a Department of Bacteriology and Medical Mycology, and b Department of Environmental Hygiene, Istituto Superiore di Sanita', Viale Regina Elena 299, 00161 Rome, Italy
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Abstract |
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Introduction |
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During a long low endemic period in Italy, N. meningitidis isolates showed a trend of slightly decreased susceptibility to rifampicin, recommended by the Health Authorities in Italy, as most of the strains isolated in the last 15 years are highly resistant to sulphonamides.6
Sequencing of the rpoB and mtr genes was performed on six rifampicin-resistant N. meningitidis isolates belonging to serogroup C and two to serogroup B. The strains were also characterized phenotypically and genotypically to determine their clonal relationship.
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Materials and methods |
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Eight strains were found to be resistant and were included in the study together with sixteen Rifs strains.
Five sets of primers were designed, referring to the sequence of N. meningitidis rpoB gene clusters I, II and III (AC# Z54353, by O. J. Nolte, unpublished), and N. gonorrhoeae mtrR and its promoter gene (AC# Z25796), to generate templates for sequencing (Table I). Analysis of the sequences was performed using the University of Wisconsin Genetics Computer Group (GCG) software package.
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For PCRRFLP analysis a 1116 bp region of the porA gene and a 910 bp region of the porB gene were amplified using two sets of primers, as described by Feavers et al.10 Ten microlitres of the PCR products were digested at 37°C for 3 h with 40 U of the following endonucleases: MspI, HaeIII, RsaI, HinfI (New England Biolabs). To define the degree of polymorphism for porA and porB genes, each pattern was compared with the others for the number of shared bands.
The following GenBank accession numbers were assigned to the rpoB genes of the eight rifampicin-resistant N. meningitidis strains: 694 (AJ270498); 745 (AJ270495); 870 (AJ270496); 888 (AJ270497); 899 (AJ270499); 901 (AJ270503); 944 (AJ270502); and 978 (AJ270494).
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Results |
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A histidine was replaced by a tyrosine residue at position 552 in strains 694, 899 and 901. At the same 552 position, an asparagine residue instead of a histidine, was found in strain 944.
Sequencing performed in the same region of three Rifs strains, used as controls, showed the absence of these mutations.
The mtr promoter region and mtrR gene showed the presence of some mutations (data not shown) that were the same in both resistant and susceptible strains (eight Rifr and 16 Rifs).
The molecular typing obtained by PFGE of the eight Rifr strains, is shown in the Figure. Two PFGE types were identified: one produced by strains 694, 899, 901, 944 and 978 (Figure
, lanes 15); the other by strains 745, 870 and 888 (Figure
, lanes 68). The two PFGE patterns were considered unrelated according to Tenover's classification and with a Dice coefficient of <0.85.
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Discussion |
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Few reports have been published concerning the mutations affecting the rpoB gene in N. meningitidis strains.3,4
The results obtained by sequencing a 1082 bp fragment in the rpoB gene of all the Rifr strains examined, showed the presence of two mutations in the region corresponding to cluster I. According to these mutations the strains could be divided into two groups. The first, including strains 745, 870 and 888 (serogroup C) and 978 (serogroup B), showed the presence of a valine residue instead of an asparagine at codon position 542. This substitution is documented in N. meningitidis for the first time.
In the second group, a histidine was replaced by a tyrosine at position 552 in strains 694, 899 and 901 (serogroup C), and by an asparagine in strain 944 (serogroup B). Both mutations have already been described by Carter et al.3
Among the resistant strains no relationship was found between MIC values for rifampicin and specific genetic mutations in the rpoB gene, as the same mutations gave two different resistance phenotypes with MICs of 64 and >256 mg/L, respectively.
To explain this difference we also investigated the role of the mtr efflux system recognized as a contributor to antimicrobial resistance in gonococci.5 However, no difference was found between mutations detected in Rifr and Rifs N. meningitidis strains examined in this study. Thus, other factors as yet unknown could contribute to the increased resistance to rifampicin, such as reduced entry of drug into the bacterium.
Molecular typing by PFGE and PCRRFLP analysis of porA and porB genes indicated that rifampicin resistance in the Italian strains may be associated with the circulation of two different clonal groups without any correlation with the specific mutation detected in the rpoB gene.
Since the role of genetic exchange mechanisms in the spread of rifampicin-resistant N. meningitidis strains cannot be ruled out, the circulation of isolates with this Rifr phenotype will be monitored carefully.
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Acknowledgments |
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Notes |
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References |
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2 . Yagupsky, P., Ashkenazi, S. & Block, C. (1993). Rifampicin-resistant meningococci causing invasive disease and failure of prophylaxis. Lancet 341, 11523.[ISI][Medline]
3 . Carter, P. E., Abadi, J. R., Yakubu, D. E. & Pennington, T. H. (1994). Molecular characterization of rifampin-resistant Neisseria meningitidis. Antimicrobial Agents and Chemotherapy 38, 125661.[Abstract]
4 . Nolte, O. (1997). Rifampicin resistance in Neisseria meningitidis: evidence from a study of sibling strains, description of new mutations and notes on population genetics. Journal of Antimicrobial Chemotherapy 39, 74755.[Abstract]
5 . Veal, W. L., Yellen, A., Balthazar, J. T., Pan, W., Spratt, B. G. & Shafer, W. M. (1998). Loss-of-function mutations in the mtr efflux system of Neisseria gonorrhoeae. Microbiology 144, 6217.[Abstract]
6 . Connolly, M. & Noah, N. (1999). Is group C meningococcal disease increasing in Europe? A report of surveillance of meningococcal infection in Europe 19936. Epidemiology and Infection 122, 419.[ISI][Medline]
7 . Abdillabi, H. & Poolman, J. T. (1987). Whole-cell Elisa for typing Neisseria meningitidis with monoclonal antibodies. FEMS Microbiology Letters 48, 36771.[ISI]
8 . Dice, L. R. (1945). Measure of the amount of ecologic associations between species. Ecology 26, 277302.
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Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 22339.
10 . Feavers, I. M., Suker, J., McKenna, A. J., Heath, A. B. & Maiden, M. C. (1992). Molecular analysis of the serotyping antigens of Neisseria meningitidis. Infection and Immunity 60, 36209.[Abstract]
Received 2 August 2000; returned 21 September 2000; revised 20 October 2000; accepted 3 November 2000