Journal of Pharmacy and Pharmaceutics | Volume 4 | Issue 2 | 2017-11-07

Molecular Identification of Staphylococcus aureus Isolated from Clinical Samples by Specific PCR Assay Targeting the Sgnal Transduction Gene

Zhanmin Liu

Abstract

Staphylococcus aureus (S. aureus ) is a common, Gram-positive species that is pathogenic in both human and animals. Rapid and direct identification of this bacterium specifically from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. Although, some molecular identification assays are reported, some false-positive cases are described; it is an urgent need to develop a specific diagnostic method. Currently, the sensitivity and specificity of polymerase chain reaction targeting a signal transduction gene ( vick ) for the diagnosis of S. aureus isolates in clinical samples was developed. The assay is sensitive enough to detect 5500 copies of a cloned fragment of the S. aureus vick gene. This PCR assay can be used for rapid clinical diagnosis of S. aureus infection and detection of the pathogen in food samples.

Keywords: PCR, Rapid identification, Signal transduction gene, Staphylococcus Aureus, Vick

Full text

Staphylococcus aureus is a type of bacteria that about 30% of people carry in their noses as normal flora. In healthcare settings, its infections can be serious or fatal disease entities such as toxic-shock syndrome and staphylococcal scarlet fever [1] . Apart from being a leading source of gastroenteritis via contaminated foods [2] , S. aureus has also been responsible for an increasing number of hospital-acquired infections because of its ability to acquire and develop resistance to antibiotics [3] . In particular, the emergence of methicillin-resistant Staphylococcus aureus (MRSA), first noted over four decades ago, has made it one of the most important human bacterial pathogens of modern times. S. aureus are routinely characterized by growth properties, specific surface constituents and their ability to coagulate blood plasma from various sources (due to its ability to produce staphylocoagulase), to produce a thermostable nuclease (DNase) and to form clumps in the presence of fibrinogen (clumping factor). Several Staphylococcus species, such as Staphylococcus epidermis (S. epidermis), Staphylococcus haemolyticus (S. haemolyticus ), and the coagulase negative species, have been isolated from ewe s milk and were found to produce one or several staphylococcus enterotoxins [2] and consequently there is a need for methods to specifically discriminate S. aureus from other staphylococci and non-staphylococci as quickly as possible. Conventional identification methods and novel assays based on immunofluorescence probe, DNA microarray, surface enhanced laser desorption and ionization time of flight mass spectrometry, and whole-cell SELEX methods are time-consuming and may yield false-positive or false-negative results, and misclassifications with automated susceptibility testing systems or commercially available latex agglutination kits have been reported recently [4-13] . Many laboratory diagnostic techniques, including those targeting femA, nuc, coa , 16S rRNA, Sa442 and other genes in S. aureus , and rational antibacterial therapies have played an important role in the control of this pathogen [6,14-16] . Especially, detection of S. aureus genes associated with antibiotic resistance including mec A, aacA-aphD, tetM, erm (A), erm (C), vat (A), vat (B), vat (C) by PCR facilitates the appropriate control of the pathogen [17,18] . However, some false positive or negative cases by previous molecular identification assays are reported [16-19] . Therefore, it is particularly important to establish species-specific detection methods, and some species- and virulence-specific genes are present and used as specific marker in many bacteria including Listeria innocua, Pasteurella multocida and Streptococcus pyogenes in the existing literature [20,21] . Recent description of the whole genome sequences of S. aureus strains provides the great opportunity to carry out detailed investigation of the molecular mechanisms, virulence and pathogenesis in the microbial genome levels. In addition, the systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of S. aureus sequences can identify coding and conserved non-coding regions, including regulatory elements and species-marker. Based on the previous strategy, the signal transduction gene, vick is described as the species-specific diagnostic marker for identification of S. aureus [17] . In the present study, we developed a new diagnostic method for rapid identification of S. aureus targeting the vick gene. Materials and Methods Samples A total of 110 strains of S. aureus were studied (Table 1). They comprised five S. aureus reference strains (ATCC29213, 6538, 25923, 12228; American Type Culture Collection, Rockville, Md. and CMCC26001; China Medical Culture Collection Center), and 64 confirmed isolates of S. aureus by biochemical test and other bacteria used as negative controls for the PCR experiments from patient samples isolated in the Laboratory of Microbiology (Peking University Health Science Center, Beiijng, China) were used to determine the PCR in vitro assay specificity and sensitivity. Table 1: List of bacteria strains examined in the study. Species Strain code Staphylococcus aureus ATCC25923 human ATCC 6538 human ATCC29213 human CMCC 26001 human 05I-043 human 05C-054 clinic 05C-256 clinic 05A-176 clinic 05A-005 clinic 05A-075 clinic 05C-280 clinic 05Q-132 clinic 05N-119 clinic 05L-189 clinic 05B-205 clinic 05B-272 clinic 05C-212 clinic 05N-071 clinic 05B-033 clinic 05A-095 clinic 05L-085 clinic 05C-077 clinic 05L-254 clinic 05L-103 clinic 05L-201 clinic 05N-075 clinic 05O-114 clinic 05Q-236 clinic 05I-073 clinic 05E-186 clinic 05C-250 clinic 05B-206 clinic 05B-008 clinic 05B-012 clinic 05B-038 clinic 05B-041 clinic 05B-043 clinic 05B-255 clinic 05B-289 clinic 05C-052 clinic 05C-136 clinic 05C-155 Food 05C-215 clinic 05C-288 clinic 05I-056 clinic 05C-299 clinic 05C-173 clinic 05I-052 clinic 05A-100 clinic 05H-150 clinic 05L-224 clinic 05F-148 clinic 05N-138 clinic 05B-085 clinic 05G-226 clinic SJTU-4 clinic SJTI-5 food 05E-024 food 05G-021 clinic 05H-150 clinic 05L-228 clinic 05M-146 clinic 05D-005 clinic 05D-261 clinic 05N-074 clinic 05G-064 clinic 05D-361 clinic 05D-154 clinic Staphylococcus epidermidis ATCC12228 clinic 05A-078 clinic 05C-088 clinic 05C-229 clinic 05F-028 clinic 05G-121 clinic 05G-157 clinic 05K-181 clinic 03A-103 clinic 03L-032 clinic 03L-029 clinic Staphylococcus haemolyticus 05C-059 clinic 05C-134 clinic 05G-077 clinic 05G-086 clinic 05G-099 clinic 05G-100 clinic 05G-191 clinic 05G-193 clinic 05G-243 clinic 05H-003 clinic Enterococcus faecalis 05K-96 clinic 05I-93 clinic 05H-216 clinic 05B-56 clinic 05H-282 clinic Enterococcus faecium 05D-54 clinic 05G-267 clinic 05C-384 clinic 05D-027 clinic 05D-034 clinic Vibiro parahaemolyticus AS1.1190 shrimp ATCC17802 shrimp CMCC50001 fish CMCC500017 fish Salmonel choleraesuis CMCC50004 chick CMCC 50035 egg 11200 chick 12002 chick Escherichia coli 05A-001 clinic 05A-008 clinic 05A-48 clinic DNA preparation Purified DNA stock was prepared in our laboratory as follows. Bacteria were grown overnight in LB broth at 37 C, and 1.5 ml cultures of the organism were centrifuged at 3000 rpm/min for 5 min, then washed twice with TE (Tris 10 mM, EDTA 1 mM, pH 8.0). The purified organism was resuspended in 456 l of TE, and lysed by adding 24 l of 50 mg/ml lysozyme and incubated at 37 C for one hour. Then 53 l of 10% sodium dodecyl sulfate was added and continued incubation for 15 min at 68 C. Finally, 87 l of 5 mol/L NaCl and 69 l 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 and 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 incubating at -20 C for 15 min. The DNA pellets were washed twice with ice chilled 70% ethanol, air dried and the DNA pellets was 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/ l for PCR analysis. Oligonucleotide primers and PCR procedure Oligonucleotide primers were designed on the basis of the signal transduction gene vicK that is S. aureus species-specific diagnostic marker in our previous research. The sequences of the primers were 5 -CTAATACTGAAAGTGAGAAACGTA-3 and 5 -TCCTGCACAATCGTACTAAA-3 ) and facilitated the amplification of a 289-bp DNA fragment from only S. aureus . Each DNA amplification was performed in 200 l microtubes using a 50 l reaction mixture containing the 10 ng genomic DNA template, 1.0 U Taq DNA polymerase (Tiangen Biotechnology, Corporation, Beijing, China), 5 l of 10 PCR buffer, 50 M dNTPs, 25 pM each primers and double-distilled water to the final volume of 50 l. The reaction mixture with no template DNA was used as a negative control. All the amplifications were carried out in a PCR system PTC-200 (Biorad, Foster city, CA, USA) with initial denaturation at 94 C for 5 min followed by 35 cycles of denaturation 94 C for 40 s, primer annealing at 50 C for 40 s and extension at 72 C for 1 min, and followed final extension at 72 C for 10 min. After completion of all cycles, 6 l 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 g/ml). The stained gels were visualized under UV light and photographed using a Las300 Fuji Film (Fuji, Japan). Amplicons identification In order to confirm the specificity of the PCR amplifications, the PCR products were purified from agarose gels and cloned into a plasmid vector (pMD18-T vector, Takara Biotechnolgy (Dalian) Corporation, Dalian, China) following the manufacturer s protocol. Escherichia coli (DH5 , Tiangen Biotechnology, Beijing, China) were transformed and colonies containing inserts were selected using blue/white screening. Plasmid DNA was isolated using Tiangen plasmid mini prep kit (Tiangen Biotechnology, Beijing, China) following the manufacturer s protocol. Recombinant plasmid DNA was sequenced bi-directionally using the primers, M13-M4: 5 -GTTTT CCCAG TCACG AC-3 and BcaBEST Primer RV-M 5 -GAGCG GATAA CAATT TCACA CAGG-3 bi-directionally using an automated DNA sequencer (ABI 3730 DNA analyzer, Foster city, CA, USA). Sensitivity test In order to determine the limit detection of the assay, the sensitivity test was explored with the copies of S. aureus vick gene. Plasmid DNA containing the 289 bp fragment of the S. aureus vick gene was quantified using spectrophotometry and was serially diluted in water. These serially diluted samples were used as template for the PCR assay for 10 reactions for each dilution. After analysis the PCR product, the sensitivity of the assay was gained. Results Specificity The PCR assay specifically amplified a 289 base pair product from S. aureus , and did not produce amplicons when other organisms DNA from Staphylococcus epidermidis, Staphylococcus haemolyticus, Enterococcus faecalis, Enterococcus faecium, Vibrio parahaemolyticus, Salmonel choleraesuis and E. coli (Figure. 1; data not shown for all organisms) were used as a template for the PCR reaction (Figure.1). To prove their identity, amplicons were extracted from the gel, purified, inserted into pMD18-T Vector and sequenced, and the DNA sequence of the amplicons from the S. aureus isolates in this study was 100% identical to the S. aureus sequences in Genbank, which indicated the discriminating power of the PCR described here. Figure 1: Electrophoretic examination of PCR products generated with S. aureus vicK gene primers. Lanes 1 13 contained amplified DNA products from S. aureus strains (ATCC12228, 25923, 6538, 29213, 26001, 05I-56, 05C-54, 05C-256, 05A-176, 05A-5, 05A-75, 05C-280 and 05Q-132 representing other 56 strains); lanes 14, S. epidermidis strains (05A-78 representing other 9 strains); lane 15, S. haemolyticus (05G-99 representing other 9 strains), lane 16, Enterococcus faecalis (05K-96 representing other 4 strains); lane 17, Enterococcus faecium (05-D54 representing other 4 strains); lane 18, Vibiro parahaemolyticus (ATCC 17802 representing other 3 strains); lane 19, Salmonel choleraesuis (CMCC50004 representing other 3 strains); lane 20, Escherichia coli (05A-001 representing other 2 strains) and lane 21, negative control with no template DNA,. On the left of lane 1 is a DNA molecular weight ladder (DNA ladder, Tiangen, Beijing, China). Sensitivity The PCR was able to detect S. aureus DNA spiked with as little as 5500 gene copies/ l in DNA isolated from the plasmids containing the target sequence (Figure. 2). The frequency of amplicon detection in the replicate reactions is presented in Table 2, and consistent detection (100%) required 5500 gene copies of the target molecule per reaction. Figure 2: Sensitivity of PCR amplification targeting vick gene. Lane 1-9 of serially diluted plasmid containing the 289 bp fragment of S. aureus vick were used as template and their concentration were 5.5 10 9 , 5.5 10 8 , 5.5 10 7 , 5.5 10 6 , 5.5 10 5 , 5.5 10 4 , 5.5 10 3 , 5.5 10 2 and 55 copies/ l, respectively. M indicates the lane containing marker DNAs, and the numbers on the left of the panel indicate the sizes of the markers Table 1: Sensitivity of S. aureus -specific PCR assay for the detection of vick gene copies diluted in S. aureus samples. Gene copies per microliter Success rate (%) (positive PCR reactions/attempts 55000000 100(10/10) 5500000 100(10/10) 550000 100(10/10) 55000 100(10/10) 5500 100(10/10) 550 30(3/10) Discussion and Conclusion Although S. aureus is not difficult to grow and is easy to identify, there is a need for the development of rapid and sensitive DNA-based assay that is suitable for the detection of S.aureus to improve the rapidity and the accuracy of the diagnosis of S. aureus infections. Since introduction of PCR over thirty years ago, PCR methods for the detection of infectious organisms have been recognized as increasingly valuable clinical diagnostic tools. The development of highly sensitive and specific PCR assays has alleviated problems typically associated with microorganisms which are found in low densities in tissue (or tissue fluids), difficult to culture, or serologically similar. There are PCR amplification assays targeting various genes, including mecA, nuc , SEs encoding enterotoxins [17] . However, all of these targets are not ubiquitously found in the species S. aureus , and consequently, these PCR assays are not suitable for the detection and identification of S. aureus [18] . The coa gene coding for the coagulase protein has also been considered a candidate for the development of DNA-based diagnostic assays for S. aureus , however, this gene is highly polymorphic and does not allow for the ubiquitous identification of all S. aureus strains [22] . Hence, there is still an urgent need for development of species-specific and rapid diagnostic method for rapid identification of S. aureus from clinical samples. In our work, 110 strains isolated from clinical samples in various hospitals were identified in biochemical test, and used to identify the validity of our novel PCR method. The PCR assay results revealed a good correlation with those identified by biochemical test. The S. aureus -specific PCR described in this study was determined to be very sensitive and was able to detect S. aureus DNA in samples containing less than 5500 gene copies per reaction. The number of target molecules, in conjunction with the detection limit of the specific PCR assay, is a critically important determinant of the clinical diagnostic sensitivity of tests used to detect the presence of pathogenic organisms. This is especially applicable to PCR assays in which a negative test may not be useful in ruling out infection with a particular pathogen that can induce persistent low-level infection. The lower percentage of PCR positive test results obtained from samples with 5500 or less gene copies/ l highlights the effect that sampling has on detection in patient samples containing low copy numbers. Since it is impossible to detect less than 5500 gene per PCR reaction, the number of target molecules that are actually placed in the reaction dictates whether or not this particular sample will result in a positive result. The probability of a target molecule being placed into the reaction is determined by Poissonian statistics since the molecules in the solution should have a Poisson Distribution (Pfaff). For this assay 5500 copies vick gene per reaction are necessary for detection of S.aureus every time. Conflict of interest: The authors have no conflict of interest. References 1. Robinson, R.K. Encyclopaedia of Food Microbiology. (2000) San Diego, CA: Academic Press. Pubmed || Crossref || Others 2. Le Loir, Y., Baron, F., Michel, G. Staphylococcus aureus and food poisoning. (2003) Gene Mol Res 2(1): 63-76. Pubmed || Crossref || Others 3. Montesinos, I., Salido, E., Delgado T., et al. Epidemiology of methicillin-resistant Staphylococcus aureus at a university hospital in the Canary Islands. (2003) Infect Control Hosp Epidemiol 24(9): 667-672. Pubmed || Crossref || Others 4. Huang, S.H. Gold nanoparticle-based immunochromatographic test for identification of Staphylococcus aureus from clinical specimens. (2006) Clin Chim Acta 373(1-2): 139-43. Pubmed || Crossref || Others 5. Yang, Y.C., Yu, H., Xiao, D.W., et al. Rapid identification of Staphylococcus aureus by surface enhanced laser desorption and ionization time of flight mass spectrometry. (2009) J Microbiol Methods 77(2): 202-206. Pubmed || Crossref || Others 6. He, Y., Liu, H.L., Xian, M.J., et al. Detection and identification of Staphylococcus aureus in raw milk by hybridization to oligonucleotide microarray. (2010) Afri J Biotec 9(15): 2284-2289. Pubmed || Crossref || Others 7. Shrestha, N.K., Scalera, N.M., Wilson, D.A., et al. Rapid Identification of Staphylococcus aureus and methicillin resistance by flow cytometry using a peptide nucleic acid probe. (2011) J Clin Microbiol. 49(9): 3383-3385. Pubmed || Crossref || Others 8. Wu, Q., Li, Y., Wang, M., et al. Rapid identification of Staphylococcus aureus directly from positive blood culture media using quantum dots as fluorescence probes. (2013) APMIS 121(4): 348-352. Pubmed || Crossref || Others 9. Idelevich, E.A., Walther, T., Molinaro, S., et al. Bacteriophage-based latex agglutination test for rapid identification of Staphylococcus aureus . (2014) J Clin Microbiol 52(9): 3394-3398. Pubmed || Crossref || Others 10. Qian, Q., Eichelberger, K., Kirby, J.E. Rapid identification of Staphylococcus aureus directly from bactec blood culture broth by the BinaxNOW S. aureus Test. (2014) J Clin Microbiol 52(1): 319-320. Pubmed || Crossref || Others 11. Heraud, S., Freydiere, A.M., Doleans-Jordheim, A., et al. Direct identification of Staphylococcus aureus and determination of methicillin susceptibility from positive blood-culture bottles in a Bact/ALERT system using Binax Now S. aureus and PBP2a tests. (2015) Ann Lab Med 35(4): 454-457. Pubmed || Crossref || Others 12. Moon, J., Kim, G., Park, S.B., et al. Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus -specific DNA aptamers. (2015) Sensors 15(4): 8884-8897. Pubmed || Crossref || Others 13. Chang, T., Wang, L., Zhao, K., et al. Duplex identification of Staphylococcus aureus by aptamer and gold nanoparticles. (2016) J nanosci nanotechnol 16(6): 5513-5519. Pubmed || Crossref || Others 14. Brakstad, O.G, Aasbakk, K., Maeland, J.A. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. (1992) J Clin Microbiol 30 (7): 1654-1660. Pubmed || Crossref || Others 15. Kobayashi, N., Wu, H., Kojima, K., et al. Detection of mecA, femA, and femB genes in clinical strains of staphylococci using polymerase chain reaction. (1994) Epidemiol Infec 113(2): 259-266. Pubmed || Crossref || Others 16. Klaassen, C.H., de Valk, H.A., Horrevorts, A.M. Clinical Staphylococcus aureus isolate negative for the Sa442 fragment. (2003) J Clin Microbiol 41(9): 4493. Pubmed || Crossref || Others 17. Liu, Z.M., Shi, X.M., Pan, F. Species-specific diagnostic marker for rapid identification of Staphylococcus aureus . (2007) Diagn Microbiol Infect Dis. 59(4): 379-382. Pubmed || Crossref || Others 18. Strommenger, B., Schmidt, C., Werner, G., et al. DNA microarray for the detection of therapeutically relevant antibiotic resistance determinants in clinical isolates of Staphylococcus aureus . (2007) Mol Cell Probes 21(3): 161-170. Pubmed || Crossref || Others 19. Sutterlin, K., Englert, R., Schmidt-Wieland, T., et al. Sporadic cases of Staphylococcus aureus organisms negative for a species-specific 442-base pair chromosomal fragment. (2003) J Clin Microbiol 41(7): 3449. Pubmed || Crossref || Others 20. Liu, D.Y., Lawrence, M.L., Austin, F.W. Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes. (2004) J Microbiol Methods 58(2): 263-267. Pubmed || Crossref || Others 21. Liu, D.Y., Hollingshead, S., Swiatlo, E., et al. Rapid identification of Streptococcus pyogenes with PCR primers from a putative transcriptional regulator gene. (2005) Res Microbiol 156(4): 564-567. Pubmed || Crossref || Others 22. Schwarzkopf, A., Karch, H. Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker. (1994) J Clin Microbiol 32(10): 2407-2412. Pubmed || Crossref || Others