Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
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Abstract |
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Introduction |
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Materials and methods |
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Strains were isolated and grown on TYEGF agar [2% typtone (Oxoid, Basingstoke, UK), 1% Yeast Extract (Oxoid), 0.5% glucose, 2 mg/L furazolidone] under anaerobic conditions at 35&°;C. Identification was as described by Marples & McGinley.2
Cutaneous propionibacterial isolates were collected from the faces of acne patients and dermatologists at clinics in Leeds (UK), Ferrara (Italy), Uppsala (Sweden), Kecskemet (Hungary), Athens (Greece) and Malaga (Spain). Swabs were plated directly onto TYEGF plates containing erythromycin (0.5 mg/L).
Antibiotics
Antibiotics were from Sigma (Poole, UK), except for pristinamycin IA and telithromycin (Aventis, Romainville, France), josamycin (Novartis, Kundl, Austria) and azithromycin (Pfizer, Sandwich, UK).
MIC determination
MICs of eight MLS antibiotics were determined by agar dilution on WilkinsChalgren agar (Oxoid) as described by the NCCLS.3 Inocula contained 105 cfu per 1 µL spot delivered by a multipoint inoculator (Denley, Billinghurst, UK). MICs were recorded after 3 days anaerobic incubation at 37&°;C. Type strains of P. acnes (NCTC 737) and P. granulosum (NCTC 11865) were included as controls.
Detection of the acquired MLS resistance determinant
A degenerate primer pair (P1; see Figure) was designed based on a PileUp (Wisconsin Genetics Computer Group) alignment of erm genes from high GC Gram-positive organisms. Sequences used, based on the revised nomenclature4 were: erm(E), GenBank M11200; erm(H), P13079; erm(O), M74717; erm(S), P45439, all from Streptomyces; erm(R) from Aeromicrobium, M11276; and erm(X) from Corynebacterium, M36726formerly ermCd. Primer sequences were: 5'-GGBCARAAYTTYCTCNBCVACC-3' and 5'-CCGGYSCGSYKSCGVGCSWYTCSHRCTG-3'. Amplification conditions were 94&°;C (3 mins) then 30 cycles of 94&°;C (30 s), 56&°;C (30 s), 72&°;C (30 s) followed by 72&°;C (5 min). The expected product size was 404 bp. Amplification products were purified by the Wizard PCR purification system (Promega, Madison, WI, USA). DNA manipulations were by standard procedures using pBluescript. E. coli transformants carrying the erm(X) gene were selected on LB agar containing erythromycin (200 mg/L). Subsequent PCR detection of the transposon employed three pairs of primers based on Tn5432. Primer pairs were (positions indicated in Figure
), P2: 5'-GTCTGCATACGGACACGG-3' and 5'-CGAGCGACTTCCCACTGC-3', P3: 5'-GAAACAACGTACGGAGC-3' and 5'-GGTTGAGGTAGACAAAC-3', and P4: 5'-GGGAAATTCTCCGAAGG-3' and 5'-GGTGATGTCGTTTCGAC-3'.
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Results |
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The resistance determinant from a Spanish strain (SP64) was cloned as a 5.2 kb PstI fragment into pBluescript in E. coli JM109. The entire 5.2 kb was sequenced in both directions and found to contain the complete sequence of the composite transposon Tn5432 (GenBank AF411029), originally described in Corynebacterium striatum.6 This element is 4524 bp in length and composed of two copies of IS1249 (of the IS256 family) each encoding a transposase (tnp1249) with erm(X) and a truncated insertion sequence of the ISL3 family now termed ISCx17 between them (Figure). A single base change (C
T) was detected between erm(X) present in propionibacteria and the published sequence,6 leading to a proline to serine substitution at position 163 of the P. acnes erm(X) sequence. This is the same change as between the sequences of erm(X) from C. striatum (Tn5432) and Corynebacterium diphtheriae (pNG2).5,6 One other base change (G
A) was detected in the transposon, leading to an arginine to histidine substitution at residue 126 of tnpCX, the protein encoded by the truncated ISCx1. The sequences external to Tn5432 in SP64 do not correspond to the sequence of pTP10, the plasmid on which this transposon was first identified, and the other resistances encoded by pTP107 are not present in any of the propionibacterial isolates. Plasmids were not detected in any of the 14 strains.
PCR assays were employed to determine whether the transposon was present in all the phenotypically ketolide-resistant strains from diverse geographical locations. Three new primer pairs based on the sequence of Tn5432 were used in PCR amplifications. One pair used sequences from erm(X) and IS1249 to show that the resistance determinant was located within Tn5432. All 45 phenotypically ketolide-resistant isolates were positive in all reactions, demonstrating that Tn5432-containing erm(X) was present in all ketolide-resistant strains. Strain SP64 (Tn5432) was used as a positive control and one sample of each band was sequenced. No products were obtained with six negative control strains containing rRNA mutations. erm(X) was expressed inducibly in its original host,8 but was found to be constitutively expressed in all three cutaneous Propionibacterium species (Table).
Attempts to introduce Tn5432 into P. acnes from E. coli S-17 using the conjugative plasmid pKmob2 containing Tn5432 as a suicide vector and published methods9,10 were unsuccessful although transfer to the restriction-deficient Corynebacterium glutamicum DM39 was accomplished at a frequency of 1 in 109 recipients. Using filter mating and P. acnes SP64 as a donor, a single P. granulosum transconjugant was obtained from several experiments (<1 in 1011 recipients). Attempts to transfer Tn5432 between P. acnes strains by filter mating were unsuccessful (data not shown).
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Discussion |
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erm(X) accounted for <10% of erythromycin resistance among the isolates tested. The determinant, however, confers a higher degree of resistance than the known 23S rRNA mutations to several MLS antibiotics especially clindamycin and telithromycin. Clindamycin is extensively used in the topical treatment of acne and mutational resistance may not have been protective in vivo against the high drug concentrations achievable via this route. We may expect the incidence of the transposon to increase if topical antibiotics for acne continue to be widely used.
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Acknowledgements |
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Notes |
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References |
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2 . Marples, R. R. & McGinley, K. J. (1974). Corynebacterium acnes and other anerobic diptheroids from human skin. Journal of Medical Microbiology 7, 34957. [ISI][Medline]
3 . National Committee for Clinical Laboratory Standards. (1997). Methods for Antimicrobial Susceptibility Testing of Anaerobic BacteriaFourth Edition: Approved Standard M11-A4. NCCLS, Villanova, PA.
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Roberts, M. C., Sutcliffe, J., Courvalin, P., Bogo Jensen, L., Rood, J. & Seppela, H. (1999). Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrobial Agents and Chemotherapy 43, 282330.
5 . Hodgson, A. L. M., Krywult, J. & Radford, A. J. (1990). Nucleotide sequence of the erythromycin resistance gene from Corynebacterium plasmid pNG2. Nucleic Acids Research 18, 1891. [ISI][Medline]
6 . Tauch, A., Kassing, F., Kalinowski, J. & Pühler, A. (1995). The Corynebacterium xerosis composite transposon Tn5432 consists of two identical insertion sequences designated IS1249, flanking the erythromycin resistance gene ermCX. Plasmid 34, 11931. [ISI][Medline]
7 . Tauch, A., Krieft, S., Kalinowski, J. & Pühler, A. (2000). The 51,409-bp R-plasmid pTP10 from the multiresistant clinical isolate Corynebacterium striatum M82B is composed of DNA segments initially identified in soil bacteria and in plant, animal and human pathogens. Molecular and General Genetics 263, 111. [ISI][Medline]
8 . Tauch, A., Kassing, F., Kalinowski, J. & Pühler, A. (1995). The erythromycin resistance gene of the Corynebacterial xerosis R-plasmid pTP10 also carrying chloramphenicol, kanamycin, and tetracycline resistances is capable of transposition in Corynebacterium glutamicum. Plasmid 33, 16879. [ISI][Medline]
9 . Schäfer, A., Kalinowski, J., Simon, R., Seep-Feldhaus, A.-H. & Pühler, A. (1990). High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria. Journal of Bacteriology 172, 16636. [ISI][Medline]
10 . Schäfer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G. & Pühler, A. (1994). Small mobilizable muti-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 6973. [ISI][Medline]
Received 19 July 2001; returned 5 October 2001; revised 22 October 2001; accepted 23 October 2001