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Antimicrobial Agents and Chemotherapy, June 2008, p. 1917-1923, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01409-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Production of Enterocins L50A, L50B, and IT, a New Enterocin, by Enterococcus faecium IT62, a Strain Isolated from Italian Ryegrass in Japan{triangledown}

Esther Izquierdo,1 Audrey Bednarczyk,2 Christine Schaeffer,2 Yimin Cai,3 Eric Marchioni,1 Alain Van Dorsselaer,2 and Saïd Ennahar1*

Laboratoire de Chimie Analytique et Sciences de l'Aliment, IPHC-DSA, ULP, CNRS, 74 route du Rhin, 67400 Illkirch-Graffenstaden, France,1 Laboratoire de Spectrométrie de Masse Bioorganique, IPHC-DSA, ULP, CNRS, 25 rue Becquerel, 67087 Strasbourg, France,2 National Institute of Livestock and Grassland Science, Functional Feed Research Team, Nasushiobara, Tochigi 329-2793, Japan3

Received 31 October 2007/ Returned for modification 27 February 2008/ Accepted 30 March 2008


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ABSTRACT
 
Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met24. The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin.


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INTRODUCTION
 
Enterococci are an essential part of the endogenous gut microbiota of humans and animals, where they are believed to play a key role in the balance of the microbiota, thereby showing great potential as probiotics (2, 5). They are also widespread in nature, found in wastewaters, feces, and slurry, as well as in soil and on plants (27), and are thus considered to be indicators for fecal contamination. Furthermore, being lactic acid bacteria (LAB), enterococci participate in the fermentation of foods such as cheese (20), black olives (4), and sausage (23), as well as in feed fermentations such as that of silage, for which they are of commercial importance.

Enterococci are also promising for the biopreservation of food, especially by means of bacteriocin production (19). Bacteriocins are ribosomally synthesized antimicrobial peptides with activities that are usually directed against species closely related to the producing bacterium (26, 29). Much focus has been directed in the past few years toward enterococci which have emerged as a prominent group of bacteriocin-producing LAB, mainly because of the diversity of the bacteriocins (enterocins) produced and the potential for their use as food biopreservatives. In fact, species of Enterococcus are quite unique in that they produce a wide array of structurally diverse antimicrobial peptides, often more than one per strain, some of which are atypical and distinct from known bacteriocins. Some of these atypical enterocins, such as enterocins L50A and L50B (8) and enterocin Q (7), could not fit into the traditional classification of bacteriocins produced by LAB, which has largely prompted the recent reclassification of class II bacteriocins into a new scheme (19).

The diversity of enterocins is attributed to the robust nature of enterococci, which allows them to survive in a wide range of ecological niches, as well as their superior genetic exchange mechanisms (19). Such attractive traits could only increase the interest in the biotechnological use of enterocins or their producers in food preservation, especially since these bacteriocins are particularly active against many food-borne pathogens, such as bacilli, clostridia, staphylococci, and Listeria species (17, 19). As a result, enterocins are being extensively investigated for possible use as food preservatives in dairy and meat products (4, 20, 23). However, enthusiasm for enterococci has been somewhat diminished by the emergence of multiple-antibiotic-resistant enterococci among species of nosocomial pathogens and the presence of virulence factors among food isolates (11, 13, 18, 31). The potential health risk linked to enterococcal strains will therefore have to be carefully evaluated prior to any food application (17). Nevertheless, as pointed out by Franz et al. (19), enterococcal bacteriocins produced by heterologous hosts or added as cell-free preparations appear not to carry particular safety concerns when it comes to applications in food preservation.

A study by De Vuyst et al. (10) into the ecological distribution of enterocins involving a large number of strains from different sources did in fact not find a correlation between the origins of the strains and the types of inhibitory spectra of the bacteriocins produced. Results from a recent investigation by Inoue et al. (24) suggested, though, that the dominant type of bacteriocin in Enterococcus faecium clinical isolates may differ from the dominant type of bacteriocin found in food-grade E. faecium isolates. It was pointed out that the anti-Listeria activity of bacteriocins from food-grade strains may provide these bacteria with a selective advantage against Listeria species in their particular ecological niche. On the other hand, bacteriocins identified in clinical isolates of E. faecium, such as bacteriocin 32, which is not active against Listeria monocytogenes, would provide a competitive advantage in the clinical environment in association with multiple-drug resistance. This idea helped explain the high incidence of bacteriocin 32 among vancomycin-resistant isolates. The suggestion that the type of bacteriocin that becomes dominant within any environment is influenced by the surrounding ecology is interesting and may have important implications for the future of the biotechnological use of enterocins. However, reports on the ecological distribution of enterocins remain scarce, and further studies are needed. For instance, while research on enterocins has focused mainly on bacteriocinogenic enterococci of food or clinical origins (9, 17), less attention has been given to isolates from plant and environmental sources (30).

In the present work, we describe the isolation and identification of three bacteriocins produced by E. faecium IT62, a strain isolated from Italian ryegrass in Japan. The spectrum of activity and the chemical structure of each antibacterial peptide are reported.


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MATERIALS AND METHODS
 
Bacterial strains and cultures. The bacteriocin producer E. faecium IT62 was isolated from Italian ryegrass (Lolium multiflorum Lam.). Lactococcus lactis RO50 (15) and E. faecium WHE 81 (14) were chosen as the indicator strains to assess bacteriocin activity during the purification process. Strains used to determine the activity spectra of purified bacteriocins are listed in Table 1. All cultures were maintained as frozen stocks held at –80°C in brain heart infusion (BHI) broth with 30% glycerol (Bio-Rad, Marnes-La-Coquette, France). For short-term preservation, the strains were subcultured on BHI agar (Bio-Rad) and kept at 4°C. Before experimental use, the strains were cultivated twice for 18 to 24 h in MRS broth (Biokar Diagnostics, Beauvais, France).


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  		TABLE 1. Antimicrobial activities of purified enterocins L50A (peptide 2), L50B (peptide 1), and IT (peptide 3)

Antibacterial activity determination. Bacteriocin activity in the cell-free culture supernatant and in fractions obtained during the purification process was tested by the spot-on-a-lawn test (14) by aliquoting a 10-µl sample onto MRS agar that was overlaid with lactobacillus agar AOAC (Difco, Le Pont-de-Claix, France) inoculated with indicator cells at approximately 106 CFU/ml. Overnight cultures of L. lactis RO50 and E. faecium WHE 81 prepared in MRS broth at 30 and 37°C, respectively, were used for inoculation. To monitor inhibitory activity during the purification process, the diameter of the inhibition zone (in millimeters) was measured. The activities of purified peptides were quantified by using serial twofold dilutions and expressed in activity units (AU) per milliliter as previously described (14).

To determine the antimicrobial spectrum of each bacteriocin, the effects of purified peptides on the growth of a wide range of microorganisms (Table 1) were assessed using a Bioscreen 200C automated turbidometer (Labsystems, Helsinki, Finland). Strains were grown for 18 h under the conditions indicated in Table 1. Cultures were diluted in fresh broth to reach a final concentration of ca. 105 CFU/ml, and 190-µl samples of each were loaded into microplate wells. Ten-microliter aliquots of purified bacteriocin solutions were added to wells containing indicator bacteria. Controls consisted of 190-µl samples of diluted cultures supplemented with 10 µl of sterile deionized water. All assays were done in duplicate. The growth of indicator strains at 30 or 37°C, depending on the species, was monitored at 600 nm for 24 h. Bacteriocin activity was expressed as the maximal difference in absorbance between control and assay samples, which was reached at the end of the exponential growth phase (between 7 and 14 h, depending on strains).

Bacteriocin purification. E. faecium IT62 was inoculated into 1 liter of MRS broth to yield an initial concentration of ca. 105 CFU/ml and incubated for 15 h at 37°C. Cells were removed by centrifugation at 4,000 x g for 15 min, and the supernatant was filter sterilized (using a 0.45-µm-pore-size Porafil cellulose acetate filter; Macherey-Nagel, Hoerdt, France). The obtained culture extract was adjusted to pH 6 with NaOH and purified by passage on a cation exchanger (SP-Sepharose HP; 100-mm length and 26-mm internal diameter [Amersham Biosciences, Orsay, France[). Equilibration and washing were done with 20 mM sodium acetate at pH 6 (buffer A), prior to elution over 60 min with an NaCl gradient from 0 to 1 M NaCl in buffer A at a flow rate of 5.0 ml/min. At this stage, two groups of active fractions (fraction group A and fraction group B) were observed, collected separately, and applied to a C8 reverse-phase column (Polaris C8-A; 250-mm length, 10-mm internal diameter, 180-Å pore size, and 5-µm particle size [Varian, Les Ulis, France]) for high-pressure liquid chromatography (HPLC). After a 20-min wash with 35% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA) for fraction group A and with 20% acetonitrile in water containing 0.1% TFA for fraction group B, elution was done with a gradient of H2O-acetonitrile containing 0.1% TFA (see Fig. 1) at a flow rate of 2 ml/min. Bacteriocins were detected at 280 nm by using a Varian ProStar photodiode array detector. Samples containing the purified bacteriocins were stored at –20°C.


Figure 1
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  		FIG. 1. Profiles of HPLC elution from a C8 column at 280 nm. (A) Fraction group A obtained after cation-exchange chromatography; (B) peptides 1 and 2 purified to homogeneity; (C) fraction group B obtained after cation-exchange chromatography. Black arrows indicate the peaks corresponding to active fractions; dashed lines indicate acetonitrile percentage.

ESI-MS analysis of intact proteins. The determination of the molecular masses of purified bacteriocins was performed by electrospray ionization mass spectrometry (ESI-MS). Samples obtained from liquid chromatography were injected directly into a 1200L quadrupole mass spectrometer (Varian) for tandem mass spectrometry (MS/MS). Data were acquired over a range of mass-to-charge ratios (m/z) of 500 to 1,500.

Proteolytic digestion, liquid chromatography-nanospray MS/MS (nano-LC/MS/MS), and sequence analysis. Purified bacteriocins were vacuum dried using a Speed Vac SC100 system (Savant, MN) and resuspended in a solution of 40 µl of trifluoroethanol and 40 µl of 10 mM DL-dithiothreitol prepared in 50 mM ammonium bicarbonate. After heating of the mixtures at 65°C for 1 h, 40 µl of 25 mM ammonium bicarbonate containing 55 mM iodoacetamide was added. The solutions were left for 1 h at room temperature in the dark, and then 40 µl of 25 mM ammonium bicarbonate containing 20 mM DL-dithiothreitol was added and the mixtures were left for one more hour at room temperature in the dark. Samples were diluted to reach a final trifluoroethanol percentage of 5%, and digestion was performed overnight at room temperature in the presence of modified porcine trypsin (Promega, Madison, WI) at protein ratios of 1 to 20. Digestion was stopped by adding 0.1% formic acid.

Tryptic digests were analyzed by nano-LC/MS/MS using an 1100 series HPLC-chip/MS system (Agilent Technologies, Palo Alto, CA) coupled to an HCT ultra electrospray ion trap (Bruker Daltonics, Bremen, Germany) with a capillary cap voltage of –1,750 V. The three most abundant peptides, preferably doubly charged ions, corresponding to each MS spectrum were selected for further isolation and fragmentation. The MS/MS scanning was performed in the ultrascan resolution mode at a rate of change in the m/z of 26.000 per s. Results from a total of six scans were averaged to obtain an MS/MS spectrum. Mass data collected during nano-LC/MS/MS analysis were processed, converted into mgf files, and compared against the NCBInr database by using a local Mascot server (Matrix Science, London, United Kingdom).

N-terminal sequencing. In instances in which a bacteriocin could not be identified through nano-LC/MS/MS analysis, N-terminal sequencing was performed on a model 473A microsequencer (Applied Biosystems, Foster City, CA). Samples were loaded onto polybrene-treated, precycled glass fiber filters. Phenylthiohydantoin amino acids were identified by chromatography on a 2.1- by 200-mm phenylthiohydantoin C18 column.


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RESULTS
 
Purification of the bacteriocins and mass analysis by ESI-MS. Following cation exchange, two well-separated groups of active fractions were observed. When activity against L. lactis RO50 was tested by the spot-on-a-lawn method, only fractions eluted between min 30 and 39 (fraction group A) were active, but when E. faecium WHE 81 was used as the indicator strain, activity was also detected in fractions eluted between min 50 and 62 (fraction group B). Fraction groups A and B were then separately applied to a reverse-phase chromatography column. Fraction group A resulted in four well-defined absorbance peaks, and activity was detected in fractions corresponding to peaks 1, 2, and 2' (Fig. 1A). Peak 1 and 2 fractions were purified to homogeneity (Fig. 1B), and the activities of the separate peptides were checked. The reverse-phase chromatography analysis of fraction group B revealed a single active peak (peak 3) (Fig. 1C). The average molecular masses of the four active peptides (those corresponding to peaks 1, 2, 2', and 3) obtained by ESI-MS were as follows: 5,206 Da for peptide 1, 5,218 Da for peptide 2, 5,234 Da for peptide 2', and 6,390 Da for peptide 3. The nonactive peptide corresponding to peak 1' (Fig. 1A) was also analyzed by ESI-MS, and the mass obtained was 5,222 Da. Molecular masses of peptides 1, 1', 2, and 2' were close to those of enterocins L50A and L50B (8). A difference of 16 Da between peptides 1 and 1' and between peptides 2 and 2' was observed, suggesting that peptides 1' and 2' may be respective oxidized forms of peptides 1 and 2.

Structural analysis. Nano-LC/MS/MS analysis of tryptic digests of peptides 1, 2, and 2' allowed the identification of enterocins L50A and L50B (Table 2). Mascot searches including the possibility of methionine oxidation and N formylation were performed, given that such modifications are common features of bacteriocins (3, 34). In fact, two forms of L50A were identified: one was formylated at the N-terminal Met (peptide 2), and the second was oxidized at Met24 and formylated at the N-terminal Met (peptide 2'). Bacteriocin L50B formylated at the N-terminal Met (peptide 1) was identified. These results were in agreement with the molecular masses of intact peptides determined by ESI-MS analysis. Peptide 1' was not analyzed by MS/MS, but the analogy in the elution profiles of peptides 1 and 1' and the difference of 16 Da between these peptides suggested that peptide 1' corresponds to the N-formylated enterocin L50B with oxidation at Met24.


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  		TABLE 2. Tryptic peptide sequences observed by nano-LC/MS/MSa

Mascot search results obtained after nano-LC/MS/MS analysis of the tryptic digest from peptide 3 allowed the identification of four fragments (Table 2) that were present in a previously identified enterococcal bacteriocin, bacteriocin 32 (24). However, the theoretical average mass of bacteriocin 32 (M = 7,998 Da) did not correspond to the mass of the purified peptide 3 determined by ESI-MS (M = 6,390 Da). In order to obtain additional sequence information, peptide 3 was sequenced by Edman degradation. The first 38 amino acids (AAQRGYIYKKYPKGAKVPNKVKMLVNIRGKQTMRTCYL) could be determined. The combination of data obtained by Edman degradation and tryptic digestion allowed the identification of 94% of the sequence of peptide 3. Given the molecular mass of peptide 3, the remaining three amino acids were deduced based on the sequence of bacteriocin 32, whose C-terminal part was identical to peptide 3. This peptide, 16 amino acids shorter than bacteriocin 32, was named enterocin IT.

Activities of the purified bacteriocins. The activities of chromatographically isolated peptides (in sample volumes of 4 ml) were assessed by titration against the indicator strains E. faecium WHE 81 and L. lactis RO50. Enterocin L50A was more active against L. lactis RO50 (1,600 AU/ml) than against E. faecium WHE 81 (800 AU/ml). The same pattern was observed with enterocin L50B, which showed activities of 400 AU/ml when tested against L. lactis RO50 and 100 AU/ml when tested against E. faecium WHE 81. Enterocin IT, however, showed strong activity against E. faecium WHE 81 (102,400 AU/ml) but was not active at all against L. Lactis RO50.

The differences observed in the activity spectra of enterocins L50A, L50B, and enterocin IT were further investigated by testing the sensitivities of a wide range of bacteria to the three bacteriocins in liquid media by using an automated turbidometer. A strain was considered sensitive when its growth was partially or completely inhibited. In most instances, sensitive strains had their growth significantly slowed down, as was the case for L. lactis 72 and E. faecium WHE 81 (Fig. 2). The data for all indicator bacteria are summarized in Table 1, which presents the highest differences in population size obtained compared to controls during incubation.


Figure 2
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  		FIG. 2. Examples of the antimicrobial effects of purified bacteriocins on indicator bacteria. (A) L. lactis 72; (B) E. faecium WHE 81. {diamond}, control; {blacktriangleup}, enterocin L50A; -, enterocin L50B; {blacksquare}, enterocin IT. Arrows show the maximal differences in absorbance at 600 nm between control and assay samples obtained during growth.

All strains tested, even to a certain extent those of gram-negative bacteria, were inhibited by enterocins L50A and L50B, which is quite surprising considering LAB bacteriocins. While LAB tested were more sensitive to L50A than to L50B, gram-negative strains showed greater sensitivity to L50B than to L50A. The activity spectrum of enterocin IT was, on the other hand, very narrow, with only 5 strains (E. faecium strains WHE 81, 25, and CIP 5855, Bacillus subtilis CIP 7718, and L. lactis 72) of 31 strains tested being sensitive. The growth inhibition caused by enterocin IT was also weaker than that caused by enterocins L50A and L50B.


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DISCUSSION
 
According to the biochemical data presented in this work, E. faecium IT62 produces three different bacteriocins, enterocins L50A and L50B and enterocin IT. Enterocins L50A and L50B could be identified, but only in formylated and/or oxidized forms. Peptides 1 and 2 corresponded to N-formylated enterocins L50B and L50A, respectively. Peptides 1' and 2', on the other hand, carried an additional modification consisting of oxidation at Met24. Unmodified peptides were not isolated, suggesting that N-terminal formylation may be a native modification during bacteriocin biosynthesis. It is remarkable that enterocin L50A is still active after oxidation at Met24 but that no activity could be detected for enterocin L50B after a similar oxidation modification.

Enterocins L50A and L50B are widespread bacteriocins that are produced by several E. faecium strains isolated from very diverse food sources, such as Spanish dry fermented sausages (8), Spanish-stye green olive fermentations (16), Malaysian mold-fermented tempeh (28), Moroccan soft cheese (1), and Mongolian airag (3). Nevertheless, in some instances (1, 3), the experimental masses obtained after the purification of these bacteriocins have differed from the theoretical ones (5,190 Da for enterocin L50A and 5,178 Da for enterocin L50B). We suspect that these discrepancies are due to chemical modifications at Met residues which are likely to complicate the identification of enterocins.

The third bacteriocin isolated in the present work was a 6,390-Da peptide made of 54 amino acids that was named enterocin IT. The amino acid sequence of enterocin IT is identical to the sequence of the C-terminal part of the previously reported bacteriocin 32 (24) and 16 amino acids shorter than the sequence of the latter. The sequence of bacteriocin 32 was obtained based on the nucleotide sequence of its structural gene bacA (24), with an L L A sequence as the signal region for the cleavage of the prepeptide, i.e., at position 19 (Fig. 3). Comparison with other bacteriocin signal peptides (22) showed, however, that L L A is a less likely recognition region for the signal peptidase than V E A, which is located at positions 33 to 35 of the prepeptide. In fact, while L L A has not been reported as the cleavage site for other bacteriocins, V E A is the cleavage site for other sec-dependent bacteriocins, such as bacteriocin 31 (33) and enterocin SE-K4 (12). Also, the sequence V X A has been found for enterolysin A, bacteriocin T8, enterocin P, dysgalacticin, and hiracin JM79 (19, 21, 32). Provided that the cleavage region of the signal peptide sequenced by Inoue et al. (24) is not L L A but V E A, the bacteriocin obtained would have exactly the same size and sequence as the enterocin IT identified in the present work. However, bacteriocin 32 would have to be purified and sequenced to determine whether the cleavage site previously determined (24) is correct or not.


Figure 3
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  		FIG. 3. Amino acid sequence deduced by Inoue et al. (24) from the structural gene of bacteriocin 32. The putative signal region according to Inoue et al. is underlined with a single solid line, and the corresponding cleavage site is indicated by a white arrow. The bacteriocin 32 sequence is indicated by underlining with a dotted line. A more likely signal region is double underlined, and the corresponding cleavage site is indicated by a black arrow.

The activity spectra of the three purified bacteriocins, L50A, L50B, and enterocin IT, were determined. Enterocins L50A and L50B showed broad antimicrobial spectra, with activity against all strains tested in this study. Interestingly, enterocins L50A and L50B inhibited the growth of gram-negative strains, which is not a typical characteristic of bacteriocins produced by gram-positive bacteria (25). This activity against gram-negative strains was observed only in liquid media and could not be detected by the spot-on-a-lawn test. The activity spectra of enterocins L50A and L50B were very similar, but some differences could be observed. As previously reported (8), enterocin L50A was more active against gram-positive bacteria than enterocin L50B. On the other hand, all gram-negative strains were more sensitive to enterocin L50B than to enterocin L50A, suggesting that the modes of action of the two peptides may be different. Enterocins L50A and L50B are known for their synergistic activity (8), and their cumulative activity spectrum has already been determined (6, 16). However, this is the first report of the individual antimicrobial spectra of these two bacteriocins. The slight differences in their structures may be responsible for the complementary roles of the two peptides and may explain their synergistic properties. In contrast with enterocins L50A and L50B, enterocin IT showed a very narrow antimicrobial spectrum, since it was active only against E. faecium strains, one strain of B. subtilis, and one strain of L. lactis. A narrow spectrum was also reported previously for bacteriocin 32 (24).

E. faecium IT62, isolated from ryegrass, produced two kinds of bacteriocins with very different activities that were previously isolated from very different sources: on the one hand, enterocins L50A and L50B, two synergistic bacteriocins with a wide antimicrobial spectrum that were identified previously in several food isolates, and on the other hand, enterocin IT, a bacteriocin with a very narrow spectrum and a structure close to that of bacteriocin 32, which was identified previously in numerous clinical isolates of E. faecium. This finding goes against the idea of a particular ecological distribution of bacteriocins in which, for instance, the anti-Listeria bacteriocins would be found in food and bacteriocins such as bacteriocin 32 or enterocin IT would be found in clinical settings, where they would be associated with drug resistance (22). A bacterial strain can, in fact, as is the case for E. faecium IT62, produce bacteriocins with very different properties and activity spectra. Nonetheless, bacteria that produce the "right" bacteriocin in a particular ecological niche stand a better chance of surviving and becoming dominant.

In the search for bacteriocins, most purification processes are based on a single indicator strain, which offers a narrow vision of the antimicrobial activities of the bacteria investigated, especially when bacteriocins with narrow spectra are produced. These bacteriocins are likely to be neglected and discarded during the purification process in favor of bacteriocins with broader spectra. This pattern may explain why enterocin IT was not detected in the numerous cases in which enterocins L50A and L50B were found. The use of multiple indicator bacteria during the purification stages is therefore highly advisable in order to detect multibacteriocin production.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Chimie Analytique et Sciences de l'Aliment, IPHC-DSA, ULP, CNRS, 74 route du Rhin, 67400 Illkirch-Graffenstaden, France. Phone and fax: 33-390-244-325. E-mail: ennahar{at}pharma.u-strasbg.fr Back

{triangledown} Published ahead of print on 7 April 2008. Back


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Antimicrobial Agents and Chemotherapy, June 2008, p. 1917-1923, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01409-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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