Development of a novel bacteriophage based biomagnetic separation method as an aid for sensitive detection of viable Escherichia coli

Wang, Ziyuan; Wang, Danhui; Chen, Juhong; Sela, David A.; Nugen, Sam R.

doi:10.1039/c5an01769f

Cited by 47 publications

(45 citation statements)

References 46 publications

(49 reference statements)

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“…So the use of the phage can help distinguish and separate the dead and living cells (26). There have been several researches involved in using phage to separate the bacteria followed by other detection methods (13,57). The detection methods for Salmonella and E.coli O157:H7 based on bacteriophage form foods matrix are commercially available.…”

Section: Bacteriophagementioning

confidence: 99%

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Wang

et al. 2016

Journal of Food Protection

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An effective and rapid method for the separation of bacteria from food matrix remains a bottleneck for rapid bacteria detection for food safety. Bacteria can strongly attach to a food surface or internalize within the matrix, making their isolation extremely difficult. Traditional methods of separating bacteria from food routinely involve stomaching, blending, and shaking. However, these methods may not be efficient at removing all the bacteria from complex matrices. Here, we investigate the benefits of using enzyme digestion followed by immunomagnetic separation to isolate Salmonella from spinach and lettuce. Enzymatic digestion using pectinase and cellulase was able to break down the structure of the leafy green vegetables, resulting in the detachment and release of Salmonella from the leaves. Immunomagnetic separation of Salmonella from the liquefied sample allowed an additional separation step to achieve a more pure sample without leaf debris that may benefit additional downstream applications. We have investigated the optimal combination of pectinase and cellulase for the digestion of spinach and lettuce to improve sample detection yields. The concentrations of enzymes used to digest the leaves were confirmed to have no significant effect on the viability of the inoculated Salmonella. Results reported that the recovery of the Salmonella from the produce after enzyme digestion of the leaves was significantly higher (P <0.05) than traditional sample preparation methods to separate bacteria (stomaching and manually shaking). The results demonstrate the potential for use of enzyme digestion prior to separation can improve the efficiency of bacteria separation and increase the likelihood of detecting pathogens in the final detection assay.

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Section: Bacteriophagementioning

confidence: 99%

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Wang

et al. 2016

Journal of Food Protection

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“…Ͼ86% of E. coli cells, resulting in a detection limit of 10 2 CFU · ml Ϫ1 (35). The limitations of phages as affinity molecules include their relatively large size in comparison to that of antibodies, the retention of enzymatic activity by their binding proteins, and their basal lytic activity for target cells, which releases cellular components that could hinder downstream detection (34).…”

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confidence: 99%

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

Denyes

Dunne

Steiner

et al. 2017

Appl Environ Microbiol

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Bacteriophage-based assays and biosensors rival traditional antibodybased immunoassays for detection of low-level Salmonella contaminations. In this study, we harnessed the binding specificity of the long tail fiber (LTF) from bacteriophage S16 as an affinity molecule for the immobilization, enrichment, and detection of Salmonella. We demonstrate that paramagnetic beads (MBs) coated with recombinant gp37-gp38 LTF complexes (LTF-MBs) are highly effective tools for rapid affinity magnetic separation and enrichment of Salmonella. Within 45 min, the LTF-MBs consistently captured over 95% of Salmonella enterica serovar Typhimurium cells from suspensions containing from 10 to 10 5 CFU · ml Ϫ1 , and they yielded equivalent recovery rates (93% Ϯ 5%, n ϭ 10) for other Salmonella strains tested. LTF-MBs also captured Salmonella cells from various food sample preenrichments, allowing the detection of initial contaminations of 1 to 10 CFU per 25 g or ml. While plating of bead-captured cells allowed ultrasensitive but time-consuming detection, the integration of LTF-based enrichment into a sandwich assay with horseradish peroxidaseconjugated LTF (HRP-LTF) as a detection probe produced a rapid and easy-to-use Salmonella detection assay. The novel enzyme-linked LTF assay (ELLTA) uses HRP-LTF to label bead-captured Salmonella cells for subsequent identification by HRP-catalyzed conversion of chromogenic 3,3=,5,5=-tetramethylbenzidine substrate. The color development was proportional for Salmonella concentrations between 10 2 and 10 7 CFU · ml Ϫ1 as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 10 2 CFU · ml Ϫ1 S. Typhimurium cells. It positively identified 21 different Salmonella strains, with no cross-reactivity for other bacteria. In conclusion, the phage-based ELLTA represents a rapid, sensitive, and specific diagnostic assay that appears to be superior to other currently available tests. IMPORTANCEThe incidence of foodborne diseases has increased over the years, resulting in major global public health issues. Conventional methods for pathogen detection can be laborious and expensive, and they require lengthy preenrichment steps. Rapid enrichment-based diagnostic assays, such as immunomagnetic separation, can reduce detection times while also remaining sensitive and specific. A critical component in these tests is implementing affinity molecules that retain the ability to specifically capture target pathogens over a wide range of in situ applications. The protein complex that forms the distal tip of the bacteriophage S16 long tail fiber is shown here to represent a highly sensitive affinity molecule for the specific enrichment and detection of Salmonella. Phage-encoded long tail fibers have huge potential for development as novel affinity molecules for robust and specific diagnostics of a vast spectrum of bacteria.

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“…The multiplicity of the infection is a natural amplification of a signal and increases the sensitivity of the method. Unfortunately, this type of phage detection is not a viable quantification method when testing natural water sources because VBNC or injured cells will not replicate the phage dependably (Vinay et al ., ; Wang et al ., ).…”

Section: Sensor Methodsmentioning

confidence: 97%

New strategies for the enumeration of enteric pathogens in water

Gorski

Rivadeneira

Cooley

2019

Environmental Microbiology Reports

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Water quality standards for drinking water and recreational waters have long been based on the enumeration of faecal coliforms in the various water supplies, with 0 CFU Escherichia coli/100 ml for drinking water and <126 CFU generic E. coli/100 ml for recreational waters. Irrigation water will soon undergo the same scrutiny in the United States. For over 50 years the most probable number method has been used by laboratories to estimate the level of viable bacteria in a sample, but this method is labour intensive and slow, especially if large numbers of samples need to be tested. In this review, we describe some recent innovations in methods to enumerate enteric pathogens in water. These methods are based on different reasoning schemes that can be categorized as biosensors and nucleic acid-based methods. All the methods described here used natural water sources. Several were also used to survey the bacterial levels in naturally contaminated samples. The different methods vary in their limits of detection, ease of use, and potential portability. Some combine very good limits of detection with the ability to overcome technical challenges; however, there is considerable room for improvement, as none of the methods are without shortcomings.

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Development of a novel bacteriophage based biomagnetic separation method as an aid for sensitive detection of viable Escherichia coli

Cited by 47 publications

References 46 publications

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

New strategies for the enumeration of enteric pathogens in water

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