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1 The Korean Journal of Microbiology, Vol. 42, No. 4, December 2006, p Copyright 2006, The Microbiological Society of Korea w ell ü l Quorum Sensing autoinducer w x xá v 1 Á 2 Á 3 Á *» w œw» w p y ƒm w» ³ w ü Ÿ ell ewš y ell l ell ü (Catheter- Associate Urinary Tract Infection; CA-UTI) w l Escherichia coli, Pseudomonas aeruginosa š Staphylococcus aureus, w. ³ w quorum sensing mechanism ³ w» ƒ ³ quorum sensing y autoinducer (AIs) w w mrna x y wš, w. ƒ ³ ³ yw 24, 30 w sample. 24 w sample ƒ š reverse transcription polymerase chain reaction (RT-PCR) ww ƒ AIs w ƒ x l y w. E. coli AIs w (ygag ) mrnaƒ x l CFU/ml, CFU/ml P. aeruginosa CFU/ml ùkû. ³ yw ygag mrnaƒ x l CFU/ml, CFU/ml CFU/ml ùkû. w 30 w sample RT-PCR,» l ƒ AIs w mrnaƒ 30 w j x y w. Real-time RT-PCR w AIs w mrna x w ƒ ³ yw AIs w x. ƒ x E. coli ygag mrna x ³ yw š 30 ƒw š, P. aeruginosa yw š 40, P. aeruginosa š 250 š yw š 5 mrna x ƒw. w ³ 4ƒ P. aeruginosa mrnaƒ ƒ x y w. Key words ý autoinducer (AIs), biofilm, catheter-associated urinary tract infection (CA-UTI), quorum sensing (QS), real-time RT-PCR ell(catheter) ( Á ) ( )Áû ( )» ( y Á Ÿ ) ü d w» w š ƒ w, ellƒ e (indwelling urethral catheter). w ell e š Rumbaugh (20) ell (Catheter-Associated Urinary Tract Infection; CA- UTI) 40% w 80% w šwš. ell ell ü l w v w Quorum sensing (QS) l y» w. QS l s w autoinducer (AIs) w s y mw p x *To whom correspondence should be addressed. Tel: , Fax: sslee@kyonggi.ac.kr w, x s w x w (8, 27). QS y AIs p y autoinducer-1 (AI-1), t l N-acyl-homoserine lactone (AHL) y w wš, l y šrk w, l œm w p universal signal autoinducer-2 (AI-2)ƒ š š (16, 21, 23, 24). l w QS mw (virulence)(11) Ÿ(bioluminescence)(10, 17), v x (6), DNA (competence)(14), w (2) w w Ti plasmid (9) w x w š š ù ell ü» x. QS w x v k s, w,» w w w ƒ w w CA-UTI e w ƒ (18). ell ü v x w l QS 277
2 278 Mi-Hye Lee et al. Kor. J. Microbiol mechanism ³ w v š. ell ü QS mechanism ³ w» w y ƒ w e ell v x ³ Escherichia coli, Pseudomonas aeruginosa Staphylococcus aureus, w. z ƒ ³ in vitro ³ yw w z RT-PCR real-time RT-PCR w QS y AIs w w, E. coli ygag (AI-2), P. aeruginosa (AI-1) (AI- 1) š (AI-2) mrnaƒ x l y ƒ ³ AIs w mrna x mw QS w ³ y w. e ell l l ell ü v x ³ Escherichia coli, Pseudomonas aeruginosa Staphylococcus aureus Ÿ ell ew y ell ü w. ell t 1 cm w š(fig. 1), ell ü v w Luria-Bertani (LB, Difco, USA) broth w z 37 o C, 24 w. E. coli E. coli direct agar (Merck, Germany), Pseudomonas cetrimide agar (Scharlau, Spain) Staphylococci mannitol salt agar (Scharlau, Spain) streakw w. w ³ xkw w š API 20 kit (20E, 20NE, 20STAPH, biomeriux, Marcy-I'Etoile, France) z Bergey's mannual of Systematic Bacteriology (3) šw w. PCR w AIs w cloning E. coli, P. aeruginosa chromosomal DNA Fig. 1. A schematic of the foley catheter used in this study. Dotted circle means the isolation region of E. coli, P. aeruginosa, and S. aureus. Silhavy (22) w, DNA Genbank l AIs w E. coli ygag, P. aeruginosa, š DNA» y w ƒƒ primer (Table 1) w PCR ww. PCR icycler thermal cycler (Bio-Rad, USA) w 95 C 5 z o 95 C 1 o, 48 o C(54 o C, 56 C) 1 30 o w, 72 C 1 30z o w z, 72 C 10 o final elongation sw. PCR 1.0% agarose gel» w» mw ƒ PCR Gel Purification Kit (Bioneer, Korea) w w. ƒ PCR pgem-t vector (Promega, USA) w E. coli DH5α x y k z blue/white screening mw clone w. clone v w z wz EcoRI NotI w ƒ target y wš, ƒ v» w. NCBI Blast Search ( v CLUSTAL X w w. ƒ ³ yw ³ sampling E. coli, P. aeruginosa LB w z, OD ¾ w. z ƒ ³ (E. coli ; EC, P. aeruginosa ; PA, Table 1. A list of oligonucleotide primers used in this study Primer specificity GenBank accession no. Gene Direction Primer sequence E. coli NC_00913 ygag forward 5'-cactgactagatgtgcagttc-3' reverse 5'-gtggctaaatgccgttgttag-3' 16S rdna forward 5'-caggtgtagcggtgaaatgc-3' reverse 5'-gggcacaacctccaagtcg-3' BA forward 5'-ggagggaattcaaaatgac-3' reverse 5'-agtggttctcaaagaattcgg-3' 16S rdna forward 5'-ccgaactgagaacaactttatggg-3' reverse 5'-cgtgctacaatggacaatacaaag-3' P. aeruginosa NC_ forward 5'-cgaggacttggtcatgatcg-3' reverse 5'-aaatcgtctgacgacctcacac-3' forward 5'-gggtccggatccaccgaaatc-3' reverse 5'-ccgacggatccccgtcatgaa-3' 16S rdna forward 5'-tggtgttccttcctatatctacgc-3' reverse 5'-gtggttcagcaagttggatgtg-3'
3 Vol. 42, No. 4 l Quorum sensing x 279 ; SA) ³ yw(yw ) LB 0.001%ƒ w 37 o C, 150 rpm w. ƒ AIs w mrnaƒ x l y w» w 0 5 ¾ 30, 5 24 ¾ 2 EC, PA, SA yw l samplingw. w» l 30 ¾» p sampling ww. Samplingw w ³ z w š, phosphate-buffered saline (PBS, ph 7.4) washingw w. z ³ w z -20 o C w RT-PCR realtime RT-PCR w AIs w mrna x y x w. RT-PCR w AIs w mrna x y w -20 C w ³ ³ o lysozyme (Sigma, USA) w 37 C 2 o lysis g RNA w. Lysozyme w lysis lysozyme TE buffer (ph 8.0) 1 mg/mlƒ w, lysis 400 U ribonuclease inhibitor (Takara, Korea) ƒ w. w x RNase free water (BIO BASIC, Canada) w. cdna w RNA 10 µl ƒ AIs w 20 pmol reverse primer z 70 o C 5, ice 5 k z, 5 reaction buffer (250 mm Tris-HCl, 150 mm KCl, 40 mm MgCl 2, ph 8.3) 5 µl, 5 mm deoxyribonucleoside triphosphates (dntps), 4 U ribonuclease inhibitor, 200 U M-MLV reverse transcriptase (Bioneer, Korea) ƒw 20 µl z 37 o C 1 g. cdna w w z ƒƒ primer (Table 1) w PCR ³ AIs w s g. PCR icycler thermal cycler (Bio-Rad, USA) w 95 o C 5 z 95 o C 1, 48 o C (ygag) (54 o C;,, 56 o C; ) 1 30, 72 o C 1 30z w, 72 o C 10 final elongation s w. PCR 1.5% agarose gel» w E. coli ygag, P. aeruginosa, mrna x y w. Real-time RT-PCR w AIs w mrna x (1) RNA cdna w 30 w samplingw ƒ l yw w l ³ ice z 5 mg (w/w) w easy-blue TM Total RNA Extraction kit (Intron, Korea) w RNA w. w RNA DEPC w z spectrophotometer (Gene Quant pro, Amersham bioscience, USA) w 260 nm 280 nm Ÿ d w RNA y w. cdnaw RNA 500 ng target 20 pmol reverse primer z 70 o C 5, ice 5 k z 5 reaction buffer (250 mm Tris-HCl, 150 mm KCl, 40 mm MgCl 2, ph 8.3), 5 mm dntps, 4 U ribonuclease inhibitor, 200 U M-MLV Reverse Transcriptase (Bioneer, Korea) ƒw 20 µl z, 37 o C 1 g. cdna w w z real-time PCR ƒƒ AIs w mrna x w. (2) Real-time RT-PCR mrna x w real-time PCR (MiniOpticon, Bio-Rad, USA). 5 pmol target forward primer 1 µl, reverse primer 1 µl, cdna 1 µl, iq SYBR Green Supermix (Bio-Rad, USA) 10 µl ƒw 20 µl. Real-time PCR w s ƒ sample w 95 C 5 z o 95 o C 30, 54 o C 20, 72 o C 30 40z w, óù z PCR 95 C ƒ wš o 55 C z o 60 C 95æ o ƒ w melting curve d w. Real-time PCR syber green (SYBR) w p s wì x primer-dimerù p s SYBR ww melting curve mw PCR p y w. (3) Real-time RT-PCR data x target mrna x d w» w w 2 - Ct (13) w ygag,, mrna x w. š ell ü v x ³ ell ü x ³ Staphylococcus, Enterococcus, Klebsiella, Enterobacter, Acinetobacter, Edwardsiella, Morganella ³ Pseudomonas aeruginosa, Escherichia coli, Proteuse mirabilis (1, 12, 15, 25). y e ell l v x ³ Escherichia coli, Pseudomonas aeruginosa Staphylococcus aureus, w. E. coli E. coli direct agar (Merck, Germany) MH1 xk rod. API 20E kit w Á yw p y w š, API 20E V6.0 w, MH1 E. coli. Pseudomonas cetrimide agar (Scharlau, Spain) MH2 xk rod. API 20NE kit w Á yw p y w š, API 20NE V6.0 w, MH2 P. aeruginosa. Staphylococci mannitol salt agar (Scharlau, Spain) MH3
4 280 Mi-Hye Lee et al. Kor. J. Microbiol xk cocci. API 20 STAPH kit w Á yw p y w š API 20 STAPH V6.0 w, MH3 ( ). v x l AIs w cloning E. coli, P. aeruginosa ³ chromosomal DNA w š PCR mw s k 530 bp (E. coli ygag), 630 bp (P. aeruginosa ), 710 bp (P. aeruginosa ) š 640 bp ( ) ¼ AIs w DNA r y w. s DNA r w ƒƒ pgem-t l cloning w, z ƒ ƒ v w wz EcoRI NotI w 3 kb vector 530 bp ygag, 630 bp, 710 bp š 640 bp y w ( ). y v DNA» w z NCBI Blast Search v CLUSTAL X w» ƒ ³ AIs w 99% y w (Fig. 2). RT-PCR w AIs w mrna x Fuqua (8) QS mechanism ƒ l j w, s AIs w l y y mw x w š šw š. ell ü v x w quorum sensing q wš ³ w» w» QS y AIs w w mrna x w. 24 w samplingw EC, PA, SA yw w ³ Fig. 2. Multiple alignments of nucleotide sequences of E. coli ygag, and P. aeruginosa and. (A) The target gene sequence of E. coli ygag NC_00913 was compared with that of E. coli ygag M which was isolated in this study. (B) and (C) In addition, the sequences from P. aeruginosa and NC_ were aligned with those from P. aeruginosa M of the isolate strain. Fig. 3. RT-PCR products of ygag (E. coli), (), and (P. aeruginosa) in 24 hr culture samples. The amplified RT-PCR products were analyzed on 2% agarose gel. RT-PCR products of ygag,,, and genes, were amplified using cdnas as template. Lane M: molecular size marker. Lane 1: RT-PCR results of single culture before reaching minimum cell density of initial mrna expression. Lane 2: RT-PCR results of mixed culture before reaching minimum cell density of initial mrna expression. Lane 3: RT-PCR results of single culture at minimum cell density of initial mrna expression. Lane 4: RT-PCR results of mixed culture at minimum cell density of initial mrna expression. (A) E. coli ygag 530 bp, (B) S. aureus 640 bp, (C) P. aeruginosa 630 bp, (D) P. aeruginosa 710 bp.
5 Vol. 42, No. 4 l Quorum sensing x 281 AIs w mrnaƒ x l RT-PCR mw y w (Fig. 3). RT-PCR E. coli ygag, AIs w mrnaƒ x l CFU/ml, CFU/ ml (1.5 ) P. aeruginosa CFU/ml (2 ) y. yw ygag, AIs w mrnaƒ x l CFU/ml, CFU/ml (2 ), CFU/ml (2.5 ) ùkû (Fig. 3). ƒ EC, PA, SA yw w AIs w mrna x ƒ l detection. ƒ EC, PA, SA yw w š» l 5 ¾ (exponential growth) š, z»(stationary phase) 24 ¾, yw j. w y ƒ ell ew m 30 ƒ ew w AIs w mrna x y w» w 30» w, 30 mrnaƒ x y w (Fig. 4). mw AIs w ƒ x l w AIs w mrna x. v x ³ QS w y (1) Real-time RT-PCR w AIs w mrna x 30 ³ yw k w» samplingw ³ target mrna x RT-PCR y w w 30 x y w. mrna x k ƒ» AIs w mrna x d mw QS w E. coli, P. aeruginosa ³ y y w» w real-time RT-PCR w x ww Fig. 5. Real-time PCR s d w (relative quantification) (absolute quantification) x, AIs w mrna x d w» w w.» (reference gene) w PCR w» w» x w x w» GAPDH, β-actin, β 2 -microglobulin, rrna w (4, 19). x» ƒ l 16S rrna w ƒ ³ 16S rrna E. coli 7, P. aeruginosa 4 š 5 copies (26). w 2 (13) w - Ct E. coli ygag, P. aeruginosa š mrna x w. Fig. 4. RT-PCR products of ygag (E. coli), (), and (P. aeruginosa) in 30 days culture samples. The amplified RT- PCR products were analyzed on 2% agarose gel. RT-PCR products of ygag,,, and genes, were amplified using cdnas as template. Lane 1-15: RT-PCR products amplified from the sample of single culture (A; controls without reverse transcriptase, B; E. coli ygag 530 bp, C; 640 bp, D; P. aeruginosa 630 bp, E; P. aeruginosa 710 bp). (2) E. coli ³ yw E. coli ygag mrna x E. coli ygag EC 1 l 9 ¾ mrna x ƒw š, z w 30 ¾ ù yw mrna x 1 l 17 ¾ ƒw w. EC yw k x w» EC w sample ygag mrna x ù 13 z l yw w sample x» w. yw EC» wš[ OD 1.0 ³ 30 mg(w/w), yw ƒ ³ OD 1.0 ƒ ³ 10 mg 30 mg ] 19 š 30 mrna x ƒ 30 ¾ yw k x w (Fig. 5A). (3) P. aeruginosa ³ yw P. aeruginosa mrna x P. aeruginosa PA 1 l 13 ¾ mrna x ƒw š, z w 30 ¾ ù yw mrna x 1 l 17 ¾ ƒ w w. PA yw k x w yw PA» wš» l PA yw w sample mrna x ƒ» w. 7 š 40 mrna x ƒ 30 ¾ yw k x w. w
6 282 Mi-Hye Lee et al. Kor. J. Microbiol mrna expression level from single culture which was inoculated with or or and mixed culture which was inoculated with, and during 30 days. Results of real-time quantitative RT-PCR of equal amounts of total RNA (500 ng) isolated from each culture sample. The relative mrna input is calculated from the threshold cycle (Ct) values obtained from real-time quantitative RT-PCR based on the modified comparative 2 method (13). All data presented are an average of three independent experiments with the standard deviation, indicated by the error bar and plotted on a logarithmic scale. (A) Transcript expression level of (B) transcript expression level of, (C) transcript expression level of, (D) transcript expression level of (E) Pure culture of each single strain transcript expression level of,, and, (F) pure culture of mixed species transcript expression level of,, and Fig. 5. E. coli E. coli P. aeruginosa, P. aeruginosa - Ct E. coli ygag, P. aeruginosa P. aeruginosa. ygag ygag 단일배양 및 세 균주의 혼합배양에서 의 mrna 발현량 분석 의 는 SA 배양과 혼합배양한 sample에서 모두 1 일부터 9일까지 mrna 발현량이 증가하였으며 11일부터 30일까 지 그 양이 감소되었으나 발현은 지속되었다. 또한 혼합배양시 SA 배양보다 초기 접종량이 더 적었음에도 불구하고 SA 배양과 혼합배양에서 mrna의 발현량을 비교하면 전반적으로 혼합배양 한 sample에서 mrna가 더 많이 발현되었으며 11일에서 최고 약 5배 이상의 mrna 발현량 증가를 보였다. 또한 SA 배양시 의 mrna 발현 양상을 다른 target 유전자인, 와 의 mrna 발현량과 비교했을 때, 이들에 비해 지속적으로 mrna 발현이 크게 증가되거나 감소되지 않았다(Fig. 5D). 기부터 PA배양보다 혼합배양한 sample에서 mrna가 더 많이 발현되기 시작하여 17일에는 최고 약 250배 이상의 mrna 발 현량 증가를 보였으며 30일까지 혼합배양 상태에서 발현량이 많 은 경향이 지속되었다(Fig. 5B, C). (4) ygag (5) 각 세 균주의 단일 배양과 세 균주의 혼합배양에서 AIs 합 성 유전자의 mrna 발현량 분석 Fig. 5A, B와 C에서 확인한 의 와 의 와 의 mrna 발현양상은 Eberhard (7)와 Nealson (17)의 연구결과와 유사하였다. 그들의 연구에 따르면 AIs의 발현은 낮 은 박테리아 농도에서는 고농도에 도달하기까지 지속적으로 발 현량이 증가되고 일정이상 고농도에 이르면 저해를 받아 발현량 이 줄어든다고 보고 하였다. EC, PA 및 SA 배양한 sample에서,, 와 의 mrna 발현량을 비교했을 때 의 와 가가 장 많은 발현량을 보였다. 초기부터 와 가 가장 많은 발 현량을 보였으며, 의 mrna 발현은 15일까지 가장 많은 발 현량을 보였으며 17일에는 보다 의 mrna 발현이 더 많 아져서 30일까지 가장 많은 발현량을 보였다(Fig. 5E). 혼합배양 상태에서 각 균주의 분포를 16S rrna copy수로 보 정 후 상대정량을 통하여 비교하였을 때 30일 동안 약 40%로 와 분포 비율이 높았으며 는 20% E. coli ygag P. aeruginosa ygag P. aeruginosa E. coli P. aeruginosa
7 Vol. 42, No. 4 l Quorum sensing x 283 ùkû (Fig. 6). yw w sample AIs w mrna x w E. coli P. aeruginosaƒ w w wš,» l P. aeruginosa ƒ ƒ x, E. coli ygagƒ ƒ x š mrna x 30 j (Fig. 5F). Real-time RT-PCR x, yw³ AIs w mrna x w w ùkû. EC, PA SA yw AIs w mrna x y w. 3 ³ 4 mrna x w yw sample P. aeruginosa x E. coli ygag w š 10,000 mrna x. w ³ ¼ AIs w mrna x ƒw» l x ƒ ƒ ù w w (Fig. 5). w x QS mw ƒ ³ y w ƒ. Miller (16) QS y l ƒ w universal signal Autoinducer- 2 (AI-2)ƒ w, furanosyl borate diester x (5, 21). AI-2 w ƒ l Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Helicobacter pylori, Haemophilus influenzae, Bacillus subtilis, Borrelia burgdorferi, Neisseria meningitidis, Yersinia pestis, Camphylobacter jejuni, Vibrio cholerae, Streptococcus pneumoniae š š (16). AI-2 l w» l globalw y l y w wš ù, AI-2» w y x (5). w š l l AIs w x w ö e ƒ w, x w l E. coli ƒ AI-2 w š. ù P. aeruginosa AI-2 w AIs p N-acyl-homoserine lactone (AHL) w š (28). ƒ ³ ³ yw AIs w mrna x ƒ QS w w AIs l y ƒ w l AIs w wì ƒ ³ AIs w mrna x ƒ. p, yw P. aeruginosa x ƒ w AIs AHL E. coli ƒ w AI-2 w ƒ AIs w. ell v x w l E. coli, P. aeruginosa quorum sensing» w AIs w ygag,, x y in vitro y w, z v x w mw CA- UTI w quorum sensing» ƒ w»» w ww. š x Fig. 6. Bacterial population rate of the mixed culture of three species at 37 o C in LB broth with shaking for 30 days. Bacterial population rate of E. coli (þ), P. aeruginosa ( G ), and ( G ) were measured by real-time RT-PCR. 1.,, š, ¼ y,, k w s ell v x w» z. w»wz 46, Bainton, N.J., P. Stead, S.R. Chhabra, B.W. Bycroft, G.P. Salmond, G.S. Stewart, and P. Williams N-(3-oxo-hexanoyl)-Lhomoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora. Biochem. J. 288, Brenner, D.J., N.R. Krieg, and J.T. Staley Bergey's manual of systematic bacteriology, Second Edition 4. Bustin, S.A Absolute quantification of mrna using realtime reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, Chen, X., S. Schauder, N. Potier, A. Van Dorsselaer, I. Pelczer, B.L. Bassler, and F.M. Hughson Structural identification of a bacterial quorum sensing signal containing boron. Nature 415, Davies, D.G., M.R. Parsek, J.P. Pearson, B.H. Iglewski, J.W. Costetton, and E.P. Greenberg The involvement of cell-tocell signals in the development of a bacterial biofilm. Science 280,
8 284 Mi-Hye Lee et al. Kor. J. Microbiol 7. Eberhard, A Inhibition and activation of bacterial luciferase synthesis. J. Bacteriol. 109, Fuqua, W.C. and S.C. Winans A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite. J. Bacteriol. 176, Fuqua, W.C., S.C. Winans, and E.P. Greenberg Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176, Hastings, J.W. and K.H. Nealson. Bacterial bioluminescence Annu. Rev. Microbiol. 31, De Kievit, T.R. and B.H. Iglewski Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 68, Kumon, H., H. Hashimoto, M. Nishimura, K. Monden, and N. Ono Catheter-associated urinary tract infections: impact of catheter materials on their management. Int. J. Urol. 17, Livark, K.J. and T.D. Schmittgen Analysis of relative gene expression data using real-time quantitative PCR and the 2 - Ct method. Methods 25, Magnuson, R., J. Solomin, and A.D. Grossman Biochemical and genetic characterization of a competence pheromone from B. subtilis. Cell 77, Merle, V., J.M. Germain, H. Bugel, M. Nouvellon, J.F. Lemeland, and P. Czernichow Nosocominal urinary tract infections in urologic patients: assessment of a prospective surveillance program including 10,000 patients. Eur. Urol. 41, Miller, M.B. and B.L. Bassler Quorum sensing in bacteria. Ann. Rev. Microbiol. 55, Nealson, K.H. and J.W. Hastings Bacterial bioluminescence: its control and ecological significance. Microbiol. Rev. 43, Parsek, M.R. and E.P. Greenberg Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in association with higher organisms. Proc. Natl. Acad. Sci. USA 97, Pfaffl, M.W A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e Rumbaugh, K.P., J.A. Griswold, and A.N. Hamood The role of quorum sensing in the in vivo virulence of Pseudomonas aeruginosa. Microbes Infect. 2, Schaefer, A.L., D.L. Val, B.L. Hanzelka, J.E. Crona, and E.P. Greenberg Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein. Proc. Natl. Acad. Sci. USA 93, Silhavy, T.J., M.L. Berman, and L.W. Enquist Experiments with gene fusions. Cold Spring Harbor Laboratory, New York, USA. 23. Surette, M.G. and B.L. Bassler Quorum sensing in Escherichia coli and Salmonella typhimurium. Proc. Natl Acad. Sci. USA 95, Surette, M.G., M.B. Miller, and B.L. Bassler Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production. Proc. Natl. Acad. Sci. USA 96, Tambyah, P.A Catheter-associated urinary tract infections: diagnosis and prophylaxix. Int. J. Antimicrob. Agents. 24, S Tourova, T.P Copy nember of ribosomal operon in prokaryotes and its effect on phylogenetic analyses. Microbiol. 72, Whitehead, N.A., A.M. Barnard, H. Slater, N.J. Simpson, and G.P. Salmond Quorum sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 25, Williams, S.C., E.K. Patter, N.L. Carty, J.A. Griswold, A.N. Hamood, and K.P. Rumbaugh Pseudomonas aeruginosa autoinducer enters and fuctions in mammalian cells. J. Bacteriol. 186, (Received November 9, 2006/Accepted December 22, 2006) ABSTRACT : The Analysis of Expression of Autoinducer Synthesis Genes Involved in Quorum Sensing among Catheter Associated Bacteria Mi-Hye Lee, Pil-Soo Seo 1, Ji-youl Lee 2, Kyong-Ran Peck 3, and Sang-Seob Lee* (Department of Biological Engineering, Kyonggi University, Suwon , Korea and 1 Department of Korea Biological Resourse Center, Kyonggi University, Suwon , Korea 2 Department of Urology, School of Medicine, The Catholic University,Bucheon , Korea, 3 Division of Infectious Diseases, Sungkyunkwan University School of Medicine, Seoul , Korea) The most biofilm forming bacteria in catheter, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were isolated and identified from a patient's catheter occuring catheter-associated urinary tract infection (CA-UTI). We examined mrna expression and its quantification of AIs synthetic genes encoding signal substance of quorum sensing from each bacterial species in order to elucidated quorum sensing mechanism. Both pure cultures for each bacterial strains and a mixed cultures with three were grown for 24 hr and 30 days. Initial densities to be able to detect mrna expression on single strains culture were shown at CFU/ml, CFU/ml of E. coli for ygag and for, and at CFU/ml of P. aeruginosa for and. Also, in mixed culture of three, initial cell densities of mrna expression were appear to at CFU/ml, CFU/ml of E. coli for ygag and for, and at CFU/ml of P. aeruginosa for and
9 Vol. 42, No. 4 l Quorum sensing x 285. Each AIs synthetic gene was expressed in initial cell density and the mrna expression of the genes were detected continously during 30 days. And then, the quantification of mrna expression level of ygag,,, and which were related AIs synthesis was done each time point by real-time RT-PCR. Interestingly, the mrna levels of ygag,,, and from the mixed culture was higher than those from each single strain culture. In the case of E. coli ygag, the amount of transcript from the mixed culture was at least 30 times for that from single culture. In the case of P. aeruginosa and, the amount of transcript from the mixed culture was at least 40 times and 250 times for that from single strain culture. In the case of, the amount of transcript from the mixed culture was at least 5 times for that from single strain culture. And specially, the mrna expression of and of P. aeruginosa showed the highest efficency among four AIs synthetic genes.
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