A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of
Staphylococcus aureus and enterococci to reduce the cost of using lysostaphin. This protocol reduces the
expenses of PFGE typing of S. aureus and enterococci as it removes the use of lysostaphin during the
spheroplast formation from these bacteria

Ghodousi, A.

Mehdi Feizabadi, M.

Nomanpour, B.

Omrani, M.

Shahcheraghi, F.

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of
Staphylococcus aureus and enterococci to reduce the cost of using lysostaphin. This protocol reduces the
expenses of PFGE typing of S. aureus and enterococci as it removes the use of lysostaphin during the
spheroplast formation from these bacteria

Mehdi Feizabadi, M

Nomanpour, B

Omrani, M

GHODOUSI, Arash

Archivio istituzionale della ricerca - Università di Palermo

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyrightAuthor's personal copyNoteDevelopment of a modiﬁed DNA extraction method for pulsed-ﬁeld gelelectrophoresis analysis of Staphylococcus aureus and enterococci withoutusing lysostaphinMohammad Mehdi Feizabadi a,⁎, Arash Ghodousi a, Bizhan Nomanpour a,Maryam Omrani a, Fereshteh Shahcheraghi ba Department for Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iranb Department of Bacteriology, Pasteur Institute of Iran, 12th of Farvardin Street, Tehran, Irana b s t r a c ta r t i c l e i n f oArticle history:Received 23 July 2010Received in revised form 25 October 2010Accepted 5 November 2010Available online 13 November 2010Keywords:Pulse-ﬁeld gel electrophoresis(PFGE)MRSAVRENosocomial infectionsA modiﬁed pulsed-ﬁeld gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates ofStaphylococcus aureus and enterococci to reduce the cost of using lysostaphin. This protocol reduces theexpenses of PFGE typing of S. aureus and enterococci as it removes the use of lysostaphin during thespheroplast formation from these bacteria.© 2010 Elsevier B.V. All rights reserved.Methicillin-resistant Staphylococcus aureus (MRSA) and vancomy-cin resistant enterococci (VRE) are pathogens often associated withnosocomial infections (Emori and Gaynes, 1993; Engemann et al.,2003; Schramm et al., 2006). Nosocomial infections have a tremen-dous impact on the morbidity, mortality and costs of hospitalization.Annually, two million patients are estimated to develop nosocomialinfections in the United States (Richards et al., 1999).Molecular typing methods are used to study the outbreakinvestigations at hospital and to trace the source of infections.Pulsed-ﬁeld gel electrophoresis (PFGE) is a gold standard and apowerful typing technique for contradistinction of bacterial strainscausing hospital-acquired infections (Birren and Lai, 1993; Leonardet al., 1995; Maslow et al., 1993; Swaminathan and Matar, 1993;Tenover et al., 1995). Problems arise when this technique is applied todifferentiate MRSA isolates since spheroplast formation from thisorganism needs lysostaphin (McDougal et al., 2003; Wenming et al.,2008), which is very expensive. Similarly, this enzyme also is used forDNA extraction from enterococci (Hogan et al., 2008; Murray et al.,1990; Saeedi et al., 2002; Sulakvelidze et al., 2000).In this study we successfully developed a novel protocol toeliminate lysostaphin from the DNA extraction.We used protocol described by Murray et al. (1990) for DNAextraction of enterococci and protocol described McDougal et al.(2003) in order to gain genomic DNA of S. aureus.The bacterial strains used in this study were isolated fromnosocomial infection specimens (clinical isolates of S. aureus andclinical isolates of Enterococcus faecalis). S. aureus ATCC29213 and E.faecalis ATCC29212 were used as controls. We used Salmonellaserotype Branderup strain H9812 as marker (Hunter et al., 2005).Enterococcous faecium strain D366 with van B phenotype (kindlyprovided by B. Murray, Texas University) was also included as controlin this experiment.Initially, one colony from each strain was sub-cultured fromplates containing trypticase soy agar with sheep blood into 5 mlBrain Heart Infusion Broth (BHI) in 15×100 mm screw-cap tubes.The tubes were then incubated for 16–18 h at 35–37 °C with gentleshaking until the optical density of suspension reached to 0.8–1.2 at600 nm for S. aureus strains and 1.3–1.7 for enterococci. From thissuspension, 350 μl was transferred into 1.5 ml micro-centrifuge tubeand the cells were spinned at 12,000 rpm (13,684.32 g) for 5 min.The supernatant was removed and the pellet was re-suspendedin 300 μl TEN buffer (100 mM EDTA, 0.15 M NaCl, 100 mM Tris pH7.5) and centrifuged at 12,000 rpm (13,684.32 g) for 5 min.Journal of Microbiological Methods 84 (2011) 144–146⁎ Corresponding author. Tel./fax: +98 21 88955810.E-mail address: mfeizabadi@tums.ac.ir (M.M. Feizabadi).0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2010.11.001Contents lists available at ScienceDirectJournal of Microbiological Methodsj ourna l homepage: www.e lsev ie r.com/ locate / jmicmethAuthor's personal copySubsequently, the sediments were suspended in 150 μl of EC buffer(100 mM EDTA, 1 M NaCl, 6 mM Tris–HCl (pH 7.6), 0.2% deoxycho-late–0.5% Sarkosyl and 0.5% Brij-58). The suspension was heated inboiling water for 5–10 min before mixing it with an equal volume of2% low melting point agarose (LMP). The LMP agarose was preparedin EC buffer and placed in a 50 °C water bath until use. The mixturewas poured into plug molds. The plugs were solidiﬁed for 5 min at4 °C. They were then transferred into 50 ml falcon tubes containing10 ml EC buffer and lysozyme (25 mg/ml) and then placed in 36 °Cwater bath overnight with gentle shaking. The plugs were trans-ferred to 9900 μl of lysis buffer II (0.5 mM EDTA, 1% Sarkosyl) plus100 μl of 20 mg/ml proteinase K (the ﬁnal concentration ofproteinase K was 0.2 mg/ml) and incubated in water bath at 50 °Cfor 4 h with gentle shaking. They were washed three times with10 ml of TE buffer for 30 min. The restriction digestion step was doneby adding 30 U SmaI enzyme for 4 h incubation at 25 °C after 30 minpreincubation at 30 °C.The plugs were placed in wells of 1.0% agarose gels (Bio-Rad)made with 0.5X TBE, sealing with the same agarose. Restrictionfragments of DNA are separated in 1% agarose in 0.5X TBE usingclamped homogeneous electric ﬁelds (CHEF DRIII, Bio-Rad). Theelectrophoretic conditions were 6 V/cm; 120° switch angle at14 °C, and a run time of 24 h divided into two different blocks. Theﬁrst block had a run time of 20 h, an initial switching time of 5 s,and a ﬁnal switching time of 35 s, whereas the second block used aswitch time ramp of 5 s–10 s for 4 h. The gel was stained with0.5 mg/ml ethidium bromide for 20 min and de-stained in waterfor 20 min.Despite omitting lysostaphin in our DNA extraction procedure,there were no differences of DNA bands using this new methodcompared to the standard protocol (Fig. 1) The results werereproducible when further experiments were performed on moreclinical strains (Fig. 2). Another advantage of our method is costsaving of proteinase K which is also expensive as we used 0.2 mg/mlof this reagent comparedwith other studies that used at least 1 mg/ml(Murray et al., 1990; Sulakvelidze et al., 2000).The PFGE patterns obtained for strains of S. aureus and enterococciare shown in Fig. 1. As the picture shows there is no signiﬁcantdifference between using lysostaphin, or boiling, in order to obtainwhole DNA. The type ability and repeatability of our new methodwere checked with further experiments using other isolates of S.aureus (n=30) and enterococci (n=30) (data is not shown).In conclusion, the protocol described in this article allows a cost-saving and reproducible alternative to previously published PFGEtyping protocols for typing of staphylococci, enterococci and,potentially, other gram-positive bacteria. We expect that theprocedure will be of value during epidemiological investigations ofMRSA and VRE outbreaks and for comparative characterization ofMRSA and VRE strains isolated in different geographic loci.This project was supported by Tehran University of MedicalSciences, project No. 8740.ReferencesBirren, B., Lai, E., 1993. Pulsed Field Gel Electrophoresis: A Practical Guide. AcademicPress, Inc., San Diego, Calif.Fig. 1. DNAﬁngerprinting of Staphylococcus aureus andenterococci after digestion of genomicDNAwith SmaI and resolution by PFGE.M: Salmonella serotypeBranderup strainH9812 asmarker.Lanes: 1, 10 E. faecalisATCC29212 and S. aureusATCC29213 (standard DNA extraction protocol); 2, 11: E. faecalisATCC29212 and S. aureusATCC29213 (newmethodwithout lysostaphin); 3, 5:clinical isolates of E. faecalis (standardDNAextraction protocol); 4, 6: clinical isolates of E. faecalis (newmethodwithout lysostaphin); 7, 12: clinical isolates of S. aureus (standardDNAextractionprotocol); 8, 13: clinical isolates of S. aureusnewmethodwithout lysostaphin), and 9: Enterococcus faeciumD366 (newmethodwithout lysostaphin).Molecular sizes are shown inkilo base pairson the right.145M.M. Feizabadi et al. / Journal of Microbiological Methods 84 (2011) 144–146Author's personal copyEmori, T.G., Gaynes, R.P., 1993. An overview of nosocomial infections, including the roleof the microbiology laboratory. Clin. Microbiol. Rev. 6, 428–442.Engemann, J.J., Carmeli, Y., Cosgrove, S.E., Fowler, V.G., Bronstein, M.Z., Trivette, S.L.,Briggs, J.P., Sexton, D.J., Kaye, K.S., 2003. Adverse clinical and economic outcomesattributable to methicillin resistance among patients with Staphylococcus aureussurgical site infection. Clin. Infect. Dis. 36, 592–598.Hogan, J.S., Petersson-Wolfe, C.S., Adams, S., Wolf, S.L., 2008. Genomic typing ofenterococci isolated from bovine mammary glands and environmental sources. J.Dairy Sci. 91, 615–619.Hunter, S.B., Vauterin, P., Lambert-Fair, M.A., Van Duyne, M.S., Kubota, K., Graves, L.,Wrigley, D., Barrett, T., Ribot, E., 2005. Establishment of a universal size standardstrain for use with the pulsenet standardized pulsed-ﬁeld gel electrophoresisprotocols: converting the national databases to the new size standard. J. Clin.Microbiol. 43, 1045–1050.Leonard, R.B., Mayer, J., Sasser, M., Woods, M.L., Mooney, B.R., Brinton, B.G., Newcomb-Gayman, P.L., Carroll, K.C., 1995. Comparison ofMIDI Sherlock system and pulsed-ﬁeld gel electrophoresis in characterizing strains of methicillin-resistant Staphy-lococcus aureus from a recent hospital outbreak. J. Clin. Microbiol. 33, 2723–2727.Maslow, J.N., Slutsky, A.M., Arbeit, R.D., 1993. Application of pulsedﬁeld gelelectrophoresis to molecular epidemiology. In: Persing, D.H., Smith, T.F., Tenover,F.C., White, T.J. (Eds.), Diagnostic Molecular Microbiology: Principles and Applica-tions. American Society for Microbiology, Washington, D.C, pp. 563–572.McDougal, L., Steward, C.D., Killgore, G.E., Chaitram, J.M., McAllister, S., Tenover, F.C.,2003. Pulsed-ﬁeld gel electrophoresis typing of oxacillin-resistant Staphylococcusaureus isolates from the United States: establishing a national database. J. Clin.Microbiol. 11, 5113–5120.Murray, B.E., Singh, K.V., Heath, J.D., Sharma, B.R., Weinstock, G.M., 1990. Comparison ofgenomic DNAs of different enterococcal isolates using restriction endonucleaseswith infrequent recognition sites. J. Clin. Microbiol. 28, 2059–2063.Richards, M.J., Edwards, J.R., Culver, D.H., Gaynes, R.P., et al., 1999. Nosocomialinfections in medical intensive care units in the United States. Crit. Care Med. 27,887–892.Saeedi, B., Hällgren, A., Jonasson, J., Nilsson, L.E., Hanberger, H., Isaksson, B., 2002.Modiﬁed pulsed-ﬁeld gel electrophoresis protocol for typing of enterococci. APMIS110 (12), 869–874.Schramm, G.E., Johnson, J.A., Doherty, J.A., et al., 2006. Methicillin-resistant Staphylococcusaureus sterile-site infection: the importance of appropriate initial antimicrobialtreatment. Crit. Care Med. 34, 2069–2074.Sulakvelidze, A., Turabelidze, D., Kotetishvili, M., Kreger, A., Morris, J.G., 2000. Improvedpulsed-ﬁeld gel electrophoresis for typing vancomycin-resistant enterococci. J.Clin. Microbiol. 4242–4245.Swaminathan, B., Matar, G.M., 1993. Molecular typingmethods. In: Persing, D.H., Smith,T.F., Tenover, F.C., White, T.J. (Eds.), Diagnostic Molecular Microbiology: Principlesand Applications. American Society for Microbiology, Washington, D.C, pp. 26–50.Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H.,Swaminathan, B., 1995. Interpreting chromosomal DNA restriction patternsproduced by pulsed-ﬁeld gel electrophoresis: criteria for bacterial strain typing. J.Clin. Microbiol. 33, 2233–2239.Wenming, Z., Clark, N.C., McDougal, L.K., Hageman, J., McDonald, L.C., Patel, J.B., 2008.Vancomycin-resistant Staphylococcus aureus isolates associated with Inc18-likevana plasmids in Michigan. J. Antimicrob. Agents Chemother. 52, 452–457.Fig. 2. DNAﬁngerprinting of Staphylococcus aureus and enterococci after digestion of genomic DNAwith SmaI and resolution by PFGE. DNAswere extractedwithout using lysostaphin. Lanes; 1:S. aureusATCC29213, 2–8: clinical isolates of S. aureus, 9–17: clinical isolates of E. faecalis, 12 and13:Repetitive isolates fromanother lung transplantedpatient atdifferent timeswhich equivalentto lane 4 of Fig. 1 (same patient), 14 and 15: Repetitive Isolates from a lung transplanted patient at different times which equivalent to lane 3 of Fig. 1 (same patient). The numbers show themolecular sizes of fragments in kilo base pairs.146 M.M. Feizabadi et al. / Journal of Microbiological Methods 84 (2011) 144–146

Development of a modified DNA extraction method for pulsed-field gel
electrophoresis analysis of Staphylococcus aureus and enterococci without
using lysostaphin

https://iris.unipa.it/retrieve/handle/10447/77582/71513/Development_of_a_modified_DNA_extraction_method_for_Pulsed-Field_Gel_Electrophoresis_analysis_of_Sta.pdf

Development of a modified DNA extraction method for pulsed-field gel
electrophoresis analysis of Staphylococcus aureus and enterococci without
using lysostaphin

Development of a modified DNA extraction method for pulsed-field gel electrophoresis analysis of Staphylococcus aureus and enterococci without using lysostaphin

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