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Antimicrobial Agents and Chemotherapy, December 2000, p. 3425-3427, Vol. 44, No. 12
Danish Veterinary Laboratory, DK-1790 Copenhagen V,
Denmark
Received 30 November 1999/Returned for modification 28 July
2000/Accepted 24 August 2000
Fragments (414 bp) of the gene-encoding ribosomal protein L16 from
Enterococcus faecium and Enterococcus faecalis
that were resistant and susceptible to the oligosaccharide antibiotics
avilamycin and evernimicin (SCH 27899) were sequenced and compared. The
susceptible E. faecalis and E. faecium isolates
had sequences that were similar to those of the type strains. All
resistant E. faecalis isolates contained the same base pair
variation [CGT (Arg-56) Multiply resistant enterococci have
emerged as increasingly important nosocomial pathogens during the
last decade (10, 12, 13). This has increased the
interest in searching for new antibiotics or modifications of older
antibiotics with activity against multiply resistant staphylococci and
enterococci. One of these agents is evernimicin (SCH 27899) (Ziracin),
an oligosaccharide antimicrobial agent belonging to the
everninomicins that has been developed by Schering-Plough. This
compound has shown excellent activity against enterococci,
staphylococci, and streptococci of human origin (5, 6, 8, 11,
14) but was recently suspended by the company from any further
clinical development. The everninomicins have been known since the
1960s (15) but have not previously gained any clinical interest.
Another oligosaccharide, avilamycin, has been used as a growth promoter
for food animals in the European Union for several years, and
resistance to avilamycin has frequently been found among
Enterococcus faecium isolates from broilers in Denmark
(2). Cross-resistance between avilamycin and
evernimicin has been detected among Enterococcus
faecalis and E. faecium isolates
(1).
The mode of action of avilamycin and evernimicin is not well
elucidated. It has been suggested that avilamycin acts by binding to
the 30S part of the ribosome and thereby inhibiting the protein synthesis (16). However, recently Adrian and Klugman
reported that single base-pair mutations in ribosomal protein L16
giving rise to an amino acid substitution (Ile-52 This study was conducted to assess the effects of variations in the L16
sequence of E. faecalis and E. faecium isolates
on susceptibility to the oligosaccharide antimicrobial agents
avilamycin and evernimicin (SCH27899).
Bacterial isolates.
The bacterial isolates chosen for sequence
analysis of L16 are shown in Table 1. The
isolates were chosen on the basis of their susceptibility or resistance
to avilamycin. Eleven avilamycin-resistant and 4 susceptible
isolates of E. faecalis and 11 resistant and 6 susceptible
E. faecium isolates were chosen. All isolates
originated from different broiler farms or pig herds and were
collected from the continuous surveillance of antimicrobial resistance
among food animals in Denmark between 1995 and 1998 (2). The following reference strains were included:
E. faecalis ATCC 19433, E. faecalis ATCC
29212, and E. faecium CCUG542.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Presence of Variations in Ribosomal Protein L16 Corresponding
to Susceptibility of Enterococci to Oligosaccharides
(Avilamycin and Evernimicin)
ABSTRACT
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References
CAT (His-56)]. The same variation
and two additional variations [ATC (Ile-52) ACC
(Thr-52) and ATC (Ile-52) AGC (Ser-52)] were found in the resistant E. faecium isolates. This study indicated
that resistance to the oligosaccharides in enterococci is associated
with variations in the ribosomal protein L16.
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Ser-52 or Thr-52) resulted in decreased susceptibility to evernimicin in
Streptococcus pneumoniae (P. V. Adrian and K. P. Klugman, Abstr. 38th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. C110, 1998). Furthermore, McNicholas et al.
(7) showed that evernimicin binds the 50S subunit and that
the binding sites for avilamycin and evernimicin overlap on the 50S subunit.
TABLE 1.
Origin, drug susceptibility, and observed mutations of
Enterococcus isolates chosen for sequencing
Susceptibility testing. Susceptibility to avilamycin was determined by culturing on Mueller-Hinton II agar plates containing twofold serial dilutions of antimicrobials (MIC determinations) at dilutions ranging from 0.25 to 128 µg/ml, according to NCCLS guidelines (9). The susceptibility to evernimicin (SCH27899) was determined by using the E-test according to the manufacturer's guidelines (AB Biodisk, Solna, Sweden).
PCR amplification and DNA sequencing.
The sequence for
L16 for Bacillus subtilis (accession number U43929) and
S. pneumoniae (accession number AF126059) was retrieved
from GenBank. The sequence from B. subtilis was used to
search the database of The Institute for Genomic Research
[TIGR] (http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi?) for
similar sequences for E. faecalis. Similarly, L16 sequences
of Staphylococcus aureus, Streptococcus mutans, and
Streptococcus pyogenes were also retrieved from the TIGR
database. Based on a similar sequence of E. faecalis (V583),
fragments (414 internal base pairs) of the L16 gene were amplified
using the primers P1 (5'-AAACGTGTAAAACACCGTCG-3') and P2
(5'CATTCGATTCACCACCCATT-3') (Fig.
1). The amplification products were
sequenced on an ABI 373A automatic sequencer using the AmpliTaq FS dye
terminator kit (Applied Biosystems, Foster City, Calif.). The sequences
were compared and analyzed using DNAsis software (Hitachi Software
Engineering Co., Ltd.).
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PFGE. Pulsed-field gel electrophoresis (PFGE) analysis of the 22 avilamycin-resistant E. faecalis and E. faecium isolates chosen for sequence analysis was performed as previously described (4).
Complete agreement between resistance and susceptibility to avilamycin and evernimicin (SCH27899) was found among the isolates. The MICs of avilamycin were between 0.5 and 2 µg/ml for the susceptible isolates and from 32 to >64 µg/ml for the resistant isolates (Table 1). For evernimicin (SCH27899) the MICs ranged from 0.125 to 0.75 µg/ml for the avilamycin-susceptible isolates and from 2 to 8 µg/ml for the avilamycin-resistant isolates. All oligosaccharide-susceptible E. faecalis isolates had a DNA sequence identical to the sequence retrieved from the TIGR database. The E. faecium type strain CCUG542 and the five oligosaccharide-susceptible E. faecium isolates all shared the same DNA sequence. This sequence showed 89% DNA homology and 95% amino acid homology to the sequence of E. faecalis. Two different mutations associated with decreased susceptibility to evernimicin have previously been observed among S. pneumoniae isolates (Adrian and Klugman, 38th ICAAC). These mutations, ATC (Ile-52) AGC (Ser-52) and ATC (Ile-52) ACC
(Thr-52), were detected among 1 and 5, respectively, of the 11 E. faecium isolates examined in this study (Table 1 and Fig. 1). In
addition, another mutation [CGT (Arg-56) CAT (His-56)] was
detected in all the 11 resistant E. faecalis isolates and in
the remaining 5 resistant E. faecium isolates.
All resistant E. faecium isolates were of different
genotypes as determined by PFGE typing. This indicates that resistance has developed among several different clones. In contrast, all E. faecalis isolates belonged to the same indistinguishable
SmaI PFGE type, even though all the isolates were from
different herds and collected during a period of 3 years.
When the DNA sequence and translated amino acid sequence of the L16
gene were compared to those of B. subtilis, E. faecalis, S. aureus, S. pneumoniae, and S. pyogenes, it was observed
that all three mutations were within an otherwise conserved
21-amino-acid area of L16. The observed conservation of this area in
several bacterial species could indicate that this region is essential for the function of L16 in gram-positive bacteria. The conservation in
amino acid sequence could perhaps indicate that
oligosaccharide-resistant variants with changes in this region
are less fit than the susceptible variants and that resistance thus
will disappear over time.
Recently, a decrease in the occurrence of avilamycin-resistant
E. faecium isolates has been detected along with a
decreased consumption (3), indicating that resistance will
decrease when the selective pressure is removed.
In conclusion, the observations in this study and the studies by Adrian
and Klugman (38th ICAAC) and McNicholas et al. (7; P. M. McNicholas, P. A. Mann, D. J. Najarian, L. Miesel,
R. S. Hare, K. J. Shaw, and T. A. Black. Abstr. 39th
Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-846,
1999) strongly suggest that oligosaccharide antimicrobial agents
such as avilamycin and evernimicin (SCH27899) act by binding to
ribosomal protein L16 and thereby probably interact with the
peptidyltransferase activity.
Nucleotide sequence accession numbers. The sequences obtained in this study have been deposited in GenBank under the accession numbers AF291861, AF291862, AF291863, AF291864, and AF291865.
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ACKNOWLEDGMENTS |
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We are grateful to René Hendriksen, Betina Elemark, Dorte Nielsen, and Christina Aaby Svendsen for technical assistance. The E-test was supplied by Schering-Plough Research Institute, Bloomfield, N.J.
This study was supported by a grant from Schering-Plough.
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FOOTNOTES |
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* Corresponding author. Mailing address: Danish Veterinary Laboratory, 27 Bülowsvej, DK-1790 Copenhagen V, Denmark. Phone: 45 35 30 01 00. Fax: 45 35 30 01 20. E-mail: faa{at}svs.dk.
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Antimicrobial Agents and Chemotherapy, December 2000, p. 3425-3427, Vol. 44, No. 12
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