ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2004, p. 4882–4885 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.12.4882–4885.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 12
VanE-Type Vancomycin-Resistant Enterococcus faecalis Clinical Isolates from Australia Lorena Abadía-Patin ˜o,1† Keryn Christiansen,2 Jan Bell,3 Patrice Courvalin,1 and Bruno Pe´richon1* Unite´ des Agents Antibacte´riens, Institut Pasteur, Paris, France,1 and Department of Microbiology and Infectious Diseases, Royal Perth Hospital, Perth, Western Australia,2 and Women’s and Children’s Hospital, Adelaide, South Australia,3 Australia Received 22 June 2004/Returned for modification 16 July 2004/Accepted 9 August 2004
the activated VanS. We have previously demonstrated in E. faecalis BM4405 (i) that the five genes are cotranscribed from a PE promoter located upstream from the operon and (ii) that VanE, VanXYE, and VanTE are sufficient to confer low-level resistance to vancomycin (1). To date, only two VanE-type strains have been isolated (7, 11, 22). We have studied three additional VanE-type resistant strains isolated from rectal swabs collected from patients screened for glycopeptide-resistant enterococci at the Royal Perth Hospital in Australia in 2001 during an outbreak of vancomycin-resistant enterococci (E. Lambert, C. McCullough, G. Coombs, J. Pearson, F. O’Brien, J. Bell, A. Berry, and K. Christiansen, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-1118, 2002) (8). These strains— BM4574, BM4575, and BM4576—were resistant to low levels of vancomycin (MICs of 6, 32, and 16 g/ml, respectively) and susceptible to teicoplanin (MIC of 0.5 g/ml). Organization of the vanE operon in these strains was determined by PCR mapping by using several pairs of primers (Table 1) specific for each gene of the prototype BM4405 vanE operon. The amplification products had the expected size (data not shown), indicating that all of the genes constituting the vanE operon were present in the three strains and in the same order as in BM4405 (1). BM4574, BM4575, and BM4576 strains were compared by contour-clamped homogeneous electric field gel electrophoresis. Genomic DNA embedded in agarose plugs was digested overnight at 27°C with 25 U of SmaI, and fragments were separated on a 0.8% agarose gel by using a CHEF-DRIII system (Bio-Rad, Hercules, Calif.) under the following conditions: total migration, 24 h; initial pulse, 60 s; final pulse, 120 s; voltage, 6 V/cm; included angle, 120°; and temperature, 14°C. SmaI-generated fragments indicated that the three isolates were different (data not shown). The cytoplasmic peptidoglycan precursors of BM4574, BM4575, and BM4576 grown in the absence or in the presence of a subinhibitory concentration (half the MIC) of vancomycin (Table 2) were analyzed as described previously (15). In the absence of induction, UDP-Mur-Nac-pentapeptide(D-Ala) was the main late precursor (89 to 92% of all precursors) synthe-
Binding to the C-terminal dipeptide of peptidoglycan intermediates by vancomycin and teicoplanin results in inhibition of the transglycosylation and transpeptidation reactions in peptidoglycan assembly (19). Six types of glycopeptide resistance have been described to date in enterococci: five are acquired (VanA, -B, -D, -E, and -G) (4, 11, 14, 18) and one, VanC, is an intrinsic property of Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens (13, 16). Glycopeptide resistance is due to the replacement of the normal peptidoglycan precursors by modified precursors terminating in D-Ala-D-lactate (D-Ala-D-Lac; VanA, -B, and -D) or in D-Ala-D-serine (D-Ala-DSer; VanC, -E, and -G) which exhibit diminished binding affinity for glycopeptides (5). In addition to production of modified peptidoglycan precursors, resistant strains are also able to eliminate the precursors normally synthesized by the host (4). VanE-type glycopeptide resistance, first described in 1999 in Enterococcus faecalis BM4405 and characterized by low-level resistance to vancomycin and susceptibility to teicoplanin, is phenotypically and biochemically similar to VanC (11). The chromosomally located vanE operon is composed of five genes encoding a ligase (VanE) which synthesizes the dipeptide DAla-D-Ser, a bifunctional D,D-peptidase (VanXYE) that possesses D,D-dipeptidase (hydrolysis of the dipeptide D-Ala-D-Ala synthesized by the host ligase) and D,D-carboxypeptidase (elimination of the C-terminal residue of peptidoglycan precursors synthesized from D-Ala-D-Ala that has escaped VanXYE hydrolysis) activities, a serine racemase (VanTE) that converts L-Ser to D-Ser, and a two-component regulatory system (VanRE/VanSE) that is implicated in the regulation of expression of the resistance genes (1, 7). VanS-type proteins are membrane-bound histidine kinases, and VanR-type proteins act as transcriptional activators that can be phosphorylated on an aspartate residue by acquisition of the phosphoryl group of
* Corresponding author. Mailing address: Unite´ des Agents Antibacte´riens, Institut Pasteur, 25, Rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: (33) (1) 45-68-83-18. Fax: (33) (1) 45-68-83-19. E-mail:
[email protected]. † Present address: IIBCA Universidad de Oriente, Biomedica, Cerro del Medio, 6101 Cumana´, Edo. Sucre, Venezuela. 4882
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Three distinct Enterococcus faecalis VanE-type isolates—BM4574, BM4575, and BM4576–obtained in Australia were studied. Expression of the resistance genes was constitutive in BM4575, probably due to a 2-bp deletion into the vanSE gene, and inducible in BM4574 and BM4576. Transcription analysis of the vanE operons suggested that the five genes were cotranscribed from an initiation site located 25 bp upstream from the ATG start codon of vanE.
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TABLE 1. Oligodeoxynucleotides used for PCR mapping Primer
Sequence
Positiona
E37 E8 E6 E15 TE3 VRM2 SE2 VRM1
5⬘-GGATCACCGAAGAAGGT 5⬘-CAAATCCACTGCTAAAC 5⬘-TGGTTTAGCAGTGGATT 5⬘-CAGAAGCTGAGCTAGT 5⬘-GGTTAGGTACAGAGT 5⬘-TAACTCTTCTGACGATAT 5⬘-GGAGTTCTTAAGTCATGT 5⬘-GTTATGGCGCATATTGCT
119–135 1345–1329 1327–1343 2576–2561 3688–3702 4319–4302 4921–4904 4365–4382
a
Amplified genes
vanE-vanXYE vanXYE-vanTE vanTE-vanRE vanRE-vanSE
Nucleotide numbering begins at the first base of the vanE gene.
TABLE 2. Cytoplasmic peptidoglycan precursors synthesized and D,D-peptidase (VanXYE) and serine racemase (VanTE) activities in extracts from the VanE-type strains % Peptidoglycan precursorsa E. faecalis strain
Culture condition
Activity (nmol/min/mg of protein)
UDPMurNactetrapeptide
UDPMurNacpentapeptide (D-Ala)
UDPMurNacpentapeptide (D-Ser)
D,D-Dipeptidase
cytoplasmic fraction
D,D-carboxypeptidase
cytoplasmic fraction
Serine racemase membrane fraction
BM4574
Uninduced Vm (3 g/ml)
7 13
89 4
4 83
0.30 ⫾ 0.12 1.5 ⫾ 0.51
0.44 ⫾ 0.28 2 ⫾ 0.43
8 33
BM4575
Uninduced Vm (16 g/ml)
12 12
10 NDb
78 89
1 ⫾ 0.32 1.45 ⫾ 0.41
0.87 ⫾ 0.21 1.1 ⫾ 0.34
22 29
BM4576
Uninduced Vm (8 g/ml)
4 12
92 5
4 83
0.57 ⫾ 0.19 2 ⫾ 0.42
0.51 ⫾ 0.2 2.16 ⫾ 0.39
9 43
a The cytoplasmic peptidoglycan precursors of strains grown with or without vancomycin were analyzed as described previously (21). Enzyme assays were performed with the S100 and C100 fractions of uninduced or induced cultures as described previously (9). Vm, vancomycin. b ND, not detectable.
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enzyme was inducible by vancomycin in BM4574 and BM4576 but constitutive in BM4575 (Table 2). The kinase and phosphatase activities of VanS-type sensors control the level of phosphorylation of VanR-type proteins (2, 6, 23) and therefore the expression level of van operons. To test whether a mutation in the VanSE sensor was likely to be responsible for constitutivity of vancomycin resistance in BM4575, the nucleotide sequence of the vanSE gene of the three strains was determined. Gene amplification was carried out with the specific primers RE1 (5⬘-CCGAGACAGCCA AAT) and E67 (5⬘-TCCTGAGCTAAGATAGCTTAG). The PCR products were ligated into pCR2.1 (Invitrogen, Groningen, The Netherlands), transformed into Escherichia coli Top10, and sequenced. A 2-bp deletion leading to a truncated protein of 271 amino acids instead of 357 was found in the constitutive BM4575 strain. The truncated VanSE (GenBank accession no. AY700375) did not possess the G1, F, and G2 domains that are conserved in, and which form a nucleotidebinding surface within the active site of, the sensors of twocomponent regulatory systems (17). Furthermore, it has been demonstrated that the G2 box plays an important role in modulating phosphatase activity (10, 12, 24). Thus, it is likely that deletion of these domains is responsible for constitutive expression of the resistance genes in BM4575. However, based on the observation of slightly increased D,D-peptidase and serine racemase activities and of UDP-Mur-Nac-pentadepeptide(D-Ser) after growth of BM4575 in presence of vancomy-
sized by BM4574 and BM4576, whereas BM4575 produced mainly UDP-Mur-Nac-pentapeptide(D-Ser) (78%) (Table 2). After incubation with vancomycin, UDP-Mur-Nac-tetrapeptide (12 to 13%) and high proportions (83 to 89%) of UDPMur-Nac-pentadepeptide(D-Ser) were detected in the three strains. These data indicate that two strains, BM4574 and BM4576, were inducibly resistant to vancomycin by production of precursors ending in D-Ala-D-Ser, whereas BM4575 was constitutively resistant. D,D-Dipeptidase and D,D-carboxypeptidase activities in the three strains were assayed in enzyme extracts prepared as described previously (3, 20). The amount of D-Ala released from hydrolysis of the D-Ala-D-Ala dipeptide (VanX activity) and from UDP-Mur-NAc-L-Ala-␥-D-Glu-L-Lys-D-Ala-D-Ala pentapeptide (VanY activity) was measured in the cytoplasmic and membrane fractions (Table 2). Very weak D,D-dipeptidase and D,D-carboxypeptidase activities were found in the cytoplasmic extracts (Table 2). No D,D-peptidase activity was found in the membrane extracts. These results are similar to those previously obtained with BM4405 (11). VanTE is a membrane-bound serine racemase required for production of D-Ser. Serine racemase activity was assayed in the three strains as described previously (9). VanTE activity was detected in the membrane fractions from uninduced or induced BM4575, whereas much larger amounts were present in the membrane fractions of induced than uninduced strains BM4574 and BM4576, thus confirming that production of the
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ANTIMICROB. AGENTS CHEMOTHER.
cin, the truncated VanSE seems to retain partial activity. No mutations relative to the prototype BM4405 VanE-type strain were found in the inducibly resistant strains BM4574 and BM4576. Transcriptional analysis of the vanE operons was performed. Total RNA of the three strains was analyzed by Northern blot and primer extension by using probes internal to every gene in the vanE operon as described previously (1). As in VanE-type BM4405, a single transcript of ca. 5,800 nucleotides was observed that hybridized with all of the probes, suggesting that the five genes were cotranscribed (data not shown). Primer extension was performed to locate the transcriptional start site for vanE in the three strains (Fig. 1). A signal located 25 bp upstream from the start codon of vanE was detected. The PE promoter contained putative ⫺35 (TTGATA) and ⫺10 (TATACT) regions that differed from those in BM4405 (1) and were separated by 22 bp. In conclusion, vancomycin resistance in BM4574, BM4575, and BM4576 was due to the presence of a typical vanE operon. Expression of the resistance genes was inducible by vancomycin in BM4574 and BM4576, whereas it was constitutive in BM4575, most probably due to synthesis of a truncated VanSE protein. Only two VanE-type E. faecalis strains have been described previously, and it is of interesting that the VanE strains reported here were isolated in 2001 during the systematic screening for glycopeptide-resistant enterococci during an outbreak. The mechanism of acquisition by E. faecalis of VanE-type resistance remains unknown. Attempts to transfer resistance to other E. faecalis or E. faecium strains were unsuccessful (7, 11). Genes for an integrase and a putative excisionase that may have been involved in the acquisition of the operon have been found in the vanE operon of strain N00-410,
which is located at a site where mobile elements have been shown to integrate (7). It appears that the vanE operon has spread in E. faecalis and that VanE-type resistance may be more prevalent than initially thought. We thank P. Reynolds for technical advice in peptidoglycan precursor determination and for reading of the manuscript. L.A.-P. received a grant from the Fondo Nacional de Ciencia y Tecnología of the Venezuelan government. REFERENCES 1. Abadía-Patin ˜ o, L., P. Courvalin, and B. Pe´richon. 2002. vanE gene cluster of vancomycin-resistant Enterococcus faecalis BM4405. J. Bacteriol. 184:6457– 6464. 2. Arthur, M., F. Depardieu, G. Gerbaud, M. Galimand, R. Leclercq, and P. Courvalin. 1997. The VanS sensor negatively controls VanR-mediated transcriptional activation of glycopeptide resistance genes of Tn1546 and related elements in the absence of induction. J. Bacteriol. 179:97–106. 3. Arthur, M., F. Depardieu, P. Reynolds, and P. Courvalin. 1996. Quantitative analysis of the metabolism of soluble cytoplasmic peptidoglycan precursors of glycopeptide-resistant enterococci. Mol. Microbiol. 21:33–44. 4. Arthur, M., P. E. Reynolds, and P. Courvalin. 1996. Glycopeptide resistance in enterococci. Trends Microbiol. 4:401–407. 5. Arthur, M., P. E. Reynolds, F. Depardieu, S. Evers, S. Dutka-Malen, R. Quintiliani, Jr., and P. Courvalin. 1996. Mechanisms of glycopeptide resistance in enterococci. J. Infect. 32:11–16. 6. Baptista, M., F. Depardieu, P. Reynolds, P. Courvalin, and M. Arthur. 1997. Mutations leading to increased levels of resistance to glycopeptide antibiotics in VanB-type enterococci. Mol. Microbiol. 25:93–105. 7. Boyd, D. A., T. Cabral, P. Van Caeseele, J. Wylie, and M. R. Mulvey. 2002. Molecular characterization of the vanE gene cluster in vancomycin-resistant Enterococcus faecalis N00–410 isolated in Canada. Antimicrob. Agents Chemother. 46:1977–1979. 8. Christiansen, K. J., P. A. Tibbett, W. Beresford, J. W. Pearman, R. C. Lee, G. W. Coombs, I. D. Kay, F. G. O’Brien, S. Palladino, C. R. Douglas, P. D. Montgomery, T. Orrell, A. M. Peterson, F. P. Kosaras, J. P. Flexman, C. H. Heath, and C. A. McCullough. 2004. Eradication of a large outbreak of a single strain of vanB vancomycin-resistant Enterococcus faecium at a major Australian teaching hospital. Infect. Control Hosp. Epidemiol. 25:384–390. 9. Depardieu, F., M. G. Bonora, P. E. Reynolds, and P. Courvalin. 2003. The
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FIG. 1. Identification of the transcriptional start site by primer extension analysis. (Left) Primer elongation product obtained with oligodeoxynucleotide PE1 (1) and total RNA from BM4574 (lane 1), BM4575 (lane 2), and BM4576 (lane 3). Lanes T, G, C, and A show the results of sequencing reactions performed with the PE1 primer. The ⫹1 transcriptional start site is indicated by an arrow. (Right) Sequence from nucleotide positions ⫺81 to 12 (numbering from the A of the ATG start codon of vanE). The ribosome-binding site, the ⫹1 transcriptional start site for the vanE, vanXYE, vanTE, vanRE, and vanSE mRNA, and the ⫺35 and ⫺10 promoter sequences located upstream are in boldface and are indicated. The ATG start codon of vanE is underlined.
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