Antibiotic resistance and molecular characteristics of Salmonella Typhimurium isolated from chicken and pork in Guangzhou, Guangdong in 2018
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摘要:
目的 调查2018年广州市鸡源和猪源鼠伤寒沙门菌的耐药性及分子特征，为合理使用抗生素和耐药菌防控提供依据。 方法 使用API 20E生化试验鉴定鼠伤寒沙门菌。 使用CMV3AGNF药敏板进行药敏试验。 利用Illumina Miseq测序平台进行全基因组测序，分析序列类型、抗生素耐药基因和点突变，以及构建系统发育树。 结果 广州市鸡源（15株）和猪源（21株）鼠伤寒沙门菌对氨苄青霉素、萘啶酸、四环素、氯霉素和磺胺异恶唑的耐药率≥60.00%，多重耐药率超过65.00%，以氨苄西林-萘啶酸-四环索-氯霉素-（甲氧苄氨嘧啶/磺胺甲恶唑-磺胺异恶唑）-庆大霉素耐药谱为主，且猪源菌耐药更为严重。 猪源菌对tet(B)、aph(3'')-Ib和aph(6)-Id的携带率高于鸡源菌（P<0.05），携带更多的耐药基因（P<0.05）。 GyrA点突变在鸡源菌和猪源菌的分布差异无统计学意义。 系统发育及序列类型分析发现，鼠伤寒沙门菌共分为4个分支，鸡源菌更具遗传多态性，C4优势分支同时包含鸡源菌和猪源菌。 结论 鼠伤寒沙门菌耐药普遍，猪源菌的耐药则更为严重。畜牧业应合理使用抗生素，应加强鼠伤寒沙门菌的病原监测尤其是对猪肉食品的监测，防控耐药菌的传播。 Abstract:Objective To investigate the antibiotic resistance and molecular characteristics of Salmonella Typhimurium isolated from chicken and pork in Guangzhou, 2018, and provide data support for the rational use of antibiotics and the prevention of drug-resistant pathogens. Methods The S. Typhimurium isolates were identified by using API 20E biochemical tests, and antibiotic susceptibility testing was performed by using Sensititre CMV3AGNF plates. The whole genome was sequenced by using Illumina Miseq platform to analyze sequence types (ST), antibiotic resistance genes (ARGs) and point mutations, and to construct phylogenetic tree. Results The antibiotic resistance rates of S. Typhimurium strains isolated from chicken (n=15) and pork (n=21) in Guangzhou to ampicillin, nalidixic acid, tetracycline, chloramphenicol, and sulfisoxazole were ≥60.00%, and the multi-drug resistance (MDR) rates were more than 65.00%. The ampicillin-nalidixic acid-tetracycline-chloramphenicol-(trimethoprim/sulfamethoxazole-chloramphenicol)-gentamicin was the predominant antibiotic resistance pattern, and the drug resistance of S. Typhimurium isolated from pork was more serious. The carriage rates of tet(B), aph(3")-Ib, and aph(6)-Id were higher in pork isolates than in chicken isolates, and more ARGs were found in pork isolates. There was no difference in the distribution of gyrA point mutations between isolates from chicken and pork. Phylogenetic analysis and sequence type analysis showed that the S. Typhimurium were divided into four clades. The chicken isolates were more genetically polymorphic, and the C4 dominant clades contained both isolates from chicken and pork. Conclusion The antibiotic resistance of S. Typhimurium is common, and the antibiotic resistance of the pathogen from pork is more serious. Antibiotics should be used rationally in animal husbandry. It is necessary to further strengthen the S. Typhimurium surveillance, especially in pork food, for the prevention of resistant pathogens. -
图 1 鸡源和猪源鼠伤寒沙门菌系统发育树
注：QRDR. 喹诺酮耐药决定区，CRO. 头孢曲松，FOX. 头孢西丁，XNL. 头孢噻呋，AMP. 氨苄西林，AUG-2. 阿莫西林/克拉维酸，CIP. 环丙沙星，NAL. 萘啶酸，AZI. 阿奇霉素，TET. 四环素，CHL. 氯霉素，SXT. 甲氧苄氨嘧啶/磺胺甲恶唑，FIS. 磺胺异恶唑，GEN. 庆大霉素，STR. 链霉素，ST. 序列类型
Figure 1. The phylogenetic tree of S. Typhimurium strains of chicken and pork
表 1 鸡源和猪源鼠伤寒沙门菌抗生素耐药率
Table 1. Antibiotic resistance rates of S. Typhimurium strains of chicken and pork
抗生素 鸡源（n=15） 猪源（n=21） P值 耐药
株数耐药率
（%）耐药
株数耐药率
（%）β-内酰胺类 头孢曲松 1 6.67 3 14.29 0.858 头孢西丁 0 0.00 0 0.00 – 头孢噻呋 1 6.67 3 14.29 0.858 氨苄青霉素 10 66.67 18 85.71 0.343 阿莫西林/克拉维酸 1 6.67 0 0.00 0.864 喹诺酮类 环丙沙星 0 0.00 0 0.00 – 萘啶酸 11 73.33 14 66.67 0.951 大环内酯类 阿奇霉素 0 0.00 2 9.52 0.623 四环素类 四环素 9 60.00 21 100.00 0.007 氯霉素类 氯霉素 9 60.00 14 66.67 0.953 磺胺类 甲氧苄氨嘧啶/磺胺
甲恶唑7 46.67 12 57.14 0.778 磺胺异恶唑 10 66.67 20 95.24 0.070 氨基糖苷类 庆大霉素 7 46.67 9 42.86 1.000 链霉素 4 26.67 13 61.90 0.080 注：–. 不符合检验条件，未统计检验 
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表 2 鸡源和猪源鼠伤寒沙门菌耐药谱
Table 2. Antibiotic resistance patterns of S. Typhimurium strains of chicken and pork
来源 耐药种类 耐药谱 菌株数 耐药率(%) 合计a 鸡源 13 （86.67） 1 3 （20.00） TET 2 13.33 NAL 1 6.67 5 4 （26.67） （CRO-XNL-AMP）-NAL-CHL-FIS-GEN 1 6.67 （AMP-AUG-2）-NAL-CHL-（SXT-FIS）-GEN 1 6.67 AMP-NAL-CHL-（SXT-FIS）-GEN 1 6.67 AMP-NAL-TET-FIS-STR 1 6.67 6 6 （40.00） AMP-NAL-TET-CHL-（SXT-FIS）-GEN 3 20.00 AMP-NAL-TET-CHL-（SXT-FIS）-STR 2 13.33 AMP-NAL-TET-CHL-FIS-（GEN-STR） 1 6.67 猪源 21 （100.00） 3 TET-FIS-STR 1 4.76 1 （4.76） 4 8 （38.10） AMP-TET-FIS-STR 3 14.29 （CRO-XNL-AMP）-TET-FIS-STR 2 9.52 （CRO-XNL-AMP）-TET-CHL-（SXT-FIS） 1 4.76 NAL-AZI-TET-CHL 1 4.76 AMP-NAL-TET-FIS 1 4.76 （CRO-XNL-AMP）-TET-CHL-（SXT-FIS） 1 4.76 5 NAL-TET-CHL-（SXT-FIS）-（GEN-STR） 1 4.76 1 （4.76） 6 10 （47.62） AMP-NAL-TET-CHL-（SXT-FIS）-GEN 5 23.81 AMP-NAL-TET-CHL-（SXT-FIS）-（GEN-STR） 3 14.29 AMP-NAL-TET-CHL-（SXT-FIS）-STR 2 9.52 7 AMP-NAL-AZI-TET-CHL-FIS-STR 1 4.76 1 （4.76） 注：CRO. 头孢曲松；XNL. 头孢噻呋；AMP. 氨苄西林；AUG-2. 阿莫西林/克拉维酸；TET. 四环素；GEN. 庆大霉素；STR. 链霉素；CHL. 氯霉素；NAL. 萘啶酸；SXT. 甲氧苄氨嘧啶/磺胺甲恶唑；FIS. 磺胺异恶唑；AZI. 阿奇霉素
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表 3 鸡源和猪源鼠伤寒沙门菌序列类型
Table 3. Sequence types of S. Typhimurium strains of chicken and pork
序列类型 鸡源（n=15） 猪源（n=21） 株数 率（%） 株数 率（%） ST19 3 20.00 1 4.76 ST34 9 60.00 20 95.24 ST36 1 6.67 0 0.00 ST99 2 13.33 0 0.00 注：ST. 序列类型
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表 4 鸡源和猪源鼠伤寒沙门菌耐药基因及点突变
Table 4. Antibiotic resistance genes and point mutations of S. Typhimurium strains of chicken and pork
耐药基因/点突变 鸡源（n=15） 猪源（n=21） P值 株数 率（%） 株数 率（%） 超广谱β-内酰胺类 blaCTX-M-27 1 6.67 0 0.00 0.864 blaCTX-M-55 0 0.00 2 9.52 0.623 blaOXA-1 4 26.67 6 28.57 1.000 blaTEM-1 2 13.33 10 47.62 0.073 喹诺酮类 aac（6'）-Ib-cr 4 26.67 6 28.57 1.000 oqxA 4 26.67 9 42.86 0.519 oqxB 4 26.67 9 42.86 0.519 qnrS1 0 0.00 2 9.52 0.623 qnrS2 0 0.00 1 4.76 1.000 四环素类 tet（B） 4 26.67 17 80.95 0.004 tet（M） 0 0.00 3 14.29 0.359 氯霉素类 catB3 4 26.67 6 28.57 1.000 cmlA1 5 33.33 11 52.38 0.427 floR 5 33.33 10 47.62 0.607 磺胺类 dfrA12 5 33.33 10 47.62 0.607 sul1 5 33.33 8 38.10 1.000 sul2 6 40.00 16 76.19 0.064 sul3 5 33.33 9 42.86 0.817 氨基糖苷类耐药基因 aac（2'）-IIa 2 13.33 0 0.00 0.325 aac（3）-IId 1 6.67 0 0.00 0.864 aac（3）-IV 5 33.33 9 42.86 0.817 aac（6'）-Iaa 15 100.00 21 100.00 – aac（6'）-Ib7 1 6.67 0 0.00 0.864 aadA2 0 0.00 3 14.29 0.359 ant（3''）-IIa 5 33.33 11 52.38 0.427 aph（3'）-Ia 5 33.33 8 38.10 1.000 aph（3''）-Ib 2 13.33 11 52.38 0.040 aph（4）-Ia 5 33.33 9 42.86 0.817 aph（6）-Id 2 13.33 11 52.38 0.040 QRDR点突变 gyrA S83F 1 6.67 1 4.76 1.000 gyrA D87N 6 40.00 5 23.81 0.501 gyrA D87Y 1 6.67 7 33.33 0.136 注：–. 不符合检验条件，未统计检验，QRDR. 喹诺酮耐药决定区
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表 5 鸡源和猪源鼠伤寒沙门菌抗生素耐药与耐药基因携带关系
Table 5. Relationship of antimicrobial susceptibility with antimicrobial resistance genes of S. Typhimurium strains of chicken and pork
抗生素 表型耐药数
(株)表型不耐药数
(株)基因
阳性基因
阴性基因
阳性基因
阴性β-内酰胺类 头孢曲松 3 1 19 13 头孢西丁 0 0 22 14 头孢噻呋 3 1 19 13 氨苄青霉素 18 10 4 4 阿莫西林/克拉维酸 0 1 22 13 喹诺酮类 环丙沙星 0 0 17 19 萘啶酸 13 12 4 7 大环内酯类 阿奇霉素 0 2 0 34 四环素类 四环素 18 12 3 3 氯霉素类 氯霉素 13 10 5 8 磺胺类 甲氧苄氨嘧啶/磺胺甲恶唑 19 0 17 0 磺胺异恶唑 30 0 6 0 氨基糖苷类 庆大霉素 9 7 15 5 链霉素 14 3 10 9
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