Anaerobe 34 (2015) 24e26
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Note
Synergistic inhibition of Clostridium difficile with nisin-lysozyme combination treatment Changhoon Chai, Kyung-Soo Lee, Se-Wook Oh* Department of Food and Nutrition, Kookmin University, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 24 March 2015 Accepted 7 April 2015 Available online 8 April 2015
Clostridium difficile vegetative cells were not inhibited completely after a 120-min treatment with 40 nM nisin or 0.8 mM lysozyme. However, these cells were completely inhibited after only a 30-min incubation with both 20 nM nisin and 0.2 mM lysozyme. © 2015 Published by Elsevier Ltd.
Keywords: Clostridium difficile C. difficile-associated disease Nisin Lysozyme Synergistic inhibition
Clostridium difficile is a gram-positive, anaerobic, spore-forming, pathogenic bacterium. C. difficile is a leading pathogen causing nosocomial diarrhea and colitis [1]. C. difficile is resistant to a wide range of antibiotics, infection occurs in approximately 20% of patients taking antibiotics [2]. C. difficile-associated colitis has been cited as a direct cause of death in 1e2% of affected patients [3]. The morbidity and mortality of C. difficile-associated disease (CDAD) has steadily increased over the past decade along with the increased use of antibiotics [4]. Nisin is an antimicrobial peptide secreted by Lactococcus latis and is generally regarded as safe for human consumption (GRAS) [5]. Nisin has a narrow spectrum of antimicrobial activity, primarily affecting gram-positive bacteria [5]. Thus, it is expected that the use of nisin in CDAD treatment to inhibit C. difficile will only minimally disrupt gut microbiota. Nisin binds to the anionic phospholipid of the cytoplasmic membrane of gram-positive bacteria. The nisin is subsequently inserted into the membrane, making a pore in the membrane, and killing the bacteria [6]. As a gram-positive bacterium, C. difficile has a thick peptidoglycan layer surrounding the cytoplasmic membrane. Accordingly, we propose that lysozyme, a natural, GRAS
* Corresponding author. Department of Food and Nutrition, Kookmin University, 77, Jeongneung-ro, Seongbuk-gu, Seoul, 136-702, Republic of Korea. E-mail address: [email protected] (S.-W. Oh). http://dx.doi.org/10.1016/j.anaerobe.2015.04.003 1075-9964/© 2015 Published by Elsevier Ltd.
antimicrobial, hydrolyzes peptidoglycan bonds between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), thereby improving the inhibitory effect of nisin on C. difficile. In this study, we investigated the inhibition of C. difficile by nisin, lysozyme, and a combination of nisin and lysozyme in vitro. C. difficile ATCC 9689, ATCC 43593, and ATCC 43601 were obtained from the Korean Collection for Type Cultures (KCTC). Each strain was cultured overnight in brain heart infusion broth (BHI broth; OXOID) at 37 C under anaerobic conditions (80% N2, 10% H2, and 10% CO2) in an anaerobic chamber (Whitley A35 Anaerobic Workstation; Don Whitley Scientific). A three-strain cocktail of vegetative C. difficile was prepared by combining 1 mL of each C. difficile culture into a sterile tube. Freshly prepared C. difficile ATCC 9689 was used to investigate the inhibitory activities of nisin, lysozyme, and a combination of nisin and lysozyme; a freshly prepared C. difficile cocktail was used to confirm the synergistic inhibition of C. difficile with the nisin-lysozyme combination. Stock solutions of nisin (SigmaeAldrich) and lysozyme (SigmaeAldrich) were prepared in deionized water (DW). Stock solutions were diluted to prepare 9.9 mL of different concentrations of nisin, lysozyme, and nisin-lysozyme solutions. Nisin and lysozyme were diluted to prepare 5e140 nM and 0.2e1.0 mM solutions, respectively. Prior to use, all solutions were equilibrated in an anaerobic chamber for at least 4 h. Freshly cultured C. difficile ATCC 9689 and the C. difficile threestrain cocktail were diluted with sterile DW to a concentration of
C. Chai et al. / Anaerobe 34 (2015) 24e26
107 CFU mL 1. Different concentrations of nisin, lysozyme, and nisin-lysozyme solutions were inoculated with C. difficile by adding 100 mL of diluted, fresh C. difficile ATCC 9689 or the C. difficile cocktail. Cultures were then incubated in an anaerobic chamber at 37 C for 120 min. One milliliter of each culture was transferred to a 1.5-mL microtube every 30 min for 120 min. Immediately after the transfer, 1.5-mL microtubes were centrifuged; the supernatant was discarded and the pelleted C. difficile was resuspended in 1 mL of sterile DW to wash out the nisin and lysozyme. The washing step was repeated 3 times. The resuspended C. difficile was cultured on TBHI agar plates [(BHI agar containing 0.1% L-cysteine (SigmaeAldrich) and 0.1% sodium taurocholate (SigmaeAldrich)]. After anaerobic incubation at 37 C for 24 h, the colonies on the TBHI agar plates were quantified. Experiments assessing the inhibition of C. difficile by nisin, lysozyme, and the nisin-lysozyme combination were performed in triplicate. Nisin and lysozyme both inhibited C. difficile ATCC 9689. The more nisin and lysozyme and the longer C. difficile ATCC 9689 was exposed to, the greater number of C. difficile ATCC 9689 was reduced (Fig. 1). Nisin was a more potent inhibitor than lysozyme; C. difficile ATCC 9689 at 105 CFU mL 1 was completely inhibited after 120 min with 40 nM nisin, but with 0.8 mM lysozyme (Fig. 1).
25
Synergistic inhibition of C. difficile ATCC 9689 was observed with the combination of nisin and lysozyme (Fig. 2a). C. difficile ATCC 9689 at 105 CFU mL 1 was not completely inhibited by 20 nM nisin or 0.2 mM lysozyme even after 120 min (Fig. 1). However, no colonies formed on TBHI agar plates inoculated with C. difficile ATCC 9689 that had been treated with solutions containing both 20 nM nisin and 0.2 mM lysozyme for 30 min (Fig. 2a). This experiment revealed a more potent inhibitory effect of nisin-lysozyme combination than the sum of the individual effects of 20 nM nisin and 0.2 mM lysozyme (Fig. 1). Moreover, nisin at concentrations less than 20 nM (10 and 15 nM) completely inhibited C. difficile ATCC 9689 at 60 min, if 0.2 mM lysozyme was also included (Fig. 2a). To investigate the inhibitory effect of the nisin-lysozyme combination on other C. difficile strains, a nisin-lysozyme mixture with different concentrations of nisin and 0.2 mM lysozyme was applied to a three-strain cocktail of C. difficile (ATCC 9689, ATCC 43593, and ATCC 43601). The C. difficile strains present in the three-strain cocktail were not completely inhibited in solutions containing 10 nM nisin and 0.2 mM lysozyme (Fig. 2b). This suggests that either C. difficile ATCC 43593 or ATCC 43601 is less susceptible to the nisin-lysozyme combination than C. difficile ATCC 9689.
Fig. 1. Inhibition of Clostridium difficile by different concentrations of nisin and lysozyme. (a) nisin (concentration: 20e120 nmol mL 0.2e1.0 mmol mL 1).
1
) and (b) lysozyme (concentration:
Fig. 2. Synergistic inhibition of Clostridium difficile by a combination of nisin-lysozyme. The concentration of nisin was varied from 5 to 20 nmol mL lysozyme was 0.2 mmol mL 1. (a) C. difficile ATCC 9689 and (b) a three-strain cocktail of C. difficile ATCC 9689, ATCC 43593, and ATCC 43601.
1
and the concentration of
26
C. Chai et al. / Anaerobe 34 (2015) 24e26
However, with 20 nM nisin and 0.2 mM lysozyme, all C. difficile in the cocktail were completely inhibited after 30 min (Fig. 2b). C. difficile, a gram-positive bacteria, has a thick peptidoglycan layer encasing its cytoplasmic membrane. Lysozyme hydrolyzes the glycosidic bond between NAM and NAG, making the bacteria vulnerable to cell lysis [7]. Nisin incorporates into the phospholipids of the cytoplasmic membrane spanning the peptidoglycan layer, generates pores in the membrane, and lyses gram-positive bacteria [6]. When nisin and lysozyme are combined to inhibit gram-positive bacteria, the hydrolysis of the NAM-NAG linkage by lysozyme might facilitate access of nisin to the cytoplasmic membrane. We propose that this mechanism underlies the synergistic inhibition of C. difficile with a combination of nisin and lysozyme (Fig. 2). Because both nisin and lysozyme are categorized as GRAS, the combined use of nisin and lysozyme may be a safe and effective means to inhibit C. difficile in the treatment of CDAD. Acknowledgments This study was supported by the National Research Foundation
of Korea (NRF) grant, funded by the Korean government (MEST) (No. 2011e0015547). References [1] M. Rupnik, M.H. Wilcox, D.N. Gerding, Clostridium difficile infection: new developments in epidemiology and pathogenesis, Nat. Rev. Microbiol. 7 (2009) 526e536. [2] J. Wistrom, S.R. Norrby, E.B. Myhre, S. Eriksson, G. Granstrom, L. Lagergren, et al., Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: a prospective study, J. Antimicrob. Chemother. 47 (2001) 43e50. [3] M.A. Miller, M. Hyland, M. Ofner-Agostini, M. Gourdeau, M. Ishak, Morbidity, mortality, and healthcare burden of nosocomial Clostridium difficile-associated diarrhea in Canadian hospitals, Infect. Control Hosp. Epidemiol. 23 (2002) 137e140. [4] K.C. Carroll, J.G. Bartlett, Biology of Clostridium difficile: implications for epidemiology and diagnosis, Annu Rev. Microbiol. 65 (2011) 501e521. [5] D. Perlman, I.L. Allen, Advances in Applied Microbiology, Elsevier, Amsterdam, 1981. [6] T.J. Montville, Y. Chen, Mechanistic action of pediocin and nisin: recent progress and unresolved questions, Appl. Microbiol. Biotechnol. 50 (1998) 511e519. [7] A.J. Kirby, The lysozyme mechanism sorted-after 50 years, Nat. Struct. Mol. Biol. 8 (2001) 737e739.
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Note
Synergistic inhibition of Clostridium difficile with nisin-lysozyme combination treatment Changhoon Chai, Kyung-Soo Lee, Se-Wook Oh* Department of Food and Nutrition, Kookmin University, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 24 March 2015 Accepted 7 April 2015 Available online 8 April 2015
Clostridium difficile vegetative cells were not inhibited completely after a 120-min treatment with 40 nM nisin or 0.8 mM lysozyme. However, these cells were completely inhibited after only a 30-min incubation with both 20 nM nisin and 0.2 mM lysozyme. © 2015 Published by Elsevier Ltd.
Keywords: Clostridium difficile C. difficile-associated disease Nisin Lysozyme Synergistic inhibition
Clostridium difficile is a gram-positive, anaerobic, spore-forming, pathogenic bacterium. C. difficile is a leading pathogen causing nosocomial diarrhea and colitis [1]. C. difficile is resistant to a wide range of antibiotics, infection occurs in approximately 20% of patients taking antibiotics [2]. C. difficile-associated colitis has been cited as a direct cause of death in 1e2% of affected patients [3]. The morbidity and mortality of C. difficile-associated disease (CDAD) has steadily increased over the past decade along with the increased use of antibiotics [4]. Nisin is an antimicrobial peptide secreted by Lactococcus latis and is generally regarded as safe for human consumption (GRAS) [5]. Nisin has a narrow spectrum of antimicrobial activity, primarily affecting gram-positive bacteria [5]. Thus, it is expected that the use of nisin in CDAD treatment to inhibit C. difficile will only minimally disrupt gut microbiota. Nisin binds to the anionic phospholipid of the cytoplasmic membrane of gram-positive bacteria. The nisin is subsequently inserted into the membrane, making a pore in the membrane, and killing the bacteria [6]. As a gram-positive bacterium, C. difficile has a thick peptidoglycan layer surrounding the cytoplasmic membrane. Accordingly, we propose that lysozyme, a natural, GRAS
* Corresponding author. Department of Food and Nutrition, Kookmin University, 77, Jeongneung-ro, Seongbuk-gu, Seoul, 136-702, Republic of Korea. E-mail address: [email protected] (S.-W. Oh). http://dx.doi.org/10.1016/j.anaerobe.2015.04.003 1075-9964/© 2015 Published by Elsevier Ltd.
antimicrobial, hydrolyzes peptidoglycan bonds between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), thereby improving the inhibitory effect of nisin on C. difficile. In this study, we investigated the inhibition of C. difficile by nisin, lysozyme, and a combination of nisin and lysozyme in vitro. C. difficile ATCC 9689, ATCC 43593, and ATCC 43601 were obtained from the Korean Collection for Type Cultures (KCTC). Each strain was cultured overnight in brain heart infusion broth (BHI broth; OXOID) at 37 C under anaerobic conditions (80% N2, 10% H2, and 10% CO2) in an anaerobic chamber (Whitley A35 Anaerobic Workstation; Don Whitley Scientific). A three-strain cocktail of vegetative C. difficile was prepared by combining 1 mL of each C. difficile culture into a sterile tube. Freshly prepared C. difficile ATCC 9689 was used to investigate the inhibitory activities of nisin, lysozyme, and a combination of nisin and lysozyme; a freshly prepared C. difficile cocktail was used to confirm the synergistic inhibition of C. difficile with the nisin-lysozyme combination. Stock solutions of nisin (SigmaeAldrich) and lysozyme (SigmaeAldrich) were prepared in deionized water (DW). Stock solutions were diluted to prepare 9.9 mL of different concentrations of nisin, lysozyme, and nisin-lysozyme solutions. Nisin and lysozyme were diluted to prepare 5e140 nM and 0.2e1.0 mM solutions, respectively. Prior to use, all solutions were equilibrated in an anaerobic chamber for at least 4 h. Freshly cultured C. difficile ATCC 9689 and the C. difficile threestrain cocktail were diluted with sterile DW to a concentration of
C. Chai et al. / Anaerobe 34 (2015) 24e26
107 CFU mL 1. Different concentrations of nisin, lysozyme, and nisin-lysozyme solutions were inoculated with C. difficile by adding 100 mL of diluted, fresh C. difficile ATCC 9689 or the C. difficile cocktail. Cultures were then incubated in an anaerobic chamber at 37 C for 120 min. One milliliter of each culture was transferred to a 1.5-mL microtube every 30 min for 120 min. Immediately after the transfer, 1.5-mL microtubes were centrifuged; the supernatant was discarded and the pelleted C. difficile was resuspended in 1 mL of sterile DW to wash out the nisin and lysozyme. The washing step was repeated 3 times. The resuspended C. difficile was cultured on TBHI agar plates [(BHI agar containing 0.1% L-cysteine (SigmaeAldrich) and 0.1% sodium taurocholate (SigmaeAldrich)]. After anaerobic incubation at 37 C for 24 h, the colonies on the TBHI agar plates were quantified. Experiments assessing the inhibition of C. difficile by nisin, lysozyme, and the nisin-lysozyme combination were performed in triplicate. Nisin and lysozyme both inhibited C. difficile ATCC 9689. The more nisin and lysozyme and the longer C. difficile ATCC 9689 was exposed to, the greater number of C. difficile ATCC 9689 was reduced (Fig. 1). Nisin was a more potent inhibitor than lysozyme; C. difficile ATCC 9689 at 105 CFU mL 1 was completely inhibited after 120 min with 40 nM nisin, but with 0.8 mM lysozyme (Fig. 1).
25
Synergistic inhibition of C. difficile ATCC 9689 was observed with the combination of nisin and lysozyme (Fig. 2a). C. difficile ATCC 9689 at 105 CFU mL 1 was not completely inhibited by 20 nM nisin or 0.2 mM lysozyme even after 120 min (Fig. 1). However, no colonies formed on TBHI agar plates inoculated with C. difficile ATCC 9689 that had been treated with solutions containing both 20 nM nisin and 0.2 mM lysozyme for 30 min (Fig. 2a). This experiment revealed a more potent inhibitory effect of nisin-lysozyme combination than the sum of the individual effects of 20 nM nisin and 0.2 mM lysozyme (Fig. 1). Moreover, nisin at concentrations less than 20 nM (10 and 15 nM) completely inhibited C. difficile ATCC 9689 at 60 min, if 0.2 mM lysozyme was also included (Fig. 2a). To investigate the inhibitory effect of the nisin-lysozyme combination on other C. difficile strains, a nisin-lysozyme mixture with different concentrations of nisin and 0.2 mM lysozyme was applied to a three-strain cocktail of C. difficile (ATCC 9689, ATCC 43593, and ATCC 43601). The C. difficile strains present in the three-strain cocktail were not completely inhibited in solutions containing 10 nM nisin and 0.2 mM lysozyme (Fig. 2b). This suggests that either C. difficile ATCC 43593 or ATCC 43601 is less susceptible to the nisin-lysozyme combination than C. difficile ATCC 9689.
Fig. 1. Inhibition of Clostridium difficile by different concentrations of nisin and lysozyme. (a) nisin (concentration: 20e120 nmol mL 0.2e1.0 mmol mL 1).
1
) and (b) lysozyme (concentration:
Fig. 2. Synergistic inhibition of Clostridium difficile by a combination of nisin-lysozyme. The concentration of nisin was varied from 5 to 20 nmol mL lysozyme was 0.2 mmol mL 1. (a) C. difficile ATCC 9689 and (b) a three-strain cocktail of C. difficile ATCC 9689, ATCC 43593, and ATCC 43601.
1
and the concentration of
26
C. Chai et al. / Anaerobe 34 (2015) 24e26
However, with 20 nM nisin and 0.2 mM lysozyme, all C. difficile in the cocktail were completely inhibited after 30 min (Fig. 2b). C. difficile, a gram-positive bacteria, has a thick peptidoglycan layer encasing its cytoplasmic membrane. Lysozyme hydrolyzes the glycosidic bond between NAM and NAG, making the bacteria vulnerable to cell lysis [7]. Nisin incorporates into the phospholipids of the cytoplasmic membrane spanning the peptidoglycan layer, generates pores in the membrane, and lyses gram-positive bacteria [6]. When nisin and lysozyme are combined to inhibit gram-positive bacteria, the hydrolysis of the NAM-NAG linkage by lysozyme might facilitate access of nisin to the cytoplasmic membrane. We propose that this mechanism underlies the synergistic inhibition of C. difficile with a combination of nisin and lysozyme (Fig. 2). Because both nisin and lysozyme are categorized as GRAS, the combined use of nisin and lysozyme may be a safe and effective means to inhibit C. difficile in the treatment of CDAD. Acknowledgments This study was supported by the National Research Foundation
of Korea (NRF) grant, funded by the Korean government (MEST) (No. 2011e0015547). References [1] M. Rupnik, M.H. Wilcox, D.N. Gerding, Clostridium difficile infection: new developments in epidemiology and pathogenesis, Nat. Rev. Microbiol. 7 (2009) 526e536. [2] J. Wistrom, S.R. Norrby, E.B. Myhre, S. Eriksson, G. Granstrom, L. Lagergren, et al., Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: a prospective study, J. Antimicrob. Chemother. 47 (2001) 43e50. [3] M.A. Miller, M. Hyland, M. Ofner-Agostini, M. Gourdeau, M. Ishak, Morbidity, mortality, and healthcare burden of nosocomial Clostridium difficile-associated diarrhea in Canadian hospitals, Infect. Control Hosp. Epidemiol. 23 (2002) 137e140. [4] K.C. Carroll, J.G. Bartlett, Biology of Clostridium difficile: implications for epidemiology and diagnosis, Annu Rev. Microbiol. 65 (2011) 501e521. [5] D. Perlman, I.L. Allen, Advances in Applied Microbiology, Elsevier, Amsterdam, 1981. [6] T.J. Montville, Y. Chen, Mechanistic action of pediocin and nisin: recent progress and unresolved questions, Appl. Microbiol. Biotechnol. 50 (1998) 511e519. [7] A.J. Kirby, The lysozyme mechanism sorted-after 50 years, Nat. Struct. Mol. Biol. 8 (2001) 737e739.
