Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells
Authors: Denyes Jenna M. , Dunne Matthew , Steiner Stanislava , Mittelviefhaus Maximilian , Weiss Agnes , Schmidt Herbert , Klumpp Jochen , Loessner Martin J. 
Published: April 2017

Bacteriophage-based assays and biosensors rival traditional antibody-based 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
−1
, and they yielded equivalent recovery rates (93% ± 5%,
n
= 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 peroxidase-conjugated 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
−1
as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 10
2
CFU · ml
−1
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.

IMPORTANCE
The 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.