Rapid Clinical Screening of Burkholderia pseudomallei Colonies by a Bacteriophage Tail Fiber-Based Latex Agglutination Assay

Abstract

Melioidosis is a life-threatening disease in humans caused by the Gram- negative bacterium Burkholderia pseudomallei. As severe septicemic melioidosis can lead to death within 24 to 48 hours, a rapid diagnosis of melioidosis is critical for ensuring an optimal antibiotic course is prescribed to patients. Here, we report the development and evaluation of a bacteriophage tail fiber-based latex agglutination assay for rapid detection of B. pseudomallei infection. Burkholderia phage E094 was isolated from rice paddy fields in northeast Thailand, and whole genome sequenced to identify its tail fiber (94TF). The 94TF complex was structurally characterized, which involved identification of a tail assembly protein that forms an essential component of the mature fiber. Recombinant 94TF was conjugated to latex beads and developed into an agglutination-based assay (94TF-LAA). 94TF-LAA was initially tested against a large library of Burkholderia and other bacterial strains before a field evaluation was performed during routine clinical testing. The sensitivity and specificity of the 94TF-LAA were assessed alongside standard biochemical analyses on 300 patient specimens collected from an endemic area of melioidosis over 11 months. The 94TF-LAA took less than 5 minutes to produce positive agglutination, demonstrating 98% (95% CI; 94.2%−99.59%) sensitivity and 83% (95% CI; 75.64%−88.35%) specificity when compared to biochemical-based detection. Overall, we show how a Burkholderia-specific phage tail fiber can be exploited for rapid detection of B. pseudomallei. The 94TF-LAA has the potential for further development as a supplementary diagnostic to assist in clinical identification of this life-threatening pathogen. IMPORTANCE Rapid diagnosis of melioidosis is essential for ensuring optimal antibiotic courses are prescribed to patients, and thus warrants the development of cost-effective and easy-to-use tests for implementation in under-resourced areas such as Northeast Thailand and other tropical regions. Phage tail fibers are an interesting alternative to antibodies for use in various diagnostic assays for different pathogenic bacteria. As exposed appendages of phages, tail fibers are physically robust, easy to manufacture, and critically many tail fibers (such as 94TF investigated here) can target a given bacterial species with remarkable specificity. Here, we demonstrate the effectiveness of a latex agglutination assay using a Burkholderia-specific tail fiber 94TF against biochemical-based detection methods that are the standard diagnostic in many endemic areas of meilodosis.

A novel lytic phage potentially effective for phage therapy against Burkholderia pseudomallei in the tropics

“…According to the canonical phage model, the specific host binding was achieved by ORF84, which was spiked on the end tail and might be used for developing a novel detection kit. A protein with a similar function in phage E094 has been experimentally utilized for distinguishing B. pseudomallei in serum samples [ 50 ], although this approach needs to be refined to minimize the occurrence of false positives. Moreover, ORF83, the enzyme responsible for digestion of the extracellular polysaccharide, can be used as another biological probe, and ORF78, an endolysin capable of dissolving the bacterial murein, might be prepared as a highly efficient bactericide against contamination of B. pseudomallei in environment and food.…”

A perfect fit: Bacteriophage receptor-binding proteins for diagnostic and therapeutic applications

“…In a field evaluation using 300 patient isolates taken from different specimens (e.g. urine and blood), the assay presented a diagnostic accuracy of 90%, sensitivity of 98%, and specificity of 83% (the latter being improved through assay buffer modification) [24]. An alternative approach developed by Shi et al leveraged the different interaction mechanisms of phage TFs and the antibiotic Polymyxin B to their respective cell wall targets to produce a dual-site detection assay for Pseudomonas aeruginosa.…”

A host recognition module shared among distantAlteromonasbacteriophage families features tail fibers with transient chaperone “caps”

“…Interestingly, other TF chaperones have been shown to remain attached to their TFs after production 22,23 and have also been proposed to play alternative roles besides being simply chaperones. For example, the tail fiber assembly (tfa) protein of Escherichia coli phage Mu remains bound to the tip of the TF and has been shown to bind the same lipopolysaccharide receptor as the TF 24 .…”

“…While the chaperone (gp27) was essential for recombinant production of functional TFs, gp27 was shown to remain weakly associated with the mature fiber after production, suggesting an alternative role besides assisting with fiber maturation. Interestingly, other TF chaperones have been shown to remain attached to their TFs after production 22, 23 and have also been proposed to play alternative roles besides being simply chaperones. For example, the tail fiber assembly (tfa) protein of Escherichia coli phage Mu remains bound to the tip of the TF and has been shown to bind the same lipopolysaccharide receptor as the TF 24 .…”