteritidis, and little has been investigated to control serovars in serogroup C, such as S. enterica serovar Thompson, despite their increasing prevalence. Here, we isolated phages targeting MDR S. Thompson and characterized the antimicrobial activities of MSP1 phage, a virulent phage with a broad host range. MSP1 phage strongly infected S. Thompson and S. Mbandaka isolates from retail chicken and also other serovars, including Dublin, Enteritidis, Heidelberg, Paratyphi, and Typhimurium. MSP1 phage was able to inhibit the biofilm formation on stainless steel and glass formation by around 42.7-47.9 %. MSP1 phage was robust to withstand wide ranges of pH (4-12) and temperature (30-60 °C), and no genes associated with antibiotic resistance and virulence were found in the phage genome, suggesting that this phage is suitable for food application. When MSP1 phage was tested on foods (chicken meat and milk), MSP1 phage significantly reduced the level of MDR S. Thompson below the detection limit. Our findings suggest that MSP1 phage is a promising antimicrobial agent for the control of food contamination by MDR S. Thompson.

Bacteriophage and endolysin engineering for biocontrol of food pathogens/pathogens in the food: recent advances and future trends.

Despite advances in modern technologies, various foodborne outbreaks have continuously threatened the food safety. The overuse of and abuse/misuse of antibiotics have escalated this threat due to the prevalence of multidrug-resistant (MDR) pathogens. Therefore, the development of new methodologies for controlling microbial contamination is extremely important to ensure the food safety. As an alternative to antibiotics, bacteriophages(phages) and derived endolysins have been proposed as novel, effective, and safe antimicrobial agents and applied for the prevention and/or eradication of bacterial contaminants even in foods and food processing facilities. In this review, we describe recent genetic and protein engineering tools for phages and endolysins. The major aim of engineering is to overcome limitations such as a narrow host range, low antimicrobial activity, and low stability of phages and endolysins. Phage engineering also aims to deter the emergence of phage resistance. In the case of endolysin engineering, enhanced antibacterial ability against Gram-negative and Gram-positive bacteria is another important goal. Here, we summarize the successful studies of phages and endolysins treatment in different types of food. Moreover, this review highlights the recent advances in engineering techniques for phages and endolysins, discusses existing challenges, and suggests technical opportunities for further development, especially in terms of antimicrobial agents in the food industry.

Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence.

Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P < 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence. IMPORTANCE Pectobacterium carotovorum subsp. carotovorum is a phytopathogen that causes soft rot disease in various crops by producing plant cell wall-degrading enzymes (PCWDEs). Although antibiotics and copper-based pesticides have been extensively applied to inhibit P. carotovorum subsp. carotovorum, the emergence of antibiotic-resistant bacteria and demand for harmless antimicrobial products have emphasized the necessity of finding alternative therapeutic strategies. To address this problem, we developed a phage cocktail consisting of three P. carotovorum subsp. carotovorum-specific phages that recognize colanic acids and flagella of P. carotovorum subsp. carotovorum. The phage cocktail treatments significantly decreased P. carotovorum subsp. carotovorum populations, as well as soft rot symptoms in napa cabbage. Simultaneously, they resulted in virulence attenuation in phage-resistant P. carotovorum subsp. carotovorum, which was represented by decreased PCWDE production and decreased flagellum-mediated swimming motility. These results suggested that preparations of phage cocktails targeting multiple receptors would be an effective approach to biocontrol of P. carotovorum subsp. carotovorum in crops.

Colanic Acid Is a Novel Phage Receptor of Pectobacterium carotovorum subsp. carotovorum Phage POP72.

The emergence and widespread nature of pathogen resistance to antibiotics and chemicals has led to the re-consideration of bacteriophages as an alternative biocontrol agent in several fields, including agriculture. In this study, we isolated and characterized a novel bacteriophage, POP72, that specifically infects Pectobacterium carotovorum subsp. carotovorum (Pcc), which frequently macerates agricultural crops. POP72 contains a 44,760 bp double-stranded DNA genome and belongs to the family Podoviridae. To determine the phage receptor for POP72, a random mutant library of Pcc was constructed using a Tn5 transposon and screened for resistance against POP72 infection. Most of the resistant clones had a Tn5 insertion in various genes associated with colanic acid (CA) biosynthesis. The phage adsorption rate and CA production decreased dramatically in the resistant clones. Complementation of the clones with the pUHE21-2 lacI q vector harboring genes associated with CA biosynthesis restored their sensitivity to POP72, as well as their ability to produce CA. These results suggest that CA functions as a novel phage receptor for POP72. The application of POP72 protected Chinese cabbage from Pcc infection, suggesting that phage POP72 would be an effective alternative antimicrobial agent to protect agricultural products from Pcc.