Keisuke Nakase

Relationship between the severity of acne vulgaris and antimicrobial resistance of bacteria isolated from acne lesions in a hospital in Japan.

Propionibacterium acnes and Staphylococcus epidermidis are normal skin inhabitants that are frequently isolated from lesions caused by acne, and these micro-organisms are considered to contribute to the inflammation of acne. In the present study, we examined the antimicrobial susceptibilities and resistance mechanisms of P. acnes and S. epidermidis isolated from patients with acne vulgaris in a university hospital in Japan from 2009 to 2010. Additionally, we analysed the relationship between the antimicrobial resistance of P. acnes and the severity of acne vulgaris. Some P. acnes strains (18.8 %; 13/69) were resistant to clindamycin. All strains had a mutation in the 23S rRNA gene, except for one strain that expressed erm(X) encoding a 23S rRNA methylase. Tetracycline-resistant P. acnes strains were found to represent 4.3 % (3/69) of the strains, and this resistance was caused by a mutation in the 16S rRNA gene. Furthermore, three strains with reduced susceptibility to nadifloxacin (MIC = 16 µg ml(-1)) were detected. When analysing the correlation between the antimicrobial resistance of P. acnes and S. epidermidis, more than 80 % of the patients who carried clindamycin-resistant P. acnes also carried clindamycin-resistant S. epidermidis. However, no epidemic strain that exhibited antimicrobial resistance was detected in the P. acnes strains when analysed by PFGE. Therefore, our results suggest that the antimicrobial resistance of P. acnes is closely related to antimicrobial therapy. Additionally, those P. acnes strains tended to be frequently found in severe acne patients rather than in mild acne patients. Consequently, the data support a relationship between using antimicrobial agents and the emergence of antimicrobial resistance.

First report of high levels of clindamycin-resistant Propionibacterium acnes carrying erm(X) in Japanese patients with acne vulgaris.

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Antimicrobial susceptibility and phylogenetic analysis of Propionibacterium acnes isolated from acne patients in Japan between 2013 and 2015

The prevalence of antimicrobial‐resistant Propionibacterium acnes strains isolated from acne patients has been increasing in Japan. Here, to estimate the current resistance rate, we tested antimicrobial susceptibility among P. acnes from acne patients having visited a specialized dermatology clinic between 2013 and 2015. Rates of resistance to macrolides and clindamycin were 44.3 (31/70) and 38.6% (27/70), respectively. erm(X), which confers high‐level clindamycin resistance (minimum inhibitory concentration ≥256 μg/mL), was detected in six isolates, whereas no resistance determinants were identified in eight strains showing high‐level resistance to clindamycin. Using single‐locus sequence typing, the P. acnes isolates were classified into five clades (A, E, F, H and K), with all high‐level clindamycin‐resistant strains lacking known clindamycin resistance determinants being grouped together (in clade F). P. acnes isolates from patients previously treated with macrolides and clindamycin showed a macrolide resistance rate (55.3%) significantly higher than that of those from patients not having received these treatments (21.7%, P < 0.05). Furthermore, strains of clade F, which were very rarely isolated from healthy individuals, were more frequently recovered from patients with severe acne (40.0%) than those with mild acne (23.3%). Our data showed an increase in macrolide‐resistant P. acnes prevalence in Japan due to the use of antimicrobial agents for acne treatment. Furthermore, we identified strains of specific phylogenetic groups frequently associated with severe acne patients.

Propionibacterium acnes is developing gradual increase in resistance to oral tetracyclines

&NA; Propionibacterium acnes is an anaerobic bacterium that causes deep infection in organs and prosthetic joints, in addition to acne vulgaris. Many tetracycline‐resistant P. acnes strains have been isolated because oral tetracyclines are frequently used as an acne treatment against P. acnes. In this study, we found a novel tetracycline resistance mechanism in P. acnes. Three doxycycline‐resistant (MIC: 16 &mgr;g ml−1) strains were isolated from 69 strains in acne patients in Japan between 2010 and 2011. Additionally, six insusceptible strains (MIC: 1‐2 &mgr;g ml−1) that had reduced susceptibility compared to susceptible strains (MIC: ≤0.5 &mgr;g ml−1) were identified. All doxycycline‐resistant strains had a G1036C mutation in the 16S rRNA gene in addition to an amino acid substitution in the ribosomal S10 protein encoded by rpsJ. By contrast, insusceptible strains had an amino acid substitution in the S10 protein but no mutation in the 16S rRNA. When the mutant with decreased susceptibility to doxycycline was obtained in vitro, only the mutated S10 protein was found (MIC: 4 &mgr;g ml−1), not the mutated 16S rRNA gene. This result shows that the S10 protein amino acid substitution contributes to reduced doxycycline susceptibility in P. acnes and suggests that tetracycline resistance is acquired through a 16S rRNA mutation after the S10 protein amino acid substitution causes reduced susceptibility.

Determination of the Mutant Prevention Concentration and the Mutant Selection Window of Topical Antimicrobial Agents against Propionibacterium acnes

Determination of the mutant prevention concentration (MPC) and the mutant selection window (MSW) of antimicrobial agents used to treat pathogenic bacteria is important in order to apply effective antimicrobial therapies. Here, we determined the MPCs of the major topical antimicrobial agents against Propionibacterium acnes and Staphylococcus aureus which cause skin infections and compared their MSWs. Among the MPCs of nadifloxacin and clindamycin, the clindamycin MPC was determined to be the lowest against P. acnes. In contrast, the nadifloxacin MPC was the lowest against S. aureus. Calculations based on the minimum inhibitory concentrations and MPCs showed that clindamycin has the lowest MSW against both P. acnes and S. aureus. Nadifloxacin MSWs were 4-fold higher against P. acnes than against S. aureus. It is more likely for P. acnes to acquire resistance to fluoroquinolones than S. aureus. Therefore, topical application of clindamycin contributes very little to the emergence of resistant P. acnes and S. aureus strains.

Long-term administration of oral macrolides for acne treatment increases macrolide-resistant Propionibacterium acnes

Macrolide‐resistant Propionibacterium acnes are frequently isolated from patients with acne vulgaris, and the most resistant isolates (>90% resistance) have the 23S rRNA mutation. An increase in resistant P. acnes with this mutation is thought to be caused by the inappropriate use of antimicrobials. Therefore, we studied the mutation frequency of macrolide resistance in P. acnes in vitro. When P. acnes mutants were exposed to clarithromycin after being incubated in broth without antimicrobials, resistant mutants with the 23S rRNA mutation were not isolated. However, the mutants were obtained at the frequency of 10−6 after being pre‐incubated with 0.03 μg/mL of antimicrobials. This is the estimated epidermal concentration of clarithromycin after p.o. administration. The resistant mutants had the 23S rRNA mutations A2058G, A2059G and C2611G. When pre‐incubated with clarithromycin, C2611G mutants which showed resistance to clarithromycin were obtained 32.1% more often than pre‐incubated with clindamycin (P < 0.01). By contrast, when pre‐incubated with clindamycin, A2058G mutants, which show high‐level resistance to both clarithromycin and clindamycin, were more frequently obtained than pre‐incubated with clarithromycin (87.5%, P < 0.01). No difference in the isolation rate of A2059G mutants, which show high‐level resistance to macrolides but low‐level resistance to clindamycin, was found with either treatment. These results indicate the possibility that long‐term use of oral macrolides for acne treatment facilitate the increase of macrolide‐resistant P. acnes.

Involvement of adenosine triphosphate-binding cassette subfamily B member 1 in the augmentation of triacylglycerol excretion by Propionibacterium acnes in differentiated hamster sebocytes

An onset of acne, a common inflammatory skin disease, is associated with excess sebum production and secretion in sebaceous glands. Because Propionibacterium acnes has been reported to augment intracellular sebum accumulation in sebaceous glands in hamsters, it remains unclear whether P. acnes influences sebum secretion from differentiated sebocytes. Both P. acnes culture media (Acnes73‐CM) and formalin‐killed P. acnes (F‐Acnes73) dose‐dependently increased the extracellular levels of triacylglycerol (TG), a major sebum component, and Rhodamine 123, a substrate of adenosine triphosphate‐binding cassette (ABC) transporter, from differentiated hamster sebocytes (DHS). In addition, the gene expression of the ABC subfamily B member 1 (ABCB1) was dose‐dependently augmented by adding Acnes73‐CM and F‐Acnes73 into DHS. Furthermore, the F‐Acnes73‐induced increase of TG excretion was suppressed by PSC833, a selective ABCB1 inhibitor. On the other hand, peptidoglycan (PGN), which is a Toll‐like receptor 2 (TLR2) ligand in P. acnes, increased extracellular TG levels, transporter activity and ABCB1 mRNA expression in DHS. The PGN‐augmented TG excretion was suppressed by PSC833. Thus, these results provide novel evidence that P. acnes facilitates sebum secretion due to the activation of ABCB1 concomitantly with the increased ABCB1 expression, which may result from the activation of the TLR2 pathway in DHS. Therefore, the ABCB1 inhibitor is likely to become a candidate as a possible therapeutic for the treatment of acne.