Staphylococcus aureus is a ubiquitous gram-positive bacterial pathogen resulting in many morbidity and mortality cases worldwide. S. aureus rapidly develops resistance to antibiotics (1). In the 1960s, the methicillin group of antibiotics (including cloxacillin) was introduced allowing the control of infections caused by S. aureus (2). However, in 1961, resistance to methicillin soon evolved by hospital-acquired methicillin resistant Staphylococcus aureus (HA-MRSA). Resistance to methicillin occurred due to the acquisition of a MecA gene carried on a mobile genetic element (Staphylococcal Cassette Chromosome mec; SCCmec) (2-3). Although, MRSA was mostly confined to hospital-acquired infections for a long period of time, in 1993, a new form of MRSA known as community acquired MRSA (CA-MRSA) appeared in Western Australia (2). The incidence of community-associated infections was thought to emerge due to the development of hypervirulent and/or highly transmissible MRSA strains (1). CA-MRSA is currently a global health problem and is epidemic in the US. In 2004, MRSA was reported as the most frequent cause of infection that was presented to emergency departments in the US (1). In 2005, in Egypt, it was reported that the percentage of MRSA from S. aureus isolates was 63% (n=243) (4). CA-MRSA is mostly transmitted via skin-to-skin contact (1). Predisposing risk factors to CA-MRSA infection includes skin trauma, injection drug use, and poor personal hygiene. Risk groups for CA-MRSA infection include professional athletes (contact sports), military personnel, children, and incarcerated individuals (1). Disease severity ranges from minor skin and soft tissue infections to severe life threatening infections; including fatal sepsis, necrotizing fasciitis and pneumonia (1). CA-MRSA is treated using oral antimicrobial agents including cotrimoxazole, clinamycin, tetracyclines (doxycycline and minocycline), rifampicin and fusidic acid (5). It is important to note that no clinically approved vaccine for the prevention of S. aureus infections is available (5).
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The DNA genome of S. aureus is 2.8 - 2.9 Mb in size; composed of core and accessory genes (6). Most of the core genes are associated with metabolism and other housekeeping functions. However, some core genes were found not be linked to growth/survival but instead found associated with S. aureus-specific virulence genes. Accessory genome usually consists of mobile genetic elements (MGEs) including bacteriophages, pathogenicity islands, chromosomal cassettes, genomic islands, and transposons (6). These are mostly responsible for virulence, drug and metal resistance, substrate utilization and miscellaneous metabolism (6). S. aureus isolates also often carry 1 or more free or integrated plasmids. These plasmids carry genes responsible for resistance to antibiotics, heavy metals, or antiseptics (6). Two main differences exist between CA-MRSA and HA-MRSA. The first is the presence of SCCmec (mobile genetic elements) types IV & V in CA-MRSA while HA-MRSA mainly harbor SCCmec types I, II, and III. Panton-Valentine leukocidin (PVL) exotoxin is found in severe skin infections and necrotizing pneumonia associated with CA-MRSA (3).
Swabs of the anterior nares, perineal region and throat of infected patients (MRSA PCR-positive) and normal controls (non-infected healthy volunteers, MRSA PCR negative) will be collected. Swabs will be done using sterile cotton or Dacron swabs that will be placed in a liquid buffered medium for transportation to the laboratory. Samples should be refrigerated while transported and analyzed within 5 days (7-8).
3. DNA extraction method
Bacterial cells from swabs will be lysed and Genomic DNA extracted using QIAGEN Genomic DNA extraction kit according to manufacturer's instructions (9). Overnight enrichment (culture) will be performed in samples prior to lysis if needed.
4. Oligotargeter design and Detection plan
The unmodified AuNP-based assay will first be optimized on PCR products obtained by a multiplex-PCR reaction performed on extracted MRSA DNA. For the design of the six primers used for the PCR reaction, NCBI bioinformatics tools will be used. The primers are designed so that the right-junction sequences of SCCmec (types I, II, III, IV and V; one primer for each type) and the conserved sequence orfX gene (highly conserved open reading frame in S. aureus (3, 10)) are amplified. The PCR product produced will be mixed with the hybridization buffer. The hybridization buffer contains NaCl as well as two oligonucleotides that detect conserved regions within the PCR product. After denaturation and annealing, unmodified AuNPs will be added. In the absence of target, the oligonucleotides adsorb onto the AuNP's surface protecting it from salt-induced aggregation and the solution remains red. In presence of target, the oligonucleotides hybridize to the PCR product and salt-induced aggregation of AuNPs occurs and the solution turns blue in color.
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After process optimization using MRSA PCR products, the AuNP-based assay will be performed on unamplified MRSA DNA. The extracted DNA will be sheared physically or treated with restriction enzymes and then analyzed using the AuNP assay in two reactions. The first reaction confirms that the causative organism is S. aureus (AuNP-based assay performed using two oligonucleotides targeting conserved sequence of orfX gene) and the second determines the presence of MRSA (using two oligonucleotides against conserved region within MexA gene) (3, 10).
5. Current diagnostic tests available for MRSA detection
Diagnostic assays for MRSA detection can be divided into culture-based methods as well as molecular assays. Culture-based methods can be further divided into conventional and rapid-culture based methods. Conventional culture-based methods depend on selective culturing in liquid and/or on solid media. These methods are time-consuming where the result is given to patient after about 2-3 days resulting in the development of severe complications as well as significant disease spread (3, 7). Also it may give false-positive or false-negative results with sensitivity and specificity of 78-80% and 99%, respectively (8). More rapid culture-based methods utilizing chromogenic agars have been developed that produce results within 1.35-2.31 days (3). These culture agars incorporate a colorless chromogenic substrate that mimics a metabolic substrate. When the colorless chromogenic substrate is cleaved by a specific target bacterial enzyme, it becomes insoluble and colored. When the cleaved chromogen accumulates within the bacterial cell, the color builds up and the colony possessing the enzyme can be easily differentiated. Currently available Chromogenic media for MRSA diagnosis include ChromID (bioMérieux, Marcy l'Etoile, France), MRSA Select (BioRad Laboratories, Belgium), CHROMagar MRSA (CHROMagar Microbiology, France; BD Diagnostics, Belgium), Chromogen oxacillin S. aureus medium (Axon Labs AG, Stuttgart, Germany), Chromogenic MRSA/Denim Blue agar (Oxoid, Basingstoke, UK), MRSA Ident agar (Heipha Gmbh, Eppelheim, Germany) and Oxacillin resistance screening agar base (ORSAB, Oxoid) (3). Although chromogenic media are about 2 to 13 times more expensive than conventional media, it spares a number of subcultures, additional tests/reagents and technologist's time that are needed to confirm diagnosis in case of conventional culture-based MRSA detection (3). Sensitivity and specificity differ according to the media used ranging from 40-100% and 44-100%, respectively (3).
A novel culture-based assay for MRSA detection has been developed (Baclite Rapid MRSA test; 3M Healthcare, Berkshire, UK). This assay detects ciprofloxacin-resistant MRSA strains by the measurement of adenylate kinase (AK) activity using bioluminescence. The total assay time is 5 hrs with assay sensitivity and specificity of 90.4% and 95.7%, respectively (3). However, the material cost of the assay is 9.5-12$/test, which is higher than conventional culture-based methods (3). Another drawback is that cases of either CA-MRSA or HA-MRSA that are not resistant to ciprofloxacin will be missed (3).
Molecular methods provide many advantages over culture methods including higher sensitivity (lower detection limits), high-throughput screening and faster detection (as low as 75 min) thus reducing risk of disease spread and progression (3, 7). Available PCR methods for MRSA include GeneXpert MRSA assay (Cepheid, Sunnyvale, CA), the GenoType MRSA Direct (Hain Lifescience, Nehren, Germany), the Hyplex StaphyloResistÂ® PCR (BAG, Lich, Germany), the IDI-MRSA (GenOhm, San Diego, CA; BD Diagnostics), and Lightcycler Staphylococcus and MRSA detection kit (LC assay, Roche Diagnostics, Mannheim, Germany) (3). Sensitivity of PCR was found to be superior to cultural methods where a sensitivity of 93% has been reported (8). Specificity reported for PCR is 96% (8). However, the major drawback in molecular methods is their cost. For example, IDI-MRSA is a FDA cleared kit for the direct detection of MRSA from nasal specimens with high sensitivity and specificity. However, it is significantly more expensive than culture-based detection methods (36.7$/test) (3).
Thus novel molecular methods that are cost-effective are highly needed to allow for the inexpensive as well as rapid detection of MRSA allowing control over disease spread and progression. The developed AuNP-based assay benefits from the unique physical properties of the AuNPs allowing for the sensitive, rapid and inexpensive detection of such deadly bacteria. It is important to note that our assay uses unmodified AuNPs which makes the assay much simpler compared to other published assays utilizing probe-modified AuNPs (9, 11).