[22] Efficient rna isolation method for analysis of transcription in sessile staphylococcus epidermidis biofilm cultures

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mixed results, but the presence and activity of the ica gene cluster indicate that it behaves as a virulence factor in at least some animal and cell culture models. It remains to be seen whether other factors can be identified that are involved in the formation or maintenance of staphylococcal biofilms in vivo, and that could be potential targets for the prevention or elimination of biomedical implant-associated infections. Acknowledgments This project is supported by the German Bundesministerium fOr Bildung, Wissenschaft, Forschung und Technologie (BMBF) (DLR: 01KI9751/1). The technical assistance of Ulrike Pfitzner is gratefully acknowledged.

[22] Efficient RNA Isolation Method for Analysis of Transcription in Sessile Staphylococcus epidermidis Biofilm Cultures B y SABINE DOBINSKY a n d DIETRICH M A C K

Introduction

Staphylococcus epidermidis is a normal inhabitant of human skin and mucous membranes. With the increasing use of foreign biomaterials in medicine these organisms have become one of the most frequently isolated pathogens in nosocomial infections.l'2 The specific pathogenicity of these skin commensals can be attributed to an unusual ability to colonize polymer surfaces in multilayered communities referred to as biofilms. 3'4 Initially, bacteria attach to a polymer surface, followed by accumulation of bacterial cells in multilayered cell aggregates encased in an amorphous glycocalyx, a,4 A polysaccharide intercellular adhesin (PIA) essential for bacterial accumulation mediates intercellular adhesion in these biofilms. 5,6 1 M. E. Rupp and G. L. Archer, Clin. Infect. Dis. 19, 231 (1994). 2 j. Huebner and D. A. Goldmann, Annu. Rev. Med. 50, 223 (1999). 3 D. Mack, J. Hosp. Infect. 43(Suppl.), S 113-S 125 (1999). 4 D. Mack, K. Bartscht, S. Dohinsky, M. A. Horstkotte, K. Kid, J. K. M. Knobloch, and P. Schtifer, in "Handbook for Studying Bacterial Adhesion: Principles, Methods, and Applications" (Y. H. An and R. J. Friedman, eds.), p. 307. Humana Press, Totowa, New Jersey, 2000. 5 D. Mack, M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs, Infect. Immun. 62, 3244 (1994). 6 D. Mack, W. Fischer, A. Krokotsch, K. Leopold, R. Hartmann, H. Egge, and R. Laufs, J. Bacteriol. 178, 175 (1996).

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Synthesis of PIA requires the expression of the i c a A D B C gene locus of S. epidermidis. 7-9 Expression of biofilm formation and PIA by S. epidermidis depends significantly on different environmental factors such as type of growth medium used, l°-12 the presence of specific carbohydrates (such as glucose) in the medium, 1°'13 and composition of the atmosphere. 11'14 At least three independent regulatory gene loci control expression of the synthetic genes for PIA synthesis on the level of transcription. 12 Apparently, expression of genes relevant for S. epidermidis biofilm formation is tightly regulated. Transcriptional activity in biofilms of the S. epidermidis i c a A D B C locus and other gene loci relevant for biofilm formation under different physiologic growth conditions are almost completely unknown at present. Analysis of transcription under these conditions requires the recovery of extremely high-quality m R N A from established S. epidermidis biofilms. Established RNA extraction procedures proved to be unreliable and inefficient for extraction of R N A from staphylococci, because of their extremely stable cell wall. 15-17 In addition, these procedures often require extended periods of enzymatic digestion with proteases or lysostaphin for facilitating lysis of staphylococcal cells, potentially leading to degradation of short-lived m R N A species. 18 Many of these drawbacks have been solved by introduction of a new method for RNA extraction from Staphylococcus aureus and mycobacteria, using cell disruption by zirconia/silica beads in a high-speed shaking apparatus (FastPrep system; Bio 101, Vista, CA) and stabilization of extracted RNA by chaotropic reagents containing acid phenol, cetyltrimethylammonium bromide (CTAB), sodium acetate, and dithiothreito117 or modifications of this solution (chaotropic R N A stabilization reagent, FastPrep system; Bio 101). By this method m R N A from planktonic 7 C. Heilmann, O. Schweitzer,C. Gerke, N. Vanittanakom,D. Mack, and E Grtz, Mol. Microbiol. 20, 1083 (1996). 8 C. Gerke, A. Kraft, R. Siissmuth,O. Schweitzer, and E Grtz, J. Biol. Chem. 273~ 18586 (1998). 9 D. Mack, J. Riedewald,H. Rohde, T. Magnus, H. H. Feucht, H. A. Elsner, R. Laufs, and M. E. Rupp, Infect. lmmun. 67, 1004 (1999). l0 G. D. Christensen,W. A. Simpson, A. L. Bisno, and E. H. Beachey, Infect. lmmun. 37, 318 (1982). II M. Hussain, M. H. Wilcox, P. J. White, M. K. Faulkner, and R. C. Spencer, J. Hosp. Infect. 20, 173 (1992). 12D. Mack, H. Rohde, S. Dobinsky, J. Riedewald, M. Nedelmann, J. K. M. Knobloch, H.-A. Eisner, and H. H. Feucht, Infect. lmmun. 68, 3799 (2000). 13D. Mack, N. Siemssen. and R. Laufs, Infect. lmmun. 611,2048 (1992). 14L. P. Barker, W. A. Simpson, and G. D. Christensen,J. Clin. Microbiol. 28, 2578 (1990). 15K. J. Reddy and M. Gilman, in "Current Protocols in Molecular Microbiology" (E M. Ausubel. R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds.), Vol. 1, pp. 4.4.1-4.4.7. John Wiley & Sons, New York, 1993. 16j. S. Kornblum,S. J. Projan, S. L. Moghazeh, and R. P. Novick, Gene 63, 75 (1988). 17A. L. Cheung, K. J. Eberhardt, and V. A. Fischetti,Anal Biochem. 222, 511 (1994). 18M. Mempel, H. Feucht, W. Ziebuhr, M. Endres, R. Laufs, and L. GrUter, Antimicrob. Agents Chemother. 38, 1251 (1994).

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TABLE I YIELD OF RNA EXTRACTIONOF SESSILE Staphylococcus epidermidis CULTURES AT VARIOUSTIME POINTS AFTERBIOFILM INDUCTION Hours after glucose induction 0 1 4 6 8

Amount of biofilm (OD570)a 0 0 1.25 2.5 2.5

RNA yield/cell density at OD578 (l~g/OD)

Absorbance ratio

41.4 39.6 21.4 3.7 3.6

2.01 2.05 2.09 1.90 1.93

(A26o/A28o)

a Biofilm production by S. epidermidis was determined by a semiquantitative adhesion assay.2° Bacteria (200 ixl/well) were grown in TSBaGlcoxoid for 15-17 hr in 96-well tissue culture plates (Nunclon Delta; Nunc, Roskilde, Denmark). Biofilm formation was then induced by adding glucose [5 g.1 of a 10% (w/v) stock solution per well] at various time points before termination of the experiment. Plates were washed and dried in ambient air, and adherent biofilms were stained with gentian violet. The optical density of stained biofilms was measured at 570 nm (using 405 nm as reference wavelength) in an automatic spectrophotometer (Behring, Marburg, Germany).

cultures of S. epidermidis 1457 and RP62A can be successfully prepared for use in Northern blotting experiments demonstrating transcription of icaADBC. 12"19 E x t r a c t i o n o f RNA f r o m S e s s i l e Staphylococcus epidermidis Biofilm Cells In tryptic soy broth lacking glucose [TSBctGlcoxoid prepared from tryptone (Oxoid, Basingstoke, England), neutralized soya peptone (Oxoid), NaC1, and dipotassium phosphate as indicated by the manufacturer] S. epidermidis 1457 displays a biofilm-negative phenotype, as biofilm formation depends on the presence of glucose in the growth medium) °'13 Induction of stationary phase cultures with glucose induces biofilm formation and PIA synthesis, and fully established biofilms are formed 6-8 hr after glucose induction (Table I). 13'20 Staphylococcus epidermidis 1457 is precultured in 10 ml of TSBaGlcoxoid for 6 - 1 0 hr with shaking at 160 rpm at 37 °. The culture is diluted 1 : 100 in the same medium and 10 ml is inoculated into 9-cm plastic tissue culture plates (Nunc, Roskilde, Denmark) and incubated at 37 ° for 15-17 hr. Biofilm formation is then induced by the addition of 19 W. Ziebuhr, V. Krimmer, S. Rachid, I. L6Bner, E G6tz, and J. Hacker, Mol. Microbiol. 32, 345 (1999). 20 D. Mack, K. Bartscht, C. Fischer, H. Rohde, C. de Grahl, S. Dobinsky, M. A. Horstkotte, K. Kiel, and J. K. M. Knobloch, Methods Enzymol. 336 [20] (2001) (this volume).

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12345

FIG. 1. RNA extraction from S. epidermidis biofilms at time points 0 (lane 1), 1 hr (lane 2), 4 hr (lane 3), 6 hr (lane 4), and 8 hr (lane 5) after glucose induction, using zirconia/silica beads and a highspeed shaking apparatus (FastPrep system; Bit 101).17 Cells were grown and induced as described in text. Cell preparation included only sonication (twice, 30 sec each) to disintegrate the biofilm before RNA extraction. Samples (10 i~g) of total cellular RNA were separated on a 1% (w/v) agaroseformaldehyde gel.

glucose [0.25% (w/v) final concentration] to the growth medium at various time points before extraction of RNA.13 Yield and composition of total cellular RNA from S. epidermidis biofilm cultures at various time points after glucose induction are compared, using the standard extraction protocol with zirconia/silica beads and a high-speed shaking apparatus as described by Cheung et al.17 for extraction of RNA from planktonic S. aureus cultures. Before the extraction procedure the biofilms are disintegrated by sonication (twice, 30 sec each). The yield of extracted RNA by this method clearly depends on the age of the biofilm analyzed (Table I). Six hours after glucose induction of biofilm formation the biofilm formed reaches a maximum value (OD570 -----2.5). Approximately 10 times less RNA is obtained as compared with the yield of a noninduced culture (OD570 = 0) or a 1-hr biofilm (OD570 = 0). No significant contamination with DNA or proteins as determined by the A26o/A28o ratio can be detected in either preparation (Table I). Significant differences in the composition of the extracted RNA are observed depending on the time after glucose induction. From cells harvested 6 or 8 hr after glucose induction the extraction procedure yields predominantly small 5S rRNA (Fig. 1, lanes 4 and 5), indicating incomplete disruption of cells from established S. epidermidis biofilms.

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1 2 3 4 5 6 m

!

m

FIG.2. RNA yield and quality produced by various cell preparation procedures preceding RNA extraction with zirconiaJsilicabeads and a high-speed shaking apparatus (FastPrep system; Bio 101) from S. epidermidisbiofilms. Samples (10 ixg) of total cellular RNA extracted from 8-hr biofilms were analyzedon a 1% (w/v) agarose-formaldehydegel. Lane 1, simplebiofilmdisintegrationby sonicating twice (30 sec each); lane 2, same preparationmethod for a 1-hrbiofilm, used as positivecontrol;lane 3, cell preparation by additional sonication (fivetimes, 2 min each); lane 4, cell preparationby additional sonication (five times, 1 min each); lane 5, cell wall lysis with lysostaphin, 150 U/ml, 2 min at 37°; lane 6, cell wall lysis with lysostaphin, 150 U/ml, 5 min at 37°.

Several different approaches have been used to overcome these problems. Increasing the processing time in the high-speed shaking apparatus using the zirconia/silica beads does not result in an increase in RNA yield (data not shown). In an attempt to more vigorously destroy the bacterial cell wall, the time of sonication before RNA extraction was prolonged. After sonicating the 8-hr biofilm an additional five times (1 min each), marginal RNA extraction is observed (Fig. 2, lane 4). Sonicating an additional 10 min (five times 2 min each) improves the recovery of 23S and 16S rRNA, but still gives unsatisfactory results as predominantly 5S rRNA is extracted (Fig. 2, lane 3). Introduction of an enzymatic lysis step with lysostaphin before RNA extraction results in complete disruption of the staphylococcal cell wall. Incubation for 2 min at 37 ° is already sufficient to allow complete extraction of RNA, including 16S and 23S rRNA species, in high yield and purity (Fig. 2, lane 5). Apparently, the S. epidermidis cell wall of biofilm cells is sufficiently disrupted by the zirconia/silica beads only after partial lysis of the peptidoglycan interpeptide bridges by lysostaphin.

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b

1

2

3

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4

3600

FIG.3. Northern blot analysis of total cellular RNA extracted from a 4-hr biofilm after various cell preparation procedures. Quantities (10 ~g) of each sample were separated on a 1% (w/v) agaroseformaldehydegel, blotted onto nylon membrane,and hybridizedwith an icaC-specificoligonucleotide. Lane 1, positive control, simple disintegration of the biofilm by sonicating twice, 30 sec each; lane 2, cell preparation by an additional five 1-min sonications; lane 3, cell wall lysis with lysostaphin, 150 U/ml, 2 min at 37°; lane 4, cell wall lysis with lysostaphin, 150 U/ml, 5 min at 37°.

Prolonged incubation times at 37 ° are a problematic step in R N A extraction procedures.15,16, 28 To verify the functional integrity of the extracted RNA, Northern blot analysis is performed with a 32p-labeled oligonucleotide probe specific for icaADBC, the gene locus encoding enzymes for the synthesis of PIA. 7,8 RNA is extracted from a 4-hr biofilm after the cells have been manipulated according to the various protocols. This preparation can serve as positive control, as at this time after biofilm induction bacterial cells are readily disrupted even by the standard method o f R N A extraction after sonication (twice, 30 sec each) to disintegrate the biofilm. Similar hybridization patterns are observed with an icaC-specific probe with all samples, even when cell wall lysis is performed with lysostaphin for 5 min at 37 ° (Fig. 3, lane 4). Prolonged sonication (five times, 1 min each) also does no harm to the extracted R N A (Fig. 3, lane 2). Although the icaADBC transcript is about 3.6 kb long no indication o f degradation of the m R N A species is observed. Because the half-lives o f some m R N A species might be even shorter than 2 min it is recommended that a similar control experiment be performed to determine the stability of the specific m R N A under investigation.

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Critical for efficient extraction of RNA from established S. epidermidis biofilms in high yield and purity is the lysostaphin lysis step before cell disruption, which is also observed with biofilms of other biofilm-producing S. epidermidis strains (data not shown). The method probably can be adapted for the analysis of transcription of relevant genes in S. aureus biofilms and in biofilms of other gram-positive bacterial species. 21'22

Optimized RNA Extraction Protocol for Staphylococcus epidermidis Biofilms Staphylococcus epidermidis 1457 is grown in TSBctGlcoxoid and induced with glucose as described above. For harvesting S. epidermidis biofilms at each time point two 9-cm tissue culture plates are placed on ice. The cells are scraped from the surface with a disposable cell scraper into the growth medium and the suspension is immediately transferred to a prechilled centrifuge tube (Falcon; Becton Dickinson, Heidelberg, Germany) on ice. All further manipulations are carried out at 4 °, using precooled solutions. Ceils are sedimented by centrifugation (6000g, 10 rain) and washed once in an equal volume of ice-cold phosphate-buffered saline. The cell pellet tends to adhere to the inner surface of glass pipettes and therefore suspension of the cells must be performed carefully. Bacteria are then suspended in 10 ml of phosphate-buffered saline and the cell aggregates are disintegrated by sonication (twice, 30 sec each) in an ice bath, using the 3-mm microtip of a sonicator disintegrator (Branson sonifier 250-D; Branson Ultrasonics, Danbury, Connecticut) at 70% of maximal amplitude. Samples are removed to determine the cell density as OD578. Cells are sedimented, suspended in 10 ml of ice-cold 0.5 M EDTA, pH 8.0, and washed by repeating sonication (twice, 30 sec each) on ice. After sedimentation, bacteria are suspended in 1 ml of TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) containing lysostaphin (150 U/ml; Sigma, Deisenhofen, Germany) and incubated for 2 min at 37 °. Immediately thereafter the reaction tube is placed on ice for 5 min. The cells are then collected by centrifugation (13,000g, 5 min) and suspended in 100 p.1 of ice-cold HEO (aqua ad injectabilia; B. Braun Melsungen, Melsungen, Germany) and 500 ixl of chaotropic RNA-stabilizing reagent (CRSR; Bio 101). The suspension is transferred to a 2-ml FastRNA tube (type blue) with zirconia/silica beads (FastPrep system; Bio 101), which already contains 500 txl of acid phenol, pH 4.5 (Amresco, Solon, OH), and 100 pA of chloroform-isoamyl alcohol (24 : 1, v/v; CIA). The tubes are processed in a high-speed shaking apparatus (FP 120 FastPrep cell disruptor; Savant Instruments, Farmingdale, NY), three times (20 sec each) at maximal speed. The phases are separated by centrifuging the samples (13,000g, 21S. E. Cramton,C. Gerke,N. F. Schnell,W.W. Nichols,and E G6tz,Infect.lmmun. 67, 5427(1999). 22D. McKenney,K. L. Pouliot,Y.Wang,V.Murthy,M. Ulrich,G. Doting,J. C. Lee,D. A. Goldmann, and G. B. Pier, Science284, 1523(1999).

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30 min). The supernatant is carefully removed and approximately 50-80 I.zl of the solution over the interphase is left behind to avoid contamination with DNA and proteins. The supernatant is reextracted with 500 I~1 of CIA and phases are separated again after vortexing for 10 sec. The upper layer is transferred to a new reaction tube and the RNA is precipitated by adding 500 tzl of 2-propanol. RNA is sedimented (13,000g, 10 rain) and washed in 500 t~1 of 70% (v/v) ethanol. Residual ethanol is removed with a micropipette and the RNA is dissolved in 100 izl of H20. To minimize carbohydrate contamination an additional lithium chloride precipitation is performed by adding 20 I~1of 12 M LiC1 and 100 I.d of 2-propanol per 100 ~1 of dissolved RNA. After 15 min of incubation on ice the precipitated RNA is sedimented, washed with 70% (v/v) ethanol, and finally dissolved in 50-200 ~1 of H20. The RNA concentration is determined spectrophotometrically at 260 rim. To assess the integrity of the purified RNA, samples (10 Izg) are analyzed on 1% (w/v) agarose-formaldehyde gels in morpholinepropanesulfonic acid (MOPS) running buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0).

Northern Blot Analysis of Isolated RNA RNA separated by electrophoresis is blotted onto Zeta-probe membranes (Bio-Rad, Munich, Germany) and fixed by baking at 80 ° for 30 min. Hybridization is performed with an icaC-specific oligonucleotide (5'-GAA ATA GCC ATA CCA TTG TCC-Y) at 52 ° overnight in modified hybridization buffer followed by two washing steps as suggested by the manufacturer [7% (w/v) sodium dodecyl sulfate (SDS), 20 mM sodium phophate (pH 7.0), 10× Denhardt's solution, 5× standard saline citrate (SSC), 10% (w/v) dextran sulfate, and denatured herring sperm DNA (100 I~g/ml)] as described. 12 The membranes are analyzed by autoradiography (Kodak X-Omat X-ray film). Acknowledgments We thank Rainer Lanfs for continuous support. The photographic work of C. Schltiter is acknowledged. This work was supported by a grant of the Deutsche Porschungsgemeinschafl to D.M.