Phage-Mediated Molecular Detection (PMMD): A Novel Rapid Method for Phage-Specific Bacterial Detection

Abstract

Bacterial infections pose a challenge to human health and burden the health care system, especially with the spread of antibiotic-resistant populations. To provide effective treatment and improved prognosis, effective diagnostic methods are of great importance. Here we present phage-mediated molecular detection (PMMD) as a novel molecular method for the detection and assessment of bacterial antibiotic resistance. This technique consists of a brief incubation, of approximately ten minutes, of the biological sample with a natural bacteriophage (phage) targeting the bacteria of interest. This is followed by total RNA extraction and RT-PCR. We applied this approach to Staphylococcus aureus (SA), a major causative agent of human bacterial infections. PMMD demonstrated a high sensitivity, rapid implementation, and specificity dependent on the phage host range. Moreover, due to the dependence of the signal on the physiological state of the bacteria, PMMD can discriminate methicillin-sensitive from methicillin-resistant SA (MSSA vs. MRSA). Finally, we extended this method to the detection and antibiotic sensitivity determination of other bacteria by proving PMMD efficacy for Bacillus anthracis.

Rapid Microscopic Detection of Bacillus anthracis by Fluorescent Receptor Binding Proteins of Bacteriophages

“…The host-mediated phage amplification/PCR amplification hybrid identification approach has recently seen methodological improvements. In an improved technique named phage-mediated molecular detection (PMMD) a short incubation period of bacterial host culture ( Staphylococcus aureus or B. anthracis ) with a species-specific phage is followed by RNA extraction and reverse transcription PCR (RT-PCR) on specific phage transcripts [ 58 ]. The authors thus took advantage of the high number (relative to DNA) of phage RNA molecules produced per infected cell, which can be expected to far exceed the number of nascent phage DNA genomes.…”

“…In this assay B. anthracis was grown prior to RT-PCR for 3 h without phage followed by an infection phase of about 13 min and RNA-preparation. A further advantage of this technique is that it can be coupled to antibiotic susceptibility testing [ 58 ]. Nevertheless, in contrast to the new RBP reporter assay, B. anthracis detection by PMMD requires growth of live bacteria not always possible, especially in field settings [ 59 ], whereas RBP reporters introduced here, may be also used on inactivated cells if required.…”

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

“…Infection-based detection: Infection of a bacterial host by lytic phage leads to rapid progeny phage production and ensuing cell lysis. (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.…”

“…Phage RNA has also been used as a biomarker. For instance, surrogate marker loci in engineered mycobacteriophages [77,78] or simply the phage's natural RNA [54] have both been detected using qPCR methods. Methods that are based on reverse transcription of phage RNA can offer enhanced sensitivity and are less prone to false-positive results than DNA amplification-based detection systems [79].…”

“…Host adsorption and phage genome injection followed by reporter gene expression is generally sufficient to produce a detectable signal, allowing reporter phages to circumvent the gamut of intracellular phage defense mechanisms that can inhibit various stages of the phage infection cycle [83,84]. n/a n/a [54] n/a, not available; DETL, detection limit, DETT; overall detection time; PMMD, phage-mediated molecular detection; RPA, recombinase polymerase amplification; * DETL and DETT are reported for individual assays as they were derived or provided from the source articles.…”