| [Objective]Acne is a chronic inflammatory hair follicle sebaceous gland disease.It occurs predominantly in Adolescence.The epidemiological report shows that the prevalence of acne is between from 60% to 90%.Corynebacterium acnes(C.ances)are an important factor in the development of acne.Using antibiotics which has become one of the main treatments in acne,but this also lead to antibioctic resistance.By collecting outpatients in acne patients,isolate and culture clinical strains of C.acnes,conduct 11 clinically commonly used antibiotic susceptibility tests in vitro,we select clinically resistant and sensitive strains for high-throughput gene sequence.In order to understand the antibiotic resistence situation of acne patients in Dongguan area we provide theoretical basis for clinician to use drugs from the molecular level.[Methods]We Collected 212 cases of acne patients who meet the research criteria.Then collecting and isolating C.acnes,we cultured them in anaerobic conditions and preliminary identified bacterial morphology by Gram staining with microscopy.Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique,we can identify strains.We also tested drug susceptibility in vitro and detected the minimum inhibitory concentrations of macrolides,lincosamides,tetracyclines,cephalosporins,quinolones,nitroimidazoles,and sulfonamides in clinical isolates by agar dilution method.Based on drug susceptibility test,57 strains including highly cross-resistant ones and sensitive ones were obtained.DNA and agarose gel electrophoresis were performed using Cetyltrimethylammonium Ammonium Bromide(CTAB)method.After DNA integrity was tested,whole high-throughput shotgun(WGS)resequencing was performed on the machine.The standard C.acnes strain ATCC 6919 was sequenced using the second generation sequencing Illuminate and three-generation sequencing PacBio RSII.The experimental data assembly was completed by SMRT Analysis(HGAP3).[Results]1.In 212 acne patients,156 strains of Corynebacterium acnes were isolated.In vitro susceptibility test showed that,156 strains of Corynebacterium acnes were 100 % sensitive to tetracycline,doxycycline,minocycline and ceftriaxone,while 100 % resistant to metronidazole and sulfamethoxazole.The remaining antibiotics including erythromycin,azithromycin,clarithromycin,clindamycin and levofloxacin have multiple levels of cross-resistance.Drug resistance result was as follows: clarithromycin 84.0 %(131/156),azithromycin 48.7 %(76/156),erythromycin 30.1 %(47/156),clindamycin 28.9%(45/156)and levofloxacin 5.1 %(8/156).Of all of them 19.4 % were double resistant,5.7 % were triple resistant,28.1 % were resistant to quadruple and 4.3 % were resistant to five.2.The complete genome of Corynebacterium acnes ATCC 6919(ID: CP023676)consists of a single circular chromosome of 2,495,001 base pairs in length(1 contig,N50 of 13023)with a GC content of 60.02% and it contains 2,450 genes.We predicted and annotated 2,437 putative genes with a sequencing depth of 100 x.We found that ATCC 6919 carries the thyA resistance gene(ID: 08890)in the CARD database.3.Among the 57 strains,4 were sensitive ones,7 strains were low-to-moderately resistant to clarithromycin,6 strains were low or highly resistant to azithromycin and the remaining 40 strains were multiple resistant ones.Of the 40 multiple resistant strains,25%(10/40)were found to have A?G mutations at the 2058 site of the 23 SrRNA fragment,and 5%(2/40)were found to have A?T mutations at the 2058 site of the 23 SrRNA fragment.There is 65%(26/40)annotated ermX resistance gene positive by the CARD and ARDB databases and remaining 5%(2/40)was found neither resistance gene nor mutation sites on 23 SrRNA fragment.There was no overlap between samples which carry ermX resistance gene and mutations at the 2058 site of the 23 SrRNA fragment.In addition,of the 40 multiple strains,6 were found five resistance to different antibiotics and 83.3%(5/6)was annotated ermX resistance gene positive by the CARD and ARDB databases.[Conclusion]This study was the first in China to use the second-generation sequencing technology to detect Corynebacterium acnes resistance genes.Corynebacterium acnes ATCC 6919 was detected by he second and third generation whole genome sequencing and sequencing results were uploaded to the NCBI database.A large simple size of 57 Corynebacterium acnes strains were used in this study.The drug resistance result showed that 65% of multidrug-resistant strains carried ermX resistance genes and 30% found AG or AT mutations at position 2058 of 23 SrRNA fragments.There is also an unknown drug resistance mechanism in 5% of multi-drug resistant strains.The proportion of this drug resistance mechanism is consistent with the clinical manifestations of extensive resistance to macrolide-lincosamide antibiotics in Corynebacterium acnes. |
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