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Letter
Rifampin-resistant Neisseria
meningitidis
Muhamed-Kheir Taha,*
Maria Leticia Zarantonelli,* Corinne Ruckly,* Dario Giorgini,* and Jean-Michel
Alonso*
*Institut Pasteur, Paris, France
Suggested
citation for this article
To the Editor: Immediate management of meningococcal disease requires
antimicrobial drug treatment of patients with β-lactams and chemoprophylaxis
of contact persons with rifampin. High-level resistance to rifampin (MIC
>32 mg/L) in Neisseria meningitidis is provoked by mutations
(most frequently at the residue His 552) in the rpoB gene encoding
the b subunit of RNA polymerase (1,2). Resistance
may lead to chemoprophylaxis failure and must be rapidly detected (3).
Concerns have been raised about the clonal spread of resistant isolates
(1); however, rifampin-resistant isolates are
rarely reported. We tested 6 N. meningitidis isolates corresponding
to 3 pairs of linked cases of meningococcal disease. In each pair, the
index case was due to a rifampin-susceptible isolate and was followed
by the secondary case due to a resistant isolate in a contact person.
Phenotyping and genotyping of the isolates showed that each pair belonged
to a different major serogroup (A, B, and C) and to a different genetic
lineage (ST-7, ST-32, and ST-2794) (Figure). We
next amplified a fragment in rpoB between codons 421 and 701 by
using oligonucleotide rpoBF1 (5´gttttcccagtcacgacgttgtaCTGTCCGAAGCCCAACAAAACTCTTGG3´)
and rpoBR1 (5´ttgtgagcggataacaatttcTTCCAAGAATGGAATCAGGGATGCTGC3´).
The 2 oligonucleotides harbor adaptors (in lower case) corresponding to
universal forward and reverse oligonucleotides that can be used for sequencing
after amplification. We also analyzed 2 cerebrospinal fluid (CSF) samples
corresponding to 2 linked culture-negative cases of meningococcal disease
in which the second case was believed to have been caused by rifampin-resistant
N. meningitidis. These 2 cases were diagnosed by polymerase chain
reaction (PCR) detection of meningococcal DNA, as previously described
(4).
The 3 rifampin-susceptible isolates harbored a wild-type rpoB
sequence (His 552), as did the first CSF sample. All 3 rifampin-resistant
isolates harbored a His→Tyr mutation, while analysis of the second
CSF sample showed a His→Asn mutation (Figure).
Both mutations have been observed in N. meningitidis (3).
No other difference in the sequence was seen among all isolates on the
amplified fragment. This approach can rapidly detect rpoB mutations
and can be applied to culture-negative clinical samples.
The virulence of the isolates was evaluated through their ability to
provoke bacteremia in mice after 6-week-old female BALB/c mice (Janvier,
France) were injected intraperitoneally. Bacteremia is a good indicator
of bacterial virulence as it reflects bacterial survival upon invasion
of the bloodstream. The experimental design was approved by the Institut
Pasteur Review Board. The rifampin-resistant clinical isolate LNP22330
showed substantially reduced bacteremia when compared to the corresponding
susceptible isolate LNP21362 (Figure). Such a reduction
was not significant for the other 2 pairs (LNP18278/LNP18378 and LNP18368/LNP18491),
but these strains were all less virulent than LNP21362, with ≈1
log10 lower blood bacterial loads. The 3 pairs of isolates
belonged to different genetic lineages according to the multilocus sequence
typing typing. Indeed, we have recently proved that virulence of meningococcal
isolates in the mouse model depends on the genetic lineage of the tested
isolate (5).
To better study the impact of rpoB mutation on meningococcal virulence
we constructed an isogenic mutant strain, NM05-08, by transforming the
susceptible isolate LNP21362 with a PCR-amplified fragment from a resistant
isolate (LNP22330), as previously described (6).
The PCR fragment corresponded to the product of amplification between
the oligonucleotides ropB1UP (5´ggccgtctgaaCTGTCCGAAGCCCAACAAAACTCTTGG3´)
and rpoBR1. The oligonucleotide RpoB1UP is the same as the upstream rpoBF1
but with a DNA uptake sequence (in lower case) that was added at the 5´
end to permit DNA transformation (7). The transformant
strain NM05-08 was resistant to rifampin (MIC >32 mg/L), and the sequence
of the rpoB gene confirmed the His→Tyr mutation. When compared
to the parental isolate (LNP21362), strain NM05-08 showed reduced virulence.
Indeed, bacterial loads were similar to those observed for the resistant
isolate LNP22330 (Figure). These results strongly
suggest a direct negative impact of rpoB mutations on meningococcal
virulence. Mutations in the rpoB gene have been reported to confer
pleiotropic phenotypes (8).
The data reported here show that rifampin-resistant isolates were not
clonal but belonged to different genetic lineages. The results of virulence
assays in mice suggest that mutations in rpoB in resistant isolates
may have a major biological cost for N. meningitidis, which can
be defined as lower bacterial fitness in terms of survival in the bloodstream.
This biological cost could explain the lack of clonal expansion of meningococcal
isolates that acquired resistance to rifampin.
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Suggested citation
for this article:
Taha M-K, Zarantonelli
ML, Ruckly C, Giorgini D, Alonso J-M. Rifampin-resistant Neisseria
meningitidis [letter]. Emerg Infect Dis [serial on the Internet].
2006 May [date cited]. Available from http://www.cdc.gov/ncidod/EID/vol12no05/05-1296.htm
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