Staphylococcus aureusis the most important pathogen associated with skin infection and foodborne disease in humans. The organism is a common microflora of human epithelia exhibiting permanent nasal colonization between 30% - 40% of the human population . Symptomatic S. aureus infections may occur following breaks in skin or mucosal barriers, potentially causing severe invasive infections. In extreme condition, S. aureus may lead to life-threatening manifestations such as bacteremia, endocarditis, and osteomyelitis  . Staphylococcus aureus is grouped into the coagulase-positive Staphylococci (CoPS) based on its ability to produce coagulase enzyme, contrasting to the less pathogenic coagulase-negative Staphylococci (CoNS). Although recent reports highlighted the increasing significance of CoNS as opportunistic pathogens, S. aureus remains a major health threat in clinical settings . In particular, S. aureus has been recognized the most common cause of nosocomial bloodstream infections, where most infections involved the healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA)  . HA-MRSA infections are usually difficult to treat due to their resistance to multiple antibiotics, contributing to a significant mortality and morbidity . The worldwide concern of S. aureus infections increases with recent emergence of community-associated MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA) which posed a significant impact and economic burden in public health and livestock industry   .
Due to the importance of S. aureus as an opportunistic pathogen, identification of this bacterium in clinical specimens is essential particularly in discriminating S. aureus from the CoNS and non-staphylococci. Rapid identification of S. aureus allows initiation of the appropriate antibiotic therapy in patients at the early stage of disease onset and prevents development of serious illness. In most clinical laboratories, phenotypic identification of S. aureus is routinely carried out based on morphological characterization on agar and biochemical tests . Presumptive isolation of mannitol-positive S. aureus is usually determined by growth on mannitol salt agar, producing yellow-coloured colonies as a result of mannitol fermentation . Nonetheless, isolation of mannitol-positive CoNS species in human nasal and clinical specimens has been previously demonstrated in Nigeria and Japan  . Coagulase production remains a standard confirmatory test for S. aureus  , however, accurate identification by this test depends on the nature and quality of the plasma used . Furthermore, prolong incubation is sometimes required to achieve reliable results of coagulase test and this represents a great disadvantage for hospital diagnostics .
Advanced molecular approaches provide rapid and reliable means of identifying S. aureus from clinical samples. Through these methods, S. aureus has been identified efficiently using DNA microarray, real-time PCR, fluorescence in situ hybridization, surface enhanced laser desorption/ionization time of flight and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry     . These methods however, require highly expensive equipments, which limit their use in healthcare diagnostics. In contrast, PCR-based approaches are more cost effective and highly efficient to be used for rapid identification of S. aureus. Standard microbial identification utilizes 16S ribosomal DNA sequence analysis, useful for identification at species level . PCR targeting constitutively expressed genes such as nuc, femA, sodA and coa allows rapid identification of S. aureus without further requirement of gene sequencing    . Moreover, several S. aureus genes associated with antibiotic resistance and enterotoxins have been shown to facilitate identification of the bacteria from various sources with high sensitivity and specificity  . Polymorphisms however occur in some of these genes which limit their use as a reliable identification marker for S. aureus  .
PCR targeting a signal transduction gene, vicK has been recently described as highly specific and sensitive of detecting a low copy genetic material of S. aureus  . vicK encodes the bacterial two-component signal transduction system (TCS) that senses and responds towards the environmental stimuli especially when invading the host . The gene regulons of TCS are species-specific, which play diverse roles in modulating of cell division, cell-wall biosynthesis and membrane integrity . This study aimed to evaluate vicK as a potential rapid identification marker for S. aureus by determining the frequency of vicK detection in S. aureus clinical isolates.
2. Materials and Methods
2.1. Bacteria Isolates
A total of 110 Staphylococci clinical isolates were obtained from the Pathology Laboratory, Hospital Sultanah Nur Zahirah (HSNZ), Kuala Terengganu, Malaysia, within five months from September 2008 until January 2009 (Table 1). The isolates comprised of one hundred S. aureus and ten Staphylococcus spp.; Staphylococcus epidermidis (4), Staphylococcus haemolyticus (2), Staphylococcus hominis (2), Staphylococcus saphrophyticus (1) and Staphylococcus cohnii (1). The organisms were isolated from various body sites of hospitalized patients contracted with the bacterial infection (Table 2). Identification of Staphylococcus spp. to species level was preceded by sequencing of the bacterial 16S rDNA. Staphylococcus aureus(ATCC 29213) was used as a positive control for PCR amplification. For non-Staphylococci negative controls, eight different types of bacteria comprising of three Gram-positives (Streptococcus uberis, Micrococcus sp., Bacillus sp.) and five Gram-negatives (Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp., Salmonella sp. and Aeromonas sp.) were obtained from
Table 1. Staphylococci clinical isolates collected from Hospital Sultanah Nur Zahirah (HSNZ), Kuala Terengganu, Malaysia, from September 2008-January 2009.
the Microbiology Laboratory, School of Fundamental Science, Universiti Malaysia Terengganu (UMT) (Table 3). These isolates were sub-cultured on nutrient agar and incubated at 37˚C overnight.
2.2. Identification of Bacteria
Primary bacterial identification was performed by basic microbiological methods using colony morphology and biochemical tests. Staphylococcus aureus were sub-cultured on blood agar for examination of morphological characteristics and haemolytic activity. The bacteria were further examined by Gram staining, catalase test, coagulase test and growth on mannitol salt agar.
2.3. DNA Extraction
DNA was prepared by boiling method as previously described with minor
Table 2. The source of Staphylococci isolated from patients of Hospital Sultanah Nur Zahirah.
Table 3. Non-Staphylococci obtained from the Microbiology Laboratory, School of Fundamental Science, Universiti Malaysia Terengganu (UMT).
modifications . In brief, 1 - 2 bacterial colonies were suspended in sterile water and boiled for 10 minutes. The suspension was then spun down at 10,000× g for 5 min. The supernatant containing bacterial DNA was kept on ice and freshly used as a template for PCR amplification.
2.4. vicK Gene Amplification
PCR was performed in 25 µl reaction using an EppendorfMastercycler® Gradient. The PCR mixtures contained 5 µl 5X PCR buffer, 4 µl MgCl2 (25 mM), 1 μl dNTPs (10 mM), 1 µl each reverse and forward vicK gene primers (10 μM), 1U Taq DNA polymerase, DNA template (1 µl) and sterile nuclease-free water to the final volume of 25 μl. The vicK primer sequences used: forward 5’-CTAATACTG AAAGTGAGAAACGTA-3’ and reverse 5’-TCCTGCACAATCGTACTAAA-3’, as established by Liu et al. (2007). Distilled water with no DNA template was used as a negative control. The thermal PCR cycling conditions consisted of initial denaturation at 95˚C for 5 min; and 30 cycles of denaturation at 95˚C for 1 min, annealing at 52.6˚C for 1 min, extension at 72˚C for 1 min, and a final extension at 72˚C for 5 min. Amplified PCR products were separated in 1.2% agarose gel stained with ethidium bromide (0.5 µg/ml), visualized under UV light and photographed using Image Master VDS was prepared by boiling method as previously described with minor modifications .
2.5. DNA Sequencing
In order to confirm that vicK gene had been successfully amplified, at least three vicK gene amplicons were sequenced-verified. The PCR products were excised from agarose gel and the DNA was purified using QIAquick Gel Extraction Kit according to the manufacturer’s protocol. Purified DNA was eluted in 20 µl of nuclease free water and centrifuged at 12,000 × g. The purified fragments were sent to First BASE Laboratories Sdn. Bhd. for DNA sequencing using both forward and reverse vicK primers. Results obtained were analyzed using Chromas in order to assess the quality of chromatogram. Nucleotide sequence obtained from the chromatogram was exported to Entrez NCBI. The sequence was submitted for BLAST search against NCBI bacterial database.
2.6. Data Analysis
The specificity and sensitivity of PCR targeting vicK were calculated according to Kateete et al. (2010) as follows:
Sensitivity (%) = [True positive/(True Positive + False Negative)] × 100
Specificity (%) = [True Negative/(True Negative +False Positive)] × 100
3.1. Identification of S. aureus
A total of one hundred S. aureus isolates collected from HSNZ, Kuala Terengganu were identified generally based on conventional phenotypic methods. Upon growth on blood agar, the organisms appeared round golden-yellow colonies and resulted in haemolytic activity. Growth was visible on mannitol salt agar, changing the colour of the medium to yellow as a result of mannitol fermentation. The isolates were catalase-positive and coagulase-positive. Isolates with uncertain coagulase activity was further tested with tube coagulase test as previously recommended , which produced a clot for positive reaction. The bacteria cells appeared as Gram-positive cocci and retained purple upon gram stain.
3.2. Amplification of vicK Gene
The specificity of PCR targeting vicK was evaluated in all bacteria isolates using vicK gene primers established previously . Results of this study demonstrated amplification of a single fragment vicK (289 bp) in 98% of S. aureus isolates (Figure 1, Table 4). This gives 98% sensitivity level of using vicK as a target in identifying S. aureus from clinical isolates. In order to confirm the vicK sequence identity, randomly picked PCR amplicons were sequenced-verified. BLAST search analysis affirms the identification of vicKS. aureus, demonstrating 100% nucleotide sequence homology. In a parallel control experiments, there was no amplification of vicK in all ten isolates of Staphylococcus spp. and eight non-Staphylococci tested (Table 4). Hence, a 100% specificity level was achieved in discriminating S. aureus from other Staphylococcus spp. and non-Staphylococci.
Figure 1. Amplification of vicK gene fragment of representative eleven S. aureus clinical isolates, resolved in 1.2% agarose gel and stained with ethidium bromide. Lane M is 100 bp marker; Lane 1 is PCR negative control (without DNA template); Lane 2 is S. aureus positive control (ATCC 29213); Lanes 3 to 13 are S. aureus clinical isolates, respectively. Arrow indicates vicK amplified fragments at 289 bp.
Table 4. Percentage of Staphylococcus aureus isolates identified by vicK amplification.
Staphylococci are colonizers of human skin and mucosal surfaces, frequently cause severe infection in humans. In the present study, 110 Staphylococci clinical isolates were obtained from HSNZ, Kuala Terengganu, Malaysia within five months (September 2008-January 2009). The highest percentage of organisms was found in pus (56.4%), followed by blood (18.2%), eye (10.9%) and cerebrospinal fluid (8.2%) (Table 2). The highly encounter of this organism in the pus is consistent with previous studies reported in many hospitals   . This could be due to the frequent association of Staphylococci with skin and soft tissue infections, which usually manifest abscesses  . The exposure of skin wounds to Staphylococci particularly S. aureus, usually arises in patients with predisposing risk factors such as burns, chronic illness and the use of medical devices  . Staphylococcus aureus may colonize almost 30% - 40% of individuals and can be easily spread through contact with an infected person or through personal belongings . This colonization significantly increases the chances of infections by providing a reservoir of the pathogen. Once the bacteria penetrate through skin, they are potential of causing severe infections, such as skin infections, wound infections and bacteremia. The latter contributes to the greatest problem in medical field as S. aureus has been recognized the most common cause of nosocomial bloodstream infections . This has also been a challenge due to the presence of heterogenous population of S. aureus colonizing a human body which severely complicate antibiotic therapy  .
Due to their public importance, rapid detection and identification of S. aureus from clinical isolates is crucial. Accurate identification of S. aureus from the less pathogenic CoNS and non-Staphylococci is essential for the appropriate therapeutic use of antibiotics and timely intervention for infection control. In this study, S. aureus isolates collected from HSNZ were identified according to the standard protocols by significant growth on culture media and biochemical tests . While single phenotypic test is inefficient for identification of S. aureus , morphological observation on blood agar, haemolytic activity, growth on mannitol salt agar, Gram staining, catalase test and coagulase test were performed. Although S. aureus can be easily grown on standard culture media and identified based on several biochemical tests, there is a need for rapid and sensitive DNA-based assay for the discrimination of S. aureus from other CoNS and non-Staphylococci. Rapid identification of this bacterium with high accuracy is important for early diagnosis of S. aureus infections which leads to treatment of patients with appropriate antibiotic therapy. Conventional PCR assays are highly versatile and commonly used for identification of pathogens within hours. It is recognized that PCR is highly sensitive and accurate, allowing for the detection of a very low copy number of genetic material  .
Most PCR methods target highly conserved genes within the species to identify S. aureus at the species level . In the present study, PCR targeting a signal transduction gene, vicK of S. aureus was evaluated as a potential marker for rapid identification of the bacteria. vicK is a unique gene that encodes the two-component signal transduction system, responsible for regulating bacterial responses to extracellular signals . The gene is highly conserved and has been shown to be species-specific . Following PCR amplification of HSNZ isolates, the vicK gene fragment has been successfully detected in 98% of S. aureus (Figure 1, Table 4). This correlates well with previous findings demonstrating high sensitivity and specificity of PCR targeting vicK  . It was also evident that vicK was undetectable in other staphylococci (S. epidermidis, S. haemolyticus, S. hominis, S. saphrophyticus and S. cohnii) and non-staphylococci tested (Streptococcus uberis, Micrococcus sp., Bacillus sp., Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp., Salmonella sp. and Aeromonas sp.). This gives a 100% specificity of vicK amplification by PCR in discriminating S. aureus from other bacteria species. Although one isolate represented each species in this study, many representative isolates of other types of bacteria have been previously tested for vicK detection, demonstrating similar observations  . The high percentage of S. aureus identified by vicK amplification indicates the gene is species-specific, thus offering a useful target for specific and rapid identification of S. aureus.
It is noteworthy that a small percentage (2%) of S. aureus was unable to be recognized by vicK amplification (Table 4). This may be contributed by several factors. One factor might be due to a very low concentration of DNA harvested from boiling of the bacterial colonies. Although this method was rapid and easy to perform, the resulted DNA varies among samples . The true limit of detecting vicK by PCR was reported above 5500 copies of plasmid DNA , thus the efficiency of harvesting DNA target and the amount of DNA yield can limit the sensitivity of PCR assay. We believe the use of appropriate DNA extraction protocols incorporating efficient cell lysis and purification steps may greatly improve the quality of harvested DNA.
Another factor that may contribute to the lack of vicK detection could be due to misidentification of S. aureus. False identification occasionally occurs by misinterpretation of results derived from phenotypic methods . A high rate of false identification by phenotypic methods has been reported in Nigeria contributing to 85% misidentification of S. aureus, whereas 49% false identification has been documented in Libya  . Although coagulase test represents the standard method for identification of S. aureus, a few CoNS produce clumping factor which can be erroneously interpreted as coagulase-positive S. aureus  . Due to budgetary and time constraints, further experiments were not carried out to verify the identity of these isolates. We believe that identification of the bacteria through 16S ribosomal DNA sequencing will be able to precisely determine the identity of these organisms to the species level.
The advantage of PCR assay described in this study is the use of only one primer set targeting vicK for specific detection of S. aureus. An increasing number of bacterial isolates collected from various clinical and geographical sources may be performed in future to support findings of this study. We also believe that rapid detection of S. aureus directly from clinical specimens is important for timely intervention and infection control. Thus, the potential use of PCR assay described here using S. aureus clinical sources requires further investigation.
PCR targeting the vicK gene had successfully identified a high percentage of S. aureus clinical isolates obtained from HSNZ. The gene is species-specific and highly discriminative of S. aureus from other organisms, thus potential to be used as a rapid diagnostic marker for identification of S. aureus.
We would like to acknowledge the Department of Pathology, Hospital Sultanah Nur Zahirah, for providing bacteria isolates. We thank the Universiti Malaysia Terengganu for providing research fund and lab facilities to carry out this project.
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