Antimicrobial susceptibilities of propionibacterium acnes isolated from patients with acne vulgaris

Microbiol Immunol 2008; 52: 621–624
Antimicrobial susceptibilities of Propionibacterium acnes
isolated from patients with acne vulgaris
Natsumi Ishida1, Hidemasa Nakaminami1, Norihisa Noguchi1, Ichiro Kurokawa2, Setsuko Nishijima3,
and Masanori Sasatsu1
1Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392,2Department of Dermatology, Nishijima Skin Clinic, 16-15 Koriminaminochou, Neyagawa, Osaka 572-0084, and 3Department of Dermatology,Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie 514-8507, Japan Antibiotic susceptibilities of Propionibacterium acnes in Japan were determined. Erythromycin-
resistance was found in 10.4% (5/48) of the strains, and four of these were cross-resistance to clindamycin.
Although the erythromycin ribosome methylase gene erm
(X) was looked for, no strain carrying erm(X)
was found. Sequencing analysis revealed that all of the erythromycin-resistant strains had a mutation in
the peptidyl transferase region of the 23S rRNA gene: G2057A, A2058G, or A2059G. Consequently, our
results show that P. acnes
resistance to macrolides is caused by a mutation in the 23S rRNA gene, and has
been increasing in Japan.

Key words acne vulgaris, macrolides, P. acnes, 23S rRNA.
Acne vulgaris is a common multifactorial skin disease After topical erythromycin and clindamycin were in- which presents mainly as seborrheic lesions on the face troduced to the market, P. acnes with high-level resis- and upper trunk (1–3). Unlike in the U.S.A. and Europe, tance to erythromycin was frequently found in the U.K.
severe acne vulgaris is not common in Japan. The skin and the U.S.A. (7). Erythromycin-resistant strains have bacterium P. acnes is a Gram-positive, anaerobic bacillus been reported among cutaneous propionibacteria in Eu- that colonizes sebaceous glands (3). This bacterium grows rope, U.S.A., Australia, and Japan (8, 5, 7). Although vigorously in a suitable environment, causing obstruction erythromycin- and/or clindamycin-resistant strains are of the pilosebaceous glands by excessive sebaceous secre- reported to colonize at least 50% of patients in Europe, a tion or keratotic plugs, and has been implicated in the study conducted between 1994 and 1995 revealed that pathogenesis of inflamed lesions (3).
erythromycin-resistant strains were found in only 4% In Japan, orally administered 14-membered ring (2/50) of P. acnes isolates from Japanese patients with macrolides, β-lactams, and fluoroquinolones are ap- proved for use in treating patients with acne vulgaris Erythromycin resistance in P. acnes is considered to (4). Of the topical antibiotics, clindamycin and nadi- be caused by mutation of the peptidyl transferase region floxacin are approved and commonly used in Japan for in the domain V of 23S rRNA, and by the target site al- the treatment of acne vulgaris (5). Macrolides are used not teration with the 23S rRNA dimethylase that is encoded only as antimicrobial agents but also as anti-inflammatory by erm(X) (10). Macrolide-resistant strains show cross- resistance to the structurally unrelated lincosamides and Correspondence
Norihisa Noguchi, Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Ha-
chioji, Tokyo 192-0392, Japan.
Tel: +81 426 76 5619; fax: +81 426 76 5647; email:
Received 17 June 2007; revised 11 July 2008, 24 July 2008; accepted 5 August 2008 List of Abbreviations: CLSI, Clinical and Laboratory Standards Institute; GAM, Gifu anaerobic medium; MIC, minimal inhibitory concentration; P.
acnes, Propionibacterium acnes.
c 2008 The Societies and Blackwell Publishing Asia Pty Ltd streptogramin B, therefore this type of resistance is termed Table 1. Antimicrobial susceptibilities for P. acnes (n = 48) isolated from
Little is known about current antimicrobial suscepti- bility and resistance mechanisms of P. acnes in Japan. In the present study, we determined the MIC of various an- timicrobial agents for P. acnes isolated from patients with acne vulgaris between 2006 and 2007 in Japan. Addition- ally, we studied the mechanisms of MLSB resistance in the resistant strains isolated in this study.
A total of 48 P. acnes samples were collected from 73 patients with acne vulgaris between 2006 and 2007 in Japan. The samples were cultured on modified GAM agar (Nissui Pharmaceutical, Tokyo, Japan) under anaerobic conditions at 35◦C for 72 hr. P. acnes was identified by Api 20 A (bioM´erieux, Marcy l’Etoile, France). P. acnes JCM6425 (ATCC6919) was used as a quality control strain for antimicrobial susceptibility testing (7), and JCM 6473 (ATCC11828) was used as a wild type strain for 23S rRNA.
Susceptibility testing was performed by an agar di- lution procedure according to the CLSI guidelines(12). Faropenem, cefaclor, cefcapene, cefditoren, clar- Resistance breakpoint of the following antimicrobial agents were de- ithromycin, clindamycin, levofloxacin, and nadifloxacin fined according to CLSI and this study: amoxicillin, ≥16 μg/ml; cefaclor, were kindly provided by their manufacturers. Amoxi- ≥16 μg/ml; cefcapene, ≥64 μg/ml; cefditoren, ≥64 μg/ml; faropenem,≥16 μg/ml; ciprofloxacin, ≥8 μg/ml; levofloxacin, ≥8 μg/ml; nadi- cillin, erythromycin, and fusidic acid were purchased floxacin, ≥8 μg/ml; erythromycin, ≥2 μg/ml; clarithromycin, ≥2 μg/ml; from Sigma-Aldrich. (Tokyo, Japan), and josamycin, josamycin, ≥4 μg/ml; clindamycin, ≥8 μg/ml; tetracycline, ≥16 μg/ml; ciprofloxacin, tetracycline, minocycline, and chloram- minocycline, ≥16 μg/ml; chloramphenicol, ≥32 μg/ml; fusidic acid, phenicol were from Wako Pure Chemical Industries.
(Osaka, Japan). Break points of antimicrobial agents weredefined by the interpretation criteria of the CLSI (12),and undefined breakpoints were defined in this study.
The macrolides-resistant strains were classified into re- of the 23S rRNA gene was performed using the follow- sistance groups I to IV according to Ross et al. (10).
ing internal 23S rRNA primers (GenBank accession No.
Group I indicates high-level resistance to 14-membered AE017283): 5 -CGATGTATACGGACTGACTCC-3 and ring macrolides and lincosamides and low-level resistance 5 -AACTACCCATCAGGCACTGT-3 . Sequencing reac- to 16-membered ring macrolides. Group II indicates high- tions and analysis were performed as previously described level resistance to all MLSB. Group III indicates low-level resistance to 14-membered ring macrolides and suscepti- In 73 patients with acne vulgaris, 48 isolates (65.8%) bility to 16-membered ring macrolides and lincosamides.
were identified as P. acnes. The MIC of 48 strains Group IV indicates high-level resistance to 14- and 16- are shown in Table 1. All strains were susceptible to membered ring macrolides and low-level resistance to lin- all tested antimicrobial agents, apart from macrolides and clindamycin. Although the MIC of fusidic acid To detect erm(X) carried by the resistance group was 16 μg/ml, this value was the same as JCM6425.
II strains (10), the following primers were designed Of the 48 strains, 10.4% (5/48) showed resistance to based on the sequence of P. acnes erm(X) lo- the 14-membered ring macrolides erythromycin and cated on the transposon Tn5432 (GenBank accession clarithromycin. Furthermore, four erythromycin- and No. AF411029): 5 -CTCACCAACCACAAGATCATC-3 clarithromycin-resistant strains were cross-resistant to and 5 -GAAGAGATCGATCCAGTCGTT-3 (product size, 710 bp). The PCR reaction was performed according to The macrolides-resistant P. acnes strains found in this Ross et al. (11). Amplification of the 23S rRNA gene, in- study were classified into resistance groups I to IV accord- cluding domain V, was performed as described by Meier ing to their susceptibilities to macrolides and clindamycin et al. (13). PCR was performed using 25 cycles of 30 s (Table 2). Strains 1, 3, and 4, with high-level resistance of denaturation at 95◦C, 30 s of annealing at 55◦C, and to 14-membered ring macrolides and lincosamides and 2 min of extension at 72◦C. Sequencing of domain V low-level resistance to 16-membered ring macrolides, c 2008 The Societies and Blackwell Publishing Asia Pty Ltd Current surveillance P. acnes in Japan Table 2. Sequencing analysis for mutations of 23S rRNA gene in P. acnes isolated from patients with acne vulgaris
were classified into group I. In the same manner, strains 2 those reported in Ross et al. (3). It has been hypothesized and 5 were classified into groups IV and III, respectively.
that changes that disrupt the base pairing of A2058 and Although erm(X), which confers high-level resistance to U2016 alter the MLSB binding site, resulting in high-level all MLSB, was looked for, no strain carrying erm(X) was erythromycin resistance, whereas the weaker rearrange- detected (data not shown). Therefore, no resistance group ment caused by disruption of the G2057-C2611 base pair- II strain was found in this study. Sequencing analysis of ing affects the binding sites of fewer antimicrobial agents domain V of the 23S rRNA gene showed that all five strains and leads to lower levels of erythromycin resistance (17).
carried a point mutation within the peptidyl transferase Our results are in agreement with this report.
region. All strains of group I had a transition of adenine Long-term macrolide antibiotic therapy is commonly to guanine at the position of 2058 (A2058G). Group III used to treat acne vulgaris. Furthermore, macrolides are and group IV strains carried a transition of G2057A and frequently used for the treatment of Helicobacter pylori infections and respiratory tract infections in Japan. One We investigated the current antimicrobial susceptibili- report has shown that the prevalence of resistant propi- ties of P. acnes isolated from patients with acne vulgaris onibacteria on the skin of untreated contacts of treated in Japan. In this paper, we are the first to provide detailed patients varied from 41% in Hungary to 86% in Spain data on current antimicrobial susceptibilities and resis- (10). Thus, the use of macrolides will be a high selective tance mechanisms of clinical P. acnes isolates in Japan to pressure for the development of resistance in P. acnes (2, antimicrobial agents used to treat acne vulgaris.
7). Our data show that mutationally caused macrolide re- In 1999, P. acnes isolated from patients with acne vul- sistance in P. acnes strains has been increasing in Japan.
garis in Japan was generally susceptible to most of the Therefore, there is concern in Japan about future emer- antimicrobial agents used in this study (5). However, in gence of strains with high-level resistance to macrolides the present study, five (10.4%) of 48 strains were resistant to macrolides, and four of them showed cross-resistanceto clindamycin. Therefore, our data suggests that MLSB-resistant P. acnes strains have been increasing in Japan.
Sequencing analysis of domain V of the 23S rRNA gene revealed that the sequences of all five strains carried a We thank K. Furui for her excellent assistance. This work point mutation in the peptidyl transferase region. The re- was supported by the Matching Fund Subsidy for Private sistance groups I, III, and IV strains had a point mutation at position 2058, 2057, and 2059, respectively. Further-more, each group showed different MLSB susceptibility REFERENCES
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