Multidrug Resistance Plasmid pTZC1 Could Be Pooled among Cutibacterium Strains on the Skin Surface

The emergence of antimicrobial resistance not only in Cutibacterium acnes strain but also other skin bacteria such as Staphylococcus epidermidis is a big concern due to antimicrobial use for the treatment of acne vulgaris. Increased prevalence of macrolides-clindamycin resistant C. acnes relates to the acquisition of exogenous antimicrobial resistance genes. erm(50) is harbored by the multidrug resistance plasmid pTZC1, which has been found in C. acnes and C. granulosum strains isolated from patients with acne vulgaris. ABSTRACT Acne vulgaris is a chronic inflammatory skin disease that is exacerbated by Cutibacterium acnes. Although antimicrobials such as macrolides, clindamycin, and tetracyclines are used to treat acne caused by C. acnes, the increasing prevalence of antimicrobial-resistant C. acnes strains has become a global concern. In this study, we investigated the mechanism by which interspecies transfer of multidrug-resistant genes can lead to antimicrobial resistance. Specifically, the transfer of pTZC1 between C. acnes and C. granulosum isolated from specimens of patients with acne was investigated. Among the C. acnes and C. granulosum isolated from 10 patients with acne vulgaris, 60.0% and 70.0% of the isolates showed resistance to macrolides and clindamycin, respectively. The multidrug resistance plasmid pTZC1, which codes for macrolide-clindamycin resistance gene erm(50) and tetracycline resistance gene tet(W), was identified in both C. acnes and C. granulosum isolated from the same patient. In addition, whole-genome sequencing revealed that the pTZC1 sequences of C. acnes and C. granulosum showed 100% identity using comparative whole-genome sequencing analysis. Therefore, we hypothesize that the horizontal transfer of pTZC1 between C. acnes and C. granulosum strains may occur on the skin surface. The plasmid transfer test revealed a bidirectional transfer of pTZC1 between C. acnes and C. granulosum, and transconjugants that obtained pTZC1 exhibited multidrug resistance. In conclusion, our results revealed that the multidrug resistance plasmid pTZC1 could be transferred between C. acnes and C. granulosum. Furthermore, since pTZC1 transfer among different species may aid in the prevalence of multidrug resistant strains, antimicrobial resistance genes may have been pooled on the skin surface. IMPORTANCE The emergence of antimicrobial resistance not only in Cutibacterium acnes strain but also other skin bacteria such as Staphylococcus epidermidis is a big concern due to antimicrobial use for the treatment of acne vulgaris. Increased prevalence of macrolides-clindamycin resistant C. acnes relates to the acquisition of exogenous antimicrobial resistance genes. erm(50) is harbored by the multidrug resistance plasmid pTZC1, which has been found in C. acnes and C. granulosum strains isolated from patients with acne vulgaris. In this study, C. acnes and C. granulosum with pTZC1 were found in the same patient, and plasmid transfer between C. acnes and C. granulosum was proved by transconjugation assay. This study showed plasmid transfer between other species and the possibility of further prevalence antimicrobial resistance between Cutibacterium species.

of hair follicles, is associated with exacerbated inflammation in acne vulgaris. For acne treatment, benzoyl peroxide and topical retinoid, including adapalene, are used to ameliorate hyperkeratinization, while antimicrobial agents target C. acnes in the acute inflammatory phase. Among the antimicrobial agents, topical clindamycin, nadifloxacin, and ozenoxacin, in addition to oral doxycycline, minocycline, and roxithromycin, are recommended by the Japanese acne treatment guidelines (3). However, the emergence and increase of antimicrobial-resistant C. acnes strains have become a great concern, and antimicrobial use for acne treatment is strongly related to these phenomena (4 to 7). Cutibacterium species, such as C. avidum, C. granulosum, C. modestum, and C. namnetense, in addition to C. acnes, are commensal skin bacteria (8,9). Cutibacterium species other than C. acnes are rarely found in acne lesions.
Antimicrobial-resistant C. avidum and C. granulosum strains are more frequently found in patients with acne vulgaris than C. acnes (10). Furthermore, antimicrobial-resistant Staphylococcus epidermidis, a major skin commensal bacterium, was isolated more frequently from patients with acne vulgaris than from healthy individuals (11). Thus, the efficient use of antimicrobial agents for acne treatment is closely related to the development of antimicrobial resistance in various skin inhabitants, including C. acnes.
C. acnes can develop antimicrobial resistance through various mechanisms. Mutations in 23S rRNA and acquisition of ribosomal methylase coding genes erm(X) and erm(50) are well-known mechanisms of resistance against the macrolides-clindamycin (12,13). erm(X) is found on chromosomes or linear plasmids and is transmitted between C. acnes strains (14,15). In contrast, erm(50) and the tetracycline resistance gene tet(W) are only found in C. acnes and are located on pTZC1. pTZC1 also carries a type-IV secretion system operon, which is associated with conjugative transfer among C. acnes strains (16).
In this study, we showed the coexistence of C. acnes and C. granulosum in acne pustules and the transference of pTZC1 between different strains of Cutibacterium species.

RESULTS
Antimicrobial susceptibility of Cutibacterium strains isolated from patients with acne vulgaris. The C. acnes and C. granulosum strains were isolated simultaneously from specimens isolated from the acne pustules of 10 patients with acne vulgaris, of which four were diagnosed with severe (40.0%) and three with moderate and mild (30.0%) acne (17). The antimicrobial susceptibility of these strains was determined (Table 1). Clarithromycinand clindamycin-resistant strains were found in 60.0% (6/10) and 70.0% (7/10) of the C. acnes strains and 40.0% (4/10) and 50% (5/10) of the C. granulosum strains, respectively. The rates of resistance to clarithromycin and clindamycin in the C. granulosum strains were higher than those in the C. acnes strains. In addition, doxycycline-resistant strains have been identified in C. acnes and C. granulosum isolated from an one patient out of ten patients.
Antimicrobial resistance factors in Cutibacterium strains. The resistance factors of macrolides-clindamycin and tetracyclines were analyzed (Table 2). While 23S rRNA mutations were found in four and two C. acnes and C. granulosum isolates, respectively, erm(X) was detected in three C. granulosum strains. Moreover, the pTZC1 harboring erm(50) and tet(W) was found in both C. acnes and C. granulosum strains isolated from the specimens isolated from two patients.
Whole-genome sequence analysis. Whole-genome sequencing revealed that both C. acnes TP-CU426 and C. granulosum TP-CG7 had a chromosome (2,495,124 bp, with a GC content of 60.0%; and 2,148,765 bp, with a GC content of 64.1%, respectively) and a plasmid (30,947 bp, with a GC content of 65.0%). The coverage levels were 1,403 and 1,633 for C. acnes TP-CU426 and C. granulosum TP-CG7, respectively. The genome sequences of the chromosomes and pTZC1 from C. acnes TP-CU426 and C. granulosum TP-CG7 were deposited in NCBI GenBank under accession numbers AP026712, AP026713, AP026710, and AP026711. The pTZC1 sequence of C. acnes TP-CU426 (accession no. AP026713) and C. granulosum TP-CG7 (accession no. AP026711) showed 100% identity (30,947/30,947 bp) ( Fig. 1 and supplemental material). In contrast to the pTZC1 from the C. acnes TP-CU389 (accession no. LC473083), these sequences contained a 500-bp deletion. This region was located on the 16,115 to 16,668 bp region of pTZC1 from the C. acnes TP-CU389 and was estimated to be a noncoding sequence using BLAST analysis.
Resistance factors conferring doxycycline resistance were analyzed in these strains (18). The C. acnes TP-CU426 and C. granulosum TP-CG7 had mutations in rpsJ (A170T, K57M; T172G, Y58D) but not in 16S rRNA. The upstream sequences of tet(W) are known to contribute to tetracycline resistance (4). When upstream sequences of tet(W) were compared,

Strains Species
Resistance factor and pTZC1 23S rRNA mutation a Acquisition of pTZC1 was confirmed by detection of three genes corded (erm(50), tet(W), and traE). ND, not detected. those sequences in the C. acnes TP-CU426 and C. granulosum TP-CG7 were identical to those of group Biii reported by Aoki et al. (4). Transconjugation of erm(50) between C. acnes and C. granulosum strains. pTZC1 found in the two species from the same patient had identical sequences with the same 500 bp deletion. This provided strong evidence that the plasmid was transferred between C. acnes and C. granulosum strains on the skin surface. Thus, the transferability of pTZC1 between C. acnes and C. granulosum strains was examined (Table 3).
Thus, we confirmed that pTZC1 could be transferred in both directions between C. acnes and C. granulosum in vitro. Furthermore, the pTZC1 was transferred between C. acnes and C. granulosum strains. Transconjugants were obtained from all the donors. The frequency of transconjugation differed according to the recipient strain. All the transconjugants showed high MIC values for clarithromycin and clindamycin. In contrast, the MIC values for doxycycline and minocycline in transconjugants were higher in the donor strains than in recipient strains and varied with the strain. These results strongly suggested that pTZC1 could be transferred between the C. acnes and C. granulosum strains on the skin and confer resistance to macrolides and clindamycin.
Growth ability of transconjugants and expression of pTZC1. The effect of pTZC1 acquisition was analyzed by calculating the doubling time and copy number of pTZC1 (Table 4). Differences in the doubling times were within 2-fold for all strains, indicating that pTZC1 acquisition did not affect the growth ability.
The copy numbers of pTZC1 in each recipient strain and the transconjugant were compared (Table 4). Although some combinations showed significant increases or decreases in the copy number depending on the strain, no relationship between the copy number of pTZC1 and the MIC values for macrolides, clindamycin, and tetracyclines was observed.

DISCUSSION
In 2008, the multidrug resistance plasmid pTZC1, which harbors erm(50) and tet(W), was found in the C. acnes strains isolated from acne patients in Osaka, Japan (16). Subsequently, C. acnes strains having pTZC1 were isolated in Tokyo and Hiroshima, and spread in Japan (12). In this study, the C. acnes and C. granulosum strains harboring pTZC1 were isolated together from two patients.
Therefore, pTZC1 was presumed to be transferred among different species. Of these two patients, one had use history of topical clindamycin and oral roxithromycin and the other had use history of oral minocycline (data not shown). Although antimicrobial use was not directly involved in the acquisition of antimicrobial resistance plasmid, increased selection pressure by antimicrobials may affect the advantage of survival in strains acquiring pTZC1.
pTZC1 sequences in C. acnes TP-CU426 and C. granulosum TP-CG7 isolated from the same patient showed 100% identity and had 500 bp deletion compared with pTZC1 in C. acnes TP-CU389 in our previous report (16). The 500-bp deletion sequence was located between the open reading frame (ORF) no. 10 and 11 in C. acnes TP-CU389. Therefore, the deleted sequence was to be a noncoding sequence that did not affect the plasmid function.
Therefore, pTZC1 was probably transferred between C. acnes TP-CU426 and C. granulosum TP-CG7 on the skin surface. In addition, the pTZC1 transfer test between C. acnes and C. granulosum showed bidirectional transconjugation. Transconjugants that acquired pTZC1 showed high-level resistance to clarithromycin and clindamycin (MICs $128 mg/mL). erm (50) encodes a 23S rRNA methylase and confers high-level resistance to macrolides and clindamycin (16). In contrast, tet(W) encodes a 16S rRNA protective protein, which is resistant to tetracyclines (16). C. acnes strains having tet(W) exhibited resistance or low susceptibility to tetracyclines, such as doxycycline and minocycline. Differences in tetracycline susceptibility may be related to tet(W) expression levels (4). Both the C. acnes TP-CU426 and C. granulosum TP-CG7 had not only acquisition of tet(W) but also rpsJ mutation (substitution of S10 protein), due to which these strains showed high MIC for doxycycline. The acquisition of antimicrobial resistance factors may affect the growth ability. However, the C. granulosum TP-CG7 clinical isolate carrying pTZC1 exhibited similar growth levels to C. granulosum ATCC25564 as a type strain, suggesting that the possession of pTZC1 has little effect on growth ability. Therefore, strains with pTZC1 are likely capable of surviving and inhabiting the skin surface, similar to those without pTZC1.
The Japanese acne treatment guidelines recommend the use of topical clindamycin, oral macrolides, and oral tetracyclines (3). In addition to antimicrobial activity, these antimicrobials have anti-inflammatory activity and have been used to treat acne for a long time (19). The increased prevalence of strains carrying the multidrug-resistant plasmid pTZC1 may cause failure in antimicrobial treatment for acne vulgaris. Unlike C. acnes, C. granulosum is not known to develop during acne exacerbation. C. granulosum occupies a lower proportion of the normal skin flora and can contribute to skin health (20). In contrast, C. granulosum, like C. acnes, becomes an opportunistic pathogen and reportedly causes pathogenesis in surgical site infections of artificial joints and endocarditis (21 to 25). Although Cutibacterium species have recently gained attention as opportunistic pathogens, little is known about antimicrobial resistance in C. granulosum (26,27). If C. granulosum causes opportunistic infections, an increase in the number of antimicrobial-resistant strains can hinder the selection of appropriate antimicrobials. Furthermore, pTZC1 transfer to C. acnes via C. granulosum may complicate the antimicrobial treatment of acne vulgaris.
In conclusion, our findings revealed that the multidrug resistance plasmid pTZC1 can be transferred between C. acnes and C. granulosum strains. Furthermore, because pTZC1 can be transferred between different species, it may aid in the prevalence of multidrug-resistant strains, and antimicrobial resistance genes may have been pooled on the skin surface. Further studies, such as in vitro skin models or ex vivo plasmid transfer studies, are necessary to confirm our findings.

MATERIALS AND METHODS
Strains and culture condition. Cutibacterium strains were collected from 212 patients with acne who visited Toranomon Hospital between 2013 and 2018, and 264 patients who visited 13 dermatology clinics between 2016 and 2017 (4,12,28,29). The colonies grown from specimens isolated from the patients under anaerobic conditions were determined using multiplex PCR for the identification of Cutibacterium species, according to our previous report (29). The strains could not be determined using multiplex PCR but were determined by BLAST homology analysis of 16S rRNA sequences. Single-locus sequence typing (SLST) of the C. acnes strains was performed using multiplex PCR according to the method described by Barnard et al. (30,31).
Other species were determined by BLAST homology analysis of 16S rRNA gene sequences (31,32). C. acnes ATCC 6919, ATCC 11828, and C. granulosum ATCC 25564 were used as the type strains for the quality control of antimicrobial susceptibility tests. All strains were incubated for 48 h at 35°C on modified GAM agar (Nissui Pharmaceutical, Tokyo, Japan) under anaerobic conditions in an anaerobic box (Hirasawa, Tokyo, Japan) and AnaeroPack-Anaero (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan).
Patients' information. A history of antimicrobial use within 6 months of occurrence of acne was obtained using a patient questionnaire. The questionnaire did not contain personally identifiable information, to protect patient privacy. The severity of acne was classified according to the establishment of grading criteria for acne severity (17).
Antimicrobial susceptibility testing. Antimicrobial susceptibility was evaluated by determining the MIC using the agar doubling dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (33). Bacterial strains were resuspended in Brucella broth (Becton, Dickinson, Franklin Lakes, NJ, USA) containing 5 mg/mL hemin (Alfa Aesar, Haverhill, MA, USA), 1 mg/mL vitamin K1 (FUJIFILM Wako Pure Chemical, Osaka, Japan), and 5% lysed defibrinated horse blood (Nippon Bio-test Laboratories, Saitama, Japan). The resuspension solution was inoculated on Brucella agar (Becton, Dickinson) containing 5 mg/mL hemin, 1 mg/mL vitamin K1, and 5% lysed defibrinated sheep blood (Nippon Bio-test Laboratories). Minocycline hydrochloride (Fujifilm Wako Pure Chemical), clarithromycin, clindamycin hydrochloride monohydrate, and doxycycline hydrate (Tokyo Chemical Industries) were used as antimicrobial agents. Resistance breakpoints of Cutibacterium species were defined based on the C. acnes strains defined by the M100 of the Clinical and Laboratory Standard Institute (13,33).
Transconjugation of erm(50) between C. acnes and C. granulosum. The conjugation test for the multidrug resistant plasmid pTZC1, which codes erm(50), was performed by modifying the filter mating method previously described by Aoki et al. (14). Donor and recipient strains were incubated in a modified GAM medium (Nissui Pharmaceutical) for 48 h with shaking, and then inoculated at 1/100 of the volume into fresh medium. Bacterial cultures were incubated until an optical density at 600 nm (OD 600 ) of 0.2 was obtained (OD 600 was measured using Multiskan procured by Thermo Fisher Scientific). Donor and recipient strains were diluted 10-fold in GAM medium and collected at a rate of 4:1 in a bacterial population on a nitrocellulose membrane filter (diameter, 13 mm; pore size, 0.45 mm; Advantec, Tokyo, Japan). These filters were placed on modified GAM agar and incubated under anaerobic conditions for 3 days at 35°C. The culture on these filters was resuspended in fresh medium and spread over modified GAM agar containing 50 mg/mL rifampicin and 2 mg/mL clarithromycin. Transconjugants containing plasmids were defined as grown colonies that tested positive for erm(50), tet(W), and traE by PCR (16). Transconjugation frequencies were presented as mean 6 standard deviation (SD) averaged across three independent experiments.
Growth ability of transconjugant. To evaluate the influence of plasmid acquisition on bacterial growth, growth curves were plotted for recipient strains and transconjugants (5). After 48 h of anaerobic incubation with shaking, the bacterial culture was inoculated into a freshly modified GAM medium, and the OD 600 of the cultures was measured over time. Doubling time was calculated as follows: [(t2 -t1) Â log 2 ]/(logOD 600 at t2 -log OD 600 at t1) (35). The data are presented as mean 6 SD averaged across three independent experiments.
Statistical analysis. Statistical comparisons between the two groups were performed using Welch's t test and Fisher's exact test in the js-STAR XR v.1.0.4j (http://www.kisnet.or.jp/nappa/software/star/) software.
Data availability. The genome sequences of the chromosomes and pTZC1 from C. acnes TP-CU426 were deposited in NCBI GenBank under accession numbers AP026712 and AP026713. The genome sequences of the chromosomes and pTZC1 from Cutibacterium granulosum were deposited in NCBI GenBank under accession numbers AP026710 and AP026711.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, TXT file, 0.1 MB.