Antimicrobial spectrum, resistance gene detection and ERIC-PCR genotyping of Vibrio scophthalmi
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摘要: 2020年6—10月，从辽宁地区患病大菱鲆 (Scophthalmus maximus) 体内共分离到394株菌株。基于16S rRNA序列鉴定分离株，采用微量稀释法分析随机挑选的18株大菱鲆弧菌 (Vibrio scophthalmi) 对8种抗生素的敏感性，检测其耐药基因携带情况，并基于ERIC-PCR进行分型研究。结果显示，分离株以弧菌属为主，共232株 (58.88%)，其中大菱鲆弧菌117株 (29.70%)。经rpoD序列比对确定的18株大菱鲆弧菌对硫酸新霉素、氟甲喹和盐酸多西环素不耐药，对其他5种抗菌药物有不同的耐药率，多重耐药率为66.7%。18株大菱鲆弧菌共检测到酰胺醇类耐药基因floR (61.11%) 和cmlA (66.67%)、磺胺类耐药基因sul2 (55.56%)、喹诺酮类耐药基因qnrA (50%) 和qnrS (5.56%) 5种耐药基因，未检测到氨基糖苷类和四环素类耐药基因。酰胺醇类耐药基因与耐药表型符合率为88.89%，有一定的相关性。磺胺类耐药基因与耐药表型符合率次之 (50%)，喹诺酮类符合率最低 (33.33%)。ERIC-PCR将该18株大菱鲆弧菌分为4个亚型，I型 (44.44%) 和III型 (44.44%) 为主要谱型，与耐药表型或耐药基因无明显相关性。根据药敏结果，防治辽宁地区大菱鲆弧菌建议首选盐酸多西环素。Abstract: From June to October of 2020, we had isolated 394 strains from diseased turbot (Scophthalmus maximus) in Liaoning Province, and identified them based on the 16S rRNA sequence. Then we randomly selected 18 Vibrio scophthalmi strains, which were further typed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), so as to analyze the susceptibility to 8 antibiotics and the presence of drug-resistance genes. The results show that most of the isolates were Vibrio with 232 strains (58.88%), among which 117 strains (29.70%) were V. scophthalmi. The 18 strains identified by rpoD sequence alignment were not resistant to neomycin sulfate, flumequine and doxycycline hydrochloride, but resistant to other five antibiotics with different ratios. The multiple drug resistance rate was 66.7%. Five drug resistance genes in the 18 strains of V. scophthalmi were detected (Amide alcohol resistance genes floR of 61.11% and cmlA of 66.67%, sulfonamide resistance gene sul2 of 55.56%, quinolone resistance genes qnrA of 50% and qnrS of 5.56%). No aminoglycosides or tetracycline resistant genes were detected. The coincidence rate of amide alcohol resistance genes and drug resistance phenotype was 88.89%, indicating a certain correlation between them. Sulfonamide resistance genes had a coincidence rate of 50% with the resistance phenotype, and quinolones resistance genes had the lowest coincidence rate of 33.33%. Finally, we clustered the 18 strains into four types by using ERIC-PCR fingerprinting. Type I (44.44%) and Type III (44.44%) were the main clusters. We observed apparent absence of correlation between ERIC-PCR fingerprinting and the resistance phenotype or resistance genes. Thus, it is conluded that doxycycline hydrochloride can be the first choice for the control of V. scophthalmi in Liaoning aquaculture farms.
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Keywords:
- Vibrio scophthalmi /
- Drug resistance /
- Microdilution method /
- Drug resistance genes /
- ERIC-PCR
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图 1 18株大菱鲆弧菌rpoD基因PCR扩增结果
M. DL2000 DNA Marker；1—18. Vs1—Vs18；N. 阴性对照。
Figure 1. PCR amplification product of rpoD in 18 strains of V. scophthalmi
M. DL2000 DNA Marker; 1 to 18. Vs1 to Vs18; N. Negative control.
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图 2 基于弧菌rpoD序列相似性构建的系统发育进化树
Figure 2. Phylogenetic tree constructed from rpoD sequence of Vibrio
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图 3 大菱鲆弧菌分离株ERIC-PCR指纹图谱与UPGMA树状图
Figure 3. Genetic diversity diagram based on ERIC-PCR and UPGMA dendrogram of V. scophthalmi strains
表 1 PCR引物
Table 1 Primers used for PCR
目的基因
Target gene引物序列 (5'—3')
Primer sequence (5'—3')产物长度
Product size/bp参考文献
Reference16S rRNA 27F: AGAGTTTGATCCTGGCTCAG
1492R: GGTTACCTTGTTACGACTT约1 500 [8] rpoD F:ATAGAAATAACCAGACGTAAGTTNGCYTCNACCATYTCYTTYT
R:ACGACTGACCCGGTACGCATGTAYATGMGNGARATGGGNACNGT780 [16] 氨基糖苷类耐药基因
Aminoglycoside resistance geneaph (3')-Ⅰa F: TGACTGGGCACAACAGACAA
R: CGGCGATACCGTAAAGCAC677 [17] aac (6') -Ⅰb F: ATGACCTTGCCATGCTCTATGA
R: CGAATGCCTGGCGTGTTT486 [17] aac (3')-Ⅰb F: ACCCTACGAGGAGACTCTGAATG
R: CCAAGCATCGGCATCTCATA384 [17] 酰胺醇类耐药基因
Amide alcohol resistance genefloR F: GCTTCACTGGCGATGGATATTTA
R: CAAAGTAATGAATATCGCCTGCC450 [18] catA F: AAAAATTATATCCGACTCTCTTA
R: CTTGAATCGATAAGGGAATATAG420 [18] cmlA F: TGCCAGCAGTGCCGTTTAT
R: CACCGCCCAAGCAGAAGTA500 [18] 四环素类耐药基因
Tetracycline resistance genetetA F: TTTCGGGTTCGGGATGGT
R: CAGGCAGAGCAAGTAGAGGG915 [19] tetC F: CTGGGCTGCTTCCTAATGC
R: AGCTGTCCCTGATGGTCGT480 [19] tetM F: GAGGTCCGTCTGAACTTTGCG
R: AGAAAGGATTTGGCGGCACT580 [19] 磺胺类耐药基因
Sulfonamide resistance genesul1 F: CATTGCCTGGTTGCTTCAT
R: ATCCGACTCGCAGCATTT238 [19] sul2 F: CATCATTTTCGGCATCGTC
R: TCTTGCGGTTTCTTTCAGC793 [19] sul3 F: AGATGTGATTGATTTGGGAGC
R: TAGTTGTTTCTGGATTAGAGCCT443 [19] 喹诺酮类耐药基因
Quinolone resistance geneqnrA F: ATTTCTCACGCCAGGATTTG
R: GATCGGCAAAGGTCAGGTCA516 [20] qnrB F: GATCGTGAAAGCCAGAAAGG
R: ACGATGCCTGGTAGTTGTCC469 [20] qnrS F: ACGACATTCGTCAACTGCAA
R: TAAATTGGCACCCTGTAGGC417 [20] aac (6')-Ⅰb-cr F: TTGCGATGCTCTATGAGTGGCTA
R: CTCGAATGCCTGGCGTGTTT523 [20] oqxAB F: CCCTGGACCGCACATAAAG
R: AAAGAACAAGATTCACCGCAAC482 [20] 
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表 2 基于16S rRNA测序的394株分离株鉴定结果
Table 2 Identification result of 394 strains based on 16S rRNA sequencing
属
Genus种
Species6月
Jun.7月
Jul.8月
Aug.9月
Sep.10月
Oct.合计
Total弧菌属 Vibrio 大菱鲆弧菌 V. scophthalmi 13 10 35 26 33 117 (29.70%) 溶藻弧菌 V. alginolyticus 0 1 23 11 0 35 (8.89%) V. atlanticus 2 3 1 6 3 15 (3.81%) 嗜环弧菌 V. cyclitrophicus 0 0 0 4 6 10 (2.54%) V. toranzoniae 1 2 1 7 4 15 (3.81%) 其他 Other 7 3 2 17 11 40 (10.15%) 假单胞菌属 Pseudomonas 8 3 8 11 16 46 (11.68%) 假交替单胞菌属 Pseudoalteromonas 0 5 12 18 0 35 (8.88%) 其他 Other 14 17 10 33 7 81 (20.56%) 合计 Total 45 44 92 133 80 394
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表 3 18株大菱鲆弧菌的药物敏感性
Table 3 Drug sensitivity of 18 V. scophthalmi strains
分离株
Isolate恩诺沙星
ENR硫酸新霉素
NEO甲砜霉素
TAP氟苯尼考
FFC盐酸多西环素
DO氟甲喹
FLU磺胺间甲氧嘧啶钠
SMM磺胺甲恶唑+甲氧苄啶
SMZ+TMPVs1 S I S S S S S S Vs2 S S R R S S R S Vs3 S S S S S S R R Vs4 S S S S S S S S Vs5 S S S S S S R S Vs6 S I S S S S S S Vs7 S S S S S S R R Vs8 S S S S S S S S Vs9 R I R R S S S S Vs10 R S R R S S S S Vs11 S I R R S S S S Vs12 R S R R S S S S Vs13 S I S S S S R R Vs14 S I S S S S S S Vs15 I S R R S S R R Vs16 R S R R S S S S Vs17 R I R R S S S S Vs18 R I R R S S S S 注：S. 敏感；I. 中敏；R. 耐药。 Note: S. Sensitive; I. Intermediary; R. Resistant.
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表 4 大菱鲆弧菌耐药基因携带情况
Table 4 Drug resistance genes status of V. scophthalmi
抗生素类型
Type of antibiotics耐药基因
Drug resistance gene携带耐药基因的分离株 (N=18)
Isolates carrying resistance gene氨基糖苷类 Aminoglycosides aac (6')-Ⅰb, aac (6')-Ⅰb, aac (3')-Ⅰb 0 (0%) 四环素类 Tetracyclines tetA, tetC, tetM 0 (0%) 酰胺醇类 Amido alcohols floR Vs2, Vs3, Vs4, Vs7, Vs9, Vs11, Vs12, Vs15, Vs16, Vs17, Vs18 (61.11%) catA 0 (0%) cmlA Vs1, Vs3, Vs4, Vs5, Vs6, Vs7, Vs8, Vs9, Vs12, Vs15, Vs16, Vs18 (66.67%) 磺胺类 Sulfonamides sul1 0 (0%) sul2 Vs3, Vs4, Vs7, Vs9, Vs11, Vs12, Vs15, Vs16, Vs17, Vs18 (55.56%) sul3 0 (0%) 喹诺酮类 Quinolones qnrA Vs1, Vs2, Vs3, Vs5, Vs7, Vs8, Vs9, Vs14, Vs18 (50%) qnrB 0 (0%) qnrS Vs11 (5.56%) oqxAB 0 (0%) aac (6')-Ⅰb-cr 0 (0%)
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表 5 大菱鲆弧菌分离株耐药基因与耐药表型符合率
Table 5 Coincidence rate of drug resistance genes and phenotype of V. scophthalmi strains
抗生素类型
Type of antibiotics耐药表型株数
Number of isolates with
drug resistance phenotype有耐药表型且携带耐药基因株数
Number of strains with drug
resistance phenotype and
drug resistance genes符合率
Coincidence rate/%氨基糖苷类 Aminoglycosides 0 0 100 四环素类 Tetracyclines 0 0 100 酰胺醇类 Amido alcohols 9 8 88.89 磺胺类 Sulfonamides 6 3 50 喹诺酮类 Quinolones 6 2 33.33
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表 6 大菱鲆弧菌分离株耐药谱、耐药基因谱和ERIC基因型
Table 6 Drug resistance spectrum, resistance gene spectrum and ERIC genotype of V. scophthalmi strains
耐药谱
Antibiotic resistance spectrum耐药基因谱
Drug resistance genes spectrumERIC基因型
ERIC genotypeVs1 无 cmlA+qnrA Ⅰ Vs2 甲砜霉素、氟苯尼考、磺胺间甲氧嘧啶钠 floR+qnrA Ⅰ Vs6 无 cmlA Ⅰ Vs7 磺胺间甲氧嘧啶钠、磺胺甲恶唑甲氧苄啶复合物 floR+cmlA+sul2+qnrA Ⅰ Vs10 甲砜霉素、氟苯尼考、恩诺沙星 无 Ⅰ Vs13 磺胺间甲氧嘧啶钠、磺胺甲恶唑甲氧苄啶复合物 无 Ⅰ Vs15 甲砜霉素、氟苯尼考、磺胺间甲氧嘧啶钠、磺胺甲恶唑甲氧苄啶复合物 floR+cmlA+sul2 Ⅰ Vs18 甲砜霉素、氟苯尼考、恩诺沙星 floR+cmlA+sul2+qnrA Ⅰ Vs11 甲砜霉素、氟苯尼考 floR+cmlA+sul2+qnrS Ⅱ Vs3 磺胺间甲氧嘧啶钠、磺胺甲恶唑甲氧苄啶复合物 floR+cmlA+sul2+qnrA Ⅲ Vs4 无 floR+cmlA+sul2 Ⅲ Vs5 磺胺间甲氧嘧啶钠 cmlA+qnrA Ⅲ Vs8 无 cmlA+qnrA Ⅲ Vs9 甲砜霉素、氟苯尼考、恩诺沙星 floR+cmlA+sul2+qnrA Ⅲ Vs14 无 qnrA Ⅲ Vs16 甲砜霉素、氟苯尼考、恩诺沙星 floR+cmlA+sul2 Ⅲ Vs17 甲砜霉素、氟苯尼考、恩诺沙星 floR+sul2 Ⅲ Vs12 甲砜霉素、氟苯尼考、恩诺沙星 floR+cmlA+sul2 Ⅳ
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