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Triton X-100-induced lipoteichoic acid release is correlated with the methicillin resistance in Staphylococcus aureus
FEMS Microbiology Letters 182 (2000) 77^79
www.fems-microbiology.org
Triton X-100-induced lipoteichoic acid release is correlated with the methicillin resistance in Staphylococcus aureus Kouji Ohta, Hitoshi Komatsuzawa *, Motoyuki Sugai, Hidekazu Suginaka Department of Microbiology, Hiroshima University School of Dentistry, Kasumi 1-2-3, Minami-ku, Hiroshima City, Hiroshima 734-8553, Japan Received 15 July 1999; accepted 10 October 1999
Abstract We previously reported that Triton X-100 (TRX) reduced methicillin resistance in Staphylococcus aureus, although the degree of reduction varied among strains. One of the biological effects of TRX on S. aureus cells was enhancement of lipoteichoic acid (LTA) release. We investigated the correlation between the amount of LTA released and the degree of reduction in methicillin resistance induced by TRX. The strains showing the greatest reduction of methicillin resistance released the largest amount of LTA, compared to those showing slight or moderate reduction. A mutant whose resistance was not affected by TRX did not increase its release of LTA. These findings suggest that LTA release is associated with a reduction in methicillin resistance in the presence of TRX. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Methicillin-resistant Staphylococcus aureus; Triton X-100; Lipoteichoic acid
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) show an intrinsic resistance to L-lactam antibiotics due to a 78-kDa penicillin binding protein (PBP) 2P(A), encoded by the mecA gene [2,5,12,15]. We reported that Triton X-100 (TRX) enhanced the in vitro susceptibility of MRSA to oxacillin [8,9]. TRX did not a¡ect the amount of PBP2P or the binding a¤nity for penicillin [9], so the sensitizing e¡ect is considered to be associated with other factor(s). During our characterization of this sensitizing e¡ect, we found that TRX stimulated lipoteichoic acid (LTA) release [9]. This ¢nding was also reported by Raychaudhuri and Chatterjee [11]. LTA is an amphiphilic molecule and anchored to the bacterial cell membrane [4]. It is considered to be responsible for the regulation of peptidoglycan hydrolases in Gram-positive bacteria [3,6]. It has been speculated that L-lactam antibiotics liberate the acylated LTA from bacterial cells, thereby activating the peptidoglycan hydrolases that ultimately cause the cells to undergo bacteriolysis [13]. However, the pre-
cise role of LTA still remains unclear. In this study, we measured the amount of LTA released with or without TRX in di¡erent strains that showed a range of resistance to oxacillin in the presence of TRX. 2. Materials and methods 2.1. Bacterial strains and growth conditions Eleven MRSA strains were used in this study (Table 1). COL was kindly provided by Alexander Tomasz. HK9445 and HK9023 were derived from KSA8, which is a clinical isolate with a high resistance to methicillin. HK9445, which has a low resistance to methicillin, was originally isolated from KSA8 by Tn551 insertion into the fmtA gene [7]. HK9023 was constructed by the transduction of Tn551 from BB742 (femA : :Tn551) [1,10] to KSA8. S. aureus strains were grown in trypticase soy broth (TSB) at 37³C. When necessary, erythromycin (30 Wg ml31 ) was added to the medium. The concentration of TRX in this study was 0.02% (v/v), which was the critical micellar concentration. 2.2. Isolation for TRX-resistant MRSA
* Corresponding author. Tel. : +81 (82) 257 5636; Fax: +81 (82) 257 5639; E-mail : [email protected]
MRSA strain KSA8 was used for isolating the sponta-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 5 6 4 - 9
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K. Ohta et al. / FEMS Microbiology Letters 182 (2000) 77^79
neous mutant. An overnight culture of KSA8 was plated on trypticase soy agar (TSA) containing 64 Wg ml31 oxacillin and 0.02% TRX, on which the majority of KSA8 cells could not grow. Colonies which grew on the plate were re-plated on the same plate, and this experiment was repeated twice. Fifty colonies were then selected, and the oxacillin susceptibility in the presence or absence of 0.02% TRX was measured. Among them, one strain, designated HK9554, showed the same susceptibility to oxacillin in the presence or absence of TRX. We checked the stability of HK9554 by measuring the minimum inhibitory concentration (MIC) of oxacillin after several passages, and found that the phenotype of HK9554 was stable. 2.3. MIC determination The MIC of oxacillin in the presence or absence of TRX was determined by the microdilution method as described elsewhere [9]. 2.4. LTA release LTA release was measured by the method described elsewhere [9]. Brie£y, a small portion of overnight culture was inoculated into fresh TSB at 37³C with shaking, and grown to OD660 0.3. Then, [2-3 H]glycerol (37 kBq ml31 ) was added and incubated for 2 h at 37³C with shaking. The cells were then collected, and washed with PBS three times. The cells were adjusted to OD660 0.15 with TSB containing 10 mM glycerol in the presence or absence of 0.02% TRX. Culture samples (1 ml) were taken at 30-min intervals for 3 h. The culture was centrifuged, then the supernatant was treated with chloroform-methanol (2:1 [v/v]). The water phase of each sample was tested for radioactivity with a scintillation counter.
2.5. Cell viability To check cell viability in the presence of TRX, we used the pour-plate method, counting the colony forming units (CFU) as the number of viable cells. A small portion of overnight culture was inoculated into fresh TSB and grown to OD660 0.2 at 37³C with shaking. Then, 0.02% TRX was added, and the sample was reincubated at 37³C with shaking. At 1-h intervals, OD660 was measured and 100-Wl aliquots were plated on TSA plates. After 1 day incubation, CFU on each plate was counted. 3. Results and discussion Fig. 1 shows that the amount of LTA released in COL was enhanced by the addition of 0.02% TRX, while that in KSAF1 was not. We investigated the cell turbidity and cell viabilities of COL and KSAF1 in the presence of TRX, and found that these strains were not lysed and killed by 0.02% TRX (data not shown). Therefore, we concluded that the enhancement of LTA release was not due to cell lysis. Both strains showed a high resistance to methicillin in the absence of TRX, but their MICs of methicillin in the presence of TRX di¡ered signi¢cantly. The COL strain showed a dramatic decrease in methicillin resistance and increase in LTA release in the presence of TRX, while KSAF1, which was resistant to the TRX sensitizing e¡ect, did not increase its LTA release (Fig. 1). We further investigated the relationship between LTA release and decrease in methicillin resistance by co-incubating TRX and six clinical MRSA strains. As the amount of LTA released with TRX increased, the reduction in methicillin resistance became more signi¢cant (Table 1). In the presence of TRX, KSA1, KSA4, and MR6, which showed drastic re-
Table 1 MIC ratio and LTA release ratio of various MRSA strains Strain
Relevant properties
KSA1 KSA4 KSA7 KSA8 KSA9 KSAF1 MR6 COL HK9445 HK9023 HK9554
clinical isolate, MCr clinical isolate, Mcr clinical isolate, Mcr clinical isolate, Mcr clinical isolate, Mcr clinical isolate, Mcr clinical isolate, Mcr laboratory strain, Mcr mutagenized fmtA: :Tn551 in KSA8 mutagenized femA : :Tn551 in KSA8 Triton X-100-resistant mutant, isolated from KSA8
MIC of oxacillin (Wg ml31 ) None
Triton X-100
64 512 512 512 128 1024 32 512 16 0.06 1024
0.25 0.5 8 16 0.06 1024 0.25 1 0.5 N.D. 1024
MIC ratioa (without Triton/with Triton)
LTA release ratiob (with Triton/ without Triton)
256 1024 64 32 2048 1 128 512 32 ^ 1
4.3 3.1 1.4 1.8 4.2 1.4 2.7 4.0 4.4 4.3 1.0
N.D., not determined due to inhibition of growth in the presence of 0.02% Triton X-100. a MIC ratio (MIC of oxacillin in the absence of Triton X-100/MIC in the presence of Triton X-100). b LTA release ratio (amount of LTA released in the presence of Triton X-100/amount of LTA released in the absence of Triton X-100).
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79
Acknowledgements This work was supported by a grant-in-aid for Encouragement of Young Scientists (Grant 10770119) from the Ministry of Education, Science, Sports and Culture of Japan. References
Fig. 1. LTA release from S. aureus cells. The release of [2-3 H]glycerollabeled LTA from S. aureus in the absence (open symbols) or presence (solid symbols) of Triton X-100 was measured as described in Section 2. Triton X-100 (¢nal 0.02%) was added at 0 min. Symbols : a and b, COL; O and R KSAF1.
ductions in methicillin resistance, released large amounts (2.7^4 times) of LTA. The amount of LTA released by KSA7 and KSA8, which showed slight reductions in resistance, increased slightly in the presence of TRX. Two Tn551 insertional mutants, HK9445 (fmtA: :Tn551) and HK9023 (femA: :Tn551), showed drastic reductions in methicillin resistance compared to the wild-type. These strains released a large amount of LTA in the presence of TRX, compared to the parent, although the MIC ratio was the same as that of the parent due to a decreased MIC in the absence of TRX (Table 1). The TRX-resistant mutant HK9554, which showed the same MIC with or without TRX, did not increase its LTA release. These ¢ndings suggest that the e¡ect of TRX on methicillin susceptibility is associated with TRX-induced LTA release. Our previous study showed that various types of Triton X which reduced methicillin resistance also promoted LTA release [14]. Some types of Triton X exerting a sensitizing e¡ect promoted LTA release, while other types of Triton X showing no e¡ect did not, which also indicates a correlation between LTA release and Triton X-sensitizing e¡ect. LTA is considered to be a regulator of peptidoglycan hydrolases, but the precise function of LTA still remains unclear. Therefore, how LTA release induced by TRX a¡ects methicillin resistance must be clari¢ed in further studies. It is noteworthy that the amount of LTA released by TRX di¡ers among strains. The chemical structure of released LTA has been identi¢ed as mostly acylated LTA, and some deacylated LTA [9]. Deacylated LTA is mainly released during growth in the absence of TRX. Acylated LTA is anchored to the membrane by the glycolipid region, and so is a cell membrane component. Therefore, acylated LTA may be released from the membrane in exchange for incorporation of TRX. The di¡ering responses to TRX among strains suggest that their membrane stabilities or compositions are di¡erent.
[1] Berger-Ba«chi, B., Barberis-Maino, L., Stra«ssle, A. and Kayser, F.H. (1989) Fem A, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization. Mol. Gen. Genet. 219, 263^269. [2] Brown, D.F. and Reynolds, P.E. (1980) Intrinsic resistance to L-lactam antibiotics in Staphylococcus aureus. FEBS Lett. 122, 275^278. [3] Cleveland, R.F., Wicken, A.J., Daneo-Moore, L. and Shockman, G.D. (1976) Inhibition of wall autolysis in Streptococcus faecalis by lipoteichoic acid and lipids. J. Bacteriol. 126, 192^197. [4] Coley, J., Duckworth, M. and Baddiley, J. (1972) The occurrence of lipoteichoic acids in the membranes of Gram-positive bacteria. J. Gen. Microbiol. 73, 587^591. [5] Fontana, R. (1985) Penicillin-binding proteins and the intrinsic resistance to L-lactams in Gram-positive cocci. J. Antimicrob. Chemother. 16, 412^416. [6] Ho«ltje, J.V. and Tomasz, A. (1975) Lipoteichoic acid: a speci¢c inhibitor of autolysin activity in Pneumococcus. Proc. Natl. Acad. Sci. USA 72, 1690^1694. [7] Komatsuzawa, H., Sugai, M., Ohta, K., Fujiwara, T., Nakashima, S., Suzuki, J., Lee, C.Y. and Suginaka, H. (1997) Cloning and characterization of the fmt gene which a¡ects the methicillin resistance level and autolysis in the presence of Triton X-100 in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 41, 2355^ 2361. [8] Komatsuzawa, H., Sugai, M., Shirai, C., Suzuki, J., Hiramatsu, K. and Suginaka, H. (1995) Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin. FEMS Microbiol. Lett. 134, 209^212. [9] Komatsuzawa, H., Suzuki, J., Sugai, M., Miyake, Y. and Suginaka, H. (1994) The e¡ect of Triton X-100 on the in-vitro susceptibility of methicillin-resistant Staphylococcus aureus to oxacillin. J. Antimicrob. Chemother. 34, 885^897. [10] Maidhof, H., Reinicke, B., Blu«mel, P., Berger-Ba«chi, B. and Labischinski, H. (1991) femA, which encodes a factor essential for expression of methicillin resistance, a¡ects glycine content of peptidoglycan in methicillin-resistant and methicillin-suscetible Staphylococcus aureus strains. J. Bacteriol. 173, 3507^3513. [11] Raychaudhuri, D. and Chatterjee, A.N. (1985) Use of resistant mutants to study the interaction of Triton X-100 with Staphylococcus aureus. J. Bacteriol. 164, 1337^1349. [12] Reynolds, P.E. and Fuller, C. (1986) Methicillin-resistant strains of Staphylococcus aureus; presence of an identical additional penicillinbinding protein in all strains examined. FEMS Microbiol. Lett. 33, 251^254. [13] Suginaka, H., Shimatani, M., Ogawa, M. and Kotani, S. (1979) Prevention of penicillin-induced lysis of Staphylococcus aureus by cellular lipoteichoic acid. J. Antibiot. 32, 73^77. [14] Suzuki, J., Komatsuzawa, H., Sugai, M., Ohta, K., Kozai, K., Nagasaka, N. and Suginaka, H. (1997) E¡ect of various types of Triton X on the susceptibilities of methicillin-resistant staphylococci to oxacillin. FEMS Microbiol. Lett. 153, 327^331. [15] Ubukata, K., Nonoguchi, R., Matsuhashi, M. and Konno, M. (1989) Expression and inducibility in Staphylococcus aureus of the mecA gene which encodes a methicillin-resistant S. aureus-speci¢c penicillin-binding protein. J. Bacteriol. 171, 2882^2885.
FEMSLE 9141 16-12-99
www.fems-microbiology.org
Triton X-100-induced lipoteichoic acid release is correlated with the methicillin resistance in Staphylococcus aureus Kouji Ohta, Hitoshi Komatsuzawa *, Motoyuki Sugai, Hidekazu Suginaka Department of Microbiology, Hiroshima University School of Dentistry, Kasumi 1-2-3, Minami-ku, Hiroshima City, Hiroshima 734-8553, Japan Received 15 July 1999; accepted 10 October 1999
Abstract We previously reported that Triton X-100 (TRX) reduced methicillin resistance in Staphylococcus aureus, although the degree of reduction varied among strains. One of the biological effects of TRX on S. aureus cells was enhancement of lipoteichoic acid (LTA) release. We investigated the correlation between the amount of LTA released and the degree of reduction in methicillin resistance induced by TRX. The strains showing the greatest reduction of methicillin resistance released the largest amount of LTA, compared to those showing slight or moderate reduction. A mutant whose resistance was not affected by TRX did not increase its release of LTA. These findings suggest that LTA release is associated with a reduction in methicillin resistance in the presence of TRX. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Methicillin-resistant Staphylococcus aureus; Triton X-100; Lipoteichoic acid
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) show an intrinsic resistance to L-lactam antibiotics due to a 78-kDa penicillin binding protein (PBP) 2P(A), encoded by the mecA gene [2,5,12,15]. We reported that Triton X-100 (TRX) enhanced the in vitro susceptibility of MRSA to oxacillin [8,9]. TRX did not a¡ect the amount of PBP2P or the binding a¤nity for penicillin [9], so the sensitizing e¡ect is considered to be associated with other factor(s). During our characterization of this sensitizing e¡ect, we found that TRX stimulated lipoteichoic acid (LTA) release [9]. This ¢nding was also reported by Raychaudhuri and Chatterjee [11]. LTA is an amphiphilic molecule and anchored to the bacterial cell membrane [4]. It is considered to be responsible for the regulation of peptidoglycan hydrolases in Gram-positive bacteria [3,6]. It has been speculated that L-lactam antibiotics liberate the acylated LTA from bacterial cells, thereby activating the peptidoglycan hydrolases that ultimately cause the cells to undergo bacteriolysis [13]. However, the pre-
cise role of LTA still remains unclear. In this study, we measured the amount of LTA released with or without TRX in di¡erent strains that showed a range of resistance to oxacillin in the presence of TRX. 2. Materials and methods 2.1. Bacterial strains and growth conditions Eleven MRSA strains were used in this study (Table 1). COL was kindly provided by Alexander Tomasz. HK9445 and HK9023 were derived from KSA8, which is a clinical isolate with a high resistance to methicillin. HK9445, which has a low resistance to methicillin, was originally isolated from KSA8 by Tn551 insertion into the fmtA gene [7]. HK9023 was constructed by the transduction of Tn551 from BB742 (femA : :Tn551) [1,10] to KSA8. S. aureus strains were grown in trypticase soy broth (TSB) at 37³C. When necessary, erythromycin (30 Wg ml31 ) was added to the medium. The concentration of TRX in this study was 0.02% (v/v), which was the critical micellar concentration. 2.2. Isolation for TRX-resistant MRSA
* Corresponding author. Tel. : +81 (82) 257 5636; Fax: +81 (82) 257 5639; E-mail : [email protected]
MRSA strain KSA8 was used for isolating the sponta-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 5 6 4 - 9
FEMSLE 9141 16-12-99
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K. Ohta et al. / FEMS Microbiology Letters 182 (2000) 77^79
neous mutant. An overnight culture of KSA8 was plated on trypticase soy agar (TSA) containing 64 Wg ml31 oxacillin and 0.02% TRX, on which the majority of KSA8 cells could not grow. Colonies which grew on the plate were re-plated on the same plate, and this experiment was repeated twice. Fifty colonies were then selected, and the oxacillin susceptibility in the presence or absence of 0.02% TRX was measured. Among them, one strain, designated HK9554, showed the same susceptibility to oxacillin in the presence or absence of TRX. We checked the stability of HK9554 by measuring the minimum inhibitory concentration (MIC) of oxacillin after several passages, and found that the phenotype of HK9554 was stable. 2.3. MIC determination The MIC of oxacillin in the presence or absence of TRX was determined by the microdilution method as described elsewhere [9]. 2.4. LTA release LTA release was measured by the method described elsewhere [9]. Brie£y, a small portion of overnight culture was inoculated into fresh TSB at 37³C with shaking, and grown to OD660 0.3. Then, [2-3 H]glycerol (37 kBq ml31 ) was added and incubated for 2 h at 37³C with shaking. The cells were then collected, and washed with PBS three times. The cells were adjusted to OD660 0.15 with TSB containing 10 mM glycerol in the presence or absence of 0.02% TRX. Culture samples (1 ml) were taken at 30-min intervals for 3 h. The culture was centrifuged, then the supernatant was treated with chloroform-methanol (2:1 [v/v]). The water phase of each sample was tested for radioactivity with a scintillation counter.
2.5. Cell viability To check cell viability in the presence of TRX, we used the pour-plate method, counting the colony forming units (CFU) as the number of viable cells. A small portion of overnight culture was inoculated into fresh TSB and grown to OD660 0.2 at 37³C with shaking. Then, 0.02% TRX was added, and the sample was reincubated at 37³C with shaking. At 1-h intervals, OD660 was measured and 100-Wl aliquots were plated on TSA plates. After 1 day incubation, CFU on each plate was counted. 3. Results and discussion Fig. 1 shows that the amount of LTA released in COL was enhanced by the addition of 0.02% TRX, while that in KSAF1 was not. We investigated the cell turbidity and cell viabilities of COL and KSAF1 in the presence of TRX, and found that these strains were not lysed and killed by 0.02% TRX (data not shown). Therefore, we concluded that the enhancement of LTA release was not due to cell lysis. Both strains showed a high resistance to methicillin in the absence of TRX, but their MICs of methicillin in the presence of TRX di¡ered signi¢cantly. The COL strain showed a dramatic decrease in methicillin resistance and increase in LTA release in the presence of TRX, while KSAF1, which was resistant to the TRX sensitizing e¡ect, did not increase its LTA release (Fig. 1). We further investigated the relationship between LTA release and decrease in methicillin resistance by co-incubating TRX and six clinical MRSA strains. As the amount of LTA released with TRX increased, the reduction in methicillin resistance became more signi¢cant (Table 1). In the presence of TRX, KSA1, KSA4, and MR6, which showed drastic re-
Table 1 MIC ratio and LTA release ratio of various MRSA strains Strain
Relevant properties
KSA1 KSA4 KSA7 KSA8 KSA9 KSAF1 MR6 COL HK9445 HK9023 HK9554
clinical isolate, MCr clinical isolate, Mcr clinical isolate, Mcr clinical isolate, Mcr clinical isolate, Mcr clinical isolate, Mcr clinical isolate, Mcr laboratory strain, Mcr mutagenized fmtA: :Tn551 in KSA8 mutagenized femA : :Tn551 in KSA8 Triton X-100-resistant mutant, isolated from KSA8
MIC of oxacillin (Wg ml31 ) None
Triton X-100
64 512 512 512 128 1024 32 512 16 0.06 1024
0.25 0.5 8 16 0.06 1024 0.25 1 0.5 N.D. 1024
MIC ratioa (without Triton/with Triton)
LTA release ratiob (with Triton/ without Triton)
256 1024 64 32 2048 1 128 512 32 ^ 1
4.3 3.1 1.4 1.8 4.2 1.4 2.7 4.0 4.4 4.3 1.0
N.D., not determined due to inhibition of growth in the presence of 0.02% Triton X-100. a MIC ratio (MIC of oxacillin in the absence of Triton X-100/MIC in the presence of Triton X-100). b LTA release ratio (amount of LTA released in the presence of Triton X-100/amount of LTA released in the absence of Triton X-100).
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79
Acknowledgements This work was supported by a grant-in-aid for Encouragement of Young Scientists (Grant 10770119) from the Ministry of Education, Science, Sports and Culture of Japan. References
Fig. 1. LTA release from S. aureus cells. The release of [2-3 H]glycerollabeled LTA from S. aureus in the absence (open symbols) or presence (solid symbols) of Triton X-100 was measured as described in Section 2. Triton X-100 (¢nal 0.02%) was added at 0 min. Symbols : a and b, COL; O and R KSAF1.
ductions in methicillin resistance, released large amounts (2.7^4 times) of LTA. The amount of LTA released by KSA7 and KSA8, which showed slight reductions in resistance, increased slightly in the presence of TRX. Two Tn551 insertional mutants, HK9445 (fmtA: :Tn551) and HK9023 (femA: :Tn551), showed drastic reductions in methicillin resistance compared to the wild-type. These strains released a large amount of LTA in the presence of TRX, compared to the parent, although the MIC ratio was the same as that of the parent due to a decreased MIC in the absence of TRX (Table 1). The TRX-resistant mutant HK9554, which showed the same MIC with or without TRX, did not increase its LTA release. These ¢ndings suggest that the e¡ect of TRX on methicillin susceptibility is associated with TRX-induced LTA release. Our previous study showed that various types of Triton X which reduced methicillin resistance also promoted LTA release [14]. Some types of Triton X exerting a sensitizing e¡ect promoted LTA release, while other types of Triton X showing no e¡ect did not, which also indicates a correlation between LTA release and Triton X-sensitizing e¡ect. LTA is considered to be a regulator of peptidoglycan hydrolases, but the precise function of LTA still remains unclear. Therefore, how LTA release induced by TRX a¡ects methicillin resistance must be clari¢ed in further studies. It is noteworthy that the amount of LTA released by TRX di¡ers among strains. The chemical structure of released LTA has been identi¢ed as mostly acylated LTA, and some deacylated LTA [9]. Deacylated LTA is mainly released during growth in the absence of TRX. Acylated LTA is anchored to the membrane by the glycolipid region, and so is a cell membrane component. Therefore, acylated LTA may be released from the membrane in exchange for incorporation of TRX. The di¡ering responses to TRX among strains suggest that their membrane stabilities or compositions are di¡erent.
[1] Berger-Ba«chi, B., Barberis-Maino, L., Stra«ssle, A. and Kayser, F.H. (1989) Fem A, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization. Mol. Gen. Genet. 219, 263^269. [2] Brown, D.F. and Reynolds, P.E. (1980) Intrinsic resistance to L-lactam antibiotics in Staphylococcus aureus. FEBS Lett. 122, 275^278. [3] Cleveland, R.F., Wicken, A.J., Daneo-Moore, L. and Shockman, G.D. (1976) Inhibition of wall autolysis in Streptococcus faecalis by lipoteichoic acid and lipids. J. Bacteriol. 126, 192^197. [4] Coley, J., Duckworth, M. and Baddiley, J. (1972) The occurrence of lipoteichoic acids in the membranes of Gram-positive bacteria. J. Gen. Microbiol. 73, 587^591. [5] Fontana, R. (1985) Penicillin-binding proteins and the intrinsic resistance to L-lactams in Gram-positive cocci. J. Antimicrob. Chemother. 16, 412^416. [6] Ho«ltje, J.V. and Tomasz, A. (1975) Lipoteichoic acid: a speci¢c inhibitor of autolysin activity in Pneumococcus. Proc. Natl. Acad. Sci. USA 72, 1690^1694. [7] Komatsuzawa, H., Sugai, M., Ohta, K., Fujiwara, T., Nakashima, S., Suzuki, J., Lee, C.Y. and Suginaka, H. (1997) Cloning and characterization of the fmt gene which a¡ects the methicillin resistance level and autolysis in the presence of Triton X-100 in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 41, 2355^ 2361. [8] Komatsuzawa, H., Sugai, M., Shirai, C., Suzuki, J., Hiramatsu, K. and Suginaka, H. (1995) Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin. FEMS Microbiol. Lett. 134, 209^212. [9] Komatsuzawa, H., Suzuki, J., Sugai, M., Miyake, Y. and Suginaka, H. (1994) The e¡ect of Triton X-100 on the in-vitro susceptibility of methicillin-resistant Staphylococcus aureus to oxacillin. J. Antimicrob. Chemother. 34, 885^897. [10] Maidhof, H., Reinicke, B., Blu«mel, P., Berger-Ba«chi, B. and Labischinski, H. (1991) femA, which encodes a factor essential for expression of methicillin resistance, a¡ects glycine content of peptidoglycan in methicillin-resistant and methicillin-suscetible Staphylococcus aureus strains. J. Bacteriol. 173, 3507^3513. [11] Raychaudhuri, D. and Chatterjee, A.N. (1985) Use of resistant mutants to study the interaction of Triton X-100 with Staphylococcus aureus. J. Bacteriol. 164, 1337^1349. [12] Reynolds, P.E. and Fuller, C. (1986) Methicillin-resistant strains of Staphylococcus aureus; presence of an identical additional penicillinbinding protein in all strains examined. FEMS Microbiol. Lett. 33, 251^254. [13] Suginaka, H., Shimatani, M., Ogawa, M. and Kotani, S. (1979) Prevention of penicillin-induced lysis of Staphylococcus aureus by cellular lipoteichoic acid. J. Antibiot. 32, 73^77. [14] Suzuki, J., Komatsuzawa, H., Sugai, M., Ohta, K., Kozai, K., Nagasaka, N. and Suginaka, H. (1997) E¡ect of various types of Triton X on the susceptibilities of methicillin-resistant staphylococci to oxacillin. FEMS Microbiol. Lett. 153, 327^331. [15] Ubukata, K., Nonoguchi, R., Matsuhashi, M. and Konno, M. (1989) Expression and inducibility in Staphylococcus aureus of the mecA gene which encodes a methicillin-resistant S. aureus-speci¢c penicillin-binding protein. J. Bacteriol. 171, 2882^2885.
FEMSLE 9141 16-12-99