Epidemiological surveillance and genetic evolution of Riemerella anatipestifer in Guangdong Province
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摘要:目的
明确广东地区鸭疫里默杆菌Riemerella anatipestifer的血清型、耐药状况及遗传进化关系。
方法从规模化鸭场分离鉴定鸭疫里默氏杆菌，通过玻片凝集试验鉴定血清型；利用试管两倍稀释法测试抗菌药物的最低抑菌浓度，分析药物的敏感性；采用全基因组测序技术分析序列特征并构建核心基因组遗传进化树。
结果共分离鉴定鸭疫里默氏杆菌168株，血清1、2、3、5、6、7、8、10型均有流行，血清1型的菌株高达54.17% (91/168)，其次为2型，占27.97%(47/168)。48株代表性菌株对庆大霉素、卡那霉素、盐酸环丙沙星表现高度耐药，耐药率均超过80%；对土霉素、盐酸四环素、盐酸金霉素、氧氟沙星、诺氟沙星、磺胺二甲嘧啶、磺胺对甲氧嘧啶的耐药率均在60%以上；对阿莫西林、头孢噻肟和大观霉素的耐药率低于30%。受试菌株对5~12种药物耐药，共有44种耐药谱型。成功获得46株菌株全基因组序列，共检出6种耐药基因，其中，耐药基因erm(F)和tet(X)的检出率较高，分别为73.91%(34/46)和82.60%(38/46)，同时携带2种以上耐药基因的菌株占95.65%(44/46)。18株(39.13%，18/46)菌株ST分型成功，分属11个ST型。所有测序菌株与数据库中来自中国的菌株在遗传进化关系上最接近，主要存在于优势克隆群系Clade 1和Clade 3中。
结论本研究鸭疫里默氏杆菌分离株的优势血清型为1型，耐药性严重，所携带的耐药基因与耐药表型具有一定相关性，ST型呈多样性，与多位点序列分型(Multi-locus sequence typing, MLST)数据库中来自我国菌株的遗传背景相近。研究结果可为鸭疫里默氏杆菌病疫苗免疫预防与药物治疗提供依据，有助于掌握鸭疫里默氏杆菌遗传进化特征。
Abstract:ObjectiveTo clarify serotype, drug resistance and genetic evolution of Rimerella anatipestifer in Guangdong.
MethodThe isolates of R. anatipestifer were isolated and identified from samples of the large-scale duck farms. Serotype was identified by glass agglutination test. The minimum inhibitory concentration was tested by double dilution method in test tube, and the drug sensitivity was analyzed. Whole genome sequencing technology was used to analyze the sequence characteristices and construct the genetic evolution tree of the core genome.
ResultA total of 168 isolates of R. anatipestifer were isolated and identified. All serotype 1, 2, 3, 5, 6, 7, 8 and 10 were prevalent. Serotype 1 was dominant, reaching 54.17%(91/168), followed by serotype 2 (27.97%, 47/168). The 48 representative isolates were used for drug sensitivty test and sequence analysis. The isolates showed high drug resistance to gentamicin, kanamycin and ciprofloxacin hydrochloride, and the drug resistance rates were all more than 80%. The drug resistance rates to oxytetracycline, tetracycline hydrochloride, chlortetracycline hydrochloride, ofloxacin, norfloxacin, sulfadimidine and sulfamethoxydiazine were all more than 60%, and the drug resistance rates to amoxicillin, cefotaxime and spectinomycin were all less than 30%. The 48 representative isolates were resistant to 5−12 kinds of drugs, and there were 44 types of drug resistance spectrum. The whole genome sequences of 46 isolates were obtained successfully, and six drug-resistant genes were detected. The detection rates of drug-resistant geneserm(F) and tet(X) were 73.91% (34/46) and 82.60% (38/46), respectively. In addition, 95.65% (44/46) of the isolates carried more than two drug-resistant genes at the same time. Total 18 isolates (39.13%, 18/46) were typed successfully with 11 kinds of ST. Further genetic tree construction based on the core genome showed that all the sequenced isolates had a high similarity with the isolates from China in the multi-locus sequence typing (MLST) database, and mainly existed in the dominant clones of Clade 1 and Clade 3.
ConclusionSerotype 1 is superior in the investigated R. anatipestifer isolates, and the drug resistance is serious. The drug-resistant genes and resistant phenotype have certain relevance. ST type has a high diversity. The genetic background of tested strains was similar with Chinese strains in MLST database. The results of this study can provide a basis for vaccine immunity prevention and drug therapy of R. anatipestife disease as well as for understanding the genetic evolution characteristics of R. anatipestife.
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Keywords:
- Riemerella anatipestife /
- Serotype /
- Drug resistance /
- Genetic evolution /
- Epidemiology
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图 1 鸭疫里默氏杆菌分离株PCR鉴定结果
M：DNA标准DL2000；1、2：鸭疫里默氏杆菌分离株；3：阳性对照；4：阴性对照
Figure 1. PCR identification results of Riemerella anatipestifer isolates
M: DNA marker DL2000; 1, 2: Riemerella anatipestifer isolates; 3: Positive control; 4: Negative control
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图 2 48株鸭疫里默氏杆菌代表性分离株多重耐药性分布
Figure 2. Results of multi-drug resistance of 48 Riemerella anatipestifer representative isolates
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图 3 105株鸭疫里默氏杆菌菌株核心基因组系统进化树
Figure 3. Phylogenetic tree of core genomes for 105 isolates of Riemerella anatipestifer
表 1 采集样本与分离鸭疫里默氏杆菌信息
Table 1 Collected samples and isolation information of Riemerella anatipestifer
地区
District采集时间
Acquisition time养殖场数
Farm number样本数
Sample number分离株数
Isolate number代表性株数
Representative isolate number韶关 Shaoguan 2015−05—2019−04 19 69 15 5 河源 Heyuan 2015−07—2019−11 16 56 13 5 云浮 Yunfu 2015−09—2019−12 30 112 25 4 肇庆 Zhaoqing 2015−04—2019−10 17 63 12 4 清远 Qingyuan 2015−03—2019−12 23 91 19 5 佛山 Foshan 2015−05—2019−12 15 57 12 5 茂名 Maoming 2015−04—2019−11 20 80 16 5 惠州 Huizhou 2015−05—2019−11 23 80 17 5 汕头 Shantou 2015−03—2019−12 25 91 21 5 湛江 Zhanjiang 2015−05—2019−12 22 82 18 5 总计 Total 2015—2019 210 781 168 48 
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表 2 各地区各血清型鸭疫里默氏杆菌分离株数量
Table 2 Isolate number of each serotype for Riemerella anatipestifer isolates from different districts
地区
District分离株数
Isolate number血清型 Serotype 1 2 3 4 5 6 7 8 9 10 未定型 Undefined 韶关 Shaoguan 15 7 2 2 0 0 1 0 1 0 0 2 河源 Heyuan 13 7 4 1 0 0 0 0 0 0 1 0 云浮 Yunfu 25 15 8 0 0 1 0 0 0 0 0 1 肇庆 Zhaoqing 12 6 3 1 0 0 0 0 1 0 1 0 清远 Qingyuan 19 10 6 1 0 1 0 0 0 0 0 1 佛山 Foshan 12 7 4 0 0 0 0 1 0 0 0 0 茂名 Maoming 16 8 5 1 0 0 0 0 1 0 1 0 惠州 Huizhou 17 12 3 0 0 0 0 1 0 0 1 0 汕头 Shantou 21 12 7 0 0 0 0 0 0 0 2 0 湛江 Zhanjiang 18 7 5 0 0 1 1 1 2 0 0 1 总计 Total 168 91 47 6 0 3 2 3 5 0 6 5
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表 3 48株鸭疫里默氏杆菌代表性分离株最低抑菌浓度(MIC)测定结果1)
Table 3 The minimum inhibitory concentration (MIC) of 48 representative isolates of Riemerella anatipestifer
药物类型
Drug type药物1)
DrugMIC/
(mg·L−1)MIC50/
(mg·L−1)MIC90/
(mg·L−1)占比/% Proportion 敏感
Sensitive中介
Intermediate耐药
Resistantβ−内酰胺类 AMX <0.125~256 1 128 75.00(36/48) 6.25(3/48) 18.75(9/48) β-lactams AMP <0.125~256 8 128 64.58(31/48) — 35.42(17/48) CTX <0.125~256 0.25 8 91.67(44/48) 4.17(2/48) 4.17(2/48) 四环素类 OXY <0.125~128 8 16 20.83(10/48) 14.58(7/48) 64.58(31/48) Tetracyclines TCY <0.125~128 8 32 18.75(9/48) 10.42(5/48) 70.83(34/48) CTE <0.125~32 8 16 18.75(9/48) 20.83(10/48) 60.42(29/48) 氨基糖苷类 GEN 0.25~>256 128 >256 8.33(4/48) — 91.67(44/48) Aminoglycosides KAN 2~>256 >256 >256 8.33(4/48) 2.08(1/48) 89.58(43/48) STP 16~256 64 128 45.83(22/48) 25.00(12/48) 29.17(14/48) 喹诺酮类 OFX 0.5~64 8 32 20.83(10/48) 6.25(3/48) 72.92(35/48) Quinolones NOR 0.25~128 32 64 14.58(7/48) 16.67(8/48) 68.75(33/48) CIP 0.5~64 16 32 14.58(7/48) 4.17(2/48) 81.25(39/48) 磺胺类 SDI 64~>256 256 >256 52.08(25/48) — 47.92(23/48) Sulfonamides SUL 32~>256 >256 >256 39.58(19/48) — 60.42(29/48) SMD 128~>256 >256 >256 33.33(16/48) — 66.67(32/48) 1)AMX：阿莫西林，AMP：氨苄西林，CTX：头孢噻肟，OXY：土霉素，TCY：盐酸四环素，CTE：盐酸金霉素，GEN：庆大霉素，KAN：卡那霉素，STP：大观霉素，OFX：氧氟沙星，NOR：诺氟沙星，CIP：盐酸环丙沙星，SDI：磺胺嘧啶，SUL：磺胺二甲嘧啶，SMD：磺胺对甲氧嘧啶；MIC50和MIC90为能抑制50%和90%细菌生长所需的MIC；“—”表示未检测出菌株
1) AMX: Amoxicillin, AMP: Ampicillin, CTX: Cefotaxime, OXY: Oxytetracycline, TCY: Tetracycline hydrochloride, CTE: Chlortetracycline hydrochloride, GEN: Gentamicin, KAN: Kanamycin, STP: Spectinomycin, OFX: Ofloxacin, NOR: Norfloxacin, CIP: Ciprofloxacin hydrochloride, SDI: Sulfadiazine, SUL: Sulfadimidine, SMD: Sulfametoxydiazine; MIC50 and MIC90 are MIC values that can inhibit 50% and 90% bacteria growth; “—” indicates no detectable isolate
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表 4 48株鸭疫里默氏杆菌代表性分离株耐药谱统计结果
Table 4 Drug resistance spectrum of 48 representative isolates of Riemerella anatipestifer
药物数量
Drug number耐药谱1)
Resistant spectrum菌株数
Isolate number占比/%
Proportion5 OFX-NOR-OXY-GEN-KAN
OFX-CTE-GEN-CIP-KAN1
12.08
2.086 OFX-NOR-TCY-GEN-CIP-KAN
OFX-NOR-GEN-CIP-KAN-SMD
NOR-GEN-CIP-KAN-SUL-SMD
CIP-KAN-STP-SUL-SMD-SDI1
1
1
12.08
2.08
2.08
2.087 OXY-TCY-CTE-GEN-KAN-SUL-NOR
OXY-GEN-KAN-SDI-SUL-SMD-CIP
OXY-TCY-CTE-GEN-KAN-OFX-CIP
OXY-TCY-CTE-GEN-KAN-NOR-CIP1
1
1
22.08
2.08
2.08
4.168 OFX-NOR-TCY-CTE-GEN-CIP-SUL-SMD
NOR-TCY-CTE-GEN-CIP-KAN-SDI-SMD
CIP-GEN-SDI-SUL-SMD-NOR-TCY-AMP
AMX-AMP-OXY-TCY-CTE-GEN-KAN-OFX
AMP-STP-SDI-SUL-SMD-GEN-KAN-OXY1
1
1
1
12.08
2.08
2.08
2.08
2.089 OFX-NOR-TCY-OXY-CTE-GEN-CIP-KAN-SUL
OFX-NOR-TCY-CIP-KAN-SDI-SUL-SMD-GEN
OFX-NOR-AMX-CTE-GEN-CIP-KAN-SUL-SMD
OXY-TCY-GEN-KAN-OFX-NOR-CIP-SDI-SUL
TCY-GEN-CIP-KAN-SDI-SMD-CTE-OFX-SUL
OXY-AMP-GEN-KAN-STP-SDI-SUL-SMD-NOR
OFX-NOR-OXY-GEN-CIP-KAN-SUL-SMD-SMD
OFX-TCY-OXY-GEN-KAN-STP-SDI-SUL-SMD
AMP-CTX-SDI-GEN-SMD-OFX-STP-KAN-CIP
AMX-AMP-OXY-TCY-STP-KAN-OFX-SDI-GEN
SMD-ERY-OFX-SDI-CIP-AMX-AMP-CTE-TCY
NOR-TCY-OXY-CTE-GEN-CIP-KAN-SUL-AMP2
1
1
2
1
1
1
1
1
1
1
14.16
2.08
2.08
4.16
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.0810 NOR-TCY-OXY-CTE-GEN-CIP-KAN-STP -SMD-SDI
AMP-OXY-TCY-CTE-GEN-KAN-OFX-NOR-CIP-SMD
OFX-NOR-TCY-CTE-GEN-CIP-KAN-SDI-SMD-SUL
OFX-NOR-TCY-OXY-CTE-GEN-CIP-KAN-SMD-SDI
OFX-NOR-AMX-AMP-GEN-CIP-KAN-SUL-SMD-CTE
OFX-NOR-TCY-AMX-OXY-CTE-GEN-CIP-KAN-SDI
OFX-NOR-TCY-OXY-CTE-GEN-CIP-KAN-SUL-SMD
TCY-AMP-GEN-CIP-KAN-STP-SDI-SUL-SMD-KAN
AMX-AMP-OXY-TCY-CTE-GEN-KAN-OFX-SDI-SUL
OFX-NOR-OXY-CTE-AMP-GEN-CIP-KAN-SUL-SMD
OFX-NOR-AMX-AMP-GEN-CIP-KAN-SDI-SMD-STP
TCY-OXY-GEN-KAN-STP-SDI-SUL-SMD-CTX-AMP1
1
1
1
1
1
1
1
1
1
1
12.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.0811 OFX-NOR-TCY-CTE-OXY-GEN-CIP-KAN-STP-SDI-SMD
OFX-NOR-TCY-OXY-CTE-AMP-GEN-CIP-KAN-STP-SUL
OFX-NOR-TCY-OXY-CTE-AMP-GEN-CIP-KAN-STP-SMD2
1
14.16
2.08
2.0812 AMX-OXY-TCY-CTE-STP-KAN-OFX-NOR-SUL-SMD-SMD-CIP
OFX-NOR-TCY-OXY-AMP-GEN-CIP-STP-SUL-SMD-SDI-CTE1
12.08
2.081) AMX：阿莫西林，AMP：氨苄西林，CTX：头孢噻肟，OXY：土霉素，TCY：盐酸四环素，CTE：盐酸金霉素，GEN：庆大霉素，KAN：卡那霉素，STP：大观霉素，OFX：氧氟沙星，NOR：诺氟沙星，CIP：盐酸环丙沙星，SDI：磺胺嘧啶，SUL：磺胺二甲嘧啶，SMD：磺胺对甲氧嘧啶
1) AMX: Amoxicillin, AMP: Ampicillin, CTX: Cefotaxime, OXY: Oxytetracycline, TCY: Tetracycline hydrochloride, CTE: Chlortetracycline hydrochloride, GEN: Gentamicin, KAN: Kanamycin, STP: Spectinomycin, OFX: Ofloxacin, NOR: Norfloxacin, CIP: Ciprofloxacin hydrochloride, SDI: Sulfadiazine, SUL: Sulfadimidine, SMD: Sulfametoxydiazine
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表 5 46株鸭疫里默氏杆菌代表性分离株耐药基因携带情况
Table 5 The resistance genes carried by 46 Riemerella anatipestifer representative isolates
药物
Drug耐药基因
Resistant gene阳性率/%
Positive rate大环内酯类 erm(F) 73.91(34/46) Macrolides ere(D) 19.57(9/46) 四环素类
Tetracyclinestet(X) 82.60(38/46) 氟苯尼考
FlorfenicolfloR 30.43(14/46) 氨基糖苷类
AminoglycosidesaadS 34.78(16/46) β−内酰胺类 β-lactams bla OXA-209 54.35(25/46)
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