A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Hinkley, Troy; Singh, Siddharth; Garing, Spencer; Ny, Anne-Laure M. Le; Nichols, Kevin P.; Peters, Joseph E.; Talbert, Joey N.; Nugen, Sam R.

doi:10.1038/s41598-018-33097-4

Cited by 48 publications

(52 citation statements)

References 25 publications

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“…Incubation of the filters allows recovery from VBNC, which is necessary for phage infection. The method was only tested on drinking water but showed a low LOD of 20 CFU/100 ml (Hinkley et al ., ). Litvinov et al .…”

Section: Additional 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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Section: Additional Methodsmentioning

confidence: 97%

New strategies for the enumeration of enteric pathogens in water

Gorski

Rivadeneira

Cooley

2019

Environmental Microbiology Reports

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“…As seen in Figure 3, only cultures infected with phage NRGp4 or NRGp5 produced luminescent signals above the limit of detection. While changes to the promoter and ribosome binding sites upstream of the reporter only served to decrease the luminescence signal during the infection of identical cultures (data not shown), removal of the pelB N-terminal secretion signal used in phage NRGp4 [48] produced nearly a half log increase in signal intensity (Figure 3) when identical cultures were infected and compared.…”

Section: Phage Characterizationmentioning

confidence: 94%

“…The luminescence during the phage infections was monitored for a negative control, T7 Select, and NRGp5, as well as a previously developed NRGp4 [48]. Luminescent signals were measured using the same microplate reader (Biotek Instruments, Winooski, VT, USA) in sterile 24-well suspension culture plates (Cellstar, Greiner Bio-One, Monroe, NC, USA) with a 0.1 s integration time.…”

Section: Phage Characterizationmentioning

confidence: 99%

“…To create the recombinant phages used in our novel detection platform, an enzyme expression cassette was inserted into a wild type phage genome to force expression of a heterologous reporter enzyme, in addition to new phage progeny upon phage infection. The NanoLuc enzyme was selected as the reporter enzyme, as it is more than 100x more active than its luminescent counterparts [33], and this highly active reporter enzyme has already ben widely deployed in a variety of detection assays [25,26,31,32], including bacteriophage-based schemes [24,48,49]. The NanoLuc enzyme was further functionalized by genetically fusing a cellulose binding module (CBM) to the C-terminus of the NanoLuc reporter gene.…”

Section: Introductionmentioning

confidence: 99%

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A Syringe-Based Biosensor to Rapidly Detect Low Levels of Escherichia Coli (ECOR13) in Drinking Water Using Engineered Bacteriophages

Hinkley

Garing

Jain

et al. 2020

Sensors

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A sanitized drinking water supply is an unconditional requirement for public health and the overall prosperity of humanity. Potential microbial and chemical contaminants of drinking water have been identified by a joint effort between the World Health Organization (WHO) and the United Nations Children’s Fund (UNICEF), who together establish guidelines that define, in part, that the presence of Escherichia coli (E. coli) in drinking water is an indication of inadequate sanitation and a significant health risk. As E. coli is a nearly ubiquitous resident of mammalian gastrointestinal tracts, no detectable counts in 100 mL of drinking water is the standard used worldwide as an indicator of sanitation. The currently accepted EPA method relies on filtration, followed by growth on selective media, and requires 24–48 h from sample to results. In response, we developed a rapid bacteriophage-based detection assay with detection limit capabilities comparable to traditional methods in less than a quarter of the time. We coupled membrane filtration with selective enrichment using genetically engineered bacteriophages to identify less than 20 colony forming units (CFU) E. coli in 100 mL drinking water within 5 h. The combination of membrane filtration with phage infection produced a novel assay that demonstrated a rapid, selective, and sensitive detection of an indicator organism in large volumes of drinking water as recommended by the leading world regulatory authorities.

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“…(D) Released progeny phages [51,52] or bacterial cell content (e.g., ATP, DNA, RNA and bacterial proteins) provide excellent markers for downstream detection of the original bacterial host [53][54][55]. Alternatively, genetically engineered phages encoding reporters such as fluorescent proteins (E) [56][57][58], luciferases (F) [59][60][61][62] or hydrolyzing enzymes (e.g., β-galactosidase) (G) [63,64] are used. These phages express the reporter proteins during host infection to produce an amplifying fluorescent or bioluminescent signal upon the addition of a substrate.…”

Section: Viruses 2020 12 X For Peer Review 3 Of 25mentioning

confidence: 99%

Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments

Meile

Kilcher

Loessner

et al. 2020

Viruses

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Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.

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A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Cited by 48 publications

References 25 publications

New strategies for the enumeration of enteric pathogens in water

New strategies for the enumeration of enteric pathogens in water

A Syringe-Based Biosensor to Rapidly Detect Low Levels of Escherichia Coli (ECOR13) in Drinking Water Using Engineered Bacteriophages

Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments

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