Lytic phage as a specific and selective probe for detection of Staphylococcus aureus—A surface plasmon resonance spectroscopic study

Abstract

Rapid and reliable detection of harmful pathogens at low levels are vital due to the related environmental and economical impact. While antibodies (monoclonal or polyclonal) are successfully employed in many immunoanalysis procedures as a biorecognition element, many of them remain costly with a comparatively short shelf life and uncertain manufacturability. Additionally, they suffer from several limitations, such as susceptibility to hostile environmental stresses such as temperature, pH, ionic strength, and cross-reactivity. The development of easy available, sensitive, and robust alternative molecular recognition elements, capable of providing a very high level of selectivity are very attractive to industry and may benefit in multiple areas. Several attempts have been made to utilize fluorescent-tagged bacteriophages and phage-displayed peptides for bacterial detection. However, involvement of complex labeling and detecting procedures make these approaches time-consuming and complicated. Here, we are reporting for the first time, the label-free detection of Staphylococcus aureus using lytic phage as highly specific and selective biorecognition element and surface plasmon resonance-based SPREETA™ sensor as a detection platform. Lytic phage was immobilized on the gold surface of SPREETA sensor via trouble-free direct physical adsorption. The detection limit was found to be 10⁴ cfu/ml. Detection specificity was investigated by an inhibition assay while selectivity was examined with Salmonella typhimurium. The preliminary results using lytic phage as a probe for bacterial detection, in combination with SPR platform are promising and hence can be employed for rapid and label-free detection of different bacterial pathogens.

Introduction

Responsible for both community acquired and hospital acquired Staphylococcus aureus infection for over 100 years, multi-drug resistant S. aureus remains a threat to the human population (Lowy, 1998, Jay, 2000, Aucken et al., 2002). It is one of the common causes of confirmed bacterial food poisoning in the US with over 50 outbreaks involving multiple cases (Labbe and Garcia, 2001). An antibiotic-resistant strain of S. aureus was also the cause of an outbreak in a UK hospital in the year 2000. Up to 30–50% of humans carry S. aureus in nostrils and on skin surfaces, thus contamination of food products is a direct result of poor handling and sanitation of processed goods. S. aureus can cause multiple illnesses such as urinary tract infections, pneumonia, gastroenteritis, respiratory diseases, bronchial diseases, abdominal cramps, and diarrhea.

S. aureus produces a variety of highly stabile toxic proteins, such as different staphylococcal enterotoxins and the toxic shock syndrome toxin (Arbuthnott et al., 1990, Jarraud et al., 2001, Lina et al., 1997). The production of these toxins occur in between 10 and 46 °C and as early as 4–6 h after contamination of food products, while most common food borne pathogens need longer incubation periods ranging from 10 h to several days (Mead et al., 1999). To elicit food poisoning, a minimum staphylococcal enterotoxin E dose of 1 μg is required and can be produced with a bacterial population as low as 10⁵ cfu/g (Jay, 2000, Honeyman et al., 2001). During major outbreaks, S. aureus was specifically identified by traditional microbiological laboratory procedures, which involve culturing the bacteria followed by either specific genotype or phenotype diagnostic assays (Stepan et al., 2004). Even though these methods are specific and sensitive, they often take 24–48 h to confirm the results. Hence, a rapid and simple method of detection is required to identify low quantities of bacteria and toxins.

The advent of modern technologies led to the development of analytical devices such as chemical and biological sensors, which changed the way pathogens are identified. These analytical devices offer near real-time monitoring coupled with rapid and specific detection of the analyte of interest. A biosensor is comprised of a physical transducer (electrochemical, optical, mass, and thermal) and a specific bio-probe (antibody, enzymes, nucleic acids, receptors, and whole cells) in which the biospecific interaction between the probe and target of interest is converted to a measurable signal by the physical transducer (Hobson et al., 1996, Ivnitski et al., 1999, Hall, 2002, Leonard et al., 2003). The sensitivity and specificity of the overall system depends on the transduction technique employed and the type of bio-probe used. Surface plasmon resonance biosensors based on optical transduction are particularly attractive for the above applications as they offer “label-free” detection and “near real-time” monitoring of interactions. Combined with high sensitivity, this technique has been widely employed for the detection of various bacteria (Koubova et al., 2001, Bokken et al., 2003, Leonard et al., 2004), toxins (Naimushin et al., 2002, Medina, 2005), and viruses (Boltovets et al., 2004).

Surface plasmon resonance (SPR) is an optical phenomenon, which occurs when TM-polarized light undergoes total internal reflection (TIR) at certain angles of incidence (resonance angle) at the interface of two media of different refractive indices (RI), generating a non-radiative evanescent wave. If the interface is modified with a thin layer of metal (Au/Ag ∼ 50 nm) and a monochromatic p-polarized light is used as a source, a minimum in reflectivity is observed at the resonance angle. The resonance angle (or position) of this minimum is very sensitive to minute changes in the RI of the adjacent medium, which is directly related to the change in surface concentration of interacting ligands. This change in RI is continuously monitored to produce a sensorgram having RIU (refractive index unit) as y-axis and time as x-axis. Typically, a response of 1000 RIU or 0.1° change in angle corresponds to a change in surface protein concentration of 1 ng/mm² (Green et al., 2000, Myszka, 2000, Rich and Myszka, 2000, Simonian et al., 2002).

The design of a robust biosensor demands the choice of the molecular probes to be specific, selective, and stable for longer period of shelf life. Most of the affinity-based biosensing assays currently utilize monoclonal or polyclonal antibody for specific antigen detection. Even though these antibodies, in many cases are specific and selective, both formats of the antibodies suffer from environmental instabilities and require laborious and expensive procedures for isolation and purification (Pancrazio et al., 1999). In addition, polyclonal antibodies are limited by their heterogeneity towards other species or strains and bring forth the need for alternative probe selections (Petrenko and Vodyanoy, 2003, Goldman et al., 2000). Bacteriophages are ever-present components of the microbial communities on earth (Paul et al., 1997). Their specificity coupled with resistance to environmental stresses make them an excellent molecular probe for the detection of pathogenic bacteria. This recognition ability has been exploited in several ways, namely in phage typing where a panel of phages were used to discriminate between different isolates of bacteria. Many strains of Salmonella, Listeria, and Staphylococcus were still identified by phage typing method as it is inexpensive and easy to use (Kutter and Sulakvelidze, 2004). Several articles demonstrate the potential for development of fluorescent-tagged bacteriophage for Escherichia coli O157:H7 (Goodridge et al., 1999) and Salmonella typhimurium LT2 (Mosier-Boss et al., 2003) detection, and phage-displayed peptides labeled with the dye Cy5 for staphylococcal enterotoxin B detection (Goldman et al., 2000). However, involvement of labeling by fluorophore dyes or more complicated labeling by the dsDNA of phage bound to specially design gold particles, with further collection of phage-infected bacteria by filters and fluorescence microscope image analysis, make these approaches multi-step and complicated.

Earlier we reported the highly sensitive detection of model antigen β-galactosidase by SPR biosensor (up to 0.41 nM) using a filamentous phage as a bio-probe selected by phage-display technique (Sokkalinga Balasubramanian et al., 2004).

In this study, we are reporting the use of lytic phage as a bio-recognition element and SPREETA™ sensor as a detection platform, for label-free direct measurement of S. aureus in low concentrations. Specificity of the detection assay was tested by an inhibition assay, while selectivity of phage–S. aureus interaction was examined using non-specific S. typhimurium.