Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water.

Rapid detection of bacteria responsible for foodborne diseases is a growing necessity for public health. Reporter bacteriophages (phages) are robust biorecognition elements uniquely suited for the rapid and sensitive detection of bacterial species. The advantages of phages include their host specificity, ability to distinguish viable and non-viable cells, low cost, and ease of genetic engineering. Upon infection with reporter phages, target bacteria express reporter enzymes encoded within the phage genome. In this study, the T7 coliphage was genetically engineered to express the newly developed luceriferase, NanoLuc (NLuc), as an indicator of bacterial contamination. While several genetic approaches were employed to optimize reporter enzyme expression, the novel achievement of this work was the successful fusion of the NanoLuc reporter to a carbohydrate binding module (CBM) with specificity to crystalline cellulose. This novel chimeric reporter (nluc::cbm) bestows the specific and irreversible immobilization of NanoLuc onto a low-cost, widely available crystalline cellulosic substrate. We have shown the possibility of detecting the immobilized fusion protein in a filter plate which resulted from a single CFU of E. coli. We then demonstrated that microcrystalline cellulose can be used to concentrate the fusion reporter from 100 mL water samples allowing a limit of detection of <10 CFU mL-1E. coli in 3 hours. Therefore, we conclude that our phage-based detection assay displays significant aptitude as a proof-of-concept drinking water diagnostic assay for the low-cost, rapid and sensitive detection of E. coli. Additional improvements in the capture efficiency of the phage-based fusion reporter should allow a limit of detection of <10 CFU per 100 mL.

Phage & phosphatase: a novel phage-based probe for rapid, multi-platform detection of bacteria.

Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection.

Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

Results of a pilot antibiotic resistance survey of Albanian poultry farms.

Global dissemination of antibiotic-resistant bacteria in food animals is a major public health concern. Whilst many countries have implemented prudent antibiotic use policies and surveillance systems both in clinical and veterinary settings, there are no such systems in place in Albania and little is known about the levels of antibiotic-resistant bacteria in food animals within the country. A total of 172 poultry samples were taken from six Albanian farms over a 3-month period and were tested for the presence of Enterobacteriaceae. In total, 91 bacterial isolates were obtained and were characterised by species (Escherichia coli, Salmonella spp. or other Enterobacteriaceae) and by susceptibility to 11 antibiotics. Resistance rates of E. coli and Salmonella isolates were, respectively: amoxicillin (86%, 64%); chloramphenicol (77%, 82%); ciprofloxacin (93%, 73%); cefotaxime (14%, 0%); gentamicin (12%, 0%); kanamycin (30%, 18%); nalidixic acid (91%, 73%); streptomycin (70%, 55%); sulphonamides (91%, 73%); tetracycline (95%, 73%); and trimethoprim (79%, 64%). Multidrug resistance to at least four antibiotics was observed in 95% of E. coli isolates and 82% of Salmonella. In conclusion, these data indicate that: (i) Salmonella and E. coli isolates from Albanian poultry farms exhibit high to extremely high levels of antibiotic resistance; (ii) Salmonella and E. coli isolates exhibit resistance to multiple antibiotics; and (iii) multidrug resistance profiles among Enterobacteriaceae are geographically widespread. Implementation of prudent antibiotic use policies in food animals and related surveillance will be necessary to reduce the emergence, spread and establishment of highly resistant strains across poultry farms in Albania.

Bioengineering bacteriophages to enhance the sensitivity of phage amplification-based paper fluidic detection of bacteria.

Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages' ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 10(3)CFU/mL of Escherichia coli in broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100CFU/100mL in inoculated river water after 9h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use.