Colorimetric detection of Staphylococcus aureus with enhanced sensitivity based on phage covalently immobilized Co3O4 nanozyme through synergistic inhibition effect.

Staphylococcus aureus (S. aureus) is a common foodborne pathogen, posing a serious threat to public health. Consequently, it is crucial to establish a platform for sensitive and specific determination of S. aureus in food. Herein, phage SapYZUH5, isolated by our lab, was covalently immobilized on Co3O4 to synthesize SapYZUH5@Co3O4. Notably, SapYZUH5@Co3O4 exhibited remarkable oxidase-like activity, enabling the catalysis of dissolved oxygen to generate superoxide anion free radicals and accelerate the TMB chromogenic reaction. Upon introduction of S. aureus, specific capture by SapYZUH5@Co3O4 resulted in inhibiting its oxidase-like activity and decelerating the 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction. Moreover, S. aureus can be lysed to release the reductive bacterial contents, which can further inhibit the TMB chromogenic reaction. Based on this principle, SapYZUH5@Co3O4 + TMB reaction system was employed for detection with enhanced sensitivity of S. aureus, yielding an equation: A = - 0.092 Log (CSA) + 0.79 (R2 = 0.987), with an ultralow limit of detection (LOD) of 28 CFU mL-1. This system exhibited remarkable specificity and anti-interfere towards S. aureus, owing to the excellent affinity of SapYZUH5 towards S. aureus. In addition, S. aureus in the actual food samples was detected using this system, yielding recoveries ranging from 96.34 to 109.43%, demonstrating its exceptional accuracy. Hence, our proposed covalent immobilization of phage on the nanozyme can realize sensitive and specific colorimetric determination of S. aureus in food samples.

Immobilization of a broad host range phage on the peroxidase-like Fe-MOF for colorimetric determination of multiple Salmonella enterica strains in food.

A broad host range phage-based nanozyme (Fe-MOF@SalmpYZU47) was prepared for colorimetric detection of multiple Salmonella enterica strains. The isolation of a broad host range phage (SalmpYZU47) capable of infecting multiple S. enterica strains was achieved. Then, it was directly immobilized onto the Fe-MOF to prepare Fe-MOF@SalmpYZU47, exhibiting peroxidase-like activity. The peroxidase-like activity can be specifically inhibited by multiple S. enterica strains, benefiting from the broad host range capture ability of Fe-MOF@SalmpYZU47. Based on it, a colorimetric detection approach was developed for S. enterica in the range from 1.0 × 102 to 1.0 × 108 CFU mL-1, achieving a low limit of detection (LOD) of 11 CFU mL-1. The Fe-MOF@SalmpYZU47 was utilized for detecting S. enterica in authentic food samples, achieving recoveries ranging from 91.88 to 105.34%. Hence, our proposed broad host range phage-based nanozyme exhibits significant potential for application in the colorimetric detection of pathogenic bacteria.

Isolation and characterization of the new isolated bacteriophage YZU-L1 against Citrobacter freundii from a package-swelling of meat product.

Citrobacter freundii is an important foodborne pathogen that can cause urethritis, bacteremia, necrotizing abscess, and meningitis in infants. In this study, a gas-producing isolate from vacuum-packed meat products was identified as C. freundii by 16S rDNA. In addition, a new virulent phage YZU-L1, which could specifically lyse C. freundii, was isolated from sewage samples in Yangzhou. Transmission electron microscopy showed that phage YZU-L1 had a polyhedral head of 73.51 nm in diameter and a long tail of 161.15 nm in length. According to phylogenetic analysis employing the terminase large subunit, phage YZU-L1 belonged to the Demerecviridae family and the Markadamsvirinae subfamily. The burst size was 96 PFU/cell after 30 min of latent period and 90 min of rising period. Phage YZU-L1 could maintain high activity at pH of 4-13, and resist 50 °C for up to 60 min. The complete genome of YZU-L1 was 115,014 bp double-stranded DNA with 39.94% G + C content, encoding 164 open reading frames (ORFs), without genes encoding for virulence, antibiotic resistance, or lysogenicity. Phage YZU-L1 treatment significantly reduced the viable bacterial count of C. freundii in a sterile fish juice model, which is expected to be a natural agent for the biocontrol of C. freundii in foods.

Isolation and genomic characterization of Vmp-1 using Vibrio mimicus as the host: A novel virulent bacteriophage capable of cross-species lysis against three Vibrio spp.

Vibrio mimicus is a zoonotic pathogen that is widely distributed in aquatic habitats/environments (marine coastal water, estuaries, etc). The development of biocontrol agents for V. mimicus is imperative for the prevention and control of aquatic animal diseases and human food-borne infections. In this study, a broad-spectrum bacteriophage Vmp-1 was isolated from dealt aquatic product in a local market by double-layer agar plate method using V. mimicus CICC21613 as the host bacteria. Results indicated that Vmp-1, which belongs to the family Podoviridae, showed good pH tolerance (pH 3.0-12.0) and thermal stability (30-50 °C). The optimal multiplicity of infection (MOI) of Vmp-1 was 0.001 for a 20-min incubation and 100-min lysis period. Vmp-1 effectively controlled V. mimicus CICC21613 in LBS model (MOI = 0.0001, 0.001, 0.01, 0.1, 1) within 8 h. The full length of the Vmp-1 genome was 43,312 bp, with average GC content of 49.5%, and a total of 44 protein-coding regions. This study provides a novel phage strain that has the highest homology with vB_VpP_HA5 (GenBank: OK585159.1, 95.96%) for the development of biocontrol agents for V. mimicus.

Comparision of biological and genomic characteristics of five virulent bacteriophages against Enterobacter hormaechei.

Enterobacter hormaechei is a zoonotic bacteria that may cause respiratory diseases in animals and neonatal sepsis in humans. Bacteriophages are increasingly considered as potential biocontrol agents to control pathogens in the food industry. In this study, five E. hormaechei virulent phages, named as Ehp-YZU08, Ehp-YZU10, Ehp-YZU9-1, Ehp-YZU9-2 and Ehp-YZU9-3, were isolated from sewage in China and analyzed for their biological and whole-genome characteristics, and a comparative genomic analysis was performed to study the functional genes and phylogenetic evolution of phages. The results showed that four of the phage strains belong to the Podoviridae family and one belongs to the Myoviridae family. The burst sizes were 70-283 PFU/cell after a latent period of 5-40 min. Phages were able to survive in a pH range of 5-10 and resist temperatures up to 60 °C for 60 min. The sequencing results showed that the full length of the genomes of the five phages ranged from 39,502 to 173,418 bp. Each phage contained multiple genes related to phage replication, and genes related to bacterial virulence or drug resistance were not found. The five phages belonged to three different groups by a construction of a phylogenetic tree, and the significant genetic evolutionary distance from each E. hormaechei phage was observed. The inhibition assay showed that all five phages could completely inhibit the growth of E. hormaechei at 37 °C within 8 h, suggesting that the phages in this study have great potential for the development of biocontrol agents against E. hormaechei in the food industry.

Bacteriophages: A weapon against mixed-species biofilms in the food processing environment.

Mixed-species biofilms represent the most frequent actual lifestyles of microorganisms in food processing environments, and they are usually more resistant to control methods than single-species biofilms. The persistence of biofilms formed by foodborne pathogens is believed to cause serious human diseases. These challenges have encouraged researchers to search for novel, natural methods that are more effective towards mixed-species biofilms. Recently, the use of bacteriophages to control mixed-species biofilms have grown significantly in the food industry as an alternative to conventional methods. This review highlights a comprehensive introduction of mixed-species biofilms formed by foodborne pathogens and their enhanced resistance to anti-biofilm removal strategies. Additionally, several methods for controlling mixed-species biofilms briefly focused on applying bacteriophages in the food industry have also been discussed. This article concludes by suggesting that using bacteriophage, combined with other 'green' methods, could effectively control mixed-species biofilms in the food industry.

Isolation and genomic characterization of P.A-5, a novel virulent bacteriophage against Enterobacter hormaechei.

Enterobacter hormaechei is a foodborne pathogen responsible for neonatal sepsis in humans and respiratory disease in animals. In this work, a new virulent phage (P.A-5) infecting E. hormaechei was isolated from domestic sewage samples and characterized. Transmission electron microscopy revealed that P.A-5 belonged to the family Myoviridae having a head size of 77.53 nm and a tail length of 72.24 nm. The burst size was 262 PFU/cell after a latent period of 20 min. Phage P.A-5 was able to survive in a pH range of 4-9 and resist temperatures up to 55 °C for 60 min. The genome sequence of P.A-5 had homology most similar to that of Shigellae phage MK-13 (GenBank: MK509462.1). Pork artificially contaminated with E. hormaechei was used as a model to evaluate the potential of P.A-5. The results clearly showed that P.A-5 treatment can completely inhibit E. hormaechei growth in pork within 8 h, indicating the potential use of P.A-5 as a biocontrol agent for E. hormaechei.

Development of a highly sensitive fluorescence method for tartrazine determination in food matrices based on carbon dots.

In this work, an ultrasensitive sensing system based on fluorescent carbon dots (CDs) was developed for the tartrazine (Tar) determination. The CDs were prepared via a simple one-pot hydrothermal method with m-phenylenediamine as the only precursor. The physical and chemical properties were in detail characterized by transmission electron microscopy (TEM), MALDI-TOF MS, UV-vis absorption and photoluminescence (PL) spectroscopy, elemental analysis, and Fourier transform infrared spectroscopy (FTIR). Upon exposure to Tar, the fluorescence of CDs was efficiently quenched via the dynamic interaction between CDs and Tar as well as the inner filter effect (IFE). With this information, the CDs were proposed as a fluorescence probe for Tar detection. It was found that CDs had high sensitivity and selectivity for Tar sensing, and the linear relationship was observed in the range of 0.01-25.0 µM with the corresponding detection limit (3σ/k) of 12.4 nM, which is much more sensitive than any of the existed CD-based sensing platform. The investigated sensing system was finally utilized for Tar sensing in various food matrices with a high degree of accuracy. The spiked recoveries were in a range of 96.4-105.2%, and the relative standard deviations (RSDs) were lower than 4.13%. This work highlights the great application prospects of CDs for Tar sensing in a rapid, simple, and sensitive way.

Isolation of virulent phages infecting dominant mesophilic aerobic bacteria in cucumber pickle fermentation.

Pickle is a type of mildly lactic acid fermented vegetable and is a traditional dish favored in China, Japan, and Korea. Corruption of spoilage bacteria and accumulation of nitrite during vegetable fermentation are common problems that affect the pickle industry and consumer health. In this work, cucumber juice was used as a vegetable model to study the dominant mesophilic aerobic bacteria (MAB) producing nitrite during pickle fermentation. Virulent phages infecting the dominant MABs combined with Lactobacillus plantarum M6 were used to control these bacteria. Enterobacter cloacae and Pseudomonas fluorescens are the dominant MABs in the fermentation of cucumber juice containing 4% or 8% NaCl, with isolation percentages reaching 30.6% and 23.1%, respectively. Virulent phages PspYZU5415 and EcpYZU01 were isolated using P. fluorescens J5415 and E. cloacae J01 as the host bacteria, respectively. These two phages show a broad host range and strong lytic activity, and their genomes contain no toxins and antibiotic resistance genes. PspYZU5415 and EcpYZU01 were combined into a cocktail (designated as Phage MIX) that effectively inhibits the growth of E. cloacae and P. fluorescens in cucumber juice with different salt concentrations. PhageMIX combined with L. plantarum M6 decreased the counts of P. mendocina and E. cloacae to undetectable levels at 48 h during the fermentation of cucumber juice artificially contaminated with P. mendocina and E. cloacae. In addition, nitrite content increased to 11.3 mg/L at 20 h and then degraded completely at 36 h. By contrast, P. mendocina and E. cloacae remained in the groups without PhageMIX during fermentation (0-48 h). Nitrite content rapidly increased to 65.7 mg/L at 12 h and then decreased to 21.6 mg/L at 48 h in the control group. This study suggests that PhageMIX combined with lactic acid bacterial strains can be used as an ecological starter for controlling the dominant MABs P. mendocina and E. cloacae and for reducing nitrate production during the early stage of pickle fermentation.