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Species-specific diagnostic marker for rapid identification of Staphylococcus aureus
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Diagnostic Microbiology and Infectious Disease 59 (2007) 379 – 382 www.elsevier.com/locate/diagmicrobio
Species-specific diagnostic marker for rapid identification of Staphylococcus aureus Zhan-min Liu, Xian-ming Shi⁎, Feng Pan Department of Food Science and Technology, Shanghai Jiaotong University, Shanghai 201101, PR China Received 31 March 2007; accepted 19 June 2007
Abstract Staphylococcus aureus is a common bacterial pathogen that has emerged as an increasingly important health concern. Following the recent publication of the genome sequences of 9 S. aureus strains (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), a gene of S. aureus that relates signal transduction as a target for rapid detection and identification of the pathogen has been investigated. By sequence analysis of S. aureus signal transduction genes from the complete genome of S. aureus ATCC N315 and their comparison with other DNA sequences using GenBank BLAST searches, we identified a unique gene, vicK. Polymerase chain reaction primers (vicK1 and vicK2) derived from this gene allowed amplification of a 289-bp DNA fragment only from S. aureus and not from other Staphylococcus species and other common bacteria tested. Besides offering an additional target for specific confirmation of S. aureus, further analysis of the signal transduction gene vicK and its related protein product may lead to new insights into the molecular mechanisms of S. aureus maintenance and pathogenicity. © 2007 Published by Elsevier Inc. Keywords: Diagnostic marker; Rapid identification; Staphylococcus aureus
1. Introduction Staphylococcus aureus is the staphylococcal species most commonly associated with human (nosocomial and community-acquired) and animal infections. The strains belonging to this species also cause food poisoning and are routinely characterized by growth properties, specific surface constituents, and their ability to coagulate blood plasma from various sources (staphylocoagulase) to produce a thermostable nuclease and to form clumps in the presence of fibrinogen (clumping factor). Several staphylococcus species, such as Staphylococcus epidermis and Staphylococcus haemolyticus, coagulase-negative species, have been isolated from ewe's milk and were found to produce one or several Staphylococcal enterotoxins (SEs) (Le Loir et al., 2003). Consequently, there is a need for developing rapid methods to specifically discriminate S. aureus from other staphylococcal species and nonstaphylococci in variety of ⁎ Corresponding author. Tel.: +86-21-64783841; fax: +86-2164783841. E-mail addresses: [email protected] (Z. Liu), [email protected] (X. Shi). 0732-8893/$ – see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2007.06.011
food commodities. Conventional identification methods are time-consuming and may yield false-positive or falsenegative results. Misidentification or misclassifications with automated susceptibility testing systems or commercially available latex agglutination kits have been reported recently (Ribeiro et al., 1999; Wilkerson et al., 1997). Development of improved laboratory diagnostic techniques, including those targeting femA (Vannuffel et al., 1995), nuc (Barski et al., 1996), coa (Martineau et al., 1998), 16S rRNA (Strommenger et al., 2003), Sa442 (Stuhlmeier and Stuhlmeier, 2003), and other genes in S. aureus, and rational use of antibacterial therapies have contributed to the control of this pathogen. Detection of S. aureus genes associated with antibiotic resistance (e.g., mecA [encoding methicillin resistance], tetM [tetracycline resistance], vat (A), vat (B), vat (C) [streptogramin A resistance]) by polymerase chain reaction (PCR) facilitates appropriate early antibiotic treatment. In addition, the use of multilocus sequence typing aids epidemiologic investigation of S. aureus infections. Recent description of the whole genome sequences of several S. aureus strains (N315, Mu50, MW2, NCTC8325, COL, RF122, USA300, MRSA252, and MSSA476) offers the opportunity to conduct detailed investigation on the
380
Z. Liu et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 379–382
mechanisms of S. aureus and survival in its niche environments as well as its pathogenicity.
Table 1 List of bacteria strains examined in the study Species
Number of strains
Amplification product size (bp)
S. aureus S. epidermidis S. haemolyticus Staphylococcus cohnii Staphylococcus hominis S. capitis Staphylococcus saprophyticus S. sciuri S. simulans S. warneri S. intermedius E. faecium E. faecalis Enterococcus casseliflavus Enterococcus durans Enterococcus avium Vibiro parahaemolyticus Salmonel choleraesuis Escherichia coli
66 11 10 1 1 1 1 1 1 1 1 5 5 1 1 1 4 4 3
289 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
molecular mechanisms behind its virulence and pathogenesis. In this report, we focus on one specialized gene group in S. aureus that encodes 2-component signal transduction system (TCS). Because of their important roles in sensing and responding to environmental conditions and in bacterial pathogenesis (Hoch, 2000; Inouye and Dutta, 2003; Stock et al., 2000), unique signal transduction genes may be required for a group of bacteria to fit in its particular ecologic niche. Previous results indicated that speciesspecific certain genes are present in a food-borne pathogen, Listeria monocytogenes (Liu et al., 2004a); therefore, TCS genes may display genus, species, or subspecies specificity. The identification of TCS genes in S. aureus will not only provide species-specific targets for diagnostic application but also lead to improved understanding of the genetic
2. Materials and methods 2.1. Bacterial strains A collection of 119 bacterial isolates were used, including 66 S. aureus, 11 S. epidermis, 10 S. haemolyticus, 8 other staphylococcus species, 5 Enterococcus faecium, 5 Enterococcus faecalis, 3 other Enterococcus species, and other Gram-positive and Gram-negative bacteria (Table 1). Among them, reference strains were also included such as those from the American Type Culture Collection (ATCC) and the China Medical Culture Collection Center (CMCC), Beijing, China, and Peking University Health Science Center, Beijing, China. S. aureus human isolates originated from Peking University Health Science Center, Beijing, China. S. epidermidis human isolates were from the Nanjing Children's Hospital, Nanjing, China, and Zhongshan Hopital of Fudan University, Shanghai, China. Other clinical bacterial strains were mainly isolated from the Jiling University Affiliated First Hospital, Jilin, China; Wuhan University Hospital, Wuhan, China; and Jiangsu Province Hospital, Nanjing, China and Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China. Other bacterial strains were kept in our laboratory. 2.2. DNA isolation The purified DNA stock was prepared in our laboratory as follows. Bacteria were grown overnight in Luria Bertani (LB) broth at 37 °C, and 1.5 mL culture of the organism was centrifuged by 3000 rpm/min for 5 min, then washed twice with TE (Tris 10 mmol/L, EDTA 1 mmol/L, pH 8.0). The purified organism was resuspended in 456 μL of TE, and lysed by adding 24 μL of 50 mg/mL lysozyme and incubating at 37 °C for 1 h. Then, 53 μL of 10% sodium
Fig. 1. Agarose gel electrophoresis of PCR products amplified with S. aureus vicK gene primers. Lanes 1–4, S. aureus strains (ATCC 29213, 25923, 6538, and CMCC 26001 representing 62 other strains); lane 5, S. epidermidis strains (ATCC 12228, representing 11 strains); lane 6, S. haemolyticus representing 10 strains; lane 7, Staphylococcus cohnii; lane 8, Staphylococcus hominis; lane 9, Staphylococcus capitis; lane 10, Staphylococcus saprophyticus; lane 11, Staphylococcus sciuri; lane 12, Staphylococcus simulans; lane 13, Staphylococcus warneri; lane 14, Staphylococcus intermedius; lane 15, E. faecalis representing 5 strains; lane 16, E. faecium representing 5 strains; lane 17, Enterococcus casseliflavus; lane 18, Enterococcus durans; lane 19, Enterococcus avium; lane 20, Vibiro parahaemolyticus (ATCC 17802 representing 4 strains); lane 21, Salmonel choleraesuis (CMCC 50004, representing 4 strains); lane 22, Escherichia coli representing 3 strains; and lane 23, negative control with no template DNA. On the left of lane M is DNA molecular weight ladder (DNA marker, Tiangen, Beijing, China).
Z. Liu et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 379–382
dodecyl sulfate was added and continued incubation for 15 min at 68 °C. Finally, 87 μL of 5 mol/L NaCl and 69 μL of 1% cetyl trimethyl ammonium bromide were added and incubated at 68 °C for an additional 15 min to release DNA. Subsequently, the samples were extracted with equal volumes of phenol/chloroform/isoamyl alcohol (25:24:1) followed by phenol/chloroform (1:1). The DNA was then precipitated by the addition of 1/10 volumes of 3 mol/L sodium acetate and 2 volumes of 100% ethanol and incubation at −20 °C for 15 min. The DNA pellets were washed twice with ice chilled with 70% ethanol, air dried, and dissolved in distillation water; and the DNA concentrations were determined at UV 260/280 nm in a DU800 UV spectrophotometer (Beckman Coulter, Fullerton, CA). A small amount of DNA from each bacterial strain was diluted in distilled water to 10 ng/μL for PCR analysis. 2.3. Identification of S. aureus-specific signal transduction gene S. aureus genes that encode signal transduction system were identified from the genome sequence of a S. aureus strain ATCC N315 and screened against other DNA sequences with GenBank's BLAST searches. Only 1 gene uniquely present in S. aureus was selected for further evaluation. Oligonucleotide primers were then designed from the gene of interest with Primer Premier 5.00 software (Premier Biosoft International, Palo Alto, CA) and synthesized with ABI 37300 high-throughput DNA synthesizer (Shanghai Sangon, Shanghai, China). 2.4. Polymerase chain reaction PCR was performed in a 50-μL volume using a PCR system PTC-200 (Bio-Rad, Foster City, CA). Each reaction contained 1.0 U Taq DNA polymerase (Tiangen Biotechnology, Beijing, China), 1× PCR buffer (containing 10 mmol/LTris–HCl [pH 9.0], 50 mmol/L KCl, and 1.5 mmol/ L MgCl2), 50 μmol/L dATP, dTTP, dCTP, and dGTP, 25 pmol/L of each primer, and 10 ng genomic DNA. The reaction mixture with no template DNA was used as a negative control. The thermal cycling conditions consisted of an initial denaturation at 94°C for 5 min, followed by 35 amplification cycles (94 °C for 40 s, 50 °C for 40 s, and 72 °C for 1 min), and final extension step at 72 °C for 10 min. After completion of all cycles, 6 μL of 10× DNA loading buffer was added to each tube, and the amplified products were examined in 1.5 % agarose gel electrophoresis in the presence of ethidium bromide (0.5 μg/mL). The stained gels were visualized under UV light and photographed using a Las300 Fuji Film (Fuji, Japan). 3. Results Based on results from BLAST searches, a S. aureusspecific gene that encodes histidine kinase was identified in the genome sequence of S. aureus N315 (GenBank
381
accession no. BA000018.3) (Kuroda et al., 2001). It was noted that a stretch of nucleotides identical to vicK (nt. 26414–26702) was also found in the complete genome of S. aureus MRSA252 strain (GenBank accession no. BX571856.1), MSSA476 (GenBank accession no. BX571857.1), NCTC8325 strain (GenBank accession no. CP000253.1) (Holden et al., 2004), COL strain (GenBank accession no. CP000046.1) (Gill et al., 2005), Mu50 strain (GenBank accession no. BA000017.4) (Kuroda et al., 2001), MW2 strain (GenBank accession no. BA000033.2) (Baba et al., 2002), RF122 strain (GenBank accession no. AJ938182.1) as well as USA300 strain (GenBank accession no. CP000255.1) (Diep et al., 2006). However, vicK gene sequence was clearly absent in other bacterial genomes available at GenBank. A pair of oligonucleotide primers was thus designed from the signal transduction gene vicK (vicK1: 5′-CTAATACTGAAAGTGAGAAACGTA-3′ and vicK2: 5′-TCCTGCACAATCGTACTAAA-3′). These primers correlated with the vicK gene sequence at nt 26414–26437 and nt 26683–26702, respectively, which facilitated the amplification of a 289-bp DNA fragment from S. aureus. Using these primers (vicK1 and vicK2) in PCR with a collection of 119 bacterial strains, it was observed that a specific DNA fragment of the expected size (289 bp) was generated from all 66 S. aureus strains only, but not from 29 non-S. aureus isolates or 24 other bacteria (Fig. 1; data not all shown). The accompanying negative (no DNA template) control did not yield any amplification product with these primers (Fig. 1). This observation suggests that the 2-component system regulator gene, vicK, is specific for S. aureus and can be one of useful diagnostic markers.
4. Discussion The current study utilized the genome sequence of S. aureus to search for a S. aureus-specific diagnostic marker for this human pathogen. Although many other important proteins have been conserved in various species, those genes may change in accordance with different environments. From the existing literature, certain speciesand virulence-specific genes are present and conserved in many bacteria including Listeria innocua and Pasteurella multocida (Liu et al., 2003; Liu et al., 2004b). By examining signal transduction genes in S. aureus, we identified the signal transduction gene, vicK, which displays species specificity for S. aureus. Many researches show that TCS are highly conserved in low-GTP + CTP content Gram-positive bacteria, which regulate various processes such as motility, sporulation, cell division, and virulence (Hendrik et al., 2005; Celine and James, 1998). Each of the species including many important human pathogens such as Streptococcus pneumoniae, S. aureus, and E. faecalis contains homologues of a single essential TCS that suggests TCS genes have a potential
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capacity of being a diagnostic marker for the identification of S. aureus, and our research in this article also prove this hypothesis. Although the complete genome sequences of S. aureus strains have only recently become available, the functions of many genes in this species remain uncharacterized. From the genome sequence data of S. aureus, most of the genes immediately upstream or downstream from vicK gene encode conserved hypothetical proteins. Hence, it is possible that this region of the chromosome is involved in speciesspecific maintenance or adaptation. To our knowledge, there is at least one well-documented example of a signal transduction gene (vicK), in a food-borne bacterial pathogen S. aureus, which regulates virulence in an in vivo model (Martin et al., 1999). Further researches on this speciesspecific marker will be carried out subsequently. Acknowledgments This research was supported by the China Postdoctoral Science Foundation (CPSF number 20060390636) and the Key Technologies Research and Application Development Program of Science and Technology Commission of Shanghai Municipality (no. 05DZ19101). References Barski P, Piechowicz L, Galinski J, Kur J (1996) Rapid assay for detection of methicillin-resistant Staphylococcus aureus using multiplex PCR. Mol Cell Probes 10:471–475. Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K, et al (2002) Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359:1819–1827. Celine F, James AH (1998) A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J Bacteriol 180:6375–6383. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH (2006) Complete genome sequence of USA300, an epidemic clone of community-acquired methicillin-resistant Staphylococcus aureus. Lancet 367:731–739. Gill SR, Fouts DE, Archer GL, Mongodin EF, Deboy RT, et al (2005) Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacteriol 187:2426–2438.
Hendrik S, Kristine N, Eun-Ja K, Marta P, James AH (2005) YycH regulates the activity of the essential yycFG two-component system in Bacillus subtilis. J Bacteriol 187:5419–5426. Hoch JA (2000) Two-component and phosphorelay signal transduction. Curr Opin Microbiol 3:165–170. Holden MT, Feil EJ, Lindsay JA, Peacock SJ, Day NP (2004) Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc Natl Acad Sci U S A 101:9786–9791. Inouye M, Dutta R (2003) Histidine kinases in signal transduction. San Diego: Academic Press. Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H (2001) Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357:1225–1240. Le Loir Y, Baron F, Gautier M (2003) Staphylococcus aureus and food poisoning. Genet Mol Res 2:63–76. Liu D, Ainsworth AJ, Austin FW, Lawrence ML (2003) Identification of Listeria innocua by PCR targeting a putative transcriptional regulator gene. FEMS Microbiol Lett 223:205–210. Liu D, Ainsworth AJ, Austin FW, Lawrence ML (2004a) Use of PCR primers derived from a putative transcriptional regulator gene for species-specific identification of Listeria monocytogenes. Int J Food Microbiol 91:297–304. Liu D, Lawrence ML, Austin FW (2004b) Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes. J Microbiol Methods 58:263–267. Martineau F, Picard FJ, Roy PH, Couellette M, Bergeron MG (1998) Species-specific and ubiquitous-DNA–based assays for rapid identification of Staphylococcus aureus. J Clin Microbiol 36:618–623. Martin PK, Li T, Sun D, Biek DP, Schmid MB (1999) Role in cell permeability of an essential two-component system in Staphylococcus aureus. J Bacteriol 181:3666–3673. Ribeiro J, Vieira FD, King T, D'Arezzo JB, Boyce JM (1999) Misclassification of susceptible strains of Staphylococcus aureus as methicillin-resistant Staphylococcus aureus by a rapid automated susceptibility testing system. J Clin Microbiol 37:1619–1620. Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69:183–215. Strommenger B, Kettlitz C, Werner G, Witte W (2003) Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol 41: 4089–4094. Stuhlmeier R, Stuhlmeier KM (2003) Fast, simultaneous, and sensitive detection of staphylococci. J Clin Pathol 56:782–785. Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, Gala JL (1995) Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J Clin Pathol 33:2864–2867. Wilkerson M, McAllister S, Miller JM, Heiter BJ, Bourbeau PP (1997) Comparison of five agglutination tests for identification of Staphylococcus aureus. J Clin Microbiol 35:148–151.
Diagnostic Microbiology and Infectious Disease 59 (2007) 379 – 382 www.elsevier.com/locate/diagmicrobio
Species-specific diagnostic marker for rapid identification of Staphylococcus aureus Zhan-min Liu, Xian-ming Shi⁎, Feng Pan Department of Food Science and Technology, Shanghai Jiaotong University, Shanghai 201101, PR China Received 31 March 2007; accepted 19 June 2007
Abstract Staphylococcus aureus is a common bacterial pathogen that has emerged as an increasingly important health concern. Following the recent publication of the genome sequences of 9 S. aureus strains (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), a gene of S. aureus that relates signal transduction as a target for rapid detection and identification of the pathogen has been investigated. By sequence analysis of S. aureus signal transduction genes from the complete genome of S. aureus ATCC N315 and their comparison with other DNA sequences using GenBank BLAST searches, we identified a unique gene, vicK. Polymerase chain reaction primers (vicK1 and vicK2) derived from this gene allowed amplification of a 289-bp DNA fragment only from S. aureus and not from other Staphylococcus species and other common bacteria tested. Besides offering an additional target for specific confirmation of S. aureus, further analysis of the signal transduction gene vicK and its related protein product may lead to new insights into the molecular mechanisms of S. aureus maintenance and pathogenicity. © 2007 Published by Elsevier Inc. Keywords: Diagnostic marker; Rapid identification; Staphylococcus aureus
1. Introduction Staphylococcus aureus is the staphylococcal species most commonly associated with human (nosocomial and community-acquired) and animal infections. The strains belonging to this species also cause food poisoning and are routinely characterized by growth properties, specific surface constituents, and their ability to coagulate blood plasma from various sources (staphylocoagulase) to produce a thermostable nuclease and to form clumps in the presence of fibrinogen (clumping factor). Several staphylococcus species, such as Staphylococcus epidermis and Staphylococcus haemolyticus, coagulase-negative species, have been isolated from ewe's milk and were found to produce one or several Staphylococcal enterotoxins (SEs) (Le Loir et al., 2003). Consequently, there is a need for developing rapid methods to specifically discriminate S. aureus from other staphylococcal species and nonstaphylococci in variety of ⁎ Corresponding author. Tel.: +86-21-64783841; fax: +86-2164783841. E-mail addresses: [email protected] (Z. Liu), [email protected] (X. Shi). 0732-8893/$ – see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2007.06.011
food commodities. Conventional identification methods are time-consuming and may yield false-positive or falsenegative results. Misidentification or misclassifications with automated susceptibility testing systems or commercially available latex agglutination kits have been reported recently (Ribeiro et al., 1999; Wilkerson et al., 1997). Development of improved laboratory diagnostic techniques, including those targeting femA (Vannuffel et al., 1995), nuc (Barski et al., 1996), coa (Martineau et al., 1998), 16S rRNA (Strommenger et al., 2003), Sa442 (Stuhlmeier and Stuhlmeier, 2003), and other genes in S. aureus, and rational use of antibacterial therapies have contributed to the control of this pathogen. Detection of S. aureus genes associated with antibiotic resistance (e.g., mecA [encoding methicillin resistance], tetM [tetracycline resistance], vat (A), vat (B), vat (C) [streptogramin A resistance]) by polymerase chain reaction (PCR) facilitates appropriate early antibiotic treatment. In addition, the use of multilocus sequence typing aids epidemiologic investigation of S. aureus infections. Recent description of the whole genome sequences of several S. aureus strains (N315, Mu50, MW2, NCTC8325, COL, RF122, USA300, MRSA252, and MSSA476) offers the opportunity to conduct detailed investigation on the
380
Z. Liu et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 379–382
mechanisms of S. aureus and survival in its niche environments as well as its pathogenicity.
Table 1 List of bacteria strains examined in the study Species
Number of strains
Amplification product size (bp)
S. aureus S. epidermidis S. haemolyticus Staphylococcus cohnii Staphylococcus hominis S. capitis Staphylococcus saprophyticus S. sciuri S. simulans S. warneri S. intermedius E. faecium E. faecalis Enterococcus casseliflavus Enterococcus durans Enterococcus avium Vibiro parahaemolyticus Salmonel choleraesuis Escherichia coli
66 11 10 1 1 1 1 1 1 1 1 5 5 1 1 1 4 4 3
289 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
molecular mechanisms behind its virulence and pathogenesis. In this report, we focus on one specialized gene group in S. aureus that encodes 2-component signal transduction system (TCS). Because of their important roles in sensing and responding to environmental conditions and in bacterial pathogenesis (Hoch, 2000; Inouye and Dutta, 2003; Stock et al., 2000), unique signal transduction genes may be required for a group of bacteria to fit in its particular ecologic niche. Previous results indicated that speciesspecific certain genes are present in a food-borne pathogen, Listeria monocytogenes (Liu et al., 2004a); therefore, TCS genes may display genus, species, or subspecies specificity. The identification of TCS genes in S. aureus will not only provide species-specific targets for diagnostic application but also lead to improved understanding of the genetic
2. Materials and methods 2.1. Bacterial strains A collection of 119 bacterial isolates were used, including 66 S. aureus, 11 S. epidermis, 10 S. haemolyticus, 8 other staphylococcus species, 5 Enterococcus faecium, 5 Enterococcus faecalis, 3 other Enterococcus species, and other Gram-positive and Gram-negative bacteria (Table 1). Among them, reference strains were also included such as those from the American Type Culture Collection (ATCC) and the China Medical Culture Collection Center (CMCC), Beijing, China, and Peking University Health Science Center, Beijing, China. S. aureus human isolates originated from Peking University Health Science Center, Beijing, China. S. epidermidis human isolates were from the Nanjing Children's Hospital, Nanjing, China, and Zhongshan Hopital of Fudan University, Shanghai, China. Other clinical bacterial strains were mainly isolated from the Jiling University Affiliated First Hospital, Jilin, China; Wuhan University Hospital, Wuhan, China; and Jiangsu Province Hospital, Nanjing, China and Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China. Other bacterial strains were kept in our laboratory. 2.2. DNA isolation The purified DNA stock was prepared in our laboratory as follows. Bacteria were grown overnight in Luria Bertani (LB) broth at 37 °C, and 1.5 mL culture of the organism was centrifuged by 3000 rpm/min for 5 min, then washed twice with TE (Tris 10 mmol/L, EDTA 1 mmol/L, pH 8.0). The purified organism was resuspended in 456 μL of TE, and lysed by adding 24 μL of 50 mg/mL lysozyme and incubating at 37 °C for 1 h. Then, 53 μL of 10% sodium
Fig. 1. Agarose gel electrophoresis of PCR products amplified with S. aureus vicK gene primers. Lanes 1–4, S. aureus strains (ATCC 29213, 25923, 6538, and CMCC 26001 representing 62 other strains); lane 5, S. epidermidis strains (ATCC 12228, representing 11 strains); lane 6, S. haemolyticus representing 10 strains; lane 7, Staphylococcus cohnii; lane 8, Staphylococcus hominis; lane 9, Staphylococcus capitis; lane 10, Staphylococcus saprophyticus; lane 11, Staphylococcus sciuri; lane 12, Staphylococcus simulans; lane 13, Staphylococcus warneri; lane 14, Staphylococcus intermedius; lane 15, E. faecalis representing 5 strains; lane 16, E. faecium representing 5 strains; lane 17, Enterococcus casseliflavus; lane 18, Enterococcus durans; lane 19, Enterococcus avium; lane 20, Vibiro parahaemolyticus (ATCC 17802 representing 4 strains); lane 21, Salmonel choleraesuis (CMCC 50004, representing 4 strains); lane 22, Escherichia coli representing 3 strains; and lane 23, negative control with no template DNA. On the left of lane M is DNA molecular weight ladder (DNA marker, Tiangen, Beijing, China).
Z. Liu et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 379–382
dodecyl sulfate was added and continued incubation for 15 min at 68 °C. Finally, 87 μL of 5 mol/L NaCl and 69 μL of 1% cetyl trimethyl ammonium bromide were added and incubated at 68 °C for an additional 15 min to release DNA. Subsequently, the samples were extracted with equal volumes of phenol/chloroform/isoamyl alcohol (25:24:1) followed by phenol/chloroform (1:1). The DNA was then precipitated by the addition of 1/10 volumes of 3 mol/L sodium acetate and 2 volumes of 100% ethanol and incubation at −20 °C for 15 min. The DNA pellets were washed twice with ice chilled with 70% ethanol, air dried, and dissolved in distillation water; and the DNA concentrations were determined at UV 260/280 nm in a DU800 UV spectrophotometer (Beckman Coulter, Fullerton, CA). A small amount of DNA from each bacterial strain was diluted in distilled water to 10 ng/μL for PCR analysis. 2.3. Identification of S. aureus-specific signal transduction gene S. aureus genes that encode signal transduction system were identified from the genome sequence of a S. aureus strain ATCC N315 and screened against other DNA sequences with GenBank's BLAST searches. Only 1 gene uniquely present in S. aureus was selected for further evaluation. Oligonucleotide primers were then designed from the gene of interest with Primer Premier 5.00 software (Premier Biosoft International, Palo Alto, CA) and synthesized with ABI 37300 high-throughput DNA synthesizer (Shanghai Sangon, Shanghai, China). 2.4. Polymerase chain reaction PCR was performed in a 50-μL volume using a PCR system PTC-200 (Bio-Rad, Foster City, CA). Each reaction contained 1.0 U Taq DNA polymerase (Tiangen Biotechnology, Beijing, China), 1× PCR buffer (containing 10 mmol/LTris–HCl [pH 9.0], 50 mmol/L KCl, and 1.5 mmol/ L MgCl2), 50 μmol/L dATP, dTTP, dCTP, and dGTP, 25 pmol/L of each primer, and 10 ng genomic DNA. The reaction mixture with no template DNA was used as a negative control. The thermal cycling conditions consisted of an initial denaturation at 94°C for 5 min, followed by 35 amplification cycles (94 °C for 40 s, 50 °C for 40 s, and 72 °C for 1 min), and final extension step at 72 °C for 10 min. After completion of all cycles, 6 μL of 10× DNA loading buffer was added to each tube, and the amplified products were examined in 1.5 % agarose gel electrophoresis in the presence of ethidium bromide (0.5 μg/mL). The stained gels were visualized under UV light and photographed using a Las300 Fuji Film (Fuji, Japan). 3. Results Based on results from BLAST searches, a S. aureusspecific gene that encodes histidine kinase was identified in the genome sequence of S. aureus N315 (GenBank
381
accession no. BA000018.3) (Kuroda et al., 2001). It was noted that a stretch of nucleotides identical to vicK (nt. 26414–26702) was also found in the complete genome of S. aureus MRSA252 strain (GenBank accession no. BX571856.1), MSSA476 (GenBank accession no. BX571857.1), NCTC8325 strain (GenBank accession no. CP000253.1) (Holden et al., 2004), COL strain (GenBank accession no. CP000046.1) (Gill et al., 2005), Mu50 strain (GenBank accession no. BA000017.4) (Kuroda et al., 2001), MW2 strain (GenBank accession no. BA000033.2) (Baba et al., 2002), RF122 strain (GenBank accession no. AJ938182.1) as well as USA300 strain (GenBank accession no. CP000255.1) (Diep et al., 2006). However, vicK gene sequence was clearly absent in other bacterial genomes available at GenBank. A pair of oligonucleotide primers was thus designed from the signal transduction gene vicK (vicK1: 5′-CTAATACTGAAAGTGAGAAACGTA-3′ and vicK2: 5′-TCCTGCACAATCGTACTAAA-3′). These primers correlated with the vicK gene sequence at nt 26414–26437 and nt 26683–26702, respectively, which facilitated the amplification of a 289-bp DNA fragment from S. aureus. Using these primers (vicK1 and vicK2) in PCR with a collection of 119 bacterial strains, it was observed that a specific DNA fragment of the expected size (289 bp) was generated from all 66 S. aureus strains only, but not from 29 non-S. aureus isolates or 24 other bacteria (Fig. 1; data not all shown). The accompanying negative (no DNA template) control did not yield any amplification product with these primers (Fig. 1). This observation suggests that the 2-component system regulator gene, vicK, is specific for S. aureus and can be one of useful diagnostic markers.
4. Discussion The current study utilized the genome sequence of S. aureus to search for a S. aureus-specific diagnostic marker for this human pathogen. Although many other important proteins have been conserved in various species, those genes may change in accordance with different environments. From the existing literature, certain speciesand virulence-specific genes are present and conserved in many bacteria including Listeria innocua and Pasteurella multocida (Liu et al., 2003; Liu et al., 2004b). By examining signal transduction genes in S. aureus, we identified the signal transduction gene, vicK, which displays species specificity for S. aureus. Many researches show that TCS are highly conserved in low-GTP + CTP content Gram-positive bacteria, which regulate various processes such as motility, sporulation, cell division, and virulence (Hendrik et al., 2005; Celine and James, 1998). Each of the species including many important human pathogens such as Streptococcus pneumoniae, S. aureus, and E. faecalis contains homologues of a single essential TCS that suggests TCS genes have a potential
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capacity of being a diagnostic marker for the identification of S. aureus, and our research in this article also prove this hypothesis. Although the complete genome sequences of S. aureus strains have only recently become available, the functions of many genes in this species remain uncharacterized. From the genome sequence data of S. aureus, most of the genes immediately upstream or downstream from vicK gene encode conserved hypothetical proteins. Hence, it is possible that this region of the chromosome is involved in speciesspecific maintenance or adaptation. To our knowledge, there is at least one well-documented example of a signal transduction gene (vicK), in a food-borne bacterial pathogen S. aureus, which regulates virulence in an in vivo model (Martin et al., 1999). Further researches on this speciesspecific marker will be carried out subsequently. Acknowledgments This research was supported by the China Postdoctoral Science Foundation (CPSF number 20060390636) and the Key Technologies Research and Application Development Program of Science and Technology Commission of Shanghai Municipality (no. 05DZ19101). References Barski P, Piechowicz L, Galinski J, Kur J (1996) Rapid assay for detection of methicillin-resistant Staphylococcus aureus using multiplex PCR. Mol Cell Probes 10:471–475. Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K, et al (2002) Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359:1819–1827. Celine F, James AH (1998) A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J Bacteriol 180:6375–6383. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH (2006) Complete genome sequence of USA300, an epidemic clone of community-acquired methicillin-resistant Staphylococcus aureus. Lancet 367:731–739. Gill SR, Fouts DE, Archer GL, Mongodin EF, Deboy RT, et al (2005) Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacteriol 187:2426–2438.
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