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Antimicrobial Agents and Chemotherapy, May 2000, p. 1365-1366, Vol. 44, No. 5
Unité des Agents Antibactériens,
Institut Pasteur, 75724 Paris Cedex 15, France
Received 2 August 1999/Returned for modification 26 November
1999/Accepted 24 January 2000
Spectinomycin resistance in clinical isolates of Neisseria
meningitidis and Neisseria gonorrhoeae was found to
be due to mutations G1064C and C1192U (Escherichia coli
numbering) in 16S rRNA genes, respectively.
Among Neisseria species,
only N. meningitidis and N. gonorrhoeae are
considered primary pathogens (8). Strains of N. gonorrhoeae are always pathogenic, whereas strains of N. meningitidis, in addition to causing acute meningitis and
septicemia, can also colonize the oro- and nasopharynx of a healthy
carrier. Development of drug resistance in N. gonorrhoeae
has led to concern about the almost inevitable increase of resistance
in N. meningitidis (8). Cephalosporins and
fluoroquinolones are the two classes of antibiotics recommended for
primary therapy (8). Resistance to alternative therapy such
as chloramphenicol for meningococcal infections (5) or
spectinomycin for gonococcal infections (1) has been reported.
In gram-positive bacteria, resistance to spectinomycin, although much
less common than resistance to other aminoglycoside-aminocyclitol antibiotics, is usually due to production of an aminoglycoside 9-O-nucleotidyltransferase of type I (9, 12).
Recently, a spectinomycin phosphotransferase has been reported for the
gram-negative Legionella pneumophila (16). In
Escherichia coli, spectinomycin resistance has been shown to
be due to mutations in helix 34 of 16S rRNA (15). This helix
consists of an upper and a lower stem separated by an internal loop
containing two uracil residues.
We report mutations responsible for spectinomycin resistance in 16S
rRNA genes of clinical isolates of N. meningitidis and N. gonorrhoeae.
Since 1988, surveillance of antibiotic resistance in clinical isolates
of Neisseria spp. has been conducted at the National Center
for Meningococci, Institut Pasteur, Paris, France, by disk-agar diffusion. Out of more than 16,800 clinical isolates, a single N. meningitidis isolate, LNP16311, and four N. gonorrhoeae
isolates were found to be resistant to spectinomycin. The five strains of Neisseria spp. remained susceptible to penicillins,
cephalosporins, tetracyclines, macrolides, rifamycins, and quinolones.
Among the three N. gonorrhoeae strains isolated from
urethritis patients in Gabon (LNP8205, Libreville, May 1989; LNP8919
and LNP8920, Franceville, March 1990), only strain LNP8205, as well as
strain LNP9455, isolated from a urethritis patient in November 1990 at Saint Louis Hospital in Paris, was studied further.
N. meningitidis LNP16311, serogroup Y, was isolated in 1998 from the rhinopharynx of a 71-year-old male in Macon, France. A
spectinomycin-resistant transformant, N. meningitidis
BM4417, obtained after transformation of strain BM4377 (5,
13) with total DNA from LNP16311, was included in the study.
Spectinomycin-susceptible N. meningitidis LNP15908 and
BM4377 and N. gonorrhoeae LNP6910 were used as controls.
Total DNA from N. meningitidis LNP16311 and BM4417 and from
N. gonorrhoeae LNP8205 and LNP9455 was transferred to
nitrocellulose sheets (Nytran; Schleicher & Schuell, Dassel, Germany)
and hybridized to 32P-labeled
ant(9)-I- and aph-specific
probes (Radiochemical Centre, Amersham, England). Lack of hybridization
suggested that spectinomycin resistance in these strains was not due to
acquisition of known genes.
In structural models of E. coli 16S rRNA, spectinomycin
resistance mutations are located in the upper stem of helix 34 (Fig. 1) (7), which is formed by
base pairing of regions 1046 to 1067 and 1189 to 1211 (E. coli numbering [2, 3]). Comparison of the
sequence of 16S rRNA rrs genes from E. coli
(GenBank accession no. V00348) to those of N. meningitidis
(Sanger Centre; http://www.sanger.ac.uk/) indicated 79% identity with
contig 407.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Spectinomycin Resistance in Neisseria
spp. Due to Mutations in 16S rRNA
ABSTRACT
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FIG. 1.
Secondary structure of the upper stem of 16S rRNA helix
34 of E. coli (2). Transversion G1064C and
transition C1192U (E. coli numbering) in
spectinomycin-resistant N. meningitidis and N. gonorrhoeae, respectively, are indicated by open letters. The
locations of other spectinomycin resistance single mutations, A1191G,C
and G1193A in Chlamydomonas reinhardtii (6),
A1191C in N. tabacum chloroplast (17), G1193A in
Nicotiana undulata chloroplast (4), and
G1064U,C,A in E. coli (3), and of double
mutations G1064U-C1192A, G1064C-C1192G, and G1064A-C1192U in E. coli (3) are indicated by circles.
A region internal to rrs genes, including helix 34, of spectinomycin-susceptible and -resistant N. meningitidis and N. gonorrhoeae was explored by PCR using a DNA Thermal Cycler (model 2400; Perkin-Elmer Cetus, Norwalk, Conn.) and total DNA as a template. Two heptadecadeoxynucleotides (F.980, 5'CTTACCTGGTCTTGACA3', and R.1353, 5'CGATTACTAGCGATTCC3'; E. coli numbering) designed from contig 407 and synthesized by the methoxy-phosphoramidite method (Unité de Chimie Organique, Institut Pasteur) allowed amplification of fragments with the predicted size (data not shown).
Double-stranded sequencing of the 373-bp PCR products was performed by
the dideoxynucleotide chain termination method (14) with a
T7 Sequenase PCR product sequencing kit (Amersham, Little Chalfont,
Buckinghamshire, England), the 17-mer primers, and
-35S-dATP (Amersham). The sequence of helix 34 (positions 1046 to 1067 and 1189 to 1211, E. coli numbering)
from Neisseria strains was identical to that of E. coli except at position 1201, located in the lower stem of helix
34, where a transversion converted the adenine present in E. coli to a cytosine in Neisseria spp.
The sequence of helix 34 in the spectinomycin-resistant N. meningitidis clinical isolate LNP16311 differed from that of
susceptible strain LNP15908 by a guanine-to-cytosine transversion at
position 1064 (E. coli numbering [Fig. 1]). An identical
substitution was found in the spectinomycin-resistant transformant
BM4417 relative to BM4377. In both cases, the sequence of the
amplification product obtained directly without cloning did not display
any ambiguity (Fig. 2). These data
indicate that, like in LNP16311, each of the three rrs genes
(10) in N. meningitidis BM4377 has been altered,
an observation compatible with the small number of rrn operons in this species. Mutations at this position conferring spectinomycin resistance have been described for E. coli
(G1064U,C,A [3]) and Nicotiana tabacum
chloroplast (G1064A [4]).
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Spectinomycin-resistant N. gonorrhoeae LNP8205 differed from susceptible LNP6910 by a cytosine-to-thymine transition at position 1192 (E. coli numbering [Fig. 1 and data not shown]). Similar mutations conferring spectinomycin resistance have been described for E. coli (C1192U [15] and C1192U,G,A [11]) and N. tabacum chloroplast (C1192U [17]).
In conclusion, spectinomycin resistance in N. meningitidis
and N. gonorrhoeae was due to mutations already found in the
16S rRNA genes. Spectinomycin alone is used in infections due to
N. gonorrhoeae, in particular in the case of a high
prevalence of 
-lactamase-producing strains (1), and is
likely to be responsible for emergence of resistance. The reason for
the mutation in N. meningitidis, against which spectinomycin
is not used, remains unknown.
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ACKNOWLEDGMENTS |
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We thank J. M. Alonso and M. Guibourdenche for the gift of strains.
This work was supported in part by a Bristol-Myers Squibb Unrestricted Biomedical Research Grant in Infectious Diseases.
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FOOTNOTES |
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* Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: (33) (1) 45 68 83 18. Fax: (33) (1) 45 68 83 19. E-mail: galimand{at}pasteur.fr.
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Antimicrobial Agents and Chemotherapy, May 2000, p. 1365-1366, Vol. 44, No. 5
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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