Because of the inherent nature of phage specificity to their host, phage diagnostics can promptly and sensitively detect their specific host in a variety of clinical and environmental samples. Certainly, a number of previous studies have reported the successful use of phage PCR methods for bacterial detection e.g., [37
]. Nevertheless, the inherent background signal associated with the input requires the propagation of the phages to produce a progeny level sufficient to overcome this issue. Therefore, upon infection, relatively long incubation times are need it. This was cleverly resolved by Mulvey and collaborators by focusing on the detection of RNA molecules synthetized by a highly-transcribed phage-borne artificial cassette instead of detecting phage DNA [10
]. Due to the fact that the number of phage RNA molecules produced per infected cell generally far exceeds the number of new phage DNA genomes, we explored the possibility of developing phage RNA detection as a reliable and rapid diagnostic tool.
The results presented support PMMD as an alternative technique for medical diagnosis of S. aureus
. First, PMMD discriminates between antibiotic sensitive and resistant bacteria. As proof of principle, we have used cefoxitin, the CDC (Centers for Disease Control and Prevention)- and CLSI (Clinical & Laboratory Standards Institute)-recommended drug for testing of MRSA strains. Although the use with other antibiotics has to be determined empirically, we cannot anticipate major problems in adapting the system for other drugs, either bactericidal or bacteriostatic, since the active growth of the bacteria is essential for the phage metabolism [40
]. However, firstly compared with PCR approaches that amplify gene resistance markers, PMMD can be used for a number of antibiotics, does not require different primer pairs for different resistance cassettes, and avoids false positives due to the presence of silent or mutant cassettes [40
]. Secondly, the LOD to obtain a strong signal is ~100 bacterial cells. It is important to note that although the background signal was almost null for the uninfected bacteria controls, there was a small amount of background signal which interfered with our LOD results when unpurified phage lysates were used. Thus, highly-purified phages, in our case obtained by a CsCl-gradient isolation procedure, are an essential requirement for high sensitivity. Thirdly, the method is fast; we estimate its implementation time to be ~ 3 hours or ~5 hours depending on whether only detection or detection plus antibiotic sensitivity is performed. This time frame could be decreased by automation of the process and adjustment of the incubation periods depending on the antibiotic and culture media used. For example, we use 1-hour incubation time in LB media to promote high bacterial metabolic rate, but it is known that significantly shorter incubation times also work [25
]. Finally, PMMD is specific and can be applied to complex mixtures. The specificity level is determined by the phage used. Phage K infects the majority of S. aureus
strains and only propagates in a few isolates of closely-related Staphylococcus
. This is the case of Staphylococcus hyicus
, used sometimes as a surrogate for phage K propagation for safety reasons [19
]. Moreover, co-infection with other phage(s) can raise the number of positive SA strains to >95% [18
]. In addition, due to the simplicity of PMMD, there is no need to genetically engineer the phage; this approach can be easily expanded to other bacterial species, as we exemplified here in the case of B. anthracis
In summary, we have developed PMMD as a novel, proof-of-concept method for the detection and drug sensitivity assessment of bacteria using an S. aureus/phage K pair. PMMD shows promising characteristics for its applicability for clinical and research diagnostic purposes. Nevertheless, further development is required to cover most bacterial strains of a given species, for example by using phage cocktails. Moreover, this methodology has the potential to be adapted for the rapid selection of phages active against clinical isolates during phage therapy.