Analysis of virulence and drug resistance of pan drug resistant Klebsiella pneumoniae
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摘要:
目的 明确2株不同来源泛耐药肺炎克雷伯菌耐药基因、毒力因子及同源性。 方法 2株泛耐药菌均于2019年1月从2位绍兴市人民医院住院患者的尿液中分离得到，采用微量肉汤稀释法对菌株进行最低抑菌浓度(MIC)检测，EDTA和PBA协同试验筛查碳青霉烯酶表型。通过三代测序平台进行基因组测序并对耐药基因和毒力因子筛选，对测序结果进行拼接、组装以及注释等。多位点序列分型(MLST)技术对菌株进行基因分型。 结果 除了替加环素和多黏菌素，2株菌对包括碳青霉烯类在内的大部分抗菌药物均耐药，EDTA和PBA协同试验提示可能携带碳青霉烯酶。2株菌均携带有1个染色体及2个质粒，染色体基因相差1 192 bp，质粒基因数量完全相同。检测到SHV-28、DHA-18、NMC、SPM、AIM等β内酰胺类耐药基因，AcrA-AcrB-TolC等外排系统及OmpK35和OmpK36外膜蛋白缺失，毒力因子包括铁摄取系统、菌毛、荚膜、内毒素、二元调控系统等。MLST分型均为ST15。 结论 β内酰胺类耐药基因合并外膜蛋白丢失是细菌对碳青霉烯类耐药的主要原因，首次在本地区肺炎克雷伯菌中检测到NMC、SPM、AIM β内酰胺类耐药基因。院内可能存在泛耐药肺炎克雷伯菌克隆传播。 Abstract:Objective To identify resistance genes, virulence factors and homology of two strains of pan drug resistant Klebsiella pneumoniae with different origins. Methods Two strains of pan drug resistant bacteria were isolated from the urine of two inpatients in early 2019. The minimal inhibitory concentration of the strain was determined by broth dilution, and the carbapenemase phenotype was screened by EDTA and PBA. Using the third-generation sequencing platform for genome sequencing and screening of drug-resistant genes and virulence factors, the sequencing Results were spliced, assembled and annotated. Multilocus sequence typing (MLST) was conducted for genotyping of strains. Results The two strains were resistant to most antibiotics including carbapenems except tigecycline and polymyxin. The synergistic test of EDTA and PBA suggested that carbapenemase might be carried. The two strains all carried one chromosome and two plasmids. The difference of chromosome genes was 1 192 bp, and the number of plasmids was the same. β-lactam resistance genes such as SHV-28, DHA-18, NMC, SPM and AIM, efflux system such as AcrA-AcrB-TolC, and outer membrane protein deletions of OmpK35 and OmpK36 were detected. The virulence factors included iron uptake system, pili, capsule, endotoxin and binary regulatory system. All MLST types were ST15. Conclusion β-lactam resistance genes combined with the loss of outer membrane proteins are the main reasons for the resistance of bacteria to carbapenems. NMC, SPM and AIM β-lactam resistance genes are detected in Klebsiella pneumoniae for the first time. There may be clonal transmission of pan drug resistant Klebsiella pneumoniae. -
Key words:
- Klebsiella pneumoniae /
- Carbapenems /
- Virulence /
- Whole genome sequencing /
- Multilocus sequence typing
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图 3 肺炎克雷伯菌SX306基因组圈图
注：图中共有6圈，从内往外第1个圈展示了GC skew，绿色为GC skew正值部分，橘色为GC skew负值部分；第2个圈展示了GC含量；第3个圈展示了rRNA及tRNA注释信息，其中紫色为rRNA信息，红色为tRNA信息；第4圈和第5圈展示CDS注释信息，颜色不同代表该CDS的COG注释分类不同，其中第4圈为CDS处于负链，第5圈为CDS处于正链；最外圈则展示了总基因组大小。
图 4 肺炎克雷伯菌SX306 COG注释分类图
注：A为RNA加工与修饰；B为染色质结构与动力学；C为能源生产和转换；D为细胞周期控制，细胞分裂，染色体分裂；E为氨基酸转运与代谢；F为核苷酸转运与代谢；G为碳水化合物运输和代谢；H为辅酶转运与代谢；I为脂质转运与代谢；J为翻译、核糖体结构与起源；K为转录；L为复制、重组和修复；M为细胞壁/膜/包膜起源；N为细胞运动；O为翻译后修饰、蛋白质转换、监督；P为无机离子转运与代谢；Q为次生代谢产物的生物合成、运输和分解代谢；R为一般功能预测；S为功能未知；T为信号转导机制；U为细胞内运输、分泌和囊泡转运；V为防御机制。
表 1 肺炎克雷伯菌药敏结果
抗菌药物 SX306 SX308 折点(μg/mL) MIC(μg/mL) 结果 MIC(μg/mL) 结果 磷霉素 128.00 中介 125.00 耐药 ≤64.00；≥256.00 氨曲南 64.00 耐药 64.00 耐药 ≤4.00；≥16.00 头孢他啶 256.00 耐药 16.00 耐药 ≤4.00；≥16.00 头孢哌酮舒巴坦 256.00 耐药 64.00 耐药 ≤16.00；≥64.00 哌拉西林他唑巴坦 256.00 耐药 32.00 中介 ≤16.00；≥128.00 左氧氟沙星 32.00 耐药 32.00 耐药 ≤2.00；≥8.00 阿米卡星 128.00 耐药 128.00 耐药 ≤16.00；≥64.00 米诺环素 32.00 耐药 2.00 敏感 ≤4.00；≥16.00 亚胺培南 32.00 耐药 4.00 耐药 ≤1.00；≥4.00 美罗培南 32.00 耐药 4.00 耐药 ≤1.00；≥4.00 替加环素 0.50 敏感 0.25 敏感 ≤1.00； > 2.00 多黏菌素 0.50 敏感 0.50 敏感 ≤2.00； > 2.00 
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表 2 SX306基因组大小和GC含量统计(bp)
片段名称 A T C G N 基因组大小 GC含量 CP040657 1 143 026 1 149 081 1 539 703 1 537 863 0 5 369 673 0.573 138 CP040658 32 788 33 299 37 572 36 075 0 139 734 0.527 051 CP040659 20 243 21 310 23 691 21 792 0 87 036 0.522 577 总数 11 969 057 1 203 690 1 600 966 1 595 730 0 5 596 443 0.571 201
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表 3 SX308基因组大小和GC含量统计(bp)
片段名称 A T C G N 基因组大小 GC含量 CP040660 1 143 212 1 149 317 1 540 048 1 538 288 0 5 370 865 0.573 155 CP040661 32 788 33 299 37 572 36 075 0 139 734 0.527 051 CP040662 20 243 21 310 23 691 21 792 0 87 036 0.522 577 总数 1 196 243 1 203 926 1 601 311 1 596 155 0 5 597 635 0.571 217
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表 4 肺炎克雷伯菌耐药基因类别及基因名称
耐药基因类别 耐药基因名称 β内酰胺类耐药基因 SHV-28、DHA-18、NMC、SPM、AIM 喹诺酮类耐药基因 gyrB、parC、parE 磷霉素类耐药基因 GlpT、UhpT、MurA 氨基糖苷类耐药基因 AAC(6')-Ib 四环素类耐药基因 RpsJ 甲氧苄氨嘧啶类耐药基因 DfrA3 氯霉素类耐药基因 CfrA、CatⅢ、Cmlv 硝基呋喃类耐药基因 NfsA 外膜蛋白丢失 OmpK35、OmpK36、LamB 外排系统 AcrA-AcrB-TolC、OqxA-OqxB、AcrR、RamA 二元调控系统 PhoPQ、BasSR、mgrB
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表 5 肺炎克雷伯菌毒力因子功能及名称
功能 毒力因子(基因)名称 功能 毒力因子(基因)名称 铁摄取系统(生长代谢) 血红素摄取蛋白 内毒素(损伤宿主细胞) 脂多糖合成蛋白 血红素转运蛋白 脂多糖装配蛋白 铁蛋白转运蛋白 胞外多糖蛋白 铁蛋白受体 脂多糖修饰酶 铁载体合成酶 脂寡糖 铁载体-分枝杆菌素、弧菌素、耶尔森菌素 O抗原 菌毛(黏附) Ⅰ型菌毛蛋白 酶类(抗吞噬) 藻酸盐合成酶 Ⅲ型菌毛蛋白 腺苷酸环化酶 Ⅳ型菌毛蛋白 谷氨酸合成酶 菌毛黏附素 脂肪酸合成酶 菌毛黏附相关蛋白Lap 尿素酶 荚膜(黏附及抗吞噬) 荚膜多糖 外膜孔蛋白(侵袭及抗吞噬) OmpA、OmpD、Omp89、HasF 分泌系统(侵袭) Ⅱ型：GspA 外排系统(抵抗宿主免疫) AdeFGH、FarAB、MtrCDE Ⅲ型：HopAN、Mlr、Mll、Rsp、RhaS、VscN 二元调控系统(抵抗宿主免疫) GacS/GacA、PmrA/PmrB、 Ⅳ：CbuG、CbuK、CoxH2/Riml、Dot/Icm、VirB/VirD PhoP/PhoQ、BvgA、BfmS Ⅵ：SCI-Ⅰ、BscS
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