1 Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
2 National Food Biotechnology Centre, University College Cork, Ireland
3 Microbiology Department, University College Cork, Ireland
Correspondence
R. Paul Ross
pross{at}moorepark.teagasc.ie
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Considerable progress has recently been made in determining the traits responsible for pathogenesis of enterococci. For example, studies have shown that phenotypes such as production of cytolysin, which has both haemolytic activity (by lysing a broad spectrum of cells, including human, horse and rabbit erythrocytes) and bactericidal activity (against Gram-positive bacteria) play a role in the progression of enterococcal infection (Ike et al., 1984; Jett et al., 1992
). Cytolysin enhances the virulence of Enterococcus faecalis in animal models, such as murine peritonitis and rabbit endophthalmitis (Ike et al., 1984
; Jett et al., 1992
; Chow et al., 1993
; Jett et al., 1994
). Other factors include aggregation substance, which is a pheromone-inducible surface protein of E. faecalis and promotes mating aggregate formation during bacterial conjugation (Clewell, 1993
). This protein has been shown to enhance enterococcal adherence to renal cells (Joyanes et al., 2000
) and to mediate internalization of E. faecalis by cultured human intestinal epithelial cells (Olmsted et al., 1994
). In contrast, gelatinase is a protease that hydrolyses gelatin, collagen, casein, haemoglobin and other bioactive peptides (Coque et al., 1995
), which suggests a role in inflammatory processes (Makinen et al., 1989
). Other variable traits associated with enterococcal virulence are the enterococcal surface protein (Esp), which may contribute to the ability of E. faecalis to evade detection by the immune system (Shankar et al., 1999
) and the EfaA proteins which are homologues of cell surface adhesions found on a number of streptococcal species (Lowe et al., 1995
; Singh et al., 1998
) and are expressed in serum. In addition, sex pheromones are peptides expressed prior to conjugation which induce genes on the plasmid of the donor strain to produce aggregation substance, thus increasing the frequency of plasmid transfer (Clewell 1990
; Simjee & Gill, 1997
).
Recently, Eaton & Gasson (2001) investigated the incidence of known virulence factors in medical, food and dairy starter Enterococcus strains. Not surprisingly, PCR and gene probe strategies revealed that medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than starter strains. All of the E. faecalis strains tested possessed multiple virulence determinants while Enterococcus faecium strains were generally free of virulence determinants. In addition, conjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. Thus, despite featuring in dairy fermentations for decades, the use of Enterococcus spp. in foods requires careful safety evaluation as the transfer of virulence determinants and antibiotic resistance to starter strains via natural conjugation mechanisms poses a potential risk in a mixed microbial environment.
In the present study, an E. faecalis strain which exhibited a broad spectrum of inhibition to Gram-positive bacteria, including Listeria, was isolated from Irish raw milk. This activity was found to be due to the production of both cytolysin and a heat-labile antimicrobial protein of 34 kDa. The latter protein was found to have cell-wall-degrading activity and N-terminal sequencing followed by genetic analysis revealed a protein which was 100 % identical to the cell-wall-degrading enzyme, enterolysin A (NCBI accession no. AF249740; Nilsen, 1999). Subsequently, it is shown that the DPC5280 strain also contains genes responsible for multiple virulence factors and multiple antibiotic resistance.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Spectrum of inhibition.
To test the sensitivity of a strain to the antimicrobial activity produced by E. faecalis, 10 µl aliquots of a fresh overnight culture of E. faecalis DPC5280 were first spotted onto GM17 agar plates and incubated overnight at 37 °C. These plates were then overlaid with 3 ml soft agar seeded with 100 µl of the indicator strain (overnight culture) (Table 2). The sensitivity of a strain to the producer was scored according to the diameter of the zone of inhibition surrounding E. faecalis DPC5280. The experiment was performed in triplicate and the mean zone diameter was calculated. The positive control used was the supernatant of E. faecalis FA2-2.pAD1 and the negative control was the supernatant of E. faecalis OG1X.
|
Electroblotting and N-terminal amino acid analysis of enterolysin A proteins.
Proteins were electrotransferred from PAGE gels onto PVDF membranes (Bio-Rad) in CAPS buffer (Sigma), pH 11, using a Trans-Blot cell (Bio-Rad), according to manufacturer's instructions. Proteins were stained with Coomassie brillant blue R250, cut off from the membrane and sequenced on a Beckman LF 3000 microsequencer (Molecular Biology Unit, University of Newcastle upon Tyne). Database searches were performed with the program BLASTP.
Lytic activity spectrum and assay.
To test for sensitivity of a strain to partially purified enterolysin A, well diffusion assays were performed as described by Ryan et al. (1996). Molten agar was cooled to 48 °C and seeded with the indicator strains (overnight culture). The inoculated medium was dispensed into sterile Petri plates, allowed to solidify and dried. Wells (
4·6 mm diameter) were made in the seeded agar plates into which 50 µl aliquots of partially purified enterolysin A were dispensed and the plates were incubated overnight. The sensitivity of a strain to enterolysin A was scored according to the diameter of the zone of inhibition surrounding the wells. The experiment was performed in triplicate and the mean zone diameter was determined. DPC5280 supernatant was used as a positive control. The effect of the lytic activity was monitored by measuring the decrease in turbidity of a cell suspension of L. lactis HP. Cells were harvested in the exponential-growth phase at an OD600 of approximately 0·2. Aliquots were supplemented with 50 000 arbitrary units (AU) ml-1 (as described by Ryan et al., 1996
) of enterolysin A in 20 mM sodium phosphate buffer (pH 7). As a control, the same volume of 20 mM sodium phosphate buffer was added to the other aliquots and the mixtures incubated at 30 °C. The OD600 of the cultures was measured spectrophotometrically at time intervals of 15 min over a 90 min period.
DNA manipulations.
Total DNA was extracted from overnight Enterococcus cultures using a modification of the method of Hoffman & Winston (1987). Briefly, enterococcal cells were subjected to vortex mixing in the presence of acid-washed glass beads, SDS, Triton X-100, phenol and chloroform. This mixture was then centrifuged to separate the DNA from cellular debris. The DNA was then precipitated using sodium acetate and ethanol, and subsequently washed using 70 % ethanol prior to resuspension in molecular standard distilled water. Plasmid DNA was isolated from E. faecalis DPC5280 using the method of Anderson & McKay (1983)
. Oligonucleotide primers for PCR were obtained from Genosys and are listed in Table 3
. PCR amplifications were performed in 50 µl reaction mixtures using 2 µg DNA, 2 mM MgCl2, 50 pmol each primer and 1 U Taq polymerase (Bioline). Negative control PCRs with no template DNA were also performed. Samples were subjected to a cycle of denaturation (94 °C for 1 min), annealing (at an appropriate temperature for 1 min) and elongation (72 °C for 1 min) for 35 cycles using a Hybaid PCR express unit. Amplified 16S rDNA was purified from a 1 % agarose gel using a QIAquick Gel Purification Kit (Qiagen).
|
DNA sequencing and analysis.
Purified 16S rDNA was sequenced with an automated DNA sequencer (MWG-BIOTECH custom DNA sequencing service, Ebersberg, Germany) using the CO1 and CO2 primers. Sequence analysis was performed using DNAStar software and the program BLASTN.
Production of gelatinase and haemolysin.
Gelatinase activity was detected by the growth of E. faecalis DPC5280 on 3 % gelatin medium as described by Su et al. (1991). Gelatinase-positive colonies were identified by a turbid halo after 2 days incubation at 37 °C. For investigation of haemolysin production, E. faecalis DPC5280 was streaked onto fresh 5 % horse blood agar plates and grown for 24 h at 37 °C. Zones of clearing surrounding isolated colonies indicated haemolysin production.
Antibiotic susceptibility testing.
The antibiotic resistance phenotype of E. faecalis DPC5280 and other control enterococcal strains, listed in Table 4, was determined by the use of antibiotic susceptibility discs obtained from Oxoid. GM17 plates were seeded with 0·5 % inoculum of each enterococcal strain after overnight growth. Six antibiotic discs were placed on each plate and after overnight incubation at 37 °C, the diameter of the zone of inhibition around each disc was measured. The experiment was carried out in triplicate and the results are shown as the mean.
|
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
DPC5280 contains the genetic determinants for cytolysin production
Cultivation of DPC5280 on equine blood agar plates revealed that it had a haemolytic phenotype. Given that the haemolytic phenotype is often associated with production of cytolysin in enterococci, the strain was tested for cyl genetic determinants using primers designed for the cylLL, cylLS and cylM genes. In repeated PCRs, a product of 2659 bp was amplified (Fig. 1a) which was sequenced and shown to be 100 % identical to the entire cylLL, cylLS and cylM gene cluster found on the cytolysin encoding plasmid pAD1.
|
|
|
Interestingly, the C terminus of enterolysin A shares weak homology (21 % identity in a 138 aa stretch) with a protein from Clostridium acetobutylicum (NC_003030; Nolling et al., 2001) containing ChW-repeats (clostridial hydrophobic, with a conserved tryptophan; Nolling et al., 2001
). ChW-repeat domains contain a distinct repetitive structure which may function in either substrate-binding or proteinprotein interactions (Nolling et al., 2001
), suggesting they may play a critical role in the cell-wall binding of enterolysin. Some of the ChW-repeat proteins contain additional domains such as glycosyl hydrolases or proteases, which implicates them in the degradation of polysaccharides and proteins. Several also contain domains that are involved in cell interactions, such as the cell adhesion domain (Kelly et al., 1999
) and the leucine-rich repeat (internalin) domain (Marino et al., 1999
). Modular structure with separate domains for catalytic activity and substrate binding is typical for lysins of Gram-positive bacteria (Riley, 1993
; Baba & Schneewind, 1996
) and similarity between bacteriophage and bacterial DNA allows shuffling of domains by recombination, restructuring both viral and bacterial genomes (Garcia et al., 1988
; Lopez et al., 1992
). A short threonine-proline (TP)-rich putative linker sequence is present between the domains of enterolysin A, whose role in the evolution of many protein families is well established by enabling a mix and match of many protein domains (Cooper & Salmond, 1993
).
In a simultaneous effort to identify enterolysin from a two-dimensional electrophoretic gel, the same non-haemolytic fraction used in one-dimensional electrophoresis was analysed and a protein of similar molecular mass (but with a pI of approximately 6·5) to enterolysin A was subjected to N-terminal sequencing. Interestingly, the sequence obtained (SEDYNLLGVKNYDQYALGAPSGCEGASLLQGLQYKGKIPDWDL) was 100 % identical to a sequence encoded by a gene directly downstream of entL which displays homology to putative transcriptional regulators from Listeria innocua (38 % identity in a 70 aa stretch; AL596163), Listeria monocytogenes (38 % identity in a 70 aa stretch; AL591973) and Listeria innocua (42 % identity in a 49 aa stretch; AL596166). Given the proximity of these genes and the lack of evidence of an obvious strong intergenic promoter it is probable that the genes are co-transcribed.
To assess the effectiveness of enterolysin A activity, the decrease in OD600 of L. lactis HP culture upon exposure to the enzyme was measured over 90 min. Fig. 4 demonstrates that the addition of enterolysin A resulted in a rapid reduction in OD600 when compared with that of comparable control cultures. A similar result was observed by Nilsen (1999)
on exposure of L. lactis IL1403 to enterolysin A. Indeed, following the 90 min period, all of the cells had been lysed by enterolysin A. Fig. 4
also shows a photo of the L. lactis HP cultures after the 90 min period, with a completely lysed culture on the left which has been treated with enterolysin A, while the untreated culture on the right remains turbid. From these results, it is evident that enterolysin A provides DPC5280 with an additional advantage against competing bacteria as lysis of other bacteria may release nutrients, which the strain can then utilize for growth.
|
In addition, enterococci have emerged as major nosocomial pathogens in part because of their resistance to multiple antibiotics, which allows them to survive and subsequently infect patients. Considering the pathogenic potential of DPC5280, the spectrum of resistance to various antibiotics was determined. The relative sensitivities of E. faecalis DPC5280 and two control strains to 30 different antibiotics are presented in Table 4. The results demonstrate that E. faecalis DPC5280 and the control E. faecalis strains were resistant to some of the cell-wall-inhibiting antibiotics such as cefotetan and cefoxitin which belong to the cephalosporin class of antibiotics. This result may be expected as most enterococci have naturally occurring or inherent resistance to such cell-wall-active agents. All strains tested were found to be sensitive to ampicillin, penicillin G (
-lactams) and vancomycin (glycopeptide). Resistance to these drugs would most likely be acquired after exposure to fixed concentrations of the antibiotics (Hodges et al., 1992
). With regards to antibiotics which inhibit protein synthesis, E. faecalis DPC5280 was found to be resistant to chloramphenicol, erythromycin, minocycline, oxytetracycline and tetracycline when compared with the controls. Natural resistance to these agents is usually plasmid- or transposon-mediated and Teuber et al. (1996)
reported a common multiple drug resistance type that included resistance to tetracycline, chloramphenicol, erythromycin and also gentamicin. All strains were found to be generally intrinsically resistant to the aminoglycosides such gentamicin and kanamycin.
The evolutionary development of resistance to many drugs has been attributed to the possession of broad host range and extremely mobile genetic elements like conjugative plasmids and transposons (e.g. pAM1 or Tn916; Clewell et al., 1995
). Since E. faecalis DPC5280 possesses the determinants for sex pheromones, aggregation substance and cytolysin, it is likely the strain possesses one or more sex pheromone plasmids. Since sex pheromone plasmids may also carry one or more antibiotic genes (Clewell, 1990
; Wirth, 1994
), the plasmid profile (Fig. 1b
) was determined which revealed the presence of two plasmids (78 and 27 kb). The smaller plasmid was partially sequenced and contained a gene for kanamycin resistance (R. M. Hickey and others, unpublished) which was 100 % identical to that found on the E. faecalis plasmid pJH1 (Trieu-Cuot & Courvalin, 1983
).
Conclusions
A raw milk isolate, E. faecalis DPC5280 was found to contain multiple genes associated with virulence, including cytolysin and gelatinase, and was resistant to antibiotics which inhibit protein synthesis. In addition, this strain produces an endopeptidase, enterolysin A encoded by entL, which is homologous to other cell wall lytic enzymes such as lysostaphin and zoocin A. However, unlike these enzymes enterolysin A has a broad spectrum of activity and can lyse a wide range of Gram-positive bacteria. It is tempting to suggest that production of this potent antimicrobial may contribute to the pathogenic potential of some enterococcal strains since it could allow them to better compete in complex microbial environments such as the human gastrointestinal tract.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, D. G. & McKay, L. L. (1983). Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol 46, 549552.[Medline]
Baba, T. & Schneewind, O. (1996). Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus. EMBO J 15, 47894797.[Abstract]
Beresford, T. & Condon, S. (1991). Cloning and partial characterization of genes for ribosomal ribonucleic acid in Lactococcus lactis subsp. lactis. FEMS Microbiol Lett 62, 319323.[Medline]
Chow, J. W., Thal, L. A., Perri, M. B., Vazquez, J. A., Donabedian, S. M., Clewell, D. B. & Zervos, M. J. (1993). Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocartitis. Antimicrob Agents Chemother 37, 24742477.[Abstract]
Clewell, D. B. (1990). Movable genetic elements and antibiotic resistance in enterococci. Eur J Clin Microbiol Infect Dis 9, 90102.[Medline]
Clewell, D. B. (1993). Bacterial sex pheromone-induced plasmid transfer. Cell 73, 912.[Medline]
Clewell, D. B., Tomich, P. K., Gawron-Burke, M. C., Franke, A. E., Yagi, Y. & An, F. Y. (1982). Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. J Bacteriol 152, 12201230.[Medline]
Clewell, D. B., Flannagan, S. E. & Jaworski, D. D. (1995). Unconstrained bacterial promiscuity: the Tn916-Tn1545 family of conjugative transposons. Trends Microbiol 3, 229236.[CrossRef][Medline]
Cooper, V. J. C. & Salmond, G. P. C. (1993). Molecular analysis of the major cellulase (CelV) of Erwinia carotovora: evidence for an evolutionary mix and match of enzyme domains. Mol Gen Genet 241, 342350.
Coque, T. M., Patterson, J. E., Steckelberg, J. M. & Murray, B. E. (1995). Incidence of hemolysin, gelatinase, and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from the feces of hospitalized and community-based persons. J Infect Dis 171, 12231229.[Medline]
Eaton, T. J. & Gasson, M. J. (2001). Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol 67, 16281635.
Facklam, R. R. & Sahm, D. F. (1995). Enterococcus. In Manual of Clinical Microbiology, pp. 308314. Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover & R. H. Yolken. Washington, DC: American Society for Microbiology.
Garcia, E., Garcia, J. L., Garcia, P., Arraras, A., Sanchez-Puelles, J. M. & Lopez, R. (1988). Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Proc Natl Acad Sci U S A 85, 914918.[Abstract]
Garcia, P., Bascaran, V., Rodriguez, A. & Suarez, J. E. (1997). Isolation and characterization of promoters from the Lactobacillus casei temperate bacteriophage A2. Can J Microbiol 43, 10631068.[Medline]
Hayashida, M., Watanabe, K., Muramatsu, T. & Goto, M. A. (1987). Further characterization of PL-1 phage-associated N-acetyl-muramidase of Lactobacillus casei. J Gen Microbiol 133, 13431349.
Hodges, T. L., Zighelboim-Daum, S., Eliopoulos, G. M., Wennersten, C. & Moellering, R. C., Jr (1992). Antimicrobial susceptibility changes in Enterococcus faecalis following various penicillin exposure regimens. Antimicrob Agents Chemother 36, 121125.[Abstract]
Hoffman, C. S. & Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267272.[CrossRef][Medline]
Ike, Y., Craig, R. A., White, B. A., Yagi, Y. & Clewell, D. B. (1983). Modification of Streptococcus faecalis sex pheromones after acquistion of plasmid DNA. Proc Natl Acad Sci U S A 92, 1205512059.
Ike, Y., Hashimoto, H. & Clewell, D. B. (1984). Hemolysin of Streptococcus faecalis subspecies zymogenes contributes to virulence in mice. Infect Immun 45, 528530.[Medline]
Jett, B. D., Jensen, H. G., Nordquist, R. E. & Gilmore, M. S. (1992). Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis. Infect Immun 60, 24452452.[Abstract]
Jett, B. D., Huyke, M. M. & Gilmore, M. S. (1994). Virulence of enterococci. Clin Microbiol Rev 7, 462478.[Abstract]
Jones, R. N., Marshall, S. A., Pfaller, M. A., Wilke, W. W., Hollis, R. J., Erwin, M. E., Edmond, M. B. & Wenzel, R. P. (1997). Nosocomial enterococcal blood stream infections in the SCOPE program: antimicrobial resistance, species occurrence, molecular testing results, and laboratory testing accuracy. SCOPE Hospital Study Group. Diagn Microbiol Infect Dis 29, 95102.[CrossRef][Medline]
Joyanes, P., Pascual, A., Martinez-Martinez, L., Hevia, A. & Perea, E. J. (2000). In vitro adherence of Enterococcus faecalis and Enterococcus faecium to urinary catheters. Eur J Clin Microbiol Infect Dis 19, 124127.[CrossRef][Medline]
Kashige, N., Nakashima, Y., Miake, F. & Watanabe, K. (2000). Cloning, sequence analysis, and expression of Lactobacillus casei phage PL-1 lysis genes. Arch Virol 145, 15211534.[CrossRef][Medline]
Kelly, G., Prasannan, S., Daniell, S., Fleming, K., Frankel, G., Dougan, G., Connerton, L. & Matthews, S. (1999). Structure of the cell-adhesion fragment of intimin from enteropathogenic Escherichia coli. Nat Struct Biol 6, 313318.[CrossRef][Medline]
Klein, G., Pack, A. & Reuter, G. (1998). Antibiotic resistance patterns of enterococci and occurrence of vancomycin-resistant enterococci in raw minced beef and pork in Germany. Appl Envir Microbiol 64, 18251830.
Knudtson, L. M. & Hartman, P. A. (1993). Antibiotic resistance among enterococcal isolates from environmental and clinical sources. J Food Prot 56, 486492.
Kuhnen, E., Richter, F., Richter, K. & Andries, L. (1988). Establishment of a typing system for group D streptococci. Zentbl Bakteriol Hygiene A 267, 322330.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Leclerc, D. & Asselin, A. (1989). Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can J Microbiol 35, 749753.[Medline]
Lopez, R., Garcia, J. L., Garcia, E., Ronda, C. & Garcia, P. (1992). Structural analysis and biological significance of the cell wall lytic enzymes of Streptococcus pneumoniae and its bacteriophage. FEMS Microbiol Lett 79, 439447.[CrossRef][Medline]
Low, D. E., Willey, B. M., Betschel, S. & Kreiswirth, B. (1994). Enterococcus: pathogens of the 90s. Eur J Surg Suppl 573, 1924.[Medline]
Lowe, A. M., Lambert, P. A. & Smith, A. W. (1995). Cloning of an Enterococcus faecalis endocarditis antigen: homology with adhesins from some oral streptococci. Infect Immun 63, 703706.[Abstract]
Makinen, P. L., Clewell, D. B., An, F. & Makinen, K. K. (1989). Purification and substrate specificity of a strongly hydrophobic extracellular metalloendopeptidase (gelatinase) from Streptococcus faecalis (strain OG1-10). J Biol Chem 264, 33253334.
Marino, M., Braun, L., Cossart, P. & Ghosh, P. (1999). Structure of the lnlB leucine-rich repeats, a domain that triggers host cell invasion by the bacterial pathogen L. monocytogenes. Mol Cell 4, 10631072.[Medline]
McKay, L. L. & Baldwin, K. A. (1984). Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage. Appl Envir Microbiol 47, 6874.[Medline]
Moellering, R. C., Jr (1992). Emergence of Enterococcus as a significant pathogen. Clin Infect Dis 14, 11731176.[Medline]
Murray, B. E. (1990). The life and times of the Enterococcus. Clin Microbiol Rev 3, 4665.[Medline]
Nilsen, T. (1999). Novel enterococcal bacteriocins; optimization of production, purification, biochemical and genetic chacterization. PhD thesis, Agricultural University of Norway, Ås, Norway.
Nolling, J., Breton, G., Omelchenko, M. V. & 16 other authors (2001). Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183, 48234838.
Olmsted, S. B., Dunny, G. M., Erlandsen, S. L. & Wells, C. L. (1994). A plasmid-encoded surface protein on Enterococcus faecalis augments its internalization by cultured intestinal epithelial cells. J Infect Dis 170, 15491556.[Medline]
Parente, E. & Hill, C. (1992). Inhibition of Listeria in buffer, broth and milk by enterocin 1146, a bacteriocin produced by Enterococcus faecium. J Food Prot 55, 503508.
Piard, J. C., Muriana, P. M., Desmazeaud, M. J. & Klaenhammer, T. R. (1992). Purification and partial characterisation of lacticin 481, a lanthionine-containing bacteriocin produced by Lactococcus lactis subsp. lactis CNRZ481. Appl Environ Microbiol 58, 279284.[Abstract]
Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli. Mol Gen Genet 214, 241248.[Medline]
Recsei, P. A., Gruss, A. D. & Novick, R. P. (1987). Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans. Proc Natl Acad Sci U S A 84, 11271131.[Abstract]
Riley, M. A. (1993). Molecular mechanisms of colicin evolution. Mol Biol Evol 10, 13801395.[Abstract]
Ryan, M. P., Rea, M. C., Hill, C. & Ross, R. P. (1996). An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147. Appl Environ Microbiol 62, 612619.[Abstract]
Schindler, C. A. & Schuhardt, V. T. (1964). Lysostaphin: a new bacteriolytic agent for the Staphylococcus. Proc Natl Acad Sci U S A 51, 414421.[Medline]
Schleifer, K. H. & Fischer, U. (1982). Description of a new species of the genus Staphylococcus: Staphylococcus carnosus. Int J Syst Bacteriol 32, 153156.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407477.[Medline]
Shankar, V., Baghdayan, A. S., Huycke, M. M., Lindahl, G. & Gilmore, M. S. (1999). Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect Immun 67, 193200.
Simjee, S. & Gill, M. J. (1997). Gene transfer, gentamicin resistance and enterococci. J Hosp Infect 36, 249259.[CrossRef][Medline]
Simmonds, R. S., Simpson, W. J. & Tagg, J. R. (1997). Cloning and sequence analysis of zooA, a Streptococcus zooepidemicus gene encoding a bacteriocin-like inhibitory substance having a domain structure similar to that of lysostaphin. Gene 189, 255261.[CrossRef][Medline]
Singh, K. V., Coque, T. M., Weinstock, G. M. & Murray, B. E. (1998). In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification. FEMS Immunol Med Microbiol 21, 323331.[CrossRef][Medline]
Su, Y. A., Sulavik, M. C., He, P., Makinen, K. K., Makinen, P. L., Fiedler, S., Wirth, R. & Clewell, D. B. (1991). Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens. Infect Immun 59, 415420.[Medline]
Sugai, M., Fujiwara, T., Akiyama, T., Ohara, M., Komatsuzawa, H., Inoue, S. & Suginaka, H. (1997). Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. J Bacteriol 179, 11931202.[Abstract]
Teuber, M., Perreten, V. & Wirsching, F. (1996). Antibiotikumresistente Bakterien: eine neue Dimension in der Lebensmittelmikrobiiologie. Lebensmittel-Technologie 29, 182199.
Trieu-Cuot, P. & Courvalin, P. (1983). Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5'-aminoglycoside phosphotransferase type III. Gene 23, 331341.[CrossRef][Medline]
Watanabe, K., Hayashida, M., Ishibashi, K. & Nakashima, Y. (1984). An N-acetylmuramidase induced by PL-1 phage infection of Lactobacillus casei. J Gen Microbiol 130, 275277.[Medline]
Wirth, R. (1994). The sex pheromone system of Enterococcus faecalis. More than just a plasmid-collection mechanism? Eur J Biochem 222, 235246.[Abstract]
Received 15 August 2002;
revised 11 November 2002;
accepted 28 November 2002.