Rifampin-resistant Neisseria meningitidis
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《传染病的形成》
Institut Pasteur, Paris, France
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.
References
Carter PE, Abadi FJ, Yakubu DE, Pennington TH. Molecular characterization of rifampin-resistant Neisseria meningitidis. Antimicrob Agents Chemother. 1994;38:1256–61.
Nolte O, Muller M, Reitz S, Ledig S, Ehrhard I, Sonntag HG. Description of new mutations in the rpoB gene in rifampin-resistant Neisseria meningitidis selected in vitro in a stepwise manner. J Med Microbiol. 2003;52:1077–81.
Stefanelli P, Fazio C, La Rosa G, Marianelli C, Muscillo M, Mastrantonio P. Rifampin-resistant meningococci causing invasive disease: detection of point mutations in the rpoB gene and molecular characterization of the strains. J Antimicrob Chemother. 2001;47:219–22.
Taha MK. Simultaneous approach for nonculture PCR-based identification and serogroup prediction of Neisseria meningitidis. J Clin Microbiol. 2000;38:855–7.
Lancellotti M, Guiyoule A, Ruckly C, Hong E, Alonso JM, Taha MK. Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex. Microbes Infect. 2006;8:191–6.
Antignac A, Kriz P, Tzanakaki G, Alonso JM, Taha MK. Polymorphism of Neisseria meningitidis penA gene associated with reduced susceptibility to penicillin. J Antimicrob Chemother. 2001;47:285–96.
Goodman SD, Scocca JJ. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A. 1988;85:6982–6.
Jin DJ, Gross CA. Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli. J Bacteriol. 1989;171:5229–31.(Muhamed-Kheir Taha, Maria)
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.
References
Carter PE, Abadi FJ, Yakubu DE, Pennington TH. Molecular characterization of rifampin-resistant Neisseria meningitidis. Antimicrob Agents Chemother. 1994;38:1256–61.
Nolte O, Muller M, Reitz S, Ledig S, Ehrhard I, Sonntag HG. Description of new mutations in the rpoB gene in rifampin-resistant Neisseria meningitidis selected in vitro in a stepwise manner. J Med Microbiol. 2003;52:1077–81.
Stefanelli P, Fazio C, La Rosa G, Marianelli C, Muscillo M, Mastrantonio P. Rifampin-resistant meningococci causing invasive disease: detection of point mutations in the rpoB gene and molecular characterization of the strains. J Antimicrob Chemother. 2001;47:219–22.
Taha MK. Simultaneous approach for nonculture PCR-based identification and serogroup prediction of Neisseria meningitidis. J Clin Microbiol. 2000;38:855–7.
Lancellotti M, Guiyoule A, Ruckly C, Hong E, Alonso JM, Taha MK. Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex. Microbes Infect. 2006;8:191–6.
Antignac A, Kriz P, Tzanakaki G, Alonso JM, Taha MK. Polymorphism of Neisseria meningitidis penA gene associated with reduced susceptibility to penicillin. J Antimicrob Chemother. 2001;47:285–96.
Goodman SD, Scocca JJ. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A. 1988;85:6982–6.
Jin DJ, Gross CA. Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli. J Bacteriol. 1989;171:5229–31.(Muhamed-Kheir Taha, Maria)