Journal of Antimicrobial Chemotherapy (2002) 49, 165-168
© 2002 The British Society for Antimicrobial Chemotherapy
Brief report |
Detection of transposon Tn5432–mediated macrolide-lincosamide-streptogramin B (MLSB) resistance in cutaneous propionibacteria from six European cities
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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Forty-five cutaneous propionibacterial isolates from six European cities were found to be highly resistant to all macrolide-lincosamide-streptogramin B antibiotics, including the ketolide telithromycin. This contrasts with previously documented phenotypes associated with 23S rRNA mutations. Sequencing of the resistance determinant showed it to be erm(X) of corynebacterial origin located on the composite transposon Tn5432.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum are ubiquitous residents of human skin. P. acnes and P. granulosum have been implicated in the pathogenesis of acne. Previous investigations classified erythromycin-resistant propionibacteria from the UK into four phenotypic classes based on their patterns of cross-resistance to macrolide-lincosamide-streptogramin B (MLS) antibiotics.1 Resistance groups I, III and IV were shown to be associated with point mutations in 23S rRNA at Escherichia coli-equivalent bases 2058, 2057 and 2059, respectively.1 The basis of resistance among group II isolates was never determined. We have collected and characterized resistant propionibacteria from acne patients attending dermatology clinics in six European cities. We wished to determine whether resistant isolates in countries where antibiotic usage patterns differ carry chromosomal mutations or whether mobile genetic elements exist in the population.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Isolation of resistant propionibacteria
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 Wilkins–Chalgren 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, M36726—formerly 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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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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A total of 486 erythromycin-resistant propionibacteria were isolated from acne patients and dermatologists in six European cities. These isolates were classified into resistance groups I–IV2 based on their resistance patterns to eight MLS antibiotics. Predictably, the majority of isolates fell into phenotypic classes associated with a 2058 or 2059 rRNA base mutation (Table
). Forty-five of the 486 erythromycin-resistant isolates were resistant at high level (MICs 
512 mg/L) to all MLS antibiotics tested, including the ketolide, telithromycin. Mutations in 23S rRNA confer no resistance, or low-level resistance to this antibiotic (Table). Nineteen of the isolates exhibiting this phenotype were from Greece (14.4% of resistant isolates), eight from Spain (13.6%), six from Hungary (12.8%), seven from Sweden (7.0%), three from Italy (3.1%) and two from the UK (2.7%). Using biochemical methods, 22 of these isolates were identified as P. acnes, 22 as P. granulosum and one as P. avidum.
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Genomic DNA was extracted from 14 ketolide-resistant propionibacteria. Degenerate primers were used in a PCR to detect a GC-rich erm gene. A 404 bp product was obtained from all 14 isolates tested. No product was obtained from a negative control group of six strains containing 2058 or 2059 ribosomal mutations. The 404 bp bands from four isolates were cloned into pGEM easy (Promega) in E. coli JM109. Sequencing showed all the inserts to be identical partial sequences of the corynebacterial erm(X) gene.5,6
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 (GA) 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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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We have discovered a transposon-based MLS resistance determinant in cutaneous propionibacteria from six European countries. This is the first time Tn5432 has been reported outside Corynebacteria. The resistance gene, erm(X), is present in all three propionibacterial species (P. acnes, P. avidum and P. granulosum), showing that it may have been mobilized into propionibacteria on at least three occasions from its original host, most likely to be one of several species of Corynebacterium that colonize moist areas of human skin, and, like propionibacteria, have GC-rich DNA. Alternatively, the transposon may have transferred between propionibacterial species. The transposon Tn5432 is non-conjugative and difficult to mobilize in the laboratory. The mechanism by which it transferred into propionibacteria has not been elucidated. Tn5432 is borne on the multi-resistance plasmid pTP10 in Corynebacterium striatum. There is no evidence of the presence of this plasmid in erm(X)-containing propionibacteria.
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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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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This work was supported by Dermik Laboratories (Aventis).
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Notes |
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* Corresponding author. Tel: +44-113-233-5631; Fax: +44-113-233-5638; E-mail: micjir{at}leeds.ac.uk
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References |
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Received 19 July 2001; returned 5 October 2001; revised 22 October 2001; accepted 23 October 2001
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